EUROPEAN PHARMACOPOEIA 8.

0 Abacavir sulfate

04/2013:2589 Time Mobile phase A Mobile phase B

(min) (per cent V/V) (per cent V/V)

ABACAVIR SULFATE 0 - 25 100 0

25 - 27 100 → 0 0 → 100

Abacaviri sulfas 27 - 37 0 100

Flow rate : 1.0 mL/min.

Detection : spectrophotometer at 286 nm.
Injection : 20 μL.
Identification of impurities : use the chromatogram supplied
with abacavir for system suitability CRS and the chromatogram
obtained with reference solution (a) to identify the peaks due
to impurities A and D.
Relative retention with reference to abacavir (retention
C28H38N12O6S Mr 671 time = about 17 min) : impurity D = about 0.8 ;
[188062-50-2] impurity A = about 0.9.
System suitability : reference solution (a) :
DEFINITION
– resolution : minimum 1.5 between the peaks due to
Bis[[(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9- impurities D and A ; minimum 1.5 between the peaks due
yl]cyclopent-2-enyl]methanol] sulfate. to impurity A and abacavir.
Content : 99.0 per cent to 101.0 per cent (anhydrous substance). Limit :
CHARACTERS – impurity A : not more than 3 times the area of the principal
peak in the chromatogram obtained with reference
Appearance : white or almost white powder. solution (b) (0.3 per cent).
Solubility : soluble in water, practically insoluble in ethanol Related substances. Liquid chromatography (2.2.29). Prepare
(96 per cent) and in methylene chloride. the solutions immediately before use and transfer them to
IDENTIFICATION low-adsorption, inert glass vials.
Carry out either tests A, B, D or tests B, C, D. Test solution. Dissolve 25 mg of the substance to be examined
in water R and dilute to 100.0 mL with the same solvent.
A. Specific optical rotation (2.2.7) : − 58.0 to − 54.0, Sonicate until dissolution is complete.
determined on solution S (see Tests).
Reference solution (a). Dissolve 2.5 mg of abacavir for peak
B. Infrared absorption spectrophotometry (2.2.24). identification CRS (containing impurities B and D) in 10.0 mL
Comparison : abacavir sulfate CRS. of water R.
C. Enantiomeric purity (see Tests). Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with water R. Dilute 1.0 mL of this solution to
D. Solution S gives reaction (a) of sulfates (2.3.1).
10.0 mL with water R.
TESTS Column :
Solution S. Dissolve 0.250 g in water R and dilute to 25.0 mL – size : l = 0.15 m, Ø = 3.9 mm ;
with the same solvent. – stationary phase : end-capped octadecylsilyl silica gel for
Enantiomeric purity. Liquid chromatography (2.2.29). chromatography R (5 μm) ;
Solution A. Mix 0.5 mL of trifluoroacetic acid R and 100 mL – temperature : 30 °C.
of methanol R. Mobile phase :
Solution B. Mix 30 volumes of methanol R, 30 volumes of – mobile phase A : dilute 0.5 mL of trifluoroacetic acid R in
2-propanol R and 40 volumes of heptane R. 1000 mL of water R ;
Test solution. Dissolve 40 mg of the substance to be examined – mobile phase B : water R, methanol R (15:85 V/V) ;
in 30 mL of solution A. Sonicate until dissolution is complete. Time Mobile phase A Mobile phase B
Add 30 mL of 2-propanol R and dilute to 100.0 mL with (min) (per cent V/V) (per cent V/V)
heptane R. 0-5 95 5
Reference solution (a). Dissolve 2 mg of abacavir for system
5 - 25 95 → 70 5 → 30
suitability CRS (containing impurities A and D) in 1.5 mL of
solution A. Sonicate until dissolution is complete. Add 1.5 mL 25 - 40 70 → 10 30 → 90
of 2-propanol R and dilute to 5.0 mL with heptane R.
Reference solution (b). Dilute 1.0 mL of the test solution to Flow rate : 1.0 mL/min.
100.0 mL with solution B. Dilute 1.0 mL of this solution to Detection : spectrophotometer at 254 nm.
10.0 mL with solution B. Injection : 20 μL.
Column : Identification of impurities : use the chromatogram
– size : l = 0.25 m, Ø = 4.6 mm ; supplied with abacavir for peak identification CRS and the
chromatogram obtained with reference solution (a) to identify
– stationary phase : amylose derivative of silica gel for chiral
the peaks due to impurities B and D.
separation R (10 μm) ;
Relative retention with reference to abacavir (retention
– temperature : 30 °C. time = about 22 min) : impurity D = about 1.04 ;
Mobile phase : impurity B = about 1.3.
– mobile phase A : diethylamine R, 2-propanol R, heptane R System suitability : reference solution (a) :
(0.1:15:85 V/V/V) ; – resolution : minimum 1.5 between the peaks due to abacavir
– mobile phase B : heptane R, 2-propanol R (50:50 V/V) ; and impurity D.

General Notices (1) apply to all monographs and other texts 1459
Acacia, spray-dried EUROPEAN PHARMACOPOEIA 8.0

Limits :
– impurity B : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (b) (0.2 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.10 per cent) ; C. [(1S,4R)-4-(2,6-diamino-9H-purin-9-yl)cyclopent-2-
enyl]methanol,
– total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.5 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
12 mL of solution S complies with test A. Prepare the reference
solution using 2 mL of lead standard solution (1 ppm Pb) R. D. [(1R,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-
Water (2.5.32) : maximum 0.5 per cent, determined on yl]cyclopent-2-enyl]methanol,
60.0 mg.
Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
1.0 g.

ASSAY
Dissolve 0.300 g in 50 mL of water R. Titrate with 0.1 M sodium
hydroxide, determining the end-point potentiometrically
(2.2.20).
E. [(1R,3S)-3-[2-amino-6-(cyclopropylamino)-9H-purin-9-
1 mL of 0.1 M sodium hydroxide is equivalent to 33.54 mg yl]cyclopentyl]methanol,
of C28H38N12O6S.

IMPURITIES
Specified impurities : A, B.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities F. 6-(cyclopropylamino)-9-[(1R,4S)-4-[[(1,1-
for demonstration of compliance. See also 5.10. Control of dimethylethyl)oxy]methyl]cyclopent-2-enyl]-9H-
impurities in substances for pharmaceutical use) : C, D, E, F. purine-2-amine.

01/2009:0308
corrected 6.8

ACACIA, SPRAY-DRIED
Acaciae gummi dispersione desiccatum
DEFINITION
A. [(1R,4S)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9- Spray-dried acacia is obtained from a solution of acacia.
yl]cyclopent-2-enyl]methanol,
CHARACTERS
It dissolves completely and rapidly, after about 20 min, in
twice its mass of water. The liquid obtained is colourless or
yellowish, dense, viscous, adhesive, translucent and weakly
acid to blue litmus paper. Spray-dried acacia is practically
insoluble in ethanol (96 per cent).
IDENTIFICATION
A. Examined under a microscope, in ethanol (96 per cent) R,
the powder is seen to consist predominantly of spheroidal
particles about 4-40 μm in diameter, with a central cavity
containing 1 or several air-bubbles ; a few minute flat
fragments are present. Only traces of starch granules are
B. 6-(cyclopropylamino)-9-[(1R,4S)-4-[[(2,5-diamino-6- visible. No vegetable tissue is seen.
chloropyrimidin-4-yl)oxy]methyl]cyclopent-2-enyl]-9H- B. Examine the chromatograms obtained in the test for
purine-2-amine, glucose and fructose.

1460 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Acamprosate calcium

Results : the chromatogram obtained with the test solution to the zone of xylose in the chromatogram obtained with the
shows 3 zones due to galactose, arabinose and rhamnose. reference solution.
No other important zones are visible, particularly in the Loss on drying (2.2.32) : maximum 10.0 per cent, determined
upper part of the chromatogram. on 1.000 g by drying in an oven at 105 °C.
C. Dissolve 1 g of the drug to be examined in 2 mL of water R Total ash (2.4.16). : maximum 4.0 per cent.
by stirring frequently for 20 min. Add 2 mL of ethanol
(96 per cent) R. After shaking a white gelatinous mucilage is Microbial contamination
formed which becomes fluid on adding 10 mL of water R. TAMC : acceptance criterion 104 CFU/g (2.6.12).
TYMC : acceptance criterion 102 CFU/g (2.6.12).
TESTS
Absence of Escherichia coli (2.6.13).
Solution S. Dissolve 3.0 g of the drug to be examined in 25 mL
of water R by stirring for 10 min. Allow to stand for 20 min Absence of Salmonella (2.6.13).
and dilute to 30 mL with water R. FUNCTIONALITY-RELATED CHARACTERISTICS
Glucose and fructose. Thin-layer chromatography (2.2.27). This section provides information on characteristics that are
Test solution. To 0.100 g in a thick-walled centrifuge tube recognised as being relevant control parameters for one or
add 2 mL of a 100 g/L solution of trifluoroacetic acid R, shake more functions of the substance when used as an excipient (see
vigorously to dissolve the forming gel, stopper the tube and chapter 5.15). This section is a non-mandatory part of the
heat the mixture at 120 °C for 1 h. Centrifuge the hydrolysate, monograph and it is not necessary to verify the characteristics
transfer the clear supernatant carefully into a 50 mL flask, add to demonstrate compliance. Control of these characteristics can
10 mL of water R and evaporate to dryness under reduced however contribute to the quality of a medicinal product by
pressure. To the resulting clear film add 0.1 mL of water R and improving the consistency of the manufacturing process and
0.9 mL of methanol R. Centrifuge to separate the amorphous the performance of the medicinal product during use. Where
precipitate. Dilute the supernatant, if necessary, to 1 mL with control methods are cited, they are recognised as being suitable
methanol R. for the purpose, but other methods can also be used. Wherever
Reference solution. Dissolve 10 mg of arabinose R, 10 mg of results for a particular characteristic are reported, the control
galactose R, 10 mg of glucose R, 10 mg of rhamnose R and method must be indicated.
10 mg of xylose R in 1 mL of water R and dilute to 10 mL The following characteristic may be relevant for spray-dried
with methanol R. acacia used as a viscosity-increasing agent and/or suspending
Plate : TLC silica gel plate R. agent in aqueous preparations.
Mobile phase : 16 g/L solution of sodium dihydrogen Apparent viscosity. Determine the dynamic viscosity using a
phosphate R, butanol R, acetone R (10:40:50 V/V/V). capillary viscometer (2.2.9) or a rotating viscometer (2.2.10)
on a 100 g/L solution of spray-dried acacia (dried substance).
Application : 10 μL as bands.
Development A : over a path of 10 cm.
Drying A : in a current of warm air for a few minutes. 01/2008:1585
corrected 6.0
Development B : over a path of 15 cm using the same mobile
phase.
Drying B : at 110 °C for 10 min.
ACAMPROSATE CALCIUM
Detection : spray with anisaldehyde solution R and heat at
110 °C for 10 min.
Acamprosatum calcicum
Results : the chromatogram obtained with the reference
solution shows 5 clearly separated coloured zones due to
galactose (greyish-green or green), glucose (grey), arabinose
(yellowish-green), xylose (greenish-grey or yellowish-grey)
and rhamnose (yellowish-green), in order of increasing
RF value. The chromatogram obtained with the test solution C10H20CaN2O8S2 Mr 400.5
shows no grey zone and no greyish-green zone between [77337-73-6]
the zones corresponding to galactose and arabinose in the
DEFINITION
chromatogram obtained with the reference solution.
Calcium bis[3-(acetylamino)propane-1-sulfonate].
Starch, dextrin and agar. To 10 mL of solution S previously
boiled and cooled add 0.1 mL of 0.05 M iodine. No blue or Content : 98.0 per cent to 102.0 per cent (dried substance).
reddish-brown colour develops. CHARACTERS
Sterculia gum Appearance : white or almost white powder.
A. Place 0.2 g in a 10 mL ground-glass-stoppered cylinder Solubility : freely soluble in water, practically insoluble in
graduated in 0.1 mL. Add 10 mL of ethanol (60 per ethanol (96 per cent) and in methylene chloride.
cent V/V) R and shake. Any gel formed occupies not more
than 1.5 mL. IDENTIFICATION
B. To 1.0 g add 100 mL of water R and shake. Add 0.1 mL A. Infrared absorption spectrophotometry (2.2.24).
of methyl red solution R. Not more than 5.0 mL of Comparison: Ph. Eur. reference spectrum of acamprosate
0.01 M sodium hydroxide is required to change the colour calcium.
of the indicator. B. It gives reaction (a) of calcium (2.3.1).
Tannins. To 10 mL of solution S add 0.1 mL of ferric chloride
solution R1. A gelatinous precipitate is formed, but neither the TESTS
precipitate nor the liquid shows a dark blue colour. Solution S. Dissolve 5.0 g in carbon dioxide-free water R and
Tragacanth. Examine the chromatograms obtained in the test dilute to 100 mL with the same solvent.
for Glucose and fructose. Appearance of solution. Solution S is clear (2.2.1) and
Results : the chromatogram obtained with the test solution colourless (2.2.2, Method II).
shows no greenish-grey or yellowish-grey zone corresponding pH (2.2.3) : 5.5 to 7.0 for solution S.

General Notices (1) apply to all monographs and other texts 1461
Acarbose EUROPEAN PHARMACOPOEIA 8.0

Impurity A. Liquid chromatography (2.2.29). 01/2008:2089

Test solution. Dissolve 0.40 g of the substance to be examined
in distilled water R and dilute to 20.0 mL with the same ACARBOSE
solvent. Dilute 10.0 mL of this solution to 100.0 mL with
borate buffer solution pH 10.4 R. Place 3.0 mL of the solution Acarbosum
obtained in a 25 mL ground-glass-stoppered tube. Add
0.15 mL of a freshly prepared 5 g/L solution of fluorescamine R
in acetonitrile R. Shake immediately and vigorously for 30 s.
Place in a water-bath at 50 °C for 30 min. Cool under a stream
of cold water. Centrifuge and filter the supernatant through a
suitable membrane filter (nominal pore size 0.45 μm), 25 mm
in diameter.
Reference solution. Dissolve 50 mg of acamprosate C25H43NO18 Mr 646
impurity A CRS in distilled water R and dilute to 200.0 mL with [56180-94-0]
the same solvent. Dilute 0.4 mL of the solution to 100.0 mL DEFINITION
with borate buffer solution pH 10.4 R. Treat 3.0 mL of this
solution in the same way as the test solution O-4,6-Dideoxy-4-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-
(hydroxymethyl)cyclohex-2-enyl]amino]-α-D-glucopyranosyl-
Column : (1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranose, which
– size : l = 0.15 m, Ø = 4.6 mm ; is produced by certain strains of Actinoplanes utahensis.
– stationary phase : spherical octadecylsilyl silica gel for Content : 95.0 per cent to 102.0 per cent (anhydrous substance).
chromatography R (5 μm) with a specific surface area of
CHARACTERS
170 m2/g and a pore size of 12 nm.
Appearance : white or yellowish, amorphous powder,
Mobile phase : acetonitrile R, methanol R, 0.1 M phosphate hygroscopic.
buffer solution pH 6.5 R (10:10:80 V/V/V).
Solubility : very soluble in water, soluble in methanol,
Flow rate : 1 mL/min. practically insoluble in methylene chloride.
Detection : spectrophotometer at 261 nm.
IDENTIFICATION
Injection : 20 μL. A. Infrared absorption spectrophotometry (2.2.24).
Run time : 6 times the retention time of impurity A Comparison: acarbose for identification CRS.
Retention times : fluorescamine = about 4 min ; B. Examine the chromatograms obtained in the assay.
impurity A = about 8 min ; acamprosate is not detected by Results : the principal peak in the chromatogram obtained
this system. with the test solution is similar in retention time and size
Limits : to the principal peak in the chromatogram obtained with
– impurity A : not more than the area of the corresponding reference solution (a).
peak in the chromatogram obtained with the reference TESTS
solution (0.05 per cent).
Solution S. Dissolve 1.00 g in carbon dioxide-free water R and
Heavy metals (2.4.8) : maximum 10 ppm. dilute to 20.0 mL with the same solvent.
Dissolve 2.0 g in distilled water R and dilute to 20 mL with pH (2.2.3) : 5.5 to 7.5 for solution S.
the same solvent. 12 mL of the solution complies with test A.
Prepare the reference solution using 10 mL of lead standard Specific optical rotation (2.2.7) : + 168 to + 183 (anhydrous
solution (1 ppm Pb) R. substance).
Dilute 2.0 mL of solution S to 10.0 mL with water R.
Loss on drying (2.2.32) : maximum 0.4 per cent, determined
on 1.000 g by drying in an oven at 105 °C. Absorbance (2.2.25): maximum 0.15 at 425 nm for solution S.
Related substances. Liquid chromatography (2.2.29).
ASSAY Test solution. Dissolve 0.200 g of the substance to be examined
To 4 g of cation-exchange resin R (75-150 μm) add 20 mL of in water R and dilute to 10.0 mL with the same solvent.
distilled water R and stir magnetically for 10 min. Introduce Reference solution (a). Dissolve the contents of a vial of
this suspension into a glass column, 45 cm long and 2.2 cm acarbose CRS in 5.0 mL of water R.
in internal diameter, equipped with a polytetrafluoroethylene Reference solution (b). Dissolve 20 mg of acarbose for peak
flow cap covered by a glass-wool plug. Allow a few millilitres identification CRS (acarbose containing impurities A, B, C, D,
of this solution to flow, then place a plug of glass wool over the E, F, G and H) in 1 mL of water R.
resin. Pass 50 mL of 1 M hydrochloric acid through the column.
The pH of the eluate is close to 1. Wash with 3 quantities, each Reference solution (c). Dilute 1.0 mL of the test solution to
of 200 mL, of distilled water R to obtain an eluate at pH 6. 100.0 mL with water R.
Dissolve 0.100 g of the substance to be examined in 15 mL Column :
of distilled water R. Pass through the column and wash with – size : l = 0.25 m, Ø = 4 mm,
3 quantities, each of 25 mL, of distilled water R, collecting – stationary phase : aminopropylsilyl silica gel for
the eluate. Allow to elute until an eluate at pH 6 is obtained. chromatography R (5 μm),
Titrate the solution obtained with 0.1 M sodium hydroxide, – temperature : 35 °C.
determining the end-point potentiometrically (2.2.20).
Mobile phase : mix 750 volumes of acetonitrile R1 and
1 mL of 0.1 M sodium hydroxide corresponds to 20.02 mg of 250 volumes of a solution containing 0.60 g/L of potassium
C10H20CaN2O8S2. dihydrogen phosphate R and 0.35 g/L of disodium hydrogen
phosphate dihydrate R.
IMPURITIES
Flow rate : 2.0 mL/min.
Detection : spectrophotometer at 210 nm.
Injection : 10 μL of the test solution and reference solutions (b)
A. 3-aminopropane-1-sulfonic acid (homotaurine). and (c).

1462 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Acarbose

Run time : 2.5 times the retention time of acarbose. Injection : test solution and reference solution (a).
Identification of impurities : use the chromatogram Calculate the percentage content of C25H43NO18 from the areas
supplied with acarbose for peak identification CRS and the of the peaks and the declared content of acarbose CRS.
chromatogram obtained with reference solution (b) to identify
the peaks due to impurities A, B, C, D, E, F, G and H. STORAGE
Relative retention with reference to acarbose (retention In an airtight container.
time = about 16 min) : impurity D = about 0.5 ;
impurity H = about 0.6 ; impurity B = about 0.8 ; IMPURITIES
impurity A = about 0.9 ; impurity C = about 1.2 ; Specified impurities : A, B, C, D, E, F, G, H.
impurity E = about 1.7 ; impurity F = about 1.9 ;
impurity G = about 2.2.
System suitability : reference solution (b) :
– peak-to-valley ratio : minimum 1.2, where Hp = height
above the baseline of the peak due to impurity A and
Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to acarbose,
– the chromatogram obtained is similar to the chromatogram
supplied with acarbose for peak identification CRS. A. O-4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-
Limits : 3-(hydroxymethyl)cyclohex-2-enyl]amino]-α-D-
glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-
– correction factors : for the calculation of contents, arabino-hex-2-ulopyranose,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity B = 0.63 ;
impurity D = 0.75 ; impurity E = 1.25 ; impurity F = 1.25 ;
impurity G = 1.25 ;
– impurity A : not more than 0.6 times the area of the
principal peak in the chromatogram obtained with
reference solution (c) (0.6 per cent) ;
– impurity B : not more than 0.5 times the area of the B. (1R,4R,5S,6R)-4,5,6-trihydroxy-2-(hydroxymethyl)cyclo-
principal peak in the chromatogram obtained with hex-2-enyl 4-O-[4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,5,6-tri-
reference solution (c) (0.5 per cent) ; hydroxy-3-(hydroxymethyl)cyclohex-2-enyl]ami-
– impurity C : not more than 1.5 times the area of the no]-α-D-glucopyranosyl]-α-D-glucopyranoside,
principal peak in the chromatogram obtained with
reference solution (c) (1.5 per cent) ;
– impurity D : not more than the area of the principal peak
in the chromatogram obtained with reference solution (c)
(1.0 per cent) ;
– impurity E : not more than 0.2 times the area of the
principal peak in the chromatogram obtained with
reference solution (c) (0.2 per cent) ;
– impurities F, G : for each impurity, not more than 0.3 times
the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.3 per cent) ;
– impurity H : not more than 0.2 times the area of the C. α-D-glucopyranosyl 4-O-[4,6-dideoxy-4-[[(1S,4R,5S,6S)-
principal peak in the chromatogram obtained with 4,5,6-trihydroxy-3-(hydroxymethyl)cyclohex-2-
reference solution (c) (0.2 per cent) ; enyl]amino]-α-D-glucopyranosyl]-α-D-glucopyranoside,
– any other impurities : for each impurity, not more than
0.2 times the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.2 per cent) ;
– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (c)
(3.0 per cent) ;
– disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (c) D. 4-O-[4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-
(0.1 per cent). 3-(hydroxymethyl)cyclohex-2-enyl]amino]-α-D-
glucopyranosyl]-D-glucopyranose,
Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 1.5 g in water R and dilute to 15 mL with the same
solvent. If the solution is not clear, carry out prefiltration and
use the filtrate. 10 mL complies with limit test E. Prepare
the reference solution using 20 mL of lead standard solution
(1 ppm Pb) R.
Water (2.5.12) : maximum 4.0 per cent, determined on 0.300 g.
Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
1.0 g. E. O-4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-
3-(hydroxymethyl)cyclohex-2-enyl]amino]-α-D-
ASSAY glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-
Liquid chromatography (2.2.29) as described in the test for α-D-glucopyranosyl-(1→4)-D-arabino-hex-2-ulopyranose
related substances with the following modification. (4-O-α-acarbosyl-D-fructopyranose),

General Notices (1) apply to all monographs and other texts 1463
Acebutolol hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Test solution. Dissolve 20.0 mg in a 0.1 per cent V/V

solution of hydrochloric acid R and dilute to 100.0 mL with
the same acid solution. Dilute 5.0 mL of this solution to
100.0 mL with a 0.1 per cent V/V solution of hydrochloric
acid R.
Spectral range : 220-350 nm.
F. O-4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,5,6-trihydroxy- Absorption maxima : at 233 nm and 322 nm.
3-(hydroxymethyl)cyclohex-2-enyl]amino]-α-D- Specific absorbance at the absorption maximum : 555 to 605
glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)- at 233 nm.
O-α-D-glucopyranosyl-(1→4)-D-glucopyranose
(4-O-α-acarbosyl-D-glucopyranose), B. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs.
Comparison: acebutolol hydrochloride CRS.
C. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 20 mg of the substance to be
examined in methanol R and dilute to 20 mL with the same
solvent.
Reference solution (a). Dissolve 20 mg of acebutolol
hydrochloride CRS in methanol R and dilute to 20 mL with
the same solvent.
Reference solution (b). Dissolve 20 mg of pindolol CRS in
methanol R and dilute to 20 mL with the same solvent. To
G. α-D-glucopyranosyl O-4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,5,6- 1 mL of this solution add 1 mL of reference solution (a).
trihydroxy-3-(hydroxymethyl)cyclohex-2-enyl]amino]-α- Plate : TLC silica gel F254 plate R.
D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-O- Mobile phase : perchloric acid R, methanol R, water R
α-D-glucopyranoside (α-D-glucopyranosyl α-acarboside), (5:395:600 V/V/V).
Application : 10 μL.
Development : over 3/4 of the plate.
Drying : in air.
Detection : examine in ultraviolet light at 254 nm.
System suitability : the chromatogram obtained with
reference solution (b) shows 2 clearly separated principal
H. O-4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,5,6-trihydroxy- spots.
3-(hydroxymethyl)cyclohex-2-enyl]amino]-α-D-
Results : the principal spot in the chromatogram obtained
glucopyranosyl-(1→4)-O-6-deoxy-α-D-glucopyranosyl-
with the test solution is similar in position and size to
(1→4)-D-glucopyranose.
the principal spot in the chromatogram obtained with
reference solution (a).
01/2008:0871 D. It gives reaction (a) of chlorides (2.3.1).
corrected 7.0
TESTS
ACEBUTOLOL HYDROCHLORIDE Appearance of solution. The solution is not more opalescent
than reference suspension II (2.2.1) and not more intensely
Acebutololi hydrochloridum coloured than reference solution BY5 (2.2.2, Method II).
Dissolve 0.5 g in water R and dilute to 10 mL with the same
solvent.
pH (2.2.3) : 5.0 to 7.0.
Dissolve 0.20 g in carbon dioxide-free water R and dilute to
20 mL with the same solvent.
Related substances. Liquid chromatography (2.2.29).
C18H29ClN2O4 Mr 372.9 Test solution. Dissolve 0.100 g of the substance to be examined
[34381-68-5] in mobile phase A and dilute to 50.0 mL with mobile phase A.
Reference solution (a). Dissolve 20.0 mg of the substance to
DEFINITION be examined in mobile phase A and dilute to 100.0 mL with
N-[3-Acetyl-4-[(2RS)-2-hydroxy-3-[(1-methylethyl)ami- mobile phase A. Dilute 0.5 mL of this solution to 50.0 mL
no]propoxy]phenyl]butanamide hydrochloride. with mobile phase A.
Content : 99.0 per cent to 101.0 per cent (dried substance). Reference solution (b). Dissolve the contents of a vial of
acebutolol impurity I CRS in 1.0 mL of mobile phase A.
CHARACTERS
Reference solution (c). Mix 2.0 mL of reference solution (a)
Appearance : white or almost white, crystalline powder. and 1.0 mL of reference solution (b) and dilute to 10.0 mL
Solubility : freely soluble in water and in ethanol (96 per cent), with mobile phase A.
very slightly soluble in acetone and in methylene chloride. Reference solution (d). Dissolve 5.0 mg of acebutolol
mp : about 143 °C. impurity C CRS in 10 mL of acetonitrile R and dilute to
25.0 mL with mobile phase A. Dilute 0.5 mL of this solution
IDENTIFICATION to 50.0 mL with mobile phase A.
First identification : B, D. Reference solution (e). Dissolve 5.0 mg of acebutolol
Second identification : A, C, D. impurity B CRS in 10.0 mL of acetonitrile R and dilute to
A. Ultraviolet and visible absorption spectrophotometry 25.0 mL with mobile phase A. Dilute 1.0 mL of this solution
(2.2.25). to 50.0 mL with mobile phase A.

1464 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Acebutolol hydrochloride

Column : IMPURITIES
– size : l = 0.125 m, Ø = 4 mm, Specified impurities : A, B, C, D, E, F, G, H, I, J, K.
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm),
– temperature : 40 °C.
Mobile phase :
– mobile phase A : mix 2.0 mL of phosphoric acid R, and
3.0 mL of triethylamine R and dilute to 1000 mL with A. N-[3-acetyl-4-[(2RS)-oxiran-2-ylmethoxy]phenyl]butana-
water R ; mide,
– mobile phase B : mix equal volumes of acetonitrile R and
mobile phase A ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-2 98 2
B. R1 = R2 = CO-CH3 : N-[3-acetyl-4-[(2RS)-2-hydroxy-
2 - 30.5 98 → 10 2 → 90
3-[(1-methylethyl)amino]propoxy]phenyl]acetamide
30.5 - 41 10 90 (diacetolol),

Flow rate : 1.2 mL/min. D. R1 = H, R2 = CO-CH3 : 1-[5-amino-2-[(2RS)-2-hydroxy-3-

[(1-methylethyl)amino]propoxy]phenyl]ethanone,
Detection : spectrophotometer at 240 nm.
E. R1 = CO-CH2-CH2-CH3, R2 = H : N-[4-[(2RS)-2-hydroxy-
Injection : 25 μL.
3-[(1-methylethyl)amino]propoxy]phenyl]butanamide,
System suitability: reference solution (c) :
J. R1 = CO-CH2-CH3, R2 = CO-CH3 : N-[3-acetyl-4-[(2RS)-
– resolution : minimum 7.0 between the peaks due to 2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenyl]-
impurity I and acebutolol. propanamide,
Limits :
K. R1 = R2 = CO-CH2-CH2-CH3 : N-[3-butano-
– impurity B : not more than the area of the principal peak yl-4-[(2RS)-2-hydroxy-3-[(1-methylethyl)amino]pro-
in the chromatogram obtained with reference solution (e) poxy]phenyl]butanamide,
(0.2 per cent) ;
– impurity C : not more than the area of the principal peak
in the chromatogram obtained with reference solution (d)
(0.1 per cent) ;
– impurity I : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.2 per cent) ; C. N-(3-acetyl-4-hydroxyphenyl)butanamide,
– any other impurity : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent) ;
– total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in F. R = OH : N-[3-acetyl-4-[(2RS)-2,3-
the chromatogram obtained with reference solution (a) dihydroxypropoxy]phenyl]butanamide,
(0.05 per cent).
I. R = NH-CH2-CH3 : N-[3-acetyl-4-[(2RS)-3-(ethylamino)-
Heavy metals (2.4.8) : maximum 20 ppm. 2-hydroxypropoxy]phenyl]butanamide,
Dissolve 0.50 g in 20.0 mL of water R. The solution complies
with test E. Prepare the reference solution by diluting 10.0 mL
of lead standard solution (1 ppm Pb) R to 20.0 mL with water R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 3 h.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
G. N,N′-[[(1-methylethyl)imino]bis[(2-hydroxypropane-1,3-
ASSAY diyl)oxy(3-acetyl-1,4-phenylene)]]dibutanamide (biamine),
Dissolve 0.300 g in 50 mL of ethanol (96 per cent) R and add
1 mL of 0.1 M hydrochloric acid. Carry out a potentiometric
titration (2.2.20), using 0.1 M sodium hydroxide. Read the
volume added between the 2 points of inflexion.
1 mL of 0.1 M sodium hydroxide is equivalent to 37.29 mg
of C18H29ClN2O4.

STORAGE H. N,N′-[(2-hydroxypropane-1,3-diyl)bis[oxy(3-acetyl-1,4-
Protected from light. phenylene)]]dibutanamide.

General Notices (1) apply to all monographs and other texts 1465
Aceclofenac EUROPEAN PHARMACOPOEIA 8.0

07/2009:1281 Reference solution (e). Mix 1.0 mL of reference solution (b)

corrected 7.7 and 1.0 mL of reference solution (d) and dilute to 100.0 mL
with the solvent mixture.
ACECLOFENAC Reference solution (f). Dissolve the contents of a vial of
diclofenac impurity A CRS (aceclofenac impurity I) in 1.0 mL
of the solvent mixture, add 1.5 mL of the solvent mixture and
Aceclofenacum mix.
Reference solution (g). Dissolve 4 mg of aceclofenac for peak
identification CRS (containing impurities B, C, D, E and G) in
2.0 mL of the solvent mixture.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : spherical end-capped octadecylsilyl silica
gel for chromatography R (5 μm) with a pore size of 10 nm
C16H13Cl2NO4 Mr 354.2
and a carbon loading of 19 per cent ;
[89796-99-6]
– temperature : 40 °C.
DEFINITION Mobile phase :
[[[2-[(2,6-Dichlorophenyl)amino]phenyl]acetyl]oxy]acetic – mobile phase A : 1.12 g/L solution of phosphoric acid R
acid. adjusted to pH 7.0 with a 42 g/L solution of sodium
Content : 99.0 per cent to 101.0 per cent (dried substance). hydroxide R ;
– mobile phase B : water R, acetonitrile R (10:90 V/V) ;
CHARACTERS
Time Mobile phase A Mobile phase B
Appearance : white or almost white, crystalline powder. (min) (per cent V/V) (per cent V/V)
Solubility : practically insoluble in water, freely soluble in 0 - 25 70 → 50 30 → 50
acetone, soluble in ethanol (96 per cent).
25 - 30 50 → 20 50 → 80
IDENTIFICATION
30 - 50 20 80
First identification : B.
Second identification : A, C. Flow rate : 1.0 mL/min.
A. Ultraviolet and visible absorption spectrophotometry Detection : spectrophotometer at 275 nm.
(2.2.25). Injection : 10 μL of the test solution and reference solutions (c),
Test solution. Dissolve 50.0 mg in methanol R and dilute (e), (f) and (g).
to 100.0 mL with the same solvent. Dilute 2.0 mL of the Identification of impurities : use the chromatogram supplied
solution to 50.0 mL with methanol R. with aceclofenac for peak identification CRS and the
Spectral range : 220-370 nm. chromatogram obtained with reference solution (g) to identify
the peaks due to impurities B, C, D, E and G.
Absorption maximum : at 275 nm.
Relative retention with reference to aceclofenac (retention
Specific absorbance at the absorption maximum : 320 to 350.time = about 11 min) : impurity A = about 0.8 ;
B. Infrared absorption spectrophotometry (2.2.24). impurity G = about 1.3 ; impurity H = about 1.5 ;
Comparison : Ph. Eur. reference spectrum of aceclofenac. impurity I = about 2.3 ; impurity D = about 3.1 ;
impurity B = about 3.2 ; impurity E = about 3.3 ;
C. Dissolve about 10 mg in 10 mL of ethanol (96 per cent) R.
impurity C = about 3.5 ; impurity F = about 3.7.
To 1 mL of the solution, add 0.2 mL of a mixture, prepared
System suitability : reference solution (c) :
immediately before use, of equal volumes of a 6 g/L solution
of potassium ferricyanide R and a 9 g/L solution of ferric – resolution : minimum 5.0 between the peaks due to
chloride R. Allow to stand protected from light for 5 min. impurity A and aceclofenac.
Add 3 mL of a 10.0 g/L solution of hydrochloric acid R. Limits :
Allow to stand protected from light for 15 min. A blue – impurity A : not more than the area of the corresponding
colour develops and a precipitate is formed. peak in the chromatogram obtained with reference
TESTS solution (c) (0.2 per cent) ;
– impurities B, C, D, E, G : for each impurity, not more than
Related substances. Liquid chromatography (2.2.29). Prepare the area of the peak due to aceclofenac in the chromatogram
the solutions immediately before use. obtained with reference solution (e) (0.2 per cent) ;
Solvent mixture : mobile phase A, mobile phase B (30:70 V/V). – impurity F : not more than the area of the corresponding
Test solution. Dissolve 50.0 mg of the substance to be peak in the chromatogram obtained with reference
examined in the solvent mixture and dilute to 25.0 mL with solution (e) (0.2 per cent) ;
the solvent mixture. – impurity H : not more than the area of the corresponding
Reference solution (a). Dissolve 21.6 mg of diclofenac peak in the chromatogram obtained with reference
sodium CRS (impurity A) in the solvent mixture and dilute to solution (e) (0.1 per cent) ;
50.0 mL with the solvent mixture. – impurity I : not more than the area of the corresponding
Reference solution (b). Dilute 2.0 mL of the test solution to peak in the chromatogram obtained with reference
10.0 mL with the solvent mixture. solution (f) (0.1 per cent) ;
Reference solution (c). Mix 1.0 mL of reference solution (a) – unspecified impurities: not more than 0.5 times the area of
and 1.0 mL of reference solution (b) and dilute to 100.0 mL the peak due to aceclofenac in the chromatogram obtained
with the solvent mixture. with reference solution (e) (0.10 per cent) ;
Reference solution (d). Dissolve 4.0 mg of aceclofenac – total : not more than 0.7 per cent ;
impurity F CRS and 2.0 mg of aceclofenac impurity H CRS in – disregard limit : 0.1 times the area of the peak due to
the solvent mixture, then dilute to 10.0 mL with the solvent aceclofenac in the chromatogram obtained with reference
mixture. solution (e) (0.02 per cent).

1466 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Acemetacin

Heavy metals (2.4.8) : maximum 10 ppm.

To 2.0 g in a silica crucible, add 2 mL of sulfuric acid R to wet
the substance. Heat progressively to ignition and continue
heating until an almost white or at most a greyish residue
is obtained. Carry out the ignition at a temperature not
exceeding 800 °C. Allow to cool. Add 3 mL of hydrochloric
acid R and 1 mL of nitric acid R. Heat and evaporate slowly
to dryness. Cool and add 1 mL of a 100 g/L solution of
E. ethyl [[[2-[(2,6-dichlorophenyl)amino]phenyl]-
hydrochloric acid R and 10.0 mL of distilled water R. Neutralise
acetyl]oxy]acetate (ethyl ester of aceclofenac),
with a 1.0 g/L solution of ammonia R using 0.1 mL of
phenolphthalein solution R as indicator. Add 2.0 mL of a
60 g/L solution of anhydrous acetic acid R and dilute to 20 mL
with distilled water R. 12 mL of the solution complies with
test A. Prepare the reference solution using lead standard
solution (1 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on F. benzyl [[[2-[(2,6-dichlorophenyl)amino]phenyl]-
1.0 g. acetyl]oxy]acetate (benzyl ester of aceclofenac),
ASSAY
Dissolve 0.300 g in 40 mL of methanol R. Titrate with
0.1 M sodium hydroxide, determining the end-point
potentiometrically (2.2.20).
1 mL of 0.1 M sodium hydroxide is equivalent to 35.42 mg
of C16H13Cl2NO4.
STORAGE
G. [[[[[2-[(2,6-dichlorophenyl)amino]phenyl]-
Protected from light.
acetyl]oxy]acetyl]oxy]acetic acid (acetic aceclofenac),
IMPURITIES
Specified impurities : A, B, C, D, E, F, G, H, I.

H. [[[[[[[2-[(2,6-dichlorophenyl)amino]phenyl]-
acetyl]oxy]acetyl]oxy]acetyl]oxy]acetic acid (diacetic
A. [2-[(2,6-dichlorophenyl)amino]phenyl]acetic acid aceclofenac),
(diclofenac),

I. 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-indol-2-one.
B. methyl [2-[(2,6-dichlorophenyl)amino]phenyl]acetate
(methyl ester of diclofenac), 04/2008:1686
corrected 7.0

ACEMETACIN
Acemetacinum

C. ethyl [2-[(2,6-dichlorophenyl)amino]phenyl]acetate (ethyl

ester of diclofenac),

C21H18ClNO6 Mr 415.8
[53164-05-9]
DEFINITION
[[[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
D. methyl [[[2-[(2,6-dichlorophenyl)amino]phenyl]- yl]acetyl]oxy]acetic acid.
acetyl]oxy]acetate (methyl ester of aceclofenac), Content : 99.0 per cent to 101.0 per cent (dried substance).

General Notices (1) apply to all monographs and other texts 1467
Acemetacin EUROPEAN PHARMACOPOEIA 8.0

CHARACTERS – use the chromatogram obtained with reference solution (b)

Appearance : yellow or greenish-yellow, crystalline powder. to identify the peak due to impurity B.
Solubility : practically insoluble in water, soluble in acetone, Relative retention with reference to acemetacin (retention
slightly soluble in anhydrous ethanol. time = about 15 min) : impurity A = about 0.7 ;
impurity B = about 0.9 ; impurity F = about 1.2 ;
It shows polymorphism (5.9). impurity C = about 1.3 ; impurity D = about 1.5 ;
impurity E = about 2.2.
IDENTIFICATION System suitability : reference solution (d) :
Infrared absorption spectrophotometry (2.2.24). – peak-to-valley ratio : minimum 15, where Hp = height above
Comparison : acemetacin CRS. the baseline of the peak due to impurity B and Hv = height
If the spectra obtained in the solid state show differences, above the baseline of the lowest point of the curve
dissolve the substance to be examined and the reference separating this peak from the peak due to acemetacin.
substance separately in acetone R, evaporate to dryness and Limits :
record new spectra using the residues. – correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
TESTS corresponding correction factor : impurity C = 1.3 ;
Related substances. Liquid chromatography (2.2.29). impurity D = 1.4 ; impurity F = 1.3 ;
Test solution. Dissolve 0.100 g of the substance to be examined – impurity E : not more than 3 times the area of the principal
in acetonitrile for chromatography R and dilute to 20.0 mL peak in the chromatogram obtained with reference
with the same solvent. solution (a) (0.3 per cent) ;
Reference solution (a). Dilute 5.0 mL of the test solution – impurity B : not more than the area of the corresponding
to 50.0 mL with acetonitrile for chromatography R. Dilute peak in the chromatogram obtained with reference
1.0 mL of this solution to 100.0 mL with acetonitrile for solution (c) (0.2 per cent) ;
chromatography R. – impurity A : not more than the area of the corresponding
Reference solution (b). Dissolve 5.0 mg of acemetacin peak in the chromatogram obtained with reference
impurity A CRS and 10.0 mg of indometacin CRS (impurity B) solution (c) (0.1 per cent) ;
in acetonitrile for chromatography R, and dilute to 50.0 mL – impurities C, D, F : for each impurity, not more than the
with the same solvent. area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent) ;
Reference solution (c). Dilute 1.0 mL of reference solution (b)
to 20.0 mL with acetonitrile for chromatography R. – unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
Reference solution (d). To 1 mL of reference solution (b), add with reference solution (a) (0.10 per cent) ;
10 mL of the test solution and dilute to 20 mL with acetonitrile
for chromatography R. – total : not more than 4 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
Reference solution (e). Dissolve the contents of a vial of (0.4 per cent) ;
acemetacin impurity mixture CRS (containing impurities C, D,
E and F) in 1.0 mL of the test solution. – disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
Column : (0.05 per cent).
– size : l = 0.25 m, Ø = 4 mm ; Heavy metals (2.4.8) : maximum 20 ppm.
– stationary phase : spherical end-capped octadecylsilyl silica Solvent mixture : methanol R, acetone R (10:90 V/V).
gel for chromatography R (5 μm) ;
0.250 g complies with test H. Prepare the reference solution
– temperature : 40 °C. using 0.5 mL of lead standard solution (10 ppm Pb) R.
Mobile phase : Loss on drying (2.2.32) : maximum 0.5 per cent, determined
– mobile phase A : dissolve 1.0 g of potassium dihydrogen on 1.000 g by drying in an oven at 105 °C.
phosphate R in 900 mL of water R, adjust to pH 6.5 with Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1 M sodium hydroxide and dilute to 1000 mL with water R ; 1.0 g.
– mobile phase B : acetonitrile for chromatography R ;
ASSAY
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
Dissolve 0.350 g in 20 mL of acetone R and add 10 mL of
0-5 95 5
water R. Titrate with 0.1 M sodium hydroxide, determining
the end-point potentiometrically (2.2.20).
5-9 95 → 65 5 → 35 1 mL of 0.1 M sodium hydroxide is equivalent to 41.58 mg
9 - 16 65 35 of C21H18ClNO6.
16 - 28 65 → 20 35 → 80 STORAGE
28 - 34 20 80 Protected from light.

Flow rate : 1.0 mL/min. IMPURITIES

Detection : spectrophotometer at 235 nm. Specified impurities : A, B, C, D, E, F.
Injection : 20 μL.
Identification of impurities :
– use the chromatogram supplied with acemetacin impurity
mixture CRS and the chromatogram obtained with
reference solution (e) to identify the peaks due to
impurities C, D, E and F ; A. 4-chlorobenzoic acid,

1468 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Acesulfame potassium

System suitability : reference solution (b) :

– the chromatogram shows 2 clearly separated zones.
Results : the principal zone in the chromatogram obtained
with the test solution is similar in position and size to
the principal zone in the chromatogram obtained with
reference solution (a).
B. R1 = R2 = R3 = H : [1-(4-chlorobenzoyl)-5-methoxy-2- C. 0.5 mL of solution S (see Tests) gives reaction (b) of
methylindol-3-yl]acetic acid (indometacin), potassium (2.3.1).
C. R1 = Cl, R2 = H, R3 = CH2-CO2H : [[[1-(3,4- TESTS
dichlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3- Solution S. Dissolve 10.0 g in carbon dioxide-free water R and
yl]acetyl]oxy]acetic acid, dilute to 50 mL with the same solvent.
D. R1 = H, R2 = C(CH3)3, R3 = CH2-CO2H : Appearance of solution. Solution S is clear (2.2.1) and
[[[1-(4-chlorobenzoyl)-6-(1,1-dimethylethyl)-5- colourless (2.2.2, Method II).
methoxy-2-methyl-1H-indol-3-yl]acetyl]oxy]acetic acid,
Acidity or alkalinity. To 20 mL of solution S add 0.1 mL
E. R1 = R2 = H, R3 = CH2-CO-O-C(CH3)3 : 1,1-dimethylethyl of bromothymol blue solution R1. Not more than 0.2 mL
[[[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3- of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is
yl]acetyl]oxy]acetate, required to change the colour of the indicator.
F. R1 = R2 = H, R3 = CH2-CO-O-CH2-CO2H : Impurity A. Thin-layer chromatography (2.2.27).
[[[[[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol- Test solution. Dissolve 0.80 g of the substance to be examined
3-yl]acetyl]oxy]acetyl]oxy]acetic acid. in water R and dilute to 10 mL with the same solvent.
Reference solution (a). Dissolve 50 mg of acetylacetamide R
01/2013:1282 (impurity A) in water R and dilute to 25 mL with the same
solvent. To 5 mL of the solution add 45 mL of water R and
ACESULFAME POTASSIUM dilute to 100 mL with methanol R.
Reference solution (b). To 10 mL of reference solution (a) add
Acesulfamum kalicum 1 mL of the test solution and dilute to 20 mL with methanol R.
Plate : TLC silica gel plate R.
Mobile phase : water R, ethanol (96 per cent) R, ethyl acetate R
(2:15:74 V/V/V).
Application : 5 μL.
Development : over 2/3 of the plate.
C4H4KNO4S Mr 201.2 Drying : in air until the solvents are completely removed.
[55589-62-3] Detection : spray with phosphoric vanillin solution R and heat
at 120 °C for about 10 min ; examine in daylight.
DEFINITION
System suitability : the chromatogram obtained with reference
Potassium 6-methyl-1,2,3-oxathiazin-4-olate 2,2-dioxide. solution (a) shows a clearly visible spot and the chromatogram
Content : 99.0 per cent to 101.0 per cent (dried substance). obtained with reference solution (b) shows 2 clearly separated
CHARACTERS spots.
Appearance : white or almost white, crystalline powder or Limit :
colourless crystals. – impurity A : any spot due to impurity A is not more intense
Solubility : soluble in water, very slightly soluble in acetone than the spot in the chromatogram obtained with reference
and in ethanol (96 per cent). solution (a) (0.125 per cent).
Impurity B. Liquid chromatography (2.2.29).
IDENTIFICATION Test solution. Dissolve 0.100 g of the substance to be examined
First identification : A, C. in water R and dilute to 10.0 mL with the same solvent.
Second identification : B, C. Reference solution (a). Dissolve 4.0 mg of acesulfame potassium
A. Infrared absorption spectrophotometry (2.2.24). impurity B CRS in water R and dilute to 100.0 mL with the
Comparison : acesulfame potassium CRS. same solvent. Dilute 1.0 mL of the solution to 200.0 mL with
B. Thin-layer chromatography (2.2.27). water R.
Test solution. Dissolve 5 mg of the substance to be examined Reference solution (b). Dissolve 0.100 g of the substance to
in water R and dilute to 5 mL with the same solvent. be examined in reference solution (a) and dilute to 10.0 mL
with the same solution.
Reference solution (a). Dissolve 5 mg of acesulfame
potassium CRS in water R and dilute to 5 mL with the same Column :
solvent. – size : l = 0.25 m, Ø = 4.6 mm ;
Reference solution (b). Dissolve 5 mg of acesulfame – stationary phase : octadecylsilyl silica gel for
potassium CRS and 5 mg of saccharin sodium R in water R chromatography R (3 μm).
and dilute to 5 mL with the same solvent. Mobile phase : mix 40 volumes of acetonitrile R and 60 volumes
Plate : cellulose for chromatography R as the coating of a 3.3 g/L solution of tetrabutylammonium hydrogen
substance. sulfate R.
Mobile phase : concentrated ammonia R, acetone R, ethyl Flow rate : 1 mL/min.
acetate R (10:60:60 V/V/V). Detection : spectrophotometer at 234 nm.
Application : 5 μL as bands. Injection : 20 μL.
Development : twice over 2/3 of the plate. Run time : twice the retention time of acesulfame.
Drying : in a current of warm air. Relative retention with reference to acesulfame (retention
Detection : examine in ultraviolet light at 254 nm. time = about 5.3 min) : impurity B = about 1.6.

General Notices (1) apply to all monographs and other texts 1469
Acetazolamide EUROPEAN PHARMACOPOEIA 8.0

System suitability : Content : 98.5 per cent to 101.0 per cent (dried substance).
– signal-to-noise ratio : minimum 10 for the peak due to
CHARACTERS
impurity B in the chromatogram obtained with reference
solution (a); Appearance : white or almost white, crystalline powder.
– peak-to-valley ratio : minimum 1.2, where Hp = height Solubility : very slightly soluble in water, slightly soluble in
above the baseline of the peak due to impurity B and ethanol (96 per cent). It dissolves in dilute solutions of alkali
Hv = height above the baseline of the lowest point of the hydroxides.
curve separating this peak from the peak due to acesulfame, It shows polymorphism (5.9).
in the chromatogram obtained with reference solution (b).
Limit : IDENTIFICATION
– impurity B : not more than the area of the principal peak First identification : A, B.
in the chromatogram obtained with reference solution (a) Second identification : A, C, D.
(20 ppm). A. Ultraviolet and visible absorption spectrophotometry
Fluorides : maximum 3 ppm. (2.2.25).
Potentiometry (2.2.36, Method I). Solution A. Dissolve 30.0 mg in 0.01 M sodium hydroxide
and dilute to 100.0 mL with the same solvent. Dilute
Test solution. Dissolve 3.000 g of the substance to 10.0 mL of the solution to 100.0 mL with 0.01 M sodium
be examined in distilled water R, add 15.0 mL of hydroxide.
total-ionic-strength-adjustment buffer R1 and dilute to 50.0 mL
with distilled water R. Solution B. Dilute 25.0 mL of solution A to 100.0 mL with
0.01 M sodium hydroxide.
Reference solutions. To 0.5 mL, 1.0 mL, 1.5 mL and 3.0 mL
of fluoride standard solution (10 ppm F) R add 15.0 mL of Spectral range : 230-260 nm for solution A ; 260-350 nm
total-ionic-strength-adjustment buffer R1 and dilute to 50.0 mL for solution B.
with distilled water R. Absorption maximum : at 240 nm for solution A ; at 292 nm
Indicator electrode : fluoride-selective. for solution B.
Reference electrode : silver-silver chloride. Specific absorbance at the absorption maximum : 162 to 176
for solution A ; 570 to 620 for solution B.
Heavy metals (2.4.8) : maximum 5 ppm.
B. Infrared absorption spectrophotometry (2.2.24).
12 mL of solution S complies with test A. Prepare the reference
Comparison: acetazolamide CRS.
solution using lead standard solution (1 ppm Pb) R.
If the spectra obtained in the solid state show differences,
Loss on drying (2.2.32) : maximum 1.0 per cent, determined dissolve the substance to be examined and the reference
on 1.000 g by drying in an oven at 105 °C for 3 h. substance separately in ethanol (96 per cent) R, evaporate to
ASSAY dryness and record new spectra using the residues.
Dissolve 0.150 g in 50 mL of anhydrous acetic acid R. Titrate C. Introduce about 20 mg into a test-tube and add 4 mL
with 0.1 M perchloric acid, determining the end-point of dilute hydrochloric acid R and 0.2 g of zinc powder R.
potentiometrically (2.2.20). Immediately place a piece of lead acetate paper R over the
mouth of the tube. The paper shows a brownish-black
1 mL of 0.1 M perchloric acid is equivalent to 20.12 mg
colour.
of C4H4KNO4S.
D. Dissolve about 25 mg in a mixture of 0.1 mL of dilute
IMPURITIES sodium hydroxide solution R and 5 mL of water R. Add
Specified impurities : A, B. 0.1 mL of copper sulfate solution R. A greenish-blue
precipitate is formed.
TESTS
Appearance of solution. The solution is not more opalescent
than reference suspension II (2.2.1) and not more intensely
coloured than reference solution Y5 or BY5 (2.2.2, Method II).
A. 3-oxobutanamide (acetylacetamide),
Dissolve 1.0 g in 10 mL of 1 M sodium hydroxide.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 40 mg of the substance to be examined
in the mobile phase and dilute to 100.0 mL with the mobile
phase.
B. 5-chloro-6-methyl-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide. Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10.0 mL with the mobile phase.
04/2009:0454
Reference solution (b). Dissolve the contents of a vial
of acetazolamide for system suitability CRS (containing
ACETAZOLAMIDE impurities A, B, C, D, E and F) in 1.0 mL of the mobile phase.
Column :
Acetazolamidum – size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : end-capped propoxybenzene silica gel for
chromatography R (4 μm).
Mobile phase : acetonitrile for chromatography R, 6.8 g/L
solution of potassium dihydrogen phosphate R (10:90 V/V).
C 4H 6N4O 3S 2 Mr 222.2 Flow rate : 1.0 mL/min.
[59-66-5]
Detection : spectrophotometer at 265 nm.
DEFINITION Injection : 25 μl.
N-(5-Sulfamoyl-1,3,4-thiadiazol-2-yl)acetamide. Run time : 3.5 times the retention time of acetazolamide.

1470 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Acetic acid, glacial

Identification of impurities : use the chromatogram supplied D. R1 = H, R2 = SO2-NH2 : 5-amino-1,3,4-thiadiazole-2-

with acetazolamide for system suitability CRS and the sulfonamide,
chromatogram obtained with reference solution (b) to identify E. R1 = CO-CH3, R2 = SO2-OH : 5-acetamido-1,3,4-
the peaks due to impurities A, B, C, D, E and F. thiadiazole-2-sulfonic acid,
Relative retention with reference to acetazolamide
(retention time = about 8 min) : impurity E = about 0.3 ;
impurity D = about 0.4 ; impurity B = about 0.6 ;
impurity C = about 1.4 ; impurity A = about 2.1 ;
impurity F = about 2.6.
F. N-[5-[(5-acetamido-1,3,4-thiadiazol-2-yl)-
System suitability : reference solution (b) : sulfonyl]sulfamoyl-1,3,4-thiadiazol-2-yl]acetamide,
– resolution : minimum 2.0 between the peaks due to
impurities E and D.
Limits :
– correction factors : for the calculation of content, multiply G. 5-amino-1,3,4-thiadiazole-2-thiol.
the peak areas of the following impurities by the
corresponding correction factor : impurity B = 2.3 ; 01/2008:0590
impurity C = 2.6 ; impurity D = 1.6 ;
– impurities A, B, C, D, E, F : for each impurity, not more ACETIC ACID, GLACIAL
than 1.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
(0.15 per cent) ;
Acidum aceticum glaciale
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
– total : not more than 6 times the area of the principal peak C 2H 4O 2 Mr 60.1
in the chromatogram obtained with reference solution (a) [64-19-7]
(0.6 per cent) ;
DEFINITION
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a) Content : 99.0 per cent m/m to 100.5 per cent m/m.
(0.05 per cent). CHARACTERS
Sulfates (2.4.13) : maximum 500 ppm. Appearance : crystalline mass or clear, colourless, volatile
To 0.4 g add 20 mL of distilled water R and dissolve by heating liquid.
to boiling. Allow to cool with frequent shaking and filter. Solubility : miscible with water, with ethanol (96 per cent) and
Heavy metals (2.4.8) : maximum 20 ppm. with methylene chloride.
1.0 g complies with test C. Prepare the reference solution using IDENTIFICATION
2 mL of lead standard solution (10 ppm Pb) R. A. A 100 g/L solution is strongly acid (2.2.4).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined B. To 0.03 mL add 3 mL of water R and neutralise with
on 1.000 g by drying in an oven at 105 °C. dilute sodium hydroxide solution R. The solution gives
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on reaction (b) of acetates (2.3.1).
1.0 g.
TESTS
ASSAY Solution S. Dilute 20 mL to 100 mL with distilled water R.
Dissolve 0.200 g in 25 mL of dimethylformamide R. Titrate Appearance. The substance to be examined is clear (2.2.1)
with 0.1 M ethanolic sodium hydroxide, determining the and colourless (2.2.2, Method II).
end-point potentiometrically (2.2.20).
Freezing point (2.2.18) : minimum 14.8 °C.
1 mL of 0.1 M ethanolic sodium hydroxide is equivalent to
22.22 mg of C4H6N4O3S2. Reducing substances. To 5.0 mL add 10.0 mL of water R and
mix. To 5.0 mL of this solution add 6 mL of sulfuric acid R,
IMPURITIES cool and add 2.0 mL of 0.0167 M potassium dichromate. Allow
Specified impurities : A, B, C, D, E, F. to stand for 1 min and add 25 mL of water R and 1 mL of
a freshly prepared 100 g/L solution of potassium iodide R.
Other detectable impurities (the following substances would, Titrate with 0.1 M sodium thiosulfate, using 1.0 mL of starch
if present at a sufficient level, be detected by one or other of solution R as indicator. Not less than 1.0 mL of 0.1 M sodium
the tests in the monograph. They are limited by the general thiosulfate solution is required.
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical Chlorides (2.4.4) : maximum 25 mg/L.
use (2034). It is therefore not necessary to identify these Dilute 10 mL of solution S to 15 mL with water R.
impurities for demonstration of compliance. See also 5.10. Sulfates (2.4.13) : maximum 50 mg/L, determined on
Control of impurities in substances for pharmaceutical use): G. solution S.
Iron (2.4.9) : maximum 5 ppm.
Dilute 5.0 mL of solution A obtained in the test for heavy
metals to 10.0 mL with water R.
A. R1 = CO-CH3, R2 = Cl : N-(5-chloro-1,3,4-thiadiazol-2- Heavy metals (2.4.8) : maximum 5 ppm.
yl)acetamide, Dissolve the residue obtained in the test for residue on
B. R1 = CO-CH3, R2 = H : N-(1,3,4-thiadiazol-2-yl)acetamide, evaporation by heating with 2 quantities, each of 15 mL,
of water R and dilute to 50.0 mL (solution A). 12 mL of
C. R1 = CO-CH3, R2 = SH : N-(5-sulfanyl-1,3,4-thiadiazol- solution A complies with test A. Prepare the reference solution
2-yl)acetamide, using lead standard solution (2 ppm Pb) R.

General Notices (1) apply to all monographs and other texts 1471
Acetone EUROPEAN PHARMACOPOEIA 8.0

Residue on evaporation : maximum 0.01 per cent. Reference solution (b). Dilute 100 μL of benzene R to 100.0 mL
Evaporate 20 g to dryness on a water-bath and dry at with the test solution. Dilute 0.20 mL of this solution to
100-105 °C. The residue weighs a maximum of 2.0 mg. 100.0 mL with the test solution.
Column :
ASSAY
– material : fused silica,
Weigh accurately a conical flask with a ground-glass stopper
containing 25 mL of water R. Add 1.0 mL of the substance – size : l = 50 m, Ø = 0.3 mm,
to be examined and weigh again accurately. Add 0.5 mL – stationary phase : macrogol 20 000 R (film thickness 1 μm).
of phenolphthalein solution R and titrate with 1 M sodium Carrier gas : helium for chromatography R.
hydroxide.
Linear velocity : 21 cm/s.
1 mL of 1 M sodium hydroxide is equivalent to 60.1 mg
of C2H4O2. Split ratio : 1:50.
Temperature :
STORAGE
Time Temperature
In an airtight container. (min) (°C)
Column 0 - 11 45 → 100

11 - 20 100
01/2008:0872
Injection port 150

ACETONE Detector 250

Detection : flame ionisation.

Acetonum Injection : 1 μL.
Retention time : impurity C = about 7.5 min.
System suitability :
– resolution : minimum 5.0 between the peak due to
C 3H 6O Mr 58.08 impurity A (2nd peak) and the peak due to impurity B
[67-64-1] (3rd peak) in the chromatogram obtained with reference
solution (a),
DEFINITION
– signal-to-noise ratio : minimum 5 for the peak due to
Propanone.
impurity C in the chromatogram obtained with reference
CHARACTERS solution (b).
Appearance : volatile, clear, colourless liquid. Limits :
Solubility : miscible with water and with ethanol (96 per cent). – impurities A, B : for each impurity, not more than the
The vapour is flammable. difference between the areas of the corresponding peaks in
the chromatogram obtained with reference solution (a) and
IDENTIFICATION the areas of the corresponding peaks in the chromatogram
obtained with the test solution (0.05 per cent V/V),
A. Relative density (see Tests).
– impurity C : not more than the difference between the
B. To 1 mL, add 3 mL of dilute sodium hydroxide solution R
area of the peak due to impurity C in the chromatogram
and 0.3 mL of a 25 g/L solution of sodium nitroprusside R.
obtained with reference solution (b) and the area of the
An intense red colour is produced which becomes violet
corresponding peak in the chromatogram obtained with
with the addition of 3.5 mL of acetic acid R.
the test solution (2 ppm V/V),
C. To 10 mL of a 0.1 per cent V/V solution of the substance
to be examined in ethanol (50 per cent V/V) R, add 1 mL – any other impurity : for each impurity, not more than the
of a 10 g/L solution of nitrobenzaldehyde R in ethanol difference between the area of the peak due to impurity A in
(50 per cent V/V) R and 0.5 mL of strong sodium hydroxide the chromatogram obtained with reference solution (a) and
solution R. Allow to stand for about 2 min and acidify with the area of the corresponding peak in the chromatogram
acetic acid R. A greenish-blue colour is produced. obtained with the test solution (0.05 per cent V/V).
Matter insoluble in water. Dilute 1.0 mL to 20 mL with
TESTS water R. The solution is clear (2.2.1).
Appearance of solution. To 10 mL add 10 mL of water R. The Residue on evaporation : maximum 50 ppm.
solution is clear (2.2.1) and colourless (2.2.2, Method II).
Evaporate 20.0 g to dryness on a water-bath and dry at
Acidity or alkalinity. To 5 mL add 5 mL of carbon dioxide-free 100-105 °C. The residue weighs a maximum of 1 mg.
water R, 0.15 mL of phenolphthalein solution R and 0.5 mL of
Water (2.5.12) : maximum 3 g/L, determined on 10.0 mL. Use
0.01 M sodium hydroxide. The solution is pink. Add 0.7 mL of
20 mL of anhydrous pyridine R as solvent.
0.01 M hydrochloric acid and 0.05 mL of methyl red solution R.
The solution is red or orange. STORAGE
Relative density (2.2.5) : 0.790 to 0.793. Protected from light.
Reducing substances. To 30 mL add 0.1 mL of 0.02 M
potassium permanganate and allow to stand in the dark for IMPURITIES
2 h. The mixture is not completely decolourised. Specified impurities : A, B, C.
Related substances. Gas chromatography (2.2.28).
A. CH3-OH : methanol,
Test solution. The substance to be examined.
Reference solution (a). To 0.5 mL of methanol R add 0.5 mL B. CH3-CHOH-CH3 : propan-2-ol (isopropanol),
of 2-propanol R and dilute to 100.0 mL with the test solution.
Dilute 1.0 mL of this solution to 10.0 mL with the test solution. C. C6H6 : benzene.

1472 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Acetylcysteine

01/2008:1485 Reference solution (c). Dissolve 20 mg of choline chloride R

corrected 6.0 in methanol R, add 0.4 mL of test solution (a) and dilute to
2.0 mL with methanol R.
ACETYLCHOLINE CHLORIDE Plate : TLC silica gel plate R.
Mobile phase : mix 20 volumes of a 40 g/L solution of
Acetylcholini chloridum ammonium nitrate R, 20 volumes of methanol R and
60 volumes of acetonitrile R.
Application : 5 μL as bands of 10 mm by 2 mm.
Development : over 2/3 of the plate.
Detection : spray with potassium iodobismuthate solution R3.
C7H16ClNO2 Mr 181.7 System suitability : the chromatogram obtained with reference
[60-31-1] solution (c) shows 2 clearly separated zones.
Limits :
DEFINITION
– any impurity : any zones in the chromatogram obtained
2-(Acetyloxy)-N,N,N-trimethylethanaminium chloride. with test solution (a), apart from the principal zone, are not
Content : 98.5 per cent to 101.5 per cent (dried substance). more intense than the principal zone in the chromatogram
obtained with reference solution (a) (1 per cent).
CHARACTERS
Appearance : white or almost white crystalline powder or Trimethylamine. Dissolve 0.1 g in 10 mL of sodium carbonate
colourless crystals, very hygroscopic. solution R and heat to boiling. No vapours appear which turn
red litmus paper R blue.
Solubility : very soluble in water, freely soluble in alcohol,
slightly soluble in methylene chloride. Heavy metals (2.4.8) : maximum 10 ppm.
12 mL of solution S complies with test A. Prepare the reference
IDENTIFICATION solution using lead standard solution (1 ppm Pb) R.
First identification : B, E. Loss on drying (2.2.32) : maximum 1.0 per cent, determined
Second identification : A, C, D, E. on 1.000 g by drying in an oven at 105 °C for 3 h.
A. Melting point (2.2.14) : 149 °C to 152 °C. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Introduce the substance to be examined into a capillary the residue obtained in the test for loss on drying.
tube. Dry in an oven at 100-105 °C for 3 h. Seal the tube ASSAY
and determine the melting point.
Dissolve 0.200 g in 20 mL of carbon dioxide-free water R.
B. Infrared absorption spectrophotometry (2.2.24). Neutralise with 0.01 M sodium hydroxide using 0.15 mL of
Comparison : acetylcholine chloride CRS. phenolphthalein solution R as indicator. Add 20.0 mL of 0.1 M
C. Examine the chromatograms obtained in the test for sodium hydroxide and allow to stand for 30 min. Titrate with
related substances. 0.1 M hydrochloric acid.
Results : the principal zone in the chromatogram obtained 1 mL of 0.1 M sodium hydroxide is equivalent to 18.17 mg of
with test solution (b) is similar in position, colour and size C7H16ClNO2.
to the principal zone in the chromatogram obtained with STORAGE
reference solution (b).
In ampoules, protected from light.
D. To 15 mg add 10 mL of dilute sodium hydroxide solution R,
2 mL of 0.02 M potassium permanganate and heat. The IMPURITIES
vapours formed change the colour of red litmus paper R
to blue.
E. 0.5 mL of solution S (see Tests) gives reaction (a) of
chlorides (2.3.1). A. 2-hydroxy-N,N,N-trimethylethanaminium chloride
(choline chloride),
TESTS
Solution S. Dissolve 5.0 g in carbon dioxide-free water R and
dilute to 50 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not B. 2-(acetyloxy)-N,N-dimethylethanaminium chloride,
more intensely coloured than reference solution Y6 or BY6
(2.2.2, Method II).
Acidity. Dilute 1 mL of solution S to 10 mL with carbon
dioxide-free water R. Add 0.05 mL of phenolphthalein C. N,N-dimethylmethanamine.
solution R. Not more than 0.4 mL of 0.01 M sodium hydroxide
is required to change the colour of the indicator to pink. 01/2008:0967
Related substances. Thin-layer chromatography (2.2.27). corrected 7.0
Prepare the solutions immediately before use.
Test solution (a). Dissolve 0.30 g of the substance to be ACETYLCYSTEINE
examined in methanol R and dilute to 3.0 mL with the same
solvent. Acetylcysteinum
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
with methanol R.
Reference solution (a). Dilute 1 mL of test solution (a) to
100 mL with methanol R.
Reference solution (b). Dissolve 20.0 mg of acetylcholine
chloride CRS in methanol R and dilute to 2.0 mL with the C5H9NO3S Mr 163.2
same solvent. [616-91-1]

General Notices (1) apply to all monographs and other texts 1473
Acetylcysteine EUROPEAN PHARMACOPOEIA 8.0

DEFINITION Flow rate : 1.0 mL/min.

(2R)-2-(Acetylamino)-3-sulfanylpropanoic acid. Detection : spectrophotometer at 220 nm.
Content : 98.0 per cent to 101.0 per cent (dried substance). Injection : 20 μL, 3 times ; inject 0.01 M hydrochloric acid as a
blank.
CHARACTERS
Run time : 5 times the retention time of acetylcysteine (about
Appearance : white or almost white, crystalline powder or 30 min).
colourless crystals.
Retention time : impurity A = about 2.2 min ; impurity B =
Solubility : freely soluble in water and in ethanol (96 per cent), about 2.4 min ; 2-methyl-2-thiazoline-4-carboxylic
practically insoluble in methylene chloride. acid, originating in test solution (c) = about 3.3 min ;
IDENTIFICATION acetylcysteine = about 6.4 min ; impurity C = about 12 min ;
impurity D = about 14 min.
First identification : A, C.
System suitability : reference solution (a) :
Second identification : A, B, D, E.
– resolution : minimum 1.5 between the peaks due to
A. Specific optical rotation (see Tests). impurities A and B and minimum 2.0 between the peaks
B. Melting point (2.2.14) : 104 °C to 110 °C. due to impurities C and D.
C. Infrared absorption spectrophotometry (2.2.24). From the chromatogram obtained with test solution (a),
Preparation : discs of potassium bromide R. calculate the percentage content of the known impurities
Comparison : acetylcysteine CRS. (T1) and the unknown impurities (T2) using the following
equations :
D. Examine the chromatograms obtained in the test for
related substances.
Results : the principal peak in the chromatogram obtained
with test solution (b) is similar in retention time and size
to the principal peak in the chromatogram obtained with
reference solution (b).
E. To 0.5 mL of solution S (see Tests) add 0.05 mL of a A1 = peak area of individual impurity (impurity A,
50 g/L solution of sodium nitroprusside R and 0.05 mL of impurity B, impurity C and impurity D) in the
concentrated ammonia R. A dark violet colour develops. chromatogram obtained with test solution (a) ;
TESTS A2 = peak area of the corresponding individual
Solution S. Dissolve 1.0 g in carbon dioxide-free water R and impurity (impurity A, impurity B, impurity C and
dilute to 20 mL with the same solvent. impurity D) in the chromatogram obtained with
reference solution (a) ;
Appearance of solution. Solution S is clear (2.2.1) and
A3 = peak area of unknown impurity in the
colourless (2.2.2, Method II).
chromatogram obtained with test solution (a) ;
pH (2.2.3) : 2.0 to 2.8.
A4 = peak area of acetylcysteine in the chromatogram
To 2 mL of solution S add 8 mL of carbon dioxide-free water R obtained with reference solution (b) ;
and mix. m1 = mass of the substance to be examined in test
Specific optical rotation (2.2.7) : + 21.0 to + 27.0 (dried solution (a) ;
substance). m2 = mass of the individual impurity in reference
In a 25 mL volumetric flask, mix 1.25 g with 1 mL of a 10 g/L solution (a) ;
solution of sodium edetate R. Add 7.5 mL of a 40 g/L solution m
of sodium hydroxide R, mix and dissolve. Dilute to 25.0 mL 3 = mass of acetylcysteine in reference solution (b).
with phosphate buffer solution pH 7.0 R2. Limits :
Related substances. Liquid chromatography (2.2.29). Except – impurities A, B, C, D : for each impurity, maximum 0.5 per
where otherwise prescribed, prepare the solutions immediately cent ;
before use. – any other impurity : for each impurity, maximum 0.5 per
Test solution (a). Suspend 0.80 g of the substance to be cent ;
examined in 1 mL of 1 M hydrochloric acid and dilute to – total : maximum 0.5 per cent ;
100.0 mL with water R. – disregard limit : 0.1 times the area of the principal peak in
Test solution (b). Dilute 5.0 mL of test solution (a) to 100.0 mL the chromatogram obtained with reference solution (b)
with water R. Dilute 5.0 mL of this solution to 50.0 mL with (0.05 per cent) ; disregard any peak with a retention time of
water R. about 3.3 min due to 2-methyl-2-thiazoline-4-carboxylic
Test solution (c). Use test solution (a) after storage for at least acid.
1 h. Heavy metals (2.4.8) : maximum 10 ppm.
Reference solution (a). Suspend 4.0 mg of acetylcysteine CRS, 2.0 g complies with test C. Prepare the reference solution using
4.0 mg of L-cystine R (impurity A), 4.0 mg of L-cysteine R 2 mL of lead standard solution (10 ppm Pb) R.
(impurity B), 4.0 mg of acetylcysteine impurity C CRS and
4.0 mg of acetylcysteine impurity D CRS in 1 mL of 1 M Zinc : maximum 10 ppm.
hydrochloric acid and dilute to 100.0 mL with water R. Atomic absorption spectrometry (2.2.23, Method II).
Reference solution (b). Suspend 4.0 mg of acetylcysteine CRS Test solution. Dissolve 1.00 g in 0.001 M hydrochloric acid and
in 1 mL of 1 M hydrochloric acid and dilute to 100.0 mL with dilute to 50.0 mL with the same acid.
water R. Reference solutions. Prepare the reference solutions using zinc
Column : standard solution (5 mg/mL Zn) R, diluting with 0.001 M
– size : l = 0.25 m, Ø = 4 mm ; hydrochloric acid.
– stationary phase : octadecylsilyl silica gel for Source : zinc hollow-cathode lamp.
chromatography R (5 μm). Wavelength : 213.8 nm.
Mobile phase : stir 3 volumes of acetonitrile R and 97 volumes Atomisation device : air-acetylene flame.
of water R in a beaker ; adjust to pH 3.0 with phosphoric acid R. Use a correction procedure for non-specific absorption.

1474 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 β-Acetyldigoxin

Loss on drying (2.2.32) : maximum 1.0 per cent, determined 01/2008:2168

on 1.000 g by drying in an oven in vacuo at 70 °C for 3 h. corrected 6.7
Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
1.0 g. β-ACETYLDIGOXIN
β-Acetyldigoxinum
ASSAY
Dissolve 0.140 g in 60 mL of water R and add 10 mL of dilute
hydrochloric acid R. After cooling in iced water, add 10 mL of
potassium iodide solution R and titrate with 0.05 M iodine,
using 1 mL of starch solution R as indicator.
1 mL of 0.05 M iodine is equivalent to 16.32 mg of C5H9NO3S.

STORAGE
Protected from light.

IMPURITIES
Specified impurities : A, B, C, D.

C43H66O15 Mr 823
[5355-48-6]
DEFINITION
3β-[(4-O-Acetyl-2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-
2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D-
ribo-hexopyranosyl)oxy]-12β,14-dihydroxy-5β-card-20(22)-
enolide.
A. 3,3′-disulfanediylbis[(2R)-2-aminopropanoic acid] Content : 97.0 per cent to 102.0 per cent (dried substance).
(L-cystine),
CHARACTERS
Appearance : white or almost white powder.
Solubility : practically insoluble in water, sparingly soluble in
methylene chloride, slightly soluble in ethanol (96 per cent).
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: β-acetyldigoxin CRS.
B. (2R)-2-amino-3-sulfanylpropanoic acid (L-cysteine), TESTS
Specific optical rotation (2.2.7) : + 26.2 to + 28.2 (dried
substance).
Dissolve 0.50 g in a mixture of equal volumes of methanol R
and methylene chloride R and dilute to 25.0 mL with the same
mixture of solvents.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
Solvent mixture. Mix equal volumes of methanol R2 and
acetonitrile for chromatography R.
Test solution. Dissolve 50.0 mg of the substance to be
examined in the solvent mixture and dilute to 100.0 mL with
the solvent mixture.
C. (2R,2′R)-3,3′-disulfanediylbis[2-(acetylamino)propanoic
acid] (N,N′-diacetyl-L-cystine), Reference solution (a). Dissolve 10.0 mg of β-acetyldigoxin CRS
in the solvent mixture and dilute to 20.0 mL with the solvent
mixture.
Reference solution (b). Dilute 1.0 mL of the test solution
to 20.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Reference solution (c). Dissolve 5 mg of gitoxin CRS
(impurity D) in the solvent mixture and dilute to 100.0 mL
with the solvent mixture. To 5.0 mL of this solution, add
0.5 mL of reference solution (a) and dilute to 100.0 mL with
the solvent mixture.
Reference solution (d). Dissolve 5.0 mg of β-acetyldigoxin for
D. (2R)-2-(acetylamino)-3-(acetylsulfanyl)propanoic acid peak identification CRS (containing impurities A and B) in
(N,S-diacetyl-L-cysteine). 10.0 mL of the solvent mixture.

General Notices (1) apply to all monographs and other texts 1475
β-Acetyldigoxin EUROPEAN PHARMACOPOEIA 8.0

Column : STORAGE
– size : l = 0.125 m, Ø = 4.0 mm ; Protected from light.
– stationary phase : octadecylsilyl silica gel for
chromatography R (4 μm). IMPURITIES
Mobile phase : Specified impurities : A, B, D.
– mobile phase A : water for chromatography R ; Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
– mobile phase B : acetonitrile for chromatography R ;
the tests in the monograph. They are limited by the general
Time Mobile phase A Mobile phase B acceptance criterion for other/unspecified impurities. It
(min) (per cent V/V) (per cent V/V) is therefore not necessary to identify these impurities for
0 - 10 70 30 demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : C, E, F, G, H.
10 - 20 70 → 35 30 → 65

20 - 20.1 35 → 70 65 → 30

20.1 - 25 70 30

Flow rate : 1.5 mL/min.

Detection : spectrophotometer at 225 nm.
Injection : 10 μL of the test solution and reference solutions (b),
(c) and (d).
Identification of impurities : use the chromatograms obtained
with reference solutions (c) and (d) to identify the peaks due
to impurities A, B and D.
Relative retention with reference to β-acetyldigoxin
(retention time = about 9 min): impurity B = about 0.3 ;
impurity A = about 0.7 ; impurity D = about 1.2.
System suitability: reference solution (c) :
– resolution : minimum 1.5 between the peaks due to
β-acetyldigoxin and impurity D ;
– symmetry factor : maximum 2.5 for the peak due to A. 3β-[(3-O-acetyl-2,6-dideoxy-β-D-ribo-hexopyranosyl-
β-acetyldigoxin. (1→4)-2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-
Limits : dideoxy-β-D-ribo-hexopyranosyl)oxy]-12β,14-dihydroxy-
5β-card-20(22)-enolide (α-acetyldigoxin),
– impurities A, B : for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent) ;
– impurity D : not more than 0.6 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.3 per cent) ;
– any other impurity : for each impurity, not more than
0.4 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.2 per cent) ;
– sum of impurities other than A, B and D : not more than
1.2 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.6 per cent) ;
– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(1.5 per cent) ;
– disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent). B. 3β-[(2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-
The thresholds indicated under Related substances dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-
(Table 2034.-1) in the general monograph Substances for D-ribo-hexopyranosyl)oxy]-12β,14-dihydroxy-5β-card-
pharmaceutical use (2034) do not apply. 20(22)-enolide (digoxin),
Loss on drying (2.2.32) : maximum 1.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
the residue obtained in the test for loss on drying.

ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : test solution and reference solution (a).
Calculate the percentage content of C43H66O15 from the C. 3β,12β,14-trihydroxy-5β-card-20(22)-enolide
declared content of β-acetyldigoxin CRS. (digoxigenin),

1476 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Acetylsalicylic acid

D. 3β-[(2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-
dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β- G. 3β-[(3-O-acetyl-2,6-dideoxy-β-D-ribo-hexopyranosyl-
D-ribo-hexopyranosyl)oxy]-14,16β-dihydroxy-5β-card- (1→4)-2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-
20(22)-enolide (gitoxin), dideoxy-β-D-ribo-hexopyranosyl)oxy]-14-hydroxy-5β-
card-20(22)-enolide (α-acetyldigitoxin),

H. 3β-[(4-O-acetyl-2,6-dideoxy-β-D-ribo-hexopyranosyl-
E. 3β-[(2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6- (1→4)-2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-
dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β- dideoxy-β-D-ribo-hexopyranosyl)oxy]-14-hydroxy-5β-
D-ribo-hexopyranosyl)oxy]-14-hydroxy-5β-card-20(22)-
card-20(22)-enolide (β-acetyldigitoxin).
enolide (digitoxin),

07/2012:0309

ACETYLSALICYLIC ACID
Acidum acetylsalicylicum

C 9H 8O 4 Mr 180.2
[50-78-2]
DEFINITION
2-(Acetyloxy)benzoic acid.
Content : 99.5 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
colourless crystals.
F. 3β-[(3,4-O-diacetyl-2,6-dideoxy-β-D-ribo-hexopyranosyl-
(1→4)-2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6- Solubility : slightly soluble in water, freely soluble in ethanol
dideoxy-β-D-ribo-hexopyranosyl)oxy]-12β,14-dihydroxy- (96 per cent).
5β-card-20(22)-enolide (diacetyldigoxin), mp : about 143 °C (instantaneous method).

General Notices (1) apply to all monographs and other texts 1477
Acetylsalicylic acid EUROPEAN PHARMACOPOEIA 8.0

IDENTIFICATION Limits :
First identification : A, B. – impurities A, B, C, D, E, F : for each impurity, not more
Second identification : B, C, D. than 1.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
A. Infrared absorption spectrophotometry (2.2.24).
(0.15 per cent);
Comparison : acetylsalicylic acid CRS.
– unspecified impurities : for each impurity, not more than
B. To 0.2 g add 4 mL of dilute sodium hydroxide solution R 0.5 times the area of the principal peak in the chromatogram
and boil for 3 min. Cool and add 5 mL of dilute sulfuric obtained with reference solution (a) (0.05 per cent) ;
acid R. A crystalline precipitate is formed. Filter, wash
the precipitate and dry at 100-105 °C. The melting point – total : not more than 2.5 times the area of the principal peak
(2.2.14) is 156 °C to 161 °C. in the chromatogram obtained with reference solution (a)
(0.25 per cent);
C. In a test tube mix 0.1 g with 0.5 g of calcium hydroxide R.
Heat the mixture and expose to the fumes produced – disregard limit : 0.3 times the area of the principal peak in
a piece of filter paper impregnated with 0.05 mL of the chromatogram obtained with reference solution (a)
nitrobenzaldehyde solution R. A greenish-blue or (0.03 per cent).
greenish-yellow colour develops on the paper. Moisten the Heavy metals (2.4.8) : maximum 20 ppm.
paper with dilute hydrochloric acid R. The colour becomes Dissolve 1.0 g in 12 mL of acetone R and dilute to 20 mL with
blue. water R. 12 mL of the solution complies with test B. Prepare
D. Dissolve with heating about 20 mg of the precipitate the reference solution using lead standard solution (1 ppm Pb)
obtained in identification test B in 10 mL of water R and obtained by diluting lead standard solution (100 ppm Pb) R
cool. The solution gives reaction (a) of salicylates (2.3.1). with a mixture of 6 volumes of water R and 9 volumes of
acetone R.
TESTS
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Appearance of solution. The solution is clear (2.2.1) and on 1.000 g by drying in vacuo.
colourless (2.2.2, Method II).
Dissolve 1.0 g in 9 mL of ethanol (96 per cent) R. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use. ASSAY
Test solution. Dissolve 0.100 g of the substance to be examined In a flask with a ground-glass stopper, dissolve 1.000 g in
in acetonitrile for chromatography R and dilute to 10.0 mL 10 mL of ethanol (96 per cent) R. Add 50.0 mL of 0.5 M sodium
with the same solvent. hydroxide. Close the flask and allow to stand for 1 h. Using
Reference solution (a). Dissolve 50.0 mg of salicylic acid R 0.2 mL of phenolphthalein solution R as indicator, titrate with
(impurity C) in the mobile phase and dilute to 50.0 mL with 0.5 M hydrochloric acid. Carry out a blank titration.
the mobile phase. Dilute 1.0 mL of the solution to 100.0 mL 1 mL of 0.5 M sodium hydroxide is equivalent to 45.04 mg
with the mobile phase. of C9H8O4.
Reference solution (b). Dissolve 10 mg of salicylic acid R
(impurity C) in the mobile phase and dilute to 10.0 mL with STORAGE
the mobile phase. To 1.0 mL of the solution add 0.2 mL of the In an airtight container.
test solution and dilute to 100.0 mL with the mobile phase.
Reference solution (c). Dissolve with the aid of ultrasound IMPURITIES
the contents of a vial of acetylsalicylic acid for peak Specified impurities : A, B, C, D, E, F.
identification CRS (containing impurities A, B, D, E and F) in
1.0 mL of acetonitrile R.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for A. 4-hydroxybenzoic acid,
chromatography R (5 μm).
Mobile phase : phosphoric acid R, acetonitrile for
chromatography R, water R (2:400:600 V/V/V).
Flow rate : 1 mL/min.
Detection : spectrophotometer at 237 nm. B. 4-hydroxybenzene-1,3-dicarboxylic acid (4-hydroxyiso-
Injection : 10 μL. phthalic acid),
Run time : 7 times the retention time of acetylsalicylic acid.
Identification of impurities : use the chromatogram
obtained with reference solution (a) to identify the peak
due to impurity C ; use the chromatogram supplied with
acetylsalicylic acid for peak identification CRS and the
chromatogram obtained with reference solution (c) to identify C. 2-hydroxybenzenecarboxylic acid (salicylic acid),
the peaks due to impurities A, B, D, E and F.
Relative retention with reference to acetylsalicylic acid
(retention time = about 5 min) : impurity A = about 0.7 ;
impurity B = about 0.8 ; impurity C = about 1.3 ;
impurity D = about 2.3 ; impurity E = about 3.2 ;
impurity F = about 6.0.
System suitability : reference solution (b) :
– resolution : minimum 6.0 between the peaks due to D. 2-[[2-(acetyloxy)benzoyl]oxy]benzoic acid (acetylsalicyl-
acetylsalicylic acid and impurity C. salicylic acid),

1478 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 N-Acetyltryptophan

Application : 2 μL.
Development : over a path of 10 cm.
Drying : in an oven at 100-105 °C for 15 min.
Detection : examine in ultraviolet light at 254 nm.
System suitability : reference solution (b) :
– the chromatogram shows 2 clearly separated spots.
E. 2-[(2-hydroxybenzoyl)oxy]benzoic acid (salsalate,
salicylsalicylic acid), Results : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to
the principal spot in the chromatogram obtained with
reference solution (a).
D. Dissolve about 2 mg in 2 mL of water R. Add 2 mL
of dimethylaminobenzaldehyde solution R6. Heat on a
water-bath. A blue or greenish-blue colour develops.
E. It gives the reaction of acetyl (2.3.1). Proceed as described
for substances hydrolysable only with difficulty.
F. 2-(acetyloxy)benzoic anhydride (acetylsalicylic anhydride). TESTS
Appearance of solution. The solution is clear (2.2.1) and not
01/2009:1383 more intensely coloured than reference solution Y7 or GY7
corrected 7.0 (2.2.2, Method II).
Dissolve 1.0 g in a 40 g/L solution of sodium hydroxide R and
N-ACETYLTRYPTOPHAN dilute to 100 mL with the same alkaline solution.
Optical rotation (2.2.7) : − 0.1° to + 0.1°.
N-Acetyltryptophanum Dissolve 2.50 g in a 40 g/L solution of sodium hydroxide R and
dilute to 25.0 mL with the same alkaline solution.
Related substances. Liquid chromatography (2.2.29). Prepare
the test and reference solutions immediately before use.
Buffer solution pH 2.3. Dissolve 3.90 g of sodium dihydrogen
phosphate R in 1000 mL of water R. Add about 700 mL of a
C13H14N2O3 Mr 246.3 2.9 g/L solution of phosphoric acid R and adjust to pH 2.3 with
[87-32-1] the same acid solution.
Solvent mixture : acetonitrile R, water R (10:90 V/V).
DEFINITION
Test solution. Dissolve 0.10 g of the substance to be examined
(RS)-2-Acetylamino-3-(1H-indol-3-yl)propanoic acid. in a mixture of 50 volumes of acetonitrile R and 50 volumes
Content : 99.0 per cent to 101.0 per cent (dried substance). of water R and dilute to 20.0 mL with the same mixture of
solvents.
PRODUCTION
Reference solution (a). Dilute 1.0 mL of the test solution to
Tryptophan used for the production of N-acetyltryptophan 100.0 mL with the solvent mixture.
complies with the test for impurity A and other related
substances in the monograph on Tryptophan (1272). Reference solution (b). Dilute 4.0 mL of reference solution (a)
to 100.0 mL with the solvent mixture.
CHARACTERS Reference solution (c). Dissolve the contents of a vial of
Appearance : white or almost white, crystalline powder, or 1,1′-ethylidenebistryptophan CRS in 1 mL of reference
colourless crystals. solution (b).
Solubility : slightly soluble in water, very soluble in ethanol Column :
(96 per cent). It dissolves in dilute solutions of alkali – size : l = 0.25 m, Ø = 4.6 mm ;
hydroxides. – stationary phase : octadecylsilyl silica gel for
mp : about 205 °C. chromatography R (5 μm) ;
IDENTIFICATION – temperature : 40 °C.
First identification : A, B. Mobile phase :
Second identification : A, C, D, E. – mobile phase A : acetonitrile R, buffer solution pH 2.3
(115:885 V/V) ;
A. Optical rotation (see Tests).
– mobile phase B : acetonitrile R, buffer solution pH 2.3
B. Infrared absorption spectrophotometry (2.2.24). (350:650 V/V) ;
Comparison : N-acetyltryptophan CRS.
Time Mobile phase A Mobile phase B
C. Thin-layer chromatography (2.2.27). (min) (per cent V/V) (per cent V/V)
Test solution. Dissolve 50 mg of the substance to be 0 - 10 100 0
examined in 0.2 mL of concentrated ammonia R and dilute
to 10 mL with water R. 10 - 45 100 → 0 0 → 100
Reference solution (a). Dissolve 50 mg of 45 - 65 0 100
N-acetyltryptophan CRS in 0.2 mL of concentrated
ammonia R and dilute to 10 mL with water R. Flow rate : 0.7 mL/min.
Reference solution (b). Dissolve 10 mg of tryptophan R in Detection : spectrophotometer at 220 nm.
the test solution and dilute to 2 mL with the test solution. Injection : 20 μL of the test solution and reference solutions (a)
Plate : TLC silica gel F254 plate R. and (c).
Mobile phase : glacial acetic acid R, water R, butanol R Retention time : N-acetyltryptophan = about 29 min ;
(25:25:40 V/V/V). 1,1′-ethylidenebis(tryptophan) = about 34 min.

General Notices (1) apply to all monographs and other texts 1479
N-Acetyltryptophan EUROPEAN PHARMACOPOEIA 8.0

System suitability: reference solution (c) :

– resolution : minimum 8.0 between the peaks due to
N-acetyltryptophan and 1,1′-ethylidenebis(tryptophan) ;
if necessary, adjust the time programme for the elution
gradient (an increase in the duration of elution with mobile
phase A produces longer retention times and a better C. R = H : (S)-2-amino-4-(2-aminophenyl)-4-oxobutanoic
resolution) ; acid (kynurenine),
– symmetry factor : maximum 3.5 for the peak due to
E. R = CHO : (S)-2-amino-4-[2-(formylamino)phenyl]-4-
1,1′-ethylidenebistryptophan in the chromatogram
oxobutanoic acid (N-formylkynurenine),
obtained with reference solution (c).
Limits :
– impurities A, B, C, D, E, F, G, H, I, J, K, L : for each impurity,
not more than 0.25 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.25 per cent) ;
– total : not more than 0.5 times the area of the principal peak D. (S)-2-amino-3-(5-hydroxy-1H-indol-3-yl)propanoic acid
in the chromatogram obtained with reference solution (a) (5-hydroxytryptophan),
(0.5 per cent) ;
– disregard limit : 0.01 times the area of the principal peak
the chromatogram obtained with reference solution (a)
(0.01 per cent).
Ammonium (2.4.1, Method B) : maximum 200 ppm,
determined on 0.10 g. F. (S)-2-amino-3-(phenylamino)propanoic acid
Prepare the standard using 0.2 mL of ammonium standard (3-phenylaminoalanine),
solution (100 ppm NH4) R.
Iron (2.4.9) : maximum 10 ppm.
Dissolve 1.0 g in 50 mL of hydrochloric acid R1, with heating
at 50 °C. Allow to cool. In a separating funnel, shake with
3 quantities, each of 10 mL, of methyl isobutyl ketone R1,
shaking for 3 min each time. To the combined organic layers G. (S)-2-amino-3-(2-hydroxy-1H-indol-3-yl)propanoic acid
add 10 mL of water R and shake for 3 min. Examine the (2-hydroxytryptophan),
aqueous layer.
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test C. Prepare the reference solution using
2 mL of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on H. R = H : (3RS)-1,2,3,4-tetrahydro-9H-β-carboline-3-
1.0 g. carboxylic acid,

ASSAY I. R = CH3 : 1-methyl-1,2,3,4-tetrahydro-9H-β-carboline-3-

Dissolve 0.200 g in 5 mL of methanol R. Add 50 mL of carboxylic acid,
anhydrous ethanol R. Titrate with 0.1 M sodium hydroxide,
determining the end-point potentiometrically (2.2.20).
1 mL of 0.1 M sodium hydroxide is equivalent to 24.63 mg
of C13H14N2O3.
STORAGE
Protected from light.
IMPURITIES J. R = CHOH-CH2-OH : (S)-2-amino-3-[2-[2,3-dihydroxy-1-
Specified impurities : A, B, C, D, E, F, G, H, I, J, K, L. (1H-indol-3-yl)propyl]-1H-indol-3-yl]propanoic acid,

K. R = H : (S)-2-amino-3-[2-(1H-indol-3-ylmethyl)-1H-indol-
3-yl]propanoic acid,

A. (S)-2-amino-3-(1H-indol-3-yl)propanoic acid
(tryptophan),

B. (S)-2-amino-3-[(3RS)-3-hydroxy-2-oxo-2,3-dihydro-1H- L. 1-(1H-indol-3-ylmethyl)-1,2,3,4-tetrahydro-9H-β-
indol-3-yl]propanoic acid (dioxyindolylalanine), carboline-3-carboxylic acid.

1480 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 N-Acetyltyrosine

07/2011:1384 Reference solution (a). Dilute 1.0 mL of the test solution to

100.0 mL with mobile phase A. Dilute 1.0 mL of this solution
N-ACETYLTYROSINE to 10.0 mL with mobile phase A.
Reference solution (b). Dissolve 20.0 mg of tyrosine CRS
(impurity A) in 2 mL of a 40 g/L solution of sodium
N-Acetyltyrosinum hydroxide R and dilute to 20.0 mL with water R. Dilute 1.0 mL
of this solution to 10.0 mL with water R.
Reference solution (c). Dilute 1.0 mL of reference solution (b)
to 10.0 mL with mobile phase A.
Reference solution (d). Dilute 1.0 mL of reference solution (b)
to 20.0 mL with the test solution.
C11H13NO4 Mr 223.2 Column :
[537-55-3] – size : l = 0.15 m, Ø = 3 mm ;
DEFINITION – stationary phase : spherical octadecylsilyl silica gel for
chromatography R (3 μm) ;
(2S)-2-(Acetylamino)-3-(4-hydroxyphenyl)propanoic acid.
– temperature : 40 °C.
Content : 98.5 per cent to 101.0 per cent (dried substance). Mobile phase :
CHARACTERS – mobile phase A : mix 1.0 mL of phosphoric acid R and
Appearance : white or almost white, crystalline powder or 1000 mL of water for chromatography R ;
colourless crystals. – mobile phase B : acetonitrile R1 ;
Solubility : freely soluble in water, practically insoluble in Time Mobile phase A Mobile phase B
cyclohexane. (min) (per cent V/V) (per cent V/V)
0-2 97 3
IDENTIFICATION
First identification : A, B. 2 - 15 97 → 62 3 → 38

Second identification : A, C, D. Flow rate : 0.7 mL/min.

A. Specific optical rotation (see Tests). Detection : spectrophotometer at 219 nm.
B. Infrared absorption spectrophotometry (2.2.24). Injection : 2 μL of the test solution and reference solutions (a),
Comparison : N-acetyltyrosine CRS. (c) and (d).
C. Thin-layer chromatography (2.2.27). Relative retention with reference to N-acetyltyrosine (retention
Test solution. Dissolve 80 mg of the substance to be time = about 6 min) : impurity A = about 0.5.
examined in a mixture of 3 volumes of glacial acetic System suitability : reference solution (d) :
acid R, 3 volumes of water R and 94 volumes of anhydrous – resolution : minimum 5.0 between the principal peak and
ethanol R, and dilute to 10 mL with the same mixture of the peak due to impurity A.
solvents. Limits :
Reference solution. Dissolve 80 mg of N-acetyltyrosine CRS – impurity A : not more than 0.8 times the area of the
in a mixture of 3 volumes of glacial acetic acid R, 3 volumes corresponding peak in the chromatogram obtained with
of water R and 94 volumes of anhydrous ethanol R, and reference solution (c) (0.8 per cent) ;
dilute to 10 mL with the same mixture of solvents.
– unspecified impurities : for each impurity, not more than the
Plate : TLC silica gel F254 plate R. area of the principal peak in the chromatogram obtained
Mobile phase : water R, glacial acetic acid R, ethyl acetate R with reference solution (a) (0.10 per cent) ;
(10:15:75 V/V/V). – total : maximum 1.0 per cent ;
Application : 5 μL. – disregard limit : 0.5 times the area of the principal peak in
Development : over 2/3 of the plate. the chromatogram obtained with reference solution (a)
Drying : in air. (0.05 per cent).
Detection : examine in ultraviolet light at 254 nm. Chlorides (2.4.4) : maximum 200 ppm.
Results : the principal spot in the chromatogram obtained Dilute 10 mL of solution S to 15 mL with water R.
with the test solution is similar in position and size to the Sulfates (2.4.13) : maximum 200 ppm.
principal spot in the chromatogram obtained with the Dissolve 1.0 g in distilled water R and dilute to 20 mL with
reference solution. the same solvent.
D. Solution S (see Tests) is strongly acid (2.2.4). Ammonium (2.4.1, Method B) : maximum 200 ppm,
TESTS determined on 0.100 g.
Prepare the standard using 0.2 mL of ammonium standard
Solution S. Dissolve 2.50 g in water R and dilute to 100.0 mL
solution (100 ppm NH4) R.
with the same solvent.
Iron (2.4.9) : maximum 20 ppm.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II). In a separating funnel, dissolve 0.5 g in 10 mL of dilute
hydrochloric acid R. Shake with 3 quantities, each of 10 mL, of
Specific optical rotation (2.2.7) : + 46 to + 49 (dried methyl isobutyl ketone R1, shaking for 3 min each time. To the
substance). combined organic layers add 10 mL of water R and shake for
Dilute 10.0 mL of solution S to 25.0 mL with water R. 3 min. The aqueous layer complies with the test.
Related substances. Liquid chromatography (2.2.29). Carry Heavy metals (2.4.8) : maximum 10 ppm.
out the test protected from light. Dissolve 2.0 g in water R and dilute to 20 mL with the same
Test solution. Dissolve 50.0 mg of the substance to be solvent. 12 mL of the solution complies with test A. Prepare
examined in mobile phase A and dilute to 50.0 mL with the reference solution using lead standard solution (1 ppm
mobile phase A. Pb) R.

General Notices (1) apply to all monographs and other texts 1481
Aciclovir EUROPEAN PHARMACOPOEIA 8.0

Loss on drying (2.2.32) : maximum 0.5 per cent, determined IDENTIFICATION

on 1.000 g by drying in an oven at 105 °C. Infrared absorption spectrophotometry (2.2.24).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Comparison: aciclovir CRS.
1.0 g.
Bacterial endotoxins (2.6.14) : less than 25 IU/g, if intended TESTS
for use in the manufacture of parenteral preparations without Appearance of solution. The solution is clear (2.2.1) and not
a further appropriate procedure for the removal of bacterial more intensely coloured than reference solution Y7 (2.2.2,
endotoxins. Method II).
ASSAY Dissolve 0.25 g in a 4 g/L solution of sodium hydroxide R and
dilute to 25 mL with the same solvent.
Dissolve 0.180 g in 50 mL of carbon dioxide-free water R.
Titrate with 0.1 M sodium hydroxide, determining the Related substances. Liquid chromatography (2.2.29). Prepare
end-point potentiometrically (2.2.20). the solutions immediately before use.
1 mL of 0.1 M sodium hydroxide is equivalent to 22.32 mg Solvent mixture : dimethyl sulfoxide R, water R (20:80 V/V).
of C11H13NO4. Phosphate buffer solution pH 2.5. Dissolve 3.48 g of
dipotassium hydrogen phosphate R in 1000 mL of water R and
STORAGE adjust to pH 2.5 with phosphoric acid R.
Protected from light. If the substance is sterile, store in a Phosphate buffer solution pH 3.1. Dissolve 3.48 g of
sterile, airtight, tamper-proof container. dipotassium hydrogen phosphate R in 1000 mL of water R and
IMPURITIES adjust to pH 3.1 with phosphoric acid R.
Specified impurities : A. Test solution. Dissolve 25 mg of the substance to be examined
in 5.0 mL of dimethyl sulfoxide R and dilute to 25.0 mL with
Other detectable impurities (the following substances would, water R.
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general Reference solution (a). Dissolve 5 mg of aciclovir for system
acceptance criterion for other/unspecified impurities and/or suitability CRS (containing impurities A, B, J, K, N, O and P) in
by the general monograph Substances for pharmaceutical 1 mL of dimethyl sulfoxide R and dilute to 5.0 mL with water R.
use (2034). It is therefore not necessary to identify these Reference solution (b). Dilute 1.0 mL of the test solution to
impurities for demonstration of compliance. See also 5.10. 100.0 mL with the solvent mixture. Dilute 1.0 mL of this
Control of impurities in substances for pharmaceutical use) : B. solution to 10.0 mL with the solvent mixture.
Reference solution (c). Dissolve the contents of a vial of
aciclovir for peak identification 1 CRS (containing impurities C
and I) in 200 μL of dimethyl sulfoxide R and dilute to 1.0 mL
with water R.
A. (2S)-2-amino-3-(4-hydroxyphenyl)propanoic acid Reference solution (d). Dissolve the contents of a vial of
(tyrosine), aciclovir for peak identification 2 CRS (containing impurities F
and G) in 1.0 mL of reference solution (a).
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
B. (2S)-2-(acetylamino)-3-[4-(acetoxy)phenyl]propanoic acid
(diacetyltyrosine). Mobile phase :
– mobile phase A : acetonitrile R, phosphate buffer solution
pH 3.1 (1:99 V/V) ;
01/2014:0968
– mobile phase B : acetonitrile R, phosphate buffer solution
pH 2.5 (50:50 V/V) ;
ACICLOVIR
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
Aciclovirum 0-5 100 0

5 - 27 100 → 80 0 → 20

27 - 40 80 20

Flow rate : 1.0 mL/min.

C8H11N5O3 Mr 225.2 Detection : spectrophotometer at 254 nm.
[59277-89-3] Injection : 10 μL of the test solution and reference solutions (b),
(c) and (d).
DEFINITION Identification of impurities : use the chromatogram supplied
2-Amino-9-[(2-hydroxyethoxy)methyl]-1,9-dihydro-6H- with aciclovir for peak identification 1 CRS and the
purin-6-one. chromatogram obtained with reference solution (c) to identify
Content : 98.5 per cent to 101.0 per cent (anhydrous substance). the peaks due to impurities C and I ; use the chromatogram
supplied with aciclovir for peak identification 2 CRS and the
CHARACTERS chromatogram obtained with reference solution (d) to identify
Appearance : white or almost white, crystalline powder. the peaks due to impurities A, B, F, G, J, K, N, O and P.
Solubility : slightly soluble in water, very slightly soluble Relative retention with reference to aciclovir (retention
in ethanol (96 per cent), practically insoluble in heptane. time = about 13 min) : impurity B = about 0.4 ;
It dissolves in dilute solutions of mineral acids and alkali impurity P = about 0.7 ; impurity C = about 0.9 ;
hydroxides. impurity N = about 1.37 ; impurities O and Q = about 1.42 ;

1482 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Aciclovir

impurity I = about 1.57 ; impurity J = about 1.62 ;

impurity F = about 1.7 ; impurity A = about 1.8 ; impurities K
and R = about 2.5 ; impurity G = about 2.6.
System suitability :
– resolution : minimum 1.5 between the peaks due to A. 2-[(2-amino-6-oxo-1,6-dihydro-9H-purin-9-
impurity C and aciclovir in the chromatogram obtained yl)methoxy]ethyl acetate,
with reference solution (c) ; minimum 1.5 between the
peaks due to impurities F and A and minimum 1.5 between
the peaks due to impurities K and G in the chromatogram
obtained with reference solution (d).
Limits :
– correction factor : for the calculation of content, multiply B. 2-amino-1,7-dihydro-6H-purin-6-one (guanine),
the peak area of impurity I by 1.5 ;
– impurity B : not more than 7 times the area of the principal
peak in the chromatogram obtained with reference
solution (b) (0.7 per cent) ;
– sum of impurities O and Q : not more than 3 times the area C. 2-amino-7-[(2-hydroxyethoxy)methyl]-1,7-dihydro-6H-
of the principal peak in the chromatogram obtained with purin-6-one,
reference solution (b) (0.3 per cent) ;
– sum of impurities K and R : not more than twice the area
of the principal peak in the chromatogram obtained with
reference solution (b) (0.2 per cent) ;
– impurities A, G, J, N, P : for each impurity, not more than
twice the area of the principal peak in the chromatogram F. N-[9-[(2-hydroxyethoxy)methyl]-6-oxo-6,9-dihydro-1H-
obtained with reference solution (b) (0.2 per cent) ; purin-2-yl]acetamide,
– impurities C, F, I : for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
reference solution (b) (0.1 per cent) ;
– unspecified impurities : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.05 per cent) ; G. 2-[[2-(acetylamino)-6-oxo-1,6-dihydro-9H-purin-9-
– total : not more than 15 times the area of the principal peak yl]methoxy]ethyl acetate,
in the chromatogram obtained with reference solution (b)
(1.5 per cent) ;
– disregard limit : 0.3 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.03 per cent).
Water (2.5.12) : maximum 6.0 per cent, determined on 0.500 g.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on I. 2-amino-7-[[2-[(2-amino-6-oxo-1,6-dihydro-9H-purin-9-
1.0 g. yl)methoxy]ethoxy]methyl]-1,7-dihydro-6H-purin-6-one,
Bacterial endotoxins (2.6.14, Method D) : less than
0.50 IU/mg, if intended for use in the manufacture of
parenteral preparations without a further appropriate
procedure for the removal of bacterial endotoxins.

ASSAY J. 9,9′-[ethylenebis(oxymethylene)]bis(2-amino-1,9-dihydro-
Dissolve 0.150 g in 60 mL of anhydrous acetic acid R. Titrate 6H-purin-6-one),
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20). Carry out a blank titration.
1 mL of 0.1 M perchloric acid is equivalent to 22.52 mg
of C8H11N5O3.

IMPURITIES K. 2,2′-(methylenediimino)bis[9-[(2-hydroxyethoxy)methyl]-
1,9-dihydro-6H-purin-6-one],
Specified impurities : A, B, C, F, G, I, J, K, N, O, P, Q, R.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of L. N-(9-acetyl-6-oxo-6,9-dihydro-1H-purin-2-yl)acetamide
impurities in substances for pharmaceutical use) : L, M. (N2,9-diacetylguanine),

General Notices (1) apply to all monographs and other texts 1483
Acitretin EUROPEAN PHARMACOPOEIA 8.0

Carry out all operations as rapidly as possible and avoid

exposure to actinic light ; use freshly prepared solutions.
IDENTIFICATION
First identification : B.
Second identification : A, C.
M. 2-[[2-(acetylamino)-6-oxo-1,6-dihydro-7H-purin-7-
yl]methoxy]ethyl acetate, A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
N. unknown structure, Test solution. Dissolve 15.0 mg in 10 mL of
O. unknown structure, tetrahydrofuran R and dilute immediately to 100.0 mL
with the same solvent. Dilute 2.5 mL of this solution to
100.0 mL with tetrahydrofuran R.
Spectral range : 300-400 nm.
Absorption maximum : at 358 nm.
Specific absorbance at the absorption maximum : 1350 to
P. 2-amino-9-(2-hydroxyethyl)-1,9-dihydro-6H-purin-6-one, 1475.
B. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs.
Comparison: acitretin CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
substance separately in 2-propanol R heating under reflux,
filter, evaporate to dryness and record new spectra using
the residues.
C. Examine the chromatograms obtained in the assay.
Results : the principal peak in the chromatogram obtained
with test solution (b) is similar in retention time to
Q. mixture of 2-amino-9-[[2-(hydroxymethoxy) the principal peak in the chromatogram obtained with
ethoxy]methyl]-1,9-dihydro-6H-purin-6-one and reference solution (a).
2-amino-9-[[2-(hydroxyethoxy)methoxy]methyl]-1,9-
dihydro-6H-purin-6-one, TESTS
Related substances. Liquid chromatography (2.2.29).
Maintain the sampler at 4 °C.
Test solution (a). Dissolve 25.0 mg of the substance to be
examined in 5 mL of tetrahydrofuran R and dilute immediately
to 100.0 mL with anhydrous ethanol R.
Test solution (b). Dilute 10.0 mL of test solution (a) to 25.0 mL
with anhydrous ethanol R.
R. 9,9′-[methylenebis(oxyethyleneoxymethylene)]bis(2-
amino-1,9-dihydro-6H-purin-6-one). Reference solution (a). Dissolve 25.0 mg of acitretin CRS in
5 mL of tetrahydrofuran R and dilute immediately to 100.0 mL
with anhydrous ethanol R. Dilute 10.0 mL of this solution to
07/2010:1385 25.0 mL with anhydrous ethanol R.
corrected 7.0 Reference solution (b). Dissolve 1.0 mg of tretinoin CRS in
anhydrous ethanol R and dilute to 20.0 mL with the same
ACITRETIN solvent. Mix 5.0 mL of this solution with 2.5 mL of reference
solution (a) and dilute to 100.0 mL with anhydrous ethanol R.
Acitretinum Reference solution (c). Dilute 2.5 mL of reference solution (a)
to 50.0 mL with anhydrous ethanol R. Dilute 3.0 mL of this
solution to 20.0 mL with anhydrous ethanol R.
Column :
– size l = 0.25 m, Ø = 4 mm ;
– stationary phase : microparticulate octadecylsilyl silica gel
for chromatography R (5 μm) with a specific surface area
C21H26O3 Mr 326.4 of 200 m2/g, a pore size of 15 nm and a carbon loading of
[55079-83-9] 20 per cent ;
DEFINITION – temperature : 25 °C.
(all-E)-9-(4-Methoxy-2,3,6-trimethylphenyl)-3,7- Mobile phase : a 0.3 per cent V/V solution of glacial acetic
dimethylnona-2,4,6,8-tetraenoic acid. acid R in a mixture of 8 volumes of water R and 92 volumes
of anhydrous ethanol R.
Content : 98.0 per cent to 102.0 per cent (dried substance).
Flow rate : 0.6 mL/min.
CHARACTERS Detection : spectrophotometer at 360 nm.
Appearance : yellow or greenish-yellow, crystalline powder. Injection : 10 μL of test solution (a) and reference solutions (b)
Solubility : practically insoluble in water, sparingly soluble in and (c).
tetrahydrofuran, slightly soluble in acetone and in ethanol Run time : 2.5 times the retention time of acitretin.
(96 per cent), very slightly soluble in cyclohexane. Retention time : impurity A = about 4.8 min ; tretinoin = about
It is sensitive to air, heat and light, especially in solution. 5.2 min ; acitretin = about 6.2 min ; impurity B = about
It shows polymorphism. 10.2 min.

1484 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Adapalene

System suitability : reference solution (b) : IMPURITIES

– resolution : minimum 2.0 between the peaks due to acitretin Specified impurities : A, B.
and tretinoin ; if necessary, adjust the concentration of
anhydrous ethanol R.
Limits :
– impurities A, B : for each impurity, not more than the area
of the peak due to acitretin in the chromatogram obtained
with reference solution (c) (0.3 per cent) ; A. (2Z,4E,6E,8E)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-
dimethylnona-2,4,6,8-tetraenoic acid,
– total : not more than the area of the peak due to acitretin
in the chromatogram obtained with reference solution (b)
(1.0 per cent) ;
– disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (c).
Palladium : maximum 10 ppm.
B. ethyl (all-E)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-
Atomic absorption spectrometry (2.2.23, Method I). dimethylnona-2,4,6,8-tetraenoate.
Test solution. Introduce 2.0 g into a quartz beaker and add
3 mL of magnesium nitrate solution R. Heat in a muffle furnace 01/2010:2445
to 350 °C at a rate of 40 °C/min to incinerate the content.
Ignite at about 450 °C for 8 h and then at 550 ± 50 °C for a
further hour. Dissolve the residue in a mixture of 0.75 mL
ADAPALENE
of hydrochloric acid R and 0.25 mL of nitric acid R, warming
gently. Cool, then transfer the solution into a volumetric Adapalenum
flask containing water R and dilute to 50.0 mL with the same
solvent.
Reference solution. Dissolve 0.163 g of heavy magnesium
oxide R in a mixture of 0.5 mL of nitric acid R, 1.5 mL of
hydrochloric acid R and 50 mL of water R, add 2.0 mL of
palladium standard solution (20 ppm Pd) R and dilute to
100.0 mL with water R.
Source : palladium hollow-cathode lamp. C28H28O3 Mr 412.5
[106685-40-9]
Wavelength : 247.6 nm.
DEFINITION
Atomisation device : air-acetylene flame.
6-(4-Methoxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)naphtha-
Heavy metals (2.4.8) : maximum 20 ppm. lene-2-carboxylic acid.
2.0 g complies with test C. Prepare the reference solution using Content : 98.0 per cent to 102.0 per cent (dried substance).
2 mL of lead standard solution (10 ppm Pb) R. CHARACTERS
Loss on drying (2.2.32) : maximum 0.5 per cent, determined Appearance : white or almost white powder.
on 1.000 g by drying in vacuo at 100 °C for 4 h. Solubility : practically insoluble in water, sparingly soluble in
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on tetrahydrofuran, practically insoluble in ethanol (96 per cent).
1.0 g.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
ASSAY
Comparison: adapalene CRS.
Carry out the assay protected from light, use amber volumetric
flasks and prepare the solutions immediately before use. TESTS
Appearance of solution. The solution is clear (2.2.1) and not
Liquid chromatography (2.2.29) as described in the test for
more intensely coloured than reference solution BY6 (2.2.2,
related substances with the following modifications.
Method II).
Injection : test solution (b) and reference solution (a). Dissolve 0.2 g in tetrahydrofuran R and dilute to 20 mL with
System suitability : the same solvent.
Related substances. Liquid chromatography (2.2.29).
– repeatability : maximum relative standard deviation of
1.0 per cent after 6 injections of reference solution (a) ; if Solvent mixture : tetrahydrofuran R, acetonitrile R, water R
necessary, adjust the integration parameters. (20:37:43 V/V/V).
Test solution (a). Dissolve 40.0 mg of the substance to be
Calculate the percentage content of C21H26O3 from the examined in 10 mL of tetrahydrofuran R, add 7 mL of the
declared content of acitretin CRS. solvent mixture and dilute to 20.0 mL with tetrahydrofuran R.
Test solution (b). Dissolve 20.0 mg of the substance to be
STORAGE examined in 50 mL of tetrahydrofuran R, add 35 mL of the
solvent mixture and dilute to 100.0 mL with tetrahydrofuran R.
In an airtight container, protected from light, at a temperature Dilute 5.0 mL of the solution to 50.0 mL with the solvent
of 2 °C to 8 °C. mixture.
It is recommended that the contents of an opened container Reference solution (a). Dilute 1.0 mL of test solution (a) to
be used as soon as possible and any unused part be protected 10.0 mL with tetrahydrofuran R. Dilute 1.0 mL of this solution
by an atmosphere of inert gas. to 100.0 mL with the solvent mixture.

General Notices (1) apply to all monographs and other texts 1485
Adapalene EUROPEAN PHARMACOPOEIA 8.0

Reference solution (b). Dissolve 2.4 mg of adapalene – total : not more than 5 times the area of the principal peak
impurity C CRS in 2 mL of tetrahydrofuran R and dilute to in the chromatogram obtained with reference solution (a)
20.0 mL with the same solvent. Dilute 2.0 mL of the solution (0.5 per cent) ;
to 20.0 mL with the solvent mixture. To 2.0 mL of this solution – disregard limit : 0.5 times the area of the principal peak in
add 2.0 mL of reference solution (a) and dilute to 20.0 mL the chromatogram obtained with reference solution (a)
with the solvent mixture. (0.05 per cent).
Reference solution (c). Dissolve the contents of a vial of Heavy metals (2.4.8) : maximum 20 ppm.
adapalene for peak identification CRS (containing impurities A,
C and D) in 0.5 mL of tetrahydrofuran R and dilute to 1.0 mL 0.250 g complies with test G. Prepare the reference solution
with the solvent mixture. using 0.5 mL of lead standard solution (10 ppm Pb) R.
Reference solution (d). Dissolve 20.0 mg of adapalene CRS in Loss on drying (2.2.32) : maximum 0.5 per cent, determined
50 mL of tetrahydrofuran R, add 35 mL of the solvent mixture on 1.000 g by drying in an oven at 105 °C for 4 h.
and dilute to 100.0 mL with tetrahydrofuran R. Dilute 5.0 mL Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
of the solution to 50.0 mL with the solvent mixture. 1.0 g.
Column :
ASSAY
– size : l = 0.25 m, Ø = 4.6 mm ;
Liquid chromatography (2.2.29) as described in the test for
– stationary phase : end-capped phenylsilyl silica gel for
related substances with the following modification.
chromatography R (5 μm) with a carbon loading of 7.5 per
cent ; Injection : test solution (b) and reference solution (d).
– temperature : 30 °C. Calculate the percentage content of adapalene from the
declared content of adapalene CRS.
Mobile phase :
– mobile phase A : glacial acetic acid R, water R (0.1:100 V/V) ; IMPURITIES
– mobile phase B : tetrahydrofuran R, acetonitrile R Specified impurities : A, C, D.
(35:65 V/V) ;
Other detectable impurities (the following substances would,
Time Mobile phase A Mobile phase B if present at a sufficient level, be detected by one or other of
(min) (per cent V/V) (per cent V/V) the tests in the monograph. They are limited by the general
0 - 2.5 50 50 acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
2.5 - 40 50 → 28 50 → 72 use (2034). It is therefore not necessary to identify these
40 - 42 28 72 impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use) : B.
Flow rate : 1.2 mL/min.
Detection : spectrophotometer at 270 nm.
Injection : 25 μL of test solution (a) and reference solutions (a),
(b) and (c).
Identification of impurities : use the chromatogram supplied
with adapalene for peak identification CRS and the
chromatogram obtained with reference solution (c) to identify
the peaks due to impurities A, C and D.
A. 2,2′-binaphthalene-6,6′-dicarboxylic acid,
Relative retention with reference to adapalene (retention
time = about 20 min) : impurity A = about 0.3 ;
impurity C = about 0.9 ; impurity D = about 1.9.
System suitability : reference solution (b) :
– resolution : minimum 4.5 between the peaks due to
impurity C and adapalene ;
– signal-to-noise ratio : minimum 10 for the peak due to
impurity C.
Limits : B. 6-[3-(3-hydroxytricyclo[3.3.1.13,7]dec-1-yl)-4-
methoxyphenyl]naphthalene-2-carboxylic acid,
– correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
corresponding correction factor : impurity A = 0.7 ;
impurity C = 7 ; impurity D = 1.4 ;
– impurity A : not more than 3 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.3 per cent) ;
C. 1-(2-methoxyphenyl)tricyclo[3.3.1.13,7]decane,
– impurity D : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.2 per cent) ;
– impurity C : not more than 1.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.15 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained D. 1,1′-[4,4′-bis(methoxy)biphenyl-3,3′-diyl]bis(tri-
with reference solution (a) (0.10 per cent) ; cyclo[3.3.1.13,7]decane).

1486 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Adenosine

01/2008:0800 Reference solution (c). Dissolve 10 mg of adenine CRS and

corrected 6.0 10 mg of adenosine R in dilute acetic acid R, with heating if
necessary, and dilute to 10 mL with the same acid.
ADENINE Apply to the plate 5 μL of each solution. Develop over a
path of 12 cm using a mixture of 20 volumes of concentrated
ammonia R, 40 volumes of ethyl acetate R and 40 volumes of
Adeninum propanol R. Dry the plate in a current of warm air and examine
in ultraviolet light at 254 nm. Any spot in the chromatogram
obtained with test solution (a), apart from the principal
spot, is not more intense than the spot in the chromatogram
obtained with reference solution (b) (0.5 per cent). The test
is not valid unless the chromatogram obtained with reference
C 5H 5N5 Mr 135.1 solution (c) shows two clearly separated spots.
[73-24-5] Chlorides (2.4.4). To 10 mL of solution S add 1 mL of
concentrated ammonia R and 3 mL of silver nitrate solution R2.
DEFINITION Filter. Wash the precipitate with a little water R and dilute the
Adenine contains not less than 98.5 per cent and not more filtrate to 15 mL with water R. The solution complies with the
than the equivalent of 101.0 per cent of 7H-purin-6-amine, limit test for chlorides (100 ppm). When carrying out the
calculated with reference to the dried substance. test, add 2 mL of dilute nitric acid R instead of 1 mL of dilute
nitric acid R.
CHARACTERS
Sulfates (2.4.13). Dilute 10 mL of solution S to 15 mL with
A white or almost white powder, very slightly soluble in water distilled water R. The solution complies with the limit test for
and in alcohol. It dissolves in dilute mineral acids and in sulfates (300 ppm).
dilute solutions of alkali hydroxides.
Ammonium. Prepare a cell consisting of two watch-glasses
IDENTIFICATION 60 mm in diameter placed edge to edge. To the inner wall
First identification : A. of the upper watch-glass stick a piece of red litmus paper R
5 mm square and wetted with a few drops of water R. Finely
Second identification : B, C. powder the substance to be examined, place 0.5 g in the
A. Examine by infrared absorption spectrophotometry lower watch-glass and suspend in 0.5 mL of water R. To the
(2.2.24), comparing with the spectrum obtained with suspension add 0.30 g of heavy magnesium oxide R. Briefly
adenine CRS. Examine the substances prepared as discs. triturate with a glass rod. Immediately close the cell by putting
B. Examine the chromatograms obtained in the test for related the two watch-glasses together. Heat at 40 °C for 15 min.
substances. The principal spot in the chromatogram The litmus paper is not more intensely blue coloured than a
obtained with test solution (b) is similar in position and standard prepared at the same time and in the same manner
size to the principal spot in the chromatogram obtained using 0.05 mL of ammonium standard solution (100 ppm
with reference solution (a). NH4) R, 0.5 mL of water R and 0.30 g of heavy magnesium
C. To 1 g add 3.5 mL of propionic anhydride R and boil for oxide R (10 ppm).
15 min with stirring. Cool. To the resulting crystalline mass Heavy metals (2.4.8). 1.0 g complies with test C for heavy
add 15 mL of light petroleum R and heat to boiling with metals (20 ppm). Prepare the reference solution using 2 mL
vigorous stirring. Cool and filter. Wash the precipitate with of lead standard solution (10 ppm Pb) R.
two quantities, each of 5 mL, of light petroleum R. Dissolve Loss on drying (2.2.32). Not more than 0.5 per cent,
the precipitate in 10 mL of water R and boil for 1 min. determined on 1.000 g by drying in an oven at 105 °C.
Filter the mixture at 30 °C to 40 °C. Allow to cool. Filter,
and dry the precipitate at 100 °C to 105 °C for 1 h. The Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
melting point (2.2.14) of the precipitate is 237 °C to 241 °C. on 1.0 g.

TESTS ASSAY
Dissolve 0.100 g in a mixture of 20 mL of acetic anhydride R
Solution S. Suspend 2.5 g in 50 mL of distilled water R and and 30 mL of anhydrous acetic acid R. Titrate with 0.1 M
boil for 3 min. Cool and dilute to 50 mL with distilled water R. perchloric acid, determining the end-point potentiometrically
Filter. Use the filtrate as solution S. (2.2.20).
Appearance of solution. Dissolve 0.5 g in dilute hydrochloric 1 mL of 0.1 M perchloric acid is equivalent to 13.51 mg of
acid R and dilute to 50 mL with the same acid. The solution is C H N .
clear (2.2.1) and colourless (2.2.2, Method II). 5 5 5

Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of

bromothymol blue solution R1 and 0.2 mL of 0.01 M sodium 01/2009:1486
hydroxide. The solution is blue. Add 0.4 mL of 0.01 M
hydrochloric acid. The solution is yellow. ADENOSINE
Related substances. Examine by thin-layer chromatography
(2.2.27), using silica gel GF254 R as the coating substance. Adenosinum
Test solution (a). Dissolve 0.10 g of the substance to be
examined in dilute acetic acid R, with heating if necessary, and
dilute to 10 mL with the same acid.
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
with dilute acetic acid R.
Reference solution (a). Dissolve 10 mg of adenine CRS in dilute
acetic acid R, with heating if necessary, and dilute to 10 mL
with the same acid.
Reference solution (b). Dilute 1 mL of test solution (b) to C10H13N5O4 Mr 267.2
20 mL with dilute acetic acid R. [58-61-7]

General Notices (1) apply to all monographs and other texts 1487
Adenosine EUROPEAN PHARMACOPOEIA 8.0

DEFINITION Limits :
9-β-D-Ribofuranosyl-9H-purin-6-amine. – correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
Content : 99.0 per cent to 101.0 per cent (dried substance). corresponding correction factor : impurity A = 0.6 ;
impurity G = 1.4 ;
CHARACTERS
– impurity A : not more than twice the area of the principal
Appearance : white or almost white, crystalline powder. peak in the chromatogram obtained with reference
Solubility : slightly soluble in water, soluble in hot water, solution (a) (0.2 per cent) ;
practically insoluble in ethanol (96 per cent) and in methylene – impurity G : not more than the area of the principal peak
chloride. It dissolves in dilute mineral acids. in the chromatogram obtained with reference solution (a)
mp : about 234 °C. (0.1 per cent) ;
– unspecified impurities : for each impurity, not more than the
IDENTIFICATION area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
Infrared absorption spectrophotometry (2.2.24).
– total : not more than 5 times the area of the principal peak
Comparison : adenosine CRS. in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
TESTS – disregard limit : 0.5 times the area of the principal peak in
Solution S. Suspend 5.0 g in 100 mL of distilled water R and the chromatogram obtained with reference solution (a)
heat to boiling. Allow to cool, filter with the aid of vacuum (0.05 per cent).
and dilute to 100 mL with distilled water R. Chlorides (2.4.4) : maximum 100 ppm.
Appearance of solution. Solution S is colourless (2.2.2, Dilute 10 mL of solution S to 15 mL with water R.
Method II). Sulfates (2.4.13) : maximum 200 ppm, determined on
Acidity or alkalinity. To 10 mL of solution S, add 0.1 mL solution S.
of bromocresol purple solution R and 0.1 mL of 0.01 M Ammonium (2.4.1, Method B) : maximum 10 ppm,
hydrochloric acid. The solution is yellow. Add 0.4 mL of determined on 0.5 g.
0.01 M sodium hydroxide. The solution is violet-blue.
Prepare the standard using 5 mL of ammonium standard
Specific optical rotation (2.2.7) : − 45 to − 49 (dried solution (1 ppm NH4) R.
substance).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Dissolve 1.25 g in 1 M hydrochloric acid and dilute to 50.0 mL on 1.000 g by drying in an oven at 105 °C.
with the same acid. Examine within 10 min of preparing the
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
solution.
1.0 g.
Related substances
ASSAY
Liquid chromatography (2.2.29).
Dissolve 0.200 g, warming slightly if necessary, in a mixture
Solvent mixture. Dissolve 6.8 g of potassium hydrogen sulfate R of 20 mL of acetic anhydride R and 30 mL of anhydrous acetic
and 3.4 g of tetrabutylammonium hydrogen sulfate R in acid R. Titrate with 0.1 M perchloric acid, determining the
water R, adjust to pH 6.5 with a 60 g/L solution of potassium end-point potentiometrically (2.2.20).
hydroxide R and dilute to 1000 mL with the same solvent. Use
1 mL of 0.1 M perchloric acid is equivalent to 26.72 mg
a freshly prepared solvent mixture.
of C10H13N5O4.
Test solution. Dissolve 20 mg of the substance to be examined
in the mobile phase and dilute to 20 mL with the mobile phase. IMPURITIES
Reference solution (a). Dilute 1.0 mL of the test solution to Specified impurities : A, G.
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution Other detectable impurities (the following substances would,
to 10.0 mL with the mobile phase. if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
Reference solution (b). Dissolve 5 mg of adenine R (impurity A) acceptance criterion for other/unspecified impurities and/or
and 5 mg of inosine R (impurity G) in the mobile phase and by the general monograph Substances for pharmaceutical use
dilute to 50 mL with the mobile phase. Dilute 4 mL of this (2034). It is therefore not necessary to identify these impurities
solution to 100 mL with the mobile phase. for demonstration of compliance. See also 5.10. Control of
Column : impurities in substances for pharmaceutical use) : F, H.
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : water R, solvent mixture (40:60 V/V).
Flow rate : 1.5 mL/min. A. 7H-purin-6-amine (adenine),
Detection : spectrophotometer at 254 nm.
Injection : 20 μL.
Run time : 1.5 times the retention time of adenosine.
Relative retention with reference to adenosine (retention
time = about 13 min) : impurity A = about 0.3 ;
impurity G = about 0.4.
System suitability : reference solution (b) :
– resolution : minimum 1.5 between the peaks due to F. 1-β-D-ribofuranosylpyrimidine-2,4(1H,3H)-dione
impurities A and G. (uridine),

1488 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Adipic acid

Reference solution (b). Dilute 1.0 mL of the test solution to

100.0 mL with the mobile phase, dilute 1.0 mL of the solution
to 10.0 mL with the mobile phase.
Column :
– size : l = 0.125 m, Ø = 4.0 mm,
– stationary phase : spherical octadecylsilyl silica gel for
chromatography R (5 μm) with a specific surface area of
350 m2/g and a pore size of 10 nm,
– temperature : 30 °C.
G. 9-β-D-ribofuranosyl-1,9-dihydro-6H-purin-6-one
(inosine), Mobile phase : mix 3 volumes of acetonitrile R and 97 volumes
of a 24.5 g/L solution of dilute phosphoric acid R.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 209 nm.
Injection : 20 μL.
Run time : 3 times the retention time of adipic acid.
System suitability : reference solution (a) :
– resolution : minimum 9.0 between the peaks due to glutaric
acid and adipic acid.
Limits :
H. 2-amino-9-β-D-ribofuranosyl-1,9-dihydro-6H-purin-6- – any impurity : not more than the area of the principal peak
one (guanosine). in the chromatogram obtained with reference solution (b)
(0.1 per cent),
– total : not more than 5 times the area of the principal peak
01/2008:1586 in the chromatogram obtained with reference solution (b)
corrected 6.0 (0.5 per cent),
– disregard limit : 0.5 times the area of the principal peak in
ADIPIC ACID the chromatogram obtained with reference solution (b)
(0.05 per cent).
Acidum adipicum Chlorides (2.4.4) : maximum 200 ppm.
Dilute 2.5 mL of solution S to 15 mL with water R.
Nitrates : maximum 30 ppm.
C6H10O4 Mr 146.1 To 1 mL of solution S add 2 mL of concentrated ammonia R,
[124-04-9] 0.5 mL of a 10 g/L solution of manganese sulfate R, 1 mL of a
10 g/L solution of sulfanilamide R and dilute to 20 mL with
DEFINITION water R. Add 0.10 g of zinc powder R and cool in iced water
Hexanedioic acid. for 30 min ; shake from time to time. Filter and cool 10 mL of
Content : 99.0 per cent to 101.0 per cent (dried substance). the filtrate in iced water. Add 2.5 mL of hydrochloric acid R1
and 1 mL of a 10 g/L solution of naphthylethylenediamine
CHARACTERS dihydrochloride R. Allow to stand at room temperature. After
Appearance : white or almost white, crystalline powder. 15 min the mixture is not more intensely coloured than a
Solubility : sparingly soluble in water, soluble in boiling water, standard prepared at the same time and in the same manner,
freely soluble in ethanol (96 per cent) and in methanol, soluble using 1.5 mL of nitrate standard solution (2 ppm NO3) R
in acetone. instead of 1 mL of solution S. The test is invalid if a blank
solution prepared at the same time and in the same manner,
IDENTIFICATION using 1 mL of water R instead of 1 mL of solution S, is more
A. Melting point (2.2.14) : 151 °C to 154 °C. intensely coloured than a 2 mg/L solution of potassium
permanganate R.
B. Infrared absorption spectrophotometry (2.2.24).
Sulfates (2.4.13) : maximum 500 ppm.
Comparison : adipic acid CRS.
Dilute 3 mL of solution S to 15 mL with distilled water R.
TESTS Iron (2.4.9) : maximum 10 ppm, determined on solution S.
Solution S. Dissolve 5.0 g with heating in distilled water R and Heavy metals (2.4.8) : maximum 10 ppm.
dilute to 50 mL with the same solvent. Allow to cool and to
crystallise. Filter through a sintered-glass filter (40) (2.1.2). 12 mL of solution S complies with test A. Prepare the reference
Wash the filter with distilled water R. Collect the filtrate and solution using lead standard solution (1 ppm Pb) R.
the washings until a volume of 50 mL is obtained. Loss on drying (2.2.32) : maximum 0.2 per cent, determined
Appearance of solution. The solution is clear (2.2.1) and on 1.000 g by drying in an oven at 105 °C.
colourless (2.2.2, Method II). Sulfated ash (2.4.14) : maximum 0.1 per cent.
Dissolve 1.0 g in methanol R and dilute to 20 mL with the Melt 1.0 g completely over a gas burner, then ignite the melted
same solvent. substance with the burner. After ignition, lower or remove the
Related substances. Liquid chromatography (2.2.29). flame in order to prevent the substance from boiling and keep
it burning until completely carbonised. Carry out the test for
Test solution. Dissolve 0.20 g of the substance to be examined sulfated ash using the residue.
in the mobile phase and dilute to 10.0 mL with the mobile
phase. ASSAY
Reference solution (a). Dissolve 20 mg of glutaric acid R in Dissolve 60.0 mg in 50 mL of water R. Add 0.2 mL of
1.0 mL of the test solution and dilute to 10.0 mL with the phenolphthalein solution R and titrate with 0.1 M sodium
mobile phase. hydroxide.

General Notices (1) apply to all monographs and other texts 1489
Adrenaline EUROPEAN PHARMACOPOEIA 8.0

1 mL of 0.1 M sodium hydroxide is equivalent to 7.31 mg of Reference solution (b). Dissolve 1.5 mg of noradrenaline
C6H10O4. tartrate CRS (impurity B) and 1.5 mg of adrenalone
hydrochloride R (impurity C) in solvent mixture B, add
IMPURITIES 1.0 mL of the test solution and dilute to 100 mL with solvent
mixture B.
Reference solution (c). Dissolve the contents of a vial of
A. R = CH2-CO2H : pentanedioic acid (glutaric acid), adrenaline impurity mixture CRS (containing impurities D
and E) in 1.0 mL of the blank solution.
B. R = CO2H : butanedioic acid (succinic acid), Reference solution (d). Dissolve 4 mg of adrenaline with
C. R = [CH2]3-CO2H : heptanedioic acid (pimelic acid). impurity F CRS in 0.5 mL of 0.1 M hydrochloric acid and
dilute to 5 mL with solvent mixture B.
Blank solution : 0.1 M hydrochloric acid, solvent mixture B
07/2008:2303 (1:9 V/V).
Column :
ADRENALINE – size : l = 0.10 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (3 μm) ;
Adrenalinum
– temperature : 50 °C.
Mobile phase :
– mobile phase A : acetonitrile R1, solvent mixture A
(5:95 V/V) ;
– mobile phase B : acetonitrile R1, solvent mixture A
C9H13NO3 Mr 183.2 (45:55 V/V) ;
[51-43-4] Time Mobile phase A Mobile phase B
DEFINITION (min) (per cent V/V) (per cent V/V)
0 - 15 92 → 50 8 → 50
4-[(1R)-1-Hydroxy-2-(methylamino)ethyl]benzene-1,2-diol.
Synthetic product. 15 - 20 50 → 92 50 → 8
Content : 99.0 per cent to 101.0 per cent (dried substance). 20 - 25 92 8

CHARACTERS Flow rate : 2.0 mL/min.

Appearance : white or almost white crystalline powder, Detection : spectrophotometer at 210 nm.
becoming coloured on exposure to air and light. Injection : 20 μL.
Solubility : practically insoluble in water, in ethanol (96 per
Identification of impurities : use the chromatogram supplied
cent) and in methylene chloride. It dissolves in hydrochloric with adrenaline impurity mixture CRS and the chromatogram
acid. obtained with reference solution (c) to identify the peaks
IDENTIFICATION due to impurities D and E ; use the chromatogram supplied
with adrenaline with impurity F CRS and the chromatogram
A. Infrared absorption spectrophotometry (2.2.24). obtained with reference solution (d) to identify the peak due
Comparison : adrenaline CRS. to impurity F.
B. Specific optical rotation (see Tests). Relative retention with reference to adrenaline
(retention time = about 4 min) : impurity F = about 0.2 ;
TESTS impurity B = about 0.8 ; impurity C = about 1.3 ;
Solution S. Dissolve 1.000 g in a 25.75 g/L solution of impurity D = about 3.3 ; impurity E = about 3.7.
hydrochloric acid R and dilute to 50.0 mL with the same System suitability : reference solution (b) :
solvent. Examine the solution immediately. – resolution : minimum 3.0 between the peaks due to
Appearance of solution. Solution S is not more opalescent impurity B and adrenaline.
than reference suspension II (2.2.1) and not more intensely Limits :
coloured than reference solution BY5 (2.2.2, Method II).
– correction factors : for the calculation of content, multiply
Specific optical rotation (2.2.7) : − 50.0 to − 54.0 (dried the peak areas of the following impurities by the
substance), determined on solution S. corresponding correction factor : impurity D = 0.7 ;
Related substances. Liquid chromatography (2.2.29). Prepare impurity E = 0.6 ;
the solutions protected from light. – impurities B, C, F : for each impurity, not more than
Solvent mixture A. Dissolve 5.0 g of potassium dihydrogen twice the area of the principal peak in the chromatogram
phosphate R and 2.6 g of sodium octanesulfonate R in water obtained with reference solution (a) (0.2 per cent) ;
for chromatography R and dilute to 1000 mL with the same – impurities D, E : for each impurity, not more than the area
solvent (it is usually necessary to stir for at least 30 min of the principal peak in the chromatogram obtained with
to achieve complete dissolution). Adjust to pH 2.8 with reference solution (a) (0.1 per cent) ;
phosphoric acid R. – unspecified impurities : for each impurity, not more than the
Solvent mixture B : acetonitrile R1, solvent mixture A area of the principal peak in the chromatogram obtained
(13:87 V/V). with reference solution (a) (0.10 per cent) ;
Test solution. Dissolve 40 mg of the substance to be examined – total : not more than 5 times the area of the principal peak
in 5 mL of 0.1 M hydrochloric acid and dilute to 50.0 mL with in the chromatogram obtained with reference solution (a)
solvent mixture B. (0.5 per cent) ;
Reference solution (a). Dilute 1.0 mL of the test solution – disregard limit : 0.5 times the area of the principal peak in
to 100.0 mL with solvent mixture B. Dilute 1.0 mL of this the chromatogram obtained with reference solution (a)
solution to 10.0 mL with solvent mixture B. (0.05 per cent).

1490 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Adrenaline tartrate

Loss on drying (2.2.32) : maximum 0.5 per cent, determined DEFINITION

on 1.000 g by drying over diphosphorus pentoxide R at a (1R)-1-(3,4-Dihydroxyphenyl)-2-(methylamino)ethanol
pressure not exceeding 0.7 kPa for 18 h. hydrogen (2R,3R)-2,3-dihydroxybutanedioate.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Content : 98.5 per cent to 101.0 per cent (dried substance).
1.0 g.
CHARACTERS
ASSAY Appearance : white or greyish-white, crystalline powder.
Dissolve 0.150 g in 50 mL of anhydrous acetic acid R. Titrate Solubility : freely soluble in water, slightly soluble in ethanol
with 0.1 M perchloric acid, determining the end-point (96 per cent).
potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 18.32 mg IDENTIFICATION
of C9H13NO3. A. Dissolve 5 g in 50 mL of a 5 g/L solution of sodium
metabisulfite R and make alkaline by addition of
STORAGE ammonia R. Keep the mixture at room temperature for at
Under nitrogen, protected from light. least 15 min and filter. Reserve the filtrate for identification
test C. Wash the precipitate with 3 quantities, each of 10 mL,
IMPURITIES of methanol R. Dry at 80 °C. The specific optical rotation
Specified impurities : B, C, D, E, F. (2.2.7) of the residue (adrenaline base) is − 53.5 to − 50,
determined using a 20.0 g/L solution in 0.5 M hydrochloric
acid.
B. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs of adrenaline base prepared as described
under identification test A.
B. (1R)-2-amino-1-(3,4-dihydroxyphenyl)ethanol Comparison: use adrenaline base prepared as described
(noradrenaline), under identification test A from 50 mg of adrenaline
tartrate CRS dissolved in 5 mL of a 5 g/L solution of sodium
metabisulfite R. Keep the mixture at room temperature for
at least 30 min. Filter through a sintered-glass filter (2.1.2).
C. 0.2 mL of the filtrate obtained in identification test A gives
reaction (b) of tartrates (2.3.1).
C. 1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanone
(adrenalone), TESTS
Appearance of solution. The solution is not more opalescent
than reference suspension II (2.2.1) and not more intensely
coloured than reference solution BY5 (2.2.2, Method II).
Dissolve 0.5 g in water R and dilute to 10 mL with the same
solvent. Examine the solution immediately.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions protected from light.
D. 4-[(1R)-2-(benzylmethylamino)-1-hydroxyethyl]benzene-
1,2-diol, Solvent mixture A. Dissolve 5.0 g of potassium dihydrogen
phosphate R and then 2.6 g of sodium octanesulfonate R in
water for chromatography R, and dilute to 1000 mL with the
same solvent (it is usually necessary to stir for at least 30 min
to achieve complete dissolution). Adjust to pH 2.8 with
phosphoric acid R.
Solvent mixture B : acetonitrile R1, solvent mixture A
(130:870 V/V).
E. 2-(benzylmethylamino)-1-(3,4-dihydroxyphenyl)ethanone, Test solution. Dissolve 75 mg of the substance to be examined
in 5 mL of 0.1 M hydrochloric acid and dilute to 50 mL with
solvent mixture B.
Reference solution (a). Dilute 1.0 mL of the test solution
to 100.0 mL with solvent mixture B. Dilute 1.0 mL of this
solution to 10.0 mL with solvent mixture B.
F. (1R)-1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanesul- Reference solution (b). Dissolve 1.5 mg of noradrenaline
fonic acid. tartrate CRS (impurity B) and 1.5 mg of adrenalone
hydrochloride R (impurity C) in solvent mixture B, add
1.0 mL of the test solution and dilute to 100.0 mL with solvent
01/2008:0254 mixture B.
Reference solution (c). Dissolve the contents of a vial of
ADRENALINE TARTRATE adrenaline impurity mixture CRS (impurities D and E) in
0.1 mL of 0.1 M hydrochloric acid and 0.9 mL of solvent
mixture B.
Adrenalini tartras
Reference solution (d). Dissolve 7.5 mg of adrenaline tartrate
with impurity A CRS in 0.5 mL of 0.1 M hydrochloric acid and
dilute to 5.0 mL with solvent mixture B.
Blank solution : 0.1 M hydrochloric acid, solvent mixture B
(1:9 V/V).
C13H19NO9 Mr 333.3 Column :
[51-42-3] – size : l = 0.10 m, Ø = 4.6 mm ;

General Notices (1) apply to all monographs and other texts 1491
Air, medicinal EUROPEAN PHARMACOPOEIA 8.0

– stationary phase : end-capped octadecylsilyl silica gel for STORAGE

chromatography R (3 μm) ; In an airtight container, or preferably in a sealed tube under
– temperature : 50 °C. vacuum or under an inert gas, protected from light.
Mobile phase : IMPURITIES
– mobile phase A : acetonitrile R1, solvent mixture A Specified impurities : A, B, C, D, E.
(5:95 V/V) ;
– mobile phase B : acetonitrile R1, solvent mixture A A. unknown structure,
(45:55 V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 15 92 → 50 8 → 50

15 - 20 50 → 92 50 → 8 B. (1R)-2-amino-1-(3,4-dihydroxyphenyl)ethanol
(noradrenaline),
20 - 25 92 8

Flow rate : 2.0 mL/min.

Detection : spectrophotometer at 210 nm.
Injection : 20 μL.
Identification of impurities : use the chromatogram supplied C. 1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanone
with adrenaline impurity mixture CRS and the chromatogram (adrenalone),
obtained with reference solution (c) to identify the peaks
due to impurities D and E ; use the chromatogram supplied
with adrenaline tartrate with impurity A CRS and the
chromatogram obtained with reference solution (d) to identify
the peak due to impurity A.
Relative retention with reference to adrenaline
(retention time = about 4 min): impurity B = about 0.8 ;
impurity C = about 1.3 ; impurity A = about 3.2 ; D. 4-[(1R)-2-(benzylmethylamino)-1-hydroxyethyl]benzene-
impurity D = about 3.3 ; impurity E = about 3.7. 1,2-diol,
System suitability : reference solution (b) :
– resolution : minimum 3.0 between the peaks due to
impurity B and adrenaline.
Limits :
– correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
corresponding correction factor : impurity D = 0.7 ;
E. 2-(benzylmethylamino)-1-(3,4-dihydroxyphenyl)ethanone.
impurity E = 0.6 ;
– impurity A : not more than 3 times the area of the principal
peak in the chromatogram obtained with reference 01/2009:1238
solution (a) (0.3 per cent) ;
– impurities B, C : for each impurity, not more than twice the AIR, MEDICINAL
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.2 per cent) ;
Aer medicinalis
– impurities D, E : for each impurity, not more than the area
of the principal peak in the chromatogram obtained with DEFINITION
reference solution (a) (0.1 per cent) ; Compressed ambient air.
– unspecified impurities : for each impurity, not more than the Content : 20.4 per cent V/V to 21.4 per cent V/V of oxygen (O2).
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ; CHARACTERS
– total : not more than 6 times the area of the principal peak Appearance : colourless gas.
in the chromatogram obtained with reference solution (a) Solubility : at 20 °C at a pressure of 101 kPa, 1 volume dissolves
(0.6 per cent) ; in about 50 volumes of water.
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a) PRODUCTION
(0.05 per cent). Carbon dioxide : maximum 500 ppm V/V, determined using
Loss on drying (2.2.32) : maximum 0.5 per cent, determined an infrared analyser (2.5.24).
on 1.000 g by drying in vacuo for 18 h. Gas to be examined. Filter the substance to be examined to
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on avoid stray light phenomena.
1.0 g. Reference gas (a). Use a mixture of 21 per cent V/V of oxygen R
and 79 per cent V/V of nitrogen R1, containing less than
ASSAY 1 ppm V/V of carbon dioxide R1.
Dissolve 0.300 g in 50 mL of anhydrous acetic acid R, heating Reference gas (b). Use a mixture of 21 per cent V/V of oxygen R
gently if necessary. Titrate with 0.1 M perchloric acid until a and 79 per cent V/V of nitrogen R1, containing 500 ppm V/V
bluish-green colour is obtained, using 0.1 mL of crystal violet of carbon dioxide R1.
solution R as indicator. Calibrate the apparatus and set the sensitivity using reference
1 mL of 0.1 M perchloric acid is equivalent to 33.33 mg gases (a) and (b). Measure the content of carbon dioxide in
of C13H19NO9. the gas to be examined.

1492 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Air, medicinal

Carbon monoxide : maximum 5 ppm V/V, determined using Reference gas (a). Use a mixture of 21 per cent V/V of oxygen R
an infrared analyser (2.5.25). and 79 per cent V/V of nitrogen R1, containing less than
Gas to be examined. Filter the substance to be examined to 0.05 ppm V/V of nitrogen monoxide and nitrogen dioxide.
avoid stray light phenomena. Reference gas (b). Use a mixture of 2 ppm V/V of nitrogen
Reference gas (a). Use a mixture of 21 per cent V/V of oxygen R monoxide R in nitrogen R1.
and 79 per cent V/V of nitrogen R1, containing less than Calibrate the apparatus and set the sensitivity using reference
1 ppm V/V of carbon monoxide R. gases (a) and (b). Measure the content of nitrogen monoxide
and nitrogen dioxide in the gas to be examined.
Reference gas (b). Use a mixture of 21 per cent V/V of oxygen R
and 79 per cent V/V of nitrogen R1, containing 5 ppm V/V Water : maximum 67 ppm V/V, determined using an
of carbon monoxide R. electrolytic hygrometer (2.5.28), except where the competent
authority decides that the following limit applies to medicinal
Calibrate the apparatus and set the sensitivity using reference air generated on-site and distributed in pipe-line systems
gases (a) and (b). Measure the content of carbon monoxide in operating at a pressure not greater than 10 bars and a
the gas to be examined. temperature not less than 5 °C : maximum 870 ppm V/V,
Sulfur dioxide : maximum 1 ppm V/V, determined using an determined using an electrolytic hygrometer (2.5.28).
ultraviolet fluorescence analyser (Figure 1238.-1). Assay. Determine the concentration of oxygen in air using a
The apparatus consists of the following : paramagnetic analyser (2.5.27).
– a system generating ultraviolet radiation with a wavelength IDENTIFICATION
of 210 nm, made up of an ultraviolet lamp, a collimator,
and a selective filter ; the beam is blocked periodically by a First identification : C.
chopper rotating at high speeds ; Second identification : A, B.
– a reaction chamber, through which flows the gas to be A. In a conical flask containing the substance to be examined,
examined ; place a glowing wood splinter. The splinter remains
glowing.
– a system that detects radiation emitted at a wavelength of B. Use a gas burette (Figure 1238.-2) of 25 mL capacity in
350 nm, made up of a selective filter, a photomultiplier the form of a chamber in the middle of which is a tube
tube and an amplifier. graduated in 0.2 per cent between 19.0 per cent and 23.0 per
Gas to be examined. Filter the substance to be examined. cent, and isolated at each end by a tap with a conical barrel.
Reference gas (a). Use a mixture of 21 per cent V/V of oxygen R The lower tap is joined to a tube with an olive-shaped
and 79 per cent V/V of nitrogen R1. nozzle and is used to introduce the gas into the apparatus. A
cylindrical funnel above the upper tap is used to introduce
Reference gas (b). Use a mixture of 21 per cent V/V of oxygen R the absorbent solution. Wash the burette with water R and
and 79 per cent V/V of nitrogen R1, containing 0.5 ppm V/V dry. Open the 2 taps. Connect the nozzle to the source of
to 2 ppm V/V of sulfur dioxide R1. the gas to be examined and set the flow rate to 1 L/min.
Calibrate the apparatus and set the sensitivity using reference Flush the burette by passing the gas to be examined
gases (a) and (b). Measure the content of sulfur dioxide in through it for 1 min. Close the lower tap of the burette and
the gas to be examined. immediately afterwards the upper tap. Rapidly disconnect
Oil : maximum 0.1 mg/m3, determined using an oil detector the burette from the source of the gas to be examined.
tube (2.1.6), when an oil-lubricated compressor is used for Rapidly give a half turn to the upper tap to eliminate any
the production. excess pressure in the burette. Keeping the burette vertical,
fill the funnel with a freshly prepared mixture of 21 mL of a
Nitrogen monoxide and nitrogen dioxide : maximum 560 g/L solution of potassium hydroxide R and 130 mL of
2 ppm V/V in total, determined using a chemiluminescence a 200 g/L solution of sodium dithionite R. Open the upper
analyser (2.5.26). tap slowly. The solution absorbs the oxygen and enters the
Gas to be examined. The substance to be examined. burette. Allow to stand for 10 min without shaking. Read

Figure 1238.-1. – UV fluorescence analyser

General Notices (1) apply to all monographs and other texts 1493
Air, synthetic medicinal EUROPEAN PHARMACOPOEIA 8.0

the level of the liquid meniscus on the graduated part of systems operating at a pressure not greater than 10 bars and
the burette. This figure represents the percentage V/V of a temperature not less than 5 °C : maximum 870 ppm V/V,
oxygen. The value read is 20.4 to 21.4. determined using a water vapour detector tube (2.1.6).
STORAGE
As a gas, in suitable containers complying with the legal
regulations or as a gas supplied by a pipe network.
LABELLING
Where applicable, the label states the production method, as
regards to the use of an oil - lubricated compression.
IMPURITIES
A. CO2 : carbon dioxide,
B. SO2 : sulfur dioxide,
C. NO : nitrogen monoxide,
D. NO2 : nitrogen dioxide,
E. oil,
F. CO : carbon monoxide,
G. H2O : water.

01/2008:1684

AIR, SYNTHETIC MEDICINAL

Aer medicinalis artificiosus
DEFINITION
Mixture of Nitrogen (1247) and Oxygen (0417).
Content : 95.0 per cent to 105.0 per cent of the nominal value
which is between 21.0 per cent V/V to 22.5 per cent V/V of
oxygen (O2).
CHARACTERS
Colourless and odourless gas.
Solubility : at a temperature of 20 °C and a pressure of 101 kPa,
1 volume dissolves in about 50 volumes of water.
PRODUCTION
Water (2.5.28) : maximum 67 ppm V/V.
Assay (2.5.27). Carry out the determination of oxygen in
gases.
IDENTIFICATION
First identification : C.
Second identification : A, B.
Figure 1238.-2. – Gas burette A. In a conical flask containing the substance to be examined,
place a glowing splinter of wood. The splinter remains
C. It complies with the limits of the assay. glowing.
B. Use a gas burette (Figure 1684.-1) of 25 mL capacity in
TESTS the form of a chamber, in the middle of which is a tube
Carbon dioxide : maximum 500 ppm V/V, determined using graduated in 0.2 per cent between 19.0 per cent and
a carbon dioxide detector tube (2.1.6). 23.0 per cent, and isolated at each end by a tap with a
Sulfur dioxide : maximum 1 ppm V/V, determined using a conical barrel. The lower tap is joined to a tube with an
sulfur dioxide detector tube (2.1.6). olive-shaped nozzle and is used to introduce the gas into
the apparatus. A cylindrical funnel above the upper tap
Oil : maximum 0.1 mg/m3, determined using an oil detector is used to introduce the absorbent solution. Wash the
tube (2.1.6), when an oil-lubricated compressor is used for burette with water R and dry. Open both taps. Connect the
the production. nozzle to the source of the substance to be examined and
Nitrogen monoxide and nitrogen dioxide : maximum set the flow rate to 1 L/min. Flush the burette by passing
2 ppm V/V, determined using a nitrogen monoxide and the substance to be examined through it for 1 min. Close
nitrogen dioxide detector tube (2.1.6). the lower tap of the burette and immediately afterwards the
upper tap. Rapidly disconnect the burette from the source
Carbon monoxide : maximum 5 ppm V/V, determined using of the substance to be examined. Rapidly give a half turn
a carbon monoxide detector tube (2.1.6). of the upper tap to eliminate any excess pressure in the
Water vapour : maximum 67 ppm V/V, determined using burette. Keeping the burette vertical, fill the funnel with a
a water vapour detector tube (2.1.6), except where the freshly prepared mixture of 21 mL of a 560 g/L solution of
competent authority decides that the following limit applies potassium hydroxide R and 130 mL of a 200 g/L solution
to medicinal air generated on-site and distributed in pipe-line of sodium dithionite R. Open the upper tap slowly. The

1494 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Alanine

solution absorbs the oxygen and enters the burette. Allow 01/2008:0752
to stand for 10 min without shaking. Read the level of the corrected 6.0
liquid meniscus on the graduated part of the burette. This
figure represents the percentage V/V of oxygen. The value ALANINE
read is 95.0 per cent to 105.0 per cent of the nominal value.
Alaninum

C3H7NO2 Mr 89.1
[56-41-7]
DEFINITION
Alanine contains not less than 98.5 per cent and not more
than the equivalent of 101.0 per cent of (S)-2-aminopropanoic
acid, calculated with reference to the dried substance.
CHARACTERS
White or almost white, crystalline powder or colourless
crystals, freely soluble in water, very slightly soluble in alcohol.
IDENTIFICATION
First identification : A, B.
Second identification : A, C, D.
A. Specific optical rotation (see Tests).
B. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
alanine CRS. Examine the substances prepared as discs.
C. Examine the chromatograms obtained in the test for
ninhydrin-positive substances. The principal spot in the
chromatogram obtained with test solution (b) is similar
in position, colour and size to the principal spot in the
chromatogram obtained with reference solution (a).
D. Dissolve 0.5 g in a mixture of 1 mL of water R, 0.5 mL
of a 100 g/L solution of sodium nitrite R and 0.25 mL of
hydrochloric acid R1. Shake. Gas is given off. Add 2 mL of
dilute sodium hydroxide solution R, followed by 0.25 mL of
iodinated potassium iodide solution R. After about 30 min,
a yellow precipitate with a characteristic odour is formed.
TESTS
Solution S. Dissolve 2.5 g in distilled water R and dilute to
50 mL with the same solvent.
Appearance of solution. Dilute 10 mL of solution S to
20 mL with water R. The solution is clear (2.2.1) and not
more intensely coloured than reference solution BY6 (2.2.2,
Method II).
Specific optical rotation (2.2.7). Dissolve 2.50 g in
hydrochloric acid R1 and dilute to 25.0 mL with the same acid.
The specific optical rotation is + 13.5 to + 15.5, calculated with
reference to the dried substance.
Ninhydrin-positive substances. Examine by thin-layer
Figure 1684.-1.– Gas burette chromatography (2.2.27), using a TLC silica gel plate R.
C. It complies with the limits of the assay. Test solution (a). Dissolve 0.10 g in water R and dilute to
10 mL with the same solvent.
TESTS
Test solution (b). Dilute 1 mL of test solution (a) to 50 mL
Water vapour: maximum 67 ppm V/V, determined using a with water R.
water vapour detector tube (2.1.6). Reference solution (a). Dissolve 10 mg of alanine CRS in
STORAGE water R and dilute to 50 mL with the same solvent.
Reference solution (b). Dilute 5 mL of test solution (b) to
As a compressed gas in suitable containers complying with the 20 mL with water R.
legal regulations or as a compressed gas supplied by a pipe
network, after mixing of the components. Reference solution (c). Dissolve 10 mg of alanine CRS and
10 mg of glycine CRS in water R and dilute to 25 mL with the
LABELLING same solvent.
The label states the nominal content of O2 in per cent V/V. Apply separately to the plate 5 μL of each solution. Allow
the plate to dry in air. Develop over a path of 15 cm with a
IMPURITIES mixture of 20 volumes of glacial acetic acid R, 20 volumes of
water R and 60 volumes of butanol R. Allow the plate to dry in
A. H2O : water. air. Spray with ninhydrin solution R. Heat the plate at 100 °C

General Notices (1) apply to all monographs and other texts 1495
Albendazole EUROPEAN PHARMACOPOEIA 8.0

to 105 °C for 15 min. Any spot in the chromatogram obtained TESTS

with test solution (a), apart from the principal spot, is not Appearance of solution. The solution is clear (2.2.1) and not
more intense than the spot in the chromatogram obtained more intensely coloured than reference solution BY6 (2.2.2,
with reference solution (b) (0.5 per cent). The test is not valid Method II).
unless the chromatogram obtained with reference solution (c)
shows two clearly separated spots. Dissolve 0.10 g in a mixture of 1 volume of anhydrous formic
acid R and 9 volumes of methylene chloride R and dilute to
Chlorides (2.4.4). Dilute 5 mL of solution S to 15 mL with 10 mL with the same mixture of solvents.
water R. The solution complies with the limit test for chlorides
(200 ppm). Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be
Sulfates (2.4.13). Dilute 10 mL of solution S to 15 mL with
examined in 5 mL of methanol R containing 1 per cent V/V of
distilled water R. The solution complies with the limit test for
sulfuric acid R and dilute to 50.0 mL with the mobile phase.
sulfates (300 ppm).
Reference solution (a). Dissolve 10.0 mg of the substance
Ammonium (2.4.1). 50 mg complies with limit test B for to be examined in 10 mL of methanol R containing 1 per
ammonium (200 ppm). Prepare the standard using 0.1 mL of cent V/V of sulfuric acid R and dilute to 100.0 mL with the
ammonium standard solution (100 ppm NH4) R. mobile phase. Dilute 0.5 mL of this solution to 20.0 mL with
Iron (2.4.9). In a separating funnel, dissolve 1.0 g in 10 mL of the mobile phase.
dilute hydrochloric acid R. Shake with three quantities, each of Reference solution (b). Dissolve 50.0 mg of the substance
10 mL, of methyl isobutyl ketone R1, shaking for 3 min each to be examined and 50 mg of oxibendazole CRS in 5 mL of
time. To the combined organic layers add 10 mL of water R methanol R containing 1 per cent V/V of sulfuric acid R and
and shake for 3 min. The aqueous layer complies with the dilute to 100.0 mL with the mobile phase.
limit test for iron (10 ppm).
Column :
Heavy metals (2.4.8). Dissolve 2.0 g in water R and dilute to – size : l = 0.25 m, Ø = 4.6 mm ;
20 mL with the same solvent. 12 mL of the solution complies
with test A for heavy metals (10 ppm). Prepare the reference – stationary phase : spherical end-capped octadecylsilyl silica
solution using lead standard solution (1 ppm Pb) R. gel for chromatography R (5 μm) with a pore size of 10 nm
and a carbon loading of 19 per cent.
Loss on drying (2.2.32). Not more than 0.5 per cent,
Mobile phase : mix 300 volumes of a 1.67 g/L solution of
determined on 1.000 g by drying in an oven at 105 °C.
ammonium dihydrogen phosphate R and 700 volumes of
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined methanol R.
on 1.0 g. Flow rate : 0.7 mL/min.
ASSAY Detection : spectrophotometer at 254 nm.
Dissolve 80.0 mg in 3 mL of anhydrous formic acid R. Injection : 20 μL.
Add 30 mL of anhydrous acetic acid R. Using 0.1 mL of Run time : 1.5 times the retention time of albendazole.
naphtholbenzein solution R as indicator, titrate with 0.1 M Relative retention with reference to albendazole :
perchloric acid, until the colour changes from brownish-yellow impurity D = about 0.40 ; impurities B and C = about 0.43 ;
to green. impurity E = about 0.47 ; impurity F = about 0.57 ;
1 mL of 0.1 M perchloric acid is equivalent to 8.91 mg of impurity A = about 0.80.
C3H7NO2.
System suitability : reference solution (b) :
STORAGE – resolution : minimum 3.0 between the peaks due to
Store protected from light. albendazole and oxibendazole.
Limits :
01/2008:1386 – impurities A, B, C, D, E, F : for each impurity, not more
corrected 6.0 than 1.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
ALBENDAZOLE (0.75 per cent);
– total : not more than 3 times the area of the principal peak
Albendazolum in the chromatogram obtained with reference solution (a)
(1.5 per cent) ;
– disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent).
C12H15N3O2S Mr 265.3 Loss on drying (2.2.32) : maximum 0.5 per cent, determined
[54965-21-8] on 1.000 g by drying in an oven at 105 °C for 4 h.
Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
DEFINITION 1.0 g.
Methyl [5-(propylsulfanyl)-1H-benzimidazol-2-yl]carbamate.
Content : 98.0 per cent to 102.0 per cent (dried substance). ASSAY
In order to avoid overheating during the titration, mix
CHARACTERS thoroughly throughout and stop the titration immediately after
Appearance : white or slightly yellowish powder. the end-point has been reached.
Solubility : practically insoluble in water, freely soluble in Dissolve 0.250 g in 3 mL of anhydrous formic acid R and add
anhydrous formic acid, very slightly soluble in methylene 40 mL of anhydrous acetic acid R. Titrate with 0.1 M perchloric
chloride, practically insoluble in ethanol (96 per cent). acid, determining the end-point potentiometrically (2.2.20).
IDENTIFICATION 1 mL of 0.1 M perchloric acid is equivalent to 26.53 mg
of C12H15N3O2S.
Infrared absorption spectrophotometry (2.2.24).
Preparation : discs. STORAGE
Comparison : albendazole CRS. Protected from light.

1496 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Alcuronium chloride

IMPURITIES Reference solution. Dissolve 10 mg of alcuronium

Specified impurities : A, B, C, D, E, F. chloride CRS in methanol R and dilute to 10 mL with the
same solvent.
Plate : TLC silica gel plate R.
Mobile phase : mix 15 volumes of a 58.4 g/L solution of
sodium chloride R, 35 volumes of dilute ammonia R2 and
A. R = S-CH2-CH2-CH3 : 5-(propylsulfanyl)-1H-benzimidazol- 50 volumes of methanol R.
2-amine, Application : 10 μL.
D. R = SO2-CH2-CH2-CH3 : 5-(propylsulfonyl)-1H- Development : over a path of 15 cm.
benzimidazol-2-amine, Drying : in air for 10 min.
Detection : spray with 0.1 M ammonium and cerium nitrate.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
B. R = SO-CH2-CH2-CH3 : methyl [5-(propylsulfinyl)-1H- the reference solution.
benzimidazol-2-yl]carbamate, C. It gives reaction (a) of chlorides (2.3.1).
C. R = SO2-CH2-CH2-CH3 : methyl [5-(propylsulfonyl)-1H- TESTS
benzimidazol-2-yl]carbamate,
Solution S. Dissolve 0.250 g in carbon dioxide-free water R
E. R = H : methyl (1H-benzimidazol-2-yl)carbamate, and dilute to 25.0 mL with the same solvent.
F. R = S-CH3 : methyl [5-(methylsulfanyl)-1H-benzimidazol- Appearance of solution. Solution S is clear (2.2.1) and not
2-yl]carbamate. more intensely coloured than reference solution Y6, BY6 or B6
(2.2.2, Method I).
01/2008:1285 Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of
methyl red solution R and 0.2 mL of 0.01 M hydrochloric acid.
ALCURONIUM CHLORIDE The solution is red. Add 0.4 mL of 0.01 M sodium hydroxide.
The solution is yellow.
Alcuronii chloridum Specific optical rotation (2.2.7) : − 430 to − 451 (anhydrous
substance), determined on solution S.
Propan-2-ol (2.4.24, System A) : maximum 1.0 per cent.
Related substances. Liquid chromatography (2.2.29).
Solvent mixture. Mix 100 mL of methanol R, 200 mL
of acetonitrile R and 200 mL of a 6.82 g/L solution of
potassium dihydrogen phosphate R. Dissolve 1.09 g of sodium
laurylsulfonate for chromatography R in the mixture and adjust
the apparent pH to 8.0 with a 100 g/L solution of sodium
hydroxide R.
Test solution. Dissolve 0.20 g of the substance to be examined
in the solvent mixture and dilute to 100.0 mL with the solvent
C44H50Cl2N4O2 Mr 738 mixture.
[15180-03-7]
Reference solution (a). Dilute 0.5 mL of the test solution to
DEFINITION 100.0 mL with the solvent mixture.
(1R,3aS,10S,11aS,12R,14aS,19aS,20bS,21S,22aS,23E,26E)- Reference solution (b). Dilute 4.0 mL of reference solution (a)
23,26-bis(2-Hydroxyethylidene)-1,12-bis(prop-2-enyl)- to 10.0 mL with the solvent mixture.
2,3,11,11a,13,14,22,22a-octahydro-10H,21H-1,21:10,12- Reference solution (c). Dilute 1.0 mL of reference solution (a)
diethano-19aH,20bH-[1,5]diazocino[1,2,3-lm:5,6,7- to 10.0 mL with the solvent mixture.
l′m′]dipyrrolo[2′,3′-d:2′′,3′′ :d′]dicarbazolediium dichloride Reference solution (d). To 5.0 mL of the test solution add
(4,4′-didesmethyl-4,4′-bis(prop-2-enyl)toxiferin I dichloride). 5.0 mg of allylstrychnine bromide CRS, dissolve in the solvent
Content : 98.0 per cent to 102.0 per cent (anhydrous substance). mixture and dilute to 100.0 mL with the solvent mixture.
CHARACTERS Column :
Appearance : white or slightly greyish-white, crystalline – size : l = 0.25 m, Ø = 4 mm ;
powder. – stationary phase : octylsilyl silica gel for chromatography R
Solubility : freely soluble in water and in methanol, soluble in (5 μm).
ethanol (96 per cent), practically insoluble in cyclohexane. Mobile phase : mix 200 mL of methanol R, 400 mL of
Carry out the identification, tests and assay as rapidly as acetonitrile R and 400 mL of a 6.82 g/L solution of
possible avoiding exposure to actinic light. potassium dihydrogen phosphate R. Dissolve 2.18 g of sodium
laurylsulfonate for chromatography R in the mixture and adjust
IDENTIFICATION the apparent pH to 5.4 with a 100 g/L solution of phosphoric
First identification : A, C. acid R.
Second identification : B, C. Flow rate : 1.2 mL/min.
A. Infrared absorption spectrophotometry (2.2.24). Detection : spectrophotometer at 254 nm.
Comparison : alcuronium chloride CRS. Injection : 10 μL.
B. Thin-layer chromatography (2.2.27). Run time : twice the retention time of alcuronium.
Test solution. Dissolve 10 mg of the substance to be System suitability : reference solution (d) :
examined in methanol R and dilute to 10 mL with the same – resolution : minimum 4.0 between the peaks due to
solvent. N-allylstrychnine and alcuronium.

General Notices (1) apply to all monographs and other texts 1497
Alfacalcidol EUROPEAN PHARMACOPOEIA 8.0

Limits : 01/2014:1286
– impurities A, B : for each impurity, not more than the
area of the principal peak in the chromatogram obtained ALFACALCIDOL
with reference solution (a) (0.5 per cent) and not more
than one of the peaks has an area greater than the area Alfacalcidolum
of the principal peak in the chromatogram obtained with
reference solution (b) (0.2 per cent) ;
– total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (a)
(1 per cent) ;
– disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (c)
(0.05 per cent).
Water (2.5.12) : maximum 5.0 per cent, determined on 0.500 g.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. C27H44O2 Mr 400.6
[41294-56-8]
ASSAY DEFINITION
(5Z,7E)-9,10-Secocholesta-5,7,10(19)-triene-1α,3β-diol.
Dissolve 0.300 g by stirring in 70 mL of acetic anhydride R
for 1 min. Titrate with 0.1 M perchloric acid until the colour Content : 97.0 per cent to 102.0 per cent.
changes from violet-blue to greenish-blue, using 0.1 mL of A reversible isomerisation to pre-alfacalcidol takes place in
crystal violet solution R as indicator. solution, depending on temperature and time. The activity is
due to both compounds (see Assay).
1 mL of 0.1 M perchloric acid is equivalent to 36.9 mg
of C44H50Cl2N4O2. CHARACTERS
Appearance : white or almost white crystals.
STORAGE Solubility : practically insoluble in water, freely soluble in
ethanol (96 per cent), soluble in fatty oils.
In an airtight container under nitrogen, protected from light,
at a temperature of 2 °C to 8 °C. It is sensitive to air, heat and light.

IDENTIFICATION
IMPURITIES
A. Infrared absorption spectrophotometry (2.2.24).
Specified impurities : A, B. Comparison: Ph. Eur. reference spectrum of alfacalcidol.
B. Examine the chromatograms obtained in the test for related
substances.
Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time and size
to the principal peak in the chromatogram obtained with
reference solution (a).
TESTS
Related substances. Liquid chromatography (2.2.29) : use
the normalisation procedure. Carry out the test as rapidly as
possible, avoiding exposure to light and air.
Test solution. Dissolve 1.0 mg of the substance to be examined
A. (1R,3aS,9R,9aR,10R,11aS,12R,14aS,19aS,20R,- without heating in 10.0 mL of the mobile phase.
20aR,20bS,21R,22aS)-1,12-bis(prop-2-enyl)- Reference solution (a). Dissolve 1.0 mg of alfacalcidol CRS
2,3,9a,11,11a,13,14,19a,20a,21,22,22a-dodecahydro- without heating in 10.0 mL of the mobile phase.
10H,20bH-1,23:12,27-dimethano-9,10:20,21- Reference solution (b). Dilute 1.0 mL of reference solution (a)
bis(epoxyprop[2]eno)-9H,20H-[1,5]diazocino[1,2,3- to 100.0 mL with the mobile phase. Dilute 1.0 mL of this
lm:5,6,7-l′m′]dipyrrolo[2′,3′-d:2′′,3′′ :d′]dicarbazolediium solution to 20.0 mL with the mobile phase.
dichloride (4,4′-diallylcaracurin V dichloride), Reference solution (c). In order to prepare pre-alfacalcidol in
situ, dissolve the contents of a vial of alfacalcidol for system
suitability CRS (containing impurities A and B) in 25 mL of
the mobile phase, heat in a water-bath at 80 °C under a reflux
condenser for 2 h and cool.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : ammonia R, water R, acetonitrile R
(1:200:800 V/V/V).
B. (4bS,7R,7aS,8aR,13R,13aR,13bS)-13-hydroxy-7-(prop-
2-enyl)-5,6,7a,8,8a,11,13,13a,13b,14-decahydro-7,9- Flow rate : 2.6 mL/min.
methano-7H-oxepino[3,4-a]pyrrolo[2,3-d]carbazolium Detection : spectrophotometer at 265 nm.
chloride ((4R,17R)-4-allyl-17,18-epoxy-17-hydroxy-19,20- Injection : 100 μL of the test solution and reference solutions (b)
didehydrocuranium chloride). and (c).

1498 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Alfadex

Run time : twice the retention time of alfacalcidol.

Identification of impurities : use the chromatogram
supplied with alfacalcidol for system suitability CRS and the
chromatogram obtained with reference solution (c) to identify
the peaks due to impurities A and B.
Relative retention with reference to alfacalcidol (retention
time = about 21 min) : pre-alfacalcidol = about 0.88 ;
impurity A = about 0.93 ; impurity B = about 1.1.
System suitability: reference solution (c) :
– resolution : minimum 1.5 between the peaks due to B. (5Z,7E)-9,10-secocholesta-5,7,10(19)-triene-1β,3β-diol
pre-alfacalcidol and impurity A and minimum 1.5 between (1β-calcidol),
the peaks due to impurity A and alfacalcidol.
Limits :
– impurities A, B : for each impurity, maximum 0.5 per cent ;
– unspecified impurities : for each impurity, maximum
0.10 per cent ;
– total : maximum 1.0 per cent ;
– disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.05 per cent) ; disregard the peak due to pre-alfacalcidol.
C. 6ξ-[(3S,5R)-3,5-dihydroxy-2-methylcyclohex-1-en-1-yl]-
ASSAY 2-phenyl-2,5,10-triaza-4,19-dinor-9ξ-cholest-7-ene-1,3-
dione.
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications. 01/2012:1487
Injection : test solution and reference solutions (a) and (c).
System suitability: reference solution (c) : ALFADEX
– repeatability : maximum relative standard deviation of 1 per Alfadexum
cent for the peak due to alfacalcidol after 6 injections.
Calculate the percentage content of C27H44O2 taking into
account the assigned content of alfacalcidol CRS and, if
necessary, the peak due to pre-alfacalcidol.

STORAGE
Under nitrogen, in an airtight container, protected from light,
at a temperature of 2 °C to 8 °C.
The contents of an opened container are to be used
immediately.

IMPURITIES
Specified impurities : A, B. [C6H10O5]6 Mr 973
[10016-20-3]
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of DEFINITION
the tests in the monograph. They are limited by the general Cyclohexakis-(1→4)-(α-D-glucopyranosyl) (cyclomaltohexaose
acceptance criterion for other/unspecified impurities and/or or α-cyclodextrin).
by the general monograph Substances for pharmaceutical Content : 97.0 per cent to 102.0 per cent (dried substance).
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10. CHARACTERS
Control of impurities in substances for pharmaceutical use): C. Appearance : white or almost white, amorphous or crystalline
powder.
Solubility : freely soluble in water and in propylene glycol,
practically insoluble in anhydrous ethanol and in methylene
chloride.
IDENTIFICATION
A. Specific optical rotation (see Tests).
B. Examine the chromatograms obtained in the assay.
Results : the principal peak in the chromatogram obtained
with test solution (b) is similar in retention time and size
to the principal peak in the chromatogram obtained with
reference solution (c).
C. Dissolve 0.2 g in 2 mL of iodine solution R4 by warming on
A. (5E,7E)-9,10-secocholesta-5,7,10(19)-triene-1α,3β-diol a water-bath, and allow to stand at room temperature ; a
(trans-alfacalcidol), yellowish-brown precipitate is formed.

General Notices (1) apply to all monographs and other texts 1499
Alfadex EUROPEAN PHARMACOPOEIA 8.0

TESTS – sum of impurities other than A and B : not more than

Solution S. Dissolve 1.000 g in carbon dioxide-free water R 0.5 times the area of the peak due to alfadex in the
and dilute to 100.0 mL with the same solvent. chromatogram obtained with reference solution (b) (0.5 per
cent).
Appearance of solution. Solution S is clear (2.2.1).
Heavy metals (2.4.8) : maximum 10 ppm.
pH (2.2.3) : 5.0 to 8.0.
2.0 g complies with test C. Prepare the reference solution using
Mix 1 mL of a 223.6 g/L solution of potassium chloride R and 2 mL of lead standard solution (10 ppm Pb) R.
30 mL of solution S.
Loss on drying (2.2.32) : maximum 11 per cent, determined
Specific optical rotation (2.2.7) : + 147 to + 152 (dried on 1.000 g by drying in an oven at 120 °C for 2 h.
substance), determined on solution S.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Reducing sugars : maximum 0.2 per cent. 1.0 g.
Test solution. To 1 mL of solution S add 1 mL of cupri-tartaric
solution R4. Heat on a water-bath for 10 min, cool to room ASSAY
temperature. Add 10 mL of ammonium molybdate reagent R1
Liquid chromatography (2.2.29) as described in the test for
and allow to stand for 15 min.
related substances with the following modifications.
Reference solution. Prepare a reference solution at the same
time and in the same manner as the test solution, using 1 mL Injection : test solution (b) and reference solutions (a) and (c).
of a 0.02 g/L solution of glucose R. System suitability :
Measure the absorbance (2.2.25) of the test solution and the – repeatability : maximum relative standard deviation of
reference solution at the absorption maximum at 740 nm using 2.0 per cent for the peak due to alfadex after 5 injections of
water R as the compensation liquid. The absorbance of the reference solution (a).
test solution is not greater than that of the reference solution.
Calculate the percentage content of [C6H10O5]6 from the
Light-absorbing impurities. Examine solution S between declared content of alfadex CRS.
230 nm and 750 nm. Between 230 nm and 350 nm, the
absorbance (2.2.25) is not greater than 0.10. Between 350 nm STORAGE
and 750 nm, the absorbance (2.2.25) is not greater than 0.05.
In an airtight container.
Related substances. Liquid chromatography (2.2.29).
Test solution (a). Dissolve 0.25 g of the substance to be IMPURITIES
examined in water R with heating, cool and dilute to 25.0 mL
with the same solvent. Specified impurities : A, B.
Test solution (b). Dilute 5.0 mL of test solution (a) to 50.0 mL
with water R.
Reference solution (a). Dissolve 25.0 mg of betadex CRS
(impurity A), 25.0 mg of gammacyclodextrin CRS (impurity B)
and 50.0 mg of alfadex CRS in water R, then dilute to 50.0 mL
with the same solvent.
Reference solution (b). Dilute 5.0 mL of reference solution (a)
to 50.0 mL with water R.
Reference solution (c). Dissolve 25.0 mg of alfadex CRS in
water R and dilute to 25.0 mL with the same solvent.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (10 μm).
A. cycloheptakis-(1→4)-(α-D-glucopyranosyl) (betadex or
Mobile phase : methanol R, water R (10:90 V/V). cyclomaltoheptaose or β-cyclodextrin),
Flow rate : 1.5 mL/min.
Detection : differential refractometer.
Equilibration : with the mobile phase for about 3 h.
Injection : 50 μL of test solution (a) and reference solutions (a)
and (b).
Run time : 3.5 times the retention time of alfadex.
Relative retention with reference to alfadex (retention
time = about 10 min) : impurity B = about 0.7 ;
impurity A = about 2.2.
System suitability: reference solution (a) :
– resolution : minimum 1.5 between the peaks due
to impurity B and alfadex ; if necessary, adjust the
concentration of methanol in the mobile phase.
Limits :
– impurities A, B : for each impurity, not more than 0.5 times
the area of the corresponding peak in the chromatogram B. cyclooctakis-(1→4)-(α-D-glucopyranosyl)
obtained with reference solution (b) (0.25 per cent) ; (cyclomaltooctaose or γ-cyclodextrin).

1500 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Alfentanil hydrochloride

01/2008:1062 Time Mobile phase A Mobile phase B

corrected 7.0 (min) (per cent V/V) (per cent V/V)
0 - 15 90 → 40 10 → 60

ALFENTANIL HYDROCHLORIDE 15 - 20 40 60

20 - 25 40 → 90 60 → 10
Alfentanili hydrochloridum Flow rate : 1.5 mL/min.
Detection : spectrophotometer at 220 nm.
Equilibration : with acetonitrile R for at least 30 min and then
with the mobile phase at the initial composition for at least
5 min.
Injection : 10 μL ; inject methanol R as a blank.
Retention time : impurity E = about 6 min ; alfentanil = about
7 min.
C21H33ClN6O3 Mr 453.0 Identification of impurities: use the chromatogram obtained
[69049-06-5] with reference solution (a) to identify the peak due to
impurity E ; disregard any other peak.
DEFINITION System suitability : reference solution (a) :
N-[1-[2-(4-Ethyl-4,5-dihydro-5-oxo-1H-tetrazol-1-yl)ethyl]- – resolution : minimum 4.0 between the peaks due to
4-(methoxymethyl)piperidin-4-yl]-N-phenylpropanamide alfentanil and impurity E ; if necessary, adjust the
hydrochloride. concentration of acetonitrile in the mobile phase or adjust
Content : 98.5 per cent to 101.5 per cent (anhydrous substance). the time programme for the linear-gradient elution.
Limits :
CHARACTERS – impurities A, B, C, D, E, F, G, H : for each impurity, not more
Appearance : white or almost white powder. than the area of the principal peak in the chromatogram
Solubility : freely soluble in water, in ethanol (96 per cent) and obtained with reference solution (b) (0.25 per cent) ;
in methanol. – total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (b)
mp : about 140 °C, with decomposition. (0.5 per cent) ;
IDENTIFICATION – disregard limit : 0.2 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
A. Infrared absorption spectrophotometry (2.2.24). (0.05 per cent) ; disregard any peak due to the blank.
Comparison : Ph. Eur. reference spectrum of alfentanil Water (2.5.12) : 3.0 per cent to 4.0 per cent, determined on
hydrochloride. 0.500 g.
B. Dissolve 50 mg in a mixture of 0.4 mL of ammonia R ASSAY
and 2 mL of water R. Mix, allow to stand for 5 min and
filter. Acidify the filtrate with dilute nitric acid R. It gives Dissolve 0.350 g in 50 mL of a mixture of 1 volume of ethanol
reaction (a) of chlorides (2.3.1). (96 per cent) R and 4 volumes of water R and add 5.0 mL of
0.01 M hydrochloric acid. Titrate with 0.1 M sodium hydroxide,
TESTS determining the end-point potentiometrically (2.2.20). Read
the volume added between the 2 points of inflexion.
Appearance of solution. The solution is clear (2.2.1) and 1 mL of 0.1 M sodium hydroxide is equivalent to 45.30 mg
colourless (2.2.2, Method II). of C21H33ClN6O3.
Dissolve 0.2 g in water R and dilute to 20 mL with the same
solvent. STORAGE
Protected from light.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.100 g of the substance to be examined IMPURITIES
in methanol R and dilute to 10.0 mL with the same solvent. Specified impurities : A, B, C, D, E, F, G, H.
Reference solution (a). In order to produce impurity E in situ,
dissolve 10 mg of the substance to be examined in 10.0 mL of
dilute hydrochloric acid R. Heat on a water-bath under a reflux
condenser for 4 h. Neutralise with 10.0 mL of dilute sodium
hydroxide solution R. Evaporate to dryness on a water-bath.
Cool and take up the residue in 10 mL of methanol R. Filter.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with methanol R. Dilute 5.0 mL of this solution to
20.0 mL with methanol R.
Column : A. cis-N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol-
1-yl)ethyl]-4-(methoxymethyl)piperidin-4-yl]-N-
– size : l = 0.1 m, Ø = 4.6 mm ; phenylpropanamide N-oxide,
– stationary phase : octadecylsilyl silica gel for
chromatography R (3 μm).
Mobile phase :
– mobile phase A : 5 g/L solution of ammonium carbonate R
in a mixture of 10 volumes of tetrahydrofuran R and B. trans-N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol-
90 volumes of water R ; 1-yl)ethyl]-4-(methoxymethyl)piperidin-4-yl]-N-
– mobile phase B : acetonitrile R ; phenylpropanamide N-oxide,

General Notices (1) apply to all monographs and other texts 1501
Alfuzosin hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Solubility : freely soluble in water, sparingly soluble in ethanol

(96 per cent), practically insoluble in methylene chloride.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
C. N-[4-(methoxymethyl)piperidin-4-yl]-N-phenylpropan-
amide, Comparison: alfuzosin hydrochloride CRS.
B. It gives reaction (a) of chlorides (2.3.1).
TESTS
pH (2.2.3) : 4.0 to 5.5.
Dissolve 0.500 g in carbon dioxide-free water R and dilute
D. N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol- to 25.0 mL with the same solvent. Use a freshly prepared
1-yl)ethyl]-4-(methoxymethyl)piperidin-4-yl]-N- solution.
phenylacetamide,
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 40 mg of the substance to be examined
in the mobile phase and dilute to 100.0 mL with the mobile
phase.
Reference solution (a). Dilute 1.0 mL of the test solution to
E. 1-ethyl-1,4-dihydro-4-[2-[[4-(methoxymethyl)-4- 100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
phenylamino]piperidin-1-yl]ethyl]-5H-tetrazol-5-one, to 10.0 mL with the mobile phase.
Reference solution (b). Dissolve 4 mg of alfuzosin for system
suitability CRS (containing impurities A and D) in the mobile
phase and dilute to 10 mL with the mobile phase.
Column :
F. N-[1-(2-hydroxyethyl)-4-(methoxymethyl)piperidin-4-yl]- – size : l = 0.15 m, Ø = 4.6 mm ;
N-phenylpropanamide, – stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : mix 1 volume of tetrahydrofuran R, 20 volumes
of acetonitrile R and 80 volumes of a solution prepared
as follows : dilute 5.0 mL of perchloric acid R in 900 mL
of water R, adjust to pH 3.5 with dilute sodium hydroxide
solution R and dilute to 1000 mL with water R.
G. N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol-1- Flow rate : 1.5 mL/min.
yl)ethyl]-4-(propanoyloxymethyl)piperidin-4-yl]-N- Detection : spectrophotometer at 254 nm.
phenylpropanamide, Injection : 10 μL.
Run time : twice the retention time of alfuzosin.
Identification of impurities : use the chromatogram supplied
with alfuzosin for system suitability CRS and the chromatogram
obtained with reference solution (b) to identify the peaks due
H. N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol- to impurities A and D.
1-yl)ethyl]-4-(methoxymethyl)piperidin-4-yl]-N- Relative retention with reference to alfuzosin (retention
phenylbutanamide. time = about 8 min) : impurity D = about 0.4 ;
impurity A = about 1.2.
04/2008:1287 System suitability : reference solution (b) :
– peak-to-valley ratio : minimum 5.0, where Hp = height
ALFUZOSIN HYDROCHLORIDE above the baseline of the peak due to impurity A and
Hv = height above the baseline of the lowest point of the
Alfuzosini hydrochloridum curve separating this peak from the peak due to alfuzosin.
Limits :
– impurity D : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.2 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
C19H28ClN5O4 Mr 425.9 with reference solution (a) (0.10 per cent) ;
[81403-68-1] – total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
DEFINITION (0.3 per cent) ;
(2RS)-N-[3-[(4-Amino-6,7-dimethoxyquinazolin-2- – disregard limit : 0.5 times the area of the principal peak in
yl)methylamino]propyl]tetrahydrofuran-2-carboxamide the chromatogram obtained with reference solution (a)
hydrochloride. (0.05 per cent).
Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
Water (2.5.12) : maximum 0.5 per cent, determined on
CHARACTERS 1.000 g.
Appearance : white or almost white, crystalline powder, slightly Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
hygroscopic. 1.0 g.

1502 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Alginic acid

ASSAY IDENTIFICATION
Dissolve 0.300 g in a mixture of 40 mL of anhydrous acetic A. To 0.2 g add 20 mL of water R and 0.5 mL of sodium
acid R and 40 mL of acetic anhydride R. Titrate with 0.1 M carbonate solution R. Shake and filter. To 5 mL of the filtrate
perchloric acid, determining the end-point potentiometrically add 1 mL of calcium chloride solution R. A voluminous
(2.2.20). gelatinous mass is formed.
1 mL of 0.1 M perchloric acid is equivalent to 42.59 mg B. To 5 mL of the filtrate obtained in identification test A add
of C19H28ClN5O4. 0.5 mL of a 123 g/L solution of magnesium sulfate R. No
voluminous gelatinous mass is formed.
STORAGE
C. To 5 mg add 5 mL of water R, 1 mL of a freshly prepared
In an airtight container, protected from light. 10 g/L solution of 1,3-dihydroxynaphthalene R in ethanol
IMPURITIES (96 per cent) R and 5 mL of hydrochloric acid R. Boil gently
for 3 min, cool, add 5 mL of water R, and shake with 15 mL
Specified impurities : D. of di-isopropyl ether R. Carry out a blank test. The upper
Other detectable impurities (the following substances would, layer obtained with the substance to be examined exhibits a
if present at a sufficient level, be detected by one or other of deeper bluish-red colour than that obtained with the blank.
the tests in the monograph. They are limited by the general
TESTS
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical useChlorides : maximum 1.0 per cent.
(2034). It is therefore not necessary to identify these impurities
To 2.50 g add 50 mL of dilute nitric acid R, shake for 1 h and
for demonstration of compliance. See also 5.10. Control ofdilute to 100.0 mL with dilute nitric acid R. Filter. To 50.0 mL
impurities in substances for pharmaceutical use): A, B, C, E.
of the filtrate add 10.0 mL of 0.1 M silver nitrate and 5 mL of
toluene R. Titrate with 0.1 M ammonium thiocyanate, using
2 mL of ferric ammonium sulfate solution R2 as indicator and
shaking vigorously towards the end-point.
1 mL of 0.1 M silver nitrate is equivalent to 3.545 mg of Cl.
Heavy metals (2.4.8) : maximum 20 ppm.
A. N-[3-[(4-amino-6,7-dimethoxyquinazolin-2- 1.0 g complies with test F. Prepare the reference solution using
yl)methylamino]propyl]furan-2-carboxamide, 2 mL of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 15.0 per cent, determined
on 0.1000 g by drying in an oven at 105 °C for 4 h.
Sulfated ash (2.4.14) : maximum 8.0 per cent (dried
substance), determined on 0.100 g.
Microbial contamination
B. R = Cl : 2-chloro-6,7-dimethoxyquinazolin-4-amine,
TAMC : acceptance criterion 102 CFU/g (2.6.12).
D. R = N(CH3)-[CH2]3-NH2 : N-(4-amino-6,7- Absence of Escherichia coli (2.6.13).
dimethoxyquinazolin-2-yl)-N-methylpropane-1,3-diamine,
Absence of Salmonella (2.6.13).
E. R = N(CH3)-[CH2]3-NH-CO-H : N-[3-[(4-amino-6,7-di-
methoxyquinazolin-2-yl)methylamino]propyl]formamide, ASSAY
To 0.2500 g add 25 mL of water R, 25.0 mL of 0.1 M sodium
hydroxide and 0.2 mL of phenolphthalein solution R. Titrate
with 0.1 M hydrochloric acid.
1 mL of 0.1 M sodium hydroxide is equivalent to 4.502 mg
of carboxyl groups (-CO2H).
FUNCTIONALITY-RELATED CHARACTERISTICS
C. (2RS)-N-[3-[(4-amino-6,7-dimethoxyquinazolin-
2-yl)amino]propyl]-N-methyltetrahydrofuran-2- This section provides information on characteristics that are
carboxamide. recognised as being relevant control parameters for one or
more functions of the substance when used as an excipient (see
chapter 5.15). This section is a non-mandatory part of the
01/2009:0591
monograph and it is not necessary to verify the characteristics
to demonstrate compliance. Control of these characteristics can
ALGINIC ACID however contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process and
Acidum alginicum the performance of the medicinal product during use. Where
control methods are cited, they are recognised as being suitable
DEFINITION for the purpose, but other methods can also be used. Wherever
Mixture of polyuronic acids [(C6H8O6)n] composed of residues results for a particular characteristic are reported, the control
of D-mannuronic and L-guluronic acids, obtained mainly from method must be indicated.
algae belonging to the Phaeophyceae. A small proportion of The following characteristics may be relevant for alginic acid
the carboxyl groups may be neutralised. used as disintegrant and/or binder.
Content : 19.0 per cent to 25.0 per cent of carboxyl groups
(-CO2H) (dried substance). Particle-size distribution (2.9.31 or 2.9.38).
Settling volume. Place 75 mL of water R in a 100 mL
CHARACTERS graduated cylinder and add 1.5 g of the substance to be
Appearance : white or pale yellowish-brown, crystalline or examined in 0.5 g portions, shaking vigorously after each
amorphous powder. addition. Dilute to 100.0 mL with water R and shake again
Solubility : very slightly soluble or practically insoluble in until the substance is homogeneously distributed. Allow to
ethanol (96 per cent), practically insoluble in organic solvents. stand for 4 h and determine the volume of the settled mass.
It swells in water but does not dissolve ; it dissolves in solutions The following characteristic may be relevant for alginic acid
of alkali hydroxides. used as gelling agent or viscosity-increasing agent.

General Notices (1) apply to all monographs and other texts 1503
Alimemazine hemitartrate EUROPEAN PHARMACOPOEIA 8.0

Apparent viscosity. Determine the dynamic viscosity using a Detection : spectrophotometer at 253 nm.
rotating viscometer (2.2.10). Injection : 20 μL.
Prepare a 20 g/L suspension of alginic acid (dried substance) Run time : twice the retention time of alimemazine.
and add 0.1 M sodium hydroxide until a solution is obtained. Identification of impurities : use the chromatogram supplied
with alimemazine for system suitability CRS and the
01/2014:2650 chromatogram obtained with reference solution (b) to identify
the peaks due to impurities A, B and C.
ALIMEMAZINE HEMITARTRATE Relative retention with reference to alimemazine
(retention time = about 27 min) : impurity A = about 0.1 ;
impurity B = about 0.5 ; impurity C = about 1.4.
Alimemazini hemitartras System suitability : reference solution (b) :
– resolution : minimum 5.0 between the peaks due to
alimemazine and impurity C.
Calculation of percentage contents:
– correction factors : multiply the peak areas of the following
impurities by the corresponding correction factor :
impurity A = 4.4 ; impurity C = 0.4 ;
C20H25N2O3S Mr 373.5 – for each impurity, use the concentration of alimemazine in
[4330-99-8] reference solution (a).
DEFINITION Limits:
– impurity B : maximum 0.3 per cent ;
(2RS)-N,N,2-Trimethyl-3-(10H-phenothiazin-10-yl)propan-1-
amine hemi[(2R,3R)-2,3-dihydroxybutanedioate]. – impurities A, C : for each impurity, maximum 0.15 per cent ;
Content : 99.0 per cent to 101.0 per cent (dried substance). – unspecified impurities : for each impurity, maximum
0.10 per cent ;
CHARACTERS – total : maximum 0.5 per cent ;
Appearance : white or very slightly yellowish powder. – reporting threshold : 0.05 per cent.
Solubility : freely soluble in water, sparingly soluble in ethanol Loss on drying (2.2.32) : maximum 0.5 per cent, determined
(96 per cent), practically insoluble in toluene. on 1.000 g by drying in an oven at 105 °C for 3 h.
It deteriorates when exposed to air and light. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24). ASSAY
Comparison : alimemazine hemitartrate CRS. Dissolve 0.300 g in 50 mL of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point
TESTS potentiometrically (2.2.20).
Appearance of solution. The solution is not more opalescent 1 mL of 0.1 M perchloric acid is equivalent to 37.35 mg of
than reference suspension II (2.2.1) and not more intensely C20H25N2O3S.
coloured than reference solution BY5 (2.2.2, Method II).
Dissolve 1.0 g in water R and dilute to 10 mL with the same STORAGE
solvent. In an airtight container, protected from light.
pH (2.2.3) : 5.0 to 6.5. Carry out the test protected from light IMPURITIES
and use a freshly prepared solution.
Specified impurities : A, B, C.
Dissolve 1.0 g in carbon dioxide-free water R and dilute to
50 mL with the same solvent.
Related substances. Liquid chromatography (2.2.29). Carry
out the test protected from light and use freshly prepared
solutions.
Solvent mixture : acetonitrile R, water R (20:80 V/V).
Test solution. Dissolve 35 mg of the substance to be examined
in the solvent mixture and dilute to 100.0 mL with the solvent A. (2RS)-N,N,2-trimethyl-3-(5-oxido-10H-phenothiazin-10-
mixture. yl)propan-1-amine,
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Reference solution (b). Dissolve 3.5 mg of alimemazine for
system suitability CRS (containing impurities A, B and C) in
the solvent mixture and dilute to 10.0 mL with the solvent
mixture. B. (2RS)-N,2-dimethyl-3-(10H-phenothiazin-10-yl)propan-
Column : 1-amine,
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : base-deactivated end-capped octadecylsilyl
silica gel for chromatography R (3 μm) ;
– temperature : 40 °C.
Mobile phase : acetonitrile R, methanol R, 3.854 g/L solution of
ammonium acetate R (10:40:50 V/V/V).
Flow rate : 1.3 mL/min. C. 10H-phenothiazine.

1504 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Allopurinol

01/2008:1288 Reference solution (a). Dissolve 10 mg of allantoin CRS in a

corrected 6.0 mixture of 1 volume of methanol R and 1 volume of water R
and dilute to 10 mL with the same mixture of solvents.
ALLANTOIN Reference solution (b). Dissolve 10 mg of urea R in 10 mL of
water R. Dilute 1 mL of this solution to 10 mL with methanol R.
Allantoinum Reference solution (c). Mix 1 mL of reference solution (a) and
1 mL of reference solution (b).
Apply to the plate 10 μL of test solution (a) and 5 μL each of
test solution (b), reference solution (a), reference solution (b)
and reference solution (c). Develop over a path of 10 cm
using a mixture of 15 volumes of glacial acetic acid R,
25 volumes of water R and 60 volumes of butanol R. Allow
C 4H 6N4O 3 Mr 158.1 the plate to dry in air. Spray the plate with a 5 g/L solution
[97-59-6] of dimethylaminobenzaldehyde R in a mixture of 1 volume
of hydrochloric acid R and 3 volumes of methanol R. Dry
DEFINITION the plate in a current of hot air. Examine in daylight after
Allantoin contains not less than 98.5 per cent and 30 min. Any spot in the chromatogram obtained with test
not more than the equivalent of 101.0 per cent of solution (a), apart from the principal spot, is not more intense
(RS)-(2,5-dioxoimidazolidin-4-yl)urea. than the spot in the chromatogram obtained with reference
solution (b) (0.5 per cent). The test is not valid unless the
CHARACTERS chromatogram obtained with reference solution (c) shows two
A white or almost white, crystalline powder, slightly soluble clearly separated principal spots.
in water, very slightly soluble in alcohol.
Loss on drying (2.2.32). Not more than 0.1 per cent,
It melts at about 225 °C, with decomposition. determined on 1.000 g by drying in an oven at 105 °C.
IDENTIFICATION Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
First identification : A. on 1.0 g.
Second identification : B, C, D. ASSAY
A. Examine by infrared absorption spectrophotometry Dissolve 120.0 mg in 40 mL of water R. Titrate with
(2.2.24), comparing with the spectrum obtained with 0.1 M sodium hydroxide, determining the end-point
allantoin CRS. potentiometrically (2.2.20).
B. Examine the chromatograms obtained in the test for related 1 mL of 0.1 M sodium hydroxide is equivalent to 15.81 mg
substances. The principal spot in the chromatogram of C4H6N4O3.
obtained with test solution (b) is similar in position,
IMPURITIES
colour and size to the principal spot in the chromatogram
obtained with reference solution (a).
C. Boil 20 mg with a mixture of 1 mL of dilute sodium
hydroxide solution R and 1 mL of water R. Allow to cool. A. glyoxylic acid,
Add 1 mL of dilute hydrochloric acid R. To 0.1 mL of the
solution add 0.1 mL of a 100 g/L solution of potassium
bromide R, 0.1 mL of a 20 g/L solution of resorcinol R and
3 mL of sulfuric acid R. Heat for 5 min to 10 min on a
water-bath. A dark blue colour develops, which becomes B. carbamide (urea).
red after cooling and pouring into about 10 mL of water R.
D. Heat about 0.5 g. Ammonia vapour is evolved, which turns 01/2008:0576
red litmus paper R blue. corrected 6.8

TESTS ALLOPURINOL
Solution S. Dissolve 0.5 g in carbon dioxide-free water R,
with heating if necessary, and dilute to 100 mL with the same Allopurinolum
solvent.
Acidity or alkalinity. To 5 mL of solution S add 5 mL of
carbon dioxide-free water R, 0.1 mL of methyl red solution R
and 0.2 mL of 0.01 M sodium hydroxide. The solution is yellow.
Add 0.4 mL of 0.01 M hydrochloric acid. The solution is red.
Optical rotation (2.2.7). The angle of optical rotation, C 5H 4N4O Mr 136.1
determined on solution S, is − 0.10° to + 0.10°. [315-30-0]
Reducing substances. Shake 1.0 g with 10 mL of water R for DEFINITION
2 min. Filter. Add 1.5 mL of 0.02 M potassium permanganate.
The solution must remain violet for at least 10 min. 1,5-Dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one.
Content : 97.0 per cent to 102.0 per cent (dried substance).
Related substances. Examine by thin-layer chromatography
(2.2.27), using a suitable cellulose for chromatography R as the CHARACTERS
coating substance. Appearance : white or almost white powder.
Test solution (a). Dissolve 0.10 g of the substance to be Solubility : very slightly soluble in water and in ethanol (96 per
examined in 5.0 mL of water R with heating. Allow to cool. cent). It dissolves in dilute solutions of alkali hydroxides.
Dilute to 10 mL with methanol R. Use the solution immediately
after preparation. IDENTIFICATION
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL with First identification : B.
a mixture of 1 volume of methanol R and 1 volume of water R. Second identification : A, C, D.

General Notices (1) apply to all monographs and other texts 1505
Allopurinol EUROPEAN PHARMACOPOEIA 8.0

A. Ultraviolet and visible absorption spectrophotometry Injection : 20 μL of test solution (a) and reference solutions (a)
(2.2.25). and (b).
Test solution. Dissolve 10 mg in 1 mL of a 4 g/L solution Run time : twice the retention time of allopurinol.
of sodium hydroxide R and dilute to 100.0 mL with a Elution order : impurity A, impurity B, impurity C, allopurinol.
10.3 g/L solution of hydrochloric acid R. Dilute 10.0 mL
Retention time : allopurinol = about 10 min.
of this solution to 100.0 mL with a 10.3 g/L solution of
hydrochloric acid R. System suitability : reference solution (b) :
Spectral range : 220-350 nm. – resolution : minimum 1.1 between the peaks due to
Absorption maximum : at 250 nm. impurities B and C.
Absorption minimum : at 231 nm. Limits :
Absorbance ratio : A231/A250 = 0.52 to 0.62. – impurity A : not more than twice the area of the principal
peak in the chromatogram obtained with reference
B. Infrared absorption spectrophotometry (2.2.24). solution (a) (0.2 per cent) ;
Comparison : allopurinol CRS.
– impurity B : not more than the area of the principal peak
C. Dissolve 0.3 g in 2.5 mL of dilute sodium hydroxide in the chromatogram obtained with reference solution (a)
solution R and add 50 mL of water R. Add slowly and (0.1 per cent) ;
with shaking 5 mL of silver nitrate solution R1. A white
precipitate is formed which does not dissolve on the – impurity C : not more than the area of the corresponding
addition of 5 mL of ammonia R. peak in the chromatogram obtained with reference
solution (b) (0.1 per cent) ;
D. Thin-layer chromatography (2.2.27).
– unspecified impurities : for each impurity, not more than the
Test solution. Dissolve 20 mg of the substance to be area of the principal peak in the chromatogram obtained
examined in concentrated ammonia R and dilute to 10 mL with reference solution (a) (0.10 per cent) ;
with the same solvent.
– sum of impurities other than A, B and C : not more than
Reference solution. Dissolve 20 mg of allopurinol CRS in 3 times the area of the principal peak in the chromatogram
concentrated ammonia R and dilute to 10 mL with the obtained with reference solution (a) (0.3 per cent) ;
same solvent.
– disregard limit : 0.5 times the area of the principal peak in
Plate : TLC silica gel F254 plate R. the chromatogram obtained with reference solution (a)
Mobile phase : anhydrous ethanol R, methylene chloride R (0.05 per cent).
(40:60 V/V).
Impurities D and E. Liquid chromatography (2.2.29). Use
Application : 10 μL. freshly prepared solutions. Store and inject them at 8 °C, using
Development : over 2/3 of the plate. a cooled autosampler.
Drying : in air. Solution A : 1.25 g/L solution of potassium dihydrogen
Detection : examine in ultraviolet light at 254 nm. phosphate R.
Results : the principal spot in the chromatogram obtained Test solution. Dissolve 50.0 mg of the substance to be
with the test solution is similar in position and size to the examined in 5.0 mL of a 4 g/L solution of sodium hydroxide R
principal spot in the chromatogram obtained with the and dilute immediately to 100.0 mL with solution A.
reference solution. Reference solution. Dissolve 5.0 mg of allopurinol
TESTS impurity D CRS and 5.0 mg of allopurinol impurity E CRS in
5.0 mL of a 4 g/L solution of sodium hydroxide R and dilute
Related substances. Liquid chromatography (2.2.29). Use immediately to 100.0 mL with solution A. Dilute 1.0 mL of
freshly prepared solutions. Store and inject them at 8 °C, using this solution to 100.0 mL with solution A.
a cooled autosampler.
Column :
Test solution (a). Dissolve 25.0 mg of the substance to be
examined in 2.5 mL of a 4 g/L solution of sodium hydroxide R – size : l = 0.05 m, Ø = 4.6 mm ;
and dilute immediately to 50.0 mL with the mobile phase. – stationary phase : base-deactivated octadecylsilyl silica gel for
Test solution (b). Dissolve 20.0 mg of the substance to be chromatography R (3 μm).
examined in 5.0 mL of a 4 g/L solution of sodium hydroxide R Mobile phase : methanol R, 1.25 g/L solution of potassium
and dilute immediately to 250.0 mL with the mobile phase. dihydrogen phosphate R (10:90 V/V).
Reference solution (a). Dilute 2.0 mL of test solution (a) to Flow rate : 2 mL/min.
100.0 mL with the mobile phase. Dilute 5.0 mL of this solution Detection : spectrophotometer at 230 nm.
to 100.0 mL with the mobile phase. Injection : 20 μL.
Reference solution (b). Dissolve 5 mg of allopurinol Run time : 1.5 times the retention time of impurity E.
impurity A CRS, 5 mg of allopurinol impurity B CRS and
5.0 mg of allopurinol impurity C CRS in 5.0 mL of a 4 g/L Retention times : impurity D = about 3.6 min ;
solution of sodium hydroxide R and dilute immediately to impurity E = about 4.5 min.
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution System suitability : reference solution :
to 100.0 mL with the mobile phase. – resolution : minimum 2.0 between the peaks due to
Reference solution (c). Dissolve 20.0 mg of allopurinol CRS in impurities D and E.
5.0 mL of a 4 g/L solution of sodium hydroxide R and dilute Limits :
immediately to 250.0 mL with the mobile phase. – impurity D : not more than the area of the corresponding
Column : peak in the chromatogram obtained with the reference
– size : l = 0.25 m, Ø = 4.6 mm ; solution (0.1 per cent) ;
– stationary phase : octadecylsilyl silica gel for – impurity E : not more than the area of the corresponding
chromatography R (5 μm). peak in the chromatogram obtained with the reference
Mobile phase : 1.25 g/L solution of potassium dihydrogen solution (0.1 per cent).
phosphate R. Impurity F. Liquid chromatography (2.2.29).
Flow rate : 1.4 mL/min. Under the following conditions, any hydrazine in the sample
Detection : spectrophotometer at 230 nm. reacts with benzaldehyde to give benzaldehyde azine.

1506 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Almagate

Solvent mixture. Mix equal volumes of dilute sodium hydroxide

solution R and methanol R.
Solution A. Dissolve 2.0 g of benzaldehyde R in the solvent
mixture and dilute to 50.0 mL with the solvent mixture.
Prepare immediately before use.
A. R1 = NH2, R2 = H : 5-amino-1H-pyrazole-4-carboxamide,
Test solution. Dissolve 250.0 mg of the substance to be
examined in 5 mL of the solvent mixture. Add 4 mL B. R1 = NH2, R2 = CHO : 5-(formylamino)-1H-pyrazole-4-
of solution A, mix and allow to stand for 2.5 h at room carboxamide,
temperature. Add 5.0 mL of hexane R and shake for 1 min. D. R1 = O-C2H5, R2 = H : ethyl 5-amino-1H-pyrazole-4-
Allow the layers to separate and use the upper layer. carboxylate,
Reference solution. Dissolve 10.0 mg of hydrazine sulfate R in E. R1 = O-C2H5, R2 = CHO : ethyl 5-(formylamino)-1H-
the solvent mixture by sonicating for about 2 min and dilute pyrazole-4-carboxylate,
to 50.0 mL with the solvent mixture. Dilute 1.0 mL to 20.0 mL
with the solvent mixture. Dilute 1.0 mL of this solution to
20.0 mL with the solvent mixture. To 5.0 mL of the solution
obtained, add 4 mL of solution A, mix and allow to stand for
2.5 h at room temperature. Add 5.0 mL of hexane R and shake
for 1 min. Allow the layers to separate and use the upper layer.
Blank solution. To 5 mL of the solvent mixture add 4 mL
of solution A, mix and allow to stand for 2.5 h at room C. 5-(4H-1,2,4-triazol-4-yl)-1H-pyrazole-4-carboxamide,
temperature. Add 5.0 mL of hexane R and shake for 1 min. F. H2N-NH2 : diazane (hydrazine).
Allow the layers to separate and use the upper layer.
Column : 01/2009:2010
corrected 7.0
– size : l = 0.25 m, Ø = 4.0 mm ;
– stationary phase : cyanosilyl silica gel for chromatography R ALMAGATE
(5 μm) with a pore size of 10 nm ;
– temperature : 30 °C. Almagatum
Mobile phase : 2-propanol R, hexane R (5:95 V/V). Al2Mg6C2O20H14,4H2O Mr 630
Flow rate : 1.5 mL/min. [66827-12-1]
Detection : spectrophotometer at 310 nm. DEFINITION
Injection : 20 μL. Hydrated aluminium magnesium hydroxycarbonate.
Relative retention with reference to benzaldehyde (retention Content :
time = about 2.8 min) : benzaldehyde azine = about 0.8. – aluminium : 15.0 per cent to 17.0 per cent (calculated
System suitability: reference solution : as Al2O3),
– magnesium : 36.0 per cent to 40.0 per cent (calculated
– resolution : minimum 2 between the peaks due to
as MgO),
benzaldehyde azine and benzaldehyde ;
– carbonic acid : 12.5 per cent to 14.5 per cent (calculated
– signal-to-noise ratio : minimum 20 for the peak due to as CO2).
benzaldehyde azine.
Limit : CHARACTERS
Appearance : white or almost white, fine, crystalline powder.
– impurity F : the area of the peak due to benzaldehyde azine
in the chromatogram obtained with the test solution is Solubility : practically insoluble in water, in ethanol (96 per
not more than the area of the corresponding peak in cent) and in methylene chloride. It dissolves with effervescence
the chromatogram obtained with the reference solution and heating in dilute mineral acids.
(10 ppm of hydrazine sulfate equivalent to 2.5 ppm of IDENTIFICATION
hydrazine). A. Infrared absorption spectrophotometry (2.2.24).
Heavy metals (2.4.8) : maximum 20 ppm. Comparison: Ph. Eur. reference spectrum of almagate.
1.0 g complies with test C. Prepare the reference solution using B. Dissolve 0.15 g in dilute hydrochloric acid R and dilute to
2 mL of lead standard solution (10 ppm Pb) R. 20 mL with the same acid. 2 mL of the solution gives the
Loss on drying (2.2.32) : maximum 0.5 per cent, determined reaction of aluminium (2.3.1).
on 1.000 g by drying in an oven at 105 °C. C. 2 mL of the solution prepared under identification test B
gives the reaction of magnesium (2.3.1).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. TESTS
pH (2.2.3) : 9.1 to 9.7.
ASSAY Disperse 4.0 g in 100 mL of carbon dioxide-free water R, stir
Liquid chromatography (2.2.29) as described in the test for for 2 min and filter.
related substances with the following modification. Neutralising capacity. Carry out the test at 37 °C. Disperse
Injection : test solution (b) and reference solution (c). 0.5 g in 100 mL of water R, heat, add 100.0 mL of 0.1 M
Calculate the percentage content of C5H4N4O from the hydrochloric acid, previously heated and stir continuously ; the
declared content of allopurinol CRS. pH (2.2.3) of the solution between 5 min and 20 min is not
less than 3.0 and not greater than 4.5. Add 10.0 mL of 0.5 M
hydrochloric acid, previously heated, stir continuously for 1 h
IMPURITIES and titrate with 0.1 M sodium hydroxide to pH 3.5 ; not more
Specified impurities : A, B, C, D, E, F. than 20.0 mL of 0.1 M sodium hydroxide is required.

General Notices (1) apply to all monographs and other texts 1507
Almond oil, refined EUROPEAN PHARMACOPOEIA 8.0

Chlorides (2.4.4) : maximum 0.1 per cent. Carrier gas : helium for chromatography R.
Dissolve 0.33 g in 5 mL of dilute nitric acid R and dilute to Flow rate : 100 mL/min.
100 mL with water R. Prepare simultaneously the standard by Temperature :
diluting 0.7 mL of dilute nitric acid R to 5 mL with water R – column : 65 °C ;
and adding 10 mL of chloride standard solution (5 ppm Cl) R.
– detector : 190 °C.
Sulfates (2.4.13) : maximum 0.4 per cent.
Detection : thermal conductivity.
Dissolve 0.25 g in 5 mL of dilute hydrochloric acid R and dilute
to 100 mL with distilled water R. Prepare simultaneously the Run time : 16 min.
standard by adding 0.8 mL of dilute hydrochloric acid R to System suitability :
15 mL of sulfate standard solution (10 ppm SO4) R. – average percentage of carbon in 5 reference samples must
Sodium : maximum 150 ppm. be within ± 0.2 per cent of the value assigned to the CRS ;
the difference between the upper and the lower values of
Atomic absorption spectrometry (2.2.23, Method I).
the percentage of carbon in these samples must be below
Test solution. Dissolve 0.25 g in 50 mL of a 103 g/L solution 0.2 per cent.
of hydrochloric acid R.
Calculate the percentage content of carbonic acid in the test
Reference solutions. Prepare the reference solutions using sample according to the following formula :
sodium standard solution (200 ppm Na) R, diluted as necessary
with a 103 g/L solution of hydrochloric acid R.
Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 1.0 g in dilute hydrochloric acid R and dilute to C = percentage content of carbonic acid in the reference
20.0 mL with the same acid. 12 mL of the solution complies sample ;
with test A. Prepare the reference solution using lead standard
solution (1 ppm Pb) R. K = mean value for the 5 reference samples of the ratio
of the mass in milligrams to the area of the peak
Loss on ignition : 43.0 per cent to 49.0 per cent, determined due to carbonic acid ;
on 1.000 g by ignition at 900 ± 50 °C.
A = area of the peak due to carbonic acid in the
Microbial contamination chromatogram obtained with the test sample ;
TAMC : acceptance criterion 103 CFU/g (2.6.12). m = sample mass, in milligrams.
TYMC : acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13). STORAGE
Absence of Pseudomonas aeruginosa (2.6.13). In an airtight container.
ASSAY
Aluminium. Dissolve 1.000 g in 5 mL of hydrochloric acid R, 01/2010:1064
heating if necessary. Allow to cool to room temperature
and dilute to 100.0 mL with water R (solution A). Introduce ALMOND OIL, REFINED
10.0 mL of solution A into a 250 mL conical flask, add 25.0 mL
of 0.05 M sodium edetate, 20 mL of buffer solution pH 3.5 R, Amygdalae oleum raffinatum
40 mL of ethanol R and 2 mL of a freshly prepared 0.25 g/L
solution of dithizone R in ethanol R. Titrate the excess of DEFINITION
sodium edetate with 0.05 M zinc sulfate until the colour Fatty oil obtained from the ripe seeds of Prunus dulcis
changes from greenish-violet to pink. (Mill.) D.A. Webb var. dulcis or Prunus dulcis (Mill.) D.A.
1 mL of 0.05 M sodium edetate is equivalent to 2.549 mg Webb var. amara (DC.) Buchheim or a mixture of both
of Al2O3. varieties by cold expression. It is then refined. A suitable
Magnesium. Introduce 10.0 mL of solution A prepared in the antioxidant may be added.
assay of aluminium into a 500 mL conical flask, add 200 mL
CHARACTERS
of water R, 20 mL of triethanolamine R with shaking, 10 mL
of ammonium chloride buffer solution pH 10.0 R and 50 mg Appearance : pale yellow, clear liquid.
of mordant black 11 triturate R. Titrate with 0.05 M sodium Solubility : slightly soluble in ethanol (96 per cent), miscible
edetate until the colour changes from violet to pure blue. with light petroleum.
1 mL of 0.05 M sodium edetate is equivalent to 2.015 mg Relative density : about 0.916.
of MgO. It solidifies at about − 18 °C.
Carbonic acid : 12.5 per cent to 14.5 per cent.
IDENTIFICATION
Test sample. Place 7.00 mg of the substance to be examined in
a tin capsule. Seal the capsule. A. Identification of fatty oils by thin-layer chromatography
(2.3.2).
Reference sample. Place 7.00 mg of almagate CRS in a tin
capsule. Seal the capsule. Results : the chromatogram obtained is similar to the
corresponding chromatogram shown in Figure 2.3.2.-1.
Introduce separately the test sample and the reference sample
into a combustion chamber of a CHN analyser purged with B. Composition of fatty acids (see Tests).
helium for chromatography R and maintained at a temperature TESTS
of 1020 °C. Simultaneously, introduce oxygen R at a pressure
of 40 kPa and a flow rate of 20 mL/min and allow complete Specific absorbance (2.2.25) : 0.2 to 6.0, determined at the
combustion of the sample. Sweep the combustion gases absorption maximum at 270 nm.
through a reduction reactor and separate the gases formed by To 0.100 g add cyclohexane R and dilute to 10.0 mL with the
gas chromatography (2.2.28). same solvent. Adapt the concentration of the solution so that
Column : the absorbance lies between 0.5 and 1.5, measured in a 1 cm
cell.
– size : l = 2 m, Ø = 4 mm ;
– stationary phase : ethylvinylbenzene-divinylbenzene Acid value (2.5.1) : maximum 0.5, determined on 5.0 g.
copolymer R1. Peroxide value (2.5.5, Method A) : maximum 5.0.

1508 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Alprazolam

Unsaponifiable matter (2.5.7) : maximum 0.9 per cent, TESTS

determined on 5.0 g. Absorbance (2.2.25) : maximum 0.2, determined at the
Composition of fatty acids (2.4.22, Method A). Use the absorption maximum at 270 nm. The ratio of the absorbance
mixture of calibrating substances in Table 2.4.22.-3. measured at 232 nm to that measured at 270 nm is greater
Composition of the fatty-acid fraction of the oil: than 7.
To 0.100 g add cyclohexane R and dilute to 10.0 mL with the
– saturated fatty acids of chain length less than C16 : maximum
0.1 per cent ; same solvent.
– palmitic acid : 4.0 per cent to 9.0 per cent ; Acid value (2.5.1) : maximum 2.0, determined on 5.0 g.
– palmitoleic acid : maximum 0.8 per cent ; Peroxide value (2.5.5, Method A) : maximum 15.0.
– margaric acid : maximum 0.2 per cent ; Unsaponifiable matter (2.5.7) : maximum 0.9 per cent,
– stearic acid : maximum 3.0 per cent ; determined on 5.0 g.
– oleic acid : 62.0 per cent to 86.0 per cent ; Composition of fatty acids. (2.4.22, Method A). Use the
mixture of calibrating substances in Table 2.4.22.-3.
– linoleic acid : 20.0 per cent to 30.0 per cent ;
Composition of the fatty-acid fraction of the oil :
– linolenic acid : maximum 0.4 per cent ;
– saturated fatty acids of chain length less than C16 : maximum
– arachidic acid : maximum 0.2 per cent ; 0.1 per cent,
– eicosenoic acid : maximum 0.3 per cent ; – palmitic acid : 4.0 per cent to 9.0 per cent,
– behenic acid : maximum 0.2 per cent ; – palmitoleic acid : maximum 0.8 per cent,
– erucic acid : maximum 0.1 per cent. – margaric acid : maximum 0.2 per cent,
Sterols (2.4.23). – stearic acid : maximum 3.0 per cent,
Composition of the sterol fraction of the oil : – oleic acid : 62.0 per cent to 86.0 per cent,
– cholesterol : maximum 0.7 per cent ; – linoleic acid : 20.0 per cent to 30.0 per cent,
– campesterol : maximum 5.0 per cent ; – linolenic acid : maximum 0.4 per cent,
– stigmasterol : maximum 4.0 per cent ; – arachidic acid : maximum 0.2 per cent,
– β-sitosterol : 73.0 per cent to 87.0 per cent ; – eicosenoic acid : maximum 0.3 per cent,
– Δ5-avenasterol : minimum 5.0 per cent ; – behenic acid : maximum 0.2 per cent,
– erucic acid : maximum 0.1 per cent.
– Δ7-stigmastenol : maximum 3.0 per cent ;
– Δ7-avenasterol : maximum 3.0 per cent ; Sterols (2.4.23).
Composition of sterol fraction of the oil :
– brassicasterol : maximum 0.3 per cent.
– cholesterol : maximum 0.7 per cent,
Water (2.5.32) : maximum 0.1 per cent, determined on 1.00 g.
– campesterol : maximum 4.0 per cent,
STORAGE – stigmasterol : maximum 3.0 per cent,
In a well-filled container, protected from light. – β-sitosterol : 73.0 per cent to 87.0 per cent,
– Δ5-avenasterol : minimum 10.0 per cent,
– Δ7-stigmastenol : maximum 3.0 per cent,
01/2010:0261 – Δ7-avenasterol : maximum 3.0 per cent,
– brassicasterol : maximum 0.3 per cent.
ALMOND OIL, VIRGIN Water (2.5.32) : maximum 0.1 per cent, determined on 1.00 g.
STORAGE
Amygdalae oleum virginale In a well-filled container, protected from light.
DEFINITION
Fatty oil obtained by cold expression from the ripe seeds of 01/2008:1065
Prunus dulcis (Mill.) D.A. Webb var. dulcis or Prunus dulcis corrected 6.0
(Mill.) D.A. Webb var. amara (DC.) Buchheim or a mixture
of both varieties. ALPRAZOLAM
CHARACTERS
Appearance : yellow, clear liquid.
Alprazolamum
Solubility : slightly soluble in ethanol (96 per cent), miscible
with light petroleum.
Relative density : about 0.916.
It solidifies at about − 18 °C.
IDENTIFICATION
First identification : A, C.
Second identification : A, B.
A. Absorbance (see Tests). C17H13ClN4 Mr 308.8
[28981-97-7]
B. Identification of fatty oils by thin-layer chromatography
(2.3.2). DEFINITION
Results : the chromatogram obtained is similar to the 8-Chloro-1-methyl-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]-
corresponding chromatogram shown in Figure 2.3.2.-1. benzodiazepine.
C. Composition of fatty acids (see Tests). Content : 99.0 per cent to 101.0 per cent (dried substance).

General Notices (1) apply to all monographs and other texts 1509
Alprazolam EUROPEAN PHARMACOPOEIA 8.0

CHARACTERS Mobile phase :

Appearance : white or almost white, crystalline powder. – mobile phase A : buffer solution, methanol R (44:56 V/V) ;
Solubility : practically insoluble in water, freely soluble in – mobile phase B : buffer solution, methanol R (5:95 V/V) ;
methylene chloride, sparingly soluble in acetone and in – temperature : 40 °C ;
ethanol (96 per cent).
Time Mobile phase A Mobile phase B
It shows polymorphism (5.9). (min) (per cent V/V) (per cent V/V)
IDENTIFICATION 0 - 15 98 2
First identification : B. 15 - 35 98 → 1 2 → 99
Second identification : A, C. 35 - 40 1 99
A. Dissolve the substance to be examined in the smallest
necessary quantity of ethyl acetate R and evaporate to Flow rate : 2 mL/min.
dryness on a water-bath. Thoroughly mix 5.0 mg of the Detection : spectrophotometer at 254 nm.
substance to be examined with 5.0 mg of alprazolam CRS. Injection : 10 μL ; inject dimethylformamide R as a blank.
The melting point (2.2.14) of the mixture does not differ
by more than 2 °C from the melting point of the substance Retention time : triazolam = about 9 min ; alprazo-
to be examined. lam = about 10 min.
B. Infrared absorption spectrophotometry (2.2.24). System suitability : reference solution (a) :
Preparation : discs. – resolution : minimum 1.5 between the peaks due to
triazolam and alprazolam.
Comparison : alprazolam CRS.
Limits :
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference – total : not more than the area of the principal peak in
substance separately in the minimum volume of ethyl the chromatogram obtained with reference solution (b)
acetate R, evaporate to dryness on a water-bath and record (0.25 per cent);
new spectra using the residues. – disregard limit : 0.2 times the area of the principal peak in
C. Thin-layer chromatography (2.2.27). the chromatogram obtained with reference solution (b)
(0.05 per cent).
Test solution. Dissolve 10 mg of the substance to be
examined in methanol R and dilute to 10 mL with the same Loss on drying (2.2.32) : maximum 0.5 per cent, determined
solvent. on 1.000 g by drying in an oven at 105 °C.
Reference solution (a). Dissolve 10 mg of alprazolam CRS Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
in methanol R and dilute to 10 mL with the same solvent. 1.0 g.
Reference solution (b). Dissolve 10 mg of alprazolam CRS ASSAY
and 10 mg of midazolam CRS in methanol R and dilute to
10 mL with the same solvent. Dissolve 0.140 g in 50 mL of a mixture of 2 volumes of acetic
anhydride R and 3 volumes of anhydrous acetic acid R.
Plate : TLC silica gel GF254 plate R. Titrate with 0.1 M perchloric acid, determining the end-point
Mobile phase : glacial acetic acid R, water R, methanol R, potentiometrically (2.2.20). Titrate to the 2nd point of
ethyl acetate R (2:15:20:80 V/V/V/V). inflexion.
Application : 5 μL. 1 mL of 0.1 M perchloric acid is equivalent to 15.44 mg
Development : over a path of 12 cm. of C17H13CIN4.
Drying : in air. STORAGE
Detection : examine in ultraviolet light at 254 nm. Protected from light.
System suitability: reference solution (b) :
– the chromatogram shows 2 clearly separately spots. IMPURITIES
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to
the principal spot in the chromatogram obtained with
reference solution (a).
TESTS
Related substances. Liquid chromatography (2.2.29). A. (4RS)-3-amino-6-chloro-2-methyl-4-phenyl-3,4-
Buffer solution. Dissolve 7.7 g of ammonium acetate R in dihydroquinazolin-4-ol,
1000 mL of water R and adjust to pH 4.2 with glacial acetic
acid R.
Test solution. Dissolve 0.100 g of the substance to be examined
in dimethylformamide R and dilute to 10.0 mL with the same
solvent.
Reference solution (a). Dissolve 2 mg of alprazolam CRS and
2 mg of triazolam CRS in dimethylformamide R and dilute to
100.0 mL with the same solvent.
Reference solution (b). Dilute 5.0 mL of the test solution to
100.0 mL with dimethylformamide R. Dilute 0.5 mL of this B. R = CH2OH : [5-chloro-2-[3-(hydroxymethyl)-5-methyl-
solution to 10.0 mL with dimethylformamide R. 4H-1,2,4-triazol-4-yl]phenyl]phenylmethanone,
Column : C. R = H : [5-chloro-2-[3-methyl-4H-1,2,4-triazol-4-
– size : l = 0.25 m, Ø = 4.6 mm ; yl]phenyl]phenylmethanone,
– stationary phase : phenylsilyl silica gel for chromatography R1 F. R = CH2Cl : [5-chloro-2-[3-(chloromethyl)-5-methyl-4H-
(5 μm). 1,2,4-triazol-4-yl]phenyl]phenylmethanone,

1510 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Alprenolol hydrochloride

04/2010:0876

ALPRENOLOL HYDROCHLORIDE
Alprenololi hydrochloridum

D. 8-chloro-1-ethenyl-6-phenyl-4H-[1,2,4]triazolo[4,3-
a][1,4]benzodiazepine,
C15H24ClNO2 Mr 285.8
[13707-88-5]
DEFINITION
(2RS)-1-[(1-Methylethyl)amino]-3-[2-(prop-2-enyl)phenoxy]-
propan-2-ol hydrochloride.
Content : 99.0 per cent to 101.0 per cent (dried substance).
E. (2-amino-5-chlorophenyl)phenylmethanone, CHARACTERS
Appearance : white or almost white, crystalline powder or
colourless crystals.
Solubility : very soluble in water, freely soluble in ethanol
(96 per cent) and in methylene chloride.
IDENTIFICATION
First identification : B, D.
Second identification : A, C, D.
A. Melting point (2.2.14) : 108 °C to 112 °C.
G. 7-chloro-1-methyl-5-phenyl[1,2,4]triazolo[4,3-a]quinolin- B. Infrared absorption spectrophotometry (2.2.24).
4-amine, Comparison: alprenolol hydrochloride CRS.
C. Examine the chromatograms obtained in the test for
impurity D.
Detection : examine in daylight, after exposure to iodine
vapour for 30 min.
Results : the principal spot in the chromatogram obtained
with test solution (b) is similar in position, colour and size
to the principal spot in the chromatogram obtained with
reference solution (a).
H. bis[[4-(2-benzoyl-4-chlorophenyl)-5-methyl-4H-1,2,4- D. It gives reaction (a) of chlorides (2.3.1).
triazol-3-yl]methyl]amine, TESTS
Solution S. Dissolve 1.0 g in carbon dioxide-free water R and
dilute to 50 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution B9 (2.2.2,
Method II).
Acidity or alkalinity. To 10 mL of solution S add 0.2 mL of
methyl red solution R and 0.2 mL of 0.01 M hydrochloric acid ;
the solution is red. Add 0.4 mL of 0.01 M sodium hydroxide ;
the solution is yellow.
Impurity C : maximum 0.1 per cent.
Dissolve 0.25 g in ethanol (96 per cent) R and dilute to 25 mL
I. [5-chloro-2-[3-[[(6RS)-8-chloro-6-hydroxy-1-methyl- with the same solvent. The absorbance (2.2.25) measured at
6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin- 297 nm is not greater than 0.20.
5(6H)-yl]methyl]-5-methyl-4H-1,2,4-triazol-4-
yl]phenyl]phenylmethanone, Impurity D. Thin-layer chromatography (2.2.27).
Test solution (a). Dissolve 0.50 g of the substance to be
examined in methanol R and dilute to 10 mL with the same
solvent.
Test solution (b). Dilute 1 mL of test solution (a) to 50 mL
with methanol R.
Reference solution (a). Dissolve 10 mg of alprenolol
hydrochloride CRS in methanol R and dilute to 10 mL with
the same solvent.
Reference solution (b). Dissolve 10 mg of alprenolol
J. 2,17-dichloro-6,13-dimethyl-18b,19a-diphenyl-8b,19a- hydrochloride CRS and 10 mg of oxprenolol hydrochloride CRS
dihydro-10H,18bH-[1,2,4]triazolo[4′′′,3′′′:1″,2″]- in methanol R and dilute to 10 mL with the same solvent.
quinolo[3″,4″:4′,5′]oxazolo[3′,2′-d]-1,2,4-triazolo[4,3-a]- Reference solution (c). Dilute 5 mL of test solution (b) to
[1,4]benzodiazepine. 50 mL with methanol R.

General Notices (1) apply to all monographs and other texts 1511
Alprostadil EUROPEAN PHARMACOPOEIA 8.0

Plate : TLC silica gel G plate R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Mobile phase : place 2 beakers each containing 30 mL of on 1.000 g by drying over diphosphorus pentoxide R at a
ammonia R at the bottom of the tank containing a mixture of pressure not exceeding 2.7 kPa.
5 volumes of methanol R and 95 volumes of ethyl acetate R. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Application : 5 μL. 1.0 g.
Development : over a path of 15 cm in a tank saturated for at ASSAY
least 1 h.
Dissolve 0.400 g in 25 mL of a mixture of equal volumes
Drying : at 100 °C for 15 min. of anhydrous ethanol R and water R. Add 10 mL of 0.01 M
Detection : expose to iodine vapour for up to 6 h. hydrochloric acid. Carry out a potentiometric titration
System suitability : reference solution (b) : (2.2.20), using 0.1 M sodium hydroxide. Read the volume
added between the 2 points of inflexion.
– the chromatogram shows 2 clearly separated spots.
1 mL of 0.1 M sodium hydroxide is equivalent to 28.58 mg
Limits : test solution (a) : of C15H24ClNO2.
– impurity D : any spot with an RF value greater than that of
the principal spot is not more intense than the principal STORAGE
spot in the chromatogram obtained with reference Protected from light.
solution (c) (0.2 per cent).
IMPURITIES
Related substances. Liquid chromatography (2.2.29).
Specified impurities : C, D.
Test solution. Dissolve 20.0 mg of the substance to be
examined in the mobile phase and dilute to 10.0 mL with the Other detectable impurities (the following substances would,
mobile phase. if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
Reference solution (a). Dissolve 4.0 mg of alprenolol acceptance criterion for other/unspecified impurities and/or
hydrochloride CRS and 0.8 mg of 4-isopropylphenol R in the by the general monograph Substances for pharmaceutical use
mobile phase and dilute to 100.0 mL with the mobile phase. (2034). It is therefore not necessary to identify these impurities
Reference solution (b). Dilute 4.0 mL of the test solution to for demonstration of compliance. See also 5.10. Control of
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution impurities in substances for pharmaceutical use) : A, B.
to 10.0 mL with the mobile phase.
Column :
– size : l = 0.15 m, Ø = 4 mm ;
– stationary phase : octylsilyl silica gel for chromatography R
(5 μm). A. R1 = OH, R2 = CH2-CH=CH2 : (2RS)-3-[2-(prop-2-
Mobile phase : mix 0.656 g of sodium octanesulfonate R with enyl)phenoxy]propan-1,2-diol,
150 mL of acetonitrile R and dilute to 500 mL with phosphate
buffer pH 2.8 prepared as follows : mix 1.78 g of phosphoric C. R1 = NH-CH(CH3)2, R2 = CH=CH-CH3 :
acid R and 15.6 g of sodium dihydrogen phosphate R and dilute (2RS)-1-[(1-methylethyl)amino]-3-[2-(prop-1-
to 2000 mL with water R. enyl)phenoxy]propan-2-ol,
Flow rate : 1 mL/min.
Detection : spectrophotometer at 280 nm.
Equilibration : with the mobile phase for about 1 h.
Injection : 20 μL. B. 2-(prop-2-enyl)phenol,
Run time : twice the retention time of alprenolol.
Retention time : alprenolol = about 11 min ; 4-isopropylphe-
nol = about 18 min.
System suitability: reference solution (a) :
– resolution : minimum 5 between the peaks due to
alprenolol and 4-isopropylphenol ; if necessary, adjust D. 1,1′-[(1-methylethyl)imino]bis[3-[2-(prop-2-
the concentration of sodium octanesulfonate and/or enyl)phenoxy]propan-2-ol].
acetonitrile in the mobile phase (increase the concentration
of sodium octanesulfonate to increase the retention time of 01/2008:1488
alprenolol and increase the concentration of acetonitrile to
decrease the retention times of both compounds). ALPROSTADIL
Limits :
– unspecified impurities : for each impurity, not more Alprostadilum
than 0.25 times the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.10 per cent) ;
– total : not more than the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.4 per
cent) ;
– disregard limit : 0.1 times the area of the principal peak in
C20H34O5 Mr 354.5
the chromatogram obtained with reference solution (b)
[745-65-3]
(0.04 per cent).
Heavy metals (2.4.8) : maximum 10 ppm. DEFINITION
Dissolve 2.0 g in 20 mL of water R. 12 mL of the solution 7-[(1R,2R,3R)-3-Hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-
complies with test A. Prepare the reference solution using lead oxocyclopentyl]heptanoic acid.
standard solution (1 ppm Pb) R. Content : 95.0 per cent to 102.5 per cent (anhydrous substance).

1512 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Alprostadil

CHARACTERS Flow rate : 1 mL/min.

Appearance : white or slightly yellowish, crystalline powder. Detection : spectrophotometer at 200 nm.
Solubility : practically insoluble in water, freely soluble in Injection : 20 μL loop injector.
alcohol, soluble in acetone, slightly soluble in ethyl acetate. System suitability :
IDENTIFICATION – retention time : alprostadil = about 63 min,
A. Specific optical rotation (2.2.7) : − 70 to − 60 (anhydrous – resolution : minimum of 1.5 between the peaks due to
substance). impurity H and alprostadil in the chromatogram obtained
with reference solution (b).
Immediately before use, dissolve 50 mg in alcohol R and
dilute to 10.0 mL with the same solvent. System B
B. Infrared absorption spectrophotometry (2.2.24). Use the same conditions as for system A with the following
mobile phase and elution programme :
Preparation : discs.
– mobile phase A. Dissolve 3.9 g of sodium dihydrogen
Comparison : alprostadil CRS. phosphate R in water R and dilute to 1000.0 mL with the
C. Examine the chromatograms obtained in the assay. same solvent ; adjust to pH 2.5 with a 2.9 g/L solution of
Results : the principal peak in the chromatogram obtained phosphoric acid R (approximately 600 mL is required) ; to
with the test solution is similar in retention time and size 600 mL of the buffer solution add 400 mL of acetonitrile R1;
to the principal peak in the chromatogram obtained with – mobile phase B. Use mobile phase B as described under
the reference solution. system A;
TESTS Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
Related substances. Liquid chromatography (2.2.29). Prepare
0 - 50 100 0
the solutions protected from light.
Test solution. Dissolve 10.0 mg of the substance to be 50 - 51 100 → 0 0 → 100
examined in a mixture of equal volumes of acetonitrile R1 51 - 61 0 100
and water R and dilute to 10.0 mL with the same mixture of
solvents. 61 - 62 0 → 100 100 → 0
Reference solution (a). Dilute 100 μL of the test solution to 62 - 72 100 0
20.0 mL with a mixture of equal volumes of acetonitrile R1
and water R. System suitability :
Reference solution (b). Dissolve 1.0 mg of dinoprostone – relative retentions with reference to alprostadil
impurity C CRS (alprostadil impurity H) and 1.0 mg of (retention time = about 7 min) : impurity A = about 2.4 ;
alprostadil CRS in a mixture of equal volumes of acetonitrile R1 impurity B = about 2.6,
and water R and dilute to 20.0 mL with the same mixture of – resolution : minimum of 1.5 between the peaks due to
solvents. impurity A and impurity B in the chromatogram obtained
Reference solution (c). In order to prepare in situ the with reference solution (c).
degradation compounds (impurity A and impurity B), dissolve Carry out the test according to system A and B.
1 mg of the substance to be examined in 100 μL of 1 M sodium Limits :
hydroxide (the solution becomes brownish-red), wait for – correction factors : multiply the areas of the corresponding
3 min and add 100 μL of 1 M phosphoric acid (yellowish-white peaks using the correction factors in Table 1488.-1 to
opalescent solution) ; dilute to 5.0 mL with a mixture of obtain the corrected areas,
equal volumes of acetonitrile R1 and water R.
Table 1488.-1
System A
Impurity Relative retention Relative retention Correction factor
Column : (system A) (system B)
– size : l = 0.25 m, Ø = 4.0 mm, impurity G 0.80 - 0.7
– stationary phase : base-deactivated octylsilyl silica gel for impurity F 0.88 - 0.8
chromatography R (4 μm) with a pore size of 6 nm,
-
– temperature : 35 °C. impurity D 0.90 1.0

Mobile phase : impurity H 0.96 - 0.7

– mobile phase A. Dissolve 3.9 g of sodium dihydrogen impurity E 1.10 - 0.7
phosphate R in water R and dilute to 1000.0 mL with the
impurity C - 1.36 1.9
same solvent ; adjust to pH 2.5 with a 2.9 g/L solution of
phosphoric acid R (approximately 600 mL is required) ; to impurity K - 1.85 0.06
740 mL of the buffer solution add 260 mL of acetonitrile R1;
impurity A - 2.32 0.7
– mobile phase B. Dissolve 3.9 g of sodium dihydrogen
phosphate R in water R and dilute to 1000.0 mL with the impurity B - 2.45 1.5
same solvent ; adjust to pH 2.5 with a 2.9 g/L solution of -
impurity I 4.00 1.0
phosphoric acid R (approximately 600 mL is required) ; to
200 mL of the buffer solution add 800 mL of acetonitrile R1; impurity J - 5.89 1.0
Time Mobile phase A Mobile phase B – impurity A (corrected area) : not more than 3 times the area
(min) (per cent V/V) (per cent V/V) of the principal peak in the chromatogram obtained with
0 - 75 100 0 reference solution (a) (1.5 per cent),
75 - 76 100 → 0 0 → 100 – impurity B (corrected area) : not more than the area of
the principal peak in the chromatogram obtained with
76 - 86 0 100
reference solution (a) (0.5 per cent),
86 - 87 0 → 100 100 → 0 – any other impurity (corrected area) : not more than 1.8 times
87 - 102 100 0 the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.9 per cent), and not

General Notices (1) apply to all monographs and other texts 1513
Alprostadil EUROPEAN PHARMACOPOEIA 8.0

more than 1 such peak has an area greater than the area
of the principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent). Evaluate impurities
appearing at relative retentions less than 1.2 by system A
and impurities appearing at relative retentions greater than
1.2 by system B,
– total (corrected area) : not more than 3 times the area of D. 7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3R)-3-hydroxyoct-
the principal peak in the chromatogram obtained with 1-enyl]-5-oxocyclopentyl]heptanoic acid
reference solution (a) (1.5 per cent), (15-epiprostaglandin E1),

– disregard limit : 0.1 times the area of the principal peak in

the chromatogram obtained with reference solution (a)
(0.05 per cent).
Water (2.5.32) : maximum 0.5 per cent, determined on 50 mg.

ASSAY
E. 7-[(1R,2R,3S)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-
Liquid chromatography (2.2.29) as described in the test for 5-oxocyclopentyl]heptanoic acid (11-epiprostaglandin E1),
related substances, system A. Prepare the solutions protected
from light.
Test solution. Dissolve 10.0 mg of the substance to be
examined in a mixture of equal volumes of acetonitrile R1
and water R and dilute to 25.0 mL with the same mixture of
solvents. Dilute 3.0 mL of the solution to 20.0 mL with a
mixture of equal volumes of acetonitrile R1 and water R.
Reference solution. Dissolve 10.0 mg of alprostadil CRS in F. 7-[(1S,2R,3R)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-
a mixture of equal volumes of acetonitrile R1 and water R 5-oxocyclopentyl]heptanoic acid (8-epiprostaglandin E1),
and dilute to 25.0 mL with the same mixture of solvents.
Dilute 3.0 mL of the solution to 20.0 mL with a mixture of
equal volumes of acetonitrile R1 and water R.
Injection : 20 μL.
Calculate the percentage content of C20H34O5.

STORAGE G. (5Z)-7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1-
enyl]-5-oxocyclopentyl]hept-5-enoic acid (dinoprostone),
At a temperature of 2 °C to 8 °C.

IMPURITIES

H. (5E)-7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3S)-3-
hydroxyoct-1-enyl]-5-oxocyclopentyl]hept-5-enoic
acid ((5E)-prostaglandin E2),

A. 7-[(1R,2S)-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-
oxocyclopent-3-enyl]heptanoic acid (prostaglandin A1),

I. R = CH2-CH3 : ethyl 7-[(1R,2R,3R)-3-hydroxy-2-[(1E,3S)-

3-hydroxyoct-1-enyl]-5-oxocyclopentyl]heptanoate
(prostaglandin E1, ethyl ester),

B. 7-[2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-oxocyclopent-1- J. R = CH(CH3)2 : 1-methylethyl 7-[(1R,2R,3R)-

enyl]heptanoic acid (prostaglandin B1), 3-hydroxy-2-[(1E,3S)-3-hydroxyoct-1-enyl]-5-
oxocyclopentyl]heptanoate (prostaglandin E1, isopropyl
ester),

C. 7-[(1R,2R,3R)-3-hydroxy-2-[(1E)-3-oxooct-1-enyl]-5-
oxocyclopentyl]heptanoic acid (15-oxoprostaglandin E1), K. triphenylphosphine oxide.

1514 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Alteplase for injection

07/2013:1170 N-terminal sequence. N-terminal sequencing is applied to

corrected 8.0 determine the correct N-terminal sequence and to determine
semiquantitatively additional cleavage sites in the alteplase
ALTEPLASE FOR INJECTION molecule, for example at position AA 275-276 or at position
AA 27-28. The N-terminal sequence must conform with the
sequence of human tissue plasminogen activator.
Alteplasum ad iniectabile Isoelectric focusing. The consistency in the
microheterogeneity of glycosylation of the alteplase
molecule can be demonstrated by isoelectric focusing (IEF).
A complex banding pattern with 10 major and several minor
bands in the pH range 6.5-8.5 is observed. Denaturing
conditions are applied to achieve a good separation of
differently charged variants of alteplase. The broad charge
distribution observed is due to a population of molecules,
which differ in the fine structure of biantenary and triantenary
complex-type carbohydrate residues, with different degrees of
substitution with sialic acids. The banding pattern of alteplase
test samples must be consistent with the pattern of alteplase
reference standard.
Single-chain alteplase content. The alteplase produced by
CHO (Chinese hamster ovary) cells in serum-free medium is
predominantly single-chain alteplase. The single-chain form
can be separated from the two-chain form by gel-permeation
liquid chromatography under reducing conditions as described
under Single-chain content (see Tests). The single-chain
alteplase content in bulk samples must be higher than 60 per
cent.
DEFINITION
Tryptic-peptide mapping. The primary structure of the
Alteplase for injection is a sterile, freeze-dried preparation alteplase molecule is verified by tryptic-peptide mapping
of alteplase, a tissue plasminogen activator produced by as described under Identification B. The reduced and
recombinant DNA technology. It has a potency of not less carboxymethylated molecule is cleaved by trypsin into about
than 500 000 IU per milligram of protein. 50 peptides, which are separated by reverse-phase liquid
Tissue plasminogen activator binds to fibrin clots and activates chromatography. A characteristic chromatogram (fingerprint)
plasminogen, leading to the generation of plasmin and to the is obtained. The identity of the tryptic-peptide map of a
degradation of fibrin clots or blood coagulates. given alteplase sample with the profile of a well-characterised
Alteplase consists of 527 amino acids with a calculated reference standard is an indirect confirmation of the
relative molecular mass of 59 050 without consideration of amino-acid sequence, because even single amino-acid
the carbohydrate moieties attached at positions Asn 117, exchanges in individual peptides can be detected by this
Asn 184 and Asn 448. The total relative molecular mass is sensitive technique. In addition, complex peaks of the
approximately 65 000. Alteplase is cleaved by plasmin between glycopeptides can be isolated from the tryptic-peptide map
amino-acids 275 and 276 into a two-chain form (A chain and and separated in a second dimension, either by reverse-phase
B chain) that are connected by a disulfide bridge between Cys liquid chromatography under modified conditions or by
264 and Cys 395. The single-chain form and the two-chain capillary electrophoresis. By this two-dimensional separation
form show comparable fibrinolytic activity in vitro. of glycopeptide variants, lot-to-lot consistency of the
microheterogeneity of glycosylation can be demonstrated.
PRODUCTION
The tryptic-peptide map of alteplase samples must be
Alteplase is produced by recombinant DNA synthesis in cell consistent with the tryptic-peptide map of alteplase reference
culture ; the fermentation takes place in serum-free medium. standard.
The purification process is designed to remove efficiently Monomer content. The monomer content of alteplase is
potential impurities, such as antibiotics, DNA and protein measured by gel-permeation liquid chromatography under
contaminants derived both from the host cell and from the non-reduced conditions as described under Monomer content
production medium, and potential viral contaminants. (see Tests). The monomer content of alteplase bulk samples
If alteplase is stored in bulk form, stability (maintenance must be higher than 95 per cent.
of potency) in the intended storage conditions must be
demonstrated. Type I/Type II alteplase content. CHO cells produce
2 glycosylation variants of alteplase. Type I alteplase contains
The production, purification and product consistency are 1 polymannose-type glycosylation at position Asn 117 and
checked by a number of analytical methods described below, 2 complex-type glycosylation sites at positions Asn 184 and
carried out routinely as in-process controls. Asn 448. Type II alteplase is only glycosylated at positions
Protein content. The protein concentration of alteplase Asn 117 and Asn 448.
solutions is determined by measuring the absorbance (2.2.25) The ratio of Type I/Type II alteplase is constant in the range of
of the protein solution at 280 nm and at 320 nm, using 45 to 65 per cent of Type I and 35 to 55 per cent of Type II.
formulation buffer as the compensation liquid. If dilution The content of alteplase Type I and Type II can be determined
of alteplase samples is necessary, the samples are diluted by a densitometric scan of SDS-PAGE (sodium dodecyl sulfate
in formulation buffer. For the calculation of the alteplase polyacrylamide gel electrophoresis) gel. Plasmin-treated
concentration, the absorbance value (A280 − A320) is divided by samples of alteplase, which are reduced and carboxymethylated
the specific absorption coefficient for alteplase of 1.9. before loading on the gel, are separated into 3 bands : Type
Potency. The potency of alteplase is determined in an in vitro I alteplase A-chain (AA 1-275), Type II alteplase A-chain
clot-lysis assay as described under Assay. The specific activity (AA 1-275) and alteplase B-chain (AA 276-527). The ratio
of bulk alteplase is approximately 580 000 IU per milligram of of Type I/Type II alteplase is determined from a calibration
alteplase. curve, which is obtained by a densitometric scan of defined

General Notices (1) apply to all monographs and other texts 1515
Alteplase for injection EUROPEAN PHARMACOPOEIA 8.0

mixtures of purified Type I alteplase and Type II alteplase Reference solution. Prepare as for the test solution using a
standards. suitable reference standard instead of the preparation to
SDS-PAGE. SDS-PAGE (silver staining) is used to demonstrate be examined.
purity of the alteplase bulk material and the integrity of the The chromatographic procedure may be carried out using :
alteplase molecule. For alteplase bulk samples, no additional
– a column 0.1 m long and 4.6 mm in internal diameter
protein bands compared to reference standard or degradation
packed with octadecylsilyl silica gel for chromatography R
products must occur in SDS-PAGE gels at a loading amount
(5 μm to 10 μm) ;
of 2.5 μg alteplase protein per lane and a limit of detection of
5 ng per protein (BSA) band. Mobile phase A. 8 g/L solution of sodium dihydrogen
Bacterial endotoxins (2.6.14) : less than 1 IU per milligram of phosphate R, adjusted to pH 2.85 with phosphoric acid R,
alteplase. filtered and degassed ;
Sialic acids. Proceed using a suitable validated method Mobile phase B. 75 per cent V/V solution of acetonitrile R
developed according to general chapter 2.2.59. Glycan analysis in mobile phase A ;
of glycoproteins. The sialic acids content for the test samples – as detector a spectrophotometer set at 210 nm.
must be in the range of 70 to 130 per cent compared to
alteplase reference standard, which contains about 3 moles of Equilibrate the system with mobile phase A at a flow rate
sialic acids per mole of alteplase. of 1 mL/min. After injection of the solution, increase the
proportion of mobile phase B at a rate of 0.44 per cent
Neutral sugars. Dilute alteplase samples and the reference per minute until the ratio of mobile phase A to mobile
standard in the assay buffer, containing 34.8 g/L of arginine R, phase B is 60:40, then increase the proportion of mobile
0.1 g/L of polysorbate 80 R and adjusted to pH 7.4 with phase B at a rate of 1.33 per cent per minute until the ratio
phosphoric acid R, to a protein concentration of 50 μg/mL. of mobile phase A to mobile phase B is 20:80 and then
Prepare the following concentrations of mannose in the continue elution with this mixture for a further 10 min.
same assay buffer for a calibration curve : 20, 30, 40, 50 and Record the chromatogram for the reference solution : the
60 μg/mL. Pipette 2 mL of alteplase samples and reference test is not valid unless the resolution of peaks 6 (peptides
standard, as well as 2 mL of each mannose concentration in 268-275) and 7 (peptides 1-7) is at least 1.5 ; wh1 and wh2
duplicate in reagent tubes. Add 50 μL of phenol R, followed are not more than 0.4 min. Inject about 100 μL of the test
by 5 mL of sulfuric acid R, in each reagent tube. Incubate solution and record the chromatogram. Verify the identity
the mixture for 30 min at room temperature. Measure the of the peaks by comparison with the chromatograms of
absorbance at 492 nm for each tube. Read the content of the reference solution. There should not be any additional
neutral sugars from the mannose calibration curve. The significant peaks or shoulders, a significant peak or
neutral sugar content is expressed in moles of neutral sugar shoulder being defined as one with an area response equal
per mole of alteplase, taking into account the dilution factor to or greater than 5 per cent of peak 19 (peptides 278-296) ;
for alteplase samples and reference standard and using a no significant peak is missing. A type chromatogram for
relative molecular mass of 180.2 for mannose and a relative identification of the peaks cited is shown in Figure 1170.-1.
molecular mass of 59 050 for the alteplase protein moiety. The
neutral sugar content of the alteplase samples must be in the TESTS
range of 70 to 130 per cent compared to alteplase reference
standard, which contains about 12 moles of neutral sugar per Appearance of solution. The reconstituted preparation is
mole of alteplase. clear (2.2.1) and not more intensely coloured than reference
solution Y7 (2.2.2, Method II).
CHARACTERS pH (2.2.3) : 7.1 to 7.5.
White or slightly yellow powder or solid friable mass. Solubility. Add the volume of the liquid stated on the label.
Reconstitute the preparation as stated on the label immediately The preparation dissolves completely within 2 min at 20 °C to
before carrying out the Identification, Tests (except those for 25 °C.
solubility and water) and Assay. Protein content. Prepare a solution of the substance to be
examined with an accurately known concentration of about
IDENTIFICATION 1 g/L. Using a 34.8 g/L solution of arginine R adjusted to pH 7.3
with phosphoric acid R, dilute an accurately measured volume
A. The assay serves also to identify the preparation.
of the solution of the substance to be examined so that the
B. Tryptic-peptide mapping. Examine by liquid absorbance measured at the maximum at about 280 nm is 0.5
chromatography (2.2.29). to 1.0 (test solution). Measure the absorbance (2.2.25) of the
Test solution. Dilute the preparation to be examined solution at the maximum at about 280 nm and at 320 nm using
with water R to obtain a solution containing about 1 mg the arginine solution as the compensation liquid. Calculate
of alteplase per millilitre. Dialyse about 2.5 mL of the the protein content in the portion of alteplase taken from the
solution for at least 12 h into a solution containing 480 g/L following expression :
of urea R, 44 g/L of tris(hydroxymethyl)aminomethane R
and 1.5 g/L of sodium edetate R and adjusted to pH 8.6,
using a membrane with a cut-off point corresponding to
a relative molecular mass of 10 000 for globular proteins. in which V is the volume of the test solution, A280 is the
Measure the volume of the solution, transfer it to a clean absorbance at the maximum at about 280 nm and A320 is the
test-tube and add per millilitre 10 μL of a 156 g/L solution absorbance at 320 nm.
of dithiothreitol R. Allow to stand for 4 h, cool in iced water
and add per millilitre of solution 25 μL of a freshly prepared Single-chain content. Examine by liquid chromatography
190 g/L solution of iodoacetic acid R. Allow to stand in the (2.2.29).
dark for 30 min. Add per millilitre 50 μL of dithiothreitol Test solution. Dissolve the preparation to be examined in
solution to stop the reaction. Dialyse for 24 h against an water R to obtain a solution containing about 1 mg of alteplase
8 g/L solution of ammonium hydrogen carbonate R. Add per millilitre. Place about 1 mL of the solution in a tube, add
1 part of trypsin for peptide mapping R to 100 parts of 3 mL of a 3 g/L solution of dithiothreitol R in the mobile phase,
the protein and allow to stand for 6 h to 8 h. Repeat the place a cap on the tube and heat at about 80 °C for 3 min to
addition of trypsin and allow to stand for a total of 24 h. 5 min.

1516 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Alteplase for injection

Figure 1170.-1. – Chromatogram for tryptic-peptide mapping of alteplase

The chromatographic procedure may be carried out using : ASSAY

– a column 0.6 m long and 7.5 mm in internal diameter The potency of alteplase is determined by comparing its
packed with silica-based, rigid, hydrophilic gel with ability to activate plasminogen to form plasmin with the
spherical particles 10 μm to 13 μm in diameter, suitable for same capacity of a reference preparation calibrated in
size-exclusion chromatography ; International Units. The formation of plasmin is measured by
– as mobile phase at a flow rate of 0.5 mL/min a solution the determination of the lysis time of a fibrin clot in given
containing 30 g/L of sodium dihydrogen phosphate R and conditions.
1 g/L of sodium dodecyl sulfate R, adjusted to pH 6.8 with The International Unit is the activity of a stated quantity of
dilute sodium hydroxide solution R ; the International Standard of alteplase. The equivalence in
– as detector a spectrophotometer set at 214 nm. International Units of the International Standard is stated by
the World Health Organization.
Inject about 50 μL of the test solution and record the
chromatogram. The chromatogram shows 2 major peaks Solvent buffer. A solution containing 1.38 g/L of sodium
corresponding to single-chain and two-chain alteplase. dihydrogen phosphate monohydrate R, 7.10 g/L of anhydrous
Calculate the relative amount of single-chain alteplase from disodium hydrogen phosphate R, 0.20 g/L of sodium azide R
the peak area values. and 0.10 g/L of polysorbate 80 R.
The test is not valid unless : the number of theoretical plates Human thrombin solution. A solution of human thrombin R
calculated on the basis of the single-chain alteplase peak is containing 33 IU/mL in solvent buffer.
at least 1000. The content of single-chain alteplase is not Human fibrinogen solution. A 2 g/L solution of fibrinogen R
less than 60 per cent of the total amount of alteplase-related in solvent buffer.
substances found. Human plasminogen solution. A 1 g/L solution of human
Monomer content. Examine by liquid chromatography plasminogen R in solvent buffer.
(2.2.29). Test solutions. Using a solution of the substance to be
Test solution. Reconstitute the preparation to be examined to examined containing 1 g/L, prepare serial dilutions using
obtain a solution containing about 1 mg per millilitre. solvent buffer, for example 1:5000, 1:10 000, 1:20 000.
The chromatographic procedure may be carried out using : Reference solutions. Using a solution of a suitable reference
– a column 0.6 m long and 7.5 mm in internal diameter standard having an accurately known concentration of about
packed with silica-based rigid, hydrophilic gel with 1 g/L (580 000 IU of alteplase per millilitre), prepare 5 serial
spherical particles 10 μm to 13 μm in diameter, suitable for dilutions using water R to obtain reference solutions having
size-exclusion chromatography ; known concentrations in the range 9.0 IU/mL to 145 IU/mL.
– as mobile phase at a flow rate of 0.5 mL/min a solution To each of a set of labelled glass test-tubes, add 0.5 mL of
containing 30 g/L of sodium dihydrogen phosphate R and human thrombin solution. Allocate each test and reference
1 g/L of sodium dodecyl sulfate R, adjusted to pH 6.8 with solution to a separate tube and add to each tube 0.5 mL of the
dilute sodium hydroxide solution R ; solution allocated to it. To each of a second set of labelled glass
tubes, add 20 μL of human plasminogen solution, and 1 mL of
– as detector a spectrophotometer set at 214 nm. human fibrinogen solution, mix and store on ice. Beginning
Inject the test solution and record the chromatogram. The test with the reference/thrombin mixture containing the lowest
is not valid unless the number of theoretical plates calculated number of International Units per millilitre, record the time
for the alteplase monomer peak is at least 1000. Measure the and separately add 200 μL of each of the thrombin mixtures
response for all peaks, i.e. peaks corresponding to alteplase to the test tubes containing the plasminogen-fibrinogen
species of different molecular masses. Calculate the relative mixture. Using a vortex mixer, intermittently mix the contents
content of monomer from the area values of these peaks. The of each tube for a total of 15 s and carefully place in a rack
monomer content for alteplase must be at least 95 per cent. in a circulating water-bath at 37 °C. A visibly turbid clot
Water (2.5.12). Not more than 4.0 per cent, determined by forms within 30 s and bubbles subsequently form within
the semi-micro determination of water. the clot. Record the clot-lysis time as the time between the
first addition of alteplase solution and the moment when
Bacterial endotoxins (2.6.14) : less than 1 IU per milligram the last bubble rises to the surface. Using a least-squares fit,
of protein. determine the equation of the line using the logarithms of the
Sterility (2.6.1). It complies with the test for sterility. concentrations of the reference preparation in International

General Notices (1) apply to all monographs and other texts 1517
Altizide EUROPEAN PHARMACOPOEIA 8.0

Units per millilitre versus the logarithms of the values of If the spectra obtained show differences, dissolve 50 mg of the
their clot-lysis times in seconds, according to the following substance to be examined and 50 mg of the reference substance
equation : separately in 2 mL of acetone R and evaporate the solvent.
Precipitate by adding 1 mL of methylene chloride R. Evaporate
to dryness and record new spectra using the residues.
in which t is the clot-lysis time, US the activity in International TESTS
Units per millilitre of the reference preparation, b is the slope
Impurity B. Thin-layer chromatography (2.2.27).
and a the y-intercept of the line. The test is not valid unless the
correlation coefficient is − 0.9900 to − 1.0000. From the line Test solution. Dissolve 0.200 g of the substance to be examined
equation and the clot-lysis time for the test solution, calculate in acetone R and dilute to 2.0 mL with the same solvent.
the logarithm of the activity UA from the following equation : Reference solution (a). Dissolve 10.0 mg of altizide
impurity B CRS in acetone R and dilute to 25.0 mL with the
same solvent.
Reference solution (b). To 1.0 mL of reference solution (a) add
Calculate the alteplase activity in International Units per 1.0 mL of the test solution.
millilitre from the following expression : Reference solution (c). Dilute 5.0 mL of reference solution (a)
to 10.0 mL with acetone R.
Plate : TLC silica gel F254 plate R.
in which D is the dilution factor for the test solution. Calculate Mobile phase : acetone R, methylene chloride R
the specific activity in the portion of the substance to be (25:75 V/V).
examined from the following expression : Application : 10 μL of the test solution and reference
solutions (b) and (c).
Development : over 2/3 of the plate.
in which P is the concentration of protein obtained in the test Drying : in air.
for protein content. Detection : spray with a mixture of equal volumes of a 10 g/L
The estimated potency is not less than 90 per cent and not solution of potassium permanganate R and a 50 g/L solution of
more than 110 per cent of the stated potency. sodium carbonate R, prepared immediately before use. Allow
to stand for 30 min and examine in daylight.
STORAGE System suitability : reference solution (b) :
Store in a colourless, glass container, under vacuum or under – the chromatogram shows 2 clearly separated spots.
an inert gas, protected from light, at a temperature of 2 °C to
30 °C. Limit : any spot due to impurity B is not more intense than the
principal spot in the chromatogram obtained with reference
LABELLING solution (c) (0.2 per cent).
The label states : Related substances. Liquid chromatography (2.2.29).
– the number of International Units per container ; Prepare the solutions immediately before use, except reference
solution (b).
– the amount of protein per container ;
Test solution. Dissolve 50 mg of the substance to be examined
– the name and volume of the liquid to be used for
in 5 mL of acetonitrile R and dilute to 25 mL with the mobile
reconstitution.
phase.
Reference solution (a). Dilute 1.0 mL of the test solution to
07/2008:2185 100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10.0 mL with the mobile phase.
ALTIZIDE Reference solution (b). In order to produce impurity A in
situ, dissolve 50 mg of the substance to be examined in 5 mL
Altizidum of acetonitrile R and dilute to 25 mL with water R. Allow to
stand for 30 min.
Reference solution (c). Dissolve 4 mg of furosemide CRS in
2 mL of acetonitrile R, add 2 mL of the test solution and dilute
to 100 mL with the mobile phase.
Column :
– size : l = 0.15 m, Ø = 3.9 mm ;
C11H14ClN3O4S3 Mr 383.9
[5588-16-9] – stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm) ;
DEFINITION – temperature : 30 °C.
(3RS)-6-Chloro-3-[(prop-2-enylsulfanyl)methyl]-3,4-dihydro- Mobile phase : acetonitrile R, water R previously adjusted to
2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide. pH 2.0 with perchloric acid R (25:75 V/V).
Content : 97.5 per cent to 102.0 per cent (anhydrous substance). Flow rate : 0.7 mL/min.
CHARACTERS Detection : spectrophotometer at 270 nm.
Appearance : white or almost white powder. Injection : 5 μL.
Solubility : practically insoluble in water, soluble in methanol, Run time : twice the retention time of altizide.
practically insoluble in methylene chloride. Relative retention with reference to altizide (retention
It shows polymorphism (5.9). time = about 25 min) : impurity A = about 0.15 ;
furosemide = about 1.05.
IDENTIFICATION System suitability : reference solution (c) :
Infrared absorption spectrophotometry (2.2.24). – resolution : minimum 1.0 between the peaks due to altizide
Comparison : altizide CRS. and furosemide.

1518 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Aluminium chloride hexahydrate

Limits : TESTS
– impurity A : not more than 3 times the area of the principal Solution S. Dissolve 2.5 g in water R and dilute to 50 mL with
peak in the chromatogram obtained with reference the same solvent.
solution (a) (0.3 per cent) ; Appearance of solution. Solution S is clear (2.2.1) and
– unspecified impurities : for each impurity, not more than the colourless (2.2.2, Method II).
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ; pH (2.2.3) : 3.0 to 3.5.
– total : not more than 5 times the area of the principal peak Dissolve 1.0 g in carbon dioxide-free water R and dilute to
in the chromatogram obtained with reference solution (a) 10 mL with the same solvent.
(0.5 per cent) ; Ammonium (2.4.1) : maximum 0.2 per cent.
– disregard limit : 0.5 times the area of the principal peak in To 1 mL of solution S add 4 mL of water R. Dilute 0.5 mL of
the chromatogram obtained with reference solution (a) this solution to 14 mL with water R.
(0.05 per cent). Iron (2.4.9) : maximum 100 ppm.
Water (2.5.32) : maximum 0.5 per cent, determined on Dilute 2 mL of solution S to 10 mL with water R. Use in this
50.0 mg. test 0.3 mL of thioglycollic acid R.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g. 12 mL of solution S complies with test A. Prepare the reference
ASSAY solution using lead standard solution (1 ppm Pb) R.
Liquid chromatography (2.2.29) as described in the test for ASSAY
related substances, with the following modifications. Dissolve 0.900 g in 20 mL of water R and carry out the
Test solution. Dissolve 25.0 mg of the substance to be complexometric titration of aluminium (2.5.11).
examined in 2 mL of acetonitrile R and dilute to 25.0 mL with 1 mL of 0.1 M sodium edetate is equivalent to 47.44 mg
the mobile phase. of AlK(SO4)2,12H2O.
Reference solution. Dissolve 25.0 mg of altizide CRS in 2 mL
of acetonitrile R and dilute to 25.0 mL with the mobile phase.
01/2008:0971
Calculate the percentage content of C11H14ClN3O4S3 from the
declared content of altizide CRS.
ALUMINIUM CHLORIDE
IMPURITIES HEXAHYDRATE
Specified impurities : A, B.
Aluminii chloridum hexahydricum
AlCl3,6H2O Mr 241.4
[7784-13-6]

A. 4-amino-6-chlorobenzene-1,3-disulfonamide, DEFINITION
Content : 95.0 per cent to 101.0 per cent.
CHARACTERS
Appearance : white or slightly yellow, crystalline powder or
B. 3-[(2,2-dimethoxyethyl)sulfanyl]prop-1-ene. colourless crystals, deliquescent.
Solubility : very soluble in water, freely soluble in ethanol
(96 per cent), soluble in glycerol.
01/2008:0006
IDENTIFICATION
ALUM A. Dilute 0.1 mL of solution S2 (see Tests) to 2 mL with
water R. The solution gives reaction (a) of chlorides (2.3.1).
Alumen B. Dilute 0.3 mL of solution S2 to 2 mL with water R. The
solution gives the reaction of aluminium (2.3.1).
AlK(SO4)2,12H2O Mr 474.4 TESTS
[7784-24-9]
Solution S1. Dissolve 10.0 g in distilled water R and dilute to
DEFINITION 100 mL with the same solvent.
Content : 99.0 per cent to 100.5 per cent of AlK(SO4)2,12H2O. Solution S2. Dilute 50 mL of solution S1 to 100 mL with
water R.
CHARACTERS
Appearance of solution. Solution S2 is clear (2.2.1) and not
Appearance : granular powder or colourless, transparent, more intensely coloured than reference solution B7 (2.2.2,
crystalline masses. Method II).
Solubility : freely soluble in water, very soluble in boiling water,
Sulfates (2.4.13) : maximum 100 ppm, determined on
soluble in glycerol, practically insoluble in ethanol (96 per
solution S1.
cent).
Iron (2.4.9) : maximum 10 ppm, determined on solution S1.
IDENTIFICATION
Alkali and alkaline-earth metals : maximum 0.5 per cent.
A. Solution S (see Tests) gives the reactions of sulfates (2.3.1).
To 20 mL of solution S2 add 100 mL of water R and heat
B. Solution S gives the reaction of aluminium (2.3.1). to boiling. To the hot solution add 0.2 mL of methyl red
C. Shake 10 mL of solution S with 0.5 g of sodium hydrogen solution R. Add dilute ammonia R1 until the colour of the
carbonate R and filter. The filtrate gives reaction (a) of indicator changes to yellow and dilute to 150 mL with water R.
potassium (2.3.1). Heat to boiling and filter. Evaporate 75 mL of the filtrate to

General Notices (1) apply to all monographs and other texts 1519
Aluminium hydroxide, hydrated, for adsorption EUROPEAN PHARMACOPOEIA 8.0

dryness on a water-bath and ignite to constant mass. The vigorously at least 5 times. Centrifuge or filter through a
residue weighs a maximum of 2.5 mg. non-protein-retaining filter. Immediately determine the
Heavy metals (2.4.8) : maximum 20 ppm. protein content (2.5.33, Method 2) of either the supernatant
or the filtrate.
12 mL of solution S1 complies with test A. Prepare the
reference solution using lead standard solution (2 ppm Pb) R. It complies with the test if no bovine albumin is detectable
in the supernatant or filtrate of the 2 mg/mL bovine
Water (2.5.12) : 42.0 per cent to 48.0 per cent, determined on albumin R solution (maximum level of adsorption) and in the
50.0 mg. supernatant or filtrate of bovine albumin R solutions of lower
ASSAY concentrations. Solutions containing 3 mg/mL, 5 mg/mL and
10 mg/mL bovine albumin R may show bovine albumin in the
Dissolve 0.500 g in 25.0 mL of water R. Carry out the supernatant or filtrate, proportional to the amount of bovine
complexometric titration of aluminium (2.5.11). Titrate with albumin in the solutions.
0.1 M zinc sulfate until the colour of the indicator changes
from greyish-green to pink. Carry out a blank titration. Sedimentation. If necessary, adjust the substance to be
examined to pH 6.0 using dilute hydrochloric acid R or dilute
1 mL of 0.1 M sodium edetate is equivalent to 24.14 mg sodium hydroxide solution R. Dilute with distilled water R
of AlCl3,6H2O. to obtain an aluminium concentration of approximately
STORAGE 5 mg/mL. If the aluminium content of the substance to
be examined is lower than 5 mg/mL, adjust to pH 6.0 and
In an airtight container.
dilute with a 9 g/L solution of sodium chloride R to obtain an
aluminium concentration of about 1 mg/mL. After shaking
04/2008:1664 for at least 30 s, place 25 mL of the preparation in a 25 mL
graduated cylinder and allow to stand for 24 h.
ALUMINIUM HYDROXIDE, It complies with the test if the volume of the clear supernatant
is less than 5 mL for the gel with an aluminium content of
HYDRATED, FOR ADSORPTION about 5 mg/mL.
It complies with the test if the volume of the clear supernatant
Aluminii hydroxidum hydricum is less than 20 mL for the gel with an aluminium content of
ad adsorptionem about 1 mg/mL.
Chlorides (2.4.4) : maximum 0.33 per cent.
[AlO(OH)],nH2O Dissolve 0.5 g in 10 mL of dilute nitric acid R and dilute to
500 mL with water R.
DEFINITION
Content : 90.0 per cent to 110.0 per cent of the content of Nitrates : maximum 100 ppm.
aluminium stated on the label. Place 5 g in a test-tube immersed in ice-water, add 0.4 mL
NOTE : shake the gel vigorously for at least 30 s immediately of a 100 g/L solution of potassium chloride R, 0.1 mL of
before examining. diphenylamine solution R and, dropwise with shaking, 5 mL
of sulfuric acid R. Transfer the tube to a water-bath at 50 °C.
CHARACTERS After 15 min, any blue colour in the solution is not more
Appearance : white or almost white, translucent, viscous, intense than that in a standard prepared at the same time and
colloidal gel. A supernatant may be formed upon standing. in the same manner using 5 mL of nitrate standard solution
(100 ppm NO3) R.
Solubility : a clear or almost clear solution is obtained with
alkali hydroxide solutions and mineral acids. Sulfates (2.4.13) : maximum 0.5 per cent.
Dilute 2 mL of solution S to 20 mL with water R.
IDENTIFICATION
Ammonium (2.4.1, Method B) : maximum 50 ppm,
Solution S (see Tests) gives the reaction of aluminium. determined on 1.0 g.
To 10 mL of solution S add about 0.5 mL of dilute hydrochloric Prepare the standard using 0.5 mL of ammonium standard
acid R and about 0.5 mL of thioacetamide reagent R. No solution (100 ppm NH4) R.
precipitate is formed. Add dropwise 5 mL of dilute sodium
hydroxide solution R. Allow to stand for 1 h. A gelatinous Arsenic (2.4.2, Method A) : maximum 1 ppm, determined on
white precipitate is formed which dissolves upon addition of 1 g.
5 mL of dilute sodium hydroxide solution R. Gradually add Iron (2.4.9) : maximum 15 ppm, determined on 0.67 g.
5 mL of ammonium chloride solution R and allow to stand for Heavy metals (2.4.8) : maximum 20 ppm.
30 min. The gelatinous white precipitate is re-formed.
Dissolve 2.0 g in 10 mL of dilute nitric acid R and dilute
TESTS to 20 mL with water R. The solution complies with test A.
Solution S. Add 1 g to 4 mL of hydrochloric acid R. Heat at Prepare the reference solution using lead standard solution
60 °C for 1 h, cool, dilute to 50 mL with distilled water R and (2 ppm Pb) R.
filter if necessary. Bacterial endotoxins (2.6.14) : less than 5 IU of endotoxin
pH (2.2.3) : 5.5 to 8.5. per milligram of aluminium, if intended for use in the
manufacture of an adsorbed product without a further
Adsorption power. Dilute the substance to be examined appropriate procedure for the removal of bacterial endotoxins.
with distilled water R to obtain an aluminium concentration
of 5 mg/mL. Prepare bovine albumin R solutions with the ASSAY
following concentrations of bovine albumin : 0.5 mg/mL, Dissolve 2.50 g in 10 mL of hydrochloric acid R, heating for
1 mg/mL, 2 mg/mL, 3 mg/mL, 5 mg/mL and 10 mg/mL. If 30 min at 100 °C on a water-bath. Cool and dilute to 20 mL
necessary, adjust the gel and the bovine albumin R solutions with water R. To 10 mL of the solution, add concentrated
to pH 6.0 with dilute hydrochloric acid R or dilute sodium ammonia R until a precipitate is obtained. Add the smallest
hydroxide solution R. quantity of hydrochloric acid R needed to dissolve the
For adsorption, mix 1 part of the diluted gel with 4 parts of precipitate and dilute to 20 mL with water R. Carry out the
each of the solutions of bovine albumin R and allow to stand at complexometric titration of aluminium (2.5.11). Carry out
room temperature for 1 h. During this time shake the mixture a blank titration.

1520 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Aluminium magnesium silicate

STORAGE Lead : maximum 15 ppm.

At a temperature not exceeding 30 °C. Do not allow to Atomic absorption spectrometry (2.2.23, Method I).
freeze. If the substance is sterile, store in a sterile, airtight,Test solution. Transfer 10.0 g to a 250 mL beaker containing
tamper-proof container. 100 mL of dilute hydrochloric acid R. Mix, cover with a
watch glass and boil for 15 min. Allow to cool to room
LABELLING temperature and allow the insoluble matter to settle. Decant
The label states the declared content of aluminium. the supernatant through a rapid-flow filter paper into a
400 mL beaker. To the insoluble matter in the 250 mL beaker
add 25 mL of hot water R. Stir, allow the insoluble matter
01/2009:1388 to settle and decant the supernatant through the filter into
corrected 7.0 the 400 mL beaker. Repeat the extraction with 2 additional
quantities, each of 25 mL, of water R, decanting each time the
ALUMINIUM MAGNESIUM SILICATE supernatant through the filter into the 400 mL beaker. Wash
the filter with 25 mL of hot water R, collecting this filtrate
Aluminii magnesii silicas in the 400 mL beaker. Concentrate the combined filtrates to
about 20 mL by gently boiling. If a precipitate appears, add
DEFINITION about 0.1 mL of nitric acid R, heat to boiling and allow to
Mixture of particles with colloidal particle size of cool to room temperature. Filter the concentrated extracts
montmorillonite and saponite, free from grit and through a rapid-flow filter paper into a 50 mL volumetric
non-swellable ore. flask. Transfer the remaining contents of the 400 mL beaker
Content : through the filter paper and into the flask with water R. Dilute
this solution to 50.0 mL with water R.
– aluminium (Al ; Ar 26.98) : 95.0 per cent to 105.0 per cent of
Reference solutions. Prepare the reference solutions using lead
the value stated on the label ;
standard solution (10 ppm Pb) R, diluted as necessary with
– magnesium (Mg ; Ar 24.30) : 95.0 per cent to 105.0 per cent water R.
of the value stated on the label.
Source : lead hollow-cathode lamp.
CHARACTERS Wavelength : 217 nm.
Appearance : almost white powder, granules or plates. Atomisation device : oxidising air-acetylene flame.
Solubility : practically insoluble in water and in organic Loss on drying (2.2.32) : maximum 8.0 per cent, determined
solvents. on 1.000 g by drying in an oven at 105 °C.
It swells in water to produce a colloidal dispersion. Microbial contamination
IDENTIFICATION TAMC : acceptance criterion 103 CFU/g (2.6.12).
2
A. Fuse 1 g with 2 g of anhydrous sodium carbonate R. Warm TYMC : acceptance criterion 10 CFU/g (2.6.12).
the residue with water R and filter. Acidify the filtrate Absence of Escherichia coli (2.6.13).
with hydrochloric acid R and evaporate to dryness on a
ASSAY
water-bath. 0.25 g of the residue gives the reaction of
silicates (2.3.1). Aluminium. Atomic absorption spectrometry (2.2.23,
B. Dissolve the remainder of the residue obtained in Method I).
identification test A in a mixture of 5 mL of dilute Test solution. In a platinum crucible mix 0.200 g with
hydrochloric acid R and 10 mL of water R. Filter and add 1.0 g of lithium metaborate R. Heat slowly at first and
ammonium chloride buffer solution pH 10.0 R. A white, ignite at 1000-1200 °C for 15 min. Allow to cool, then
gelatinous precipitate is formed. Centrifuge and keep the place the crucible in a 100 mL beaker containing 25 mL of
supernatant for identification C. Dissolve the remaining dilute nitric acid R and add an additional 50 mL of dilute
precipitate in dilute hydrochloric acid R. The solution gives nitric acid R, filling and submerging the crucible. Place a
the reaction of aluminium (2.3.1). polytetrafluoroethylene-coated magnetic stirring bar in the
C. The supernatant obtained after centrifugation in crucible and stir gently with a magnetic stirrer until dissolution
identification test B gives the reaction of magnesium (2.3.1). is complete. Pour the contents into a 250 mL beaker and
remove the crucible. Warm the solution and transfer through
TESTS a rapid-flow filter paper into a 250 mL volumetric flask, wash
the filter and beaker with water R and dilute to 250.0 mL with
pH (2.2.3) : 9.0 to 10.0.
water R (solution A). To 20.0 mL of solution A add 20 mL of a
Disperse 5.0 g in 100 mL of carbon dioxide-free water R. 10 g/L solution of sodium chloride R and dilute to 100.0 mL
Arsenic (2.4.2, Method A) : maximum 3 ppm. with water R.
Transfer 16.6 g to a 250 mL beaker containing 100 mL of Reference solutions. Dissolve, with gentle heating, 1.000 g of
dilute hydrochloric acid R. Mix, cover with a watch glass and aluminium R in a mixture of 10 mL of hydrochloric acid R
boil gently, with occasional stirring, for 15 min. Allow the and 10 mL of water R. Allow to cool, then dilute to 1000.0 mL
insoluble matter to settle and decant the supernatant through with water R (1 mg of aluminium per millilitre). Into 3
a rapid-flow filter paper into a 250 mL volumetric flask, identical volumetric flasks, each containing 0.20 g of sodium
retaining as much sediment as possible in the beaker. To the chloride R, introduce 2.0 mL, 5.0 mL and 10.0 mL of this
residue in the beaker add 25 mL of hot dilute hydrochloric solution respectively, and dilute to 100.0 mL with water R.
acid R, stir, heat to boiling, allow the insoluble matter to Source : aluminium hollow-cathode lamp.
settle and decant the supernatant through the filter into the Wavelength : 309 nm.
volumetric flask. Repeat the extraction with 4 additional Atomisation device : oxidising acetylene-nitrous oxide flame.
quantities, each of 25 mL, of hot dilute hydrochloric acid R,
decanting each supernatant through the filter into the Magnesium. Atomic absorption spectrometry (2.2.23,
volumetric flask. At the last extraction, transfer as much of Method I).
the insoluble matter as possible onto the filter. Allow the Test solution. Dilute 25.0 mL of solution A, prepared in the
combined filtrates to cool to room temperature and dilute to assay for aluminium, to 50.0 mL with water R. To 5.0 mL of
250.0 mL with dilute hydrochloric acid R. Dilute 5.0 mL of this this solution add 20.0 mL of lanthanum nitrate solution R and
solution to 25.0 mL with dilute hydrochloric acid R. dilute to 100.0 mL with water R.

General Notices (1) apply to all monographs and other texts 1521
Aluminium oxide, hydrated EUROPEAN PHARMACOPOEIA 8.0

Reference solutions. Place 1.000 g of magnesium R in a 250 mL solution complies with test A. Prepare the reference solution
beaker containing 20 mL of water R and carefully add 20 mL of using 10 mL of lead standard solution (1 ppm Pb) R.
hydrochloric acid R, warming if necessary to dissolve. Transfer Microbial contamination
the solution to a volumetric flask and dilute to 1000.0 mL with
water R (1 mg of magnesium per millilitre). Dilute 5.0 mL TAMC : acceptance criterion 103 CFU/g (2.6.12).
of this solution to 250.0 mL with water R. Into 4 identical TYMC : acceptance criterion 102 CFU/g (2.6.12).
volumetric flasks, introduce 5.0 mL, 10.0 mL, 15.0 mL and Absence of bile-tolerant gram-negative bacteria (2.6.13).
20.0 mL of the solution respectively. To each flask add 20.0 mL Absence of Escherichia coli (2.6.13).
of lanthanum nitrate solution R and dilute to 100.0 mL with
water R. ASSAY
Source : magnesium hollow-cathode lamp. Dissolve 0.800 g in 10 mL of hydrochloric acid R1, heating
Wavelength : 285 nm. on a water-bath. Cool and dilute to 50.0 mL with water R.
To 10.0 mL of the solution add dilute ammonia R1 until a
Atomisation device : reducing air-acetylene flame. precipitate begins to appear. Add the smallest quantity of dilute
LABELLING hydrochloric acid R needed to dissolve the precipitate and
dilute to 20 mL with water R. Carry out the complexometric
The label states the content of aluminium and magnesium. titration of aluminium (2.5.11).
1 mL of 0.1 M sodium edetate is equivalent to 5.098 mg
of Al2O3.
01/2011:0311
STORAGE
ALUMINIUM OXIDE, HYDRATED In an airtight container, at a temperature not exceeding 30 °C.
FUNCTIONALITY-RELATED CHARACTERISTICS
Aluminii oxidum hydricum This section provides information on characteristics that are
DEFINITION recognised as being relevant control parameters for one or
Content : 47.0 per cent to 60.0 per cent of Al2O3 (Mr 102.0). more functions of the substance when used as an excipient (see
chapter 5.15). This section is a non-mandatory part of the
CHARACTERS monograph and it is not necessary to verify the characteristics
Appearance : white or almost white, amorphous powder. to demonstrate compliance. Control of these characteristics can
however contribute to the quality of a medicinal product by
Solubility : practically insoluble in water. It dissolves in dilute improving the consistency of the manufacturing process and
mineral acids and in solutions of alkali hydroxides. the performance of the medicinal product during use. Where
IDENTIFICATION control methods are cited, they are recognised as being suitable
for the purpose, but other methods can also be used. Wherever
Solution S (see Tests) gives the reaction of aluminium (2.3.1). results for a particular characteristic are reported, the control
TESTS method must be indicated.
Solution S. Dissolve 2.5 g in 15 mL of hydrochloric acid R, The following characteristics may be relevant for hydrated
heating on a water-bath. Dilute to 100 mL with distilled aluminium oxide used as adsorbent.
water R. Particle-size distribution (2.9.31).
Appearance of solution. Solution S is not more opalescent Specific surface area (2.9.26).
than reference suspension II (2.2.1) and not more intensely
coloured than reference solution GY6 (2.2.2, Method II). 01/2009:2166
Alkaline impurities. Shake 1.0 g with 20 mL of carbon
dioxide-free water R for 1 min and filter. To 10 mL of the ALUMINIUM PHOSPHATE GEL
filtrate add 0.1 mL of phenolphthalein solution R. Any
pink colour disappears on the addition of 0.3 mL of 0.1 M
hydrochloric acid.
Aluminii phosphatis liquamen
Neutralising capacity. Carry out the test at 37 °C. Disperse
0.5 g in 100 mL of water R, heat, add 100.0 mL of 0.1 M
hydrochloric acid, previously heated, and stir continuously ; DEFINITION
the pH (2.2.3) of the solution after 10 min, 15 min and 20 min Hydrated AlPO4 in gel form.
is not less than 1.8, 2.3 and 3.0 respectively and is at no time Content : 19.0 per cent to 21.0 per cent of AlPO4.
greater than 4.5. Add 10.0 mL of 0.5 M hydrochloric acid,
previously heated, stir continuously for 1 h and titrate with CHARACTERS
0.1 M sodium hydroxide to pH 3.5 ; not more than 35.0 mL of Appearance : gel.
0.1 M sodium hydroxide is required.
Solubility : practically insoluble in water, in ethanol (96 per
Chlorides (2.4.4) : maximum 1 per cent. cent) and in methylene chloride. It dissolves in dilute solutions
Dissolve 0.1 g with heating in 10 mL of dilute nitric acid R and of mineral acids.
dilute to 100 mL with water R. Dilute 5 mL of the solution
to 15 mL with water R. IDENTIFICATION
A. Solution S (see Tests) gives reaction (b) of phosphates
Sulfates (2.4.13) : maximum 1 per cent. (2.3.1).
Dilute 4 mL of solution S to 100 mL with distilled water R. B. Solution S gives the reaction of aluminium (2.3.1).
Arsenic (2.4.2, Method A) : maximum 4 ppm, determined on C. It complies with the assay.
10 mL of solution S.
Heavy metals (2.4.8) : maximum 60 ppm. TESTS
Neutralise 20 mL of solution S with concentrated ammonia R, Solution S. Dissolve 2.00 g in dilute hydrochloric acid R and
using metanil yellow solution R as an external indicator. Filter, dilute to 100 mL with the same acid.
if necessary, and dilute to 30 mL with water R. 12 mL of the pH (2.2.3) : 6.0 to 8.0.

1522 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Aluminium phosphate, hydrated

Peroxides : maximum 150 ppm, expressed as hydrogen maintain boiling for 3 min. Cool, then add 25 mL of ethanol
peroxide. (96 per cent) R. Titrate with 0.1 M zinc sulfate, determining
Test solution. Dissolve with heating 1.0 g of the substance to the end-point potentiometrically (2.2.20).
be examined in 5 mL of dilute hydrochloric acid R, then add 1 mL of 0.1 M zinc sulfate is equivalent to 12.2 mg of AlPO4.
5 mL of water R and 2 mL of divanadium pentoxide solution
in sulfuric acid R. STORAGE
Reference solution. Dilute 1.0 mL of dilute hydrogen peroxide In an airtight container.
solution R to 200.0 mL with water R. To 1 mL of this solution
add 9 mL of water R and 2 mL of divanadium pentoxide
01/2008:1598
solution in sulfuric acid R.
corrected 6.0
The test solution is not more intensely coloured than the
reference solution.
ALUMINIUM PHOSPHATE,
Chlorides (2.4.4): maximum 500 ppm.
HYDRATED
Dissolve 1.3 g in 5 mL of dilute nitric acid R and dilute to
200 mL with water R.
Aluminii phosphas hydricus
Soluble phosphates : maximum 0.5 per cent, expressed as PO4.
Test solution. Centrifuge 10.0 g until a clear supernatant is AlPO4,xH2O Mr 122.0 (anhydrous substance)
obtained. To 2.00 mL of the supernatant add 20.0 mL of a
10.3 g/L solution of hydrochloric acid R and dilute to 100.0 mL DEFINITION
with water R. To 10.0 mL of this solution add 10.0 mL of Content : 94.0 per cent to 102.0 per cent of AlPO4 (Mr 122.0)
nitro-molybdovanadic reagent R and dilute to 50.0 mL with (ignited substance).
water R. Allow to stand protected from light for 15 min.
Reference solution. Add 10.0 mL of nitro-molybdovanadic CHARACTERS
reagent R to 10.0 mL of a 143 mg/L solution of potassium Appearance : white or almost white powder.
dihydrogen phosphate R and dilute to 50.0 mL with water R. Solubility : very slightly soluble in water, practically insoluble
Allow to stand protected from light for 15 min. in ethanol (96 per cent). It dissolves in dilute solutions of
Measure the absorbances (2.2.25) of the 2 solutions at 400 nm. mineral acids and alkali hydroxides.
The absorbance of the test solution is not greater than that of
the reference solution. IDENTIFICATION
A. Solution S (see Tests) gives reaction (b) of phosphates
Sulfates (2.4.13) : maximum 0.2 per cent. (2.3.1).
Dilute 25 mL of solution S to 100 mL with distilled water R. B. Solution S gives the reaction of aluminium (2.3.1).
Soluble aluminium : maximum 50 ppm.
TESTS
To 16.0 g add 50 mL of water R. Heat to boiling for 5 min.
Cool and centrifuge. Separate the supernatant. Wash the Solution S. Dissolve 2.00 g in dilute hydrochloric acid R and
residue with 20 mL of water R and centrifuge. Separate the dilute to 100 mL with the same acid.
supernatant and add to the first supernatant. To the combined Appearance of solution. Solution S is clear (2.2.1) and
supernatants add 5 mL of hydrochloric acid R and 20 mL of colourless (2.2.2, Method II).
water R. Introduce all of this solution into a 500 mL conical pH (2.2.3) : 5.5 to 7.2
flask and carry out the complexometric titration of aluminium
(2.5.11) using 0.01 M sodium edetate. Shake 4.0 g with carbon dioxide-free water R and dilute to
100 mL with the same solvent.
Arsenic (2.4.2, Method A) : maximum 1 ppm, determined on
1.0 g. Chlorides (2.4.4) : maximum 1.3 per cent.
Dissolve 50.0 mg in 10 mL of dilute nitric acid R and dilute to
Heavy metals (2.4.8) : maximum 10 ppm.
200 mL with water R.
Dissolve 4.0 g in dilute hydrochloric acid R and dilute to
20 mL with the same acid. 12 mL of the solution complies Soluble phosphates : maximum 1.0 per cent, calculated as
with test A. Prepare the reference solution using lead standard PO43-.
solution (2 ppm Pb) R. Test solution. Stir 5.0 g with 150 mL of water R for 2 h. Filter
and wash the filter with 50 mL of water R. Combine the filtrate
Acid neutralising capacity. Add 2.0 g to 30 mL of 0.1 M and the washings and dilute to 250.0 mL with water R. Dilute
hydrochloric acid heated to 37 °C and maintain at 37 °C while 10.0 mL of this solution to 100.0 mL with water R.
shaking. Determine the pH after 15 min. The pH (2.2.3) of
the mixture is 2.0 to 2.5. Reference solution (a). Dissolve 2.86 g of potassium dihydrogen
phosphate R in water R and dilute to 100 mL with the same
Residue on ignition : 19.0 per cent to 23.0 per cent. solvent.
Heat 0.500 g at 50 °C for 5 hours, then ignite at 500 ± 50 °C Reference solution (b). Dilute 1 mL of reference solution (a)
until constant mass. to 5 mL with water R.
Microbial contamination Reference solution (c). Dilute 3 mL of reference solution (a)
TAMC : acceptance criterion 103 CFU/g (2.6.12). to 5 mL with water R.
TYMC : acceptance criterion 102 CFU/g (2.6.12). Treat each solution as follows. To 5.0 mL add 4 mL of dilute
sulfuric acid R, 1 mL of ammonium molybdate solution R,
Absence of bile-tolerant gram-negative bacteria (2.6.13). 5 mL of water R and 2 mL of a solution containing 0.10 g of
Absence of Escherichia coli (2.6.13). 4-methylaminophenol sulfate R, 0.5 g of anhydrous sodium
sulfite R and 20.0 g of sodium metabisulfite R in 100 mL of
ASSAY water R. Shake and allow to stand for 15 min. Dilute to 25.0 mL
Dissolve with heating 0.300 g in 5 mL of dilute hydrochloric with water R and allow to stand for a further 15 min. Measure
acid R. Add 45 mL of water R, 10.0 mL of 0.1 M sodium edetate the absorbance (2.2.25) at 730 nm. Calculate the content of
and 30 mL of a mixture of equal volumes of ammonium soluble phosphates from a calibration curve prepared using
acetate solution R and dilute acetic acid R. Heat to boiling and reference solutions (a), (b) and (c) after treatment.

General Notices (1) apply to all monographs and other texts 1523
Aluminium sodium silicate EUROPEAN PHARMACOPOEIA 8.0

Sulfates (2.4.13) : maximum 0.6 per cent. gently, with occasional stirring, for 15 min. Centrifuge, and
Dilute 8 mL of solution S to 100 mL with distilled water R. decant the supernatant through a rapid-flow filter paper
into a 250 mL volumetric flask. To the residue in the beaker,
Arsenic (2.4.2): maximum 1 ppm. add 25 mL of hot dilute hydrochloric acid R, stir, centrifuge,
1.0 g complies with limit test A. and decant the supernatant through the same filter into the
Heavy metals (2.4.8) : maximum 20 ppm. volumetric flask. Repeat the extraction with 3 additional
Dissolve 1.0 g in dilute hydrochloric acid R and dilute to quantities, each of 25 mL, of hot dilute hydrochloric acid R,
20 mL with the same acid. 12 mL of the solution complies filtering each supernatant through this filter into the
with test A. Prepare the reference solution using lead standard volumetric flask. Allow the combined filtrates to cool to room
solution (1 ppm Pb) R. temperature and dilute to 250.0 mL with dilute hydrochloric
acid R. Dilute 10.0 mL of the solution to 25.0 mL with water R.
Loss on ignition. 10.0 per cent to 20.0 per cent, determined
on 1.000 g at 800 ± 50 °C. Lead : maximum 5 ppm.
Neutralising capacity. Add 0.50 g to 30 mL of 0.1 M Atomic absorption spectrometry (2.2.23, Method I).
hydrochloric acid previously heated to 37 °C and maintain at Test solution. Transfer 5.0 g to a 250 mL beaker containing
this temperature for 15 min while stirring. The pH (2.2.3) of 50 mL of dilute hydrochloric acid R. Mix, cover with a watch
the mixture after 15 min at 37 °C is 2.0 to 2.5. glass and boil for 15 min. Allow to cool to room temperature.
Centrifuge, and decant the supernatant through a rapid-flow
ASSAY filter paper into a 250 mL beaker. To the insoluble matter add
Dissolve 0.400 g in 10 mL of dilute hydrochloric acid R and 25 mL of hot water R. Stir vigorously, centrifuge, and decant
dilute to 100.0 mL with water R. To 10.0 mL of the solution, the supernatant through the same filter into the beaker. Repeat
add 10.0 mL of 0.1 M sodium edetate and 30 mL of a mixture the extraction with 2 additional quantities, each of 25 mL, of
of equal volumes of ammonium acetate solution R and dilute hot water R, decanting each supernatant through the filter
acetic acid R. Boil for 3 min, then cool. Add 25 mL of ethanol into the beaker. Wash the filter with 25 mL of hot water R,
(96 per cent) R and 1 mL of a freshly prepared 0.25 g/L solution collecting the filtrate in the beaker. Concentrate the combined
of dithizone R in alcohol R. Titrate the excess of sodium edetate filtrates by gently boiling to about 15 mL. Add about 0.05 mL
with 0.1 M zinc sulfate until the colour changes to pink. of heavy metal-free nitric acid R, heat to boiling and allow to
cool to room temperature. Filter the concentrated extracts
1 mL of 0.1 M sodium edetate is equivalent to 12.20 mg of
through a rapid-flow filter paper into a 25 mL volumetric
AlPO4.
flask. Transfer the remaining contents of the beaker through
STORAGE the filter paper and into the volumetric flask with water R and
dilute to 25.0 mL with the same solvent.
In an airtight container.
Reference solutions. Into 4 separate 100 mL volumetric flasks,
introduce respectively 3.0 mL, 5.0 mL, 10.0 mL and 15.0 mL
01/2009:1676 of lead standard solution (10 ppm Pb) R, add 0.20 mL of heavy
corrected 7.0 metal-free nitric acid R and dilute to 100.0 mL with water R.
Source : lead hollow-cathode lamp.
ALUMINIUM SODIUM SILICATE Wavelength : 217.0 nm.
Atomisation device : air-acetylene flame.
Aluminii natrii silicas
Loss on drying (2.2.32) : maximum 8.0 per cent, determined
DEFINITION on 1.000 g by drying in an oven at 105 °C for 4 h.
Silicic acid aluminium sodium salt of synthetic origin. Loss on ignition : 5.0 per cent to 11.0 per cent (dried
Content : substance), determined on 1.000 g by ignition in a platinum
– aluminium (Al ; Mr 26.98) : 2.7 per cent to 7.9 per cent crucible to constant mass at 1000 ± 25 °C.
(dried substance) ; Microbial contamination
– sodium (Na ; Mr 22.99): 3.7 per cent to 6.3 per cent (dried TAMC : acceptance criterion 103 CFU/g (2.6.12).
substance). TYMC : acceptance criterion 102 CFU/g (2.6.12).
CHARACTERS Absence of Escherichia coli (2.6.13).
Appearance : white or almost white, fine, light, amorphous ASSAY
powder.
Aluminium. Atomic absorption spectrometry (2.2.23,
Solubility : practically insoluble in water and in organic
Method I).
solvents.
Acid mixture. Add 50 mL of nitric acid R to 500 mL of water R.
IDENTIFICATION Dissolve in this solution 17 g of tartaric acid R and dilute to
A. Transfer 1.0 g to a 100 mL beaker and add 10 mL of dilute 1000 mL with water R.
hydrochloric acid R. Mix, cover with a watch glass and boil Blank solution. Dissolve 1.4 g of anhydrous lithium
for 15 min. Allow to cool to room temperature, mix and metaborate R in 60 mL of the acid mixture and dilute to
centrifuge the solution. 2 mL of the supernatant gives the 200 mL with water R.
reaction of aluminium (2.3.1). Test solution. In a platinum crucible mix 0.200 g with 1.4 g of
B. 2 mL of the supernatant obtained in identification test A anhydrous lithium metaborate R. Heat slowly at first and ignite
gives reaction (a) of sodium (2.3.1). at 1100 ± 25 °C for 15 min. Cool, then place the crucible in a
C. 0.2 g gives the reaction of silicates (2.3.1). 100 mL beaker containing 60 mL of the acid mixture. Place
a polytetrafluoroethylene-coated magnetic stirring bar in
TESTS the crucible and stir gently with a magnetic stirrer for 16 h.
pH (2.2.3) : 9.5 to 11.5. Transfer the contents of the crucible into a 200 mL volumetric
Disperse 5.0 g in 100 mL of carbon dioxide-free water R. flask. Wash the crucible, the magnetic stirring bar and the
beaker with water R and dilute to 200.0 mL with the same
Arsenic (2.4.2, Method A) : maximum 3 ppm. solvent (solution A). To 10.0 mL of this solution, add 1.0 mL
Transfer 8.3 g to a 250 mL beaker containing 50 mL of dilute of lanthanum chloride solution R and dilute to 50.0 mL with
hydrochloric acid R. Mix, cover with a watch glass and boil water R.

1524 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Aluminium stearate

Reference solutions. Into 5 separate 50 mL volumetric flasks, Allow to cool. In a separating funnel, separate the aqueous
introduce respectively 1.0 mL, 2.5 mL, 5.0 mL, 7.5 mL and layer and shake the ether layer with 2 quantities, each of 4 mL,
10.0 mL of aluminium standard solution (100 ppm Al) R, add of distilled water R. Combine the aqueous layers, wash with
1 mL of lanthanum chloride solution R and 10 mL of the blank 15 mL of peroxide-free ether R and dilute to 50.0 mL with
solution, and dilute to 50.0 mL with water R. distilled water R (solution S). Evaporate the ether layer to
Source : aluminium hollow-cathode lamp. dryness and dry the residue at 100-105 °C. Keep the residue
for identification tests A and B.
Wavelength : 309.3 nm.
Atomisation device : acetylene-nitrous oxide flame. Acidity or alkalinity. To 1.0 g add 20 mL of carbon
dioxide-free water R and boil for 1 min with continuous
Sodium. Atomic emission spectrometry (2.2.22, Method I). shaking. Cool and filter. To 10 mL of the filtrate add 0.05 mL
Test solution. To 2.0 mL of solution A, prepared in the assay of bromothymol blue solution R4. Not more than 0.05 mL of
of aluminium, add 1 mL of a 12.5 g/L solution of caesium 0.1 M hydrochloric acid or 0.1 M sodium hydroxide is required
chloride R and dilute to 20.0 mL with water R. to change the colour of the indicator.
Reference solutions. Into 5 separate 200 mL volumetric Chlorides (2.4.4) : maximum 0.1 per cent.
flasks, each containing 10 mL of a 12.5 g/L solution of
Dilute 0.5 mL of solution S to 15 mL with water R.
caesium chloride R, introduce respectively 1.0 mL, 2.0 mL,
4.0 mL, 6.0 mL and 10.0 mL of sodium standard solution Sulfates (2.4.13) : maximum 0.5 per cent.
(200 ppm Na) R and dilute to 200.0 mL with water R. Dilute 0.3 mL of solution S to 15 mL with distilled water R.
Wavelength : 589.0 nm. Cadmium : maximum 3 ppm.
Atomic absorption spectrometry (2.2.23, Method II).
07/2012:1663 For the preparation of all aqueous solutions and for the rinsing
of glassware before use, employ water that has been passed
through a strong-acid, strong-base, mixed-bed ion-exchange
ALUMINIUM STEARATE resin before use. Select all reagents to have as low a content of
cadmium, lead and nickel as practicable and store all reagent
Aluminii stearas solutions in containers of borosilicate glass. Clean glassware
before use by soaking in warm 8 M nitric acid for 30 min and
DEFINITION by rinsing with deionised water.
Aluminium salts of a mixture of solid organic acids consisting Blank solution. Dilute 25 mL of cadmium- and lead-free nitric
mainly of variable proportions of aluminium stearate and acid R to 100.0 mL with water R.
aluminium palmitate. The organic acids are obtained from
sources of vegetable or animal origin. Modifier solution. Dissolve 20 g of ammonium dihydrogen
phosphate R and 1 g of magnesium nitrate R in water R and
Content :
dilute to 100 mL with the same solvent. Alternatively, use
– aluminium (Al ; Ar 26.98) : 3.0 per cent to 9.0 per cent an appropriate matrix modifier as recommended by the
(dried substance) ; graphite furnace atomic absorption (GFAA) spectrometer
– stearic acid in the fatty acid fraction : minimum 40.0 per manufacturer.
cent ; Test solution. Place 0.100 g of the substance to be examined
– sum of stearic acid and palmitic acid in the fatty acid in a polytetrafluoroethylene digestion bomb and add 2.5 mL
fraction : minimum 90.0 per cent. of cadmium- and lead-free nitric acid R. Close and seal the
bomb according to the manufacturer’s operating instructions.
CHARACTERS When using a digestion bomb, be thoroughly familiar with the
Appearance : white or almost white, very fine, light powder. safety and operating instructions. Carefully follow the bomb
Solubility : practically insoluble in water and in anhydrous manufacturer’s instructions regarding care and maintenance
ethanol. of these digestion bombs. Do not use metal-jacketed bombs
or liners that have been used with hydrochloric acid due
IDENTIFICATION to contamination from corrosion of the metal jacket by
First identification : C, D. hydrochloric acid. Heat the bomb in an oven at 170 °C for 3 h.
Second identification : A, B, D. Cool the bomb slowly in air to room temperature according
to the bomb manufacturer’s instructions. Place the bomb in a
A. Freezing point (2.2.18) : minimum 53 °C, determined on fume cupboard and open carefully as corrosive gases may be
the residue obtained in the preparation of solution S (see expelled. Dissolve the residue in water R and dilute to 10.0 mL
Tests). with the same solvent.
B. Acid value (2.5.1) : 195 to 210. Reference solution. Prepare a solution containing
Dissolve 0.200 g of the residue obtained in the preparation 0.00165 μg/mL of cadmium nitrate tetrahydrate R in the blank
of solution S in 25 mL of the prescribed mixture of solvents. solution (equivalent to 0.006 μg/mL of Cd).
C. Examine the chromatograms obtained in the assay of Dilute 1.0 mL of the test solution to 10.0 mL with the blank
stearic acid and palmitic acid. solution. Prepare mixtures of this solution, the reference
Results : the 2 principal peaks in the chromatogram solution and the blank solution in the following proportions :
obtained with the test solution are similar in retention time (1.0:0:1.0 V/V/V), (1.0:0.25:0.75 V/V/V), (1.0:0.5:0.5 V/V/V),
to the 2 principal peaks in the chromatogram obtained (1.0:0.75:0.25 V/V/V). To each mixture add 50 μL of
with the reference solution. the modifier solution and mix. These solutions contain
D. 1 mL of solution S gives the reaction of aluminium (2.3.1). respectively 0 μg, 0.0015 μg, 0.0030 μg and 0.0045 μg of
The addition of 0.5 mL of dilute hydrochloric acid R cadmium per millilitre from the reference solution. Keep the
described in the general method is omitted. remaining test solution for use in the test for lead and nickel.
Source : cadmium hollow-cathode lamp.
TESTS
Wavelength : 228.8 nm.
Solution S. To 5.0 g add 50 mL of peroxide-free ether R, 20 mL
of dilute nitric acid R and 20 mL of distilled water R and heat Atomisation device : furnace.
gently under a reflux condenser until dissolution is complete. Platform : pyrolytically coated with integrated tube.

General Notices (1) apply to all monographs and other texts 1525
Aluminium stearate EUROPEAN PHARMACOPOEIA 8.0

Operating conditions : use the temperature programme Reference solution. Prepare a solution of 0.050 μg/mL of Ni by
recommended for cadmium by the GFAA manufacturer. An suitable dilutions of a 0.2477 μg/mL solution of nickel nitrate
example of temperature parameters for GFAA analysis of hexahydrate R with the blank solution.
cadmium is shown below. Prepare mixtures of the test solution, the reference solution
Stage Final temperature Ramp time Hold time and the blank solution in the following proportions :
(°C) (s) (s) (1.0:0:1.0 V/V/V), (1.0:0.5:0.5 V/V/V), (1.0:1.0:0 V/V/V). To
Drying 110 10 20 each mixture add 50 μL of the modifier solution and mix.
These solutions contain respectively 0 μg, 0.0125 μg and
Ashing 600 10 30 0.025 μg of nickel per millilitre from the reference solution.
Atomisation 1800 0 5 Source : nickel hollow-cathode lamp.
Wavelength : 232.0 nm.
Lead : maximum 10 ppm.
Atomic absorption spectrometry (2.2.23, Method II). Atomisation device : furnace.
For the preparation of all aqueous solutions and for the rinsing Platform : pyrolytically coated with integrated tube.
of glassware before use, employ water that has been passed Operating conditions: use the temperature programme
through a strong-acid, strong-base, mixed-bed ion-exchange recommended for nickel by the GFAA manufacturer. An
resin before use. Select all reagents to have as low a content of example of temperature parameters for GFAA analysis of
cadmium, lead and nickel as practicable and store all reagent nickel is shown below.
solutions in containers of borosilicate glass. Clean glassware Stage Ramp time
Final temperature Hold time
before use by soaking in warm 8 M nitric acid for 30 min and
(°C) (s) (s)
by rinsing with deionised water.
Drying 110 10 20
Blank solution. Use the solution described in the test for
cadmium. Ashing 1000 20 30
Modifier solution. Use the solution described in the test for Atomisation 2300 0 5
cadmium.
Test solution. Use the solution described in the test for Loss on drying (2.2.32) : maximum 6.0 per cent, determined
cadmium. on 1.000 g by drying in an oven at 105 °C.
Reference solution. Prepare a solution of 0.100 μg/mL of Pb by Microbial contamination
suitable dilutions of lead standard solution (100 ppm Pb) R
TAMC : acceptance criterion 103 CFU/g (2.6.12).
with the blank solution.
Prepare mixtures of the test solution, the reference solution TYMC : acceptance criterion 102 CFU/g (2.6.12).
and the blank solution in the following proportions : Absence of Escherichia coli (2.6.13).
(1.0:0:1.0 V/V/V), (1.0:0.5:0.5 V/V/V), (1.0:1.0:0 V/V/V). To Absence of Salmonella (2.6.13).
each mixture add 50 μL of the modifier solution and mix.
These solutions contain respectively 0 μg, 0.025 μg and 0.05 μg ASSAY
of lead per millilitre from the reference solution. Aluminium. To 0.250 g in a 250 mL conical flask add 20 mL
Source : lead hollow-cathode lamp. of methanol R and, slowly, 2 mL of sulfuric acid R. Heat the
Wavelength : 283.3 nm. solution for 30 min under reflux on a water-bath, swirling
Atomisation device : furnace. frequently. Allow to cool. Add 100 mL of water R and adjust
Platform : pyrolytically coated with integrated tube. to about pH 1 by adding approximately 12 mL of dilute
sodium hydroxide solution R. Add 20.0 mL of 0.1 M sodium
Operating conditions : use the temperature programme edetate and adjust to between pH 5 and pH 6 by the addition
recommended for lead by the GFAA manufacturer. An of sodium acetate R. Add 70 mg of xylenol orange triturate R
example of temperature parameters for GFAA analysis of lead and titrate immediately and quickly with 0.1 M zinc sulfate
is shown below. until the colour changes from yellow to pinkish-violet.
Stage Final temperature Ramp time Hold time 1 mL of 0.1 M sodium edetate is equivalent to 2.698 mg of Al.
(°C) (s) (s)
Drying 110 10 20
Stearic acid and palmitic acid. Gas chromatography (2.2.28) :
use the normalisation procedure.
450 10 30
Ashing Test solution. In a conical flask fitted with a reflux condenser,
Atomisation 2000 0 5 dissolve 0.100 g of the substance to be examined in 5 mL of
boron trifluoride-methanol solution R. Boil under a reflux
Nickel : maximum 5 ppm. condenser for 10 min. Add 4 mL of heptane R through
Atomic absorption spectrometry (2.2.23, Method II). the condenser and boil again under a reflux condenser for
For the preparation of all aqueous solutions and for the rinsing 10 min. Allow to cool. Add 20 mL of saturated sodium
of glassware before use, employ water that has been passed chloride solution R. Shake and allow the layers to separate.
through a strong-acid, strong-base, mixed-bed ion-exchange Dry the organic layer over 0.1 g of anhydrous sodium sulfate R
resin before use. Select all reagents to have as low a content of previously washed with heptane R. Dilute 1.0 mL of the
cadmium, lead and nickel as practicable and store all reagent solution to 10.0 mL with heptane R.
solutions in containers of borosilicate glass. Clean glassware Reference solution. Prepare the reference solution in the same
before use by soaking in warm 8 M nitric acid for 30 min and manner as the test solution using 50.0 mg of palmitic acid CRS
by rinsing with deionised water. and 50.0 mg of stearic acid CRS instead of the substance to
Blank solution. Use the solution described in the test for be examined.
cadmium. Column :
Modifier solution. Dissolve 20 g of ammonium dihydrogen – material : fused silica ;
phosphate R in water R and dilute to 100 mL with the same – size : l = 30 m, Ø = 0.32 mm ;
solvent. Alternatively, use an appropriate matrix modifier as
recommended by the GFAA spectrometer manufacturer. – stationary phase : macrogol 20 000 R (film thickness 0.5 μm).
Test solution. Use the solution described in the test for Carrier gas : helium for chromatography R.
cadmium. Flow rate : 2.4 mL/min.

1526 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Alverine citrate

Temperature : Heavy metals (2.4.8) : maximum 50 ppm.

Time Temperature Dilute 8 mL of solution S to 20 mL with water R. 12 mL of the
(min) (°C) solution complies with test A. Prepare the reference solution
Column 0-2 70 using lead standard solution (1 ppm Pb) R.
2 - 36 70 → 240 ASSAY
36 - 41 240 Dissolve 0.500 g in 20 mL of water R. Carry out the
complexometric titration of aluminium (2.5.11).
Injection port 220
1 mL of 0.1 M sodium edetate is equivalent to 17.11 mg
Detector 260 of Al2(SO4)3.

Detection : flame ionisation. STORAGE

Injection : 1 μL. In an airtight container.
Relative retention with reference to methyl stearate : methyl
palmitate = about 0.9. 01/2008:2156
System suitability: reference solution :
– resolution : minimum 5.0 between the peaks due to methyl ALVERINE CITRATE
palmitate and methyl stearate ;
– repeatability : maximum relative standard deviation of Alverini citras
3.0 per cent for the areas of the peaks due to methyl
palmitate and methyl stearate after 6 injections ; maximum
relative standard deviation of 1.0 per cent for the ratio of
the areas of the peaks due to methyl palmitate to the areas
of the peaks due to methyl stearate after 6 injections.

01/2008:0165 C26H35NO7 Mr 473.6

[5560-59-8]
ALUMINIUM SULFATE DEFINITION
N-Ethyl-3-phenyl-N-(3-phenylpropyl)propan-1-amine
Aluminii sulfas dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate.
Content : 99.0 per cent to 101.0 per cent (dried substance).
Al2(SO4)3,xH2O Mr 342.1 (anhydrous substance)
CHARACTERS
DEFINITION
Appearance : white or almost white, crystalline powder.
Content : 51.0 per cent to 59.0 per cent of Al2(SO4)3. Solubility : slightly soluble in water and in methylene chloride,
It contains a variable quantity of water of crystallisation. sparingly soluble in ethanol (96 per cent).
CHARACTERS mp : about 104 °C.
Appearance : colourless, lustrous crystals or crystalline masses. IDENTIFICATION
Solubility : soluble in cold water, freely soluble in hot water, Infrared absorption spectrophotometry (2.2.24).
practically insoluble in ethanol (96 per cent).
Comparison: alverine citrate CRS.
IDENTIFICATION
TESTS
A. Solution S (see Tests) gives reaction (a) of sulfates (2.3.1).
pH (2.2.3) : 3.5 to 4.5.
B. Solution S gives the reaction of aluminium (2.3.1).
Dissolve 0.250 g in carbon dioxide-free water R and dilute to
TESTS 50.0 mL with the same solvent.
Solution S. Dissolve 2.5 g in water R and dilute to 50 mL with Related substances. Gas chromatography (2.2.28) : use the
the same solvent. normalisation procedure. Use freshly prepared solutions.
Appearance of solution. Solution S is not more opalescent Test solution. Dissolve 0.250 g of the substance to be examined
than reference suspension III (2.2.1) and is colourless (2.2.2, in water R and dilute to 20 mL with the same solvent. Add
Method II). 2 mL of concentrated ammonia R and shake with 3 quantities,
each of 15 mL, of methylene chloride R. To the combined
pH (2.2.3) : 2.5 to 4.0.
lower layers add anhydrous sodium sulfate R, shake, filter, and
Dissolve 0.5 g in carbon dioxide-free water R and dilute to evaporate the filtrate at a temperature not exceeding 30 °C,
25 mL with the same solvent. using a rotary evaporator. Take up the residue with methylene
Alkali and alkaline-earth metals : maximum 0.4 per cent. chloride R and dilute to 10.0 mL with the same solvent.
To 20 mL of solution S add 100 mL of water R, heat and add Reference solution (a). Dissolve 5 mg of alverine
0.1 mL of methyl red solution R. Add dilute ammonia R1 until impurity D CRS (impurity D citrate) in 5 mL of water R, add
the colour of the indicator changes to yellow. Dilute to 150 mL 1 mL of concentrated ammonia R and shake with 3 quantities,
with water R, heat to boiling and filter. Evaporate 75 mL of each of 5 mL, of methylene chloride R. To the combined lower
the filtrate to dryness on a water-bath and ignite. The residue layers add anhydrous sodium sulfate R, shake, filter, and
weighs a maximum of 2 mg. evaporate the filtrate at a temperature not exceeding 30 °C,
Ammonium (2.4.1) : maximum 500 ppm. using a rotary evaporator. Take up the residue with methylene
chloride R, add 0.2 mL of the test solution and dilute to 2 mL
Dilute 0.4 mL of solution S to 14 mL with water R. with methylene chloride R.
Iron (2.4.9) : maximum 100 ppm. Reference solution (b). Dilute 1.0 mL of the test solution to
Dilute 2 mL of solution S to 10 mL with water R. Use 0.3 mL 100.0 mL with methylene chloride R. Dilute 1.0 mL of this
of thioglycollic acid R in this test. solution to 20.0 mL with methylene chloride R.

General Notices (1) apply to all monographs and other texts 1527
Amantadine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Reference solution (c). Dissolve the contents of a vial of IMPURITIES

alverine for peak identification CRS (containing impurities C Specified impurities : A, B, C, D, E.
and E) in 1 mL of methylene chloride R.
Column :
– material : fused silica ;
– size : l = 25 m, Ø = 0.32 mm ; A. R = Cl : 1-chloro-3-phenylpropane,
– stationary phase : poly(dimethyl)(diphenyl)siloxane R (film
B. R = OH : 3-phenylpropan-1-ol,
thickness 0.45 μm).
Carrier gas : helium for chromatography R. C. R = NH-C2H5 : N-ethyl-3-phenylpropan-1-amine,
Flow rate : 2.2 mL/min.
Split ratio : 1:11.
Temperature :
D. N-(3-cyclohexylpropyl)-N-ethyl-3-phenylpropan-1-amine,
Time Temperature
(min) (°C)
Column 0-7 120

7 - 13 120 → 240

13 - 21 240

21 - 24 240 → 290

24 - 39 290 E. 3-phenyl-N,N-bis(3-phenylpropyl)propan-1-amine.
Injection port 290
07/2012:0463
Detector 290

Detection : flame ionisation. AMANTADINE HYDROCHLORIDE

Injection : 1 μL.
Identification of impurities : use the chromatogram Amantadini hydrochloridum
supplied with alverine for peak identification CRS and the
chromatogram obtained with reference solution (c) to identify
the peaks due to impurities C and E.
Relative retention with reference to alverine (retention
time = about 16 min) : impurity A = about 0.28 ; C10H18ClN Mr 187.7
impurity B = about 0.29 ; impurity C = about 0.46 ; [665-66-7]
impurity D = about 0.97 ; impurity E = about 1.7.
DEFINITION
System suitability: reference solution (a) :
Tricyclo[3.3.1.13,7]decan-1-amine hydrochloride.
– resolution : minimum 3.0 between the peaks due to
impurity D and alverine. Content : 98.5 per cent to 101.0 per cent (anhydrous substance).
Limits : CHARACTERS
– impurities A, B : for each impurity, maximum 0.1 per cent ; Appearance : white or almost white, crystalline powder.
– impurity C : maximum 0.2 per cent ; Solubility : freely soluble in water and in ethanol (96 per cent).
– impurities D, E : for each impurity, maximum 0.3 per cent ; It sublimes on heating.
– unspecified impurities : for each impurity, maximum IDENTIFICATION
0.10 per cent ;
First identification : A, D.
– total : maximum 1.0 per cent ;
Second identification : B, C, D.
– disregard limit : the area of the principal peak in the
A. Infrared absorption spectrophotometry (2.2.24).
chromatogram obtained with reference solution (b)
(0.05 per cent). Comparison: amantadine hydrochloride CRS.
Heavy metals (2.4.8) : maximum 20 ppm. B. To 0.1 g add 1 mL of pyridine R, mix and add 0.1 mL of
acetic anhydride R. Heat to boiling for about 10 s. Pour the
0.5 g complies with test G. Prepare the reference solution hot solution into 10 mL of dilute hydrochloric acid R, cool
using 1 mL of lead standard solution (10 ppm Pb) R. to 5 °C and filter. The precipitate, washed with water R and
Loss on drying (2.2.32) : maximum 0.5 per cent, determined dried in vacuo at 60 °C for 1 h, melts (2.2.14) at 147 °C to
on 1.000 g by drying in an oven at 80 °C for 2 h. 151 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on C. Dissolve 0.2 g in 1 mL of 0.1 M hydrochloric acid. Add
1.0 g. 1 mL of a 500 g/L solution of sodium nitrite R. A white
precipitate is formed.
ASSAY D. 1 mL of solution S (see Tests) gives reaction (a) of chlorides
Dissolve 0.375 g in 50 mL of anhydrous acetic acid R. Titrate (2.3.1).
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20). TESTS
1 mL of 0.1 M perchloric acid is equivalent to 47.36 mg Solution S. Dissolve 2.5 g in carbon dioxide-free water R and
of C26H35NO7. dilute to 25 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
STORAGE more intensely coloured than reference solution Y7 (2.2.2,
Protected from light. Method II).

1528 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ambroxol hydrochloride

Acidity or alkalinity. Dilute 2 mL of solution S to 10 mL – disregard limit : calculate the ratio (R3) of 0.5 times the area
with carbon dioxide-free water R. Add 0.1 mL of methyl red of the peak due to amantadine to the area of the peak due
solution R and 0.2 mL of 0.01 M sodium hydroxide. The to the internal standard from the chromatogram obtained
solution is yellow. Add 0.4 mL of 0.01 M hydrochloric acid. with the reference solution ; from the chromatogram
The solution is red. obtained with the test solution, calculate the ratio of the
Related substances. Gas chromatography (2.2.28). area of any peak, apart from the principal peak and the
peak due to the internal standard, to the area of the peak
Internal standard solution. Dissolve 0.500 g of adamantane R due to the internal standard : disregard any peak with a
in methylene chloride R and dilute to 10.0 mL with the same ratio less than R3 (0.05 per cent).
solvent.
Test solution. Weigh 0.5 g of the substance to be examined Heavy metals (2.4.8) : maximum 20 ppm.
into a centrifuge tube. Add 9 mL of methylene chloride R and 12 mL of solution S complies with test A. Prepare the reference
10 mL of a 210 g/L solution of sodium hydroxide R. Shake for solution using lead standard solution (2 ppm Pb) R.
10 min. Discard the upper layer. Dry the lower layer over Water (2.5.12) : maximum 0.5 per cent, determined on 2.00 g.
anhydrous sodium sulfate R. Filter and collect the filtrate in a
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
volumetric flask. Add 0.1 mL of the internal standard solution
1.0 g.
and dilute to 10.0 mL with methylene chloride R.
Reference solution. Weigh 5 mg of amantadine ASSAY
hydrochloride CRS into a centrifuge tube. Add 9 mL of Dissolve 0.150 g in a mixture of 5.0 mL of 0.01 M hydrochloric
methylene chloride R and 10 mL of a 210 g/L solution of acid and 50 mL of ethanol (96 per cent) R. Carry out a
sodium hydroxide R. Shake for 10 min. Discard the upper potentiometric titration (2.2.20), using 0.1 M sodium
layer. Dry the lower layer over anhydrous sodium sulfate R. hydroxide. Read the volume added between the 2 points of
Filter and collect the filtrate in a volumetric flask. Add 1.0 mL inflexion.
of the internal standard solution and dilute to 100.0 mL with 1 mL of 0.1 M sodium hydroxide is equivalent to 18.77 mg
methylene chloride R. of C10H18ClN.
Column :
– material : fused silica ; IMPURITIES
– size : l = 30 m, Ø = 0.53 mm ; Other detectable impurities (the following substances would,
– stationary phase : base-deactivated poly(dimethyl)(diphen- if present at a sufficient level, be detected by one or other of
yl)siloxane R (film thickness 1 μm). the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
Carrier gas : helium for chromatography R.
by the general monograph Substances for pharmaceutical use
Flow rate : 4 mL/min. (2034). It is therefore not necessary to identify these impurities
Split ratio : 1:50. for demonstration of compliance. See also 5.10. Control of
Temperature : impurities in substances for pharmaceutical use) : A, B.
Time Temperature
(min) (°C)
Column 0-5 70

5 - 23 70 → 250 A. 1-chlorotricyclo[3.3.1.13,7]decane,
23 - 40 250

Injection port 220

Detector 300

Detection : flame ionisation. B. N-(tricyclo[3.3.1.13,7]dec-1-yl)acetamide.

Injection : 1 μL.
Relative retention with reference to amantadine (retention 01/2011:1489
time = about 14 min) : internal standard = about 0.8.
System suitability: reference solution : AMBROXOL HYDROCHLORIDE
– resolution : minimum 5.0 between the peaks due to the
internal standard and amantadine. Ambroxoli hydrochloridum
Limits :
– unspecified impurities : calculate the ratio (R1) of the area
of the peak due to amantadine to the area of the peak due
to the internal standard from the chromatogram obtained
with the reference solution ; from the chromatogram
obtained with the test solution, calculate the ratio of the
area of any peak, apart from the principal peak and the
peak due to the internal standard, to the area of the peak C13H19Br2ClN2O Mr 414.6
due to the internal standard : this ratio is not greater than [23828-92-4]
R1 (0.10 per cent) ;
DEFINITION
– total : calculate the ratio (R2) of 3 times the area of the
peak due to amantadine to the area of the peak due to the trans-4-[(2-Amino-3,5-dibromobenzyl)amino]cyclohexanol
internal standard from the chromatogram obtained with hydrochloride.
the reference solution ; from the chromatogram obtained Content : 99.0 per cent to 101.0 per cent (dried substance).
with the test solution, calculate the ratio of the sum of the
areas of any peaks, apart from the principal peak and the CHARACTERS
peak due to the internal standard, to the area of the peak Appearance : white or yellowish, crystalline powder.
due to the internal standard : this ratio is not greater than Solubility : sparingly soluble in water, soluble in methanol,
R2 (0.3 per cent) ; practically insoluble in methylene chloride.

General Notices (1) apply to all monographs and other texts 1529
Ambroxol hydrochloride EUROPEAN PHARMACOPOEIA 8.0

IDENTIFICATION Mobile phase : a mixture of equal volumes of acetonitrile R and

First identification : B, D. a solution prepared as follows : dissolve 1.32 g of ammonium
phosphate R in 900 mL of water R, adjust to pH 7.0 with
Second identification : A, C, D. phosphoric acid R and dilute to 1000 mL with water R.
A. Ultraviolet and visible absorption spectrophotometry Flow rate : 1 mL/min.
(2.2.25). Detection : spectrophotometer at 248 nm.
Test solution. Dissolve 20.0 mg in 0.05 M sulfuric acid and Injection : 20 μL.
dilute to 100.0 mL with the same acid. Dilute 2.0 mL of the
solution to 10.0 mL with 0.05 M sulfuric acid. Run time : 3 times the retention time of ambroxol.
Identification of impurities: use the chromatogram obtained
Spectral range : 200-350 nm. with reference solution (b) to identify the peak due to
Absorption maxima : at 245 nm and 310 nm. impurity B.
Absorbance ratio : A245/A310 = 3.2 to 3.4. Relative retention with reference to ambroxol (retention
B. Infrared absorption spectrophotometry (2.2.24). time = about 9 min) : impurity B = about 0.6.
Comparison : ambroxol hydrochloride CRS. System suitability : reference solution (b) :
– resolution : minimum 4.0 between the peaks due to
C. Thin-layer chromatography (2.2.27).
impurity B and ambroxol.
Test solution. Dissolve 50 mg of the substance to be Limits :
examined in methanol R and dilute to 5 mL with the same
solvent. – unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
Reference solution. Dissolve 50 mg of ambroxol with reference solution (a) (0.10 per cent),
hydrochloride CRS in methanol R and dilute to 5 mL with
– total : not more than 3 times the area of the principal peak
the same solvent.
in the chromatogram obtained with reference solution (a)
Plate : TLC silica gel F254 plate R. (0.3 per cent),
Mobile phase : concentrated ammonia R, propanol R, ethyl – disregard limit : 0.5 times the area of the principal peak in
acetate R, hexane R (1:10:20:70 V/V/V/V). the chromatogram obtained with reference solution (a)
Application : 10 μL. (0.05 per cent).
Development : over 2/3 of the plate. Heavy metals (2.4.8) : maximum 20 ppm.
Drying : in air. 1.0 g complies with test C. Prepare the reference solution using
2 mL of lead standard solution (10 ppm Pb) R.
Detection : examine in ultraviolet light at 254 nm.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Results : the principal spot in the chromatogram obtained on 1.000 g by drying in an oven at 105 °C.
with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
reference solution. 1.0 g.
D. Dissolve 25 mg in 2.5 mL of water R, mix with 1.0 mL of ASSAY
dilute ammonia R1 and allow to stand for 5 min. Filter and Dissolve 0.300 g in 70 mL of ethanol (96 per cent) R and add
acidify the filtrate with dilute nitric acid R. The filtrate gives
5 mL of 0.01 M hydrochloric acid. Carry out a potentiometric
reaction (a) of chlorides (2.3.1). titration (2.2.20), using 0.1 M sodium hydroxide. Read
the volume added between the 2 points of inflexion.
TESTS
1 mL of 0.1 M sodium hydroxide is equivalent to 41.46 mg of
Solution S. Dissolve 0.75 g in methanol R and dilute to 15 mL C H Br ClN O.
13 19 2 2
with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not STORAGE
more intensely coloured than reference solution Y6 (2.2.2, Protected from light.
Method II). IMPURITIES
pH (2.2.3) : 4.5 to 6.0. Other detectable impurities (the following substances would,
Dissolve 0.2 g in carbon dioxide-free water R and dilute to if present at a sufficient level, be detected by one or other of
20 mL with the same solvent. the tests in the monograph. They are limited by the general
Related substances. Liquid chromatography (2.2.29). Prepare acceptance criterion for other/unspecified impurities and/or
the solutions immediately before use. by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
Test solution. Dissolve 50 mg of the substance to be examined for demonstration of compliance. See also 5.10. Control of
in water R and dilute to 50.0 mL with the same solvent. impurities in substances for pharmaceutical use) : A, B, C, D, E.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with water R. Dilute 1.0 mL of this solution to
10.0 mL with the mobile phase.
Reference solution (b). In order to prepare impurity B in situ,
dissolve 5 mg of the substance to be examined in 0.2 mL
of methanol R, add 0.04 mL of a mixture of 1 volume of A. Ar-CH2OH : (2-amino-3,5-dibromophenyl)methanol,
formaldehyde solution R and 99 volumes of water R. Heat at
60 °C for 5 min. Evaporate to dryness under a current of
nitrogen. Dissolve the residue in 5 mL of water R and dilute to
20.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.0 mm ;
– stationary phase : octadecylsilyl silica gel for B. trans-4-(6,8-dibromo-1,4-dihydroquinazolin-3(2H)-
chromatography R (5 μm). yl)cyclohexanol,

1530 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Amidotrizoic acid dihydrate

Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on

1.0 g.
ASSAY
C. trans-4-[[(E)-2-amino-3,5-dibromobenzyliden]amino]cy-
clohexanol, Dissolve 0.300 g in 30 mL of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 36.85 mg
of C18H28N2O4S.
D. cis-4-[(2-amino-3,5-dibromobenzyl)amino]cyclohexanol, STORAGE
E. Ar-CH=O : 2-amino-3,5-dibromobenzaldehyde. Protected from light.

01/2008:0368 07/2012:0873
corrected 6.0
AMIDOTRIZOIC ACID DIHYDRATE
AMFETAMINE SULFATE
Acidum amidotrizoicum dihydricum
Amfetamini sulfas

C18H28N2O4S Mr 368.5
[60-13-9] C11H9I3N2O4,2H2O Mr 650
[50978-11-5]
DEFINITION
Bis[(2RS)-1-phenylpropan-2-amine] sulfate. DEFINITION
Content : 99.0 per cent to 100.5 per cent (dried substance). 3,5-Bis(acetylamino)-2,4,6-triiodobenzoic acid dihydrate.
CHARACTERS Content : 98.5 per cent to 101.0 per cent (dried substance).
Appearance : white or almost white powder. CHARACTERS
Solubility : freely soluble in water, slightly soluble in ethanol Appearance : white or almost white, crystalline powder.
(96 per cent). Solubility : very slightly soluble in water and in ethanol (96 per
IDENTIFICATION cent). It dissolves in dilute solutions of alkali hydroxides.
First identification : A, B, E. IDENTIFICATION
Second identification : A, C, D, E. First identification : A.
A. Optical rotation (2.2.7) : − 0.04° to + 0.04° (measured in a Second identification : B, C.
2 dm tube), determined on solution S (see Tests). A. Infrared absorption spectrophotometry (2.2.24).
B. Infrared absorption spectrophotometry (2.2.24). Comparison: amidotrizoic acid dihydrate CRS.
Preparation : mulls in liquid paraffin R. B. Thin-layer chromatography (2.2.27).
Comparison : Ph. Eur. reference spectrum of amfetamine Test solution. Dissolve 25 mg of the substance to be
sulfate. examined in a 3 per cent V/V solution of ammonia R in
C. To 50 mL of solution S add 5 mL of strong sodium hydroxide methanol R and dilute to 5 mL with the same solution.
solution R and 0.5 mL of benzoyl chloride R and shake. Reference solution. Dissolve 25 mg of amidotrizoic acid
Continue to add benzoyl chloride R in portions of 0.5 mL dihydrate CRS in a 3 per cent V/V solution of ammonia R
until no further precipitate is formed. Filter, wash the in methanol R and dilute to 5 mL with the same solution.
precipitate with water R, recrystallise twice from a mixture
of equal volumes of ethanol (96 per cent) R and water R, Plate : TLC silica gel GF254 plate R.
then dry at 100-105 °C. The crystals melt (2.2.14) at 131 °C Mobile phase : anhydrous formic acid R, methyl ethyl
to 135 °C. ketone R, toluene R (20:25:60 V/V/V).
D. To about 2 mg add 1 mL of sulfuric acid-formaldehyde Application : 2 μL.
reagent R. An orange colour develops and quickly becomes Development : over 2/3 of the plate.
dark-brown. Drying : in air until the solvents have evaporated.
E. Solution S gives reaction (a) of sulfates (2.3.1). Detection : in ultraviolet light at 254 nm.
TESTS Results : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to the
Solution S. Dissolve 2.0 g in carbon dioxide-free water R and
principal spot in the chromatogram obtained with the
dilute to 100 mL with the same solvent.
reference solution.
Appearance of solution. Solution S is clear (2.2.1) and C. Heat 50 mg gently in a small porcelain dish over a naked
colourless (2.2.2, Method II). flame. Violet vapour is evolved.
Acidity or alkalinity. To 25 mL of solution S add 0.1 mL
of methyl red solution R. Not more than 0.1 mL of 0.01 M TESTS
hydrochloric acid or 0.01 M sodium hydroxide is required to Appearance of solution. The solution is clear (2.2.1) and
change the colour of the indicator. colourless (2.2.2, Method II).
Loss on drying (2.2.32) : maximum 1.0 per cent, determined Dissolve 1.0 g in dilute sodium hydroxide solution R and dilute
on 1.00 g by drying in an oven at 105 °C. to 20 mL with the same solution.

General Notices (1) apply to all monographs and other texts 1531
Amidotrizoic acid dihydrate EUROPEAN PHARMACOPOEIA 8.0

Related substances. Liquid chromatography (2.2.29). and 12 mL of dilute hydrochloric acid R. Shake gently and allow
Solvent mixture. Dissolve 0.250 g of sodium hydroxide R and to stand for exactly 2 min after adding the hydrochloric acid.
0.860 g of sodium dihydrogen phosphate R in 50 mL of water R Add 10 mL of a 20 g/L solution of ammonium sulfamate R.
and dilute to 1000 mL with the same solvent. Allow to stand for 5 min, shaking frequently, and add 0.15 mL
of a 100 g/L solution of α-naphthol R in ethanol (96 per cent) R.
Test solution. Dissolve 40.0 mg of the substance to be
Shake and allow to stand for 5 min. Add 3.5 mL of buffer
examined in 10.0 mL of the solvent mixture with the aid of
solution pH 10.9 R, mix and dilute to 50.0 mL with water R.
ultrasound.
The absorbance (2.2.25), measured within 20 min at 485 nm
Reference solution (a). Dilute 1.0 mL of the test solution to using as the compensation liquid a solution prepared at the
100.0 mL with the solvent mixture. Dilute 1.0 mL of this same time and in the same manner but omitting the substance
solution to 10.0 mL with the solvent mixture. to be examined, is not greater than 0.30.
Reference solution (b). Dilute 1.0 mL of reference solution (a) Heavy metals (2.4.8) : maximum 20 ppm.
to 10.0 mL with the solvent mixture.
Dissolve 2.0 g in 4 mL of dilute sodium hydroxide solution R
Reference solution (c). Dissolve the contents of a vial of and dilute to 20 mL with water R. 12 mL of the solution
amidotrizoic acid for system suitability CRS (impurities A, B, complies with test A. Prepare the reference solution using lead
C and D) in 1.0 mL of the solvent mixture. standard solution (2 ppm Pb) R.
Column : Loss on drying (2.2.32) : 4.5 per cent to 7.0 per cent,
– size : l = 0.25 m, Ø = 4.6 mm ; determined on 0.500 g by drying in an oven at 105 °C.
– stationary phase : end-capped octadecylsilyl silica gel for Sulfated ash (2.4.14) : maximum 0.1 per cent, determined
chromatography R (5 μm). on 1.0 g.
Mobile phase. Dissolve 3.4 g of tetrabutylammonium hydrogen
sulfate R in a mixture of 230 mL of acetonitrile R and 770 mL ASSAY
of water R. To 0.150 g in a 250 mL round-bottomed flask add 5 mL
Flow rate : 1.0 mL/min. of strong sodium hydroxide solution R, 20 mL of water R,
1 g of zinc powder R and a few glass beads. Boil under a
Detection : spectrophotometer at 236 nm.
reflux condenser for 30 min. Allow to cool and rinse the
Injection : 20 μL. condenser with 20 mL of water R, adding the rinsings to
Run time : 4 times the retention time of amidotrizoic acid. the flask. Filter through a sintered-glass filter (2.1.2) and
Identification of impurities : use the chromatogram supplied wash the filter with several quantities of water R. Collect the
with amidotrizoic acid for system suitability CRS and the filtrate and washings. Add 40 mL of dilute sulfuric acid R
chromatogram obtained with reference solution (c) to identify and titrate immediately with 0.1 M silver nitrate. Determine
the peaks due to impurities A, B, C and D. the end-point potentiometrically (2.2.20), using a suitable
electrode system such as silver/mercurous sulfate.
Relative retention with reference to amidotrizoic acid
(retention time = about 5 min): impurity B = about 0.8 ; 1 mL of 0.1 M silver nitrate is equivalent to 20.47 mg of
impurity C = about 0.9 ; impurity A = about 1.4 ; C11H9I3N2O4.
impurity D = about 1.8.
STORAGE
System suitability :
Protected from light.
– resolution : minimum 1.5 between the peaks due to
impurities B and C in the chromatogram obtained with IMPURITIES
reference solution (c) ; Specified impurities : A, B, D.
– signal-to-noise ratio : minimum 25 for the principal peak in Other detectable impurities (the following substances would,
the chromatogram obtained with reference solution (b). if present at a sufficient level, be detected by one or other of
Limits : the tests in the monograph. They are limited by the general
– impurity B : not more than the area of the principal peak acceptance criterion for other/unspecified impurities and/or
in the chromatogram obtained with reference solution (a) by the general monograph Substances for pharmaceutical use
(0.1 per cent) ; (2034). It is therefore not necessary to identify these impurities
– impurities A, D : for each impurity, not more than the area for demonstration of compliance. See also 5.10. Control of
of the principal peak in the chromatogram obtained with impurities in substances for pharmaceutical use) : C, E.
reference solution (b) (0.01 per cent) ;
– unspecified impurities : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.05 per cent) ;
– total : not more than 1.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.15 per cent) ; A. 3-(acetylamino)-5-amino-2,4,6-triiodobenzoic acid,
– disregard limit : 0.3 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.03 per cent), except for the peaks due to impurities A
and D.
Halides expressed as chlorides (2.4.4) : maximum 150 ppm.
Dissolve 0.55 g in a mixture of 4 mL of dilute sodium hydroxide B. 3,5-bis(acetylamino)-2,4-diiodobenzoic acid,
solution R and 15 mL of water R. Add 6 mL of dilute nitric
acid R and filter.
Free aromatic amines. Maintain the solutions and reagents
in iced water, protected from bright light. To 0.50 g in a 50 mL
volumetric flask add 15 mL of water R. Shake and add 1 mL
of dilute sodium hydroxide solution R. Cool in iced water, add
5 mL of a freshly prepared 5 g/L solution of sodium nitrite R C. 3,5-bis(acetylamino)-2,6-diiodobenzoic acid,

1532 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Amikacin

Detection : spray with ninhydrin solution R1 and heat at

110 °C for 5 min.
System suitability : reference solution (b) :
– the chromatogram shows 2 clearly separated spots.
Results : the principal spot in the chromatogram obtained
D. 3-(acetylamino)-5-[(iodoacetyl)amino]-2,4,6- with the test solution is similar in position, colour and size
triiodobenzoic acid, to the principal spot in the chromatogram obtained with
reference solution (a).
TESTS
pH (2.2.3) : 9.5 to 11.5.
Dissolve 0.1 g in carbon dioxide-free water R and dilute to
10 mL with the same solvent.
Specific optical rotation (2.2.7) : + 97 to + 105 (anhydrous
E. 3-(acetylamino)-5-(diacetylamino)-2,4,6-triiodobenzoic substance).
acid.
Dissolve 0.50 g in water R and dilute to 25.0 mL with the same
solvent.
07/2012:1289 Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 25 mg of the substance to be examined
AMIKACIN in mobile phase A and dilute to 50.0 mL with mobile phase A.
Reference solution (a). Dilute 1.0 mL of the test solution to
Amikacinum 100.0 mL with mobile phase A.
Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 10.0 mL with mobile phase A.
Reference solution (c). Dissolve 5 mg of amikacin for system
suitability CRS (containing impurities A, B, F and H) in mobile
phase A and dilute to 10 mL with mobile phase A.
Reference solution (d). Dissolve 5.0 mg of amikacin
impurity I CRS in mobile phase A and dilute to 20.0 mL with
mobile phase A. Dilute 1.0 mL of the solution to 100.0 mL
with mobile phase A.
Column :
C22H43N5O13 Mr 585.6
[37517-28-5] – size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
DEFINITION chromatography R (5 μm) ;
6-O-(3-Amino-3-deoxy-α-D-glucopyranosyl)-4-O-(6- – temperature : 40 °C.
amino-6-deoxy-α-D-glucopyranosyl)-1-N-[(2S)-4-amino-2- Mobile phase :
hydroxybutanoyl]-2-deoxy-D-streptamine.
– mobile phase A : a mixture prepared with carbon dioxide-free
Antimicrobial substance obtained from kanamycin A. water R, containing 1.8 g/L of sodium octanesulfonate R,
Semi-synthetic product derived from a fermentation product. 20 g/L of anhydrous sodium sulfate R1, 1.4 per cent V/V of
Content : 96.5 per cent to 102.0 per cent (anhydrous substance). tetrahydrofuran R, and 5 per cent V/V of 0.2 M potassium
dihydrogen phosphate R previously adjusted to pH 3.0 with
CHARACTERS dilute phosphoric acid R ; degas ;
Appearance : white or almost white powder. – mobile phase B : a mixture prepared with carbon dioxide-free
Solubility : sparingly soluble in water, slightly soluble in water R, containing 1.8 g/L of sodium octanesulfonate R,
methanol, practically insoluble in acetone and in ethanol 28 g/L of anhydrous sodium sulfate R1, 1.4 per cent V/V of
(96 per cent). tetrahydrofuran R, and 5 per cent V/V of 0.2 M potassium
dihydrogen phosphate R previously adjusted to pH 3.0 with
IDENTIFICATION dilute phosphoric acid R ; degas ;
A. Infrared absorption spectrophotometry (2.2.24). Time Mobile phase A Mobile phase B
Comparison : amikacin CRS. (min) (per cent V/V) (per cent V/V)
B. Thin-layer chromatography (2.2.27). 0-3 100 0
Test solution. Dissolve 25 mg of the substance to be 3 - 38.0 100 → 30 0 → 70
examined in water R and dilute to 10 mL with the same
solvent. 38.0 - 38.1 30 → 0 70 → 100

Reference solution (a). Dissolve 25 mg of amikacin CRS in 38.1 - 68 0 100

water R and dilute to 10 mL with the same solvent.
Reference solution (b). Dissolve 5 mg of kanamycin Flow rate : 1.0 mL/min.
monosulfate CRS in 1 mL of the test solution and dilute to Post-column solution : mixture of 1 volume of carbonate-free
10 mL with water R. sodium hydroxide solution R and 24 volumes of previously
degassed carbon dioxide-free water R, which is added in
Plate : TLC silica gel plate R.
a pulseless manner to the column effluent using a 375 μL
Mobile phase : methylene chloride R, ammonia R, methanol R polymeric mixing coil.
(25:30:40 V/V/V).
Flow rate of post-column solution : 0.3 mL/min.
Application : 5 μL.
Detection : pulsed amperometric detector or equivalent with
Development : over 3/4 of the plate. a gold indicator electrode, a silver-silver chloride reference
Drying : in air. electrode, and a stainless steel auxiliary electrode which is the

General Notices (1) apply to all monographs and other texts 1533
Amikacin EUROPEAN PHARMACOPOEIA 8.0

cell body, held at respectively + 0.05 V detection, + 0.75 V Run time : 1.3 times the retention time of amikacin.
oxidation and − 0.15 V reduction potentials, with pulse Retention time : amikacin = about 30 min.
durations according to the instrument used.
System suitability : reference solution :
Injection : 20 μL.
– symmetry factor : maximum 1.5 for the peak due to
Identification of impurities : use the chromatogram amikacin ; if necessary, adjust the amount of acetonitrile R1
supplied with amikacin for system suitability CRS and the in the mobile phase ; peak splitting may be observed when
chromatogram obtained with reference solution (c) to the retention time becomes too short ;
identify the peaks due to impurities A, B, F and H ; use the
chromatogram obtained with reference solution (d) to identify – repeatability : maximum relative standard deviation of
the peak due to impurity I. 1.5 per cent after 6 injections.
Relative retention with reference to amikacin (retention Calculate the percentage content of C22H43N5O13 taking into
time = about 28 min) : impurity I = about 0.13 ; account the assigned content of amikacin CRS.
impurity F = about 0.92 ; impurity B = about 0.95 ;
impurity A = about 1.62 ; impurity H = about 1.95. IMPURITIES
System suitability: reference solution (c) : Specified impurities : A, B, F, H, I.
– peak-to-valley ratio : minimum 5, where Hp = height Other detectable impurities (the following substances would,
above the baseline of the peak due to impurity B and if present at a sufficient level, be detected by one or other of
Hv = height above the baseline of the lowest point of the the tests in the monograph. They are limited by the general
curve separating this peak from the peak due to amikacin ; acceptance criterion for other/unspecified impurities and/or
if necessary, adjust the volume of tetrahydrofuran in the by the general monograph Substances for pharmaceutical use
mobile phase. (2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
Limits : impurities in substances for pharmaceutical use) : C, D, E, G.
– impurities A, B, F, H : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.5 per cent) ;
– impurity I : not more than the area of the principal peak
in the chromatogram obtained with reference solution (d)
(0.5 per cent) ;
– any other impurity : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.5 per cent) ;
– total : not more than 1.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(1.5 per cent) ;
– disregard limit : the area of the principal peak in the A. 4-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-6-O-(6-
chromatogram obtained with reference solution (b) (0.1 per amino-6-deoxy-α-D-glucopyranosyl)-1-N-[(2S)-4-amino-
cent). 2-hydroxybutanoyl]-2-deoxy-L-streptamine,
Water (2.5.12) : maximum 8.5 per cent, determined on 0.200 g.
Sulfated ash (2.4.14) : maximum 0.5 per cent, determined on
1.0 g.

ASSAY
Liquid chromatography (2.2.29).
Test solution. Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 10.0 mL with the
mobile phase.
Reference solution. Dissolve 50.0 mg of amikacin CRS in the
mobile phase and dilute to 10.0 mL with the mobile phase.
B. 4-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-6-O-(6-
Column : amino-6-deoxy-α-D-glucopyranosyl)-1,3-N-bis[(2S)-4-
– size : l = 0.25 m, Ø = 4.6 mm ; amino-2-hydroxybutanoyl]-2-deoxy-L-streptamine,
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm) ;
– temperature : 40 °C.
Mobile phase : a mixture prepared with carbon dioxide-free
water R, containing 1.8 g/L of sodium octanesulfonate R,
20 g/L of anhydrous sodium sulfate R1, 5.8 per cent V/V
of acetonitrile R1, and 5 per cent V/V of 0.2 M potassium
dihydrogen phosphate R previously adjusted to pH 3.0 with
dilute phosphoric acid R ; degas.
Flow rate : 1.0 mL/min.
C. 4-O-(6-amino-6-deoxy-α-D-glucopyranosyl)-6-O-[3-
Detection : spectrophotometer at 200 nm. [[(2S)-4-amino-2-hydroxybutanoyl]amino]-3-deoxy-α-D-
Injection : 20 μL. glucopyranosyl]-2-deoxy-D-streptamine,

1534 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Amikacin sulfate

07/2012:1290

AMIKACIN SULFATE
Amikacini sulfas

D. 6-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-4-O-
(6-amino-6-deoxy-α-D-glucopyranosyl)-2-deoxy-D-
streptamine (kanamycin),

C22H47N5O21S2 Mr 782
[39831-55-5]
DEFINITION
6-O-(3-Amino-3-deoxy-α-D-glucopyranosyl)-4-O-(6-
amino-6-deoxy-α-D-glucopyranosyl)-1-N-[(2S)-4-amino-2-
hydroxybutanoyl]-2-deoxy-D-streptamine sulfate.
E. 4-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-6-O-[6- Antimicrobial substance obtained from kanamycin A.
[[(2S)-4-amino-2-hydroxybutanoyl]amino]-6-deoxy-α-D- Semi-synthetic product derived from a fermentation product.
glucopyranosyl]-2-deoxy-L-streptamine, Content : 96.5 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white powder.
Solubility : freely soluble in water, practically insoluble in
acetone and in ethanol (96 per cent).
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: amikacin sulfate CRS.
B. Thin-layer chromatography (2.2.27).
F. 6-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-4-O-[6- Test solution. Dissolve 25 mg of the substance to be
[(2S)-4-amino-2-hydroxybutanoyl]amino-6-deoxy-α-D- examined in water R and dilute to 10 mL with the same
glucopyranosyl]-1-N-[(2S)-4-amino-2-hydroxybutanoyl]- solvent.
2-deoxy-D-streptamine, Reference solution (a). Dissolve 25 mg of amikacin
sulfate CRS in water R and dilute to 10 mL with the same
solvent.
Reference solution (b). Dissolve 5 mg of kanamycin
monosulfate CRS in 1 mL of the test solution and dilute to
10 mL with water R.
Plate : TLC silica gel plate R.
Mobile phase : methylene chloride R, ammonia R, methanol R
(25:30:40 V/V/V).
Application : 5 μL.
G. 6-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-4-O-(6- Development : over 3/4 of the plate.
amino-6-deoxy-α-D-glucopyranosyl)-1-N-[(2R)-4-amino- Drying : in air.
2-hydroxybutanoyl]-2-deoxy-D-streptamine, Detection : spray with ninhydrin solution R1 and heat at
110 °C for 5 min.
System suitability : reference solution (b) :
– the chromatogram shows 2 clearly separated spots.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
reference solution (a).
C. It gives reaction (a) of sulfates (2.3.1).
TESTS
H. 6-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-1-N-[(2S)- pH (2.2.3) : 2.0 to 4.0.
4-amino-2-hydroxybutanoyl]-4-O-(2,6-diamino-2,6- Dissolve 0.1 g in carbon dioxide-free water R and dilute to
dideoxy-α-D-glucopyranosyl)-2-deoxy-D-streptamine, 10 mL with the same solvent.
Specific optical rotation (2.2.7) : + 76 to + 84 (dried
substance).
Dissolve 0.50 g in water R and dilute to 25.0 mL with the same
I. (2S)-4-amino-2-hydroxybutanoic acid. solvent.

General Notices (1) apply to all monographs and other texts 1535
Amikacin sulfate EUROPEAN PHARMACOPOEIA 8.0

Related substances. Liquid chromatography (2.2.29). curve separating this peak from the peak due to amikacin ;
Test solution. Dissolve 33 mg of the substance to be examined if necessary, adjust the volume of tetrahydrofuran in the
in mobile phase A and dilute to 50.0 mL with mobile phase A. mobile phase.
Reference solution (a). Dilute 1.0 mL of the test solution to Limits :
100.0 mL with mobile phase A. – impurities A, B, F, H : for each impurity, not more than
Reference solution (b). Dilute 1.0 mL of reference solution (a) 0.5 times the area of the principal peak in the chromatogram
to 10.0 mL with mobile phase A. obtained with reference solution (a) (0.5 per cent) ;
Reference solution (c). Dissolve 5 mg of amikacin for system – impurity I : not more than the area of the principal peak
suitability CRS (containing impurities A, B, F and H) in mobile in the chromatogram obtained with reference solution (d)
phase A and dilute to 10 mL with mobile phase A. (0.5 per cent) ;
Reference solution (d). Dissolve 6.6 mg of amikacin – any other impurity : for each impurity, not more than
impurity I CRS in mobile phase A and dilute to 20.0 mL with 0.5 times the area of the principal peak in the chromatogram
mobile phase A. Dilute 1.0 mL of the solution to 100.0 mL obtained with reference solution (a) (0.5 per cent) ;
with mobile phase A. – total : not more than 1.5 times the area of the principal peak
Column : in the chromatogram obtained with reference solution (a)
– size : l = 0.25 m, Ø = 4.6 mm ; (1.5 per cent) ;
– stationary phase : end-capped octadecylsilyl silica gel for – disregard limit : the area of the principal peak in the
chromatography R (5 μm) ; chromatogram obtained with reference solution (b) (0.1 per
cent).
– temperature : 40 °C.
Mobile phase : Sulfate : 23.3 per cent to 25.8 per cent (dried substance).
– mobile phase A : a mixture prepared with carbon dioxide-free Dissolve 0.250 g in 100 mL of water R and adjust the solution
water R, containing 1.8 g/L of sodium octanesulfonate R, to pH 11 using concentrated ammonia R. Add 10.0 mL of
20 g/L of anhydrous sodium sulfate R1, 1.4 per cent V/V of 0.1 M barium chloride and about 0.5 mg of phthalein purple R.
tetrahydrofuran R, and 5 per cent V/V of 0.2 M potassium Titrate with 0.1 M sodium edetate adding 50 mL of ethanol
dihydrogen phosphate R previously adjusted to pH 3.0 with (96 per cent) R when the colour of the solution begins to
dilute phosphoric acid R; degas ; change and continue the titration until the violet-blue colour
disappears.
– mobile phase B : a mixture prepared with carbon dioxide-free
water R, containing 1.8 g/L of sodium octanesulfonate R, 1 mL of 0.1 M barium chloride is equivalent to 9.606 mg of
28 g/L of anhydrous sodium sulfate R1, 1.4 per cent V/V of sulfate (SO4).
tetrahydrofuran R, and 5 per cent V/V of 0.2 M potassium Loss on drying (2.2.32) : maximum 13.0 per cent, determined
dihydrogen phosphate R previously adjusted to pH 3.0 with on 0.500 g by drying in an oven at 105 °C at a pressure not
dilute phosphoric acid R; degas ; exceeding 0.7 kPa for 3 h.
Time Mobile phase A Mobile phase B Pyrogens (2.6.8). If intended for use in the manufacture
(min) (per cent V/V) (per cent V/V) of parenteral preparations without a further appropriate
0-3 100 0 procedure for the removal of pyrogens, it complies with the
test for pyrogens. Inject per kilogram of the rabbit’s mass
3 - 38.0 100 → 30 0 → 70 5 mL of a solution containing 25 mg of the substance to be
38.0 - 38.1 30 → 0 70 → 100 examined in water for injections R.
38.1 - 68 0 100 ASSAY
Liquid chromatography (2.2.29).
Flow rate : 1.0 mL/min.
Test solution. Dissolve 50.0 mg of the substance to be
Post-column solution : mixture of 1 volume of carbonate-free
examined in the mobile phase and dilute to 10.0 mL with the
sodium hydroxide solution R and 24 volumes of previously
mobile phase.
degassed carbon dioxide-free water R, which is added in
a pulseless manner to the column effluent using a 375 μL Reference solution. Dissolve 50.0 mg of amikacin sulfate CRS in
polymeric mixing coil. the mobile phase and dilute to 10.0 mL with the mobile phase.
Flow rate of post-column solution : 0.3 mL/min. Column :
Detection : pulsed amperometric detector or equivalent with – size : l = 0.25 m, Ø = 4.6 mm ;
a gold indicator electrode, a silver-silver chloride reference – stationary phase : end-capped octadecylsilyl silica gel for
electrode, and a stainless steel auxiliary electrode which is the chromatography R (5 μm) ;
cell body, held at respectively + 0.05 V detection, + 0.75 V – temperature : 40 °C.
oxidation and − 0.15 V reduction potentials, with pulse Mobile phase : a mixture prepared with carbon dioxide-free
durations according to the instrument used. water R, containing 1.8 g/L of sodium octanesulfonate R,
Injection : 20 μL. 20 g/L of anhydrous sodium sulfate R1, 5.8 per cent V/V
Identification of impurities : use the chromatogram of acetonitrile R1, and 5 per cent V/V of 0.2 M potassium
supplied with amikacin for system suitability CRS and the dihydrogen phosphate R previously adjusted to pH 3.0 with
chromatogram obtained with reference solution (c) to dilute phosphoric acid R; degas.
identify the peaks due to impurities A, B, F and H; use the Flow rate : 1.0 mL/min.
chromatogram obtained with reference solution (d) to identify
Detection : spectrophotometer at 200 nm.
the peak due to impurity I.
Injection : 20 μL.
Relative retention with reference to amikacin (retention
time = about 28 min) : impurity I = about 0.13 ; Run time : 1.3 times the retention time of amikacin.
impurity F = about 0.92 ; impurity B = about 0.95 ; Retention time : amikacin = about 30 min.
impurity A = about 1.62 ; impurity H = about 1.95. System suitability : reference solution :
System suitability: reference solution (c) : – symmetry factor : maximum 1.5 for the peak due to
– peak-to-valley ratio : minimum 5, where Hp = height amikacin ; if necessary, adjust the amount of acetonitrile R1
above the baseline of the peak due to impurity B and in the mobile phase; peak splitting may be observed when
Hv = height above the baseline of the lowest point of the the retention time becomes too short ;

1536 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Amikacin sulfate

– repeatability : maximum relative standard deviation of

1.5 per cent after 6 injections.
Calculate the percentage content of C22H47N5O21S2 taking into
account the assigned content of amikacin sulfate CRS.

STORAGE
If the substance is sterile, store in a sterile, airtight,
tamper-proof container.
D. 6-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-4-O-
(6-amino-6-deoxy-α-D-glucopyranosyl)-2-deoxy-D-
IMPURITIES
streptamine (kanamycin),
Specified impurities : A, B, F, H, I.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : C, D, E, G.

E. 4-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-6-O-[6-
[[(2S)-4-amino-2-hydroxybutanoyl]amino]-6-deoxy-α-D-
glucopyranosyl]-2-deoxy-L-streptamine,

A. 4-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-6-O-(6- F. 6-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-4-O-[6-
amino-6-deoxy-α-D-glucopyranosyl)-1-N-[(2S)-4-amino- [(2S)-4-amino-2-hydroxybutanoyl]amino-6-deoxy-α-D-
2-hydroxybutanoyl]-2-deoxy-L-streptamine, glucopyranosyl]-1-N-[(2S)-4-amino-2-hydroxybutanoyl]-
2-deoxy-D-streptamine,

G. 6-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-4-O-(6-
amino-6-deoxy-α-D-glucopyranosyl)-1-N-[(2R)-4-amino-
2-hydroxybutanoyl]-2-deoxy-D-streptamine,
B. 4-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-6-O-(6-
amino-6-deoxy-α-D-glucopyranosyl)-1,3-N-bis[(2S)-4-
amino-2-hydroxybutanoyl]-2-deoxy-L-streptamine,

H. 6-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-1-N-[(2S)-
4-amino-2-hydroxybutanoyl]-4-O-(2,6-diamino-2,6-
dideoxy-α-D-glucopyranosyl)-2-deoxy-D-streptamine,

C. 4-O-(6-amino-6-deoxy-α-D-glucopyranosyl)-6-O-[3-
[[(2S)-4-amino-2-hydroxybutanoyl]amino]-3-deoxy-α-D-
glucopyranosyl]-2-deoxy-D-streptamine, I. (2S)-4-amino-2-hydroxybutanoic acid.

General Notices (1) apply to all monographs and other texts 1537
Amiloride hydrochloride EUROPEAN PHARMACOPOEIA 8.0

04/2010:0651 Reference solution (b). Dilute 1.0 mL of reference solution (a)

to 10.0 mL with a mixture of 1 volume of acetonitrile R and
AMILORIDE HYDROCHLORIDE 3 volumes of water R.
Reference solution (c). Dissolve 5.0 mg of amiloride
Amiloridi hydrochloridum impurity A CRS in a mixture of 1 volume of acetonitrile R
and 3 volumes of water R and dilute to 5.0 mL with the same
mixture of solvents. Dilute 1.0 mL of this solution to 100.0 mL
with a mixture of 1 volume of acetonitrile R and 3 volumes
of water R.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
C6H9Cl2N7O,2H2O Mr 302.1 – stationary phase : octadecylsilyl silica gel for
[17440-83-4] chromatography R (5 μm).
DEFINITION Mobile phase : mix 5 volumes of tetramethylammonium
3,5-Diamino-N-carbamimidoyl-6-chloropyrazine-2- hydroxide solution R, 250 volumes of acetonitrile R and
carboxamide hydrochloride dihydrate. 745 volumes of water R ; adjust to pH 7.0 with a mixture of
1 volume of phosphoric acid R and 9 volumes of water R.
Content : 98.0 per cent to 101.0 per cent (anhydrous substance). Adjust the concentration of acetonitrile in the mobile phase so
CHARACTERS that the retention time of impurity A is 5-6 min (an increase in
the concentration of acetonitrile results in a shorter retention
Appearance : pale yellow or greenish-yellow powder. time). Adjust the concentration of tetramethylammonium
Solubility : slightly soluble in water and in anhydrous ethanol. hydroxide and of phosphoric acid keeping the pH at 7.0 so
that the retention time of amiloride is 9-12 min (an increase
IDENTIFICATION in the concentration results in a shorter retention time for
First identification : A, D. amiloride).
Second identification : B, C, D. Flow rate : 1 mL/min.
A. Infrared absorption spectrophotometry (2.2.24). Detection : spectrophotometer at 254 nm.
Comparison : amiloride hydrochloride CRS. Injection : 20 μL.
B. Thin-layer chromatography (2.2.27). Run time : 5 times the retention time of amiloride.
Test solution. Dissolve 40 mg of the substance to be System suitability : reference solution (b) :
examined in methanol R and dilute to 10 mL with the same – signal-to-noise ratio : minimum 5.0 for the peak due to
solvent. amiloride.
Reference solution. Dissolve 40 mg of amiloride Limits :
hydrochloride CRS in methanol R and dilute to 10 mL with
the same solvent. – unspecified impurities : for each impurity, not more than
0.2 times the area of the peak due to impurity A in the
Plate : TLC silica gel plate R. chromatogram obtained with reference solution (c)
Mobile phase : dilute ammonia R1, water R, dioxan R (0.10 per cent);
(6:6:88 V/V/V) ; freshly prepared mixture. – total : not more than the area of the peak due to impurity A
Application : 5 μL. in the chromatogram obtained with reference solution (c)
Development : over 2/3 of the plate. (0.5 per cent) ;
Drying : in air. – disregard limit : 0.1 times the area of the peak due to
Detection : examine in ultraviolet light at 365 nm. impurity A in the chromatogram obtained with reference
Results : the principal spot in the chromatogram obtained solution (c) (0.05 per cent).
with the test solution is similar in position, fluorescence Water (2.5.12) : 11.0 per cent to 13.0 per cent, determined on
and size to the principal spot in the chromatogram obtained 0.200 g.
with the reference solution. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
C. Dissolve about 10 mg in 10 mL of water R. Add 10 mL of a 1.0 g.
200 g/L solution of cetrimide R, 0.25 mL of dilute sodium
hydroxide solution R and 1 mL of bromine water R. A ASSAY
greenish-yellow colour is produced. Add 2 mL of dilute Dissolve 0.200 g in a mixture of 5.0 mL of 0.01 M hydrochloric
hydrochloric acid R. The solution becomes deep yellow and acid and 50 mL of ethanol (96 per cent) R. Carry out a
shows blue fluorescence in ultraviolet light at 365 nm. potentiometric titration (2.2.20), using 0.1 M sodium
D. It gives reaction (b) of chlorides (2.3.1). hydroxide. Read the volume added between the 2 points of
inflexion.
TESTS 1 mL of 0.1 M sodium hydroxide is equivalent to 26.61 mg
Free acid. Dissolve 1.0 g in a mixture of 50 mL of methanol R of C6H9Cl2N7O.
and 50 mL of water R and titrate with 0.1 M sodium hydroxide,
determining the end-point potentiometrically (2.2.20). Not STORAGE
more than 0.3 mL of 0.1 M sodium hydroxide is required to Protected from light.
reach the end-point.
IMPURITIES
Related substances. Liquid chromatography (2.2.29). Other detectable impurities (the following substances would,
Test solution. Dissolve 20.0 mg of the substance to be if present at a sufficient level, be detected by one or other of
examined in a mixture of 1 volume of acetonitrile R and the tests in the monograph. They are limited by the general
3 volumes of water R and dilute to 10.0 mL with the same acceptance criterion for other/unspecified impurities and/or
mixture of solvents. by the general monograph Substances for pharmaceutical
Reference solution (a). Dilute 1.0 mL of the test solution to use (2034). It is therefore not necessary to identify these
100.0 mL with a mixture of 1 volume of acetonitrile R and impurities for demonstration of compliance. See also 5.10.
3 volumes of water R. Control of impurities in substances for pharmaceutical use) : A.

1538 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 4-Aminobenzoic acid

Reference solution. Dissolve 25.0 mg of 4-nitrobenzoic acid R

and 25.0 mg of benzocaine R in methanol R and dilute to
100.0 mL with the same solvent. Dilute 1.0 mL to 50.0 mL
with the mobile phase. Dilute 1.0 mL of this solution to
10.0 mL with the mobile phase.
A. methyl 3,5-diamino-6-chloropyrazine-2-carboxylate.
Column :
– size : l = 0.12 m, Ø = 4.0 mm,
01/2008:1687
– stationary phase : octylsilyl silica gel for chromatography R
(5 μm).
4-AMINOBENZOIC ACID Mobile phase : mix 20 volumes of a mixture of 70 volumes of
acetonitrile R and 80 volumes of methanol R, and 80 volumes
Acidum 4-aminobenzoicum of a solution containing 1.5 g/L of potassium dihydrogen
phosphate R and 2.5 g/L of sodium octanesulfonate R adjusted
to pH 2.2 with phosphoric acid R.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 270 nm.
C7H7NO2 Mr 137.1
[150-13-0] Injection : 20 μL.
Run time : 11 times the retention time of 4-aminobenzoic acid.
DEFINITION Relative retention with reference to 4-aminobenzoic acid
4-Aminobenzoic acid. (retention time = about 3 min) : impurity A = about 4 ;
Content : 99.0 per cent to 101.0 per cent (anhydrous substance). impurity B = about 9.
Limits :
CHARACTERS
– impurity A : not more than the area of the corresponding
Appearance : white or slightly yellow, crystalline powder.
peak in the chromatogram obtained with the reference
Solubility : slightly soluble in water, freely soluble in alcohol. It solution (0.2 per cent),
dissolves in dilute solutions of alkali hydroxides.
– impurity B : not more than the area of the corresponding
IDENTIFICATION peak in the chromatogram obtained with the reference
First identification : B. solution (0.2 per cent),
Second identification : A, C. – any other impurity : not more than 0.5 times the area of the
peak due to impurity A in the chromatogram obtained with
A. Melting point (2.2.14) : 186 °C to 189 °C. the reference solution (0.1 per cent),
B. Infrared absorption spectrophotometry (2.2.24). – total : not more than 2.5 times the area of the peak due
Comparison : 4-aminobenzoic acid CRS. to impurity A in the chromatogram obtained with the
C. Thin-layer chromatography (2.2.27). reference solution (0.5 per cent),
Test solution. Dissolve 20 mg of the substance to be – disregard limit : 0.1 times the area of the peak due to
examined in methanol R and dilute to 20 mL with the same impurity A in the chromatogram obtained with the
solvent. reference solution (0.02 per cent).
Reference solution (a). Dissolve 20 mg of 4-aminobenzoic Impurity C and impurity D. Gas chromatography (2.2.28).
acid CRS in methanol R and dilute to 20 mL with the same Internal standard solution. Dissolve 20.0 mg of lauric acid R
solvent. in methylene chloride R and dilute to 100.0 mL with the same
Reference solution (b). Dissolve 10 mg of 4-nitrobenzoic solvent.
acid R in 10 mL of reference solution (a). Test solution. Dissolve 1.000 g of the substance to be examined
Plate : suitable silica gel with a fluorescent indicator having in 10.0 mL of an 84 g/L solution of sodium hydroxide R
an optimal intensity at 254 nm as the coating substance. and extract with 2 quantities, each of 10 mL, of methylene
Mobile phase : glacial acetic acid R, hexane R, methylene chloride R. Combine and wash with 5 mL of water R ; filter
chloride R (5:20:75 V/V/V). through anhydrous sodium sulfate R. Wash the filter with
Application : 1 μL. methylene chloride R. Evaporate in a water-bath at 50-60 °C to
obtain a volume of about 1-5 mL. Add 1.0 mL of the internal
Development : over a path of 10 cm.
standard solution and dilute to 10.0 mL with methylene
Drying : in air. chloride R.
Detection : examine in ultraviolet light at 254 nm. Reference solution (a). Dissolve 20.0 mg of aniline R in
System suitability : the chromatogram obtained with methylene chloride R and dilute to 100.0 mL with the same
reference solution (b) shows 2 clearly separated spots. solvent.
Results : the principal spot in the chromatogram obtained Reference solution (b). Dissolve 20.0 mg of p-toluidine R in
with the test solution is similar in position and size to methylene chloride R and dilute to 100.0 mL with the same
the principal spot in the chromatogram obtained with solvent.
reference solution (a). Reference solution (c). Dilute 0.50 mL of reference solution (a),
TESTS 0.50 mL of reference solution (b) and 10.0 mL of the internal
standard solution to 100.0 mL with methylene chloride R.
Appearance of solution. The solution is clear (2.2.1) and not
Column :
more intensely coloured than reference solution B5 (2.2.2,
Method II). – material : fused silica,
Dissolve 1.0 g in alcohol R and dilute to 20 mL with the same – size : l = 30 m, Ø = 0.32 mm,
solvent. – stationary phase : poly[methyl(95)phenyl(5)]siloxane R (film
Related substances. Liquid chromatography (2.2.29). thickness 0.5 μm).
Test solution. Dissolve 25.0 mg of the substance to be Carrier gas : helium for chromatography R.
examined in the mobile phase and dilute to 100.0 mL with Flow rate : 1.0 mL/min.
the mobile phase. Split ratio : 1:10.

General Notices (1) apply to all monographs and other texts 1539
Aminocaproic acid EUROPEAN PHARMACOPOEIA 8.0

Temperature : 01/2008:0874
corrected 6.0
Time Temperature
(min) (°C)
Column 0-4 130 AMINOCAPROIC ACID
4 - 6.5 130 → 180
Acidum aminocaproicum
6.5 - 11.5 180

Injection port 280

Detector 300 C6H13NO2 Mr 131.2

[60-32-2]
Detection : flame ionisation. DEFINITION
Injection : 2 μL ; inject the test solution and reference Aminocaproic acid contains not less than 98.5 per cent and not
solution (c). more than the equivalent of 101.0 per cent of 6-aminohexanoic
Retention time : internal standard = about 9.5 min. acid, calculated with reference to the dried substance.
Limits : CHARACTERS
– impurity C : calculate the ratio (R) of the area of the peak A white or almost white, crystalline powder or colourless
due to impurity C to the area of the peak due to the internal crystals, freely soluble in water, slightly soluble in alcohol.
standard from the chromatogram obtained with reference It melts at about 205 °C with decomposition.
solution (c) ; calculate the ratio of the area of the peak due
to impurity C to the area of the peak due to the internal IDENTIFICATION
standard from the chromatogram obtained with the test First identification : A.
solution : this ratio is not greater than R (10 ppm), Second identification : B, C, D.
– impurity D : calculate the ratio (R) of the area of the peak A. Examine by infrared absorption spectrophotometry
due to impurity D to the area of the peak due to the internal (2.2.24), comparing with the spectrum obtained with
standard from the chromatogram obtained with reference aminocaproic acid CRS. Examine the substances prepared
solution (c) ; calculate the ratio of the area of the peak due as discs.
to impurity D to the area of the peak due to the internal B. Examine the chromatograms obtained in the test for
standard from the chromatogram obtained with the test ninhydrin-positive substances. The principal spot in the
solution : this ratio is not greater than R (10 ppm). chromatogram obtained with the test solution (b) is similar
Iron (2.4.9) : maximum 40 ppm. in position, colour and size to the principal spot in the
Dissolve 0.250 g in 3 mL of alcohol R and dilute to 10.0 mL chromatogram obtained with reference solution (a).
with water R. C. Dissolve 0.5 g in 4 mL of a mixture of equal volumes of
dilute hydrochloric acid R and water R. Evaporate to dryness
Heavy metals (2.4.8) : maximum 20 ppm. by heating on a water-bath. Dry the residue in a desiccator.
1.0 g complies with test C. Prepare the reference solution using Dissolve the residue in about 2 mL of boiling ethanol R.
2 mL of lead standard solution (10 ppm Pb) R. Allow to cool and maintain at 4 °C to 8 °C for 3 h. Filter
Water (2.5.12) : maximum 0.2 per cent, determined on 1.00 g. under reduced pressure. The residue washed with about
10 mL of acetone R and dried at 60 °C for 30 min, melts
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on (2.2.14) at 131 °C to 133 °C.
1.0 g. D. Dissolve about 5 mg in 0.5 mL of distilled water R. Add
3 mL of dimethylformamide R and 2 mL of ascorbic acid
ASSAY solution R. Heat on a water-bath. An orange colour
Dissolve 0.100 g with heating in 50 mL of carbon dioxide-free develops.
water R. Titrate with 0.1 M sodium hydroxide determining the
end-point potentiometrically (2.2.20). TESTS
1 mL of 0.1 M sodium hydroxide is equivalent to 13.71 mg Solution S. dissolve 10.0 g in carbon dioxide-free water R and
of C7H7NO2. dilute to 50.0 mL with the same solvent.
Appearance of solution. Solution S is colourless (2.2.2,
STORAGE Method II) and remains clear (2.2.1) on standing for 24 h.
Protected from light. pH (2.2.3). The pH of solution S is 7.5 to 8.0.
Absorbance (2.2.25).
IMPURITIES A. The absorbance of solution S at 287 nm is not more
than 0.10 and at 450 nm is not more than 0.03.
B. Place 2.0 g in an even layer in a shallow dish 9 cm in
diameter, cover and allow to stand at 98 °C to 102 °C for
72 h. Dissolve in water R and dilute to 10.0 mL with the
same solvent. The absorbance of the solution at 287 nm is
A. R = CO2H, R′ = NO2 : 4-nitrobenzoic acid, not more than 0.15 and at 450 nm is not more than 0.03.
Ninhydrin-positive substances. Examine by thin-layer
B. R = CO-O-C2H5, R′ = NH2 : ethyl 4-aminobenzoate chromatography (2.2.27), using a suitable silica gel as the
(benzocaine), coating substance.
Test solution (a). Dissolve 0.10 g of the substance to be
examined in water R and dilute to 10 mL with the same
C. R = H, R′ = NH2 : aniline, solvent.
Test solution (b). Dilute 1 mL of test solution (a) to 50 mL
D. R = CH3, R′ = NH2 : 4-methylaniline (p-toluidine). with water R.

1540 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Aminoglutethimide

Reference solution (a). Dissolve 10 mg of aminocaproic Reference solution (a). Dissolve 25 mg of

acid CRS in water R and dilute to 50 mL with the same solvent. aminoglutethimide CRS in acetone R and dilute to
Reference solution (b). Dilute 5 mL of test solution (b) to 5 mL with the same solvent.
20 mL with water R. Reference solution (b). Dissolve 25 mg of
Reference solution (c). Dissolve 10 mg of aminocaproic aminoglutethimide CRS and 25 mg of glutethimide CRS in
acid CRS and 10 mg of leucine CRS in water R and dilute to acetone R and dilute to 5 mL with the same solvent.
25 mL with the same solvent. Plate : TLC silica gel F254 plate R.
Apply separately to the plate 5 μL of each solution. Allow Mobile phase : glacial acetic acid R, methanol R, ethyl
the plate to dry in air. Develop over a path of 15 cm using acetate R (0.5:15:85 V/V/V).
a mixture of 20 volumes of glacial acetic acid R, 20 volumes
Application : 5 μL.
of water R and 60 volumes of butanol R. Dry the plate in a
current of warm air. Spray with ninhydrin solution R and heat Development : over 3/4 of the plate.
at 100 °C to 105 °C for 15 min. Any spot in the chromatogram Drying : in air.
obtained with the test solution (a), apart from the principal Detection : examine in ultraviolet light at 254 nm.
spot, is not more intense than the spot in the chromatogram
obtained with reference solution (b) (0.5 per cent). The test System suitability : reference solution (b) :
is not valid unless the chromatogram obtained with reference – the chromatogram shows 2 clearly separed spots.
solution (c) shows two clearly separated principal spots. Results : the principal spot in the chromatogram obtained
Heavy metals (2.4.8). 12 mL of solution S complies with test A with the test solution is similar in position and size to
for heavy metals (10 ppm). Prepare the reference solution the principal spot in the chromatogram obtained with
using lead standard solution (2 ppm Pb) R. reference solution (a).
Loss on drying (2.2.32). Not more than 0.5 per cent, TESTS
determined on 1.000 g by drying in an oven at 105 °C.
Solution S. Dissolve 1.0 g in methanol R and dilute to 20.0 mL
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined with the same solvent.
on 1.0 g.
Appearance of solution. Solution S is clear (2.2.1) and not
ASSAY more intensely coloured than reference solution Y7 (2.2.2,
Dissolve 0.100 g in 20 mL of anhydrous acetic acid R. Method II).
Using 0.1 mL of crystal violet solution R as indicator, titrate Optical rotation (2.2.7): − 0.10° to + 0.10°, determined on
with 0.1 M perchloric acid until the colour changes from solution S.
bluish-violet to bluish-green.
Related substances. Liquid chromatography (2.2.29).
1 mL of 0.1 M perchloric acid is equivalent to 13.12 mg of
C6H13NO2. Solvent mixture : methanol R, acetate buffer solution pH 5.0 R
(50:50 V/V).
Test solution. Dissolve 0.100 g of the substance to be examined
01/2011:1291 in the solvent mixture and dilute to 50.0 mL with the solvent
mixture.
AMINOGLUTETHIMIDE Reference solution (a). Dissolve 5.0 mg of aminoglutethimide
impurity A CRS in the solvent mixture and dilute to 25.0 mL
Aminoglutethimidum with the solvent mixture.
Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 10.0 mL with the solvent mixture.
Reference solution (c). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture.
Reference solution (d). Dilute 1.0 mL of the test solution to
10.0 mL with reference solution (a).
C13H16N2O2 Mr 232.3 Column :
[125-84-8] – size : l = 0.15 m, Ø = 3.9 mm ;
DEFINITION – stationary phase : octadecylsilyl silica gel for
(3RS)-3-(4-Aminophenyl)-3-ethylpiperidine-2,6-dione. chromatography R (4 μm) ;
Content : 98.0 per cent to 101.5 per cent (dried substance). – temperature : 40 °C.
Mobile phase : mix 27 volumes of methanol R and 73 volumes
CHARACTERS of acetate buffer solution pH 5.0 R.
Appearance : white or slightly yellow, crystalline powder.
Flow rate : 1.3 mL/min.
Solubility : practically insoluble in water, freely soluble in
acetone, soluble in methanol. Detection : spectrophotometer at 240 nm.
Injection : 10 μL of the test solution and reference solutions (b),
IDENTIFICATION (c) and (d).
First identification : B. Run time : 4 times the retention time of aminoglutethimide.
Second identification : A, C. Identification of impurities: use the chromatogram obtained
A. Melting point (2.2.14) : 150 °C to 154 °C. with reference solution (b) to identify the peak due to
B. Infrared absorption spectrophotometry (2.2.24). impurity A.
Comparison : aminoglutethimide CRS. Relative retention with reference to aminoglutethimide
C. Thin-layer chromatography (2.2.27). (retention time = about 9 min) : impurity A = about 1.3.
Test solution. Dissolve 25 mg of the substance to be System suitability : reference solution (d) :
examined in acetone R and dilute to 5 mL with the same – resolution : minimum 2.0 between the peaks due to
solvent. aminoglutethimide and impurity A.

General Notices (1) apply to all monographs and other texts 1541
Amiodarone hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Limits : IMPURITIES
– impurity A : not more than twice the area of the principal Specified impurities : A, D.
peak in the chromatogram obtained with reference Other detectable impurities (the following substances would,
solution (b) (2.0 per cent) ; if present at a sufficient level, be detected by one or other of
– unspecified impurities : for each impurity, not more than the tests in the monograph. They are limited by the general
0.1 times the area of the principal peak in the chromatogram acceptance criterion for other/unspecified impurities and/or
obtained with reference solution (c) (0.10 per cent) ; by the general monograph Substances for pharmaceutical use
– sum of impurities other than A : not more than the area (2034). It is therefore not necessary to identify these impurities
of the principal peak in the chromatogram obtained with for demonstration of compliance. See also 5.10. Control of
reference solution (c) (1.0 per cent) ; impurities in substances for pharmaceutical use) : B, C.
– total : maximum 2.0 per cent for the sum of the contents
of all impurities ;
– disregard limit : 0.05 times the area of the principal peak
in the chromatogram obtained with reference solution (c)
(0.05 per cent).
Impurity D. Liquid chromatography (2.2.29). Carry out the A. R3 = NH2, R4 = H : (3RS)-3-(3-aminophenyl)-3-
test protected from light. Use shaking, not sonication or heat, ethylpiperidine-2,6-dione (3-aminoglutethimide),
to dissolve the reference substance and the substance to be
B. R3 = NO2, R4 = H : (3RS)-3-ethyl-3-(3-nitrophenyl)-
examined.
piperidine-2,6-dione,
Test solution. Dissolve 0.100 g of the substance to be examined
in dimethyl sulfoxide R and dilute to 100.0 mL with the same C. R3 = H, R4 = NO2 : (3RS)-3-ethyl-3-(4-nitrophenyl)-
solvent. piperidine-2,6-dione,
Reference solution. Dissolve 3.0 mg of aminoglutethimide
impurity D CRS in dimethyl sulfoxide R and dilute to 100.0 mL
with the same solvent. Dilute 1.0 mL of this solution to
100.0 mL with dimethyl sulfoxide R.
Column :
– size : l = 0.12 m, Ø = 4 mm ;
– stationary phase : octadecylsilyl silica gel for D. 3,3′-[diazenediylbis(4,1-phenylene)]bis(3-ethylpiperidine-
chromatography R (5 μm). 2,6-dione) (azoglutethimide).
Mobile phase : dissolve 0.285 g of sodium edetate R in water R,
add 7.5 mL of dilute acetic acid R and 50 mL of 0.1 M 01/2008:0803
potassium hydroxide and dilute to 1000 mL with water R ; corrected 7.5
adjust to pH 5.0 with glacial acetic acid R ; mix 350 mL of this
solution with 650 mL of methanol R. AMIODARONE HYDROCHLORIDE
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 328 nm. Amiodaroni hydrochloridum
Injection : 10 μL.
System suitability : test solution :
– number of theoretical plates : minimum 3300, calculated
for the principal peak ;
– mass distribution ratio : 2.0 to 5.0 for the principal peak ;
– symmetry factor : maximum 1.2 for the principal peak.
Limit : C25H30ClI2NO3 Mr 682
[19774-82-4]
– impurity D : not more than the area of the principal peak
in the chromatogram obtained with the reference solution DEFINITION
(300 ppm). (2-Butylbenzofuran-3-yl)[4-[2-(diethylamino)ethoxy]-3,5-
Sulfates (2.4.13) : maximum 500 ppm. diiodophenyl]methanone hydrochloride.
Dilute 6 mL of solution S to 15 mL with distilled water R. Content : 98.5 per cent to 101.0 per cent (dried substance).
Heavy metals (2.4.8) : maximum 10 ppm. CHARACTERS
Dissolve 2.0 g in 15 mL of acetone R and dilute to 20 mL with Appearance : white or almost white, fine, crystalline powder.
water R. 12 mL of the solution complies with test B. Prepare Solubility : very slightly soluble in water, freely soluble in
the reference solution using lead standard solution (1 ppm Pb) methylene chloride, soluble in methanol, sparingly soluble in
obtained by diluting lead standard solution (100 ppm Pb) R ethanol (96 per cent).
with a mixture of 5 mL of water R and 15 mL of acetone R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined IDENTIFICATION
on 1.000 g by drying in an oven at 105 °C. A. Infrared absorption spectrophotometry (2.2.24).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Comparison: amiodarone hydrochloride CRS.
1.0 g. B. It gives reaction (b) of chlorides (2.3.1).
ASSAY TESTS
Dissolve 0.180 g in 50 mL of anhydrous acetic acid R and Appearance of solution. The solution is clear (2.2.1) and not
titrate with 0.1 M perchloric acid, determining the end-point more intensely coloured than reference solution GY5 or BY5
potentiometrically (2.2.20). (2.2.2, Method II).
1 mL of 0.1 M perchloric acid is equivalent to 23.23 mg Dissolve 1.0 g in methanol R and dilute to 20 mL with the
of C13H16N2O2. same solvent.

1542 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Amiodarone hydrochloride

pH (2.2.3) : 3.2 to 3.8. Limits :

Dissolve 1.0 g in carbon dioxide-free water R, heating at 80 °C, – impurities A, B, C, D, E, F, G : for each impurity, not
cool and dilute to 20 mL with the same solvent. more than the area of the peak due to amiodarone in
Impurity H. Thin-layer chromatography (2.2.27). Prepare the chromatogram obtained with the reference solution
the solutions immediately before use and keep protected from (0.2 per cent) ;
bright light. – unspecified impurities : for each impurity, not more than
Test solution. Dissolve 0.500 g of the substance to be examined 0.5 times the area of the peak due to amiodarone in
in methylene chloride R and dilute to 5.0 mL with the same the chromatogram obtained with the reference solution
solvent. (0.10 per cent);
Reference solution (a). Dissolve 10.0 mg of (2-chloroeth- – total : not more than 2.5 times the area of the peak due
yl)diethylamine hydrochloride R (impurity H) in methylene to amiodarone in the chromatogram obtained with the
chloride R and dilute to 50.0 mL with the same solvent. Dilute reference solution (0.5 per cent) ;
2.0 mL of the solution to 20.0 mL with methylene chloride R. – disregard limit : 0.25 times the area of the peak due to
Reference solution (b). Mix 2.0 mL of the test solution and amiodarone in the chromatogram obtained with the
2.0 mL of reference solution (a). reference solution (0.05 per cent).
Plate : TLC silica gel F254 plate R. Iodides : maximum 150 ppm.
Mobile phase : anhydrous formic acid R, methanol R, methylene Prepare the test and reference solutions simultaneously.
chloride R (5:10:85 V/V/V).
Solution A. Add 1.50 g of the substance to be examined
Application : 50 μL of the test solution and reference to 40 mL of water R at 80 °C and shake until completely
solution (a); 100 μL of reference solution (b). dissolved. Cool and dilute to 50.0 mL with water R.
Development : over 2/3 of the plate. Test solution. To 15.0 mL of solution A add 1.0 mL of
Drying : in a current of cold air. 0.1 M hydrochloric acid and 1.0 mL of 0.05 M potassium iodate.
Detection : spray with potassium iodobismuthate solution R1 Dilute to 20.0 mL with water R. Allow to stand protected from
and then with dilute hydrogen peroxide solution R ; examine light for 4 h.
immediately in daylight. Reference solution. To 15.0 mL of solution A add 1.0 mL of
System suitability: reference solution (b): 0.1 M hydrochloric acid, 1.0 mL of an 88.2 mg/L solution of
– the spot due to impurity H is clearly visible. potassium iodide R and 1.0 mL of 0.05 M potassium iodate.
Dilute to 20.0 mL with water R. Allow to stand protected from
Limit :
light for 4 h.
– impurity H : any spot with the same RF as the spot due to
impurity H in the chromatogram obtained with reference Measure the absorbances (2.2.25) of the solutions at 420 nm,
solution (b) is not more intense than the spot in the using a mixture of 15.0 mL of solution A and 1.0 mL of
chromatogram obtained with reference solution (a) 0.1 M hydrochloric acid diluted to 20.0 mL with water R as the
(0.02 per cent). compensation liquid. The absorbance of the test solution is
not greater than half the absorbance of the reference solution.
Related substances. Liquid chromatography (2.2.29).
Heavy metals (2.4.8) : maximum 20 ppm.
Buffer solution pH 4.9. To 800 mL of water R add 3.0 mL of
glacial acetic acid R, adjust to pH 4.9 with dilute ammonia R1 1.0 g complies with test C. Prepare the reference solution using
and dilute to 1000 mL with water R. 2 mL of lead standard solution (10 ppm Pb) R.
Test solution. Dissolve 0.125 g of the substance to be examined Loss on drying (2.2.32) : maximum 0.5 per cent, determined
in a mixture of equal volumes of acetonitrile R and water R on 1.000 g by drying at 50 °C at a pressure not exceeding
and dilute to 25.0 mL with the same mixture of solvents. 0.3 kPa for 4 h.
Reference solution. Dissolve 5 mg of amiodarone Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
impurity D CRS, 5 mg of amiodarone impurity E CRS and 1.0 g.
5.0 mg of amiodarone hydrochloride CRS in methanol R and
dilute to 25.0 mL with the same solvent. Dilute 1.0 mL of ASSAY
the solution to 20.0 mL with a mixture of equal volumes of Dissolve 0.600 g in a mixture of 5.0 mL of 0.01 M hydrochloric
acetonitrile R and water R. acid and 75 mL of ethanol (96 per cent) R. Carry out a
Column : potentiometric titration (2.2.20), using 0.1 M sodium
– size : l = 0.15 m, Ø = 4.6 mm ; hydroxide. Read the volume added between the 2 points of
inflexion.
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm) ; 1 mL of 0.1 M sodium hydroxide is equivalent to 68.18 mg
– temperature : 30 °C. of C25H30ClI2NO3.
Mobile phase : buffer solution pH 4.9, methanol R, acetonitrile R STORAGE
(30:30:40 V/V/V).
Protected from light, at a temperature not exceeding 30 °C.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 240 nm. IMPURITIES
Injection : 10 μL. Specified impurities : A, B, C, D, E, F, G, H.
Run time : twice the retention time of amiodarone.
Relative retention with reference to amiodarone (retention
time = about 24 min) : impurity A = about 0.26 ;
impurity D = about 0.29 ; impurity E = about 0.37 ;
impurity B = about 0.49 ; impurity C = about 0.55 ;
impurity G = about 0.62 ; impurity F = about 0.69.
System suitability: reference solution :
– resolution : minimum 3.5 between the peaks due to A. (2-butylbenzofuran-3-yl)[4-[2-(diethyl-
impurities D and E. amino)ethoxy]phenyl]methanone,

General Notices (1) apply to all monographs and other texts 1543
Amisulpride EUROPEAN PHARMACOPOEIA 8.0

04/2013:1490
corrected 8.0

AMISULPRIDE
Amisulpridum
B. (2-butylbenzofuran-3-yl)[4-[2-(ethylamino)ethoxy]-3,5-
diiodophenyl]methanone,

C17H27N3O4S Mr 369.5
[71675-85-9]
DEFINITION
4-Amino-N-[[(2RS)-1-ethylpyrrolidin-2-yl]methyl]-5-
C. (2-butylbenzofuran-3-yl)[4-[2-(diethylamino)ethoxy]-3- (ethylsulfonyl)-2-methoxybenzamide.
iodophenyl]methanone,
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, freely soluble in
methylene chloride, sparingly soluble in anhydrous ethanol.
mp : about 126 °C.
IDENTIFICATION
D. (2-butylbenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)- Infrared absorption spectrophotometry (2.2.24).
methanone, Comparison: amisulpride CRS.
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution Y6 (2.2.2,
Method II).
Dissolve 1.0 g in 3 mL of a mixture of 1 volume of acetic acid R
and 4 volumes of water R, and dilute to 20 mL with water R.
Impurity A. Thin-layer chromatography (2.2.27).
E. (2-butylbenzofuran-3-yl)(4-hydroxyphenyl)methanone, Test solution. Dissolve 0.20 g of the substance to be examined
in methanol R and dilute to 10 mL with the same solvent.
Reference solution (a). Dissolve 5 mg of sulpiride
impurity A CRS (amisulpride impurity A) in methanol R and
dilute to 25 mL with the same solvent. Dilute 2 mL of the
solution to 20 mL with methanol R.
Reference solution (b). Dilute 1 mL of the test solution to
10 mL with reference solution (a).
Plate : TLC silica gel G plate R.
F. (2-butylbenzofuran-3-yl)(4-hydroxy-3-iodophenyl)metha- Mobile phase : 50 per cent V/V solution of concentrated
none, ammonia R, anhydrous ethanol R, di-isopropyl ether R
(10:25:65 V/V/V) ; use the upper layer obtained after shaking
the mixture.
Application : 10 μL.
Development : over 2/3 of the plate.
Drying : in air.
Detection : spray with ninhydrin solution R and heat at
100-105 °C for 15 min.
Retardation factors : impurity A = about 0.2 ;
amisulpride = about 0.5.
G. [4-[2-(diethylamino)ethoxy]-3,5-diiodophenyl][2-[(1RS)- System suitability : the chromatogram obtained with reference
1-methoxybutyl]benzofuran-3-yl]methanone, solution (b) shows 2 clearly separated spots.
Limit :
– impurity A : any spot due to impurity A is not more intense
than the corresponding spot in the chromatogram obtained
with reference solution (a) (0.1 per cent).
Related substances. Liquid chromatography (2.2.29).
H. 2-chloro-N,N-diethylethanamine (2-chlorotriethylamine, Solvent mixture : acetonitrile R1, methanol R2, mobile phase A
(2-chloroethyl)diethylamine). (12:16:72 V/V/V).

1544 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Amisulpride

Test solution. Dissolve 0.10 g of the substance to be examined IMPURITIES

in 16 mL of methanol R2, add 12 mL of acetonitrile R1 and Specified impurities : A.
dilute to 100.0 mL with mobile phase A. Other detectable impurities (the following substances would,
Reference solution (a). Dilute 1.0 mL of the test solution to if present at a sufficient level, be detected by one or other of
100.0 mL with the solvent mixture. Dilute 1.0 mL of this the tests in the monograph. They are limited by the general
solution to 10.0 mL with the solvent mixture. acceptance criterion for other/unspecified impurities and/or
Reference solution (b). Dissolve the contents of a vial of by the general monograph Substances for pharmaceutical
amisulpride for system suitability CRS (containing impurity B) use (2034). It is therefore not necessary to identify these
in 1.0 mL of the solvent mixture. impurities for demonstration of compliance. See also 5.10.
Column : Control of impurities in substances for pharmaceutical use) : B,
C, D, E, F, G, H.
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : base-deactivated octylsilyl silica gel for
chromatography R (5 μm) ;
– temperature : 40 °C.
Mobile phase :
A. [(2RS)-1-ethylpyrrolidin-2-yl]methanamine,
– mobile phase A : dissolve 0.7 g of sodium octanesulfonate R
in 930 mL of water R, add 45.0 mL of a 5 per cent V/V
solution of dilute sulfuric acid R, adjust to pH 2.3 with a
5 per cent V/V solution of dilute sulfuric acid R, and dilute
to 1000 mL with water R ;
– mobile phase B : methanol R2 ;
– mobile phase C : acetonitrile R1 ; B. 4-amino-N-[[(2RS)-1-ethylpyrrolidin-2-yl]methyl]-5-
(ethylsulfonyl)-2-hydroxybenzamide,
Time Mobile phase A Mobile phase B Mobile phase C
(min) (per cent V/V/V) (per cent V/V/V) (per cent V/V/V)
0 - 18 72 16 12

18 - 35 72 → 50 16 → 38 12

Flow rate : 1.5 mL/min.

Detection : spectrophotometer at 225 nm. C. 4-amino-N-[[(2RS)-1-ethylpyrrolidin-2-yl]methyl]-5-
Injection : 10 μL. iodo-2-methoxybenzamide,
Identification of impurities : use the chromatogram obtained
with reference solution (b) to identify the peak due to
impurity B.
Relative retention with reference to amisulpride (retention
time = about 17 min) : impurity B = about 1.1.
System suitability : reference solution (b) : D. 4-amino-N-[[(2RS)-1-ethylpyrrolidin-2-yl]methyl]-2-
– peak-to-valley ratio : minimum 2.0, where Hp = height above methoxy-5-(methylsulfonyl)benzamide,
the baseline of the peak due to impurity B and Hv = height
above the baseline of the lowest point of the curve
separating this peak from the peak due to amisulpride.
Calculation of percentage contents : use the concentration of
amisulpride in reference solution (a).
E. 4-amino-5-(ethylsulfonyl)-2-methoxybenzoic acid,
Limits :
– unspecified impurities : for each impurity, maximum
0.10 per cent ;
– total : maximum 0.3 per cent ;
– reporting threshold : 0.05 per cent.
Heavy metals (2.4.8) : maximum 10 ppm. F. 4-amino-N-[[(2RS)-1-ethyl-1-oxidopyrrolidin-2-
Dissolve 4.0 g by gently heating in 5 mL of dilute acetic acid R. yl]methyl]-5-(ethylsulfonyl)-2-methoxybenzamide,
Allow to cool and dilute to 20 mL with water R. 12 mL of the
solution complies with test A. Prepare the reference solution
using lead standard solution (2 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 3 h.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on G. 4-amino-N-[(3RS)-1-ethylpiperidin-3-yl]-5-
1.0 g. (ethylsulfonyl)-2-methoxybenzamide,
ASSAY
Dissolve 0.300 g with shaking in a mixture of 5 mL of acetic
anhydride R and 50 mL of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 36.95 mg of H. 4-amino-N-[[(2RS)-1-ethylpyrrolidin-2-yl]methyl]-5-
C17H27N3O4S. (ethylsulfonyl)-2-methoxy-N-methylbenzamide.

General Notices (1) apply to all monographs and other texts 1545
Amitriptyline hydrochloride EUROPEAN PHARMACOPOEIA 8.0

01/2008:0464 Relative retention with reference to amitriptyline

corrected 6.3 (retention time = about 14 min) : impurity B = about 0.9 ;
impurity A = about 2.2.
AMITRIPTYLINE HYDROCHLORIDE System suitability : reference solution (a) :
– resolution : minimum 2.0 between the peaks due to
Amitriptylini hydrochloridum impurity B and amitriptyline.
Limits :
– impurity B : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (b) (0.1 per cent) ;
– impurity A : not more than 0.5 times the area of the
corresponding peak in the chromatogram obtained with
reference solution (b) (0.05 per cent) ;
– unspecified impurities : for each impurity, not more than the
C20H24ClN Mr 313.9 area of the peak due to amitriptyline in the chromatogram
[549-18-8] obtained with reference solution (b) (0.10 per cent) ;
DEFINITION – total : not more than 3 times the area of the peak due to
amitriptyline in the chromatogram obtained with reference
3-(10,11-Dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)-
solution (b) (0.3 per cent) ;
N,N-dimethylpropan-1-amine hydrochloride.
– disregard limit : 0.5 times the area of the peak due to
Content : 99.0 per cent to 101.0 per cent (dried substance).
amitriptyline in the chromatogram obtained with reference
CHARACTERS solution (b) (0.05 per cent).
Appearance : white or almost white powder or colourless Heavy metals (2.4.8) : maximum 20 ppm.
crystals. 1.0 g complies with test F. Prepare the reference solution using
Solubility : freely soluble in water, in ethanol (96 per cent) and 2 mL of lead standard solution (10 ppm Pb) R.
in methylene chloride. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 2 h.
IDENTIFICATION
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
A. Infrared absorption spectrophotometry (2.2.24). 1.0 g.
Comparison : amitriptyline hydrochloride CRS.
ASSAY
B. 20 mg gives reaction (a) of chlorides (2.3.1).
Dissolve 0.250 g in 30 mL of ethanol (96 per cent) R. Titrate
TESTS with 0.1 M sodium hydroxide, determining the end-point
Appearance of solution. The solution is clear (2.2.1) and not potentiometrically (2.2.20).
more intensely coloured than reference solution B7 (2.2.2, 1 mL of 0.1 M sodium hydroxide is equivalent to 31.39 mg
Method II). of C20H24ClN.
Dissolve 1.25 g in water R and dilute to 25 mL with the same STORAGE
solvent.
Protected from light.
Acidity or alkalinity. Dissolve 0.20 g in carbon dioxide-free
water R and dilute to 10 mL with the same solvent. Add IMPURITIES
0.1 mL of methyl red solution R and 0.2 mL of 0.01 M sodium
Specified impurities : A, B.
hydroxide. The solution is yellow. Add 0.4 mL of 0.01 M
hydrochloric acid. The solution is red. Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
Related substances. Liquid chromatography (2.2.29). the tests in the monograph. They are limited by the general
Test solution. Dissolve 50.0 mg of the substance to be acceptance criterion for other/unspecified impurities and/or
examined in the mobile phase and dilute to 50.0 mL with the by the general monograph Substances for pharmaceutical use
mobile phase. (2034). It is therefore not necessary to identify these impurities
Reference solution (a). Dissolve 5.0 mg of dibenzosuberone CRS for demonstration of compliance. See also 5.10. Control of
(impurity A) and 5.0 mg of cyclobenzaprine hydrochloride CRS impurities in substances for pharmaceutical use) : C, D, E, F, G.
(impurity B) in 5.0 mL of the test solution and dilute to
100.0 mL with the mobile phase.
Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 50.0 mL with the mobile phase.
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : end-capped polar-embedded octadecylsilyl A. 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-one
amorphous organosilica polymer R (5 μm) ; (dibenzosuberone),
– temperature : 40 °C.
Mobile phase : mix 35 volumes of acetonitrile R and 65 volumes
of a 5.23 g/L solution of dipotassium hydrogen phosphate R
previously adjusted to pH 7.0 with phosphoric acid R.
Flow rate : 1.2 mL/min.
Detection : spectrophotometer at 220 nm.
Injection : 10 μL. B. 3-(5H-dibenzo[a,d][7]annulen-5-ylidene)-N,N-
Run time : 3 times the retention time of amitriptyline. dimethylpropan-1-amine (cyclobenzaprine),

1546 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Amlodipine besilate

TESTS
Optical rotation (2.2.7) : − 0.10° to + 0.10°.
Dissolve 0.250 g in methanol R and dilute to 25.0 mL with
the same solvent.
Related substances. Liquid chromatography (2.2.29). Carry
out the test protected from light.
C. 3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)-
N-methylpropan-1-amine (nortriptyline), Test solution (a). Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with the
mobile phase.
Test solution (b). Dilute 5.0 mL of test solution (a) to 100.0 mL
with the mobile phase.
Reference solution (a). Dilute 1.0 mL of test solution (a) to
10.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 100.0 mL with the mobile phase.
D. R = CH2-CH2-CH2-N(CH3)2 : 5-[3-(dimethylamino)prop- Reference solution (b). Dissolve 5 mg of amlodipine
yl]-10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ol, impurity B CRS and 5 mg of amlodipine impurity G CRS in
G. R = H : 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ol the mobile phase and dilute to 50.0 mL with the mobile phase.
(dibenzosuberol), Dilute 1.0 mL of the solution to 10.0 mL with the mobile phase.
Reference solution (c). Dissolve 5 mg of amlodipine for peak
identification CRS (containing impurities D, E and F) in 10 mL
of the mobile phase.
Reference solution (d). Dissolve 5.0 mg of amlodipine
impurity A CRS in acetonitrile R and dilute to 5.0 mL with the
same solvent. Dilute 1.0 mL of the solution to 100.0 mL with
the mobile phase. Dilute 1.0 mL of this solution to 10.0 mL
E. N,N-dimethyl-3-(1,2,3,4,4a,10,11,11a-octahydro-5H- with the mobile phase.
dibenzo[a,d][7]annulen-5-ylidene)propan-1-amine, Reference solution (e). Dissolve 50.0 mg of amlodipine
besilate CRS in the mobile phase and dilute to 50.0 mL with
the mobile phase. Dilute 5.0 mL of the solution to 100.0 mL
with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.0 mm ;
– stationary phase : octadecylsilyl silica gel for
F. (5EZ,10RS)-5-[3-(dimethylamino)propylidene]-10,11- chromatography R (5 μm) ;
dihydro-5H-dibenzo[a,d][7]annulen-10-ol. – temperature : 30 °C.
Mobile phase : 2.3 g/L solution of ammonium acetate R,
04/2012:1491 methanol R (30:70 V/V).
Flow rate : 1.5 mL/min.
AMLODIPINE BESILATE Detection : spectrophotometer at 237 nm.
Injection : 20 μL of test solution (a) and reference solutions (a),
Amlodipini besilas (b), (c) and (d).
Run time : twice the retention time of amlodipine.
Identification of impurities : use the chromatogram supplied
with amlodipine for peak identification CRS and the
chromatogram obtained with reference solution (c) to identify
the peaks due to impurities D, E and F ; use the chromatogram
obtained with reference solution (d) to identify the peak due
to impurity A.
Relative retention with reference to amlodipine (retention
C26H31ClN2O8S Mr 567.1 time = about 20 min) : impurity G = about 0.21 ;
[111470-99-6] impurity B = about 0.25 ; impurity D = about 0.5 ;
impurity F = about 0.8 ; impurity E = about 1.3.
DEFINITION System suitability : reference solution (b) :
3-Ethyl 5-methyl (4RS)-2-[(2-aminoethoxy)methyl]- – resolution : minimum 2.0 between the peaks due to
4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5- impurities G and B.
dicarboxylate benzenesulfonate.
Limits :
Content : 97.0 per cent to 102.0 per cent (anhydrous substance).
– correction factors : for the calculation of content, multiply
CHARACTERS the peak areas of the following impurities by the
Appearance : white or almost white powder. corresponding correction factor : impurity D = 1.7 ;
Solubility : slightly soluble in water, freely soluble in methanol, impurity F = 0.7 ;
sparingly soluble in anhydrous ethanol, slightly soluble in – impurity D : not more than 3 times the area of the principal
2-propanol. peak in the chromatogram obtained with reference
solution (a) (0.3 per cent) ;
IDENTIFICATION – impurity A : not more than 1.5 times the area of the
Infrared absorption spectrophotometry (2.2.24). corresponding peak in the chromatogram obtained with
Comparison : amlodipine besilate CRS. reference solution (d) (0.15 per cent) ;

General Notices (1) apply to all monographs and other texts 1547
Ammonia solution, concentrated EUROPEAN PHARMACOPOEIA 8.0

– impurities E, F : for each impurity, not more than 1.5 times

the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.15 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
– total : maximum 0.8 per cent ; D. 3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2-
– disregard limit : 0.5 times the area of the principal peak in chlorophenyl)-6-methylpyridine-3,5-dicarboxylate,
the chromatogram obtained with reference solution (a)
(0.05 per cent) ; disregard any peak due to benzene sulfonate
(relative retention = about 0.14).
Water (2.5.12) : maximum 0.5 per cent, determined on 1.000 g.
Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
1.0 g.

ASSAY E. diethyl (4RS)-2-[(2-aminoethoxy)methyl]-4-(2-

Liquid chromatography (2.2.29) as described in the test for chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-
related substances with the following modification. dicarboxylate,
Injection : test solution (b), reference solution (e).
Calculate the percentage content of C26H31ClN2O8S from the
declared content of amlodipine besilate CRS.

STORAGE
In an airtight container, protected from light.

IMPURITIES F. dimethyl (4RS)-2-[(2-aminoethoxy)methyl]-4-(2-

chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-
Specified impurities : A, D, E, F. dicarboxylate,
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : B, G, H.
G. dimethyl 4-(2-chlorophenyl)-2,6-dimethyl-1,4-
dihydropyridine-3,5-dicarboxylate,

A. 3-ethyl 5-methyl (4RS)-4-(2-chlorophenyl)-2-[[2-(1,3- H. 2-[[2-[[(4RS)-4-(2-chlorophenyl)-3-(ethoxycarbonyl)-

dioxo-1,3-dihydro-2H-isoindol-2-yl)ethoxy]methyl]-6- 5-(methoxycarbonyl)-6-methyl-1,4-dihydropyridin-2-
methyl-1,4-dihydropyridine-3,5-dicarboxylate, yl]methoxy]ethyl]carbamoyl]benzoic acid.

01/2008:0877

AMMONIA SOLUTION,
CONCENTRATED
Ammoniae solutio concentrata
NH3 Mr 17.03
DEFINITION
Content : 25.0 per cent m/m to 30.0 per cent m/m.

B. 3-ethyl 5-methyl (4RS)-4-(2-chlorophenyl)-6- CHARACTERS

methyl-2-[[2-[[2-(methylcarbamoyl)benzoyl]amino]- Appearance : clear, colourless liquid, very caustic.
ethoxy]methyl]-1,4-dihydropyridine-3,5-dicarboxylate, Solubility : miscible with water and with ethanol (96 per cent).

1548 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ammonio methacrylate copolymer (type A)

IDENTIFICATION 01/2008:2081
A. Relative density (2.2.5) : 0.892 to 0.910.
AMMONIO METHACRYLATE
B. It is strongly alkaline (2.2.4).
COPOLYMER (TYPE A)
C. To 0.5 mL add 5 mL of water R. Bubble air through
the solution and lead the gaseous mixture obtained Ammonio methacrylatis copolymerum A
over the surface of a solution containing 1 mL of 0.1 M
hydrochloric acid and 0.05 mL of methyl red solution R. The
colour changes from red to yellow. Add 1 mL of sodium
cobaltinitrite solution R. A yellow precipitate is formed.

TESTS
Solution S. Evaporate 220 mL almost to dryness on a
water-bath. Cool, add 1 mL of dilute acetic acid R and dilute
to 20 mL with distilled water R.
Appearance of solution. The solution is clear (2.2.1) and
colourless (2.2.2, Method II). DEFINITION
To 2 mL add 8 mL of water R. Poly(ethyl propenoate-co-methyl 2-methylpropenoate-co-
2-(trimethylammonio)ethyl 2-methylpropenoate) chloride
Oxidisable substances. Cautiously add, whilst cooling, having a mean relative molecular mass of about 150 000.
8.8 mL to 100 mL of dilute sulfuric acid R. Add 0.75 mL of
0.002 M potassium permanganate. Allow to stand for 5 min. The ratio of ethyl propenoate groups to methyl
The solution remains faintly pink. 2-methylpropenoate groups to 2-(trimethylammonio)ethyl
2-methylpropenoate groups is about 1:2:0.2.
Pyridine and related substances : maximum 2 ppm,
Content of ammonio methacrylate groups : 8.9 per cent to
calculated as pyridine.
12.3 per cent (dried substance).
Measure the absorbance (2.2.25) at 252 nm using water R
as the compensation liquid. The absorbance is not greater CHARACTERS
than 0.06. Appearance : colourless to white or almost white granules or
powder.
Carbonates : maximum 60 ppm.
Solubility : practically insoluble in water, freely soluble in
To 10 mL in a test-tube with a ground-glass neck add 10 mL anhydrous ethanol and in methylene chloride giving clear
of calcium hydroxide solution R. Stopper immediately and to cloudy solutions. Due to the polymeric nature of the
mix. Any opalescence in the solution is not more intense substance, a stirring time of up to 5 h may be necessary.
than that in a standard prepared at the same time and in the
same manner using 10 mL of a 0.1 g/L solution of anhydrous IDENTIFICATION
sodium carbonate R. A. Infrared absorption spectrophotometry (2.2.24).
Chlorides (2.4.4) : maximum 1 ppm. Comparison: Ph. Eur. reference spectrum of ammonio
methacrylate copolymer (type A).
Dilute 5 mL of solution S to 15 mL with water R.
B. Viscosity (see Tests).
Sulfates (2.4.13) : maximum 5 ppm. C. It complies with the limits of the assay.
Dilute 3 mL of solution S to 15 mL with distilled water R.
TESTS
Iron (2.4.9) : maximum 0.25 ppm. Solution S. Dissolve a quantity of the substance to be
Dilute 4 mL of solution S to 10 mL with water R. examined corresponding to 12.5 g of the dried substance in a
mixture of 35.0 g of acetone R and 52.5 g of 2-propanol R.
Heavy metals (2.4.8) : maximum 1 ppm.
Viscosity (2.2.10) : maximum 15 mPa·s, determined on
Dilute 4 mL of solution S to 20 mL with water R. 12 mL of the solution S.
solution complies with test A. Prepare the reference solution Apparatus : rotating viscometer.
using lead standard solution (2 ppm Pb) R.
Dimensions:
Residue on evaporation : maximum 20 mg/L.
– spindle : diameter = 25.15 mm ; height = 90.74 mm ; shaft
Evaporate 50 mL to dryness on a water-bath and dry at diameter = 4.0 mm ;
100-105 °C for 1 h. The residue weighs a maximum of 1 mg. – cylinder : diameter = 27.62 mm ; height = 0.135 m.
Stirring speed : 30 r/min.
ASSAY Volume of solution : 16 mL of solution S.
Weigh accurately a flask with a ground-glass neck containing Temperature : 20 °C.
50.0 mL of 1 M hydrochloric acid. Add 2 mL of the substance Appearance of a film. Spread 2 mL of solution S evenly on a
to be examined and re-weigh. Add 0.1 mL of methyl red glass plate. Upon drying a clear film is formed.
solution R as indicator. Titrate with 1 M sodium hydroxide Monomers. Liquid chromatography (2.2.29).
until the colour changes from red to yellow.
Solution A. Dissolve 3.5 g of sodium perchlorate R in water for
1 mL of 1 M hydrochloric acid is equivalent to 17.03 mg of chromatography R and dilute to 100 mL with the same solvent.
NH3. Test solution. Dissolve 5.00 g of the substance to be examined
in methanol R and dilute to 50.0 mL with the same solvent. To
STORAGE 10.0 mL of this solution add 5.0 mL of solution A, dropwise,
while continuously stirring. Remove the precipitated polymer
Protected from air, at a temperature not exceeding 20 °C. by centrifugation. Use the clear supernatant solution.

General Notices (1) apply to all monographs and other texts 1549
Ammonio methacrylate copolymer (type B) EUROPEAN PHARMACOPOEIA 8.0

Reference solution. Dissolve 50.0 mg of ethyl acrylate R and DEFINITION

10.0 mg of methyl methacrylate R in methanol R and dilute to Poly(ethyl propenoate-co-methyl 2-methylpropenoate-co-
50.0 mL with the same solvent. Dilute 1.0 mL of the solution 2-(trimethylammonio)ethyl 2-methylpropenoate) chloride
to 100.0 mL with methanol R. Add 10 mL of this solution to having a mean relative molecular mass of about 150 000.
5 mL of solution A.
The ratio of ethyl propenoate groups to methyl
Column : 2-methylpropenoate groups to 2-(trimethylammonio)ethyl
– size : l = 0.12 m, Ø = 4.6 mm ; 2-methylpropenoate groups is about 1:2:0.1.
– stationary phase : octadecylsilyl silica gel for Content of ammonio methacrylate groups : 4.5 per cent to
chromatography R (7 μm). 7.0 per cent (dried substance).
Mobile phase : dilute phosphoric acid R with water for
chromatography R to obtain a solution at pH 2.0 ; mix 800 mL CHARACTERS
of this solution and 200 mL of methanol R, filter and degas. Appearance : colourless to white or almost white granules or
Flow rate : 2.0 mL/min. powder.
Detection : spectrophotometer at 202 nm. Solubility : practically insoluble in water, freely soluble in
anhydrous ethanol and in methylene chloride giving clear
Injection : 50 μL. to cloudy solutions. Due to the polymeric nature of the
System suitability: reference solution : substance, a stirring time of up to 5 h may be necessary.
– resolution : minimum 1.5 between the peaks due to
impurity A and impurity B. IDENTIFICATION
Limits : A. Infrared absorption spectrophotometry (2.2.24).
– impurity A : not more than the area of the corresponding Comparison: Ph. Eur. reference spectrum of ammonio
peak in the chromatogram obtained with the reference methacrylate copolymer (type B).
solution (100 ppm); B. Viscosity (see Tests).
– impurity B : not more than 2.5 times the area of the C. It complies with the limits of the assay.
corresponding peak in the chromatogram obtained with
TESTS
the reference solution (50 ppm).
Solution S. Dissolve a quantity of the substance to be
Methanol (2.4.24, System A) : maximum 1.5 per cent.
examined corresponding to 12.5 g of the dried substance in a
Heavy metals (2.4.8) : maximum 20 ppm. mixture of 35.0 g of acetone R and 52.5 g of 2-propanol R.
1.0 g complies with test C. Prepare the reference solution using
Viscosity (2.2.10) : maximum 15 mPa·s, determined on
2.0 mL of lead standard solution (10 ppm Pb) R. solution S.
Loss on drying (2.2.32) : maximum 3.0 per cent, determined Apparatus : rotating viscometer.
on 1.000 g by drying in vacuo at 80 °C for 5 h. Dimensions:
ASSAY – spindle : diameter = 25.15 mm ; height = 90.74 mm ; shaft
Dissolve 1.000 g in a mixture of 3 mL of anhydrous formic diameter = 4.0 mm ;
acid R and 30 mL of anhydrous acetic acid R and heat to – cylinder : diameter = 27.62 mm ; height = 0.135 m.
dissolve. Add 20 mL of acetic anhydride R. Titrate with 0.1 M Stirring speed : 30 r/min.
perchloric acid, determining the end-point potentiometrically Volume of solution : 16 mL of solution S.
(2.2.20). Temperature : 20 °C.
1 mL of 0.1 M perchloric acid is equivalent to 20.77 mg
of C9H18O2NCl (ammonio methacrylate groups). Appearance of a film. Spread 2 mL of solution S evenly on a
glass plate. Upon drying a clear film is formed.
IMPURITIES Monomers. Liquid chromatography (2.2.29).
Specified impurities : A, B. Solution A. Dissolve 3.5 g of sodium perchlorate R in water for
chromatography R and dilute to 100 mL with the same solvent.
Test solution. Dissolve 5.00 g of the substance to be examined
in methanol R and dilute to 50.0 mL with the same solvent. To
10.0 mL of this solution add 5.0 mL of solution A, dropwise,
A. R = H, R′ = C2H5 : ethyl propenoate (ethyl acrylate), while continuously stirring. Remove the precipitated polymer
by centrifugation. Use the clear supernatant solution.
B. R = R′ = CH3 : methyl 2-methylpropenoate (methyl
methacrylate). Reference solution. Dissolve 50.0 mg of ethyl acrylate R and
10.0 mg of methyl methacrylate R in methanol R and dilute to
50.0 mL with the same solvent. Dilute 1.0 mL of the solution
01/2008:2082 to 100.0 mL with methanol R. Add 10 mL of this solution to
5 mL of solution A.
AMMONIO METHACRYLATE Column :
COPOLYMER (TYPE B) – size : l = 0.12 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
Ammonio methacrylatis copolymerum B chromatography R (7 μm).
Mobile phase : dilute phosphoric acid R with water for
chromatography R to obtain a solution at pH 2.0 ; mix 800 mL
of this solution and 200 mL of methanol R, filter and degas.
Flow rate : 2.0 mL/min.
Detection : spectrophotometer at 202 nm.
Injection : 50 μL.
System suitability : reference solution :
– resolution : minimum 1.5 between the peaks due to
impurity A and impurity B.

1550 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ammonium bromide

Limits : Bromates. To 10 mL of solution S add 1 mL of starch

– impurity A : not more than the area of the corresponding solution R, 0.1 mL of a 100 g/L solution of potassium iodide R
peak in the chromatogram obtained with the reference and 0.25 mL of 0.5 M sulfuric acid and allow to stand protected
solution (100 ppm); from light for 5 min. No blue or violet colour develops.
– impurity B : not more than 2.5 times the area of the Chlorides and sulfates. Liquid chromatography (2.2.29).
corresponding peak in the chromatogram obtained with Test solution (a). Dissolve 0.400 g of the substance to be
the reference solution (50 ppm). examined in 50 mL of water for chromatography R and dilute
Methanol (2.4.24, System A) : maximum 1.5 per cent. to 100.0 mL with the same solvent.
Heavy metals (2.4.8) : maximum 20 ppm. Test solution (b). Dilute 25.0 mL of test solution (a) to 50.0 mL
with water for chromatography R.
1.0 g complies with test C. Prepare the reference solution using
2.0 mL of lead standard solution (10 ppm Pb) R. Reference solution (a). To 25.0 mL of test solution (a) add
1.0 mL of sulfate standard solution (10 ppm SO4) R and
Loss on drying (2.2.32) : maximum 3.0 per cent, determined 12.0 mL of chloride standard solution (50 ppm Cl) R and dilute
on 1.000 g by drying in vacuo at 80 °C for 5 h. to 50.0 mL with water for chromatography R.
ASSAY Reference solution (b). Dilute 10.0 mL of test solution (a) to
Dissolve 2.000 g in a mixture of 3 mL of anhydrous formic 100.0 mL with water for chromatography R. To 2.0 mL of this
acid R and 30 mL of anhydrous acetic acid R and heat to solution add 8.0 mL of chloride standard solution (50 ppm
dissolve. Add 20 mL of acetic anhydride R. Titrate with 0.1 M Cl) R and dilute to 20.0 mL with water for chromatography R.
perchloric acid, determining the end-point potentiometrically Blank solution : water for chromatography R.
(2.2.20). Column :
1 mL of 0.1 M perchloric acid is equivalent to 20.77 mg – size : l = 0.25 m, Ø = 2 mm ;
of C9H18O2NCl (ammonio methacrylate groups). – stationary phase : strongly basic anion-exchange resin for
IMPURITIES chromatography R (13 μm).
Specified impurities : A, B. Mobile phase : dissolve 0.600 g of potassium hydroxide R in
water for chromatography R and dilute to 1000.0 mL with the
same solvent.
Flow rate : 0.4 mL/min.
Detection : conductivity detector equipped with a suitable ion
suppressor.
A. R = H, R′ = C2H5 : ethyl propenoate (ethyl acrylate),
Injection : 50 μL of test solution (b), reference solutions (a)
B. R = R′ = CH3 : methyl 2-methylpropenoate (methyl and (b) and the blank solution.
methacrylate). Run time : 2.5 times the retention time of bromide.
Retention time : chloride = about 5 min ; bromide = about
8 min ; sulfate = about 16 min.
07/2012:1389
System suitability : reference solution (b) :
AMMONIUM BROMIDE – resolution : minimum 8.0 between the peaks due to chloride
and bromide.
Ammonii bromidum Limits : correct the areas of the peaks obtained with test
solution (b) and reference solution (a) using the areas of the
peaks obtained with the blank solution :
NH4Br Mr 97.9
[12124-97-9] – chlorides : the area of the peak due to chloride in test
solution (b) is not more than the difference between the
DEFINITION areas of the peaks due to chloride in the chromatograms
Content : 98.5 per cent to 101.0 per cent (dried substance). obtained with test solution (b) and reference solution (a)
(0.6 per cent) ;
CHARACTERS – sulfates : the area of the peak due to sulfate in test
Appearance : white or almost white, crystalline powder or solution (b) is not more than the difference between the
colourless crystals, hygroscopic. areas of the peaks due to sulfate in the chromatograms
Solubility : freely soluble in water, sparingly soluble in ethanol obtained with test solution (b) and reference solution (a)
(96 per cent). (100 ppm).
It becomes yellow when exposed to light or air. Iodides. To 5 mL of solution S add 0.15 mL of ferric chloride
solution R1 and 2 mL of methylene chloride R. Shake and allow
IDENTIFICATION to separate. The lower layer is colourless (2.2.2, Method I).
A. It gives reaction (a) of bromides (2.3.1). Iron (2.4.9) : maximum 20 ppm.
B. 10 mL of solution S (see Tests) gives the reaction of Dilute 5 mL of solution S to 10 mL with water R.
ammonium salts (2.3.1).
Magnesium and alkaline-earth metals (2.4.7) : maximum
TESTS 200 ppm, calculated as Ca.
Solution S. Dissolve 10.0 g in carbon dioxide-free water R and 10.0 g complies with the test for magnesium and alkaline-earth
dilute to 100 mL with the same solvent. metals. The volume of 0.01 M sodium edetate used does not
exceed 5.0 mL.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II). Heavy metals (2.4.8) : maximum 10 ppm.
Acidity or alkalinity. To 10 mL of solution S add 0.05 mL 12 mL of solution S complies with test A. Prepare the reference
of methyl red solution R. Not more than 0.5 mL of 0.01 M solution using lead standard solution (1 ppm Pb) R.
hydrochloric acid or 0.01 M sodium hydroxide is required to Loss on drying (2.2.32) : maximum 1.0 per cent, determined
change the colour of the indicator. on 1.000 g by drying in an oven at 105 °C.

General Notices (1) apply to all monographs and other texts 1551
Ammonium chloride EUROPEAN PHARMACOPOEIA 8.0

Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Loss on drying (2.2.32) : maximum 1.0 per cent, determined
1.0 g. on 1.00 g by drying in an oven at 105 °C for 2 h.
ASSAY Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
2.0 g.
Dissolve 80.0 mg in water R, add 5 mL of dilute nitric acid R
and dilute to 50 mL with water R. Titrate with 0.1 M silver ASSAY
nitrate, determining the end-point potentiometrically (2.2.20).
Dissolve 1.000 g in 20 mL of water R and add a mixture of
1 mL of 0.1 M silver nitrate is equivalent to 9.794 mg of NH4Br. 5 mL of formaldehyde solution R, previously neutralised to
Calculate the percentage content of NH4Br using the following phenolphthalein solution R, and 20 mL of water R. After
expression : 1-2 min, titrate slowly with 1 M sodium hydroxide, using a
further 0.2 mL of the same indicator.
1 mL of 1 M sodium hydroxide is equivalent to 53.49 mg
a = percentage content of NH4Br and NH4Cl obtained of NH4Cl.
in the assay and calculated as NH4Br ;
b = percentage content of Cl obtained in the test for
chlorides. 01/2008:1772
corrected 7.0
STORAGE
In an airtight container, protected from light. AMMONIUM GLYCYRRHIZATE
01/2008:0007 Ammonii glycyrrhizas
corrected 6.0

AMMONIUM CHLORIDE
Ammonii chloridum
NH4Cl Mr 53.49
[12125-02-9]
DEFINITION
Content : 99.0 per cent to 100.5 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
colourless crystals. C42H65NO16 Mr 840
[53956-04-0]
Solubility : freely soluble in water.
DEFINITION
IDENTIFICATION
Mixture of ammonium 18α- and 18β-glycyrrhizate
A. It gives the reactions of chlorides (2.3.1). (ammonium salt of (20β)-3β-[[2-O-(β-D-glucopyranosyluronic
B. 10 mL of solution S (see Tests) gives the reaction of acid)-α-D-glucopyranosyluronic acid]oxy]-11-oxoolean-12-
ammonium salts (2.3.1). en-29-oic acid), the 18β-isomer being the main component.
TESTS Content : 98.0 per cent to 102.0 per cent (anhydrous substance).
Solution S. Dissolve 10.0 g in carbon dioxide-free water R CHARACTERS
prepared from distilled water R and dilute to 100 mL with the Appearance : white or yellowish-white, hygroscopic powder.
same solvent.
Solubility : slightly soluble in water, very slightly soluble
Appearance of solution. Solution S is clear (2.2.1) and in anhydrous ethanol, practically insoluble in acetone. It
colourless (2.2.2, Method II). dissolves in dilute solutions of acids and of alkali hydroxides.
Acidity or alkalinity. To 10 mL of solution S add 0.05 mL
of methyl red solution R. Not more than 0.5 mL of 0.01 M IDENTIFICATION
hydrochloric acid or 0.01 M sodium hydroxide is required to A. Infrared absorption spectrophotometry (2.2.24).
change the colour of the indicator. Comparison: ammonium glycyrrhizate CRS.
Bromides and iodides. To 10 mL of solution S add 0.1 mL B. Dissolve 0.1 g in 20 mL of water R, add 2 mL of dilute
of dilute hydrochloric acid R and 0.05 mL of chloramine sodium hydroxide solution R and heat cautiously. On
solution R. After 1 min, add 2 mL of chloroform R and shake heating, the solution gives off vapours that may be identified
vigorously. The chloroform layer remains colourless (2.2.2, by the alkaline reaction of wet litmus paper (2.3.1).
Method I).
Sulfates (2.4.13) : maximum 150 ppm. TESTS
Dilute 10 mL of solution S to 15 mL with distilled water R. Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution BY7 (2.2.2,
Calcium (2.4.3) : maximum 200 ppm. Method I).
Dilute 5 mL of solution S to 15 mL with distilled water R.
Dissolve 1.0 g in ethanol (20 per cent V/V) R and dilute to
Iron (2.4.9) : maximum 20 ppm. 100.0 mL with the same solvent.
Dilute 5 mL of solution S to 10 mL with water R. Specific optical rotation (2.2.7) : + 49.0 to + 54.0 (anhydrous
Heavy metals (2.4.8) : maximum 10 ppm. substance).
12 mL of solution S complies with test A. Prepare the reference Dissolve 0.5 g in ethanol (50 per cent V/V) R and dilute to
solution using lead standard solution (1 ppm Pb) R. 50.0 mL with the same solvent.

1552 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ammonium hydrogen carbonate

Related substances. Liquid chromatography (2.2.29). IMPURITIES

Test solution. Dissolve 0.100 g of the substance to be examined
in the mobile phase and dilute to 100.0 mL with the mobile
phase.
Reference solution (a). Dilute 1.0 mL of the test solution to
20.0 mL with the mobile phase.
Reference solution (b). Dissolve 50 mg of ammonium
glycyrrhizate CRS in the mobile phase and dilute to 50.0 mL
with the mobile phase. Dilute 1.0 mL of the solution to
20.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.0 mm,
A. (4β,20β)-3β-[[2-O-(β-D-glucopyranosyluronic
– stationary phase : octadecylsilyl silica gel for acid)-α-D-glucopyranosyluronic acid]oxy]-23-hydroxy-
chromatography R (5-10 μm). 11-oxoolean-12-en-29-oic acid (24-hydroxyglycyrrhizinic
Mobile phase : glacial acetic acid R, acetonitrile R, water R acid).
(6:380:614 V/V/V).
Flow rate : 1.2 mL/min. 01/2008:1390
corrected 6.0
Detection : spectrophotometer at 254 nm.
Injection : 10 μL. AMMONIUM HYDROGEN
Run time : 3 times the retention time of 18β-glycyrrhizic acid. CARBONATE
Relative retention with reference to 18β-glycyrrhizic acid
(retention time = about 8 min) : impurity A = about 0.8 ; Ammonii hydrogenocarbonas
18α-glycyrrhizic acid = about 1.2.
System suitability : reference solution (b) : NH4HCO3 Mr 79.1
[1066-33-7]
– resolution : minimum 2.0 between the peaks due to
18β-glycyrrhizic acid and 18α-glycyrrhizic acid. DEFINITION
Limits : Content : 98.0 per cent to 101.0 per cent.
– 18α-glycyrrhizic acid : not more than twice the sum of the CHARACTERS
areas of the peaks in the chromatogram obtained with Appearance : fine, white or almost white, crystalline powder or
reference solution (a) (10.0 per cent), white or almost white crystals, slightly hygroscopic.
– impurity A : not more than the sum of the areas of the peaks Solubility : freely soluble in water, practically insoluble in
in the chromatogram obtained with reference solution (a) ethanol (96 per cent).
(5.0 per cent), It volatilises rapidly at 60 °C. The volatilisation takes place
– any other impurity : for each impurity, not more than slowly at ambient temperatures if the substance is slightly
0.4 times the sum of the areas of the peaks in the moist. It is in a state of equilibrium with ammonium
chromatogram obtained with reference solution (a) (2.0 per carbamate.
cent), IDENTIFICATION
– sum of other impurities : not more than 1.4 times the sum of A. It gives the reaction of carbonates and bicarbonates (2.3.1).
the areas of the peaks in the chromatogram obtained with B. Dissolve 50 mg in 2 mL of water R. The solution gives the
reference solution (a) (7.0 per cent), reaction of ammonium salts (2.3.1).
– disregard limit : 0.04 times the sum of the areas of the peaks
TESTS
in the chromatogram obtained with reference solution (a)
(0.2 per cent). Solution S. Dissolve 14.0 g in 100 mL of distilled water R. Boil
to remove the ammonia, allow to cool and dilute to 100.0 mL
Heavy metals (2.4.8) : maximum 20 ppm. with distilled water R.
1.0 g complies with limit test C. Prepare the reference solution Chlorides (2.4.4) : maximum 70 ppm.
using 2 mL of lead standard solution (10 ppm Pb) R.
Dilute 5 mL of solution S to 15 mL with water R.
Water (2.5.12) : maximum 6.0 per cent, determined on 0.250 g. Sulfates (2.4.13) : maximum 70 ppm, determined on
Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on solution S.
1.0 g. Iron (2.4.9) : maximum 40 ppm.
Dilute 1.8 mL of solution S to 10 mL with water R.
ASSAY
Heavy metals (2.4.8) : maximum 10 ppm.
Dissolve 0.600 g in 60 mL of anhydrous acetic acid R heating Dissolve cautiously 2.5 g in 25 mL of 1 M hydrochloric acid.
at 80 °C if necessary. Cool. Titrate with 0.1 M perchloric acid, 12 mL of the solution complies with test A. Prepare the
determining the end-point potentiometrically (2.2.20). reference solution using lead standard solution (1 ppm Pb) R.
1 mL of 0.1 M perchloric acid is equivalent to 84.0 mg
of C42H65NO16. ASSAY
Dissolve cautiously 1.0 g in 20.0 mL of 0.5 M sulfuric acid and
dilute to 50 mL with water R. Boil, cool and titrate the excess
STORAGE
of acid with 1 M sodium hydroxide, using 0.1 mL of methyl red
In an airtight container. solution R as indicator.

General Notices (1) apply to all monographs and other texts 1553
Amobarbital EUROPEAN PHARMACOPOEIA 8.0

1 mL of 0.5 M sulfuric acid is equivalent to 79.1 mg Acidity or alkalinity. To 1.0 g add 50 mL of water R and boil
of NH4HCO3. for 2 min. Allow to cool and filter. To 10 mL of the filtrate
add 0.15 mL of methyl red solution R and 0.1 mL of 0.01 M
STORAGE sodium hydroxide. The solution is yellow. Add 0.2 mL of
In an airtight container. 0.01 M hydrochloric acid. The solution is red.
Related substances. Examine by thin-layer chromatography
(2.2.27), using silica gel GF254 R as the coating substance.
01/2008:0594 Test solution. Dissolve 1.0 g of the substance to be examined
corrected 6.0 in alcohol R and dilute to 100 mL with the same solvent.
Reference solution. Dilute 0.5 mL of the test solution to 100 mL
with alcohol R.
AMOBARBITAL Apply separately to the plate 20 μL of each solution. Develop
over a path of 15 cm using the lower layer from a mixture
Amobarbitalum of 5 volumes of concentrated ammonia R, 15 volumes of
alcohol R and 80 volumes of chloroform R. Examine the plate
immediately in ultraviolet light at 254 nm. Any spot in the
chromatogram obtained with the test solution, apart from
the principal spot, is not more intense than the spot in the
chromatogram obtained with the reference solution. Spray
with diphenylcarbazone mercuric reagent R. Allow the plate to
dry in air and spray with freshly prepared alcoholic potassium
hydroxide solution R diluted 1 in 5 with aldehyde-free
C11H18N2O3 Mr 226.3 alcohol R. Heat at 100 °C to 105 °C for 5 min and examine
[57-43-2] immediately. Any spot in the chromatogram obtained with
DEFINITION the test solution, apart from the principal spot, is not more
intense than the spot in the chromatogram obtained with the
Amobarbital contains not less than 99.0 per cent and reference solution (0.5 per cent).
not more than the equivalent of 101.0 per cent of
5-ethyl-5-(3-methylbutyl)pyrimidin-2,4,6(1H,3H,5H)-trione, Loss on drying (2.2.32). Not more than 0.5 per cent,
calculated with reference to the dried substance. determined on 1.000 g by drying in an oven at 105 °C.
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
CHARACTERS on 1.0 g.
A white or almost white, crystalline powder, very slightly
soluble in water, freely soluble in alcohol, soluble in methylene ASSAY
chloride. It forms water-soluble compounds with alkali Dissolve 0.100 g in 5 mL of pyridine R. Add 0.5 mL of
hydroxides and carbonates and with ammonia. thymolphthalein solution R and 10 mL of silver nitrate solution
in pyridine R. Titrate with 0.1 M ethanolic sodium hydroxide
IDENTIFICATION until a pure blue colour is obtained. Carry out a blank
First identification : A, B. titration.
Second identification : A, C, D. 1 mL of 0.1 M ethanolic sodium hydroxide is equivalent to
11.31 mg of C11H18N2O3.
A. Determine the melting point (2.2.14) of the substance to be
examined. Mix equal parts of the substance to be examined
and amobarbital CRS and determine the melting point of 01/2008:0166
the mixture. The difference between the melting points corrected 6.0
(which are about 157 °C) is not greater than 2 °C.
B. Examine by infrared absorption spectrophotometry
AMOBARBITAL SODIUM
(2.2.24), comparing with the spectrum obtained with
amobarbital CRS. Amobarbitalum natricum
C. Examine by thin-layer chromatography (2.2.27), using
silica gel GF254 R as the coating substance.
Test solution. Dissolve 0.1 g of the substance to be examined
in alcohol R and dilute to 100 mL with the same solvent.
Reference solution. Dissolve 0.1 g of amobarbital CRS in
alcohol R and dilute to 100 mL with the same solvent.
Apply separately to the plate 10 μL of each solution. C11H17N2NaO3 Mr 248.3
Develop over a path of 18 cm using the lower layer from [64-43-7]
a mixture of 5 volumes of concentrated ammonia R,
DEFINITION
15 volumes of alcohol R and 80 volumes of chloroform R.
Examine immediately in ultraviolet light at 254 nm. The Amobarbital sodium contains not less than 98.5 per cent
principal spot in the chromatogram obtained with the test and not more than the equivalent of 102.0 per cent of
solution is similar in position and size to the principal spot sodium derivative of 5-ethyl-5-(3-methylbutyl)pyrimidin-
in the chromatogram obtained with the reference solution. 2,4,6(1H,3H,5H)-trione, calculated with reference to the dried
substance.
D. It gives the reaction of non-nitrogen substituted
barbiturates (2.3.1). CHARACTERS
A white or almost white, granular powder, hygroscopic, very
TESTS
soluble in carbon dioxide-free water (a small fraction may be
Appearance of solution. Dissolve 1.0 g in a mixture of 4 mL insoluble), freely soluble in alcohol.
of dilute sodium hydroxide solution R and 6 mL of water R.
The solution is clear (2.2.1) and not more intensely coloured IDENTIFICATION
than reference solution Y6 (2.2.2, Method II). First identification : A, B, E.

1554 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Amoxicillin sodium

Second identification : A, C, D, E. ASSAY

A. Acidify 10 mL of solution S (see Tests) with dilute Dissolve 0.200 g in 5 mL of ethanol R. Add 0.5 mL of
hydrochloric acid R and shake with 20 mL of ether R. thymolphthalein solution R and 10 mL of silver nitrate solution
Separate the ether layer, wash with 10 mL of water R, dry in pyridine R. Titrate with 0.1 M ethanolic sodium hydroxide
over anhydrous sodium sulfate R and filter. Evaporate until a pure blue colour is obtained. Carry out a blank
the filtrate to dryness and dry the residue at 100 °C to titration.
105 °C (test residue). Repeat the operations using 0.1 g of 1 mL of 0.1 M ethanolic sodium hydroxide is equivalent to
amobarbital sodium CRS (reference residue). Determine 24.83 mg of C11H17N2NaO3.
the melting point (2.2.14) of the test residue. Mix equal
parts of the test residue and the reference residue and STORAGE
determine the melting point of the mixture. The difference Store in an airtight container.
between the melting points (which are about 157 °C) is not
greater than 2 °C. 01/2008:0577
B. Examine by infrared absorption spectrophotometry corrected 6.0
(2.2.24), comparing the spectrum obtained with the
reference residue prepared from amobarbital sodium CRS AMOXICILLIN SODIUM
with that obtained with the test residue (see identification
test A). Amoxicillinum natricum
C. Examine by thin-layer chromatography (2.2.27), using
silica gel GF254 R as the coating substance.
Test solution. Dissolve 0.1 g of the substance to be examined
in alcohol R and dilute to 100 mL with the same solvent.
Reference solution. Dissolve 0.1 g of amobarbital
sodium CRS in alcohol R and dilute to 100 mL with the
same solvent. C16H18N3NaO5S Mr 387.4
Apply separately to the plate 10 μL of each solution. [34642-77-8]
Develop over a path of 18 cm using the lower layer of
a mixture of 5 volumes of concentrated ammonia R, DEFINITION
15 volumes of alcohol R and 80 volumes of chloroform R. Sodium (2S,5R,6R)-6-[[(2R)-2-amino-2-(4-hydroxyphenyl)-
Examine immediately in ultraviolet light at 254 nm. The acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo-
principal spot in the chromatogram obtained with the test [3.2.0]heptane-2-carboxylate.
solution is similar in position and size to the principal spot Semi-synthetic product derived from a fermentation product.
in the chromatogram obtained with the reference solution. Content : 89.0 per cent to 102.0 per cent (anhydrous substance).
D. It gives the reaction of non-nitrogen substituted
barbiturates (2.3.1). CHARACTERS
E. It gives reaction (a) of sodium (2.3.1). Appearance : white or almost white, very hygroscopic, powder.
Solubility : very soluble in water, sparingly soluble in anhydrous
TESTS ethanol, very slightly soluble in acetone.
Solution S. Dissolve 5.0 g in alcohol (50 per cent V/V) R and IDENTIFICATION
dilute to 50 mL with the same solvent.
First identification : A, D.
Appearance of solution. Solution S is clear (2.2.1) and not Second identification : B, C, D.
more intensely coloured than reference solution Y7 (2.2.2,
Method II). A. Infrared absorption spectrophotometry (2.2.24).
Preparation : dissolve 0.250 g in 5 mL of water R, add
pH (2.2.3). Dissolve 5.0 g in carbon dioxide-free water R and 0.5 mL of dilute acetic acid R, swirl and allow to stand for
dilute to 50 mL with the same solvent. Disregard any slight 10 min in iced water. Filter the crystals and wash with
residue. The pH of the solution is not more than 11.0. 2-3 mL of a mixture of 1 volume of water R and 9 volumes
Related substances. Examine by thin-layer chromatography of acetone R, then dry in an oven at 60 °C for 30 min.
(2.2.27), using silica gel GF254 R as the coating substance. Comparison: amoxicillin trihydrate CRS.
Test solution. Dissolve 1.0 g of the substance to be examined B. Thin-layer chromatography (2.2.27).
in alcohol R and dilute to 100 mL with the same solvent. Test solution. Dissolve 25 mg of the substance to be
Reference solution. Dilute 0.5 mL of the test solution to 100 mL examined in 10 mL of sodium hydrogen carbonate
with alcohol R. solution R.
Apply separately to the plate 20 μL of each solution. Develop Reference solution (a). Dissolve 25 mg of amoxicillin
over a path of 15 cm using the lower layer of a mixture trihydrate CRS in 10 mL of sodium hydrogen carbonate
of 5 volumes of concentrated ammonia R, 15 volumes of solution R.
alcohol R and 80 volumes of chloroform R. Examine the Reference solution (b). Dissolve 25 mg of amoxicillin
plate immediately in ultraviolet light at 254 nm. Spray with trihydrate CRS and 25 mg of ampicillin trihydrate CRS in
diphenylcarbazone mercuric reagent R. Allow the plate to dry 10 mL of sodium hydrogen carbonate solution R.
in air and spray with freshly prepared alcoholic potassium Plate : TLC silanised silica gel plate R.
hydroxide solution R diluted 1 in 5 with aldehyde-free alcohol R.
Heat at 100 °C to 105 °C for 5 min and examine immediately. Mobile phase : mix 10 volumes of acetone R and 90 volumes
When examined in ultraviolet light and after spraying, any of a 154 g/L solution of ammonium acetate R previously
spot in the chromatogram obtained with the test solution, adjusted to pH 5.0 with glacial acetic acid R.
apart from the principal spot, is not more intense than the spot Application : 1 μL.
in the chromatogram obtained with the reference solution Development : over a path of 15 cm.
(0.5 per cent). Disregard any spot at the point of application. Drying : in air.
Loss on drying (2.2.32). Not more than 3.0 per cent, Detection : expose to iodine vapour until the spots appear
determined on 0.50 g by drying in an oven at 130 °C. and examine in daylight.

General Notices (1) apply to all monographs and other texts 1555
Amoxicillin sodium EUROPEAN PHARMACOPOEIA 8.0

System suitability: reference solution (b) : Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
– the chromatogram shows 2 clearly separated spots.
0 - tR 92 8
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size tR - (tR + 25) 92 → 0 8 → 100
to the principal spot in the chromatogram obtained with (tR + 25) - (tR + 40) 0 100
reference solution (a).
(tR + 40) - (tR + 55) 92 8
C. Place about 2 mg in a test-tube about 150 mm long and
about 15 mm in diameter. Moisten with 0.05 mL of water R tR = retention time of amoxicillin determined with reference solution (c)
and add 2 mL of sulfuric acid-formaldehyde reagent R.
Mix the contents of the tube by swirling ; the solution is If the mobile phase has been adjusted to achieve the required
practically colourless. Place the test-tube in a water-bath resolution, the adjusted composition will apply at time zero in
for 1 min ; a dark yellow colour develops. the gradient and in the assay.
D. It gives reaction (a) of sodium (2.3.1). Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 254 nm.
TESTS Injection : 50 μL of reference solutions (b) and (c) with
Appearance of solution. The solution is not more opalescent isocratic elution at the initial mobile phase composition and
than reference suspension II (2.2.1), it may show an initial, but 50 μL of test solution (b) and reference solution (d) according
transient, pink colour, and after 5 min, its absorbance (2.2.25) to the elution gradient described under Mobile phase ; inject
at 430 nm is not greater than 0.20. mobile phase A as a blank according to the elution gradient
described under Mobile phase.
Dissolve 1.0 g in water R and dilute to 10.0 mL with the same
solvent. Examine immediately after dissolution. Identification of impurities: use the chromatogram obtained
with reference solution (d) to identify the 3 principal peaks
pH (2.2.3) : 8.0 to 10.0. eluted after the main peak corresponding to impurity C,
Dissolve 2.0 g in carbon dioxide-free water R and dilute to amoxicillin dimer (impurity J ; n = 1) and amoxicillin trimer
20 mL with the same solvent. (impurity J ; n = 2).
Specific optical rotation (2.2.7) : + 240 to + 290 (anhydrous Relative retention with reference to amoxicillin :
substance). impurity C = about 3.4 ; impurity J (n = 1) = about 4.1 ;
impurity J (n = 2) = about 4.5.
Dissolve 62.5 mg in a 4 g/L solution of potassium hydrogen
phthalate R and dilute to 25.0 mL with the same solution. System suitability : reference solution (b) :
– resolution : minimum 2.0 between the peaks due to
Related substances. Liquid chromatography (2.2.29). amoxicillin and cefadroxil ; if necessary, adjust the ratio
Test solution (a). Dissolve 30.0 mg of the substance to be A:B of the mobile phase.
examined in mobile phase A and dilute to 50.0 mL with Limits :
mobile phase A.
– impurity J (n = 1) : not more than 3 times the area of
Test solution (b). Dissolve 30.0 mg of the substance to be the principal peak in the chromatogram obtained with
examined in mobile phase A and dilute to 20.0 mL with reference solution (c) (3 per cent) ;
mobile phase A. Prepare immediately before use. – any other impurity : for each impurity, not more than
Reference solution (a). Dissolve 30.0 mg of amoxicillin twice the area of the principal peak in the chromatogram
trihydrate CRS in mobile phase A and dilute to 50.0 mL with obtained with reference solution (c) (2 per cent) ;
mobile phase A. – total : not more than 9 times the area of the principal peak
Reference solution (b). Dissolve 4.0 mg of cefadroxil CRS in in the chromatogram obtained with reference solution (c)
mobile phase A and dilute to 50 mL with mobile phase A. To (9 per cent) ;
5.0 mL of this solution add 5.0 mL of reference solution (a) – disregard limit : 0.1 times the area of the principal peak in
and dilute to 100 mL with mobile phase A. the chromatogram obtained with reference solution (c)
Reference solution (c). Dilute 2.0 mL of reference solution (a) (0.1 per cent).
to 20.0 mL with mobile phase A. Dilute 5.0 mL of this solution N,N-Dimethylaniline (2.4.26, Method A or B) : maximum
to 20.0 mL with mobile phase A. 20 ppm.
Reference solution (d). To 0.20 g of amoxicillin trihydrate R 2-Ethylhexanoic acid (2.4.28) : maximum 0.8 per cent m/m.
add 1.0 mL of water R. Shake and add dropwise dilute sodium Heavy metals (2.4.8) : maximum 20 ppm.
hydroxide solution R to obtain a solution. The pH of the
solution is about 8.5. Store the solution at room temperature 1.0 g complies with test C. Prepare the reference solution using
for 4 h. Dilute 0.5 mL of this solution to 50.0 mL with mobile 2 mL of lead standard solution (10 ppm Pb) R.
phase A. Water (2.5.12) : maximum 3.0 per cent, determined on 0.400 g.
Column : Bacterial endotoxins (2.6.14) : less than 0.25 IU/mg, if
– size : l = 0.25 m, Ø = 4.6 mm ; intended for use in the manufacture of parenteral preparations
without a further appropriate procedure for the removal of
– stationary phase : octadecylsilyl silica gel for bacterial endotoxins.
chromatography R (5 μm).
Mobile phase : ASSAY
Liquid chromatography (2.2.29) as described in the test for
– mobile phase A : mix 1 volume of acetonitrile R and related substances with the following modifications.
99 volumes of a 25 per cent V/V solution of 0.2 M potassium
dihydrogen phosphate R adjusted to pH 5.0 with dilute Mobile phase : initial composition of the mixture of mobile
sodium hydroxide solution R ; phases A and B, adjusted where applicable.
Injection : test solution (a) and reference solution (a).
– mobile phase B : mix 20 volumes of acetonitrile R and
80 volumes of a 25 per cent V/V solution of 0.2 M potassium System suitability : reference solution (a) :
dihydrogen phosphate R adjusted to pH 5.0 with dilute – repeatability : maximum relative standard deviation of
sodium hydroxide solution R ; 1.0 per cent after 6 injections.

1556 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Amoxicillin trihydrate

Calculate the percentage content of amoxicillin sodium by

multiplying the percentage content of amoxicillin by 1.060.

STORAGE
In an airtight container. If the substance is sterile, store in a
sterile, airtight, tamper-proof container.

IMPURITIES G. (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-(4-hydroxy-
phenyl)acetyl]amino]2-(4-hydroxyphenyl)acetyl]ami-
no]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-
2-carboxylic acid (D-(4-hydroxyphenyl)glycylamoxicillin),

A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylic acid
(6-aminopenicillanic acid),
H. (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-(4-
hydroxyphenyl)acetic acid,

I. (2R)-2-amino-2-(4-hydroxyphenyl)acetic acid,
B. (2S,5R,6R)-6-[[(2S)-2-amino-2-(4-hydroxyphenyl)-
acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo-
[3.2.0]heptane-2-carboxylic acid (L-amoxicillin),

C. (4S)-2-[5-(4-hydroxyphenyl)-3,6-dioxopiperazin-2-yl]-5,5-
dimethylthiazolidine-4-carboxylic acid (amoxicillin J. co-oligomers of amoxicillin and penicilloic acids of
diketopiperazines), amoxicillin,

D. (4S)-2-[[[(2R)-2-amino-2-(4-
hydroxyphenyl)acetyl]amino]carboxymethyl]-5,5-dime- K. oligomers of penicilloic acids of amoxicillin.
thylthiazolidine-4-carboxylic acid (penicilloic acids of
amoxicillin),
01/2013:0260

AMOXICILLIN TRIHYDRATE
Amoxicillinum trihydricum

E. (2RS,4S)-2-[[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]-
amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid
(penilloic acids of amoxicillin),

C16H19N3O5S,3H2O Mr 419.4
[61336-70-7]
DEFINITION
(2S,5R,6R)-6-[[(2R)-2-Amino-2-(4-hydroxyphenyl)acetyl]-
amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]hep-
F. 3-(4-hydroxyphenyl)pyrazin-2-ol, tane-2-carboxylic acid trihydrate.

General Notices (1) apply to all monographs and other texts 1557
Amoxicillin trihydrate EUROPEAN PHARMACOPOEIA 8.0

Semi-synthetic product derived from a fermentation product. Reference solution (b). Dissolve 4.0 mg of cefadroxil CRS in
Content : 95.0 per cent to 102.0 per cent (anhydrous substance). mobile phase A and dilute to 50 mL with mobile phase A. To
5.0 mL of this solution add 5.0 mL of reference solution (a)
CHARACTERS and dilute to 100 mL with mobile phase A.
Appearance : white or almost white, crystalline powder. Reference solution (c). Dilute 2.0 mL of reference solution (a)
Solubility : slightly soluble in water, very slightly soluble to 20.0 mL with mobile phase A. Dilute 5.0 mL of this solution
in ethanol (96 per cent), practically insoluble in fatty oils. to 20.0 mL with mobile phase A.
It dissolves in dilute acids and dilute solutions of alkali Column :
hydroxides. – size : l = 0.25 m, Ø = 4.6 mm ;
IDENTIFICATION – stationary phase : octadecylsilyl silica gel for
First identification : A. chromatography R (5 μm).
Second identification : B, C. Mobile phase :
A. Infrared absorption spectrophotometry (2.2.24). – mobile phase A : acetonitrile R, buffer solution pH 5.0
Comparison : amoxicillin trihydrate CRS. (1:99 V/V) ;
B. Thin-layer chromatography (2.2.27). – mobile phase B : acetonitrile R, buffer solution pH 5.0
Test solution. Dissolve 25 mg of the substance to be (20:80 V/V) ;
examined in 10 mL of sodium hydrogen carbonate Time Mobile phase A Mobile phase B
solution R. (min) (per cent V/V) (per cent V/V)
Reference solution (a). Dissolve 25 mg of amoxicillin 0 - tR 92 8
trihydrate CRS in 10 mL of sodium hydrogen carbonate
tR - (tR + 25) 92 → 0 8 → 100
solution R.
Reference solution (b). Dissolve 25 mg of amoxicillin (tR + 25) - (tR + 40) 0 100
trihydrate CRS and 25 mg of ampicillin trihydrate CRS in (tR + 40) - (tR + 55) 92 8
10 mL of sodium hydrogen carbonate solution R.
tR = retention time of amoxicillin determined with reference solution (c)
Plate : TLC silanised silica gel plate R.
Mobile phase : mix 10 volumes of acetone R and 90 volumes If the mobile phase composition has been adjusted to achieve
of a 154 g/L solution of ammonium acetate R previously the required resolution, the adjusted composition will apply at
adjusted to pH 5.0 with glacial acetic acid R. time zero in the gradient and in the assay.
Application : 1 μL. Flow rate : 1.0 mL/min.
Development : over a path of 15 cm. Detection : spectrophotometer at 254 nm.
Drying : in air. Injection : 50 μL of reference solutions (b) and (c) with
Detection : expose to iodine vapour until the spots appear isocratic elution at the initial mobile phase composition and
and examine in daylight. 50 μL of test solution (b) according to the elution gradient
System suitability: reference solution (b) : described under Mobile phase ; inject mobile phase A as
– the chromatogram shows 2 clearly separated spots. a blank according to the elution gradient described under
Results : the principal spot in the chromatogram obtained Mobile phase.
with the test solution is similar in position, colour and size System suitability : reference solution (b) :
to the principal spot in the chromatogram obtained with – resolution : minimum 2.0 between the peaks due to
reference solution (a). amoxicillin and cefadroxil ; if necessary, adjust the ratio
C. Place about 2 mg in a test-tube about 150 mm long and A:B of the mobile phase.
about 15 mm in diameter. Moisten with 0.05 mL of water R Limit :
and add 2 mL of sulfuric acid-formaldehyde reagent R. – any impurity : for each impurity, not more than the area
Mix the contents of the tube by swirling ; the solution is of the principal peak in the chromatogram obtained with
practically colourless. Place the test-tube in a water-bath reference solution (c) (1 per cent).
for 1 min ; a dark yellow colour develops.
N,N-Dimethylaniline (2.4.26, Method A or B) : maximum
TESTS 20 ppm.
Solution S. With the aid of ultrasound or gentle heating, Water (2.5.12) : 11.5 per cent to 14.5 per cent, determined on
dissolve 0.100 g in carbon dioxide-free water R and dilute to 0.100 g.
50.0 mL with the same solvent.
Sulfated ash (2.4.14) : maximum 1.0 per cent, determined on
pH (2.2.3): 3.5 to 5.5 for solution S. 1.0 g.
Specific optical rotation (2.2.7) : + 290 to + 315 (anhydrous
substance), determined on solution S. ASSAY
Liquid chromatography (2.2.29) as described in the test for
Related substances. Liquid chromatography (2.2.29).
related substances with the following modifications.
Buffer solution pH 5.0. To 250 mL of 0.2 M potassium
dihydrogen phosphate R add dilute sodium hydroxide Mobile phase : initial composition of the mixture of mobile
solution R to pH 5.0 and dilute to 1000.0 mL with water R. phases A and B, adjusted where applicable.
Test solution (a). Dissolve 30.0 mg of the substance to be Injection : test solution (a) and reference solution (a).
examined in mobile phase A and dilute to 50.0 mL with System suitability : reference solution (a) :
mobile phase A. – repeatability : maximum relative standard deviation of
Test solution (b). Dissolve 30.0 mg of the substance to be 1.0 per cent after 6 injections.
examined in mobile phase A and dilute to 20.0 mL with Calculate the percentage content of C16H19N3O5S taking into
mobile phase A. Prepare immediately before use. account the assigned content of amoxicillin trihydrate CRS.
Reference solution (a). Dissolve 30.0 mg of amoxicillin
trihydrate CRS in mobile phase A and dilute to 50.0 mL with STORAGE
mobile phase A. In an airtight container.

1558 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Amoxicillin trihydrate

IMPURITIES

G. (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-(4-
hydroxyphenyl)acetyl]amino]-2-(4-hydroxy-
A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia- phenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylic acid 1-azabicyclo[3.2.0]heptane-2-carboxylic acid
(6-aminopenicillanic acid), (D-(4-hydroxyphenyl)glycylamoxicillin),

H. (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-(4-
B. (2S,5R,6R)-6-[[(2S)-2-amino-2-(4-hydroxyphenyl)- hydroxyphenyl)acetic acid,
acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo-
[3.2.0]heptane-2-carboxylic acid (L-amoxicillin),

I. (2R)-2-amino-2-(4-hydroxyphenyl)acetic acid,

C. (4S)-2-[5-(4-hydroxyphenyl)-3,6-dioxopiperazin-2-yl]-
5,5-dimethylthiazolidine-4-carboxylic acid (amoxicillin
diketopiperazines),

J. co-oligomers of amoxicillin and of penicilloic acids of

amoxicillin,

D. (4S)-2-[[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]-
amino]carboxymethyl]-5,5-dimethylthiazolidine-4-
carboxylic acid (penicilloic acids of amoxicillin),

K. oligomers of penicilloic acids of amoxicillin,

E. (2RS,4S)-2-[[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]-
amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid
(penilloic acids of amoxicillin),

L. (2S,5R,6R)-6-[[(2S,5R,6R)-6-[[(2R)-2-amino-2-(4-
hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-
thia-1-azabicyclo[3.2.0]heptane-2-carbonyl]amino]-3,3-
dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-
F. 3-(4-hydroxyphenyl)pyrazin-2-ol, carboxylic acid (6-APA amoxicillin amide).

General Notices (1) apply to all monographs and other texts 1559
Amphotericin B EUROPEAN PHARMACOPOEIA 8.0

01/2009:1292 D. Examine the chromatograms obtained in the test for

corrected 6.6 related substances.
Results : the principal peak in the chromatogram obtained
AMPHOTERICIN B with the test solution at 383 nm is similar in retention time
to the principal peak in the chromatogram obtained with
Amphotericinum B reference solution (a).
TESTS
Related substances. Liquid chromatography (2.2.29). Protect
the solutions from light and use within 24 h of preparation,
except for reference solution (c) which should be injected
immediately after its preparation.
Solvent mixture : 10 g/L solution of ammonium acetate R,
N-methylpyrrolidone R, methanol R (1:1:2 V/V/V).
Test solution. Dissolve 20.0 mg of the substance to be
examined in 15 mL of N-methylpyrrolidone R and within 2 h
dilute to 50.0 mL with the solvent mixture. Dilute 5.0 mL of
this solution to 25.0 mL with the solvent mixture.
Reference solution (a). Dissolve 20.0 mg of amphotericin B CRS
in 15 mL of N-methylpyrrolidone R and within 2 h dilute
C47H73NO17 Mr 924 to 50.0 mL with the solvent mixture. Dilute 5.0 mL of this
[1397-89-3] solution to 25.0 mL with the solvent mixture.
Reference solution (b). Dilute 1.0 mL of reference solution (a)
DEFINITION to 100.0 mL with the solvent mixture.
Mixture of antifungal polyenes produced by the growth of Reference solution (c). Dissolve 20.0 mg of nystatin CRS
certain strains of Streptomyces nodosus or obtained by any in 15 mL of N-methylpyrrolidone R and within 2 h dilute
other means. It consists mainly of amphotericin B which to 50.0 mL with the solvent mixture. Dilute 5.0 mL of the
is (1R,3S,5R,6R,9R,11R,15S,16R,17R,18S,19E,21E,23E,- solution to 25.0 mL with reference solution (a). Dilute 2.0 mL
25E,27E,29E,31E,33R,35S,36R,37S)-33-[(3-amino-3,6- of this solution to 100.0 mL with the solvent mixture.
dideoxy-β-D-mannopyranosyl)oxy]-1,3,5,6,9,11,17,37-
octahydroxy-15,16,18-trimethyl-13-oxo-14,39-dioxa- Reference solution (d). In order to prepare impurities B and
bicyclo[33.3.1]nonatriaconta-19,21,23,25,27,29,31-heptaene- C, dissolve 10 mg of the substance to be examined in 5 mL of
36-carboxylic acid. N-methylpyrrolidone R and within 2 h add 35 mL of a mixture
of 1 volume of methanol R and 4 volumes of anhydrous
Content : minimum 750 IU/mg (dried substance). ethanol R. Add 0.10 mL of dilute hydrochloric acid R, mix and
CHARACTERS incubate at 25 °C for 2.5 h. Add 10 mL of 10 g/L solution of
ammonium acetate R and mix.
Appearance : yellow or orange, hygroscopic powder.
Reference solution (e). Dissolve 4 mg of amphotericin B for
Solubility : practically insoluble in water, soluble in dimethyl
peak identification CRS (containing impurities A and B) in
sulfoxide and in propylene glycol, slightly soluble in
5 mL of N-methylpyrrolidone R and within 2 h dilute to 50 mL
dimethylformamide, very slightly soluble in methanol,
with the solvent mixture.
practically insoluble in ethanol (96 per cent).
Blank solution. The solvent mixture.
It is sensitive to light in dilute solutions.
Column :
IDENTIFICATION – size : l = 0.15 m, Ø = 4.6 mm ;
First identification : B, D. – stationary phase : base-deactivated end-capped octadecylsilyl
Second identification : A, C. silica gel for chromatography R (3 μm) ;
A. Ultraviolet and visible absorption spectrophotometry – temperature : 20 °C.
(2.2.25). Mobile phase :
Test solution. Dissolve 25 mg in 5 mL of dimethyl – mobile phase A : mix 1 volume of methanol R, 3 volumes of
sulfoxide R and dilute to 50 mL with methanol R. Dilute acetonitrile R and 6 volumes of a 4.2 g/L solution of citric
2 mL of the solution to 200 mL with methanol R. acid R previously adjusted to pH 4.7 using concentrated
Spectral range : 300-450 nm. ammonia R ;
Absorption maxima : at 362 nm, 381 nm and 405 nm. – mobile phase B : mix 12 volumes of methanol R, 20 volumes
Absorbance ratios : of a 4.2 g/L solution of citric acid R previously adjusted to
pH 3.9 using concentrated ammonia R and 68 volumes of
– A362/A381 = 0.57 to 0.61 ;
acetonitrile R ;
– A381/A405 = 0.87 to 0.93.
Time Mobile phase A Mobile phase B
B. Infrared absorption spectrophotometry (2.2.24). (min) (per cent V/V) (per cent V/V)
Comparison : amphotericin B CRS. 0-3 100 0
If the spectra obtained show differences, dry the substance
3 - 23 100 → 70 0 → 30
to be examined and reference substance at 60 °C at a
pressure not exceeding 0.7 kPa for 1 h and record new 23 - 33 70 → 0 30 → 100
spectra.
33 - 40 0 100
C. To 1 mL of a 0.5 g/L solution in dimethyl sulfoxide R, add
5 mL of phosphoric acid R to form a lower layer, avoiding Flow rate : 0.8 mL/min.
mixing the 2 liquids. A blue ring is immediately produced
Detection : spectrophotometer :
at the junction of the liquids. Mix, an intense blue colour
is produced. Add 15 mL of water R and mix ; the solution – at 303 nm : detection of tetraenes ;
becomes pale yellow. – at 383 nm : detection of heptaenes.

1560 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ampicillin, anhydrous

Injection : 20 μL of the test solution and reference solutions (b), IMPURITIES

(c), (d) and (e). Specified impurities : A, B.
Identification of impurities : use the chromatograms supplied Other detectable impurities (the following substances would,
with amphotericin B for peak identification CRS and the if present at a sufficient level, be detected by one or other of
chromatograms obtained with reference solution (e) to the tests in the monograph. They are limited by the general
identify the peaks due to impurities A and B. acceptance criterion for other/unspecified impurities and/or
Relative retention with reference to amphotericin B by the general monograph Substances for pharmaceutical
(retention time = about 16 min): impurity B = about 0.75 ; use (2034). It is therefore not necessary to identify these
impurity A = about 0.8 ; nystatin = about 0.85. impurities for demonstration of compliance. See also 5.10.
System suitability at 383 nm : reference solution (d) : Control of impurities in substances for pharmaceutical use) : C.
– resolution : minimum 1.5 between the 2 peaks presenting a
relative retention of about 0.7.
Limits :
– impurity A at 303 nm : not more than 2.5 times the area
of the principal peak in the chromatogram obtained with
reference solution (c) (5.0 per cent) ; if intended for use
in the manufacture of parenteral preparations : not more
than the area of the principal peak in the chromatogram
obtained with reference solution (c) (2.0 per cent) ;
– any other impurity at 303 nm : for each impurity, not
more than 0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (c) (1.0 per
cent) ;
– impurity B at 383 nm : not more than 4 times the area of A. amphotericin A (28,29-dihydro-amphotericin B),
the principal peak in the chromatogram obtained with
reference solution (b) (4.0 per cent) ;
– any other impurity at 383 nm : for each impurity, not
more than 2 times the area of the principal peak in the
chromatogram obtained with reference solution (b) (2.0 per
cent) ;
– total at 303 and 383 nm : maximum 15.0 per cent ;
– disregard limit at 303 nm : 0.05 times the area of the
principal peak in the chromatogram obtained with
reference solution (c) (0.1 per cent) ;
– disregard limit at 383 nm : 0.1 times the area of the principal
peak in the chromatogram obtained with reference
solution (b) (0.1 per cent).
B. amphotericin X1 (13-O-methyl-amphotericin B),
Loss on drying (2.2.32) : maximum 5.0 per cent, determined
on 1.000 g by drying in an oven at 60 °C at a pressure not
exceeding 0.7 kPa.
Sulfated ash (2.4.14) : maximum 3.0 per cent, determined on
1.0 g ; if intended for use in the manufacture of parenteral
preparations : maximum 0.5 per cent.
Bacterial endotoxins (2.6.14) : less than 1.0 IU/mg, if intended
for use in the manufacture of parenteral preparations without
a further appropriate procedure for the removal of bacterial
endotoxins.
ASSAY
Protect all solutions from light throughout the assay. Dissolve
25.0 mg in dimethyl sulfoxide R and dilute, with shaking,
to 25.0 mL with the same solvent. Under constant stirring C. amphotericin X2 (13-O-ethyl-amphotericin B).
of this stock solution, dilute with dimethyl sulfoxide R to
obtain solutions of appropriate concentrations (the following 01/2008:0167
concentrations have been found suitable : 44.4, 66.7 and corrected 6.0
100 IU/mL). Prepare final solutions by diluting 1:20 with
0.2 M phosphate buffer solution pH 10.5 so that they all
contain 5 per cent V/V of dimethyl sulfoxide. Prepare the AMPICILLIN, ANHYDROUS
reference and the test solutions simultaneously. Carry out the
microbiological assay of antibiotics (2.7.2). Ampicillinum anhydricum
STORAGE
Protected from light, at a temperature of 2 °C to 8 °C in an
airtight container. If the substance is sterile, store in a sterile,
tamper-proof container.
LABELLING
The label states, where applicable, that the substance is suitable C16H19N3O4S Mr 349.4
for use in the manufacture of parenteral preparations. [69-53-4]

General Notices (1) apply to all monographs and other texts 1561
Ampicillin, anhydrous EUROPEAN PHARMACOPOEIA 8.0

DEFINITION Related substances. Liquid chromatography (2.2.29).

(2S,5R,6R)-6-[[(2R)-2-Amino-2-phenylacetyl]amino]- Test solution (a). Dissolve 27.0 mg of the substance to be
3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2- examined in mobile phase A and dilute to 50.0 mL with
carboxylic acid. mobile phase A.
Semi-synthetic product derived from a fermentation product. Test solution (b). Dissolve 27.0 mg of the substance to be
Content : 96.0 per cent to 102.0 per cent (anhydrous substance). examined in mobile phase A and dilute to 10.0 mL with
mobile phase A. Prepare immediately before use.
CHARACTERS Reference solution (a). Dissolve 27.0 mg of anhydrous
Appearance : white or almost white, crystalline powder. ampicillin CRS in mobile phase A and dilute to 50.0 mL with
Solubility : sparingly soluble in water, practically insoluble in mobile phase A.
acetone, in ethanol (96 per cent) and in fatty oils. It dissolves Reference solution (b). Dissolve 2.0 mg of cefradine CRS in
in dilute solutions of acids and of alkali hydroxides. mobile phase A and dilute to 50 mL with mobile phase A. To
It shows polymorphism (5.9). 5.0 mL of this solution add 5.0 mL of reference solution (a).
Reference solution (c). Dilute 1.0 mL of reference solution (a)
IDENTIFICATION
to 20.0 mL with mobile phase A.
First identification : A, D.
Column :
Second identification : B, C, D.
– size : l = 0.25 m, Ø = 4.6 mm ;
A. Infrared absorption spectrophotometry (2.2.24).
– stationary phase : octadecylsilyl silica gel for
Preparation : discs of potassium bromide R. chromatography R (5 μm).
Comparison : anhydrous ampicillin CRS. Mobile phase :
B. Thin-layer chromatography (2.2.27). – mobile phase A : mix 0.5 mL of dilute acetic acid R, 50 mL
Test solution. Dissolve 25 mg of the substance to be of 0.2 M potassium dihydrogen phosphate R and 50 mL of
examined in 10 mL of sodium hydrogen carbonate acetonitrile R, then dilute to 1000 mL with water R ;
solution R. – mobile phase B : mix 0.5 mL of dilute acetic acid R, 50 mL
Reference solution (a). Dissolve 25 mg of anhydrous of 0.2 M potassium dihydrogen phosphate R and 400 mL of
ampicillin CRS in 10 mL of sodium hydrogen carbonate acetonitrile R, then dilute to 1000 mL with water R ;
solution R.
Time Mobile phase A Mobile phase B
Reference solution (b). Dissolve 25 mg of amoxicillin (min) (per cent V/V) (per cent V/V)
trihydrate CRS and 25 mg of anhydrous ampicillin CRS in
0 - tR 85 15
10 mL of sodium hydrogen carbonate solution R.
Plate : TLC silanised silica gel plate R. tR - (tR + 30) 85 → 0 15 → 100
Mobile phase : mix 10 volumes of acetone R and 90 volumes (tR + 30) - (tR + 45) 0 100
of a 154 g/L solution of ammonium acetate R previously
85 15
adjusted to pH 5.0 with glacial acetic acid R. (tR + 45) - (tR + 60)

Application : 1 μL. tR = retention time of ampicillin determined with reference solution (c)
Development : over a path of 15 cm.
If the mobile phase composition has been adjusted to achieve
Drying : in air. the required resolution, the adjusted composition will apply at
Detection : expose to iodine vapour until the spots appear time zero in the gradient and in the assay.
and examine in daylight. Flow rate : 1.0 mL/min.
System suitability: reference solution (b) : Detection : spectrophotometer at 254 nm.
– the chromatogram shows 2 clearly separated spots.
Injection : 50 μL of reference solutions (b) and (c) with
Results : the principal spot in the chromatogram obtained isocratic elution at the initial mobile phase composition and
with the test solution is similar in position, colour and size 50 μL of test solution (b) according to the elution gradient
to the principal spot in the chromatogram obtained with described under Mobile phase ; inject mobile phase A as
reference solution (a). a blank according to the elution gradient described under
C. Place about 2 mg in a test-tube about 150 mm long and Mobile phase.
about 15 mm in diameter. Moisten with 0.05 mL of water R System suitability : reference solution (b) :
and add 2 mL of sulfuric acid-formaldehyde reagent R.
Mix the contents of the tube by swirling ; the solution is – resolution : minimum 3.0 between the peaks due to
practically colourless. Place the test-tube in a water-bath ampicillin and cefradin ; if necessary, adjust the ratio A:B
for 1 min ; a dark yellow colour develops. of the mobile phase.
D. Water (see Tests). Limit :
– any impurity : for each impurity, not more than the area
TESTS of the principal peak in the chromatogram obtained with
Appearance of solution. The solutions are not more reference solution (c) (1.0 per cent).
opalescent than reference suspension II (2.2.1). N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm.
Dissolve 1.0 g in 10 mL of 1 M hydrochloric acid. Separately Water (2.5.12) : maximum 2.0 per cent, determined on 0.300 g.
dissolve 1.0 g in 10 mL of dilute ammonia R2. Examine
immediately after dissolution. Sulfated ash (2.4.14) : maximum 0.5 per cent, determined on
1.0 g.
pH (2.2.3) : 3.5 to 5.5.
Dissolve 0.1 g in carbon dioxide-free water R and dilute to ASSAY
40 mL with the same solvent. Liquid chromatography (2.2.29) as described in the test for
Specific optical rotation (2.2.7) : + 280 to + 305 (anhydrous related substances with the following modifications.
substance). Mobile phase : initial composition of the mixture of mobile
Dissolve 62.5 mg in water R and dilute to 25.0 mL with the phases A and B, adjusted where applicable.
same solvent. Injection : test solution (a) and reference solution (a).

1562 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ampicillin, anhydrous

System suitability: reference solution (a) :

– repeatability : maximum relative standard deviation of
1.0 per cent after 6 injections.
Calculate the percentage content of C16H19N3O4S from the
declared content of anhydrous ampicillin CRS.

STORAGE G. (3R,6R)-3,6-diphenylpiperazine-2,5-dione,
In an airtight container, at a temperature not exceeding 30 °C.

IMPURITIES

H. 3-phenylpyrazin-2-ol,

A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylic acid
(6-aminopenicillanic acid),

I. (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-phenylacetyl]-
amino]-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-
4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid
(D-phenylglycylampicillin),
B. (2S,5R,6R)-6-[[(2S)-2-amino-2-phenylacetyl]amino]-
3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-
carboxylic acid (L-ampicillin),

J. (2S,5R,6R)-6-[(2,2-dimethylpropanoyl)amino]-3,3-
dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-
carboxylic acid,

C. (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5-
dimethylthiazolidine-4-carboxylic acid (diketopiperazines
of ampicillin),

K. (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-phenylacetic
acid,

D. R = CO2H : (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]-
carboxymethyl]-5,5-dimethylthiazolidine-4-carboxylic acid
(penicilloic acids of ampicillin),
L. (2R)-2-amino-2-phenylacetic acid (D-phenylglycine),
F. R = H : (2RS,4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]-
methyl]-5,5-dimethylthiazolidine-4-carboxylic acid
(penilloic acids of ampicillin),

E. (2R)-2-[[[(2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]-
amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]-
hept-2-yl]carbonyl]amino]-2-phenylacetic acid M. co-oligomers of ampicillin and of penicilloic acids of
(ampicillinyl-D-phenylglycine), ampicillin.

General Notices (1) apply to all monographs and other texts 1563
Ampicillin sodium EUROPEAN PHARMACOPOEIA 8.0

01/2008:0578 Mix the contents of the tube by swirling ; the solution is

corrected 6.0 practically colourless. Place the test-tube in a water-bath
for 1 min ; a dark yellow colour develops.
AMPICILLIN SODIUM D. It gives reaction (a) of sodium (2.3.1).
TESTS
Ampicillinum natricum Appearance of solution. Solutions A and B are not more
opalescent than reference suspension II (2.2.1) and the
absorbance (2.2.25) of solution B at 430 nm is not greater
than 0.15.
Place 1.0 g in a conical flask and add slowly and with
continuous swirling 10 mL of 1 M hydrochloric acid
(solution A). Separately dissolve 1.0 g in water R and dilute
C16H18N3NaO4S Mr 371.4 to 10.0 mL with the same solvent (solution B). Examine
[69-52-3] immediately after dissolution.
pH (2.2.3) : 8.0 to 10.0.
DEFINITION Dissolve 2.0 g in carbon dioxide-free water R and dilute
Sodium (2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]- to 20 mL with the same solvent. Measure 10 min after
3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2- dissolution.
carboxylate. Specific optical rotation (2.2.7) : + 258 to + 287 (anhydrous
Semi-synthetic product derived from a fermentation product. substance).
Content : 91.0 per cent to 102.0 per cent (anhydrous substance). Dissolve 62.5 mg in a 4 g/L solution of potassium hydrogen
phthalate R and dilute to 25.0 mL with the same solvent.
CHARACTERS
Related substances. Liquid chromatography (2.2.29).
Appearance : white or almost white powder, hygroscopic.
Test solution (a). Dissolve 31.0 mg of the substance to be
Solubility : freely soluble in water, sparingly soluble in acetone, examined in mobile phase A and dilute to 50.0 mL with
practically insoluble in fatty oils and in liquid paraffin. mobile phase A.
IDENTIFICATION Test solution (b). Dissolve 31.0 mg of the substance to be
First identification : A, D. examined in mobile phase A and dilute to 10.0 mL with
mobile phase A. Prepare immediately before use.
Second identification : B, C, D.
Reference solution (a). Dissolve 27.0 mg of anhydrous
A. Infrared absorption spectrophotometry (2.2.24). ampicillin CRS in mobile phase A and dilute to 50.0 mL with
Preparation : dissolve 0.250 g in 5 mL of water R, add mobile phase A.
0.5 mL of dilute acetic acid R, swirl and allow to stand for Reference solution (b). Dissolve 2.0 mg of cefradine CRS in
10 min in iced water. Filter the crystals through a small mobile phase A and dilute to 50 mL with mobile phase A. To
sintered-glass filter (40) (2.1.2), applying suction, wash with 5.0 mL of this solution add 5.0 mL of reference solution (a).
2-3 mL of a mixture of 1 volume of water R and 9 volumes
Reference solution (c). Dilute 1.0 mL of reference solution (a)
of acetone R, then dry in an oven at 60 °C for 30 min.
to 20.0 mL with mobile phase A.
Comparison : ampicillin trihydrate CRS. Reference solution (d). To 0.20 g of the substance to be
B. Thin-layer chromatography (2.2.27). examined add 1.0 mL of water R. Heat the solution at 60 °C
Test solution. Dissolve 25 mg of the substance to be for 1 h. Dilute 0.5 mL of this solution to 50.0 mL with mobile
examined in 10 mL of sodium hydrogen carbonate phase A.
solution R. Column :
Reference solution (a). Dissolve 25 mg of ampicillin – size : l = 0.25 m, Ø = 4.6 mm ;
trihydrate CRS in 10 mL of sodium hydrogen carbonate – stationary phase : octadecylsilyl silica gel for
solution R. chromatography R (5 μm).
Reference solution (b). Dissolve 25 mg of amoxicillin Mobile phase :
trihydrate CRS and 25 mg of ampicillin trihydrate CRS in – mobile phase A : mix 0.5 mL of dilute acetic acid R, 50 mL
10 mL of sodium hydrogen carbonate solution R. of 0.2 M potassium dihydrogen phosphate R and 50 mL of
Plate : TLC silanised silica gel plate R. acetonitrile R, then dilute to 1000 mL with water R ;
Mobile phase : mix 10 volumes of acetone R and 90 volumes – mobile phase B : mix 0.5 mL of dilute acetic acid R, 50 mL
of a 154 g/L solution of ammonium acetate R previously of 0.2 M potassium dihydrogen phosphate R and 400 mL of
adjusted to pH 5.0 with glacial acetic acid R. acetonitrile R, then dilute to 1000 mL with water R ;
Application : 1 μL. Time Mobile phase A Mobile phase B
Development : over a path of 15 cm. (min) (per cent V/V) (per cent V/V)
Drying : in air. 0 - tR 85 15
Detection : expose to iodine vapour until the spots appear tR - (tR + 30) 85 → 0 15 → 100
and examine in daylight.
(tR + 30) - (tR + 45) 0 100
System suitability: reference solution (b) :
– the chromatogram shows 2 clearly separated spots. (tR + 45) - (tR + 60) 85 15

Results : the principal spot in the chromatogram obtained tR = retention time of ampicillin determined with reference solution (c)
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with If the mobile phase composition has been adjusted to achieve
reference solution (a). the required resolution, the adjusted composition will apply at
C. Place about 2 mg in a test-tube about 150 mm long and time zero in the gradient and in the assay.
about 15 mm in diameter. Moisten with 0.05 mL of water R Flow rate : 1.0 mL/min.
and add 2 mL of sulfuric acid-formaldehyde reagent R. Detection : spectrophotometer at 254 nm.

1564 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ampicillin sodium

Injection : 50 μL of reference solutions (b) and (c) with ASSAY

isocratic elution at the initial mobile phase composition and Liquid chromatography (2.2.29) as described in the test for
50 μL of test solution (b) and reference solution (d) according related substances with the following modifications.
to the elution gradient described under Mobile phase ; inject
Mobile phase : initial composition of the mixture of mobile
mobile phase A as a blank according to the elution gradient
phases A and B, adjusted where applicable.
described under Mobile phase.
Injection : test solution (a) and reference solution (a).
Identification of peaks : use the chromatogram obtained with
reference solution (d) to identify the peaks due to ampicillin System suitability : reference solution (a) :
and ampicillin dimer. – repeatability : maximum relative standard deviation of
Relative retention with reference to ampicillin : ampicillin 1.0 per cent after 6 injections.
dimer = about 2.8. Calculate the percentage content of ampicillin sodium by
System suitability : reference solution (b) : multiplying the percentage content of ampicillin by 1.063.
– resolution : minimum 3.0 between the peaks due to STORAGE
ampicillin and cefradin ; if necessary adjust the ratio A:B In an airtight container. If the substance is sterile, store in a
of the mobile phase. sterile, airtight, tamper-proof container.
Limits :
IMPURITIES
– ampicillin dimer : not more than 4.5 times the area of
the principal peak in the chromatogram obtained with
reference solution (c) (4.5 per cent) ;
– any other impurity : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (c) (2 per cent).
A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-
N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm. 1-azabicyclo[3.2.0]heptane-2-carboxylic acid
2-Ethylhexanoic acid (2.4.28) : maximum 0.8 per cent m/m. (6-aminopenicillanic acid),
Methylene chloride. Gas chromatography (2.2.28).
Internal standard solution. Dissolve 1.0 mL of ethylene
chloride R in water R and dilute to 500.0 mL with the same
solvent.
Test solution (a). Dissolve 1.0 g of the substance to be
examined in water R and dilute to 10.0 mL with the same
B. (2S,5R,6R)-6-[[(2S)-2-amino-2-phenylacetyl]amino]-
solvent.
3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-
Test solution (b). Dissolve 1.0 g of the substance to be carboxylic acid (L-ampicillin),
examined in water R, add 1.0 mL of the internal standard
solution and dilute to 10.0 mL with water R.
Reference solution. Dissolve 1.0 mL of methylene chloride R
in water R and dilute to 500.0 mL with the same solvent. To
1.0 mL of this solution add 1.0 mL of the internal standard
solution and dilute to 10.0 mL with water R.
Column :
– material : glass ;
C. (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5-
– size : l = 1.5 m, Ø = 4 mm ; dimethylthiazolidine-4-carboxylic acid (diketopiperazines
– stationary phase : diatomaceous earth for gas of ampicillin),
chromatography R impregnated with 10 per cent m/m of
macrogol 1000 R.
Carrier gas : nitrogen for chromatography R.
Flow rate : 40 mL/min.
Temperature :
– column : 60 °C ;
D. R = CO2H : (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]-
– injection port : 100 °C ; carboxymethyl]-5,5-dimethylthiazolidine-4-carboxylic acid
– detector : 150 °C. (penicilloic acids of ampicillin),
Detection : flame ionisation. F. R = H : (2RS,4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]-
Calculate the content of methylene chloride taking its density methyl]-5,5-dimethylthiazolidine-4-carboxylic acid
at 20 °C to be 1.325 g/mL. (penilloic acids of ampicillin),
Limit :
– methylene chloride : maximum 0.2 per cent m/m.
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test C. Prepare the reference solution using
2 mL of lead standard solution (10 ppm Pb) R.
Water (2.5.12) : maximum 2.0 per cent, determined on 0.300 g.
Bacterial endotoxins (2.6.14) : less than 0.15 IU/mg, if E. (2R)-2-[[[(2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]-
intended for use in the manufacture of parenteral preparations amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo-
without a further appropriate procedure for the removal of [3.2.0]hept-2-yl]carbonyl]amino]-2-phenylacetic acid
bacterial endotoxins. (ampicillinyl-D-phenylglycine),

General Notices (1) apply to all monographs and other texts 1565
Ampicillin trihydrate EUROPEAN PHARMACOPOEIA 8.0

G. (3R,6R)-3,6-diphenylpiperazine-2,5-dione,
N. oligomers of penicilloic acids of ampicillin.

01/2008:0168
corrected 6.0

AMPICILLIN TRIHYDRATE
H. 3-phenylpyrazin-2-ol,
Ampicillinum trihydricum

C16H19N3O4S,3H2O Mr 403.5
I. (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-phenylacetyl]- [7177-48-2]
amino]-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylic acid DEFINITION
(D-phenylglycylampicillin), (2S,5R,6R)-6-[[(2R)-2-Amino-2-phenylacetyl]amino]-
3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-
carboxylic acid trihydrate.
Semi-synthetic product derived from a fermentation product.
Content : 96.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
J. (2S,5R,6R)-6-[(2,2-dimethylpropanoyl)amino]-3,3- Solubility : slightly soluble in water, practically insoluble in
dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2- ethanol (96 per cent) and in fatty oils. It dissolves in dilute
carboxylic acid, solutions of acids and of alkali hydroxides.
IDENTIFICATION
First identification : A, D.
Second identification : B, C, D.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: ampicillin trihydrate CRS.
B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 25 mg of the substance to be
K. (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-phenylacetic examined in 10 mL of sodium hydrogen carbonate
acid, solution R.
Reference solution (a). Dissolve 25 mg of ampicillin
trihydrate CRS in 10 mL of sodium hydrogen carbonate
solution R.
Reference solution (b). Dissolve 25 mg of amoxicillin
trihydrate CRS and 25 mg of ampicillin trihydrate CRS in
L. (2R)-2-amino-2-phenylacetic acid (D-phenylglycine), 10 mL of sodium hydrogen carbonate solution R.
Plate : TLC silanised silica gel plate R.
Mobile phase : mix 10 volumes of acetone R and 90 volumes
of a 154 g/L solution of ammonium acetate R previously
adjusted to pH 5.0 with glacial acetic acid R.
Application : 1 μL.
Development : over a path of 15 cm.
Drying : in air.
Detection : expose to iodine vapour until the spots appear
and examine in daylight.
System suitability : reference solution (b) :
– the chromatogram shows 2 clearly separated spots.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
M. co-oligomers of ampicillin and of penicilloic acids of to the principal spot in the chromatogram obtained with
ampicillin, reference solution (a).

1566 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ampicillin trihydrate

C. Place about 2 mg in a test-tube about 150 mm long and Injection : 50 μL of reference solutions (b) and (c) with
about 15 mm in diameter. Moisten with 0.05 mL of water R isocratic elution at the initial mobile phase composition and
and add 2 mL of sulfuric acid-formaldehyde reagent R. 50 μL of test solution (b) according to the elution gradient
Mix the contents of the tube by swirling ; the solution is described under Mobile phase ; inject mobile phase A as
practically colourless. Place the test-tube in a water-bath a blank according to the elution gradient described under
for 1 min ; a dark yellow colour develops. Mobile phase.
D. Water (see Tests). System suitability : reference solution (b) :
– resolution : minimum 3.0 between the peaks due to
TESTS ampicillin and cefradin ; if necessary, adjust the ratio A:B
Appearance of solution. The solutions are not more of the mobile phase.
opalescent than reference suspension II (2.2.1). Limit :
Dissolve 1.0 g in 10 mL of 1 M hydrochloric acid. Separately – any impurity : for each impurity, not more than the area
dissolve 1.0 g in 10 mL of dilute ammonia R2. Examine of the principal peak in the chromatogram obtained with
immediately after dissolution. reference solution (c) (1.0 per cent).
pH (2.2.3) : 3.5 to 5.5. N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm.
Dissolve 0.1 g in carbon dioxide-free water R and dilute to Water (2.5.12) : 12.0 per cent to 15.0 per cent, determined on
40 mL with the same solvent. 0.100 g.
Specific optical rotation (2.2.7) : + 280 to + 305 (anhydrous Sulfated ash (2.4.14) : maximum 0.5 per cent, determined on
substance). 1.0 g.
Dissolve 62.5 mg in water R and dilute to 25.0 mL with the ASSAY
same solvent. Liquid chromatography (2.2.29) as described in the test for
Related substances. Liquid chromatography (2.2.29). related substances with the following modifications.
Test solution (a). Dissolve 31.0 mg of the substance to be Mobile phase : initial composition of the mixture of mobile
examined in mobile phase A and dilute to 50.0 mL with phases A and B, adjusted where applicable.
mobile phase A. Injection : test solution (a) and reference solution (a).
Test solution (b). Dissolve 31.0 mg of the substance to be System suitability : reference solution (a) :
examined in mobile phase A and dilute to 10.0 mL with
– repeatability : maximum relative standard deviation of
mobile phase A. Prepare immediately before use.
1.0 per cent after 6 injections.
Reference solution (a). Dissolve 27.0 mg of anhydrous Calculate the percentage content of ampicillin from the
ampicillin CRS in mobile phase A and dilute to 50.0 mL with declared content of anhydrous ampicillin CRS.
mobile phase A.
Reference solution (b). Dissolve 2 mg of cefradine CRS in STORAGE
mobile phase A and dilute to 50 mL with mobile phase A. In an airtight container.
To 5 mL of this solution, add 5 mL of reference solution (a).
Reference solution (c). Dilute 1.0 mL of reference solution (a) IMPURITIES
to 20.0 mL with mobile phase A.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-
Mobile phase : 1-azabicyclo[3.2.0]heptane-2-carboxylic acid
– mobile phase A : mix 0.5 mL of dilute acetic acid R, 50 mL (6-aminopenicillanic acid),
of 0.2 M potassium dihydrogen phosphate R and 50 mL of
acetonitrile R, then dilute to 1000 mL with water R ;
– mobile phase B : mix 0.5 mL of dilute acetic acid R, 50 mL
of 0.2 M potassium dihydrogen phosphate R and 400 mL of
acetonitrile R, then dilute to 1000 mL with water R ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V) B. (2S,5R,6R)-6-[[(2S)-2-amino-2-phenylacetyl]amino]-
0 - tR 85 15 3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-
carboxylic acid (L-ampicillin),
tR - (tR + 30) 85 → 0 15 → 100

(tR + 30) - (tR + 45) 0 100

(tR + 45) - (tR + 60) 85 15

tR = retention time of ampicillin determined with reference solution (c)

If the mobile phase composition has been adjusted to achieve

the required resolution, the adjusted composition will apply at
time zero in the gradient and in the assay.
C. (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5-
Flow rate : 1.0 mL/min.
dimethylthiazolidine-4-carboxylic acid (diketopiperazines
Detection : spectrophotometer at 254 nm. of ampicillin),

General Notices (1) apply to all monographs and other texts 1567
Amylmetacresol EUROPEAN PHARMACOPOEIA 8.0

L. (2R)-2-amino-2-phenylacetic acid (D-phenylglycine),

D. R = CO2H : (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]-
carboxymethyl]-5,5-dimethylthiazolidine-4-carboxylic acid
(penicilloic acids of ampicillin),

F. R = H : (2RS,4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]-
methyl]-5,5-dimethylthiazolidine-4-carboxylic acid
(penilloic acids of ampicillin),

M. co-oligomers of ampicillin and of penicilloic acids of

ampicillin,

E. (2R)-2-[[[(2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]-
amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo-
[3.2.0]hept-2-yl]carbonyl]amino]-2-phenylacetic acid
(ampicillinyl-D-phenylglycine),

N. (3S)-6-[[(2R)-2-amino-2-phenylacetyl]amino]-2,2-
dimethyl-7-oxo-2,3,4,7-tetrahydro-1,4-thiazepine-3-
carboxylic acid.

01/2011:2405
G. (3R,6R)-3,6-diphenylpiperazine-2,5-dione,
AMYLMETACRESOL
Amylmetacresolum

H. 3-phenylpyrazin-2-ol,
C12H18O Mr 178.3
[1300-94-3]
DEFINITION
5-Methyl-2-pentylphenol.
Content : 98.0 per cent to 102.0 per cent.
CHARACTERS
I. (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-phenylacetyl]- Appearance : clear or almost clear liquid, or solid crystalline
amino]-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo- mass, colourless or slightly yellow when freshly prepared. The
4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid substance changes colour during storage by darkening and/or
(D-phenylglycylampicillin), discolouration to dark yellow, brownish-yellow or pink.
Solubility : practically insoluble in water, very soluble in
acetone and in ethanol (96 per cent).
It solidifies at about 22 °C.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
J. (2S,5R,6R)-6-[(2,2-dimethylpropanoyl)amino]-3,3- Preparation : film between 2 plates of potassium bromide R.
dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2- Comparison: amylmetacresol CRS.
carboxylic acid,
TESTS
Related substances. Gas chromatography (2.2.28) : use the
normalisation procedure.
Internal standard solution. Dissolve 0.100 g of
butylhydroxytoluene R in 2-propanol R and dilute to 10.0 mL
with the same solvent.
Test solution (a). Dissolve 0.1000 g of the substance to be
K. (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-phenylacetic examined in 2-propanol R and dilute to 10.0 mL with the
acid, same solvent.

1568 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Amylmetacresol

Test solution (b). To 2.0 mL of test solution (a) add 2.0 mL STORAGE
of the internal standard solution and dilute to 10.0 mL with In an airtight, non-metallic container, protected from light.
2-propanol R.
Reference solution (a). Dissolve 10 mg of m-cresol R IMPURITIES
(impurity B) and 10 mg of p-cresol R (impurity D) in Specified impurities : A, G, K.
2-propanol R and dilute to 100.0 mL with the same solvent. Other detectable impurities (the following substances would,
Reference solution (b). Dissolve the contents of a vial of if present at a sufficient level, be detected by one or other of
amylmetacresol for peak identification CRS (containing the tests in the monograph. They are limited by the general
impurities A, G and K) in 1.0 ml of 2-propanol R. acceptance criterion for other/unspecified impurities and/or
Reference solution (c). Dissolve 0.1000 g of amylmetacresol CRS by the general monograph Substances for pharmaceutical
in 2-propanol R and dilute to 10.0 mL with the same solvent. use (2034). It is therefore not necessary to identify these
To 2.0 mL of this solution add 2.0 mL of the internal standard impurities for demonstration of compliance. See also 5.10.
solution and dilute to 10.0 mL with 2-propanol R. Control of impurities in substances for pharmaceutical use) : B,
Reference solution (d). Dilute 1.0 mL of test solution (a) to C, D, E, F, H, I, J.
100.0 mL with 2-propanol R. Dilute 1.0 mL of this solution to
20.0 mL with 2-propanol R.
Column :
– material : fused silica ;
– size : l = 30 m, Ø = 0.25 mm ; A. 4-methyl-2-pentylphenol,
– stationary phase : macrogol 20 000 R (film thickness 0.5 μm).
Carrier gas : helium for chromatography R.
Linear velocity : 33 cm/s.
Split ratio : 1:30. B. 3-methylphenol (m-cresol),
Temperature :
Time Temperature
(min) (°C)
Column 0 - 17.5 100 → 240
C. 5-methyl-2-[(2RS)-2-methylbutyl]phenol,
17.5 - 32.5 240

Injection port 250

Detector 250

Detection : flame ionisation. D. 4-methylphenol (p-cresol),

Injection : 1.0 μL of test solution (a) and reference solutions (a),
(b) and (d).
Identification of impurities : use the chromatogram supplied
with amylmetacresol for peak identification CRS and the
chromatogram obtained with reference solution (b) to identify
the peaks due to impurities A, G and K. E. 1-(2-hydroxy-4-methylphenyl)pentan-1-one,
Relative retention with reference to amylmetacresol (retention
time = about 16 min) : impurity G (diastereoisomer 1) = about
0.51 ; impurity G (diastereoisomer 2) = about 0.53 ;
impurity D = about 0.77 ; impurity B = about 0.78 ;
impurity K = about 0.95 ; impurity A = about 0.99.
System suitability: reference solution (a) : F. 1-(2-hydroxy-5-methylphenyl)pentan-1-one,
– resolution : minimum 1.5 between the peaks due to
impurities D and B.
Limits :
– impurity A : maximum 0.6 per cent ;
– impurities G (sum of the 2 diastereoisomers), K : for each G. 5-methyl-2-pentylcyclohexanone,
impurity, maximum 0.15 per cent ;
– unspecified impurities : for each impurity, maximum
0.10 per cent ;
– total : maximum 1.0 per cent ; H. ethyl pentanoate,
– disregard limit : the area of the peak due to amylmetacresol
in the chromatogram obtained with reference solution (d)
(0.05 per cent).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. I. 3-methylphenyl pentanoate,
ASSAY
Gas chromatography (2.2.28) as described in the test for
related substances with the following modification.
Injection : 1.0 μL of test solution (b) and reference solution (c).
J. 4-methylphenyl pentanoate,
Calculate the percentage content of C12H18O from the declared
content of amylmetacresol CRS. K. unknown structure.

General Notices (1) apply to all monographs and other texts 1569
Anastrozole EUROPEAN PHARMACOPOEIA 8.0

04/2013:2406 Time Mobile phase A Mobile phase B

(min) (per cent V/V) (per cent V/V)

ANASTROZOLE 0-2 95 5

2 - 54 95 → 35 5 → 65

Anastrozolum Flow rate : 1.0 mL/min.

Detection : spectrophotometer at 215 nm.
Injection : 20 μL of test solution (a) and reference solutions (a)
and (b).
Identification of impurities: use the chromatogram obtained
with reference solution (b) to identify the peak due to
impurity E.
Relative retention with reference to anastrozole (retention
C17H19N5 Mr 293.4 time = about 29 min) : impurity E = about 1.05.
[120511-73-1] System suitability : reference solution (b) :
– resolution : minimum 3.5 between the peaks due to
DEFINITION anastrozole and impurity E.
2,2′-[5-(1H-1,2,4-Triazol-1-ylmethyl)benzene-1,3-diyl]bis(2- Calculation of percentage contents :
methylpropanenitrile). – for each impurity, use the concentration of anastrozole in
Content : 98.0 per cent to 102.0 per cent (anhydrous substance). reference solution (a).
Limits :
CHARACTERS – unspecified impurities : for each impurity, maximum
Appearance : white or almost white powder. 0.10 per cent ;
Solubility : very slightly soluble in water, freely soluble in – total : maximum 0.2 per cent ;
anhydrous ethanol, practically insoluble in cyclohexane. – reporting threshold : 0.05 per cent.
It shows polymorphism (5.9). Water (2.5.32) : maximum 0.3 per cent, determined on
50.0 mg.
IDENTIFICATION Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Infrared absorption spectrophotometry (2.2.24). 1.0 g.
Comparison : anastrozole CRS. ASSAY
If the spectra obtained in the solid state show differences, Liquid chromatography (2.2.29) as described in the test for
dissolve the substance to be examined and the reference related substances with the following modification.
substance separately in acetone R, evaporate to dryness and Injection : test solution (b) and reference solution (c).
record new spectra using the residues.
Calculate the percentage content of C17H19N5 taking into
account the assigned content of anastrozole CRS.
TESTS
Related substances. Liquid chromatography (2.2.29). IMPURITIES
Solvent mixture : acetonitrile R1, water for chromatography R Other detectable impurities (the following substances would,
(50:50 V/V). if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
Test solution (a). Dissolve 25 mg of the substance to be acceptance criterion for other/unspecified impurities and/or
examined in the solvent mixture and dilute to 100.0 mL with by the general monograph Substances for pharmaceutical
the solvent mixture. use (2034). It is therefore not necessary to identify these
Test solution (b). Dissolve 25.0 mg of the substance to be impurities for demonstration of compliance. See also 5.10.
examined in the solvent mixture and dilute to 200.0 mL with Control of impurities in substances for pharmaceutical use):
the solvent mixture. A, B, C, D, E, F, G, H, I.
Reference solution (a). Dilute 1.0 mL of test solution (a) to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Reference solution (b). Dissolve 2.5 mg of anastrozole
impurity E CRS in 20.0 mL of the solvent mixture. Dilute
1.0 mL of the solution to 50.0 mL with test solution (a).
Reference solution (c). Dissolve 25.0 mg of anastrozole CRS in A. 2-[3-[(1RS)-1-cyanoethyl]-5-(1H-1,2,4-triazol-1-
the solvent mixture and dilute to 200.0 mL with the solvent ylmethyl)phenyl]-2-methylpropanenitrile,
mixture.
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : end-capped polar-embedded octadecylsilyl
amorphous organosilica polymer R (3.5 μm).
Mobile phase :
– mobile phase A : phosphoric acid R, water for
chromatography R (0.1 :100 V/V) ;
– mobile phase B : phosphoric acid R, acetonitrile R1 B. (2RS)-2,3-bis[3-(1-cyano-1-methylethyl)-5-(1H-1,2,4-
(0.1 :100 V/V) ; triazol-1-ylmethyl)phenyl]-2-methylpropanenitrile,

1570 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Antazoline hydrochloride

01/2008:0972
corrected 6.0

ANTAZOLINE HYDROCHLORIDE
Antazolini hydrochloridum
C. 2,2′-[5-(bromomethyl)benzene-1,3-diyl]bis(2-
methylpropanenitrile),

C17H20ClN3 Mr 301.8
[2508-72-7]
DEFINITION
D. 2,2′-[5-(dibromomethyl)benzene-1,3-diyl]bis(2- Antazoline hydrochloride contains not less than 99.0 per
methylpropanenitrile), cent and not more than the equivalent of 101.0 per cent of
N-benzyl-N-[(4,5-dihydro-1H-imidazol-2-yl)methyl]aniline
hydrochloride, calculated with reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble
in water, soluble in alcohol, slightly soluble in methylene
chloride.
It melts at about 240 °C, with decomposition.
E. 2,2′-[5-(hydroxymethyl)benzene-1,3-diyl]bis(2-
methylpropanenitrile), IDENTIFICATION
First identification : A, D.
Second identification : B, C, D.
A. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
antazoline hydrochloride CRS. Examine the substances as
discs prepared using potassium chloride R.
F. 4-methylbenzenesulfonic acid,
B. Examine the chromatograms obtained in the test for related
substances in daylight after spraying. The principal spot in
the chromatogram obtained with test solution (b) is similar
in position, colour and size to the principal spot in the
chromatogram obtained with reference solution (b).
C. To 5 mL of solution S (see Tests) add, drop by drop, dilute
sodium hydroxide solution R until an alkaline reaction
is produced. Filter. The precipitate, washed with two
quantities, each of 10 mL, of water R and dried in a
G. 2,2′-[5-(4H-1,2,4-triazol-4-ylmethyl)benzene-1,3- desiccator under reduced pressure, melts (2.2.14) at 119 °C
diyl]bis(2-methylpropanenitrile), to 123 °C.
D. It gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S. Dissolve 2.0 g in carbon dioxide-free water R
prepared from distilled water R, heating at 60 °C if necessary.
Allow to cool and dilute to 100 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y7 (2.2.2,
Method II).
H. 2,2′-(5-methylbenzene-1,3-diyl)bis(2-methylpropaneni- Acidity or alkalinity. To 10 mL of solution S add 0.2 mL
trile), of methyl red solution R. Not more than 0.1 mL of 0.01 M
hydrochloric acid or 0.01 M sodium hydroxide is required to
change the colour of the indicator.
Related substances. Examine by thin-layer chromatography
(2.2.27), using silica gel GF254 R as the coating substance. Heat
the plate at 110 °C for 15 min before using.
Test solution (a). Dissolve 0.10 g of the substance to be
examined in methanol R and dilute to 5 mL with the same
solvent.
I. 2,2′-[5-(chloromethyl)benzene-1,3-diyl]bis(2- Test solution (b). Dilute 1 mL of test solution (a) to 5 mL with
methylpropanenitrile). methanol R.

General Notices (1) apply to all monographs and other texts 1571
Anticoagulant and preservative solutions for human blood EUROPEAN PHARMACOPOEIA 8.0

Reference solution (a). Dilute 0.5 mL of test solution (a) to below. Subject to agreement by the competent authority, other
100 mL with methanol R. substances, such as red-cell preservatives, may be included in
Reference solution (b). Dissolve 20 mg of antazoline the formula provided that their name and concentration are
hydrochloride CRS in methanol R and dilute to 5 mL with the stated on the label.
same solvent. Anticoagulant and preservative solutions for human blood are
Reference solution (c). Dissolve 20 mg of xylometazoline presented in airtight, tamper-proof containers of glass (3.2.1)
hydrochloride CRS in 1 mL of test solution (a) and dilute to or plastic (3.2.3).
5 mL with methanol R.
Apply to the plate 5 μL of each solution. Develop over a path Anticoagulant acid-citrate-glucose solutions
of 15 cm using a mixture of 5 volumes of diethylamine R, (ACD)
10 volumes of methanol R and 85 volumes of ethyl acetate R.
Dry the plate in a current of warm air for 15 min. Examine A B
in ultraviolet light at 254 nm. The test is not valid unless the
Sodium citrate (0412) 22.0 g 13.2 g
chromatogram obtained with reference solution (c) shows
two clearly separated principal spots. Spray with a mixture Citric acid monohydrate (0456) 8.0 g 4.8 g
of equal volumes of a 200 g/L solution of ferric chloride R
or Citric acid, anhydrous (0455) 7.3 g 4.4 g
and a 5 g/L solution of potassium ferricyanide R. Examine
immediately in daylight. Any spot in the chromatogram Glucose monohydrate (0178)* 24.5 g 14.7 g
obtained with test solution (a), apart from the principal
or Glucose, anhydrous (0177)* 22.3 g 13.4 g
spot, is not more intense than the spot in the chromatogram
obtained with reference solution (a) (0.5 per cent). Water for injections (0169) to 1000.0 mL 1000.0 mL
Heavy metals (2.4.8). 1.0 g complies with test C for heavy Volume to be used per 100 mL of blood 15.0 mL 25.0 mL
metals (20 ppm). Prepare the reference solution using 2 mL
of lead standard solution (10 ppm Pb) R. *The competent authority may require that the substances comply
with the test for pyrogens given in the monographs on Glucose
Loss on drying (2.2.32). Not more than 0.5 per cent, monohydrate (0178) and Glucose, anhydrous (0177), respectively.
determined on 1.000 g by drying in an oven at 105 °C for 3 h.
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined CHARACTERS
on the residue obtained in the test for loss on drying. A colourless or faintly yellow, clear liquid, practically free
from particles.
ASSAY
Dissolve 0.250 g in 100 mL of alcohol R. Add 0.1 mL of IDENTIFICATION
phenolphthalein solution R1. Titrate with 0.1 M alcoholic A. Examine by thin-layer chromatography (2.2.27), using
potassium hydroxide. silica gel G R as the coating substance.
1 mL of 0.1 M alcoholic potassium hydroxide is equivalent to Test solution. Dilute 2 mL of the solution to be examined
30.18 mg of C17H20ClN3. (for formula A) or 3 mL (for formula B) to 100 mL with
a mixture of 2 volumes of water R and 3 volumes of
IMPURITIES methanol R.
Reference solution (a). Dissolve 10 mg of glucose CRS
in a mixture of 2 volumes of water R and 3 volumes of
methanol R and dilute to 20 mL with the same mixture of
solvents.
Reference solution (b). Dissolve 10 mg each of glucose CRS,
lactose CRS, fructose CRS and sucrose CRS in a mixture of
A. N-(2-aminoethyl)-2-(benzylphenylamino)acetamide. 2 volumes of water R and 3 volumes of methanol R and
dilute to 20 mL with the same mixture of solvents.
Apply separately to the plate 2 μL of each solution and
thoroughly dry the points of application. Develop over a
01/2008:0209 path of 15 cm using a mixture of 10 volumes of water R,
15 volumes of methanol R, 25 volumes of anhydrous acetic
ANTICOAGULANT AND acid R and 50 volumes of ethylene chloride R. The volumes
of solvents have to be measured accurately since a slight
PRESERVATIVE SOLUTIONS FOR excess of water produces cloudiness. Dry the plate in a
HUMAN BLOOD current of warm air. Repeat the development immediately,
after renewing the mobile phase. Dry the plate in a current
of warm air and spray evenly with a solution of 0.5 g of
Solutiones anticoagulantes et sanguinem thymol R in a mixture of 5 mL of sulfuric acid R and 95 mL
humanum conservantes of alcohol R. Heat at 130 °C for 10 min. The principal spot
in the chromatogram obtained with the test solution is
DEFINITION similar in position, colour and size to the principal spot in
Anticoagulant and preservative solutions for human blood the chromatogram obtained with reference solution (a).
are sterile and pyrogen-free solutions prepared with water The test is not valid unless the chromatogram obtained
for injections, filtered, distributed in the final containers and with reference solution (b) shows 4 clearly separated spots.
sterilised. The content of sodium citrate (C6H5Na3O7,2H2O), B. To 2 mL add 5 mL of cupri-citric solution R. Heat to boiling.
glucose monohydrate (C6H12O6,H2O) or anhydrous glucose An orange precipitate is formed and the solution becomes
(C6H12O6) and sodium dihydrogen phosphate dihydrate yellow.
(NaH2PO4,2H2O) is not less than 95.0 per cent and not more
than 105.0 per cent of that stated in the formulae below. C. To 2 mL (for formula A) add 3 mL of water R or to 4 mL
The content of citric acid monohydrate (C6H8O7,H2O) or (for formula B) add 1 mL of water R. The solution gives the
anhydrous citric acid (C6H8O7) is not less than 90.0 per cent reaction of citrates (2.3.1).
and not more than 110.0 per cent of that stated in the formulae D. 0.5 mL gives reaction (b) of sodium (2.3.1).

1572 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Anticoagulant and preservative solutions for human blood

TESTS sodium thiosulfate using 0.5 mL of starch solution R, added

pH (2.2.3). The pH of the solution to be examined is 4.7 to 5.3. towards the end of the titration, as indicator (n1 mL). Carry
out a blank titration using 25.0 mL of water R (n2 mL).
Hydroxymethylfurfural. To 2.0 mL add 5.0 mL of a 100 g/L
Calculate the content of reducing sugars as anhydrous glucose
solution of p-toluidine R in 2-propanol R containing 10 per
or as glucose monohydrate, as appropriate, from Table 0209.-1.
cent V/V of glacial acetic acid R and 1.0 mL of a 5 g/L solution
of barbituric acid R. The absorbance (2.2.25), determined Table 0209.-1
at 550 nm after allowing the mixture to stand for 2 min to Volume of 0.1 M Anhydrous glucose Glucose mono-
3 min, is not greater than that of a standard prepared at the sodium thiosulfate in milligrams hydrate in
same time in the same manner using 2.0 mL of a solution (n2− n1 mL) milligrams
containing 5 ppm of hydroxymethylfurfural R for formula A
or 3 ppm of hydroxymethylfurfural R for formula B. 8 19.8 21.6

Sterility (2.6.1). They comply with the test for sterility. 9 22.4 24.5

Pyrogens (2.6.8). They comply with the test for pyrogens. 10 25.0 27.2
Dilute with a pyrogen-free, 9 g/L solution of sodium chloride R 11 27.6 30.2
to obtain a solution containing approximately 5 g/L of sodium
citrate. Inject 10 mL of the diluted solution per kilogram of 12 30.3 33.1
the rabbit’s mass. 13 33.0 36.1
ASSAY 14 35.7 39.0
Citric acid. To 10.0 mL (for formula A) or to 20.0 mL (for 15 38.3 42.1
formula B) add 0.1 mL of phenolphthalein solution R1. Titrate
with 0.2 M sodium hydroxide until a pink colour is obtained. 16 41.3 45.2
1 mL of 0.2 M sodium hydroxide is equivalent to 14.01 mg of
C6H8O7,H2O or to 12.81 mg of C6H8O7. STORAGE
Sodium citrate. Prepare a chromatography column 0.10 m Store in an airtight, tamper-proof container, protected from
long and 10 mm in internal diameter and filled with strongly light.
acidic ion-exchange resin R (300 μm to 840 μm). Maintain a LABELLING
1 cm layer of liquid above the resin at all times. Wash the
column with 50 mL of de-ionised water R at a flow rate of The label states :
12-14 mL/min. – the composition and volume of the solution,
Dilute 10.0 mL of the solution to be examined (for formula A) – the maximum amount of blood to be collected in the
or 15.0 mL (for formula B) to about 40 mL with de-ionised container.
water R in a beaker and transfer to the column reservoir,
washing the beaker 3 times with a few millilitres of de-ionised Anticoagulant citrate-phosphate-glucose
water R. Allow the solution to run through the column at
a flow rate of 12-14 mL/min and collect the eluate. Wash solution (CPD)
the column with 2 quantities, each of 30 mL, and with one Sodium citrate (0412) 26.3 g
quantity of 50 mL, of de-ionised water R. The column can be
used for 3 successive determinations before regeneration with Citric acid monohydrate (0456) 3.27 g
3 times its volume of dilute hydrochloric acid R. Titrate the or Citric acid, anhydrous (0455) 2.99 g
combined eluate and washings (about 150 mL) with 0.2 M
sodium hydroxide, using 0.1 mL of phenolphthalein solution R1 Glucose monohydrate (0178)* 25.5 g
as indicator. or Glucose, anhydrous (0177)* 23.2 g
Calculate the content of sodium citrate in grams per litre from Sodium dihydrogen phosphate dihydrate (0194) 2.51 g
the following expressions :
Water for injections (0169) to 1000.0 mL
For formula A :
Volume to be used per 100 mL of blood 14.0 mL
or *The competent authority may require that the substances comply
with the test for pyrogens given in the monographs on Glucose
monohydrate (0178) and Glucose, anhydrous (0177), respectively.
For formula B :
or CHARACTERS
A colourless or faintly yellow, clear liquid, practically free
n = number of millilitres of 0.2 M sodium hydroxide from particles.
used in the titration, IDENTIFICATION
C = content of citric acid monohydrate in grams per A. Examine by thin-layer chromatography (2.2.27), using
litre determined as prescribed above, silica gel G R as the coating substance.
C′ = content of anhydrous citric acid in grams per litre Test solution. Dilute 2 mL of the solution to be examined
determined as prescribed above. to 100 mL with a mixture of 2 volumes of water R and
Reducing sugars. Dilute 5.0 mL (for formula A) or 10.0 mL 3 volumes of methanol R.
(for formula B) to 100.0 mL with water R. Introduce 25.0 mL Reference solution (a). Dissolve 10 mg of glucose CRS
of the solution into a 250 mL conical flask with ground-glass in a mixture of 2 volumes of water R and 3 volumes of
neck and add 25.0 mL of cupri-citric solution R1. Add a few methanol R and dilute to 20 mL with the same mixture of
pieces of porous material, attach a reflux condenser, heat so solvents.
that boiling begins within 2 min and boil for exactly 10 min. Reference solution (b). Dissolve 10 mg each of glucose CRS,
Cool and add 3 g of potassium iodide R dissolved in 3 mL of lactose CRS, fructose CRS and sucrose CRS in a mixture of
water R. Add 25 mL of a 25 per cent m/m solution of sulfuric 2 volumes of water R and 3 volumes of methanol R and
acid R with caution and in small quantities. Titrate with 0.1 M dilute to 20 mL with the same mixture of solvents.

General Notices (1) apply to all monographs and other texts 1573
Anticoagulant and preservative solutions for human blood EUROPEAN PHARMACOPOEIA 8.0

Apply separately to the plate 2 μL of each solution and Citric acid. To 20.0 mL add 0.1 mL of phenolphthalein
thoroughly dry the starting points. Develop over a path of solution R1 and titrate with 0.2 M sodium hydroxide.
15 cm using a mixture of 10 volumes of water R, 15 volumes
of methanol R, 25 volumes of anhydrous acetic acid R and Calculate the content of citric acid monohydrate (C), or
50 volumes of ethylene chloride R. The volumes of solvents anhydrous citric acid (C′ ), in grams per litre from the
have to be measured accurately since a slight excess of equations :
water produces cloudiness. Dry the plate in a current
of warm air. Repeat the development immediately, after
renewing the mobile phase. Dry the plate in a current
of warm air and spray evenly with a solution of 0.5 g of
thymol R in a mixture of 5 mL of sulfuric acid R and 95 mL
of alcohol R. Heat at 130 °C for 10 min. The principal spot n = number of millilitres of 0.2 M sodium hydroxide
in the chromatogram obtained with the test solution is used in the titration,
similar in position, colour and size to the principal spot in P = content of sodium dihydrogen phosphate dihydrate
the chromatogram obtained with reference solution (a). in grams per litre determined as prescribed above.
The test is not valid unless the chromatogram obtained
with reference solution (b) shows 4 clearly separated spots.Sodium citrate. Prepare a chromatography column 0.10 m
long and 10 mm in internal diameter and filled with strongly
B. To 2 mL add 5 mL of cupri-citric solution R. Heat to boiling. acidic ion-exchange resin R (300 μm to 840 μm). Maintain a
An orange precipitate is formed and the solution becomes 1 cm layer of liquid above the resin at all times. Wash the
yellow. column with 50 mL of de-ionised water R at a flow rate of
12-14 mL/min.
C. To 2 mL add 3 mL of water R. The solution gives the
reaction of citrates (2.3.1). Dilute 10.0 mL of the solution to be examined to about 40 mL
with de-ionised water R in a beaker and transfer to the column
D. 1 mL gives reaction (b) of phosphates (2.3.1). reservoir, washing the beaker 3 times with a few millilitres
of de-ionised water R. Allow the solution to run through the
E. 0.5 mL gives reaction (b) of sodium (2.3.1). column at a flow rate of 12-14 mL/min and collect the eluate.
Wash the column with 2 quantities, each of 30 mL, and with
one quantity of 50 mL, of de-ionised water R. The column can
TESTS be used for 3 successive determinations before regeneration
with 3 times its volume of dilute hydrochloric acid R. Titrate
pH (2.2.3). The pH of the solution is 5.3 to 5.9. the combined eluate and washings (about 150 mL) with 0.2 M
Hydroxymethylfurfural. To 2.0 mL add 5.0 mL of a 100 g/L sodium hydroxide, using 0.1 mL of phenolphthalein solution R1
solution of p-toluidine R in 2-propanol R containing 10 per as indicator.
cent V/V of glacial acetic acid R and 1.0 mL of a 5 g/L solution Calculate the content of sodium citrate in grams per litre from
of barbituric acid R. The absorbance (2.2.25), determined the following expressions :
at 550 nm after allowing the mixture to stand for 2 min to
3 min, is not greater than that of a standard prepared at the
same time in the same manner using 2.0 mL of a solution
containing 5 ppm of hydroxymethylfurfural R.
Sterility (2.6.1). They comply with the test for sterility.
Pyrogens (2.6.8). They comply with the test for pyrogens. n = number of millilitres of 0.2 M sodium hydroxide
Dilute with a pyrogen-free, 9 g/L solution of sodium chloride R used in the titration,
to obtain a solution containing approximately 5 g/L of sodium P = content of sodium dihydrogen phosphate
citrate. Inject 10 mL of the diluted solution per kilogram of dihydrate in grams per litre determined as
the rabbit’s mass. prescribed above,
C = content of citric acid monohydrate in grams per
litre determined as prescribed above,
ASSAY
C′ = content of anhydrous citric acid in grams per litre
Sodium dihydrogen phosphate. Dilute 10.0 mL to 100.0 mL determined as prescribed above.
with water R. To 10.0 mL of this solution add 10.0 mL of
nitro-molybdovanadic reagent R. Mix and allow to stand at Reducing sugars. Dilute 5.0 mL to 100.0 mL with water R.
20 °C to 25 °C for 30 min. At the same time and in the same Introduce 25.0 mL of the solution into a 250 mL conical
manner, prepare a reference solution using 10.0 mL of a flask with ground-glass neck and add 25.0 mL of cupri-citric
standard solution containing 0.219 g of potassium dihydrogen solution R1. Add a few pieces of porous material, attach a
phosphate R per litre. Measure the absorbance (2.2.25) of the reflux condenser, heat so that boiling begins within 2 min
2 solutions at 450 nm using as the compensation liquid a and boil for exactly 10 min. Cool and add 3 g of potassium
solution prepared in the same manner using 10 mL of water R. iodide R dissolved in 3 mL of water R. Add 25 mL of a 25 per
Calculate the content of sodium dihydrogen phosphate cent m/m solution of sulfuric acid R with caution and in small
dihydrate (P) in grams per litre from the expression : quantities. Titrate with 0.1 M sodium thiosulfate using 0.5 mL
of starch solution R, added towards the end of the titration, as
indicator (n1 mL). Carry out a blank titration using 25.0 mL of
water R (n2 mL).
Calculate the content of reducing sugars as anhydrous glucose
C = concentration of potassium dihydrogen or as glucose monohydrate, as appropriate, from Table 0209.-1.
phosphate R in the standard solution in grams per
litre,
STORAGE
A1 = absorbance of the test solution,
Store in an airtight, tamper-proof container, protected from
A2 = absorbance of the reference solution. light.

1574 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Anti-T lymphocyte immunoglobulin for human use, animal

LABELLING animals. The feed originates from a controlled source and

The label states : no animal proteins are added. The suppliers of animals are
– the composition and volume of the solution, certified by the competent authority.
– the maximum amount of blood to be collected in the If the animals are treated with antibiotics, a suitable withdrawal
container. period is allowed before collection of blood or plasma. The
animals are not treated with penicillin antibiotics. If a live
07/2013:1928 vaccine is administered, a suitable waiting period is imposed
between vaccination and collection of serum or plasma for
immunoglobulin production.
ANTI-T LYMPHOCYTE The species, origin and identification number of the animals
IMMUNOGLOBULIN FOR HUMAN are specified.
USE, ANIMAL IMMUNISATION
The antigens used are identified and characterised, where
Immunoglobulinum anti-T lymphocytorum appropriate. They are identified by their names and a batch
ex animale ad usum humanum number ; information on the source and preparation are
recorded.
DEFINITION The selected animals are isolated for at least 1 week before
Sterile liquid or freeze-dried preparation containing being immunised according to a defined schedule with booster
immunoglobulins, obtained from serum or plasma of animals, injections at suitable intervals. Adjuvants may be used.
mainly rabbits or horses, immunised with human lymphocytic Animals are kept under general health surveillance and
antigens. specific antibody production is controlled at each cycle of
The immunoglobulin has the property of diminishing the immunisation.
number and function of immunocompetent cells, in particular Animals are thoroughly examined before collection of blood
T-lymphocytes. The preparation contains principally or plasma. If an animal shows any pathological lesion not
immunoglobulin G. It may contain antibodies against other related to the immunisation process, it is not used, nor are
lymphocyte subpopulations and against other cells. The any other of the animals in the group concerned, unless it is
preparation is intended for intravenous administration, evident that their use will not impair the safety of the product.
after dilution with a suitable diluent where applicable. The
preparation may contain excipients such as stabilisers. Human antigens such as continuously growing T-lymphocyte
cell lines or thymocytes are used to immunise the animals.
Applicable provisions of the monograph on Immunosera for Cells may be subjected to a sorting procedure. The
human use, animal (0084) are stated below. immunising antigens are shown to be free from infectious
PRODUCTION agents by validated methods for relevant blood-borne
pathogens, notably hepatitis B virus (HBV), hepatitis C
GENERAL PROVISIONS virus (HCV) and human immunodeficiency virus (HIV)
The production method has been shown to yield consistently and other relevant adventitious agents originating from the
immunoglobulins of acceptable safety, potency in man and preparation of the antigen. The cells used comply with defined
stability. requirements for purity of the cell population and freedom
Any reagent of biological origin used in production shall be from adventitious agents.
free of contamination with bacteria, fungi and viruses. The
COLLECTION OF BLOOD OR PLASMA
method of preparation includes a step or steps that have been
shown to remove or inactivate known agents of infection. Collection of blood is made by venepuncture or
plasmapheresis. The puncture area is shaved, cleaned
During development studies, it shall be demonstrated that the and disinfected. The animals may be anaesthetised under
production method yields a product that : conditions that do not influence the quality of the product.
– does not transmit infectious agents,
No antimicrobial preservative is added to the plasma and
– is characterised by a defined pattern of immunological serum samples. The blood or plasma is collected in such a
activity, notably : antigen binding, complement-dependent manner as to maintain sterility of the product. The blood or
and independent cytotoxicity, cytokine release, induction plasma collection is conducted at a site separate from the area
of T-cell activation, cell death, where the animals are kept or bred and the area where the
– does not contain antibodies that cross-react with human immunoglobulin is purified. If the serum or plasma is stored
tissues to a degree that would impair clinical safety, before further processing, precautions are taken to avoid
– has a defined maximum content of anti-thrombocyte microbial contamination.
antibody activity, Several single plasma or serum samples may be pooled before
– has a defined maximum content of haemoglobin. purification. The single or pooled samples are tested before
The product has been shown, by suitable tests in animals and purification for the following tests.
evaluation during clinical trials, to be well tolerated. Tests for contaminating viruses. Each pool is tested for
Reference preparation. A batch shown to be suitable for contaminating viruses by suitable in vitro tests including
checking the validity of the assay and whose efficacy has inoculation to cell cultures capable of detecting a wide range of
been demonstrated in clinical trials, or a batch representative viruses relevant for the particular product. Where applicable,
thereof. in vitro tests for contaminating viruses are carried out on
ANIMALS the adsorbed pool, after the last production stage that may
introduce viral contaminants.
The animals used are of a species approved by the competent
authority, are healthy and exclusively reserved for production PURIFICATION AND VIRAL INACTIVATION
of anti-T lymphocyte immunoglobulin. They are tested and The immunoglobulins are concentrated and purified by
shown to be free from a defined list of infectious agents. The fractional precipitation, chromatography, immuno-adsorption
introduction of animals into a closed herd follows specified or by other suitable chemical or physical methods. The
procedures, including definition of quarantine measures. methods are selected and validated to avoid contamination
Where appropriate, tests for additional specific agents are at all steps of processing and to avoid formation of protein
considered depending on the geographical localisation of the aggregates that effect immunobiological characteristics of
establishment used for the breeding and production of the the product.

General Notices (1) apply to all monographs and other texts 1575
Anti-T lymphocyte immunoglobulin for human use, animal EUROPEAN PHARMACOPOEIA 8.0

Unless otherwise justified and authorised, validated Extractable volume (2.9.17). It complies with the requirement
procedures are applied for removal and/or inactivation of for extractable volume.
viruses. pH (2.2.3). The pH is within the limits approved for the
After purification and treatment for removal and/or particular product.
inactivation of viruses, a stabiliser may be added to the
intermediate product, which may be stored for a period Osmolality (2.2.35) : minimum 240 mosmol/kg after dilution,
defined in the light of stability data. where applicable.
Only an intermediate product that complies with the following Total protein (2.5.33) : 90 per cent to 110 per cent of the
requirements may be used in the preparation of the final bulk. amount stated on the label.
If the method of preparation includes a step for adsorption Stabiliser. Determine the amount of stabiliser by a suitable
of cross-reacting anti-human antibodies using material from physico-chemical method. The preparation contains not
human tissues and/or red blood cells, the human materials less than 80 per cent and not more than 120 per cent of the
are submitted to a validated procedure for inactivation of quantity stated on the label.
infectious agents, unless otherwise justified and authorised. Distribution of molecular size. Size-exclusion
If erythrocytes are used for adsorption, the donors for chromatography (2.2.30).
such materials comply with the requirements for donors
of blood and plasma of the monograph on Human plasma Test solution. Dilute the preparation to be examined with a
for fractionation (0853). If other human material is used, 9 g/L solution of sodium chloride R to a concentration suitable
it is shown by validated methods to be free from relevant for the chromatographic system used. A concentration in the
blood-borne pathogens, notably HBV, HCV and HIV. If range 2-20 g/L is usually suitable.
substances are used for inactivation or removal of viruses, it Reference solution. Dilute human immunoglobulin (molecular
shall have been shown that any residues present in the final size) BRP with a 9 g/L solution of sodium chloride R to the
product have no adverse effects on the patients treated with same protein concentration as the test solution.
the anti-T lymphocyte immunoglobulin. Column :
FINAL BULK – size : l = 0.6 m, Ø = 7.5 mm,
The final bulk is prepared from a single intermediate product – stationary phase : silica gel for size-exclusion
or from a pool of intermediate products obtained from chromatography R, a grade suitable for fractionation of
animals of the same species. No antimicrobial preservative globular proteins in the molecular mass range of 20 000
is added either during the manufacturing procedure or for to 200 000.
preparation of the final bulk solution. During manufacturing,
the solution is passed through a bacteria-retentive filter. Mobile phase : dissolve 4.873 g of disodium hydrogen phosphate
dihydrate R, 1.741 g of sodium dihydrogen phosphate
FINAL LOT monohydrate R and 11.688 g of sodium chloride R in 1 L of
The final bulk of anti-T-lymphocyte immunoglobulin is water R.
distributed aseptically into sterile, tamper-proof containers.
The containers are closed as to prevent contamination. Flow rate : 0.5 mL/min.
Only a final lot that complies with the requirements prescribed Detection : spectrophotometer at 280 nm.
below under Identification, Tests and Assay may be released Injection : 50-600 μg of protein.
for use.
Retention time : identify the peaks in the chromatogram
CHARACTERS obtained with the test solution by comparison with the
Appearance : chromatogram obtained with the reference solution ; any peak
with a retention time shorter than that of dimer corresponds
– liquid preparation : clear or slightly opalescent, colourless to polymers and aggregates.
or pale yellow liquid ;
– freeze-dried preparation : white or slightly yellow powder or System suitability :
solid friable mass, which after reconstitution gives a liquid – reference solution : the principal peak corresponds to IgG
preparation corresponding to the description above. monomer and there is a peak corresponding to dimer with
a retention time relative to monomer of 0.85 ± 0.05,
IDENTIFICATION
– test solution : the relative retentions of monomer and dimer
A. Using a suitable range of species-specific antisera, carry out are 1 ± 0.05 with reference to the corresponding peaks in
precipitation tests on the preparation to be examined. It is the chromatogram obtained with the reference solution.
recommended that the test be carried out using antisera
specific to the plasma proteins of each species of domestic Limits :
animal commonly used in the preparation of materials of – total monomer and dimer : at least 95 per cent of the total
biological origin in the country concerned and antisera area of the peaks ;
specific to human plasma proteins. The preparation is – total polymers and aggregates : maximum 5 per cent of the
shown to contain proteins originating from the animal used total area of the peaks.
for the anti-T lymphocyte immunoglobulin production.
Purity. Polyacrylamide gel electrophoresis (2.2.31), under
B. Examine by a suitable immunoelectrophoresis technique. non-reducing and reducing conditions.
Using antiserum to normal serum of the animal used for
production, compare this serum and the preparation to be Resolving gel. Non-reducing conditions : 8 per cent acrylamide ;
examined, both diluted to a concentration that will allow reducing conditions : 12 per cent acrylamide.
a clear gammaglobulin precipitation arc to be obtained Test solution. Dilute the preparation to be examined to a
on the gel. The main component of the preparation to be protein concentration of 0.5-2 mg/mL.
examined corresponds to the IgG component of normal Reference solution. Dilute the reference preparation to the
serum of the animal used for production. same protein concentration as the test solution.
C. The preparation complies with the assay. Application : 10 μL.
TESTS Detection : Coomassie staining.
Solubility. For the freeze-dried preparation, to a container add Results : compared with the electropherogram of the
the volume of the liquid stated on the label. The preparation reference solution, no additional bands are found in the
dissolves completely within the time stated on the label. electropherogram of the test solution.

1576 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Anti-T lymphocyte immunoglobulin for human use, animal

Anti-A and anti-B haemagglutinins (2.6.20, Method A). The using a haemocytometer. Cell viability of at least 90 per cent
1 to 64 dilution does not show agglutination. is required. Adjust the cell number to 7 × 106/mL by adding
Where applicable, dilute the preparation to be examined as buffer solution for flow cytometry. Store the cell suspension
prescribed for use before preparing the dilutions for the test. at 4 °C and use within 12 h.
Haemolysins. Prepare a 1 to 64 dilution of the preparation If necessary, the first PBMC pellet may be resuspended in
to be examined, diluted if necessary as stated on the label. buffered salt solution pH 7.2 containing 20 per cent foetal
Take 6 aliquots of the 1 to 64 dilution. To 1 volume of 3 of calf serum and stored overnight at 2 °C. Centrifuge at 400 g
the aliquots, add 1 volume of a 10 per cent V/V suspension at 2-8 °C for 10 min. Discard the supernatant. Suspend the
of group A1, group B and group O erythrocytes in a 9 g/L cell pellet in buffer solution for flow cytometry. Determine
solution of sodium chloride R, respectively. To 1 volume of the number and vitality of the cells using a haemocytometer.
the remaining 3 aliquots, add 1 volume of a 10 per cent V/V Cell viability of at least 90 per cent is required. Adjust the
suspension of group A1, group B and group O erythrocytes cell number to 7 × 106/mL by adding buffer solution for flow
in a 9 g/L solution of sodium chloride R, respectively, and to cytometry.
each aliquot 1 volume of fresh group AB serum (as a source of It is also possible for cells to be immediately frozen and stored
complement). Mix and incubate at 37 °C for 1 h. Examine the in nitrogen using the following method.
supernatant liquids for haemolysis. No signs of haemolysis Buffer solution for freezing. To 20 mL of cell culture medium,
are present. add 25 mL of foetal calf serum and 5 mL of dimethyl
Thrombocyte antibodies. Examined by a suitable method, sulfoxide (DMSO). Store this solution at 2-8 °C and use within
the level of thrombocyte antibodies is shown to be below that 3 h.
approved for the specific product. 20 × 106 cells per ampoule are frozen. These ampoules are
Water (2.5.12): maximum 3 per cent. stored in liquid nitrogen.
Sterility (2.6.1). It complies with the test. Buffer solution for thawing. To 450 mL of cell culture medium,
add 50 mL of foetal calf serum. Store this solution at 2-8 °C
Pyrogens (2.6.8). Unless otherwise justified and authorised, and use within 3 h.
it complies with the test for pyrogens. Unless otherwise Each ampoule is thawed in a water-bath at 37 °C with
prescribed, inject 1 mL per kilogram of the rabbit’s body mass. shaking. Cell suspension is repeated in a buffer solution for
ASSAY thawing. Centrifuge at 200 g at 2-8 °C for 10 min. Discard
the supernatant. Suspend the cell pellet in buffer solution for
The biological activity is determined by measuring the
flow cytometry. Repeat the procedure for centrifugation and
complement-dependent cytotoxicity on target cells. Flow
resuspension of cells once. After the second centrifugation,
cytometry is performed with read-out of dead cells stained
resuspend the cells pellet in 1 mL of buffer solution for flow
using propidium iodide. The activity is expressed as the
cytometry. Determine the number and vitality of the cells
concentration of anti-T lymphocyte immunoglobulin
using a haemocytometer. Cell viability of at least 90 per cent
in milligrams per millilitre which mediates 50 per cent
is required. Adjust the cell number to 7 × 106/mL by adding
cytotoxicity.
buffer solution for flow cytometry. Store the cell suspension at
Lymphocyte separation medium. Commercial separation 4 °C and use within 3 h.
media with low viscosity and a density of 1.077 g/mL.
Test solutions. For freeze-dried preparations, reconstitute as
Complement. Commercial complement is suitable. stated on the label. Prepare 3 independent series of not fewer
Buffered salt solution pH 7.2. Dissolve 8.0 g of sodium than 7 dilutions using buffer solution for flow cytometry as
chloride R, 0.2 g of potassium chloride R, 3.18 g of disodium diluent.
hydrogen phosphate R and 0.2 g of potassium dihydrogen Reference solutions. For freeze-dried preparations, reconstitute
phosphate R in water R and dilute to 1000.0 mL with the same according to the instructions for use. Prepare 3 independent
solvent. dilution series of not fewer than 7 dilutions using buffer
Buffer solution for flow cytometry. Add 40 mL of 0.1 per solution for flow cytometry as diluent.
cent V/V sodium azide R and 10 mL of foetal calf serum to Distribute 75 μL of each of the dilutions of the test solution or
440 mL of buffered salt solution pH 7.2. The foetal calf serum reference solution to each of a series of wells of a microtitre
is inactivated at 56 °C for 30 min prior to use. Store at 4 °C. plate. Add 25 μL of the cell suspension of PBMC into each
Propidium iodide solution. Dissolve propidium iodide R in well. Add 25 μL of rabbit complement to each of the wells.
buffered salt solution pH 7.2, to a concentration of 1 mg/mL. Incubate at 37 °C for 30 min.
Store this stock solution at 2-8 °C and use within 1 month. Centrifuge the plates at 200 g at 4 °C for 8 min, discard the
For the assay, dilute this solution with buffer solution for supernatant and keep the plate on ice. Preparation for flow
flow cytometry, to obtain a concentration of 5 μg/mL. Store cytometry measurement is done step-wise by using a certain
at 2-8 °C and use within 3 h. number of wells in order to allow labelling with propidium
Microtitre plates. Plates used to prepare immunoglobulin iodide R solution and measurement within a defined time
dilutions are U- or V-bottomed polystyrene or poly(vinyl period. Resuspend carefully the cell pellet of a certain number
chloride) plates without surface treatment. of wells with 200 μL of propidium iodide solution. Transfer
Micronic tubes. Suitable for flow cytometry measurement. the suspension into tubes. Incubate at 25 °C for 10 min then
place immediately on ice.
Cell suspension. Collect blood in anticoagulant from at least
one healthy donor. Immediately isolate the peripheral blood Proceed with fluorescence measurement in a flow cytometer.
mononuclear cells (PBMC) by gradient centrifugation in Define a region including all propidium iodide-positive
lymphocyte separation medium so that the PBMC form a cells on the basis of Forward-Scattered, light (FSC) and
visible clean interface between the plasma and the separation flourescence (FL2 or FL3 for propidium iodide). Measure the
medium. Collect the layer containing the cells and dispense percentage of propidium iodide-positive cells, without gating
into centrifuge tubes containing buffered salt solution but excluding debris. Analyse at least 3000 cells for each of the
pH 7.2. Centrifuge at 400 g at 2-8 °C for 10 min. Discard test and reference solutions.
the supernatant. Suspend the cell pellet in buffer solution for Use the percentages of dead cells to estimate the potency as the
flow cytometry. Repeat the centrifugation and resuspension concentration in milligrams per millilitre of the preparation
procedure of the cells twice. After the third centrifugation, to be examined necessary to induce 50 per cent of cytotoxicity
resuspend the cell pellet in 1 mL of buffer solution for flow by fitting a sigmoidal dose response curve to the data obtained
cytometry. Determine the number and vitality of the cells with the test and the reference preparations and by using a

General Notices (1) apply to all monographs and other texts 1577
Apomorphine hydrochloride hemihydrate EUROPEAN PHARMACOPOEIA 8.0

4-parameter logistic model (see, for example, chapter 5.3) and A. Ultraviolet and visible absorption spectrophotometry
suitable software. The test is not valid unless the percentage of (2.2.25).
propidium iodide-positive cells at the lower asymptote of the Test solution. Dissolve 10.0 mg in a 10.3 g/L solution of
curve is less then 15 per cent and the percentage of propidium hydrochloric acid R and dilute to 100.0 mL with the same
iodide-positive cells at the upper asymptote of the curve is at acid solution. Dilute 10.0 mL of the solution to 100.0 mL
least 80 per cent. with a 10.3 g/L solution of hydrochloric acid R.
The estimated activity is 70 per cent to 130 per cent of the Spectral range : 230-350 nm
activity approved for the particular product.
Absorption maximum : at 273 nm.
The confidence limits (P = 0.95) are not less than 80 per cent
and not more than 125 per cent of the estimated potency. Shoulder : at 300-310 nm.
Specific absorbance at the absorption maximum : 530 to 570.
STORAGE B. Infrared absorption spectrophotometry (2.2.24).
Protected from light at the temperature stated on the label. Comparison: apomorphine hydrochloride hemihydrate CRS.
Expiry date. The expiry date is calculated from the beginning C. To 5 mL of solution S (see Tests) add a few millilitres of
of the assay. sodium hydrogen carbonate solution R until a permanent,
white precipitate is formed. The precipitate slowly becomes
LABELLING greenish. Add 0.25 mL of 0.05 M iodine and shake. The
The label states : precipitate becomes greyish-green. Collect the precipitate.
– for liquid preparations, the volume of the preparation in The precipitate dissolves in methylene chloride R giving a
the container and the protein content, violet-blue solution and in ethanol (96 per cent) R giving
a blue solution.
– for freeze-dried preparations :
D. To 2 mL of solution S (see Tests) add 0.1 mL of nitric
– the name and the volume of the reconstitution liquid acid R. Mix and filter. The filtrate gives reaction (a) of
to be added, chlorides (2.3.1).
– the quantity of protein in the container,
– that the immunoserum is to be used immediately TESTS
after reconstitution, Solution S. Dissolve 0.25 g without heating in carbon
– the time required for complete dissolution, dioxide-free water R and dilute to 25 mL with the same solvent.
– the animal species of origin, Appearance of solution. Solution S is clear (2.2.1) and
not more intensely coloured than reference solution BY5 or
– the name and amount of stabiliser, where applicable, GY5(2.2.2, Method II).
– the dilution to be made before use of the product.
pH (2.2.3) : 4.0 to 5.0 for solution S.
Specific optical rotation (2.2.7) : − 52 to − 48 (dried
substance).
07/2012:0136 Dissolve 0.25 g in a 2.06 g/L solution of hydrochloric acid R
and dilute to 25.0 mL with the same acid solution.
APOMORPHINE HYDROCHLORIDE Related substances. Liquid chromatography (2.2.29).
HEMIHYDRATE Test solution. Dissolve 50.0 mg of the substance to be
examined in a 1 per cent V/V solution of glacial acetic acid R
and dilute to 20.0 mL with the same solution.
Apomorphini hydrochloridum
Reference solution (a). Dilute 1.0 mL of the test solution to
hemihydricum 100.0 mL with a 1 per cent V/V solution of glacial acetic
acid R. Dilute 1.0 mL of this solution to 10.0 mL with a 1 per
cent V/V solution of glacial acetic acid R.
Reference solution (b). Dissolve 12.5 mg of apomorphine
impurity B CRS in a 1 per cent V/V solution of glacial acetic
acid R and dilute to 10.0 mL with the same solution.
Reference solution (c). Dilute 2.0 mL of reference solution (b)
to 10.0 mL with a 1 per cent V/V solution of glacial acetic
C17H18ClNO2,½H2O Mr 312.8 acid R. Dilute 2.0 mL of this solution to 100.0 mL with a 1 per
[41372-20-7] cent V/V solution of glacial acetic acid R.
DEFINITION Reference solution (d) . Dissolve 25 mg of boldine R in a 1 per
cent V/V solution of glacial acetic acid R and dilute to 10.0 mL
(6aR)-6-Methyl-5,6,6a,7-tetrahydro-4H-diben- with the same solution. To 1 mL of this solution add 1 mL of
zo[de,g]quinoline-10,11-diol hydrochloride hemihydrate. the test solution and dilute to 10.0 mL with a 1 per cent V/V
Content : 98.5 per cent to 101.5 per cent (dried substance). solution of glacial acetic acid R.
Column :
CHARACTERS
– size : l = 0.15 m, Ø = 4.6 mm ;
Appearance : white or slightly yellowish-brown or green-tinged
greyish, crystalline powder or crystals ; on exposure to air and – stationary phase : end-capped octadecylsilyl silica gel for
light, the green tinge becomes more pronounced. chromatography R (5 μm) ;
Solubility : sparingly soluble in water and in ethanol (96 per – temperature : 35 °C.
cent), practically insoluble in toluene. Mobile phase :
– mobile phase A : 1.1 g/L solution of sodium octanesulfonate R,
IDENTIFICATION adjusted to pH 2.2 with a 50 per cent m/m solution of
First identification : B, D. phosphoric acid R ;
Second identification : A, C, D. – mobile phase B : acetonitrile R ;

1578 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Aprotinin

Time Mobile phase A Mobile phase B

(min) (per cent V/V) (per cent V/V)
0-2 85 15

2 - 32 85 → 68 15 → 32

32 - 37 68 32

Flow rate : 1.5 mL/min. B. 7,8-didehydro-4,5α-epoxy-17-methylmorphinan-3,6α-diol

Detection : spectrophotometer at 280 nm. (morphine),
Injection : 10 μL.
Identification of impurities : use the chromatogram obtained
with reference solution (b) to identify the peak due to
impurity B.
Relative retention with reference to apomorphine
(retention time = about 18 min) : impurity B = about 0.4 ;
boldine = about 0.9.
System suitability : reference solution (d) :
C. (6aR)-9-[7,8-didehydro-4,5α-epoxy-3-hydroxy-
– resolution : minimum 2.5 between the peaks due to boldine 17-methylmorphinan-6α-yl]-6-methyl-5,6,6a,7-
and apomorphine. tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol
Limits : (morphine-apomorphine dimer).
– impurity B : not more than 0.75 times the area of the
corresponding peak in the chromatogram obtained with 01/2011:0580
reference solution (c) (0.15 per cent) ;
– unspecified impurities : for each impurity, not more than the APROTININ
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ; Aprotininum
– total : maximum 0.5 per cent ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent).
Loss on drying (2.2.32) : 2.5 per cent to 4.2 per cent,
determined on 1.000 g by drying in an oven at 105 °C for 2 h.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
C284H432N84O79S7 Mr 6511
ASSAY
Dissolve 0.250 g in a mixture of 5.0 mL of 0.01 M hydrochloric DEFINITION
acid and 50 mL of ethanol (96 per cent) R. Carry out a Aprotinin is a polypeptide consisting of a chain of 58 amino
potentiometric titration (2.2.20), using 0.1 M sodium acids. It inhibits stoichiometrically the activity of several
hydroxide. Read the volume added between the first 2 points proteolytic enzymes such as chymotrypsin, kallikrein, plasmin
of inflexion. and trypsin. It contains not less than 3.0 Ph. Eur. U. of
1 mL of 0.1 M sodium hydroxide is equivalent to 30.38 mg aprotinin activity per milligram, calculated with reference to
of C17H18ClNO2. the dried substance.
PRODUCTION
STORAGE
The animals from which aprotinin is derived must fulfil the
In an airtight container, protected from light. requirements for the health of animals suitable for human
consumption.
IMPURITIES
The method of manufacture is validated to demonstrate that
Specified impurities : B. the product, if tested, would comply with the following tests.
Other detectable impurities (the following substances would, Abnormal toxicity (2.6.9). Inject into each mouse a quantity
if present at a sufficient level, be detected by one or other of of the substance to be examined containing 2 Ph. Eur. U.
the tests in the monograph. They are limited by the general dissolved in a sufficient quantity of water for injections R to
acceptance criterion for other/unspecified impurities and/or give a volume of 0.5 mL.
by the general monograph Substances for pharmaceutical use Histamine (2.6.10) : maximum 0.2 μg of histamine base per
(2034). It is therefore not necessary to identify these impurities 3 Ph. Eur. U.
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : A, C. CHARACTERS
Appearance : almost white hygroscopic powder.
Solubility : soluble in water and in isotonic solutions,
practically insoluble in organic solvents.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solution. Solution S (see Tests).
A. (6aR)-10-methoxy-6-methyl-5,6,6a,7-tetrahydro-4H- Reference solution. Dilute aprotinin solution BRP in water R
dibenzo[de,g]quinolin-11-ol (apocodeine), to obtain a concentration of 15 Ph. Eur. U./mL.

General Notices (1) apply to all monographs and other texts 1579
Aprotinin EUROPEAN PHARMACOPOEIA 8.0

Plate : TLC silica gel G plate R. Run time : 30 min.

Mobile phase : water R, glacial acetic acid R (80:100 V/V) Identification of impurities : use the electropherogram supplied
containing 100 g/L of sodium acetate R. with aprotinin solution BRP and the electropherogram
Application : 10 μL. obtained with the reference solution to identify the peaks due
to impurities A and B.
Development : over a path of 12 cm.
Relative migration with reference to aprotinin (migration
Drying : in air. time = about 22 min) : impurity A = about 0.98 ;
Detection : spray with a solution of 0.1 g of ninhydrin R in a impurity B = about 0.99.
mixture of 6 mL of a 10 g/L solution of cupric chloride R, System suitability : reference solution after at least 6 injections :
21 mL of glacial acetic acid R and 70 mL of anhydrous
ethanol R. Dry the plate at 60 °C. – migration time : aprotinin = 19.0 min to 25.0 min ;
Results : the principal spot in the chromatogram obtained – resolution : minimum 0.8 between the peaks due to
with the test solution is similar in position, colour and size impurities A and B ; minimum 0.5 between the peaks due
to the principal spot in the chromatogram obtained with to impurity B and aprotinin ;
the reference solution. – peak distribution : the electrophoregram obtained
B. Determine the ability of the substance to be examined to is qualitatively and quantitatively similar to the
inhibit trypsin activity using the method described below. electropherogram supplied with aprotinin solution BRP ;
Test solution. Dilute 1 mL of solution S to 50 mL with – height of the principal peak : at least 1000 times the height
buffer solution pH 7.2 R. of the baseline noise. If necessary, adjust the sample load to
give peaks of sufficient height.
Trypsin solution. Dissolve 10 mg of trypsin BRP in 0.002 M
Limits :
hydrochloric acid and dilute to 100 mL with the same acid.
– impurity A : maximum 8.0 per cent ;
Casein solution. Dissolve 0.2 g of casein R in buffer solution
pH 7.2 R and dilute to 100 mL with the same buffer solution. – impurity B : maximum 7.5 per cent.
Precipitating solution : glacial acetic acid R, water R, Pyroglutamyl-aprotinin and related compounds. Liquid
anhydrous ethanol R (1:49:50 V/V/V). chromatography (2.2.29) : use the normalisation procedure.
Mix 1 mL of the test solution with 1 mL of the trypsin Test solution. Prepare a solution of the substance to
solution. Allow to stand for 10 min and add 1 mL of the be examined in mobile phase A, containing about
casein solution. Incubate at 35 °C for 30 min. Cool in iced 5 Ph. Eur. U./mL.
water and add 0.5 mL of the precipitating solution. Shake Reference solution. Dissolve the contents of a vial of aprotinin
and allow to stand at room temperature for 15 min. The for system suitability CRS in 2.0 mL of mobile phase A.
solution is cloudy. Carry out a blank test under the same Column :
conditions using buffer solution pH 7.2 R instead of the test – size : l = 0.075 m, Ø = 7.5 mm ;
solution. The solution is not cloudy.
– stationary phase : strong cation-exchange silica gel for
TESTS chromatography R (10 μm) ;
Solution S. Prepare a solution of the substance to be examined – temperature : 40 °C.
containing 15 Ph. Eur. U./mL, calculated from the activity Mobile phase :
stated on the label. – mobile phase A : dissolve 3.52 g of potassium dihydrogen
Appearance of solution. Solution S is clear (2.2.1). phosphate R and 7.26 g of disodium hydrogen phosphate
dihydrate R in 1000 mL of water ; filter and degas ;
Absorbance (2.2.25) : maximum 0.80 by measuring at the
absorption maximum at 277 nm. – mobile phase B : dissolve 3.52 g of potassium dihydrogen
phosphate R, 7.26 g of disodium hydrogen phosphate
Prepare a solution of the substance to be examined containing dihydrate R and 66.07 g of ammonium sulfate R in 1000 mL
3.0 Ph. Eur. U./ mL. of water ; filter and degas ;
Des-Ala-aprotinin and des-Ala-des-Gly-aprotinin.
Time Mobile phase A Mobile phase B
Capillary zone electrophoresis (2.2.47) : use the normalisation
(min) (per cent V/V) (per cent V/V)
procedure.
0 - 21 92 → 64 8 → 36
Test solution. Prepare a solution of the substance to be
examined in water R containing not less than 1 Ph. Eur. U./mL. 21 - 30 64 → 0 36 → 100
Reference solution. Dilute aprotinin solution BRP in water R to
Flow rate : 1.0 mL/min.
obtain the same concentration as the test solution.
Detection : spectrophotometer at 210 nm.
Capillary :
Injection : 40 μL.
– material : uncoated fused silica ;
Relative retention with reference to aprotinin (retention
– size : effective length = 45-60 cm, Ø = 75 μm. time = 17.0 min to 20.0 min): impurity C = about 0.9.
Temperature : 25 °C. System suitability : reference solution :
CZE buffer. Dissolve 8.21 g of potassium dihydrogen – resolution : minimum 1.5 between the peaks due to
phosphate R in 400 mL of water R, adjust to pH 3.0 with impurity C and aprotinin ;
phosphoric acid R, dilute to 500.0 mL with water R and filter
through a membrane filter (nominal pore size 0.45 μm). – symmetry factor : maximum 1.3 for the peak due to
aprotinin.
Detection : spectrophotometer at 214 nm.
Limits :
Between-run rinsing : rinse the capillary for at least 1 min with
– impurity C : maximum 1.0 per cent ;
0.1 M sodium hydroxide filtered through a membrane filter
(nominal pore size 0.45 μm) and for 2 min with the CZE – any other impurity : maximum 0.5 per cent ;
buffer. – sum of impurities other than C : maximum 1.0 per cent.
Injection : under pressure or vacuum (for example, 3 s at a Aprotinin oligomers. Size-exclusion chromatography
differential pressure of 3.5 kPa). (2.2.30) : use the normalisation procedure.
Migration : apply a field strength of 0.2 kV/cm, using the CZE Test solution. Prepare a solution of the substance to be
buffer as the electrolyte in both buffer reservoirs. examined in water R containing about 5 Ph. Eur. U./mL.

1580 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Aprotinin concentrated solution

Reference solution. Treat the substance to be examined to Dilute trypsin solution. Dilute 0.5 mL of the trypsin solution
obtain about 2 per cent aprotinin oligomers. For example, to 10.0 mL with 0.0015 M borate buffer solution pH 8.0 R.
heat freeze-dried aprotinin at about 110 °C for about 4 h. Allow to stand at room temperature for 10 min and then keep
Then dissolve in water R to obtain a concentration of about in iced water.
5 Ph. Eur. U./mL. Maintain an atmosphere of nitrogen in the reaction flask
Column : 3 columns coupled in series : and stir continuously ; introduce 9.0 mL of 0.0015 M borate
– size : l = 0.30 m, Ø = 7.8 mm ; buffer solution pH 8.0 R and 1.0 mL of a freshly prepared
– stationary phase : hydrophilic silica gel for chromatography R 6.9 g/L solution of benzoylarginine ethyl ester hydrochloride R.
of a grade suitable for fractionation of globular proteins in Adjust to pH 8.0 with 0.1 M sodium hydroxide. When the
the relative molecular mass range of 20 000 to 10 000 000 temperature has reached equilibrium at 25 ± 0.1 °C, add
(8 μm). 1.0 mL of the trypsin and aprotinin solution and start a timer.
Maintain at pH 8.0 by the addition of 0.1 M sodium hydroxide
Mobile phase : acetonitrile R, glacial acetic acid R, water R and note the volume added every 30 s. Continue the reaction
(2:2:6 V/V/V) ; filter and degas. for 6 min. Determine the number of millilitres of 0.1 M
Flow rate : 1.0 mL/min. sodium hydroxide used per second (n1 mL). Carry out, under
Detection : spectrophotometer at 277 nm. the same conditions, a titration using 1.0 mL of the dilute
Injection : 100 μL. trypsin solution. Determine the number of millilitres of 0.1 M
sodium hydroxide used per second (n2 mL).
Run time : 40 min.
Calculate the aprotinin activity in European Pharmacopoeia
Relative retention with reference to aprotinin monomer Units per milligram using the following expression :
(retention time = 24.5 min to 25.5 min) : aprotinin
dimer = about 0.9.
System suitability: reference solution :
– resolution : minimum 1.3 between the peaks due to The estimated activity is not less than 90 per cent and not
aprotinin dimer and monomer ; more than 110 per cent of the activity stated on the label.
– symmetry factor : maximum 2.5 for the peak due to
aprotinin monomer. STORAGE
Limit : In an airtight, tamper-proof container, protected from light.
– total : maximum 1.0 per cent. LABELLING
Loss on drying (2.2.32) : maximum 6.0 per cent, determined The label states :
on 0.100 g by drying in vacuo. – the number of European Pharmacopoeia Units of aprotinin
Bacterial endotoxins (2.6.14) : less than 0.14 IU per European activity per milligram ;
Pharmacopoeia Unit of aprotinin, if intended for use in the – where applicable, that the substance is suitable for use in
manufacture of parenteral preparations without a further the manufacture of parenteral preparations.
appropriate procedure for the removal of bacterial endotoxins.
IMPURITIES
ASSAY
The activity of aprotinin is determined by measuring its
inhibitory action on a solution of trypsin of known activity.
The inhibiting activity of the aprotinin is calculated from the
difference between the initial activity and the residual activity
of the trypsin.
The inhibiting activity of aprotinin is expressed in European
Pharmacopoeia Units. 1 Ph. Eur. U. inhibits 50 per cent of the
enzymatic activity of 2 microkatals of trypsin.
Use a reaction vessel with a capacity of about 30 mL, provided A. Ra = H, Rb = OH : aprotinin-(1-56)-peptide,
with :
B. Ra = H, Rb = Gly-OH : aprotinin-(1-57)-peptide,
– a device that will maintain a temperature of 25 ± 0.1 °C ;
– a stirring device, such as a magnetic stirrer ; C. Ra = Glp, Rb = Gly-Ala-OH : (5-oxoprolyl)aprotinin
– a lid with 5 holes for accommodating the electrodes, the tip (pyroglutamylaprotinin).
of a burette, a tube for the admission of nitrogen and the
introduction of the reagents.
01/2011:0579
An automatic or manual titration apparatus may be used. In
the latter case the burette is graduated in 0.05 mL and the
pH-meter is provided with a wide reading scale and glass and APROTININ CONCENTRATED
calomel or glass-silver-silver chloride electrodes. SOLUTION
Test solution. Prepare a solution of the substance to be
examined in 0.0015 M borate buffer solution pH 8.0 R expected Aprotinini solutio concentrata
to contain 1.67 Ph. Eur. U./mL (about 0.6 mg (m mg) per
millilitre).
Trypsin solution. Prepare a solution of trypsin BRP containing
about 0.8 microkatals per millilitre (about 1 mg/mL), using
0.001 M hydrochloric acid as the solvent. Use a freshly
prepared solution and keep in iced water.
Trypsin and aprotinin solution. To 4.0 mL of the trypsin
solution add 1.0 mL of the test solution. Dilute immediately to
40.0 mL with 0.0015 M borate buffer solution pH 8.0 R. Allow
to stand at room temperature for 10 min and then keep in
iced water. Use within 6 h of preparation. C284H432N84O79S7 Mr 6511

General Notices (1) apply to all monographs and other texts 1581
Aprotinin concentrated solution EUROPEAN PHARMACOPOEIA 8.0

DEFINITION Absorbance (2.2.25) : maximum 0.80 by measuring at the

Aprotinin concentrated solution is a solution of aprotinin, a absorption maximum at 277 nm.
polypeptide consisting of a chain of 58 amino acids, which Prepare a solution containing 3.0 Ph. Eur. U./mL.
inhibits stoichiometrically the activity of several proteolytic Des-Ala-aprotinin and des-Ala-des-Gly-aprotinin.
enzymes such as chymotrypsin, kallikrein, plasmin and Capillary zone electrophoresis (2.2.47) : use the normalisation
trypsin. It contains not less than 15.0 Ph. Eur. U. of aprotinin procedure.
activity per millilitre.
Test solution. Dilute the preparation to be examined in water R
PRODUCTION to obtain a concentration of not less than 1 Ph Eur. U./mL.
The animals from which aprotinin is derived must fulfil the Reference solution. Dilute aprotinin solution BRP in water R to
requirements for the health of animals suitable for human obtain the same concentration as the test solution.
consumption. Capillary :
The method of manufacture is validated to demonstrate that – material : uncoated fused silica ;
the product, if tested, would comply with the following tests. – size : effective length = 45-60 cm, Ø = 75 μm.
Abnormal toxicity (2.6.9). Inject into each mouse a quantity Temperature : 25 °C.
of the preparation to be examined containing 2 Ph. Eur. U.
diluted with a sufficient quantity of water for injections R to CZE buffer. Dissolve 8.21 g of potassium dihydrogen
give a volume of 0.5 mL. phosphate R in 400 mL of water R, adjust to pH 3.0 with
phosphoric acid R, dilute to 500.0 mL with water R and filter
Histamine (2.6.10) : maximum 0.2 μg of histamine base per through a membrane filter (nominal pore size 0.45 μm).
3 Ph. Eur. U. Detection : spectrophotometer at 214 nm.
CHARACTERS Between-run rinsing : rinse the capillary for at least 1 min with
Appearance : clear, colourless liquid. 0.1 M sodium hydroxide filtered through a membrane filter
(nominal pore size 0.45 μm) and for 2 min with the CZE
IDENTIFICATION buffer.
A. Thin-layer chromatography (2.2.27). Injection : under pressure or vacuum (for example, 3 s at a
Test solution. Solution S (see Tests). differential pressure of 3.5 kPa).
Reference solution. Dilute aprotinin solution BRP in water R Migration : apply a field strength of 0.2 kV/cm, using the CZE
to obtain a concentration of 15 Ph. Eur. U./mL. buffer as the electrolyte in both buffer reservoirs.
Plate : TLC silica gel G plate R. Run time : 30 min.
Mobile phase : water R, glacial acetic acid R (80:100 V/V) Identification of impurities : use the electropherogram supplied
containing 100 g/L of sodium acetate R. with aprotinin solution BRP and the electropherogram
obtained with the reference solution to identify the peaks due
Application : 10 μL. to impurities A and B.
Development : over a path of 12 cm. Relative migration with reference to aprotinin (migration
Drying : in air. time = about 22 min) : impurity A = about 0.98 ;
Detection : spray with a solution of 0.1 g of ninhydrin R in a impurity B = about 0.99.
mixture of 6 mL of a 10 g/L solution of cupric chloride R, System suitability : reference solution after at least 6 injections :
21 mL of glacial acetic acid R and 70 mL of anhydrous – migration time : aprotinin = 19.0 min to 25.0 min ;
ethanol R. Dry the plate at 60 °C.
– resolution : minimum 0.8 between the peaks due to
Results : the principal spot in the chromatogram obtained impurities A and B ; minimum 0.5 between the peaks due
with the test solution is similar in position, colour and size to impurity B and aprotinin ;
to the principal spot in the chromatogram obtained with
the reference solution. – peak distribution : the electrophoregram obtained
is qualitatively and quantitatively similar to the
B. Determine the ability of the preparation to be examined to electropherogram supplied with aprotinin solution BRP ;
inhibit trypsin activity using the method described below.
– height of the principal peak : at least 1000 times the height
Test solution. Dilute 1 mL of solution S to 50 mL with of the baseline noise. If necessary, adjust the sample load to
buffer solution pH 7.2 R. give peaks of a sufficient height.
Trypsin solution. Dissolve 10 mg of trypsin BRP in 0.002 M Limits :
hydrochloric acid and dilute to 100 mL with the same acid.
– impurity A : maximum 8.0 per cent ;
Casein solution. Dissolve 0.2 g of casein R in buffer solution
pH 7.2 R and dilute to 100 mL with the same buffer solution. – impurity B : maximum 7.5 per cent.
Precipitating solution : glacial acetic acid R, water R, Pyroglutamyl-aprotinin and related compounds. Liquid
anhydrous ethanol R (1:49:50 V/V/V). chromatography (2.2.29) : use the normalisation procedure.
Mix 1 mL of the test solution with 1 mL of the trypsin Test solution. Dilute the preparation to be examined in mobile
solution. Allow to stand for 10 min and add 1 mL of the phase A to a concentration of about 5 Ph. Eur. U./mL.
casein solution. Incubate at 35 °C for 30 min. Cool in iced Reference solution. Dissolve the contents of a vial of aprotinin
water and add 0.5 mL of the precipitating solution. Shake for system suitability CRS in 2.0 mL of mobile phase A.
and allow to stand at room temperature for 15 min. The Column :
solution is cloudy. Carry out a blank test under the same – size : l = 0.075 m, Ø = 7.5 mm ;
conditions using buffer solution pH 7.2 R instead of the test
solution. The solution is not cloudy. – stationary phase : strong cation-exchange silica gel for
chromatography R (10 μm) ;
TESTS – temperature : 40 °C.
Solution S. Prepare a solution containing 15 Ph. Eur. U./mL, Mobile phase :
if necessary by dilution, on the basis of the activity stated on – mobile phase A : dissolve 3.52 g of potassium dihydrogen
the label. phosphate R and 7.26 g of disodium hydrogen phosphate
Appearance of solution. Solution S is clear (2.2.1). dihydrate R in 1000 mL of water ; filter and degas ;

1582 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Aprotinin concentrated solution

– mobile phase B : dissolve 3.52 g of potassium dihydrogen ASSAY

phosphate R, 7.26 g of disodium hydrogen phosphate The activity of aprotinin is determined by measuring its
dihydrate R and 66.07 g of ammonium sulfate R in 1000 mL inhibitory action on a solution of trypsin of known activity.
of water ; filter and degas ; The inhibiting activity of the aprotinin is calculated from the
Time Mobile phase A Mobile phase B difference between the initial activity and the residual activity
(min) (per cent V/V) (per cent V/V) of the trypsin.
0 - 21 92 → 64 8 → 36 The inhibiting activity of aprotinin is expressed in European
Pharmacopoeia Units. 1 Ph. Eur. U. inhibits 50 per cent of the
21 - 30 64 → 0 36 → 100
enzymatic activity of 2 microkatals of trypsin.
Flow rate : 1.0 mL/min. Use a reaction vessel with a capacity of about 30 mL, provided
Detection : spectrophotometer at 210 nm. with :
Injection : 40 μL. – a device that will maintain a temperature of 25 ± 0.1 °C ;
Relative retention with reference to aprotinin (retention – a stirring device, such as a magnetic stirrer ;
time = 17.0 min to 20.0 min) : impurity C = about 0.9. – a lid with 5 holes for accommodating the electrodes, the tip
System suitability: reference solution : of a burette, a tube for the admission of nitrogen and the
– resolution : minimum 1.5 between the peaks due to introduction of the reagents.
impurity C and aprotinin ; An automatic or manual titration apparatus may be used. In
– symmetry factor : maximum 1.3 for the peak due to the latter case the burette is graduated in 0.05 mL and the
aprotinin. pH-meter is provided with a wide reading scale and glass and
calomel or glass-silver-silver chloride electrodes.
Limits :
Test solution. With 0.0015 M borate buffer solution pH 8.0 R
– impurity C : maximum 1.0 per cent ; prepare an appropriate dilution (D) of the aprotinin
– any other impurity : maximum 0.5 per cent ; concentrated solution expected, on the basis of the stated
– sum of impurities other than C : maximum 1.0 per cent. potency, to contain 1.67 Ph. Eur. U./mL.
Aprotinin oligomers. Size-exclusion chromatography Trypsin solution. Prepare a solution of trypsin BRP containing
(2.2.30) : use the normalisation procedure. about 0.8 microkatals per millilitre (about 1 mg/mL), using
Test solution. Dilute the preparation to be examined in water R 0.001 M hydrochloric acid as the solvent. Use a freshly
to obtain a concentration of about 5 Ph. Eur. U./mL. prepared solution and keep in iced water.
Reference solution. Treat the substance to be examined to Trypsin and aprotinin solution. To 4.0 mL of the trypsin
obtain about 2 per cent aprotinin oligomers. For example, solution add 1.0 mL of the test solution. Dilute immediately to
heat freeze-dried aprotinin at about 110 °C for about 4 h. 40.0 mL with 0.0015 M borate buffer solution pH 8.0 R. Allow
Then dissolve in water R to obtain a concentration of about to stand at room temperature for 10 min and then keep in
5 Ph. Eur. U./mL. iced water. Use within 6 h of preparation.
Column : 3 columns coupled in series : Dilute trypsin solution. Dilute 0.5 mL of the trypsin solution
to 10.0 mL with 0.0015 M borate buffer solution pH 8.0 R.
– size : l = 0.30 m, Ø = 7.8 mm ;
Allow to stand at room temperature for 10 min and then keep
– stationary phase : hydrophilic silica gel for chromatography R in iced water.
of a grade suitable for fractionation of globular proteins in
the relative molecular mass range of 20 000 to 10 000 000 Maintain an atmosphere of nitrogen in the reaction flask
(8 μm). and stir continuously ; introduce 9.0 mL of 0.0015 M borate
buffer solution pH 8.0 R and 1.0 mL of a freshly prepared
Mobile phase : acetonitrile R, glacial acetic acid R, water R 6.9 g/L solution of benzoylarginine ethyl ester hydrochloride R.
(2:2:6 V/V/V) ; filter and degas. Adjust to pH 8.0 with 0.1 M sodium hydroxide. When the
Flow rate : 1.0 mL/min. temperature has reached equilibrium at 25 ± 0.1 °C, add
Detection : spectrophotometer at 277 nm. 1.0 mL of the trypsin and aprotinin solution and start a timer.
Injection : 100 μL. Maintain at pH 8.0 by the addition of 0.1 M sodium hydroxide
and note the volume added every 30 s. Continue the reaction
Run time : 40 min. for 6 min. Determine the number of millilitres of 0.1 M
Relative retention with reference to aprotinin monomer sodium hydroxide used per second (n1 mL). Carry out, under
(retention time = 24.5 min to 25.5 min) : aprotinin the same conditions, a titration using 1.0 mL of the dilute
dimer = about 0.9. trypsin solution. Determine the number of millilitres of 0.1 M
System suitability: reference solution : sodium hydroxide used per second (n2 mL).
– resolution : minimum 1.3 between the peaks due to Calculate the aprotinin activity in European Pharmacopoeia
aprotinin dimer and monomer ; Units per millilitre using the following expression :
– symmetry factor : maximum 2.5 for the peak due to
aprotinin monomer.
Limit : D = dilution factor of the aprotinin concentrated
– total : maximum 1.0 per cent. solution to be examined in order to obtain a
Specific activity of the dry residue : minimum 3.0 Ph. Eur. U. solution containing 1.67 Ph. Eur. U./mL.
of aprotinin activity per milligram of dry residue. The estimated activity is not less than 90 per cent and not
Evaporate 25.0 mL to dryness in a water-bath, dry the residue more than 110 per cent of the activity stated on the label.
at 110 °C for 15 h and weigh. From the mass of the residue
and the activity determined as described below, calculate the STORAGE
number of European Pharmacopoeia Units per milligram of In an airtight, tamper-proof container, protected from light.
dry residue.
LABELLING
Bacterial endotoxins (2.6.14) : less than 0.14 IU per European
Pharmacopoeia Unit of aprotinin, if intended for use in the The label states :
manufacture of parenteral preparations without a further – the number of European Pharmacopoeia Units of aprotinin
appropriate procedure for the removal of bacterial endotoxins. activity per millilitre ;

General Notices (1) apply to all monographs and other texts 1583
Arachis oil, hydrogenated EUROPEAN PHARMACOPOEIA 8.0

– where applicable, that the substance is suitable for use in Split ratio : 1:100.
the manufacture of parenteral preparations. Temperature :
IMPURITIES – column : 180 °C for 20 min ;
– injection port and detector : 250 °C.
Detection : flame ionisation.
Composition of the fatty-acid fraction of the oil :
– saturated fatty acids of chain length less than C14 : maximum
0.5 per cent ;
– myristic acid : maximum 0.5 per cent ;
– palmitic acid : 7.0 per cent to 16.0 per cent ;
– stearic acid : 3.0 per cent to 19.0 per cent ;
A. Ra = H, Rb = OH : aprotinin-(1-56)-peptide, – oleic acid and isomers : 54.0 per cent to 78.0 per cent ;
B. Ra = H, Rb = Gly-OH : aprotinin-(1-57)-peptide, – linoleic acid and isomers : maximum 10.0 per cent ;
C. Ra = Glp, Rb = Gly-Ala-OH : (5-oxoprolyl)aprotinin – arachidic acid : 1.0 per cent to 3.0 per cent ;
(pyroglutamylaprotinin). – eicosenoic acids : maximum 2.1 per cent ;
– behenic acid : 1.0 per cent to 5.0 per cent ;
07/2010:1171 – erucic acid and isomers : maximum 0.5 per cent ;
corrected 7.0
– lignoceric acid : 0.5 per cent to 3.0 per cent.
ARACHIS OIL, HYDROGENATED Nickel : maximum 1 ppm.
Atomic absorption spectrometry (2.2.23, Method II).
Arachidis oleum hydrogenatum Test solution. Into a platinum or silica crucible previously
tared after ignition introduce 5.0 g. Cautiously heat and
DEFINITION introduce into the substance a wick formed from twisted
Oil obtained by refining, bleaching, hydrogenating and ashless filter paper. Ignite the wick. When the substance has
deodorising oil obtained from the shelled seeds of Arachis ignited stop heating. After combustion, ignite in a muffle
hypogaea L. Each type of hydrogenated arachis oil is furnace at about 600 ± 50 °C. Continue ignition until white
characterised by its nominal drop point. ash is obtained. After cooling, take up the residue with
2 quantities, each of 2 mL, of dilute hydrochloric acid R and
CHARACTERS transfer into a 25 mL graduated flask. Add 0.3 mL of nitric
Appearance : white or faintly yellowish, soft mass which melts acid R and dilute to 25.0 mL with water R.
to a clear, pale yellow liquid when heated. Reference solutions. Prepare 3 reference solutions by adding
Solubility : practically insoluble in water, freely soluble in 1.0 mL, 2.0 mL and 4.0 mL of nickel standard solution (0.2 ppm
methylene chloride and in light petroleum (bp : 65-70 °C), Ni) R to 2.0 mL of the test solution and diluting to 10.0 mL
very slightly soluble in ethanol (96 per cent). with water R.
IDENTIFICATION Source : nickel hollow-cathode lamp.
First identification : A, B. Wavelength : 232 nm.
Second identification : A, C. Atomisation device : graphite furnace.
A. Drop point (see Tests). Carrier gas : argon R.
B. Identification of fatty oils by thin-layer chromatography STORAGE
(2.3.2). Protected from light.
Results : the chromatogram obtained is similar to the
chromatogram for arachis oil shown in Figure 2.3.2.-1. LABELLING
C. Composition of fatty acids (see Tests). The label states the nominal drop point.
TESTS 07/2011:0263
Drop point (2.2.17) : 32 °C to 43 °C, and within 3 °C of the
nominal value. ARACHIS OIL, REFINED
Acid value (2.5.1) : maximum 0.5.
Dissolve 10.0 g in 50 mL of the prescribed solvent by heating Arachidis oleum raffinatum
on a water-bath.
DEFINITION
Peroxide value (2.5.5, Method A) : maximum 5.0.
The refined fatty oil obtained from the shelled seeds of Arachis
Dissolve 5.0 g in 30 mL of the prescribed solvent by heating hypogaea L. A suitable antioxidant may be added.
on a water-bath.
Unsaponifiable matter (2.5.7) : maximum 1.0 per cent. CHARACTERS
Alkaline impurities (2.4.19). It complies with the test. Appearance : clear, yellowish, viscous liquid.
Composition of fatty acids (2.4.22, Method A). Use the Solubility : very slightly soluble in ethanol (96 per cent),
mixture of calibrating substances in Table 2.4.22.-3. miscible with light petroleum.
Column : Relative density : about 0.915.
– material : fused silica ; It solidifies at about 2 °C.
– size : l = 25 m, Ø = 0.25 mm ; IDENTIFICATION
– stationary phase : poly(cyanopropyl)siloxane R (film Identification of fatty oils by thin-layer chromatography
thickness 0.2 μm). (2.3.2).
Carrier gas : helium for chromatography R. Results : the chromatogram obtained is similar to the
Flow rate : 0.7 mL/min. corresponding chromatogram shown in Figure 2.3.2.-1.

1584 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Arginine

TESTS E. Dissolve about 25 mg in 2 mL of water R. Add 1 mL

Acid value (2.5.1) : maximum 0.5, determined on 10.0 g. of α-naphthol solution R and 2 mL of a mixture of
equal volumes of strong sodium hypochlorite solution R and
Peroxide value (2.5.5, Method A) : maximum 5.0. water. A red colour develops.
Unsaponifiable matter (2.5.7) : maximum 1.0 per cent,
determined on 5.0 g.
TESTS
Alkaline impurities (2.4.19). It complies with the test.
Solution S. Dissolve 2.5 g in distilled water R and dilute to
Composition of fatty acids. (2.4.22, Method A). Use the 50 mL with the same solvent.
mixture of calibrating substances in Table 2.4.22.-3.
Appearance of solution. Solution S is clear (2.2.1) and not
Composition of the fatty-acid fraction of the oil: more intensely coloured than reference solution BY6 (2.2.2,
– saturated fatty acids of chain length less than C16 : maximum Method II).
0.4 per cent ;
Specific optical rotation (2.2.7). Dissolve 2.00 g in
– palmitic acid : 5.0 per cent to 14.0 per cent ; hydrochloric acid R1 and dilute to 25.0 mL with the same acid.
– stearic acid : 1.3 per cent to 6.5 per cent ; The specific optical rotation is + 25.5 to + 28.5, calculated with
– oleic acid : 35.0 per cent to 72.0 per cent ; reference to the dried substance.
– linoleic acid : 12.0 per cent to 43.0 per cent ; Ninhydrin-positive substances. Examine by thin-layer
– linolenic acid : maximum 0.6 per cent ; chromatography (2.2.27), using a TLC silica gel plate R.
– arachidic acid : 0.5 per cent to 3.0 per cent ; Test solution (a). Dissolve 0.10 g of the substance to be
– eicosenoic acid : 0.5 per cent to 3.0 per cent ; examined in dilute hydrochloric acid R and dilute to 10 mL
with the same acid.
– behenic acid : 1.0 per cent to 5.0 per cent ;
– erucic acid : maximum 0.5 per cent ; Test solution (b). Dilute 1 mL of test solution (a) to 50 mL
with water R.
– lignoceric acid : 0.5 per cent to 3.0 per cent.
Water (2.5.32) : maximum 0.1 per cent, determined on 1.00 g. Reference solution (a). Dissolve 10 mg of arginine CRS in 0.1 M
hydrochloric acid and dilute to 50 mL with the same acid.
STORAGE Reference solution (b). Dilute 5 mL of test solution (b) to
In a well-filled container, protected from light. 20 mL with water R.
Reference solution (c). Dissolve 10 mg of arginine CRS and
10 mg of lysine hydrochloride CRS in 0.1 M hydrochloric acid
01/2008:0806 and dilute to 25 mL with the same acid.
corrected 6.0
Apply to the plate 5 μL of each solution. Allow the plate to
dry in air. Develop over a path of 15 cm using a mixture
ARGININE of 30 volumes of concentrated ammonia R and 70 volumes
of 2-propanol R. Dry the plate at 100 °C to 105 °C until
Argininum the ammonia disappears completely. Spray with ninhydrin
solution R and heat at 100 °C to 105 °C for 15 min. Any spot in
the chromatogram obtained with test solution (a), apart from
the principal spot, is not more intense than the spot in the
chromatogram obtained with reference solution (b) (0.5 per
cent). The test is not valid unless the chromatogram obtained
with reference solution (c) shows two clearly separated spots.
C6H14N4O2 Mr 174.2
[74-79-3] Chlorides (2.4.4). To 5 mL of solution S add 0.5 mL of dilute
nitric acid R and dilute to 15 mL with water R. The solution
DEFINITION complies with the limit test for chlorides (200 ppm).
Arginine contains not less than 98.5 per cent and Sulfates (2.4.13). To 10 mL of solution S, add 1.7 mL of
not more than the equivalent of 101.0 per cent of dilute hydrochloric acid R and dilute to 15 mL with distilled
(S)-2-amino-5-guanidinopentanoic acid, calculated with water R. The solution complies with the limit test for sulfates
reference to the dried substance. (300 ppm).
CHARACTERS Ammonium (2.4.1). 50 mg complies with limit test B for
A white or almost white, crystalline powder or colourless ammonium (200 ppm). Prepare the standard using 0.1 mL of
crystals, freely soluble in water, very slightly soluble in alcohol. ammonium standard solution (100 ppm NH4) R.
Iron (2.4.9). In a separating funnel, dissolve 1.0 g in 10 mL of
IDENTIFICATION dilute hydrochloric acid R. Shake with three quantities, each of
First identification : A, C. 10 mL, of methyl isobutyl ketone R1, shaking for 3 min each
Second identification : A, B, D, E. time. To the combined organic layers add 10 mL of water R
and shake for 3 min. The aqueous layer complies with the
A. Specific optical rotation (see Tests).
limit test for iron (10 ppm).
B. Solution S (see Tests) is strongly alkaline (2.2.4).
Heavy metals (2.4.8). Dissolve 2.0 g in water R and dilute to
C. Examine by infrared absorption spectrophotometry 20 mL with the same solvent. 12 mL of the solution complies
(2.2.24), comparing with the spectrum obtained with with test A for heavy metals (10 ppm). Prepare the reference
arginine CRS. Examine the substances prepared as discs. solution using lead standard solution (1 ppm Pb) R.
D. Examine the chromatograms obtained in the test for
ninhydrin-positive substances. The principal spot in the Loss on drying (2.2.32). Not more than 0.5 per cent,
chromatogram obtained with test solution (b) is similar determined on 1.000 g by drying in an oven at 105 °C.
in position, colour and size to the principal spot in the Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
chromatogram obtained with reference solution (a). on 1.0 g.

General Notices (1) apply to all monographs and other texts 1585
Arginine aspartate EUROPEAN PHARMACOPOEIA 8.0

ASSAY Reference solution (b). Dilute 2 mL of reference solution (a)

Dissolve 0.150 g in 50 mL of water R. Using 0.2 mL of to 50 mL with water R.
methyl red mixed solution R as indicator, titrate with 0.1 M Plate : TLC silica gel G plate R.
hydrochloric acid until the colour changes from green to Mobile phase : ammonia R, propanol R (36:64 V/V).
violet-red. Application : 5 μL.
1 mL of 0.1 M hydrochloric acid is equivalent to 17.42 mg of Development : over 2/3 of the plate.
C6H14N4O2.
Drying : at 100-105 °C for 10 min.
STORAGE Detection : spray with ninhydrin solution R and heat at
Store protected from light. 100-105 °C for 10 min.
System suitability : reference solution (b) :
– the chromatogram shows 2 clearly separated principal
01/2008:2096 spots.
corrected 6.0
Limit : test solution (a) :
– any impurity : any spots, apart from the 2 principal spots,
ARGININE ASPARTATE are not more intense than each of the 2 principal spots in
the chromatogram obtained with reference solution (b)
Arginini aspartas (0.2 per cent).
Chlorides (2.4.4) : maximum 200 ppm.
Dilute 2.5 mL of solution S to 15 mL with water R.
Sulfates (2.4.13) : maximum 300 ppm.
To 0.5 g add 2.5 mL of dilute hydrochloric acid R and dilute to
C10H21N5O6 Mr 307.3 15 mL with distilled water R. Examine after 30 min.
[7675-83-4] Ammonium (2.4.1) : maximum 100 ppm, determined on
DEFINITION 100 mg.
(2S)-2-Amino-5-guanidinopentanoic acid (2S)-2- Heavy metals (2.4.8) : maximum 20 ppm.
aminobutanedioate. 12 mL of solution S complies with test A. Prepare the reference
Content : 99.0 per cent to 101.0 per cent (dried substance). solution using lead standard solution (2 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
CHARACTERS on 1.000 g by drying in an oven at 60 °C for 24 h.
Appearance : white or almost white granules or powder. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Solubility : very soluble in water, practically insoluble in 1.0 g.
alcohol and in methylene chloride.
ASSAY
IDENTIFICATION Dissolve 80.0 mg in 2 mL of anhydrous formic acid R. Add
A. Specific optical rotation (see Tests). 50 mL of anhydrous acetic acid R. Titrate with 0.1 M perchloric
B. Infrared absorption spectrophotometry (2.2.24). acid, determining the end-point potentiometrically (2.2.20).
Comparison : arginine aspartate CRS. 1 mL of 0.1 M perchloric acid is equivalent to 10.24 mg
C. Examine the chromatograms obtained in the test for of C10H21N5O6.
ninhydrin-positive substances.
Results : the 2 principal spots in the chromatogram obtained 01/2008:0805
with test solution (b) are similar in position, colour and corrected 6.0
size to the 2 principal spots in the chromatogram obtained
with reference solution (a). ARGININE HYDROCHLORIDE
TESTS
Arginini hydrochloridum
Solution S. Dissolve 5.0 g in carbon dioxide-free water R and
dilute to 50 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y7 (2.2.2,
Method II).
pH (2.2.3): 6.0 to 7.0 for solution S. C6H15ClN4O2 Mr 210.7
[1119-34-2]
Specific optical rotation (2.2.7) : + 25 to + 27 (dried
substance). DEFINITION
Dissolve 2.50 g in dilute hydrochloric acid R and dilute to Arginine hydrochloride contains not less than 98.5 per cent
25.0 mL with the same acid. and not more than the equivalent of 101.0 per cent of the
hydrochloride of (S)-2-amino-5-guanidinopentanoic acid,
Ninhydrin-positive substances. Thin-layer chromatography
calculated with reference to the dried substance.
(2.2.27).
Test solution (a). Dissolve 0.20 g of the substance to be CHARACTERS
examined in water R and dilute to 10 mL with the same A white or almost white, crystalline powder or colourless
solvent. crystals, freely soluble in water, very slightly soluble in alcohol.
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
with water R. IDENTIFICATION
Reference solution (a). Dissolve 25 mg of arginine R and 25 mg First identification : A, B, E.
of aspartic acid R in water R and dilute to 25 mL with the Second identification : A, C, D, E.
same solvent. A. Specific optical rotation (see Tests).

1586 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Argon

B. Examine by infrared absorption spectrophotometry Loss on drying (2.2.32). Not more than 0.5 per cent,
(2.2.24), comparing with the spectrum obtained with determined on 1.000 g by drying in an oven at 105 °C.
arginine hydrochloride CRS. Examine the substances Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
prepared as discs. on 1.0 g.
C. Examine the chromatograms obtained in the test for
ninhydrin-positive substances. The principal spot in the ASSAY
chromatogram obtained with test solution (b) is similar Dissolve 0.180 g in 3 mL of anhydrous formic acid R.
in position, colour and size to the principal spot in the Add 30 mL of anhydrous acetic acid R. Using 0.1 mL of
chromatogram obtained with reference solution (a). naphtholbenzein solution R as indicator, titrate with 0.1 M
D. Dissolve about 25 mg in 2 mL of water R. Add 1 mL perchloric acid until the colour changes from brownish-yellow
of α-naphthol solution R and 2 mL of a mixture of to green.
equal volumes of strong sodium hypochlorite solution R and 1 mL of 0.1 M perchloric acid is equivalent to 21.07 mg of
water R. A red colour develops. C6H15ClN4O2.
E. It gives reaction (a) of chlorides (2.3.1). STORAGE
TESTS Store protected from light.
Solution S. Dissolve 2.5 g in distilled water R and dilute to
50 mL with the same solvent. 07/2010:2407
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution BY6 (2.2.2, ARGON
Method II).
Specific optical rotation (2.2.7). Dissolve 2.00 g in Argon
hydrochloric acid R1 and dilute to 25.0 mL with the same acid.
The specific optical rotation is + 21.0 to + 23.5, calculated with Ar Ar 39.95
reference to the dried substance. [7440-37-1]
Ninhydrin-positive substances. Examine by thin-layer DEFINITION
chromatography (2.2.27), using a TLC silica gel plate R.
Gas obtained by fractional distillation of ambient air.
Test solution (a). Dissolve 0.10 g of the substance to be
examined in water R and dilute to 10 mL with the same Content : minimum 99.995 per cent V/V of Ar, calculated by
solvent. deduction of the sum of impurities found when performing
the test for impurities and the water content.
Test solution (b). Dilute 1 mL of test solution (a) to 50 mL
with water R. This monograph applies to argon for medicinal use.
Reference solution (a). Dissolve 10 mg of arginine CHARACTERS
hydrochloride CRS in water R and dilute to 50 mL with the Appearance : colourless gas.
same solvent.
Solubility : at 20 °C and at a pressure of 101 kPa, 1 volume
Reference solution (b). Dilute 5 mL of test solution (b) to dissolves in about 29 volumes of water.
20 mL with water R.
Reference solution (c). Dissolve 10 mg of arginine IDENTIFICATION
hydrochloride CRS and 10 mg of lysine hydrochloride CRS in A. Verify that the gas is not oxygen using a paramagnetic
water R and dilute to 25 mL with the same solvent. analyser (2.5.27).
Apply to the plate 5 μL of each solution. Allow the plate to B. Gas chromatography (2.2.28).
dry in air. Develop over a path of 15 cm using a mixture Gas to be examined. The substance to be examined.
of 30 volumes of concentrated ammonia R and 70 volumes Reference gas. Use the following mixture of gases
of 2-propanol R. Dry the plate at 100 °C to 105 °C until in argon R1 : methane R1 (5 ppm V/V), nitrogen R1
the ammonia disappears completely. Spray with ninhydrin (5 ppm V/V), oxygen R (5 ppm V/V).
solution R and heat at 100 °C to 105 °C for 15 min. Any spot in
Column :
the chromatogram obtained with test solution (a), apart from
the principal spot, is not more intense than the spot in the – material : stainless steel ;
chromatogram obtained with reference solution (b) (0.5 per – size : l = 2 m, Ø = 3 mm ;
cent). The test is not valid unless the chromatogram obtained – stationary phase : molecular sieve for chromatography R
with reference solution (c) shows two clearly separated spots. (particle size 150-180 μm, pore size 0.5 nm).
Sulfates (2.4.13). Dilute 10 mL of solution S to 15 mL with Carrier gas : helium for chromatography R.
distilled water R. The solution complies with the limit test for Flow rate : 10 mL/min.
sulfates (300 ppm).
Temperature :
Ammonium (2.4.1). 50 mg complies with limit test B for – column : 50 °C ;
ammonium (200 ppm). Prepare the standard using 0.1 mL of
ammonium standard solution (100 ppm NH4) R. – detector : 150 °C.
Detection : thermal conductivity.
Iron (2.4.9). In a separating funnel, dissolve 1.0 g in 10 mL of
dilute hydrochloric acid R. Shake with three quantities, each of Injection : 25 μL.
10 mL, of methyl isobutyl ketone R1, shaking for 3 min each System suitability : reference gas :
time. To the combined organic layers add 10 mL of water R – resolution : minimum 3.0 between the peaks due to
and shake for 3 min. The aqueous layer complies with the argon/oxygen and nitrogen and minimum 2.0 between
limit test for iron (10 ppm). the peaks due to nitrogen and methane.
Heavy metals (2.4.8). Dissolve 2.0 g in water R and dilute to Results : the principal peak in the chromatogram obtained
20 mL with the same solvent. 12 mL of the solution complies with the gas to be examined is similar in retention time
with test A for heavy metals (10 ppm). Prepare the reference to the principal peak in the chromatogram obtained with
solution using lead standard solution (1 ppm Pb) R. the reference gas.

General Notices (1) apply to all monographs and other texts 1587
Articaine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

TESTS Content : 98.5 per cent to 101.0 per cent (dried substance).
Impurities. Gas chromatography (2.2.28). CHARACTERS
Gas to be examined. The substance to be examined. Appearance : white or almost white, crystalline powder.
Reference gas. Use the following mixture of gases in argon R1 : Solubility : freely soluble in water and in ethanol (96 per cent).
methane R1 (5 ppm V/V), nitrogen R1 (5 ppm V/V), oxygen R
(5 ppm V/V). IDENTIFICATION
Column : First identification : B, D.
– material : stainless steel ; Second identification : A, C, D.
– size : l = 4 m, Ø = 4 mm ; A. Dissolve 50.0 mg in a 1 g/L solution of hydrochloric
– stationary phase : molecular sieve for chromatography R acid R and dilute to 100.0 mL with the same acid. Dilute
(particle size 150-180 μm, pore size 0.5 nm). 5.0 mL of the solution to 100.0 mL with a 1 g/L solution of
Carrier gas : argon R1. hydrochloric acid R. Examined between 200 nm and 350 nm
(2.2.25), the solution shows an absorption maximum at
Flow rate : 70 mL/min.
272 nm. The specific absorbance at the maximum is 290
Temperature : to 320.
– column : 80 °C ; B. Infrared absorption spectrophotometry (2.2.24).
– detector : 40 °C. Preparation : place dropwise 20 μL of the test solution on
Detection : discharge ionisation. 300 mg discs.
Injection : 1 mL. Test solution. Dissolve 0.1 g in 5 mL of water R, add 3 mL
Sample rate : 100 mL/min. of a saturated solution of sodium hydrogen carbonate R and
Relative retention with reference to impurity C (retention shake twice with 2 mL of methylene chloride R. Combine the
time = about 4.7 min) : impurity A = about 0.4 ; methylene chloride layers, dilute to 5.0 mL with methylene
impurity B = about 0.7. chloride R and dry over anhydrous sodium sulfate R.
System suitability : reference gas : Comparison: articaine hydrochloride CRS.
– resolution : minimum 3.0 between the peaks due to C. Thin-layer chromatography (2.2.27).
impurities A and B and minimum 2.0 between the peaks Test solution. Dissolve 20 mg of the substance to be
due to impurities B and C. examined in 5 mL of ethanol (96 per cent) R.
Limits : Reference solution. Dissolve 20 mg of articaine
– impurity A : not more than the area of the corresponding hydrochloride CRS in 5 mL of ethanol (96 per cent) R.
peak in the chromatogram obtained with the reference gas Plate : TLC silica gel F254 plate R.
(5.0 ppm V/V) ; Mobile phase : triethylamine R, ethyl acetate R, heptane R
– total : maximum 0.0040 per cent of the sum of the areas of (10:35:65 V/V/V).
all the peaks (40.0 ppm V/V). Application : 5 μL.
Water (2.5.28) : maximum 10.0 ppm V/V, determined using Development : over a path of 15 cm.
an electrolytic hygrometer. Drying : in air.
STORAGE Detection : examine in ultraviolet light at 254 nm.
In gaseous or liquid state, in suitable containers, complying Results : the principal spot in the chromatogram obtained
with the legal regulations. with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the
IMPURITIES reference solution.
Specified impurities : A, D. D. It gives reaction (a) of chlorides (2.3.1).
Other detectable impurities: B, C. TESTS
A. oxygen, Solution S. Dissolve 0.50 g in water R and dilute to 10 mL
B. nitrogen, with the same solvent.
C. methane, Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution BY6 (2.2.2,
D. water. Method I).
pH (2.2.3) : 4.2 to 5.2.
04/2012:1688
Dissolve 0.20 g in carbon dioxide-free water R and dilute to
ARTICAINE HYDROCHLORIDE 20.0 mL with the same solvent.
Related substances. Liquid chromatography (2.2.29).
Articaini hydrochloridum Test solution. Dissolve 10.0 mg of the substance to be
examined in the mobile phase and dilute to 10.0 mL with the
mobile phase.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10.0 mL with the mobile phase.
Reference solution (b). Dissolve 10.0 mg of articaine
impurity A CRS and 5.0 mg of articaine impurity E CRS in the
C13H21ClN2O3S Mr 320.8 mobile phase and dilute to 100.0 mL with the mobile phase.
[23964-57-0]
Reference solution (c). Add 1.0 mL of reference solution (b) to
DEFINITION 50.0 mg of articaine hydrochloride CRS and dilute to 50 mL
Methyl 4-methyl-3-[[(2RS)-2- with the mobile phase.
(propylamino)propanoyl]amino]thiophene-2-carboxylate Reference solution (d). Dilute 1.0 mL of reference solution (b)
hydrochloride. to 50.0 mL with the mobile phase.

1588 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Articaine hydrochloride

Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : spherical end-capped octadecylsilyl silica
gel for chromatography R (5 μm) with a specific surface area
of 335 m2/g and a carbon loading of 19 per cent ;
– temperature : 45 °C. A. methyl 3-[[2-(propylamino)acetyl]amino]-4-
Mobile phase : mix 25 volumes of acetonitrile R and 75 volumes methylthiophene-2-carboxylate (acetamidoarticaine),
of a solution prepared as follows : dissolve 2.02 g of sodium
heptanesulfonate R and 4.08 g of potassium dihydrogen
phosphate R in water R and dilute to 1000 mL with the same
solvent. Adjust to pH 2.0 with phosphoric acid R.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 276 nm. B. 4-methyl-3-[[(2RS)-2-(propylamino)propanoyl]-
Injection : 10 μL of the test solution and reference solutions (a), amino]thiophene-2-carboxylic acid (articaine acid),
(c) and (d).
Run time : 5 times the retention time of articaine.
Relative retention with reference to articaine (retention
time = about 9 min) : impurity A = about 0.8 ;
impurity E = about 0.86.
System suitability: reference solution (c) :
– resolution : minimum 1.2 between the peaks due to C. 1-methylethyl 4-methyl-3-[[(2RS)-2-(propylamino)propa-
impurities A and E. noyl]amino]thiophene-2-carboxylate (articaine isopropyl
Limits : ester),
– impurity A : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (d) (0.2 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
– total of unspecified impurities : not more than 5 times the D. methyl 3-[[(2RS)-2-(ethylamino)propanoyl]amino]-4-
area of the principal peak in the chromatogram obtained methylthiophene-2-carboxylate (ethylarticaine),
with reference solution (a) (0.5 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent).
Heavy metals (2.4.8) : maximum 5 ppm.
Dissolve 4.0 g in 20.0 mL of water R. 12 mL of the solution
complies with test A. Prepare the reference solution using lead
E. methyl 4-methyl-3-[[(2RS)-2-[(1-methylethyl)amino]prop-
standard solution (1 ppm Pb) R.
anoyl]amino]thiophene-2-carboxylate (isopropylarticaine),
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 5 h.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY
Dissolve 0.250 g in a mixture of 5.0 mL of 0.01 M hydrochloric
acid and 50 mL of ethanol (96 per cent) R. Carry out F. 4-methyl-N-propyl-3-[[(2RS)-2-(propylamino)propa-
a potentiometric titration (2.2.20) using 0.1 M sodium noyl]amino]thiophene-2-carboxamide (articaine acid
hydroxide. Read the volume added between the 2 points of propionamide),
inflexion.
1 mL of 0.1 M sodium hydroxide is equivalent to 32.08 mg
of C13H21ClN2O3S.
STORAGE
Protected from light.
IMPURITIES G. methyl 3-[[(2RS)-2-(butylamino)propanoyl]amino]-4-
Specified impurities : A. methylthiophene-2-carboxylate (butylarticaine),
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use) : H. methyl 3-[[(2RS)-2-(dipropylamino)propanoyl]amino]-4-
B, C, D, E, F, G, H, I, J. methylthiophene-2-carboxylate (dipropylarticaine),

General Notices (1) apply to all monographs and other texts 1589
Ascorbic acid EUROPEAN PHARMACOPOEIA 8.0

Specific optical rotation (2.2.7) : + 20.5 to + 21.5.

Dissolve 2.50 g in water R and dilute to 25.0 mL with the same
solvent.
Impurity E : maximum 0.2 per cent.
Test solution. Dissolve 0.25 g in 5 mL of water R. Neutralise
I. methyl 3-amino-4-methylthiophene-2-carboxylate using dilute sodium hydroxide solution R and add 1 mL of
(3-aminoarticaine), dilute acetic acid R and 0.5 mL of calcium chloride solution R.
Reference solution. Dissolve 70 mg of oxalic acid R in water R
and dilute to 500 mL with the same solvent ; to 5 mL of this
solution add 1 mL of dilute acetic acid R and 0.5 mL of calcium
chloride solution R.
Allow the solutions to stand for 1 h. Any opalescence in the
J. methyl 3-[[(2RS)-2-bromopropanoyl]amino]-4- test solution is not more intense than that in the reference
methylthiophene-2-carboxylate (bromo compound). solution.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
01/2011:0253
Phosphate buffer solution. Dissolve 6.8 g of potassium
dihydrogen phosphate R in water R and dilute to about 175 mL
ASCORBIC ACID with the same solvent. Filter through a membrane filter
(nominal pore size 0.45 μm) and dilute to 1000 mL with
Acidum ascorbicum water R.
Test solution. Dissolve 0.500 g of the substance to be examined
in the mobile phase and dilute to 10.0 mL with the mobile
phase.
Reference solution (a). Dissolve 10.0 mg of ascorbic acid
impurity C CRS in the mobile phase and dilute to 5.0 mL with
the mobile phase.
C 6H 8O 6 Mr 176.1
[50-81-7] Reference solution (b). Dissolve 5.0 mg of ascorbic acid
impurity D CRS and 5.0 mg of ascorbic acid CRS in the mobile
DEFINITION phase, add 2.5 mL of reference solution (a) and dilute to
(5R)-5-[(1S)-1,2-Dihydroxyethyl]-3,4-dihydroxyfuran-2(5H)- 100.0 mL with the mobile phase.
one. Reference solution (c). Dilute 1.0 mL of the test solution to
Content : 99.0 per cent to 100.5 per cent. 200.0 mL with the mobile phase. Mix 1.0 mL of this solution
with 1.0 mL of reference solution (a).
CHARACTERS Column :
Appearance : white or almost white, crystalline powder or – size : l = 0.25 m, Ø = 4.6 mm ;
colourless crystals, becoming discoloured on exposure to air – stationary phase : aminopropylsilyl silica gel for
and moisture. chromatography R (5 μm) ;
Solubility : freely soluble in water, sparingly soluble in ethanol
– temperature : 45 °C.
(96 per cent).
Mobile phase : phosphate buffer solution, acetonitrile R1
mp : about 190 °C, with decomposition.
(25:75 V/V).
IDENTIFICATION Flow rate : 1.0 mL/min.
First identification : B, C. Detection : spectrophotometer at 210 nm.
Second identification : A, C, D. Injection : 20 μL of the test solution and reference solutions (b)
A. Ultraviolet and visible absorption spectrophotometry and (c).
(2.2.25). Run time : 2.5 times the retention time of ascorbic acid.
Test solution. Dissolve 0.10 g in water R and dilute Identification of impurities: use the chromatogram obtained
immediately to 100.0 mL with the same solvent. Add with reference solution (b) to identify the peaks due to
1.0 mL of this solution to 10 mL of 0.1 M hydrochloric acid impurities C and D.
and dilute to 100.0 mL with water R. Relative retention with reference to ascorbic acid
Absorption maximum : at 243 nm, determined immediately (retention time = about 11 min) : impurity D = about 0.4 ;
after dissolution. impurity C = about 1.7.
Specific absorbance at the absorption maximum : 545 to 585. System suitability :
B. Infrared absorption spectrophotometry (2.2.24). – resolution : minimum 3.0 between the peaks due to ascorbic
Comparison : ascorbic acid CRS. acid and impurity C in the chromatogram obtained with
C. pH (2.2.3) : 2.1 to 2.6 for solution S (see Tests). reference solution (c) ;
D. To 1 mL of solution S add 0.2 mL of dilute nitric acid R – signal-to-noise ratio : minimum 20 for the peak due to
and 0.2 mL of silver nitrate solution R2. A grey precipitate impurity C in the chromatogram obtained with reference
is formed. solution (b).
Limits :
TESTS – impurities C, D : for each impurity, not more than 1.5 times
Solution S. Dissolve 1.0 g in carbon dioxide-free water R and the area of the corresponding peak in the chromatogram
dilute to 20 mL with the same solvent. obtained with reference solution (b) (0.15 per cent) ;
Appearance of solution. Solution S is clear (2.2.1) and not – unspecified impurities : for each impurity, not more than the
more intensely coloured than reference solution BY7 (2.2.2, area of the peak due to ascorbic acid in the chromatogram
Method II). obtained with reference solution (b) (0.10 per cent) ;

1590 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ascorbyl palmitate

– total of impurities other than C and D : not more than

twice the area of the peak due to ascorbic acid in the
chromatogram obtained with reference solution (b) (0.2 per
cent) ;
– disregard limit : 0.5 times the area of the peak due to D. methyl D-xylo-hex-2-ulosonate (methyl D-sorbosonate),
ascorbic acid in the chromatogram obtained with reference
solution (b) (0.05 per cent).
Copper : maximum 5 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
E. oxalic acid,
Test solution. Dissolve 2.0 g in 0.1 M nitric acid and dilute to
25.0 mL with the same acid.
Reference solutions. Prepare the reference solutions (0.2 ppm,
0.4 ppm and 0.6 ppm) by diluting copper standard solution
(10 ppm Cu) R with 0.1 M nitric acid.
Source : copper hollow-cathode lamp.
F. (5R)-5-[(1R)-1,2-dihydroxyethyl]-3,4-dihydroxyfuran-
Wavelength : 324.8 nm. 2(5H)-one,
Atomisation device : air-acetylene flame.
Adjust the zero of the apparatus using 0.1 M nitric acid.
Iron : maximum 2 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Dissolve 5.0 g in 0.1 M nitric acid and dilute to
25.0 mL with the same acid. G. (2R)-2-[(2R)-3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl]-
Reference solutions. Prepare the reference solutions (0.2 ppm, 2-hydroxyacetic acid,
0.4 ppm and 0.6 ppm) by diluting iron standard solution
(20 ppm Fe) R with 0.1 M nitric acid.
Source : iron hollow-cathode lamp.
Wavelength : 248.3 nm.
Atomisation device : air-acetylene flame.
Adjust the zero of the apparatus using 0.1 M nitric acid.
H. methyl (2R)-2-[(2R)-3,4-dihydroxy-5-oxo-2,5-
Heavy metals (2.4.8) : maximum 10 ppm. dihydrofuran-2-yl]-2-hydroxyacetate.
Dissolve 2.0 g in water R and dilute to 20 mL with the same
solvent. 12 mL of the solution complies with test A. Prepare 04/2013:0807
the reference solution using lead standard solution (1 ppm
Pb) R. ASCORBYL PALMITATE
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. Ascorbylis palmitas
ASSAY
Dissolve 0.150 g in a mixture of 10 mL of dilute sulfuric
acid R and 80 mL of carbon dioxide-free water R. Add 1 mL of
starch solution R. Titrate with 0.05 M iodine until a persistent
violet-blue colour is obtained.
1 mL of 0.05 M iodine is equivalent to 8.81 mg of C6H8O6.
C22H38O7 Mr 414.5
STORAGE [137-66-6]
In a non-metallic container, protected from light.
DEFINITION
IMPURITIES (2S)-2-[(2R)-3,4-Dihydroxy-5-oxo-2,5-dihydrofuran-2-yl]-2-
Specified impurities : C, D, E. hydroxyethyl hexadecanoate.
Other detectable impurities (the following substances would, Content : 98.0 per cent to 100.5 per cent (dried substance).
if present at a sufficient level, be detected by one or other of
CHARACTERS
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or Appearance : white or yellowish-white powder.
by the general monograph Substances for pharmaceutical use Solubility : practically insoluble in water, freely soluble in
(2034). It is therefore not necessary to identify these impurities ethanol (96 per cent) and in methanol, practically insoluble
for demonstration of compliance. See also 5.10. Control of in methylene chloride and in fatty oils.
impurities in substances for pharmaceutical use) : A, F, G, H.
IDENTIFICATION
A. Specific optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: ascorbyl palmitate CRS.
A. 2-furaldehyde,
C. Dissolve about 10 mg in 5 mL of methanol R. The solution
decolourises dichlorophenolindophenol standard solution R.
TESTS
Solution S. Dissolve 2.50 g in methanol R and dilute to
C. D-xylo-hex-2-ulosonic acid (D-sorbosonic acid), 25.0 mL with the same solvent.

General Notices (1) apply to all monographs and other texts 1591
Asparagine monohydrate EUROPEAN PHARMACOPOEIA 8.0

Appearance of solution. Solution S is clear (2.2.1) and not pH (2.2.3) : 4.0 to 6.0 for solution S.
more intensely coloured than reference solution BY4 (2.2.2, Specific optical rotation (2.2.7) : + 33.7 to + 36.0 (dried
Method I). substance).
Specific optical rotation (2.2.7) : + 21 to + 24 (dried Dissolve 2.50 g in a 309.0 g/L solution of hydrochloric acid R
substance), determined on solution S. and dilute to 25.0 mL with the same acid.
Related substances. The thresholds indicated under Related Ninhydrin-positive substances. Thin-layer chromatography
substances (Table 2034.-1) in the general monograph (2.2.27).
Substances for pharmaceutical use (2034) do not apply. Test solution (a). Dissolve 0.25 g of the substance to be
Heavy metals (2.4.8) : maximum 10 ppm. examined in water R, heating to not more than 40 °C, and
2.0 g complies with test C. Prepare the reference solution using dilute to 10 mL with the same solvent.
2 mL of lead standard solution (10 ppm Pb) R. Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
Loss on drying (2.2.32) : maximum 1.0 per cent, determined with water R.
on 1.000 g by drying in vacuo at 60 °C for 5 h. Reference solution (a). Dilute 1.0 mL of test solution (a) to
200 mL with water R.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. Reference solution (b). Dissolve 25 mg of glutamic acid R in
water R, add 1 mL of test solution (a) and dilute to 10 mL
ASSAY with water R.
Dissolve 0.200 g in 50 mL of ethanol (96 per cent) R. Add Reference solution (c). Dissolve 25 mg of asparagine
30 mL of water R and titrate with 0.05 M iodine until a yellow monohydrate CRS in water R and dilute to 10 mL with the
colour is obtained. same solvent.
1 mL of 0.05 M iodine is equivalent to 20.73 mg of C22H38O7. Plate : TLC silica gel G plate R.
Mobile phase : glacial acetic acid R, water R, butanol R
STORAGE (25:25:50 V/V/V).
In an airtight container, protected from light. Application : 5 μL.
Development : over half of the plate.
Drying : at 110 °C for 15 min.
07/2010:2086
Detection : spray with ninhydrin solution R and heat at 110 °C
for 10 min.
ASPARAGINE MONOHYDRATE System suitability : reference solution (b) :
– the chromatogram shows 2 clearly separated principal
Asparaginum monohydricum spots.
Limit : test solution (a) :
– any impurity : any spot, apart from the principal spot, is not
more intense than the principal spot in the chromatogram
obtained with reference solution (a) (0.5 per cent).
C4H8N2O3,H2O Mr 150.1
[5794-13-8] Chlorides (2.4.4) : maximum 200 ppm.
Dilute 12.5 mL of solution S to 15 mL with water R.
DEFINITION Sulfates (2.4.13) : maximum 200 ppm.
(2S)-2,4-Diamino-4-oxobutanoic acid monohydrate. To 0.75 g add 2.5 mL of dilute hydrochloric acid R and dilute
Content : 99.0 per cent to 101.0 per cent (dried substance). to 15 mL with distilled water R. Examine after 30 min.
CHARACTERS Ammonium (2.4.1, Method B) : maximum 0.1 per cent,
determined on 10 mg.
Appearance : white or almost white, crystalline powder or
colourless crystals. Iron (2.4.9) : maximum 10 ppm.
Solubility : slightly soluble in water, practically insoluble in Dissolve 1.0 g in dilute hydrochloric acid R and dilute to 10 mL
ethanol (96 per cent) and in methylene chloride. with the same acid. Shake 3 times with 10 mL of methyl
isobutyl ketone R1 for 3 min. Wash the combined organic
IDENTIFICATION phases with 10 mL of water R for 3 min. The aqueous phase
First identification : A, B. complies with the limit test for iron.
Second identification : A, C. Heavy metals (2.4.8) : maximum 10 ppm.
A. Specific optical rotation (see Tests). Dissolve 2.0 g in a mixture of 3 mL of dilute hydrochloric
acid R and 15 mL of water R with gentle warming if necessary.
B. Infrared absorption spectrophotometry (2.2.24). Dilute to 20 mL with water R. 12 mL of the solution complies
Comparison : asparagine monohydrate CRS. with test A. Prepare the reference solution using lead standard
C. Examine the chromatograms obtained in the test for solution (1 ppm Pb) R.
ninhydrin-positive substances. Loss on drying (2.2.32) : 10.5 per cent to 12.5 per cent,
Results : the principal spot in the chromatogram obtained determined on 1.000 g by drying in an oven at 130 °C for 3 h.
with test solution (b) is similar in position, colour and size Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
to the principal spot in the chromatogram obtained with 1.0 g.
reference solution (c).
ASSAY
TESTS
Dissolve 0.110 g in 5 mL of anhydrous formic acid R. Add
Solution S. Dissolve with heating 2.0 g in carbon dioxide-free 50 mL of anhydrous acetic acid R. Titrate with 0.1 M perchloric
water R and dilute to 100 mL with the same solvent. acid, determining the end-point potentiometrically (2.2.20).
Appearance of solution. Solution S is clear (2.2.1) and 1 mL of 0.1 M perchloric acid is equivalent to 13.21 mg
colourless (2.2.2, Method II). of C4H8N2O3.

1592 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Aspartame

IMPURITIES Detection : spray with ninhydrin solution R and heat at

Specified impurities : A, B. 100-105 °C for 15 min.
Results : the spot in the chromatogram obtained with the
test solution is similar in position, colour and size to the
spot in the chromatogram obtained with the reference
solution.
A. (2S)-2-aminobutanedioic acid (aspartic acid),
D. Dissolve about 20 mg in 5 mL of methanol R and add
1 mL of alkaline hydroxylamine solution R1. Heat on a
water-bath for 15 min. Allow to cool and adjust to about
pH 2 with dilute hydrochloric acid R. Add 0.1 mL of ferric
B. (2S)-2-aminopentanedioic acid (glutamic acid). chloride solution R1. A brownish-red colour is produced.
TESTS
01/2008:0973
corrected 6.0 Solution S. Dissolve 0.8 g in carbon dioxide-free water R and
dilute to 100 mL with the same solvent.
ASPARTAME Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution GY6 (2.2.2,
Method II).
Aspartamum
Conductivity (2.2.38) : maximum 30 μS·cm− 1.
Dissolve 0.80 g in carbon dioxide-free water R prepared from
distilled water R and dilute to 100.0 mL with the same solvent.
Measure the conductivity of the solution (C1) and that of the
water used for preparing the solution (C2). The readings must
be stable within 1 per cent over a period of 30 s.
Calculate the conductivity of the solution of the substance to
be examined using the following expression :
C14H18N2O5 Mr 294.3
[22839-47-0]
Specific optical rotation (2.2.7) : + 14.5 to + 16.5 (dried
DEFINITION substance).
(3S)-3-Amino-4-[[(2S)-1-methoxy-1-oxo-3-phenylpropan- Dissolve 2.00 g in a 690 g/L solution of anhydrous formic
2-yl]amino]-4-oxobutanoic acid (methyl α-L-aspartyl-L- acid R and dilute to 50.0 mL with the same solution. Measure
phenylalaninate). within 30 min of preparation.
Content : 98.0 per cent to 102.0 per cent (dried substance). Related substances. Liquid chromatography (2.2.29).
CHARACTERS Test solution. Dissolve 0.60 g of the substance to be examined
Appearance : white or almost white, slightly hygroscopic, in a mixture of 1.5 volumes of glacial acetic acid R and
crystalline powder. 98.5 volumes of water R and dilute to 100.0 mL with the same
mixture of solvents.
Solubility : sparingly soluble or slightly soluble in water and
in ethanol (96 per cent), practically insoluble in hexane and Reference solution (a). Dissolve 4.5 mg of aspartame
in methylene chloride. impurity A CRS in a mixture of 1.5 volumes of glacial acetic
acid R and 98.5 volumes of water R and dilute to 50.0 mL with
IDENTIFICATION the same mixture of solvents.
First identification : B. Reference solution (b). Dissolve 30.0 mg of phenylalanine R
Second identification : A, C, D. (impurity C) in a mixture of 15 volumes of glacial acetic acid R
A. Ultraviolet and visible absorption spectrophotometry and 85 volumes of water R and dilute to 100.0 mL with the
(2.2.25). same mixture of solvents. Dilute 1.0 mL of this solution to
10.0 mL with water R.
Test solution. Dissolve 0.1 g in ethanol (96 per cent) R and
dilute to 100 mL with the same solvent. Reference solution (c). Dilute 5.0 mL of the test solution
to 10.0 mL with water R. Dilute 3.0 mL of this solution to
Spectral range : 230-300 nm. 100.0 mL with water R.
Absorption maxima : at 247 nm, 252 nm, 258 nm and Reference solution (d). Dissolve 30.0 mg of L-aspartyl-L-
264 nm. phenylalanine R (impurity B) in a mixture of 15 volumes of
B. Infrared absorption spectrophotometry (2.2.24). glacial acetic acid R and 85 volumes of water R and dilute to
Preparation : discs. 100.0 mL with the same mixture of solvents. Dilute 1.0 mL
Comparison : aspartame CRS. of the solution to 10.0 mL with water R. Mix 1.0 mL of this
C. Thin-layer chromatography (2.2.27). solution with 1.0 mL of reference solution (b).
Test solution. Dissolve 15 mg of the substance to be Column
examined in 2.5 mL of water R and dilute to 10 mL with – size : l = 0.25 m, Ø = 4.0 mm ;
acetic acid R. – stationary phase : octadecylsilyl silica gel for
Reference solution. Dissolve 15 mg of aspartame CRS in chromatography R (5-10 μm).
2.5 mL of water R and dilute to 10 mL with acetic acid R. Mobile phase : mix 10 volumes of acetonitrile R and 90 volumes
Plate : TLC silica gel G plate R. of a 6.8 g/L solution of potassium dihydrogen phosphate R
Mobile phase : water R, anhydrous formic acid R, methanol R, previously adjusted to pH 3.7 with phosphoric acid R.
methylene chloride R (2:4:30:64 V/V/V/V). Flow rate : 1 mL/min.
Application : 20 μL. Detection : spectrophotometer at 220 nm.
Development : over a path of 15 cm. Injection : 20 μL.
Drying : in air. Run time : twice the retention time of aspartame.

General Notices (1) apply to all monographs and other texts 1593
Aspartic acid EUROPEAN PHARMACOPOEIA 8.0

System suitability : reference solution (d) : 01/2008:0797

– resolution : minimum 3.5 between the peaks due to corrected 6.0
impurities B and C.
Limits : ASPARTIC ACID
– impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (a)
Acidum asparticum
(1.5 per cent) ;
– impurity C : not more than the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.5 per cent) ; C4H7NO4 Mr 133.1
– sum of impurities other than A and C : not more than the [56-84-8]
area of the principal peak in the chromatogram obtained DEFINITION
with reference solution (c) (1.5 per cent) ;
Aspartic acid contains not less than 98.5 per cent and
– disregard limit : disregard any peak due to the solvent. not more than the equivalent of 101.5 per cent of
Heavy metals (2.4.8) : maximum 10 ppm. (2S)-2-aminobutanedioic acid, calculated with reference to the
1.0 g complies with test C. Prepare the reference solution using dried substance.
1 mL of lead standard solution (10 ppm Pb) R. CHARACTERS
Loss on drying (2.2.32) : maximum 4.5 per cent, determined A white or almost white, crystalline powder or colourless
on 1.000 g by drying in an oven at 105 °C. crystals, slightly soluble in water, practically insoluble in
Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on alcohol. It dissolves in dilute mineral acids and in dilute
1.0 g. solutions of alkali hydroxides.
IDENTIFICATION
ASSAY
First identification : A, C.
Dissolve 0.250 g in 1.5 mL of anhydrous formic acid R and
60 mL of anhydrous acetic acid R. Titrate immediately Second identification : A, B, D.
with 0.1 M perchloric acid, determining the end-point A. Specific optical rotation (see Tests).
potentiometrically (2.2.20). B. A suspension of 1 g in 10 mL of water R is strongly acid
1 mL of 0.1 M perchloric acid is equivalent to 29.43 mg (2.2.4).
of C14H18N2O5. C. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
STORAGE aspartic acid CRS. Examine the substances prepared as
In an airtight container. discs.
D. Examine the chromatograms obtained in the test for
IMPURITIES ninhydrin-positive substances. The principal spot in the
Specified impurities : A, C. chromatogram obtained with test solution (b) is similar
in position, colour and size to the principal spot in the
Other detectable impurities (the following substances would, chromatogram obtained with reference solution (a).
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general TESTS
acceptance criterion for other/unspecified impurities and/or Appearance of solution. Dissolve 0.5 g in 1 M hydrochloric
by the general monograph Substances for pharmaceutical acid and dilute to 10 mL with the same acid. The solution is
use (2034). It is therefore not necessary to identify these clear (2.2.1) and not more intensely coloured than reference
impurities for demonstration of compliance. See also 5.10. solution BY6 (2.2.2, Method II).
Control of impurities in substances for pharmaceutical use) : B.
Specific optical rotation (2.2.7). Dissolve 2.000 g in
hydrochloric acid R1 and dilute to 25.0 mL with the same acid.
The specific optical rotation is + 24.0 to + 26.0, calculated with
reference to the dried substance.
Ninhydrin-positive substances. Examine by thin-layer
chromatography (2.2.27), using a TLC silica gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be
A. 2-[(2S,5S)-5-benzyl-3,6-dioxopiperazin-2-yl]acetic acid, examined in 2 mL of ammonia R and dilute to 10 mL with
water R.
Test solution (b). Dilute 1 mL of test solution (a) to 50 mL
with water R.
Reference solution (a). Dissolve 10 mg of aspartic acid CRS in
2 mL of dilute ammonia R1 and dilute to 50 mL with water R.
Reference solution (b). Dilute 5 mL of test solution (b) to
20 mL with water R.
Reference solution (c). Dissolve 10 mg of aspartic acid CRS
B. (3S)-3-amino-4-[[(1S)-1-carboxy-2-phenylethyl]amino]-4- and 10 mg of glutamic acid CRS in 2 mL of dilute ammonia R1
oxobutanoic acid (α-L-aspartyl-L-phenylalanine), and dilute to 25 mL with water R.
Apply separately to the plate 5 μL of each solution. Allow
the plate to dry in air. Develop over a path of 15 cm using a
mixture of 20 volumes of glacial acetic acid R, 20 volumes of
water R and 60 volumes of butanol R. Allow the plate to dry
in air, spray with ninhydrin solution R. Heat at 100-105 °C
C. (2S)-2-amino-3-phenylpropanoic acid (L-phenylalanine). for 15 min. Any spot in the chromatogram obtained with test

1594 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Atenolol

solution (a), apart from the principal spot, is not more intense B. Ultraviolet and visible absorption spectrophotometry
than the spot in the chromatogram obtained with reference (2.2.25).
solution (b) (0.5 per cent). The test is not valid unless the Test solution. Dissolve 0.100 g in methanol R and dilute
chromatogram obtained with reference solution (c) shows 2 to 100 mL with the same solvent. Dilute 10.0 mL of this
clearly separated principal spots. solution to 100 mL with methanol R.
Chlorides (2.4.4). Dissolve 0.25 g in 3 mL of dilute nitric Spectral range : 230-350 nm.
acid R and dilute to 15 mL with water R. The solution, to Absorption maxima : at 275 nm and 282 nm.
which 1 mL of water R is added instead of dilute nitric acid R,
Absorbance ratio : A275/A282 = 1.15 to 1.20.
complies with the limit test for chlorides (200 ppm).
C. Infrared absorption spectrophotometry (2.2.24).
Sulfates (2.4.13). Dissolve 0.5 g in 4 mL of hydrochloric
acid R and dilute to 15 mL with distilled water R. The solution Comparison: atenolol CRS.
complies with the limit test for sulfates (300 ppm). Carry out D. Thin-layer chromatography (2.2.27).
the evaluation of the test after 30 min. Test solution. Dissolve 10 mg of the substance to be
Ammonium.(2.4.1) 50 mg complies with limit test B examined in 1 mL of methanol R.
(200 ppm). Prepare the standard using 0.1 mL of ammonium Reference solution. Dissolve 10 mg of atenolol CRS in 1 mL
standard solution (100 ppm NH4) R. of methanol R.
Iron (2.4.9). In a separating funnel, dissolve 1.0 g in 10 mL of Plate : TLC silanised silica gel F254 plate R.
dilute hydrochloric acid R. Shake with 3 quantities, each of Mobile phase : concentrated ammonia R1, methanol R
10 mL, of methyl isobutyl ketone R1, shaking for 3 min each (1:99 V/V).
time. To the combined organic layers add 10 mL of water R Application : 10 μL.
and shake for 3 min. The aqueous layer complies with the Drying : in air.
limit test for iron (10 ppm).
Detection : examine in ultraviolet light at 254 nm.
Heavy metals (2.4.8). 2.0 g complies with test D (10 ppm). Results : the principal spot in the chromatogram obtained
Prepare the reference solution using 2 mL of lead standard with the test solution is similar in position and size to the
solution (10 ppm Pb) R. principal spot in the chromatogram obtained with the
Loss on drying (2.2.32). Not more than 0.5 per cent, reference solution.
determined on 1.000 g by drying in an oven at 105 °C.
TESTS
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
on 1.0 g. Solution S. Dissolve 0.10 g in water R and dilute to 10 mL
with the same solvent.
ASSAY Appearance of solution. Solution S is clear (2.2.1) and not
Dissolve 0.100 g in 50 mL of carbon dioxide-free water R, more intensely coloured than degree 6 of the range of reference
with slight heating if necessary. Cool and add 0.1 mL of solutions of the most appropriate colour (2.2.2, Method II).
bromothymol blue solution R1. Titrate with 0.1 M sodium
hydroxide until the colour changes from yellow to blue. Optical rotation (2.2.7): + 0.10° to − 0.10°, determined on
solution S.
1 mL of 0.1 M sodium hydroxide is equivalent to 13.31 mg
of C4H7NO4. Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50 mg of the substance to be examined
STORAGE in 20 mL of the mobile phase and dilute to 25.0 mL with the
Protected from light. mobile phase.
Reference solution (a). Dissolve 2 mg of atenolol for system
04/2009:0703 suitability CRS (containing impurities B, F, G, I and J) in
1.0 mL of the mobile phase.
Reference solution (b). Dilute 1.0 mL of the test solution to
ATENOLOL 100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10.0 mL with the mobile phase.
Atenololum Column :
– size : l = 0.125 m, Ø = 4.0 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : dissolve 1.0 g of sodium octanesulfonate R and
C14H22N2O3 Mr 266.3 0.4 g of tetrabutylammonium hydrogen sulfate R in 1 L of a
[29122-68-7] mixture of 20 volumes of tetrahydrofuran R, 180 volumes
of methanol R2, and 800 volumes of a 3.4 g/L solution of
DEFINITION potassium dihydrogen phosphate R ; adjust the apparent pH
2-[4-[(2RS)-2-Hydroxy-3-[(1-methylethyl)amino]propoxy]- to 3.0 with phosphoric acid R.
phenyl]acetamide. Flow rate : 0.6 mL/min.
Content : 99.0 per cent to 101.0 per cent (dried substance). Detection : spectrophotometer at 226 nm.
Injection : 10 μL.
CHARACTERS
Run time : 5 times the retention time of atenolol.
Appearance : white or almost white powder. Identification of impurities : use the chromatogram supplied
Solubility : sparingly soluble in water, soluble in anhydrous with atenolol for system suitability CRS and the chromatogram
ethanol, slightly soluble in methylene chloride. obtained with reference solution (a) to identify the peaks due
to impurities B, F, G, I and J.
IDENTIFICATION
Relative retention with reference to atenolol (retention
First identification : C. time = about 8 min) : impurity B = about 0.3 ; impurity J = about
Second identification : A, B, D. 0.7 ; impurity I = about 0.8 ; impurity F = about 2.0 (pair of
A. Melting point (2.2.14) : 152 °C to 155 °C. peaks) ; impurity G = about 3.5.

General Notices (1) apply to all monographs and other texts 1595
Atomoxetine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

System suitability: reference solution (a) :

– resolution : minimum 1.4 between the peaks due to
impurities J (unidentified impurity) and I.
Limits : F. 2,2′-[[(1-methylethyl)imino]bis[(2-hydroxypropane-3,1-
– correction factor : for the calculation of content, multiply diyl)oxy-4,1-phenylene]]diacetamide,
the peak area of impurity I by 1.5 ;
– impurity B : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (b) (0.2 per cent) ;
– impurities F, G, I : for each impurity, not more than 1.5 times G. 2-[4-[(2RS)-2-hydroxy-3-[(1-methylethyl)amino]-
the area of the principal peak in the chromatogram propoxy]phenyl]acetic acid,
obtained with reference solution (b) (0.15 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.10 per cent) ;
– total : not more than 5 times the area of the principal peak H. 2-[4-[(2RS)-2-hydroxy-3-[(1-methylethyl)amino]-
in the chromatogram obtained with reference solution (b) propoxy]phenyl]acetonitrile,
(0.5 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent). I. 2-[4-[(2RS)-3-(ethylamino)-2-hydroxypropoxy]-
phenyl]acetamide.
Chlorides (2.4.4) : maximum 0.1 per cent.
Dissolve 50 mg in a mixture of 1 mL of dilute nitric acid R and 01/2014:2640
15 mL of water R. The solution, without further addition of
dilute nitric acid R, complies with the test.
ATOMOXETINE HYDROCHLORIDE
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C. Atomoxetini hydrochloridum
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY
Dissolve 0.200 g in 80 mL of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
C17H22ClNO Mr 291.8
1 mL of 0.1 M perchloric acid is equivalent to 26.63 mg of
[82248-59-7]
C14H22N2O3.
DEFINITION
IMPURITIES
(3R)-N-Methyl-3-(2-methylphenoxy)-3-phenylpropan-1-
Specified impurities : B, F, G, I. amine hydrochloride.
Other detectable impurities (the following substances would, Content : 98.0 per cent to 102.0 per cent (dried substance).
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general CHARACTERS
acceptance criterion for other/unspecified impurities and/or Appearance : white or almost white powder.
by the general monograph Substances for pharmaceutical use Solubility : sparingly soluble in water, soluble in anhydrous
(2034). It is therefore not necessary to identify these impurities ethanol, practically insoluble in heptane.
for demonstration of compliance. See also 5.10. Control of
It shows polymorphism (5.9).
impurities in substances for pharmaceutical use): A, D, E, H.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: atomoxetine hydrochloride CRS.
If the spectra obtained in the solid state show differences,
A. R-H : 2-(4-hydroxyphenyl)acetamide, dissolve the substance to be examined and the reference
substance separately in anhydrous ethanol R, evaporate to
dryness and record new spectra using the residues.
B. Isomeric purity (see Tests).
B. 2-[4-[(2RS)-2,3-dihydroxypropoxy]phenyl]acetamide, C. It gives reaction (a) of chlorides (2.3.1).
TESTS
Isomeric purity. Liquid chromatography (2.2.29) : use the
normalisation procedure.
D. 2-[4-[(2RS)-3-chloro-2-hydroxypropoxy]phenyl]acet- Test solution. Dissolve 35.0 mg of the substance to be
amide, examined in 2.5 mL of anhydrous ethanol R, sonicate until
dissolution is complete and dilute to 10.0 mL with heptane R.
Reference solution (a). Dissolve 3.5 mg of atomoxetine
impurity B CRS and 1 mg of atomoxetine impurity D CRS in
E. 2,2′-[(2-hydroxypropane-1,3-diyl)bis(oxy-4,1- 5 mL of anhydrous ethanol R, sonicate until dissolution is
phenylene)]diacetamide, complete and dilute to 20.0 mL with heptane R.

1596 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Atomoxetine hydrochloride

Reference solution (b). Dissolve 35.0 mg of the substance to be Detection : spectrophotometer at 215 nm.
examined in 2.5 mL of anhydrous ethanol R. Add 1.0 mL of Injection : 10 μL of test solution (a) and reference solutions (a),
reference solution (a) and dilute to 10.0 mL with heptane R. (b) and (c).
Reference solution (c). Dilute 1.0 mL of reference solution (a) Run time : 2.5 times the retention time of atomoxetine.
to 100.0 mL with heptane R. Identification of impurities: use the chromatogram obtained
Column : with reference solution (b) to identify the peaks due to
– size : l = 0.25 m, Ø = 4.6 mm ; impurities E and H ; use the chromatogram supplied with
– stationary phase : cellulose derivative of silica gel for chiral atomoxetine for impurity A identification CRS and the
separation R (5 μm). chromatogram obtained with reference solution (c) to identify
Mobile phase : mix 1.5 mL of diethylamine R, 2.0 mL of the peak due to impurity A.
trifluoroacetic acid R and 150.0 mL of 2-propanol R and dilute Relative retention with reference to atomoxetine
to 1000 mL with heptane R. (retention time = about 10 min) : impurity E = about 0.2 ;
Flow rate : 1.0 mL/min. impurity H = about 0.3 ; impurity A = about 0.7.
Detection : spectrophotometer at 273 nm. System suitability : reference solution (b) :
Injection : 10 μL of the test solution and reference solutions (b) – resolution : minimum 5.0 between the peaks due to
and (c). impurities E and H.
Run time : 1.3 times the retention time of atomoxetine. Calculation of percentage contents :
Identification of impurities : use the chromatogram obtained – for each impurity, use the concentration of atomoxetine
with reference solution (b) to identify the peaks due to hydrochloride in reference solution (a).
impurities B and D. Limits :
Relative retention with reference to atomoxetine – impurity A : maximum 0.3 per cent ;
(retention time = about 12 min) : impurity B = about 0.5 ; – unspecified impurities : for each impurity, maximum
impurity D = about 0.6. 0.10 per cent ;
System suitability : reference solution (b) : – total : maximum 0.5 per cent ;
– resolution : minimum 1.8 between the peaks due to – reporting threshold : 0.05 per cent.
impurities B and D.
Heavy metals (2.4.8) : maximum 10 ppm.
Limits :
Solvent mixture : water R, methanol R (20:80 V/V).
– impurity B : maximum 0.5 per cent ;
0.250 g complies with test H. Prepare the reference solution
– impurity D : maximum 0.15 per cent ; using 0.25 mL of lead standard solution (10 ppm Pb) R.
– unspecified impurities : for each impurity, maximum Loss on drying (2.2.32) : maximum 0.5 per cent, determined
0.10 per cent ; on 1.000 g by drying in vacuo at 105 °C for 2 h.
– disregard limit : the area of the peak due to impurity B in
the chromatogram obtained with reference solution (c) Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
(0.05 per cent); disregard any peak with a relative retention 1.0 g.
with reference to atomoxetine of about 0.7 (impurity A). ASSAY
Related substances. Liquid chromatography (2.2.29). Liquid chromatography (2.2.29) as described in the test for
Solution A. Dissolve 5.9 g of sodium octanesulfonate related substances with the following modification.
monohydrate R in 1000 mL of a 2.9 g/L solution of phosphoric Injection : test solution (b) and reference solution (d).
acid R previously adjusted to pH 2.5 with a 280 g/L solution of
Calculate the percentage content of C17H22ClNO taking
potassium hydroxide R.
into account the assigned content of atomoxetine
Test solution (a). Dissolve 25 mg of the substance to be hydrochloride CRS.
examined in the mobile phase and dilute to 10.0 mL with the
mobile phase. IMPURITIES
Test solution (b). Dissolve 25.0 mg of the substance to be Specified impurities : A, B, D.
examined in the mobile phase and dilute to 100.0 mL with Other detectable impurities (the following substances would,
the mobile phase. if present at a sufficient level, be detected by one or other of
Reference solution (a). Dilute 1.0 mL of test solution (a) to the tests in the monograph. They are limited by the general
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution acceptance criterion for other/unspecified impurities and/or
to 10.0 mL with the mobile phase. by the general monograph Substances for pharmaceutical use
Reference solution (b). Dissolve 7.5 mg of 3-(methylamino)- (2034). It is therefore not necessary to identify these impurities
1-phenylpropan-1-ol R (impurity H) and 5 mg of mandelic for demonstration of compliance. See also 5.10. Control of
acid R (impurity E) in test solution (b) and dilute to 50 mL impurities in substances for pharmaceutical use) : C, E, F, G, H.
with test solution (b).
Reference solution (c). Dissolve 5 mg of atomoxetine for
impurity A identification CRS in the mobile phase and dilute
to 20 mL with the mobile phase.
Reference solution (d). Dissolve 25.0 mg of atomoxetine
hydrochloride CRS in the mobile phase and dilute to 100.0 mL
with the mobile phase. A. N-methyl-3-phenoxy-3-phenylpropan-1-amine,
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : end-capped octylsilyl silica gel for
chromatography R (3.5 μm) ;
– temperature : 40 °C.
Mobile phase : propanol R, solution A (27:73 V/V). B. (3S)-N-methyl-3-(2-methylphenoxy)-3-phenylpropan-1-
Flow rate : 1.0 mL/min. amine,

General Notices (1) apply to all monographs and other texts 1597
Atorvastatin calcium trihydrate EUROPEAN PHARMACOPOEIA 8.0

Content : 97.0 per cent to 102.0 per cent (anhydrous substance).

CHARACTERS
Appearance : white or almost white powder.
Solubility : very slightly soluble in water, slightly soluble in
C. (3R)-N-methyl-3-(4-methylphenoxy)-3-phenylpropan-1- ethanol (96 per cent), practically insoluble in methylene
amine, chloride.
It shows polymorphism (5.9).
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: atorvastatin calcium trihydrate CRS.
D. (3R)-N-methyl-3-(3-methylphenoxy)-3-phenylpropan-1- If the spectra obtained in the solid state show differences,
amine, dissolve the substance to be examined and the reference
substance separately in methanol R, evaporate to dryness
and record new spectra using the residues.
B. Enantiomeric purity (see Tests).
C. Water (see Tests).
D. Ignite. The residue gives reaction (b) of calcium (2.3.1).
E. (2S)-2-hydroxy-2-phenylacetic acid (L-mandelic acid), Filtration may be necessary in case the residue does not
completely dissolve.
TESTS
Enantiomeric purity. Liquid chromatography (2.2.29).
Solvent mixture : anhydrous ethanol R, methanol R (50:50 V/V).
Test solution. Dissolve 10 mg of the substance to be examined
F. (3S)-3-(3-fluoro-2-methylphenoxy)-N-methyl-3- in 4 mL of the solvent mixture and dilute to 10.0 mL with
phenylpropan-1-amine, hexane R.
Reference solution (a). Dissolve 2 mg of atorvastatin
impurity E CRS in methanol R and dilute to 20.0 mL with the
same solvent (solution A). Dissolve 10 mg of the substance
to be examined in 1.25 mL of methanol R, add 0.75 mL of
solution A and 2 mL of anhydrous ethanol R and dilute to
10.0 mL with hexane R.
Reference solution (b). To 2.0 mL of the test solution add
G. 3,3′-[(2-methylbenzene-1,3-diyl)bis(oxy)]bis(N-methyl-
40.0 mL of the solvent mixture and dilute to 100.0 mL with
3-phenylpropan-1-amine),
hexane R. To 3.0 mL of this solution add 5 mL of the solvent
mixture and dilute to 20.0 mL with hexane R.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : amylose derivative of silica gel for
chromatography R (10 μm).
H. 3-(methylamino)-1-phenylpropan-1-ol. Mobile phase : trifluoroacetic acid R, anhydrous ethanol R,
hexane R (0.1:6:94 V/V/V).
Flow rate : 1.0 mL/min.
04/2011:2191 Detection : spectrophotometer at 244 nm.
Injection : 20 μL.
ATORVASTATIN CALCIUM Run time : 1.2 times the retention time of atorvastatin.
TRIHYDRATE Relative retention with reference to atorvastatin (retention
time = about 44 min): impurity E = about 0.8.
Atorvastatinum calcicum trihydricum System suitability : reference solution (a) :
– resolution : minimum 2.0 between the peaks due to
impurity E and atorvastatin.
Limit :
– impurity E : not more than the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.3 per cent).
Related substances. Liquid chromatography (2.2.29).
Test solution (a). Dissolve 40.0 mg of the substance to be
examined in dimethylformamide R and dilute to 100.0 mL
C66H68CaF2N4O10,3H2O Mr 1209 with the same solvent.
[344423-98-9] Test solution (b). Dissolve 50 mg of the substance to be
examined in dimethylformamide R and dilute to 50.0 mL with
DEFINITION the same solvent.
Calcium (3R,5R)-7-[2-(4-fluorophenyl)-5-(1-methylethyl)- Reference solution (a). Dissolve 40.0 mg of atorvastatin
3-phenyl-4-(phenylcarbamoyl)-1H-pyrrol-1-yl]-3,5- calcium trihydrate CRS in dimethylformamide R and dilute to
dihydroxyheptanoate trihydrate. 100.0 mL with the same solvent.

1598 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Atorvastatin calcium trihydrate

Reference solution (b). Dilute 1.0 mL of test solution (b) to Sodium : maximum 0.4 per cent (anhydrous substance).
100.0 mL with dimethylformamide R. Dilute 1.0 mL of this Atomic absorption spectrometry (2.2.23, Method I).
solution to 10.0 mL with dimethylformamide R.
Solvent mixture: hydrochloric acid R, water R, methanol R
Reference solution (c). Dissolve 2.5 mg of atorvastatin (2:25:75 V/V/V).
impurity A CRS, 2.5 mg of atorvastatin impurity B CRS,
2.5 mg of atorvastatin impurity C CRS, 2.5 mg of atorvastatin Test solution. Dissolve 5.0 mg in the solvent mixture and
impurity D CRS and 2.5 mg of the substance to be examined dilute to 100.0 mL with the solvent mixture.
in dimethylformamide R and dilute to 50.0 mL with the same Reference solutions. Prepare the reference solutions using
solvent. sodium standard solution (50 ppm Na) R, diluting with the
Column : solvent mixture.
– size : l = 0.25 m, Ø = 4.6 mm ; Source : sodium hollow-cathode lamp.
– stationary phase : octylsilyl silica gel for chromatography R Wavelength : 589.0 nm.
(5 μm) ; Atomisation device : air-acetylene flame.
– temperature : 35 °C. Heavy metals (2.4.8) : maximum 20 ppm.
Mobile phase : Solvent mixture : water R, methanol R (10:90 V/V).
– mobile phase A : tetrahydrofuran R, acetonitrile R, 3.9 g/L It complies with test H with the following modifications.
solution of ammonium acetate R adjusted to pH 5.0 with Test solution. Dissolve 0.250 g of the substance to be examined
glacial acetic acid R (12:21:67 V/V/V) ; in 30 mL of the solvent mixture.
– mobile phase B : tetrahydrofuran R, 3.9 g/L solution of Reference solution. Dilute 0.5 mL of lead standard solution
ammonium acetate R adjusted to pH 5.0 with glacial acetic (10 ppm Pb) R to 30 mL with the solvent mixture.
acid R, acetonitrile R (12:27:61 V/V/V) ;
Blank solution : 30 mL of the solvent mixture.
Time Mobile phase A Mobile phase B
Water (2.5.12) : 3.5 per cent to 5.5 per cent, determined on
(min) (per cent V/V) (per cent V/V)
0.130 g.
0 - 40 100 0

40 - 70 100 → 20 0 → 80 ASSAY
70 - 85 20 → 0 80 → 100 Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Flow rate : 1.5 mL/min. Injection : test solution (a) and reference solution (a).
Detection : spectrophotometer at 244 nm. Calculate the percentage content of C66H68CaF2N4O10 from the
Injection : 20 μL of test solution (b) and reference solutions (b) declared content of atorvastatin calcium trihydrate CRS.
and (c).
IMPURITIES
Identification of impurities : use the chromatogram obtained
with reference solution (c) to identify the peaks due to Specified impurities : A, B, C, D, E.
impurities A, B, C and D. Other detectable impurities (the following substances would,
Relative retention with reference to atorvastatin if present at a sufficient level, be detected by one or other of
(retention time = about 33 min) : impurity A = about 0.8 ; the tests in the monograph. They are limited by the general
impurity B = about 0.9 ; impurity C = about 1.2 ; acceptance criterion for other/unspecified impurities and/or
impurity D = about 2.1. by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
If necessary, adjust the mobile phase by increasing or for demonstration of compliance. See also 5.10. Control of
decreasing the percentage of acetonitrile or the pH of the impurities in substances for pharmaceutical use) : F, G, H.
ammonium acetate solution to achieve a retention time of
about 33 min for atorvastatin. For example, raising the pH
would decrease the retention time of atorvastatin.
System suitability: reference solution (c) :
– resolution : minimum 1.5 between the peaks due to
impurity B and atorvastatin.
Limits :
– impurities A, B : for each impurity, not more than 3 times
the area of the principal peak in the chromatogram A. (3R,5R)-3,5-dihydroxy-7-[5-(1-methylethyl)-2,3-diphenyl-
obtained with reference solution (b) (0.3 per cent) ; 4-(phenylcarbamoyl)-1H-pyrrol-1-yl]heptanoic acid
– impurities C, D : for each impurity, not more than 1.5 times (desfluoroatorvastatin),
the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.15 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.10 per cent) ;
– total : not more than 15 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(1.5 per cent) ;
– disregard limit : 0.5 times the area of the principal
peak in the chromatogram obtained with reference B. (3RS,5SR)-7-[2-(4-fluorophenyl)-5-(1-methylethyl)-
solution (b) (0.05 per cent) ; disregard the peak due to 3-phenyl-4-(phenylcarbamoyl)-1H-pyrrol-1-yl]-3,5-
dimethylformamide. dihydroxyheptanoic acid,

General Notices (1) apply to all monographs and other texts 1599
Atovaquone EUROPEAN PHARMACOPOEIA 8.0

C. (3R,5R)-7-[2,3-bis(4-fluorophenyl)-5-(1-methylethyl)-
4-(phenylcarbamoyl)-1H-pyrrol-1-yl]-3,5- H. (4R,6R)-6-[2-[2-(4-fluorophenyl)-5-(1-methylethyl)-3-
dihydroxyheptanoic acid (fluoroatorvastatin), phenyl-4-(phenylcarbamoyl)-1H-pyrrol-1-yl]ethyl]-4-
hydroxytetrahydro-2H-pyran-2-one.

07/2013:2192

ATOVAQUONE
Atovaquonum
D. 3-[(4-fluorophenyl)carbonyl]-2-(2-methylpropanoyl)-N,3-
diphenyloxirane-2-carboxamide,

C22H19ClO3 Mr 366.8
[95233-18-4]
DEFINITION
2-[trans-4-(4-Chlorophenyl)cyclohexyl]-3-hydroxynaphtha-
lene-1,4-dione.
Content : 97.5 per cent to 102.0 per cent (anhydrous substance).

E. (3S,5S)-7-[2-(4-fluorophenyl)-5-(1-methylethyl)-3- CHARACTERS
phenyl-4-(phenylcarbamoyl)-1H-pyrrol-1-yl]-3,5- Appearance : yellow, crystalline powder.
dihydroxyheptanoic acid (ent-atorvastatin), Solubility : practically insoluble in water, sparingly soluble in
methylene chloride, very slightly soluble in methanol.
It shows polymorphism (5.9).
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: atovaquone CRS.
If the spectra obtained show differences, dissolve 0.1 g of the
substance to be examined and 0.1 g of the reference substance
separately in 2.5 mL of a 50 g/L solution of potassium
hydroxide R in methanol R. Filter the solutions and add each
filtrate dropwise to a mixture of 0.8 mL of acetic acid R and
1.5 mL of methanol R, stirring continuously. Filter, wash the
residues with methanol R and then with water R, and dry
F. (3R,5R)-7-[[(3R,5R)-7-[2-(4-fluorophenyl)-5-(1- under vacuum at 55 °C. Record new spectra using the residues.
methylethyl)-3-phenyl-4-(phenylcarbamoyl)-1H- TESTS
pyrrol-1-yl]-3,5-dihydroxyheptanoyl]amino]-3,5-
dihydroxyheptanoic acid, Related substances. Liquid chromatography (2.2.29). Carry
out the test protected from light.
Solvent mixture : water R, acetonitrile R1 (20:80 V/V).
Test solution. Dissolve 25.0 mg of the substance to be
examined in the solvent mixture and dilute to 100.0 mL with
the solvent mixture.
Reference solution (a). Dissolve 25.0 mg of atovaquone CRS
in the solvent mixture and dilute to 100.0 mL with the solvent
mixture.
Reference solution (b). Dissolve 2.5 mg of atovaquone for
system suitability CRS (containing impurities B and C) in the
solvent mixture and dilute to 10.0 mL with the solvent mixture.
G. (3R,5R)-7-[2-(4-fluorophenyl)-5-(1-methylethyl)-3- Reference solution (c). Dilute 1.0 mL of the test solution to
phenyl-4-(phenylcarbamoyl)-1H-pyrrol-1-yl]-5-hydroxy- 100.0 mL with the solvent mixture. Dilute 1.0 mL of this
3-methoxyheptanoic acid (3-O-methylatorvastatin), solution to 10.0 mL with the solvent mixture.

1600 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Atracurium besilate

Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : phosphoric acid R, methanol R2, water for
chromatography R, acetonitrile R1 (0.5:17.5:30:52.5 V/V/V/V). B. 2-[cis-4-(4-chlorophenyl)cyclohexyl]-3-hydroxynaphtha-
Flow rate : 2.5 mL/min. lene-1,4-dione,
Detection : spectrophotometer at 220 nm.
Injection : 20 μL of the test solution and reference solutions (b)
and (c).
Run time : twice the retention time of atovaquone.
Identification of impurities : use the chromatogram supplied
with atovaquone for system suitability CRS and the
chromatogram obtained with reference solution (b) to identify C. 2-[(1RS)-4-(4-chlorophenyl)cyclohex-3-en-1-yl]-3-
the peaks due to impurities B and C. hydroxynaphthalene-1,4-dione,
Relative retention with reference to atovaquone (retention
time = about 15 min): impurity B = about 0.85 ;
impurity C = about 0.90.
System suitability : reference solution (b) :
– resolution : minimum 2.0 between the peaks due to
impurity C and atovaquone ;
D. 2-[trans-4-(4-chlorophenyl)cyclohexyl]-3-methoxy-
– peak-to-valley ratio : minimum 1.5, where Hp = height naphthalene-1,4-dione.
above the baseline of the peak due to impurity C and
Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to impurity B. 04/2013:1970
Calculation of percentage contents :
– for each impurity, use the concentration of atovaquone in ATRACURIUM BESILATE
reference solution (c).
Limits : Atracurii besilas
– impurity B : maximum 0.5 per cent ;
– impurity C : maximum 0.2 per cent ;
– unspecified impurities : for each impurity, maximum
0.10 per cent ;
– total : maximum 0.6 per cent ;
– reporting threshold : 0.05 per cent.
Water (2.5.32) : maximum 0.3 per cent, determined on 0.100 g
using the evaporation technique :
– temperature : 160 °C ;
– heating time : 3 min ;
– flow rate : 50 mL/min.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY C65H82N2O18S2 Mr 1243
Liquid chromatography (2.2.29) as described in the test for [64228-81-5]
related substances with the following modification.
DEFINITION
Injection : test solution and reference solution (a).
Mixture of the cis-cis, cis-trans and trans-trans isomers of
Calculate the percentage content of C22H19ClO3 taking into 2,2′-[pentane-1,5-diylbis[oxy(3-oxopropane-1,3-diyl)]]bis[1-
account the assigned content of atovaquone CRS. (3,4-dimethoxybenzyl)-6,7-dimethoxy-2-methyl-1,2,3,4-
IMPURITIES tetrahydroisoquinolinium] dibenzenesulfonate.
Specified impurities : B, C. Content : 96.0 per cent to 102.0 per cent (anhydrous substance).
Other detectable impurities (the following substances would, CHARACTERS
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general Appearance : white or yellowish-white, slightly hygroscopic
acceptance criterion for other/unspecified impurities and/or powder.
by the general monograph Substances for pharmaceutical use Solubility : soluble in water, very soluble in acetonitrile, in
(2034). It is therefore not necessary to identify these impurities ethanol (96 per cent) and in methylene chloride.
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : A, D. IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: atracurium besilate CRS.
B. Examine the chromatograms obtained in the assay.
Results : the 3 principal isomeric peaks in the chromatogram
obtained with test solution (a) are similar in retention time
A. 2-[trans-4-(4-chlorophenyl)cyclohexyl]-1-oxo-1H-indene- to those in the chromatogram obtained with reference
3-carboxylic acid, solution (a).

General Notices (1) apply to all monographs and other texts 1601
Atracurium besilate EUROPEAN PHARMACOPOEIA 8.0

TESTS Relative retention with reference to the atracurium cis-cis

Solution S. Dissolve 1.00 g in water R and dilute to 100 mL isomer (retention time = about 30 min) : impurity E = about 0.2 ;
with the same solvent. impurity F = about 0.25 ; impurity G = about 0.3 ;
impurity D1 = about 0.45 ; impurity D2 = about 0.5 ;
Appearance of solution. Solution S is clear (2.2.1) and not atracurium trans-trans isomer = about 0.8 ; atracurium
more intensely coloured than reference solution Y7 (2.2.2, cis-trans isomer = about 0.9 ; impurity A1 = about 1.04 ;
Method II). impurity I1 = about 1.07 ; impurity H1 = about 1.07
Related substances. Liquid chromatography (2.2.29). (shoulder on the front of peak A2) ; impurity A2 (major
isomer) = about 1.08 ; impurity K1 = about 1.09 (shoulder on
Test solution (a). Dissolve 50.0 mg of the substance to be the tail of peak A2) ; impurity I2 (major isomer) = about 1.12 ;
examined in mobile phase A and dilute to 50.0 mL with impurity H2 (major isomer) = about 1.12 ; impurity K2
mobile phase A. (major isomer) = about 1.12 ; impurity B = about 1.15 ;
Test solution (b). Dissolve 0.100 g of the substance to be impurity C1 = about 1.2 ; impurity C2 (major
examined in mobile phase A and dilute to 10.0 mL with isomer) = about 1.3.
mobile phase A. System suitability :
Reference solution (a). Dissolve 50.0 mg of atracurium – resolution : minimum 1.5 between the peaks due to the
besilate CRS in mobile phase A and dilute to 50.0 mL with atracurium trans-trans isomer and the atracurium cis-trans
mobile phase A. isomer, and minimum 1.5 between the peaks due to the
Reference solution (b). Dilute 1.0 mL of test solution (a) to atracurium cis-trans isomer and the atracurium cis-cis
100.0 mL with mobile phase A. isomer in the chromatogram obtained with reference
solution (a);
Reference solution (c). Dissolve 20.0 mg of methyl
benzenesulfonate R in acetonitrile R and dilute to 100.0 mL – peak-to-valley ratio : minimum 1.2, where Hp = height
with the same solvent. Dilute 50 μL of the solution to 100.0 mL above the baseline of the peak due to impurity A1 and
with mobile phase A. Hv = height above the baseline of the lowest point of
the curve separating this peak from the peak due to the
Reference solution (d). Dissolve 2.0 mg of atracurium for peak atracurium cis-cis isomer in the chromatogram obtained
identification CRS (containing impurities A1, A2, B, C1, C2, with reference solution (d).
D1, D2, E, G and K) in 2.0 mL of mobile phase A.
Limits :
Reference solution (e). Dissolve 2.0 mg of atracurium for
impurity F identification CRS in 2.0 mL of mobile phase A. – correction factor : for the calculation of content, multiply
the peak area of impurity G by 0.5 ;
Column :
– impurity E : not more than 1.5 times the sum of the areas
– size : l = 0.25 m, Ø = 4.6 mm ; of the peaks due to the atracurium cis-cis, trans-trans and
– stationary phase : base-deactivated end-capped octadecylsilyl cis-trans isomers in the chromatogram obtained with
silica gel for chromatography R (5 μm). reference solution (b) (1.5 per cent) ;

Mobile phase : – impurities A, D : for each impurity, for the sum of the areas
of the 2 isomer peaks, not more than 1.5 times the sum
– mobile phase A : mix 5 volumes of methanol R, 20 volumes of the areas of the peaks due to the atracurium cis-cis,
of acetonitrile R and 75 volumes of a 10.2 g/L solution of trans-trans and cis-trans isomers in the chromatogram
potassium dihydrogen phosphate R previously adjusted to obtained with reference solution (b) (1.5 per cent) ;
pH 3.1 with phosphoric acid R ;
– impurity C : for the sum of the areas of the 2 isomer peaks,
– mobile phase B : mix 20 volumes of acetonitrile R, not more than the sum of the areas of the peaks due to
30 volumes of methanol R and 50 volumes of a 10.2 g/L the atracurium cis-cis, trans-trans and cis-trans isomers in
solution of potassium dihydrogen phosphate R previously the chromatogram obtained with reference solution (b)
adjusted to pH 3.1 with phosphoric acid R ; (1.0 per cent) ;
Time Mobile phase A Mobile phase B – impurities F, G : for each impurity, not more than the sum
(min) (per cent V/V) (per cent V/V) of the areas of the peaks due to the atracurium cis-cis,
0-5 80 20 trans-trans and cis-trans isomers in the chromatogram
obtained with reference solution (b) (1.0 per cent) ;
5 - 15 80 → 40 20 → 60
– impurities H, I, K : for the sum of the areas of the isomer
15 - 25 40 60 peaks of these impurities, not more than the sum of the
25 - 30 40 → 0 60 → 100 areas of the peaks due to the atracurium cis-cis, trans-trans
and cis-trans isomers in the chromatogram obtained with
30 - 45 0 100 reference solution (b) (1.0 per cent) ;

Flow rate : 1 mL/min. – unspecified impurities : for each impurity, not more than
0.1 times the sum of the areas of the peaks due to the
Detection : spectrophotometer at 280 nm. atracurium cis-cis, trans-trans and cis-trans isomers in
the chromatogram obtained with reference solution (b)
Injection : 20 μL of test solution (a) and reference solutions (a), (0.10 per cent);
(b), (d) and (e).
– total : not more than 3.5 times the sum of the areas of the
Identification of impurities : use the chromatogram obtained peaks due to the atracurium cis-cis, trans-trans and cis-trans
with reference solution (d) and the chromatogram supplied isomers in the chromatogram obtained with reference
with atracurium for peak identification CRS to identify solution (b) (3.5 per cent) ;
the peaks due to impurities A1, A2, B, C1, C2, D1, D2, E,
G and K ; use the chromatogram obtained with reference – disregard limit : 0.05 times the sum of the areas of the peaks
solution (e) and the chromatogram supplied with atracurium due to the atracurium cis-cis, trans-trans and cis-trans
for impurity F identification CRS to identify the peak due to isomers in the chromatogram obtained with reference
impurity F. solution (b) (0.05 per cent).

1602 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Atracurium besilate

Impurity J. Liquid chromatography (2.2.29) as described in

the test for related substances with the following modifications.
Mobile phase :
A. 1-(3,4-dimethoxybenzyl)-2-[13-[1-(3,4-dimethoxybenzyl)-
Time Mobile phase A Mobile phase B 6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl]-3,11-
(min) (per cent V/V) (per cent V/V) dioxo-4,10-dioxatridecyl]-6,7-dimethoxy-2-methyl-
0-5 80 20 1,2,3,4-tetrahydroisoquinolinium (A1 = cis-trans isomer,
A2 = cis-cis isomer),
5 - 15 80 → 75 20 → 25

15 - 25 75 25

25 - 30 75 → 55 25 → 45

30 - 38 55 → 0 45 → 100 B. pentane-1,5-diyl bis[3-[1-(3,4-dimethoxybenzyl)-6,7-

dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl]propanoate],
38 - 45 0 100

Detection : spectrophotometer at 217 nm.

Injection : 100 μL of test solution (b) and reference solution (c).
Retention time : impurity J = about 25 min ; atracurium C. 1-(3,4-dimethoxybenzyl)-2-(3,11-dioxo-4,10-
trans-trans isomer = about 38 min. dioxatridec-12-enyl)-6,7-dimethoxy-2-methyl-1,2,3,4-
tetrahydroisoquinolinium (C1 = trans isomer, C2 = cis
Limit : isomer),
– impurity J : not more than the area of the principal peak
in the chromatogram obtained with reference solution (c)
(10 ppm).
Isomer composition. Liquid chromatography (2.2.29) as D. 1-(3,4-dimethoxybenzyl)-2-[3-[(5-hydroxypentyl)oxy]-
described in the test for related substances with the following 3-oxopropyl]-6,7-dimethoxy-2-methyl-1,2,3,4-
modifications. Use the normalisation procedure. tetrahydroisoquinolinium (D1 = trans isomer, D2 = cis
Injection : test solution (a). isomer),
Limits :
– atracurium cis-cis isomer : 55.0 per cent to 60.0 per cent,
– atracurium cis-trans isomer : 34.5 per cent to 38.5 per cent,
E. 2-(2-carboxyethyl)-1-(3,4-dimethoxybenzyl)-6,7-
– atracurium trans-trans isomer : 5.0 per cent to 6.5 per cent. dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinolinium,
Water (2.5.12) : maximum 5.0 per cent, determined on 1.000 g.
F. R+-CH3 : 1-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2,2-
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on dimethyl-1,2,3,4-tetrahydroisoquinolinium,
1.0 g.
G. R-CH3 : 1-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2-
ASSAY methyl-1,2,3,4-tetrahydroisoquinoline,
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : test solution (a) and reference solution (a).
Calculate the percentage content of C65H82N2O18S2 from the
sum of the areas of the peaks due to the 3 isomers. H. 2,2′-[hexane-1,6-diylbis[oxy(3-oxopropane-1,3-
diyl)]]bis[1-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2-
STORAGE methyl-1,2,3,4-tetrahydroisoquinolinium] (H1 = cis-trans
In an airtight container, protected from light, at a temperature isomer, H2 = cis-cis isomer),
of 2 °C to 8 °C.

IMPURITIES
Specified impurities : A, C, D, E, F, G, H, I, J, K. I. 2,2′-[(3-methylpentane-1,5-diyl)bis[oxy(3-oxopropane-
Other detectable impurities (the following substances would, 1,3-diyl)]]bis[1-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2-
if present at a sufficient level, be detected by one or other of methyl-1,2,3,4-tetrahydroisoquinolinium] (I1 = cis-trans
the tests in the monograph. They are limited by the general isomer, I2 = cis-cis isomer),
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use): B.
J. methyl benzenesulfonate,

K. 2,2′-[hexane-1,5-diylbis[oxy(3-oxopropane-1,3-
diyl)]]bis[1-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2-
methyl-1,2,3,4-tetrahydroisoquinolinium].

General Notices (1) apply to all monographs and other texts 1603
Atropine EUROPEAN PHARMACOPOEIA 8.0

07/2010:2056 Reference solution (b). Dissolve 5 mg of atropine

impurity B CRS in the test solution and dilute to 20.0 mL with
ATROPINE the test solution. Dilute 5.0 mL of this solution to 25.0 mL
with mobile phase A.
Atropinum Reference solution (c). Dissolve the contents of a vial of
atropine for peak identification CRS (containing impurities A,
D, E, F, G and H) in 1.0 mL of mobile phase A.
Reference solution (d). Dissolve 5 mg of tropic acid R
(impurity C) in mobile phase A and dilute to 10.0 mL with
mobile phase A. Dilute 1.0 mL of the solution to 100.0 mL
with mobile phase A. Dilute 1.0 mL of this solution to 10.0 mL
with mobile phase A.
C17H23NO3 Mr 289.4 Column :
[51-55-8] – size : l = 0.10 m, Ø = 4.6 mm ;
DEFINITION – stationary phase : octadecylsilyl silica gel for
(1R,3R,5S)-8-Methyl-8-azabicyclo[3.2.1]oct-3-yl chromatography R (3 μm).
(2RS)-3-hydroxy-2-phenylpropanoate. Mobile phase :
Content : 99.0 per cent to 101.0 per cent (dried substance). – mobile phase A : dissolve 3.5 g of sodium dodecyl sulfate R
in 606 mL of a 7.0 g/L solution of potassium dihydrogen
CHARACTERS phosphate R previously adjusted to pH 3.3 with a 5.8 g/L
Appearance : white or almost white, crystalline powder or solution of phosphoric acid R, and mix with 320 mL of
colourless crystals. acetonitrile R1 ;
Solubility : very slightly soluble in water, freely soluble in – mobile phase B : acetonitrile R1 ;
ethanol (96 per cent) and in methylene chloride. Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
IDENTIFICATION
0-2 95 5
First identification : A, B, E.
Second identification : A, C, D, E. 2 - 20 95 → 70 5 → 30
A. Melting point (2.2.14) : 115 °C to 119 °C. Flow rate : 1 mL/min.
B. Infrared absorption spectrophotometry (2.2.24). Detection : spectrophotometer at 210 nm.
Comparison : atropine CRS. Injection : 10 μL.
C. Thin-layer chromatography (2.2.27). Identification of impurities : use the chromatogram
Test solution. Dissolve 10 mg of the substance to be supplied with atropine for peak identification CRS and
examined in methanol R and dilute to 10 mL with the same the chromatogram obtained with reference solution (c) to
solvent. identify the peaks due to impurities A, D, E, F, G and H ; use
Reference solution. Dissolve 10 mg of atropine CRS in the chromatogram obtained with reference solution (b) to
methanol R and dilute to 10 mL with the same solvent. identify the peak due to impurity B ; use the chromatogram
Plate : TLC silica gel plate R. obtained with reference solution (d) to identify the peak due
Mobile phase : concentrated ammonia R, water R, acetone R to impurity C.
(3:7:90 V/V/V). Relative retention with reference to atropine (retention
Application : 10 μL. time = about 11 min) : impurity C = about 0.2 ;
impurity E = about 0.67 ; impurity D = about 0.73 ;
Development : over half of the plate. impurity F = about 0.8 ; impurity B = about 0.89 ;
Drying : at 100-105 °C for 15 min. impurity H = about 0.93 ; impurity G = about 1.1 ;
Detection : after cooling, spray with dilute potassium impurity A = about 1.7.
iodobismuthate solution R. System suitability : reference solution (b) :
Results : the principal spot in the chromatogram obtained – resolution : minimum 2.5 between the peaks due to
with the test solution is similar in position, colour and size impurity B and atropine.
to the principal spot in the chromatogram obtained with Limits :
the reference solution.
– correction factors : for the calculation of content, multiply
D. Place about 3 mg in a porcelain crucible and add 0.2 mL of the peak areas of the following impurities by the
fuming nitric acid R. Evaporate to dryness on a water-bath. corresponding correction factor : impurity A = 0.6 ;
Dissolve the residue in 0.5 mL of a 30 g/L solution of impurity C = 0.6 ;
potassium hydroxide R in methanol R ; a violet colour
develops. – impurities E, H : for each impurity, not more than 3 times
the area of the principal peak in the chromatogram
E. Optical rotation (see Tests). obtained with reference solution (a) (0.3 per cent) ;
TESTS – impurities A, B, C, D, F, G : for each impurity, not more than
twice the area of the principal peak in the chromatogram
Optical rotation (2.2.7) : − 0.70° to + 0.05° (measured in a
obtained with reference solution (a) (0.2 per cent) ;
2 dm tube).
– unspecified impurities : for each impurity, not more than the
Dissolve 1.25 g in ethanol (96 per cent) R and dilute to 25.0 mL
area of the principal peak in the chromatogram obtained
with the same solvent.
with reference solution (a) (0.10 per cent) ;
Related substances. Liquid chromatography (2.2.29). – total : not more than 5 times the area of the principal peak
Test solution. Dissolve 24 mg of the substance to be examined in the chromatogram obtained with reference solution (a)
in mobile phase A and dilute to 100.0 mL with mobile phase A. (0.5 per cent) ;
Reference solution (a). Dilute 1.0 mL of the test solution to – disregard limit : 0.5 times the area of the principal peak in
100.0 mL with mobile phase A. Dilute 1.0 mL of this solution the chromatogram obtained with reference solution (a)
to 10.0 mL with mobile phase A. (0.05 per cent).

1604 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Atropine sulfate

Loss on drying (2.2.32) : maximum 0.2 per cent, determined

on 1.000 g by drying in an oven at 105 °C for 2 h.
ASSAY
Dissolve 0.250 g in 40 mL of anhydrous acetic acid R, heating
if necessary, and allow to cool. Titrate with 0.1 M perchloric G. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
acid, determining the end-point potentiometrically (2.2.20). (2RS)-2-hydroxy-3-phenylpropanoate (littorine),
1 mL of 0.1 M perchloric acid is equivalent to 28.94 mg H. unknown structure.
of C17H23NO3.
04/2008:0068
STORAGE
corrected 7.0
Protected from light.
IMPURITIES
ATROPINE SULFATE
Specified impurities : A, B, C, D, E, F, G, H. Atropini sulfas

A. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
2-phenylpropenoate (apoatropine), C34H48N2O10S,H2O Mr 695
[5908-99-6]
DEFINITION
Bis[(1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
(2RS)-3-hydroxy-2-phenylpropanoate] sulfate monohydrate.
Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
CHARACTERS
B. (1R,3r,5S)-8-azabicyclo[3.2.1]oct-3-yl (2RS)-3-hydroxy-2-
phenylpropanoate (noratropine), Appearance : white or almost white, crystalline powder or
colourless crystals.
Solubility : very soluble in water, freely soluble in ethanol
(96 per cent).
IDENTIFICATION
First identification : A, B, E.
Second identification : C, D, E, F.
C. (2RS)-3-hydroxy-2-phenylpropanoic acid (tropic acid), A. Optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: atropine sulfate CRS.
C. Dissolve about 50 mg in 5 mL of water R and add 5 mL of
picric acid solution R. The precipitate, washed with water R
and dried at 100-105 °C for 2 h, melts (2.2.14) at 174 °C
to 179 °C.
D. (1R,3S,5R,6RS)-6-hydroxy-8-methyl-8-azabicy- D. To about 1 mg add 0.2 mL of fuming nitric acid R and
clo[3.2.1]oct-3-yl (2S)-3-hydroxy-2-phenylpropanoate evaporate to dryness in a water-bath. Dissolve the residue
(6-hydroxyhyoscyamine), in 2 mL of acetone R and add 0.1 mL of a 30 g/L solution
of potassium hydroxide R in methanol R. A violet colour
develops.
E. It gives the reactions of sulfates (2.3.1).
F. It gives the reaction of alkaloids (2.3.1).
TESTS
pH (2.2.3) : 4.5 to 6.2.
E. (1S,3R,5S,6RS)-6-hydroxy-8-methyl-8-azabicy- Dissolve 0.6 g in carbon dioxide-free water R and dilute to
clo[3.2.1]oct-3-yl (2S)-3-hydroxy-2-phenylpropanoate 30 mL with the same solvent.
(7-hydroxyhyoscyamine), Optical rotation (2.2.7) : − 0.50° to + 0.05° (measured in a
2 dm tube).
Dissolve 2.50 g in water R and dilute to 25.0 mL with the same
solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 24 mg of the substance to be examined
in mobile phase A and dilute to 100.0 mL with mobile phase A.
F. (1R,2R,4S,5S,7s)-9-methyl-3-oxa-9-azatricy- Reference solution (a). Dilute 1.0 mL of the test solution to
clo[3.3.1.02,4]non-7-yl (2S)-3-hydroxy-2-phenylpropanoate 100.0 mL with mobile phase A. Dilute 1.0 mL of this solution
(hyoscine), to 10.0 mL with mobile phase A.

General Notices (1) apply to all monographs and other texts 1605
Atropine sulfate EUROPEAN PHARMACOPOEIA 8.0

Reference solution (b). Dissolve 5 mg of atropine Water (2.5.12) : 2.0 per cent to 4.0 per cent, determined on
impurity B CRS in the test solution and dilute to 20 mL with 0.500 g.
the test solution. Dilute 5 mL of this solution to 25 mL with Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
mobile phase A. 1.0 g.
Reference solution (c). Dissolve the contents of a vial of
atropine for peak identification CRS (containing impurities A, ASSAY
D, E, F, G and H) in 1 mL of mobile phase A. Dissolve 0.500 g in 30 mL of anhydrous acetic acid R, warming
Reference solution (d). Dissolve 5 mg of tropic acid R if necessary. Cool the solution. Titrate with 0.1 M perchloric
(impurity C) in mobile phase A and dilute to 10 mL with acid, determining the end-point potentiometrically (2.2.20).
mobile phase A. Dilute 1 mL of the solution to 100 mL with 1 mL of 0.1 M perchloric acid is equivalent to 67.68 mg
mobile phase A. Dilute 1 mL of this solution to 10 mL with of C34H48N2O10S.
mobile phase A.
Column : STORAGE
– size : l = 0.10 m, Ø = 4.6 mm ; Protected from light.
– stationary phase : octadecylsilyl silica gel for
chromatography R (3 μm). IMPURITIES
Mobile phase : Specified impurities : A, B, C, D, E, F, G, H.
– mobile phase A : dissolve 3.5 g of sodium dodecyl
sulfate R in 606 mL of a 7.0 g/L solution of potassium
dihydrogen phosphate R previously adjusted to pH 3.3
with 0.05 M phosphoric acid, and mix with 320 mL of
acetonitrile R1 ;
– mobile phase B : acetonitrile R1 ;
Time
A. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl
Mobile phase A Mobile phase B
2-phenylpropenoate (apoatropine),
(min) (per cent V/V) (per cent V/V)
0-2 95 5

2 - 20 95 → 70 5 → 30

Flow rate : 1 mL/min.

Detection : spectrophotometer at 210 nm.
Injection : 10 μL.
B. (1R,3r,5S)-8-azabicyclo[3.2.1]oct-3-yl (2RS)-3-hydroxy-2-
Identification of impurities : use the chromatogram
phenylpropanoate (noratropine),
supplied with atropine for peak identification CRS and the
chromatogram obtained with reference solution (c) to identify
the peaks due to impurities A, D, E, F, G and H. Use the
chromatogram obtained with reference solution (b) to identify
the peak due to impurity B, and use the chromatogram
obtained with reference solution (d) to identify the peak due
to impurity C.
Relative retention with reference to atropine (retention C. (2RS)-3-hydroxy-2-phenylpropanoic acid (tropic acid),
time = about 11 min) : impurity C = about 0.2 ;
impurity E = about 0.67 ; impurity D = about 0.73 ;
impurity F = about 0.8 ; impurity B = about 0.89 ;
impurity H = about 0.93 ; impurity G = about 1.1 ;
impurity A = about 1.7.
System suitability : reference solution (b) :
– resolution : minimum 2.5 between the peaks due to D. (1R,3S,5R,6RS)-6-hydroxy-8-methyl-8-azabicyclo-
impurity B and atropine. [3.2.1]oct-3-yl (2S)-3-hydroxy-2-phenylpropanoate
Limits : (6-hydroxyhyoscyamine),
– correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
corresponding correction factor : impurity A = 0.6 ;
impurity C = 0.6 ;
– impurities E, H : for each impurity, not more than 3 times
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.3 per cent) ;
– impurities A, B, C, D, F, G : for each impurity, not more than E. (1S,3R,5S,6RS)-6-hydroxy-8-methyl-8-azabicy-
twice the area of the principal peak in the chromatogram clo[3.2.1]oct-3-yl (2S)-3-hydroxy-2-phenylpropanoate
obtained with reference solution (a) (0.2 per cent) ; (7-hydroxyhyoscyamine),
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
– total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in F. (1R,2R,4S,5S,7s)-9-methyl-3-oxa-9-azatricy-
the chromatogram obtained with reference solution (a) clo[3.3.1.02,4]non-7-yl (2S)-3-hydroxy-2-phenylpropanoate
(0.05 per cent). (hyoscine),

1606 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Azaperone for veterinary use

– temperature : 25 °C.
Mobile phase :
– mobile phase A : dissolve 1.4 g of anhydrous sodium sulfate R
in 900 mL of water R, add 16.0 mL of 0.01 M sulfuric acid
and dilute to 1000 mL with water R ;
G. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl – mobile phase B : methanol R ;
(2RS)-2-hydroxy-3-phenylpropanoate (littorine).
Time Mobile phase A Mobile phase B
H. unknown structure. (min) (per cent V/V) (per cent V/V)
0 - 15 95 → 20 5 → 80

15 - 20 20 80
04/2010:1708
corrected 7.0 Flow rate : 1.5 mL/min.
Detection : spectrophotometer at 230 nm.
AZAPERONE FOR VETERINARY USE Injection : 10 μL.
Relative retention with reference to azaperone (retention
Azaperonum ad usum veterinarium time = about 9 min) : impurity A = about 0.9 ;
impurity B = about 1.1 ; impurity C = about 1.15.
System suitability : reference solution (a) :
– resolution : minimum 8.0 between the peaks due to
azaperone and to benperidol.
Limits :
– impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (b)
C19H22FN3O Mr 327.4 (0.25 per cent);
[1649-18-9]
– unspecified impurities : for each impurity, not more than
DEFINITION 0.8 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.20 per cent) ;
1-(4-Fluorophenyl)-4-[4-(pyridin-2-yl)piperazin-1-yl]butan-
1-one. – sum of impurities B and C : not more than 3 times the area
of the principal peak in the chromatogram obtained with
Content : 99.0 per cent to 101.0 per cent (dried substance). reference solution (b) (0.75 per cent) ;
CHARACTERS – total : not more than 4 times the area of the principal peak
Appearance : white or almost white powder. in the chromatogram obtained with reference solution (b)
(1.0 per cent) ;
Solubility : practically insoluble in water, freely soluble in
acetone and in methylene chloride, soluble in ethanol (96 per – disregard limit : 0.2 times the area of the principal peak in
cent). the chromatogram obtained with reference solution (b)
(0.05 per cent).
It shows polymorphism (5.9).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
IDENTIFICATION on 1.000 g by drying in vacuo at 60 °C for 4 h.
Infrared absorption spectrophotometry (2.2.24). Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Preparation : discs. 1.0 g.
Comparison : azaperone CRS. ASSAY
If the spectra obtained show differences, dissolve the substance Dissolve 0.130 g in 70 mL of a mixture of 1 volume of
to be examined and the reference substance separately in anhydrous acetic acid R and 7 volumes of methyl ethyl
acetone R, evaporate to dryness and record new spectra using ketone R. Titrate with 0.1 M perchloric acid, using 0.2 mL of
the residues. naphtholbenzein solution R as indicator.
TESTS 1 mL of 0.1 M perchloric acid is equivalent to 16.37 mg of
Appearance of solution. The solution is clear (2.2.1) and not C19H22FN3O.
more intensely coloured than reference solution Y6 (2.2.2, STORAGE
Method II).
Protected from light.
Dissolve 1.0 g in 25 mL of a 14 g/L solution of tartaric acid R.
Related substances. Liquid chromatography (2.2.29). IMPURITIES
Test solution. Dissolve 0.100 g of the substance to be examined Specified impurities : A, B, C.
in methanol R and dilute to 10.0 mL with the same solvent.
Reference solution (a). Dissolve 5.0 mg of azaperone CRS
and 6.0 mg of benperidol CRS in methanol R and dilute to
200.0 mL with the same solvent.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with methanol R. Dilute 5.0 mL of the solution to
20.0 mL with methanol R.
Column :
– size : l = 0.10 m, Ø = 4.6 mm ;
– stationary phase : base-deactivated octadecylsilyl silica gel for A. 1-(2-fluorophenyl)-4-[4-(pyridin-2-yl)piperazin-1-
chromatography R (3 μm) ; yl]butan-1-one,

General Notices (1) apply to all monographs and other texts 1607
Azathioprine EUROPEAN PHARMACOPOEIA 8.0

– stationary phase : phenylsilyl silica gel for chromatography R

(5 μm) ;
– temperature : 30 °C.
Mobile phase :
B. 4-[4-(pyridin-2-yl)piperazin-1-yl]-1-[4-[4-(pyridin-2- – mobile phase A : methanol R, solution A (5:95 V/V) ;
yl)piperazin-1-yl]phenyl]butan-1-one,
– mobile phase B : solution A, methanol R (40:60 V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-5 100 0

5 - 15 100 → 0 0 → 100
C. 4-hydroxy-1-[4-[4-(pyridin-2-yl)piperazin-1-
yl]phenyl]butan-1-one. 15 - 20 0 100

Flow rate : 1.0 mL/min.

07/2010:0369
Detection : spectrophotometer at 240 nm.
corrected 7.0
Injection : 20 μL.
AZATHIOPRINE Identification of impurities: use the chromatogram obtained
with reference solution (a) to identify the peaks due to
impurities A and B. Use the chromatogram obtained with
Azathioprinum reference solution (b) to identify the peak due to impurity G.
Relative retention with reference to azathioprine
(retention time = about 15 min) : impurity A = about 0.3 ;
impurity B = about 0.4 ; impurity G = about 0.97.
System suitability :
– resolution : minimum 2.0 between the peaks due to
impurities A and B in the chromatogram obtained with
reference solution (a) ; minimum 2.0 between the peaks
C 9H 7N7O 2S Mr 277.3 due to impurity G and azathioprine in the chromatogram
[446-86-6] obtained with reference solution (b).
DEFINITION Limits :
6-[(1-Methyl-4-nitro-1H-imidazol-5-yl)sulfanyl]-7H-purine. – impurities A, B : for each impurity, not more than 1.5 times
Content : 98.5 per cent to 101.0 per cent (dried substance). the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.15 per cent) ;
CHARACTERS – unspecified impurities : for each impurity, not more than the
Appearance : pale-yellow powder. area of the principal peak in the chromatogram obtained
Solubility : practically insoluble in water and in ethanol (96 per with reference solution (c) (0.10 per cent) ;
cent). It is soluble in dilute solutions of alkali hydroxides and – total : not more than 5 times the area of the principal peak
sparingly soluble in dilute mineral acids. in the chromatogram obtained with reference solution (c)
(0.5 per cent) ;
IDENTIFICATION
– disregard limit : 0.5 times the area of the principal peak in
Infrared absorption spectrophotometry (2.2.24). the chromatogram obtained with reference solution (c)
Comparison : azathioprine CRS. (0.05 per cent).
TESTS Loss on drying (2.2.32) : maximum 1.0 per cent, determined
on 0.500 g by drying in an oven at 105 °C.
Related substances. Liquid chromatography (2.2.29).
Solution A. 2.76 g/L solution of sodium dihydrogen phosphate Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
monohydrate R adjusted to pH 2.5 with phosphoric acid R. 1.0 g.
Test solution. Dissolve 10 mg of the substance to be examined ASSAY
in 35 mL of a 0.8 g/L solution of sodium hydroxide R and
Dissolve 0.250 g in 25 mL of dimethylformamide R. Titrate
dilute to 100.0 mL with solution A.
with 0.1 M tetrabutylammonium hydroxide, determining the
Reference solution (a). Dissolve 5 mg of azathioprine end-point potentiometrically (2.2.20).
impurity A CRS and 5 mg of mercaptopurine R (impurity B) in
8.75 mL of a 0.8 g/L solution of sodium hydroxide R and dilute 1 mL of 0.1 M tetrabutylammonium hydroxide is equivalent to
to 25.0 mL with solution A. To 1.0 mL of this solution, add 27.73 mg of C9H7N7O2S.
35 mL of a 0.8 g/L solution of sodium hydroxide R and dilute STORAGE
to 100.0 mL with solution A.
Protected from light.
Reference solution (b). Dissolve 2.5 mg of azathioprine
impurity G CRS and 2.5 mg of the substance to be examined IMPURITIES
in 8.8 mL of a 0.8 g/L solution of sodium hydroxide R and Specified impurities : A, B.
dilute to 25.0 mL with solution A. To 1.0 mL of this solution,
add 17.5 mL of a 0.8 g/L solution of sodium hydroxide R and Other detectable impurities (the following substances would,
dilute to 50.0 mL with solution A. if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
Reference solution (c). Dilute 1.0 mL of the test solution to acceptance criterion for other/unspecified impurities and/or
100.0 mL with solution A. Dilute 1.0 mL of this solution to by the general monograph Substances for pharmaceutical use
10.0 mL with solution A. (2034). It is therefore not necessary to identify these impurities
Column : for demonstration of compliance. See also 5.10. Control of
– size : l = 0.15 m, Ø = 4.6 mm ; impurities in substances for pharmaceutical use) : C, D, E, F, G.

1608 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Azelastine hydrochloride

Solubility : sparingly soluble in water, soluble in ethanol and

in methylene chloride.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
A. 1-methyl-4-nitro-1H-imidazol-5-amine, Comparison: azelastine hydrochloride CRS.
B. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S. Dissolve 1.0 g in carbon dioxide-free water R and
dilute to 100 mL with the same solvent.
B. 7H-purine-6-thiol (mercaptopurine),
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Acidity or alkalinity. To 10 mL of solution S add 0.2 mL
of bromothymol blue solution R1. Not more than 0.1 mL
of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is
C. 5-chloro-1-methyl-4-nitro-1H-imidazole, required to change the colour of the solution.
Related substances. Liquid chromatography (2.2.29).
Solvent mixture : acetonitrile for chromatography R, water R
(45:55 V/V).
Test solution. Dissolve 0.125 g of the substance to be examined
in the solvent mixture and dilute to 50.0 mL with the solvent
D. 1-methyl-4-nitro-1H-imidazole-5-thiol, mixture.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Reference solution (b). Dissolve 1 mg of azelastine
impurity B CRS, 1 mg of azelastine impurity D CRS and 1 mg
E. 1-methyl-4-nitro-1H-imidazol-5-ol, of azelastine impurity E CRS in the test solution and dilute to
20 mL with the test solution.
Column :
– size : l = 0.25 m, Ø = 4.6 mm,
– stationary phase : nitrile silica gel for chromatography R
F. 1,7-dihydro-6H-purin-6-one (hypoxanthine), (10 μm),
– temperature : 30°C.
Mobile phase : dissolve 2.16 g of sodium octanesulfonate R
and 0.68 g of potassium dihydrogen phosphate R in 740 mL
of water for chromatography R, adjust to pH 3.0-3.1 with
dilute phosphoric acid R, add 260 mL of acetonitrile for
chromatography R and mix.
Flow rate : 2.0 mL/min.
G. 6-[(1-methyl-4-nitro-1H-imidazol-5-yl)sulfanyl]-7H- Detection : spectrophotometer at 210 nm.
purin-2-amine (thiamiprine). Injection : 10 μL.
Run time : twice the retention time of azelastine.
01/2008:1633
corrected 6.0 Relative retention with reference to azelastine (retention
time = about 8-9 min) : impurity A = about 0.2 ;
AZELASTINE HYDROCHLORIDE impurity B = about 0.3 ; impurity C = about 0.4 ;
impurity D = about 0.6 ; impurity E = about 1.4.
System suitability : reference solution (b) :
Azelastini hydrochloridum
– resolution : minimum 4.0 between the peaks due to
impurities B and D,
– the peaks due to impurities D and E are baseline separated
from the principal peak.
Limits :
– correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
corresponding correction factor : impurity B = 3.6 ;
impurity D = 0.7 ; impurity E = 2.1 ;
C22H25Cl2N3O Mr 418.4
– impurities A, B, C, D, E : for each impurity, not more
[79307-93-0]
than the area of the principal peak in the chromatogram
DEFINITION obtained with reference solution (a) (0.1 per cent) ;
4-(4-Chlorobenzyl)-2-[(4RS)-1-methylhexahydro-1H-azepin- – any other impurity : for each impurity, not more than the
4-yl]phthalazin-1(2H)-one hydrochloride. area of the principal peak in the chromatogram obtained
Content : 99.0 per cent to 101.0 per cent (dried substance). with reference solution (a) (0.1 per cent) ;
– total : not more than twice the area of the principal peak
CHARACTERS in the chromatogram obtained with reference solution (a)
Appearance : white or almost white, crystalline powder. (0.2 per cent) ;

General Notices (1) apply to all monographs and other texts 1609
Azithromycin EUROPEAN PHARMACOPOEIA 8.0

– disregard limit : 0.5 times the area of the principal peak in 01/2011:1649
the chromatogram obtained with reference solution (a)
(0.05 per cent).
AZITHROMYCIN
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Azithromycinum
ASSAY
In order to avoid overheating in the reaction medium, mix
thoroughly throughout and stop the titration immediately after
the end-point has been reached.
Dissolve 0.300 g in 5 mL of anhydrous formic acid R. Add
30 mL of acetic anhydride R. Titrate quickly with 0.1 M
perchloric acid, determining the end-point potentiometrically
(2.2.20).
1.0 mL of 0.1 M perchloric acid is equivalent to 41.84 mg of
C22H25Cl2N3O.

IMPURITIES C38H72N2O12,xH2O Mr 749 (anhydrous substance)

Specified impurities : A, B, C, D, E. with x = 1 or 2
Azithromycin monohydrate : [121470-24-4]
Azithromycin dihydrate : [117772-70-0]
DEFINITION
(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-
C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl-
A. benzoyldiazane (benzohydrazide), 3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-
trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-1-
oxa-6-azacyclopentadecan-15-one. The degree of hydration is
1 or 2.
Semi-synthetic product derived from a fermentation product.
Content : 96.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
B. 1-benzoyl-2-[(4RS)-1-methylhexahydro-1H-azepin-4-
yl]diazane, Appearance : white or almost white powder.
Solubility : practically insoluble in water, freely soluble in
anhydrous ethanol and in methylene chloride.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: azithromycin CRS.
If the spectra obtained in the solid state show differences,
prepare further spectra using 90 g/L solutions in methylene
chloride R.
C. 2-[(4-chlorophenyl)acetyl]benzoic acid,
TESTS
Solution S. Dissolve 0.500 g in anhydrous ethanol R and dilute
to 50.0 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
pH (2.2.3) : 9.0 to 11.0.
Dissolve 0.100 g in 25.0 mL of methanol R and dilute to
50.0 mL with carbon dioxide-free water R.
D. 4-(4-chlorobenzyl)phthalazin-1(2H)-one, Specific optical rotation (2.2.7) : − 45 to − 49 (anhydrous
substance), determined on solution S.
Related substances. Liquid chromatography (2.2.29).
Solvent mixture. Prepare a 1.73 g/L solution of ammonium
dihydrogen phosphate R adjusted to pH 10.0 with ammonia R.
Transfer 350 mL of this solution to a suitable container. Add
300 mL of acetonitrile R1 and 350 mL of methanol R1. Mix
well.
Test solution. Dissolve 0.200 g of the substance to be examined
in the solvent mixture and dilute to 25.0 mL with the solvent
mixture.
Reference solution (a). Dilute 1.0 mL of the test solution to
E. 3-(4-chlorobenzylidene)isobenzofuran-1(3H)-one. 100.0 mL with the solvent mixture.

1610 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Azithromycin

Reference solution (b). Dissolve the contents of a vial of – impurity G : not more than 0.2 times the area of the
azithromycin for system suitability CRS (containing impurities principal peak in the chromatogram obtained with
F, H and J) in 1.0 mL of the solvent mixture and sonicate for reference solution (a) (0.2 per cent) ;
5 min. – any other impurity : for each impurity, not more than
Reference solution (c). Dissolve 8.0 mg of azithromycin for 0.2 times the area of the principal peak in the chromatogram
peak identification CRS (containing impurities A, B, C, E, F, G, obtained with reference solution (a) (0.2 per cent) ;
I, J, L, M, N, O and P) in 1.0 mL of the solvent mixture. – total : not more than 3 times the area of the principal peak
Column : in the chromatogram obtained with reference solution (a)
– size : l = 0.25 m, Ø = 4.6 mm ; (3.0 per cent) ;
– stationary phase : end-capped octadecylsilyl amorphous – disregard limit : 0.1 times the area of the principal peak in
organosilica polymer for mass spectrometry R (5 μm) ; the chromatogram obtained with reference solution (a)
(0.1 per cent) ; disregard the peaks eluting before impurity L
– temperature : 60 °C. and after impurity B.
Mobile phase :
Heavy metals (2.4.8) : maximum 25 ppm.
– mobile phase A : 1.80 g/L solution of anhydrous disodium Dissolve 2.0 g in a mixture of 15 volumes of water R and
hydrogen phosphate R adjusted to pH 8.9 with dilute 85 volumes of anhydrous ethanol R and dilute to 20 mL with
phosphoric acid R or with dilute sodium hydroxide the same mixture of solvents. 12 mL of the solution complies
solution R ; with test B. Prepare the reference solution using lead standard
– mobile phase B : methanol R1, acetonitrile R1 (250:750 V/V) ; solution (2.5 ppm Pb) obtained by diluting lead standard
Time Mobile phase A Mobile phase B solution (100 ppm Pb) R with a mixture of 15 volumes of
(min) (per cent V/V) (per cent V/V) water R and 85 volumes of anhydrous ethanol R.
0 - 25 50 → 45 50 → 55 Water (2.5.12) : 1.8 per cent to 6.5 per cent, determined on
0.200 g.
25 - 30 45 → 40 55 → 60
Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
30 - 80 40 → 25 60 → 75 1.0 g.
80 - 81 25 → 50 75 → 50
ASSAY
81 - 93 50 50 Liquid chromatography (2.2.29).
Solution A. Mix 60 volumes of acetonitrile R1 and 40 volumes
Flow rate : 1.0 mL/min.
of a 6.7 g/L solution of dipotassium hydrogen phosphate R
Detection : spectrophotometer at 210 nm. adjusted to pH 8.0 with phosphoric acid R.
Injection : 50 μL. Test solution. Dissolve 53.0 mg of the substance to be
Identification of impurities : use the chromatogram supplied examined in 2 mL of acetonitrile R1 and dilute to 100.0 mL
with azithromycin for peak identification CRS and the with solution A.
chromatogram obtained with reference solution (c) to identify Reference solution (a). Dissolve 53.0 mg of azithromycin CRS
the peaks due to impurities A, B, C, E, F, G, I, J, L, M, N, O in 2 mL of acetonitrile R1 and dilute to 100.0 mL with
and P ; use the chromatogram supplied with azithromycin for solution A.
system suitability CRS and the chromatogram obtained with Reference solution (b). Dissolve 5 mg of the substance to be
reference solution (b) to identify the peak due to impurity H. examined and 5 mg of azithromycin impurity A CRS in 0.5 mL
Relative retention with reference to azithromycin of acetonitrile R1 and dilute to 10 mL with solution A.
(retention time = 45-50 min) : impurity L = about 0.29 ; Column :
impurity M = about 0.37 ; impurity E = about 0.43 ;
impurity F = about 0.51 ; impurity D = about 0.54 ; – size : l = 0.25 m, Ø = 4.6 mm ;
impurity J = about 0.54 ; impurity I = about 0.61 ; – stationary phase : octadecylsilyl vinyl polymer for
impurity C = about 0.73 ; impurity N = about 0.76 ; chromatography R (5 μm) ;
impurity H = about 0.79 ; impurity A = about 0.83 ; – temperature : 40 °C.
impurity P = about 0.92 ; impurity O = about 1.23 ; Mobile phase : mix 60 volumes of acetonitrile R1 and
impurity G = about 1.26 ; impurity B = about 1.31. 40 volumes of a 6.7 g/L solution of dipotassium hydrogen
System suitability : reference solution (b) : phosphate R adjusted to pH 11.0 with a 560 g/L solution of
– peak-to-valley ratio : minimum 1.4, where Hp = height potassium hydroxide R.
above the baseline of the peak due to impurity J and Flow rate : 1.0 mL/min.
Hv = height above the baseline of the lowest point of the Detection : spectrophotometer at 210 nm.
curve separating this peak from the peak due to impurity F. Injection : 10 μL.
Limits : Run time : 1.5 times the retention time of azithromycin.
– correction factors : for the calculation of content, Retention time : azithromycin = about 10 min.
multiply the peak areas of the following impurities by System suitability : reference solution (b) :
the corresponding correction factor : impurity F = 0.3 ;
impurity G = 0.2 ; impurity H = 0.1 ; impurity L = 2.3 ; – resolution : minimum 3.0 between the peaks due to
impurity M = 0.6 ; impurity N = 0.7 ; impurity A and azithromycin.
– impurity B : not more than twice the area of the principal Calculate the percentage content of C38H72N2O12 from the
peak in the chromatogram obtained with reference declared content of azithromycin CRS.
solution (a) (2.0 per cent) ; STORAGE
– impurities A, C, E, F, H, I, L, M, N, O, P : for each impurity, In an airtight container.
not more than 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a) IMPURITIES
(0.5 per cent) ; Specified impurities : A, B, C, D, E, F, G, H, I, J, L, M, N, O, P.
– sum of impurities D and J : not more than 0.5 times the area Other detectable impurities (the following substances would,
of the principal peak in the chromatogram obtained with if present at a sufficient level, be detected by one or other of
reference solution (a) (0.5 per cent) ; the tests in the monograph. They are limited by the general

General Notices (1) apply to all monographs and other texts 1611
Azithromycin EUROPEAN PHARMACOPOEIA 8.0

acceptance criterion for other/unspecified impurities and/or

by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use) : K.

H. 3′-N-[[4-(acetylamino)phenyl]sulfonyl]-3′-N-
demethylazithromycin,

J. 13-O-decladinosylazithromycin,
A. R1 = OH, R2 = R6 = H, R3 = R4 = R5 = CH3 :
6-demethylazithromycin,

B. R1 = R6 = H, R2 = R3 = R4 = R5 = CH3 : 3-deoxyazithromycin
(azithromycin B),

C. R1 = OH, R2 = R3 = R5 = CH3, R4 = R6 = H :
3″-O-demethylazithromycin (azithromycin C),

L. azithromycin 3′-N-oxide,
D. R1 = OH, R2 = R3 = R4 = CH3, R5 = CH2OH, R6 = H :
14-demethyl-14-(hydroxymethyl)azithromycin
(azithromycin F),

F. R1 = OH, R2 = R4 = R5 = CH3, R3 = CHO, R6 = H :

3′-N-demethyl-3′-N-formylazithromycin,

I. R1 = OH, R2 = R4 = R5 = CH3, R3 = R6 = H :
3′-N-demethylazithromycin,
M. 3′-(N,N-didemethyl)-3′-N-formylazithromycin,

O. R1 = OH, R2 = R3 = R4 = R5 = R6 = CH3 :
2-desethyl-2-propylazithromycin,

N. 3′-de(dimethylamino)-3′-oxoazithromycin,

E. 3′-(N,N-didemethyl)azithromycin (aminoazithromycin),

K. C14,1″-epoxyazithromycin (azithromycin E),

G. 3′-N-demethyl-3′-N-[(4-methylphenyl)sulfonyl]azithro-
mycin, P. unknown structure.

1612 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Bacampicillin hydrochloride

01/2008:0808 silver nitrate solution R1. A white precipitate is formed.

corrected 6.1 Add 0.5 mL of concentrated ammonia R. The precipitate
dissolves.
BACAMPICILLIN HYDROCHLORIDE TESTS
Appearance of solution. Dissolve 0.200 g in 20 mL of
Bacampicillini hydrochloridum water R ; the solution is not more opalescent than reference
suspension II (2.2.1). Dissolve 0.500 g in 10 mL of water R ;
the absorbance (2.2.25) of the solution at 430 nm is not greater
than 0.10.
pH (2.2.3) : 3.0 to 4.5.
Dissolve 1.0 g in carbon dioxide-free water R and dilute to
50 mL with the same solvent.
C21H28ClN3O7S Mr 502.0 Specific optical rotation (2.2.7) : + 175 to + 195 (anhydrous
[37661-08-8] substance).
Dissolve 0.250 g in water R and dilute to 25.0 mL with the
DEFINITION same solvent.
(1RS)-1-[(Ethoxycarbonyl)oxy]ethyl (2S,5R,6R)-6-[[(2R)-2-
amino-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1- Related substances. Liquid chromatography (2.2.29). Prepare
azabicyclo[3.2.0]heptane-2-carboxylate hydrochloride. the test solution and reference solutions (a), (b) and (d)
immediately before use.
Semi-synthetic product derived from a fermentation product.
Phosphate buffer A. Dissolve 1.4 g of sodium dihydrogen
Content : 95.0 per cent to 102.0 per cent (anhydrous substance). phosphate monohydrate R in water R and dilute to about
CHARACTERS 800 mL with the same solvent. Adjust to pH 3.0 with dilute
phosphoric acid R and dilute to 1000.0 mL with water R.
Appearance : white or almost white powder or granules,
hygroscopic. Phosphate buffer B. Dissolve 2.75 g of sodium dihydrogen
phosphate monohydrate R and 2.3 g of disodium hydrogen
Solubility : soluble in water, freely soluble in ethanol (96 per
phosphate dihydrate R in water R and dilute to about 1800 mL
cent), soluble in methylene chloride.
with the same solvent. Adjust to pH 6.8, if necessary, using
IDENTIFICATION dilute phosphoric acid R or dilute sodium hydroxide solution R
First identification : A, D. and dilute to 2000.0 mL with water R.
Second identification : B, C, D. Test solution. Dissolve 30.0 mg of the substance to be
examined in phosphate buffer A and dilute to 100.0 mL with
A. Infrared absorption spectrophotometry (2.2.24). phosphate buffer A.
Comparison : bacampicillin hydrochloride CRS. Reference solution (a). Dissolve 30.0 mg of bacampicillin
B. Thin-layer chromatography (2.2.27). hydrochloride CRS in phosphate buffer A and dilute to
Test solution. Dissolve 10 mg of the substance to be 100.0 mL with phosphate buffer A.
examined in 2 mL of methanol R. Reference solution (b). Dilute 1.0 mL of reference solution (a)
Reference solution (a). Dissolve 10 mg of bacampicillin to 100.0 mL with phosphate buffer A.
hydrochloride CRS in 2 mL of methanol R. Reference solution (c). Dissolve 30 mg of the substance to be
Reference solution (b). Dissolve 10 mg of bacampicillin examined in phosphate buffer B and dilute to 100 mL with
hydrochloride CRS, 10 mg of talampicillin hydrochloride CRS phosphate buffer B. Heat at 80 °C for about 30 min.
and 10 mg of pivampicillin CRS in 2 mL of methanol R. Reference solution (d). Dissolve 20 mg of ampicillin
Plate : TLC silanised silica gel plate R. trihydrate CRS (impurity I) in phosphate buffer A and dilute
Mobile phase : mix 10 volumes of a 272 g/L solution of to 250 mL with phosphate buffer A. Dilute 5 mL of this
sodium acetate R adjusted to pH 5.0 with glacial acetic solution to 100 mL with phosphate buffer A.
acid R, 40 volumes of water R and 50 volumes of ethanol Column :
(96 per cent) R.
– size : l = 0.05 m, Ø = 3.9 mm ;
Application : 1 μL.
– stationary phase : octadecylsilyl silica gel for
Development : over a path of 15 cm. chromatography R (5 μm).
Drying : in a current of warm air. Mobile phase : mix 30 volumes of acetonitrile R1 and 70 volumes
Detection : spray with ninhydrin solution R1 and heat at of a 0.06 per cent m/m solution of tetrahexylammonium
60 °C for 10 min. hydrogen sulfate R in phosphate buffer B.
System suitability: reference solution (b) : Flow rate : 1.0 mL/min.
– the chromatogram shows 3 clearly separated spots. Detection : spectrophotometer at 220 nm.
Results : the principal spot in the chromatogram obtained Injection : 20 μL of the test solution and reference solutions (b),
with the test solution is similar in position, colour and size (c) and (d).
to the principal spot in the chromatogram obtained with
reference solution (a). Run time : 3.5 times the retention time of bacampicillin.
C. Place about 2 mg in a test-tube about 150 mm long and System suitability :
15 mm in diameter. Moisten with 0.05 mL of water R and – the peak due to impurity I is separated from the peaks due
add 2 mL of sulfuric acid-formaldehyde reagent R. Mix the to the solvent in the chromatogram obtained with reference
contents of the tube by swirling ; the solution is practically solution (d) ;
colourless. Place the test-tube on a water-bath for 1 min ; a – relative retention with reference to bacampicillin :
dark yellow colour develops. degradation product eluting just after bacampicillin = 1.12
D. Dissolve about 25 mg in 2 mL of water R. Add 2 mL of to 1.38 in the chromatogram obtained with reference
dilute sodium hydroxide solution R and shake. Wait a few solution (c) ; if necessary, adjust the concentration of
minutes and add 3 mL of dilute nitric acid R and 0.5 mL of tetrahexylammonium hydrogen sulfate in the mobile phase.

General Notices (1) apply to all monographs and other texts 1615
Bacampicillin hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Limits :
– any impurity : for each impurity, not more than 1.5 times
the area of the principal peak in the chromatogram
obtained with reference solution (b) (1.5 per cent) ;
– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b) C. (2RS,4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]-
(3 per cent) ; methyl]-5,5-dimethylthiazolidine-4-carboxylic acid
(penilloic acids of ampicillin),
– disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.1 per cent).
Butyl acetate and ethyl acetate (2.4.24, System A) : maximum
2.0 per cent of butyl acetate, maximum 4.0 per cent of ethyl
acetate and maximum 5.0 per cent for the sum of the contents.
Sample solution. Dissolve 50.0 mg of the substance to be
examined in water R and dilute to 10.0 mL with the same D. (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino]-
solvent. carboxymethyl]-5,5-dimethylthiazolidine-4-carboxylic acid
Use the method of standard additions. (penicilloic acids of ampicillin),

Static head-space conditions that may be used :

– equilibration temperature : 60 °C ;
– equilibration time : 20 min.
N,N-Dimethylaniline (2.4.26, Method A) : maximum 20 ppm.
Water (2.5.12) : maximum 0.8 per cent, determined on 0.300 g.
Sulfated ash (2.4.14) : maximum 1.5 per cent, determined on
1.0 g. E. (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5-
dimethylthiazolidine-4-carboxylic acid (diketopiperazines
of ampicillin),
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications.
Injection : test solution and reference solution (a).
System suitability: reference solution (a) : F. (2RS)-2-amino-3-methyl-3-sulfanylbutanoic acid
– repeatability : maximum relative standard deviation of (DL-penicillamine),
1.0 per cent after 6 injections.
Calculate the percentage content of C21H28ClN3O7S from the
declared content of bacampicillin hydrochloride CRS.

STORAGE
G. methyl (2R)-2-amino-2-phenylacetate (methyl
In an airtight container. D-phenylglycinate),

IMPURITIES

H. (1RS)-1-[(ethoxycarbonyl)oxy]ethyl (2S,5R,6R)-6-[[(2R)-
2-(acetylamino)-2-phenylacetyl]amino]-3,3-dimethyl-
7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate
A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia- (N-acetylbacampicillin),
1-azabicyclo[3.2.0]heptane-2-carboxylic acid
(6-aminopenicillanic acid),

I. (2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]-
3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-
B. (2R)-2-amino-2-phenylacetic acid (D-phenylglycine), carboxylic acid (ampicillin).

1616 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Bacitracin

01/2008:0465 Application : 10 μL.

Development : over half of the plate.
BACITRACIN Drying : at 100-105 °C.
Detection : spray with ninhydrin solution R1 and heat at
Bacitracinum 110 °C for 5 min.
Results : the spots in the chromatogram obtained with the
test solution are similar in position, size and colour to the
spots in the chromatogram obtained with the reference
solution.
B. Composition (see Tests).
C. Ignite 0.2 g. An insignificant residue remains which is not
yellow at high temperature. Allow to cool. Dissolve the
residue in 0.1 mL of dilute hydrochloric acid R. Add 5 mL of
water R and 0.2 mL of strong sodium hydroxide solution R.
No white precipitate is formed.
TESTS
Solution S. Dissolve 0.25 g in carbon dioxide-free water R and
dilute to 25 mL with the same solvent.
DEFINITION Appearance of solution. Solution S is clear (2.2.1).
Mixture of antimicrobial polypeptides produced by certain pH (2.2.3) : 6.0 to 7.0 for solution S.
strains of Bacillus licheniformis or Bacillus subtilis, the main Composition. Liquid chromatography (2.2.29) : use the
components being bacitracins A, B1, B2 and B3. normalisation procedure. Prepare the solutions immediately
Content : minimum 60 IU/mg (dried substance). before use.
Test solution. Dissolve 0.100 g of the substance to be examined
CHARACTERS
in 50.0 mL of the mobile phase.
Appearance : white or almost white powder, hygroscopic.
Reference solution (a). Suspend 20.0 mg of bacitracin zinc CRS
Solubility : freely soluble in water and in ethanol (96 per cent). in water R, add 0.2 mL of dilute hydrochloric acid R and dilute
to 10.0 mL with water R.
IDENTIFICATION
Reference solution (b). Dilute 5.0 mL of the test solution to
First identification : B, C. 100.0 mL with the mobile phase.
Second identification : A, C. Reference solution (c). Dilute 1.0 mL of reference solution (b)
A. Thin-layer chromatography (2.2.27). to 10.0 mL with the mobile phase.
Test solution. Dissolve 10 mg of the substance to be Reference solution (d). Dissolve 50.0 mg of the substance
examined in a 3.4 g/L solution of hydrochloric acid R and to be examined in 25.0 mL of a 40 g/L solution of sodium
dilute to 1.0 mL with the same solution. edetate R adjusted to pH 7.0 with dilute sodium hydroxide
Reference solution. Dissolve 10 mg of bacitracin zinc CRS solution R. Heat in a boiling water-bath for 30 min. Cool to
in a 3.4 g/L solution of hydrochloric acid R and dilute to room temperature.
1.0 mL with the same solution. Blank solution. A 40 g/L solution of sodium edetate R adjusted
Plate : TLC silica gel plate R. to pH 7.0 with dilute sodium hydroxide solution R.
Mobile phase : glacial acetic acid R, water R, butanol R Column :
(1:2:4 V/V/V). – size : l = 0.25 m, Ø = 4.6 mm ;

1. impurity A 3. impurity C 5. bacitracin B2 7. bacitracin A

2. impurity B 4. bacitracin B1 6. bacitracin B3

Figure 0465.-1. – Chromatogram of the test for composition in bacitracin obtained with the test solution at 254 nm

General Notices (1) apply to all monographs and other texts 1617
Bacitracin EUROPEAN PHARMACOPOEIA 8.0

– stationary phase : end-capped octadecylsilyl silica gel for Limit :

chromatography R (5 μm).
– sum of the areas of all peaks eluting before the peak due to
Mobile phase : add 40 volumes of acetonitrile R, 300 volumes bacitracin B1 : maximum 20.0 per cent.
of water R and 520 volumes of methanol R1 to 100 volumes
of a 34.8 g/L solution of dipotassium hydrogen phosphate R Impurity E. Liquid chromatography (2.2.29) as described in
adjusted to pH 6.0 with a 27.2 g/L solution of potassium the test for composition.
dihydrogen phosphate R. See Figure 0465.-2.
Flow rate : 1.0 mL/min. Detection : spectrophotometer at 254 nm ; spectrophotometer
Detection : spectrophotometer at 254 nm. at 300 nm for reference solution (d).
Injection : 100 μL ; inject the blank, the test solution and Injection : test solution and reference solutions (b) and (d).
reference solutions (a) and (c).
Limit :
Run time : 3 times the retention time of bacitracin A.
Relative retention with reference to bacitracin A (retention – impurity E : not more than 1.2 times the area of the
time = 15 min to 25 min) : bacitracin B1 = about 0.6 ; principal peak in the chromatogram obtained with
bacitracin B3 = about 0.8 ; impurity E = about 2.5. reference solution (b) (6.0 per cent).
If necessary, adjust the composition of the mobile phase by Loss on drying (2.2.32) : maximum 5.0 per cent, determined
changing the amount of organic modifier whilst keeping the on 1.000 g by drying at 60 °C over diphosphorus pentoxide R at
ratio constant between methanol and acetonitrile. a pressure not exceeding 0.1 kPa for 3 h.
System suitability: reference solution (a) : Sulfated ash (2.4.14) : maximum 1.0 per cent, determined on
1.0 g.
– peak-to-valley ratio : minimum of 1.2, where Hp = height
above the baseline of the peak due to bacitracin B1 Sterility (2.6.1). If intended for the preparation of ophthalmic
and Hv = height above the baseline of the lowest point dosage forms without a further appropriate sterilisation
of the curve separating this peak from the peak due to procedure, it complies with the test for sterility.
bacitracin B2. Bacterial endotoxins (2.6.14) : less than 0.8 IU/mg, if
Limits : intended for use in the manufacture of ophthalmic dosage
– bacitracin A : minimum 40.0 per cent ; forms without a further appropriate procedure for the removal
of bacterial endotoxins.
– sum of bacitracins A, B1, B2 and B3 : minimum 70.0 per
cent ;
ASSAY
– disregard limit : the area of the peak due to bacitracin A
in the chromatogram obtained with reference solution (c) Carry out the microbiological assay of antibiotics (2.7.2). Use
(0.5 per cent) ; disregard any peak observed in the blank bacitracin zinc CRS as the reference substance.
run.
Related peptides. Liquid chromatography (2.2.29) as STORAGE
described in the test for composition. In an airtight container at 2 °C to 8 °C. If the substance is
See Figure 0465.-1. sterile, store in a sterile, airtight, tamper-proof container.

1. bacitracin B1 2. bacitracin B3 3. bacitracin A 4. impurity E

Figure 0465.-2. – Chromatogram of the test for impurity E in bacitracin obtained with reference solution (d) at 300 nm

1618 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Bacitracin zinc

IMPURITIES Results : the spots in the chromatogram obtained with the

test solution are similar in position, size and colour to the
spots in the chromatogram obtained with the reference
solution.
B. Composition (see Tests).
C. Ignite about 0.15 g, allow to cool and dissolve the residue
in 1 mL of dilute hydrochloric acid R. Add 4 mL of water R.
The solution gives the reaction of zinc (2.3.1).

A. X = L-Val, Y = L-Ile, R = H : bacitracin C1, TESTS

pH (2.2.3) : 6.0 to 7.5.
B. X = L-Ile, Y = L-Val, R = H : bacitracin C2,
Shake 1.0 g for about 1 min with 10 mL of carbon dioxide-free
C. X = Y = L-Val, R = CH3 : bacitracin C3, water R and filter.
D. X = Y = L-Val, R = H : bacitracin E, Composition. Liquid chromatography (2.2.29) : use the
normalisation procedure. Prepare the solutions immediately
before use.
Test solution. Dissolve 0.100 g of the substance to be examined
in 50.0 mL of a 40 g/L solution of sodium edetate R adjusted to
pH 7.0 with dilute sodium hydroxide solution R.
Reference solution (a). Dissolve 20.0 mg of bacitracin zinc CRS
in 10.0 mL of a 40 g/L solution of sodium edetate R adjusted to
pH 7.0 with dilute sodium hydroxide solution R.
E. X = Y = L-Ile, R = CH3 : bacitracin F, Reference solution (b). Dilute 5.0 mL of the test solution to
F. X = Y = L-Ile, R = H : bacitracin H1, 100.0 mL with water R.
G. X = L-Val, Y = L-Ile, R = CH3 : bacitracin H2, Reference solution (c). Dilute 1.0 mL of reference solution (b)
to 10.0 mL with water R.
H. X = L-Ile, Y = L-Val, R = CH3 : bacitracin H3,
Reference solution (d). Dissolve 50.0 mg of the substance
I. X = L-Val, Y = L-Ile, R = H : bacitracin I1, to be examined in 25.0 mL of a 40 g/L solution of sodium
J. X = L-Ile, Y = L-Val, R = H : bacitracin I2, edetate R adjusted to pH 7.0 with dilute sodium hydroxide
solution R. Heat in a boiling water-bath for 30 min. Cool to
K. X = Y = L-Val, R = CH3 : bacitracin I3. room temperature.
01/2008:0466 Blank solution. A 40 g/L solution of sodium edetate R adjusted
to pH 7.0 with dilute sodium hydroxide R.
BACITRACIN ZINC Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
Bacitracinum zincum – stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
DEFINITION
Mobile phase : add 520 volumes of methanol R1, 40 volumes
Zinc complex of bacitracin, which consists of a mixture of of acetonitrile R and 300 volumes of water R to 100 volumes
antimicrobial polypeptides produced by certain strains of of a 34.8 g/L solution of dipotassium hydrogen phosphate R,
Bacillus licheniformis or Bacillus subtilis, the main components adjusted to pH 6.0 with a 27.2 g/L solution of potassium
being bacitracins A, B1, B2 and B3. dihydrogen phosphate R.
Content : minimum 60 IU/mg (dried substance). Flow rate : 1.0 mL/min.
CHARACTERS Detection : spectrophotometer at 254 nm.
Appearance : white or light yellowish-grey powder, Injection : 100 μL ; inject the blank, the test solution and
hygroscopic. reference solutions (a) and (c).
Solubility : slightly soluble in water and in ethanol (96 per Run time : 3 times the retention time of bacitracin A.
cent). Relative retention with reference to bacitracin A (retention
time = 15 min to 25 min) : bacitracin B1 = about 0.6 ;
IDENTIFICATION
bacitracin B3 = about 0.8 ; impurity E = about 2.5.
First identification : B, C.
If necessary, adjust the composition of the mobile phase by
Second identification : A, C. changing the amount of organic modifier whilst keeping the
A. Thin-layer chromatography (2.2.27). ratio constant between methanol and acetonitrile.
Test solution. Dissolve 10 mg of the substance to be System suitability : reference solution (a) :
examined in 0.5 mL of dilute hydrochloric acid R and dilute – peak-to-valley ratio : minimum of 1.2, where Hp = height
to 1.0 mL with water R. above the baseline of the peak due to bacitracin B1
Reference solution. Dissolve 10 mg of bacitracin zinc CRS and Hv = height above the baseline of the lowest point
in 0.5 mL of dilute hydrochloric acid R and dilute to 1.0 mL of the curve separating this peak from the peak due to
with water R. bacitracin B2.
Plate : TLC silica gel plate R. Limits :
Mobile phase : glacial acetic acid R, water R, butanol R – bacitracin A : minimum 40.0 per cent ;
(1:2:4 V/V/V).
– sum of bacitracins A, B1, B2 and B3 : minimum 70.0 per
Application : 10 μL. cent ;
Development : over half of the plate. – disregard limit : the area of the peak due to bacitracin A
Drying : at 100-105 °C. in the chromatogram obtained with reference solution (c)
Detection : spray with ninhydrin solution R1 and heat at (0.5 per cent) ; disregard any peak observed in the blank
110 °C for 5 min. run.

General Notices (1) apply to all monographs and other texts 1619
Bacitracin zinc EUROPEAN PHARMACOPOEIA 8.0

1. impurity A 3. impurity C 5. bacitracin B2 7. bacitracin A

2. impurity B 4. bacitracin B1 6. bacitracin B3

Figure 0466.-1. – Chromatogram of the test for composition in bacitracin zinc obtained with the test solution at 254 nm

1. bacitracin B1 2. bacitracin B3 3. bacitracin A 4. impurity E

Figure 0466.-2. – Chromatogram of the test for impurity E in bacitracin zinc obtained with reference solution (d) at 300 nm
Related peptides. Liquid chromatography (2.2.29) as Detection : spectrophotometer at 254 nm ; spectrophotometer
described in the test for composition. at 300 nm for reference solution (d).
See Figure 0466.-1. Injection : test solution and reference solutions (b) and (d).
Limit : Limit :
– impurity E : not more than 1.2 times the area of the
– sum of the areas of all peaks eluting before the peak due to principal peak in the chromatogram obtained with
bacitracin B1 : maximum 20.0 per cent. reference solution (b) (6.0 per cent).
Impurity E. Liquid chromatography (2.2.29) as described in Zinc : 4.0 per cent to 6.0 per cent (dried substance).
the test for composition. Dissolve 0.200 g in a mixture of 2.5 mL of dilute acetic acid R
See Figure 0466.-2. and 2.5 mL of water. Add 50 mL of water R, 50 mg of xylenol

1620 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Baclofen

orange triturate R and sufficient hexamethylenetetramine R to 01/2008:0653

produce a red colour. Add 2 g of hexamethylenetetramine R
in excess. Titrate with 0.01 M sodium edetate until a yellow BACLOFEN
colour is obtained.
1 mL of 0.01 M sodium edetate is equivalent to 0.654 mg of Zn. Baclofenum
Loss on drying (2.2.32) : maximum 5.0 per cent, determined
on 1.000 g by drying at 60 °C over diphosphorus pentoxide R at
a pressure not exceeding 0.1 kPa for 3 h.
Sterility (2.6.1). If intended for administration by spraying
into internal body cavities without a further appropriate
sterilisation procedure, it complies with the test for sterility.
C10H12ClNO2 Mr 213.7
Pyrogens (2.6.8). If intended for administration by spraying
[1134-47-0]
into internal body cavities without a further appropriate
procedure for the removal of pyrogens, it complies with the DEFINITION
test for pyrogens. Inject per kilogram of the rabbit’s mass 1 mL (3RS)-4-Amino-3-(4-chlorophenyl)butanoic acid.
of the supernatant obtained by centrifuging a suspension
containing 11 mg per millilitre in a 9 g/L solution of sodium Content : 98.0 per cent to 101.0 per cent (anhydrous substance).
chloride R. CHARACTERS
ASSAY Appearance : white or almost white powder.
Suspend 50.0 mg in 5 mL of water R, add 0.5 mL of dilute Solubility : slightly soluble in water, very slightly soluble in
hydrochloric acid R and dilute to 100.0 mL with water R. Allow ethanol (96 per cent), practically insoluble in acetone. It
the solution to stand for 30 min. Carry out the microbiological dissolves in dilute mineral acids and in dilute solutions of
assay of antibiotics (2.7.2). alkali hydroxides.
It shows polymorphism (5.9).
STORAGE
IDENTIFICATION
In an airtight container. If the substance is sterile, store in a
sterile, airtight, tamper-proof container. First identification : B.
Second identification : A, C.
IMPURITIES A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Dissolve 70 mg in water R and dilute to
100.0 mL with the same solvent.
Spectral range : 220-320 nm.
Absorption maxima : at 259 nm, 266 nm and 275 nm.
Resolution (2.2.25) : minimum 1.5 for the absorbance ratio.
Specific absorbance at the absorption maxima :
– at 259 nm : 9.8 to 10.8 ;
A. X = L-Val, Y = L-Ile, R = H : bacitracin C1, – at 266 nm : 11.5 to 12.7 ;
– at 275 nm : 8.4 to 9.3.
B. X = L-Ile, Y = L-Val, R = H : bacitracin C2, B. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs prepared using 3 mg of substance and
C. X = Y = L-Val, R = CH3 : bacitracin C3, 300 mg of potassium bromide R.
Comparison: baclofen CRS.
D. X = Y = L-Val, R = H : bacitracin E,
If the spectra obtained in the solid state show differences,
dissolve 0.1 g of each of the substances separately in 1 mL
of dilute sodium hydroxide solution R and add 10 mL of
ethanol (96 per cent) R and 1 mL of dilute acetic acid R.
Allow to stand for 1 h. Filter, wash the precipitate with
ethanol (96 per cent) R and dry in vacuo. Prepare new discs
and record the spectra.
C. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 10 mg of the substance to be
examined in the mobile phase and dilute to 10 mL with
E. X = Y = L-Ile, R = CH3 : bacitracin F, the mobile phase.
Reference solution. Dissolve 10 mg of baclofen CRS in the
F. X = Y = L-Ile, R = H : bacitracin H1, mobile phase and dilute to 10 mL with the mobile phase.
Plate : TLC silica gel G plate R.
G. X = L-Val, Y = L-Ile, R = CH3 : bacitracin H2,
Mobile phase : anhydrous formic acid R, water R, methanol R,
H. X = L-Ile, Y = L-Val, R = CH3 : bacitracin H3, chloroform R, ethyl acetate R (5:5:20:30:40 V/V/V/V/V).
Application : 5 μL.
I. X = L-Val, Y = L-Ile, R = H : bacitracin I1, Development : over a path of 12 cm.
Drying : allow the solvents to evaporate.
J. X = L-Ile, Y = L-Val, R = H : bacitracin I2, Detection : spray with ninhydrin solution R3 until the plate
is slightly wet. Place in an oven maintained at 100 °C for
K. X = Y = L-Val, R = CH3 : bacitracin I3. 10 min. Examine in daylight.

General Notices (1) apply to all monographs and other texts 1621
Bambuterol hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Results : the principal spot in the chromatogram obtained

with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
the reference solution.
TESTS
Appearance of solution. The solution is not more opalescent A. (4RS)-4-(4-chlorophenyl)pyrrolidin-2-one,
than reference suspension II (2.2.1) and not more intensely
coloured than reference solution BY5 (2.2.2, Method II).
Dissolve 0.50 g in 1 M sodium hydroxide and dilute to 25 mL
with the same solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be
examined in the mobile phase and dilute to 10.0 mL with the B. (3RS)-5-amino-3-(4-chlorophenyl)-5-oxopentanoic acid.
mobile phase.
Reference solution (a). Dissolve 25.0 mg of baclofen 01/2008:1293
impurity A CRS in the mobile phase and dilute to 10.0 mL
with the mobile phase.
BAMBUTEROL HYDROCHLORIDE
Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 100.0 mL with the mobile phase.
Reference solution (c). Dilute 2.0 mL of the test solution to
Bambuteroli hydrochloridum
100.0 mL with the mobile phase.
Reference solution (d). Dilute 2.0 mL of the test solution and
2.0 mL of reference solution (a) to 100.0 mL with the mobile
phase.
Column :
– size : l = 0.25 m, Ø = 4.0 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (10 μm). C18H30ClN3O5 Mr 403.9
Mobile phase : dissolve 1.822 g of sodium hexanesulfonate R in [81732-46-9]
1 L of a mixture of 560 volumes of water R, 440 volumes of
methanol R and 5 volumes of glacial acetic acid R. DEFINITION
Flow rate : 2.0 mL/min. 5-[(1RS)-2-[(1,1-Dimethylethyl)amino]-1-hydroxyethyl]-1,3-
phenylene bis(dimethylcarbamate) hydrochloride.
Detection : spectrophotometer at 266 nm.
Content : 98.5 per cent to 101.5 per cent (anhydrous substance).
Injection : 20 μL of the test solution and reference solutions (b),
(c) and (d). CHARACTERS
Run time : 5 times the retention time of baclofen. Appearance : white or almost white, crystalline powder.
System suitability : reference solution (d) : Solubility : freely soluble in water, soluble in ethanol (96 per
– resolution : minimum 2.0 between the peaks due to baclofen cent).
and impurity A. It shows polymorphism (5.9).
Limits :
IDENTIFICATION
– impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (b) A. Infrared absorption spectrophotometry (2.2.24).
(1.0 per cent) ; Preparation : discs.
– total : not more than the area of the principal peak in the Comparison: bambuterol hydrochloride CRS.
chromatogram obtained with reference solution (c) (2.0 per If the spectra obtained show differences, dissolve the
cent). substance to be examined and the reference substance
Water (2.5.12) : maximum 1.0 per cent, determined on 1.000 g. separately in a mixture of 1 volume of water R and
6 volumes of acetone R, cool in ice to precipitate and dry
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on both precipitates in vacuo at 50 °C to constant weight.
1.0 g. Record new spectra using the residues.
ASSAY B. It gives reaction (a) of chlorides (2.3.1).
Dissolve 0.1500 g in 50 mL of anhydrous acetic acid R.
TESTS
Titrate with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20). Solution S. Dissolve 4.0 g in carbon dioxide-free water R and
1 mL of 0.1 M perchloric acid is equivalent to 21.37 mg dilute to 20.0 mL with the same solvent.
of C10H12ClNO2. Acidity or alkalinity. To 10 mL of solution S add 0.2 mL of
methyl red solution R and 0.2 mL of 0.01 M hydrochloric acid.
IMPURITIES The solution is red. Add 0.4 mL of 0.01 M sodium hydroxide.
Specified impurities : A. The solution is yellow.
Other detectable impurities (the following substances would, Optical rotation (2.2.7) : − 0.10° to + 0.10°.
if present at a sufficient level, be detected by one or other of Dilute 1 mL of solution S to 10 mL with carbon dioxide-free
the tests in the monograph. They are limited by the general water R.
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical Related substances. Liquid chromatography (2.2.29).
use (2034). It is therefore not necessary to identify these Test solution. Dissolve 5.0 mg of the substance to be examined
impurities for demonstration of compliance. See also 5.10. in the mobile phase and dilute to 10.0 mL with the mobile
Control of impurities in substances for pharmaceutical use): B. phase.

1622 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Barbital

Reference solution (a). Dissolve 1.0 mg of formoterol fumarate D. R1 = H, R2 = R3 = CO-N(CH3)2 : 5-[(1RS)-1-hydroxyethyl]-

dihydrate CRS in the mobile phase and dilute to 10.0 mL with 1,3-phenylene bis(dimethylcarbamate),
the mobile phase. Mix 0.8 mL of this solution with 0.4 mL of
the test solution and dilute to 100.0 mL with the mobile phase.
Reference solution (b). Dilute 1.0 mL of the test solution to
50.0 mL with the mobile phase. Dilute 2.0 mL of this solution
to 20.0 mL with the mobile phase.
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : base-deactivated octadecylsilyl silica gel for E. R = H : 5-acetyl-1,3-phenylene bis(dimethylcarbamate),
chromatography R (5 μm).
Mobile phase : dissolve 1.3 g of sodium octanesulfonate R in F. R = NH-C(CH3)3 : 5-[[(1,1-dimethylethyl)amino]acetyl]-
430 mL of a mixture of 25 volumes of acetonitrile R1 and 1,3-phenylene bis(dimethylcarbamate).
75 volumes of methanol R ; then mix this solution with 570 mL
of 0.050 M phosphate buffer pH 3.0 prepared as follows :
dissolve 6.90 g of sodium dihydrogen phosphate monohydrate R 01/2008:0170
in water R and dilute to 1000 mL with water R, adjust to corrected 6.0
pH 3.0 with a 50 g/L solution of dilute phosphoric acid R.
Flow rate : 1.5 mL/min. BARBITAL
Detection : spectrophotometer at 214 nm.
Injection : 20 μL; inject the mobile phase as a blank. Barbitalum
Run time : 1.5 times the retention time of bambuterol.
Retention time : formoterol = about 7 min ; bambuterol = about
9 min. If necessary, adjust the composition of the mobile
phase ; increase the content of phosphate buffer to increase
the retention time.
System suitability: reference solution (a) : C8H12N2O3 Mr 184.2
– resolution : minimum 5.0 between the peaks due to [57-44-3]
bambuterol and formoterol. DEFINITION
Limits : Barbital contains not less than 99.0 per cent and
– impurities A, B, C, D, E, F : for each impurity, not more not more than the equivalent of 101.0 per cent of
than the area of the principal peak in the chromatogram 5,5-diethylpyrimidine-2,4,6(1H,3H,5H)-trione, calculated
obtained with reference solution (b) (0.2 per cent) ; with reference to the dried substance.
– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b) CHARACTERS
(0.6 per cent) ; A white or almost white, crystalline powder or colourless
– disregard limit : 0.25 times the area of the principal peak crystals, slightly soluble in water, soluble in boiling water and
in the chromatogram obtained with reference solution (b) in alcohol. It forms water-soluble compounds with alkali
(0.05 per cent) ; disregard any peak due to the mobile phase. hydroxides and carbonates and with ammonia.
Water (2.5.12) : maximum 0.5 per cent, determined on 0.500 g. IDENTIFICATION
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on First identification : A, B.
1.0 g. Second identification : A, C, D.
ASSAY A. Determine the melting point (2.2.14) of the substance to be
Dissolve 0.320 g in 50 mL of ethanol (96 per cent) R and add examined. Mix equal parts of the substance to be examined
5 mL of 0.01 M hydrochloric acid. Carry out a potentiometric and barbital CRS and determine the melting point of the
titration (2.2.20), using 0.1 M sodium hydroxide. Read mixture. The difference between the melting points (which
the volume added between the 2 points of inflexion. are about 190 °C) is not greater than 2 °C.
1 mL of 0.1 M sodium hydroxide is equivalent to 40.39 mg B. Examine by infrared absorption spectrophotometry
of C18H30ClN3O5. (2.2.24), comparing with the spectrum obtained with
barbital CRS.
IMPURITIES C. Examine by thin-layer chromatography (2.2.27), using
Specified impurities : A, B, C, D, E, F. silica gel GF254 R as the coating substance.
Test solution. Dissolve 75 mg of the substance to be
examined in alcohol R and dilute to 25 mL with the same
solvent.
Reference solution. Dissolve 75 mg of barbital CRS in
alcohol R and dilute to 25 mL with the same solvent.
Apply separately to the plate 10 μL of each solution.
A. R1 = NH-C(CH3)3, R2 = R3 = H : (1RS)-1-(3,5- Develop over a path of 18 cm using the lower layer of
dihydroxyphenyl)-2-[(1,1-dimethylethyl)amino]ethanol a mixture of 5 volumes of concentrated ammonia R,
(terbutaline), 15 volumes of alcohol R and 80 volumes of chloroform R.
Examine immediately in ultraviolet light at 254 nm. The
B. R1 = OH, R2 = R3 = CO-N(CH3)2 : 5-[(1RS)-1,2- principal spot in the chromatogram obtained with the test
dihydroxyethyl]-1,3-phenylene bis(dimethylcarbamate), solution is similar in position and size to the principal spot
C. R1 = NH-C(CH3)3, R2 = H, R3 = CO-N(CH3)2 : in the chromatogram obtained with the reference solution.
3-[(1RS)-2-[(1,1-dimethylethyl)amino]-1-hydroxyethyl]-5- D. It gives the reaction of non-nitrogen substituted
hydroxyphenyl dimethylcarbamate, barbiturates (2.3.1).

General Notices (1) apply to all monographs and other texts 1623
Barium sulfate EUROPEAN PHARMACOPOEIA 8.0

TESTS filtrate 0.3 mL of dilute sulfuric acid R. A white precipitate

Appearance of solution. Dissolve 1.0 g in a mixture of 4 mL is formed that is insoluble in dilute sodium hydroxide
of dilute sodium hydroxide solution R and 6 mL of water R. solution R.
The solution is clear (2.2.1) and not more intensely coloured TESTS
than reference solution Y6 (2.2.2, Method II).
Solution S. To 20.0 g add 40 mL of distilled water R and 60 mL
Acidity. Boil 1.0 g with 50 mL of water R for 2 min, allow to of dilute acetic acid R. Boil for 5 min, filter and dilute the
cool and filter. To 10 mL of the filtrate add 0.15 mL of methyl cooled filtrate to 100 mL with distilled water R.
red solution R. The solution is orange-yellow. Not more than
0.1 mL of 0.1 M sodium hydroxide is required to produce a Acidity or alkalinity. Heat 5.0 g with 20 mL of carbon
pure yellow colour. dioxide-free water R on a water-bath for 5 min and filter.
To 10 mL of the filtrate add 0.05 mL of bromothymol blue
Related substances. Examine by thin-layer chromatography solution R1. Not more than 0.5 mL of 0.01 M hydrochloric
(2.2.27), using silica gel GF254 R as the coating substance. acid or 0.01 M sodium hydroxide is required to change the
Test solution. Dissolve 1.0 g of the substance to be examined colour of the indicator.
in alcohol R and dilute to 100 mL with the same solvent. Acid-soluble substances : maximum 0.3 per cent.
Reference solution. Dilute 0.5 mL of the test solution to 100 mLEvaporate 25 mL of solution S to dryness on a water-bath
with alcohol R. and dry to constant mass at 100-105 °C. The residue weighs
Apply separately to the plate 20 μL of each solution. Develop a maximum of 15 mg.
over a path of 15 cm using the lower layer of a mixture of
5 volumes of concentrated ammonia R, 15 volumes of alcohol R Oxidisable sulfur compounds. Shake 1.0 g with 5 mL of
and 80 volumes of chloroform R. Examine immediately in water R for 30 s and filter. To the filtrate add 0.1 mL of
ultraviolet light at 254 nm. Spray with diphenylcarbazone starch solution R, dissolve 0.1 g of potassium iodide R in the
mercuric reagent R. Allow the plate to dry in air and spray mixture, add 1.0 mL of a freshly prepared 3.6 mg/L solution
with freshly prepared alcoholic potassium hydroxide solution R of potassium iodate R and 1 mL of 1 M hydrochloric acid and
diluted 1 in 5 with aldehyde-free alcohol R. Heat at 100 °C shake well. The colour of the solution is more intense than
to 105 °C for 5 min and examine immediately. When that of a standard prepared at the same time and in the same
examined in ultraviolet light and after spraying, any spot in manner, but omitting the potassium iodate.
the chromatogram obtained with the test solution, apart from Soluble barium salts : maximum 10 ppm.
the principal spot, is not more intense than the spot in the To 2.5 mL of a 0.2 mg/L solution of barium nitrate R in
chromatogram obtained with the reference solution (0.5 per a mixture of 30 volumes of ethanol (96 per cent) R and
cent). 70 volumes of water R, add 10 mL of dilute sulfuric acid R.
Loss on drying (2.2.32). Not more than 0.5 per cent, Shake and allow to stand for 5 min. To 1 mL of this solution
determined on 1.00 g by drying in an oven at 105 °C. add 10 mL of solution S. Prepare a standard in the same
manner using 10 mL of barium standard solution (2 ppm
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined Ba) R instead of solution S.
on 1.0 g.
After 10 min, any opalescence in the test solution is not more
ASSAY intense than that in the standard.
Dissolve 85.0 mg in 5 mL of pyridine R. Add 0.5 mL of Heavy metals (2.4.8) : maximum 10 ppm.
thymolphthalein solution R and 10 mL of silver nitrate solution Dilute 10 mL of solution S to 20 mL with water R. 12 mL of the
in pyridine R. Titrate with 0.1 M ethanolic sodium hydroxide solution complies with test A. Prepare the reference solution
until a pure blue colour is obtained. Carry out a blank using lead standard solution (1 ppm Pb) R.
titration.
Loss on ignition : maximum 2.0 per cent, determined on 1.0 g
1 mL of 0.1 M ethanolic sodium hydroxide is equivalent to at 600 ± 50 °C.
9.21 mg of C8H12N2O3.

01/2013:1975
01/2008:0010
corrected 7.0
BASIC BUTYLATED METHACRYLATE
BARIUM SULFATE COPOLYMER
Barii sulfas Copolymerum methacrylatis butylati
basicum
BaSO4 Mr 233.4
[7727-43-7] DEFINITION
Copolymer of 2-(dimethylamino)ethyl methacrylate, butyl
CHARACTERS methacrylate and methyl methacrylate having a mean
Appearance : fine, white or almost white powder, free from relative molecular mass of about 150 000. The ratio of
gritty particles. 2-(dimethylamino)ethyl methacrylate groups to butyl
Solubility : practically insoluble in water and in organic methacrylate and methyl methacrylate groups is about 2:1:1.
solvents. It is very slightly soluble in acids and in solutions of Content of dimethylaminoethyl groups : 20.8 per cent to
alkali hydroxides. 25.5 per cent (dried substance).
IDENTIFICATION CHARACTERS
A. Boil a suspension of 0.2 g with 5 mL of a 500 g/L solution of Appearance : colourless or yellowish granules or white or
sodium carbonate R for 5 min, add 10 mL of water R, filter almost white powder, slightly hygroscopic.
and acidify a part of the filtrate with dilute hydrochloric Solubility : practically insoluble in water, freely soluble in
acid R. The solution gives the reactions of sulfates (2.3.1). methylene chloride. It dissolves slowly in ethanol (96 per cent).
B. Wash the residue collected in the preceding test with
3 successive small quantities of water R. To the residue add IDENTIFICATION
5 mL of dilute hydrochloric acid R, filter and add to the A. Infrared absorption spectrophotometry (2.2.24).

1624 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Basic butylated methacrylate copolymer

Comparison : basic butylated methacrylate copolymer CRS. B. 2-(Dimethylamino)ethyl methacrylate. Liquid

B. It complies with the limits of the assay. chromatography (2.2.29).
Test solution. Dissolve 1.00 g of the substance to be
TESTS examined in tetrahydrofuran R and dilute to 50.0 mL with
Solution S. Dissolve 12.5 g in a mixture of 35.0 g of acetone R the same solvent.
and 52.5 g of 2-propanol R. Reference solution. Dissolve 10.0 mg of 2-(dimethyl-
amino)ethyl methacrylate CRS (impurity C) in
Viscosity (2.2.10) : 3 mPa·s to 6 mPa·s, determined on tetrahydrofuran R and dilute to 50.0 mL with the same
solution S. solvent. Dilute 2.0 mL of the solution to 50.0 mL with
Apparatus : rotating viscometer. tetrahydrofuran R.
Dimensions : Column :
– spindle : diameter = 25.15 mm, height = 90.74 mm, shaft – size : l = 0.125 m, Ø = 4.6 mm ;
diameter = 4 mm ; – stationary phase : aminopropylsilyl silica gel for
– cylinder : diameter = 27.62 mm, height = 0.135 m. chromatography R (7 μm).
Rotating speed : 30 r/min. Mobile phase : mix 25 volumes of a 3.404 g/L solution
of potassium dihydrogen phosphate R and 75 volumes of
Volume of solution : 16 mL of solution S. tetrahydrofuran R.
Temperature : 20 °C. Flow rate : 2.0 mL/min.
Absorbance (2.2.25): maximum 0.30 at 420 nm, determined Detection : spectrophotometer at 215 nm.
on solution S. Injection : 50 μL.
Appearance of a film. Spread 1.0 mL of solution S evenly on Calculate the percentage content of impurity C as described
a glass plate. Upon drying a clear film is formed. under procedure A.
Monomers : maximum 0.1 per cent for each monomer Heavy metals (2.4.8) : maximum 20 ppm.
(butyl methacrylate, methyl methacrylate and 2.0 g complies with test C. Prepare the reference solution using
2-(dimethylamino)ethyl methacrylate), determined 4.0 mL of lead standard solution (10 ppm Pb) R.
by procedures A and B. Loss on drying (2.2.32) : maximum 2.0 per cent, determined
A. Butyl methacrylate and methyl methacrylate. Liquid on 1.000 g by drying in an oven at 110 °C for 3 h.
chromatography (2.2.29). Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Solvent mixture : acetonitrile R1, phosphate buffer solution 1.0 g.
pH 2.0 R (40:60 V/V).
ASSAY
Test solution. Dissolve 1.00 g of the substance to be
examined in the solvent mixture and dilute to 50.0 mL with Dissolve 0.200 g in a mixture of 4 mL of water R and 96 mL
the solvent mixture. of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid,
determining the end-point potentiometrically (2.2.20).
Reference solution. Dissolve 20.0 mg of butyl
methacrylate CRS (impurity A) and 10.0 mg of methyl 1 mL of 0.1 M perchloric acid is equivalent to 7.21 mg of
methacrylate CRS (impurity B) in 3.0 mL of butanol R and C4H10N.
dilute to 10.0 mL with the solvent mixture. Dilute 1.0 mL STORAGE
of the solution to 250.0 mL with the solvent mixture.
In an airtight container.
Column :
– size : l = 0.125 m, Ø = 4.6 mm ; IMPURITIES
– stationary phase : octadecylsilyl silica gel for
chromatography R (7 μm).
Mobile phase : phosphate buffer solution pH 2.0 R,
methanol R (45:55 V/V). A. butyl 2-methylprop-2-enoate (butyl methacrylate),
Flow rate : 2.0 mL/min.
Detection : spectrophotometer at 205 nm.
Injection : 50 μL.
System suitability: reference solution :
– resolution : minimum 5 between the peaks due to B. methyl 2-methylprop-2-enoate (methyl methacrylate),
impurities A and B.
Calculate the percentage content of each monomer using
the following expression :

C. 2-(dimethylamino)ethyl 2-methylprop-2-enoate
(2-(dimethylamino)ethyl methacrylate).
C = concentration of the monomer in the reference FUNCTIONALITY-RELATED CHARACTERISTICS
solution, in micrograms per millilitre ; This section provides information on characteristics that are
M = mass of substance to be examined in the test recognised as being relevant control parameters for one or
solution, in grams ; more functions of the substance when used as an excipient
AT = area of the peak due to the monomer in the (see chapter 5.15). Some of the characteristics described in
the Functionality-related characteristics section may also be
chromatogram obtained with the test solution ;
present in the mandatory part of the monograph since they
AR = area of the peak due to the monomer in the also represent mandatory quality criteria. In such cases, a
chromatogram obtained with the reference cross-reference to the tests described in the mandatory part is
solution. included in the Functionality-related characteristics section.

General Notices (1) apply to all monographs and other texts 1625
Beclometasone dipropionate, anhydrous EUROPEAN PHARMACOPOEIA 8.0

Reference solution (a). Dilute 5.0 mL of test solution (b) to

Control of the characteristics can contribute to the quality
of a medicinal product by improving the consistency of the
100.0 mL with the solvent mixture.
manufacturing process and the performance of the medicinal
Reference solution (b). Dissolve 5 mg of beclometasone
product during use. Where control methods are cited, they are
dipropionate for system suitability CRS (containing impurity D)
recognised as being suitable for the purpose, but other methods
in 3 mL of mobile phase B and dilute to 5 mL with mobile
can also be used. Wherever results for a particular characteristic
phase A.
are reported, the control method must be indicated.
Reference solution (c). Dissolve 5 mg of beclometasone
The following characteristics may be relevant for basic butylated
dipropionate for peak identification CRS (containing impurities
methacrylate copolymer used as film former in tablets.
A, B, C, L and M) in 3 mL of mobile phase B and dilute to
Viscosity (see Tests). 5 mL with mobile phase A. Use 1 mL of this solution to
Appearance of a film (see Tests). dissolve the contents of a vial of beclometasone dipropionate
impurities F and N CRS.
Solubility of a film. Take the film obtained in the test for
Reference solution (d). Dissolve 50.0 mg of anhydrous
appearance of a film (see Tests), place it in a flask containing
beclometasone dipropionate CRS in 28 mL of mobile phase B
0.1 M hydrochloric acid and stir. It dissolves within 1 h. Take
and dilute to 50.0 mL with mobile phase A. Dilute 1.0 mL of
another film, place it in a flask containing phosphate buffer
this solution to 50.0 mL with the solvent mixture.
solution pH 6.8 R and stir. It does not dissolve within 2 h.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
01/2009:0654
corrected 7.0 – stationary phase : spherical difunctional bonded end-capped
octadecylsilyl silica gel for chromatography R (5 μm) ;
BECLOMETASONE DIPROPIONATE, – temperature : 50 °C.
ANHYDROUS Mobile phase :
– mobile phase A : 2.72 g/L solution of potassium dihydrogen
Beclometasoni dipropionas anhydricus phosphate R adjusted to pH 2.35 with phosphoric acid R ;
– mobile phase B : tetrahydrofuran R, acetonitrile R,
methanol R (5:23:25 V/V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-4 40 60

4 - 12 40 → 45 60 → 55

12 - 59 45 55

C28H37ClO7 Mr 521.0 Flow rate : 1.4 mL/min.

[5534-09-8] Detection : spectrophotometer at 254 nm.
Injection : 20 μl of test solution (a) and reference solutions (a),
DEFINITION (b) and (c).
9-Chloro-11β-hydroxy-16β-methyl-3,20-dioxopregna-1,4- Identification of impurities : use the chromatogram supplied
diene-17,21-diyl dipropanoate. with beclometasone dipropionate for peak identification CRS
Content : 96.0 per cent to 102.0 per cent (dried substance). and the chromatogram obtained with reference solution (c) to
identify the peaks due to impurities A, B, C, F, L, M and N ; use
CHARACTERS the chromatogram supplied with beclometasone dipropionate
Appearance : white or almost white, crystalline powder. for system suitability CRS and the chromatogram obtained with
Solubility : practically insoluble in water, freely soluble in reference solution (b) to identify the peak due to impurity D.
acetone, sparingly soluble in ethanol (96 per cent). Relative retention with reference to beclometasone
IDENTIFICATION dipropionate (retention time = about 25 min) :
impurity A = about 0.3 ; impurity B = about 0.6 ;
A. Infrared absorption spectrophotometry (2.2.24). impurity D = about 1.1 ; impurity M = about 1.2 ;
Comparison : anhydrous beclometasone dipropionate CRS. impurity L = about 1.3 ; impurity C = about 1.8 ;
B. Treat 25 mg by the oxygen-flask method (2.5.10). Use a impurity N = about 2.0 ; impurity F = about 2.2.
mixture of 1 mL of 1 M sodium hydroxide and 20 mL of System suitability : reference solution (b) :
water R to absorb the combustion products. The solution – peak-to-valley ratio : minimum 1.5, where Hp = height
gives reaction (a) of chlorides (2.3.1). above the baseline of the peak due to impurity D and
C. Loss on drying (see Tests). Hv = height above the baseline of the lowest point of
the curve separating this peak from the peak due to
TESTS
beclometasone dipropionate.
Specific optical rotation (2.2.7) : + 108 to + 115 (dried Limits :
substance).
– correction factors : for the calculation of content,
Dissolve 0.100 g in ethanol (96 per cent) R and dilute to multiply the peak areas of the following impurities by
10.0 mL with the same solvent.
the corresponding correction factor : impurity F = 1.3 ;
Related substances. Liquid chromatography (2.2.29). impurity M = 2.0 ;
Solvent mixture : mobile phase A, mobile phase B (45:55 V/V). – impurity L : not more than 6 times the area of the principal
Test solution (a). Dissolve 50.0 mg of the substance to be peak in the chromatogram obtained with reference
examined in 28 mL of mobile phase B and dilute to 50.0 mL solution (a) (0.6 per cent) ;
with mobile phase A. – impurities B, F, M : for each impurity, not more than
Test solution (b). Dilute 1.0 mL of test solution (a) to 50.0 mL 5 times the area of the principal peak in the chromatogram
with the solvent mixture. obtained with reference solution (a) (0.5 per cent) ;

1626 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Beclometasone dipropionate, anhydrous

– impurities A, D, N : for each impurity, not more than

twice the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent) ;
– impurity C : not more than 1.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.15 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ; E. R1 = Cl, R2 = CO-C2H5 : 6α,9-dichloro-11β-hydroxy-
– total : not more than 15 times the area of the principal peak 16β-methyl-3,20-dioxopregna-1,4-diene-17,21-diyl
in the chromatogram obtained with reference solution (a) dipropanoate,
(1.5 per cent) ;
H. R1 = R2 = H : 9-chloro-11β,21-dihydroxy-16β-methyl-3,20-
– disregard limit : 0.5 times the area of the principal peak in dioxopregna-1,4-dien-17-yl propanoate (beclometasone
the chromatogram obtained with reference solution (a) 17-propionate),
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 3 h.

ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : test solution (b) and reference solution (d).
Calculate the percentage content of C28H37ClO7 from
the declared content of anhydrous beclometasone I. 16β-methyl-3,20-dioxopregna-1,4,9(11)-triene-17,21-diyl
dipropionate CRS. dipropanoate,
IMPURITIES
Specified impurities : A, B, C, D, F, L, M, N.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10. J. R1 = R2 = CO-C2H5 : 9,11β-epoxy-16β-methyl-3,20-dioxo-
Control of impurities in substances for pharmaceutical use): E, 9β-pregna-1,4-diene-17,21-diyl dipropanoate,
H, I, J, O, Q, R, S, U, V.
R. R1 = R2 = H : 9,11β-epoxy-17,21-dihydroxy-16β-methyl-
9β-pregna-1,4-diene-3,20-dione,

U. R1 = CO-C2H5, R2 = H : 9,11β-epoxy-21-hydroxy-16β-
methyl-3,20-dioxo-9β-pregna-1,4-dien-17-yl propanoate,

V. R1 = H, R2 = CO-C2H5 : 9,11β-epoxy-17-hydroxy-16β-
methyl-3,20-dioxo-9β-pregna-1,4-dien-21-yl propanoate,

A. R1 = R3 = H, R2 = Cl, R4 = CO-C2H5 : 9-chloro-11β,17-

dihydroxy-16β-methyl-3,20-dioxopregna-1,4-dien-21-yl
propanoate (beclometasone 21-propionate),

B. R1 = H, R2 = Cl, R3 = CO-C2H5, R4 = CO-CH3 :

21-(acetyloxy)-9-chloro-11β-hydroxy-16β-methyl-3,20-
dioxopregna-1,4-dien-17-yl propanoate (beclometasone
21-acetate 17-propionate),
L. 9-chloro-11β-hydroxy-16β-methyl-3,20-dioxopregn-4-ene-
17,21-diyl dipropanoate,
C. R1 = H, R2 = Cl, R3 = CO-C2H5, R4 = CO-CH2-CH2-CH3 :
9-chloro-11β-hydroxy-16β-methyl-3,20-dioxo-17-
(propanoyloxy)-pregna-1,4-dien-21-yl butanoate
(beclometasone 21-butyrate 17-propionate),

D. R1 = H, R2 = Br, R3 = R4 = CO-C2H5 : 9-bromo-11β-

hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-17,21-
diyl dipropanoate,

F. R1 = Br, R2 = Cl, R3 = R4 = CO-C2H5 : 6α-bromo-9-chloro-

11β-hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene- M. 9-chloro-11β-hydroxy-16β-methyl-3,20-dioxopregna-4,6-
17,21-diyl dipropanoate, diene-17,21-diyl dipropanoate,

General Notices (1) apply to all monographs and other texts 1627
Beclometasone dipropionate monohydrate EUROPEAN PHARMACOPOEIA 8.0

Related substances. Liquid chromatography (2.2.29).

Solvent mixture : mobile phase A, mobile phase B (45:55 V/V).
Test solution (a). Dissolve 50.0 mg of the substance to be
examined in 28 mL of mobile phase B and dilute to 50.0 mL
with mobile phase A.
Test solution (b). Dilute 1.0 mL of test solution (a) to 50.0 mL
with the solvent mixture.
Reference solution (a). Dilute 5.0 mL of test solution (b) to
N. 2-bromo-9-chloro-11β-hydroxy-16β-methyl-3,20- 100.0 mL with the solvent mixture.
dioxopregna-1,4-diene-17,21-diyl dipropanoate, Reference solution (b). Dissolve 5 mg of beclometasone
dipropionate for system suitability CRS (containing impurity D)
in 3 mL of mobile phase B and dilute to 5 mL with mobile
phase A.
Reference solution (c). Dissolve 5 mg of beclometasone
dipropionate for peak identification CRS (containing impurities
B, C and L) in 3 mL of mobile phase B and dilute to 5 mL
with mobile phase A. Use 1 mL of this solution to dissolve the
contents of a vial of beclometasone dipropionate impurities
F and N CRS.
O. R1 = R2 = Cl : 9,11β-dichloro-16β-methyl-3,20-
dioxopregna-1,4-diene-17,21-diyl dipropanoate, Reference solution (d). Dissolve 50.0 mg of anhydrous
beclometasone dipropionate CRS in 28 mL of mobile phase B
Q. R1 = R2 = H : 16β-methyl-3,20-dioxopregna-1,4-diene- and dilute to 50.0 mL with mobile phase A. Dilute 1.0 mL of
17,21-diyl dipropanoate, this solution to 50.0 mL with the solvent mixture.
S. R1 = O-CO-C2H5, R2 = Cl : 9-chloro-16β-methyl-3,20- Column :
dioxopregna-1,4-diene-11β,17,21-triyl tripropanoate – size : l = 0.25 m, Ø = 4.6 mm ;
(beclometasone tripropionate). – stationary phase : spherical difunctional bonded end-capped
octadecylsilyl silica gel for chromatography R (5 μm) ;
01/2009:1709
corrected 7.0 – temperature : 50 °C.
Mobile phase :
BECLOMETASONE DIPROPIONATE – mobile phase A : 2.72 g/L solution of potassium dihydrogen
phosphate R adjusted to pH 2.35 with phosphoric acid R ;
MONOHYDRATE – mobile phase B : tetrahydrofuran R, acetonitrile R,
methanol R (5:23:25 V/V/V) ;
Beclometasoni dipropionas monohydricus
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-4 40 60

4 - 12 40 → 45 60 → 55

12 - 59 45 55

Flow rate : 1.4 mL/min.

Detection : spectrophotometer at 254 nm.
Injection : 20 μl of test solution (a) and reference solutions (a),
C28H37ClO7,H2O Mr 539.1
(b) and (c).
DEFINITION Identification of impurities : use the chromatogram supplied
9-Chloro-11β-hydroxy-16β-methyl-3,20-dioxopregna-1,4- with beclometasone dipropionate for peak identification CRS
diene-17,21-diyl dipropanoate monohydrate. and the chromatogram obtained with reference solution (c)
to identify the peaks due to impurities B, C, F and L ; use the
Content : 97.0 per cent to 102.0 per cent (dried substance).
chromatogram supplied with beclometasone dipropionate for
CHARACTERS system suitability CRS and the chromatogram obtained with
Appearance : white or almost white powder. reference solution (b) to identify the peak due to impurity D.
Solubility : practically insoluble in water, freely soluble in Relative retention with reference to beclometasone
acetone, sparingly soluble in ethanol (96 per cent). dipropionate (retention time = about 25 min) :
impurity B = about 0.6 ; impurity D = about 1.1 ;
IDENTIFICATION impurity L = about 1.3 ; impurity C = about 1.8 ;
A. Infrared absorption spectrophotometry (2.2.24). impurity F = about 2.2.
Comparison : beclometasone dipropionate monohydrate CRS. System suitability : reference solution (b) :
B. Treat 25 mg by the oxygen-flask method (2.5.10). Use a – peak-to-valley ratio : minimum 1.5, where Hp = height
mixture of 1 mL of 1 M sodium hydroxide and 20 mL of above the baseline of the peak due to impurity D and
water R to absorb the combustion products. The solution Hv = height above the baseline of the lowest point of
gives reaction (a) of chlorides (2.3.1). the curve separating this peak from the peak due to
C. Loss on drying (see Tests). beclometasone dipropionate.
Limits :
TESTS – correction factor : for the calculation of content, multiply
Specific optical rotation (2.2.7) : + 108 to + 115 (dried the peak area of impurity F by 1.3 ;
substance). – impurity B : not more than 5 times the area of the principal
Dissolve 0.100 g in ethanol (96 per cent) R and dilute to peak in the chromatogram obtained with reference
10.0 mL with the same solvent. solution (a) (0.5 per cent) ;

1628 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Beclometasone dipropionate monohydrate

– impurities C, F, L : for each impurity, not more than C. R1 = H, R2 = CO-CH2-CH2-CH3 : 9-chloro-11β-hydroxy-

1.5 times the area of the principal peak in the chromatogram 16β-methyl-3,20-dioxo-17-(propanoyloxy)-pregna-
obtained with reference solution (a) (0.15 per cent) ; 1,4-dien-21-yl butanoate (beclometasone 21-butyrate
– unspecified impurities : for each impurity, not more than the 17-propionate),
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
F. R1 = Br, R2 = CO-C2H5 : 6α-bromo-9-chloro-11β-hydroxy-
– total : not more than 10 times the area of the principal peak 16β-methyl-3,20-dioxopregna-1,4-diene-17,21-diyl
in the chromatogram obtained with reference solution (a) dipropanoate,
(1.0 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent).
Loss on drying (2.2.32) : 2.8 per cent to 3.8 per cent,
determined on 1.000 g by drying in an oven at 105 °C for 3 h.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : test solution (b) and reference solution (d).
I. 16β-methyl-3,20-dioxopregna-1,4,9(11)-triene-17,21-diyl
Calculate the percentage content of C28H37ClO7 from dipropanoate,
the declared content of anhydrous beclometasone
dipropionate CRS.
IMPURITIES
Specified impurities : B, C, F, L.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these J. R1 = R2 = CO-C2H5 : 9,11β-epoxy-16β-methyl-3,20-dioxo-
impurities for demonstration of compliance. See also 5.10. 9β-pregna-1,4-diene-17,21-diyl dipropanoate,
Control of impurities in substances for pharmaceutical use): A,
D, E, H, I, J, M, N, O, Q, R, S, U, V.
R. R1 = R2 = H : 9,11β-epoxy-17,21-dihydroxy-16β-methyl-
9β-pregna-1,4-diene-3,20-dione,

U. R1 = CO-C2H5, R2 = H : 9,11β-epoxy-21-hydroxy-16β-
methyl-3,20-dioxo-9β-pregna-1,4-dien-17-yl propanoate,

V. R1 = H, R2 = CO-C2H5 : 9,11β-epoxy-17-hydroxy-16β-
A. R1 = R3 = H, R2 = Cl, R4 = CO-C2H5 : 9-chloro-11β,17- methyl-3,20-dioxo-9β-pregna-1,4-dien-21-yl propanoate,
dihydroxy-16β-methyl-3,20-dioxopregna-1,4-dien-21-yl
propanoate (beclometasone 21-propionate),
D. R1 = H, R2 = Br, R3 = R4 = CO-C2H5 : 9-bromo-11β-
hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-17,21-
diyl dipropanoate,
E. R1 = R2 = Cl, R3 = R4 = CO-C2H5 : 6α,9-dichloro-11β-
hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-17,21-
diyl dipropanoate,
H. R1 = R4 = H, R2 = Cl, R3 = CO-C2H5 : 9-chloro-11β,21- L. 9-chloro-11β-hydroxy-16β-methyl-3,20-dioxopregn-4-ene-
dihydroxy-16β-methyl-3,20-dioxopregna-1,4-dien-17-yl 17,21-diyl dipropanoate,
propanoate (beclometasone 17-propionate),

B. R1 = H, R2 = CO-CH3 : 21-(acetyloxy)-9-chloro-11β-
hydroxy-16β-methyl-3,20-dioxopregna-1,4-dien-17-yl M. 9-chloro-11β-hydroxy-16β-methyl-3,20-dioxopregna-4,6-
propanoate (beclometasone 21-acetate 17-propionate), diene-17,21-diyl dipropanoate,

General Notices (1) apply to all monographs and other texts 1629
Beeswax, white EUROPEAN PHARMACOPOEIA 8.0

Titrate the hot solution immediately with 0.5 M hydrochloric

acid, using 1 mL of phenolphthalein solution R1 as indicator
(n1 mL). Reheat the solution to boiling several times during
the course of the titration. Carry out a blank test (n2 mL).

Ceresin, paraffins and certain other waxes. To 3.0 g, in a

N. R1 = Br, R2 = OH, R3 = Cl : 2-bromo-9-chloro-11β-hydroxy- 100 mL round-bottomed flask, add 30 mL of a 40 g/L solution
16β-methyl-3,20-dioxopregna-1,4-diene-17,21-diyl of potassium hydroxide R in aldehyde-free alcohol R and
dipropanoate, boil gently under a reflux condenser for 2 h. Remove the
condenser and immediately insert a thermometer. Place the
O. R1 = H, R2 = R3 = Cl : 9,11β-dichloro-16β-methyl-3,20- flask in a water-bath at 80 °C and allow to cool, swirling the
dioxopregna-1,4-diene-17,21-diyl dipropanoate, solution continuously. No precipitate is formed until 65 °C,
Q. R1 = R2 = R3 = H : 16β-methyl-3,20-dioxopregna-1,4- although the solution may be slightly opalescent. Beginning at
diene-17,21-diyl dipropanoate, 65 °C, the solution may become cloudy and precipitates may
be formed. At 59 °C, the solution is cloudy.
S. R1 = H, R2 = O-CO-C2H5, R3 = Cl : 9-chloro-16β-methyl-
3,20-dioxopregna-1,4-diene-11β,17,21-triyl tripropanoate Glycerol and other polyols : maximum 0.5 per cent m/m,
(beclometasone tripropionate). calculated as glycerol.
To 0.20 g add 10 mL of alcoholic potassium hydroxide
01/2008:0069 solution R and heat on a water-bath under a reflux condenser
for 30 min. Add 50 mL of dilute sulfuric acid R, cool and
BEESWAX, WHITE filter. Rinse the flask and the filter with dilute sulfuric acid R.
Combine the filtrate and washings and dilute to 100.0 mL
with dilute sulfuric acid R. Place 1.0 mL of the solution in
Cera alba a test-tube, add 0.5 mL of a 10.7 g/L solution of sodium
DEFINITION periodate R, mix and allow to stand for 5 min. Add 1.0 mL
Wax obtained by bleaching yellow beeswax. of decolorised fuchsin solution R and mix. Any precipitate
disappears. Place the tube in a beaker containing water at
CHARACTERS 40 °C. During cooling observe for 10-15 min. Any violet-blue
Appearance : white or yellowish-white pieces or plates, colour in the solution is not more intense than that in a
translucent when thin, with a fine-grained, matt and standard prepared at the same time and in the same manner
non-crystalline fracture ; when warmed in the hand they using 1.0 mL of a 10 mg/L solution of glycerol R in dilute
become soft and malleable. sulfuric acid R.
It has an odour similar to that of yellow beeswax, though
fainter and never rancid. It is tasteless and does not stick to 01/2008:0070
the teeth.
Solubility : practically insoluble in water, partially soluble in BEESWAX, YELLOW
hot ethanol (90 per cent V/V) and completely soluble in fatty
and essential oils. Cera flava
Relative density : about 0.960.
DEFINITION
TESTS Wax obtained by melting the walls of the honeycomb made by
Drop point (2.2.17) : 61 °C to 66 °C. the honey-bee, Apis mellifera L., with hot water and removing
Melt the beeswax by heating on a water-bath, pour onto a glass foreign matter.
plate and allow to cool to a semi-solid mass. Fill the metal cup
by inserting the wider end into the beeswax and repeating the CHARACTERS
procedure until beeswax extrudes from the narrow opening. Appearance : yellow or light brown pieces or plates with a
Remove the excess with a spatula and insert the thermometer fine-grained, matt and non-crystalline fracture ; when warmed
immediately. Remove the beeswax displaced. Allow to stand in the hand they become soft and malleable.
at room temperature for at least 12 h before determining the It has a faint odour, characteristic of honey. It is tasteless and
drop point. does not stick to the teeth.
Acid value : 17.0 to 24.0. Solubility : practically insoluble in water, partially soluble in
To 2.00 g (m g), in a 250 mL conical flask fitted with a reflux hot ethanol (90 per cent V/V) and completely soluble in fatty
condenser, add 40 mL of xylene R and a few glass beads. Heat and essential oils.
until the substance is dissolved. Add 20 mL of ethanol (96 per Relative density : about 0.960.
cent) R and 0.5 mL of phenolphthalein solution R1 and titrate
the hot solution with 0.5 M alcoholic potassium hydroxide TESTS
until a red colour persists for at least 10 s (n1 mL). Carry out a Drop point (2.2.17) : 61 °C to 66 °C.
blank test (n2 mL). Melt the beeswax by heating on a water-bath, pour onto a glass
plate and allow to cool to a semi-solid mass. Fill the metal cup
by inserting the wider end into the beeswax and repeating the
procedure until beeswax extrudes from the narrow opening.
Ester value (2.5.2) : 70 to 80. Remove the excess with a spatula and insert the thermometer
Saponification value : 87 to 104. immediately. Remove the beeswax displaced. Allow to stand
at room temperature for at least 12 h before determining the
To 2.00 g (m g), in a 250 mL conical flask fitted with a reflux drop point.
condenser, add 30 mL of a mixture of equal volumes of ethanol
(96 per cent) R and xylene R and a few glass beads. Heat until Acid value : 17.0 to 22.0.
the substance is dissolved. Add 25.0 mL of 0.5 M alcoholic To 2.00 g (m g), in a 250 mL conical flask fitted with a reflux
potassium hydroxide and heat under a reflux condenser for 3 h. condenser, add 40 mL of xylene R and a few glass beads. Heat

1630 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Benazepril hydrochloride

until the substance is dissolved. Add 20 mL of ethanol (96 per Content : 97.5 per cent to 102.0 per cent (dried substance).
cent) R and 0.5 mL of phenolphthalein solution R1 and titrate
the hot solution with 0.5 M alcoholic potassium hydroxide CHARACTERS
until a red colour persists for at least 10 s (n1 mL). Carry out a Appearance : white or almost white, crystalline powder,
blank test (n2 mL). hygroscopic.
Solubility : slightly soluble in water, freely soluble in anhydrous
ethanol, very slightly soluble in ethyl acetate, practically
insoluble in cyclohexane.
Ester value (2.5.2) : 70 to 80. It shows polymorphism (5.9).
Saponification value : 87 to 102.
IDENTIFICATION
To 2.00 g (m g), in a 250 mL conical flask fitted with a reflux
condenser, add 30 mL of a mixture of equal volumes of ethanol Carry out either tests A, B, D or tests B, C, D.
(96 per cent) R and xylene R and a few glass beads. Heat until A. Specific optical rotation (2.2.7) : − 141 to − 136 (dried
the substance is dissolved. Add 25.0 mL of 0.5 M alcoholic substance).
potassium hydroxide and heat under a reflux condenser for 3 h. Dissolve 1.000 g in anhydrous ethanol R and dilute to
Titrate the hot solution immediately with 0.5 M hydrochloric 50.0 mL with the same solvent.
acid, using 1 mL of phenolphthalein solution R1 as indicator
(n1 mL). Reheat the solution to boiling several times during B. Infrared absorption spectrophotometry (2.2.24).
the course of the titration. Carry out a blank test (n2 mL). Comparison: benazepril hydrochloride CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
substance separately in methanol R, evaporate to dryness
Ceresin, paraffins and certain other waxes. To 3.0 g, in a and record new spectra using the residues.
100 mL round-bottomed flask, add 30 mL of a 40 g/L solution C. Enantiomeric purity (see Tests).
of potassium hydroxide R in aldehyde-free alcohol R and D. It gives reaction (a) of chlorides (2.3.1).
boil gently under a reflux condenser for 2 h. Remove the
condenser and immediately insert a thermometer. Place the TESTS
flask in a water-bath at 80 °C and allow to cool, swirling the
Related substances. Liquid chromatography (2.2.29).
solution continuously. No precipitate is formed until 65 °C,
although the solution may be slightly opalescent. Beginning at Test solution (a). Dissolve 50.0 mg of the substance to be
65 °C, the solution may become cloudy and precipitates may examined in the mobile phase and dilute to 50.0 mL with the
be formed. At 59 °C, the solution is cloudy. mobile phase.
Glycerol and other polyols : maximum 0.5 per cent m/m, Test solution (b). Dilute 10.0 mL of test solution (a) to
calculated as glycerol. 100.0 mL with the mobile phase.
To 0.20 g add 10 mL of alcoholic potassium hydroxide Reference solution (a). Dissolve 50.0 mg of benazepril
solution R and heat on a water-bath under a reflux condenser hydrochloride CRS in the mobile phase and dilute to 50.0 mL
for 30 min. Add 50 mL of dilute sulfuric acid R, cool and with the mobile phase. Dilute 10.0 mL of this solution to
filter. Rinse the flask and the filter with dilute sulfuric acid R. 100.0 mL with the mobile phase.
Combine the filtrate and washings and dilute to 100.0 mL Reference solution (b). Dissolve the contents of a vial of
with dilute sulfuric acid R. Place 1.0 mL of the solution in benazepril for system suitability CRS (containing impurities B,
a test-tube, add 0.5 mL of a 10.7 g/L solution of sodium C, D, E, F and G) in 1.0 mL of test solution (a).
periodate R, mix and allow to stand for 5 min. Add 1.0 mL Reference solution (c). Dilute 1.0 mL of reference solution (a)
of decolorised fuchsin solution R and mix. Any precipitate to 50.0 mL with the mobile phase.
disappears. Place the tube in a beaker containing water at Column :
40 °C. During cooling observe for 10-15 min. Any violet-blue
colour in the solution is not more intense than that in a – size : l = 0.30 m, Ø = 3.9 mm ;
standard prepared at the same time and in the same manner – stationary phase : end-capped octadecylsilyl silica gel for
using 1.0 mL of a 10 mg/L solution of glycerol R in dilute chromatography R (10 μm).
sulfuric acid R. Mobile phase : add 0.2 mL of glacial acetic acid R to 1000 mL
of a mixture of 360 volumes of water R and 640 volumes of
methanol R2 ; add 0.81 g of tetrabutylammonium bromide R
01/2011:2388 and stir to dissolve.
Flow rate : 1.0 mL/min.
BENAZEPRIL HYDROCHLORIDE Detection : spectrophotometer at 240 nm.
Injection : 25 μL of test solution (a) and reference solutions (b)
Benazeprili hydrochloridum and (c).
Run time : 3 times the retention time of benazepril.
Relative retention with reference to benazepril (retention
time = about 6 min) : impurity E = about 0.3 ; impurity F = about
0.4 ; impurity C = about 0.5 ; impurity B = about 1.8 ;
impurity D = about 2.0 ; impurity G = about 2.5.
Identification of impurities : use the chromatogram
supplied with benazepril for system suitability CRS and the
C24H29ClN2O5 Mr 461.0 chromatogram obtained with reference solution (b) to identify
[86541-74-4] the peaks due to impurities B, C, D, E, F and G.
DEFINITION System suitability : reference solution (b) :
[(3S)-3-[[(1S)-1-(Ethoxycarbonyl)-3-phenylpropyl]amino]- – resolution : minimum 2.5 between the peaks due to
2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]acetic acid benazepril and impurity B and minimum 1.5 between the
hydrochloride. peaks due to impurities E and F.

General Notices (1) apply to all monographs and other texts 1631
Benazepril hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Limits : Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on

– correction factors : for the calculation of content, 1.0 g.
multiply the peak areas of the following impurities by
ASSAY
the corresponding correction factor : impurity E = 0.5 ;
impurity F = 0.7 ; Liquid chromatography (2.2.29) as described in the test for
– impurity B : not more than 2.5 times the area of the related substances with the following modification.
principal peak in the chromatogram obtained with Injection : test solution (b) and reference solution (a).
reference solution (c) (0.5 per cent) ; Calculate the percentage content of C24H29ClN2O5 from the
– impurity C : not more than 1.5 times the area of the declared content of benazepril hydrochloride CRS.
principal peak in the chromatogram obtained with
reference solution (c) (0.3 per cent) ; STORAGE
– impurities D, E, F, G : for each impurity, not more than the Protected from light, in an airtight container.
area of the principal peak in the chromatogram obtained IMPURITIES
with reference solution (c) (0.2 per cent) ;
Specified impurities : A, B, C, D, E, F, G.
– unspecified impurities : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.10 per cent) ;
– total : not more than 10 times the area of the principal peak
in the chromatogram obtained with reference solution (c)
(2.0 per cent) ;
– disregard limit : 0.25 times the area of the principal peak
in the chromatogram obtained with reference solution (c) A. [(3R)-3-[[(1R)-1-(ethoxycarbonyl)-3-phenylpropyl]ami-
(0.05 per cent). no]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]acetic
Enantiomeric purity. Liquid chromatography (2.2.29). acid,
Buffer solution pH 6.0. Dissolve 3.58 g of disodium hydrogen
phosphate R and 9.66 g of potassium dihydrogen phosphate R
in water R and dilute to 1000.0 mL with the same solvent.
Test solution. Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with the
mobile phase.
Reference solution (a). Dissolve 5.0 mg of benazepril
impurity A CRS in the mobile phase and dilute to 50.0 mL B. [(3RS)-3-[[(1SR)-1-(ethoxycarbonyl)-3-phenylpropyl]-
with the mobile phase. amino]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-1-
yl]acetic acid,
Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 100.0 mL with the mobile phase.
Reference solution (c). Dilute 1.0 mL of reference solution (a)
to 10.0 mL with the mobile phase. Dilute 1.0 mL of this
solution to 10.0 mL with the test solution.
Column :
– size : l = 0.10 m, Ø = 4.0 mm ;
– stationary phase : spherical silica gel AGP for chiral C. R = H : (2S)-2-[[(3S)-1-(carboxymethyl)-2-oxo-
chromatography R (5 μm) ; 2,3,4,5-tetrahydro-1H-1-benzazepin-3-yl]amino]-4-
– temperature : 30 °C. phenylbutanoic acid,
Mobile phase : methanol R2, buffer solution pH 6.0 (20:80 V/V). G. R = C2H5 : ethyl (2S)-2-[[(3S)-1-(2-ethoxy-2-oxoethyl)-2-
Flow rate : 0.9 mL/min. oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-3-yl]amino]-4-
Detection : spectrophotometer at 240 nm. phenylbutanoate,
Injection : 50 μL of the test solution and reference solutions (b)
and (c).
Run time : 3.5 times the retention time of benazepril.
Relative retention with reference to benazepril (retention
time = about 6 min) : impurity A = about 1.9.
System suitability: reference solution (c) :
– peak-to-valley ratio : minimum 2.5, where Hp = height D. [(3S)-3-[[(1S)-3-cyclohexyl-1-(ethoxycarbonyl)propyl]-
above the baseline of the peak due to impurity A and amino]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-1-
Hv = height above the baseline of the lowest point of the yl]acetic acid,
curve separating this peak from the peak due to benazepril.
Limit :
– impurity A : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (b) (0.1 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test C. Prepare the reference solution using E. R = H : [(3S)-3-amino-2-oxo-2,3,4,5-tetrahydro-1H-1-
2 mL of lead standard solution (10 ppm Pb) R. benzazepin-1-yl]acetic acid,
Loss on drying (2.2.32) : maximum 1.5 per cent, determined F. R = C(CH3)3 : 1,1-dimethylethyl [(3S)-3-amino-2-oxo-
on 1.000 g by drying in vacuo at 105 °C for 3 h. 2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]acetate.

1632 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Benperidol

01/2008:0370 – unspecified impurities : for each impurity, not more than the
corrected 6.0 area of the principal peak in the chromatogram obtained
with reference solution (b) (0.10 per cent) ;
BENDROFLUMETHIAZIDE – total : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (b) (0.2 per cent) ;
Bendroflumethiazidum – disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
C15H14F3N3O4S2 Mr 421.4 1.0 g.
[73-48-3]
ASSAY
DEFINITION
Dissolve 0.150 g in 50 mL of dimethyl sulfoxide R. Titrate to
(3RS)-3-Benzyl-6-(trifluoromethyl)-3,4-dihydro-2H-1,2,4- the 2nd point of inflexion with 0.1 M tetrabutylammonium
benzothiadiazine-7-sulfonamide 1,1-dioxide. hydroxide in 2-propanol, determining the end-point
Content : 98.0 per cent to 102.0 per cent (dried substance). potentiometrically (2.2.20). Carry out a blank titration.
1 mL of 0.1 M tetrabutylammonium hydroxide in 2-propanol
CHARACTERS
is equivalent to 21.07 mg of C15H14F3N3O4S2.
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, freely soluble in IMPURITIES
acetone, soluble in ethanol (96 per cent). Specified impurities : A.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : bendroflumethiazide CRS.
TESTS A. 4-amino-6-(trifluoromethyl)benzene-1,3-disulfonamide.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use. 07/2011:1172
Solvent mixture. Mix 40 volumes of methanol R and
60 volumes of a 2.0 g/L solution of citric acid R. BENPERIDOL
Test solution. Dissolve 10.0 mg of the substance to be
examined in the solvent mixture and dilute to 50.0 mL with Benperidolum
the solvent mixture.
Reference solution (a). Dissolve 2 mg of bendroflumethiazide
impurity A CRS and 2.5 mg of altizide CRS in the solvent
mixture and dilute to 10 mL with the solvent mixture. Mix
1 mL of this solution with 1 mL of the test solution and dilute
to 100 mL with the solvent mixture.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this C22H24FN3O2 Mr 381.4
solution to 10.0 mL with the solvent mixture. [2062-84-2]
Column :
DEFINITION
– size : l = 0.15 m, Ø = 3.0 mm ;
1-[1-[4-(4-Fluorophenyl)-4-oxobutyl]piperidin-4-yl]-1,3-
– stationary phase : end-capped octadecylsilyl silica gel for dihydro-2H-benzimidazol-2-one.
chromatography R (5 μm) ;
Content : 99.0 per cent to 101.0 per cent (dried substance).
– temperature : 40 °C.
Mobile phase : mix 15 volumes of tetrahydrofuran R, CHARACTERS
25 volumes of methanol R and 60 volumes of a 2.0 g/L solution Appearance : white or almost white powder.
of citric acid R. Solubility : practically insoluble in water, freely soluble in
Flow rate : 0.8 mL/min. dimethylformamide, soluble in methylene chloride, slightly
Detection : spectrophotometer at 273 nm. soluble in ethanol (96 per cent).
Injection : 20 μL. It shows polymorphism (5.9).
Run time : twice the retention time of bendroflumethiazide. IDENTIFICATION
Relative retention with reference to bendroflumethiazide First identification : A.
(retention time = about 8 min) : impurity A = about 0.2 ; Second identification : B, C, D.
altizide = about 0.5. A. Infrared absorption spectrophotometry (2.2.24).
System suitability: reference solution (a) : Comparison: benperidol CRS.
– resolution : minimum 10 between the peaks due to altizide If the spectra obtained in the solid state show differences,
and bendroflumethiazide. dissolve the substance to be examined and the reference
Limits : substance separately in the minimum volume of methyl
– impurity A : not more than the area of the principal peak isobutyl ketone R, evaporate to dryness and record new
in the chromatogram obtained with reference solution (b) spectra using the residues.
(0.1 per cent) ; B. Thin-layer chromatography (2.2.27).

General Notices (1) apply to all monographs and other texts 1633
Benperidol EUROPEAN PHARMACOPOEIA 8.0

Test solution. Dissolve 30 mg of the substance to be Relative retention with reference to benperidol (retention
examined in the mobile phase and dilute to 10 mL with time = about 6.5 min) : impurity A = about 0.2 ;
the mobile phase. impurity B = about 0.9 ; droperidol = about 1.1 ;
Reference solution (a). Dissolve 30 mg of benperidol CRS impurity D = about 1.2 ; impurity E = about 1.3 ;
in the mobile phase and dilute to 10 mL with the mobile impurity C = about 1.5.
phase. System suitability : reference solution (a) :
Reference solution (b). Dissolve 30 mg of benperidol CRS – resolution : minimum 2.0 between the peaks due to
and 30 mg of droperidol CRS in the mobile phase and dilute benperidol and droperidol.
to 10 mL with the mobile phase. Limits :
Plate : TLC silica gel F254 plate R. – impurities A, B, C, D, E : for each impurity, not more
Mobile phase : acetone R, methanol R (10:90 V/V). than the area of the principal peak in the chromatogram
Application : 10 μL. obtained with reference solution (b) (0.25 per cent) ;
Development : over 3/4 of the plate. – unspecified impurities : for each impurity, not more than
0.4 times the area of the principal peak in the chromatogram
Drying : in air. obtained with reference solution (b) (0.10 per cent) ;
Detection : examine in ultraviolet light at 254 nm. – total : not more than twice the area of the principal peak
System suitability: reference solution (b) : in the chromatogram obtained with reference solution (b)
– the chromatogram shows 2 clearly separated spots. (0.5 per cent) ;
Results : the principal spot in the chromatogram obtained – disregard limit : 0.2 times the area of the principal peak in
with the test solution is similar in position and size to the chromatogram obtained with reference solution (b)
the principal spot in the chromatogram obtained with (0.05 per cent).
reference solution (a). Loss on drying (2.2.32) : maximum 0.5 per cent, determined
C. Dissolve about 10 mg in 5 mL of anhydrous ethanol R. on 1.000 g by drying in an oven at 105 °C.
Add 0.5 mL of dinitrobenzene solution R and 0.5 mL of Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
2 M alcoholic potassium hydroxide R. A violet colour is 1.0 g in a platinum crucible.
produced which becomes brownish-red after 20 min.
D. Mix about 5 mg with 45 mg of heavy magnesium oxide R ASSAY
and ignite in a crucible until an almost white residue is Dissolve 0.300 g in 50 mL of a mixture of 1 volume of
obtained (usually less than 5 min). Allow to cool, add anhydrous acetic acid R and 7 volumes of methyl ethyl
1 mL of water R, 0.05 mL of phenolphthalein solution R1 ketone R. Titrate with 0.1 M perchloric acid, using 0.2 mL of
and about 1 mL of dilute hydrochloric acid R to render the naphtholbenzein solution R as indicator.
solution colourless. Filter. To a freshly prepared mixture 1 mL of 0.1 M perchloric acid is equivalent to 38.14 mg
of 0.1 mL of alizarin S solution R and 0.1 mL of zirconyl of C22H24FN3O2.
nitrate solution R, add 1.0 mL of the filtrate. Mix, allow
to stand for 5 min and compare the colour of the solution STORAGE
with that of a blank prepared in the same manner. The test Protected from light.
solution is yellow and the blank is red.
IMPURITIES
TESTS Specified impurities : A, B, C, D, E.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
Test solution. Dissolve 0.10 g of the substance to be examined
in dimethylformamide R and dilute to 10.0 mL with the same
solvent.
Reference solution (a). Dissolve 2.5 mg of benperidol CRS and A. 1-(piperidin-4-yl)-1,3-dihydro-2H-benzimidazol-2-one,
2.5 mg of droperidol CRS in dimethylformamide R and dilute
to 100.0 mL with the same solvent.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with dimethylformamide R. Dilute 5.0 mL of this
solution to 20.0 mL with dimethylformamide R.
Column :
– size : l = 0.1 m, Ø = 4.6 mm ;
– stationary phase : base-deactivated octadecylsilyl silica gel for B. 1-[1-[4-(2-fluorophenyl)-4-oxobutyl]piperidin-4-yl]-1,3-
chromatography R (3 μm). dihydro-2H-benzimidazol-2-one,
Mobile phase :
– mobile phase A : 10 g/L solution of tetrabutylammonium
hydrogen sulfate R ;
– mobile phase B : acetonitrile R ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 15 100 → 60 0 → 40

15 - 20 60 40

20 - 25 100 0

Flow rate : 1.5 mL/min. C. 1-[1-[4-oxo-4-[4-[4-(2-oxo-2,3-dihydro-1H-benzimidazol-

Detection : spectrophotometer at 275 nm. 1-yl)piperidin-1-yl]phenyl]butyl]piperidin-4-yl]-1,3-
Injection : 10 μL. dihydro-2H-benzimidazol-2-one,

1634 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Benserazide hydrochloride

Reference solution (a). Dissolve 5.0 mg of benserazide

impurity A CRS, 5.0 mg of benserazide impurity C CRS and
5.0 mg of benserazide hydrochloride CRS in methanol R2 and
dilute to 50.0 mL with the same solvent. Dilute 5.0 mL of this
solution to 50.0 mL with methanol R2.
Reference solution (b). Dilute 2.0 mL of reference solution (a)
to 10.0 mL with methanol R2.
D. cis-1-[1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl Reference solution (c). Dissolve 5 mg of benserazide for peak
1-oxide]-1,3-dihydro-2H-benzimidazol-2-one, identification CRS (containing impurities A, B and C) in
methanol R2 and dilute to 5.0 mL with the same solvent.
Column :
– size : l = 0.25 m, Ø = 4 mm ;
– stationary phase : octylsilyl silica gel for chromatography R
(5 μm) ;
– temperature : 30 °C.
E. trans-1-[1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl Mobile phase :
1-oxide]-1,3-dihydro-2H-benzimidazol-2-one. – mobile phase A : dissolve 2.2 g of sodium heptanesulfonate
monohydrate R and 6.8 g of potassium dihydrogen
phosphate R in 900 mL of water R, add 50 mL of
methanol R2 and adjust to pH 3.5 with phosphoric acid R ;
04/2009:1173
– mobile phase B : dissolve 2.2 g of sodium heptanesulfonate
monohydrate R and 6.8 g of potassium dihydrogen
BENSERAZIDE HYDROCHLORIDE phosphate R in 500 mL of water R, adjust to pH 3.5 with
phosphoric acid R and add 500 mL of methanol R2 ;
Benserazidi hydrochloridum Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 15 100 → 0 0 → 100

15 - 25 0 100

Flow rate : 1.3 mL/min.

Detection : spectrophotometer at 210 nm.
C10H16ClN3O5 Mr 293.7 Injection : 5 μL.
[14919-77-8]
Identification of impurities : use the chromatogram supplied
DEFINITION with benserazide for peak identification CRS and the
(2RS)-2-Amino-3-hydroxy-2′-(2,3,4-trihydroxy- chromatogram obtained with reference solution (c) to identify
benzyl)propanohydrazide hydrochloride. the peaks due to impurities A, B and C ; doubling of the peak
due to impurity C, related to separation of the (EZ)-isomers,
Content : 98.5 per cent to 101.0 per cent (anhydrous substance). may be observed.
CHARACTERS Relative retention with reference to benserazide
(retention time = about 9 min) : impurity A = about 0.6 ;
Appearance : white or yellowish-white or orange-white,
impurity C = about 1.2 ; impurity B = about 1.5.
crystalline powder.
System suitability : reference solution (a) :
Solubility : freely soluble in water, very slightly soluble in
anhydrous ethanol, practically insoluble in acetone. – resolution : minimum 5.0 between the peaks due to
benserazide and impurity C ; use the 1st peak of impurity C
It shows polymorphism (5.9).
if 2 peaks occur.
IDENTIFICATION Limits :
A. Infrared absorption spectrophotometry (2.2.24). – correction factor : for the calculation of content, multiply
Comparison : benserazide hydrochloride CRS. the peak area of impurity B by 0.7 ;
If the spectra obtained show differences, dissolve the – impurity A : not more than the area of the corresponding
substance to be examined and the reference substance peak in the chromatogram obtained with reference
separately in hot methanol R, evaporate to dryness and solution (a) (0.5 per cent) ;
record new spectra using the residues. – impurity B : not more than the area of the peak due to
B. Solution S (see Tests) gives reaction (b) of chlorides (2.3.1). benserazide in the chromatogram obtained with reference
solution (a) (0.5 per cent) ;
TESTS – impurity C : not more than the area of the corresponding
Solution S. Dissolve 1.0 g in carbon dioxide-free water R and peak or pair of peaks in the chromatogram obtained with
dilute to 100 mL with the same solvent. reference solution (a) (0.5 per cent) ;
Appearance of solution. Solution S is clear (2.2.1) and not – unspecified impurities : for each impurity, not more than the
more intensely coloured than reference solution BY6 (2.2.2, area of the peak due to benserazide in the chromatogram
Method II). obtained with reference solution (b) (0.10 per cent) ;
pH (2.2.3): 4.0 to 5.0 for solution S. – sum of impurities other than A : not more than twice the
area of the peak due to benserazide in the chromatogram
Related substances. Liquid chromatography (2.2.29). obtained with reference solution (a) (1.0 per cent) ;
All solutions must be injected immediately or stored at 4 °C. – disregard limit : 0.5 times the area of the peak due to
Test solution. Dissolve 0.100 g of the substance to be examined benserazide in the chromatogram obtained with reference
in methanol R2 and dilute to 50.0 mL with the same solvent. solution (b) (0.05 per cent).

General Notices (1) apply to all monographs and other texts 1635
Bentonite EUROPEAN PHARMACOPOEIA 8.0

Heavy metals (2.4.8) : maximum 20 ppm. water R, mix and filter. Wash the insoluble residue with
1.0 g complies with test C. Prepare the reference solution using 50 mL of water R. To this residue add 1 mL of hydrochloric
2 mL of lead standard solution (10 ppm Pb) R. acid R and 5 mL of water R. Filter. To the filtrate add 1 mL
of strong sodium hydroxide solution R and filter. To this
Water (2.5.12) : maximum 1.0 per cent, determined on 0.500 g. filtrate add 3 mL of ammonium chloride solution R. A
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on gelatinous white precipitate is formed.
1.0 g.
B. Add 2.0 g in 20 portions to 100 mL of a 10 g/L solution
ASSAY of sodium laurilsulfate R in a 100 mL graduated cylinder
In order to avoid overheating during the titration, mix about 30 mm in diameter. Allow 2 min between additions
thoroughly throughout and stop the titration immediately after for each portion to settle. Allow to stand for 2 h. The
the end-point has been reached. apparent volume of the sediment is not less than 22 mL.
Dissolve 0.250 g in 5 mL of anhydrous formic acid R. Add C. 0.25 g gives the reaction of silicates (2.3.1).
70 mL of anhydrous acetic acid R. Titrate immediately
with 0.1 M perchloric acid, determining the end-point TESTS
potentiometrically (2.2.20).
Alkalinity. To 2 g add 100 mL of carbon dioxide-free water R
1 mL of 0.1 M perchloric acid is equivalent to 29.37 mg of
and shake for 5 min. To 5 mL of this suspension add 0.1 mL of
C10H16ClN3O5.
thymolphthalein solution R. The liquid becomes bluish. Add
STORAGE 0.1 mL of 0.1 M hydrochloric acid. The liquid is decolourised
Protected from light. within 5 min.
Coarse particles : maximum 0.5 per cent.
IMPURITIES
Specified impurities : A, B, C. To 20 g add 1000 mL of water R and mix for 15 min using
a high-speed mixer capable of operating at not less than
5000 r/min. Transfer the suspension to a wet sieve (75), tared
after drying at 100-105 °C. Wash with 3 quantities, each of
500 mL, of water R, ensuring that any agglomerates have
been dispersed. Dry the sieve at 100-105 °C and weigh. The
particles on the sieve weigh a maximum of 0.1 g.
Heavy metals (2.4.8) : maximum 50 ppm.
To 5.0 g add 7.5 mL of dilute hydrochloric acid R and 27.5 mL of
A. (2RS)-2-amino-3-hydroxypropanohydrazide, water R. Boil for 5 min. Centrifuge and filter the supernatant.
Wash the centrifugation residue with water R and filter. Dilute
the combined filtrates to 50.0 mL with water R. To 5 mL of this
solution add 5 mL of water R, 10 mL of hydrochloric acid R
and 25 mL of methyl isobutyl ketone R and shake for 2 min.
Separate the layers. Evaporate the aqueous layer to dryness on
a water-bath. Dissolve the residue in 1 mL of acetic acid R,
B. (2RS)-2-amino-3-hydroxy-2′,2′-bis(2,3,4-trihydroxy-
dilute to 25 mL with water R and filter. 12 mL of the filtrate
benzyl)propanohydrazide,
complies with test A. Prepare the reference solution using lead
standard solution (1 ppm Pb) R.
Loss on drying (2.2.32): maximum 15 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
C. (2RS)-2-amino-3-hydroxy-2′-[(1EZ)-(2,3,4-trihydroxy- Microbial contamination
benzylidene)]propanohydrazide. TAMC : acceptance criterion 103 CFU/g (2.6.12).

04/2009:0467 FUNCTIONALITY-RELATED CHARACTERISTICS

This section provides information on characteristics that are
BENTONITE recognised as being relevant control parameters for one or
more functions of the substance when used as an excipient (see
Bentonitum chapter 5.15). This section is a non-mandatory part of the
monograph and it is not necessary to verify the characteristics
DEFINITION to demonstrate compliance. Control of these characteristics can
Natural clay containing a high proportion of montmorillonite, however contribute to the quality of a medicinal product by
a native hydrated aluminium silicate in which some aluminium improving the consistency of the manufacturing process and
and silicon atoms may be replaced by other atoms such as the performance of the medicinal product during use. Where
magnesium and iron. control methods are cited, they are recognised as being suitable
for the purpose, but other methods can also be used. Wherever
CHARACTERS results for a particular characteristic are reported, the control
Appearance : very fine, homogeneous, greyish-white powder method must be indicated.
with a more or less yellowish or pinkish tint.
The following characteristics may be relevant for bentonite used
Solubility : practically insoluble in water and in aqueous as viscosity-increasing agent or suspending agent.
solutions.
Sedimentation volume. To 6.0 g add 200 mL of water R and
It swells with a little water forming a malleable mass.
mix for 20 min using a high-speed mixer capable of operating
IDENTIFICATION at 10 000 r/min. Transfer 100 mL of this suspension to a
A. To 0.5 g in a metal crucible add 1 g of potassium nitrate R graduated cylinder. Allow to stand for 24 h. The volume of
and 3 g of sodium carbonate R and heat until the mixture the clear supernatant is not greater than 2 mL.
melts. Allow to cool. To this residue add 20 mL of boiling Swelling power with water : see Identification B.

1636 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Benzalkonium chloride

04/2009:0372 Acidity or alkalinity. To 50 mL of solution S add 0.1 mL of

corrected 7.1 bromocresol purple solution R. Not more than 0.1 mL of 0.1 M
hydrochloric acid or 0.1 M sodium hydroxide is required to
BENZALKONIUM CHLORIDE change the colour of the indicator.
Average relative molecular mass and ratio of alkyl
Benzalkonii chloridum components. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.400 g of the substance to be examined
in water R and dilute to 100.0 mL with the same solvent.
Reference solution. Dissolve the contents of a vial of
benzalkonium chloride for system suitability CRS in 5.0 mL
[8001-54-5] of water R.
DEFINITION Column :
Mixture of alkylbenzyldimethylammonium chlorides, the – size : l = 0.25 m, Ø = 4.6 mm ;
alkyl groups mainly having chain lengths of C12, C14 and C16. – stationary phase : end-capped nitrile silica gel for
Content : 95.0 per cent to 104.0 per cent of alkylbenzyldi- chromatography R (5 μm).
methylammonium chlorides (anhydrous substance) calculated Mobile phase : mix 45 volumes of acetonitrile R and 55 volumes
using the average relative molecular mass (see Tests). of a 13.6 g/L solution of sodium acetate R previously adjusted
CHARACTERS to pH 5.0 with glacial acetic acid R.
Appearance : white or yellowish-white powder or gelatinous, Flow rate : 2.0 mL/min.
yellowish-white fragments, hygroscopic. On heating it forms a Detection : spectrophotometer at 254 nm.
clear molten mass. Injection : 10 μL.
Solubility : very soluble in water and in ethanol (96 per cent).
An aqueous solution froths copiously when shaken. Identification of homologues : use the chromatogram supplied
with benzalkonium chloride for system suitability CRS and the
IDENTIFICATION chromatogram obtained with the reference solution to identify
First identification : B, E. the peaks due to C12, C14 and C16.
Second identification : A, C, D, E. Relative retention with reference to C12 homologue
A. Ultraviolet and visible absorption spectrophotometry (retention time = about 6 min) : C14 homologue = about 1.3 ;
(2.2.25). C 16 homologue = about 1.7.

Test solution. Dissolve 80 mg in water R and dilute to System suitability : reference solution :
100.0 mL with the same solvent. – resolution : minimum 1.5 between the peaks due to the C12
Spectral range : 220-350 nm. and C14 homologues.
Absorption maxima : at 257 nm, 263 nm and 269 nm. Calculate the average relative molecular mass of the sample
Shoulder : at about 250 nm. by summing the products for each homologue, using the
following expression :
B. Examine the chromatograms obtained in the test for average
relative molecular mass and ratio of alkyl components.
Results : the principal peaks in the chromatogram obtained
with the test solution are similar in retention time to the
principal peaks in the chromatogram obtained with the A = area of the peak due to the given homologue in the
reference solution. chromatogram obtained with the test solution ;
C. To 2 mL of solution S (see Tests) add 0.1 mL of glacial acetic B = sum of the areas of the peaks due to all homologues
acid R and, dropwise, 1 mL of sodium tetraphenylborate in the chromatogram obtained with the test
solution R. A white precipitate is formed. Filter. Dissolve solution ;
the precipitate in a mixture of 1 mL of acetone R and 5 mL
of ethanol (96 per cent) R, heating to not more than 70 °C. W = relative molecular mass for the given homologue :
Add water R dropwise to the warm solution until a slight 340, 368 and 396 for the C12, C14 and C16
opalescence forms. Heat gently until the solution is clear homologues, respectively.
and allow to cool. White crystals separate. Filter, wash with Calculate the percentage of each homologue, using the
3 quantities, each of 10 mL, of water R and dry in vacuo following expression :
over diphosphorus pentoxide R or anhydrous silica gel R
at a temperature not exceeding 50 °C. The crystals melt
(2.2.14) at 127 °C to 133 °C.
D. To 5 mL of dilute sodium hydroxide solution R add 0.1 mL
of bromophenol blue solution R1 and 5 mL of methylene C = product of the relative molecular mass of the given
chloride R and shake. The methylene chloride layer is homologue and the area of the corresponding
colourless. Add 0.1 mL of solution S and shake. The peak in the chromatogram obtained with the test
methylene chloride layer becomes blue. solution ;
E. To 2 mL of solution S add 1 mL of dilute nitric acid R. A D = sum of the C values for all homologues quantified.
white precipitate is formed which dissolves on the addition
of 5 mL of ethanol (96 per cent) R. The solution gives Limits :
reaction (a) of chlorides (2.3.1). – C12 homologue : minimum 40 per cent ;
TESTS – C14 homologue : minimum 20 per cent ;
Solution S. Dissolve 1.0 g in carbon dioxide-free water R and – sum of C12 and C14 homologues : minimum 70 per cent.
dilute to 100 mL with the same solvent. Impurities A, B and C. Liquid chromatography (2.2.29).
Appearance of solution. Solution S is clear (2.2.1) and not Prepare the solutions immediately before use.
more intensely coloured than reference solution Y6 (2.2.2, Test solution. Dissolve 0.50 g of the substance to be examined
Method II). in methanol R1 and dilute to 10.0 mL with the same solvent.

General Notices (1) apply to all monographs and other texts 1637
Benzalkonium chloride solution EUROPEAN PHARMACOPOEIA 8.0

Reference solution (a). Dissolve 25.0 mg of benzyl alcohol CRS If the titration curve after addition of 12.0 mL of the titrant
(impurity A) in methanol R1 and dilute to 100.0 mL with the shows only 1 point of inflexion, the substance to be examined
same solvent. does not comply with the test.
Reference solution (b). Dissolve 75.0 mg of benzaldehyde CRS Water (2.5.12) : maximum 10 per cent, determined on 0.300 g.
(impurity B) in methanol R1 and dilute to 100.0 mL with the Sulfated ash (2.4.14) : maximum 0.1 per cent, determined
same solvent. Dilute 1.0 mL of this solution to 10.0 mL with on 1.0 g.
methanol R1.
Reference solution (c). Dilute 1.0 mL of reference solution (a) ASSAY
to 10.0 mL with methanol R1. Dissolve 2.00 g in water R and dilute to 100.0 mL with the
Column : same solvent. Transfer 25.0 mL of the solution to a separating
funnel, add 25 mL of methylene chloride R, 10 mL of 0.1 M
– size : l = 0.15 m, Ø = 4.6 mm ;
sodium hydroxide and 10.0 mL of a freshly prepared 50 g/L
– stationary phase : end-capped octadecylsilyl silica gel for solution of potassium iodide R. Shake well, allow to separate
chromatography R (5 μm) ; and discard the methylene chloride layer. Shake the aqueous
– temperature : 30 °C. layer with 3 quantities, each of 10 mL, of methylene chloride R
Mobile phase : and discard the methylene chloride layers. To the aqueous
layer add 40 mL of hydrochloric acid R, allow to cool and titrate
– mobile phase A : dissolve 1.09 g of sodium hexanesulfonate R
with 0.05 M potassium iodate until the deep-brown colour
and 6.9 g of sodium dihydrogen phosphate monohydrate R
is almost discharged. Add 5 mL of methylene chloride R and
in water R ; adjust to pH 3.5 with phosphoric acid R and continue the titration, shaking vigorously, until the methylene
dilute to 1000.0 mL with the same solvent ;
chloride layer no longer changes colour. Carry out a blank
– mobile phase B : methanol R1 ; titration on a mixture of 10.0 mL of the freshly prepared
Time Mobile phase A Mobile phase B 50 g/L solution of potassium iodide R, 20 mL of water R and
(min) (per cent V/V) (per cent V/V) 40 mL of hydrochloric acid R.
0 - 10 80 20 1 mL of 0.05 M potassium iodate is equivalent to mg
of benzalkonium chloride where x is the average relative
10 - 14 80 → 50 20 → 50 molecular mass of the sample.
14 - 35 50 50
STORAGE
35 - 36 50 → 20 50 → 80 In an airtight container.
36 - 55 20 80
IMPURITIES
Flow rate : 1.0 mL/min. Specified impurities : A, B, C.
Detection : spectrophotometer at 210 nm for impurities A and
C, and at 257 nm for impurity B.
Injection : 20 μL.
Relative retention with reference to impurity A (retention A. benzyl alcohol,
time = about 10 min) : impurity B = about 1.3 ;
impurity C = about 2.4.
System suitability : at 210 nm :
– signal-to-noise ratio : minimum 10 for the principal peak in B. benzaldehyde,
the chromatogram obtained with reference solution (c) ;
– symmetry factor : minimum 0.6 for the peak due to
impurity A in the chromatogram obtained with reference
solution (a). C. (chloromethyl)benzene.
Limits :
– correction factor : for the calculation of content, multiply 04/2009:0371
the peak area of impurity C by 1.3 ; corrected 7.1
– impurity A : not more than the area of the corresponding
peak in the chromatogram obtained with reference BENZALKONIUM CHLORIDE
solution (a) (0.5 per cent) ; SOLUTION
– impurity B : not more than the area of the corresponding
peak in the chromatogram obtained with reference Benzalkonii chloridi solutio
solution (b) (0.15 per cent) ;
– impurity C : not more than 0.1 times the area of the DEFINITION
principal peak in the chromatogram obtained with Aqueous solution of a mixture of alkylbenzyldimethyl-
reference solution (a) (0.05 per cent). ammonium chlorides, the alkyl groups mainly having chain
Amines and amine salts. Dissolve 5.0 g with heating in lengths of C12, C14 and C16.
20 mL of a mixture of 3 volumes of 1 M hydrochloric acid and Content : 475 g/L to 525 g/L of alkylbenzyldimethylammonium
97 volumes of methanol R and add 100 mL of 2-propanol R. chlorides, calculated using the average relative molecular mass
Pass a stream of nitrogen R slowly through the solution. Titrate (see Tests). The solution may contain ethanol (96 per cent).
with up to 12.0 mL of 0.1 M tetrabutylammonium hydroxide
CHARACTERS
and record the potentiometric titration curve (2.2.20). If the
curve shows 2 points of inflexion, the volume of titrant added Appearance : clear, colourless or slightly yellowish liquid.
between the 2 points is not greater than 5.0 mL. If the curve Solubility : miscible with water and with ethanol (96 per cent).
shows no point of inflexion, the substance to be examined It froths copiously when shaken.
does not comply with the test. If the curve shows 1 point of
inflexion, repeat the test but add 3.0 mL of a 25.0 g/L solution IDENTIFICATION
of dimethyldecylamine R in 2-propanol R before the titration. First identification : B, E.

1638 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Benzalkonium chloride solution

Second identification : A, C, D, E. Identification of homologues : use the chromatogram supplied

A. Ultraviolet and visible absorption spectrophotometry with benzalkonium chloride for system suitability CRS and the
(2.2.25). chromatogram obtained with the reference solution to identify
the peaks due to homologues C12, C14 and C16.
Test solution. Dilute 0.3 mL to 100.0 mL with water R.
Relative retention with reference to C12 homologue
Spectral range : 220-350 nm. (retention time = about 6 min) : C14 homologue = about 1.3 ;
Absorption maxima : at 257 nm, 263 nm and 269 nm. C16 homologue = about 1.7.
Shoulder : at about 250 nm. System suitability : reference solution :
B. Examine the chromatograms obtained in the test for average – resolution : minimum 1.5 between the peaks due to the C12
relative molecular mass and ratio of alkyl components. and C14 homologues.
Results : the principal peaks in the chromatogram obtained Calculate the average relative molecular mass of the sample
with the test solution are similar in retention time to the by summing the products for each homologue, using the
principal peaks in the chromatogram obtained with the following expression :
reference solution.
C. To 0.05 mL add 2 mL of water R, 0.1 mL of glacial acetic
acid R and, dropwise, 1 mL of sodium tetraphenylborate
solution R. A white precipitate is formed. Filter. Dissolve A = area of the peak due to the given homologue in the
the precipitate in a mixture of 1 mL of acetone R and 5 mL chromatogram obtained with the test solution ;
of ethanol (96 per cent) R, heating to not more than 70 °C.
B = sum of the areas of the peaks due to all homologues
Add water R dropwise to the warm solution until a slight
opalescence forms. Heat gently until the solution is clear in the chromatogram obtained with the test
and allow to cool. White crystals separate. Filter, wash with solution ;
3 quantities, each of 10 mL, of water R and dry in vacuo W = relative molecular mass for the given homologue :
over diphosphorus pentoxide R or anhydrous silica gel R 340, 368 and 396 for the C12, C14 and C16
at a temperature not exceeding 50 °C. The crystals melt homologues, respectively.
(2.2.14) at 127 °C to 133 °C. Calculate the percentage of each homologue, using the
D. To 5 mL of dilute sodium hydroxide solution R add 0.1 mL following expression :
of bromophenol blue solution R1 and 5 mL of methylene
chloride R and shake. The methylene chloride layer is
colourless. Add 0.05 mL of the solution to be examined
and shake. The methylene chloride layer becomes blue.
E. To 0.05 mL add 1 mL of dilute nitric acid R. A white C = product of the relative molecular mass of the given
precipitate is formed which dissolves on the addition homologue and the area of the corresponding
of 5 mL of ethanol (96 per cent) R. The solution gives peak in the chromatogram obtained with the test
reaction (a) of chlorides (2.3.1). solution ;
D = sum of the C values for all homologues quantified.
TESTS
Limits :
Solution S. Dilute 2.0 g to 100 mL with carbon dioxide-free
– C12 homologue : minimum 40 per cent ;
water R.
– C14 homologue : minimum 20 per cent ;
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y6 (2.2.2, – sum of C12 and C14 homologues : minimum 70 per cent.
Method II). Impurities A, B and C. Liquid chromatography (2.2.29).
Acidity or alkalinity. To 50 mL of solution S add 0.1 mL of Prepare the solutions immediately before use.
bromocresol purple solution R. Not more than 0.1 mL of 0.1 M Test solution. Determine the density (2.2.5) of the solution to
hydrochloric acid or 0.1 M sodium hydroxide is required to be examined. Dilute a quantity of the solution to be examined
change the colour of the indicator. equivalent to 2.5 g of benzalkonium chloride to 50.0 mL with
Average relative molecular mass and ratio of alkyl methanol R1.
components. Liquid chromatography (2.2.29). Reference solution (a). Dissolve 25.0 mg of benzyl alcohol CRS
Test solution. Determine the density (2.2.5) of the solution to (impurity A) in methanol R1 and dilute to 100.0 mL with the
be examined. Dilute a quantity of the solution to be examined same solvent.
equivalent to about 0.400 g of benzalkonium chloride to Reference solution (b). Dissolve 75.0 mg of benzaldehyde CRS
100.0 mL with water R. (impurity B) in methanol R1 and dilute to 100.0 mL with the
same solvent. Dilute 1.0 mL of this solution to 10.0 mL with
Reference solution. Dissolve the contents of a vial of
methanol R1.
benzalkonium chloride for system suitability CRS in 5.0 mL
of water R. Reference solution (c). Dilute 1.0 mL of reference solution (a)
to 10.0 mL with methanol R1.
Column :
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : end-capped nitrile silica gel for
chromatography R (5 μm). – stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm) ;
Mobile phase : mix 45 volumes of acetonitrile R and 55 volumes
of a 13.6 g/L solution of sodium acetate R previously adjusted – temperature : 30 °C.
to pH 5.0 with glacial acetic acid R. Mobile phase :
Flow rate : 2.0 mL/min. – mobile phase A : dissolve 1.09 g of sodium hexanesulfonate R
and 6.9 g of sodium dihydrogen phosphate monohydrate R
Detection : spectrophotometer at 254 nm. in water R ; adjust to pH 3.5 with phosphoric acid R and
Injection : 10 μL. dilute to 1000.0 mL with the same solvent ;

General Notices (1) apply to all monographs and other texts 1639
Benzbromarone EUROPEAN PHARMACOPOEIA 8.0

– mobile phase B : methanol R1 ; changes colour. Carry out a blank titration on a mixture of
10.0 mL of the freshly prepared 50 g/L solution of potassium
Time Mobile phase A Mobile phase B
iodide R, 20 mL of water R and 40 mL of hydrochloric acid R.
(min) (per cent V/V) (per cent V/V)
0 - 10 80 20
1 mL of 0.05 M potassium iodate is equivalent to mg
of benzalkonium chloride where x is the average relative
10 - 14 80 → 50 20 → 50 molecular mass of the sample.
14 - 35 50 50 LABELLING
35 - 36 50 → 20 50 → 80 The label states the content of ethanol (96 per cent), if any.
36 - 55 20 80 IMPURITIES
Flow rate : 1.0 mL/min. Specified impurities : A, B, C.
Detection : spectrophotometer at 210 nm for impurities A and
C, and at 257 nm for impurity B.
Injection : 20 μL.
Relative retention with reference to impurity A (retention A. benzyl alcohol,
time = about 10 min) : impurity B = about 1.3 ;
impurity C = about 2.4.
System suitability : at 210 nm :
– signal-to-noise ratio : minimum 10 for the principal peak in B. benzaldehyde,
the chromatogram obtained with reference solution (c) ;
– symmetry factor : minimum 0.6 for the peak due to
impurity A in the chromatogram obtained with reference
solution (a).
C. (chloromethyl)benzene.
Limits :
– correction factor : for the calculation of content, multiply
the peak area of impurity C by 1.3 ; 01/2008:1393
– impurity A : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (a) (0.5 per cent) ;
BENZBROMARONE
– impurity B : not more than the area of the corresponding
peak in the chromatogram obtained with reference
Benzbromaronum
solution (b) (0.15 per cent) ;
– impurity C : not more than 0.1 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.05 per cent).
Amines and amine salts. Mix 10.0 g, while heating, with
20 mL of a mixture of 3 volumes of 1 M hydrochloric acid and
97 volumes of methanol R and add 100 mL of 2-propanol R. C17H12Br2O3 Mr 424.1
Pass a stream of nitrogen R slowly through the solution. Titrate [3562-84-3]
with up to 12.0 mL of 0.1 M tetrabutylammonium hydroxide
and record the potentiometric titration curve (2.2.20). If the DEFINITION
curve shows 2 points of inflexion, the volume of titrant added (3,5-Dibromo-4-hydroxyphenyl)(2-ethylbenzofuran-3-yl)-
between the 2 points is not greater than 5.0 mL. If the curve methanone.
shows no point of inflexion, the solution to be examined Content : 98.0 per cent to 101.0 per cent (dried substance).
does not comply with the test. If the curve shows 1 point of
inflexion, repeat the test but add 3.0 mL of a 25.0 g/L solution CHARACTERS
of dimethyldecylamine R in 2-propanol R before the titration. Appearance : white or almost white, crystalline powder.
If the titration curve after the addition of 12.0 mL of the titrant Solubility : practically insoluble in water, freely soluble in
shows only 1 point of inflexion, the solution to be examined acetone and in methylene chloride, sparingly soluble in
does not comply with the test. ethanol (96 per cent).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on mp : about 152 °C.
1.0 g.
IDENTIFICATION
ASSAY A. Infrared absorption spectrophotometry (2.2.24).
Determine the density (2.2.5) of the solution to be examined. Comparison: benzbromarone CRS.
Dilute 4.00 g to 100.0 mL with water R. Transfer 25.0 mL of
the solution to a separating funnel, add 25 mL of methylene B. By means of a copper wire, previously ignited, introduce
chloride R, 10 mL of 0.1 M sodium hydroxide and 10.0 mL of a a small amount of the substance to be examined into the
freshly prepared 50 g/L solution of potassium iodide R. Shake non-luminous part of a flame. The colour of the flame
well, allow to separate and discard the methylene chloride becomes green.
layer. Shake the aqueous layer with 3 quantities, each of 10 mL, TESTS
of methylene chloride R and discard the methylene chloride
layers. To the aqueous layer add 40 mL of hydrochloric acid R, Appearance of solution. The solution is clear (2.2.1) and not
allow to cool and titrate with 0.05 M potassium iodate until more intensely coloured than reference solution Y5 (2.2.2,
the deep-brown colour is almost discharged. Add 5 mL of Method II).
methylene chloride R and continue the titration, shaking Dissolve 1.25 g in dimethylformamide R and dilute to 25 mL
vigorously, until the methylene chloride layer no longer with the same solvent.

1640 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Benzethonium chloride

Acidity or alkalinity. Shake 0.5 g with 10 mL of carbon Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
dioxide-free water R for 1 min and filter. To 2.0 mL of the 1.0 g.
filtrate add 0.1 mL of methyl red solution R and 0.1 mL of
0.01 M hydrochloric acid. The solution is red. Add 0.3 mL of ASSAY
0.01 M sodium hydroxide. The solution is yellow. Dissolve 0.300 g in 60 mL of methanol R. Stir until completely
Related substances. Liquid chromatography (2.2.29). dissolved and add 10 mL of water R. Titrate with 0.1 M sodium
hydroxide, determining the end-point potentiometrically
Test solution. Dissolve 0.125 g of the substance to be examined (2.2.20).
in 30 mL of methanol R and dilute to 50.0 mL with the mobile
phase. 1 mL of 0.1 M sodium hydroxide is equivalent to 42.41 mg
of C17H12Br2O3.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution STORAGE
to 10.0 mL with the mobile phase. Protected from light.
Reference solution (b). Dissolve 10 mg of benzarone CRS
(impurity C) in the mobile phase and dilute to 20 mL with the IMPURITIES
mobile phase. To 5 mL of this solution add 1 mL of the test Specified impurities : A, B.
solution and dilute to 100 mL with the mobile phase. Other detectable impurities (the following substances would,
Column : if present at a sufficient level, be detected by one or other of
– size : l = 0.25 m, Ø = 4.6 mm ; the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
– stationary phase : octadecylsilyl silica gel for by the general monograph Substances for pharmaceutical
chromatography R (5 μm). use (2034). It is therefore not necessary to identify these
Mobile phase : glacial acetic acid R, acetonitrile R, water R, impurities for demonstration of compliance. See also 5.10.
methanol R (5:25:300:990 V/V/V/V). Control of impurities in substances for pharmaceutical use) : C.
Flow rate : 1.5 mL/min.
Detection : spectrophotometer at 231 nm.
Injection : 20 μL.
Run time : 2.5 times the retention time of benzbromarone.
Relative retention with reference to benzbromarone :
impurity A = about 0.6 ; impurity B = about 2.
System suitability : reference solution (b) :
A. R1 = R2 = H, R3 = Br : (3-bromo-4-hydroxyphenyl)(2-
– resolution : minimum 10.0 between the peaks due to
ethylbenzofuran-3-yl)methanone,
impurity C (1st peak) and benzbromarone (2nd peak).
Limits : B. R1 = R2 = R3 = Br : (6-bromo-2-ethylbenzofuran-3-yl)(3,5-
dibromo-4-hydroxyphenyl)methanone,
– impurity A : not more than 4 times the area of the principal
peak in the chromatogram obtained with reference C. R1 = R2 = R3 = H : (2-ethylbenzofuran-3-yl)(4-
solution (a) (0.4 per cent) ; hydroxyphenyl)methanone (benzarone).
– impurity B : not more than 10 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (1.0 per cent) ; 01/2008:0974
corrected 6.0
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ; BENZETHONIUM CHLORIDE
– sum of impurities other than A and B : not more than
twice the area of the principal peak in the chromatogram Benzethonii chloridum
obtained with reference solution (a) (0.2 per cent) ;
– disregard limit : 0.2 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.02 per cent).
Halides expressed as chlorides (2.4.4) : maximum 400 ppm.
Shake 1.25 g with a mixture of 5 mL of dilute nitric acid R
and 15 mL of water R. Filter. Rinse the filter with water R C27H42ClNO2 Mr 448.1
and dilute the filtrate to 25 mL with the same solvent. Dilute [121-54-0]
2.5 mL of this solution to 15 mL with water R.
DEFINITION
Iron (2.4.9) : maximum 125 ppm.
N-Benzyl-N,N-dimethyl-2-[2-[4-(1,1,3,3-tetramethylbutyl)-
Moisten the residue obtained in the test for sulfated ash with phenoxy]ethoxy]ethanaminium chloride.
2 mL of hydrochloric acid R and evaporate to dryness on a
water-bath. Add 0.05 mL of hydrochloric acid R and 10 mL of Content : 97.0 per cent to 103.0 per cent (dried substance).
water R, heat to boiling and maintain boiling for 1 min. Allow CHARACTERS
to cool. Rinse the crucible with water R, collect the rinsings
and dilute to 25 mL with water R. Dilute 2 mL of this solution Appearance : white or yellowish-white powder.
to 10 mL with water R. Solubility : very soluble in water and in ethanol (96 per cent),
freely soluble in methylene chloride.
Heavy metals (2.4.8) : maximum 20 ppm.
An aqueous solution froths copiously when shaken.
0.5 g complies with test C. Prepare the reference solution using
1 mL of lead standard solution (10 ppm Pb) R. IDENTIFICATION
Loss on drying (2.2.32) : maximum 0.5 per cent, determined A. Melting point (2.2.14): 158 °C to 164 °C, after drying at
on 1.000 g by drying in vacuo at 50 °C for 4 h. 105 °C for 4 h.

General Notices (1) apply to all monographs and other texts 1641
Benzocaine EUROPEAN PHARMACOPOEIA 8.0

B. Thin-layer chromatography (2.2.27). STORAGE

Test solution. Dissolve 25 mg of the substance to be Protected from light.
examined in water R and dilute to 5 mL with the same
solvent. 01/2008:0011
Reference solution. Dissolve 25 mg of benzethonium corrected 6.0
chloride CRS in water R and dilute to 5 mL with the same
solvent. BENZOCAINE
Plate : TLC silica gel F254 plate R.
Mobile phase : glacial acetic acid R, water R, methanol R Benzocainum
(5:5:100 V/V/V).
Application : 20 μL.
Development : over a path of 12 cm.
Drying : in a current of warm air.
Detection : examine in ultraviolet light at 254 nm. C9H11NO2 Mr 165.2
Results : the principal spot in the chromatogram obtained [94-09-7]
with the test solution is similar in position and size to the DEFINITION
principal spot in the chromatogram obtained with the
reference solution. Ethyl 4-aminobenzoate.
Content : 99.0 per cent to 101.0 per cent (dried substance).
C. To 5 mL of dilute sodium hydroxide solution R add 0.1 mL
of bromophenol blue solution R1 and 5 mL of methylene CHARACTERS
chloride R and shake. The lower layer is colourless. Add Appearance : white or almost white, crystalline powder or
0.1 mL of solution S (see Tests) and shake. A blue colour colourless crystals.
develops in the lower layer.
Solubility : very slightly soluble in water, freely soluble in
D. To 2 mL of solution S add 1 mL of dilute nitric acid R. A ethanol (96 per cent).
white precipitate is formed which dissolves upon addition
of 5 mL of ethanol (96 per cent) R. The solution gives IDENTIFICATION
reaction (a) of chlorides (2.3.1). First identification : A, B.
TESTS Second identification : A, C, D.
A. Melting point (2.2.14) : 89 °C to 92 °C.
Solution S. Dissolve 5.0 g in carbon dioxide-free water R and
dilute to 50 mL with the same solvent. B. Infrared absorption spectrophotometry (2.2.24).
Comparison: benzocaine CRS.
Appearance of solution. Solution S is clear (2.2.1) and
not more intensely coloured than reference solution Y6 C. To about 50 mg in a test tube add 0.2 mL of a 500 g/L
(2.2.2, Method II). solution of chromium trioxide R. Cover the mouth of the
tube with a piece of filter paper moistened with a freshly
Acidity or alkalinity. To 25 mL of solution S add 0.1 mL of prepared mixture of equal volumes of a 50 g/L solution of
phenolphthalein solution R. The solution is colourless. Add sodium nitroprusside R and a 200 g/L solution of piperazine
0.3 mL of 0.01 M sodium hydroxide. The solution is pink. hydrate R. Boil gently for at least 30 s. A blue colour
Add 0.1 mL of methyl red solution R and 0.5 mL of 0.01 M develops on the filter paper.
hydrochloric acid. The solution is orange-red. D. Dissolve about 50 mg in ethanol (96 per cent) R and dilute
Volatile bases and salts of volatile bases (2.4.1, Method B) : to 100 mL with the same solvent. 2 mL of the solution gives
maximum 50 ppm, determined on 0.20 g. the reaction of primary aromatic amines (2.3.1).
Prepare the standard using 0.1 mL of ammonium standard TESTS
solution (100 ppm NH4) R. Replace heavy magnesium oxide by
2.0 mL of strong sodium hydroxide solution R. Appearance of solution. The solution is clear (2.2.1) and
colourless (2.2.2, Method II).
Loss on drying (2.2.32) : maximum 5.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 4 h. Dissolve 1.0 g in ethanol (96 per cent) R and dilute to 20 mL
with the same solvent.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. Acidity or alkalinity. Dissolve 0.5 g in 10 mL of ethanol
(96 per cent) R previously neutralised to 0.05 mL of
ASSAY phenolphthalein solution R. Add 10 mL of carbon dioxide-free
water R. The solution remains colourless and not more than
Dissolve 2.000 g in water R and dilute to 100.0 mL with the
0.5 mL of 0.01 M sodium hydroxide is required to change the
same solvent. Transfer 25.0 mL of the solution to a separating
colour of the indicator.
funnel, add 10 mL of a 4 g/L solution of sodium hydroxide R,
10.0 mL of a freshly prepared 50 g/L solution of potassium Loss on drying (2.2.32) : maximum 0.5 per cent, determined
iodide R and 25 mL of methylene chloride R. Shake vigorously, on 1.00 g by drying in vacuo.
allow to separate and discard the lower layer. Shake the Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
upper layer with 3 quantities, each of 10 mL, of methylene 1.0 g.
chloride R and discard the lower layers. To the upper layer add
40 mL of hydrochloric acid R, allow to cool and titrate with ASSAY
0.05 M potassium iodate until the deep brown colour is almost Carry out the determination of primary aromatic
discharged. Add 4 mL of methylene chloride R and continue amino-nitrogen (2.5.8), using 0.400 g dissolved in a mixture
the titration, shaking vigorously, until the lower layer is no of 25 mL of hydrochloric acid R and 50 mL of water R.
longer brown. Carry out a blank titration using a mixture of 1 mL of 0.1 M sodium nitrite is equivalent to 16.52 mg
10.0 mL of a freshly prepared 50 g/L solution of potassium of C9H11NO2.
iodide R, 20 mL of water R and 40 mL of hydrochloric acid R.
1 mL of 0.05 M potassium iodate is equivalent to 44.81 mg STORAGE
of C27H42ClNO2. Protected from light.

1642 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Benzoyl peroxide, hydrous

01/2008:0066 Heavy metals (2.4.8) : maximum 10 ppm.

corrected 6.4 12 mL of solution S complies with test B. Prepare the reference
solution using a mixture of 5 mL of lead standard solution
BENZOIC ACID (1 ppm Pb) R and 5 mL of ethanol (96 per cent) R.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Acidum benzoicum 1.0 g.
ASSAY
Dissolve 0.200 g in 20 mL of ethanol (96 per cent) R and titrate
with 0.1 M sodium hydroxide, using 0.1 mL of phenol red
C 7H 6O 2 Mr 122.1 solution R as indicator, until the colour changes from yellow
[65-85-0] to violet-red.
1 mL of 0.1 M sodium hydroxide is equivalent to 12.21 mg
DEFINITION of C7H6O2.
Benzenecarboxylic acid.
Content : 99.0 per cent to 100.5 per cent. 01/2008:0704
CHARACTERS corrected 7.0
Appearance : white or almost white, crystalline powder or
colourless crystals. BENZOYL PEROXIDE, HYDROUS
Solubility : slightly soluble in water, soluble in boiling water,
freely soluble in ethanol (96 per cent) and in fatty oils. Benzoylis peroxidum cum aqua
IDENTIFICATION
A. Melting point (2.2.14) : 121 °C to 124 °C.
B. Solution S (see Tests) gives reaction (a) of benzoates (2.3.1).
TESTS
C14H10O4 Mr 242.2 (anhydrous substance)
Solution S. Dissolve 5.0 g in ethanol (96 per cent) R and dilute Anhydrous benzoyl peroxide : [94-36-0]
to 100 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and DEFINITION
colourless (2.2.2, Method II). Content :
Carbonisable substances. Dissolve 0.5 g with shaking in – dibenzoyl peroxide : 70.0 per cent to 77.0 per cent ;
5 mL of sulfuric acid R. After 5 min, the solution is not more – water : minimum 20.0 per cent.
intensely coloured than reference solution Y5 (2.2.2, Method I).
CHARACTERS
Oxidisable substances. Dissolve 0.2 g in 10 mL of boiling Appearance : white or almost white, amorphous or granular
water R. Cool, shake and filter. To the filtrate add 1 mL powder.
of dilute sulfuric acid R and 0.2 mL of 0.02 M potassium
permanganate. After 5 min, the solution is still coloured pink. Solubility : practically insoluble in water, soluble in acetone,
soluble in methylene chloride with the separation of water,
Halogenated compounds and halides : maximum 300 ppm. slightly soluble in ethanol (96 per cent).
All glassware used must be chloride-free and may be prepared It loses water rapidly on exposure to air with a risk of
by soaking overnight in a 500 g/L solution of nitric acid R, rinsed explosion.
with water R and stored full of water R. It is recommended that
Mix the entire sample thoroughly before carrying out the
glassware be reserved for this test.
following tests.
Solution (a). Dissolve 6.7 g in a mixture of 40 mL of 1 M
sodium hydroxide and 50 mL of ethanol (96 per cent) R and IDENTIFICATION
dilute to 100.0 mL with water R. To 10.0 mL of this solution First identification : B
add 7.5 mL of dilute sodium hydroxide solution R and 0.125 g Second identification : A, C, D.
of nickel-aluminium alloy R and heat on a water-bath for
10 min. Allow to cool to room temperature, filter into a 25 mL A. Ultraviolet and visible absorption spectrophotometry
volumetric flask and wash with 3 quantities, each of 2 mL, (2.2.25).
of ethanol (96 per cent) R. Dilute the filtrate and washings Solution A. Dissolve 80.0 mg in ethanol (96 per cent) R and
to 25.0 mL with water R. This solution is used to prepare dilute to 100.0 mL with the same solvent. Dilute 10.0 mL of
solution A. the solution to 100.0 mL with ethanol (96 per cent) R.
Solution (b). In the same manner, prepare a similar solution Solution B. Dilute 10.0 mL of solution A to 100.0 mL with
without the substance to be examined. This solution is used ethanol (96 per cent) R.
to prepare solution B. Spectral ranges : 250-300 nm for solution A ; 220-250 nm
In four 25 mL volumetric flasks, place separately 10 mL of for solution B.
solution (a), 10 mL of solution (b), 10 mL of chloride standard Absorption maxima : at 274 nm for solution A ; at 235 nm
solution (8 ppm Cl) R (used to prepare solution C) and 10 mL for solution B.
of water R. To each flask add 5 mL of ferric ammonium sulfate Shoulder : at about 282 nm for solution A.
solution R5, mix and add dropwise and with swirling 2 mL Absorbance ratio : A235/A274 = 1.17 to 1.21.
of nitric acid R and 5 mL of mercuric thiocyanate solution R.
Shake. Dilute the contents of each flask to 25.0 mL with B. Infrared absorption spectrophotometry (2.2.24).
water R and allow the solutions to stand in a water-bath at Comparison: Ph. Eur. reference spectrum of hydrous benzoyl
20 °C for 15 min. Measure at 460 nm the absorbance (2.2.25) peroxide.
of solution A using solution B as the compensation liquid, and C. Dissolve about 25 mg in 2 mL of acetone R. Add 1 mL of
the absorbance of solution C using the solution obtained with a 10 g/L solution of diethylphenylenediamine sulfate R and
10 mL of water R as the compensation liquid. The absorbance mix. A red colour develops which quickly darkens and
of solution A is not greater than that of solution C. becomes dark violet within 5 min.

General Notices (1) apply to all monographs and other texts 1643
Benzoyl peroxide, hydrous EUROPEAN PHARMACOPOEIA 8.0

D. To 1 g add 5 mL of ethanol (96 per cent) R, 5 mL of dilute – unspecified impurities : for each impurity, not more than the
sodium hydroxide solution R and 10 mL of water R. Boil the area of the principal peak in the chromatogram obtained
mixture under reflux for 20 min. Cool. The solution gives with reference solution (a) (0.10 per cent) ;
reaction (c) of benzoates (2.3.1). – disregard limit : 0.2 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
TESTS
(0.02 per cent).
Acidity. Dissolve a quantity of the substance to be examined
Chlorides (2.4.4) : maximum 0.4 per cent.
containing the equivalent of 1.0 g of dibenzoyl peroxide in
25 mL of acetone R, add 75 mL of water R and filter. Wash Dissolve a quantity of the substance to be examined containing
the residue with two quantities, each of 10 mL, of water R. the equivalent of 0.5 g of dibenzoyl peroxide in 15 mL of
Combine the filtrate and the washings and add 0.25 mL of acetone R. Add, while stirring, 50 mL of 0.05 M nitric acid.
phenolphthalein solution R1. Not more than 1.25 mL of 0.1 M Allow to stand for 10 min and filter. Wash the residue with 2
sodium hydroxide is required to change the colour of the quantities, each of 10 mL, of 0.05 M nitric acid. Combine the
indicator. Carry out a blank test. filtrate and the washings and dilute to 100 mL with 0.05 M
nitric acid. Dilute 2.5 mL of the solution to 15.0 mL with
Related substances. Liquid chromatography (2.2.29). Prepare
water R.
the solutions immediately before use.
Test solution. Dissolve a quantity of the substance to be ASSAY
examined containing the equivalent of 0.10 g of dibenzoyl
peroxide in acetonitrile R and dilute to 50 mL with the same Solution (a). Dissolve 2.500 g immediately before use in
solvent. 75 mL of dimethylformamide R and dilute to 100.0 mL with
the same solvent.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with acetonitrile R. Dilute 1.0 mL of this solution to Dibenzoyl peroxide. To 5.0 mL of solution (a) add 20 mL of
10.0 mL with acetonitrile R. acetone R and 3 mL of a 500 g/L solution of potassium iodide R
and mix. Allow to stand for 1 min. Titrate with 0.1 M sodium
Reference solution (b). Dissolve 30.0 mg of benzoic acid R in thiosulfate using 1 mL of starch solution R, added towards the
the mobile phase and dilute to 100.0 mL with the mobile end of the titration, as indicator. Carry out a blank titration.
phase. Dilute 1.0 mL of the solution to 10.0 mL with the
mobile phase. 1 mL of 0.1 M sodium thiosulfate is equivalent to 12.11 mg of
C14H10O4.
Reference solution (c). Dissolve 50.0 mg of ethyl benzoate R
in the mobile phase and dilute to 100.0 mL with the mobile Water (2.5.12). Carry out the semi-micro determination of
phase. Dilute 1.0 mL of the solution to 100.0 mL with the water, using 5.0 mL of solution (a). Use as the solvent a mixture
mobile phase. of 20.0 mL of anhydrous methanol R and 3.0 mL of a 100 g/L
solution of potassium iodide R in dimethylformamide R. After
Reference solution (d). Dissolve 50.0 mg of benzaldehyde R adding solution (a), stir for 5 min before starting the titration.
in the mobile phase and dilute to 100.0 mL with the mobile Carry out a blank determination.
phase. Dilute 1.0 mL of the solution to 100.0 mL with the
mobile phase. Calculate the percentage content of water using the following
expression :
Reference solution (e). Dissolve 30.0 mg of benzoic acid R and
30.0 mg of benzaldehyde R in the mobile phase and dilute to
100.0 mL with the mobile phase. Dilute 1.0 mL of the solution
to 10.0 mL with the mobile phase.
Column : n1 = number of millilitres of iodosulfurous reagent R
– size : l = 0.25 m, Ø = 4.6 mm ; used in the sample determination,
n2 = number of millilitres of iodosulfurous reagent R
– stationary phase : octadecylsilyl silica gel for
chromatography R (10 μm). used in the blank determination,
w = water equivalent of iodosulfurous reagent R in
Mobile phase : glacial acetic acid R, acetonitrile R, water R
(1:500:500 V/V/V). milligrams of water per millilitre of reagent,
m = mass of the substance to be examined used for the
Flow rate : 1 mL/min.
preparation of solution (a) in grams,
Detection : spectrophotometer at 235 nm. p = percentage content of dibenzoyl peroxide.
Injection : 20 μL loop injector.
Run time : 2 times the retention time of dibenzoyl peroxide. STORAGE
Relative retention with reference to dibenzoyl peroxide In a container that has been treated to reduce static discharge
(retention time = about 28.4 min) : impurity B = about 0.15 ; and that has a device for release of excess pressure, at a
impurity A = about 0.2 ; impurity C = about 0.4. temperature of 2 °C to 8 °C, protected from light.
System suitability: reference solution (e) :
– resolution : minimum 6 between the peaks due to benzoic IMPURITIES
acid and benzaldehyde.
Limits :
– impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (d)
(0.25 per cent) ;
– impurity B : not more than the area of the principal peak A. R = H : benzaldehyde,
in the chromatogram obtained with reference solution (b)
(1.5 per cent) ;
B. R = OH : benzoic acid,
– impurity C : not more than the area of the principal peak
in the chromatogram obtained with reference solution (c)
(0.25 per cent) ; C. R = O-CH2-CH3 : ethyl benzoate.

1644 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Benzyl alcohol

01/2013:0256 Temperature :
Time Temperature
BENZYL ALCOHOL (min) (°C)
Column 0 - 34 50 → 220

Alcohol benzylicus 34 - 69 220

Injection port 200

Detector 310

Detection : flame ionisation.

C 7H 8O Mr 108.1 Benzyl alcohol not intended for parenteral administration
[100-51-6] Injection : without air-plug, 0.1 μL of the test solution and
reference solution (a).
DEFINITION
Relative retention with reference to benzyl alcohol
Phenylmethanol. (retention time = about 26 min) : ethylbenzene = about 0.28 ;
Content : 98.0 per cent to 100.5 per cent. dicyclohexyl = about 0.59 ; impurity A = about 0.68 ;
impurity B = about 0.71.
CHARACTERS System suitability : reference solution (a) :
Appearance : clear, colourless, oily liquid. – resolution : minimum 3.0 between the peaks due to
impurities A and B.
Solubility : soluble in water, miscible with ethanol (96 per cent)
and with fatty and essential oils. If any peaks in the chromatogram obtained with the test
solution have the same retention time as the peaks due
Relative density : 1.043 to 1.049. to ethyl benzene or dicyclohexyl, substract the areas of
any such peaks from the peak areas at these retention
IDENTIFICATION times in the chromatograms obtained with reference
Infrared absorption spectrophotometry (2.2.24). solutions (a) or (b) (corrected peak areas of ethyl benzene
and dicyclohexyl). Any such peaks in the chromatogram
Comparison : benzyl alcohol CRS. obtained with the test solution are to be included in the
assessments for the sum of other peaks.
TESTS
Limits :
Appearance of solution. Shake 2.0 mL with 60 mL of water R.
– impurity A : not more than the difference between the
It dissolves completely. The solution is clear (2.2.1) and
area of the peak due to impurity A in the chromatogram
colourless (2.2.2, Method II).
obtained with reference solution (a) and the area of the
Acidity. To 10 mL add 10 mL of ethanol (96 per cent) R and peak due to impurity A in the chromatogram obtained
1 mL of phenolphthalein solution R. Not more than 1 mL of with the test solution (0.15 per cent) ;
0.1 M sodium hydroxide is required to change the colour of – impurity B : not more than the difference between the
the indicator to pink. area of the peak due to impurity B in the chromatogram
Refractive index (2.2.6) : 1.538 to 1.541. obtained with reference solution (a) and the area of the
peak due to impurity B in the chromatogram obtained
Peroxide value (2.5.5) : maximum 5.
with the test solution (0.10 per cent) ;
Related substances. Gas chromatography (2.2.28). – sum of other peaks with a relative retention less than that
Test solution. The substance to be examined. of benzyl alcohol : not more than 4 times the area of the
Standard solution (a). Dissolve 0.100 g of ethylbenzene R in peak due to ethylbenzene in the chromatogram obtained
the test solution and dilute to 10.0 mL with the same solution. with reference solution (a) corrected if necessary as
Dilute 2.0 mL of this solution to 20.0 mL with the test solution. described above (0.04 per cent) ;
– sum of peaks with a relative retention greater than that
Standard solution (b). Dissolve 2.000 g of dicyclohexyl R in the of benzyl alcohol : not more than the area of the peak
test solution and dilute to 10.0 mL with the same solution. due to dicyclohexyl in the chromatogram obtained with
Dilute 2.0 mL of this solution to 20.0 mL with the test solution. reference solution (a) corrected if necessary as described
Reference solution (a). Dissolve 0.750 g of benzaldehyde R above (0.3 per cent) ;
and 0.500 g of cyclohexylmethanol R in the test solution and – disregard limit : 0.01 times the area of the peak due
dilute to 25.0 mL with the test solution. Add 1.0 mL of this to ethylbenzene in the chromatogram obtained with
solution to a mixture of 2.0 mL of standard solution (a) and reference solution (a) corrected if necessary as described
3.0 mL of standard solution (b) and dilute to 20.0 mL with above (0.0001 per cent).
the test solution.
Benzyl alcohol intended for parenteral administration
Reference solution (b). Dissolve 0.250 g of benzaldehyde R Injection : without air-plug, 0.1 μL of the test solution and
and 0.500 g of cyclohexylmethanol R in the test solution and reference solution (b).
dilute to 25.0 mL with the test solution. Add 1.0 mL of this
solution to a mixture of 2.0 mL of standard solution (a) and Relative retention with reference to benzyl alcohol
2.0 mL of standard solution (b) and dilute to 20.0 mL with (retention time = about 26 min) : ethylbenzene = about 0.28 ;
the test solution. dicyclohexyl = about 0.59 ; impurity A = about 0.68 ;
impurity B = about 0.71.
Column : System suitability : reference solution (b) :
– material : fused silica ; – resolution : minimum 3.0 between the peaks due to
– size : l = 30 m, Ø = 0.32 mm ; impurities A and B.
– stationary phase : macrogol 20 000 R (film thickness 0.5 μm). If any peaks in the chromatogram obtained with the test
solution have the same retention times as the peaks due
Carrier gas : helium for chromatography R. to ethyl benzene or dicyclohexyl, substract the areas of
Linear velocity : 25 cm/s. any such peaks from the peak areas at these retention

General Notices (1) apply to all monographs and other texts 1645
Benzyl benzoate EUROPEAN PHARMACOPOEIA 8.0

times in the chromatograms obtained with reference

solutions (a) or (b) (corrected peak areas of ethyl benzene
and dicyclohexyl). Any such peaks in the chromatogram
obtained with the test solution are to be included in the B. cyclohexylmethanol.
assessments for the sum of other peaks.
Limits : 01/2008:0705
– impurity A : not more than the difference between the
area of the peak due to impurity A in the chromatogram BENZYL BENZOATE
obtained with reference solution (b) and the area of the
peak due to impurity A in the chromatogram obtained Benzylis benzoas
with the test solution (0.05 per cent) ;
– impurity B : not more than the difference between the
area of the peak due to impurity B in the chromatogram
obtained with reference solution (b) and the area of the
peak due to impurity B in the chromatogram obtained
with the test solution (0.10 per cent) ; C14H12O2 Mr 212.2
– sum of other peaks with a relative retention less than that [120-51-4]
of benzyl alcohol : not more than twice the area of the
peak due to ethylbenzene in the chromatogram obtained DEFINITION
with reference solution (b) corrected if necessary as Phenylmethyl benzoate.
described above (0.02 per cent) ; Content : 99.0 per cent to 100.5 per cent.
– sum of peaks with a relative retention greater than that
CHARACTERS
of benzyl alcohol : not more than the area of the peak
due to dicyclohexyl in the chromatogram obtained with Appearance : colourless or almost colourless crystals or
reference solution (b) corrected if necessary as described colourless or almost colourless, oily liquid.
above (0.2 per cent) ; Solubility : practically insoluble in water, miscible with ethanol
– disregard limit : 0.01 times the area of the peak due (96 per cent), with methylene chloride and with fatty and
to ethylbenzene in the chromatogram obtained with essential oils.
reference solution (b) corrected if necessary as described Eb : about 320 °C.
above (0.0001 per cent).
IDENTIFICATION
Residue on evaporation : maximum 0.05 per cent. First identification : A.
After ensuring that the substance to be examined complies Second identification : B, C.
with the test for peroxide value, evaporate 10.0 g to dryness in A. Infrared absorption spectrophotometry (2.2.24).
a tared quartz or porcelain crucible or platinum dish on a hot
plate at a temperature not exceeding 200 °C. Ensure that the Comparison: Ph. Eur. reference spectrum of benzyl benzoate.
substance to be examined does not boil during evaporation. B. To 2 g add 25 mL of alcoholic potassium hydroxide
Dry the residue on the hot plate for 1 h and allow to cool in a solution R and boil under a reflux condenser for 2 h.
desiccator. The residue weighs a maximum of 5 mg. Remove the ethanol on a water-bath, add 50 mL of water R
and distill. Collect about 25 mL of distillate and use it for
ASSAY identification test C. Acidify the liquid remaining in the
distillation flask with dilute hydrochloric acid R. A white
To 0.900 g (m g) add 15.0 mL of a freshly prepared mixture of
precipitate is formed that, when washed with water R and
1 volume of acetic anhydride R and 7 volumes of anhydrous
dried in vacuo melts (2.2.14) at 121 °C to 124 °C.
pyridine R and heat under a reflux condenser on a boiling
water-bath for 30 min. Cool and add 25 mL of water R. Using C. To the distillate obtained in identification test B add 2.5 g
0.25 mL of phenolphthalein solution R as indicator, titrate with of potassium permanganate R and 5 mL of dilute sodium
1 M sodium hydroxide (n1 mL). Carry out a blank titration hydroxide solution R. Boil under a reflux condenser for
(n2 mL). 15 min, cool and filter. Acidify the filtrate with dilute
hydrochloric acid R. A white precipitate is formed that,
Calculate the percentage content of C7H8O using the following when washed with water R and dried in vacuo, melts
expression : (2.2.14) at 121 °C to 124 °C.
TESTS
Acidity. Dissolve 2.0 g in ethanol (96 per cent) R and dilute
to 10 mL with the same solvent. Titrate with 0.1 M sodium
STORAGE hydroxide using phenolphthalein solution R as indicator. Not
In an airtight container, under nitrogen, protected from light more than 0.2 mL is required to change the colour of the
and at a temperature between 2 °C and 8 °C. indicator to pink.
Relative density (2.2.5) : 1.118 to 1.122.
LABELLING
Refractive index (2.2.6) : 1.568 to 1.570.
The label states, where applicable, that the substance is suitable
for use in the manufacture of parenteral preparations. Freezing point (2.2.18) : minimum 17.0 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
IMPURITIES 1.0 g.
Specified impurities : A, B.
ASSAY
To 2.000 g add 50.0 mL of 0.5 M alcoholic potassium hydroxide
and boil gently under a reflux condenser for 1 h. Titrate
the hot solution with 0.5 M hydrochloric acid using 1 mL of
phenolphthalein solution R as indicator. Carry out a blank
A. benzaldehyde, determination.

1646 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Benzylpenicillin, benzathine

1 mL of 0.5 M alcoholic potassium hydroxide is equivalent to Results : the 2 principal spots in the chromatogram obtained
106.1 mg of C14H12O2. with the test solution are similar in position, colour and
size to the 2 principal spots in the chromatogram obtained
STORAGE with the reference solution.
In an airtight, well-filled container, protected from light. C. Place about 2 mg in a test-tube about 150 mm long and
15 mm in diameter. Moisten with 0.05 mL of water R and
add 2 mL of sulfuric acid-formaldehyde reagent R. Mix the
01/2008:0373 contents of the tube by swirling ; the solution is practically
corrected 6.0 colourless. Place the test-tube on a water-bath for 1 min ; a
reddish-brown colour develops.
BENZYLPENICILLIN, BENZATHINE D. To 0.1 g add 2 mL of 1 M sodium hydroxide and shake
for 2 min. Shake the mixture with 2 quantities, each of
Benzylpenicillinum benzathinum 3 mL, of ether R. Evaporate the combined ether layers to
dryness and dissolve the residue in 1 mL of ethanol (50 per
cent V/V) R. Add 5 mL of picric acid solution R, heat at
90 °C for 5 min and allow to cool slowly. Separate the
crystals and recrystallise from ethanol (25 per cent V/V) R
containing 10 g/L of picric acid R. The crystals melt (2.2.14)
at about 214 °C.
TESTS
C48H56N6O8S2 Mr 909 Acidity or alkalinity. To 0.50 g add 100 mL of carbon
[1538-09-6] dioxide-free water R and shake for 5 min. Filter through a
sintered-glass filter (2.1.2). To 20 mL of the filtrate add 0.1 mL
DEFINITION of bromothymol blue solution R1. The solution is green or
N,N′-Dibenzylethane-1,2-diamine compound (1:2) with yellow. Not more than 0.2 mL of 0.02 M sodium hydroxide is
(2S,5R,6R)-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4- required to change the colour of the indicator to blue.
thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid. Related substances. Liquid chromatography (2.2.29). Prepare
Substance produced by the growth of certain strains of the solutions immediately before use, using sonication (for
Penicillium notatum or related organisms, or obtained by any about 2 min) to dissolve the samples. Avoid any overheating
other means. during the sample preparation.
Content : Test solution. Dissolve 70.0 mg of the substance to be
– benzathine benzylpenicillin : 96.0 per cent to 102.0 per cent examined in 25 mL of methanol R and dilute to 50.0 mL
(anhydrous substance) ; with a solution containing 6.8 g/L of potassium dihydrogen
– N,N′-dibenzylethylenediamine (benzathine C16H20N2 ; phosphate R and 1.02 g/L of disodium hydrogen phosphate R.
Mr 240.3) : 24.0 per cent to 27.0 per cent (anhydrous Reference solution (a). Dissolve 70.0 mg of benzathine
substance). benzylpenicillin CRS in 25 mL of methanol R and dilute to
It contains a variable quantity of water. Dispersing or 50.0 mL with a solution containing 6.8 g/L of potassium
suspending agents may be added. dihydrogen phosphate R and 1.02 g/L of disodium hydrogen
phosphate R.
CHARACTERS Reference solution (b). Dilute 1.0 mL of reference solution (a)
Appearance : white or almost white powder. to 100.0 mL with mobile phase A.
Solubility : very slightly soluble in water, freely soluble in Column :
dimethylformamide and in formamide, slightly soluble in – size : l = 0.25 m, Ø = 4.0 mm ;
ethanol (96 per cent).
– stationary phase : end-capped octadecylsilyl silica gel for
IDENTIFICATION chromatography R (5 μm) ;
First identification : A. – temperature : 40 °C.
Second identification : B, C, D. Mobile phase :
A. Infrared absorption spectrophotometry (2.2.24). – mobile phase A : mix 10 volumes of a 34 g/L solution of
Comparison : benzathine benzylpenicillin CRS. potassium dihydrogen phosphate R adjusted to pH 3.5
B. Thin-layer chromatography (2.2.27). with phosphoric acid R, 30 volumes of methanol R and
60 volumes of water R ;
Test solution. Dissolve 25 mg of the substance to be
examined in 5 mL of methanol R. – mobile phase B : mix 10 volumes of a 34 g/L solution of
potassium dihydrogen phosphate R adjusted to pH 3.5 with
Reference solution. Dissolve 25 mg of benzathine phosphoric acid R, 30 volumes of water R and 60 volumes
benzylpenicillin CRS in 5 mL of methanol R. of methanol R ;
Plate : TLC silanised silica gel plate R.
Time Mobile phase A Mobile phase B
Mobile phase : mix 30 volumes of acetone R and 70 volumes (min) (per cent V/V) (per cent V/V)
of a 154 g/L solution of ammonium acetate R adjusted to 0 - 10 75 25
pH 7.0 with ammonia R.
Application : 1 μL. 10 - 20 75 → 0 25 → 100

Development : over a path of 15 cm. 20 - 55 0 100

Drying : in air. 55 - 70 75 25
Detection : expose to iodine vapour until the spots appear
and examine in daylight. Flow rate : 1 mL/min.
System suitability: reference solution : Detection : spectrophotometer at 220 nm.
– the chromatogram shows 2 clearly separated spots. Injection : 20 μL.

General Notices (1) apply to all monographs and other texts 1647
Benzylpenicillin potassium EUROPEAN PHARMACOPOEIA 8.0

System suitability: reference solution (a) :

– relative retention with reference to benzylpenicillin :
benzathine = 0.3 to 0.4 ; impurity C = about 2.4 ; if necessary,
adjust the concentration of methanol in the mobile phase.
Limits :
– impurity C : not more than twice the sum of the areas of
the 2 principal peaks in the chromatogram obtained with
reference solution (b) (2 per cent) ;
C. benzylpenicilloic acids benzathide,
– any other impurity : for each impurity, not more than
the sum of the areas of the 2 principal peaks in the
chromatogram obtained with reference solution (b) (1 per
cent) ;
– disregard limit : 0.05 times the sum of the areas of the
2 principal peaks in the chromatogram obtained with
reference solution (b) (0.05 per cent).
D. (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7a-
Water (2.5.12) : 5.0 per cent to 8.0 per cent, determined on
tetrahydroimidazo[5,1-b]thiazole-3,7-dicarboxylic
0.300 g.
acid (penillic acid of benzylpenicillin),
Bacterial endotoxins (2.6.14, Method E) : less than
0.13 IU/mL, if intended for use in the manufacture of
parenteral preparations without a further appropriate
procedure for the removal of bacterial endotoxins.
Suspend 20 mg in 20 mL of a solution of 0.1 M sodium
hydroxide diluted 1 to 100, shake thoroughly and centrifuge.
Examine the supernatant. E. (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5-
dimethylthiazolidine-4-carboxylic acid (penicilloic acids of
ASSAY benzylpenicillin),
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications.
Mobile phase : phosphate buffer solution pH 3.5 R, methanol R,
water R (10:35:55 V/V/V).
Injection : test solution and reference solution (a).
Calculate the percentage contents of benzathine and F. (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5-
benzathine benzylpenicillin. Calculate the percentage content dimethylthiazolidine-4-carboxylic acid (penilloic acids of
of benzathine benzylpenicillin by multiplying the percentage benzylpenicillin).
content of benzylpenicillin by 1.36.
01/2008:0113
STORAGE corrected 6.0
In an airtight container. If the substance is sterile, store in a
sterile, airtight, tamper-proof container.
BENZYLPENICILLIN POTASSIUM
Benzylpenicillinum kalicum
IMPURITIES
Specified impurities : C.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use C16H17KN2O4S Mr 372.5
(2034). It is therefore not necessary to identify these impurities [113-98-4]
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : A, B, D, E, F. DEFINITION
Potassium (2S,5R,6R)-3,3-dimethyl-7-oxo-6-[(phenylacetyl)-
amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate.
Substance produced by the growth of certain strains of
Penicillium notatum or related organisms, or obtained by any
other means.
Content : 96.0 per cent to 102.0 per cent (dried substance).
CHARACTERS
A. monobenzylethylenediamine,
Appearance : white or almost white, crystalline powder.
Solubility : very soluble in water, practically insoluble in fatty
oils and in liquid paraffin.
IDENTIFICATION
First identification : A, D.
B. phenylacetic acid, Second identification : B, C, D.

1648 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Benzylpenicillin potassium

A. Infrared absorption spectrophotometry (2.2.24). – stationary phase : octadecylsilyl silica gel for
Comparison : benzylpenicillin potassium CRS. chromatography R (5 μm).
B. Thin-layer chromatography (2.2.27). Mobile phase :
Test solution. Dissolve 25 mg of the substance to be – mobile phase A : mix 10 volumes of a 68 g/L solution of
examined in 5 mL of water R. potassium dihydrogen phosphate R adjusted to pH 3.5 with
Reference solution (a). Dissolve 25 mg of benzylpenicillin a 500 g/L solution of dilute phosphoric acid R, 30 volumes
potassium CRS in 5 mL of water R. of methanol R and 60 volumes of water R ;
Reference solution (b). Dissolve 25 mg of benzylpenicillin – mobile phase B : mix 10 volumes of a 68 g/L solution of
potassium CRS and 25 mg of phenoxymethylpenicillin potassium dihydrogen phosphate R adjusted to pH 3.5 with
potassium CRS in 5 mL of water R. a 500 g/L solution of dilute phosphoric acid R, 40 volumes
of water R and 50 volumes of methanol R ;
Plate : TLC silanised silica gel plate R.
Mobile phase : mix 30 volumes of acetone R and 70 volumes Time Mobile phase A Mobile phase B
of a 154 g/L solution of ammonium acetate R previously (min) (per cent V/V) (per cent V/V)
adjusted to pH 5.0 with glacial acetic acid R. 0 - tR 70 30
Application : 1 μL. tR - (tR + 20) 70 → 0 30 → 100
Development : over a path of 15 cm. (tR + 20) - (tR + 35) 0 100
Drying : in air.
(tR + 35) - (tR + 50) 70 30
Detection : expose to iodine vapour until the spots appear
and examine in daylight. tR = retention time of benzylpenicillin determined with reference
System suitability: reference solution (b) : solution (c)
– the chromatogram shows 2 clearly separated spots. If the mobile phase composition has been adjusted to achieve
Results : the principal spot in the chromatogram obtained the required resolution, the adjusted composition will apply at
with the test solution is similar in position, colour and size time zero in the gradient and in the assay.
to the principal spot in obtained with reference solution (a). Flow rate : 1.0 mL/min.
C. Place about 2 mg in a test-tube about 150 mm long and Detection : spectrophotometer at 225 nm.
15 mm in diameter. Moisten with 0.05 mL of water R and Injection : 20 μL of reference solutions (b) and (c) with isocratic
add 2 mL of sulfuric acid-formaldehyde reagent R. Mix the elution at the initial mobile phase composition and 20 μL of
contents of the tube by swirling ; the solution is practically test solution (b) according to the elution gradient described
colourless. Place the test-tube on a water-bath for 1 min ; a under Mobile phase ; inject water R as a blank according to the
reddish-brown colour develops. elution gradient described under Mobile phase.
D. It gives reaction (a) of potassium (2.3.1). System suitability : reference solution (b) :
TESTS – resolution : minimum 6.0 between the peaks due to
impurity B and benzylpenicillin ; if necessary, adjust the
pH (2.2.3) : 5.5 to 7.5. ratio A:B of the mobile phase.
Dissolve 2.0 g in carbon dioxide-free water R and dilute to Limit :
20 mL with the same solvent.
– any impurity : for each impurity, not more than the area
Specific optical rotation (2.2.7) : + 270 to + 300 (dried of the principal peak in the chromatogram obtained with
substance). reference solution (c) (1 per cent).
Dissolve 0.500 g in carbon dioxide-free water R and dilute to Loss on drying (2.2.32) : maximum 1.0 per cent, determined
25.0 mL with the same solvent. on 1.000 g by drying in an oven at 105 °C.
Absorbance (2.2.25). Dissolve 94.0 mg in water R and dilute Bacterial endotoxins (2.6.14, Method E) : less than
to 50.0 mL with the same solvent. Measure the absorbance 0.16 IU/mg, if intended for use in the manufacture of
of the solution at 325 nm, 280 nm and at the absorption parenteral preparations without a further appropriate
maximum at 264 nm, diluting the solution, if necessary, for procedure for the removal of bacterial endotoxins.
the measurement at 264 nm. The absorbances at 325 nm
and 280 nm do not exceed 0.10 and that at the absorption ASSAY
maximum at 264 nm is 0.80 to 0.88, calculated on the basis of Liquid chromatography (2.2.29) as described in the test for
the undiluted (1.88 g/L) solution. Verify the resolution of the related substances with the following modifications.
apparatus (2.2.25) ; the ratio of the absorbances is at least 1.7. Mobile phase : initial composition of the mixture of mobile
Related substances. Liquid chromatography (2.2.29). Prepare phases A and B, adjusted where applicable.
the solutions immediately before use. Injection : test solution (a) and reference solution (a).
Test solution (a). Dissolve 50.0 mg of the substance to be Calculate the percentage content of C16H17KN2O4S by
examined in water R and dilute to 50.0 mL with the same multiplying the percentage content of benzylpenicillin sodium
solvent. by 1.045.
Test solution (b). Dissolve 80.0 mg of the substance to be
examined in water R and dilute to 20.0 mL with the same STORAGE
solvent. In an airtight container. If the substance is sterile, store in a
Reference solution (a). Dissolve 50.0 mg of benzylpenicillin sterile, airtight, tamper-proof container.
sodium CRS in water R and dilute to 50.0 mL with the same
solvent. IMPURITIES
Reference solution (b). Dissolve 10 mg of benzylpenicillin
sodium CRS and 10 mg of phenylacetic acid R (impurity B) in
water R, then dilute to 50 mL with the same solvent.
Reference solution (c). Dilute 4.0 mL of reference solution (a)
to 100.0 mL with water R. A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-
Column : 1-azabicyclo[3.2.0]heptane-2-carboxylic acid
– size : l = 0.25 m, Ø = 4.6 mm ; (6-aminopenicillanic acid),

General Notices (1) apply to all monographs and other texts 1649
Benzylpenicillin, procaine EUROPEAN PHARMACOPOEIA 8.0

Dispersing or suspending agents (for example, lecithin and

polysorbate 80) may be added.

B. phenylacetic acid, CHARACTERS

Appearance : white or almost white, crystalline powder.
Solubility : slightly soluble in water, sparingly soluble in
ethanol (96 per cent).
IDENTIFICATION
First identification : A.
C. (2S,5R,6R)-6-[[(4-hydroxyphenyl)acetyl]amino]-3,3- Second identification : B, C, D.
dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2- A. Infrared absorption spectrophotometry (2.2.24).
carboxylic acid, Comparison: procaine benzylpenicillin CRS.
B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 25 mg of the substance to be
examined in 5 mL of acetone R.
Reference solution. Dissolve 25 mg of procaine
benzylpenicillin CRS in 5 mL of acetone R.
Plate : TLC silanised silica gel plate R.
D. (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7a- Mobile phase : mix 30 volumes of acetone R and 70 volumes
tetrahydroimidazo[5,1-b]thiazole-3,7-dicarboxylic of a 154 g/L solution of ammonium acetate R previously
acid (penillic acid of benzylpenicillin), adjusted to pH 7.0 with ammonia R.
Application : 1 μL.
Development : over a path of 15 cm.
Drying : in air.
Detection : expose to iodine vapour until the spots appear
and examine in daylight.
E. (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5- System suitability : reference solution :
dimethylthiazolidine-4-carboxylic acid (penicilloic acids of – the chromatogram shows 2 clearly separated spots.
benzylpenicillin), Results : the 2 principal spots in the chromatogram obtained
with the test solution are similar in position, colour and
size to the 2 principal spots in the chromatogram obtained
with the reference solution.
C. Place about 2 mg in a test-tube about 150 mm long and
15 mm in diameter. Moisten with 0.05 mL of water R and
add 2 mL of sulfuric acid-formaldehyde reagent R. Mix the
F. (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5- contents of the tube by swirling ; the solution is practically
dimethylthiazolidine-4-carboxylic acid (penilloic acids of colourless. Place the test-tube on a water-bath for 1 min ; a
benzylpenicillin). reddish-brown colour develops.
D. Dissolve 0.1 g in 2 mL of dilute hydrochloric acid R and use
01/2008:0115 the solution which may be turbid. The solution gives the
corrected 6.0 reaction of primary aromatic amines (2.3.1).
BENZYLPENICILLIN, PROCAINE TESTS
pH (2.2.3) : 5.0 to 7.5.
Benzylpenicillinum procainum Dissolve 50 mg in carbon dioxide-free water R and dilute
to 15 mL with the same solvent. Shake until dissolution is
complete.
Specific optical rotation (2.2.7) : + 165 to + 180 (anhydrous
substance).
Dissolve 0.250 g in a mixture of 2 volumes of water R and
3 volumes of acetone R, then dilute to 25.0 mL with the same
mixture of solvents.
C29H38N4O6S,H2O Mr 588.7
[6130-64-9] Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
DEFINITION Test solution (a). Dissolve 70.0 mg of the substance to be
(2S,5R,6R)-3,3-Dimethyl-7-oxo-6-[(phenylacetyl)amino]-4- examined in the mobile phase and dilute to 50.0 mL with the
thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid compound mobile phase.
with 2-(diethylamino)ethyl 4-aminobenzoate monohydrate. Test solution (b). Dissolve 70.0 mg of the substance to be
Substance produced by the growth of certain strains of examined in the mobile phase and dilute to 100.0 mL with
Penicillium notatum or related organisms, or obtained by any the mobile phase.
other means. Reference solution (a). Dissolve 70.0 mg of procaine
Content : benzylpenicillin CRS in the mobile phase and dilute to
– procaine benzylpenicillin: 96.0 per cent to 102.0 per cent 100.0 mL with the mobile phase.
(anhydrous substance) ; Reference solution (b). Dissolve 4 mg of 4-aminobenzoic acid R
– procaine (C13H20N2O2 ; Mr 236.3) : 39.0 per cent to 42.0 per (impurity A) in reference solution (a) and dilute to 25 mL with
cent (anhydrous substance). reference solution (a).

1650 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Benzylpenicillin sodium

Reference solution (c). Dissolve 16.8 mg of 4-aminobenzoic

acid R (impurity A) in water R and dilute to 50.0 mL with
the same solvent. Dilute 1.0 mL of the solution to 10.0 mL
with water R. To 1.0 mL of this solution, add 1.0 mL of test
solution (a) and dilute to 100.0 mL with the mobile phase.
Column :
C. (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5-
– size : l = 0.25 m, Ø = 4.6 mm ; dimethylthiazolidine-4-carboxylic acid (penilloic acids of
– stationary phase : octadecylsilyl silica gel for benzylpenicillin),
chromatography R (5 μm).
Mobile phase : mix 250 mL of acetonitrile R1, 250 mL of water R
and 500 mL of a solution containing 14 g/L of potassium
dihydrogen phosphate R and 6.5 g/L of tetrabutylammonium
hydroxide solution (400 g/L) R adjusted to pH 7.0 with 1 M
potassium hydroxide ; if necessary, adjust the mixture to pH 7.2
with dilute phosphoric acid R.
Flow rate : 1.75 mL/min. D. (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7a-
Detection : spectrophotometer at 225 nm. tetrahydroimidazo[5,1-b]thiazole-3,7-dicarboxylic
acid (penillic acid of benzylpenicillin),
Injection : 10 μL of test solution (a) and reference solutions (b)
and (c).
Run time : 1.5 time the retention time of benzylpenicillin.
Elution order : impurity A, procaine, benzylpenicillin.
E. phenylacetic acid.
System suitability : reference solution (b) :
– resolution : minimum 2.0 between the peaks due to
01/2008:0114
impurity A and procaine ; if necessary, adjust the
corrected 6.0
concentration of acetonitrile in the mobile phase.
Limits :
BENZYLPENICILLIN SODIUM
– impurity A : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (c) (0.024 per cent) ; Benzylpenicillinum natricum
– any other impurity : for each impurity, not more than
the area of the peak due to benzylpenicillin in the
chromatogram obtained with reference solution (c) (1 per
cent).
Water (2.5.12) : 2.8 per cent to 4.2 per cent, determined on
0.500 g.
C16H17N2NaO4S Mr 356.4
Bacterial endotoxins (2.6.14, Method E) : less than [69-57-8]
0.10 IU/mg, if intended for use in the manufacture of
parenteral preparations without a further appropriate DEFINITION
procedure for the removal of bacterial endotoxins. Sodium (2S,5R,6R)-3,3-dimethyl-7-oxo-6-[(phenylacetyl)-
ASSAY amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate.
Liquid chromatography (2.2.29) as described in the test for Substance produced by the growth of certain strains of
related substances with the following modifications. Penicillium notatum or related organisms, or obtained by any
other means.
Injection : test solution (b) and reference solution (a).
Content : 96.0 per cent to 102.0 per cent (dried substance).
System suitability: reference solution (a) :
– repeatability : maximum relative standard deviation of CHARACTERS
1.0 per cent for the 2 principal peaks after 6 injections. Appearance : white or almost white, crystalline powder.
Calculate the percentage contents of procaine and procaine Solubility : very soluble in water, practically insoluble in fatty
benzylpenicillin. oils and in liquid paraffin.
STORAGE IDENTIFICATION
In an airtight container. If the substance is sterile, store in a First identification : A, D.
sterile, airtight, tamper-proof container. Second identification : B, C, D.
IMPURITIES A. Infrared absorption spectrophotometry (2.2.24).
Comparison: benzylpenicillin sodium CRS.
B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 25 mg of the substance to be
examined in 5 mL of water R.
A. 4-aminobenzoic acid,
Reference solution (a). Dissolve 25 mg of benzylpenicillin
sodium CRS in 5 mL of water R.
Reference solution (b). Dissolve 25 mg of benzylpenicillin
sodium CRS and 25 mg of phenoxymethylpenicillin
potassium CRS in 5 mL of water R.
Plate : TLC silanised silica gel plate R.
B. (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5- Mobile phase : mix 30 volumes of acetone R and 70 volumes
dimethylthiazolidine-4-carboxylic acid (penicilloic acids of of a 154 g/L solution of ammonium acetate R previously
benzylpenicillin), adjusted to pH 5.0 with glacial acetic acid R.

General Notices (1) apply to all monographs and other texts 1651
Benzylpenicillin sodium EUROPEAN PHARMACOPOEIA 8.0

Application : 1 μL. – mobile phase B : mix 10 volumes of a 68 g/L solution of

Development : over a path of 15 cm. potassium dihydrogen phosphate R adjusted to pH 3.5 with
a 500 g/L solution of dilute phosphoric acid R, 40 volumes
Drying : in air. of water R and 50 volumes of methanol R ;
Detection : expose to iodine vapour until the spots appear Time Mobile phase A Mobile phase B
and examine in daylight. (min) (per cent V/V) (per cent V/V)
System suitability: reference solution (b) : 0 - tR 70 30

– the chromatogram shows 2 clearly separated spots. tR - (tR + 20) 70 → 0 30 → 100

Results : the principal spot in the chromatogram obtained (tR + 20) - (tR + 35) 0 100
with the test solution is similar in position, colour and size (tR + 35) - (tR + 50) 70 30
to the principal spot in the chromatogram obtained with
reference solution (a). tR = retention time of benzylpenicillin determined with reference
solution (c)
C. Place about 2 mg in a test-tube about 150 mm long and
15 mm in diameter. Moisten with 0.05 mL of water R and If the mobile phase composition has been adjusted to achieve
add 2 mL of sulfuric acid-formaldehyde reagent R. Mix the the required resolution, the adjusted composition will apply at
contents of the tube by swirling ; the solution is practically time zero in the gradient and in the assay.
colourless. Place the test-tube on a water-bath for 1 min ; a Flow rate : 1.0 mL/min.
reddish-brown colour develops. Detection : spectrophotometer at 225 nm.
D. It gives reaction (a) of sodium (2.3.1). Injection : 20 μL of reference solutions (b) and (c) with isocratic
elution at the initial mobile phase composition and 20 μL of
TESTS test solution (b) according to the elution gradient described
pH (2.2.3) : 5.5 to 7.5. under Mobile phase ; inject water R as a blank according to the
elution gradient described under Mobile phase.
Dissolve 2.0 g in carbon dioxide-free water R and dilute to System suitability : reference solution (b) :
20 mL with the same solvent.
– resolution : minimum 6.0 between the peaks due to
Specific optical rotation (2.2.7) : + 285 to + 310 (dried impurity B and benzylpenicillin ; if necessary, adjust the
substance). ratio A:B of the mobile phase.
Dissolve 0.500 g in carbon dioxide-free water R and dilute to Limit :
25.0 mL with the same solvent. – any impurity : for each impurity, not more than the area
Absorbance (2.2.25). Dissolve 90.0 mg in water R and dilute of the principal peak in the chromatogram obtained with
to 50.0 mL with the same solvent. Measure the absorbance reference solution (c) (1 per cent).
of the solution at 325 nm, at 280 nm and at the absorption 2-Ethylhexanoic acid (2.4.28) : maximum 0.5 per cent m/m.
maximum at 264 nm, diluting the solution, if necessary, for
the measurement at 264 nm. The absorbances at 325 nm and Loss on drying (2.2.32) : maximum 1.0 per cent, determined
280 nm are not greater than 0.10 and the absorbance at the on 1.000 g by drying in an oven at 105 °C.
absorption maximum at 264 nm is 0.80 to 0.88, calculated Bacterial endotoxins (2.6.14, Method E) : less than
on the basis of the undiluted (1.80 g/L) solution. Verify 0.16 IU/mg, if intended for use in the manufacture of
the resolution of the apparatus (2.2.25) ; the ratio of the parenteral preparations without a further appropriate
absorbances is at least 1.7. procedure for the removal of bacterial endotoxins.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use. ASSAY
Liquid chromatography (2.2.29) as described in the test for
Test solution (a). Dissolve 50.0 mg of the substance to be related substances with the following modifications.
examined in water R and dilute to 50.0 mL with the same
solvent. Mobile phase : initial composition of the mixture of mobile
phases A and B, adjusted where applicable.
Test solution (b). Dissolve 80.0 mg of the substance to be
Injection : test solution (a) and reference solution (a).
examined in water R and dilute to 20.0 mL with the same
solvent. Calculate the percentage content of C16H17N2NaO4S from the
declared content of benzylpenicillin sodium CRS.
Reference solution (a). Dissolve 50.0 mg of benzylpenicillin
sodium CRS in water R and dilute to 50.0 mL with the same STORAGE
solvent. In an airtight container. If the substance is sterile, store in a
Reference solution (b). Dissolve 10 mg of benzylpenicillin sterile, airtight, tamper-proof container.
sodium CRS and 10 mg of phenylacetic acid R (impurity B) in
water R, then dilute to 50 mL with the same solvent. IMPURITIES
Reference solution (c). Dilute 4.0 mL of reference solution (a)
to 100.0 mL with water R.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-
chromatography R (5 μm). 1-azabicyclo[3.2.0]heptane-2-carboxylic acid
(6-aminopenicillanic acid),
Mobile phase :
– mobile phase A : mix 10 volumes of a 68 g/L solution of
potassium dihydrogen phosphate R adjusted to pH 3.5 with
a 500 g/L solution of dilute phosphoric acid R, 30 volumes
of methanol R and 60 volumes of water R ; B. phenylacetic acid,

1652 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Betadex

Carry out all operations as rapidly as possible avoiding exposure

to actinic light ; use freshly prepared solutions.
IDENTIFICATION
Ultraviolet and visible absorption spectrophotometry (2.2.25).
Test solution (a). Dissolve 50.0 mg in 10 mL of chloroform R
C. (2S,5R,6R)-6-[[(4-hydroxyphenyl)acetyl]amino]-3,3- and dilute immediately to 100.0 mL with cyclohexane R. Dilute
dimethyl-7-oxo-4-thia- 1-azabicyclo[3.2.0]heptane-2- 5.0 mL of this solution to 100.0 mL with cyclohexane R.
carboxylic acid, Test solution (b). Dilute 5.0 mL of test solution (a) to 50.0 mL
with cyclohexane R.
Absorption maximum : at 455 nm for test solution (b).
Absorbance ratio : A455 / A483 = 1.14 to 1.18 for test solution (b).
TESTS
Related substances. Determine the absorbance (2.2.25) of
test solutions (b) and (a) used in Identification, at 455 nm and
D. (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7a- at 340 nm respectively.
tetrahydroimidazo[5,1-b]thiazole-3,7-dicarboxylic
acid (penillic acid of benzylpenicillin), Absorbance ratio : A455 / A340 : minimum 1.5.
The thresholds indicated under Related substances
(Table 2034.-1) in the general monograph Substances for
pharmaceutical use (2034) do not apply.
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test D. Prepare the reference solution
using 2 mL of lead standard solution (10 ppm Pb) R.
E. (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5- Loss on drying (2.2.32) : maximum 0.2 per cent, determined
dimethylthiazolidine-4-carboxylic acid (penicilloic acids of on 1.000 g by drying in vacuo over diphosphorus pentoxide R
benzylpenicillin), at 40 °C for 4 h.
Sulfated ash (2.4.14) : maximum 0.2 per cent, determined
on 1.0 g, moistened with a mixture of 2 mL of dilute sulfuric
acid R and 5 mL of ethanol (96 per cent) R.
ASSAY
Measure the absorbance (2.2.25) of test solution (b) used in
F. (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5- Identification at the absorption maximum at 455 nm, using
dimethylthiazolidine-4-carboxylic acid (penilloic acids of cyclohexane R as the compensation liquid.
benzylpenicillin). Calculate the content of C40H56 taking the specific absorbance
to be 2500.

01/2008:1069 STORAGE
In an airtight container, protected from light, at a temperature
BETACAROTENE not exceeding 25 °C.

01/2008:1070
Betacarotenum corrected 7.0

BETADEX
Betadexum

C40H56 Mr 536.9
[7235-40-7]
DEFINITION
(all-E)-3,7,12,16-Tetramethyl-1,18-bis(2,6,6-trimethylcyclo-
hex-1-enyl)octadeca-1,3,5,7,9,11,13,15,17-nonaene.
Content : 96.0 per cent to 101.0 per cent (dried substance). [C6H10O5]7 Mr 1135
CHARACTERS [7585-39-9]
Appearance : brown-red or brownish-red, crystalline powder. DEFINITION
Solubility : practically insoluble in water, slightly soluble in Cycloheptakis-(1→4)-(α-D-glucopyranosyl) (cyclomaltohep-
cyclohexane, practically insoluble in anhydrous ethanol. taose or β-cyclodextrin).
It is sensitive to air, heat and light, especially in solution. Content : 98.0 per cent to 101.0 per cent (dried substance).

General Notices (1) apply to all monographs and other texts 1653
Betadex EUROPEAN PHARMACOPOEIA 8.0

CHARACTERS Injection : 50 μL of test solution (a) and reference solutions (a)

Appearance : white or almost white, amorphous or crystalline and (b).
powder. Run time : 1.5 times the retention time of betadex.
Solubility : sparingly soluble in water, freely soluble in Relative retention with reference to betadex (retention
propylene glycol, practically insoluble in anhydrous ethanol time = about 10 min) : impurity B = about 0.3 ;
and in methylene chloride. impurity A = about 0.45.
IDENTIFICATION System suitability : reference solution (a) :
A. Specific optical rotation (see Tests). – resolution : minimum 1.5 between the peaks due to
impurities B and A ; if necessary, adjust the concentration
B. Examine the chromatograms obtained in the assay. of methanol in the mobile phase.
Results : the principal peak in the chromatogram obtained Limits :
with test solution (b) is similar in retention time and size
to the principal peak in the chromatogram obtained with – impurities A, B : for each impurity, not more than 0.5 times
reference solution (c). the area of the corresponding peak in the chromatogram
obtained with reference solution (b) (0.25 per cent) ;
C. Dissolve 0.2 g in 2 mL of iodine solution R4 by warming on
a water-bath, and allow to stand at room temperature. A – sum of impurities other than A and B : not more than
yellowish-brown precipitate is formed. 0.5 times the area of the peak due to betadex in the
chromatogram obtained with reference solution (b) (0.5 per
TESTS cent).
Solution S. Dissolve 1.000 g in carbon dioxide-free water R Residual solvents. Head-space gas chromatography (2.2.28) :
with heating, allow to cool and dilute to 100.0 mL with the use the standard additions method.
same solvent. Internal standard : ethylene chloride R.
Appearance of solution. Solution S is clear (2.2.1). Test solutions. In each of 4 identical 20 mL flasks, dissolve
pH (2.2.3) : 5.0 to 8.0. 0.5 g of the substance to be examined in water R and add
0.10 g of calcium chloride R and 30 μL of α-amylase solution R.
To 10 mL of solution S add 0.1 mL of a saturated solution of Add 1 mL of reference solutions (a), (b), (c) and (d), adding a
potassium chloride R. different solution to each flask. Dilute to 10 mL with water R.
Specific optical rotation (2.2.7) : + 160 to + 164 (dried Reference solutions. Prepare a 10 μL/L solution of ethylene
substance), determined on solution S. chloride R (reference solution (a)). Prepare reference
Reducing sugars : maximum 0.2 per cent. solutions (b), (c) and (d) from reference solution (a) to
Test solution. To 1 mL of solution S add 1 mL of cupri-tartaric contain respectively, per litre, 5 μL, 10 μL and 15 μL of both
solution R4. Heat on a water-bath for 10 min, cool to room trichloroethylene R and toluene R.
temperature. Add 10 mL of ammonium molybdate reagent R1 Column :
and allow to stand for 15 min. – material : fused silica ;
Reference solution. Prepare a reference solution at the same – size : l = 25 m, Ø = 0.32 mm ;
time and in the same manner as the test solution, using 1 mL – stationary phase : macrogol 20 000 R (film thickness 1 μm).
of a 0.02 g/L solution of glucose R.
Carrier gas : helium for chromatography R.
Measure the absorbance (2.2.25) of the test solution and the
reference solution at the absorption maximum at 740 nm using Static head-space conditions which may be used :
water R as the compensation liquid. The absorbance of the – equilibration temperature : 45 °C ;
test solution is not greater than that of the reference solution. – equilibration time : 2 h.
Light-absorbing impurities. Examine solution S between Temperature :
230 nm and 750 nm. Between 230 nm and 350 nm, the – column : 50 °C ;
absorbance (2.2.25) is not greater than 0.10. Between 350 nm
and 750 nm, the absorbance (2.2.25) is not greater than 0.05. – injection port : 140 °C ;
– detector : 280 °C.
Related substances. Liquid chromatography (2.2.29).
Detection : flame ionisation.
Test solution (a). Dissolve 0.25 g of the substance to be
examined in water R with heating, cool and dilute to 25.0 mL Injection : 200 μL of the head space, at least 3 times.
with the same solvent. Retention time : toluene = about 10 min.
Test solution (b). Dilute 5.0 mL of test solution (a) to 50.0 mL System suitability :
with water R. – resolution : minimum 1.1 between the peaks due to
Reference solution (a). Dissolve 25.0 mg of alfadex CRS trichloroethylene and toluene ; minimum 1.1 between the
(impurity A), 25.0 mg of gammacyclodextrin CRS (impurity B) peaks due to toluene and ethylene chloride ;
and 50.0 mg of betadex CRS in water R, then dilute to 50.0 mL – repeatability : maximum relative standard deviations of the
with the same solvent. ratios of the areas of the peaks due to trichloroethylene
Reference solution (b). Dilute 5.0 mL of reference solution (a) and toluene to that of the peak due to ethylene chloride
to 50.0 mL with water R. of 5 per cent.
Reference solution (c). Dissolve 25.0 mg of betadex CRS in Calculate the content of trichloroethylene and of toluene
water R and dilute to 25.0 mL with the same solvent. taking their relative densities to be 1.46 and 0.87, respectively.
Column : Limits :
– size : l = 0.25 m, Ø = 4.6 mm ; – trichloroethylene : maximum 10 ppm ;
– stationary phase : octadecylsilyl silica gel for – toluene : maximum 10 ppm.
chromatography R (10 μm). Heavy metals (2.4.8) : maximum 10 ppm.
Mobile phase : methanol R, water R (10:90 V/V). 1.0 g complies with test C. Prepare the reference solution using
Flow rate : 1.5 mL/min. 1 mL of lead standard solution (10 ppm Pb) R.
Detection : differential refractometer. Loss on drying (2.2.32) : maximum 16.0 per cent, determined
Equilibration : with the mobile phase for about 3 h. on 1.000 g by drying in an oven at 120 °C for 2 h.

1654 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Betahistine dihydrochloride

Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on CHARACTERS

1.0 g. Appearance : white or slightly yellow powder, very hygroscopic.
ASSAY Solubility : very soluble in water, soluble in ethanol (96 per
cent), practically insoluble in 2-propanol.
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications. IDENTIFICATION
Injection : test solution (b) and reference solutions (a) and (c). First identification : B, D.
System suitability: reference solution (a) : Second identification : A, C, D.
– repeatability : maximum relative standard deviation of the A. Melting point (2.2.14) : 150 °C to 154 °C.
area of the peak due to betadex of 2.0 per cent.
B. Infrared absorption spectrophotometry (2.2.24).
Calculate the percentage content of [C6H10O5]7 from the
Comparison: betahistine dihydrochloride CRS.
declared content of betadex CRS.
C. Thin-layer chromatography (2.2.27).
STORAGE Test solution. Dissolve 10 mg of the substance to be
In an airtight container. examined in 2 mL of ethanol (96 per cent) R.
Reference solution. Dissolve 10 mg of betahistine
IMPURITIES
dihydrochloride CRS in 2 mL of ethanol (96 per cent) R.
Specified impurities : A, B.
Plate : TLC silica gel GF254 plate R.
Mobile phase : concentrated ammonia R, ethyl acetate R,
methanol R (0.75:15:30 V/V/V).
Application : 2 μL.
Development : over 2/3 of the plate.
Drying : at 110 °C for 10 min.
Detection : examine in ultraviolet light at 254 nm.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the
reference solution.
D. It gives reaction (a) of chlorides (2.3.1).
A. cyclohexakis-(1→4)-(α-D-glucopyranosyl) (alfadex or TESTS
cyclomaltohexaose or α-cyclodextrin), Solution S. Dissolve 5.0 g in carbon dioxide-free water R, and
dilute to 50 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution B8 (2.2.2,
Method II).
pH (2.2.3) : 2.0 to 3.0 for solution S.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 25 mg of the substance to be examined
in the mobile phase and dilute to 25.0 mL with the mobile
phase.
Reference solution (a). Dissolve 10 mg of betahistine
dihydrochloride CRS and 10 mg of 2-vinylpyridine R in the
mobile phase and dilute to 50.0 mL with the mobile phase.
Dilute 2.0 mL of the solution to 50.0 mL with the mobile phase.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase.
B. cyclooctakis-(1→4)-(α-D-glucopyranosyl) Reference solution (c). Dilute 2.0 mL of reference solution (b)
(cyclomaltooctaose or γ-cyclodextrin).
to 10.0 mL with the mobile phase.
Column :
01/2008:1665 – size : l = 0.15 m, Ø = 3.0 mm;
corrected 6.0 – stationary phase : base-deactivated end-capped octadecylsilyl
silica gel for chromatography R (5 μm).
BETAHISTINE DIHYDROCHLORIDE Mobile phase : dissolve 2.0 g of sodium dodecyl sulfate R in a
mixture of 15 mL of a 10 per cent V/V solution of sulfuric
Betahistini dihydrochloridum acid R, 35 mL of a 17 g/L solution of tetrabutylammonium
hydrogen sulfate R and 650 mL of water R ; adjust to pH 3.3
using dilute sodium hydroxide solution R and mix with 300 mL
of acetonitrile R.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 260 nm.
C8H14Cl2N2 Mr 209.1
[5579-84-0] Injection : 20 μL.
Run time : 4 times the retention time of betahistine.
DEFINITION Relative retention with reference to betahistine
N-Methyl-2-(pyridin-2-yl)ethanamine dihydrochloride. (retention time = about 7 min) : impurity B = about 0.2 ;
Content : 99.0 per cent to 101.0 per cent (dried substance). impurity A = about 0.3 ; impurity C = about 3.

General Notices (1) apply to all monographs and other texts 1655
Betahistine mesilate EUROPEAN PHARMACOPOEIA 8.0

System suitability: reference solution (a) : Content : 98.0 per cent to 101.0 per cent (anhydrous substance).
– resolution : minimum 3.5 between the peaks due to PRODUCTION
2-vinylpyridine and betahistine.
It is considered that alkylsulfonate esters are genotoxic
Limits : and are potential impurities in betahistine mesilate. The
– correction factor : for the calculation of content, multiply manufacturing process should be developed taking into
the peak area of impurity B by 0.4 ; consideration the principles of quality risk management,
– impurities A, B, C : for each impurity, not more than the together with considerations of the quality of starting
area of the principal peak in the chromatogram obtained materials, process capability and validation. The general
with reference solution (c) (0.2 per cent) ; methods 2.5.37. Methyl, ethyl and isopropyl methanesulfonate
– unspecified impurities : for each impurity, not more in methanesulfonic acid, 2.5.38. Methyl, ethyl and
than 0.5 times of the area of the principal peak in the isopropyl methanesulfonate in active substances and 2.5.39.
chromatogram obtained with reference solution (c) Methanesulfonyl chloride in methanesulfonic acid are available
(0.10 per cent) ; to assist manufacturers.
– total : not more than 0.5 times the area of the principal peak CHARACTERS
in the chromatogram obtained with reference solution (b) Appearance : white or almost white, crystalline powder, very
(0.5 per cent) ; hygroscopic.
– disregard limit : 0.25 times the area of the principal peak Solubility : very soluble in water, freely soluble in ethanol
in the chromatogram obtained with reference solution (c) (96 per cent), very slightly soluble in 2-propanol.
(0.05 per cent).
Loss on drying (2.2.32) : maximum 1.0 per cent, determined IDENTIFICATION
on 1.000 g by drying in an oven at 105 °C. First identification : B.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Second identification : A, C, D.
1.0 g. A. Melting point (2.2.14) : 108 °C to 112 °C.
B. Infrared absorption spectrophotometry (2.2.24).
ASSAY
Comparison: betahistine mesilate CRS.
Dissolve 80.0 mg in 50 mL of ethanol (96 per cent) R. Titrate
C. Thin-layer chromatography (2.2.27).
with 0.1 M sodium hydroxide, determining the end-point
potentiometrically (2.2.20). Read the volume added to reach Test solution. Dissolve 10 mg of the substance to be
the second point of inflexion. examined in ethanol (96 per cent) R and dilute to 2 mL
with the same solvent.
1 mL of 0.1 M sodium hydroxide is equivalent to 10.46 mg
of C8H14Cl2N2. Reference solution. Dissolve 10 mg of betahistine
mesilate CRS in ethanol (96 per cent) R and dilute to 2 mL
STORAGE with the same solvent.
In an airtight container. Plate : TLC silica gel F254 plate R.
Mobile phase : concentrated ammonia R, ethyl acetate R,
IMPURITIES methanol R (0.75:15:30 V/V/V).
Specified impurities : A, B, C. Application : 2 μL.
Development : over 3/4 of the plate.
Drying : at 110 °C for 10 min.
Detection : examine in ultraviolet light at 254 nm.
A. 2-ethenylpyridine (2-vinylpyridine), Results : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the
reference solution.
D. To 0.1 g add 5 mL of dilute hydrochloric acid R and shake
B. 2-(pyridin-2-yl)ethanol, for about 5 min. Add 1 mL of barium chloride solution R1.
The solution remains clear. To a further 0.1 g add 0.5 g
of anhydrous sodium carbonate R, mix and ignite until a
white residue is obtained. Allow to cool and dissolve the
residue in 7 mL of water R. The solution gives reaction (a)
of sulfates (2.3.1).
C. N-methyl-2-(pyridin-2-yl)-N-[2-(pyridin-2-
yl)ethyl]ethanamine. TESTS
Solution S. Dissolve 5.0 g in carbon dioxide-free water R
07/2013:1071 prepared from distilled water R, and dilute to 50 mL with the
same solvent.
BETAHISTINE MESILATE Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Betahistini mesilas pH (2.2.3) : 2.0 to 3.0 for solution S.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50 mg of the substance to be examined
in the mobile phase and dilute to 10.0 mL with the mobile
phase.
C10H20N2O6S2 Mr 328.4 Reference solution (a). Dissolve 10 mg of betahistine
[54856-23-4] mesilate CRS and 10 mg of 2-vinylpyridine R (impurity A) in
the mobile phase and dilute to 50.0 mL with the mobile phase.
DEFINITION Dilute 2.0 mL of this solution to 50.0 mL with the mobile
N-Methyl-2-(pyridin-2-yl)ethanamine bis(methanesulfonate). phase.

1656 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Betamethasone

Reference solution (b). Dilute 1.0 mL of the test solution to 01/2008:0312

100.0 mL with the mobile phase. corrected 6.0
Reference solution (c). Dilute 2.0 mL of reference solution (b)
to 10.0 mL with the mobile phase. BETAMETHASONE
Column :
– size : l = 0.25 m, Ø = 4.6 mm ; Betamethasonum
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : dissolve 2.0 g of sodium dodecyl sulfate R in a
mixture of 15 volumes of a 10 per cent V/V solution of sulfuric
acid R, 35 volumes of a 17 g/L solution of tetrabutylammonium
hydrogen sulfate R and 650 volumes of water R ; adjust to
pH 3.3 using dilute sodium hydroxide solution R and mix with
300 volumes of acetonitrile R. C22H29FO5 Mr 392.5
Flow rate : 1 mL/min. [378-44-9]
Detection : spectrophotometer at 260 nm. DEFINITION
Injection : 20 μL. 9-Fluoro-11β,17,21-trihydroxy-16β-methylpregna-1,4-diene-
Run time : 3 times the retention time of betahistine mesilate. 3,20-dione.
Retention time : betahistine mesilate = about 8 min. Content : 97.0 per cent to 103.0 per cent (dried substance).
System suitability: reference solution (a) : CHARACTERS
– resolution : minimum 3.5 between the peaks due to Appearance : white or almost white, crystalline powder.
impurity A and betahistine mesilate.
Solubility : practically insoluble in water, sparingly soluble in
Limits : anhydrous ethanol, very slightly soluble in methylene chloride.
– impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (c) IDENTIFICATION
(0.2 per cent) ; First identification : B, C.
– unspecified impurities : for each impurity, not more than Second identification : A, C, D, E.
0.1 times the area of the principal peak in the chromatogram A. Dissolve 10.0 mg in anhydrous ethanol R and dilute
obtained with reference solution (b) (0.10 per cent) ; to 100.0 mL with the same solvent. Place 2.0 mL
– total : not more than 0.5 times the area of the principal peak of this solution in a stoppered tube, add 10.0 mL of
in the chromatogram obtained with reference solution (b) phenylhydrazine-sulfuric acid solution R, mix and heat in
(0.5 per cent) ; a water-bath at 60 °C for 20 min. Cool immediately. The
absorbance (2.2.25) measured at 419 nm is not greater than
– disregard limit : 0.05 times the area of the principal peak 0.10.
in the chromatogram obtained with reference solution (b)
(0.05 per cent). B. Infrared absorption spectrophotometry (2.2.24).
2-Propanol (2.4.24) : maximum 0.5 per cent. Comparison: betamethasone CRS.
If the spectra obtained in the solid state show differences,
Chlorides (2.4.4) : maximum 35 ppm.
dissolve the substance to be examined and the reference
To 14 mL of solution S add 1 mL of water R. substance separately in the minimum volume of methylene
Sulfates (2.4.13) : maximum 250 ppm. chloride R, evaporate to dryness on a water-bath and record
new spectra using the residues.
Dilute 6 mL of solution S to 15 mL with distilled water R.
C. Thin-layer chromatography (2.2.27).
Heavy metals (2.4.8) : maximum 20 ppm.
Solvent mixture : methanol R, methylene chloride R
12 mL of solution S complies with test A. Prepare the reference (1:9 V/V).
solution using lead standard solution (2 ppm Pb) R.
Test solution. Dissolve 10 mg of the substance to be
Water (2.5.12) : maximum 2.0 per cent, determined on 0.50 g. examined in the solvent mixture and dilute to 10 mL with
the solvent mixture.
ASSAY
Reference solution (a). Dissolve 20 mg of betamethasone CRS
Dissolve 0.140 g in 50 mL of a mixture of 1 volume of in the solvent mixture and dilute to 20 mL with the solvent
anhydrous acetic acid R and 7 volumes of acetic anhydride R. mixture.
Titrate with 0.1 M perchloric acid, determining the end-point
Reference solution (b). Dissolve 10 mg of
potentiometrically (2.2.20).
dexamethasone CRS in reference solution (a) and
1 mL of 0.1 M perchloric acid is equivalent to 16.42 mg dilute to 10 mL with reference solution (a).
of C10H20N2O6S2. Plate : TLC silica gel F254 plate R.
STORAGE Mobile phase : butanol R saturated with water R, toluene R,
In an airtight container. ether R (5:10:85 V/V/V).
Application : 5 μL.
IMPURITIES Development : over a path of 15 cm.
Specified impurities : A. Drying : in air.
Detection A : examine in ultraviolet light at 254 nm.
Results A : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to
the principal spot in the chromatogram obtained with
A. 2-ethenylpyridine (2-vinylpyridine). reference solution (a).

General Notices (1) apply to all monographs and other texts 1657
Betamethasone EUROPEAN PHARMACOPOEIA 8.0

Detection B : spray with alcoholic solution of sulfuric acid R. Injection : 20 μL ; inject the mixture of equal volumes of
Heat at 120 °C for 10 min or until the spots appear. Allow to acetonitrile R and methanol R as a blank.
cool. Examine in daylight and in ultraviolet light at 365 nm. Retention time : methylprednisolone = about 11.5 min ;
Results : the principal spot in the chromatogram obtained betamethasone = about 12.5 min.
with the test solution is similar in position, colour in System suitability : reference solution (a) :
daylight, fluorescence in ultraviolet light at 365 nm and
size to the principal spot in the chromatogram obtained – resolution : minimum 1.5 between the peaks due to
with reference solution (a). methylprednisolone and betamethasone ; if necessary,
adjust the concentration of acetonitrile in mobile phase A.
System suitability: reference solution (b) :
Limits :
– the chromatogram shows 2 spots which may, however,
not be completely separated. – impurities A, B, C, D, E, F, G, H, I, J : for each impurity,
not more than the area of the principal peak in the
D. Mix about 5 mg with 45 mg of heavy magnesium oxide R
chromatogram obtained with reference solution (b)
and ignite in a crucible until an almost white residue is
(1.0 per cent), and not more than 1 such peak has an area
obtained (usually less than 5 min). Allow to cool, add
greater than 0.5 times the area of the principal peak in
1 mL of water R, 0.05 mL of phenolphthalein solution R1
the chromatogram obtained with reference solution (b)
and about 1 mL of dilute hydrochloric acid R to render the
(0.5 per cent) ;
solution colourless. Filter. Add 1.0 mL of the filtrate to a
freshly prepared mixture of 0.1 mL of alizarin S solution R – total : not more than twice the area of the principal peak
and 0.1 mL of zirconyl nitrate solution R. Mix, allow to in the chromatogram obtained with reference solution (b)
stand for 5 min and compare the colour of the solution (2.0 per cent) ;
with that of a blank prepared in the same manner. The test – disregard limit : 0.05 times the area of the principal peak
solution is yellow and the blank is red. in the chromatogram obtained with reference solution (b)
E. Add about 2 mg to 2 mL of sulfuric acid R and shake to (0.05 per cent).
dissolve. Within 5 min, a deep reddish-brown colour Loss on drying (2.2.32) : maximum 0.5 per cent, determined
develops. Add this solution to 10 mL of water R and mix. on 0.500 g by drying in an oven at 105 °C.
The colour is discharged and a clear solution remains.
ASSAY
TESTS
Dissolve 0.100 g in ethanol (96 per cent) R and dilute to
Specific optical rotation (2.2.7) : + 118 to + 126 (dried 100.0 mL with the same solvent. Dilute 2.0 mL of this
substance). solution to 100.0 mL with ethanol (96 per cent) R. Measure the
Dissolve 0.125 g in methanol R and dilute to 25.0 mL with absorbance (2.2.25) at the absorption maximum at 238.5 nm.
the same solvent. Calculate the content of C22H29FO5 taking the specific
Related substances. Liquid chromatography (2.2.29). absorbance to be 395.
Test solution. Dissolve 25.0 mg of the substance to be
examined in a mixture of equal volumes of acetonitrile R and STORAGE
methanol R and dilute to 10.0 mL with the same mixture of Protected from light.
solvents.
Reference solution (a). Dissolve 2 mg of betamethasone CRS IMPURITIES
and 2 mg of methylprednisolone CRS in mobile phase A, then Specified impurities : A, B, C, D, E, F, G, H, I, J.
dilute to 100.0 mL with mobile phase A.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with mobile phase A.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm) ;
– temperature : 45 °C. A. 9-fluoro-11β,17,21-trihydroxy-16α-methylpregna-1,4-
Mobile phase : diene-3,20-dione (dexamethasone),
– mobile phase A : in a 1000 mL volumetric flask mix 250 mL
of acetonitrile R with 700 mL of water R and allow to
equilibrate ; dilute to 1000 mL with water R and mix again ;
– mobile phase B : acetonitrile R ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 15 100 0
B. 21-chloro-9-fluoro-11β,17-dihydroxy-16β-methylpregna-
15 - 40 100 → 0 0 → 100 1,4-diene-3,20-dione,
40 - 41 0 → 100 100 → 0

41 - 46 100 0

Flow rate : 2.5 mL/min.

Detection : spectrophotometer at 254 nm.
Equilibration : with mobile phase B for at least 30 min
and then with mobile phase A for 5 min. For subsequent
chromatograms, use the conditions described from 40 min C. 17,21-dihydroxy-16β-methylpregna-1,4,9(11)-triene-3,20-
to 46 min. dione,

1658 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Betamethasone acetate

01/2008:0975

BETAMETHASONE ACETATE
Betamethasoni acetas
D. 9-fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregna-
1,4-dien-21-yl ethoxycarboxylate,

C24H31FO6 Mr 434.5
[987-24-6]
DEFINITION
9-Fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregna-
E. 9,11β-epoxy-17,21-dihydroxy-16β-methyl-9β-pregna-1,4- 1,4-diene-21-yl acetate.
diene-3,20-dione, Content : 97.0 per cent to 103.0 per cent (anhydrous substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, freely soluble in
acetone, soluble in ethanol (96 per cent) and in methylene
chloride.
It shows polymorphism (5.9).
IDENTIFICATION
F. 17,21-dihydroxy-16β-methylpregna-1,4,11-triene-3,20-
dione, First identification : B, C.
Second identification : A, C, D, E, F.
A. Dissolve 10.0 mg in anhydrous ethanol R and dilute to
100.0 mL with the same solvent. Place 2.0 mL of this
solution in a ground-glass-stoppered tube, add 10.0 mL of
phenylhydrazine-sulfuric acid solution R, mix and heat in
a water-bath at 60 °C for 20 min. Cool immediately. The
absorbance (2.2.25) measured at 419 nm is not greater than
0.10.
B. Infrared absorption spectrophotometry (2.2.24).
G. 11α,17,21-trihydroxy-16β-methylpregna-1,4-diene-3,20-
dione, Comparison: betamethasone acetate CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
substance separately in the minimum volume of
methanol R, evaporate to dryness on a water-bath and
record new spectra using the residues.
C. Thin-layer chromatography (2.2.27).
Solvent mixture : methanol R, methylene chloride R
(1:9 V/V).
H. 14-fluoro-11β,17,21-trihydroxy-16β-methyl-8α,9β,14β- Test solution. Dissolve 10 mg of the substance to be
pregna-1,4-diene-3,20-dione, examined in the solvent mixture and dilute to 10 mL with
the solvent mixture.
Reference solution (a). Dissolve 20 mg of betamethasone
acetate CRS in the solvent mixture and dilute to 20 mL
with the solvent mixture.
Reference solution (b). Dissolve 10 mg of prednisolone
acetate CRS in reference solution (a) and dilute to 10 mL
with reference solution (a).
Plate : TLC silica gel F254 plate R.
I. 8-fluoro-11β,17,21-trihydroxy-16β-methyl-8α,9β-pregna- Mobile phase : add a mixture of 1.2 volumes of water R and
1,4-diene-3,20-dione, 8 volumes of methanol R to a mixture of 15 volumes of
ether R and 77 volumes of methylene chloride R.
Application : 5 μL.
Development : over a path of 15 cm.
Drying : in air.
Detection A : examine in ultraviolet light at 254 nm.
Results A : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to
the principal spot in the chromatogram obtained with
J. 17,21-dihydroxy-16β-methylpregna-1,4-diene-3,20-dione. reference solution (a).

General Notices (1) apply to all monographs and other texts 1659
Betamethasone acetate EUROPEAN PHARMACOPOEIA 8.0

Detection B : spray with alcoholic solution of sulfuric acid R. Limits :

Heat at 120 °C for 10 min or until the spots appear. Allow to – impurities A, B, C, D : for each impurity, not more than
cool. Examine in daylight and in ultraviolet light at 365 nm. 0.5 times the area of the principal peak in the chromatogram
Results B : the principal spot in the chromatogram obtained obtained with reference solution (b) (0.5 per cent) ;
with the test solution is similar in position, colour in – total : not more than 1.25 times the area of the principal
daylight, fluorescence in ultraviolet light at 365 nm and peak in the chromatogram obtained with reference
size to the principal spot in the chromatogram obtained solution (b) (1.25 per cent);
with reference solution (a).
– disregard limit : 0.05 times the area of the principal peak
System suitability: reference solution (b) : in the chromatogram obtained with reference solution (b)
– the chromatogram shows 2 clearly separated spots. (0.05 per cent).
D. Add about 2 mg to 2 mL of sulfuric acid R and shake to Water (2.5.12) : maximum 4.0 per cent, determined on 0.100 g.
dissolve. Within 5 min, a deep reddish-brown colour
develops. Add this solution to 10 mL of water R and mix. ASSAY
The colour is discharged and a clear solution remains. Dissolve 0.100 g in ethanol (96 per cent) R and dilute to
E. Mix about 5 mg with 45 mg of heavy magnesium oxide R 100.0 mL with the same solvent. Dilute 2.0 mL of this
and ignite in a crucible until an almost white residue is solution to 100.0 mL with ethanol (96 per cent) R. Measure the
obtained (usually less than 5 min). Allow to cool, add absorbance (2.2.25) at the absorption maximum at 240 nm.
1 mL of water R, 0.05 mL of phenolphthalein solution R1 Calculate the content of C24H31FO6 taking the specific
and about 1 mL of dilute hydrochloric acid R to render the absorbance to be 350.
solution colourless. Filter. To a freshly prepared mixture
of 0.1 mL of alizarin S solution R and 0.1 mL of zirconyl STORAGE
nitrate solution R, add 1.0 mL of the filtrate. Mix, allow
to stand for 5 min and compare the colour of the solution Protected from light.
with that of a blank prepared in the same manner. The test
solution is yellow and the blank is red. IMPURITIES
F. About 10 mg gives the reaction of acetyl (2.3.1). Specified impurities : A, B, C, D.

TESTS
Specific optical rotation (2.2.7) : + 120 to + 128 (anhydrous
substance).
Dissolve 0.250 g in dioxan R and dilute to 25.0 mL with the
same solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be A. 9-fluoro-11β,17,21-trihydroxy-16β-methylpregna-1,4-
examined in 4 mL of acetonitrile R and dilute to 10.0 mL with diene-3,20-dione (betamethasone),
the same solvent.
Reference solution (a). Dissolve 2 mg of betamethasone
acetate CRS and 2 mg of dexamethasone acetate CRS
(impurity B) in the mobile phase, then dilute to 100.0 mL with
the mobile phase.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase.
Column : B. 9-fluoro-11β,17-dihydroxy-16α-methyl-3,20-dioxopregna-
– size : l = 0.25 m, Ø = 4.6 mm ; 1,4-dien-21-yl acetate (dexamethasone acetate),
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : in a 1000 mL volumetric flask mix 380 mL of
acetonitrile R with 550 mL of water R and allow to equilibrate ;
dilute to 1000 mL with water R and mix again.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 254 nm.
C. 9-fluoro-17-hydroxy-16β-methyl-3,20-dioxopregna-
Equilibration : with the mobile phase for about 30 min. 1,4-diene-11β,21-diyl diacetate (betamethasone
Injection : 20 μL. 11,21-diacetate),
Run time : 2.5 times the retention time of betamethasone
acetate.
Retention time : betamethasone acetate = about 19 min ;
impurity B = about 22 min.
System suitability: reference solution (a) :
– resolution : minimum 3.3 between the peaks due to
betamethasone acetate and impurity B ; if necessary, adjust
slightly the concentration of acetonitrile in the mobile D. 9,11β-epoxy-17-hydroxy-16β-methyl-3,20-dioxo-9β-
phase. pregna-1,4-diene-21-yl acetate.

1660 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Betamethasone dipropionate

04/2012:0809 Development : over 3/4 of the plate.

Drying : in air.
BETAMETHASONE DIPROPIONATE Detection A : examine in ultraviolet light at 254 nm.
Results A : the principal spot in each of the chromatograms
Betamethasoni dipropionas obtained with the test solutions is similar in position and
size to the principal spot in the chromatogram obtained
with the corresponding reference solution.
Detection B : spray with alcoholic solution of sulfuric acid R,
heat at 120 °C for 10 min or until the spots appear, and
allow to cool ; examine in daylight and in ultraviolet light
at 365 nm.
Results B : the principal spot in each of the chromatograms
obtained with the test solutions is similar in position,
colour in daylight, fluorescence in ultraviolet light at
C28H37FO7 Mr 504.6 365 nm and size to the principal spot in the chromatogram
[5593-20-4] obtained with the corresponding reference solution ; the
principal spot in each of the chromatograms obtained with
DEFINITION test solution (b) and reference solution (b) has an RF value
9-Fluoro-11β-hydroxy-16β-methyl-3,20-dioxopregna-1,4- distinctly lower than that of the principal spot in each of
diene-17,21-diyl dipropanoate. the chromatograms obtained with test solution (a) and
reference solution (a).
Content : 97.0 per cent to 102.0 per cent (dried substance).
D. Add about 2 mg to 2 mL of sulfuric acid R and shake to
CHARACTERS dissolve. Within 5 min, a deep reddish-brown colour
Appearance : white or almost white, crystalline powder. develops. Add this solution to 10 mL of water R and mix.
The colour is discharged and a clear solution remains.
Solubility : practically insoluble in water, freely soluble in
acetone and in methylene chloride, sparingly soluble in E. Mix about 5 mg with 45 mg of heavy magnesium oxide R
ethanol (96 per cent). and ignite in a crucible until an almost white residue is
obtained (usually less than 5 min). Allow to cool, add
IDENTIFICATION 1 mL of water R, 0.05 mL of phenolphthalein solution R1
First identification : B. and about 1 mL of dilute hydrochloric acid R to render the
solution colourless. Filter. Add 1.0 mL of the filtrate to a
Second identification : A, C, D, E. freshly prepared mixture of 0.1 mL of alizarin S solution R
A. Dissolve 10.0 mg in anhydrous ethanol R and dilute to and 0.1 mL of zirconyl nitrate solution R. Mix, allow to
100.0 mL with the same solvent. Place 2.0 mL of the stand for 5 min and compare the colour of the solution
solution in a ground-glass-stoppered tube, add 10.0 mL of with that of a blank prepared in the same manner. The test
phenylhydrazine-sulfuric acid solution R, mix and heat in solution is yellow and the blank is red.
a water-bath at 60 °C for 20 min. Cool immediately. The
absorbance (2.2.25) measured at 419 nm is not more than TESTS
0.10. Specific optical rotation (2.2.7) : + 84 to + 88 (dried
B. Infrared absorption spectrophotometry (2.2.24). substance).
Comparison : betamethasone dipropionate CRS. Dissolve 0.250 g in anhydrous ethanol R and dilute to 25.0 mL
C. Thin-layer chromatography (2.2.27). with the same solvent.
Test solution (a). Dissolve 25 mg of the substance to be Related substances. Liquid chromatography (2.2.29).
examined in methanol R with gentle heating and dilute to Test solution (a). Dissolve 60.0 mg of the substance to be
5 mL with the same solvent (solution A). Dilute 2 mL of examined in the mobile phase and dilute to 25.0 mL with the
solution A to 10 mL with methylene chloride R. mobile phase.
Test solution (b). Transfer 2 mL of solution A to Test solution (b). Dilute 1.0 mL of test solution (a) to 10.0 mL
a 15 mL glass tube with a ground-glass stopper or with the mobile phase.
a polytetrafluoroethylene cap. Add 10 mL of saturated Reference solution (a). Dissolve 5 mg of betamethasone
methanolic potassium hydrogen carbonate solution R and dipropionate for system suitability CRS (containing impurities
immediately pass a current of nitrogen R briskly through the B, C, D, E and G) in the mobile phase and dilute to 2.0 mL
solution for 5 min. Stopper the tube. Heat in a water-bath with the mobile phase.
at 45 °C, protected from light, for 2 h. Allow to cool.
Reference solution (b). Dilute 1.0 mL of test solution (a) to
Reference solution (a). Dissolve 25 mg of betamethasone 100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
dipropionate CRS in methanol R with gentle heating and to 10.0 mL with the mobile phase.
dilute to 5 mL with the same solvent (solution B). Dilute
2 mL of solution B to 10 mL with methylene chloride R. Reference solution (c). Dissolve 60.0 mg of betamethasone
dipropionate CRS in the mobile phase and dilute to 25.0 mL
Reference solution (b). Transfer 2 mL of solution B with the mobile phase. Dilute 1.0 mL of the solution to
to a 15 mL glass tube with a ground-glass stopper or
10.0 mL with the mobile phase.
a polytetrafluoroethylene cap. Add 10 mL of saturated
methanolic potassium hydrogen carbonate solution R and Reference solution (d). Dissolve 5 mg of betamethasone
immediately pass a current of nitrogen R briskly through the dipropionate for peak identification CRS (containing
solution for 5 min. Stopper the tube. Heat in a water-bath impurity H) in the mobile phase and dilute to 2.0 mL with
at 45 °C, protected from light, for 2 h. Allow to cool. the mobile phase.
Plate : TLC silica gel F254 plate R. Column :
Mobile phase : add a mixture of 1.2 volumes of water R and – size : l = 0.10 m, Ø = 2.0 mm ;
8 volumes of methanol R to a mixture of 15 volumes of – stationary phase : octadecylsilyl silica gel for
ether R and 77 volumes of methylene chloride R. chromatography R (2.5 μm) ;
Application : 5 μL. – temperature : 20 ± 2 °C.

General Notices (1) apply to all monographs and other texts 1661
Betamethasone dipropionate EUROPEAN PHARMACOPOEIA 8.0

Mobile phase : mix 35 mL of water R and 56 mL of acetonitrile R by the general monograph Substances for pharmaceutical use
and allow to equilibrate ; dilute to 100 mL with water R and (2034). It is therefore not necessary to identify these impurities
mix. for demonstration of compliance. See also 5.10. Control of
Flow rate : 0.2 mL/min. impurities in substances for pharmaceutical use) : A, F.
Detection : spectrophotometer at 254 nm.
Injection : 5 μL of test solution (a) and reference solutions (a),
(b) and (d).
Run time : 3 times the retention time of betamethasone
dipropionate.
Identification of impurities : use the chromatogram supplied
with betamethasone dipropionate for system suitability CRS
and the chromatogram obtained with reference solution (a) to A. 9-fluoro-11β,17,21-trihydroxy-16β-methylpregna-1,4-
identify the peaks due to impurities B, C, D, E and G ; use the diene-3,20-dione (betamethasone),
chromatogram supplied with betamethasone dipropionate for
peak identification CRS and the chromatogram obtained with
reference solution (d) to identify the peak due to impurity H.
Relative retention with reference to betamethasone
dipropionate (retention time = about 10 min):
impurity B = about 0.4 ; impurity C = about 0.5 ;
impurity D = about 0.7 ; impurity E = about 1.2 ;
impurity H = about 1.7 ; impurity G = about 2.1.
B. 9-fluoro-11β,21-dihydroxy-16β-methyl-3,20-dioxopregna-
System suitability: reference solution (a) : 1,4-dien-17-yl propanoate (betamethasone 17-propionate),
– peak-to-valley ratio : minimum 4.0, where Hp = height above
the baseline of the peak due to impurity E and Hv = height
above the baseline of the lowest point of the curve
separating this peak from the peak due to betamethasone
dipropionate.
Limits :
– correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
corresponding correction factor : impurity G = 1.3 ;
impurity H = 1.4 ; C. 9-fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregna-
– impurity C : not more than 5 times the area of the principal 1,4-dien-21-yl propanoate (betamethasone 21-propionate),
peak in the chromatogram obtained with reference
solution (b) (0.5 per cent) ;
– impurities B, H : for each impurity, not more than 3 times
the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.3 per cent) ;
– impurities D, E, G : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.2 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained D. 21-(acetyloxy)-9-fluoro-11β-hydroxy-16β-methyl-3,20-
with reference solution (b) (0.10 per cent) ; dioxopregna-1,4-dien-17-yl propanoate (betamethasone
– total : not more than 10 times the area of the principal peak 21-acetate 17-propionate),
in the chromatogram obtained with reference solution (b)
(1.0 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 1.0 per cent, determined
on 0.500 g by drying in an oven at 105 °C.
ASSAY
Liquid chromatography (2.2.29) as described in the test for E. 9-chloro-11β-hydroxy-16β-methyl-3,20-dioxopregna-
related substances with the following modification. 1,4-diene-17,21-diyl dipropanoate (beclometasone
Injection : test solution (b) and reference solution (c). dipropionate),
Calculate the percentage content of C28H37FO7 from the
declared content of betamethasone dipropionate CRS.
STORAGE
Protected from light.
IMPURITIES
Specified impurities : B, C, D, E, G, H.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of F. 9,11β-epoxy-16β-methyl-3,20-dioxo-9β-pregna-1,4-diene-
the tests in the monograph. They are limited by the general 17,21-diyl dipropanoate (9β,11β-epoxybetamethasone
acceptance criterion for other/unspecified impurities and/or dipropionate),

1662 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Betamethasone sodium phosphate

C. Thin-layer chromatography (2.2.27).

Test solution. Dissolve 10 mg of the substance to be
examined in methanol R and dilute to 10 mL with the same
solvent.
Reference solution (a). Dissolve 10 mg of betamethasone
sodium phosphate CRS in methanol R and dilute to 10 mL
with the same solvent.
Reference solution (b). Dissolve 10 mg of prednisolone
G. 9-fluoro-16β-methyl-3,20-dioxopregna-1,4-diene- sodium phosphate CRS in methanol R and dilute to 10 mL
11β,17,21-triyl tripropanoate (betamethasone with the same solvent. Dilute 5 mL of this solution to
tripropionate), 10 mL with reference solution (a).
Plate : TLC silica gel F254 plate R.
Mobile phase : glacial acetic acid R, water R, butanol R
(20:20:60 V/V/V).
Application : 5 μL.
Development : over a path of 15 cm.
Drying : in air.
Detection A : examine in ultraviolet light at 254 nm.
Results A : the principal spot in the chromatogram obtained
H. 6α-bromo-9-fluoro-11β-hydroxy-16β-methyl-3,20- with the test solution is similar in position and size to
dioxopregna-1,4-diene-17,21-diyl dipropanoate the principal spot in the chromatogram obtained with
(6α-bromobetamethasone dipropionate). reference solution (a).
Detection B : spray with alcoholic solution of sulfuric acid R.
01/2008:0810 Heat at 120 °C for 10 min or until the spots appear. Allow to
cool. Examine in daylight and in ultraviolet light at 365 nm.
BETAMETHASONE SODIUM Results B : the principal spot in the chromatogram obtained
PHOSPHATE with the test solution is similar in position, colour in
daylight, fluorescence in ultraviolet light at 365 nm and
size to the principal spot in the chromatogram obtained
Betamethasoni natrii phosphas with reference solution (a).
System suitability : reference solution (b) :
– the chromatogram shows 2 spots which may, however,
not be completely separated.
D. Add about 2 mg to 2 mL of sulfuric acid R and shake to
dissolve. Within 5 min, an intense reddish-brown colour
develops. Add the solution to 10 mL of water R and mix.
The colour is discharged and a clear solution remains.
C22H28FNa2O8P Mr 516.4
[151-73-5] E. Mix about 5 mg with 45 mg of heavy magnesium oxide R
and ignite in a crucible until an almost white residue is
DEFINITION obtained (usually less than 5 min). Allow to cool, add
9-Fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregna- 1 mL of water R, 0.05 mL of phenolphthalein solution R1
1,4-dien-21-yl disodium phosphate. and about 1 mL of dilute hydrochloric acid R to render the
solution colourless. Filter. Add 1.0 mL of the filtrate to a
Content : 96.0 per cent to 103.0 per cent (anhydrous substance).
freshly prepared mixture of 0.1 mL of alizarin S solution R
CHARACTERS and 0.1 mL of zirconyl nitrate solution R. Mix, allow to
Appearance : white or almost white powder, very hygroscopic. stand for 5 min and compare the colour of the solution
with that of a blank prepared in the same manner. The test
Solubility : freely soluble in water, slightly soluble in ethanol solution is yellow and the blank is red.
(96 per cent), practically insoluble in methylene chloride.
F. To about 40 mg add 2 mL of sulfuric acid R and heat gently
IDENTIFICATION until white fumes are evolved. Add nitric acid R dropwise,
First identification : B, C. continue the heating until the solution is almost colourless
and cool. Add 2 mL of water R, heat until white fumes are
Second identification : A, C, D, E, F.
again evolved, cool, add 10 mL of water R and neutralise to
A. Dissolve 10.0 mg in 5 mL of water R and dilute to red litmus paper R with dilute ammonia R1. The solution
100.0 mL with anhydrous ethanol R. Place 2.0 mL of this gives reaction (a) of sodium (2.3.1) and reaction (b) of
solution in a ground-glass-stoppered tube, add 10.0 mL of phosphates (2.3.1).
phenylhydrazine-sulfuric acid solution R, mix and heat in
a water-bath at 60 °C for 20 min. Cool immediately. The TESTS
absorbance (2.2.25) measured at the absorption maximum
at 450 nm is not more than 0.10. Solution S. Dissolve 1.0 g in carbon dioxide-free water R and
dilute to 20 mL with the same solvent.
B. Infrared absorption spectrophotometry (2.2.24).
Appearance of solution. Solution S is clear (2.2.1) and not
Comparison : betamethasone sodium phosphate CRS.
more intensely coloured than reference solution B7 (2.2.2,
If the spectra obtained in the solid state show differences, Method II).
dissolve the substance to be examined and the reference
substance separately in the minimum volume of ethanol pH (2.2.3) : 7.5 to 9.0.
(96 per cent) R, evaporate to dryness on a water-bath and Dilute 1 mL of solution S to 5 mL with carbon dioxide-free
record new spectra using the residues. water R.

General Notices (1) apply to all monographs and other texts 1663
Betamethasone valerate EUROPEAN PHARMACOPOEIA 8.0

Specific optical rotation (2.2.7) : + 98 to + 104 (anhydrous STORAGE

substance). In an airtight container, protected from light.
Dissolve 0.250 g in water R and dilute to 25.0 mL with the
same solvent.
01/2009:0811
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 62.5 mg of the substance to be
examined in the mobile phase and dilute to 25.0 mL with the BETAMETHASONE VALERATE
mobile phase.
Reference solution (a). Dissolve 25 mg of betamethasone Betamethasoni valeras
sodium phosphate CRS and 25 mg of dexamethasone sodium
phosphate CRS in the mobile phase and dilute to 25.0 mL with
the mobile phase. Dilute 1.0 mL of this solution to 25.0 mL
with the mobile phase.
Reference solution (b). Dilute 1.0 mL of the test solution to
50.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ; C27H37FO6 Mr 476.6
– stationary phase : octadecylsilyl silica gel for [2152-44-5]
chromatography R (5 μm).
DEFINITION
Mobile phase : in a 250 mL conical flask, weigh 1.360 g
of potassium dihydrogen phosphate R and 0.600 g of 9-Fluoro-11β,21-dihydroxy-16β-methyl-3,20-dioxopregna-
hexylamine R, mix and allow to stand for 10 min and then 1,4-dien-17-yl pentanoate.
dissolve in 185 mL of water R ; add 65 mL of acetonitrile R, Content : 97.0 per cent to 103.0 per cent (dried substance).
mix and filter (0.45 μm).
CHARACTERS
Flow rate : 1 mL/min.
Appearance : white or almost white, crystalline powder.
Detection : spectrophotometer at 254 nm.
Solubility : practically insoluble in water, freely soluble in
Equilibration : with the mobile phase for about 45 min. acetone and in methylene chloride, soluble in ethanol (96 per
Injection : 20 μL. cent).
Run time : twice the retention time of betamethasone sodium mp : about 192 °C, with decomposition.
phosphate.
Retention time : betamethasone sodium phosphate = about IDENTIFICATION
14 min ; dexamethasone sodium phosphate = about 15.5 min. A. Infrared absorption spectrophotometry (2.2.24).
System suitability: reference solution (a) : Comparison: betamethasone 17-valerate CRS.
– resolution : minimum 2.0 between the peaks due to If the spectra obtained in the solid state show differences,
betamethasone sodium phosphate and dexamethasone dissolve the substance to be examined and the reference
sodium phosphate ; if necessary, increase the concentration substance separately in the minimum volume of methylene
of acetonitrile or increase the concentration of water in the chloride R, evaporate to dryness on a water-bath and record
mobile phase. new spectra using the residues.
Limits : B. Examine the chromatograms obtained in the test for related
– any impurity : for each impurity, not more than the area substances.
of the principal peak in the chromatogram obtained with Results : the principal peak in the chromatogram obtained
reference solution (b) (2 per cent), and not more than with the test solution is similar in retention time and size
1 such peak has an area greater than 0.5 times the area to the principal peak in the chromatogram obtained with
of the principal peak in the chromatogram obtained with reference solution (b).
reference solution (b) (1 per cent) ;
TESTS
– total : not more than 1.5 times the area of the principal peak
in the chromatogram obtained with reference solution (b) Specific optical rotation (2.2.7) : + 77 to + 83 (dried
(3 per cent) ; substance).
– disregard limit : 0.025 times the area of the principal peak Dissolve 0.250 g in anhydrous ethanol R and dilute to 25.0 mL
in the chromatogram obtained with reference solution (b) with the same solvent.
(0.05 per cent). Related substances. Liquid chromatography (2.2.29).
Inorganic phosphate : maximum 1 per cent. Carry out the test protected from light. Prepare the solutions
Dissolve 50 mg in water R and dilute to 100 mL with the same immediately before use.
solvent. To 10 mL of this solution add 5 mL of molybdovanadic Solvent mixture : glacial acetic acid R, mobile phase
reagent R, mix and allow to stand for 5 min. Any yellow colour (1:1000 V/V).
in the solution is not more intense than that in a standard Test solution. Dissolve 50 mg of the substance to be examined
prepared at the same time and in the same manner using in the solvent mixture and dilute to 20.0 mL with the solvent
10 mL of phosphate standard solution (5 ppm PO4) R. mixture.
Water (2.5.12) : maximum 8.0 per cent, determined on 0.200 g. Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
ASSAY solution to 10.0 mL with the solvent mixture.
Dissolve 0.100 g in water R and dilute to 100.0 mL with the Reference solution (b). Dissolve 12.5 mg of betamethasone
same solvent. Dilute 5.0 mL of this solution to 250.0 mL with valerate for system suitability CRS (containing impurities D
water R. Measure the absorbance (2.2.25) at the absorption and G) in 5.0 mL of the solvent mixture. Use 1.0 mL of this
maximum at 241 nm. solution to dissolve the contents of a vial of betamethasone
Calculate the content of C22H28FNa2O8P taking the specific valerate impurity mixture CRS (containing impurities C, H
absorbance to be 297. and I).

1664 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Betamethasone valerate

Reference solution (c). Dissolve 6 mg of betamethasone CRS IMPURITIES

(impurity A) and 3 mg of betamethasone 21-valerate CRS Specified impurities : A, C, E, G, H, I.
(impurity E) in 30.0 mL of the solvent mixture. Dilute 1.0 mL
of this solution to 10.0 mL with the solvent mixture. Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
Column : the tests in the monograph. They are limited by the general
– size : l = 0.25 m, Ø = 4.6 mm ; acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
– stationary phase : end-capped octadecylsilyl silica gel for (2034). It is therefore not necessary to identify these impurities
chromatography R (5 μm) ; for demonstration of compliance. See also 5.10. Control of
– temperature : 20 °C. impurities in substances for pharmaceutical use) : B, D, F.
Mobile phase : acetonitrile R, water R (50:50 V/V).
Flow rate : 1 mL/min.
Detection : spectrophotometer at 239 nm.
Injection : 20 μL.
Run time : 2.5 times the retention time of betamethasone
valerate.
Identification of impurities : use the chromatogram supplied
with betamethasone valerate for system suitability CRS and A. R1 = R3 = R5 = R6 = H, R2 = F, R4 = CH3 :
the chromatogram obtained with reference solution (b) to 9-fluoro-11β,17,21-trihydroxy-16β-methylpregna-1,4-
identify the peaks due to impurities C, D, G, H and I ; use the diene-3,20-dione (betamethasone),
chromatogram obtained with reference solution (c) to identify
the peaks due to impurities A and E. C. R1 = R4 = R6 = H, R2 = F, R3 = CH3, R5 = CO-[CH2]3-CH3 :
Relative retention with reference to betamethasone valerate 9-fluoro-11β,21-dihydroxy-16α-methyl-3,20-dioxopregna-
(retention time = about 20 min) : impurity A = about 0.3 ; 1,4-dien-17-yl pentanoate (dexamethasone 17-valerate),
impurity I = about 0.6 ; impurity C = about 0.8 ;
impurity H = about 1.3 ; impurity D = about 1.4 ; E. R1 = R3 = R5 = H, R2 = F, R4 = CH3, R6 = CO-[CH2]3-CH3 :
impurity E = about 1.6 ; impurity G = about 2.0. 9-fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregna-
System suitability : reference solution (b) : 1,4-dien-21-yl pentanoate (betamethasone 21-valerate),

– resolution : minimum 1.7 between the peaks due to G. R1 = Br, R2 = F, R3 = R6 = H, R4 = CH3, R5 =

impurities H and D. CO-[CH2]3-CH3 : 6α-bromo-9-fluoro-11β,21-dihydroxy-
Limits : 16βmethyl-3,20-dioxopregna-1,4-dien-17-yl pentanoate
(6α-bromo-betamethasone valerate),
– impurity A : not more than 7 times the area of the principal
peak in the chromatogram obtained with reference H. R1 = R3 = R6 = H, R2 = Cl, R4 = CH3, R5 = CO-[CH2]3-CH3 :
solution (a) (0.7 per cent) ; 9-chloro-11β,21-dihydroxy-16β-methyl-3,20-dioxopregna-
– impurities E, G : for each impurity, not more than 3 times 1,4-dien-17-yl pentanoate (beclomethasone 17-valerate),
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.3 per cent) ; I. R1 = R3 = R4 = R6 = H, R2 = F, R5 = CO-[CH2]3-CH3 :
– impurities C, H, I : for each impurity, not more than 9-fluoro-11β,21-dihydroxy-3,20-dioxopregna-1,4-dien-17-
1.5 times the area of the principal peak in the chromatogram yl pentanoate (9-fluoro-prednisolone 17-valerate),
obtained with reference solution (a) (0.15 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
– total : not more than 15 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(1.5 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in B. R1 = F, R2 = R3 = H : 9-fluoro-11β,17-dihydroxy-
the chromatogram obtained with reference solution (a) 16β-methylpregna-1,4-diene-3,20-dione (21-deoxy-
(0.05 per cent). betamethasone),
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C. D. R1 = Br, R2 = CO-[CH2]3-CH3, R3 = OH : 9-bromo-11β,21-
dihydroxy-16β-methyl-3,20-dioxopregna-1,4-dien-17-yl
ASSAY pentanoate (9-bromo-betamethasone valerate),
Dissolve 50.0 mg in ethanol (96 per cent) R and dilute to
100.0 mL with the same solvent. Dilute 2.0 mL of this
solution to 50.0 mL with ethanol (96 per cent) R. Measure the
absorbance (2.2.25) at the absorption maximum at 240 nm.
Calculate the content of C27H37FO6 taking the specific
absorbance to be 325.

STORAGE F. 21-hydroxy-16β-methyl-3,20-dioxopregna-1,4,9(11)-trien-
Protected from light. 17-yl pentanoate (betamethasone valerate δ-9(11)).

General Notices (1) apply to all monographs and other texts 1665
Betaxolol hydrochloride EUROPEAN PHARMACOPOEIA 8.0

07/2011:1072 Acidity or alkalinity. Dissolve 0.20 g in carbon dioxide-free

water R and dilute to 20 mL with the same solvent. Add 0.2 mL
BETAXOLOL HYDROCHLORIDE of methyl red solution R and 0.2 mL of 0.01 M hydrochloric
acid. The solution is red. Add 0.4 mL of 0.01 M sodium
hydroxide. The solution is yellow.
Betaxololi hydrochloridum Related substances. Liquid chromatography (2.2.29). Prepare
reference solutions (c) and (d) immediately before use.
Test solution. Dissolve 10 mg of the substance to be examined
in the mobile phase and dilute to 5.0 mL with the mobile
phase.
Reference solution (a). Dissolve 8 mg of the substance to be
examined and 4 mg of betaxolol impurity A CRS in 20.0 mL of
C18H30ClNO3 Mr 343.9
the mobile phase.
[63659-19-8]
Reference solution (b). Dilute 1.0 mL of the test solution to
DEFINITION 100.0 mL with the mobile phase.
(2RS)-1-[4-[2-(Cyclopropylmethoxy)ethyl]phenoxy]-3-[(1- Reference solution (c). Dissolve 2 mg of betaxolol
methylethyl)amino]propan-2-ol hydrochloride. impurity C CRS in 50 mL of the mobile phase. Dilute 5 mL of
Content : 98.5 per cent to 101.5 per cent (dried substance). the solution to 20 mL with the mobile phase.
Reference solution (d). Dissolve 10 mg of betaxolol for peak
CHARACTERS identification CRS (containing impurities B, D and E) in 5 mL
Appearance : white or almost white, crystalline powder. of reference solution (c).
Solubility : very soluble in water, freely soluble in ethanol Column :
(96 per cent), soluble in methylene chloride. – size : l = 0.25 m, Ø = 4 mm ;
IDENTIFICATION – stationary phase : octylsilyl silica gel for chromatography R
First identification : B, D. (5 μm).
Second identification : A, C, D. Mobile phase : mix 175 mL of acetonitrile R and 175 mL
of methanol R and dilute to 1 L with a 3.4 g/L solution of
A. Melting point (2.2.14) : 113 °C to 117 °C. potassium dihydrogen phosphate R, previously adjusted to
B. Infrared absorption spectrophotometry (2.2.24). pH 3.0 with phosphoric acid R.
Comparison : betaxolol hydrochloride CRS. Flow rate : 1.5 mL/min.
C. Thin-layer chromatography (2.2.27). Detection : spectrophotometer at 273 nm.
Test solution. Dissolve 10 mg of the substance to be Injection : 20 μL of the test solution and reference solutions (a),
examined in 1 mL of methanol R. (b) and (d).
Reference solution (a). Dissolve 20 mg of betaxolol Run time : 4.5 the retention time of betaxolol.
hydrochloride CRS in 2 mL of methanol R.
Identification of impurities: use the chromatogram obtained
Reference solution (b). Dissolve 10 mg of oxprenolol with reference solution (a) to identify the peak due to
hydrochloride CRS in 1 mL of reference solution (a). impurity A ; use the chromatogram supplied with betaxolol for
Plate : TLC octadecylsilyl silica gel F254 plate R. peak identification CRS and the chromatogram obtained with
Mobile phase : perchloric acid R, methanol R, water R reference solution (d) to identify the peaks due to impurities B,
(0.5:50:50 V/V/V). C, D and E.
Application : 2 μL. Relative retention with reference to betaxolol (retention
time = about 8 min) : impurity B = about 0.3 ;
Development : over a path of 10 cm.
impurity A = about 0.8 ; impurity D = about 1.5 ;
Drying : in air. impurity E = about 2.2 ; impurity C = about 4.1.
System suitability: reference solution (b) : System suitability : reference solution (a) :
– the chromatogram shows 2 clearly separated spots. – resolution : minimum 2.0 between the peaks due to
Detection A : examine in ultraviolet light at 254 nm. impurity A and betaxolol.
Results A : the principal spot in the chromatogram obtained Limits :
with the test solution is similar in position and size to – impurities A, B, C, D, E : for each impurity, not more than
the principal spot in the chromatogram obtained with 0.3 times the area of the principal peak in the chromatogram
reference solution (a). obtained with reference solution (b) (0.3 per cent) ;
Detection B : spray with a 50 g/L solution of vanillin R in – unspecified impurities : for each impurity, not more than
a mixture of 5 volumes of sulfuric acid R, 10 volumes of 0.1 times the area of the principal peak in the chromatogram
glacial acetic acid R and 85 volumes of methanol R, heat at obtained with reference solution (b) (0.10 per cent) ;
100-105 °C until the colour of the spots reaches maximum
intensity (10-15 min), and examine in daylight. – total : not more than the area of the principal peak in the
chromatogram obtained with reference solution (b) (1.0 per
Results B : the principal spot in the chromatogram obtained cent) ;
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with – disregard limit : 0.05 times the area of the principal peak
reference solution (a). in the chromatogram obtained with reference solution (b)
(0.05 per cent).
D. It gives reaction (a) of chlorides (2.3.1).
Heavy metals (2.4.8) : maximum 10 ppm.
TESTS Dissolve 2.0 g in 20 mL of water R. 12 mL of the solution
Appearance of solution. The solution is clear (2.2.1) and complies with test A. Prepare the reference solution using
colourless (2.2.2, Method II). 10 mL of lead standard solution (1 ppm Pb) R.
Dissolve 0.5 g in water R and dilute to 25 mL with the same Loss on drying (2.2.32) : maximum 0.5 per cent, determined
solvent. on 1.000 g by drying in an oven at 105 °C.

1666 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Bezafibrate

Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Content : 98.0 per cent to 102.0 per cent (dried substance).
1.0 g.
CHARACTERS
ASSAY Appearance : white or almost white, crystalline powder.
Dissolve 0.300 g in a mixture of 10.0 mL of 0.01 M hydrochloric Solubility : practically insoluble in water, freely soluble in
acid and 50 mL of ethanol (96 per cent) R. Carry out a dimethylformamide, sparingly soluble in acetone and in
potentiometric titration (2.2.20), using 0.1 M sodium ethanol (96 per cent). It dissolves in dilute solutions of alkali
hydroxide. Read the volume added between the 2 points of hydroxides.
inflexion. It shows polymorphism (5.9).
1 mL of 0.1 M sodium hydroxide is equivalent to 34.39 mg
of C18H30ClNO3. IDENTIFICATION
First identification : A, B.
STORAGE Second identification : A, C.
Protected from light. A. Melting point (2.2.14) : 181 °C to 185 °C.
IMPURITIES B. Infrared absorption spectrophotometry (2.2.24).
Specified impurities : A, B, C, D, E. Comparison: bezafibrate CRS.
If the spectra obtained show differences, dissolve the
substance to be examined and the reference substance
separately in methanol R and evaporate to dryness. Dry the
residues in vacuo at 80 °C for 1 h and record new spectra
using the residues.
A. (2RS)-1-(4-ethylphenoxy)-3-[(1-methylethyl)amino]- C. Thin-layer chromatography (2.2.27).
propan-2-ol, Test solution. Dissolve 10 mg of the substance to be
examined in methanol R and dilute to 5 mL with the same
solvent.
Reference solution. Dissolve 10 mg of bezafibrate CRS in
methanol R and dilute to 5 mL with the same solvent.
Plate : TLC silica gel F254 plate R.
B. (2RS)-1-[4-(2-hydroxyethyl)phenoxy]-3-[(1-methylethyl)-
amino]propan-2-ol, Mobile phase : glacial acetic acid R, methyl ethyl ketone R,
xylene R (2.7:30:60 V/V/V).
Application : 5 μL.
Development : over half of the plate.
Drying : at 120 °C for at least 15 min.
Detection : examine in ultraviolet light at 254 nm.
C. (2RS)-2-[[4-[2-(cyclopropylmethoxy)ethyl]phenoxy]- Results : the principal spot in the chromatogram obtained
methyl]oxirane, with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the
reference solution.
TESTS
Solution S. Dissolve 1.0 g in dimethylformamide R and dilute
D. 4-[2-(cyclopropylmethoxy)ethyl]phenol, to 20 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution BY5 (2.2.2,
Method II).
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50.0 mg of the substance to be
E. (2RS)-1-[4-(2-butoxyethyl)phenoxy]-3-[(1-methylethyl)- examined in the mobile phase and dilute to 100.0 mL with
amino]propan-2-ol. the mobile phase.
Reference solution (a). Dilute 10.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 5.0 mL of this solution
07/2010:1394 to 100.0 mL with the mobile phase.
Reference solution (b). Dilute 5.0 mL of reference solution (a)
BEZAFIBRATE to 50.0 mL with the mobile phase.
Reference solution (c). To 1 mL of the test solution, add 1 mL
Bezafibratum of 0.1 M hydrochloric acid and evaporate to dryness on a hot
plate. Dissolve the residue in 20 mL of the mobile phase.
Column :
– size : l = 0.125 m, Ø = 4 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : mix 40 volumes of a 2.72 g/L solution of
C19H20ClNO4 Mr 361.8 potassium dihydrogen phosphate R adjusted to pH 2.3 with
[41859-67-0] phosphoric acid R, and 60 volumes of methanol R.
DEFINITION Flow rate : 1 mL/min.
2-[4-[2-[(4-Chlorobenzoyl)amino]ethyl]phenoxy]-2- Detection : spectrophotometer at 228 nm.
methylpropanoic acid. Injection : 20 μL.

General Notices (1) apply to all monographs and other texts 1667
Bicalutamide EUROPEAN PHARMACOPOEIA 8.0

Run time : the time necessary to detect the ester, which,

depending on the route of synthesis, may be impurity C, D
or E.
Relative retention with reference to bezafibrate (retention
time = about 6.0 min) : impurity A = about 0.5 ;
impurity B = about 0.6 ; impurity C = about 1.5 ;
impurity D = about 2.3 ; impurity E = about 6.2. C. methyl 2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]phenoxy]-
2-methylpropanoate,
System suitability :
– resolution : minimum 5.0 between the 2 principal peaks in
the chromatogram obtained with reference solution (c) ;
– signal-to-noise ratio : minimum 5 for the principal peak in
the chromatogram obtained with reference solution (b).
Limits :
– impurities A, B, C, D, E : for each impurity, not more D. ethyl 2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]-
than the area of the principal peak in the chromatogram phenoxy]-2-methylpropanoate,
obtained with reference solution (a) (0.5 per cent) ;
– unspecified impurities : for each impurity, not more than
0.2 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent) ;
– total : not more than 1.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.75 per cent) ; E. butyl 2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]-
– disregard limit : 0.1 times the area of the principal peak in phenoxy]-2-methylpropanoate.
the chromatogram obtained with reference solution (a)
(0.05 per cent).
04/2012:2196
Chlorides (2.4.4): maximum 300 ppm.
Dilute 10 mL of solution S to 50 mL with water R. Filter the BICALUTAMIDE
resultant suspension through a wet filter previously washed
with water R until free from chlorides. Prepare the standard
using 9 mL of chloride standard solution (5 ppm Cl) R and Bicalutamidum
6 mL of water R.
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test C. Prepare the reference solution using
2 mL of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined C18H14F4N2O4S Mr 430.4
on 1.000 g by drying in an oven at 105 °C. [90357-06-5]
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on DEFINITION
1.0 g. (2RS)-N-[4-Cyano-3-(trifluoromethyl)phenyl]-3-[(4-
fluorophenyl)sulfonyl]-2-hydroxy-2-methylpropanamide.
ASSAY Content : 97.5 per cent to 102.0 per cent (dried substance).
Dissolve 0.300 g in 50 mL of a mixture of 25 volumes of
water R and 75 volumes of ethanol (96 per cent) R. Using CHARACTERS
0.1 mL of phenolphthalein solution R as indicator, titrate with Appearance : white or almost white powder.
0.1 M sodium hydroxide until a pink colour is obtained. Carry Solubility : practically insoluble in water, freely soluble
out a blank titration. in acetone, slightly soluble in anhydrous ethanol and in
1 mL of 0.1 M sodium hydroxide is equivalent to 36.18 mg methylene chloride.
of C19H20ClNO4. It shows polymorphism (5.9).

IMPURITIES IDENTIFICATION
Specified impurities : A, B, C, D, E. Infrared absorption spectrophotometry (2.2.24).
Comparison: bicalutamide CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
substance separately in acetone R, evaporate to dryness and
record new spectra using the residues.
TESTS
A. 4-chloro-N-[2-(4-hydroxyphenyl)ethyl]benzamide Related substances. Liquid chromatography (2.2.29).
(chlorobenzoyltyramine), Solvent mixture : phosphoric acid R, acetonitrile R1, water R
(0.05:50:50 V/V/V).
Test solution (a). Dissolve 25.0 mg of the substance to be
examined in the solvent mixture and dilute to 25.0 mL with
the solvent mixture.
Test solution (b). Dilute 5.0 mL of test solution (a) to 25.0 mL
B. 4-chlorobenzoic acid, with the solvent mixture.

1668 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Bicalutamide

Reference solution (a). Dilute 1.0 mL of test solution (a) to ASSAY

100.0 mL with the solvent mixture. Dilute 1.0 mL of this Liquid chromatography (2.2.29) as described in the test for
solution to 10.0 mL with the solvent mixture. related substances with the following modification.
Reference solution (b). Dissolve 5 mg of bicalutamide for Injection : test solution (b) and reference solution (c).
system suitability CRS (containing impurities B and C) in the
solvent mixture and dilute to 5.0 mL with the solvent mixture. Calculate the percentage content of C18H14F4N2O4S taking into
account the assigned content of bicalutamide CRS.
Reference solution (c). Dissolve 25.0 mg of bicalutamide CRS
in the solvent mixture and dilute to 25.0 mL with the solvent IMPURITIES
mixture. Dilute 5.0 mL of the solution to 25.0 mL with the
solvent mixture. Specified impurities : C.
Column : Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
– size : l = 0.25 m, Ø = 4.0 mm ; the tests in the monograph. They are limited by the general
– stationary phase : spherical end-capped octadecylsilyl silica acceptance criterion for other/unspecified impurities and/or
gel for chromatography R (5 μm) ; by the general monograph Substances for pharmaceutical
– temperature : 50 °C. use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Mobile phase :
Control of impurities in substances for pharmaceutical use) : A,
– mobile phase A : phosphoric acid R, acetonitrile R1, water R B, D, E, F, H, J, K, L, M.
(1.9:100:1900 V/V/V) ;
– mobile phase B : phosphoric acid R, water R, acetonitrile R1
(1.9:100:1900 V/V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-3 92 8 A. (2RS)-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-
3 - 23 92 → 67 8 → 33
2-methyl-3-(phenylsulfonyl)propanamide,

23 - 43 67 → 50 33 → 50

43 - 50 50 50

Flow rate : 1.0 mL/min.

Detection : spectrophotometer at 210 nm. B. (2RS)-N-[4-cyano-3-(trifluoromethyl)phenyl]-3-[(2-
Injection : 10 μL of test solution (a) and reference solutions (a) fluorophenyl)sulfonyl]-2-hydroxy-2-methylpropanamide,
and (b).
Identification of impurities : use the chromatogram supplied
with bicalutamide for system suitability CRS and the
chromatogram obtained with reference solution (b) to identify
the peaks due to impurities B and C.
Relative retention with reference to bicalutamide C. (2RS)-N-[4-cyano-3-(trifluoromethyl)phenyl]-3-[(4-
(retention time = about 38 min): impurity B = about 0.98 ; fluorophenyl)sulfonyl]-2-methylpropanamide,
impurity C = about 1.1.
System suitability : reference solution (b) :
– peak-to-valley ratio : minimum 2.5, where Hp = height above
the baseline of the peak due to impurity B and Hv = height
above the baseline of the lowest point of the curve D. 4-amino-2-(trifluoromethyl)benzonitrile,
separating this peak from the peak due to bicalutamide.
Limits :
– impurity C : not more than 1.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.15 per cent) ;
– unspecified impurities : for each impurity, not more than the
E. (2RS)-N-[4-cyano-3-(trifluoromethyl)phenyl]-3-[(RS)-(4-
area of the principal peak in the chromatogram obtained
fluorophenyl)sulfinyl]-2-hydroxy-2-methylpropanamide,
with reference solution (a) (0.10 per cent) ;
– total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent). F. (2SR)-N-[4-cyano-3-(trifluoromethyl)phenyl]-3-[(RS)-(4-
Heavy metals (2.4.8) : maximum 20 ppm. fluorophenyl)sulfinyl]-2-hydroxy-2-methylpropanamide,
Solvent mixture : water R, acetone R (10:90 V/V).
0.500 g complies with test H. Prepare the reference solution
using 1 mL of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 4 h.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on H. (2RS)-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-[(4-
1.0 g in a platinum crucible. fluorophenyl)sulfonyl]-3-hydroxy-2-methylpropanamide,

General Notices (1) apply to all monographs and other texts 1669
Bifonazole EUROPEAN PHARMACOPOEIA 8.0

Test solution. Dissolve 50.0 mg of the substance to be

examined in 25 mL of acetonitrile R and dilute to 50.0 mL
with buffer solution pH 3.2.
Reference solution (a). Dilute 1.0 mL of the test solution to
J. (2RS)-N-[4-cyano-3-(trifluoromethyl)phenyl]-3-[(4- 100.0 mL with buffer solution pH 3.2. Dilute 1.0 mL of this
fluorophenyl)sulfanyl]-2-hydroxy-2-methylpropanamide, solution to 10.0 mL with buffer solution pH 3.2.
Reference solution (b). Dissolve 2 mg of bifonazole for system
suitability CRS (containing impurities A, B, C, D and E)
in 2 mL of acetonitrile R and dilute to 10.0 mL with buffer
solution pH 3.2.
Column :
K. (2R,2′S)-3,3′-sulfonylbis[N-[4-cyano-3-(trifluoro- – size : l = 0.125 m, Ø = 4.0 mm ;
methyl)phenyl]-2-hydroxy-2-methylpropanamide],
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm) ;
– temperature : 40 °C.
Mobile phase :
– mobile phase A : acetonitrile R1, buffer solution pH 3.2
(20:80 V/V) ;
L. (2RS,2′RS)-3,3′-sulfonylbis[N-[4-cyano-3-(trifluoro- – mobile phase B : buffer solution pH 3.2, acetonitrile R1
methyl)phenyl]-2-hydroxy-2-methylpropanamide], (20:80 V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-8 60 40

8 - 12 60 → 10 40 → 90
M. (2RS)-3-[(4-fluorophenyl)sulfonyl]-2-hydroxy-2- 12 - 30 10 90
methylpropanoic acid.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 210 nm.
04/2012:1395
Injection : 50 μL.
BIFONAZOLE Identification of impurities : use the chromatogram
supplied with bifonazole for system suitability CRS and the
chromatogram obtained with reference solution (b) to identify
Bifonazolum the peaks due to impurities A, B, C, D and E.
Relative retention with reference to bifonazole (retention
time = about 4 min) : impurity C = about 0.2 ;
impurity B = about 0.7 ; impurity A = about 3.2 ;
impurity D = about 3.6 ; impurity E = about 5.8.
System suitability : reference solution (b) :
– resolution : minimum 2.5 between the peaks due to
C22H18N2 Mr 310.4 impurity B and bifonazole.
[60628-96-8] Limits :
DEFINITION – correction factor : for the calculation of content, multiply
the peak area of impurity C by 2 ;
1-[(RS)-(Biphenyl-4-yl)phenylmethyl]-1H-imidazole.
– impurities B, D : for each impurity, not more than 5 times
Content : 98.0 per cent to 100.5 per cent (dried substance). the area of the principal peak in the chromatogram
CHARACTERS obtained with reference solution (a) (0.5 per cent) ;
Appearance : white or almost white, crystalline powder. – impurities A, C : for each impurity, not more than twice the
area of the principal peak in the chromatogram obtained
Solubility : practically insoluble in water, sparingly soluble in with reference solution (a) (0.2 per cent) ;
anhydrous ethanol.
– impurity E : not more than 1.5 times the area of the
It shows polymorphism (5.9). principal peak in the chromatogram obtained with
IDENTIFICATION reference solution (a) (0.15 per cent) ;
Infrared absorption spectrophotometry (2.2.24). – unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
Comparison : bifonazole CRS. with reference solution (a) (0.10 per cent) ;
If the spectra obtained in the solid state show differences, – total : not more than 10 times the area of the principal peak
dissolve the substance to be examined and the reference in the chromatogram obtained with reference solution (a)
substance separately in the minimum volume of 2-propanol R, (1.0 per cent) ;
evaporate to dryness and record new spectra using the
residues. – disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
TESTS (0.05 per cent).
Related substances. Liquid chromatography (2.2.29). Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Buffer solution pH 3.2. Mix 2.0 mL of phosphoric acid R with on 1.000 g by drying in an oven at 105 °C.
980 mL of water R, adjust to pH 3.2 (2.2.3) with triethylamine R Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
and dilute to 1000.0 mL with water R. 1.0 g.

1670 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Biotin

ASSAY DEFINITION
Dissolve 0.250 g in 80 mL of anhydrous acetic acid R. Titrate Biotin contains not less than 98.5 per cent and not more
with 0.1 M perchloric acid, determining the end-point than the equivalent of 101.0 per cent of 5-[(3aS,4S,6aR)-2-
potentiometrically (2.2.20). oxohexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid,
1 mL of 0.1 M perchloric acid is equivalent to 31.04 mg calculated with reference to the dried substance.
of C22H18N2. CHARACTERS
IMPURITIES A white or almost white, crystalline powder or colourless
crystals, very slightly soluble in water and in alcohol,
Specified impurities : A, B, C, D, E. practically insoluble in acetone. It dissolves in dilute solutions
of alkali hydroxides.
IDENTIFICATION
First identification : A.
Second identification : B, C.
A. (RS)-(biphenyl-4-yl)phenylmethanol, A. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
biotin CRS.
B. Examine the chromatograms obtained in the test for
related substances (see Tests). The principal spot in the
chromatogram obtained with test solution (b) is similar in
position and size to the principal spot in the chromatogram
obtained with reference solution (a).
B. 4-[(RS)-(biphenyl-4-yl)phenylmethyl]-1H-imidazole, C. Dissolve about 10 mg in 20 mL of water R with heating.
Allow to cool. Add 0.1 mL of bromine water R. The
bromine water is decolourised.
TESTS
Solution S. Dissolve 0.250 g in a 4 g/L solution of sodium
C. 1H-imidazole,
hydroxide R and dilute to 25.0 mL with the same alkaline
solution.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Specific optical rotation (2.2.7). The specific optical rotation
is + 89 to + 93, determined on solution S and calculated with
reference to the dried substance.
Related substances. Examine by thin-layer chromatography
(2.2.27), using as the coating substance a suitable silica gel
(5 μm). Prepare the solutions immediately before use and keep
D. 1,3-bis[(biphenyl-4-yl)phenylmethyl]-1H-imidazolium protected from bright light.
ion, Test solution (a). Dissolve 50 mg of the substance to be
examined in glacial acetic acid R and dilute to 10 mL with the
same solvent.
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
with glacial acetic acid R.
Reference solution (a). Dissolve 5 mg of biotin CRS in glacial
acetic acid R and dilute to 10 mL with the same solvent.
Reference solution (b). Dilute 1 mL of test solution (b) to
20 mL with glacial acetic acid R.
Reference solution (c). Dilute 1 mL of test solution (b) to
40 mL with glacial acetic acid R.
E. 1,4-bis[(biphenyl-4-yl)phenylmethyl]-1H-imidazole. Apply to the plate 10 μL of each solution. Develop over a
path of 15 cm using a mixture of 5 volumes of methanol R,
25 volumes of glacial acetic acid R and 75 volumes of toluene R.
Dry the plate in a current of warm air. Allow to cool and spray
01/2008:1073 with 4-dimethylaminocinnamaldehyde solution R. Examine
corrected 6.0 immediately in daylight. Any spot in the chromatogram
obtained with test solution (a), apart from the principal spot, is
BIOTIN not more intense than the spot in the chromatogram obtained
with reference solution (b) (0.5 per cent) and at most one
such spot is more intense than the spot in the chromatogram
Biotinum obtained with reference solution (c) (0.25 per cent).
Heavy metals (2.4.8). 1.0 g complies with test C for heavy
metals (10 ppm). Prepare the reference solution using 10 mL
of lead standard solution (1 ppm Pb) R.
Loss on drying (2.2.32). Not more than 1.0 per cent,
determined on 1.000 g by drying in an oven at 105 °C.
C10H16N2O3S Mr 244.3 Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
[58-85-5] on 1.0 g.

General Notices (1) apply to all monographs and other texts 1671
Biperiden hydrochloride EUROPEAN PHARMACOPOEIA 8.0

ASSAY DEFINITION
Suspend 0.200 g in 5 mL of dimethylformamide R. Heat (1RS)-1-[(1RS,2SR,4RS)-Bicyclo[2.2.1]hept-5-en-2-yl]-1-
until the substance has dissolved completely. Add 50 mL phenyl-3-(piperidin-1-yl)propan-1-ol hydrochloride.
of ethanol R and titrate with 0.1 M tetrabutylammonium Content : 99.0 per cent to 101.0 per cent (dried substance).
hydroxide, determining the end-point potentiometrically
(2.2.20). CHARACTERS
1 mL of 0.1 M tetrabutylammonium hydroxide is equivalent Appearance : white or almost white, crystalline powder.
to 24.43 mg of C10H16N2O3S.
Solubility : slightly soluble in water and in alcohol, very slightly
STORAGE soluble in methylene chloride.
Store protected from light. mp : about 280 °C, with decomposition.
IMPURITIES IDENTIFICATION
First identification : A, D.
Second identification : B, C, D.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: biperiden hydrochloride CRS.
B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 25 mg of the substance to be
A. di[3-[(3aS,4S,6aR)-2-oxohexahydrothieno[3,4-d]imidazol- examined in methanol R and dilute to 5 mL with the same
4-yl]propyl]acetic acid, solvent.
Reference solution (a). Dissolve 25 mg of biperiden
hydrochloride CRS in methanol R and dilute to 5 mL with
the same solvent.
B. 4-[(3aS,4S,6aR)-2-oxohexahydrothieno[3,4-d]imidazol-4- Reference solution (b). Dissolve 5 mg of biperiden
yl]butane-1,1-dicarboxylic acid, impurity A CRS in reference solution (a) and dilute to 2 mL
with the same solution.
Plate : TLC silica gel F254 plate R.
Mobile phase : diethylamine R, methanol R, toluene R
(1:1:20 V/V/V).
C. 5-(3,4-diamino-2-thienyl)pentanoic acid,
Application : 5 μL.
Development : over a path of 15 cm.
Drying : in air.
D. 2-methyl-5-[(3aS,4S,6aR)-2-oxohexahydrothieno[3,4- Detection A : examine in ultraviolet light at 254 nm.
d]imidazol-4-yl]pentanoic acid, Results A : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to
the principal spot in the chromatogram obtained with
reference solution (a).
Detection B : spray with dilute potassium iodobismuthate
solution R and then with sodium nitrite solution R and
examine in daylight.
Results B : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
reference solution (a).
System suitability : reference solution (b) :
E. 5-[(3aS,4S,6aR)-3-benzyl-2-oxohexahydrothieno[3,4- – the chromatogram shows 2 clearly separated spots.
d]imidazol-4-yl]pentanoic acid and 5-[(3aS,4S,6aR)-
1-benzyl-2-oxohexahydrothieno[3,4-d]imidazol-4- C. To about 20 mg add 5 mL of phosphoric acid R. A green
yl]pentanoic acid. colour develops.
D. It gives reaction (a) of chlorides (2.3.1).
01/2008:1074
corrected 6.0 TESTS
Solution S. Dissolve 0.10 g in carbon dioxide-free water R,
BIPERIDEN HYDROCHLORIDE heating gently if necessary, and dilute to 50 mL with the same
solvent.
Biperideni hydrochloridum Appearance of solution. Solution S is not more opalescent
than reference suspension II (2.2.1) and is colourless (2.2.2,
Method II).
pH (2.2.3) : 5.0 to 6.5 for solution S.
Related substances. Gas chromatography (2.2.28).
Test solution. Dissolve 0.10 g of the substance to be examined
in methanol R and dilute to 10 mL with the same solvent.
Reference solution (a). Dilute 0.5 mL of the test solution to
C21H30ClNO Mr 347.9 100 mL with methanol R. Dilute 10 mL of this solution to
[1235-82-1] 50 mL with methanol R.

1672 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Bisacodyl

Reference solution (b). Dissolve 5 mg of the substance to be acceptance criterion for other/unspecified impurities and/or
examined and 5 mg of biperiden impurity A CRS in methanol R by the general monograph Substances for pharmaceutical use
and dilute to 5 mL with the same solvent. Dilute 1 mL of the (2034). It is therefore not necessary to identify these impurities
solution to 10 mL with methanol R. for demonstration of compliance. See also 5.10. Control of
Column : impurities in substances for pharmaceutical use) : D, E.
– material : fused silica,
– size : l = 50 m, Ø = 0.25 mm,
– stationary phase : poly(dimethyl)(diphenyl)(divinyl)silox-
ane R (film thickness 0.25 μm).
Carrier gas : nitrogen for chromatography R.
Flow rate : 0.4 mL/min.
Split ratio : 1:250.
Temperature : A. (1RS)-1-[(1SR,2SR,4SR)-bicyclo[2.2.1]hept-5-en-2-yl]-1-
phenyl-3-(piperidin-1-yl)propan-1-ol (endo form),
Time Temperature
(min) (°C)
Column 0-5 200

5 - 40 200 → 270

Injection port 250

Detector 300

Detection : flame ionisation. B. (1RS)-1-[(1SR,2RS,4SR)-bicyclo[2.2.1]hept-5-en-2-yl]-1-

Injection : 2 μL. phenyl-3-(piperidin-1-yl)propan-1-ol,
Run time : twice the retention time of biperiden.
Relative retention with reference to biperiden : impurities A, B
and C = between 0.95 and 1.05.
System suitability :
– resolution : minimum 2.5 between the peak due to biperiden
(1st peak) and the peak due to impurity A (2nd peak) in the
chromatogram obtained with reference solution (b),
– signal-to-noise ratio : minimum 6 for the principal peak in C. (1RS)-1-[(1RS,2RS,4RS)-bicyclo[2.2.1]hept-5-en-2-yl]-1-
the chromatogram obtained with reference solution (a). phenyl-3-(piperidin-1-yl)propan-1-ol,
Limits :
– impurities A, B, C : for each impurity, maximum 0.50 per
cent of the area of the principal peak,
– any other impurity : for each impurity, maximum 0.10 per
cent of the area of the principal peak,
– total of impurities A, B and C : maximum 1.0 per cent of the
area of the principal peak, D. 1-[(1RS,2SR,4RS)-bicyclo[2.2.1]hept-5-en-2-yl]-3-
– total of impurities other than A, B and C : maximum 0.50 per (piperidin-1-yl)propan-1-one,
cent of the area of the principal peak,
– disregard limit : 0.05 per cent of the area of the principal
peak.
Impurity F (2.4.24) : maximum 2 ppm.
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test D. Prepare the reference solution
using 2 mL of lead standard solution (10 ppm Pb) R. E. 1-[(1RS,2RS,4RS)-bicyclo[2.2.1]hept-5-en-2-yl]-3-
(piperidin-1-yl)propan-1-one,
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 2 h. F. benzene.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. 01/2008:0595
ASSAY corrected 6.0
Dissolve 0.200 g in 60 mL of alcohol R. In a closed vessel,
titrate with 0.1 M alcoholic potassium hydroxide, determining BISACODYL
the end-point potentiometrically (2.2.20).
1 mL of 0.1 M alcoholic potassium hydroxide is equivalent to Bisacodylum
34.79 mg of C21H30ClNO.
STORAGE
In an airtight container, protected from light.
IMPURITIES
Specified impurities : A, B, C, F.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of C22H19NO4 Mr 361.4
the tests in the monograph. They are limited by the general [603-50-9]

General Notices (1) apply to all monographs and other texts 1673
Bisacodyl EUROPEAN PHARMACOPOEIA 8.0

DEFINITION Reference solution (b). Dissolve 2.0 mg of bisacodyl for system

4,4′-(Pyridin-2-ylmethylene)diphenyl diacetate. suitability CRS (containing impurities A, B, C, D and E) in
1.0 mL of acetonitrile R and dilute to 2.0 mL with the solvent
Content : 98.0 per cent to 101.0 per cent (dried substance).
mixture.
CHARACTERS Reference solution (c). Dissolve 5.0 mg of bisacodyl for peak
Appearance : white or almost white, crystalline powder. identification CRS (containing impurity F) in 2.5 mL of
acetonitrile R and dilute to 5.0 mL with the solvent mixture.
Solubility : practically insoluble in water, soluble in acetone,
sparingly soluble in ethanol (96 per cent). It dissolves in dilute Column :
mineral acids. – size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
IDENTIFICATION
chromatography R (5 μm).
First identification : C.
Mobile phase : mix 45 volumes of acetonitrile R and 55 volumes
Second identification : A, B, D. of a 1.58 g/L solution of ammonium formate R previously
A. Melting point (2.2.14) : 131 °C to 135 °C. adjusted to pH 5.0 with anhydrous formic acid R.
B. Ultraviolet and visible absorption spectrophotometry Flow rate : 1.5 mL/min.
(2.2.25). Detection : spectrophotometer at 265 nm.
Test solution. Dissolve 10.0 mg in a 6 g/L solution of Injection : 20 μL.
potassium hydroxide R in methanol R and dilute to 100.0 mL Run time : 3.5 times the retention time of bisacodyl.
with the same solution. Dilute 10.0 mL of this solution to
100.0 mL with a 6 g/L solution of potassium hydroxide R in Identification of impurities : use the chromatogram supplied
methanol R. with bisacodyl for system suitability CRS and the chromatogram
obtained with reference solution (b) to identify the peaks due
Spectral range : 220-350 nm. to impurities A, B, C, D and E.
Absorption maximum : at 248 nm. Relative retention with reference to bisacodyl (retention
Shoulder : at 290 nm. time = about 13 min) : impurity A = about 0.2 ;
Specific absorbance at the absorption maximum : 632 to 672. impurity B = about 0.4 ; impurity C = about 0.45 ;
C. Infrared absorption spectrophotometry (2.2.24). impurity D = about 0.8 ; impurity E = about 0.9 ;
impurity F = about 2.6.
Comparison : bisacodyl CRS.
System suitability : reference solution (b) :
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference – peak-to-valley ratio : minimum 1.5, where Hp = height
substance separately in chloroform R, evaporate to dryness above the baseline of the peak due to impurity E and
and record new spectra using the residues. Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to bisacodyl.
D. Thin-layer chromatography (2.2.27).
Limits :
Test solution. Dissolve 20 mg of the substance to be
examined in acetone R and dilute to 10 mL with the same – correction factor : for the calculation of content, multiply
solvent. the peak area of impurity A by 0.7 ;
Reference solution. Dissolve 20 mg of bisacodyl CRS in – impurities A, B : for each impurity, not more than the area
acetone R and dilute to 10 mL with the same solvent. of the principal peak in the chromatogram obtained with
reference solution (a) (0.1 per cent) ;
Plate : TLC silica gel GF254 plate R.
– impurities C, E : for each impurity, not more than 5 times
Mobile phase : methyl ethyl ketone R, xylene R (50:50 V/V). the area of the principal peak in the chromatogram
Application : 10 μL. obtained with reference solution (a) (0.5 per cent) ;
Development : over a path of 10 cm. – impurity D : not more than twice the area of the principal
Drying : in air, if necessary heating at 100-105 °C. peak in the chromatogram obtained with reference
Detection : spray with a mixture of equal volumes of 0.05 M solution (a) (0.2 per cent) ;
iodine and dilute sulfuric acid R. – impurity F : not more than 3 times the area of the principal
Results : the principal spot in the chromatogram obtained peak in the chromatogram obtained with reference
with the test solution is similar in position and size to the solution (a) (0.3 per cent) ;
principal spot in the chromatogram obtained with the – unspecified impurities : for each impurity, not more than the
reference solution. area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
TESTS – total : not more than 10 times the area of the principal peak
Acidity or alkalinity. To 1.0 g add 20 mL of carbon in the chromatogram obtained with reference solution (a)
dioxide-free water R, shake, heat to boiling, cool and filter. (1.0 per cent) ;
Add 0.2 mL of 0.01 M sodium hydroxide and 0.1 mL of methyl – disregard limit : 0.5 times the area of the principal peak in
red solution R. The solution is yellow. Not more than 0.4 mL the chromatogram obtained with reference solution (a)
of 0.01 M hydrochloric acid is required to change the colour (0.05 per cent).
of the indicator to red.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Related substances. Liquid chromatography (2.2.29). Prepare on 0.500 g by drying in an oven at 105 °C.
the solutions immediately before use.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Solvent mixture : glacial acetic acid R, acetonitrile R, water R 1.0 g.
(4:30:66 V/V/V).
Test solution. Dissolve 50 mg of the substance to be examined ASSAY
in 25 mL of acetonitrile R and dilute to 50.0 mL with the Dissolve 0.300 g in 60 mL of anhydrous acetic acid R.
solvent mixture. Titrate with 0.1 M perchloric acid determining the end-point
Reference solution (a). Dilute 1.0 mL of the test solution to potentiometrically (2.2.20).
100.0 mL with the solvent mixture. Dilute 1.0 mL of this 1 mL of 0.1 M perchloric acid is equivalent to 36.14 mg
solution to 10.0 mL with the solvent mixture. of C22H19NO4.

1674 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Bismuth subcarbonate

STORAGE Alkali and alkaline-earth metals : maximum 1.0 per cent.

Protected from light. To 1.0 g add 10 mL of water R and 10 mL of acetic acid R.
Boil for 2 min, cool and filter. Wash the residue with 20 mL
IMPURITIES of water R. To the combined filtrate and washings add 2 mL
Specified impurities : A, B, C, D, E, F. of dilute hydrochloric acid R and 20 mL of water R. Boil and
pass hydrogen sulfide R through the boiling solution until
no further precipitate is formed. Filter, wash the residue
with water R, evaporate the combined filtrate and washings
to dryness on a water-bath and add 0.5 mL of sulfuric
acid R. Ignite gently and allow to cool. The residue weighs
a maximum of 10 mg.
Arsenic (2.4.2, Method A) : maximum 5 ppm.
A. R1 = R3 = OH, R2 = H : 4,4′-(pyridin-2-ylmethylene)di- To 0.5 g in a distillation flask add 5 mL of water R and 7 mL of
phenol, sulfuric acid R, allow to cool and add 5 g of reducing mixture R
and 10 mL of hydrochloric acid R. Heat the contents of the
B. R1 = H, R2 = R3 = OH : 2-[(RS)-(4-hydroxyphenyl)(pyridin- flask to boiling gradually over 15-30 min and continue heating
2-yl)methyl]phenol, at such a rate that the distillation proceeds steadily until
the volume in the flask is reduced by half or until 5 min after
C. R1 = OH, R2 = H, R3 = O-CO-CH3 : 4-[(RS)-(4- the air-condenser has become full of steam. It is important
hydroxyphenyl)(pyridin-2-yl)methyl]phenyl acetate, that distillation be discontinued before fumes of sulfur trioxide
appear. Collect the distillate in a tube containing 15 mL of
E. R1 = H, R2 = R3 = O-CO-CH3 : 2-[(RS)-[4-(acetyloxy)- water R cooled in ice-water. Wash down the condenser with
phenyl](pyridin-2-yl)methyl]phenyl acetate, water R and dilute the distillate to 25 mL with the same
solvent. Prepare the standard using a mixture of 2.5 mL of
D. unknown structure, arsenic standard solution (1 ppm As) R and 22.5 mL of water R.
Copper : maximum 50 ppm.
F. unknown structure. To 5 mL of solution S, add 2 mL of ammonia R and dilute
to 50 mL with water R. Filter. To 10 mL of the filtrate add
1 mL of a 1 g/L solution of sodium diethyldithiocarbamate R.
The solution is not more intensely coloured than a standard
01/2008:0012 prepared at the same time in the same manner using a mixture
corrected 7.0 of 0.25 mL of copper standard solution (10 ppm Cu) R and
9.75 mL of water R instead of 10 mL of the filtrate.
BISMUTH SUBCARBONATE Lead : maximum 20 ppm.
Atomic absorption spectrometry (2.2.23, Method II).
Bismuthi subcarbonas Test solution. Dissolve 12.5 g in 75 mL of a mixture of
DEFINITION equal volumes of lead-free nitric acid R and water R. Boil for
1 min, cool and dilute to 100.0 mL with water R.
Content : 80.0 per cent to 82.5 per cent of Bi (Ar 209.0) (dried
substance). Reference solutions. Prepare the reference solutions using
appropriate quantities of lead standard solution and a 37 per
CHARACTERS cent V/V solution of lead-free nitric acid R.
Appearance : white or almost white powder. Source : lead hollow-cathode lamp.
Solubility : practically insoluble in water and in ethanol (96 per Wavelength : 283.3 nm (depending on the apparatus, the line
cent). It dissolves with effervescence in mineral acids. at 217.0 nm may be used).
IDENTIFICATION Atomisation device : air-acetylene flame.
A. It gives the reaction of carbonates (2.3.1). Silver: maximum 25 ppm.
B. It gives the reactions of bismuth (2.3.1). To 2.0 g add 1 mL of water R and 4 mL of nitric acid R. Heat
gently until dissolved and dilute to 11 mL with water R.
TESTS Cool and add 2 mL of 1 M hydrochloric acid. Allow to stand
Solution S. Shake 5.0 g with 10 mL of water R and add 20 mL protected from light for 5 min. Any opalescence in the
of nitric acid R. Heat to dissolve, cool and dilute to 100 mL solution is not more intense than that in a standard prepared
with water R. at the same time in the same manner using a mixture of 10 mL
of silver standard solution (5 ppm Ag) R, 1 mL of nitric acid R
Appearance of solution. Solution S is not more opalescent and 2 mL of 1 M hydrochloric acid.
than reference suspension II (2.2.1) and is colourless (2.2.2,
Method II). Loss on drying (2.2.32) : maximum 1.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Chlorides (2.4.4): maximum 500 ppm.
To 6.6 mL of solution S add 4 mL of nitric acid R and dilute ASSAY
to 50 mL with water R.
Dissolve 0.500 g in 3 mL of nitric acid R and dilute to 250 mL
Nitrates : maximum 0.4 per cent. with water R. Carry out the complexometric titration of
To 0.25 g in a 125 mL conical flask, add 20 mL of water R, bismuth (2.5.11).
0.05 mL of indigo carmine solution R1 and then, as a single 1 mL of 0.1 M sodium edetate is equivalent to 20.90 mg of Bi.
addition but with caution, 30 mL of sulfuric acid R. Titrate
immediately with indigo carmine solution R1 until a stable
blue colour is obtained. Not more than n mL of the titrant is STORAGE
required, n being the volume corresponding to 1 mg of NO3. Protected from light.

General Notices (1) apply to all monographs and other texts 1675
Bismuth subgallate EUROPEAN PHARMACOPOEIA 8.0

01/2008:1493 Lead : maximum 20 ppm.

corrected 7.0 Atomic absorption spectrometry (2.2.23, Method II).
Test solution. Solution S.
BISMUTH SUBGALLATE Reference solutions. Prepare the reference solutions using lead
standard solution (10 ppm Pb) R and diluting with a 6.5 per
Bismuthi subgallas cent V/V solution of lead-free nitric acid R.
Source : lead hollow-cathode lamp.
Wavelength : 283.3 nm (depending on the apparatus, the line
at 217.0 nm may be used).
Atomisation device : air-acetylene flame.
Silver: maximum 25 ppm.
C7H5BiO6 Mr 394.1
[99-26-3] Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Solution S.
DEFINITION Reference solutions. Prepare the reference solutions using
Complex of bismuth and gallic acid. silver standard solution (5 ppm Ag) R and diluting with a
Content : 48.0 per cent to 51.0 per cent of Bi (Ar 209.0) (dried 6.5 per cent V/V solution of lead-free nitric acid R.
substance). Source : silver hollow-cathode lamp.
CHARACTERS Wavelength : 328.1 nm.
Appearance : yellow powder. Atomisation device : air-acetylene flame.
Solubility : practically insoluble in water and in ethanol (96 per Substances not precipitated by ammonia : maximum 1.0 per
cent). It dissolves in mineral acids with decomposition and cent.
in solutions of alkali hydroxides, producing a reddish-brown In a porcelain or quartz dish, ignite 2.0 g, increasing the
liquid. temperature very gradually to 600 ± 50 °C ; allow to cool.
Moisten the residue with 2 mL of nitric acid R, evaporate to
IDENTIFICATION dryness on a water-bath and carefully heat and ignite once
A. Mix 0.1 g with 5 mL of water R and 0.1 mL of phosphoric more at 600 ± 50 °C. After cooling, dissolve the residue in 5 mL
acid R. Heat to boiling and maintain boiling for 2 min. of nitric acid R and dilute to 20 mL with water R. To 10 mL
Cool and filter. To the filtrate, add 1.5 mL of ferric chloride of this solution, add concentrated ammonia R until alkaline
solution R1 ; a blackish-blue colour develops. and filter. Wash the residue with water R and evaporate the
B. It gives reaction (b) of bismuth (2.3.1). combined filtrate and washings to dryness on a water-bath.
Add 0.3 mL of dilute sulfuric acid R and ignite. The residue
TESTS weighs a maximum of 10 mg.
Solution S. In a porcelain or quartz dish, ignite 1.0 g, Loss on drying (2.2.32) : maximum 7.0 per cent, determined
increasing the temperature very gradually. Heat in a muffle on 1.000 g by drying in an oven at 105 °C for 3 h.
furnace at 600 ± 50 °C for 2 h. Cool and dissolve the residue
with warming in 4 mL of a mixture of equal volumes of ASSAY
lead-free nitric acid R and water R and dilute to 20 mL with To 0.300 g add 10 mL of a mixture of equal volumes of nitric
water R. acid R and water R, heat to boiling and maintain boiling for
Acidity. Shake 1.0 g with 20 mL of water R for 1 min and 2 min. Add 0.1 g of potassium chlorate R, heat to boiling and
filter. To the filtrate add 0.1 mL of methyl red solution R. Not maintain boiling for 1 min. Add 10 mL of water R and heat
more than 0.15 mL of 0.1 M sodium hydroxide is required to until the solution becomes colourless. To the hot solution, add
change the colour of the indicator to yellow. 200 mL of water R and 50 mg of xylenol orange triturate R.
Chlorides (2.4.4) : maximum 200 ppm. Titrate with 0.1 M sodium edetate until a yellow colour is
obtained.
To 0.5 g add 10 mL of dilute nitric acid R. Heat on a water-bath
for 5 min and filter. Dilute 5 mL of the filtrate to 15 mL with 1 mL of 0.1 M sodium edetate is equivalent to 20.90 mg of Bi.
water R. STORAGE
Nitrates : maximum 0.2 per cent. Protected from light.
To 1.0 g add 25 mL of water R then 25 mL of a mixture of
2 volumes of sulfuric acid R and 9 volumes of water R. Heat at
about 50 °C for 1 min with stirring and filter. To 10 mL of the 01/2008:1494
filtrate, carefully add 30 mL of sulfuric acid R. The solution is corrected 7.0
not more intensely brownish-yellow than a reference solution
prepared at the same time as follows : to 0.4 g of gallic acid R, BISMUTH SUBNITRATE, HEAVY
add 20 mL of nitrate standard solution (100 ppm NO3) R
and 30 mL of a mixture of 2 volumes of sulfuric acid R and Bismuthi subnitras ponderosus
9 volumes of water R, then filter ; to 10 mL of the filtrate,
carefully add 30 mL of sulfuric acid R.
4[BiNO3(OH)2],BiO(OH) Mr 1462
Copper : maximum 50 ppm. [1304-85-4]
Atomic absorption spectrometry (2.2.23, Method I).
DEFINITION
Test solution. Solution S.
Content : 71.0 per cent to 74.0 per cent of Bi (Ar 209.0) (dried
Reference solutions. Prepare the reference solutions using
substance).
copper standard solution (10 ppm Cu) R and diluting with a
6.5 per cent V/V solution of lead-free nitric acid R. CHARACTERS
Source : copper hollow-cathode lamp. Appearance : white or almost white powder.
Wavelength : 324.7 nm. Solubility : practically insoluble in water and in ethanol (96 per
Atomisation device : air-acetylene flame. cent). It dissolves in mineral acids with decomposition.

1676 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Bismuth subsalicylate

IDENTIFICATION Loss on drying (2.2.32) : maximum 3.0 per cent, determined

A. Dilute 1 mL of solution S1 (see Tests) to 5 mL with water R on 1.000 g by drying in an oven at 105 °C.
and add 0.3 mL of potassium iodide solution R. A black
ASSAY
precipitate is formed which dissolves into an orange
solution with the addition of 2 mL of potassium iodide Dissolve with heating 0.250 g in 10 mL of a mixture of
solution R. 2 volumes of perchloric acid R and 5 volumes of water R. To
the hot solution, add 200 mL of water R and 50 mg of xylenol
B. It gives reaction (b) of bismuth (2.3.1).
orange triturate R. Titrate with 0.1 M sodium edetate until a
C. It gives the reaction of nitrates (2.3.1). yellow colour is obtained.
D. pH (2.2.3): maximum 2.0 for solution S2 (see Tests). 1 mL of 0.1 M sodium edetate is equivalent to 20.90 mg of Bi.
TESTS
01/2008:1495
Solution S1. Shake 5.0 g by gently heating in 10 mL of water R corrected 7.0
and add 20 mL of nitric acid R. Heat until dissolution, cool
and dilute to 100 mL with water R.
BISMUTH SUBSALICYLATE
Solution S2. Place 1.00 g in a 20 mL volumetric flask and
add 2.0 mL of lead-free nitric acid R. Allow acid attack to Bismuthi subsalicylas
take place without heating and if necessary warm slightly at
the end to completely dissolve the test sample. Add 10 mL of C7H5BiO4 Mr 362.1
water R, shake and add, in small fractions, 4.5 mL of lead-free [14882-18-9]
ammonia R ; shake and allow to cool. Dilute to 20.0 mL with
water R, shake again and allow the solids to settle. The clear DEFINITION
supernatant solution is solution S2. Complex of bismuth and salicylic acid.
Acidity. Suspend 1.0 g in 15 mL of water R and shake Content : 56.0 per cent to 59.4 per cent of Bi (Ar 209.0) (dried
several times. Allow to stand for 5 min and filter. To 10 mL substance).
of the filtrate, add 0.5 mL of phenolphthalein solution R1. Not
more than 0.5 mL of 0.1 M sodium hydroxide is required to CHARACTERS
change the colour of the indicator to pink. Appearance : white or almost white powder.
Chlorides (2.4.4): maximum 200 ppm. Solubility : practically insoluble in water and in alcohol. It
To 5.0 mL of solution S1, add 3 mL of nitric acid R and dilute dissolves in mineral acids with decomposition.
to 15 mL with water R. IDENTIFICATION
Copper : maximum 50 ppm. A. To 0.5 g add 10 mL of hydrochloric acid R1. Heat on a
Atomic absorption spectrometry (2.2.23, Method I). boiling water-bath for 5 min. Cool and filter. Retain the
Test solution. Solution S2. filtrate for identification test B. Wash the residue with dilute
Reference solutions. Prepare the reference solutions using hydrochloric acid R and then with water R. Dissolve the
copper standard solution (10 ppm Cu) R and diluting with a residue in 0.5-1 mL of dilute sodium hydroxide solution R.
37 per cent V/V solution of lead-free nitric acid R. Add 15 mL of water R. Neutralise with dilute hydrochloric
acid R. The solution gives reaction (a) of salicylates (2.3.1).
Source : copper hollow-cathode lamp.
B. The filtrate obtained in identification test A gives
Wavelength : 324.7 nm. reaction (b) of bismuth (2.3.1).
Atomisation device : air-acetylene flame.
Lead : maximum 20 ppm. TESTS
Atomic absorption spectrometry (2.2.23, Method II). Solution S. In a porcelain or quartz dish, ignite 1.0 g,
increasing the temperature very gradually. Heat in a muffle
Test solution. Solution S2.
furnace at 600 ± 25 °C for 2 h. Cool and dissolve the residue
Reference solutions. Prepare the reference solutions using lead with warming in 4 mL of a mixture of equal volumes of
standard solution (10 ppm Pb) R and diluting with a 37 per lead-free nitric acid R and water R and dilute to 20 mL with
cent V/V solution of lead-free nitric acid R. water R.
Source : lead hollow-cathode lamp. Acidity. Shake 2.0 g with 30 mL of ether R for 1 min and filter.
Wavelength : 283.3 nm (depending on the apparatus, the line To the filtrate add 30 mL of alcohol R and 0.1 mL of thymol
at 217.0 nm may be used). blue solution R. Not more than 0.35 mL of 0.1 M sodium
Atomisation device : air-acetylene flame. hydroxide is required to change the colour of the indicator
Silver : maximum 25 ppm. to blue.
Atomic absorption spectrometry (2.2.23, Method I). Chlorides (2.4.4) : maximum 200 ppm.
Test solution. Solution S2. Dissolve 0.250 g in a mixture of 2 mL of nitric acid R, 5 mL of
Reference solutions. Prepare the reference solutions using water R and 8 mL of methanol R.
silver standard solution (5 ppm Ag) R and diluting with a Nitrates : maximum 0.4 per cent.
37 per cent V/V solution of lead-free nitric acid R. To 0.1 g add 10 mL of water R and, with caution, 20 mL of
Source : silver hollow-cathode lamp. sulfuric acid R and stir. The solution is not more intensely
Wavelength : 328.1 nm. yellow coloured than a reference solution prepared at the
Atomisation device : air-acetylene flame. same time using 0.1 g of salicylic acid R, 6 mL of water R,
4 mL of nitrate standard solution (100 ppm NO3) R and 20 mL
Substances not precipitated by ammonia : maximum 1.0 per of sulfuric acid R.
cent.
Copper : maximum 50 ppm.
To 20 mL of solution S1, add concentrated ammonia R until an
alkaline reaction is produced and filter. Wash the residue with Atomic absorption spectrometry (2.2.23, Method I).
water R, and evaporate the combined filtrate and washings to Test solution. Solution S.
dryness on a water-bath. To the residue, add 0.3 mL of dilute Reference solutions. Prepare the reference solutions using
sulfuric acid R and ignite. The residue weighs a maximum copper standard solution (10 ppm Cu) R and diluting with a
of 10 mg. 6.5 per cent V/V solution of lead-free nitric acid R.

General Notices (1) apply to all monographs and other texts 1677
Bisoprolol fumarate EUROPEAN PHARMACOPOEIA 8.0

Source : copper hollow-cathode lamp. DEFINITION

Wavelength : 324.7 nm. (2RS)-1-[4-[[2-(1-Methylethoxy)ethoxy]methyl]phenoxy]-3-
Atomisation device : air-acetylene flame. [(1-methylethyl)amino]propan-2-ol fumarate.
Lead : maximum 20 ppm. Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
Atomic absorption spectrometry (2.2.23, Method II). CHARACTERS
Test solution. Solution S. Appearance : white or almost white, slightly hygroscopic
Reference solutions. Prepare the reference solutions using lead powder.
standard solution (10 ppm Pb) R and diluting with a 6.5 per Solubility : very soluble in water, freely soluble in methanol.
cent V/V solution of lead-free nitric acid R. It shows polymorphism (5.9).
Source : lead hollow-cathode lamp.
IDENTIFICATION
Wavelength : 283.3 nm (depending on the apparatus, the line
at 217.0 nm may be used). Infrared absorption spectrophotometry (2.2.24).
Atomisation device : air-acetylene flame. Comparison: bisoprolol fumarate CRS.
If the spectra obtained in the solid state show differences,
Silver : maximum 25 ppm.
dissolve the substance to be examined and the reference
Atomic absorption spectrometry (2.2.23, Method I). substance separately in methanol R, evaporate and dry the
Test solution. Solution S. residues at 60 °C at a pressure not exceeding 0.7 kPa and
Reference solutions. Prepare the reference solutions using record new spectra using the residues.
silver standard solution (5 ppm Ag) R and diluting with a
TESTS
6.5 per cent V/V solution of lead-free nitric acid R.
Source : silver hollow-cathode lamp. Related substances. Liquid chromatography (2.2.29).
Wavelength : 328.1 nm. Solvent mixture : acetonitrile R1, water for chromatography R
(20:80 V/V).
Atomisation device : air-acetylene flame.
Test solution. Dissolve 25 mg of the substance to be examined
Soluble bismuth : maximum 40 ppm. in the solvent mixture and dilute to 25.0 mL with the solvent
Atomic absorption spectrometry (2.2.23, Method I). mixture.
Test solution. Suspend 5.0 g in 100 mL of water R. Stir Reference solution (a). Dilute 1.0 mL of the test solution to
constantly for 2 h at 20-23 °C. Filter through filter paper (slow 100.0 mL with the solvent mixture. Dilute 2.0 mL of this
filtration) then through a cellulose micropore membrane filter solution to 10.0 mL with the solvent mixture.
(0.1 μm). To 10.0 mL of clear filtrate, add 0.1 mL of nitric Reference solution (b). Dissolve the contents of a vial of
acid R. bisoprolol for peak identification CRS (containing impurities A
Reference solutions. Prepare the reference solutions using and E) in 1.0 mL of the solvent mixture.
bismuth standard solution (100 ppm Bi) R and diluting with a Reference solution (c). Dissolve the contents of a vial of
mixture of equal volumes of dilute nitric acid R and water R. bisoprolol for system suitability CRS (containing impurity G)
Source : bismuth hollow-cathode lamp. in 1.0 mL of the solvent mixture.
Wavelength : 223.06 nm. Column :
Atomisation device : air-acetylene flame. – size : l = 0.25 m, Ø = 4.6 mm ;
Loss on drying (2.2.32) : maximum 1.0 per cent, determined – stationary phase : octadecylsilyl silica gel for
on 1.000 g by drying in an oven at 105 °C. chromatography R (5 μm) ;
– temperature : 20 ± 2 °C.
ASSAY
Mobile phase :
Dissolve with heating 0.300 g in 10 mL of a mixture of – mobile phase A : 10 g/L solution of phosphoric acid R ;
2 volumes of perchloric acid R and 5 volumes of water R. To
the hot solution, add 200 mL of water R and 50 mg of xylenol – mobile phase B : 10 g/L solution of phosphoric acid R in
orange triturate R. Titrate with 0.1 M sodium edetate until a acetonitrile R1 ;
yellow colour is obtained. Time Mobile phase A Mobile phase B
1 mL of 0.1 M sodium edetate is equivalent to 20.90 mg of Bi. (min) (per cent V/V) (per cent V/V)
0-4 95 5
STORAGE
4-8 95 → 80 5 → 20
Protected from light.
8 - 15 80 20

15 - 34 80 → 20 20 → 80
01/2012:1710
34 - 36 20 80
BISOPROLOL FUMARATE Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 225 nm.
Bisoprololi fumaras
Injection : 10 μL.
Identification of impurities : use the chromatogram
supplied with bisoprolol for peak identification CRS and
the chromatogram obtained with reference solution (b) to
identify the peaks due to fumaric acid and impurities A and
E ; use the chromatogram supplied with bisoprolol for system
suitability CRS and the chromatogram obtained with reference
solution (c) to identify the peak due to impurity G.
Relative retention with reference to bisoprolol (retention
C40H66N2O12 Mr 767 time = about 18 min) : impurity A = about 0.5 ;
[104344-23-2] impurity G = about 1.1 ; impurity E = about 1.2.

1678 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Bisoprolol fumarate

System suitability: reference solution (c) :

– peak-to-valley ratio : minimum 2.5, where Hp = height
above the baseline of the peak due to impurity G and
Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to bisoprolol.
Limits :
– impurity G : not more than 2.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent) ;
– impurity A : not more than 1.5 times the area of the
principal peak in the chromatogram obtained with C. 1-[4-[4-(2-hydroxy-3-isopropylamino-propoxy)-
reference solution (a) (0.3 per cent) ; benzyl]phenoxy]-3-isopropylaminopropan-2-ol,
– impurity E : not more than the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.2 per cent) ;
– unspecified impurities : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent) ;
– total : not more than 2.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
– disregard limit : 0.25 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent); disregard the peak due to fumaric acid. D. 1-[4-[4-(2-hydroxy-3-isopropylaminopropoxy)benzyloxyl-
methyl]phenoxy]-3-isopropylaminopropan-2-ol,
Water (2.5.12) : maximum 0.5 per cent, determined on 1.000 g.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.

ASSAY
Dissolve 0.300 g in 50 mL of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point E. (EZ)-[3-[4-(2-isopropoxy-ethoxymethyl)phenoxy]allyl]-
potentiometrically (2.2.20). isopropylamine,
1 mL of 0.1 M perchloric acid is equivalent to 38.35 mg
of C40H66N2O12.

STORAGE
In an airtight container, protected from light.

IMPURITIES
Specified impurities : A, E, G. F. (2RS)-2-[4-(2-isopropoxy-ethoxymethyl)phenoxy]-3-
isopropylaminopropan-2-ol,
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use) : B,
C, D, F, K, L, N, Q, R, S, T, U. G. (2RS)-1-[4-[[(2-isopropoxyethoxy)methoxy]methyl]phe-
noxy]-3-isopropylaminopropan-2-ol,

A. (2RS)-1-(4-hydroxymethyl-phenoxy)-3-isopropylamino-
propan-2-ol,
K. 2-isopropoxyethyl 4-[[(2RS)-2-hydroxy-3-
(isopropylamino)propyl]oxy]benzoate,

B. (2RS)-1-isopropylamino-3-[4-(2-propoxy-ethoxymethyl)- L. 4-[[(2RS)-2-hydroxy-3-(isopropylamino)propyl]oxy]-
phenoxy]propan-2-ol, benzaldehyde,

General Notices (1) apply to all monographs and other texts 1679
Bleomycin sulfate EUROPEAN PHARMACOPOEIA 8.0

01/2008:0976
corrected 7.8

BLEOMYCIN SULFATE
Bleomycini sulfas
N. (2RS)-1-[4-[(2-ethoxyethoxy)methyl]phenoxy]-3-
isopropylaminopropan-2-ol,

Q. (2RS)-1-(isopropylamino)-3-[4-(2-
methoxyethoxy)methyl]phenoxypropan-2-ol,

[9041-93-4]
DEFINITION
Sulfate of a mixture of glycopeptides produced by
Streptomyces verticillus or by any other means ; the
2 principal components of the mixture are N-[3-
(dimethylsulfonio)propyl]bleomycinamide (bleomycin A2)
R. (2RS)-1-(isopropylamino)-3-(4-methylphenoxy)propan- and N-[4-(carbamimidoylamino)butyl]bleomycinamide
2-ol, (bleomycin B2).
Potency : minimum 1500 IU/mg (dried substance).
CHARACTERS
Appearance : white or yellowish-white, very hygroscopic
powder.
Solubility : very soluble in water, slightly soluble in anhydrous
ethanol, practically insoluble in acetone.
IDENTIFICATION
S. 4-hydroxybenzaldehyde, A. Examine the chromatograms obtained in the test for
composition.
Results : the 2 principal peaks in the chromatogram
obtained with the test solution are similar in retention time
and size to the 2 principal peaks in the chromatogram
obtained with reference solution (a).
B. It gives the reactions of sulfates (2.3.1).
TESTS
Appearance of solution. The solution is clear (2.2.1) and its
absorbance (2.2.25) at 430 nm is not greater than 0.10.
Dissolve 0.200 g in water R and dilute to 10.0 mL with the
T. 4-[(3-isopropyl-2-oxo-1,3-oxazolidin-5-yl)methoxy]- same solvent.
benzaldehyde, pH (2.2.3) : 4.5 to 6.0.
Dissolve 50 mg in carbon dioxide-free water R and dilute to
10 mL with the same solvent.
Composition. Liquid chromatography (2.2.29) : use the
normalisation procedure.
Test solution. Dissolve 25.0 mg of the substance to be
examined in water R and dilute to 50.0 mL with the same
solvent.
Reference solution (a). Dissolve the contents of a vial of
bleomycin sulfate CRS in water R and dilute to 10.0 mL with
the same solvent.
U. 5-[[4-(hydroxymethyl)phenoxy]methyl]-3-isopropyl-1,3- Reference solution (b). Dilute 1.5 mL of reference solution (a)
oxazolidin-2-one. to 100.0 mL with water R.

1680 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Borage (starflower) oil, refined

Column : Other detectable impurities (the following substances would,

– size : l = 0.25 m, Ø = 4.6 mm ; if present at a sufficient level, be detected by one or other of
– stationary phase : octadecylsilyl silica gel for the tests in the monograph. They are limited by the general
chromatography R (7 μm). acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
Mobile phase : (2034). It is therefore not necessary to identify these impurities
– mobile phase A : methanol R ; for demonstration of compliance. See also 5.10. Control of
– mobile phase B : dissolve 0.960 g of sodium impurities in substances for pharmaceutical use): A, B, C.
pentanesulfonate R in 900 mL of acetic acid (4.8 g/L
C2H4O2), add 1.86 g of sodium edetate R, dilute to 1000 mL
with the same solvent and adjust to pH 4.3 with ammonia R ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 60 10 → 40 90 → 60

60 - end 40 60

Flow rate : 1.2 mL/min.

Detection : spectrophotometer at 254 nm.
Injection : 20 μL.
Run time : until impurity D is eluted (about 80 min).
Relative retention with reference to bleomycin A2 :
impurity D = 1.5 to 2.5.
System suitability :
– resolution : minimum 5 between the peaks due to bleomycin
A2 (1st principal peak) and bleomycin B2 (2nd principal peak)
in the chromatogram obtained with reference solution (a); A. R = OH : bleomycinic acid,
– signal-to-noise ratio : minimum 20 for the principal peak in B. R = NH-[CH2]3-NH-[CH2]4-NH2 : bleomycin A5,
the chromatogram obtained with reference solution (b) ;
– repeatability : maximum relative standard deviation of 2 per C. R = NH-[CH2]4-NH-C(=NH)-NH-[CH2]4-NH-C(=NH)-
cent for the principal peak after 6 injections of reference NH2 : bleomycin B4,
solution (a). D. R = NH-[CH2]3-S-CH3 : demethylbleomycin A2.
Limits :
01/2010:2105
– bleomycin A2 : 55 per cent to 70 per cent ;
– bleomycin B2 : 25 per cent to 32 per cent ;
BORAGE (STARFLOWER) OIL,
– sum of bleomycin A2 and B2 : minimum 85 per cent ;
– impurity D : maximum 5.5 per cent ;
REFINED
– sum of impurities other than D : maximum 9.5 per cent ;
Boraginis officinalis oleum raffinatum
– disregard limit : 0.1 per cent of the total.
Copper : maximum 200 ppm. DEFINITION
Atomic absorption spectrometry (2.2.23, Method I). Fatty oil obtained from seeds of Borago officinalis L. by
extraction and/or expression. It is then refined. A suitable
Test solution. Dissolve 50 mg in water R and dilute to 10.0 mL
antioxidant may be added.
with the same solvent.
Reference solution. Dilute 1.0 mL of copper standard solution CHARACTERS
(10 ppm Cu) R to 10.0 mL with water R. Appearance : clear, light yellow or yellow liquid.
Source : copper hollow-cathode lamp. Solubility : practically insoluble in water and in ethanol (96 per
Wavelength : 324.7 nm. cent), miscible with light petroleum.
Atomisation device : air-acetylene flame. Relative density : about 0.921.
Loss on drying (2.2.32) : maximum 3.0 per cent, determined Refractive index : about 1.476.
on 50 mg by drying at 60 °C at a pressure not exceeding IDENTIFICATION
0.67 kPa for 3 h.
First identification : B.
Bacterial endotoxins (2.6.14): less than 5 IU/mg, if intended Second identification : A.
for use in the manufacture of parenteral preparations without
a further appropriate procedure for the removal of bacterial A. Identification of fatty oils by thin-layer chromatography
endotoxins. (2.3.2).
Results : the chromatogram obtained is similar to the
ASSAY corresponding chromatogram shown in Figure 2.3.2.-1.
Carry out the microbiological assay of antibiotics (2.7.2), B. Composition of fatty acids (see Tests).
using the diffusion method. Use bleomycin sulfate CRS as the
chemical reference substance. TESTS
Acid value (2.5.1) : maximum 0.5, or maximum 0.3 if intended
STORAGE for use in the manufacture of parenteral preparations.
In an airtight container, at a temperature of 2 °C to 8 °C. If the
substance is sterile, store in a sterile, airtight, tamper-proof Peroxide value (2.5.5, Method A) : maximum 10.0, or
container. maximum 5.0 if intended for use in the manufacture of
parenteral preparations.
IMPURITIES Unsaponifiable matter (2.5.7) : maximum 2.0 per cent,
Specified impurities : D. determined on 5.0 g.

General Notices (1) apply to all monographs and other texts 1681
Borax EUROPEAN PHARMACOPOEIA 8.0

Alkaline impurities (2.4.19). It complies with the test. Sulfates (2.4.13) : maximum 50 ppm, determined on
Composition of fatty acids (2.4.22, Method A). Use the solution S.
mixture of calibrating substances in Table 2.4.22.-3. Use in this test 1.0 mL of acetic acid R. Prepare the standard
Composition of the fatty-acid fraction of the oil: using a mixture of 3 mL of sulfate standard solution
(10 ppm SO4) R and 12 mL of distilled water R.
– saturated fatty acids of chain length less than C16 : maximum
0.3 per cent, Ammonium (2.4.1) : maximum 10 ppm.
– palmitic acid : 9.0 per cent to 12.0 per cent, Dilute 6 mL of solution S to 14 mL with water R. Prepare the
standard using a mixture of 2.5 mL of ammonium standard
– palmitoleic acid : maximum 0.6 per cent, solution (1 ppm NH4) R and 7.5 mL of water R.
– stearic acid : 2.0 per cent to 6.0 per cent,
Arsenic (2.4.2, Method A) : maximum 5 ppm, determined on
– oleic acid : 12.0 per cent to 22.0 per cent, 5 mL of solution S.
– linoleic acid : 30.0 per cent to 41.0 per cent, Calcium (2.4.3) : maximum 100 ppm, determined on
– gamma-linolenic acid : 17.0 per cent to 27.0 per cent, solution S.
– alpha-linolenic acid : maximum 0.5 per cent, Prepare the standard using a mixture of 6 mL of calcium
– arachidic acid : maximum 0.5 per cent, standard solution (10 ppm Ca) R and 9 mL of distilled water R.
– eicosenoic acid : 2.8 per cent to 4.4 per cent, Heavy metals (2.4.8) : maximum 25 ppm.
– erucic acid : maximum 3.0 per cent, 12 mL of solution S complies with test A. Prepare the reference
– nervonic acid : maximum 4.5 per cent. solution using lead standard solution (1 ppm Pb) R.
Brassicasterol (2.4.23) : maximum 0.3 per cent in the sterol ASSAY
fraction of the oil. Dissolve 20 g of mannitol R in 100 mL of water R, heating if
Water (2.5.32) : maximum 0.1 per cent, determined on 1.00 g. necessary, cool and add 0.5 mL of phenolphthalein solution R
and neutralise with 0.1 M sodium hydroxide until a pink colour
STORAGE is obtained. Add 3.00 g of the substance to be examined, heat
Under an inert gas, in a well-filled, airtight container, until dissolution is complete, cool, and titrate with 1 M sodium
protected from light. hydroxide until the pink colour reappears.
1 mL of 1 M sodium hydroxide is equivalent to 0.1907 g
LABELLING of Na2B4O7,10H2O.
The label states, where applicable, that the oil is suitable for
use in the manufacture of parenteral preparations. 01/2008:0001
corrected 6.0
01/2008:0013
corrected 6.0 BORIC ACID

BORAX Acidum boricum

H3BO3 Mr 61.8
Borax [10043-35-3]

Na2B4O7,10H2O Mr 381.4 DEFINITION

[1303-96-4] Content : 99.0 per cent to 100.5 per cent.
DEFINITION CHARACTERS
Disodium tetraborate decahydrate. Appearance : white or almost white, crystalline powder,
Content : 99.0 per cent to 103.0 per cent of Na2B4O7,10H2O. colourless, shiny plates greasy to the touch, or white or almost
white crystals.
CHARACTERS Solubility : soluble in water and in ethanol (96 per cent), freely
Appearance : white or almost white, crystalline powder, soluble in boiling water and in glycerol (85 per cent).
colourless crystals or crystalline masses, efflorescent. IDENTIFICATION
Solubility : soluble in water, very soluble in boiling water, freely A. Dissolve 0.1 g by gently heating in 5 mL of methanol R,
soluble in glycerol. add 0.1 mL of sulfuric acid R and ignite the solution. The
IDENTIFICATION flame has a green border.
A. To 1 mL of solution S (see Tests) add 0.1 mL of sulfuric B. Solution S (see Tests) is acid (2.2.4).
acid R and 5 mL of methanol R and ignite. The flame has a TESTS
green border.
Solution S. Dissolve 3.3 g in 80 mL of boiling distilled water R,
B. To 5 mL of solution S add 0.1 mL of phenolphthalein cool and dilute to 100 mL with carbon dioxide-free water R
solution R. The solution is red. On the addition of 5 mL of prepared from distilled water R.
glycerol (85 per cent) R the colour disappears.
C. Solution S gives the reactions of sodium (2.3.1). Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
TESTS pH (2.2.3) : 3.8 to 4.8 for solution S.
Solution S. Dissolve 4.0 g in carbon dioxide-free water R Solubility in ethanol (96 per cent). The solution is not
prepared from distilled water R and dilute to 100 mL with the more opalescent than reference suspension II (2.2.1) and is
same solvent. colourless (2.2.2, Method II).
Appearance of solution. Solution S is clear (2.2.1) and Dissolve 1.0 g in 10 mL of boiling ethanol (96 per cent) R.
colourless (2.2.2, Method II). Organic matter. It does not darken on progressive heating to
pH (2.2.3): 9.0 to 9.6 for solution S. dull redness.

1682 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Botulinum toxin type A for injection

Sulfates (2.4.13) : maximum 450 ppm. F), with known origin and history, is grown using suitable
Dilute 10 mL of solution S to 15 mL with distilled water R. media. The bacterial strain, used for the master seed lot, shall
be identified by historical records that include information on
Heavy metals (2.4.8) : maximum 15 ppm. its origin and the tests used to characterise the strain. These
12 mL of solution S complies with test A. Prepare the reference will include morphological, cultural, biochemical, genetic and
solution using a mixture of 2.5 mL of lead standard solution serological properties of the strain. The master seed lot and
(2 ppm Pb) R and 7.5 mL of water R. the working seed lot, where applicable, must be demonstrated
ASSAY to have identical profiles. Only a seed lot that complies with
the following requirements may be used.
Dissolve 1.000 g with heating in 100 mL of water R containing
15 g of mannitol R. Titrate with 1 M sodium hydroxide, using Identification. Each seed lot is identified as containing pure
0.5 mL of phenolphthalein solution R as indicator, until a pink cultures of C. botulinum type A bacteria with no extraneous
colour is obtained. bacterial or fungal contamination.
1 mL of 1 M sodium hydroxide is equivalent to 61.8 mg Microbial purity. Each seed lot complies with the
of H3BO3. requirements for absence of contaminating micro-organisms.
The purity of bacterial cultures is verified by methods of
01/2012:2113 suitable sensitivity. These may include inoculation into
suitable media and examination of colony morphology.
BOTULINUM TOXIN TYPE A FOR Phenotypic parameters. Each seed lot must have a known
INJECTION fatty acid profile, sugar fermentation profile (glucose, lactose,
mannose, etc.) and proteolytic activity and must demonstrate
Toxinum botulinicum A ad iniectabile relevant lipase, lecithinase and gelatinase activity.
Genetic purity. Each seed lot must have information on the
DEFINITION toxin gene sequence and comply with requirements for the
Botulinum toxin type A for injection is a dried preparation absence of other genes encoding other toxin serotypes.
containing purified botulinum neurotoxin type A, which may
be present in the form of a complex with haemagglutinins Production of active toxin. A bacterial strain producing a
and non-toxic proteins. Botulinum neurotoxin type A or its high yield of active toxin, as determined by an acute toxicity
haemagglutinin complex is prepared by a suitable purification assay, is suitable. Seed lots demonstrate a capability of
process of the liquid supernatant from a broth-culture of a producing at least a minimum toxicity level appropriate for
suitable strain of Clostridium botulinum type A. the manufacturing process and scale.
The purified complexes consist of several proteins and can be MANUFACTURER’S REFERENCE PREPARATIONS
of various sizes. The largest complex (relative molecular mass During development, reference preparations are established
of about 900 000) consists of a 150 000 relative molecular for subsequent verification of batch consistency during
mass neurotoxin, a 130 000 relative molecular mass non-toxic production and for control of the bulk purified toxin and
protein and various haemagglutinins ranging between relative finished product. They are derived from representative
molecular mass 14 000 and 43 000. The purified toxin moiety batches of botulinum toxin type A that are characterised as
is composed of only the same 150 000 relative molecular mass described under Bulk Purified Toxin.
neurotoxin as is found in the 900 000 relative molecular mass The reference preparations are suitably characterised for their
neurotoxin complex, which is initially produced as a single intended purpose and are stored in suitably sized aliquots
chain and further cleaved (nicked) by endogenous proteases under conditions ensuring their suitability.
into a fully active, disulfide-linked, 54 000 relative molecular
mass light chain and a 97 000 relative molecular mass heavy BULK PURIFIED TOXIN
chain. C. botulinum type A strain is grown anaerobically, in
The preparation is reconstituted before use, as stated on the suitable media, from which cultures are selected for step-up
label. incubations under a suitably controlled anaerobic atmosphere
through the seed culture and bulk fermentation stages to
PRODUCTION allow maximum production of toxin. The toxin is purified by
GENERAL PROVISIONS suitable methods to remove nucleic acids and components
likely to cause adverse reactions.
Production of the toxin is based on seed cultures, managed
in a defined seed-lot system in which the ability to produce Only a purified toxin that complies with the following
toxin is conserved. The production method must be shown to requirements may be used in the preparation of the final bulk.
yield consistently product of activity and profile comparable For each test and for each product, limits of acceptance are
to that of lots shown in clinical studies to be of adequate safety established and each new purified toxin must comply with
and efficacy. these limits.
The production method is validated to demonstrate that Residual reagents. Removal of residual reagents used in
the product, if tested, would comply with the general test of purification steps is confirmed by suitable limit tests or by
abnormal toxicity (2.6.9) using not less than the maximum validation of the process.
human clinical dose, in the presence of a suitable amount of
specific botulinum type A antitoxin used for neutralisation. Nucleic acids. Removal of nucleic acids is confirmed by
suitable limit tests or by validation of the process.
The production method and stability of the finished product
and relevant intermediates are evaluated using the tests below. Immunological identity. The presence of specific type A toxin
Such tests include the specific toxin activity per milligram of is confirmed by a suitable immunochemical method (2.7.1).
protein of purified toxin in an appropriate functional model Specific activity. The specific activity is confirmed in a mouse
of toxin activity and may be supported by tests confirming model of toxicity or by in vivo/ex vivo methods validated with
the presence of botulinum toxin type A, and, if appropriate, respect to the LD50 assay and expressed in mouse LD50 units
associated non-toxic proteins. per milligram of protein. Specific activity must not be less
BACTERIAL SEED LOTS than 1 × 108 mouse LD50 units per milligram of protein for the
A highly toxigenic strain of C. botulinum of known toxin 150 000 relative molecular mass neurotoxin and must not be
type A and confirmed absence of genes encoding other less than 1 × 107 mouse LD50 units per milligram of protein
botulinum toxins (particularly botulinum toxin types B and for the 900 000 relative molecular mass neurotoxin complex.

General Notices (1) apply to all monographs and other texts 1683
Botulinum toxin type B for injection EUROPEAN PHARMACOPOEIA 8.0

Protein. The total protein concentration is determined by a For alternative replacement methods the potency is calculated
suitable method. An acceptable value is established for the with respect to a suitable reference preparation calibrated in
product and each batch must be shown to comply with the mouse LD50 units.
limits. The estimated potency is not less than 80 per cent and not
Protein profile. Identity and protein composition are more than 125 per cent of the stated potency. The confidence
determined by polyacrylamide gel electrophoresis (2.2.31) limits (P = 0.95) are not less than 80 per cent and not more
under reducing or non-reducing conditions or by other than 125 per cent of the estimated potency.
suitable physicochemical methods such as size-exclusion The test may be repeated but when more than 1 test is
chromatography (2.2.30), comparing with suitable reference performed, the results of all valid tests must be combined in
standards. the estimate of potency.
Total viable count. It complies with the limits approved for
the particular product. LABELLING
FINAL BULK The label states :
The final bulk is prepared by adding approved excipients to – the number of units of toxin per vial with a statement
the bulk purified toxin. The solution is filtered through a that units are product specific and not applicable to other
bacteria-retentive filter. If human albumin is added, it complies preparations containing botulinum toxin type A ;
with the monograph Human albumin solution (0255). – the name and the volume of the diluent to be added for
FINAL LOT reconstitution of the dried product.
The final bulk is distributed aseptically into sterile,
tamper-proof containers. Uniformity of fill is verified
during filling and the test for uniformity of content (2.9.6)
is not required. The containers are closed so as to prevent 07/2011:2581
contamination.
Only a final lot that is within the limits approved for the BOTULINUM TOXIN TYPE B FOR
particular product and is satisfactory with respect to each of
the requirements given below under Identification, Tests and
INJECTION
Assay may be released for use.
pH (2.2.3). The pH of the reconstituted product is within Toxinum botulinicum B ad iniectabile
± 0.5 pH units of the limit approved for the particular product. DEFINITION
Water: not more than the limit approved for the particular Botulinum toxin type B for injection is a liquid preparation
product. containing purified botulinum neurotoxin type B, which may
be present in the form of a complex with haemagglutinins
IDENTIFICATION
and non-toxic proteins. Botulinum neurotoxin type B or its
The presence of botulinum toxin type A is confirmed by a haemagglutinin complex is prepared by a suitable purification
suitable immunochemical method (2.7.1). process of the liquid supernatant from a broth-culture of
a suitable strain of Clostridium botulinum type B. Suitable
TESTS stabilisers may be added.
Sterility (2.6.1). It complies with the test for sterility. The toxin is present in its native form as a complex of
Bacterial endotoxins (2.6.14) : less than 10 IU per vial. neurotoxin and non-toxin proteins and haemagglutinins with
a total relative molecular mass of approximately 700 000. The
ASSAY neurotoxin is synthesised by the bacterium as a single-chain
polypeptide of approximately 150 000 relative molecular
In accordance with the provisions of the European mass that is activated during the fermentation process via
Convention for the Protection of Vertebrate Animals Used a proteolytic cleavage (nicking) by endogenous proteases.
for Experimental and Other Scientific Purposes, tests must The nicked protein is a fully active double-chain polypeptide
be carried out in such a way as to use the minimum number consisting of a heavy chain (100 000 relative molecular mass)
of animals and to cause the least pain, suffering, distress or and a light chain (50 000 relative molecular mass), connected
lasting harm. The LD50 assay is associated with severe suffering by a disulfide bond.
of animals and manufacturers are strongly encouraged to
develop and validate assays that will reduce the number of PRODUCTION
animals used, or refine or replace the test procedure with the
goal of promoting animal welfare. GENERAL PROVISIONS
Production of the toxin is based on seed cultures, managed
The potency of the reconstituted product is determined by an in a defined seed-lot system in which the ability to produce
LD50 assay in mice or by a method validated with respect to toxin is conserved. The production method must be shown to
the LD50 assay. The potency is expressed in terms of the LD50 yield consistently product of activity and profile comparable
for mice or relative to the reference preparation. to that of lots shown in clinical studies to be of adequate safety
For determination of the LD50, graded doses of the product and efficacy.
are injected intraperitoneally into groups of mice and the LD50 The production method is validated to demonstrate that
is calculated by the usual statistical methods (5.3) from the the product, if tested, would comply with the general test of
mouse lethality in each group. A suitable reference preparation abnormal toxicity (2.6.9) using not less than the maximum
is assayed in parallel ; the potency of the toxin is expressed human clinical dose, in the presence of a suitable amount of
relative to the reference or the value found for the reference is specific botulinum type B antitoxin used for neutralisation.
within suitable limits defined in terms of the assigned potency. The production method and stability of the finished product
After validation with respect to the LD50 assay (reference and relevant intermediates are evaluated using the tests below.
method), the product may also be assayed by other methods Such tests include the specific toxin activity per milligram of
that are preferable in terms of animal welfare, for example protein of purified toxin in an appropriate functional model
mouse bioassays using paralysis as the end-point, ex vivo of toxin activity and may be supported by tests confirming
assays using mouse phrenic nerve diaphragm, endopeptidase the presence of botulinum toxin type B, and, if appropriate,
assays in vitro and cell-based assays. associated non-toxic proteins.

1684 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Botulinum toxin type B for injection

BACTERIAL SEED LOTS Protein. The total protein concentration is determined by a

A highly toxigenic strain of C. botulinum of known toxin suitable method. An acceptable value is established for the
type B and confirmed absence of genes encoding other product and each batch must be shown to comply with the
botulinum toxins (particularly botulinum toxin types A limits.
and F), with known origin and history, is grown using suitable Protein profile. Identity and protein composition are
media. The bacterial strain, used for the master seed lot, shall determined by polyacrylamide gel electrophoresis (2.2.31)
be identified by historical records that include information on under reducing or non-reducing conditions or by other
its origin and the tests used to characterise the strain. These suitable physicochemical methods such as size-exclusion
will include morphological, cultural, biochemical, genetic and chromatography (2.2.30), comparing with suitable reference
serological properties of the strain. The master seed lot and standards.
the working seed lot, where applicable, must be demonstrated
to have identical profiles. Only a seed lot that complies with Total viable count. It complies with the limits approved for
the following requirements may be used. the particular product.
Identification. Each seed lot is identified as containing pure FINAL BULK
cultures of C. botulinum type B bacteria with no extraneous The final bulk is prepared by adding approved excipients to
bacterial or fungal contamination. the bulk purified toxin. The solution is filtered through a
bacteria-retentive filter. If human albumin is added, it complies
Microbial purity. Each seed lot complies with the with the monograph Human albumin solution (0255).
requirements for absence of contaminating micro-organisms.
The purity of bacterial cultures is verified by methods of FINAL LOT
suitable sensitivity. These may include inoculation into The final bulk is distributed aseptically into sterile,
suitable media and examination of colony morphology. tamper-proof containers. Uniformity of fill is verified
Phenotypic parameters. Each seed lot must have a known during filling and the test for uniformity of content (2.9.6)
fatty acid profile, sugar fermentation profile (glucose, lactose, is not required. The containers are closed so as to prevent
mannose, etc.) and proteolytic activity and must demonstrate contamination.
relevant lipase, lecithinase and gelatinase activity. Only a final lot that is within the limits approved for the
particular product and is satisfactory with respect to each of
Genetic purity. Each seed lot must have information on the the requirements given below under Identification, Tests and
toxin gene genomic location and on the toxin gene sequence, Assay may be released for use.
and comply with requirements for the absence of other genes
encoding other toxin serotypes. pH (2.2.3). The pH of the product is within ± 0.5 pH units of
the limit approved for the particular product.
Production of active toxin. A bacterial strain producing a
high yield of active toxin, as determined by an acute toxicity IDENTIFICATION
assay, is suitable. Seed lots demonstrate a capability of
The presence of botulinum toxin type B is confirmed by a
producing at least a minimum toxicity level appropriate for
suitable immunochemical method (2.7.1).
the manufacturing process and scale.
MANUFACTURER’S REFERENCE PREPARATIONS TESTS
During development, reference preparations are established Sterility (2.6.1). It complies with the test for sterility.
for subsequent verification of batch consistency during
production and for control of the bulk purified toxin and Bacterial endotoxins (2.6.14) : less than 10 IU per vial.
finished product. They are derived from representative ASSAY
batches of botulinum toxin type B that are characterised as
described under Bulk Purified Toxin. In accordance with the provisions of the European
The reference preparations are suitably characterised for their Convention for the Protection of Vertebrate Animals Used
intended purpose and are stored in suitably sized aliquots for Experimental and Other Scientific Purposes, tests must
under conditions ensuring their suitability. be carried out in such a way as to use the minimum number
of animals and to cause the least pain, suffering, distress or
BULK PURIFIED TOXIN lasting harm. The LD50 assay is associated with severe suffering
C. botulinum type B strain is grown anaerobically, in of animals and manufacturers are strongly encouraged to
suitable media, from which cultures are selected for step-up develop and validate assays that will reduce the number of
incubations under a suitably controlled anaerobic atmosphere animals used, or refine or replace the test procedure with the
through the seed culture and bulk fermentation stages to goal of promoting animal welfare.
allow maximum production of toxin. The toxin is purified by
suitable methods to remove nucleic acids and components The potency of the product is determined by an LD50 assay in
likely to cause adverse reactions. mice or by a method validated with respect to the LD50 assay.
Only a purified toxin that complies with the following The potency is expressed in terms of the LD50 for mice or
requirements may be used in the preparation of the final bulk. relative to the reference preparation.
For each test and for each product, limits of acceptance are For determination of the LD50, graded doses of the product
established and each new purified toxin must comply with are injected intraperitoneally into groups of mice and the LD50
these limits. is calculated by the usual statistical methods (5.3) from the
mouse lethality in each group. A suitable reference preparation
Residual reagents. Removal of residual reagents used in is assayed in parallel ; the potency of the toxin is expressed
purification steps is confirmed by suitable limit tests or by relative to the reference or the value found for the reference is
validation of the process. within suitable limits defined in terms of the assigned potency.
Nucleic acids. Removal of nucleic acids is confirmed by After validation with respect to the LD50 assay (reference
suitable limit tests or by validation of the process. method), the product may also be assayed by other methods
Immunological identity. The presence of specific type B toxin that are preferable in terms of animal welfare, for example
is confirmed by a suitable immunochemical method (2.7.1). mouse bioassays using paralysis as the end-point, ex vivo
Specific activity. The specific activity is confirmed in a mouse assays using mouse phrenic nerve diaphragm, endopeptidase
model of toxicity or by in vivo/ex vivo methods validated with assays in vitro and cell-based assays.
respect to the LD50 assay and expressed in mouse LD50 units For alternative replacement methods the potency is calculated
per milligram of protein. Specific activity must not be less with respect to a suitable reference preparation calibrated in
than 1 × 108 mouse LD50 units per milligram of protein. mouse LD50 units.

General Notices (1) apply to all monographs and other texts 1685
Bovine serum EUROPEAN PHARMACOPOEIA 8.0

The estimated potency is not less than 80 per cent and not Serum is obtained by separation of the serum from blood cells
more than 125 per cent of the stated potency. The confidence and clot under conditions designed to minimise microbial
limits (P = 0.95) are not less than 80 per cent and not more contamination. Serum from a number of animals is pooled
than 125 per cent of the estimated potency. and a batch number is allocated to the pool. Appropriate steps
The test may be repeated but when more than 1 test is are taken to ensure homogeneity of the harvested material,
performed, the results of all valid tests must be combined in intermediate pools and the final batch. Suitable measures
the estimate of potency. (for example filtration) are taken to ensure sterility or a low
bioburden. Before further processing, the serum is tested for
LABELLING sterility or bioburden. General and specific tests for viral
The label states the number of units of toxin per vial with a contaminants are carried out as described below.
statement that units are product specific and not applicable to A step or steps for virus inactivation/removal are applied to
other preparations containing botulinum toxin type B. serum intended for production of immunological veterinary
medicinal products. Unless otherwise justified and authorised
for a particular medicinal product, a step or steps for virus
01/2008:2262 inactivation/removal are applied to serum intended for
production of human and non-immunological veterinary
BOVINE SERUM medicinal products.
INACTIVATION
Serum bovinum The inactivation procedure applied is validated with respect
to a suitable representative range of viruses covering different
DEFINITION types (enveloped, non-enveloped, DNA, RNA viruses). The
Liquid fraction of blood obtained from the ox (Bos taurus L.) optimal choice of relevant and model viruses depends strongly
and from which cells, fibrin and clotting factors have been on the specific inactivation/removal procedure ; representative
removed. viruses with different degrees of resistance to the type of
treatment must be included. Bovine viral diarrhoea virus
Different types of bovine serum are used :
must be included in the viruses used for validation. Serum
– adult bovine serum obtained at slaughter from cattle that free from antibodies against bovine viral diarrhoea virus is
are declared fit for human consumption ; used in part or all of the validation studies.
– calf serum obtained at slaughter from animals, fit for human For bovine serum intended for use in immunological
consumption, before the age of 12 months ; veterinary medicinal products, for inactivation by gamma
– new-born calf serum obtained at slaughter from animals irradiation a minimum dose of 30 kGy is applied, unless
before the age of 20 days ; otherwise justified and authorised.
– foetal bovine serum obtained from normal foetuses from Critical parameters for the method of virus inactivation/re-
dams fit for human consumption ; moval are established and the parameters used in the
– donor bovine serum obtained by repeated bleeding of donor validation study are strictly adhered to during subsequent
animals from controlled donor herds. application of the procedures to each batch of serum.
This monograph provides a general quality specification For inactivation by gamma irradiation, critical parameters
for bovine serum. Various measures are applied during the include :
production of bovine serum aimed at obtaining a product that – the temperature ;
is acceptable as regards viral safety. No single measure, nor – packaging configuration ;
the combination of measures outlined below can guarantee – distribution of dosimeters to assess the effective dose
complete viral safety but they rather reduce the risk involved in received by the product whatever its position ;
the use of serum in the manufacture of medicinal products. It is
therefore necessary for the manufacturer of a medicinal product – the minimum and maximum dose received.
to take account of this when choosing the serum for a particular QUALITY CONTROL TESTS APPLIED TO EACH BATCH
use by making a risk assessment. A suitable sample size for each batch is established. Specific
tests for viral contaminants are validated with respect to
PRODUCTION sensitivity and specificity. The cell cultures used for general
All stages of serum production are submitted to a suitable tests for viral contaminants are shown to be sensitive to a
quality management system. suitable range of potential contaminants. Control cells used in
Traceability of serum is maintained from the final container the tests are cultivated, where relevant, with a bovine serum
to the abattoir of origin (for blood collected from slaughtered controlled and inactivated as described in this monograph.
animals) or to the herd of origin (for blood collected from Serum free from antibodies to bovine viral diarrhoea virus
donor animals). is required for validation of the effect of antibodies on the
Further guarantee of the safety and quality of serum may be detection limits for bovine viral diarrhoea virus.
ensured by the use of a controlled donor herd. Where serum Tests carried out on the batch prior to treatment
is obtained from such a herd, the animals are subjected to The following tests are carried out on the serum (before any
regular veterinary examination to ascertain their health status. virus inactivation/removal steps, where applicable).
Animals introduced into the herd are traceable as regards
source, breeding and rearing history. The introduction of Tests for viral contaminants. General tests supplemented by
animals into the herd follows specified procedures, including specific tests are carried out.
defined quarantine measures. During the quarantine period General tests. Validated tests are carried out by inoculation of
the animals are observed and tested to establish that they are the serum on at least 2 distinct cell lines, one of which is of
free from all agents and antibodies from which the donor herd bovine origin. The cell lines used are suitable for detecting
is claimed to be free. It may be necessary to test the animals haemadsorbing viruses such as bovine parainfluenza virus 3
in quarantine for freedom from additional agents, depending and cytopathic agents such as bovine herpesvirus 1.
on factors such as information available on their breeding Specific tests for viral contaminants (if not detected by general
and rearing history. It is recommended that animals in the tests), where relevant in view of the country of origin of
herd should not be vaccinated against bovine viral diarrhoea the serum : bluetongue virus, bovine adenovirus, bovine
virus. Tests are carried out for any agent and/or antibody from parvovirus, bovine respiratory syncytial virus, bovine viral
which the herd is claimed to be free. diarrhoea virus, rabies virus and reovirus. Depending on

1686 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Bromazepam

the country of origin, specific tests for other viruses may be 01/2008:0879
needed. The animal health status of countries is defined by the
‘Office International des Epizooties’ (OIE).
BROMAZEPAM
For serum to be subjected to a virus inactivation/removal
procedure, if evidence of viral contamination is found in any
of the tests described above, the serum is acceptable only if Bromazepamum
the virus is identified and shown to be present in an amount
that has been shown in a validation study to be effectively
inactivated.
For serum that is not to be subjected to a virus
inactivation/removal procedure, if evidence of viral
contamination is found in any of the tests described above,
the serum is not acceptable.
A test for bovine viral diarrhoea virus antibodies is carried
out ; an acceptance criterion for the titre is established taking C14H10BrN3O Mr 316.2
account of the risk assessment. [1812-30-2]
Composition. The content of a suitable selection of the
DEFINITION
following components is determined and shown to be
within the expected range for the type of serum : cholesterol, 7-Bromo-5-(pyridin-2-yl)-1,3-dihydro-2H-1,4-
α-, β- and γ-globulin, albumin, creatinine, bilirubin, glucose, benzodiazepin-2-one.
serum aspartate transaminase (SAST, formerly SGOT - Content : 99.0 per cent to 101.0 per cent (dried substance).
serum glutamic-oxaloacetic transaminase), serum alanine
transaminase (SALT, formerly SGPT - glutamic-pyruvic CHARACTERS
transaminase), phosphorus, potassium, calcium, sodium and Appearance : white or yellowish, crystalline powder.
pH. Solubility : practically insoluble in water, slightly soluble or
Tests carried out on the batch post-treatment sparingly soluble in ethanol (96 per cent) and in methylene
If bovine viral diarrhoea virus was detected before virus chloride.
inactivation/removal, the following test for bovine viral
diarrhoea virus is carried out after virus inactivation/removal. IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Test for bovine viral diarrhoea virus. A validated test for bovine
viral diarrhoea virus is carried out, for example by inoculation Comparison: bromazepam CRS.
into susceptible cell cultures, followed by not fewer than
3 subcultures and detection by immunostaining. No evidence TESTS
of the presence of bovine viral diarrhoea virus is found. Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
IDENTIFICATION Test solution. Dissolve 10.0 mg of the substance to be
A. The electrophoretic pattern corresponds to that for serum examined in 9 mL of a mixture of 1 volume of acetonitrile R
and is consistent with the type (foetal or other) of bovine and 8 volumes of methanol R. Dilute to 20.0 mL with an
serum. 11.33 g/L solution of potassium dihydrogen phosphate R
B. Bovine origin is confirmed by a suitable immunochemical previously adjusted to pH 7.0 with a 100 g/L solution of
method (2.7.1). potassium hydroxide R.
Reference solution (a). Dilute 1.0 mL of the test solution to
TESTS 100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
Osmolality (2.2.35) : 280 mosmol/kg to 365 mosmol/kg for to 10.0 mL with the mobile phase.
foetal bovine serum and 240 mosmol/kg to 340 mosmol/kg Reference solution (b). Dissolve 5 mg of bromazepam for system
for other types. suitability CRS (containing impurities A, B, C, D and E) in
Total protein (2.5.33) : 30 mg/mL to 45 mg/mL for foetal 5 mL of a mixture of 1 volume of acetonitrile R and 8 volumes
bovine serum and minimum 35 mg/mL for other types. of methanol R. Dilute to 10.0 mL with an 11.33 g/L solution
of potassium dihydrogen phosphate R previously adjusted to
Haemoglobin : maximum 4 mg/mL, determined by a pH 7.0 with a 100 g/L solution of potassium hydroxide R.
validated method, such as spectrophotometry.
Column :
Bacterial endotoxins (2.6.14) : less than 10 IU/mL for donor
bovine serum, less than 25 IU/mL for foetal bovine serum, – size : l = 0.15 m, Ø = 4.6 mm ;
less than 100 IU/mL for other types. – stationary phase : end-capped octadecylsilyl silica gel for
Sterility (2.6.1). It complies with the test. Use 10 mL for each chromatography R (3.5 μm) ;
medium. – temperature : 50 °C.
Mycoplasmas (2.6.7). It complies with the test. Mobile phase : mix 5 volumes of acetonitrile R, 45 volumes
of methanol R and 50 volumes of an 11.33 g/L solution of
STORAGE potassium dihydrogen phosphate R previously adjusted to
Frozen at − 10 °C or below. pH 7.0 with a 100 g/L solution of potassium hydroxide R.
Flow rate : 1.0 mL/min.
LABELLING Detection : spectrophotometer at 235 nm.
The label states : Injection : 20 μL.
– the type of serum ; Run time : 4 times the retention time of bromazepam.
– where applicable, that the serum has been inactivated and Identification of impurities : use the chromatogram supplied
the inactivation method ; with bromazepam for system suitability CRS and the
– where the serum has been inactivated by gamma irradiation, chromatogram obtained with reference solution (b) to identify
the target minimum dose of the irradiation procedure. the peaks due to impurities A, B, C, D and E.

General Notices (1) apply to all monographs and other texts 1687
Bromhexine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Relative retention with reference to bromazepam

(retention time = about 5 min) : impurity D = about 1.4 ;
impurity A = about 1.5 ; impurity C = about 1.6 ;
impurity E = about 2.1 ; impurity B = about 2.2.
System suitability : reference solution (b) :
– resolution : minimum 4.0 between the peaks due to
bromazepam and impurity D and minimum 1.2 between
the peaks due to impurities A and C. C. 7-bromo-5-(6-methylpyridin-2-yl)-1,3-dihydro-2H-1,4-
benzodiazepin-2-one,
Limits :
– correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
corresponding correction factor : impurity A = 1.3 ;
impurity B = 1.8 ; impurity E = 2.1 ;
– impurities A, B, E : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent) ;
D. 3-amino-6-bromo-4-(pyridin-2-yl)quinolin-2(1H)-one.
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
01/2008:0706
with reference solution (a) (0.10 per cent) ; corrected 6.0
– total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.2 per cent) ;
BROMHEXINE HYDROCHLORIDE
– disregard limit : 0.5 times the area of the principal peak in Bromhexini hydrochloridum
the chromatogram obtained with reference solution (a)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.2 per cent, determined
on 1.000 g by drying at 80 °C at a pressure not exceeding
2.7 kPa for 4 h.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
C14H21Br2ClN2 Mr 412.6
ASSAY [611-75-6]
Dissolve 0.250 g in 20 mL of anhydrous acetic acid R. Add DEFINITION
50 mL of acetic anhydride R. Titrate with 0.1 M perchloric N-(2-Amino-3,5-dibromobenzyl)-N-methylcyclohexanamine
acid, determining the end-point potentiometrically (2.2.20). hydrochloride.
1 mL of 0.1 M perchloric acid is equivalent to 31.62 mg Content : 98.5 per cent to 101.5 per cent (dried substance).
of C14H10BrN3O.
CHARACTERS
STORAGE Appearance : white or almost white, crystalline powder.
Protected from light. Solubility : very slightly soluble in water, slightly soluble in
alcohol and in methylene chloride.
IMPURITIES It shows polymorphism (5.9).
Specified impurities : A, B, E.
IDENTIFICATION
Other detectable impurities (the following substances would, First identification : A, E.
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general Second identification : B, C, D, E.
acceptance criterion for other/unspecified impurities and/or A. Infrared absorption spectrophotometry (2.2.24).
by the general monograph Substances for pharmaceutical use Comparison: bromhexine hydrochloride CRS.
(2034). It is therefore not necessary to identify these impurities If the spectra obtained in the solid state show differences,
for demonstration of compliance. See also 5.10. Control of dissolve the substance to be examined and the reference
impurities in substances for pharmaceutical use) : C, D. substance separately in methanol R, evaporate to dryness
and record new spectra using the residues.
B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 20 mg of the substance to be
examined in methanol R and dilute to 10 mL with the same
solvent.
Reference solution. Dissolve 20 mg of bromhexine
hydrochloride CRS in methanol R and dilute to 10 mL with
the same solvent.
A. R = H : (2-amino-5-bromophenyl)(pyridin-2- Plate : TLC silica gel F254 plate R.
yl)methanone,
Mobile phase : glacial acetic acid R, water R, butanol R
(17:17:66 V/V/V).
B. R = CO-CH2-Cl : N-[4-bromo-2-(pyridin-2-
ylcarbonyl)phenyl]-2-chloroacetamide, Application : 20 μL.
Development : over 3/4 of the plate.
E. R = CO-CH2-Br : 2-bromo-N-[4-bromo-2-(pyridin-2- Drying : in air.
ylcarbonyl)phenyl]acetamide, Detection : examine in ultraviolet light at 254 nm.

1688 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Bromocriptine mesilate

Results : the principal spot in the chromatogram obtained ASSAY

with the test solution is similar in position and size to the Dissolve 0.300 g in 70 mL of alcohol R and add 1 mL of
principal spot in the chromatogram obtained with the 0.1 M hydrochloric acid. Carry out a potentiometric titration
reference solution. (2.2.20), using 0.1 M sodium hydroxide. Read the volume
C. Dissolve about 25 mg in a mixture of 1 mL of dilute sulfuric between the 2 points of inflexion.
acid R and 50 mL of water R. Add 2 mL of methylene 1 mL of 0.1 M sodium hydroxide is equivalent to 41.26 mg
chloride R and 5 mL of chloramine solution R and shake. A of C14H21Br2ClN2.
brownish-yellow colour develops in the lower layer.
D. Dissolve about 1 mg in 3 mL of 0.1 M hydrochloric acid. STORAGE
The solution gives the reaction of primary aromatic amines Protected from light.
(2.3.1).
E. Dissolve about 20 mg in 1 mL of methanol R and add 1 mL IMPURITIES
of water R. The solution gives reaction (a) of chlorides Specified impurities : A, B, C, D.
(2.3.1). Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
TESTS the tests in the monograph. They are limited by the general
Appearance of solution. The solution is clear (2.2.1) and not acceptance criterion for other/unspecified impurities and/or
more intensely coloured than reference solution Y6 (2.2.2, by the general monograph Substances for pharmaceutical
Method II). use (2034). It is therefore not necessary to identify these
Dissolve 0.6 g in methanol R and dilute to 20 mL with the impurities for demonstration of compliance. See also 5.10.
same solvent. Control of impurities in substances for pharmaceutical use) : E.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50 mg of the substance to be examined
in methanol R and dilute to 10.0 mL with the same solvent.
Reference solution (a). Dissolve 5 mg of bromhexine
impurity C CRS in methanol R, add 1.0 mL of the test solution
and dilute to 10.0 mL with the same solvent.
Reference solution (b). Dilute 1.0 mL of the test solution to A. R = CH2OH : (2-amino-3,5-dibromophenyl)methanol,
100.0 mL with methanol R. Dilute 1.0 mL of this solution to B. R = CHO : 2-amino-3,5-dibromobenzaldehyde,
10.0 mL with methanol R.
Column :
– size : l = 0.12 m, Ø = 4.6 mm,
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (3 μm).
Mobile phase : mix 0.50 mL of phosphoric acid R in 950 mL of
water R, adjust to pH 7.0 with triethylamine R (about 1.5 mL)
and dilute to 1000 mL with water R ; mix 20 volumes of this C. R = H : N-(2-aminobenzyl)-N-methylcyclohexanamine,
solution with 80 volumes of acetonitrile R. D. R = Br : N-(2-amino-5-bromobenzyl)-N-
Flow rate : 1.0 mL/min. methylcyclohexanamine,
Detection : spectrophotometer at 248 nm.
Injection : 10 μL.
Run time : 2.5 times the retention time of bromhexine.
Relative retention with reference to bromhexine
(retention time = about 11 min) : impurity A = about 0.1 ;
impurity B = about 0.2 ; impurity C = about 0.4 ;
impurity D = about 0.5.
System suitability: reference solution (a) : E. (3RS)-6,8-dibromo-3-cyclohexyl-3-methyl-1,2,3,4-
tetrahydroquinazolin-3-ium.
– resolution : minimum 12.0 between the peaks due to
impurity C and bromhexine.
Limits :
– any impurity : not more than twice the area of the principal 07/2013:0596
peak in the chromatogram obtained with reference
solution (b) (0.2 per cent), and not more than 1 such peak BROMOCRIPTINE MESILATE
has an area greater than the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.1 per cent), Bromocriptini mesilas
– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.3 per cent),
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 1.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on C33H44BrN5O8S Mr 751
1.0 g. [22260-51-1]

General Notices (1) apply to all monographs and other texts 1689
Bromocriptine mesilate EUROPEAN PHARMACOPOEIA 8.0

DEFINITION Results : the principal spot in the chromatogram obtained

(6aR,9R)-5-Bromo-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2- with the test solution is similar in position, colour and size
(1-methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro- to the principal spot in the chromatogram obtained with
8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl- the reference solution.
4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide D. To 0.1 g add 5 mL of dilute hydrochloric acid R and shake
monomethanesulfonate. for about 5 min. Filter and add 1 mL of barium chloride
Content : 98.0 per cent to 101.0 per cent (dried substance). solution R1. The filtrate remains clear. To a further 0.1 g
add 0.5 g of anhydrous sodium carbonate R, mix and ignite
PRODUCTION until a white residue is obtained. Allow to cool and dissolve
the residue in 7 mL of water R (solution A). Solution A
It is considered that alkylsulfonate esters are genotoxic and gives reaction (a) of sulfates (2.3.1).
are potential impurities in bromocriptine mesilate. The
manufacturing process should be developed taking into E. Solution A obtained in identification test D gives
consideration the principles of quality risk management, reaction (a) of bromides (2.3.1).
together with considerations of the quality of starting TESTS
materials, process capability and validation. The general
methods 2.5.37. Methyl, ethyl and isopropyl methanesulfonate Appearance of solution. The solution is clear (2.2.1) and not
in methanesulfonic acid, 2.5.38. Methyl, ethyl and more intensely coloured than reference solution B5, BY5 or
isopropyl methanesulfonate in active substances and 2.5.39. Y5 (2.2.2, Method II).
Methanesulfonyl chloride in methanesulfonic acid are available Dissolve 0.25 g in methanol R and dilute to 25 mL with the
to assist manufacturers. same solvent.
pH (2.2.3) : 3.1 to 3.8.
CHARACTERS
Dissolve 0.2 g in a mixture of 2 volumes of methanol R and
Appearance : white or slightly coloured, fine crystalline 8 volumes of carbon dioxide-free water R and dilute to 20 mL
powder. with the same mixture of solvents.
Solubility : practically insoluble in water, freely soluble in Specific optical rotation (2.2.7) : + 95 to + 105 (dried
methanol, soluble in ethanol (96 per cent), sparingly soluble substance).
in methylene chloride. Dissolve 0.100 g in a mixture of equal volumes of methanol R
It is very sensitive to light. and methylene chloride R and dilute to 10.0 mL with the same
The identification, tests and assay are to be carried out as mixture of solvents.
rapidly as possible, protected from light. Related substances. Liquid chromatography (2.2.29).
Solvent mixture : buffer solution pH 2.0 R, methanol R
IDENTIFICATION (50:50 V/V).
First identification : B. Test solution. Dissolve 0.500 g of the substance to be examined
Second identification : A, C, D, E. in 5.0 mL of methanol R and dilute to 10.0 mL with buffer
A. Ultraviolet and visible absorption spectrophotometry solution pH 2.0 R.
(2.2.25). Reference solution (a). Dilute 1.0 mL of the test solution to
Test solution. Dissolve 10.0 mg in 10 mL of methanol R and 100.0 mL with the solvent mixture.
dilute to 200.0 mL with 0.01 M hydrochloric acid. Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 10.0 mL with the solvent mixture.
Spectral range : 250-380 nm.
Reference solution (c). Dissolve the contents of a vial of
Absorption maximum : at 305 nm. bromocriptine mesilate for system suitability CRS (containing
Absorption minimum : at 270 nm. impurities A and B) in 1.0 mL of the solvent mixture.
Specific absorbance at the absorption maximum : 120 to 135 Column :
(dried substance). – size : l = 0.12 m, Ø = 4 mm ;
B. Infrared absorption spectrophotometry (2.2.24). – stationary phase : octadecylsilyl silica gel for
Comparison : bromocriptine mesilate CRS. chromatography R (5 μm).
Mobile phase :
C. Thin-layer chromatography (2.2.27). Prepare the solutions
immediately before use. – mobile phase A : 0.791 g/L solution of ammonium
carbonate R ;
Solvent mixture : ethanol (96 per cent) R, methanol R,
methylene chloride R (30:30:40 V/V/V). – mobile phase B : acetonitrile R ;
Time Mobile phase A Mobile phase B
Test solution. Dissolve 10 mg of the substance to be
examined in the solvent mixture and dilute to 10 mL with (min) (per cent V/V) (per cent V/V)
the solvent mixture. 0 - 30 90 → 40 10 → 60

Reference solution. Dissolve 10 mg of bromocriptine 30 - 45 40 60

mesilate CRS in the solvent mixture and dilute to 10 mL
with the solvent mixture. Flow rate : 2 mL/min.
Plate : TLC silica gel G plate R. Detection : spectrophotometer at 300 nm.
Injection : 20 μL.
Mobile phase : concentrated ammonia R, water R,
2-propanol R, methylene chloride R, ether R (0.1:1.5:3:88:100 Identification of impurities : use the chromatogram supplied
V/V/V/V/V). with bromocriptine mesilate for system suitability CRS and the
chromatogram obtained with reference solution (c) to identify
Application : 10 μL. the peaks due to impurities A and B.
Development : immediately in an unsaturated tank, over Relative retention with reference to bromocriptine :
a path of 15 cm. impurity C = about 1.2.
Drying : in a current of cold air for 2 min. System suitability : reference solution (c) :
Detection : spray with ammonium molybdate solution R3 – resolution : minimum 1.1 between the peaks due to
and dry at 100 °C until the spots appear (about 10 min). impurities A and B.

1690 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Bromocriptine mesilate

Limits :
– impurity A : not more than 0.2 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.02 per cent) ;
– impurity C : not more than 4 times the area of the principal
peak in the chromatogram obtained with reference
solution (b) (0.4 per cent) ;
– impurities B, D, E, F, G : for each impurity, not more than C. (6aR,9S)-5-bromo-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-
twice the area of the principal peak in the chromatogram (1-methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro-
obtained with reference solution (b) (0.2 per cent) and not 8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
more than 1 such peak has an area greater than the area 4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-
of the principal peak in the chromatogram obtained with carboxamide ((9S)-2-bromo-α-ergocriptine),
reference solution (b) (0.1 per cent) ;
– total : not more than 1.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(1.5 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent), apart from the peak due to impurity A.
Loss on drying (2.2.32) : maximum 3.0 per cent, determined
on 0.500 g by drying in vacuo at 80 °C for 5 h.
D. (6aR,9R)-5-bromo-7-methyl-4,6,6a,7,8,9-
ASSAY hexahydroindolo[4,3-fg]quinoline-9-carboxylic
acid,
Dissolve 0.500 g in 80 mL of a mixture of 10 volumes of
anhydrous acetic acid R and 70 volumes of acetic anhydride R.
Titrate with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 75.1 mg
of C33H44BrN5O8S.

STORAGE
In an airtight container, protected from light, at a temperature
not exceeding − 15 °C. E. (6aR,9R)-5-bromo-7-methyl-4,6,6a,7,8,9-
hexahydroindolo[4,3-fg]quinoline-9-carboxamide,
IMPURITIES
Specified impurities : A, B, C, D, E, F, G.

F. (6aR,9R)-5-bromo-N-[(2S,5S,10aS,10bS)-10b-hydroxy-2-
A. (6aR,9R)-5-bromo-N-[(2R,5S)-2-(1-methylethyl)-5-(2- (1-methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro-
methylpropyl)-3,6-dioxo-2,3,5,6,9,10-hexahydro-8H- 8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl- 4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-
4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9- carboxamide ((2′S)-2-bromo-α-ergocriptine),
carboxamide (2-bromodehydro-α-ergocriptine),

B. (6aR,9R)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-(1- G. (6aR,9R)-5-bromo-N-[(2R,5S,10aS,10bS)-10b-methoxy-2-
methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro- (1-methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro-
8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl- 8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9- 4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-
carboxamide (α-ergocriptine), carboxamide (2-bromo-10′b-O-methyl-α-ergocriptine).

General Notices (1) apply to all monographs and other texts 1691
Bromperidol EUROPEAN PHARMACOPOEIA 8.0

07/2011:1178 TESTS
Appearance of solution. The solution is clear (2.2.1) and not
BROMPERIDOL more intensely coloured than reference solution Y7 (2.2.2,
Method II).
Dissolve 0.2 g in 20 mL of a 1 per cent V/V solution of lactic
Bromperidolum acid R.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.100 g of the substance to be examined
in methanol R and dilute to 10.0 mL with the same solvent.
Reference solution (a). Dissolve 2.5 mg of bromperidol CRS
and 5.0 mg of haloperidol CRS in methanol R and dilute to
50.0 mL with the same solvent.
C21H23BrFNO2 Mr 420.3 Reference solution (b). Dilute 5.0 mL of the test solution to
[10457-90-6] 100.0 mL with methanol R. Dilute 1.0 mL of this solution to
10.0 mL with methanol R.
DEFINITION
Column :
4-[4-(4-Bromophenyl)-4-hydroxypiperidin-1-yl]-1-(4-
fluorophenyl)butan-1-one. – size : l = 0.1 m, Ø = 4.0 mm ;
– stationary phase : base-deactivated octadecylsilyl silica gel for
Content : 99.0 per cent to 101.0 per cent (dried substance).
chromatography R (3 μm).
CHARACTERS Mobile phase :
Appearance : white or almost white powder. – mobile phase A : 17 g/L solution of tetrabutylammonium
hydrogen sulfate R ;
Solubility : practically insoluble in water, sparingly soluble
in methanol and in methylene chloride, slightly soluble in – mobile phase B : acetonitrile R ;
ethanol (96 per cent). Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
IDENTIFICATION 0 - 15 90 → 50 10 → 50
First identification : B, E.
15 - 20 50 50
Second identification : A, C, D, E.
20 - 25 90 10
A. Melting point (2.2.14) : 156 °C to 159 °C.
B. Infrared absorption spectrophotometry (2.2.24). Flow rate : 1.5 mL/min.
Comparison : bromperidol CRS. Detection : spectrophotometer at 230 nm.
C. Thin-layer chromatography (2.2.27). Injection : 10 μL.
Test solution. Dissolve 10 mg of the substance to be Relative retention with reference to bromperidol
examined in methanol R and dilute to 10 mL with the same (retention time = about 6 min) : impurity A = about 0.5 ;
solvent. impurity B = about 0.8 ; haloperidol = about 0.9 ;
impurity C = about 1.4 ; impurity D = about 1.5 ;
Reference solution (a). Dissolve 10 mg of bromperidol CRS impurity E = about 1.8 ; impurity F = about 1.85.
in methanol R and dilute to 10 mL with the same solvent.
System suitability : reference solution (a) :
Reference solution (b). Dissolve 10 mg of bromperidol CRS
– resolution : minimum 3.0 between the peaks due to
and 10 mg of haloperidol CRS in methanol R and dilute to
haloperidol and bromperidol.
10 mL with the same solvent.
Limits :
Plate : TLC octadecylsilyl silica gel plate R.
– impurities A, B, C, D, E, F : for each impurity, not more
Mobile phase : tetrahydrofuran R, methanol R, 58 g/L than the area of the principal peak in the chromatogram
solution of sodium chloride R (10:45:45 V/V/V). obtained with reference solution (b) (0.5 per cent) ;
Application : 1 μL. – unspecified impurities : for each impurity, not more than
Development : in an unsaturated tank, over 3/4 of the plate. 0.2 times the area of the principal peak in the chromatogram
Drying : in air. obtained with reference solution (b) (0.10 per cent) ;
– total : not more than twice the area of the principal peak
Detection : examine in ultraviolet light at 254 nm.
in the chromatogram obtained with reference solution (b)
System suitability: reference solution (b) : (1 per cent) ;
– the chromatogram shows 2 spots which may, however, – disregard limit : 0.1 times the area of the principal peak in
not be completely separated. the chromatogram obtained with reference solution (b)
Results : the principal spot in the chromatogram obtained (0.05 per cent).
with the test solution is similar in position and size to Loss on drying (2.2.32) : maximum 0.5 per cent, determined
the principal spot in the chromatogram obtained with on 1.000 g by drying in an oven at 105 °C.
reference solution (a). Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
D. Dissolve about 10 mg in 5 mL of anhydrous ethanol R. 1.0 g in a platinum crucible.
Add 0.5 mL of dinitrobenzene solution R and 0.5 mL of
2 M alcoholic potassium hydroxide R. A violet colour is ASSAY
produced that becomes brownish-red after 20 min. Dissolve 0.300 g in 50 mL of a mixture of 1 volume of
E. To 0.1 g in a porcelain crucible add 0.5 g of anhydrous anhydrous acetic acid R and 7 volumes of methyl ethyl
sodium carbonate R. Heat over an open flame for 10 min. ketone R. Titrate with 0.1 M perchloric acid, using 0.2 mL of
Allow to cool. Take up the residue with 5 mL of dilute naphtholbenzein solution R as indicator.
nitric acid R and filter. To 1 mL of the filtrate add 1 mL of 1 mL of 0.1 M perchloric acid is equivalent to 42.03 mg
water R. The solution gives reaction (a) of bromides (2.3.1). of C21H23BrFNO2.

1692 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Bromperidol decanoate

STORAGE 07/2011:1397
Protected from light.
BROMPERIDOL DECANOATE
IMPURITIES
Specified impurities : A, B, C, D, E, F. Bromperidoli decanoas

A. 1-(4-fluorophenyl)-4-(4-hydroxy-4-phenylpiperidin-1- C31H41BrFNO3 Mr 574.6

yl)butan-1-one, [75067-66-2]
DEFINITION
4-(4-Bromophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-
piperidin-4-yl decanoate.
Content : 98.5 per cent to 101.0 per cent (dried substance).
CHARACTERS
B. 4-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-1-(2- Appearance : white or almost white powder.
fluorophenyl)butan-1-one, Solubility : practically insoluble in water, very soluble in
methylene chloride, soluble in ethanol (96 per cent).
mp : about 60 °C.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: bromperidol decanoate CRS.
B. To 0.1 g in a porcelain crucible add 0.5 g of anhydrous
sodium carbonate R. Heat over an open flame for 10 min.
C. 4-[4-(biphenyl-4-yl)-4-hydroxypiperidin-1-yl]-1-(4- Allow to cool. Take up the residue with 5 mL of dilute
fluorophenyl)butan-1-one, nitric acid R and filter. To 1 mL of the filtrate add 1 mL of
water R. The solution gives reaction (a) of bromides (2.3.1).
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution B5 (2.2.2,
Method II).
Dissolve 2.0 g in methylene chloride R and dilute to 20 mL
with the same solvent.
D. 4-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-1-(3-
ethyl-4-fluorophenyl)butan-1-one, Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use and protect from light.
Test solution. Dissolve 0.100 g of the substance to be examined
in methanol R and dilute to 10.0 mL with the same solvent.
Reference solution (a). Dissolve 2.5 mg of bromperidol
decanoate CRS and 2.5 mg of haloperidol decanoate CRS in
methanol R and dilute to 50.0 mL with the same solvent.
Reference solution (b). Dilute 5.0 mL of the test solution to
100.0 mL with methanol R. Dilute 1.0 mL of this solution to
10.0 mL with methanol R.
Column :
– size : l = 0.1 m, Ø = 4.0 mm ;
– stationary phase : base-deactivated octadecylsilyl silica gel for
chromatography R (3 μm).
E. 4-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-1-[4-[4-
(4-bromophenyl)-4-hydroxypiperidin-1-yl]phenyl]butan- Mobile phase :
1-one, – mobile phase A : 27 g/L solution of tetrabutylammonium
hydrogen sulfate R ;
– mobile phase B : acetonitrile R ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 30 80 → 40 20 → 60

30 - 35 40 60

35 - 40 40 → 80 60 → 20
F. 4-[4-(4′-bromobiphenyl-4-yl)-4-hydroxypiperidin-1-yl]-1-
(4-fluorophenyl)butan-1-one. Flow rate : 1.5 mL/min.

General Notices (1) apply to all monographs and other texts 1693
Bromperidol decanoate EUROPEAN PHARMACOPOEIA 8.0

Detection : spectrophotometer at 230 nm.

Injection : 10 μL.
Relative retention with reference to bromperidol decanoate
(retention time = about 24 min) : impurity G = about 0.10 ;
impurity L = about 0.15 ; impurity H = about 0.8 ;
impurity A = about 0.89 ; impurity I = about 0.91 ;
impurity B = about 0.96 ; haloperidol decanoate = about 0.98 ;
impurity F = about 1.10 ; impurity C = about 1.15 ; B. 4-(4-bromophenyl)-1-[4-(2-fluorophenyl)-4-oxobutyl]-
impurity K = about 1.2 ; impurity E = about 1.23 ; piperidin-4-yl decanoate,
impurity D = about 1.25.
System suitability: reference solution (a) :
– resolution : minimum 1.5 between the peaks due to
haloperidol decanoate and bromperidol decanoate.
Limits :
– impurities A, B, C, D, E, F, G, H, I, J, K : for each impurity,
not more than the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.5 per C. 4-(4-bromophenyl)-1-[4-(3-ethyl-4-fluorophenyl)-4-
cent) ; oxobutyl]-piperidin-4-yl decanoate,
– unspecified impurities : for each impurity, not more than
0.2 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.10 per cent) ;
– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(1.5 per cent) ;
– disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in vacuo at 30 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g in a platinum crucible.
D. 4-(4-bromophenyl)-1-[4-[4-[4-(4-bromophenyl)-4-
ASSAY hydroxypiperidin-1-yl]phenyl]-4-oxobutyl]piperidin-4-yl
Dissolve 0.450 g in 50 mL of a mixture of 1 volume of decanoate,
anhydrous acetic acid R and 7 volumes of methyl ethyl
ketone R. Titrate with 0.1 M perchloric acid, using 0.2 mL of
naphtholbenzein solution R as indicator.
1 mL of 0.1 M perchloric acid is equivalent to 57.46 mg
of C31H41BrFNO3.

STORAGE
Protected from light, at a temperature below 25 °C.
E. 4-(4′-bromobiphenyl-4-yl)-1-[4-(4-fluorophenyl)-4-
IMPURITIES oxobutyl]piperidin-4-yl decanoate,
Specified impurities : A, B, C, D, E, F, G, H, I, J, K.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use) : L.
F. 4-(biphenyl-4-yl)-1-[4-(4-fluorophenyl)-4-
oxobutyl]piperidin-4-yl decanoate,

A. 1-[4-(4-fluorophenyl)-4-oxobutyl]-4-phenylpiperidin-4-yl G. 4-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-1-(4-
decanoate, fluorophenyl)butan-1-one (bromperidol),

1694 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Brompheniramine maleate

Solubility : soluble in water, freely soluble in ethanol (96 per

cent), in methanol and in methylene chloride.
IDENTIFICATION
First identification : C, F.
Second identification : A, B, D, E, F.
A. Melting point (2.2.14) : 130 °C to 135 °C.
H. 4-(4-bromophenyl)-1-[4-(4-fluorophenyl)-4- B. Ultraviolet and visible absorption spectrophotometry
oxobutyl]piperidin-4-yl octanoate, (2.2.25).
Test solution. Dissolve 65 mg in a 10.3 g/L solution of
hydrochloric acid R and dilute to 100.0 mL with the same
solution. Dilute 5.0 mL of this solution to 100.0 mL with a
10.3 g/L solution of hydrochloric acid R.
Spectral range : 220-320 nm.
Absorption maximum : at 265 nm.
Specific absorbance at the absorption maximum : 190 to 210.
I. 4-(4-bromophenyl)-1-[4-(4-fluorophenyl)-4- C. Infrared absorption spectrophotometry (2.2.24).
oxobutyl]piperidin-4-yl nonanoate,
Comparison: brompheniramine maleate CRS.
D. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 0.10 g of the substance to be
examined in methanol R and dilute to 5.0 mL with the
same solvent.
Reference solution. Dissolve 56 mg of maleic acid R in
methanol R and dilute to 10 mL with the same solvent.
J. 4-(4-bromophenyl)-1-[4-(4-fluorophenyl)-4- Plate : TLC silica gel F254 plate R.
oxobutyl]piperidin-4-yl undecanoate, Mobile phase : water R, anhydrous formic acid R, methanol R,
di-isopropyl ether R (3:7:20:70 V/V/V/V).
Application : 5 μL.
Development : over 2/3 of the plate.
Drying : in a current of air for 5 min.
Detection : examine in ultraviolet light at 254 nm.
Results : the chromatogram obtained with the test solution
shows 2 clearly separated spots; the upper spot is similar in
K. 4-(4-bromophenyl)-1-[4-(4-fluorophenyl)-4- position and size to the spot in the chromatogram obtained
oxobutyl]piperidin-4-yl dodecanoate, with the reference solution.
E. To 0.15 g in a porcelain crucible add 0.5 g of anhydrous
sodium carbonate R. Heat over an open flame for 10 min.
Allow to cool. Take up the residue in 10 mL of dilute
nitric acid R and filter. To 1 mL of the filtrate add 1 mL of
water R. The solution gives reaction (a) of bromides (2.3.1).
L. 1-(4-fluorophenyl)ethanone. F. Optical rotation (see Tests).
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
01/2014:0977 more intensely coloured than reference solution BY (2.2.2,
6
Method II).
BROMPHENIRAMINE MALEATE Dissolve 2.0 g in methanol R and dilute to 20 mL with the
same solvent.
Brompheniramini maleas pH (2.2.3) : 4.0 to 5.0.
Dissolve 0.20 g in 20 mL of carbon dioxide-free water R.
Optical rotation (2.2.7) : − 0.20° to + 0.20° (measured in a
2 dm tube).
Dissolve 2.5 g in water R and dilute to 25.0 mL with the same
solvent.
Related substances. Gas chromatography (2.2.28).
C20H23BrN2O4 Mr 435.3 Test solution. Dissolve 0.100 g of the substance to be examined
[980-71-2] in 10.0 mL of methylene chloride R.
Reference solution (a). Dilute 1.0 mL of the test solution to
DEFINITION
100.0 mL with methylene chloride R. Dilute 1.0 mL of this
(3RS)-3-(4-Bromophenyl)-N,N-dimethyl-3-(pyridin-2- solution to 10.0 mL with methylene chloride R.
yl)propan-1-amine (Z)-butenedioate. Reference solution (b). Dissolve 10 mg of chlorphenamine
Content : 98.0 per cent to 101.0 per cent (dried substance). maleate CRS (impurity A) and 10 mg of pheniramine
maleate CRS (impurity C) in methylene chloride R and dilute
CHARACTERS to 5 mL with the same solvent. To 2.5 mL of the solution add
Appearance : white or almost white, crystalline powder. 2.5 mL of the test solution.

General Notices (1) apply to all monographs and other texts 1695
Brotizolam EUROPEAN PHARMACOPOEIA 8.0

Column :
– material : fused silica ;
– size : l = 30 m, Ø = 0.32 mm ;
– stationary phase : polymethylphenylsiloxane R (film
thickness 0.5 μm).
Carrier gas : nitrogen for chromatography R.
Flow rate : 1.0 mL/min.
Split ratio : 1:5. C. (3RS)-N,N-dimethyl-3-phenyl-3-(pyridin-2-yl)propan-1-
Temperature : amine (pheniramine).
– column : 205 °C ;
– injection port and detector : 250 °C.
Detection : flame ionisation. 01/2008:2197
Injection : 1 μL. corrected 7.0
Run time : 1.2 times the retention time of brompheniramine.
Identification of impurities : use the chromatogram obtained BROTIZOLAM
with reference solution (b) to identify the peaks due to
impurities A and C.
Brotizolamum
Relative retention with reference to brompheniramine
(retention time = about 34 min) : impurity C = about 0.4 ;
impurity A = about 0.7.
System suitability : reference solution (b) :
– resolution : minimum 5.0 between the peaks due to
impurity A and brompheniramine.
Limits :
– impurities A, C : for each impurity, not more than 4 times
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.4 per cent) ; C15H10BrClN4S Mr 393.7
– unspecified impurities : for each impurity, not more than the [57801-81-7]
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ; DEFINITION
– total : not more than 10 times the area of the principal peak 2-Bromo-4-(2-chlorophenyl)-9-methyl-6H-thieno-[3,2-
in the chromatogram obtained with reference solution (a) f][1,2,4]-triazolo[4,3-a][1,4]diazepine.
(1.0 per cent) ; Content : 99.0 per cent to 101.0 per cent (dried substance).
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a) CHARACTERS
(0.05 per cent). Appearance : white or yellowish powder.
Heavy metals (2.4.8) : maximum 20 ppm. Solubility : practically insoluble in water, sparingly soluble or
1.0 g complies with test C. Prepare the reference solution using slightly soluble in methanol, slightly soluble in ethanol (96 per
2 mL of lead standard solution (10 ppm Pb) R. cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
IDENTIFICATION
on 1.000 g by drying in an oven at 105 °C for 3 h.
Infrared absorption spectrophotometry (2.2.24).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. Comparison: brotizolam CRS.
ASSAY TESTS
Dissolve 0.260 g in 50 mL of anhydrous acetic acid R. Titrate Related substances. Liquid chromatography (2.2.29). Carry
with 0.1 M perchloric acid, determining the end-point out the test protected from light and prepare the solutions
potentiometrically (2.2.20). immediately before use.
1 mL of 0.1 M perchloric acid is equivalent to 21.77 mg Test solution. Dissolve 50.0 mg of the substance to be
of C20H23BrN2O4. examined in acetonitrile R and dilute to 50.0 mL with the
STORAGE same solvent.
Protected from light. Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL of acetonitrile R. Dilute 1.0 mL of this solution to
IMPURITIES 10.0 mL with acetonitrile R.
Specified impurities : A, C. Reference solution (b). Dissolve 5 mg of the substance to be
examined and 5 mg of brotizolam impurity B CRS in 50 mL
of acetonitrile R. Dilute 2 mL of this solution to 20 mL with
acetonitrile R.
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : octylsilyl silica gel for chromatography R
A. (3RS)-3-(4-chlorophenyl)-N,N-dimethyl-3-(pyridin-2- (5 μm) ;
yl)propan-1-amine (chlorphenamine), – temperature : 40 °C.

1696 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Budesonide

Mobile phase :
– mobile phase A : 2 g/L solution of sodium heptanesulfonate
monohydrate R ;
– mobile phase B : mix 25 volumes of a 2 g/L solution of
sodium heptanesulfonate R and 75 volumes of acetonitrile R ;

Time Mobile phase A Mobile phase B

(min) (per cent V/V) (per cent V/V) A. R1 = CH3, R2 = H : 4-(2-chlorophenyl)-9-methyl-
0-4 63 37 6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine
4 - 15 63 → 12 37 → 88 (desbromobrotizolam),
B. R1 = H, R2 = Br : 2-bromo-4-(2-chlorophenyl)-
Flow rate : 2.0 mL/min. 6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine
(desmethylbrotizolam).
Detection : spectrophotometer at 242 nm.
Injection : 5 μL. 01/2010:1075
Relative retention with reference to brotizolam (retention
time = about 7.4 min) : impurity A = about 0.5 ; BUDESONIDE
impurity B = about 0.9.
System suitability : reference solution (b) :
Budesonidum
– resolution : minimum 5.0 between the peaks due to
impurity B and brotizolam.
Limits :
– impurity B : not more than the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.1 per cent) ;
C25H34O6 Mr 430.5
– unspecified impurities : for each impurity, not more than the [51333-22-3]
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ; DEFINITION
Mixture of the C-22S (epimer A) and the C-22R (epimer B)
– total : not more than twice the area of the principal peak epimers of 16α,17-[(1RS)-butylidenebis(oxy)]-11β,21-
in the chromatogram obtained with reference solution (a) dihydroxypregna-1,4-diene-3,20-dione.
(0.2 per cent) ;
Content : 97.5 per cent to 102.0 per cent (dried substance).
– disregard limit : 0.5 times the area of the principal peak in
CHARACTERS
the chromatogram obtained with reference solution (a)
(0.05 per cent). Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, freely soluble in
Chlorides (2.4.4): maximum 100 ppm.
methylene chloride, sparingly soluble in ethanol (96 per cent).
Dissolve 0.67 g in 20.0 mL of methanol R, mix and filter.
IDENTIFICATION
Loss on drying (2.2.32) : maximum 0.5 per cent, determined First identification : A.
on 1.000 g by drying in an oven at 105 °C.
Second identification : B, C, D.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on A. Infrared absorption spectrophotometry (2.2.24).
1.0 g.
Comparison: budesonide CRS.
B. Thin-layer chromatography (2.2.27).
ASSAY Solvent mixture : methanol R, methylene chloride R
Dissolve 0.150 g in a mixture of 25 mL of glacial acetic acid R (10:90 V/V).
and 50 mL of acetic anhydride R. Titrate to the second point Test solution. Dissolve 25 mg of the substance to be
of inflexion with 0.1 M perchloric acid, determining the examined in the solvent mixture and dilute to 10 mL with
end-point potentiometrically (2.2.20). the solvent mixture.
1 mL of 0.1 M perchloric acid is equivalent to 19.68 mg Reference solution (a). Dissolve 25 mg of budesonide CRS
of C15H10BrClN4S. in the solvent mixture and dilute to 10 mL with the solvent
mixture.
Reference solution (b). Dissolve 12.5 mg of triamcinolone
IMPURITIES acetonide CRS in reference solution (a) and dilute to 5 mL
with reference solution (a).
Specified impurities : B.
Plate : TLC silica gel F254 plate R.
Other detectable impurities (the following substances would, Mobile phase : add a mixture of 1.2 volumes of water R and
if present at a sufficient level, be detected by one or other of 8 volumes of methanol R to a mixture of 15 volumes of
the tests in the monograph. They are limited by the general ether R and 77 volumes of methylene chloride R.
acceptance criterion for other/unspecified impurities and/or Application : 5 μL.
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these Development : over a path of 15 cm.
impurities for demonstration of compliance. See also 5.10. Drying : in air.
Control of impurities in substances for pharmaceutical use): A. Detection A : examine in ultraviolet light at 254 nm.

General Notices (1) apply to all monographs and other texts 1697
Budesonide EUROPEAN PHARMACOPOEIA 8.0

Results A : the principal spot in the chromatogram obtained Injection : 20 μL of test solution (a) and reference solutions (a)
with the test solution is similar in position and size to and (b).
the principal spot in the chromatogram obtained with Identification of impurities : use the chromatogram
reference solution (a). supplied with budesonide for system suitability CRS and the
Detection B : spray with alcoholic solution of sulfuric acid R ; chromatogram obtained with reference solution (b) to identify
heat at 120 °C for 10 min or until the spots appear and the peaks due to impurities A, D, G, K and L.
allow to cool ; examine the chromatograms in daylight and Relative retention with reference to budesonide epimer B
in ultraviolet light at 365 nm. (retention time = about 17 min) : impurity A = about 0.1 ;
Results B : the principal spot in the chromatogram obtained epimers of impurity D = about 0.63 and 0.67 ;
with the test solution is similar in position, colour in impurity L = about 0.95 ; epimers of impurity G = about 1.2
daylight, fluorescence in ultraviolet light at 365 nm and and 1.3 ; epimers of impurity K = about 2.9 and 3.0.
size to the principal spot in the chromatogram obtained System suitability : reference solution (b) :
with reference solution (a). – peak-to-valley ratio : minimum 2.5, where Hp = height above
System suitability: reference solution (b) : the baseline of the 1st of the 2 peaks due to impurity G and
– the chromatogram shows 2 clearly separated spots. Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to budesonide
C. Dissolve about 2 mg in 2 mL of sulfuric acid R. Within
epimer A (the 2nd of the 2 principal peaks); and minimum
5 min a yellow colour develops. Within 30 min the colour
3, where Hp = height above the baseline of the peak due to
changes to brown or reddish-brown. Cautiously add the
impurity L and Hv = height above the baseline of the lowest
solution to 10 mL of water R and mix. The colour fades
point of the curve separating this peak from the peak due
and a clear solution remains.
to budesonide epimer B (the 1st of the 2 principal peaks).
D. Dissolve about 1 mg in 2 mL of a solution containing 2 g Limits :
of phosphomolybdic acid R dissolved in a mixture of 10 mL
of dilute sodium hydroxide solution R, 15 mL of water R – correction factors : for the calculation of content, multiply
and 25 mL of glacial acetic acid R. Heat for 5 min on a the peak areas of the following impurities by the
water-bath. Cool in iced water for 10 min and add 3 mL of corresponding correction factor : impurity D = 1.8 ;
dilute sodium hydroxide solution R. The solution is blue. impurity K = 1.3 ;
– impurities A, L : for each impurity, not more than twice
TESTS the sum of the areas of the 2 peaks due to the budesonide
Related substances. Liquid chromatography (2.2.29). Carry epimers in the chromatogram obtained with reference
out the test protected from light. solution (a) (0.2 per cent) ;
Solvent mixture : acetonitrile R, phosphate buffer solution – impurities D, K : for each impurity, for the sum of the areas
pH 3.2 R (32:68 V/V). of the 2 epimer peaks, not more than twice the sum of the
areas of the 2 peaks due to the budesonide epimers in the
Test solution (a). Dissolve 50 mg of the substance to be chromatogram obtained with reference solution (a) (0.2 per
examined in 15 mL of acetonitrile R and dilute to 50 mL with cent) ;
phosphate buffer solution pH 3.2 R.
– unspecified impurities : for each individual peak, not
Test solution (b). Dissolve 25.0 mg of the substance to be more than the sum of the areas of the 2 peaks due to the
examined in 15 mL of acetonitrile R and dilute to 50.0 mL budesonide epimers in the chromatogram obtained with
with phosphate buffer solution pH 3.2 R. reference solution (a) (0.10 per cent) ;
Reference solution (a). Dilute 1.0 mL of test solution (a) to – total : not more than 5 times the sum of the areas of the
10.0 mL with the solvent mixture. Dilute 1.0 mL of this 2 peaks due to the budesonide epimers in the chromatogram
solution to 100.0 mL with the solvent mixture. obtained with reference solution (a) (0.5 per cent) ;
Reference solution (b). Dissolve 5 mg of budesonide for system – disregard limit : 0.5 times the sum of the areas of the 2 peaks
suitability CRS (containing impurities A, D, G, K and L) in due to the budesonide epimers in the chromatogram
1.5 mL of acetonitrile R and dilute to 5 mL with phosphate obtained with reference solution (a) (0.05 per cent).
buffer solution pH 3.2 R.
Epimer A. Liquid chromatography (2.2.29) as described in the
Reference solution (c). Dissolve 25.0 mg of budesonide CRS in test for related substances with the following modifications.
15 mL of acetonitrile R and dilute to 50.0 mL with phosphate
Mobile phase :
buffer solution pH 3.2 R.
Time Mobile phase A Mobile phase B
Column :
(min) (per cent V/V) (per cent V/V)
– size : l = 0.15 m, Ø = 4.6 mm ; 0 - 21 100 0
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (3 μm) ; 21 - 22 100 → 0 0 → 100

– temperature : 50 °C. 22 - 31 0 100

Mobile phase :
Injection : 20 μL of test solution (b) and reference solutions (b)
– mobile phase A : anhydrous ethanol R, acetonitrile R, and (c).
phosphate buffer solution pH 3.2 R (2:32:68 V/V/V) ; Retention time : budesonide epimer B = about 17 min ;
– mobile phase B : acetonitrile R, phosphate buffer solution budesonide epimer A = about 19 min.
pH 3.2 R (50:50 V/V) ; System suitability :
Time Mobile phase A Mobile phase B – resolution : minimum 1.5 between the 2 principal peaks
(min) (per cent V/V) (per cent V/V) (budesonide epimers A and B) in the chromatogram
0 - 38 100 0 obtained with reference solution (c) ;
38 - 50 100 → 0 0 → 100 – peak-to-valley ratio : minimum 3, where Hp = height
above the baseline of the peak due to impurity L and
50 - 60 0 100 Hv = height above the baseline of the lowest point of
the curve separating this peak from the peak due to
Flow rate : 1 mL/min. budesonide epimer B (the 1st of the 2 principal peaks) in
Detection : spectrophotometer at 240 nm. the chromatogram obtained with reference solution (b).

1698 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Bufexamac

Limit :
– epimer A : 40.0 per cent to 51.0 per cent of the sum of the
areas of the 2 peaks due to the budesonide epimers.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.

ASSAY
E. 16α,17-[(1RS)-butylidenebis(oxy)]-11β,21-
Liquid chromatography (2.2.29). Examine the chromatograms dihydroxypregna-1,4,14-triene-3,20-dione,
obtained in the test for epimer A.
Calculate the percentage content of C25H34O6 from the sum of
the areas of the 2 peaks due to the budesonide epimers and
the declared content of budesonide CRS.

IMPURITIES
Specified impurities : A, D, K, L.
Other detectable impurities (the following substances would, G. 16α,17-[(1RS)-butylidenebis(oxy)]-11β,21-
if present at a sufficient level, be detected by one or other of dihydroxypregn-4-ene-3,20-dione.
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use) : B,
C, E, F, G, H, I, J.

H. 16α,17-[(1RS)-butylidenebis(oxy)]-21-hydroxypregna-
1,4,9(11)-triene-3,20-dione,

A. 11β,16α,17,21-tetrahydroxypregna-1,4-diene-3,20-dione,

I. 11β,17,21-trihydroxy-3,20-dioxopregna-1,4-dien-16α-yl
butanoate,

B. R = H : 16α,17-[(1RS)-ethylidenebis(oxy)]-11β,21-
dihydroxypregna-1,4-diene-3,20-dione,
J. 16α,17-[(1RS)-butylidenebis(oxy)]-9α-bromo-11β,21-
F. R = CH3 : 16α,17-[1-methylethylidenebis(oxy)]-11β,21- dihydroxypregna-1,4-diene-3,20-dione,
dihydroxypregna-1,4-diene-3,20-dione,

C. 16α,17-[(1RS)-butylidenebis(oxy)]-11β-hydroxy-17- L. 16α,17-[(1RS)-butylidenebis(oxy)]-21-hydroxypregna-1,4-
(hydroxymethyl)-D-homoandrosta-1,4-diene-3,17a-dione, diene-3,11,20-trione.

01/2008:1179

BUFEXAMAC
Bufexamacum

D. R = CHO : 16α,17-[(1RS)-butylidenebis(oxy)]-11β-
hydroxy-3,20-dioxopregna-1,4-dien-21-al,

K. R = CH2-O-CO-CH3 : 16α,17-[(1RS)-butylidenebis(oxy)]- C12H17NO3 Mr 223.3

11β,21-dihydroxypregna-1,4-diene-3,20-dione-21-acetate, [2438-72-4]

General Notices (1) apply to all monographs and other texts 1699
Buflomedil hydrochloride EUROPEAN PHARMACOPOEIA 8.0

DEFINITION Flow rate : 1 mL/min.

2-(4-Butoxyphenyl)-N-hydroxyacetamide. Detection : spectrophotometer at 275 nm.
Content : 98.5 per cent to 101.5 per cent (dried substance). Injection : 20 μL.
CHARACTERS Run time : 4 times the retention time of bufexamac.
Appearance : white or almost white, crystalline powder. System suitability : reference solution (b) :
Solubility : practically insoluble in water, soluble in – resolution : minimum 2.0 between the peaks due to salicylic
dimethylformamide, slightly soluble in ethyl acetate and in acid and bufexamac.
methanol. Limits :
– impurities A, B, C, D : for each impurity, not more than the
IDENTIFICATION area of the principal peak in the chromatogram obtained
First identification : B. with reference solution (a) (0.2 per cent) ;
Second identification : A, C. – total : not more than 2.5 times the area of the principal peak
A. Ultraviolet and visible absorption spectrophotometry in the chromatogram obtained with reference solution (a)
(2.2.25). (0.5 per cent) ;
Test solution. Dissolve 20 mg in methanol R and dilute to – disregard limit : 0.05 times the area of the principal peak
20 mL with the same solvent. Dilute 1 mL of this solution in the chromatogram obtained with reference solution (a)
to 50 mL with methanol R. (0.01 per cent).
Spectral range : 210-360 nm. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Absorption maxima : at 228 nm, 277 nm and 284 nm. on 1.000 g by drying in vacuo at 80 °C for 3 h.
B. Infrared absorption spectrophotometry (2.2.24). Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Preparation : discs. 1.0 g.
Comparison : bufexamac CRS. ASSAY
C. Thin-layer chromatography (2.2.27). Dissolve 0.200 g in 50 mL of dimethylformamide R. Titrate
Test solution. Dissolve 10 mg of the substance to be with 0.1 M lithium methoxide, determining the end-point
examined in methanol R and dilute to 5 mL with the same potentiometrically (2.2.20).
solvent. 1 mL of 0.1 M lithium methoxide is equivalent to 22.33 mg
Reference solution (a). Dissolve 20 mg of bufexamac CRS in of C12H17NO3.
methanol R and dilute to 10 mL with the same solvent.
Reference solution (b). Dissolve 10 mg of salicylic acid R STORAGE
in reference solution (a) and dilute to 5 mL with the same Protected from light.
solution.
IMPURITIES
Plate : TLC silica gel F254 plate R.
Specified impurities : A, B, C, D.
Mobile phase : glacial acetic acid R, dioxan R, toluene R
(4:20:90 V/V/V).
Application : 10 μL.
Development : over a path of 15 cm.
Drying : in a current of warm air. A. R = OH : 2-(4-butoxyphenyl)acetic acid,
Detection : examine in ultraviolet light at 254 nm. B. R = OCH3 : methyl 2-(4-butoxyphenyl)acetate,
System suitability: reference solution (b) :
C. R = OC4H9 : butyl 2-(4-butoxyphenyl)acetate,
– the chromatogram shows 2 clearly separated spots.
Results : the principal spot in the chromatogram obtained D. R = NH2 : 2-(4-butoxyphenyl)acetamide.
with the test solution is similar in position and size to
the principal spot in the chromatogram obtained with 04/2013:1398
reference solution (a).
TESTS BUFLOMEDIL HYDROCHLORIDE
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50.0 mg of the substance to be Buflomedili hydrochloridum
examined in the mobile phase and dilute to 20.0 mL with the
mobile phase.
Reference solution (a). Dilute 5.0 mL of the test solution to
25.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 100.0 mL with the mobile phase.
Reference solution (b). Dissolve 5 mg of bufexamac CRS and C17H26ClNO4 Mr 343.9
5 mg of salicylic acid R in the mobile phase and dilute to [35543-24-9]
10 mL with the mobile phase. Dilute 1 mL of this solution to
10 mL with the mobile phase. DEFINITION
Column : 4-(Pyrrolidin-1-yl)-1-(2,4,6-trimethoxyphenyl)butan-1-one
– size : l = 0.25 m, Ø = 4.6 mm ; hydrochloride.
– stationary phase : octadecylsilyl silica gel for Content : 98.5 per cent to 101.5 per cent (dried substance).
chromatography R (5 μm) with a specific surface CHARACTERS
area of 350 m2/g and a pore size of 10 nm.
Mobile phase : mix 30 volumes of a 1.4 g/L solution of Appearance : white or almost white, microcrystalline powder.
dipotassium hydrogen phosphate R and 70 volumes of Solubility : freely soluble in water, soluble in ethanol (96 per
methanol R, then adjust to pH 3.6 with dilute phosphoric cent), very slightly soluble in acetone.
acid R. mp : about 195 °C, with decomposition.

1700 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Buflomedil hydrochloride

IDENTIFICATION Reference solution (c). Dissolve the contents of a vial of

buflomedil for peak identification CRS (containing impurities A
First identification : B, D.
and C) in 1.0 mL of reference solution (b).
Second identification : A, C, D. Column :
A. Ultraviolet and visible absorption spectrophotometry – size : l = 0.25 m, Ø = 4.6 mm ;
(2.2.25).
– stationary phase : end-capped octadecylsilyl silica gel for
Test solution. Dissolve 25.0 mg in ethanol (96 per cent) R chromatography R (5 μm) ;
and dilute to 50.0 mL with the same solvent. Dilute 2.0 mL
of the solution to 20.0 mL with ethanol (96 per cent) R. – temperature : 40 °C.
Spectral range : 220-350 nm. Mobile phase : mix 45 volumes of acetonitrile R1 and
55 volumes of a 9.25 g/L solution of potassium dihydrogen
Absorption maximum : at 275 nm. phosphate R adjusted to pH 2.5 with phosphoric acid R.
Specific absorbance at the absorption maximum : 143 to 149. Flow rate : 1 mL/min.

B. Infrared absorption spectrophotometry (2.2.24). Detection : spectrophotometer at 210 nm.

Comparison : buflomedil hydrochloride CRS. Injection : 10 μL of the test solution and reference solutions (a)
and (c).
C. Thin-layer chromatography (2.2.27).
Run time : twice the retention time of buflomedil.
Test solution. Dissolve 40 mg of the substance to be
examined in methanol R and dilute to 2 mL with the same Identification of impurities : use the chromatogram supplied
solvent. with buflomedil for peak identification CRS and the
chromatogram obtained with reference solution (c) to identify
Reference solution. Dissolve 40 mg of buflomedil the peaks due to impurities A, B and C.
hydrochloride CRS in methanol R and dilute to 2 mL with
the same solvent. Relative retention with reference to buflomedil
(retention time = about 5 min) : impurity B = about 0.6 ;
Plate : TLC silica gel F254 plate R. impurity C = about 0.7 ; impurity A = about 1.5.

Mobile phase : triethylamine R, 2-propanol R, toluene R System suitability : reference solution (c) :
(5:50:50 V/V/V). – resolution : minimum 1.5 between the peaks due to
impurity B and impurity C.
Application : 10 μL.
Limits :
Development : over 3/4 of the plate.
– impurities A, B, C : for each impurity, not more than the
Drying : in air. area of the principal peak in the chromatogram obtained
with reference solution (a) (0.25 per cent) ;
Detection : examine in ultraviolet light at 254 nm.
– unspecified impurities : for each impurity, not more than
Results : the principal spot in the chromatogram obtained 0.4 times the area of the principal peak in the chromatogram
with the test solution is similar in position and size to the obtained with reference solution (a) (0.10 per cent) ;
principal spot in the chromatogram obtained with the
reference solution. – total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (a)
D. It gives reaction (a) of chlorides (2.3.1). (0.5 per cent) ;
– disregard limit : 0.2 times the area of the principal peak in
TESTS the chromatogram obtained with reference solution (a)
(0.05 per cent).
Solution S. Dissolve 2.5 g in carbon dioxide-free water R and
dilute to 50 mL with the same solvent. Heavy metals (2.4.8) : maximum 10 ppm.

Appearance of solution. Solution S is clear (2.2.1) and 2.0 g complies with test C. Prepare the reference solution using
colourless (2.2.2, Method II). 2 mL of lead standard solution (10 ppm Pb) R.
pH (2.2.3): 5.0 to 6.5 for solution S. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 2 h.
Related substances. Liquid chromatography (2.2.29).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Test solution. Dissolve 0.10 g of the substance to be examined 1.0 g.
in the mobile phase and dilute to 10.0 mL with the mobile
phase.
ASSAY
Reference solution (a). Dilute 0.5 mL of the test solution to
100.0 mL with the mobile phase. Dilute 5.0 mL of this solution Dissolve 0.300 g in 15 mL of anhydrous acetic acid R and add
to 10.0 mL with the mobile phase. 35 mL of acetic anhydride R. Titrate with 0.1 M perchloric
acid, determining the end-point potentiometrically (2.2.20).
Reference solution (b). Dissolve 2 mg of buflomedil
impurity B CRS in the mobile phase, add 0.5 mL of the test 1 mL of 0.1 M perchloric acid is equivalent to 34.39 mg of
solution and dilute to 100.0 mL with the mobile phase. C17H26ClNO4.

General Notices (1) apply to all monographs and other texts 1701
Bumetanide EUROPEAN PHARMACOPOEIA 8.0

IMPURITIES TESTS
Specified impurities : A, B, C. Appearance of solution. The solution is clear (2.2.1) and
colourless (2.2.2, Method II).
Dissolve 0.1 g in a 6 g/L solution of potassium hydroxide R and
dilute to 20 mL with the same solution.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50 mg of the substance to be examined
in the mobile phase and dilute to 25.0 mL with the mobile
A. 1-(2-hydroxy-4,6-dimethoxyphenyl)-4-(pyrrolidin-1- phase.
yl)butan-1-one, Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10.0 mL with the mobile phase.
Reference solution (b). Dissolve 2 mg of bumetanide
impurity A CRS and 2 mg of bumetanide impurity B CRS in
the mobile phase and dilute to 10.0 mL with the mobile phase.
Dilute 1.0 mL of this solution to 100.0 mL with the mobile
B. 1-(4-hydroxy-2,6-dimethoxyphenyl)-4-(pyrrolidin-1- phase.
yl)butan-1-one, Column :
– size : l = 0.15 m, Ø = 4.6 mm,
– stationary phase : end-capped octylsilyl silica gel for
chromatography R (3.5 μm).
Mobile phase : mix 70 volumes of methanol R, 25 volumes
of water for chromatography R and 5 volumes of a 27.2 g/L
solution of potassium dihydrogen phosphate R previously
C. 1-(2,4-dihydroxy-6-methoxyphenyl)-4-(pyrrolidin-1- adjusted to pH 7.0 with a 280 g/L solution of potassium
yl)butan-1-one. hydroxide R ; add tetrahexylammonium bromide R to this
mixture to obtain a concentration of 2.17 g/L.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 254 nm.
01/2008:1076 Injection : 10 μL.
corrected 6.0
Run time : 5 times the retention time of bumetanide.
Relative retention with reference to bumetanide
BUMETANIDE (retention time = about 6 min) : impurity B = about 0.4 ;
impurity A = about 0.6 ; impurity D = about 2.5 ;
impurity C = about 4.4.
Bumetanidum
System suitability : reference solution (b) :
– resolution : minimum 2.0 between the peaks due to
impurity A and impurity B.
Limits :
– impurities A, B, C, D : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent),
– other impurities : for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
C17H20N2O5S Mr 364.4
reference solution (a) (0.1 per cent),
[28395-03-1]
– total : not more than twice the area of the principal peak
DEFINITION in the chromatogram obtained with reference solution (a)
(0.2 per cent),
3-(Butylamino)-4-phenoxy-5-sulfamoylbenzoic acid.
– disregard limit : 0.5 times the area of the principal peak in
Content : 99.0 per cent to 101.0 per cent (dried substance). the chromatogram obtained with reference solution (a)
(0.05 per cent).
CHARACTERS Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Appearance : white or almost white, crystalline powder. on 1.000 g by drying in an oven at 105 °C for 4 h.
Solubility : practically insoluble in water, soluble in acetone Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
and in alcohol, slightly soluble in methylene chloride. It 1.0 g.
dissolves in dilute solutions of alkali hydroxides.
ASSAY
It shows polymorphism (5.9).
Dissolve 0.300 g in 50 mL of alcohol R. Add 0.1 mL of phenol
mp : about 233 °C. red solution R. Titrate with 0.1 M sodium hydroxide until a
violet-red colour is obtained. Carry out a blank titration.
IDENTIFICATION 1 mL of 0.1 M sodium hydroxide is equivalent to 36.44 mg of
Infrared absorption spectrophotometry (2.2.24). C17H20N2O5S.
Comparison : bumetanide CRS. STORAGE
If the spectra obtained in the solid state show differences, Protected from light.
dissolve the substance to be examined and the reference
substance separately in acetone R, evaporate to dryness and IMPURITIES
record new spectra using the residues. Specified impurities : A, B, C, D.

1702 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Bupivacaine hydrochloride

Detection : spray with dilute potassium iodobismuthate

solution R.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
the reference solution.
C. Dissolve 0.1 g in 10 mL of water R, add 2 mL of dilute
A. R1 = H, R2 = NO2 : 3-nitro-4-phenoxy-5-sulfamoylbenzoic sodium hydroxide solution R and shake with 2 quantities,
acid, each of 15 mL, of 1,1-dimethylethyl methyl ether R. Dry the
combined upper layers over anhydrous sodium sulfate R
B. R1 = H, R2 = NH2 : 3-amino-4-phenoxy-5-sulfamoylbenzoic and filter. Evaporate the filtrate, recrystallise the residue
acid, from ethanol (90 per cent V/V) R and dry under reduced
C. R1 = C4H9, R2 = NH-C4H9 : butyl 3-(butylamino)-4- pressure. The crystals melt (2.2.14) at 105 °C to 108 °C.
phenoxy-5-sulfamoylbenzoate, D. It gives reaction (a) of chlorides (2.3.1).
E. Optical rotation (see Tests).
TESTS
Solution S. Dissolve 1.0 g in carbon dioxide-free water R and
dilute to 50 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Acidity or alkalinity. To 10 mL of solution S add 0.2 mL of
D. 3-[[(2RS)-2-ethylhexyl]amino]-4-phenoxy-5- 0.01 M sodium hydroxide ; the pH (2.2.3) is not less than 4.7.
sulfamoylbenzoic acid. Add 0.4 mL of 0.01 M hydrochloric acid ; the pH is not greater
than 4.7.
04/2013:0541 Optical rotation (2.2.7) : − 0.10° to + 0.10°.
Dissolve 1.0 g in methanol R and dilute to 20.0 mL with the
BUPIVACAINE HYDROCHLORIDE same solvent.
Related substances. Gas chromatography (2.2.28).
Bupivacaini hydrochloridum Internal standard solution. Dissolve 25 mg of methyl
behenate R in methylene chloride R and dilute to 500 mL with
the same solvent.
Test solution. Dissolve 50.0 mg of the substance to be
examined in 2.5 mL of water R, add 2.5 mL of dilute sodium
hydroxide solution R and extract with 2 quantities, each of
5 mL, of the internal standard solution. Filter the lower layer.
C18H29ClN2O,H2O Mr 342.9 Reference solution (a). Dissolve 10 mg of the substance to be
[73360-54-0] examined, 10 mg of bupivacaine impurity B CRS and 10 mg
DEFINITION of bupivacaine impurity E CRS in 2.5 mL of water R, add
2.5 mL of dilute sodium hydroxide solution R and extract with
(2RS)-1-Butyl-N-(2,6-dimethylphenyl)piperidine-2-
2 quantities, each of 5 mL, of the internal standard solution.
carboxamide hydrochloride monohydrate.
Filter the lower layer and dilute to 20 mL with the internal
Content : 98.5 per cent to 101.0 per cent (dried substance). standard solution.
CHARACTERS Reference solution (b). Dilute 1.0 mL of the test solution to
Appearance : white or almost white, crystalline powder or 100.0 mL with the internal standard solution.
colourless crystals. Reference solution (c). Dilute 5.0 mL of reference solution (b)
Solubility : soluble in water, freely soluble in ethanol (96 per to 10.0 mL with the internal standard solution.
cent). Reference solution (d). Dilute 1.0 mL of reference solution (b)
to 10.0 mL with the internal standard solution.
IDENTIFICATION Column :
First identification : A, D, E. – material : fused silica ;
Second identification : B, C, D, E. – size : l = 30 m, Ø = 0.32 mm ;
A. Infrared absorption spectrophotometry (2.2.24). – stationary phase : poly(dimethyl)(diphenyl)siloxane R (film
Comparison : bupivacaine hydrochloride CRS. thickness 0.25 μm).
B. Thin-layer chromatography (2.2.27). Carrier gas : helium for chromatography R.
Test solution. Dissolve 25 mg of the substance to be Flow rate : 2.5 mL/min.
examined in methanol R and dilute to 5 mL with the same Split ratio : 1:12.
solvent. Temperature :
Reference solution. Dissolve 25 mg of bupivacaine Time Temperature
hydrochloride CRS in methanol R and dilute to 5 mL with (min) (°C)
the same solvent. 0 180
Plate : TLC silica gel G plate R.
Column 0 - 10 180 → 230
Mobile phase : concentrated ammonia R, methanol R
(0.1:100 V/V). 10 - 15 230
Application : 5 μL. Injection port 250
Development : over a path of 10 cm.
Detector 250
Drying : in air.

General Notices (1) apply to all monographs and other texts 1703
Bupivacaine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Detection : flame ionisation. – mobile phase B : acetonitrile R ;

Injection : 1 μL. Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
Identification of impurities : use the chromatogram obtained 0 - 10 100 0
with reference solution (a) to identify the peaks due to
impurities B and E. 10 - 15 100 → 80 0 → 20

15 - 25 80 20
Relative retention with reference to bupivacaine
(retention time = about 10 min) : impurity B = about 0.7 ;
Flow rate : 1.0 mL/min.
impurity E = about 1.1 ; internal standard = about 1.4.
Detection : spectrophotometer at 240 nm.
System suitability: reference solution (a) : Injection : 50 μL.
– resolution : minimum 3.0 between the peaks due to Identification of impurities: use the chromatogram obtained
bupivacaine and impurity E. with reference solution (a) to identify the peak due to
impurity F.
Limits : Relative retention with reference to bupivacaine (retention
– impurity B : calculate the ratio (R1) of the area of the time = about 20 min) : impurity F = about 0.3 ; methyl
principal peak to the area of the peak due to the internal benzoate = about 0.4.
standard from the chromatogram obtained with reference System suitability :
solution (c) ; from the chromatogram obtained with the – resolution : minimum 4.0 between the peaks due to
test solution, calculate the ratio of the area of the peak due impurity F and methyl benzoate in the chromatogram
to impurity B to the area of the peak due to the internal obtained with reference solution (b) ;
standard : this ratio is not greater than R1 (0.5 per cent) ;
– signal-to-noise ratio : minimum 40 for the principal peak in
– unspecified impurities : calculate the ratio (R2) of the area of the chromatogram obtained with reference solution (a).
the principal peak to the area of the peak due to the internal Limit :
standard from the chromatogram obtained with reference – impurity F : not more than the area of the principal peak
solution (d) ; from the chromatogram obtained with the test in the chromatogram obtained with reference solution (a)
solution, calculate for each impurity the ratio of the area of (10 ppm).
any peak, apart from the principal peak, the peak due to
impurity B and the peak due to the internal standard, to Heavy metals (2.4.8) : maximum 10 ppm.
the area of the peak due to the internal standard : this ratio Dissolve 2.0 g in a mixture of 15 volumes of water R and
is not greater than R2 (0.10 per cent) ; 85 volumes of methanol R and dilute to 20 mL with the same
mixture of solvents. 12 mL of the solution complies with
– total : calculate the ratio (R3) of the area of the principal peak test B. Prepare the reference solution using lead standard
to the area of the peak due to the internal standard from solution (1 ppm Pb) obtained by diluting lead standard
the chromatogram obtained with reference solution (b) ; solution (100 ppm Pb) R with a mixture of 15 volumes of
from the chromatogram obtained with the test solution, water R and 85 volumes of methanol R.
calculate the ratio of the sum of the areas of any peaks,
apart from the principal peak and the peak due to the Loss on drying (2.2.32) : 4.5 per cent to 6.0 per cent,
internal standard, to the area of the peak due to the internal determined on 1.000 g by drying in an oven at 105 °C.
standard : this ratio is not greater than R3 (1.0 per cent) ; Sulfated ash (2.4.14) : maximum 0.1 per cent, determined
on 1.0 g.
– disregard limit : ratio less than 0.05 times R3 (0.05 per cent).
Impurity F. Liquid chromatography (2.2.29). Prepare the ASSAY
solutions immediately before use. Dissolve 0.250 g in a mixture of 20 mL of water R and 25 mL
of ethanol (96 per cent) R. Add 5.0 mL of 0.01 M hydrochloric
Test solution. Dissolve 50 mg of the substance to be examined acid. Carry out a potentiometric titration (2.2.20), using 0.1 M
in mobile phase A and dilute to 10.0 mL with mobile phase A. ethanolic sodium hydroxide. Read the volume added between
Reference solution (a). Dissolve 5.0 mg of bupivacaine the 2 points of inflexion.
impurity F CRS in mobile phase A and dilute to 100.0 mL with 1 mL of 0.1 M ethanolic sodium hydroxide is equivalent to
mobile phase A. Dilute 1.0 mL of the solution to 100.0 mL 32.49 mg of C18H29ClN2O.
with mobile phase A. Dilute 1.0 mL of this solution to 10.0 mL
STORAGE
with mobile phase A.
Protected from light.
Reference solution (b). Dissolve 20 mg of methyl benzoate R
and 25 mg of bupivacaine impurity F CRS in mobile phase A IMPURITIES
and dilute to 50.0 mL with mobile phase A. Dilute 3.0 mL of Specified impurities : B, F.
the solution to 50.0 mL with mobile phase A. Dilute 1.0 mL of Other detectable impurities (the following substances would,
this solution to 10.0 mL with mobile phase A. if present at a sufficient level, be detected by one or other of
Column : the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
– size : l = 0.25 m, Ø = 4.6 mm ; by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
– stationary phase : end-capped octadecylsilyl silica gel for for demonstration of compliance. See also 5.10. Control of
chromatography R (5 μm). impurities in substances for pharmaceutical use) : A, C, D, E.
Mobile phase :
– mobile phase A : dissolve 0.23 g of sodium dihydrogen
phosphate monohydrate R and 3.626 g of disodium hydrogen
phosphate dihydrate R in water R and dilute to 1000 mL
with the same solvent ; mix equal volumes of this solution
(pH 8.0) and acetonitrile R ; A. N-(2,6-dimethylphenyl)pyridine-2-carboxamide,

1704 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Buprenorphine

Appearance of solution. Solution S is clear (2.2.1) and

colourless (2.2.2, Method II).
Specific optical rotation (2.2.7) : − 103 to − 107 (dried
substance), determined on solution S.
B. (2RS)-N-(2,6-dimethylphenyl)piperidine-2-carboxamide, Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50.0 mg of the substance to be
examined in methanol R and dilute to 10.0 mL with the same
solvent.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with methanol R. Dilute 1.0 mL of this solution to
10.0 mL with methanol R.
C. 1-(2,6-dimethylphenyl)-1,5,6,7-tetrahydro-2H-azepin-2- Reference solution (b). Dissolve 5 mg of buprenorphine for
one, system suitability CRS (containing impurities A, B, F, G, H
and J) in 1.0 mL of methanol R.
Column :
– size : l = 0.05 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (3.5 μm) ;
D. (2RS)-2,6-dichloro-N-(2,6-dimethylphenyl)hexanamide,
– temperature : 30 °C.
Mobile phase :
– mobile phase A : mix 10 volumes of acetonitrile R and
90 volumes of the following solution : dissolve 5.44 g of
potassium dihydrogen phosphate R in 900 mL of water R,
E. 6-(butylamino)-N-(2,6-dimethylphenyl)hexanamide, adjust to pH 4.5 with a 5 per cent V/V solution of phosphoric
acid R and dilute to 1000 mL with water R ;
– mobile phase B : acetonitrile R ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-2 89 11
F. 2,6-dimethylaniline. 2 - 12 89 → 64 11 → 36

12 - 15 64 → 41 36 → 59
07/2009:1180
corrected 7.0 15 - 20 41 → 39 59 → 61

Flow rate : 1.3 mL/min.

BUPRENORPHINE Detection : spectrophotometer at 240 nm.
Injection : 5 μL.
Buprenorphinum Identification of impurities : use the chromatogram supplied
with buprenorphine for system suitability CRS and the
chromatogram obtained with reference solution (b) to identify
the peaks due to impurities A, B, F, G, H and J.
Relative retention with reference to buprenorphine
(retention time = about 8.5 min) : impurity B = about 0.4 ;
impurity J = about 1.1 ; impurity F = about 1.27 ;
impurity H = about 1.33 ; impurity A = about 1.40 ;
C29H41NO4 Mr 467.6 impurity G = about 1.8.
[52485-79-7] System suitability : reference solution (b) :
DEFINITION – resolution : minimum 1.5 between the peaks due to
buprenorphine and impurity J.
(2S)-2-[17-(Cyclopropylmethyl)-4,5α-epoxy-3-hydroxy-
6-methoxy-6α,14-ethano-14α-morphinan-7α-yl]-3,3- Limits :
dimethylbutan-2-ol. – correction factor : for the calculation of content, multiply
Content : 98.5 per cent to 101.5 per cent (dried substance). the peak area of impurity G by 0.3 ;
– impurity H : not more than 2.5 times the area of the
CHARACTERS principal peak in the chromatogram obtained with
Appearance : white or almost white, crystalline powder. reference solution (a) (0.25 per cent) ;
Solubility : very slightly soluble in water, freely soluble in – impurities A, B, F, J : for each impurity, not more than
acetone, soluble in methanol, slightly soluble in cyclohexane. twice the area of the principal peak in the chromatogram
It dissolves in dilute solutions of acids. obtained with reference solution (a) (0.2 per cent) ;
mp : about 217 °C. – impurity G : not more than 1.5 times the area of the
principal peak in the chromatogram obtained with
IDENTIFICATION reference solution (a) (0.15 per cent) ;
Infrared absorption spectrophotometry (2.2.24). – unspecified impurities : for each impurity, not more than the
Comparison : buprenorphine CRS. area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
TESTS – total : not more than 7 times the area of the principal peak
Solution S. Dissolve 0.250 g in anhydrous ethanol R and dilute in the chromatogram obtained with reference solution (a)
to 25.0 mL with the same solvent. (0.7 per cent) ;

General Notices (1) apply to all monographs and other texts 1705
Buprenorphine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

– disregard limit : 0.5 times the area of the principal peak in

the chromatogram obtained with reference solution (a)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 1.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
ASSAY
F. 17-(cyclopropylmethyl)-4,5α-epoxy-6-methoxy-7α-[1-(1,1-
Dissolve 0.400 g in 40 mL of anhydrous acetic acid R. Titrate dimethylethyl)ethenyl]-6α,14-ethano-14α-morphinan-3-ol,
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 46.76 mg of
C29H41NO4.
STORAGE
Protected from light.
G. R-R : 17,17′-di(cyclopropylmethyl)-4,5α ;4′,5α′-diepoxy-
IMPURITIES 7α,7α′-di[(1S)-1-hydroxy-1,2,2-trimethylpropyl]-6,6′-
Specified impurities : A, B, F, G, H, J. dimethoxy-2,2′-bi(6α,14-ethano-14α-morphinan)-3,3′-diol
Other detectable impurities (the following substances would, (2,2′-bibuprenorphine),
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : C, D, E, I.

I. 17-(cyclopropylmethyl)-4″,4″,5″,5″-tetramethyl-4″,5″-
dihydro-(7βH)-6α,14-ethano-(5βH)-difurano-
[2′,3′,4′,5′:4,12,13,5 ;2″,3″:6,7]-14α-morphinan-3-ol,

A. R = CH2-CH2-CH=CH2 : (2S)-2-[17-(but-3-enyl)-
4,5α-epoxy-3-hydroxy-6-methoxy-6α,14-ethano-14α-
morphinan-7α-yl]-3,3-dimethylbutan-2-ol,

B. R = H : (2S)-2-(4,5α-epoxy-3-hydroxy-6-methoxy-6α,14- J. (2S)-2-[17-(cyclopropylmethyl)-4,5α-epoxy-3-hydroxy-
ethano-14α-morphinan-7α-yl)-3,3-dimethylbutan-2-ol 6-methoxy-6α,14-etheno-14α-morphinan-7α-yl]-3,3-
(norbuprenorphine), dimethylbutan-2-ol.

H. R = CH2-CH2-CH2-CH3 : (2S)-2-[17-butyl-4,5α-epoxy-3-
hydroxy-6-methoxy-6α,14-ethano-14α-morphinan-7α-yl]- 07/2009:1181
3,3-dimethylbutan-2-ol, corrected 6.6

BUPRENORPHINE HYDROCHLORIDE
Buprenorphini hydrochloridum

C. 4,5α-epoxy-7α-[(1S)-1-hydroxy-1,2,2-trimethylpropyl]-
3,6-dimethoxy-6α,14-ethano-14α-morphinan-17-
carbonitrile,
C29H42ClNO4 Mr 504.1
[53152-21-9]
DEFINITION
(2S)-2-[17-(Cyclopropylmethyl)-4,5α-epoxy-3-hydroxy-
6-methoxy-6α,14-ethano-14α-morphinan-7α-yl]-3,3-
dimethylbutan-2-ol hydrochloride.
D. R1 = R2 = CH3 : (2S)-2-[17-(cyclopropylmethyl)-4,5α- Content : 98.5 per cent to 101.5 per cent (dried substance).
epoxy-3,6-dimethoxy-6α,14-ethano-14α-morphinan-7α-
yl]-3,3-dimethylbutan-2-ol (3-O-methylbuprenorphine), CHARACTERS
Appearance : white or almost white, crystalline powder.
E. R1 = R2 = H : (2S)-2-[17-(cyclopropylmethyl)-4,5α-epoxy- Solubility : sparingly soluble in water, freely soluble in
3,6-dihydroxy-6α,14-ethano-14α-morphinan-7α-yl]-3,3- methanol, soluble in ethanol (96 per cent), practically
dimethylbutan-2-ol (6-O-desmethylbuprenorphine), insoluble in cyclohexane.

1706 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Buprenorphine hydrochloride

IDENTIFICATION System suitability : reference solution (b) :

A. Infrared absorption spectrophotometry (2.2.24). – resolution : minimum 1.5 between the peaks due to
buprenorphine and impurity J.
Comparison : buprenorphine hydrochloride CRS.
Limits :
B. 3 mL of solution S (see Tests) gives reaction (a) of chlorides
(2.3.1). – correction factor : for the calculation of content, multiply
the peak area of impurity G by 0.3 ;
TESTS – impurity H : not more than 2.5 times the area of the
principal peak in the chromatogram obtained with
Solution S. Dissolve 0.250 g in 5.0 mL of methanol R and,
reference solution (a) (0.25 per cent) ;
while stirring, dilute to 25.0 mL with carbon dioxide-free
water R. – impurities A, B, F, J : for each impurity, not more than
twice the area of the principal peak in the chromatogram
Appearance of solution. Solution S is clear (2.2.1) and obtained with reference solution (a) (0.2 per cent) ;
colourless (2.2.2, Method II).
– impurity G : not more than 1.5 times the area of the
Acidity or alkalinity. To 10.0 mL of solution S add 0.05 mL principal peak in the chromatogram obtained with
of methyl red solution R. Not more than 0.2 mL of 0.02 M reference solution (a) (0.15 per cent) ;
sodium hydroxide or 0.02 M hydrochloric acid is required to
change the colour of the indicator. – unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
Specific optical rotation (2.2.7) : − 92 to − 98 (dried with reference solution (a) (0.10 per cent) ;
substance).
– total : not more than 7 times the area of the principal peak
Dissolve 0.200 g in methanol R and dilute to 20.0 mL with in the chromatogram obtained with reference solution (a)
the same solvent. (0.7 per cent) ;
Related substances. Liquid chromatography (2.2.29). – disregard limit : 0.5 times the area of the principal peak in
Test solution. Dissolve 50.0 mg of the substance to be the chromatogram obtained with reference solution (a)
examined in methanol R and dilute to 10.0 mL with the same (0.05 per cent).
solvent. Loss on drying (2.2.32) : maximum 1.0 per cent, determined
Reference solution (a). Dilute 1.0 mL of the test solution to on 1.000 g by heating in an oven at 115-120 °C.
100.0 mL with methanol R. Dilute 1.0 mL of this solution to
ASSAY
10.0 mL with methanol R.
Dissolve 0.400 g in a mixture of 5 mL of 0.01 M hydrochloric
Reference solution (b). Dissolve 5 mg of buprenorphine for acid and 50 mL of ethanol (96 per cent) R. Carry out a
system suitability CRS (containing impurities A, B, F, G, H potentiometric titration (2.2.20), using 0.1 M sodium
and J) in 1.0 mL of methanol R. hydroxide. Read the volume added between the 2 points of
Column : inflexion. Carry out a blank titration.
– size : l = 0.05 m, Ø = 4.6 mm ; 1 mL of 0.1 M sodium hydroxide is equivalent to 50.41 mg
of C29H42ClNO4.
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (3.5 μm) ; STORAGE
– temperature : 30 °C. Protected from light.
Mobile phase :
IMPURITIES
– mobile phase A : mix 10 volumes of acetonitrile R and
90 volumes of the following solution : dissolve 5.44 g of Specified impurities : A, B, F, G, H, J.
potassium dihydrogen phosphate R in 900 mL of water R, Other detectable impurities (the following substances would,
adjust to pH 4.5 with a 5 per cent V/V solution of phosphoric if present at a sufficient level, be detected by one or other of
acid R and dilute to 1000 mL with water R ; the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
– mobile phase B : acetonitrile R ;
by the general monograph Substances for pharmaceutical use
Time Mobile phase A Mobile phase B (2034). It is therefore not necessary to identify these impurities
(min) (per cent V/V) (per cent V/V) for demonstration of compliance. See also 5.10. Control of
0-2 89 11 impurities in substances for pharmaceutical use) : C, D, E, I.
2 - 12 89 → 64 11 → 36

12 - 15 64 → 41 36 → 59

15 - 20 41 → 39 59 → 61

Flow rate : 1.3 mL/min.

Detection : spectrophotometer at 240 nm.
Injection : 5 μL. A. R = CH2-CH2-CH=CH2 : (2S)-2-[17-(but-3-enyl)-
4,5α-epoxy-3-hydroxy-6-methoxy-6α,14-ethano-14α-
Identification of impurities : use the chromatogram supplied morphinan-7α-yl]-3,3-dimethylbutan-2-ol,
with buprenorphine for system suitability CRS and the
chromatogram obtained with reference solution (b) to identify B. R = H : (2S)-2-(4,5α-epoxy-3-hydroxy-6-methoxy-6α,14-
the peaks due to impurities A, B, F, G, H and J. ethano-14α-morphinan-7α-yl)-3,3-dimethylbutan-2-ol
Relative retention with reference to buprenorphine (norbuprenorphine),
(retention time = about 8.5 min) : impurity B = about 0.4 ;
impurity J = about 1.1 ; impurity F = about 1.27 ; H. R = CH2-CH2-CH2-CH3 : (2S)-2-[17-butyl-4,5α-epoxy-3-
impurity H = about 1.33 ; impurity A = about 1.40 ; hydroxy-6-methoxy-6α,14-ethano-14α-morphinan-7α-yl]-
impurity G = about 1.8. 3,3-dimethylbutan-2-ol,

General Notices (1) apply to all monographs and other texts 1707
Buserelin EUROPEAN PHARMACOPOEIA 8.0

07/2011:1077

BUSERELIN
Buserelinum
C. 4,5α-epoxy-7α-[(1S)-1-hydroxy-1,2,2-trimethylpropyl]-
3,6-dimethoxy-6α,14-ethano-14α-morphinan-17-
carbonitrile,

C60H86N16O13 Mr 1239
[57982-77-1]
DEFINITION
5-Oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-O-
(1,1-dimethylethyl)-D-seryl-L-leucyl-L-arginyl-N-ethyl-L-
D. R1 = R2 = CH3 : (2S)-2-[17-(cyclopropylmethyl)-4,5α- prolinamide.
epoxy-3,6-dimethoxy-6α,14-ethano-14α-morphinan-7α- Synthetic nonapeptide analogue of human gonadotrophin-
yl]-3,3-dimethylbutan-2-ol (3-O-methylbuprenorphine), releasing hormone GnRH with agonistic activity to
gonadorelin. It is obtained by chemical synthesis and is
E. R1 = R2 = H : (2S)-2-[17-(cyclopropylmethyl)-4,5α-epoxy- available as an acetate.
3,6-dihydroxy-6α,14-ethano-14α-morphinan-7α-yl]-3,3- Content : 95.0 per cent to 102.0 per cent (anhydrous, acetic
dimethylbutan-2-ol (6-O-desmethylbuprenorphine), acid-free substance).
CHARACTERS
Appearance : white or slightly yellowish powder, hygroscopic.
Solubility : sparingly soluble in water and in dilute acids.
IDENTIFICATION
Carry out either tests A and B or tests A and C.
A. Examine the chromatograms obtained in the assay.
F. 17-(cyclopropylmethyl)-4,5α-epoxy-6-methoxy-7α-[1-(1,1- Results : the principal peak in the chromatogram obtained
dimethylethyl)ethenyl]-6α,14-ethano-14α-morphinan-3-ol, with the test solution is similar in retention time and size
to the principal peak in the chromatogram obtained with
reference solution (b).
B. Nuclear magnetic resonance spectrometry (2.2.64).
Preparation : 4 mg/mL solution in a mixture of 20 volumes
of deuterated acetic acid R and 80 volumes of deuterium
oxide R.
Comparison: 4 mg/mL solution of buserelin CRS in a
G. R-R : 17,17′-di(cyclopropylmethyl)-4,5α ;4′,5α′-diepoxy- mixture of 20 volumes of deuterated acetic acid R and
7α,7α′-di[(1S)-1-hydroxy-1,2,2-trimethylpropyl]-6,6′- 80 volumes of deuterium oxide R (dissolve the contents of a
dimethoxy-2,2′-bi(6α,14-ethano-14α-morphinan)-3,3′-diol vial of buserelin CRS in this solvent mixture to obtain the
(2,2′-bibuprenorphine), desired concentration).
Operating conditions :
– field strength : minimum 300 MHz ;
– temperature : 27 °C.
Results : examine the 1H NMR spectrum from 0 to 9 ppm.
The 1H NMR spectrum obtained is qualitatively similar to
the 1H NMR spectrum obtained with buserelin CRS.
C. Amino acid analysis (2.2.56). Method 1 for hydrolysis and
method 1 for analysis are suitable.
Express the content of each amino acid in moles. Calculate
I. 17-(cyclopropylmethyl)-4″,4″,5″,5″-tetramethyl-4″,5″- the relative proportions of the amino acids, taking 1/6 of
dihydro-(7βH)-6α,14-ethano-(5βH)-difurano- the sum of the number of moles of glutamic acid, histidine,
[2′,3′,4′,5′:4,12,13,5 ;2″,3″:6,7]-14α-morphinan-3-ol, tyrosine, leucine, arginine and proline as equal to 1. The
values fall within the following limits : serine 1.4 to 2.0 ;
proline 0.8 to 1.2 ; glutamic acid 0.9 to 1.1 ; leucine 0.9 to 1.1 ;
tyrosine 0.9 to 1.1 ; histidine 0.9 to 1.1 ; arginine 0.9 to 1.1.
Not more than traces of other amino acids are present.
TESTS
Appearance of solution. A 10 g/L solution is clear (2.2.1)
and not more intensely coloured than reference solution Y7
J. (2S)-2-[17-(cyclopropylmethyl)-4,5α-epoxy-3-hydroxy- (2.2.2, Method II).
6-methoxy-6α,14-etheno-14α-morphinan-7α-yl]-3,3- Specific optical rotation (2.2.7) : − 49 to − 58 (anhydrous,
dimethylbutan-2-ol. acetic acid-free substance), determined on a 10 g/L solution.

1708 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Buspirone hydrochloride

Specific absorbance (2.2.25) : 49 to 56, measured at the Calculate the content of buserelin (C60H86N16O13) using the
absorption maximum at 278 nm (anhydrous, acetic acid-free areas of the peaks in the chromatograms obtained and the
substance). declared content of C60H86N16O13 in buserelin CRS.
Dissolve 10.0 mg in 100.0 mL of 0.01 M hydrochloric acid. STORAGE
Related substances. Liquid chromatography (2.2.29). In an airtight container, protected from light, at a temperature
Test solution. Dissolve 5.0 mg of the substance to be examined of 2 °C to 8 °C. If the substance is sterile, store in an airtight,
in 5.0 mL of the mobile phase. sterile, tamper-proof container.
Reference solution (a). Dissolve the contents of a vial LABELLING
of D-His-buserelin CRS in the mobile phase. Dilute an
appropriate volume of this solution in the mobile phase to The label states :
obtain a final concentration of 1 mg/mL. Add 1.0 mL of the – the mass of peptide in the container ;
test solution to 1.0 mL of this solution. – where applicable, that the substance is suitable for use in
Reference solution (b). Dissolve the contents of a the manufacture of parenteral preparations.
vial of buserelin CRS in the mobile phase. Dilute an
IMPURITIES
appropriate volume of this solution in the mobile phase to
obtain a final concentration of 1.0 mg/mL. Specified impurities : A, B, C, D, E.
Reference solution (c). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm). A. [2-D-histidine]buserelin,
Mobile phase : mix 200 mL of acetonitrile R and 700 mL of an
11.2 g/L solution of phosphoric acid R and adjust to pH 2.5
with triethylamine R.
Flow rate : 0.8 mL/min.
Detection : spectrophotometer at 220 nm.
Injection : 10 μL of the test solution, reference solution (a) and
reference solution (c). B. [4-D-serine]buserelin,
Relative retention with reference to buserelin (retention
time = about 36 min): impurity B = about 0.76 ;
impurity C = about 0.83 ; impurity A = about 0.90 ;
impurity D = about 0.94 ; impurity E = about 0.94.
System suitability: reference solution (a) : C. buserelin-(3-9)-peptide,
– resolution : minimum 1.5 between the peaks due to
impurity A and buserelin.
Limits :
– sum of impurities D and E : not more than 3 times the area
of the principal peak in the chromatogram obtained with
reference solution (c) (3 per cent) ;
– any other impurity : for each impurity, not more than D. [5-D-tyrosine]buserelin,
3 times the area of the principal peak in the chromatogram
obtained with reference solution (c) (3 per cent) ;
– total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (c)
(5 per cent) ;
– disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (c) E. [1-(5-oxo-D-proline)]buserelin.
(0.1 per cent).
Acetic acid (2.5.34) : 3.0 per cent to 7.0 per cent. 01/2008:1711
Test solution. Dissolve 20.0 mg of the substance to be corrected 6.0
examined in a mixture of 5 volumes of mobile phase B and
95 volumes of mobile phase A and dilute to 10.0 mL with the BUSPIRONE HYDROCHLORIDE
same mixture of solvents.
Water (2.5.12) : maximum 4.0 per cent, determined on Buspironi hydrochloridum
80.0 mg.
Bacterial endotoxins (2.6.14) : less than 55.5 IU/mg, if
intended for use in the manufacture of parenteral preparations
without a further appropriate procedure for the removal of
bacterial endotoxins.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification. C21H32ClN5O2 Mr 422.0
Injection : test solution and reference solution (b). [33386-08-2]

General Notices (1) apply to all monographs and other texts 1709
Buspirone hydrochloride EUROPEAN PHARMACOPOEIA 8.0

DEFINITION Relative retention at 240 nm with reference to buspirone

8-[4-[4-(Pyrimidin-2-yl)piperazin-1-yl]butyl]-8- (retention time = about 25 min) : impurity A = about 0.2 ;
azaspiro[4.5]decane-7,9-dione hydrochloride. impurity B = about 0.3 ; impurity C = about 0.6 ;
Content : 99.0 per cent to 101.0 per cent (dried substance). impurity D = about 0.7 ; impurity E = about 0.8 ;
impurity F = about 0.9 ; impurity G = about 1.05 ;
CHARACTERS impurity H = about 1.1 ; impurity I = about 1.2 ;
Appearance : white or almost white, crystalline powder. impurity J = about 1.5.
Solubility : freely soluble in water and in methanol, practically Relative retention at 210 nm with reference to buspirone
insoluble in acetone. (retention time = about 25 min) : impurity K = about 0.6 ;
It shows polymorphism (5.9). impurity L = about 1.7 ; impurity M = about 1.8 ;
impurity N = about 1.9.
IDENTIFICATION System suitability : reference solution (b) :
A. Infrared absorption spectrophotometry (2.2.24). – peak-to-valley ratio at 240 nm : minimum 5.0, where
Comparison : buspirone hydrochloride CRS. Hp = height above the baseline of the peak due to impurity G
If the spectra obtained in the solid state show differences, and Hv = height above the baseline of the lowest point of the
dissolve the substance to be examined and the reference curve separating this peak from the peak due to buspirone;
substance separately in methanol R, evaporate to dryness – resolution at 210 nm : minimum 4.0 between the peaks due
on a water-bath and record new spectra using the residues. to impurity L and impurity N;
B. It gives reaction (a) of chlorides (2.3.1). – the chromatograms obtained are similar to the
TESTS chromatograms supplied with buspirone for system
suitability CRS.
Related substances. Liquid chromatography (2.2.29).
Limits : spectrophotometer at 240 nm :
Test solution. Dissolve 25.0 mg of the substance to be
examined in mobile phase A and dilute to 25.0 mL with – correction factor : for the calculation of content, multiply
mobile phase A. the peak area of impurity J by 2,
Reference solution (a). Dilute 1.0 mL of the test solution to – impurity E : not more than 3 times the area of the principal
100.0 mL with mobile phase A. Dilute 1.0 mL of this solution peak in the chromatogram obtained with reference
to 10.0 mL with mobile phase A. solution (a) (0.3 per cent),
Reference solution (b). Dissolve the contents of a vial of – impurity J : not more than twice the area of the principal
buspirone for system suitability CRS (containing impurities E, peak in the chromatogram obtained with reference
G, J, L and N) in 2.0 ml of mobile phase A and sonicate for solution (a) (0.2 per cent),
10 min. – any other impurity : for each impurity, not more than the
Column : area of the principal peak in the chromatogram obtained
– size : l = 0.15 m, Ø = 4.6 mm, with reference solution (a) (0.1 per cent),
– stationary phase : octadecylsilyl silica gel for – total : not more than 4 times the area of the principal peak
chromatography R (5 μm), in the chromatogram obtained with reference solution (a)
– temperature : 40 °C. (0.4 per cent),
Mobile phase : – disregard limit : 0.5 times the area of the principal
peak in the chromatogram obtained with reference
– mobile phase A : mix 950 volumes of a solution containing
solution (a) (0.05 per cent).
6.8 g/L of potassium dihydrogen phosphate R and 0.93 g/L
of sodium hexanesulfonate monohydrate R, previously Limits : spectrophotometer at 210 nm :
adjusted to pH 3.4 with phosphoric acid R and 50 volumes – impurity K : not more than the area of the principal peak
of acetonitrile R1; in the chromatogram obtained with reference solution (a)
– mobile phase B : mix 250 volumes of a solution containing (0.1 per cent),
3.4 g/L of potassium dihydrogen phosphate R and 3.52 g/L – any other impurity eluting with a relative retention greater
of sodium hexanesulfonate monohydrate R, previously than 1.6 : for each impurity, not more than the area of
adjusted to pH 2.2 with phosphoric acid R and 750 volumes the principal peak in the chromatogram obtained with
of acetonitrile R1, reference solution (a) (0.1 per cent),
Time Mobile phase A Mobile phase B – total : not more than twice the area of the principal peak
(min) (per cent V/V) (per cent V/V) in the chromatogram obtained with reference solution (a)
0-6 90 10 (0.2 per cent),
6 - 34 90 → 42 10 → 58 – disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
34 - 45 42 58 (0.05 per cent).
45 - 55 42 → 0 58 → 100 Loss on drying (2.2.32) : maximum 0.5 per cent, determined
55 - 56 0 → 100 100 → 0
on 1.000 g by drying at 105 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined
56 - 60 100 0
on 1.0 g.
60 - 61 100 → 90 0 → 10
ASSAY
Flow rate : 1 mL/min. Dissolve 0.150 g in 10 mL of glacial acetic acid R and add
Detection : variable wavelength spectrophotometer capable of 50 mL of acetic anhydride R. Titrate with 0.1 M perchloric
operating at 240 nm and at 210 nm. acid, determining the end-point potentiometrically (2.2.20).
Injection : 20 μL. 1 mL of 0.1 M perchloric acid is equivalent to 21.10 mg
Identification of impurities : use the chromatogram of C21H32ClN5O2.
supplied with buspirone for system suitability CRS and the
chromatogram obtained with reference solution (b) to identify STORAGE
the peaks due to impurities E, G, J, L and N. Protected from light.

1710 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Busulfan

IMPURITIES
Specified impurities : E, J, K.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these I. 8-[4-[4-(5-chloropyrimidin-2-yl)piperazin-1-yl]butyl]-8-
impurities for demonstration of compliance. See also 5.10. azaspiro[4.5]decane-7,9-dione,
Control of impurities in substances for pharmaceutical use) :
A, B, C, D, F, G, H, I, L, M, N.

A. 2-(piperazin-1-yl)pyrimidine,
J. 4-(7,9-dioxo-8-azaspiro[4.5]dec-8-yl)butyl [1-[2-oxo-2-
[[4-[4-(pyrimidin-2-yl)piperazin-1-yl]butyl]amino]ethyl]-
cyclopentyl]acetate,

B. 8-(pyrimidin-2-yl)-8-aza-5-azoniaspiro[4.5]decane,
K. R = H : 8-azaspiro[4.5]decane-7,9-dione,
L. R = [CH2]4-Cl : 8-(4-chlorobutyl)-8-azaspiro[4.5]decane-
7,9-dione,
M. R = [CH2]4-Br : 8-(4-bromobutyl)-8-azaspiro[4.5]decane-
7,9-dione,
C. X = [CH2]4 : 2,2′-[butane-1,4-diylbis(piperazine-1,4-
diyl)]dipyrimidine,
D. X = [CH2]4-O-[CH2]4 : 2,2′-[oxybis[butane-1,4-
diyl(piperazine-1,4-diyl)]]dipyrimidine,

N. 8,8′-(butane-1,4-diyl)bis(8-azaspiro[4.5]decane-7,9-dione).

01/2008:0542

BUSULFAN
E. [1-[2-oxo-2-[[4-[4-(pyrimidin-2-yl)piperazin-1- Busulfanum
yl]butyl]amino]ethyl]cyclopentyl]acetic acid,

C6H14O6S2 Mr 246.3
[55-98-1]
DEFINITION
Butane-1,4-diyl di(methanesulfonate).
Content : 99.0 per cent to 100.5 per cent (dried substance).
F. X = NH : 4-[4-(pyrimidin-2-yl)piperazin-1-yl]butyl CHARACTERS
[1-[2-oxo-2-[[4-[4-(pyrimidin-2-yl)piperazin-1-
Appearance : white or almost white, crystalline powder.
yl]butyl]amino]ethyl]cyclopentyl]acetate,
Solubility : very slightly soluble in water, freely soluble in
H. X = O : bis[4-[4-(pyrimidin-2-yl)piperazin-1-yl]butyl] acetone and in acetonitrile, very slightly soluble in ethanol
(cyclopentane-1,1-diyl)diacetate, (96 per cent).
mp : about 116 °C.
IDENTIFICATION
First identification : A.
Second identification : B, C, D.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: busulfan CRS.
G. 2,2′-(piperazine-1,4-diyl)dipyrimidine, B. Thin-layer chromatography (2.2.27).

General Notices (1) apply to all monographs and other texts 1711
Butyl parahydroxybenzoate EUROPEAN PHARMACOPOEIA 8.0

Test solution. Dissolve 20 mg of the substance to be DEFINITION

examined in 2 mL of acetone R. Butyl 4-hydroxybenzoate.
Reference solution. Dissolve 20 mg of busulfan CRS in 2 mL Content : 98.0 per cent to 102.0 per cent.
of acetone R.
Plate : TLC silica gel G plate R. CHARACTERS
Mobile phase : acetone R, toluene R (50:50 V/V). Appearance : white or almost white, crystalline powder or
Application : 5 μL. colourless crystals.
Development : over a path of 15 cm. Solubility : very slightly soluble in water, freely soluble in
ethanol (96 per cent) and in methanol.
Drying : in a current of warm air.
Detection : spray with anisaldehyde solution R and heat at IDENTIFICATION
120 °C. First identification : A, B.
Results : the principal spot in the chromatogram obtained Second identification : A, C.
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with A. Melting point (2.2.14) : 68 °C to 71 °C.
the reference solution. B. Infrared absorption spectrophotometry (2.2.24).
C. To 0.1 g add 5 mL of 1 M sodium hydroxide. Heat until a Comparison: butyl parahydroxybenzoate CRS.
clear solution is obtained. Allow to cool. To 2 mL of the C. Thin-layer chromatography (2.2.27).
solution add 0.1 mL of potassium permanganate solution R. Test solution (a). Dissolve 0.10 g of the substance to be
The colour changes from purple through violet to blue and examined in acetone R and dilute to 10 mL with the same
finally to green. Filter and add 1 mL of ammoniacal silver solvent.
nitrate solution R. A precipitate is formed.
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
D. To 0.1 g add 0.1 g of potassium nitrate R and 0.25 g of
with acetone R.
sodium hydroxide R, mix and heat to fusion. Allow to cool
and dissolve the residue in 5 mL of water R. Adjust to Reference solution (a). Dissolve 10 mg of butyl
pH 1-2 using dilute hydrochloric acid R. The solution gives parahydroxybenzoate CRS in acetone R and dilute to 10 mL
reaction (a) of sulfates (2.3.1). with the same solvent.
Reference solution (b). Dissolve 10 mg of propyl
TESTS parahydroxybenzoate R in 1 mL of test solution (a) and
Appearance of solution. The solution is clear (2.2.1) and not dilute to 10 mL with acetone R.
more intensely coloured than reference solution B7 (2.2.2, Plate : TLC octadecylsilyl silica gel F254 plate R.
Method II).
Mobile phase : glacial acetic acid R, water R, methanol R
Dissolve 0.25 g in 20 mL of acetonitrile R, dilute to 25 mL with (1:30:70 V/V/V).
water R and examine immediately.
Application : 2 μL of test solution (b) and reference
Acidity. Dissolve 0.20 g with heating in 50 mL of anhydrous solutions (a) and (b).
ethanol R. Add 0.1 mL of methyl red solution R. Not more than
Development : over 2/3 of the plate.
0.05 mL of 0.1 M sodium hydroxide is required to change the
colour of the indicator. Drying : in air.
Loss on drying (2.2.32) : maximum 2.0 per cent, determined Detection : examine in ultraviolet light at 254 nm.
on 1.000 g by drying in vacuo at 60 °C. System suitability : reference solution (b) :
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on – the chromatogram shows 2 clearly separated principal
1.0 g. spots.
Results : the principal spot in the chromatogram obtained
ASSAY
with test solution (b) is similar in position and size to
To 0.250 g add 50 mL of water R. Shake. Boil under a reflux the principal spot in the chromatogram obtained with
condenser for 30 min and, if necessary, make up to the reference solution (a).
initial volume with water R. Allow to cool. Using 0.3 mL of
phenolphthalein solution R as indicator, titrate with 0.1 M TESTS
sodium hydroxide until a pink colour is obtained. Solution S. Dissolve 1.0 g in ethanol (96 per cent) R and dilute
1 mL of 0.1 M sodium hydroxide is equivalent to 12.32 mg to 10 mL with the same solvent.
of C6H14O6S2.
Appearance of solution. Solution S is clear (2.2.1) and
STORAGE not more intensely coloured than reference solution BY6
(2.2.2, Method II).
In an airtight container, protected from light.
Acidity. To 2 mL of solution S add 3 mL of ethanol (96 per
cent) R, 5 mL of carbon dioxide-free water R and 0.1 mL of
07/2011:0881 bromocresol green solution R. Not more than 0.1 mL of 0.1 M
sodium hydroxide is required to change the colour of the
indicator to blue.
BUTYL PARAHYDROXYBENZOATE
Related substances. Liquid chromatography (2.2.29).
Butylis parahydroxybenzoas Test solution. Dissolve 50.0 mg of the substance to be
examined in 2.5 mL of methanol R and dilute to 50.0 mL with
the mobile phase. Dilute 10.0 mL of the solution to 100.0 mL
with the mobile phase.
Reference solution (a). Dissolve 5 mg of 4-hydroxybenzoic
acid R (impurity A), 5 mg of propyl parahydroxybenzoate R
(impurity D) and 5 mg of the substance to be examined in the
C11H14O3 Mr 194.2 mobile phase and dilute to 100.0 mL with the mobile phase.
[94-26-8] Dilute 1.0 mL of the solution to 10.0 mL with the mobile phase.

1712 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Butylhydroxyanisole

Reference solution (b). Dissolve 50.0 mg of butyl IMPURITIES

parahydroxybenzoate CRS in 2.5 mL of methanol R and dilute Specified impurities : A.
to 50.0 mL with the mobile phase. Dilute 10.0 mL of the Other detectable impurities (the following substances would,
solution to 100.0 mL with the mobile phase. if present at a sufficient level, be detected by one or other of
Reference solution (c). Dilute 1.0 mL of the test solution to the tests in the monograph. They are limited by the general
20.0 mL with the mobile phase. Dilute 1.0 mL of this solution acceptance criterion for other/unspecified impurities and/or
to 10.0 mL with the mobile phase. by the general monograph Substances for pharmaceutical use
Reference solution (d). Dissolve 5 mg of butyl (2034). It is therefore not necessary to identify these impurities
parahydroxybenzoate impurity E CRS (iso-butyl for demonstration of compliance. See also 5.10. Control of
parahydroxybenzoate) in the mobile phase and dilute to impurities in substances for pharmaceutical use) : B, C, D, E.
100.0 mL with the mobile phase.
Reference solution (e). Dilute 0.5 mL of reference solution (d)
to 50.0 mL with reference solution (b).
Column : A. 4-hydroxybenzoic acid,
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm) ;
– temperature : 35 °C.
Mobile phase : 6.8 g/L solution of potassium dihydrogen B. methyl 4-hydroxybenzoate (methyl parahydroxybenzoate),
phosphate R, methanol R (50:50 V/V).
Flow rate : 1.3 mL/min.
Detection : spectrophotometer at 272 nm.
Injection : 10 μL of the test solution and reference solutions (a),
(c) and (e).
C. ethyl 4-hydroxybenzoate (ethyl parahydroxybenzoate),
Run time : 1.5 times the retention time of butyl
parahydroxybenzoate.
Identification of impurities : use the chromatogram obtained
with reference solution (a) to identify the peaks due to
impurities A and D ; use the chromatogram obtained with
reference solution (e) to identify the peak due to impurity E. D. propyl 4-hydroxybenzoate (propyl parahydroxybenzoate),
Relative retention with reference to butyl parahydroxybenzoate
(retention time = about 22 min) : impurity A = about 0.1 ;
impurity D = about 0.5 ; impurity E = about 0.9.
System suitability :
– resolution :
E. 2-methylpropyl 4-hydroxybenzoate (iso-butyl
– minimum 5.0 between the peaks due to impurity D and parahydroxybenzoate).
butyl parahydroxybenzoate in the chromatogram obtained
with reference solution (a) ; 01/2008:0880
– minimum 1.5 between the peaks due to impurity E and
butyl parahydroxybenzoate in the chromatogram obtained
with reference solution (e).
BUTYLHYDROXYANISOLE
Limits : Butylhydroxyanisolum
– correction factor : for the calculation of content, multiply
the peak area of impurity A by 1.4 ;
– impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (c)
(0.5 per cent) ;
– unspecified impurities : for each impurity, not more than the C11H16O2 Mr 180.3
area of the principal peak in the chromatogram obtained [25013-16-5]
with reference solution (c) (0.5 per cent) ;
DEFINITION
– total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (c) Butylhydroxyanisole is 2-(1,1-dimethylethyl)-4-
(1.0 per cent) ; methoxyphenol containing not more than 10 per cent of
3-(1,1-dimethylethyl)-4-methoxyphenol.
– disregard limit : 0.2 times the area of the principal peak in
the chromatogram obtained with reference solution (c) CHARACTERS
(0.1 per cent). A white, yellowish or slightly pinkish, crystalline powder,
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on practically insoluble in water, very soluble in methylene
1.0 g. chloride, freely soluble in alcohol and in fatty oils. It dissolves
in dilute solutions of alkali hydroxides.
ASSAY
IDENTIFICATION
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification. A. Examine the chromatograms obtained in the test for
related substances. The principal spot in the chromatogram
Injection : test solution and reference solution (b). obtained with test solution (b) is similar in position,
Calculate the percentage content of C11H14O3 from the colour and size to the principal spot in the chromatogram
declared content of butyl parahydroxybenzoate CRS. obtained with reference solution (a).

General Notices (1) apply to all monographs and other texts 1713
Butylhydroxytoluene EUROPEAN PHARMACOPOEIA 8.0

B. To 0.5 mL of solution S (see Tests) add 10 mL of 01/2008:0581

aminopyrazolone solution R and 1 mL of potassium
ferricyanide solution R. Mix and add 10 mL of methylene
chloride R. Shake vigorously. After separation, the organic BUTYLHYDROXYTOLUENE
layer is red.
C. Dissolve about 10 mg in 2 mL of alcohol R. Add 1 mL of Butylhydroxytoluenum
a 1 g/L solution of testosterone propionate R in alcohol R
and 2 mL of dilute sodium hydroxide solution R. Heat in a
water-bath at 80 °C for 10 min and allow to cool. A red
colour develops.

TESTS
Solution S. Dissolve 2.5 g in alcohol R and dilute to 25 mL
with the same solvent. C15H24O Mr 220.4
Appearance of solution. Solution S is clear (2.2.1) and not [128-37-0]
more intensely coloured than intensity 5 of the range of
reference solutions of the most appropriate colour (2.2.2, DEFINITION
Method II). Butylhydroxytoluene is 2,6-bis(1,1-dimethylethyl)-4-
Related substances. Examine by thin-layer chromatography methylphenol.
(2.2.27), using silica gel G R as the coating substance.
CHARACTERS
Test solution (a). Dissolve 0.25 g of the substance to be
examined in methylene chloride R and dilute to 10 mL with A white or yellowish-white, crystalline powder, practically
the same solvent. insoluble in water, very soluble in acetone, freely soluble in
alcohol and in vegetable oils.
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
with methylene chloride R. IDENTIFICATION
Reference solution (a). Dissolve 25 mg of butylhydroxyani- First identification : A, C.
sole CRS in methylene chloride R and dilute to 10 mL with the Second identification : A, B, D.
same solvent.
A. Freezing-point (see Tests).
Reference solution (b). Dilute 1 mL of reference solution (a) to
20 mL with methylene chloride R. B. Dissolve 0.500 g in ethanol R and dilute to 100.0 mL with
the same solvent. Dilute 1.0 mL of this solution to 100.0 mL
Reference solution (c). Dissolve 50 mg of hydroquinone R with ethanol R. Examined between 230 nm and 300 nm
in 5 mL of alcohol R and dilute to 100 mL with methylene (2.2.25), the solution shows an absorption maximum at
chloride R. Dilute 1 mL of this solution to 10 mL with 278 nm. The specific absorbance at the maximum is 80
methylene chloride R. to 90.
Apply separately to the plate 5 μL of each solution. Develop C. Examine by infrared absorption spectrophotometry
over a path of 10 cm using methylene chloride R. Allow the (2.2.24), comparing with the spectrum obtained with
plate to dry in air and spray with a freshly prepared mixture of butylhydroxytoluene CRS.
10 volumes of potassium ferricyanide solution R, 20 volumes D. Dissolve about 10 mg in 2 mL of alcohol R. Add 1 mL of
of ferric chloride solution R1 and 70 volumes of water R. In the a 1 g/L solution of testosterone propionate R in alcohol R
chromatogram obtained with test solution (a) : any violet-blue and 2 mL of dilute sodium hydroxide solution R. Heat in a
spot with an RF value of about 0.35 (corresponding to water-bath at 80 °C for 10 min and allow to cool. A blue
3-(1,1-dimethylethyl)-4-methoxyphenol) is not more intense colour develops.
than the principal spot in the chromatogram obtained with
reference solution (a) (10 per cent) ; any spot corresponding to TESTS
hydroquinone is not more intense than the principal spot in
the chromatogram obtained with reference solution (c) (0.2 per Appearance of solution. Dissolve 1.0 g in methanol R and
cent) ; any spot, apart from the principal spot and any spots dilute to 10 mL with the same solvent. The solution is clear
corresponding to 3-(1,1-dimethylethyl)-4-methoxyphenol and (2.2.1) and not more intensely coloured than reference
hydroquinone, is not more intense than the principal spot solution Y5 or BY5 (2.2.2, Method II).
in the chromatogram obtained with reference solution (b) Freezing-point (2.2.18) : 69 °C to 70 °C.
(0.5 per cent). Related substances. Examine by thin-layer chromatography
Heavy metals (2.4.8). 1.0 g complies with test C for heavy (2.2.27), using silica gel G R as the coating substance.
metals (10 ppm). Prepare the reference solution using 1 mL Test solution. Dissolve 0.2 g of the substance to be examined
of lead standard solution (10 ppm Pb) R. in methanol R and dilute to 10.0 mL with the same solvent.
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined Reference solution. Dilute 1 mL of the test solution to 200 mL
on 1.0 g. with methanol R.
STORAGE Apply separately to the plate 10 μL of each solution. Develop
over a path of 15 cm using methylene chloride R. Dry the plate
Store protected from light. in air and spray with a freshly prepared mixture of 10 volumes
of potassium ferricyanide solution R, 20 volumes of ferric
IMPURITIES chloride solution R1 and 70 volumes of water R. Any spot in
the chromatogram obtained with the test solution, apart from
the principal spot, is not more intense than the spot in the
chromatogram obtained with the reference solution (0.5 per
cent).
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
A. benzene-1,4-diol (hydroquinone). on 1.0 g.

1714 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cabergoline

01/2008:1773 Column :
– size : l = 0.25 m, Ø = 4.6 mm,
CABERGOLINE – stationary phase : octadecylsilyl silica gel for
chromatography R (10 μm).
Cabergolinum Mobile phase : mix 16 volumes of acetonitrile R and 84 volumes
of a freshly prepared 6.8 g/L solution of potassium dihydrogen
phosphate R previously adjusted to pH 2.0 with phosphoric
acid R. Add 0.2 volumes of triethylamine R.
Flow rate : 1.2 mL/min.
Detection : spectrophotometer at 280 nm.
Injection : 20 μL of the test solution and reference solutions (b)
and (c).
Run time : 4 times the retention time of cabergoline.
C26H37N5O2 Mr 451.6 Relative retention with reference to cabergoline (retention
[81409-90-7] time = about 12 min) : impurity D = about 0.3 ;
impurity B = about 0.6 ; impurity A = about 0.8 ;
DEFINITION impurity C = about 2.9.
1-Ethyl-3-[3-(dimethylamino)propyl]-3-[[(6aR,9R,10aR)- System suitability : reference solution (c) :
7-(prop-2-enyl)-4,6,6a,7,8,9,10,10a-octahydroindolo[4,3- – resolution : minimum 3.0 between the peaks due to
fg]quinolin-9-yl]carbonyl]urea. cabergoline and impurity A.
Content : 98.0 per cent to 102.0 per cent (anhydrous substance). Limits :
– impurities A, C : for each impurity, not more than 1.5 times
CHARACTERS the area of the principal peak in the chromatogram
Appearance : white or almost white, crystalline powder. obtained with reference solution (b) (0.3 per cent) ;
Solubility : practically insoluble in water, freely soluble in – impurities B, D : for each impurity, not more than 0.5 times
ethanol (96 per cent), very slightly soluble in hexane. It is the area of the principal peak in the chromatogram
slightly soluble in 0.1 M hydrochloric acid. obtained with reference solution (b) (0.1 per cent) ;

It shows polymorphism (5.9). – any other impurity : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.1 per cent) ;
IDENTIFICATION
– total : not more than 4 times the area of the principal peak
A. Specific optical rotation (see Tests). in the chromatogram obtained with reference solution (b)
B. Infrared absorption spectrophotometry (2.2.24). (0.8 per cent) ;

Comparison : cabergoline CRS. – disregard limit : 0.25 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
If the spectra obtained in the solid state show differences, (0.05 per cent).
dissolve 50 mg of the substance to be examined and 50 mg Water (2.5.12) : maximum 0.5 per cent, determined on 1.000 g.
of the reference substance separately in 1 mL of ethanol
(96 per cent) R, evaporate to dryness and record new
spectra using the residues. ASSAY
Liquid chromatography (2.2.29) as described in the test for
TESTS related substances with the following modification.
Specific optical rotation (2.2.7) : − 77 to − 83 (anhydrous Injection : test solution and reference solution (a).
substance). Calculate the percentage content of C26H37N5O2 from the areas
Dissolve 0.100 g in ethanol (96 per cent) R and dilute to of the peaks and the declared content of cabergoline CRS.
50.0 mL with the same solvent.
Related substances. Liquid chromatography (2.2.29). Prepare STORAGE
the solutions immediately before use and protected from light. Protected from light.
Test solution. Dissolve 30.0 mg of the substance to be
examined in the mobile phase and dilute to 25.0 mL with the IMPURITIES
mobile phase. Specified impurities : A, B, C, D.
Reference solution (a). Dissolve 30.0 mg of cabergoline CRS in
the mobile phase and dilute to 25.0 mL with the mobile phase.
Reference solution (b). Dilute 1.0 mL of the test solution
to 100.0 mL with the mobile phase. Dilute 10.0 mL of this
solution to 50.0 mL with the mobile phase.
Reference solution (c). Suspend 50 mg of the substance to
be examined in 10 mL of 0.1 M sodium hydroxide. Stir for
about 15 min. To 1 mL of the suspension add 1 mL of 0.1 M
hydrochloric acid and dilute to 10 mL with the mobile phase.
Sonicate until dissolution is complete. The main degradation A. (6aR,9R,10aR)-7-(prop-2-enyl)-4,6,6a,7,8,9,10,10a-
product obtained is impurity A. octahydroindolo[4,3-fg]quinoline-9-carboxylic acid,

General Notices (1) apply to all monographs and other texts 1717
Caffeine EUROPEAN PHARMACOPOEIA 8.0

Appearance of solution. Solution S is clear (2.2.1) and

colourless (2.2.2, Method II).
Acidity. To 10 mL of solution S add 0.05 mL of bromothymol
blue solution R1 ; the solution is green or yellow. Not more
than 0.2 mL of 0.01 M sodium hydroxide is required to change
the colour of the indicator to blue.
Related substances. Liquid chromatography (2.2.29).
B. R = CO-NH-C2H5, R′ = H : (6aR,9R,10aR)-N9-[3-(dimethyl- Test solution. Dissolve 0.100 g of the substance to be examined
amino)propyl]-N4-ethyl-7-(prop-2-enyl)-6a,7,8,9,10,10a- in the mobile phase and dilute to 50.0 mL with the mobile
hexahydroindolo[4,3-fg]quinoline-4,9(6H)-dicarboxamide, phase. Dilute 1.0 mL of this solution to 10.0 mL with the
C. R = R’ = CO-NH-C2H5 : (6aR,9R,10aR)-N9-[3- mobile phase.
(dimethylamino)propyl]-N4-ethyl-N9-(ethylcarbamoyl)- Reference solution (a). Dilute 2.0 mL of the test solution to
7-(prop-2-enyl)-6a,7,8,9,10,10a-hexahydroindolo[4,3- 100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
fg]quinoline-4,9(6H)-dicarboxamide, to 10.0 mL with the mobile phase.
D. R = R′ = H : (6aR,9R,10aR)-N-[3-(dimethylamino)propyl]- Reference solution (b). Dissolve 5 mg of caffeine for system
7-(prop-2-enyl)-4,6,6a,7,8,9,10,10a-octahydroindolo[4,3- suitability CRS (containing impurities A, C, D and F) in the
fg]quinoline-9-carboxamide. mobile phase and dilute to 5 mL with the mobile phase. Dilute
2 mL of this solution to 10 mL with the mobile phase.
04/2008:0267 Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
CAFFEINE – stationary phase : base-deactivated end-capped octadecylsilyl
silica gel for chromatography R (5 μm).
Coffeinum Mobile phase : mix 20 volumes of tetrahydrofuran R,
25 volumes of acetonitrile R and 955 volumes of a solution
containing 0.82 g/L of anhydrous sodium acetate R previously
adjusted to pH 4.5 with glacial acetic acid R.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 275 nm.
Injection : 10 μL.
C8H10N4O2 Mr 194.2
Run time : 1.5 times the retention time of caffeine.
[58-08-2]
Identification of impurities : use the chromatogram supplied
DEFINITION with caffeine for system suitability CRS and the chromatogram
1,3,7-Trimethyl-3,7-dihydro-1H-purine-2,6-dione. obtained with reference solution (b) to identify the peaks due
Content : 98.5 per cent to 101.5 per cent (dried substance). to impurities A, C, D and F.
Retention time : caffeine = about 8 min.
CHARACTERS System suitability : reference solution (b) :
Appearance : white or almost white, crystalline powder or – resolution : minimum 2.5 between the peaks due to
silky, white or almost white, crystals. impurities C and D and minimum 2.5 between the peaks
Solubility : sparingly soluble in water, freely soluble in boiling due to impurities F and A.
water, slightly soluble in ethanol (96 per cent). It dissolves in Limits :
concentrated solutions of alkali benzoates or salicylates.
– unspecified impurities : for each impurity, not more than
It sublimes readily. 0.5 times the area of the principal peak in the chromatogram
IDENTIFICATION obtained with reference solution (a) (0.10 per cent) ;
First identification : A, B, E. – total : not more than 0.5 times the area of the principal peak
Second identification : A, C, D, E, F. in the chromatogram obtained with reference solution (a)
(0.1 per cent) ;
A. Melting point (2.2.14) : 234 °C to 239 °C.
– disregard limit : 0.25 times the area of the principal peak
B. Infrared absorption spectrophotometry (2.2.24). in the chromatogram obtained with reference solution (a)
Comparison : caffeine CRS. (0.05 per cent).
C. To 2 mL of a saturated solution add 0.05 mL of iodinated Sulfates (2.4.13) : maximum 500 ppm, determined on 15 mL
potassium iodide solution R. The solution remains clear. of solution S.
Add 0.1 mL of dilute hydrochloric acid R ; a brown
Prepare the standard using a mixture of 7.5 mL of sulfate
precipitate is formed. Neutralise with dilute sodium
standard solution (10 ppm SO4) R and 7.5 mL of distilled
hydroxide solution R ; the precipitate dissolves.
water R.
D. In a ground-glass-stoppered tube, dissolve about 10 mg
in 0.25 mL of a mixture of 0.5 mL of acetylacetone R and Heavy metals (2.4.8) : maximum 20 ppm.
5 mL of dilute sodium hydroxide solution R. Heat in a 1.0 g complies with test C. Prepare the reference solution using
water-bath at 80 °C for 7 min. Cool and add 0.5 mL of 2 mL of lead standard solution (10 ppm Pb) R.
dimethylaminobenzaldehyde solution R2. Heat again in a Loss on drying (2.2.32) : maximum 0.5 per cent, determined
water-bath at 80 °C for 7 min. Allow to cool and add 10 mL on 1.000 g by drying in an oven at 105 °C for 1 h.
of water R ; an intense blue colour develops.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
E. Loss on drying (see Tests). 1.0 g.
F. It gives the reaction of xanthines (2.3.1).
ASSAY
TESTS Dissolve 0.170 g with heating in 5 mL of anhydrous acetic
Solution S. Dissolve 0.5 g with heating in 50 mL of carbon acid R. Allow to cool, add 10 mL of acetic anhydride R
dioxide-free water R prepared from distilled water R, cool and and 20 mL of toluene R. Titrate with 0.1 M perchloric acid,
dilute to 50 mL with the same solvent. determining the end-point potentiometrically (2.2.20).

1718 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Caffeine monohydrate

1 mL of 0.1 M perchloric acid is equivalent to 19.42 mg 07/2009:0268

of C8H10N4O2.
CAFFEINE MONOHYDRATE
IMPURITIES
Other detectable impurities (the following substances would, Coffeinum monohydricum
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use): A,
B, C, D, E, F.
C8H10N4O2,H2O Mr 212.2
[5743-12-4]
DEFINITION
1,3,7-Trimethyl-3,7-dihydro-1H-purine-2,6-dione
monohydrate.
Content : 98.5 per cent to 101.5 per cent (dried substance).

A. 1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione CHARACTERS
(theophylline), Appearance : white or almost white, crystalline powder or
silky, white or almost white crystals.
Solubility : sparingly soluble in water, freely soluble in boiling
water, slightly soluble in ethanol (96 per cent). It dissolves in
concentrated solutions of alkali benzoates or salicylates.
It sublimes readily.
IDENTIFICATION
First identification : A, B, E.
B. N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro- Second identification : A, C, D, E, F.
pyrimidin-5-yl)formamide, A. Melting point (2.2.14) : 234 °C to 239 °C, determined after
drying at 100-105 °C.
B. Infrared absorption spectrophotometry (2.2.24).
Preparation : dry the substance to be examined at
100-105 °C before use.
Comparison: caffeine CRS.
C. To 2 mL of a saturated solution add 0.05 mL of iodinated
potassium iodide solution R ; the solution remains clear. Add
C. 1,3,9-trimethyl-3,9-dihydro-1H-purine-2,6-dione 0.1 mL of dilute hydrochloric acid R ; a brown precipitate
(isocaffeine), is formed. Neutralise with dilute sodium hydroxide
solution R ; the precipitate dissolves.
D. In a glass-stoppered tube, dissolve about 10 mg in
0.25 mL of a mixture of 0.5 mL of acetylacetone R and
5 mL of dilute sodium hydroxide solution R. Heat in a
water-bath at 80 °C for 7 min. Cool and add 0.5 mL of
dimethylaminobenzaldehyde solution R2. Heat again in a
water-bath at 80 °C for 7 min. Allow to cool and add 10 mL
of water R ; an intense blue colour develops.
D. 3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione
(theobromine), E. Loss on drying (see Tests).
F. It gives the reaction of xanthines (2.3.1).
TESTS
Solution S. Dissolve 0.5 g with heating in 50 mL of carbon
dioxide-free water R prepared from distilled water R, cool, and
dilute to 50 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
E. N,1-dimethyl-4-(methylamino)-1H-imidazole-5-
carboxamide (caffeidine), Acidity. To 10 mL of solution S add 0.05 mL of bromothymol
blue solution R1 ; the solution is green or yellow. Not more
than 0.2 mL of 0.01 M sodium hydroxide is required to change
the colour of the indicator to blue.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.110 g of the substance to be examined
in the mobile phase and dilute to 50.0 mL with the mobile
phase. Dilute 1.0 mL of this solution to 10.0 mL with the
F. 1,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione. mobile phase.

General Notices (1) apply to all monographs and other texts 1719
Calcifediol EUROPEAN PHARMACOPOEIA 8.0

Reference solution (a). Dilute 2.0 mL of the test solution to impurities for demonstration of compliance. See also 5.10.
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution Control of impurities in substances for pharmaceutical use) : A,
to 10.0 mL with the mobile phase. B, C, D, E, F.
Reference solution (b). Dissolve 5 mg of caffeine for system
suitability CRS (containing impurities A, C, D and F) in the
mobile phase and dilute to 5.0 mL with the mobile phase.
Dilute 2.0 mL of this solution to 10.0 mL with the mobile
phase.
Column :
– size : l = 0.15 m, Ø = 4.6 mm ; A. 1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
– stationary phase : base-deactivated end-capped octadecylsilyl (theophylline),
silica gel for chromatography R (5 μm).
Mobile phase. Mix 20 volumes of tetrahydrofuran R,
25 volumes of acetonitrile R and 955 volumes of a solution
containing 0.82 g/L of anhydrous sodium acetate R previously
adjusted to pH 4.5 with glacial acetic acid R.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 275 nm. B. N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-
Injection : 10 μL. pyrimidin-5-yl)formamide,
Run time : 1.5 times the retention time of caffeine.
Identification of impurities : use the chromatogram supplied
with caffeine for system suitability CRS and the chromatogram
obtained with reference solution (b) to identify the peaks due
to impurities A, C, D and F.
Retention time : caffeine = about 8 min.
System suitability : reference solution (b) : C. 1,3,9-trimethyl-3,9-dihydro-1H-purine-2,6-dione
(isocaffeine),
– resolution : minimum 2.5 between the peaks due to
impurities C and D ; minimum 2.5 between the peaks due
to impurities F and A.
Limits :
– unspecified impurities : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent) ; D. 3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione
– total : not more than 0.5 times the area of the principal peak (theobromine),
in the chromatogram obtained with reference solution (a)
(0.1 per cent) ;
– disregard limit : 0.25 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Sulfates (2.4.13) : maximum 500 ppm, determined on 15 mL
of solution S. E. N,1-dimethyl-4-(methylamino)-1H-imidazole-5-
Prepare the standard using a mixture of 7.5 mL of sulfate carboxamide (caffeidine),
standard solution (10 ppm SO4) R and 7.5 mL of distilled
water R.
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test C. Prepare the reference solution using
2 mL of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : 5.0 per cent to 9.0 per cent, F. 1,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione.
determined on 1.000 g by drying in an oven at 105 °C for 1 h.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on 01/2013:1295
1.0 g.
CALCIFEDIOL
ASSAY
Dissolve 0.170 g, previously dried at 100-105 °C, with heating Calcifediolum
in 5 mL of anhydrous acetic acid R. Allow to cool, and
add 10 mL of acetic anhydride R and 20 mL of toluene R.
Titrate with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 19.42 mg
of C8H10N4O2.
IMPURITIES
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical C27H44O2,H2O Mr 418.7
use (2034). It is therefore not necessary to identify these [63283-36-3]

1720 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcifediol

DEFINITION – disregard limit : 0.5 times the area of the principal peak in
(5Z,7E)-9,10-Secocholesta-5,7,10(19)-triene-3β,25-diol the chromatogram obtained with reference solution (b)
monohydrate. (0.05 per cent) ; disregard the peak due to pre-calcifediol.
Content : 97.0 per cent to 102.0 per cent (anhydrous substance). Water (2.5.32) : 3.8 per cent to 5.0 per cent, determined on
10.0 mg.
A reversible isomerisation to pre-calcifediol takes place in
solution, depending on temperature and time. The activity is ASSAY
due to both compounds (see Assay). Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications.
CHARACTERS
Injection : test solution and reference solutions (a) and (c).
Appearance : white or almost white crystals.
System suitability : reference solution (c) :
Solubility : practically insoluble in water, freely soluble in – repeatability : maximum relative standard deviation of 1 per
ethanol (96 per cent), soluble in fatty oils. cent for the peak due to calcifediol after 6 injections.
It is sensitive to air, heat and light. Calculate the percentage content of C27H44O2 using the
chromatogram obtained with reference solution (a) and taking
IDENTIFICATION into account the assigned content of calcifediol CRS and, if
A. Infrared absorption spectrophotometry (2.2.24). necessary, the peak due to pre-calcifediol.
Preparation : mix 2 mg of the substance to be examined STORAGE
and 225 mg of potassium bromide R.
Under nitrogen, in an airtight container, protected from light,
Comparison : Ph. Eur. reference spectrum of calcifediol. at a temperature of 2 °C to 8 °C.
B. Examine the chromatograms obtained in the assay. The contents of an opened container are to be used
Results : the principal peak in the chromatogram obtained immediately.
with the test solution is similar in retention time and size
to the principal peak in the chromatogram obtained with IMPURITIES
reference solution (a). Specified impurities : A, B, C, D.

TESTS
Related substances. Liquid chromatography (2.2.29) : use
the normalisation procedure. Carry out the test as rapidly as
possible, avoiding exposure to actinic light and air.
Test solution. Dissolve 1.00 mg of the substance to be
examined without heating in 10.0 mL of the mobile phase.
Reference solution (a). Dissolve 1.00 mg of calcifediol CRS
A. 9β,10α-cholesta-5,7-diene-3β,25-diol,
without heating in 10.0 mL of the mobile phase.
Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 100.0 mL with the mobile phase. Dilute 1.0 mL of this
solution to 10.0 mL with the mobile phase.
Reference solution (c). Heat 2 mL of reference solution (a) in a
water-bath at 80 °C under a reflux condenser for 2 h and cool.
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
B. cholesta-5,7-diene-3β,25-diol,
– stationary phase : octylsilyl silica gel for chromatography R1
(5 μm).
Mobile phase : water R, methanol R (20:80 V/V).
Flow rate : 1.5 mL/min.
Detection : spectrophotometer at 265 nm.
Injection : 50 μL of the test solution and reference solutions (b)
and (c).
Run time : twice the retention time of calcifediol.
Relative retention with reference to calcifediol (retention
time = about 11 min) : impurity D = about 0.85 ;
impurity B = about 1.1 ; impurity C = about 1.2 ; C. (6E)-9,10-secocholesta-5(10),6,8-triene-3β,25-diol,
pre-calcifediol = about 1.3 ; impurity A = about 1.6.
System suitability: reference solution (c) :
– resolution : minimum 5.0 between the peaks due to
calcifediol and pre-calcifediol ; if necessary, adjust the
proportions of the constituents in the mobile phase.
Limits :
– impurities A, B, C, D : for each impurity, maximum 0.5 per
cent ;
– unspecified impurities : for each impurity, maximum
0.10 per cent ;
– total : maximum 1.0 per cent ; D. (5E,7E)-9,10-secocholesta-5,7,10(19)-triene-3β,25-diol.

General Notices (1) apply to all monographs and other texts 1721
Calcipotriol, anhydrous EUROPEAN PHARMACOPOEIA 8.0

04/2013:2011 Detection : spray the hot plate with an alcoholic solution of

sulfuric acid R, dry at 140 °C for not more than 1 min and
CALCIPOTRIOL, ANHYDROUS examine in ultraviolet light at 366 nm.
Relative retention with reference to calcipotriol
(RF = about 0.4): impurity G = about 0.4 ;
Calcipotriolum anhydricum impurity H = about 0.4 ; pre-calcipotriol = about 0.9 ;
impurity A = about 1.2.
System suitability : reference solution (d) :
– the chromatogram shows a secondary spot due to
pre-calcipotriol.
Limits :
– impurity A : any spot due to impurity A is not more
intense than the spot in the chromatogram obtained
with reference solution (b) (0.25 per cent) ;
– impurities G, H : any spot due to impurity G or H is
not more intense than the spot in the chromatogram
obtained with reference solution (b) (0.25 per cent for
C27H40O3 Mr 412.6 the sum) ;
[112965-21-6]
– unspecified impurities: any other spot is not more
DEFINITION intense than the spot in the chromatogram obtained
(5Z,7E,22E,24S)-24-Cyclopropyl-9,10-secochola- with reference solution (c) (0.1 per cent).
5,7,10(19),22-tetraene-1α,3β,24-triol. B. Liquid chromatography (2.2.29).
Content : 95.5 per cent to 102.0 per cent (dried substance). Solution A. Dissolve 1.32 g of ammonium phosphate R in
A reversible isomerisation to pre-calcipotriol takes place in water R and dilute to 10.0 mL with the same solvent.
solution, depending on temperature and time. The activity is Solvent mixture: solution A, water R, methanol R
due to both compounds. (0.3:29.7:70 V/V/V).
Test solution (a). Dissolve 2 mg of the substance to be
CHARACTERS examined in the solvent mixture and dilute to 5.0 mL with
Appearance : white or almost white, crystalline powder. the solvent mixture.
Solubility : practically insoluble in water, freely soluble in Test solution (b). Dissolve 2.00 mg of the substance to be
ethanol (96 per cent), slightly soluble in methylene chloride. examined in the solvent mixture and dilute to 20.0 mL with
It is sensitive to heat and light. the same solvent mixture.
Reference solution (a). Dilute 1.0 mL of test solution (a) to
IDENTIFICATION 100.0 mL with the solvent mixture.
A. Infrared absorption spectrophotometry (2.2.24). Reference solution (b). Dilute 1.0 mL of reference
Comparison : Ph. Eur. reference spectrum of anhydrous solution (a) to 10.0 mL with the solvent mixture.
calcipotriol. Reference solution (c). Dissolve 1 mg of calcipotriol
B. Loss on drying (see Tests). monohydrate CRS (containing impurities B, C and D) in
the solvent mixture and dilute to 2.5 mL with the solvent
mixture.
TESTS
Reference solution (d). Dissolve 2.00 mg of calcipotriol
Carry out the tests for related substances and the assay as monohydrate CRS in the solvent mixture and dilute to
rapidly as possible and protected from actinic light and air. 20.0 mL with the solvent mixture.
Related substances Column :
A. Thin-layer chromatography (2.2.27). – size : l = 0.10 m, Ø = 4.0 mm ;
Solution A. To 1 mL of triethylamine R add 9 mL of – stationary phase : octadecylsilyl silica gel for
chloroform R. chromatography R (3 μm).
Test solution. Dissolve 1 mg of the substance to be Mobile phase : water R, methanol R (30:70 V/V).
examined in 100 μL of solution A.
Flow rate : 1.0 mL/min.
Reference solution (a). To 10 μL of the test solution add
Detection : spectrophotometer at 264 nm.
990 μL of solution A.
Injection : 20 μL of test solution (a) and reference
Reference solution (b). To 250 μL of reference solution (a)
solutions (a), (b) and (c).
add 750 μL of solution A.
Run time : twice the retention time of calcipotriol.
Reference solution (c). To 100 μL of reference solution (a)
add 900 μL of solution A. Relative retention with reference to calcipotriol (retention
Reference solution (d). Place 2 mg of the substance to be time = about 13.5 min) : impurity B = about 0.86 ;
examined in a vial and dissolve in 200 μL of solution A. impurity C = about 0.92 ; impurity D = about 1.3.
Close the vial and keep it in a water bath at 60 °C for 2 h. System suitability : reference solution (c):
Plate : TLC silica gel F254 plate R. – peak-to-valley ratio : minimum 1.5, where Hp = height
Mobile phase : 2-methylpropanol R, methylene chloride R above the baseline of the peak due to impurity C and
(20:80 V/V). Hv = height above the baseline of the lowest point of
the curve separating this peak from the peak due to
Application : 10 μL of the test solution and reference calcipotriol ;
solutions (b), (c) and (d).
– the chromatogram obtained is similar to the
Development : over 2/3 of the plate. chromatogram supplied with calcipotriol
Drying : in air, then at 140 °C for 10 min. monohydrate CRS.

1722 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcipotriol, anhydrous

Limits :
– impurity B : not more than 0.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent) ;
– impurities C, D : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (1.0 per cent) ;
– unspecified impurities : for each impurity, not more than
the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.10 per cent) ;
– total : not more than 2.5 times the area of the principal C. (5E,7E,22E,24S)-24-cyclopropyl-9,10-secochola-
peak in the chromatogram obtained with reference 5,7,10(19),22-tetraene-1α,3β,24-triol ((5E)-calcipotriol),
solution (a) (2.5 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Loss on drying : maximum 1.0 per cent, determined on 5 mg
by thermogravimetry (2.2.34). Heat to 105 °C at a rate of
10 °C/min and maintain at 105 °C for 60 min.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : test solution (b) and reference solution (d). D. (5Z,7E,22E,24R)-24-cyclopropyl-9,10-secochola-
5,7,10(19),22-tetraene-1α,3β,24-triol (24-epi-calcipotriol),
Calculate the percentage content of C27H40O3 taking into
account the assigned content of calcipotriol monohydrate CRS.
STORAGE
In an airtight container, protected from light, at − 20 °C or
below.
IMPURITIES
Specified impurities : A, B, C, D, G, H.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or E. rac-(5Z,7E,24S)-24-cyclopropyl-9,10-secochola-5,7,10(19)-
by the general monograph Substances for pharmaceutical use triene-1α,3β,24-triol,
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : E, F, I.
By thin-layer chromatography : A, G, H, I.
By liquid chromatography : B, C, D, E, F.

F. (5Z,7E,22E,24S)-24-cyclopropyl-1α,3β-bis[[(1,1-
dimethylethyl)dimethylsilyl]oxy]-9,10-secochola-
5,7,10(19),22-tetraen-24-ol,

A. (5Z,7E,22E)-24-cyclopropyl-1α,3β-dihydroxy-9,10-
secochola-5,7,10(19),22-tetraen-24-one,

B. (5Z,7Z,22E,24S)-24-cyclopropyl-9,10-secochola- G. 24,24′-oxybis[(5Z,7E,22E,24S)-24-cyclopropyl-9,10-
5,7,10(19),22-tetraene-1α,3β,24-triol ((7Z)-calcipotriol), secochola-5,7,10(19),22-tetraene-1α,3β-diol],

General Notices (1) apply to all monographs and other texts 1723
Calcipotriol monohydrate EUROPEAN PHARMACOPOEIA 8.0

B. Water (see Tests).

TESTS
Carry out the tests for related substances and the assay as
rapidly as possible and protected from actinic light and air.
Related substances
A. Thin-layer chromatography (2.2.27).
Solution A. To 1 mL of triethylamine R add 9 mL of
chloroform R.
Test solution. Dissolve 1 mg of the substance to be
examined in 100 μL of solution A.
Reference solution (a). To 10 μL of the test solution add
990 μL of solution A.
Reference solution (b). To 250 μL of reference solution (a)
H. (5Z,7E,22E,24R)-24-cyclopropyl-24-[[(5Z,7E,22E,24S)- add 750 μL of solution A.
24-cyclopropyl-1α,3β-dihydroxy-9,10-secochola- Reference solution (c). To 100 μL of reference solution (a)
5,7,10(19),22-tetraen-24-yl]oxy]-9,10-secochola- add 900 μL of solution A.
5,7,10(19),22-tetraene-1α,3β-diol,
Reference solution (d). Place 2 mg of the substance to be
examined in a vial and dissolve in 200 μL of solution A.
Close the vial and keep it in a water bath at 60 °C for 2 h.
Plate : TLC silica gel F254 plate R.
Mobile phase : 2-methylpropanol R, methylene chloride R
(20:80 V/V).
Application : 10 μL of the test solution and reference
solutions (b), (c) and (d).
Development : over 2/3 of the plate.
Drying : in air, then at 140 °C for 10 min.
I. (6S,7R,8R,22E,24S)-24-cyclopropyl-6,8:7,19-dicyclo-9,10- Detection : spray the hot plate with an alcoholic solution of
secochola-5(10),22-diene-1α,3β,24-triol (suprasterol of sulfuric acid R, dry at 140 °C for not more than 1 min and
calcipotriol). examine in ultraviolet light at 366 nm.
Relative retention with reference to calcipotriol
04/2013:2284 (RF = about 0.4): impurity G = about 0.4 ;
impurity H = about 0.4 ; pre-calcipotriol = about 0.9 ;
CALCIPOTRIOL MONOHYDRATE impurity A = about 1.2.
System suitability : reference solution (d) :
Calcipotriolum monohydricum – the chromatogram shows a secondary spot due to
pre-calcipotriol.
Limits :
– impurity A : any spot due to impurity A is not more
intense than the spot in the chromatogram obtained
with reference solution (b) (0.25 per cent) ;
– impurities G, H : any spot due to impurity G or H is
not more intense than the spot in the chromatogram
obtained with reference solution (b) (0.25 per cent for
the sum) ;
– unspecified impurities: any other spot is not more
intense than the spot in the chromatogram obtained
C27H40O3,H2O Mr 430.6 with reference solution (c) (0.1 per cent).
[147657-22-5] B. Liquid chromatography (2.2.29).
DEFINITION Solution A. Dissolve 1.32 g of ammonium phosphate R in
water R and dilute to 10.0 mL with the same solvent.
(5Z,7E,22E,24S)-24-Cyclopropyl-9,10-secochola-
5,7,10(19),22-tetraene-1α,3β,24-triol monohydrate. Solvent mixture: solution A, water R, methanol R
(0.3:29.7:70 V/V/V).
Content : 95.5 per cent to 102.0 per cent (anhydrous substance).
Test solution (a). Dissolve 2 mg of the substance to be
A reversible isomerisation to pre-calcipotriol takes place in
examined in the solvent mixture and dilute to 5.0 mL with
solution, depending on temperature and time. The activity is
the solvent mixture.
due to both compounds.
Test solution (b). Dissolve 2.00 mg of the substance to be
CHARACTERS examined in the solvent mixture and dilute to 20.0 mL with
Appearance : white or almost white, crystalline powder. the same solvent mixture.
Solubility : practically insoluble in water, freely soluble in Reference solution (a). Dilute 1.0 mL of test solution (a) to
ethanol (96 per cent), slightly soluble in methylene chloride. 100.0 mL with the solvent mixture.
It is sensitive to light. Reference solution (b). Dilute 1.0 mL of reference
solution (a) to 10.0 mL with the solvent mixture.
IDENTIFICATION Reference solution (c). Dissolve 1 mg of calcipotriol
A. Infrared absorption spectrophotometry (2.2.24). monohydrate CRS (containing impurities B, C and D) in
Comparison : Ph. Eur. reference spectrum of calcipotriol the solvent mixture and dilute to 2.5 mL with the solvent
monohydrate. mixture.

1724 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcipotriol monohydrate

Reference solution (d). Dissolve 2.00 mg of calcipotriol By liquid chromatography : B, C, D, E, F.

monohydrate CRS in the solvent mixture and dilute to
20.0 mL with the solvent mixture.
Column :
– size : l = 0.10 m, Ø = 4.0 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (3 μm).
Mobile phase : water R, methanol R (30:70 V/V).
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 264 nm.
Injection : 20 μL of test solution (a) and reference
solutions (a), (b) and (c). A. (5Z,7E,22E)-24-cyclopropyl-1α,3β-dihydroxy-9,10-
Run time : twice the retention time of calcipotriol. secochola-5,7,10(19),22-tetraen-24-one,
Relative retention with reference to calcipotriol (retention
time = about 13.5 min) : impurity B = about 0.86 ;
impurity C = about 0.92 ; impurity D = about 1.3.
System suitability: reference solution (c):
– peak-to-valley ratio : minimum 1.5, where Hp = height
above the baseline of the peak due to impurity C and
Hv = height above the baseline of the lowest point of
the curve separating this peak from the peak due to
calcipotriol ;
– the chromatogram obtained is similar to the
chromatogram supplied with calcipotriol
monohydrate CRS. B. (5Z,7Z,22E,24S)-24-cyclopropyl-9,10-secochola-
Limits : 5,7,10(19),22-tetraene-1α,3β,24-triol ((7Z)-calcipotriol),
– impurity B : not more than 0.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent) ;
– impurities C, D : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (1.0 per cent) ;
– unspecified impurities : for each impurity, not more than
the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.10 per cent) ;
– total : not more than 2.5 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (2.5 per cent) ; C. (5E,7E,22E,24S)-24-cyclopropyl-9,10-secochola-
5,7,10(19),22-tetraene-1α,3β,24-triol ((5E)-calcipotriol),
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Water (2.5.12) : 3.3 per cent to 5.0 per cent, determined on
0.100 g .
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : test solution (b) and reference solution (d).
Calculate the percentage content of C27H40O3 taking into
account the assigned content of calcipotriol monohydrate CRS.
D. (5Z,7E,22E,24R)-24-cyclopropyl-9,10-secochola-
STORAGE 5,7,10(19),22-tetraene-1α,3β,24-triol (24-epi-calcipotriol),
In an airtight container, protected from light.
IMPURITIES
Specified impurities : A, B, C, D, G, H.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : E, F, I. E. rac-(5Z,7E,24S)-24-cyclopropyl-9,10-secochola-5,7,10(19)-
By thin-layer chromatography : A, G, H, I. triene-1α,3β,24-triol,

General Notices (1) apply to all monographs and other texts 1725
Calcitonin (salmon) EUROPEAN PHARMACOPOEIA 8.0

01/2008:0471

CALCITONIN (SALMON)
Calcitoninum salmonis

F. (5Z,7E,22E,24S)-24-cyclopropyl-1α,3β-bis[[(1,1- C145H240N44O48S2 Mr 3432

dimethylethyl)dimethylsilyl]oxy]-9,10-secochola-
5,7,10(19),22-tetraen-24-ol, DEFINITION
Polypeptide having the structure determined for salmon
calcitonin I. It lowers the calcium concentration in plasma
of mammals by diminishing the rate of bone resorption. It
is obtained by chemical synthesis or by a method based on
recombinant DNA (rDNA) technology. It is available as an
acetate.
Content : 90.0 per cent to 105.0 per cent of the peptide
C145H240N44O48S2 (anhydrous and acetic acid-free substance).
By convention, for the purpose of labelling calcitonin (salmon)
preparations, 1 mg of calcitonin (salmon) (C145H240N44O48S2) is
equivalent to 6000 IU of biological activity.
PRODUCTION
The following requirements apply only to calcitonin (salmon)
produced by a method based on rDNA technology.
Prior to release the following tests are carried out on each
batch of final bulk product unless exemption has been granted
G. 24,24′-oxybis[(5Z,7E,22E,24S)-24-cyclopropyl-9,10- by the competent authority.
secochola-5,7,10(19),22-tetraene-1α,3β-diol], Host-cell-derived proteins. The limit is approved by the
competent authority.
Host-cell or vector-derived DNA. The limit is approved by
the competent authority.
CHARACTERS
Appearance : white or almost white powder.
Solubility : freely soluble in water.
IDENTIFICATION
A. Examine the chromatograms obtained in the assay.
Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time and size
to the principal peak in the chromatogram obtained with
the reference solution.
The following requirement applies only to calcitonin (salmon)
obtained by chemical synthesis.
B. Amino acid analysis (2.2.56).
H. (5Z,7E,22E,24R)-24-cyclopropyl-24-[[(5Z,7E,22E,24S)- Express the content of each amino acid in moles. Calculate
24-cyclopropyl-1α,3β-dihydroxy-9,10-secochola- the relative proportions of the amino acids taking as
5,7,10(19),22-tetraen-24-yl]oxy]-9,10-secochola- equivalent to 1 the sum, divided by 20, of the number
5,7,10(19),22-tetraene-1α,3β-diol, of moles of aspartic acid, glutamic acid, proline, glycine,
valine, leucine, histidine, arginine and lysine. The values
fall within the following limits : aspartic acid : 1.8 to 2.2 ;
glutamic acid : 2.7 to 3.3 ; proline : 1.7 to 2.3 ; glycine : 2.7 to
3.3 ; valine : 0.9 to 1.1 ; leucine : 4.5 to 5.3 ; histidine : 0.9 to
1.1 ; arginine : 0.9 to 1.1 ; lysine : 1.8 to 2.2 ; serine : 3.2 to
4.2 ; threonine : 4.2 to 5.2 ; tyrosine : 0.7 to 1.1 ; half-cystine :
1.4 to 2.1.
The following requirement applies only to calcitonin (salmon)
produced by a method based on rDNA technology.
C. Peptide mapping (2.2.55).
SELECTIVE CLEAVAGE OF THE PEPTIDE BONDS
Test solution. Prepare a 1 mg/mL solution of the substance
I. (6S,7R,8R,22E,24S)-24-cyclopropyl-6,8:7,19-dicyclo-9,10- to be examined. Transfer 1.0 mL to a clean tube. Add
secochola-5(10),22-diene-1α,3β,24-triol (suprasterol of 100 μL of 1 M tris-hydrochloride buffer solution pH 8.0 R
calcipotriol). and 20 μL of a freshly prepared 1.0 mg/mL solution of

1726 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcitonin (salmon)

trypsin for peptide mapping R. Allow to stand at 2-8 °C Resolution solution. Dissolve the contents of a vial of
for 16-20 h. Stop the reaction by adding 10 μL of a 50 per N-acetyl-Cys1-calcitonin CRS in 400 μL of mobile phase A
cent V/V solution of trifluoroacetic acid R. Cap the vial and and add 100 μL of the test solution.
mix. Centrifuge the vials to remove air bubbles. Column :
Reference solution. Prepare at the same time and in the – size : l = 0.25 m, Ø = 4.6 mm ;
same manner as for the test solution but using calcitonin – stationary phase : octadecylsilyl silica gel for
(salmon) CRS instead of the substance to be examined. chromatography R (5 μm) ;
CHROMATOGRAPHIC SEPARATION. Liquid chromatography – temperature : 65 °C.
(2.2.29).
Mobile phase :
Column :
– mobile phase A : dissolve 3.26 g of tetramethylammonium
– size : l = 0.25 m, Ø = 4.6 mm ; hydroxide R in 900 mL of water R, adjust to pH 2.5 with
– stationary phase : octadecylsilyl silica gel for phosphoric acid R and mix with 100 mL of acetonitrile
chromatography R (5 μm) with a pore size of 30 nm. for chromatography R ; filter and degas ;
Mobile phase : – mobile phase B : dissolve 1.45 g of tetramethylammonium
– mobile phase A : mix 1 mL of trifluoroacetic acid R and hydroxide R in 400 mL of water R, adjust to pH 2.5 with
1000 mL of water R ; filter and degas ; phosphoric acid R and mix with 600 mL of acetonitrile
for chromatography R ; filter and degas ;
– mobile phase B : mix 0.850 mL of trifluoroacetic acid R,
200 mL of water R and 800 mL of acetonitrile for Time Mobile phase A Mobile phase B
chromatography R ; filter and degas ; (min) (per cent V/V) (per cent V/V)
0 - 30 72 → 48 28 → 52
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V) 30 - 32 48 → 72 52 → 28
0 - 50 100 → 65 0 → 35 32 - 55 72 28
50 - 60 65 → 40 35 → 60
Flow rate : 1.0 mL/min.
60 - 60.1 40 → 0 60 → 100 Detection : spectrophotometer at 220 nm.
60.1 - 65.1 0 100 Injection : 20 μL.
65.1 - 65.2 0 → 100 100 → 0 Relative retention with reference to calcitonin (salmon)
(retention time = about 20 min) : impurity B = about 0.8 ;
65.2 - 80.2 100 0 impurity C = about 0.9 ; impurity D = about 1.05 ;
impurity A = about 1.15.
Flow rate : 1.2 mL/min.
System suitability : resolution solution :
Detection : spectrophotometer at 214 nm.
– resolution : minimum 5.0 between the peaks due to
Equilibration : at initial conditions for at least 15 min. calcitonin (salmon) and impurity A,
Carry out a blank run using the above-mentioned gradient.
– symmetry factor : maximum 2.5 for the peak due to
Injection : 20 μL. impurity A.
System suitability: the chromatograms obtained with the Limits :
test solution and the reference solution are qualitatively – impurities A, B, C, D : for each impurity, maximum
similar to the chromatogram of calcitonin (salmon) digest 3.0 per cent ; other unidentified, specified impurities
supplied with calcitonin (salmon) CRS. may occur that co-elute with impurities A, B, C and D ;
Results : the profile of the chromatogram obtained with the acceptance criterion applies irrespective of whether
the test solution corresponds to that of the chromatogram these impurities co-elute ;
obtained with the reference solution : the retention times – total : maximum 5.0 per cent ;
of the fragment peaks in the chromatogram obtained with
the test solution are within 5 per cent of the retention times – disregard limit : 0.1 per cent.
of the fragments obtained with the reference solution ; the The following requirement applies only to calcitonin (salmon)
peak area ratios of the fragment peaks in the chromatogram produced by a method based on rDNA technology.
obtained with the test solution, normalised to the area of B. Test solution. Prepare a 0.5 mg/mL solution of the
peak T2, are within 5 per cent of the corresponding peak substance to be examined. To 1.0 mL of this solution add
ratios in the chromatogram obtained with the reference 100 μL of 0.25 M citrate buffer solution pH 3.0 R.
solution. Resolution solution. Prepare a 1 mg/mL solution of the
substance to be examined. Mix 1 volume of the solution and
TESTS
1 volume of calcitonin-Gly CRS. To 1.0 mL of this mixture
Acetic acid (2.5.34) : 4.0 per cent to 15.0 per cent. add 100 μL of 0.25 M citrate buffer solution pH 3.0 R.
Test solution. Dissolve 10.0 mg of the substance to be Column :
examined in a mixture of 5 volumes of mobile phase B and – size : l = 0.20 m, Ø = 4.6 mm ;
95 volumes of mobile phase A and dilute to 10.0 mL with the
– stationary phase : a suitable polysulfoethylaspartamide
same mixture of mobile phases.
ion-exchange gel (5 μm).
Related substances. Liquid chromatography (2.2.29) : use the Mobile phase :
normalisation procedure.
– mobile phase A : mix 15 volumes of acetonitrile for
The following requirement applies to calcitonin (salmon), chromatography R and 85 volumes of a 2.72 g/L solution
whether obtained by chemical synthesis or by a method based of potassium dihydrogen phosphate R adjusted to pH 5.0
on rDNA technology. with a 600 g/L solution of potassium hydroxide R ;
A. Test solution. Prepare a 1.0 mg/mL solution of the – mobile phase B : mix 15 volumes of acetonitrile for
substance to be examined in mobile phase A. chromatography R and 85 volumes of a solution
Reference solution. Dissolve the contents of a vial of containing 2.72 g/L of potassium dihydrogen phosphate R
calcitonin (salmon) CRS in mobile phase A to obtain a and 29.22 g/L of sodium chloride R adjusted to pH 4.6
concentration of 1.0 mg/mL. with a 600 g/L solution of potassium hydroxide R ;

General Notices (1) apply to all monographs and other texts 1727
Calcitriol EUROPEAN PHARMACOPOEIA 8.0

Time Mobile phase A Mobile phase B

(min) (per cent V/V) (per cent V/V)
0 - 10 100 → 0 0 → 100
10 - 15 0 100
15 - 15.1 0 → 100 100 → 0 C. des-22-tyrosine-calcitonin (salmon),
15.1 - 22.1 100 0
D. O-acetylated calcitonin (salmon),
Flow rate : 1.2 mL/min.
Detection : spectrophotometer at 220 nm.
Injection : 50 μL ; rinse the injector with a 40 per cent V/V
solution of acetonitrile for chromatography R.
Relative retention with reference to calcitonin (salmon)
(retention time = about 9 min) : impurity G = about 0.4 ;
impurity F = about 0.6 ; impurity E = about 0.9. F. R = NH2 : [1,7-bis(3-sulfo-L-alanine)]calcitonin (salmon),
System suitability: resolution solution : G. R = NH-CH2-CO2H : [1,7-bis(3-sulfo-L-alanine)]calcitoni-
– resolution : minimum 3.0 between the peaks due to nylglycine (salmon).
impurity E and calcitonin (salmon).
Limits :
01/2013:0883
– impurity E : maximum 0.6 per cent ;
– impurities F, G : for each impurity, maximum 0.2 per CALCITRIOL
cent.
Water (2.5.32) : maximum 10.0 per cent. Calcitriolum
Acetic acid and water : maximum 20 per cent, calculated by
adding together the percentage contents of acetic acid and
water determined by the methods described above.
Bacterial endotoxins (2.6.14) : less than 25 IU/mg, if intended
for use in the manufacture of parenteral preparations without
a further appropriate procedure for the removal of bacterial
endotoxins.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances. Use method A for calcitonin (salmon)
obtained by chemical synthesis and method B for calcitonin
C27H44O3 Mr 416.6
(salmon) obtained by a method based on rDNA technology.
[32222-06-3]
Calculate the content of calcitonin (salmon) (C145H240N44O48S2)
from the area of the principal peak in each of the DEFINITION
chromatograms obtained with the test solution and the (5Z,7E)-9,10-Secocholesta-5,7,10(19)-triene-1α,3β,25-triol.
reference solution and the declared content of C145H240N44O48S2 Content : 97.0 per cent to 103.0 per cent.
in calcitonin (salmon) CRS. Proceed with tangential integration
of the peak areas. A reversible isomerisation to pre-calcitriol takes place in
solution, depending on temperature and time. The activity is
STORAGE due to both compounds (see Assay).
Protected from light at a temperature between 2 °C and 8 °C. If CHARACTERS
the substance is sterile, store in a sterile, airtight, tamper-proof
container. Appearance : white or almost white crystals.
Solubility : practically insoluble in water, freely soluble in
LABELLING ethanol (96 per cent), soluble in fatty oils.
The label states : It is sensitive to air, heat and light.
– the calcitonin peptide content (C145H240N44O48S2) ; IDENTIFICATION
– the origin : synthetic or rDNA technology. A. Infrared absorption spectrophotometry (2.2.24).
IMPURITIES Comparison: Ph. Eur. reference spectrum of calcitriol.
Specified impurities : A, B, C, D, E, F, G. B. Examine the chromatograms obtained in the assay.
Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time and size
to the principal peak in the chromatogram obtained with
reference solution (a).
TESTS
A. R1 = CO-CH3, R2 = NH2, X = L-Leu : acetylcalcitonin Related substances. Liquid chromatography (2.2.29) : use
(salmon), the normalisation procedure. Carry out the test as rapidly as
possible, avoiding exposure to actinic light and air.
B. R1 = H, R2 = NH2, X = D-Leu : [9-D-leucine]calcitonin Test solution. Dissolve 1.00 mg of the substance to be
(salmon), examined without heating in 10.0 mL of the mobile phase.
E. R1 = H, R2 = NH-CH2-CO2H, X = L-Leu : salmon Reference solution (a). Dissolve 1.00 mg of calcitriol CRS
calcitoninylglycine, without heating in 10.0 mL of the mobile phase.

1728 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcium acetate, anhydrous

Reference solution (b). Dilute 1.0 mL of reference solution (a)

to 100.0 mL with the mobile phase. Dilute 1.0 mL of this
solution to 10.0 mL with the mobile phase.
Reference solution (c). Heat 2 mL of reference solution (a) at
80 °C for 30 min.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octylsilyl silica gel for chromatography R1
(5 μm) ;
A. (5E,7E)-9,10-secocholesta-5,7,10(19)-triene-1α,3β,25-triol
– temperature : 40 °C. (trans-calcitriol),
Mobile phase : mix 450 volumes of a 1.0 g/L solution of
tris(hydroxymethyl)aminomethane R adjusted to pH 7.0-7.5
with phosphoric acid R, and 550 volumes of acetonitrile R.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 230 nm.
Injection : 50 μL.
Run time : twice the retention time of calcitriol.
Relative retention with reference to calcitriol (retention
time = about 14 min) : impurity C = about 0.4 ;
pre-calcitriol = about 0.88 ; impurity A = about 0.95 ; B. (5Z,7E)-9,10-secocholesta-5,7,10(19)-triene-1β,3β,25-triol
impurity B = about 1.1. (1β-calcitriol),
System suitability :
– resolution : minimum 3.5 between the peaks due to
pre-calcitriol and calcitriol in the chromatogram obtained
with reference solution (c) ;
– number of theoretical plates : minimum 10 000, calculated
for the peak due to calcitriol in the chromatogram obtained
with reference solution (a).
Limits :
– impurities A, B, C : for each impurity, maximum 0.5 per
cent ; C. (6aR,7R,9aR)-11-[(3S,5R)-3,5-dihydroxy-2-
methylcyclohex-1-enyl]-7-[(1R)-5-hydroxy-1,5-
– unspecified impurities : for each impurity, maximum
dimethylhexyl]-6a-methyl-2-phenyl-5,6,6a,7,8,9,9a,11-
0.10 per cent ;
octahydro-1H,4aH-cyclopenta[f][1,2,4]triazolo[1,2-
– total : maximum 1.0 per cent ; a]cinnoline-1,3(2H)-dione (triazoline adduct of
pre-calcitriol).
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent) ; disregard the peak due to pre-calcitriol.
01/2011:2128
corrected 7.3
ASSAY
Liquid chromatography (2.2.29) as described in the test for CALCIUM ACETATE, ANHYDROUS
related substances with the following modifications.
Injection : test solution and reference solution (a). Calcii acetas anhydricus
System suitability: reference solution (a) :
– repeatability : maximum relative standard deviation of 1 per
cent for the peak due to calcitriol after 6 injections. C4H6CaO4 Mr 158.2
Calculate the percentage content of C27H44O3 taking into [62-54-4]
account the assigned content of calcitriol CRS and, if necessary, DEFINITION
the peak due to pre-calcitriol.
Calcium diacetate.
Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
STORAGE
Under nitrogen, in an airtight container, protected from light, CHARACTERS
at a temperature of 2 °C to 8 °C. Appearance : white or almost white, hygroscopic powder.
The contents of an opened container are to be used Solubility : freely soluble in water, slightly soluble in ethanol
immediately. (96 per cent).
IDENTIFICATION
IMPURITIES A. It gives reaction (b) of calcium (2.3.1).
Specified impurities : A, B, C. B. It gives reaction (b) of acetates (2.3.1).

General Notices (1) apply to all monographs and other texts 1729
Calcium acetate, anhydrous EUROPEAN PHARMACOPOEIA 8.0

TESTS Wavelength : 455.4 nm.

Solution S. Dissolve 5.0 g in carbon dioxide-free water R and Iron (2.4.9) : maximum 20 ppm, if intended for use in the
dilute to 50.0 mL with the same solvent. manufacture of parenteral preparations or haemodialysis
Appearance of solution. Solution S is clear (2.2.1) and solutions.
colourless (2.2.2, Method II). Dilute 5 mL of solution S to 10 mL of water R.
pH (2.2.3) : 7.2 to 8.2. Magnesium : maximum 500 ppm.
Dilute 5.0 mL of solution S to 10.0 mL with carbon dioxide-free Atomic absorption spectrometry (2.2.23, Method II).
water R. Test solution. Dissolve 50.0 mg of the substance to be
Readily oxidisable substances. Dissolve 2.0 g in boiling examined in water R and dilute to 100.0 mL with the same
water R and dilute to 100 mL with boiling water R, add a few solvent.
glass beads, 6 mL of 5 M sulfuric acid and 0.3 mL of 0.02 M Reference solutions. Prepare the reference solutions using
potassium permanganate, mix, boil gently for 5 min and allow magnesium standard solution (0.1 per cent Mg) R, diluted as
the precipitate to settle. The pink colour in the supernatant necessary with water R.
is not completely discharged. Source : magnesium hollow-cathode lamp.
Chlorides (2.4.4): maximum 330 ppm. Wavelength : 285.2 nm.
Dissolve 0.15 g in water R and dilute to 15 mL with the same Atomisation device : air-acetylene flame.
solvent.
Potassium : maximum 500 ppm, if intended for use in the
Fluorides : maximum 50 ppm. manufacture of parenteral preparations or haemodialysis
Potentiometry (2.2.36, Method I). solutions.
Test solution. In a 50 mL volumetric flask, dissolve 0.200 g Atomic emission spectrometry (2.2.22, Method II).
in a 10.3 g/L solution of hydrochloric acid R, add 5.0 mL of Test solution. Dissolve 1.00 g of the substance to be examined
fluoride standard solution (1 ppm F) R and dilute to 50.0 mL in water R and dilute to 25.0 mL with the same solvent.
with a 10.3 g/L solution of hydrochloric acid R. To 20.0 mL of Reference solutions. Prepare the reference solutions using
the solution add 20.0 mL of total-ionic-strength-adjustment potassium standard solution (0.2 per cent K) R, diluted as
buffer R and 3 mL of an 82 g/L solution of anhydrous sodium necessary with water R.
acetate R. Adjust to pH 5.2 with ammonia R and dilute to
50.0 mL with distilled water R. Wavelength : 766.5 nm.
Reference solutions. To 0.25 mL, 0.5 mL, 0.75 mL and 1.0 mL Sodium : maximum 500 ppm, if intended for use in the
of fluoride standard solution (10 ppm F) R add 20.0 mL of manufacture of parenteral preparations or haemodialysis
total-ionic-strength-adjustment buffer R and dilute to 50.0 mL solutions.
with distilled water R. Atomic emission spectrometry (2.2.22, Method II).
Indicator electrode : fluoride selective. Test solution. Dissolve 1.00 g of the substance to be examined
in water R and dilute to 100.0 mL with the same solvent.
Reference electrode : silver-silver chloride.
Reference solutions. Prepare the reference solutions using
Take into account the addition of fluoride to the test solution sodium standard solution (200 ppm Na) R, diluted as necessary
for the calculation. with water R.
Nitrates. To 10.0 mL of solution S add 5 mg of sodium Wavelength : 589 nm.
chloride R, 0.05 mL of indigo carmine solution R and add with
stirring, 10 mL of nitrogen-free sulfuric acid R. The blue colour Strontium : maximum 500 ppm, if intended for use in the
remains for at least 10 min. manufacture of parenteral preparations or haemodialysis
solutions.
Sulfates (2.4.13) : maximum 600 ppm.
Atomic emission spectrometry (2.2.22, Method II).
Dissolve 0.25 g in distilled water R and dilute to 15 mL with
the same solvent. Test solution. Dissolve 2.00 g of the substance to be examined
in water R and dilute to 100.0 mL with the same solvent.
Aluminium (2.4.17) : maximum 1 ppm, if intended for
Reference solutions. Prepare the reference solutions using
use in the manufacture of peritoneal dialysis solutions,
strontium standard solution (1.0 per cent Sr) R, diluted as
haemofiltration solutions or haemodialysis solutions.
necessary with water R.
Test solution. Dissolve 4.0 g of the substance to be examined Wavelength : 460.7 nm.
in 100 mL of water R and add 10 mL of acetate buffer solution
pH 6.0 R. Heavy metals (2.4.8) : maximum 10 ppm.
Reference solution. Mix 2 mL of aluminium standard solution Dissolve 4.0 g in water R and dilute to 20 mL with the
(2 ppm Al) R, 10 mL of acetate buffer solution pH 6.0 R and same solvent. 12 mL of the solution complies with test A.
98 mL of water R. Prepare the reference solution using lead standard solution
(2 ppm Pb) R.
Blank solution. Mix 10 mL of acetate buffer solution pH 6.0 R
and 100 mL of water R. Water (2.5.12) : maximum 7.0 per cent, determined on 0.100 g.
Add 2 mL of anhydrous acetic acid R to the titration vessel in
Arsenic (2.4.2): maximum 3 ppm. addition to the methanol. Clean the titration vessel after each
3.3 mL of solution S complies with test A. determination.
Barium : maximum 50 ppm. ASSAY
Inductively coupled plasma-atomic emission spectrometry Dissolve 0.150 g in 100 mL of water R and carry out the
(2.2.57). complexometric titration of calcium (2.5.11).
Test solution. Dissolve 5.00 g of the substance to be examined 1 mL of 0.1 M sodium edetate is equivalent to 15.82 mg
in water R and dilute to 100.0 mL with the same solvent. of C4H6CaO4.
Reference solutions. Prepare the reference solutions using
barium standard solution (0.1 per cent Ba) R, diluted as STORAGE
necessary with water R. In an airtight container.

1730 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcium carbonate

LABELLING buffer R and 3 mL of an 82 g/L solution of anhydrous sodium

The label states, where applicable, that the substance is acetate R. Adjust to pH 5.2 with ammonia R and dilute to
suitable for use in the manufacture of parenteral preparations, 50.0 mL with distilled water R.
peritoneal dialysis solutions, haemofiltration solutions or Reference solutions. To 0.25 mL, 0.5 mL, 1.0 mL, 2.0 mL
haemodialysis solutions. and 5.0 mL of fluoride standard solution (10 ppm F) R add
20.0 mL of total-ionic-strength-adjustment buffer R and dilute
to 50.0 mL with distilled water R.
01/2008:1182
corrected 7.0 Indicator electrode : fluoride selective.
Reference electrode : silver-silver chloride.
CALCIUM ASCORBATE Take into account the addition of fluoride to the test solution
for the calculation.
Calcii ascorbas Copper : maximum 5 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Dissolve 2.0 g in a 9.7 g/L solution of nitric
acid R and dilute to 25.0 mL with the same acid solution.
Reference solutions. Prepare the reference solutions using
copper standard solution (10 ppm Cu) R, diluting with a 9.7 g/L
solution of nitric acid R.
Source : copper hollow-cathode lamp.
C12H14CaO12,2H2O Mr 426.3 Wavelength : 324.8 nm.
[5743-28-2] Atomisation device : air-acetylene flame.
DEFINITION Iron : maximum 2 ppm.
Calcium di[(R)-2-[(S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo- Atomic absorption spectrometry (2.2.23, Method I).
2H-furan-3-olate] dihydrate. Test solution. Dissolve 5.0 g in a 9.7 g/L solution of nitric
Content : 99.0 per cent to 100.5 per cent of C12H14CaO12,2H2O. acid R and dilute to 25.0 mL with the same acid solution.
Reference solutions. Prepare the reference solutions using
CHARACTERS iron standard solution (10 ppm Fe) R, diluting with a 9.7 g/L
Appearance : white or slightly yellowish, crystalline powder. solution of nitric acid R.
Solubility : freely soluble in water, practically insoluble in Source : iron hollow-cathode lamp.
ethanol (96 per cent). Wavelength : 248.3 nm.
IDENTIFICATION Atomisation device : air-acetylene flame.
First identification : A, B, E. Heavy metals (2.4.8) : maximum 10 ppm.
Second identification : A, C, D, E. 2.0 g complies with test D. Prepare the reference solution
A. Specific optical rotation (see Tests). using 2.0 mL of lead standard solution (10 ppm Pb) R.
B. Infrared absorption spectrophotometry (2.2.24). Loss on drying (2.2.32) : maximum 0.1 per cent, determined
Comparison : Ph. Eur. reference spectrum of calcium on 1.000 g by drying in an oven at 105 °C for 2 h.
ascorbate. ASSAY
C. Dilute 1 mL of solution S (see Tests) to 10 mL with water R. Dissolve 80.0 mg in a mixture of 10 mL of dilute sulfuric
To 2 mL of the solution add 0.2 mL of a 100 g/L solution of acid R and 80 mL of carbon dioxide-free water R. Add 1 mL of
ferrous sulfate R. A deep violet colour develops. starch solution R. Titrate with 0.05 M iodine until a persistent
D. To 1 mL of solution S add 0.2 mL of dilute nitric acid R violet-blue colour is obtained.
and 0.2 mL of silver nitrate solution R2. A grey precipitate 1 mL of 0.05 M iodine is equivalent to 10.66 mg
is formed. of C12H14CaO12,2H2O.
E. The substance gives reaction (b) of calcium (2.3.1).
STORAGE
TESTS In a non-metallic container, protected from light.
Solution S. Dissolve 5.00 g in carbon dioxide-free water R and
dilute to 50.0 mL with the same solvent. 07/2008:0014
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y6 (2.2.2, CALCIUM CARBONATE
Method II). Examine the colour of the solution immediately
after preparation of the solution. Calcii carbonas
pH (2.2.3): 6.8 to 7.4 for solution S.
CaCO3 Mr 100.1
Specific optical rotation (2.2.7) : + 95 to + 97 (dried [471-34-1]
substance), determined using freshly prepared solution S.
DEFINITION
Related substances. The thresholds indicated under Related
substances (Table 2034.-1) in the general monograph Content : 98.5 per cent to 100.5 per cent (dried substance).
Substances for pharmaceutical use (2034) do not apply. CHARACTERS
Fluorides : maximum 10 ppm. Appearance : white or almost white powder.
Potentiometry (2.2.36, Method I). Solubility : practically insoluble in water.
Test solution. In a 50 mL volumetric flask, dissolve 1.000 g
in a 10.3 g/L solution of hydrochloric acid R, add 5.0 mL of IDENTIFICATION
fluoride standard solution (1 ppm F) R and dilute to 50.0 mL A. It gives the reaction of carbonates (2.3.1).
with a 10.3 g/L solution of hydrochloric acid R. To 20.0 mL of B. 0.2 mL of solution S (see Tests) gives the reactions of
the solution add 20.0 mL of total-ionic-strength-adjustment calcium (2.3.1).

General Notices (1) apply to all monographs and other texts 1731
Calcium chloride dihydrate EUROPEAN PHARMACOPOEIA 8.0

TESTS 01/2008:0015
Solution S. Dissolve 5.0 g in 80 mL of dilute acetic acid R. corrected 6.0
When the effervescence ceases, boil for 2 min. Allow to
cool, dilute to 100 mL with dilute acetic acid R and filter, if CALCIUM CHLORIDE DIHYDRATE
necessary, through a sintered-glass filter (2.1.2).
Substances insoluble in acetic acid : maximum 0.2 per cent. Calcii chloridum dihydricum
Wash any residue obtained during the preparation of solution S
with 4 quantities, each of 5 mL, of hot water R and dry at CaCl2,2H2O Mr 147.0
100-105 °C for 1 h. The residue weighs a maximum of 10 mg. [10035-04-8]
Chlorides (2.4.4): maximum 330 ppm. DEFINITION
Dilute 3 mL of solution S to 15 mL with water R. Content : 97.0 per cent to 103.0 per cent of CaCl2,2H2O.
Sulfates (2.4.13) : maximum 0.25 per cent.
CHARACTERS
Dilute 1.2 mL of solution S to 15 mL with distilled water R.
Appearance : white or almost white, crystalline powder,
Arsenic (2.4.2, Method A) : maximum 4 ppm, determined on hygroscopic.
5 mL of solution S. Solubility : freely soluble in water, soluble in ethanol (96 per
Barium. To 10 mL of solution S add 10 mL of calcium sulfate cent).
solution R. After at least 15 min, any opalescence in the
solution is not more intense than that in a mixture of 10 mL IDENTIFICATION
of solution S and 10 mL of distilled water R. A. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
Iron (2.4.9) : maximum 200 ppm. B. It gives the reactions of calcium (2.3.1).
Dissolve 50 mg in 5 mL of dilute hydrochloric acid R and C. It complies with the limits of the assay.
dilute to 10 mL with water R. TESTS
Magnesium and alkali metals : maximum 1.5 per cent. Solution S. Dissolve 10.0 g in carbon dioxide-free water R
Dissolve 1.0 g in 12 mL of dilute hydrochloric acid R. Boil the prepared from distilled water R and dilute to 100 mL with the
solution for about 2 min and add 20 mL of water R, 1 g of same solvent.
ammonium chloride R and 0.1 mL of methyl red solution R. Appearance of solution. Solution S is clear (2.2.1) and not
Add dilute ammonia R1 until the colour of the indicator more intensely coloured than reference solution Y6 (2.2.2,
changes and then add 2 mL in excess. Heat to boiling and add Method II).
50 mL of hot ammonium oxalate solution R. Allow to stand for
4 h, dilute to 100 mL with water R and filter through a suitable Acidity or alkalinity. To 10 mL of freshly prepared solution S
filter. To 50 mL of the filtrate add 0.25 mL of sulfuric acid R. add 0.1 mL of phenolphthalein solution R. If the solution is red,
Evaporate to dryness on a water-bath and ignite to constant not more than 0.2 mL of 0.01 M hydrochloric acid is required
mass at 600 ± 50 °C. The residue weighs a maximum of 7.5 mg. to discharge the colour and if the solution is colourless, not
more than 0.2 mL of 0.01 M sodium hydroxide is required to
Heavy metals (2.4.8) : maximum 20 ppm. turn it red.
12 mL of solution S complies with test A. Prepare the reference Sulfates (2.4.13) : maximum 300 ppm.
solution using lead standard solution (1 ppm Pb) R.
Dilute 5 mL of solution S to 15 mL with distilled water R.
Loss on drying (2.2.32) : maximum 2.0 per cent, determined
on 1.000 g by drying in an oven at 200 ± 10 °C. Aluminium. To 10 mL of solution S add 2 mL of ammonium
chloride solution R and 1 mL of dilute ammonia R1 and boil
ASSAY the solution. No turbidity or precipitate is formed.
Dissolve 0.150 g in a mixture of 3 mL of dilute hydrochloric If intended for use in the manufacture of dialysis solutions,
acid R and 20 mL of water R. Boil for 2 min, allow to cool and the above test is replaced by the following test for aluminium
dilute to 50 mL with water R. Carry out the complexometric (2.4.17) : maximum 1 ppm.
titration of calcium (2.5.11). Prescribed solution. Dissolve 4 g in 100 mL of water R and add
1 mL of 0.1 M sodium edetate is equivalent to 10.01 mg 10 mL of acetate buffer solution pH 6.0 R.
of CaCO3. Reference solution. Mix 2 mL of aluminium standard solution
(2 ppm Al) R, 10 mL of acetate buffer solution pH 6.0 R and
FUNCTIONALITY-RELATED CHARACTERISTICS 98 mL of water R.
This section provides information on characteristics that are Blank solution. Mix 10 mL of acetate buffer solution pH 6.0 R
recognised as being relevant control parameters for one or and 100 mL of water R.
more functions of the substance when used as an excipient (see Barium. To 10 mL of solution S add 1 mL of calcium sulfate
chapter 5.15). This section is a non-mandatory part of the solution R. After at least 15 min, any opalescence in the
monograph and it is not necessary to verify the characteristics solution is not more intense than that in a mixture of 1 mL of
to demonstrate compliance. Control of these characteristics can distilled water R and 10 mL of solution S.
however contribute to the quality of a medicinal product by
Iron (2.4.9) : maximum 10 ppm, determined on solution S.
improving the consistency of the manufacturing process and
the performance of the medicinal product during use. Where Magnesium and alkali metals : maximum 0.5 per cent.
control methods are cited, they are recognised as being suitable To a mixture of 20 mL of solution S and 80 mL of water R add
for the purpose, but other methods can also be used. Wherever 2 g of ammonium chloride R and 2 mL of dilute ammonia R1,
results for a particular characteristic are reported, the control heat to boiling and pour into the boiling solution a hot
method must be indicated. solution of 5 g of ammonium oxalate R in 75 mL of water R.
The following characteristics may be relevant for calcium Allow to stand for 4 h, dilute to 200 mL with water R and filter
carbonate used as filler in tablets and capsules. through a suitable filter. To 100 mL of the filtrate add 0.5 mL
of sulfuric acid R. Evaporate to dryness on a water-bath and
Particle-size distribution (2.9.31 or 2.9.38). ignite to constant mass at 600 ± 50 °C. The residue weighs a
Powder flow (2.9.36). maximum of 5 mg.

1732 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcium dobesilate monohydrate

Heavy metals (2.4.8) : maximum 20 ppm. Barium. To 10 mL of solution S add 1 mL of calcium sulfate
12 mL of solution S complies with test A. Prepare the reference solution R. After at least 15 min, any opalescence in the
solution using lead standard solution (2 ppm Pb) R. solution is not more intense than that in a mixture of 1 mL of
distilled water R and 10 mL of solution S.
ASSAY
Iron (2.4.9) : maximum 7 ppm, determined on solution S.
Dissolve 0.280 g in 100 mL of water R and carry out the
complexometric titration of calcium (2.5.11). Magnesium and alkali metals : maximum 0.3 per cent.
1 mL of 0.1 M sodium edetate is equivalent to 14.70 mg To a mixture of 20 mL of solution S and 80 mL of water R add
of CaCl2,2H2O. 2 g of ammonium chloride R and 2 mL of dilute ammonia R1,
heat to boiling and pour into the boiling solution a hot
LABELLING solution of 5 g of ammonium oxalate R in 75 mL of water R.
The label states, where applicable, that the substance is suitable Allow to stand for 4 h, dilute to 200 mL with water R and filter
for use in the manufacture of dialysis solutions. through a suitable filter. To 100 mL of the filtrate add 0.5 mL
of sulfuric acid R. Evaporate to dryness on a water-bath and
STORAGE ignite to constant mass at 600 ± 50 °C. The residue weighs a
In an airtight container. maximum of 5 mg.
Heavy metals (2.4.8) : maximum 15 ppm.
01/2008:0707 12 mL of solution S complies with test A. Prepare the reference
corrected 6.0 solution using lead standard solution (2 ppm Pb) R.

CALCIUM CHLORIDE HEXAHYDRATE ASSAY

Dissolve 0.200 g in 100 mL of water R. Carry out the
Calcii chloridum hexahydricum complexometric titration of calcium (2.5.11).
1 mL of 0.1 M sodium edetate is equivalent to 21.91 mg
CaCl2,6H2O Mr 219.1 of CaCl2,6H2O.
[7774-34-7]
LABELLING
DEFINITION The label states, where applicable, that the substance is suitable
Content : 97.0 per cent to 103.0 per cent of CaCl2,6H2O. for use in the manufacture of dialysis solutions.
CHARACTERS
Appearance : white or almost white, crystalline mass or 07/2008:1183
colourless crystals. corrected 7.0
Solubility : very soluble in water, freely soluble in ethanol
(96 per cent). CALCIUM DOBESILATE
It solidifies at about 29 °C. MONOHYDRATE
IDENTIFICATION
Calcii dobesilas monohydricus
A. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
B. It gives the reactions of calcium (2.3.1).
C. It complies with the limits of the assay.
TESTS
Solution S. Dissolve 15.0 g in carbon dioxide-free water R C12H10CaO10S2,H2O Mr 436.4
prepared from distilled water R and dilute to 100 mL with the [20123-80-2]
same solvent.
DEFINITION
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y6 (2.2.2, Calcium di(2,5-dihydroxybenzenesulfonate) monohydrate.
Method II). Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
Acidity or alkalinity. To 10 mL of freshly prepared solution S CHARACTERS
add 0.1 mL of phenolphthalein solution R. If the solution is red, Appearance : white or almost white, hygroscopic powder.
not more than 0.2 mL of 0.01 M hydrochloric acid is required
to discharge the colour and if the solution is colourless, not Solubility : very soluble in water, freely soluble in anhydrous
more than 0.2 mL of 0.01 M sodium hydroxide is required to ethanol, very slightly soluble in 2-propanol, practically
turn it red. insoluble in methylene chloride.
Sulfates (2.4.13) : maximum 200 ppm. IDENTIFICATION
Dilute 5 mL of solution S to 15 mL with distilled water R. A. Ultraviolet and visible absorption spectrophotometry
Aluminium. To 10 mL of solution S add 2 mL of ammonium (2.2.25).
chloride solution R and 1 mL of dilute ammonia R1. Heat to Test solution. Dissolve 0.100 g in water R and dilute to
boiling. No turbidity or precipitate is formed. 200.0 mL with the same solvent. Dilute 5.0 mL of this
If intended for use in the manufacture of dialysis solutions, solution to 100.0 mL with water R.
the above test is replaced by the following test for aluminium Spectral range : 210-350 nm.
(2.4.17) : maximum 1 ppm. Absorption maxima : at 221 nm and 301 nm.
Prescribed solution. Dissolve 6 g in 100 mL of water R and add Specific absorbance at the absorption maximum at 301 nm :
10 mL of acetate buffer solution pH 6.0 R. 174 to 181.
Reference solution. Mix 2 mL of aluminium standard solution B. Mix 1 mL of ferric chloride solution R2, 1 mL of a freshly
(2 ppm Al) R, 10 mL of acetate buffer solution pH 6.0 R and prepared 10 g/L solution of potassium ferricyanide R
98 mL of water R. and 0.1 mL of nitric acid R. To this mixture add 5 mL of
Blank solution. Mix 10 mL of acetate buffer solution pH 6.0 R freshly prepared solution S (see Tests) : a blue colour and a
and 100 mL of water R. precipitate are immediately produced.

General Notices (1) apply to all monographs and other texts 1733
Calcium folinate EUROPEAN PHARMACOPOEIA 8.0

C. 2 mL of freshly prepared solution S gives reaction (b) of 1 mL of 0.1 M cerium sulfate is equivalent to 10.45 mg of
calcium (2.3.1). C12H10CaO10S2.
TESTS STORAGE
Solution S. Dissolve 10.0 g in carbon dioxide-free water R and In an airtight container, protected from light.
dilute to 100 mL with the same solvent.
IMPURITIES
Appearance of solution. Solution S, when freshly prepared, Specified impurities : A.
is clear (2.2.1) and colourless (2.2.2, Method II).
pH (2.2.3): 4.5 to 6.0 for solution S.
Related substances. Liquid chromatography (2.2.29). Keep
all solutions at 2-8 °C.
Buffer solution. Dissolve 1.2 g of anhydrous sodium dihydrogen A. benzene-1,4-diol (hydroquinone).
phosphate R in 900 mL of water for chromatography R, adjust
to pH 6.5 with disodium hydrogen phosphate solution R and
dilute to 1000 mL with water for chromatography R. 01/2009:0978
corrected 7.0
Test solution. Dissolve 0.100 g of the substance to be examined
in water R and dilute to 10.0 mL with the same solvent.
Reference solution (a). Dilute 1.0 mL of the test solution to
CALCIUM FOLINATE
100.0 mL with water R. Dilute 1.0 mL of this solution to
10.0 mL with water R. Calcii folinas
Reference solution (b). Dissolve 10 mg of the substance to
be examined and 10 mg of hydroquinone R (impurity A) in
water R and dilute to 10 mL with the same solvent. Dilute
1 mL of this solution to 100 mL with water R.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : spherical end-capped octadecylsilyl silica
gel for chromatography R (5 μm). C20H21CaN7O7,xH2O Mr 511.5 (anhydrous substance)
Mobile phase : acetonitrile R1, buffer solution (10:90 V/V). DEFINITION
Flow rate : 0.8 mL/min. Calcium (2S)-2-[[4-[[[(6RS)-2-amino-5-formyl-4-oxo-
Detection : spectrophotometer at 220 nm. 1,4,5,6,7,8-hexahydropteridin-6-yl]methyl]amino]-
Injection : 10 μL. benzoyl]amino]pentanedioate.
Run time : 2.5 times the retention time of dobesilate. Content :
Relative retention with reference to dobesilate (retention – calcium folinate (C20H21CaN7O7) : 97.0 per cent to 102.0 per
time = about 6 min) : impurity A = about 1.7. cent (anhydrous substance) ;
System suitability : reference solution (b) : – calcium (Ca ; Ar 40.08) : 7.54 per cent to 8.14 per cent
(anhydrous substance).
– resolution : minimum 8.0 between the peaks due to
dobesilate and impurity A. It contains a variable quantity of water.
Limits : CHARACTERS
– correction factor : for the calculation of content, multiply Appearance : white or light yellow, amorphous or crystalline,
the peak area of impurity A by 0.6 ; hygroscopic powder.
– impurity A : not more than the area of the principal peak Solubility : sparingly soluble in water, practically insoluble in
in the chromatogram obtained with reference solution (a) acetone and in ethanol (96 per cent).
(0.1 per cent) ; The amorphous form may produce supersaturated solutions
– unspecified impurities : for each impurity, not more than the in water.
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ; IDENTIFICATION
– total : not more than twice the area of the principal peak First identification : A, B, D.
in the chromatogram obtained with reference solution (a) Second identification : A, C, D.
(0.2 per cent) ; A. Specific optical rotation (see Tests).
– disregard limit : 0.5 times the area of the principal peak in B. Infrared absorption spectrophotometry (2.2.24).
the chromatogram obtained with reference solution (a) Preparation : discs.
(0.05 per cent).
Comparison: calcium folinate CRS.
Heavy metals (2.4.8) : maximum 15 ppm.
If the spectra obtained show differences, dissolve the
1.0 g complies with test C. Prepare the reference solution using substance to be examined and the reference substance
1.5 mL of lead standard solution (10 ppm Pb) R. separately in the minimum volume of water R and add
Iron (2.4.9) : maximum 10 ppm, determined on 10 mL of dropwise sufficient acetone R to produce a precipitate.
solution S. Allow to stand for 15 min, collect the precipitate by
centrifugation, wash the precipitate with 2 small quantities
Water (2.5.12) : 4.0 per cent to 6.0 per cent, determined on
of acetone R and dry. Record new spectra using the
0.500 g.
residues.
ASSAY C. Thin-layer chromatography (2.2.27).
Dissolve 0.200 g in a mixture of 10 mL of water R and 40 mL Test solution. Dissolve 15 mg of the substance to be
of dilute sulfuric acid R. Titrate with 0.1 M cerium sulfate, examined in a 3 per cent V/V solution of ammonia R and
determining the end-point potentiometrically (2.2.20). dilute to 5 mL with the same solvent.

1734 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcium folinate

Reference solution. Dissolve 15 mg of calcium folinate CRS Related substances. Liquid chromatography (2.2.29).
in a 3 per cent V/V solution of ammonia R and dilute to Test solution. Dissolve 10.0 mg of the substance to be
5 mL with the same solvent. examined in water R and dilute to 10.0 mL with the same
Plate : cellulose for chromatography F254 R as the coating solvent.
substance. Reference solution (a). Dissolve 10.0 mg of calcium folinate CRS
Mobile phase : the lower layer of a mixture of 1 volume of in water R and dilute to 10.0 mL with the same solvent.
isoamyl alcohol R and 10 volumes of a 50 g/L solution of Reference solution (b). Dilute 1.0 mL of reference solution (a)
citric acid R previously adjusted to pH 8 with ammonia R. to 100.0 mL with water R.
Application : 5 μL. Reference solution (c). Dissolve 10.0 mg of formylfolic acid CRS
Development : over a path of 15 cm. (impurity D) in the mobile phase and dilute to 100.0 mL with
the mobile phase. Dilute 1.0 mL of this solution to 10.0 mL
Drying : in air. with water R.
Detection : examine in ultraviolet light at 254 nm. Reference solution (d). Dilute 1.0 mL of reference solution (b)
Results : the principal spot in the chromatogram obtained to 10.0 mL with water R.
Reference solution (e). Dilute 5.0 mL of reference solution (c)
with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the to 10.0 mL with reference solution (b).
reference solution. Column :
D. It gives reaction (b) of calcium (2.3.1). – size : l = 0.25 m, Ø = 4 mm ;
Carry out the tests and the assay as rapidly as possible, protected
– stationary phase : octadecylsilyl silica gel for
from actinic light. chromatography R (5 μm) ;
– temperature : 40 °C.
TESTS
Mobile phase : mix 220 mL of methanol R and 780 mL
Solution S. Dissolve 1.25 g in carbon dioxide-free water R, of a solution containing 2.0 mL of tetrabutylammonium
heating at 40 °C if necessary, and dilute to 50.0 mL with the hydroxide solution (400 g/L) R and 2.2 g of disodium hydrogen
same solvent. phosphate R, previously adjusted to pH 7.8 with phosphoric
Appearance of solution. Solution S is clear (2.2.1) and its acid R.
absorbance (2.2.25) at 420 nm is not greater than 0.60. Use Flow rate : 1 mL/min.
water R as the compensation liquid. Detection : spectrophotometer at 280 nm.
pH (2.2.3): 6.8 to 8.0 for solution S. Injection : 10 μL of the test solution and reference solutions (b),
Specific optical rotation (2.2.7) : + 14.4 to + 18.0 (anhydrous (c), (d) and (e).
substance), determined on solution S. Run time : 2.5 times the retention time of folinate.
Acetone, ethanol and methanol. Head-space gas System suitability : reference solution (e) :
chromatography (2.2.28) : use the standard additions method. – resolution : minimum 2.2 between the peaks due to folinate
Test solution. Dissolve 0.25 g of the substance to be examined and impurity D.
in water R and dilute to 10.0 mL with the same solvent. Limits :
Reference solution. Dilute 0.125 g of acetone R, 0.750 g of – impurity D : not more than the area of the principal peak
anhydrous ethanol R and 0.125 g of methanol R in water R and in the chromatogram obtained with reference solution (c)
dilute to 1000.0 mL with water R. (1 per cent) ;
Column : – impurities A, B, C, E, F, G : for each impurity, not more
than the area of the principal peak in the chromatogram
– material : fused silica ; obtained with reference solution (b) (1 per cent) ;
– size : l = 10 m, Ø = 0.32 mm ; – sum of impurities other than D : not more than 2.5 times the
– stationary phase : styrene-divinylbenzene copolymer R. area of the principal peak in the chromatogram obtained
Carrier gas : nitrogen for chromatography R. with reference solution (b) (2.5 per cent) ;
– disregard limit : the area of the principal peak in the
Flow rate : 4 mL/min. chromatogram obtained with reference solution (d) (0.1 per
Static head-space conditions that may be used : cent).
– equilibration temperature : 80 °C ; Chlorides : maximum 0.5 per cent.
– equilibration time : 20 min ; Dissolve 0.300 g in 50 mL of water R heating at 40 °C
– pressurisation time : 30 s. if necessary. Add 10 mL of 2 M nitric acid and titrate
with 0.005 M silver nitrate determining the end-point
Temperature :
potentiometrically (2.2.20).
Time Temperature 1 mL of 0.005 M silver nitrate is equivalent to 0.177 mg of Cl.
(min) (°C)
Heavy metals (2.4.8) : maximum 50 ppm.
Column 0-6 125 → 185
1.0 g complies with test F. Prepare the reference solution using
6 - 15 185 5 mL of lead standard solution (10 ppm Pb) R.
Injection port 250 Platinum : maximum 20 ppm.
Detector 250 Atomic absorption spectrometry (2.2.23, Method II).
Test solution. Dissolve 1.00 g in water R and dilute to 100.0 mL
Detection : flame ionisation. with the same solvent.
Injection : at least 3 times. Reference solutions. Prepare the reference solutions using
platinum standard solution (30 ppm Pt) R, diluted as necessary
Limits :
with a mixture of 1 volume of nitric acid R and 99 volumes
– acetone : maximum 0.5 per cent ; of water R.
– ethanol : maximum 3.0 per cent ; Source : platinum hollow-cathode lamp.
– methanol : maximum 0.5 per cent. Wavelength : 265.9 nm.

General Notices (1) apply to all monographs and other texts 1735
Calcium glucoheptonate EUROPEAN PHARMACOPOEIA 8.0

Water (2.5.12) : maximum 17.0 per cent.

Dissolve 0.100 g in a mixture of 50 mL of the titration solvent
and 15 mL of formamide R. Stir for about 6 min before titrating
and use a suitable titrant that does not contain pyridine.
Bacterial endotoxins (2.6.14) : less than 0.5 IU/mg, if intended
for use in the manufacture of parenteral preparations without E. 4-[[[(6RS)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8-
a further appropriate procedure for the removal of bacterial hexahydropteridin-6-yl]methyl]amino]benzoic acid
endotoxins. (5-formyltetrahydropteroic acid),
ASSAY
Calcium. Dissolve 0.400 g in 150 mL of water R and dilute to
300 mL with the same solvent. Carry out the complexometric
titration of calcium (2.5.11).
1 mL of 0.1 M sodium edetate is equivalent to 4.008 mg of Ca.
Calcium folinate. Liquid chromatography (2.2.29) as
described in the test for related substances with the following F. R = CHO : (2S)-2-[[4-[[(2-amino-4-oxo-1,4,7,8-tetra-
modifications. hydropteridin-6-yl)methyl]formylamino]benzoyl]-
amino]pentanedioic acid (10-formyldihydrofolic acid),
Injection : test solution and reference solution (a).
System suitability : G. R = H : (2S)-2-[[4-[[(2-amino-4-oxo-1,4,7,8-tetrahydro-
pteridin-6-yl)methyl]amino]benzoyl]amino]pentanedioic
– repeatability : maximum relative standard deviation of acid (dihydrofolic acid).
2.0 per cent after 6 injections of reference solution (a).
Calculate the percentage content of C20H21CaN7O7 from the
01/2008:1399
declared content of calcium folinate CRS. corrected 6.8
STORAGE
In an airtight container, protected from light. If the substance CALCIUM GLUCOHEPTONATE
is sterile, store in a sterile, airtight, tamper-proof container.
Calcii glucoheptonas
IMPURITIES
Specified impurities : A, B, C, D, E, F, G.

A. (2S)-2[(4-aminobenzoyl)amino]pentanedioic acid, C14H26CaO16 Mr 490.4

DEFINITION
Mixture in variable proportions, of calcium di(D-glycero-D-
gulo-heptonate) and calcium di(D-glycero-D-ido-heptonate).
Content : 98.0 per cent to 102.0 per cent of calcium
2,3,4,5,6,7-hexahydroxyheptanoate (dried substance).
CHARACTERS
B. (2S)-2-[[4-[[[(6RS)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8- Appearance : white or very slightly yellow, amorphous powder,
hexahydropteridin-6-yl]methyl]formylamino]benzoyl]- hygroscopic.
amino]pentanedioic acid (5,10-diformyltetrahydrofolic Solubility : very soluble in water, practically insoluble in
acid), acetone and in ethanol (96 per cent).
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 20 mg of the substance to be
examined in 1 mL of water R.
Reference solution (a). Dissolve 20 mg of calcium
glucoheptonate CRS in 1 mL of water R.
Reference solution (b). Dissolve 10 mg of calcium
C. (2S)-2-[[4-[[(2-amino-4-oxo-1,4-dihydropteridin-6-
gluconate CRS in 0.5 mL of the test solution and dilute to
yl)methyl]amino]benzoyl]amino]pentanedioic acid (folic
1 mL with water R.
acid),
Plate : cellulose for chromatography R1 as the coating
substance.
Mobile phase : anhydrous formic acid R, water R, acetone R,
butanol R (20:20:30:30 V/V/V/V) ; use a freshly prepared
mixture.
Application : 10 μL as bands of 20 mm by 2 mm.
Development : in a tank previously allowed to saturate for
D. (2S)-2-[[4-[[(2-amino-4-oxo-1,4-dihydropteridin-6- 10 min, over a path of 12 cm.
yl)methyl]formylamino]benzoyl]amino]pentanedioic acid Drying : in air.
(10-formylfolic acid), Detection : spray with 0.02 M potassium permanganate.

1736 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcium gluconate

System suitability: reference solution (b) : STORAGE

– the chromatogram shows 2 clearly separated spots. In an airtight container. If the substance is sterile, store in a
Results : the principal spot in the chromatogram obtained sterile, airtight, tamper-proof container.
with the test solution is similar in position and size to
the principal spot in the chromatogram obtained with 01/2013:0172
reference solution (a).
B. 0.2 mL of solution S (see Tests) gives reaction (b) of calcium
(2.3.1).
CALCIUM GLUCONATE
TESTS Calcii gluconas
Solution S. Dissolve 10.0 g in carbon dioxide-free water R
prepared from distilled water R and dilute to 100 mL with the
same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y6 (2.2.2,
Method II).
C12H22CaO14,H2O Mr 448.4
pH (2.2.3): 6.0 to 8.0 for solution S. [18016-24-5]
Reducing sugars : maximum 1 per cent, expressed as glucose.
Dissolve 1.0 g in 5 mL of water R with the aid of gentle heat. DEFINITION
Cool and add 20 mL of cupri-citric solution R and a few glass Calcium bis[(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoate]
beads. Heat so that boiling begins after 4 min and maintain monohydrate (calcium di(D-gluconate) monohydrate).
boiling for 3 min. Cool rapidly and add 100 mL of a 2.4 per Content : 98.5 per cent to 102.0 per cent of C12H22CaO14,H2O.
cent V/V solution of glacial acetic acid R and 20.0 mL of
0.025 M iodine. With continuous shaking, add 25 mL of a CHARACTERS
mixture of 6 volumes of hydrochloric acid R and 94 volumes Appearance : white or almost white, crystalline or granular
of water R until the precipitate dissolves, titrate the excess of powder.
iodine with 0.05 M sodium thiosulfate using 1 mL of starch Solubility : sparingly soluble in water, freely soluble in boiling
solution R added towards the end of the titration, as indicator. water.
Not less than 12.6 mL of 0.05 M sodium thiosulfate is required.
Cyanide. Dissolve 5.0 g in 50 mL of water R and add 2.0 g of IDENTIFICATION
tartaric acid R. Place this solution in a distillation apparatus A. Thin-layer chromatography (2.2.27).
(2.2.11). The plain bend adapter attached to the end of the Test solution. Dissolve 20 mg of the substance to be
condenser has a vertical part that is long enough to extend to examined in 1 mL of water R, heating if necessary in a
1 cm from the bottom of a 50 mL test-tube used as a receiver. water-bath at 60 °C.
Place 10 mL of water R and 2 mL of 0.1 M sodium hydroxide Reference solution. Dissolve 20 mg of calcium gluconate CRS
into the receiver. Distil, collect 25 mL of distillate and dilute in 1 mL of water R, heating if necessary in a water-bath
to 50 mL with water R. To 25 mL of this solution add 25 mg at 60 °C.
of ferrous sulfate R and boil for a short time. After cooling to
Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel
about 70 °C add 10 mL of hydrochloric acid R1. After 30 min,
plate R (2-10 μm)].
filter the solution and wash the filter. A yellow spot appears
on the filter ; there is no blue or green spot. Mobile phase : concentrated ammonia R, ethyl acetate R,
water R, ethanol (96 per cent) R (10:10:30:50 V/V/V/V).
Chlorides (2.4.4): maximum 100 ppm.
Application : 1 μL.
To 5 mL of solution S, add 10 mL of water R.
Development : over 2/3 of the plate.
Sulfates (2.4.13) : maximum 100 ppm, determined on
Drying : at 100 °C for 20 min ; allow to cool.
solution S.
Detection : spray with a solution containing 10 g/L of cerium
Iron (2.4.9) : maximum 40 ppm. sulfate R and 25 g/L of ammonium molybdate R in dilute
Dilute 2.5 mL of solution S to 10 mL with water R. sulfuric acid R and heat at 105 °C for about 10 min.
Heavy metals (2.4.8) : maximum 10 ppm. Results : after 5 min, the principal spot in the chromatogram
Dissolve 2.0 g in 10 mL of buffer solution pH 3.5 R and dilute obtained with the test solution is similar in position,
to 20 mL with water R. 12 mL of the solution complies with colour and size to the principal spot in the chromatogram
test A. Prepare the reference solution using lead standard obtained with the reference solution.
solution (1 ppm Pb) R. B. Solution S (see Tests) gives the reactions of calcium (2.3.1).
Loss on drying (2.2.32) : maximum 5.0 per cent, determined TESTS
on 1.000 g by drying in an oven at 105 °C for 3 h.
Solution S. Dissolve 1.0 g in water R heated to 60 °C and
Bacterial endotoxins (2.6.14) : less than 167 IU/g, if intendeddilute to 50 mL with the same solvent.
for use in the manufacture of parenteral preparations without
a further appropriate procedure for the removal of bacterial Appearance of solution. At 60 °C, solution S is not
endotoxins. more intensely coloured than reference solution Y6 (2.2.2,
Method II). After cooling, it is not more opalescent than
ASSAY reference suspension II (2.2.1).
Dissolve 0.800 g in a mixture of 2 mL of 3 M hydrochloric acid Organic impurities and boric acid. Introduce 0.5 g into a
and 150 mL of water R. While stirring, add 12.5 mL of 0.1 M porcelain dish previously rinsed with sulfuric acid R and placed
sodium edetate, 15 mL of 1 M sodium hydroxide and 0.3 g in a bath of iced water. Add 2 mL of cooled sulfuric acid R
of hydroxynaphthol blue, sodium salt R. Titrate with 0.1 M and mix. No yellow or brown colour develops. Add 1 mL of
sodium edetate until the colour changes from violet to pure chromotrope II B solution R. A violet colour develops and does
blue. not become dark blue. The solution is not more intensely
1 mL of 0.1 M sodium edetate is equivalent to 49.04 mg coloured than that of a mixture of 1 mL of chromotrope II B
of C14H26CaO16. solution R and 2 mL of cooled sulfuric acid R.

General Notices (1) apply to all monographs and other texts 1737
Calcium gluconate, anhydrous EUROPEAN PHARMACOPOEIA 8.0

Sucrose and reducing sugars. Dissolve 0.5 g in a mixture of Reference solution. Dissolve 20 mg of calcium gluconate CRS
2 mL of hydrochloric acid R1 and 10 mL of water R. Boil for in 1 mL of water R, heating if necessary in a water-bath
5 min, allow to cool, add 10 mL of sodium carbonate solution R at 60 °C.
and allow to stand. Dilute to 25 mL with water R and filter. To Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel
5 mL of the filtrate add 2 mL of cupri-tartaric solution R and plate R (2-10 μm)].
boil for 1 min. Allow to stand for 2 min. No red precipitate Mobile phase : concentrated ammonia R, ethyl acetate R,
is formed. water R, ethanol (96 per cent) R (10:10:30:50 V/V/V/V).
Chlorides (2.4.4) : maximum 200 ppm. Application : 1 μL.
Dilute 12.5 mL of solution S to 15 mL with water R. Development : over 2/3 of the plate.
Sulfates (2.4.13) : maximum 100 ppm. Drying : at 100 °C for 20 min, then allow to cool.
Dissolve 10.0 g with heating in a mixture of 10 mL of acetic Detection : spray with a solution containing 25 g/L of
acid R and 90 mL of distilled water R. ammonium molybdate R and 10 g/L of cerium sulfate R
in dilute sulfuric acid R, and heat at 100-105 °C for about
Magnesium and alkali metals : maximum 0.4 per cent. 10 min.
Dissolve 1.00 g in 100 mL of boiling water R, add 10 mL of Results : the principal spot in the chromatogram obtained
ammonium chloride solution R, 1 mL of ammonia R and, with the test solution is similar in position, colour and size
dropwise, 50 mL of hot ammonium oxalate solution R. Allow to the principal spot in the chromatogram obtained with
to stand for 4 h, dilute to 200 mL with water R and filter. the reference solution.
Evaporate 100 mL of the filtrate to dryness and ignite. The
residue weighs a maximum of 2 mg. B. Solution S (see Tests) gives the reactions of calcium (2.3.1).
C. Loss on drying (see Tests).
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test D. Heat the substance to be examined TESTS
gradually and with care until it is almost completely Solution S. Dissolve 1.0 g in water R heated to 60 °C and
transformed into a white mass and then ignite. Prepare dilute to 50 mL with the same solvent.
the reference solution using 2 mL of lead standard solution Appearance of solution. At 60 °C, solution S is not
(10 ppm Pb) R. more intensely coloured than reference solution Y6 (2.2.2,
Microbial contamination Method II). After cooling, it is not more opalescent than
TAMC : acceptance criterion 103 CFU/g (2.6.12). reference suspension II (2.2.1).
TYMC : acceptance criterion 102 CFU/g (2.6.12). Organic impurities and boric acid. Place 0.5 g in a porcelain
dish previously rinsed with sulfuric acid R and placed in a bath
ASSAY of iced water. Add 2 mL of cooled sulfuric acid R and mix. No
Dissolve 0.8000 g in 20 mL of hot water R, allow to cool and yellow or brown colour develops. Add 1 mL of chromotrope II
dilute to 300 mL with water R. Carry out the complexometric B solution R. A violet colour develops and does not become
titration of calcium (2.5.11). dark blue. Compare the colour obtained with that of a mixture
of 1 mL of chromotrope II B solution R and 2 mL of cooled
1 mL of 0.1 M sodium edetate is equivalent to 44.84 mg sulfuric acid R.
of C12H22CaO14,H2O.
Sucrose and reducing sugars. Dissolve 0.5 g in a mixture of
2 mL of hydrochloric acid R1 and 10 mL of water R. Boil for
5 min, allow to cool, add 10 mL of sodium carbonate solution R
01/2009:2364 and allow to stand for 10 min. Dilute to 25 mL with water R
and filter. To 5 mL of the filtrate add 2 mL of cupri-tartaric
solution R and boil for 1 min. Allow to stand for 2 min. No
CALCIUM GLUCONATE, ANHYDROUS red precipitate is formed.
Chlorides (2.4.4) : maximum 200 ppm.
Calcii gluconas anhydricus Dilute 12.5 mL of solution S to 15 mL with water R.
Sulfates (2.4.13) : maximum 100 ppm.
Dissolve 10.0 g with heating in a mixture of 10 mL of acetic
acid R and 90 mL of distilled water R.
Magnesium and alkali metals : maximum 0.4 per cent
(expressed as MgO).
C12H22CaO14 Mr 430.4 Dissolve 1.00 g in 100 mL of boiling water R, add 10 mL of
ammonium chloride solution R, 1 mL of ammonia R and,
DEFINITION dropwise, 50 mL of hot ammonium oxalate solution R. Allow
to stand for 4 h, dilute to 200 mL with water R and filter.
Anhydrous calcium D-gluconate. Evaporate 100 mL of the filtrate to dryness and ignite. The
Content : 98.0 per cent to 102.0 per cent (dried substance). residue weighs a maximum of 2 mg.
CHARACTERS Heavy metals (2.4.8) : maximum 10 ppm.
Appearance : white or almost white, crystalline or granular 2.0 g complies with test D. Heat the substance to be examined
powder. gradually and with care until it is almost completely
transformed into a white mass, and then ignite. Prepare
Solubility : sparingly soluble in water, freely soluble in boiling the reference solution using 2 mL of lead standard solution
water. (10 ppm Pb) R.
IDENTIFICATION Loss on drying (2.2.32) : maximum 2.0 per cent, determined
A. Thin-layer chromatography (2.2.27). on 1.000 g by drying in an oven at 105 °C for 16 h.
Test solution. Dissolve 20 mg of the substance to be Microbial contamination
examined in 1 mL of water R, heating if necessary in a TAMC : acceptance criterion 103 CFU/g (2.6.12).
water-bath at 60 °C. TYMC : acceptance criterion 102 CFU/g (2.6.12).

1738 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcium gluconate for injection

ASSAY Organic impurities and boric acid. Introduce 0.5 g into a

Dissolve 0.350 g in 20 mL of hot water R, allow to cool and porcelain dish previously rinsed with sulfuric acid R and placed
dilute to 300 mL with water R. Carry out the complexometric in a bath of iced water. Add 2 mL of cooled sulfuric acid R
titration of calcium (2.5.11). and mix. No yellow or brown colour develops. Add 1 mL of
1 mL of 0.1 M sodium edetate is equivalent to 43.04 mg chromotrope II B solution R. A violet colour develops and does
of C12H22CaO14. not become dark blue. The solution is not more intensely
coloured than that of a mixture of 1 mL of chromotrope II B
solution R and 2 mL of cooled sulfuric acid R.
01/2013:0979
Oxalates. Liquid chromatography (2.2.29).
CALCIUM GLUCONATE FOR Test solution. Dissolve 1.00 g of the substance to be examined
in water for chromatography R and dilute to 100.0 mL with
INJECTION the same solvent.
Reference solution. Dissolve 1.00 g of the substance to
Calcii gluconas ad iniectabile be examined in water for chromatography R, add 0.5 mL
of a 0.152 g/L solution of sodium oxalate R in water for
chromatography R and dilute to 100.0 mL with the same
solvent.
Precolumn :
– size : l = 30 mm, Ø = 4 mm ;
– stationary phase : suitable strong anion-exchange resin
C12H22CaO14,H2O Mr 448.4 (30-50 μm).
[18016-24-5]
Columns 1 and 2 :
DEFINITION – size : l = 0.25 m, Ø = 4 mm ;
Calcium bis[(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoate] – stationary phase : suitable strong anion-exchange resin
monohydrate (calcium di(D-gluconate) monohydrate). (30-50 μm).
Content : 99.0 per cent to 101.0 per cent of C12H22CaO14,H2O. Anion-suppresser column : connected in series with the
precolumn and analytical columns and equipped with a
CHARACTERS micromembrane that separates the mobile phase from the
Appearance : white or almost white, crystalline or granular suppressor regeneration solution, flowing countercurrent to
powder. the mobile phase.
Solubility : sparingly soluble in water, freely soluble in boiling Mobile phase : dissolve 0.212 g of anhydrous sodium
water. carbonate R and 63 mg of sodium hydrogen carbonate R in
IDENTIFICATION water for chromatography R and dilute to 1000.0 mL with the
same solvent.
A. Thin-layer chromatography (2.2.27).
Flow rate of the mobile phase : 2 mL/min.
Test solution. Dissolve 20 mg of the substance to be
examined in 1 mL of water R, heating if necessary in a Suppressor regeneration solution : 1.23 g/L solution of sulfuric
water-bath at 60 °C. acid R in water for chromatography R.
Reference solution. Dissolve 20 mg of calcium gluconate CRS Flow rate of the suppressor regeneration solution : 4 mL/min.
in 1 mL of water R, heating if necessary in a water-bath Detection : conductance.
at 60 °C. Injection : 50 μL.
Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel System suitability : reference solution :
plate R (2-10 μm)]. – repeatability : maximum relative standard deviation of
Mobile phase : concentrated ammonia R, ethyl acetate R, 2.0 per cent for the area of the peak due to oxalate after
water R, ethanol (96 per cent) R (10:10:30:50 V/V/V/V). 5 injections.
Application : 1 μL. Inject 50 μL of each solution 3 times. Calculate the content of
Development : over 2/3 of the plate. oxalates in parts per million using the following expression :
Drying : at 100 °C for 20 min ; allow to cool.
Detection : spray with a solution containing 10 g/L of cerium
sulfate R and 25 g/L of ammonium molybdate R in dilute
sulfuric acid R and heat at 105 °C for about 10 min. ST = area of the peak due to oxalate in the chromatogram
Results : after 5 min, the principal spot in the chromatogram obtained with the test solution ;
obtained with the test solution is similar in position, SR = area of the peak due to oxalate in the chromatogram
colour and size to the principal spot in the chromatogram obtained with the reference solution.
obtained with the reference solution.
Limit :
B. About 20 mg gives reaction (b) of calcium (2.3.1).
– oxalates : maximum 100 ppm.
TESTS Sucrose and reducing sugars. Dissolve 0.5 g in a mixture of
Solution S. To 10.0 g add 90 mL of boiling distilled water R 2 mL of hydrochloric acid R1 and 10 mL of water R. Boil for
and boil with stirring, for not more than 10 s, until completely 5 min, allow to cool, add 10 mL of sodium carbonate solution R
dissolved, then dilute to 100.0 mL with the same solvent. and allow to stand for 10 min. Dilute to 25 mL with water R
and filter. To 5 mL of the filtrate add 2 mL of cupri-tartaric
Appearance of solution. At 60 °C, solution S is not solution R and boil for 1 min. Allow to stand for 2 min. No
more intensely coloured than reference solution B7 (2.2.2, red precipitate is formed.
Method II). After cooling to 20 °C, it is not more opalescent
than reference suspension II (2.2.1). Chlorides (2.4.4) : maximum 50 ppm.
pH (2.2.3) : 6.4 to 8.3. To 10 mL of previously filtered solution S add 5 mL of water R.
Dissolve 1.0 g in 20 mL of carbon dioxide-free water R, heating Phosphates (2.4.11) : maximum 100 ppm.
on a water-bath. Dilute 1 mL of solution S to 100 mL with water R.

General Notices (1) apply to all monographs and other texts 1739
Calcium glycerophosphate EUROPEAN PHARMACOPOEIA 8.0

Sulfates (2.4.13) : maximum 50 ppm, determined on IDENTIFICATION

previously filtered solution S. A. Mix 1 g with 1 g of potassium hydrogen sulfate R in
Prepare the standard using a mixture of 7.5 mL of sulfate a test tube fitted with a glass tube. Heat strongly and
standard solution (10 ppm SO4) R and 7.5 mL of distilled direct the white vapour towards a piece of filter paper
water R. impregnated with a freshly prepared 10 g/L solution of
Iron : maximum 5 ppm. sodium nitroprusside R. The filter paper develops a blue
colour in contact with piperidine R.
Atomic absorption spectrometry (2.2.23, Method I).
B. Ignite 0.1 g in a crucible. Take up the residue with 5 mL of
Test solution. Introduce 2.0 g into a 100 mL nitric acid R and heat on a water-bath for 1 min. Filter. The
polytetrafluoroethylene beaker and add 5 mL of nitric filtrate gives reaction (b) of phosphates (2.3.1).
acid R. Boil, evaporating almost to dryness. Add 1 mL of
strong hydrogen peroxide solution R and evaporate again C. It gives reaction (b) of calcium (2.3.1).
almost to dryness. Repeat the hydrogen peroxide treatment TESTS
until a clear solution is obtained. Using 2 mL of nitric acid R,
transfer the solution into a 25 mL volumetric flask. Dilute to Solution S. Dissolve 1.5 g at room temperature in carbon
25.0 mL with dilute hydrochloric acid R. In the same manner, dioxide-free water R prepared from distilled water R and dilute
prepare a compensation solution using 0.65 g of calcium to 150 mL with the same solvent.
chloride R1 instead of the substance to be examined. Appearance of solution. Solution S is not more opalescent
Reference solutions. Prepare the reference solutions from than reference suspension III (2.2.1).
iron standard solution (20 ppm Fe) R, diluting with dilute Acidity or alkalinity. To 100 mL of solution S add 0.1 mL of
hydrochloric acid R. phenolphthalein solution R. Not more than 1.5 mL of 0.1 M
Source : iron hollow-cathode lamp. hydrochloric acid or 0.5 mL of 0.1 M sodium hydroxide is
Wavelength : 248.3 nm. required to change the colour of the indicator.
Atomisation device : air-acetylene flame. Citric acid. Shake 5.0 g with 20 mL of carbon dioxide-free
water R and filter. To the filtrate add 0.15 mL of sulfuric acid R
Carry out a basic correction using a deuterium lamp. and filter again. To the filtrate add 5 mL of mercuric sulfate
Magnesium and alkali metals : maximum 0.4 per cent. solution R and heat to boiling. Add 0.5 mL of a 3.2 g/L solution
To 0.50 g add a mixture of 1.0 mL of dilute acetic acid R and of potassium permanganate R and again heat to boiling. No
10.0 mL of water R and rapidly boil, whilst shaking, until precipitate is formed.
completely dissolved. To the boiling solution add 5.0 mL of Glycerol and ethanol (96 per cent)-soluble substances :
ammonium oxalate solution R and allow to stand for at least maximum 0.5 per cent.
6 h. Filter through a sintered-glass filter (1.6) (2.1.2) into aShake 1.000 g with 25 mL of ethanol (96 per cent) R for 1 min.
porcelain crucible. Carefully evaporate the filtrate to dryness Filter. Evaporate the filtrate on a water-bath and dry the
and ignite. The residue weighs not more than 2 mg. residue at 70 °C for 1 h. The residue weighs a maximum of
Heavy metals (2.4.8) : maximum 10 ppm. 5 mg.
12 mL of solution S complies with test A. Prepare the referenceChlorides (2.4.4) : maximum 500 ppm.
solution using lead standard solution (1 ppm Pb) R. Dissolve 0.1 g in a mixture of 2 mL of acetic acid R and 8 mL
Bacterial endotoxins (2.6.14) : less than 167 IU/g. of water R and dilute to 15 mL with water R.
Microbial contamination Phosphates (2.4.11) : maximum 400 ppm.
TAMC : acceptance criterion 102 CFU/g (2.6.12). Dilute 2.5 mL of solution S to 100 mL with water R.
ASSAY Sulfates (2.4.13) : maximum 0.1 per cent, determined on
solution S.
Dissolve 0.350 g in 20 mL of hot water R, allow to cool and
dilute to 300 mL with water R. Carry out the complexometric Arsenic (2.4.2, Method A) : maximum 3 ppm.
titration of calcium (2.5.11). Use 50 mg of calconecarboxylic Dissolve 0.33 g in water R and dilute to 25 mL with the same
acid triturate R. solvent.
1 mL of 0.1 M sodium edetate is equivalent to 44.84 mg Iron (2.4.9) : maximum 50 ppm, detemined on 0.20 g.
of C12H22CaO14,H2O. Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 2.0 g in 10 mL of buffer solution pH 3.5 R and dilute
01/2008:0980 to 20 mL with water R. 12 mL of the solution complies with
corrected 6.0 test A. Prepare the reference solution using lead standard
solution (2 ppm Pb) R.
CALCIUM GLYCEROPHOSPHATE Loss on drying (2.2.32) : maximum 12.0 per cent, determined
on 1.000 g by drying in an oven at 150 °C for 4 h.
Calcii glycerophosphas ASSAY
Dissolve 0.200 g in water R. Carry out the complexometric
C3H7CaO6P Mr 210.1 titration of calcium (2.5.11).
DEFINITION 1 mL of 0.1 M sodium edetate is equivalent to 4.008 mg of Ca.
Mixture in variable proportions of the calcium
salt of (RS)-2,3-dihydroxypropyl phosphate and of 01/2013:0981
2-hydroxy-1-(hydroxymethyl)ethyl phosphate which may be
hydrated. CALCIUM HYDROGEN PHOSPHATE,
Content : 18.6 per cent to 19.4 per cent of Ca (dried substance). ANHYDROUS
CHARACTERS
Calcii hydrogenophosphas anhydricus
Appearance : white or almost white powder, hygroscopic.
Solubility : sparingly soluble in water, practically insoluble in CaHPO4 Mr 136.1
ethanol (96 per cent). [7757-93-9]

1740 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcium hydrogen phosphate, anhydrous

DEFINITION Carry out the measurement on 20.0 mL of the test solution.

Content : 98.0 per cent to 103.0 per cent. Add at least 3 times 0.10 mL of the reference solution and
carry out the measurement after each addition. Calculate the
concentration of fluorides using the calibration curve.
CHARACTERS
Sulfates : maximum 0.5 per cent.
Appearance : white or almost white, crystalline powder, or
colourless crystals. Test solution. Dissolve 0.5 g in a mixture of 5 mL of water R
and 5 mL of dilute hydrochloric acid R and dilute to 100 mL
Solubility : practically insoluble in water and in ethanol (96 per with water R. Filter if necessary. To 20 mL of this solution,
cent). It dissolves in dilute hydrochloric acid and in dilute add 1 mL of dilute hydrochloric acid R and dilute to 50 mL
nitric acid. with water R.
Reference solution. To 1.0 mL of 0.005 M sulfuric acid, add
IDENTIFICATION 1 mL of dilute hydrochloric acid R and dilute to 50 mL with
A. Dissolve with heating 0.1 g in 10 mL of dilute hydrochloric water R. Filter if necessary.
acid R. Add 2.5 mL of dilute ammonia R1, shake, and add To the test solution and to the reference solution, add 2 mL
5 mL of a 35 g/L solution of ammonium oxalate R. A white of a 120 g/L solution of barium chloride R and allow to stand
precipitate is produced. for 10 min. Any opalescence in the test solution is not more
B. Dissolve 0.1 g in 5 mL of dilute nitric acid R, add 2 mL intense than that in the reference solution.
of ammonium molybdate solution R and heat at 70 °C for Arsenic (2.4.2, Method A): maximum 10 ppm, determined
2 min. A yellow precipitate is produced. on 2 mL of solution S.
C. It complies with the limits of the assay. Barium. To 0.5 g, add 10 mL of water R and heat to boiling.
While stirring, add 1 mL of hydrochloric acid R dropwise.
TESTS Allow to cool and filter if necessary. Add 2 mL of a 10 g/L
solution of dipotassium sulfate R and allow to stand for 10 min.
Solution S. Dissolve 2.5 g in 20 mL of dilute hydrochloric No turbidity is produced.
acid R, filter if necessary and add dilute ammonia R1 until a
precipitate is formed. Add just sufficient dilute hydrochloric Iron (2.4.9) : maximum 400 ppm.
acid R to dissolve the precipitate and dilute to 50 mL with Dilute 0.5 mL of solution S to 10 mL with water R.
distilled water R. Heavy metals (2.4.8) : maximum 40 ppm.
Acid-insoluble substances : maximum 0.2 per cent. Dilute 10 mL of solution S to 20 mL with water R. 12 mL of the
Dissolve 5.0 g in 40 mL of water R, add 10 mL of hydrochloric solution complies with test A. Prepare the reference solution
acid R and heat to boiling for 5 min. Cool, then collect the using lead standard solution (1 ppm Pb) R.
insoluble substances using ashless filter paper. Wash with Loss on ignition : 6.6 per cent to 8.5 per cent, determined on
water R until turbidity is no longer produced when silver 1.000 g to constant mass at 800-825 °C.
nitrate solution R2 is added. Ignite at 600 ± 50 °C. The residue
weighs not more than 10 mg. ASSAY
Carbonates. Shake 0.5 g with 5 mL of carbon dioxide-free Dissolve 0.4 g in 12 mL of dilute hydrochloric acid R by heating
water R and add 1 mL of hydrochloric acid R. No effervescence on a water bath if necessary and dilute to 200 mL with water R.
is produced. To 20.0 mL of this solution add 25.0 mL of 0.02 M sodium
Chlorides : maximum 0.25 per cent. edetate, 50 mL of water R, 5 mL of ammonium chloride buffer
solution pH 10.7 R and about 25 mg of mordant black 11
Test solution. Dissolve 0.20 g in a mixture of 20 mL of water R triturate R. Titrate the excess of sodium edetate with 0.02 M
and 13 mL of dilute nitric acid R by warming if necessary, zinc sulfate. Carry out a blank titration.
dilute to 100 mL with water R and filter if necessary. Use 1 mL of 0.02 M sodium edetate is equivalent to 2.72 mg
50 mL of this solution. of CaHPO4.
Reference solution. To 0.70 mL of 0.01 M hydrochloric acid, add
6 mL of dilute nitric acid R and dilute to 50 mL with water R. FUNCTIONALITY-RELATED CHARACTERISTICS
Add 1 mL of silver nitrate solution R2 to the test solution and This section provides information on characteristics that are
to the reference solution and mix. After standing for 5 min recognised as being relevant control parameters for one or
protected from light, any opalescence in the test solution is more functions of the substance when used as an excipient
not more intense than that in the reference solution. (see chapter 5.15). Some of the characteristics described in
the Functionality-related characteristics section may also be
Fluorides : maximum 100 ppm. present in the mandatory part of the monograph since they
Potentiometry (2.2.36, Method II). also represent mandatory quality criteria. In such cases, a
cross-reference to the tests described in the mandatory part is
Chelating solution. Dissolve 45 g of cyclohexylenedinitrilotetra- included in the Functionality-related characteristics section.
acetic acid R in 75 mL of sodium hydroxide solution R and Control of the characteristics can contribute to the quality
dilute to 250 mL with water R. of a medicinal product by improving the consistency of the
Test solution. Dissolve 1.000 g in 4 mL of hydrochloric acid R1, manufacturing process and the performance of the medicinal
add 20 mL of chelating solution, 2.7 mL of glacial acetic acid R product during use. Where control methods are cited, they are
and 2.8 g of sodium chloride R, adjust to pH 5-6 with sodium recognised as being suitable for the purpose, but other methods
hydroxide solution R and dilute to 50.0 mL with water R. can also be used. Wherever results for a particular characteristic
are reported, the control method must be indicated.
Reference solution. Dissolve 4.42 g of sodium fluoride R,
previously dried at 300 °C for 12 h, in water R and dilute The following characteristics may be relevant for anhydrous
to 1000.0 mL with the same solvent. Dilute 50.0 mL of this calcium hydrogen phosphate used as filler in tablets and
solution to 500.0 mL with total-ionic-strength-adjustment capsules.
buffer R (200 ppm F). Particle-size distribution (2.9.31 or 2.9.38).
Indicator electrode : fluoride-selective. Bulk and tapped density (2.9.34).
Reference electrode : silver-silver chloride. Powder flow (2.9.36).

General Notices (1) apply to all monographs and other texts 1741
Calcium hydrogen phosphate dihydrate EUROPEAN PHARMACOPOEIA 8.0

01/2013:0116 Reference solution. Dissolve 4.42 g of sodium fluoride R,

previously dried at 300 °C for 12 h, in water R and dilute
CALCIUM HYDROGEN PHOSPHATE to 1000.0 mL with the same solvent. Dilute 50.0 mL of this
solution to 500.0 mL with total-ionic-strength-adjustment
DIHYDRATE buffer R (200 ppm F).
Indicator electrode : fluoride-selective.
Calcii hydrogenophosphas dihydricus Reference electrode : silver-silver chloride.
Carry out the measurement on 20.0 mL of the test solution.
CaHPO4,2H2O Mr 172.1 Add at least 3 times 0.10 mL of the reference solution and
[7789-77-7] carry out the measurement after each addition. Calculate the
concentration of fluorides using the calibration curve.
DEFINITION Sulfates : maximum 0.5 per cent.
Content : 98.0 per cent to 105.0 per cent. Test solution. Dissolve 0.5 g in a mixture of 5 mL of water R
and 5 mL of dilute hydrochloric acid R and dilute to 100 mL
CHARACTERS with water R. Filter if necessary. To 20 mL of this solution,
Appearance : white or almost white, crystalline powder. add 1 mL of dilute hydrochloric acid R and dilute to 50 mL
Solubility : practically insoluble in water and in ethanol (96 per with water R.
cent). It dissolves in dilute hydrochloric acid and in dilute Reference solution. To 1.0 mL of 0.005 M sulfuric acid, add
nitric acid. 1 mL of dilute hydrochloric acid R and dilute to 50 mL with
water R. Filter if necessary.
IDENTIFICATION To the test solution and to the reference solution, add 2 mL
A. Dissolve with heating 0.1 g in 10 mL of dilute hydrochloric of a 120 g/L solution of barium chloride R and allow to stand
acid R. Add 2.5 mL of dilute ammonia R1, shake and add for 10 min. Any opalescence in the test solution is not more
5 mL of a 35 g/L solution of ammonium oxalate R. A white intense than that in the reference solution.
precipitate is produced. Arsenic (2.4.2, Method A): maximum 10 ppm, determined
B. Dissolve 0.1 g in 5 mL of dilute nitric acid R, add 2 mL on 2 mL of solution S.
of ammonium molybdate solution R and heat at 70 °C for Barium. To 0.5 g, add 10 mL of water R and heat to boiling.
2 min. A yellow precipitate is produced. While stirring, add 1 mL of hydrochloric acid R dropwise.
C. It complies with the limits of the assay. Allow to cool and filter if necessary. Add 2 mL of a 10 g/L
solution of dipotassium sulfate R and allow to stand for 10 min.
TESTS No turbidity is produced.
Solution S. Dissolve 2.5 g in 20 mL of dilute hydrochloric Iron (2.4.9) : maximum 400 ppm.
acid R, filter if necessary and add dilute ammonia R1 until a Dilute 0.5 mL of solution S to 10 mL with water R.
precipitate is formed. Add just sufficient dilute hydrochloric
acid R to dissolve the precipitate and dilute to 50 mL with Heavy metals (2.4.8) : maximum 40 ppm.
distilled water R. Dilute 10 mL of solution S to 20 mL with water R. 12 mL of the
Acid-insoluble substances : maximum 0.2 per cent. solution complies with test A. Prepare the reference solution
using lead standard solution (1 ppm Pb) R.
Dissolve 5.0 g in 40 mL of water R, add 10 mL of hydrochloric
acid R and heat to boiling for 5 min. Cool, then collect the Loss on ignition : 24.5 per cent to 26.5 per cent, determined
insoluble substances using ashless filter paper. Wash with on 1.000 g by ignition to constant mass at 800-825 °C.
water R until turbidity is no longer produced when silver
ASSAY
nitrate solution R2 is added to the filtrate. Ignite at 600 ± 50 °C.
The residue weighs not more than 10 mg. Dissolve 0.4 g in 12 mL of dilute hydrochloric acid R by heating
on a water bath if necessary and dilute to 200 mL with water R.
Carbonates. Shake 0.5 g with 5 mL of carbon dioxide-free
To 20.0 mL of this solution add 25.0 mL of 0.02 M sodium
water R and add 1 mL of hydrochloric acid R. No effervescence
edetate, 50 mL of water R, 5 mL of ammonium chloride buffer
is produced.
solution pH 10.7 R and about 25 mg of mordant black 11
Chlorides : maximum 0.25 per cent. triturate R. Titrate the excess of sodium edetate with 0.02 M
Test solution. Dissolve 0.20 g in a mixture of 20 mL of water R zinc sulfate. Carry out a blank titration.
and 13 mL of dilute nitric acid R by warming if necessary, 1 mL of 0.02 M sodium edetate is equivalent to 3.44 mg
dilute to 100 mL with water R and filter if necessary. Use of CaHPO4,2H2O.
50 mL of this solution.
FUNCTIONALITY-RELATED CHARACTERISTICS
Reference solution. To 0.70 mL of 0.01 M hydrochloric acid, add
6 mL of dilute nitric acid R and dilute to 50 mL with water R. This section provides information on characteristics that are
recognised as being relevant control parameters for one or
Add 1 mL of silver nitrate solution R2 to the test solution and more functions of the substance when used as an excipient
to the reference solution and mix. After standing for 5 min (see chapter 5.15). Some of the characteristics described in
protected from light, any opalescence in the test solution is the Functionality-related characteristics section may also be
not more intense than that in the reference solution. present in the mandatory part of the monograph since they
Fluorides : maximum 100 ppm. also represent mandatory quality criteria. In such cases, a
Potentiometry (2.2.36, Method II). cross-reference to the tests described in the mandatory part is
included in the Functionality-related characteristics section.
Chelating solution. Dissolve 45 g of cyclohexylenedinitrilotetra- Control of the characteristics can contribute to the quality
acetic acid R in 75 mL of sodium hydroxide solution R and of a medicinal product by improving the consistency of the
dilute to 250 mL with water R. manufacturing process and the performance of the medicinal
Test solution. Dissolve 1.000 g in 4 mL of hydrochloric acid R1, product during use. Where control methods are cited, they are
add 20 mL of chelating solution, 2.7 mL of glacial acetic acid R recognised as being suitable for the purpose, but other methods
and 2.8 g of sodium chloride R, adjust to pH 5-6 with sodium can also be used. Wherever results for a particular characteristic
hydroxide solution R and dilute to 50.0 mL with water R. are reported, the control method must be indicated.

1742 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcium lactate, anhydrous

The following characteristics may be relevant for calcium Heavy metals (2.4.8) : maximum 20 ppm.
hydrogen phosphate dihydrate used as filler in tablets and Dissolve 1.0 g in 10 mL of hydrochloric acid R1 and evaporate
capsules. to dryness on a water-bath. Dissolve the residue in 20 mL of
Particle-size distribution (2.9.31 or 2.9.38). water R and filter. 12 mL of the filtrate complies with test A.
Prepare the reference solution using lead standard solution
Bulk and tapped density (2.9.34).
(1 ppm Pb) R.
Powder flow (2.9.36).
ASSAY
To 1.500 g in a mortar, add 20-30 mL of water R and 0.5 mL of
01/2008:1078 phenolphthalein solution R. Titrate with 1 M hydrochloric acid
by triturating the substance until the red colour disappears.
CALCIUM HYDROXIDE The final solution is used in the tests for carbonates.
1 mL of 1 M hydrochloric acid is equivalent to 37.05 mg
of Ca(OH)2.
Calcii hydroxidum
01/2008:2118
Ca(OH)2 Mr 74.1 corrected 6.0
[1305-62-0]
DEFINITION CALCIUM LACTATE, ANHYDROUS
Content : 95.0 per cent to 100.5 per cent.
Calcii lactas anhydricus
CHARACTERS
Appearance : white or almost white, fine powder.
Solubility : practically insoluble in water.
IDENTIFICATION C6H10CaO6 Mr 218.2
A. To 0.80 g in a mortar, add 10 mL of water R and 0.5 mL of
phenolphthalein solution R and mix. The suspension turns DEFINITION
red. On addition of 17.5 mL of 1 M hydrochloric acid, the Calcium bis(2-hydroxypropanoate) or mixture of calcium
suspension becomes colourless without effervescing. The (2R)-, (2S)- and (2RS)-2-hydroxypropanoates.
red colour occurs again when the mixture is triturated for Content : 98.0 per cent to 102.0 per cent (dried substance).
1 min. On addition of a further 6 mL of 1 M hydrochloric
acid and triturating, the solution becomes colourless. CHARACTERS
B. Dissolve about 0.1 g in dilute hydrochloric acid R and Appearance : white or almost white, crystalline or granular
dilute to 10 mL with water R. 5 mL of the solution give powder.
reaction (b) of calcium (2.3.1). Solubility : soluble in water, freely soluble in boiling water, very
slightly soluble in ethanol (96 per cent).
TESTS
Matter insoluble in hydrochloric acid : maximum 0.5 per IDENTIFICATION
cent. A. Loss on drying (see Tests).
Dissolve 2.0 g in 30 mL of hydrochloric acid R. Boil the B. It gives the reaction of lactates (2.3.1).
solution and filter. Wash the residue with hot water R. The C. It gives reaction (b) of calcium (2.3.1).
residue weighs a maximum of 10 mg.
TESTS
Carbonates : maximum 5.0 per cent of CaCO3.
Solution S. Dissolve 5.0 g with heating in carbon dioxide-free
Add 5.0 mL of 1 M hydrochloric acid to the titrated solution
water R prepared from distilled water R, allow to cool and
obtained under Assay and titrate with 1 M sodium hydroxide
dilute to 100 mL with the same solvent.
using 0.5 mL of methyl orange solution R as indicator.
1 mL of 1 M hydrochloric acid is equivalent to 50.05 mg Appearance of solution. Solution S is not more opalescent
of CaCO3. than reference suspension II (2.2.1) and not more intensely
coloured than reference solution BY6 (2.2.2, Method II).
Chlorides (2.4.4): maximum 330 ppm.
Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of
Dissolve 0.30 g in a mixture of 2 mL of nitric acid R and 10 mL phenolphthalein solution R and 0.5 mL of 0.01 M hydrochloric
of water R and dilute to 30 mL with water R. acid. The solution is colourless. Not more than 2.0 mL of
Sulfates (2.4.13) : maximum 0.4 per cent. 0.01 M sodium hydroxide is required to change the colour of
Dissolve 0.15 g in a mixture of 5 mL of dilute hydrochloric the indicator to pink.
acid R and 10 mL of distilled water R and dilute to 60 mL with Chlorides (2.4.4) : maximum 200 ppm.
distilled water R. Dilute 5 mL of solution S to 15 mL with water R.
Arsenic (2.4.2, Method A) : maximum 4 ppm. Sulfates (2.4.13) : maximum 400 ppm.
Dissolve 0.50 g in 5 mL of brominated hydrochloric acid R and Dilute 7.5 mL of solution S to 15 mL with distilled water R.
dilute to 50 mL with water R. Use 25 mL of this solution.
Barium. To 10 mL of solution S add 1 mL of calcium sulfate
Magnesium and alkali metals : maximum 4.0 per cent solution R. Allow to stand for 15 min. Any opalescence in the
calculated as sulfates. solution is not more intense than that in a mixture of 1 mL of
Dissolve 1.0 g in a mixture of 10 mL of hydrochloric acid R distilled water R and 10 mL of solution S.
and 40 mL of water R. Boil and add 50 mL of a 63 g/L solution Iron (2.4.9) : maximum 50 ppm.
of oxalic acid R. Neutralise with ammonia R and dilute to Dilute 4 mL of solution S to 10 mL with water R.
200 mL with water R. Allow to stand for 1 h and filter through
a suitable filter. To 100 mL of the filtrate, add 0.5 mL of Magnesium and alkali salts : maximum 1 per cent.
sulfuric acid R. Cautiously evaporate to dryness and ignite. To 20 mL of solution S add 20 mL of water R, 2 g of ammonium
The residue weighs a maximum of 20 mg. chloride R and 2 mL of dilute ammonia R1. Heat to boiling

General Notices (1) apply to all monographs and other texts 1743
Calcium lactate monohydrate EUROPEAN PHARMACOPOEIA 8.0

and rapidly add 40 mL of hot ammonium oxalate solution R. Barium. To 10 mL of solution S add 1 mL of calcium sulfate
Allow to stand for 4 h, dilute to 100.0 mL with water R and solution R. Allow to stand for 15 min. Any opalescence in the
filter. To 50.0 mL of the filtrate add 0.5 mL of sulfuric acid R. solution is not more intense than that in a mixture of 1 mL of
Evaporate to dryness and ignite the residue to constant mass distilled water R and 10 mL of solution S.
at 600 ± 50 °C. The residue weighs a maximum of 5 mg. Iron (2.4.9) : maximum 50 ppm.
Heavy metals (2.4.8) : maximum 10 ppm. Dilute 4 mL of solution S to 10 mL with water R.
Dissolve 2.0 g in water R and dilute to 20 mL with the same Magnesium and alkali salts : maximum 1 per cent.
solvent. 12 mL of the solution complies with test A. Prepare
the reference solution using lead standard solution (1 ppm To 20 mL of solution S add 20 mL of water R, 2 g of ammonium
Pb) R. chloride R and 2 mL of dilute ammonia R1. Heat to boiling
and rapidly add 40 mL of hot ammonium oxalate solution R.
Loss on drying (2.2.32) : maximum 3.0 per cent, determined Allow to stand for 4 h, dilute to 100.0 mL with water R and
on 0.500 g by drying in an oven at 125 °C. filter. To 50.0 mL of the filtrate add 0.5 mL of sulfuric acid R.
ASSAY Evaporate to dryness and ignite the residue to constant mass
at 600 ± 50 °C. The residue weighs a maximum of 5 mg.
Dissolve 0.200 g in water R and dilute to 300 mL with the
same solvent. Carry out the complexometric titration of Heavy metals (2.4.8) : maximum 10 ppm.
calcium (2.5.11). Dissolve a quantity equivalent to 2.0 g of the dried substance
1 mL of 0.1 M sodium edetate is equivalent to 21.82 mg in water R and dilute to 20 mL with the same solvent. 12 mL
of C6H10CaO6. of the solution complies with test A. Prepare the reference
solution using lead standard solution (1 ppm Pb) R.
Loss on drying (2.2.32) : 5.0 per cent to 8.0 per cent,
determined on 0.500 g by drying in an oven at 125 °C.
01/2008:2117
corrected 6.0 ASSAY
Dissolve a quantity equivalent to 0.200 g of the dried substance
CALCIUM LACTATE MONOHYDRATE in water R and dilute to 300 mL with the same solvent. Carry
out the complexometric titration of calcium (2.5.11).
Calcii lactas monohydricus 1 mL of 0.1 M sodium edetate is equivalent to 21.82 mg
of C6H10CaO6.

01/2008:0468
C6H10CaO6,H2O Mr 236.0 corrected 6.0
DEFINITION
CALCIUM LACTATE PENTAHYDRATE
Calcium bis(2-hydroxypropanoate) or mixture of calcium
(2R)-, (2S)- and (2RS)-2-hydroxypropanoates monohydrates.
Content : 98.0 per cent to 102.0 per cent (dried substance).
Calcii lactas pentahydricus
CHARACTERS
Appearance : white or almost white, crystalline or granular
powder.
Solubility : soluble in water, freely soluble in boiling water, very C6H10CaO6,5H2O Mr 308.3
slightly soluble in ethanol (96 per cent).
DEFINITION
IDENTIFICATION Calcium bis(2-hydroxypropanoate) or mixture of calcium
A. Loss on drying (see Tests). (2R)-, (2S)- and (2RS)-2-hydroxypropanoates pentahydrates.
B. It gives the reaction of lactates (2.3.1). Content : 98.0 per cent to 102.0 per cent (dried substance).
C. It gives reaction (b) of calcium (2.3.1).
CHARACTERS
TESTS Appearance : white or almost white, crystalline or granular
Solution S. Dissolve 5.4 g (equivalent to 5.0 g of the dried powder, slightly efflorescent.
substance) with heating in carbon dioxide-free water R Solubility : soluble in water, freely soluble in boiling water, very
prepared from distilled water R, allow to cool and dilute to slightly soluble in ethanol (96 per cent).
100 mL with the same solvent.
IDENTIFICATION
Appearance of solution. Solution S is not more opalescent
than reference suspension II (2.2.1) and not more intensely A. Loss on drying (see Tests).
coloured than reference solution BY6 (2.2.2, Method II). B. It gives the reaction of lactates (2.3.1).
Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of C. It gives reaction (b) of calcium (2.3.1).
phenolphthalein solution R and 0.5 mL of 0.01 M hydrochloric
acid. The solution is colourless. Not more than 2.0 mL of TESTS
0.01 M sodium hydroxide is required to change the colour of Solution S. Dissolve 7.1 g (equivalent to 5.0 g of the dried
the indicator to pink. substance) with heating in carbon dioxide-free water R
Chlorides (2.4.4) : maximum 200 ppm. prepared from distilled water R, allow to cool and dilute to
100 mL with the same solvent.
Dilute 5 mL of solution S to 15 mL with water R.
Appearance of solution. Solution S is not more opalescent
Sulfates (2.4.13) : maximum 400 ppm. than reference suspension II (2.2.1) and not more intensely
Dilute 7.5 mL of solution S to 15 mL with distilled water R. coloured than reference solution BY6 (2.2.2, Method II).

1744 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcium levofolinate pentahydrate

Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of C. It gives reaction (b) of calcium (2.3.1).
phenolphthalein solution R and 0.5 mL of 0.01 M hydrochloric
acid. The solution is colourless. Not more than 2.0 mL of TESTS
0.01 M sodium hydroxide is required to change the colour of Solution S. Dissolve 6.2 g (equivalent to 5.0 g of the dried
the indicator to pink. substance) with heating in carbon dioxide-free water R
Chlorides (2.4.4) : maximum 200 ppm. prepared from distilled water R, allow to cool and dilute to
100 mL with the same solvent.
Dilute 5 mL of solution S to 15 mL with water R.
Appearance of solution. Solution S is not more opalescent
Sulfates (2.4.13) : maximum 400 ppm. than reference suspension II (2.2.1) and not more intensely
Dilute 7.5 mL of solution S to 15 mL with distilled water R. coloured than reference solution BY6 (2.2.2, Method II).
Barium. To 10 mL of solution S add 1 mL of calcium sulfate Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of
solution R. Allow to stand for 15 min. Any opalescence in the phenolphthalein solution R and 0.5 mL of 0.01 M hydrochloric
solution is not more intense than that in a mixture of 1 mL of acid. The solution is colourless. Not more than 2.0 mL of
distilled water R and 10 mL of solution S. 0.01 M sodium hydroxide is required to change the colour of
Iron (2.4.9) : maximum 50 ppm. the indicator to pink.
Dilute 4 mL of solution S to 10 mL with water R. Chlorides (2.4.4) : maximum 200 ppm.
Magnesium and alkali salts : maximum 1 per cent. Dilute 5 mL of solution S to 15 mL with water R.
To 20 mL of solution S add 20 mL of water R, 2 g of ammonium Sulfates (2.4.13) : maximum 400 ppm.
chloride R and 2 mL of dilute ammonia R1. Heat to boiling Dilute 7.5 mL of solution S to 15 mL with distilled water R.
and rapidly add 40 mL of hot ammonium oxalate solution R. Barium. To 10 mL of solution S add 1 mL of calcium sulfate
Allow to stand for 4 h, dilute to 100.0 mL with water R and solution R. Allow to stand for 15 min. Any opalescence in the
filter. To 50.0 mL of the filtrate add 0.5 mL of sulfuric acid R. solution is not more intense than that in a mixture of 1 mL of
Evaporate to dryness and ignite the residue to constant mass distilled water R and 10 mL of solution S.
at 600 ± 50 °C. The residue weighs a maximum of 5 mg.
Iron (2.4.9) : maximum 50 ppm.
Heavy metals (2.4.8) : maximum 10 ppm.
Dilute 4 mL of solution S to 10 mL with water R.
Dissolve a quantity equivalent to 2.0 g of the dried substance
in water R and dilute to 20 mL with the same solvent. 12 mL Magnesium and alkali salts : maximum 1 per cent.
of the solution complies with test A. Prepare the reference To 20 mL of solution S add 20 mL of water R, 2 g of ammonium
solution using lead standard solution (1 ppm Pb) R. chloride R and 2 mL of dilute ammonia R1. Heat to boiling
Loss on drying (2.2.32) : 22.0 per cent to 27.0 per cent, and rapidly add 40 mL of hot ammonium oxalate solution R.
determined on 0.500 g by drying in an oven at 125 °C. Allow to stand for 4 h, dilute to 100.0 mL with water R and
filter. To 50.0 mL of the filtrate add 0.5 mL of sulfuric acid R.
ASSAY Evaporate to dryness and ignite the residue to constant mass
at 600 ± 50 °C. The residue weighs a maximum of 5 mg.
Dissolve a quantity equivalent to 0.200 g of the dried substance
in water R and dilute to 300 mL with the same solvent. Carry Heavy metals (2.4.8) : maximum 10 ppm.
out the complexometric titration of calcium (2.5.11). Dissolve a quantity equivalent to 2.0 g of the dried substance
1 mL of 0.1 M sodium edetate is equivalent to 21.82 mg in water R and dilute to 20 mL with the same solvent. 12 mL
of C6H10CaO6. of the solution complies with test A. Prepare the reference
solution using lead standard solution (1 ppm Pb) R.
Loss on drying (2.2.32) : 15.0 per cent to 20.0 per cent,
determined on 0.500 g by drying in an oven at 125 °C.
01/2008:0469
corrected 6.0 ASSAY
Dissolve a quantity equivalent to 0.200 g of the dried substance
CALCIUM LACTATE TRIHYDRATE in water R and dilute to 300 mL with the same solvent. Carry
out the complexometric titration of calcium (2.5.11).
Calcii lactas trihydricus 1 mL of 0.1 M sodium edetate is equivalent to 21.82 mg
of C6H10CaO6.

01/2008:1606
corrected 7.0
C6H10CaO6,3H2O Mr 272.3
DEFINITION CALCIUM LEVOFOLINATE
Calcium bis(2-hydroxypropanoate) or mixture of calcium PENTAHYDRATE
(2R)-, (2S)- and (2RS)-2-hydroxypropanoates trihydrates.
Content : 98.0 per cent to 102.0 per cent (dried substance). Calcii levofolinas pentahydricus
CHARACTERS
Appearance : white or almost white, crystalline or granular
powder.
Solubility : soluble in water, freely soluble in boiling water, very
slightly soluble in ethanol (96 per cent).
IDENTIFICATION
A. Loss on drying (see Tests). C20H21CaN7O7,5H2O Mr 511.5 (anhydrous substance)
B. It gives the reaction of lactates (2.3.1). [80433-71-2]

General Notices (1) apply to all monographs and other texts 1745
Calcium levofolinate pentahydrate EUROPEAN PHARMACOPOEIA 8.0

DEFINITION hydroxide solution R or hydrochloric acid R1 and dilute to

Calcium (2S)-2-[[4-[[[(6S)-2-amino-5-formyl-4- 20.0 mL with the same solvent.
oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl]- Acetone and ethanol. Head-space gas chromatography
amino]benzoyl]amino]pentanedioate pentahydrate. (2.2.28) : use the standard additions method.
Content : Test solution. Dissolve 0.25 g of the substance to be examined
– calcium levofolinate (C20H21CaN7O7 ; Mr 511.5) : 97.0 per in water R and dilute to 10.0 mL with the same solvent.
cent to 102.0 per cent (anhydrous substance); Reference solution. Dissolve 0.125 g of acetone R and 0.750 g
– calcium (Ca ; Ar 40.08) : 7.54 per cent to 8.14 per cent of anhydrous ethanol R in water R and dilute to 1000.0 mL
(anhydrous substance). with water R.

CHARACTERS Column :
Appearance : white or light yellow, amorphous or crystalline – material : fused silica ;
powder, hygroscopic. – size : l = 10 m, Ø = 0.32 mm ;
Solubility : slightly soluble in water, practically insoluble in – stationary phase : styrene-divinylbenzene copolymer R.
acetone and in ethanol (96 per cent).
Carrier gas : nitrogen for chromatography R.
IDENTIFICATION Flow rate : 4 mL/min.
First identification : A, B, D. Static head-space conditions which may be used :
Second identification : A, C, D. – equilibration temperature : 80 °C ;
A. Specific optical rotation (see Tests). – equilibration time : 20 min ;
B. Infrared absorption spectrophotometry (2.2.24). – pressurisation time : 30 s.
Preparation : discs. Temperature :
Comparison : calcium folinate CRS.
Time Temperature
If the spectra obtained show differences, dissolve the (min) (°C)
substance to be examined and the reference substance Column 0 - 14 80 → 220
separately in the minimum quantity of water R and add
dropwise sufficient acetone R to produce a precipitate. Injection port 110
Allow to stand for 15 min, collect the precipitate by Detector 270
centrifugation, wash the precipitate twice with a minimum
quantity of acetone R and dry. Record new spectra using Detection : flame ionisation.
the residues. Injection : at least 3 times.
C. Thin-layer chromatography (2.2.27). Limits :
Test solution. Dissolve 15 mg of the substance to be – acetone : maximum 0.5 per cent,
examined in a 3 per cent V/V solution of ammonia R and
dilute to 5 mL with the same solvent. – ethanol : maximum 3.0 per cent.
Reference solution. Dissolve 15 mg of calcium folinate CRS Related substances. Liquid chromatography (2.2.29).
in a 3 per cent V/V solution of ammonia R and dilute to Test solution. Dissolve 10.0 mg of the substance to be
5 mL with the same solvent. examined in water R and dilute to 10.0 mL with the same
Plate : cellulose for chromatography F254 R as the coating solvent.
substance. Reference solution (a). Dissolve 10.0 mg of calcium folinate CRS
Mobile phase : the lower layer of a mixture of 1 volume of in water R and dilute to 10.0 mL with the same solvent.
isoamyl alcohol R and 10 volumes of a 50 g/L solution of Reference solution (b). Dilute 1.0 mL of reference solution (a)
citric acid R previously adjusted to pH 8 with ammonia R. to 100.0 mL with water R.
Application : 5 μL. Reference solution (c). Dissolve 10.0 mg of formylfolic acid CRS
Development : over a path of 15 cm. in the mobile phase and dilute to 100.0 mL with the mobile
Drying : in air. phase. Dilute 1.0 mL of this solution to 10.0 mL with water R.
Detection : examine in ultraviolet light at 254 nm. Reference solution (d). Dilute 1.0 mL of reference solution (b)
to 20.0 mL with water R.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to the Reference solution (e). Dilute 5.0 mL of reference solution (c)
principal spot in the chromatogram obtained with the to 10.0 mL with reference solution (b).
reference solution. Column :
D. It gives reaction (b) of calcium (2.3.1). – size : l = 0.25 m, Ø = 4 mm ;
Carry out the tests and the assay as rapidly as possible, protected – stationary phase : octadecylsilyl silica gel for
from bright light. chromatography R (5 μm) ;
– temperature : 40 °C.
TESTS
Mobile phase : mix 220 mL of methanol R and 780 mL
Solution S. Dissolve 0.40 g in carbon dioxide-free water R, of a solution containing 2.0 mL of tetrabutylammonium
heating at 40 °C if necessary, and dilute to 50.0 mL with the hydroxide solution (400 g/L) R and 2.2 g of disodium hydrogen
same solvent. phosphate R previously adjusted to pH 7.8 with phosphoric
Appearance of solution. Solution S is clear (2.2.1) and its acid R. If necessary adjust the concentration of methanol R to
absorbance (2.2.25) at 420 nm has a maximum of 0.25. achieve the prescribed resolution.
pH (2.2.3): 7.5 to 8.5 for solution S. Flow rate : 1 mL/min.
Specific optical rotation (2.2.7) : − 10 to − 15 (anhydrous Detection : spectrophotometer at 280 nm.
substance), measured at 25 °C. Injection : 10 μL.
Dissolve 0.200 g in tris(hydroxymethyl)aminomethane Run time : 2.5 times the retention time of the principal peak in
solution R previously adjusted to pH 8.1 with sodium the chromatogram obtained with the test solution.

1746 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcium levofolinate pentahydrate

System suitability: reference solution (e) : Wavelength : 265.9 nm.

– resolution : minimum of 2.2 between the peaks due to Heavy metals (2.4.8) : maximum 50 ppm.
folinate and to impurity D. 1.0 g complies with test F. Prepare the reference solution using
Limits : 5 mL of lead standard solution (10 ppm Pb) R.
– impurity D : not more than 0.8 times the area of the Water (2.5.12) : 10.0 per cent to 17.0 per cent, determined on
principal peak in the chromatogram obtained with 0.200 g (ground to a very fine powder). Stir the substance to
reference solution (c) (0.8 per cent) ; be examined in the titration solvent for about 15 min before
– any other impurity : not more than 0.8 times the area of titrating and use iodosulfurous reagent R as titrant.
the principal peak in the chromatogram obtained with Bacterial endotoxins (2.6.14) : less than 0.5 IU/mg, if intended
reference solution (b) (0.8 per cent) ; for use in the manufacture of parenteral preparations without
– sum of other impurities : not more than twice the area of a further appropriate procedure for the removal of bacterial
the principal peak in the chromatogram obtained with endotoxins.
reference solution (b) (2.0 per cent) ;
– disregard limit : area of the principal peak in the ASSAY
chromatogram obtained with reference solution (d) Calcium. Dissolve 0.400 g in 150 mL of water R and dilute to
(0.05 per cent). 300 mL with the same solvent. Carry out the complexometric
Impurity H. Liquid chromatography (2.2.29) : use the titration of calcium (2.5.11).
normalisation procedure. 1 mL of 0.1 M sodium edetate is equivalent to 4.008 mg of Ca.
Test solution. Dissolve 50.0 mg of the substance to be Calcium folinate. Liquid chromatography (2.2.29) as
examined in water R and dilute to 100.0 mL with the same described in the test for related substances.
solvent. Calculate the percentage content of C20H21CaN7O7 from the
Reference solution (a). Dissolve 10.0 mg of calcium folinate CRS areas of the peaks in the chromatograms obtained with the test
in water R and dilute to 20.0 mL with the same solvent. solution and reference solution (a) and the declared content of
Reference solution (b). Dilute 1.0 mL of reference solution (a) calcium folinate CRS.
to 100.0 mL with water R. STORAGE
Column :
In an airtight container, protected from light. If the substance
– size : l = 0.15 m, Ø = 4 mm ; is sterile, store in a sterile, airtight, tamper-proof container.
– stationary phase : human albumin coated silica gel for
chromatography R (5 μm) ; IMPURITIES
– temperature : 40 °C.
Mobile phase : dissolve 9.72 g of sodium dihydrogen phosphate R
in 890 mL of water R and adjust to pH 5.0 with sodium
hydroxide solution R ; add 100 mL of 2-propanol R and 10 mL
of acetonitrile R.
Flow rate : 1 mL/min. A. (2S)-2-[(4-aminobenzoyl)amino]pentanedioic acid,
Detection : spectrophotometer at 286 nm.
Injection : 10 μL.
Retention times : levofolinate = about 9 min ; impurity H = about
19 min.
System suitability :
– resolution : minimum of 5.0 between the peaks due to
levofolinate and to impurity H in the chromatogram B. (2S)-2-[[4-[[[(6R)-2-amino-5-formyl-4-oxo-
obtained with reference solution (a). The sum of the areas 1,4,5,6,7,8-hexahydropteridin-6-yl]methyl]-
of the 2 peaks is 100 per cent. The peak area of impurity H formylamino]benzoyl]amino]pentanedioic acid
is 48 per cent to 52 per cent. In the chromatogram obtained (5,10-diformyltetrahydrofolic acid),
with reference solution (b) 2 clearly visible peaks are
obtained.
Limit :
– impurity H : maximum 0.5 per cent.
Chlorides : maximum 0.5 per cent.
Dissolve 0.300 g in 50 mL of water R heating at 40 °C
if necessary. Add 10 mL of 2 M nitric acid and titrate
with 0.005 M silver nitrate determining the end-point C. (2S)-2-[[4-[[(2-amino-4-oxo-1,4-dihydropteridin-6-
potentiometrically (2.2.20). yl)methyl]amino]benzoyl]amino]pentanedioic acid (folic
1 mL of 0.005 M silver nitrate is equivalent to 0.177 mg of Cl. acid),
Platinum : maximum 10 ppm.
Atomic absorption spectrometry (2.2.23, Method II).
Test solution. Dissolve 1.0 g in water R and dilute to 100.0 mL
with the same solvent.
Reference solutions. Prepare the reference solutions using
platinum standard solution (30 ppm Pt) R, diluted as necessary
with a mixture of 1 volume of nitric acid R and 99 volumes D. (2S)-2-[[4-[[(2-amino-4-oxo-1,4-dihydropteridin-6-
of water R. yl)methyl]formylamino]benzoyl]amino]pentanedioic acid
Source : platinum hollow-cathode lamp. (10-formylfolic acid),

General Notices (1) apply to all monographs and other texts 1747
Calcium levulinate dihydrate EUROPEAN PHARMACOPOEIA 8.0

Plate : TLC silica gel plate R.

Mobile phase : concentrated ammonia R, ethyl acetate R,
water R, ethanol (96 per cent) R (10:10:30:50 V/V/V/V).
Application : 10 μL.
Development : over a path of 10 cm.
E. 4-[[[(6S)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8-
hexahydropteridin-6-yl]methyl]amino]benzoic acid Drying : at 100-105 °C for 20 min and allow to cool.
(5-formyltetrahydropteroic acid), Detection : spray with a 30 g/L solution of potassium
permanganate R. Dry in a current of warm air for about
5 min or until the spots become yellow. Examine in
daylight.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
the reference solution.
F. R = CHO : (2S)-2-[[4-[[(2-amino-4-oxo-1,4,7,8-tetrahydro- C. To 1 mL of solution S (see Tests), add 20 mL of a 2.5 g/L
pteridin-6-yl)methyl]formylamino]benzoyl]amino]penta- solution of dinitrophenylhydrazine R in dilute hydrochloric
nedioic acid (10-formyldihydrofolic acid), acid R. Allow to stand for 15 min. Filter, wash the
precipitate with water R. Dry the precipitate in an oven at
G. R = H : (2S)-2-[[4-[[(2-amino-4-oxo-1,4,7,8-tetrahydrop- 100-105 °C. The melting point (2.2.14) is 203 °C to 210 °C.
teridin-6-yl)methyl]amino]benzoyl]amino]pentanedioic
acid (dihydrofolic acid), D. It gives reaction (b) of calcium (2.3.1).
E. Loss on drying (see Tests).
TESTS
Solution S. Dissolve 10.0 g in carbon dioxide-free water R
prepared from distilled water R and dilute to 100.0 mL with
the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
H. (2S)-2-[[4-[[[(6R)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8- more intensely coloured than reference solution Y6 (2.2.2,
hexahydropteridin-6-yl]methyl]amino]benzoyl]amino]- Method II).
pentanedioic acid. pH (2.2.3) : 6.8 to 7.8 for solution S.
Oxidisable substances. To 1 mL of solution S, add 10 mL of
01/2008:1296 water R, 1 mL of dilute sulfuric acid R and 0.25 mL of a 3.0 g/L
corrected 6.0 solution of potassium permanganate R. Mix. After 5 min, the
violet colour of the mixture is still visible.
CALCIUM LEVULINATE DIHYDRATE Sucrose and reducing sugars. To 5 mL of solution S add
2 mL of hydrochloric acid R1 and dilute to 10 mL with water R.
Calcii laevulinas dihydricus Heat to boiling for 5 min and allow to cool. Add 10 mL of
sodium carbonate solution R. Allow to stand for 5 min, dilute
to 25 mL with water R and filter. To 5 mL of the filtrate add
2 mL of cupri-tartaric solution R and heat to boiling for 1 min.
No red precipitate is formed.
Chlorides (2.4.4) : maximum 50 ppm.
C10H14CaO6,2H2O Mr 306.3
[5743-49-7] Dilute 10 mL of solution S to 15 mL with water R.
Sulfates (2.4.13) : maximum 200 ppm.
DEFINITION
Calcium di(4-oxopentanoate) dihydrate. Dilute 7.5 mL of solution S to 15 mL with distilled water R.
Content : 98.0 per cent to 101.0 per cent (dried substance). Magnesium and alkali metals : maximum 1.0 per cent.
To 10 mL of solution S, add 80 mL of water R, 10 mL of
CHARACTERS ammonium chloride solution R and 1 mL of ammonia R. Heat
Appearance : white or almost white, crystalline powder. to boiling. To the boiling solution, add dropwise 50 mL of
Solubility : freely soluble in water, very slightly soluble in warm ammonium oxalate solution R. Allow to stand for 4 h,
ethanol (96 per cent), practically insoluble in methylene then dilute to 200 mL with water R and filter. To 100 mL of
chloride. the filtrate, add 0.5 mL of sulfuric acid R. Evaporate to dryness
on a water-bath and ignite to constant mass at 600 ± 50 °C.
IDENTIFICATION The residue weighs a maximum of 5.0 mg.
First identification : A, D, E. Heavy metals (2.4.8) : maximum 10 ppm.
Second identification : B, C, D, E. 12 mL of solution S complies with test A. Prepare the reference
A. Infrared absorption spectrophotometry (2.2.24). solution using lead standard solution (1 ppm Pb) R.
Comparison : calcium levulinate dihydrate CRS. Loss on drying (2.2.32) : 11.0 per cent to 12.5 per cent,
B. Thin-layer chromatography (2.2.27). determined on 0.200 g by drying at 105 °C.
Test solution. Dissolve 60 mg of the substance to be Pyrogens (2.6.8). If intended for use in the manufacture
examined in water R and dilute to 1 mL with the same of parenteral preparations without a further appropriate
solvent. procedure for the removal of pyrogens, it complies with the
Reference solution. Dissolve 60 mg of calcium levulinate test for pyrogens. Inject per kilogram of the rabbit’s mass 4 mL
dihydrate CRS in water R and dilute to 1 mL with the same of a solution containing per millilitre 50 mg of the substance
solvent. to be examined.

1748 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcium phosphate

ASSAY Apply separately to the plate 5 μL of each solution. Develop

over a path of 12 cm using a mixture of 35 volumes of water R
Dissolve 0.240 g in 50 mL of water R. Carry out the
complexometric titration of calcium (2.5.11). and 65 volumes of ethanol R. Dry the plate in a current of
air and spray with ninhydrin solution R1. Heat at 110 °C for
1 mL of 0.1 M sodium edetate is equivalent to 27.03 mg
10 min. Any spot corresponding to 3-aminopropionic acid
of C10H14CaO6.
in the chromatogram obtained with test solution (a) is not
STORAGE more intense than the spot in the chromatogram obtained
Protected from light. with reference solution (b) (0.5 per cent).
Chlorides (2.4.4). 5 mL of solution S diluted to 15 mL with
water R complies with the limit test for chlorides (200 ppm).
01/2008:0470 Heavy metals (2.4.8). 12 mL of solution S complies with test A
corrected 6.0 for heavy metals (20 ppm). Prepare the reference solution
using lead standard solution (1 ppm Pb) R.
CALCIUM PANTOTHENATE Loss on drying (2.2.32). Not more than 3.0 per cent,
determined on 1.000 g by drying in an oven at 105 °C.
Calcii pantothenas ASSAY
Dissolve 0.180 g in 50 mL of anhydrous acetic acid R.
Titrate with 0.1 M perchloric acid determining the end-point
potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 23.83 mg of
C18H32CaN2O10.
C18H32CaN2O10 Mr 476.5
[137-08-6] STORAGE
Store in an airtight container.
DEFINITION
Calcium pantothenate contains not less than 98.0 per 04/2009:1052
cent and not more than the equivalent of 101.0 per
cent of calcium bis[3-[[(2R)-2,4-dihydroxy-3,3-
dimethylbutanoyl]amino]propanoate], calculated with
CALCIUM PHOSPHATE
reference to the dried substance.
Tricalcii phosphas
CHARACTERS
DEFINITION
A white or almost white powder, slightly hygroscopic, freely
soluble in water, slightly soluble in alcohol. Mixture of calcium phosphates.
Content : 35.0 per cent to 40.0 per cent of Ca (Ar 40.08).
IDENTIFICATION
A. Specific optical rotation (see Tests). CHARACTERS
B. Examine the chromatograms obtained in the test for Appearance : white or almost white powder.
3-aminopropionic acid. The principal spot in the Solubility : practically insoluble in water. It dissolves in dilute
chromatogram obtained with test solution (b) is similar hydrochloric acid and in dilute nitric acid.
in position, colour and size to the principal spot in the
chromatogram obtained with reference solution (a). IDENTIFICATION
C. To 1 mL of solution S (see Tests) add 1 mL of dilute A. Dissolve 0.1 g in 5 mL of a 25 per cent V/V solution of
sodium hydroxide solution R and 0.1 mL of copper sulfate nitric acid R. The solution gives reaction (b) of phosphates
solution R. A blue colour develops. (2.3.1).
D. It gives reaction (a) of calcium (2.3.1). B. It gives reaction (b) of calcium (2.3.1). Filter before adding
potassium ferrocyanide solution R.
TESTS C. It complies with the limits of the assay.
Solution S. Dissolve 2.50 g in carbon dioxide-free water R and TESTS
dilute to 50.0 mL with the same solvent.
Solution S. Dissolve 2.50 g in 20 mL of dilute hydrochloric
Appearance of solution. Solution S is clear (2.2.1) and acid R. If the solution is not clear, filter it. Add dilute
colourless (2.2.2, Method II). ammonia R1 dropwise until a precipitate is formed. Dissolve
pH (2.2.3). The pH of solution S is 6.8 to 8.0. the precipitate by adding dilute hydrochloric acid R and dilute
Specific optical rotation (2.2.7) : + 25.5 to + 27.5, determined to 50 mL with distilled water R.
on solution S and calculated with reference to the dried Chlorides (2.4.4) : maximum 0.15 per cent.
substance. Dissolve 0.22 g in a mixture of 1 mL of nitric acid R and 10 mL
3-Aminopropionic acid. Examine by thin-layer of water R and dilute to 100 mL with water R.
chromatography (2.2.27), using silica gel G R as the coating Fluorides : maximum 75 ppm.
substance. Potentiometry (2.2.36, Method II).
Test solution (a). Dissolve 0.2 g of the substance to be Test solution. Dissolve 0.250 g in 0.1 M hydrochloric acid,
examined in water R and dilute to 5 mL with the same solvent. add 5.0 mL of fluoride standard solution (1 ppm F) R and
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL dilute to 50.0 mL with 0.1 M hydrochloric acid. To 20.0 mL of
with water R. this solution add 20.0 mL of total-ionic-strength-adjustment
Reference solution (a). Dissolve 20 mg of calcium buffer R and 3 mL of an 82 g/L solution of anhydrous sodium
pantothenate CRS in water R and dilute to 5 mL with the same acetate R. Adjust to pH 5.2 with ammonia R and dilute to
solvent. 50.0 mL with distilled water R.
Reference solution (b). Dissolve 10 mg of 3-aminopropionic Reference solution. Fluoride standard solution (10 ppm F) R.
acid R in water R and dilute to 50 mL with the same solvent. Indicator electrode : fluoride-selective.

General Notices (1) apply to all monographs and other texts 1749
Calcium stearate EUROPEAN PHARMACOPOEIA 8.0

Reference electrode : silver-silver chloride. Content :

Carry out the measurements on the test solution, then add at – calcium : 6.4 per cent to 7.4 per cent (Ar 40.08) (dried
least 3 quantities, each of 0.5 mL, of the reference solution, substance) ;
carrying out a measurement after each addition. Calculate the – stearic acid in the fatty acid fraction : minimum 40.0 per
concentration of fluorides using the calibration curve, taking cent ;
into account the addition of fluoride to the test solution.
– sum of stearic acid and palmitic acid in the fatty acid
Sulfates (2.4.13) : maximum 0.5 per cent. fraction : minimum 90.0 per cent.
Dilute 1 mL of solution S to 25 mL with distilled water R.
CHARACTERS
Arsenic (2.4.2, Method A) : maximum 4 ppm, determined on
5 mL of solution S. Appearance : fine, white or almost white, crystalline powder.
Iron (2.4.9) : maximum 400 ppm. Solubility : practically insoluble in water and in ethanol (96 per
cent).
Dilute 0.5 mL of solution S to 10 mL with water R.
Heavy metals (2.4.8) : maximum 30 ppm. IDENTIFICATION
Dilute 13 mL of solution S to 20 mL with water R. 12 mL of the First identification : C, D.
solution complies with test A. Prepare the reference solution Second identification : A, B, D.
using lead standard solution (1 ppm Pb) R.
A. Freezing point (2.2.18) : minimum 53 °C, for the residue
Acid-insoluble matter: maximum 0.2 per cent. obtained in the preparation of solution S (see Tests).
Dissolve 5.0 g in a mixture of 10 mL of hydrochloric acid R B. Acid value (2.5.1) : 195 to 210.
and 30 mL of water R. Filter, wash the residue with water R
and dry to constant mass at 100-105 °C. The residue weighs Dissolve 0.200 g of the residue obtained in the preparation
a maximum of 10 mg. of solution S in 25 mL of the prescribed mixture of solvents.
Loss on ignition : maximum 8.0 per cent, determined on C. Examine the chromatograms obtained in the test for fatty
1.000 g by ignition at 800 ± 50 °C for 30 min. acid composition.
Results : the retention times of the principal peaks in
ASSAY the chromatogram obtained with the test solution are
Dissolve 0.200 g in a mixture of 1 mL of hydrochloric acid R1 approximately the same as those of the principal peaks in
and 5 mL of water R. Add 25.0 mL of 0.1 M sodium edetate the chromatogram obtained with the reference solution.
and dilute to 200 mL with water R. Adjust to about pH 10 with D. Neutralise 5 mL of solution S to red litmus paper R using
concentrated ammonia R. Add 10 mL of ammonium chloride strong sodium hydroxide solution R. The solution gives
buffer solution pH 10.0 R and a few milligrams of mordant reaction (b) of calcium (2.3.1).
black 11 triturate R. Titrate the excess sodium edetate with
0.1 M zinc sulfate until the colour changes from blue to violet. TESTS
1 mL of 0.1 M sodium edetate is equivalent to 4.008 mg of Ca. Solution S. To 5.0 g add 50 mL of peroxide-free ether R, 20 mL
of dilute nitric acid R and 20 mL of distilled water R. Boil
FUNCTIONALITY-RELATED CHARACTERISTICS
under a reflux condenser until dissolution is complete. Allow
This section provides information on characteristics that are to cool. In a separating funnel, separate the aqueous layer
recognised as being relevant control parameters for one or and shake the ether layer with 2 quantities, each of 5 mL,
more functions of the substance when used as an excipient (see of distilled water R. Combine the aqueous layers, wash with
chapter 5.15). This section is a non-mandatory part of the 15 mL of peroxide-free ether R and dilute the aqueous layer to
monograph and it is not necessary to verify the characteristics 50 mL with distilled water R (solution S). Evaporate the ether
to demonstrate compliance. Control of these characteristics can layer to dryness and dry the residue at 100-105 °C. Keep the
however contribute to the quality of a medicinal product by residue for identification tests A and B.
improving the consistency of the manufacturing process and
the performance of the medicinal product during use. Where Acidity or alkalinity. To 1.0 g add 20 mL of carbon
control methods are cited, they are recognised as being suitable dioxide-free water R and boil for 1 min with continuous
for the purpose, but other methods can also be used. Wherever shaking. Cool and filter. To 10 mL of the filtrate add 0.05 mL
results for a particular characteristic are reported, the control of bromothymol blue solution R1. Not more than 0.5 mL
method must be indicated. of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is
required to change the colour of the indicator.
The following characteristics may be relevant for calcium
phosphate is used as a filler in tablets and capsules. Chlorides (2.4.4) : maximum 0.1 per cent.
Particle-size distribution (2.9.31 or 2.9.38). Dilute 0.5 mL of solution S to 15 mL with water R.
Bulk and tapped density (2.9.34). Sulfates (2.4.13) : maximum 0.3 per cent.
Powder flow (2.9.36). Dilute 0.5 mL of solution S to 15 mL with distilled water R.
Cadmium : maximum 3 ppm.
07/2010:0882 Atomic absorption spectrometry (2.2.23, Method II).
corrected 7.0 Test solution. Place 50.0 mg in a polytetrafluoroethylene
digestion bomb and add 0.5 mL of a mixture of 1 volume of
CALCIUM STEARATE hydrochloric acid R and 5 volumes of cadmium- and lead-free
nitric acid R. Allow to digest at 170 °C for 5 h. Allow to cool.
Calcii stearas Dissolve the residue in water R and dilute to 5.0 mL with the
same solvent.
[1592-23-0] Reference solutions. Prepare the reference solutions using
cadmium standard solution (10 ppm Cd) R, diluted if necessary
DEFINITION
with a 1 per cent V/V solution of hydrochloric acid R.
Mixture of calcium salts of different fatty acids consisting Source : cadmium hollow-cathode lamp.
mainly of stearic (octadecanoic) acid [(C17H35COO)2Ca ;
Mr 607] and palmitic (hexadecanoic) acid [(C15H31COO)2Ca ; Wavelength : 228.8 nm.
Mr 550.9] with minor proportions of other fatty acids. Atomisation device : graphite furnace.

1750 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Calcium sulfate dihydrate

Lead : maximum 10 ppm. Temperature :

Atomic absorption spectrometry (2.2.23, Method II). Time Temperature
(min) (°C)
Test solution. Use the solution described in the test for
Column 0-2 70
cadmium.
2 - 36 70 → 240
Reference solutions. Prepare the reference solutions using lead
standard solution (10 ppm Pb) R, diluted if necessary with 36 - 41 240
water R.
Injection port 220
Source : lead hollow-cathode lamp.
Detector 260
Wavelength : 283.3 nm ; 217.0 nm may be used depending on
the apparatus. Detection : flame ionisation.
Atomisation device : graphite furnace. Injection : 1 μL.
Nickel : maximum 5 ppm. Relative retention with reference to methyl stearate : methyl
palmitate = about 0.9.
Atomic absorption spectrometry (2.2.23, Method II). System suitability : reference solution :
Test solution. Use the solution described in the test for – resolution : minimum 5.0 between the peaks due to methyl
cadmium. palmitate and methyl stearate.
Reference solutions. Prepare the reference solutions using Calculate the content of palmitic acid and stearic acid.
nickel standard solution (10 ppm Ni) R, diluted if necessary Disregard the peak due to the solvent.
with water R.
FUNCTIONALITY-RELATED CHARACTERISTICS
Source : nickel hollow-cathode lamp. This section provides information on characteristics that are
Wavelength : 232.0 nm. recognised as being relevant control parameters for one or
more functions of the substance when used as an excipient (see
Atomisation device : graphite furnace. chapter 5.15). This section is a non-mandatory part of the
Loss on drying (2.2.32) : maximum 6.0 per cent, determined monograph and it is not necessary to verify the characteristics
on 1.000 g by drying in an oven at 105 °C. to demonstrate compliance. Control of these characteristics can
Microbial contamination however contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process and
TAMC : acceptance criterion 103 CFU/g (2.6.12). the performance of the medicinal product during use. Where
TYMC : acceptance criterion 102 CFU/g (2.6.12). control methods are cited, they are recognised as being suitable
for the purpose, but other methods can also be used. Wherever
Absence of Escherichia coli (2.6.13). results for a particular characteristic are reported, the control
Absence of Salmonella (2.6.13). method must be indicated.
The following characteristics may be relevant for calcium
ASSAY stearate used as a lubricant in tablets and capsules.
Calcium. To 0.500 g in a 250 mL conical flask add 50 mL Particle-size distribution (2.9.31).
of a mixture of equal volumes of anhydrous ethanol R Specific surface area (2.9.26, Method I). Determine the
and butanol R, 5 mL of concentrated ammonia R, 3 mL of specific surface area in the P/Po range of 0.05 to 0.15.
ammonium chloride buffer solution pH 10.0 R, 30.0 mL Sample outgassing : 2 h at 40 °C.
of 0.1 M sodium edetate and 15 mg of mordant black 11
triturate R. Heat to 45-50 °C until the solution is clear. Cool
and titrate with 0.1 M zinc sulfate until the colour changes 04/2009:0982
from blue to violet. Carry out a blank titration.
1 mL of 0.1 M sodium edetate is equivalent to 4.008 mg of Ca. CALCIUM SULFATE DIHYDRATE
Composition of fatty acids. Gas chromatography (2.2.28) :
use the normalisation procedure. Calcii sulfas dihydricus
Test solution. In a conical flask fitted with a reflux condenser,
dissolve 0.10 g of the substance to be examined in 5 mL of CaSO4,2H2O Mr 172.2
boron trifluoride-methanol solution R. Boil under a reflux [10101-41-4]
condenser for 10 min. Add 4 mL of heptane R through the DEFINITION
condenser. Boil under a reflux condenser for 10 min. Allow to
cool. Add 20 mL of saturated sodium chloride solution R. Shake Content : 98.0 per cent to 102.0 per cent of CaSO4,2H2O.
and allow the layers to separate. Remove about 2 mL of the CHARACTERS
organic layer and dry over 0.2 g of anhydrous sodium sulfate R.
Dilute 1.0 mL of the solution to 10.0 mL with heptane R. Appearance : white or almost white fine powder.
Solubility : very slightly soluble in water, practically insoluble
Reference solution. Prepare the reference solution in the same in ethanol (96 per cent).
manner as the test solution using 50.0 mg of palmitic acid CRS
and 50.0 mg of stearic acid CRS instead of calcium stearate. IDENTIFICATION
Column : A. Loss on ignition (see Tests).
– material : fused silica ; B. Solution S (see Tests) gives reaction (a) of sulfates (2.3.1).
C. Solution S gives reaction (a) of calcium (2.3.1).
– size : l = 30 m, Ø = 0.32 mm ;
– stationary phase : macrogol 20 000 R (film thickness 0.5 μm). TESTS
Solution S. Dissolve 1.0 g in 50 mL of a 10 per cent V/V
Carrier gas : helium for chromatography R.
solution of hydrochloric acid R by heating at 50 °C for 5 min.
Flow rate : 2.4 mL/min. Allow to cool.

General Notices (1) apply to all monographs and other texts 1751
D-Camphor EUROPEAN PHARMACOPOEIA 8.0

Acidity or alkalinity. Shake 1.5 g with 15 mL of carbon DEFINITION

dioxide-free water R for 5 min. Allow to stand for 5 min and (1R,4R)-1,7,7-Trimethylbicyclo[2.2.1]heptan-2-one.
filter. To 10 mL of the filtrate add 0.1 mL of phenolphthalein
solution R and 0.25 mL of 0.01 M sodium hydroxide. The
solution is red. Add 0.30 mL of 0.01 M hydrochloric acid. The CHARACTERS
solution is colourless. Add 0.2 mL of methyl red solution R. Appearance : white or almost white, crystalline powder or
The solution is reddish-orange. friable, crystalline masses.
Chlorides (2.4.4): maximum 300 ppm. Highly volatile even at room temperature.
Shake 0.5 g with 15 mL of water R for 5 min. Allow to stand
for 15 min and filter. Dilute 5 mL of the filtrate to 15 mL with Solubility : slightly soluble in water, very soluble in alcohol
water R. and in light petroleum, freely soluble in fatty oils, very slightly
soluble in glycerol.
Arsenic (2.4.2, Method A) : maximum 10 ppm, determined
on 5 mL of solution S.
IDENTIFICATION
Iron (2.4.9) : maximum 100 ppm.
To 0.25 g add a mixture of 5 mL of hydrochloric acid R and First identification : A, C.
20 mL of water R. Heat to boiling, cool and filter. Second identification : A, B, D.
Heavy metals (2.4.8) : maximum 20 ppm. A. Specific optical rotation (see Tests).
To 2.5 g add a mixture of 2 mL of hydrochloric acid R and
15 mL of water R. Heat to boiling. Cool and then add 0.5 mL B. Melting point (2.2.14) : 175 °C to 179 °C.
of phenolphthalein solution R. Cautiously add concentrated C. Infrared absorption spectrophotometry (2.2.24).
ammonia R until the colour changes to pink. Add 0.5 mL
of glacial acetic acid R and dilute to 25 mL with water R. Comparison: racemic camphor CRS.
Filter. 12 mL of the filtrate complies with test A. Prepare the D. Dissolve 1.0 g in 30 mL of methanol R. Add 1.0 g of
reference solution using lead standard solution (2 ppm Pb) R. hydroxylamine hydrochloride R and 1.0 g of anhydrous
Loss on ignition : 18.0 per cent to 22.0 per cent, determined sodium acetate R. Boil under a reflux condenser for 2 h.
on 1.000 g by ignition to constant mass at 800 ± 50 °C. Allow to cool and add 100 mL of water R. Filter, wash the
precipitate obtained with 10 mL of water R and recrystallise
ASSAY from 10 mL of a mixture of 4 volumes of alcohol R and
Dissolve 0.150 g in 120 mL of water R. Carry out the 6 volumes of water R. The crystals, dried in vacuo, melt
complexometric titration of calcium (2.5.11). (2.2.14) at 118 °C to 121 °C.
1 mL of 0.1 M sodium edetate is equivalent to 17.22 mg
of CaSO4,2H2O. TESTS
FUNCTIONALITY-RELATED CHARACTERISTICS Carry out the weighings and dissolution rapidly.
This section provides information on characteristics that are Solution S. Dissolve 2.50 g in 10 mL of alcohol R and dilute to
recognised as being relevant control parameters for one or 25.0 mL with the same solvent.
more functions of the substance when used as an excipient (see Appearance of solution. Solution S is clear (2.2.1) and
chapter 5.15). This section is a non-mandatory part of the colourless (2.2.2, Method II).
monograph and it is not necessary to verify the characteristics
to demonstrate compliance. Control of these characteristics can Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of
however contribute to the quality of a medicinal product by phenolphthalein solution R1. The solution is colourless. Not
improving the consistency of the manufacturing process and more than 0.2 mL of 0.1 M sodium hydroxide is required to
the performance of the medicinal product during use. Where change the colour of the indicator.
control methods are cited, they are recognised as being suitable Specific optical rotation (2.2.7) : + 40.0 to + 43.0, determined
for the purpose, but other methods can also be used. Wherever on solution S.
results for a particular characteristic are reported, the control
method must be indicated. Related substances. Gas chromatography (2.2.28).
The following characteristics may be relevant for calcium Test solution. Dissolve 2.50 g of the substance to be examined
sulfate dihydrate used as filler in tablets and capsules. in heptane R and dilute to 25.0 mL with the same solvent.
Particle-size distribution (2.9.31 or 2.9.38). Reference solution (a). Dilute 1.0 mL of the test solution to
Bulk and tapped density (2.9.34). 100.0 mL with heptane R.
Powder flow (2.9.36). Reference solution (b). Dilute 10.0 mL of reference solution (a)
to 20.0 mL with heptane R.
01/2008:1400 Reference solution (c). Dissolve 0.50 g of borneol R in heptane R
corrected 7.0 and dilute to 25.0 mL with the same solvent. Dilute 5.0 mL of
the solution to 50.0 mL with heptane R.
D-CAMPHOR Reference solution (d). Dissolve 50 mg of linalol R and 50 mg
of bornyl acetate R in heptane R and dilute to 100.0 mL with
D-Camphora the same solvent.
Column :
– size : l = 30 m, Ø = 0.25 mm,
– stationary phase : macrogol 20 000 R (0.25 μm).
Carrier gas : helium for chromatography R.
Split ratio : 1:70.
C10H16O Mr 152.2
[464-49-3] Flow rate : 45 cm/s.

1752 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Camphor, racemic

Temperature :
Time Temperature
(min) (°C)
Column 0 - 10 50
10 - 35 50 → 100
C. 6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane
35 - 45 100 → 200 (β-pinene),
45 - 55 200
Injection port 220
Detector 250

Detection : flame ionisation. D. 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane (cineole),

Injection : 1 μL.
System suitability: reference solution (d).
– resolution : minimum 3.0 between the peaks due to bornyl
acetate and to linalol.
Limits :
E. R1 = CH3, R2 + R3 = O : 1,3,3-trimethylbicyclo[2.2.1]hep-
– borneol : not more than the area of the principal peak in tan-2-one (fenchone),
the chromatogram obtained with reference solution (c)
(2.0 per cent), F. R1 = CH3, R2 = OH, R3 = H : exo-1,3,3-trimethyl-
bicyclo[2.2.1]heptan-2-ol (fenchol),
– any other impurity : not more than half of the area of
the principal peak in the chromatogram obtained with G. R1 = H, R2 = OH, R3 = CH3 : exo-2,3,3-trimethyl-
reference solution (a) (0.5 per cent), bicyclo[2.2.1]heptan-2-ol (camphene hydrate),
– total of other impurities: not more than 4 times the area H. R1 = H, R2 = CH3, R3 = OH : endo-2,3,3-trimethyl-
of the principal peak in the chromatogram obtained with bicyclo[2.2.1]heptan-2-ol (methylcamphenilol),
reference solution (a) (4.0 per cent),
– disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Halogens : maximum 100 ppm.
Dissolve 1.0 g in 10 mL of 2-propanol R in a distillation flask. I. R = OH, R′ = H : exo-1,7,7-trimethylbicyclo[2.2.1]heptan-
Add 1.5 mL of dilute sodium hydroxide solution R and 50 mg 2-ol (exo-borneol),
of nickel-aluminium alloy R. Heat on a water-bath until the J. R = H, R′ = OH : endo-1,7,7-trimethylbicyclo[2.2.1]heptan-
2-propanol R has evaporated. Allow to cool and add 5 mL 2-ol (endo-borneol).
of water R. Mix and filter through a wet filter previously
washed with water R until free from chlorides. Dilute the 01/2008:0655
filtrate to 10.0 mL with water R. To 5.0 mL of the solution, corrected 6.0
add nitric acid R dropwise until the precipitate which forms
is redissolved and dilute to 15 mL with water R. The solution
complies with the limit test for chlorides (2.4.4). CAMPHOR, RACEMIC
Residue on evaporation (2.8.9) : maximum 0.05 per cent. Camphora racemica
Evaporate 2.0 g on a water-bath and dry in an oven at
100-105 °C for 1 h. The residue weighs a maximum of 1 mg.
Water. Dissolve 1 g in 10 mL of light petroleum R. The
solution is clear (2.2.1).

IMPURITIES
C10H16O Mr 152.2
[76-22-2]
DEFINITION
(1RS,4RS)-1,7,7-Trimethylbicyclo[2.2.1]heptan-2-one.
CHARACTERS
Appearance : white or almost white, crystalline powder or
A. 2,6,6-trimethylbicyclo[3.1.1]hept-2-ene (α-pinene), friable, crystalline masses, highly volatile even at room
temperature.
Solubility : slightly soluble in water, very soluble in ethanol
(96 per cent) and in light petroleum, freely soluble in fatty
oils, very slightly soluble in glycerol.
IDENTIFICATION
First identification : A, C.
B. 2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane Second identification : A, B, D.
(camphene), A. Optical rotation (see Tests).

General Notices (1) apply to all monographs and other texts 1753
Candesartan cilexetil EUROPEAN PHARMACOPOEIA 8.0

B. Melting point (2.2.14) : 172 °C to 180 °C. washed with water R until free from chlorides. Dilute the
C. Infrared absorption spectrophotometry (2.2.24). filtrate to 10.0 mL with water R. To 5.0 mL of this solution,
Preparation : mulls in liquid paraffin R. add nitric acid R dropwise until the precipitate which forms
is redissolved and dilute to 15 mL with water R. The solution
Comparison : racemic camphor CRS. complies with the limit test for chlorides (2.4.4).
D. Dissolve 1.0 g in 30 mL of methanol R. Add 1.0 g of
hydroxylamine hydrochloride R and 1.0 g of anhydrous Water. Dissolve 1 g in 10 mL of light petroleum R. The
sodium acetate R. Boil under a reflux condenser for 2 h. solution is clear (2.2.1).
Allow to cool and add 100 mL of water R. A precipitate is Residue on evaporation : maximum 0.05 per cent.
formed. Filter, wash with 10 mL of water R and recrystallise Evaporate 2.0 g on a water-bath and dry at 100-105 °C for 1 h.
from 10 mL of a mixture of 4 volumes of ethanol (96 per The residue weighs not more than 1 mg.
cent) R and 6 volumes of water R. The crystals, dried in
vacuo, melt (2.2.14) at 118 °C to 121 °C.
01/2012:2573
TESTS
Carry out the weighings rapidly.
CANDESARTAN CILEXETIL
Solution S. Dissolve 2.50 g in 10 mL of ethanol (96 per cent) R
and dilute to 25.0 mL with the same solvent.
Candesartanum cilexetili
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Acidity or alkalinity. Dissolve 1.0 g in 10 mL of ethanol
(96 per cent) R and add 0.1 mL of phenolphthalein solution R1.
The solution is colourless. Not more than 0.2 mL of 0.1 M
sodium hydroxide is required to change the colour of the
indicator.
Optical rotation (2.2.7) : − 0.15° to + 0.15°, determined on
solution S.
Related substances. Gas chromatography (2.2.28).
Test solution. Dissolve 50 mg of the substance to be examined
in hexane R and dilute to 50.0 mL with the same solvent. C33H34N6O6 Mr 611
Reference solution (a). Dissolve 50 mg of the substance to [145040-37-5]
be examined and 50 mg of bornyl acetate R in hexane R and DEFINITION
dilute to 50.0 mL with the same solvent.
Reference solution (b). Dilute 1.0 mL of the test solution to (1RS)-1-[[(Cyclohexyloxy)carbonyl]oxy]ethyl
200.0 mL with hexane R. 2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-
benzimidazole-7-carboxylate.
Column :
Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
– size : l = 2 m, Ø = 2 mm ;
– stationary phase : diatomaceous earth for gas CHARACTERS
chromatography R impregnated with 10 per cent m/m of Appearance : white or almost white powder.
macrogol 20 000 R. Solubility : practically insoluble in water, freely soluble in
Carrier gas : nitrogen for chromatography R. methylene chloride and slightly soluble in anhydrous ethanol.
Flow rate : 30 mL/min. It shows polymorphism (5.9).
Temperature :
– column : 130 °C ; IDENTIFICATION
– injection port and detector : 200 °C. Infrared absorption spectrophotometry (2.2.24).
Detection : flame ionisation. Comparison: candesartan cilexetil CRS.
Injection : 1 μL. If the spectra obtained show differences, dissolve the substance
Run time : 3 times the retention time of camphor. to be examined and the reference substance separately in
anhydrous ethanol R, evaporate to dryness and record new
System suitability : spectra using the residues.
– resolution : minimum 1.5 between the peaks due to camphor
and bornyl acetate in the chromatogram obtained with TESTS
reference solution (a) ; Related substances. Liquid chromatography (2.2.29). Prepare
– signal-to-noise ratio : minimum 5 for the principal peak in the solutions immediately before use.
the chromatogram obtained with reference solution (b). Solvent mixture : water R, acetonitrile R (40:60 V/V).
Limits : Test solution. Dissolve 20 mg of the substance to be examined
– any impurity : for each impurity, not more than 2 per cent in 50.0 mL of the solvent mixture.
of the area of the principal peak ; Reference solution (a). Dilute 1.0 mL of the test solution to
– total : not more than 4 per cent of the area of the principal 100.0 mL with the solvent mixture. Dilute 1.0 mL of this
peak ; solution to 10.0 mL with the solvent mixture.
– disregard limit : the area of the principal peak in the Reference solution (b). Dissolve 5 mg of candesartan cilexetil
chromatogram obtained with reference solution (b). for system suitability CRS (containing impurities A, B and F)
Halogens : maximum 100 ppm. in the solvent mixture and dilute to 10.0 mL with the solvent
Dissolve 1.0 g in 10 mL of 2-propanol R in a distillation flask. mixture.
Add 1.5 mL of dilute sodium hydroxide solution R and 50 mg Reference solution (c). Dissolve 2.5 mg of candesartan cilexetil
of nickel-aluminium alloy R. Heat on a water-bath until the for peak identification CRS (containing impurities G and H)
2-propanol R has evaporated. Allow to cool and add 5 mL in the solvent mixture and dilute to 5.0 mL with the solvent
of water R. Mix and filter through a wet filter previously mixture.

1754 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Candesartan cilexetil

Column : 1 mL of 0.1 M perchloric acid is equivalent to 61.1 mg of

– size : l = 0.15 m, Ø = 3.9 mm ; C33H34N6O6.
– stationary phase : octadecylsilyl silica gel for IMPURITIES
chromatography R (4 μm).
Specified impurities : A, B, F, G, H.
Mobile phase :
– mobile phase A : glacial acetic acid R, water R, acetonitrile R Other detectable impurities (the following substances would,
(1:43:57 V/V/V) ; if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
– mobile phase B : glacial acetic acid R, water R, acetonitrile R acceptance criterion for other/unspecified impurities and/or
(1:10:90 V/V/V) ; by the general monograph Substances for pharmaceutical use
Time Mobile phase A Mobile phase B (2034). It is therefore not necessary to identify these impurities
(min) (per cent V/V) (per cent V/V)
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : C, D, E, I.
0-3 100 0
3 - 33 100 → 0 0 → 100
33 - 40 0 100

Flow rate : 0.8 mL/min.

Detection : spectrophotometer at 254 nm.
Injection : 10 μL.
Identification of impurities : use the chromatogram supplied
with candesartan cilexetil for system suitability CRS and the
chromatogram obtained with reference solution (b) to identify A. ethyl 2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-
the peaks due to impurities A, B and F ; use the chromatogram yl]methyl]-1H-benzimidazole-7-carboxylate,
supplied with candesartan cilexetil for peak identification CRS
and the chromatogram obtained with reference solution (c) to
identify the peaks due to impurities G and H.
Relative retention with reference to candesartan cilexetil
(retention time = about 11 min) : impurity G = about 0.2 ;
impurity A = about 0.4 ; impurity B = about 0.5 ;
impurity F = about 2.0 ; impurity H = about 3.5.
System suitability : reference solution (b) :
– resolution : minimum 4.0 between the peaks due to
impurities A and B.
Limits :
– correction factors : for the calculation of content, multiply B. (1RS)-1-[[(cyclohexyloxy)carbonyl]oxy]ethyl
the peak areas of the following impurities by the 2-oxo-3-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-2,3-
corresponding correction factor : impurities A and G = 0.7 ; dihydro-1H-benzimidazole-4-carboxylate,
impurity H = 1.6 ;
– impurity B : not more than 3 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.3 per cent) ;
– impurities F, G : for each impurity, not more than twice the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.2 per cent) ;
– impurities A, H : for each impurity, not more than 1.5 times
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.15 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained C. (1RS)-1-[[(cyclohexyloxy)carbonyl]oxy]ethyl
with reference solution (a) (0.10 per cent) ; 3-[[2′-(1-ethyl-1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-2-
– total : not more than 6 times the area of the principal peak oxo-2,3-dihydro-1H-benzimidazole-4-carboxylate,
in the chromatogram obtained with reference solution (a)
(0.6 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent).
Water (2.5.32) : maximum 0.3 per cent, determined on
60.0 mg.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.

ASSAY
Dissolve 0.500 g in 60 mL of glacial acetic acid R. Titrate D. (1RS)-1-[[(cyclohexyloxy)carbonyl]oxy]ethyl
immediately with 0.1 M perchloric acid, determining the 3-[[2′-(2-ethyl-2H-tetrazol-5-yl)biphenyl-4-yl]methyl]-2-
end-point potentiometrically (2.2.20) at the 1st inflexion point. oxo-2,3-dihydro-1H-benzimidazole-4-carboxylate,

General Notices (1) apply to all monographs and other texts 1755
Caprylic acid EUROPEAN PHARMACOPOEIA 8.0

01/2008:1401

CAPRYLIC ACID
Acidum caprylicum

C8H16O2 Mr 144.2
[124-07-2]
E. (1RS)-1-[[(cyclohexyloxy)carbonyl]oxy]ethyl DEFINITION
2-ethoxy-1-[[2′-(1-ethyl-1H-tetrazol-5-yl)biphenyl-4- Octanoic acid.
yl]methyl]-1H-benzimidazole-7-carboxylate,
Content : 99.0 per cent to 100.5 per cent (anhydrous substance).
CHARACTERS
Appearance : clear, colourless or slightly yellowish, oily liquid.
Solubility : very slightly soluble in water, very soluble in
acetone and in ethanol (96 per cent). It dissolves in dilute
solutions of alkali hydroxides.
IDENTIFICATION
A. Relative density (see Tests).
B. Examine the chromatograms obtained in the test for related
substances.
Results : the principal peak in the chromatogram obtained
F. (1RS)-1-[[(cyclohexyloxy)carbonyl]oxy]ethyl with the test solution is similar in retention time and size
2-ethoxy-1-[[2′-(2-ethyl-2H-tetrazol-5-yl)biphenyl-4- to the principal peak in the chromatogram obtained with
yl]methyl]-1H-benzimidazole-7-carboxylate, reference solution (a).
TESTS
Appearance. The substance to be examined is clear (2.2.1)
and not more intensely coloured than reference solution Y5
(2.2.2, Method II).
Relative density (2.2.5) : 0.909 to 0.912.
Related substances. Gas chromatography (2.2.28) : use the
normalisation procedure.
Test solution. Dissolve 0.10 g of the substance to be examined
G. 2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]- in ethyl acetate R and dilute to 10.0 mL with the same solvent.
1H-benzimidazole-7-carboxylic acid (candesartan), Reference solution (a). Dissolve 0.10 g of caprylic acid CRS in
ethyl acetate R and dilute to 10.0 mL with the same solvent.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with ethyl acetate R. Dilute 5.0 mL of this solution
to 50.0 mL with ethyl acetate R.
Column :
– material : fused silica ;
– size : l = 30 m, Ø = 0.25 mm ;
– stationary phase : macrogol 20 000 2-nitroterephthalate R
(film thickness 0.25 μm).
Carrier gas : helium for chromatography R.
Flow rate : 1.5 mL/min.
H. (1RS)-1-[[(cyclohexyloxy)carbonyl]oxy]ethyl Split ratio : 1:100.
2-ethoxy-1-[[2′-[1-(triphenylmethyl)-1H-tetrazol-5- Temperature :
yl]biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylate Time Temperature
(N-tritylcandesartan), (min) (°C)
Column 0-1 100
1 - 25 100 → 220
25 - 35 220
Injection port 250
Detector 250

Detection : flame ionisation.

Injection : 1 μL.
I. methyl 2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4- System suitability : reference solution (b) :
yl]methyl]-1H-benzimidazole-7-carboxylate. – signal-to-noise ratio : minimum 5 for the principal peak.

1756 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Caprylocaproyl macrogolglycerides

Limits : CHARACTERS
– any impurity : for each impurity, maximum 0.3 per cent ; Appearance : pale-yellow, oily liquid.
– total : maximum 0.5 per cent ; Solubility : dispersible in hot water, freely soluble in methylene
– disregard limit : 0.5 times the area of the principal peak in chloride.
the chromatogram obtained with reference solution (b) Density : about 1.0 at 20 °C.
(0.05 per cent).
Refractive index : about 1.4 at 20 °C.
Heavy metals (2.4.8) : maximum 10 ppm.
Dissolve 2.0 g in ethanol (96 per cent) R and dilute to 20 mL IDENTIFICATION
with the same solvent. 12 mL of the solution complies with A. Thin-layer chromatography (2.2.27).
test B. Prepare the reference solution using 1 mL of lead Test solution. Dissolve 1.0 g of the substance to be examined
standard solution (10 ppm Pb) R and 9 mL of ethanol (96 per in methylene chloride R and dilute to 20 mL with the same
cent) R. solvent.
Water (2.5.12) : maximum 0.7 per cent, determined on 1.000 g. Plate : TLC silica gel plate R.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Mobile phase : hexane R, ether R (30:70 V/V).
1.0 g.
Application : 50 μL.
ASSAY Development : over a path of 15 cm.
Dissolve 0.125 g in 25 mL of ethanol (96 per cent) R. Titrate Drying : in air.
with 0.1 M sodium hydroxide, determining the end-point
potentiometrically (2.2.20). Detection : spray with a 0.1 g/L solution of rhodamine B R
in ethanol (96 per cent) R and examine in ultraviolet light
1 mL of 0.1 M sodium hydroxide is equivalent to 14.42 mg at 365 nm.
of C8H16O2.
Results : the chromatogram shows a spot due to triglycerides
IMPURITIES with an RF value of about 0.9 (Rst 1) and spots due to
1,3-diglycerides (Rst 0.7), to 1,2-diglycerides (Rst 0.6), to
monoglycerides (Rst 0.1) and to esters of macrogol (Rst 0).
A. n = 4 : hexanoic acid, B. Hydroxyl value (see Tests).
B. n = 5 : heptanoic acid, C. Saponification value (see Tests).
D. Composition of fatty acids (see Tests).
C. n = 7 : nonanoic acid,
D. n = 8 : decanoic acid, TESTS
Viscosity (2.2.9). Carry out the determination at 20 ± 0.5 °C.
Ethylene oxide units Type of macrogol Viscosity
per molecule (mPa·s)
(nominal value)
E. 2-propylpentanoic acid (valproic acid),
4 200 30 to 50
6 300 60 to 80
8 400 80 to 110
F. R = OCH3, n = 6 : methyl octanoate,
Acid value (2.5.1) : maximum 2.0, determined on 2.0 g.
G. R = OC2H5, n = 6 : ethyl octanoate,
Hydroxyl value (2.5.3, Method A). Use 1.0 g.
H. R = OCH3, n = 8 : methyl decanoate,
Ethylene oxide units Type of macrogol Hydroxyl value
I. R = CH3, n = 8 : undecan-2-one, per molecule
(nominal value)
4 200 80 to 120
6 300 140 to 180
J. 5-butyltetrahydrofuran-2-one (γ-hydroxyoctanoic acid
lactone). 8 400 170 to 205

Peroxide value (2.5.5, Method A) : maximum 6.0, determined

01/2008:1184 on 2.0 g.
corrected 6.0
Saponification value (2.5.6). Use 2.0 g.
CAPRYLOCAPROYL Ethylene oxide units Type of macrogol Saponification value
MACROGOLGLYCERIDES per molecule
(nominal value)
4 200 265 to 285
Macrogolglyceridorum caprylocaprates 6 300 170 to 190
DEFINITION 8 400 85 to 105
Mixtures of monoesters, diesters and triesters of glycerol and
monoesters and diesters of macrogols with a mean relative Alkaline impurities. Introduce 5.0 g into a test-tube and
molecular mass between 200 and 400. carefully add a mixture, neutralised if necessary with 0.01 M
They are obtained by partial alcoholysis of medium-chain hydrochloric acid or with 0.01 M sodium hydroxide, of 0.05 mL
triglycerides using macrogol or by esterification of glycerol of a 0.4 g/L solution of bromophenol blue R in ethanol (96 per
and macrogol with caprylic (octanoic) acid and capric cent) R, 0.3 mL of water R and 10 mL of ethanol (96 per
(decanoic) acid or a mixture of glycerol esters and condensates cent) R. Shake and allow to stand. Not more than 1.0 mL of
of ethylene oxide with caprylic acid and capric acid. They may 0.01 M hydrochloric acid is required to change the colour of
contain free macrogols. the upper layer to yellow.

General Notices (1) apply to all monographs and other texts 1757
Captopril EUROPEAN PHARMACOPOEIA 8.0

Free glycerol : maximum 5.0 per cent. Specific optical rotation (2.2.7) : − 132 to − 127 (dried
Dissolve 1.20 g in 25.0 mL of methylene chloride R. Heat if substance).
necessary. After cooling, add 100 mL of water R. Shake and Dissolve 0.250 g in anhydrous ethanol R and dilute to 25.0 mL
add 25.0 mL of periodic acetic acid solution R. Shake and with the same solvent.
allow to stand for 30 min. Add 40 mL of a 75 g/L solution Impurity F. Gas chromatography (2.2.28).
of potassium iodide R. Allow to stand for 1 min. Add 1 mL
of starch solution R. Titrate the iodine with 0.1 M sodium Reagent solution. Add 2.8 mL of acetyl chloride R dropwise to
thiosulfate. Carry out a blank titration. 17.2 mL of anhydrous methanol R at 0 °C and mix. Allow to
stand for 20 min at room temperature before use.
1 mL of 0.1 M sodium thiosulfate is equivalent to 2.3 mg of
glycerol. Test solution. Introduce 20.0 mg of the substance to be
examined into a vial and add 1.0 mL of the reagent solution.
Composition of fatty acids (2.4.22, Method A). Mix and heat at 60 °C for 30 min. Evaporate to dryness under
Composition of the fatty-acid fraction of the substance : a stream of nitrogen R. Dissolve the residue in 0.5 mL of ethyl
– caproic acid : maximum 2.0 per cent ; acetate R, add 0.5 mL of pentafluoropropionic anhydride R,
– caprylic acid : 50.0 per cent to 80.0 per cent ; mix and heat at 60 °C for 30 min. Evaporate to dryness under
a stream of nitrogen R. Dissolve the residue in 1.0 mL of butyl
– capric acid : 20.0 per cent to 50.0 per cent ; acetate R.
– lauric acid : maximum 3.0 per cent ; Reference solution (a). Dissolve the contents of a vial of
– myristic acid : maximum 1.0 per cent. captopril for system suitability CRS (containing impurity F) in
Ethylene oxide and dioxan (2.4.25) : maximum 1 ppm of 1.0 mL of the reagent solution. Prepare as described for the
ethylene oxide and maximum 10 ppm of dioxan. test solution.
Heavy metals (2.4.8) : maximum 10 ppm. Reference solution (b). Mix 0.25 mL of reference solution (a)
and 0.75 mL of butyl acetate R.
2.0 g complies with test C. Prepare the reference solution using
2 mL of lead standard solution (10 ppm Pb) R. Column :
Water (2.5.12) : maximum 1.0 per cent, determined on 1.0 g. – material : fused silica ;
Use a mixture of 30 volumes of anhydrous methanol R and – size : l = 25 m, Ø = 0.32 mm ;
70 volumes of methylene chloride R as solvent. – stationary phase : poly(dimethyl)(diphenyl)siloxane R (film
Total ash (2.4.16) : maximum 0.1 per cent. thickness 1 μm).
Carrier gas : helium for chromatography R.
LABELLING Flow rate : 1.2 mL/min.
The label states the type of macrogol used (mean relative Split ratio : 1:20.
molecular mass) or the number of ethylene oxide units per
molecule (nominal value). Temperature :
Time Temperature
(min) (°C)
04/2012:1079
Column 0 - 10 200

CAPTOPRIL 10 - 14 200 → 240

14 - 34 240
Captoprilum Injection port 270
Detector 300

Detection : flame ionisation.

Injection : 1 μL.
C9H15NO3S Mr 217.3 Relative retention with reference to captopril (retention
[62571-86-2] time = about 6 min) : impurity F = about 0.96.
DEFINITION System suitability :
(2S)-1-[(2S)-2-Methyl-3-sulfanylpropanoyl]pyrrolidine-2- – resolution : minimum 1.5 between the peaks due to
carboxylic acid. impurity F and captopril in the chromatogram obtained
with reference solution (a) ;
Content : 98.0 per cent to 101.5 per cent (dried substance).
– signal-to-noise ratio : minimum 10 for the peak due to
CHARACTERS impurity F in the chromatogram obtained with reference
Appearance : white or almost white, crystalline powder. solution (b).
Solubility : soluble in water, freely soluble in methanol and in Calculate the percentage content of impurity F using the
methylene chloride. It dissolves in dilute solutions of alkali following expression :
hydroxides.
IDENTIFICATION
A. Specific optical rotation (see Tests).
A = area of the peak due to impurity F in the
B. Infrared absorption spectrophotometry (2.2.24). chromatogram obtained with the test solution ;
Comparison : captopril CRS. B = area of the peak due to captopril in the
TESTS chromatogram obtained with the test solution.
Solution S. Dissolve 0.5 g in carbon dioxide-free water R and Limit :
dilute to 25 mL with the same solvent. – impurity F : maximum 0.2 per cent.
Appearance of solution. Solution S is clear (2.2.1) and Related substances. Liquid chromatography (2.2.29).
colourless (2.2.2, Method II). Solvent mixture : phosphoric acid R, acetonitrile R1, water R
pH (2.2.3): 2.0 to 2.6 for solution S. (0.08:10:90 V/V/V).

1758 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Captopril

Test solution. Dissolve 0.125 g of the substance to be examined – impurity J : not more than 2.5 times the area of the
in the solvent mixture and dilute to 25.0 mL with the solvent corresponding peak in the chromatogram obtained with
mixture. reference solution (a) (0.2 per cent) ;
Reference solution (a). Dissolve 4.0 mg of captopril – impurities B, C, D : for each impurity, not more than
impurity J CRS, 5.0 mg of captopril impurity B CRS, 1.5 times the area of the corresponding peak in the
5.0 mg of captopril impurity C CRS and 5.0 mg of captopril chromatogram obtained with reference solution (a)
impurity D CRS in the solvent mixture and dilute to 50.0 mL (0.15 per cent);
with the solvent mixture. Dilute 1.0 mL of the solution to – impurity E : not more than 1.5 times the area of the
20.0 mL with the solvent mixture. Prepare immediately before principal peak in the chromatogram obtained with
use. reference solution (d) (0.15 per cent) ;
Reference solution (b). Dissolve 5 mg of the substance to – unspecified impurities : for each impurity, not more than the
be examined and 5 mg of captopril impurity E CRS in area of the principal peak in the chromatogram obtained
acetonitrile R and dilute to 25.0 mL with the same solvent. with reference solution (d) (0.10 per cent) ;
Dilute 4 mL of the solution to 50.0 mL with the solvent
mixture. – total : maximum 1.2 per cent ;
Reference solution (c). In order to prepare impurity A in – disregard limit : 0.5 times the area of the principal peak in
situ, introduce 1.0 mL of the test solution into a volumetric the chromatogram obtained with reference solution (d)
flask and add 230 μL of 0.05 M iodine. If the solution is not (0.05 per cent).
colourless, add 0.1 M sodium thiosulfate dropwise until it Heavy metals (2.4.8) : maximum 20 ppm.
becomes colourless, and dilute to 50.0 mL with the solvent Solvent : water R.
mixture. Dilute 5.0 mL of this solution to 20.0 mL with the
solvent mixture. 0.50 g complies with test H. Prepare the reference solution
using 1 mL of lead standard solution (10 ppm Pb) R.
Reference solution (d). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this Loss on drying (2.2.32) : maximum 1.0 per cent, determined
solution to 10.0 mL with the solvent mixture. on 1.000 g by drying under high vacuum at 60 °C for 3 h.
Column : Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
1.0 g.
– size : l = 0.3 m, Ø = 3.9 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for ASSAY
chromatography R (10 μm) ; Dissolve 0.150 g in 30 mL of water R. Titrate with 0.05 M
– temperature : 50 °C. iodine, determining the end-point potentiometrically (2.2.20).
Mobile phase : Use a combined platinum electrode.
– mobile phase A : phosphoric acid R, water R (0.08:100 V/V) ; 1 mL of 0.05 M iodine is equivalent to 21.73 mg of C9H15NO3S.
– mobile phase B : phosphoric acid R, acetonitrile R1, water R IMPURITIES
(0.08:50:50 V/V/V) ; Specified impurities : A, B, C, D, E, F, J.
Time Mobile phase A Mobile phase B Other detectable impurities (the following substances would,
(min) (per cent V/V) (per cent V/V) if present at a sufficient level, be detected by one or other of
0-5 90 10 the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
5 - 20 90 → 50 10 → 50
by the general monograph Substances for pharmaceutical
20 - 45 50 50 use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Flow rate : 1.5 mL/min. Control of impurities in substances for pharmaceutical use) : G,
Detection : spectrophotometer at 210 nm. H, I, L, M, N, O.
Injection : 25 μL.
Identification of impurities : use the chromatogram obtained
with reference solution (a) to identify the peaks due to
impurities B, C, D and J ; use the chromatogram obtained with
reference solution (b) to identify the peak due to impurity E ; A. 1,1′-[disulfanediylbis[(2S)-2-methyl-1-oxopropane-3,1-
use the chromatogram obtained with reference solution (c) to diyl]]bis[(2S)-pyrrolidine-2-carboxylic] acid (captopril
identify the peak due to impurity A. disulfide),
Relative retention with reference to captopril (retention
time = about 15 min) : impurity C = about 0.6 ;
impurity D = about 0.8 ; impurity E = about 0.9 ;
impurity B = about 1.17 ; impurity J = about 1.22 ;
impurity A = about 1.7.
System suitability : B. (2S)-1-[(2S)-3-bromo-2-methylpropanoyl]-
– resolution : minimum 1.5 between the peaks due to pyrrolidine-2-carboxylic acid,
impurities B and J in the chromatogram obtained with
reference solution (a) ;
– resolution : minimum 2.0 between the peaks due to
impurity E and captopril in the chromatogram obtained
C. (2RS)-2-methyl-3-sulfanylpropanoic acid,
with reference solution (b).
Limits :
– impurity A : not more than 10 times the area of the principal
peak in the chromatogram obtained with reference
solution (d) (1.0 per cent) ; D. (2RS)-3-bromo-2-methylpropanoic acid,

General Notices (1) apply to all monographs and other texts 1759
Carbachol EUROPEAN PHARMACOPOEIA 8.0

01/2008:1971
corrected 6.0

CARBACHOL
E. (2S)-1-(2-methylpropanoyl)pyrrolidine-2-carboxylic acid,
Carbacholum

F. (2S)-1-[(2R)-2-methyl-3-sulfanylpropanoyl]pyrrolidine-2- C6H15ClN2O2 Mr 182.7

carboxylic acid (epi-captopril), [51-83-2]
DEFINITION
2-(Carbamoyloxy)-N,N,N-trimethylethanaminium chloride.
Content : 99.0 per cent to 101.5 per cent (dried substance).
G. (2RS)-3-(acetylsulfanyl)-2-methylpropanoic acid, CHARACTERS
Appearance : white or almost white, crystalline, hygroscopic
powder.
Solubility : very soluble in water, sparingly soluble in alcohol,
practically insoluble in acetone.
H. (2S)-1-[(2S)-3-[[(2R)-3-(acetylsulfan- IDENTIFICATION
yl)-2-methylpropanoyl]sulfanyl]-2-methylpropano- First identification : A, C.
yl]pyrrolidine-2-carboxylic acid,
Second identification : B, C.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: carbachol CRS.
B. Examine the chromatograms obtained in the test for related
substances.
I. (2S)-1-[(2S)-3-[[[(2S)-1-[(2S)-2-methyl-3- Results : the principal spot in the chromatogram obtained
sulfanylpropanoyl]pyrrolidin-2-yl]carbonyl]sulfanyl]-2- with test solution (b) is similar in position, colour and size
methylpropanoyl]pyrrolidine-2-carboxylic acid, to the principal spot in the chromatogram obtained with
reference solution (a).
C. 0.5 mL of solution S (see Tests) gives reaction (a) of
chlorides (2.3.1).
TESTS
J. (2S)-1-[(2S)-3-(acetylsulfanyl)-2-methylpropanoyl]pyrroli- Solution S. Dissolve 2.5 g in carbon dioxide-free water R and
dine-2-carboxylic acid (acetylcaptopril), dilute to 25 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Acidity or alkalinity. To 2.0 mL of solution S, add 0.05 mL of
methyl red mixed solution R. Not more than 0.2 mL of 0.01 M
hydrochloric acid or 0.01 M sodium hydroxide is required to
L. 1,1′-[methylenebis[sulfanediyl[(2S)-2-methyl-1- change the colour of the indicator.
oxopropane-3,1-diyl]]]bis[(2S)-pyrrolidine-2-carboxylic]
acid, Related substances. Thin-layer chromatography (2.2.27).
Prepare the solutions immediately before use.
Test solution (a). Dissolve 0.20 g of the substance to be
examined in methanol R and dilute to 5.0 mL with the same
solvent.
Test solution (b). Dilute 2.0 mL of test solution (a) to 20.0 mL
M. (2S)-1-[(2S)-3-[[(2S)-2-carboxypropyl]disulfanyl]-2- with methanol R.
methylpropanoyl]pyrrolidine-2-carboxylic acid,
Reference solution (a). Dissolve 20 mg of carbachol CRS in
methanol R and dilute to 5.0 mL with the same solvent.
Reference solution (b). Dissolve 8 mg of choline chloride R and
8 mg of acetylcholine chloride CRS in methanol R and dilute
to 10.0 mL with the same solvent. Dilute 5.0 mL to 10.0 mL
N. 3,3′-disulfanediylbis[(2S)-2-methylpropanoic] acid, with methanol R.
Plate : cellulose for chromatography R as the coating substance.
Mobile phase : water R, methanol R (10:90 V/V).
Application : 10 μL.
Development : over 2/3 of the plate.
O. 1,1′-[propane-2,2-diylbis[sulfanediyl[(2S)-2-methyl-1- Detection : spray with potassium iodobismuthate solution R3.
oxopropane-3,1-diyl]]]bis[(2S)-pyrrolidine-2-carboxylic] System suitability : the chromatogram obtained with reference
acid. solution (b) shows 2 clearly separated spots.

1760 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Carbamazepine

Limits : in the chromatogram obtained with test solution (a) : TESTS

– any impurity : any spot, apart from the principal spot, is Acidity or alkalinity. To 1.0 g add 20 mL of carbon
not more intense than one or other of the 2 principal spots dioxide-free water R, shake for 15 min and filter. To 10 mL of
in the chromatogram obtained with reference solution (b) the filtrate add 0.05 mL of phenolphthalein solution R1 and
(1 per cent). Compare the spots with the spot of the most 0.5 mL of 0.01 M sodium hydroxide ; the solution is red. Add
appropriate colour in the chromatogram obtained with 1.0 mL of 0.01 M hydrochloric acid ; the solution is colourless.
reference solution (b). Add 0.15 mL of methyl red solution R ; the solution is red.
Heavy metals (2.4.8) : maximum 20 ppm. Related substances. Liquid chromatography (2.2.29).
12 mL of solution S complies with test A. Prepare the reference Test solution (a). Dissolve 60.0 mg of the substance to be
solution using lead standard solution (2 ppm Pb) R. examined in methanol R2 and dilute to 20.0 mL with the same
Loss on drying (2.2.32) : maximum 1.0 per cent, determined solvent. Sonicate. Dilute 10.0 mL of this solution to 20.0 mL
on 1.000 g by drying in an oven at 105 °C for 2 h. with water R.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Test solution (b). Dilute 10.0 mL of test solution (a) to 50.0 mL
1.0 g of the residue obtained in the test for loss on drying. with a mixture of equal volumes of methanol R2 and water R.
Reference solution (a). Dissolve 7.5 mg of carbamazepine CRS,
ASSAY 7.5 mg of carbamazepine impurity A CRS and 7.5 mg of
Dissolve 0.150 g in a mixture of 10 mL of anhydrous acetic iminodibenzyl R (impurity E) in methanol R2 and dilute to
acid R and 40 mL of acetic anhydride R. Titrate with 0.1 M 100.0 mL with the same solvent. Dilute 1.0 mL of this solution
perchloric acid. Determine the end-point potentiometrically to 50.0 mL with a mixture of equal volumes of methanol R2
(2.2.20). and water R.
1 mL of 0.1 M perchloric acid is equivalent to 18.27 mg of Reference solution (b). Dissolve 60.0 mg of carbamazepine CRS
C6H15ClN2O2. in methanol R2 and dilute to 20.0 mL with the same solvent.
Sonicate. Dilute 5.0 mL of this solution to 50.0 mL with a
STORAGE mixture of equal volumes of methanol R2 and water R.
In an airtight container, protected from light. Column :
IMPURITIES – size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : nitrile silica gel for chromatography R1
(10 μm).
Mobile phase : tetrahydrofuran R, methanol R2, water R
A. 2-hydroxy-N,N,N-trimethylethanaminium chloride (3:12:85 V/V/V) ; to 1000 mL of this solution add 0.2 mL of
(choline chloride). anhydrous formic acid R and 0.5 mL of triethylamine R.
Flow rate : 2.0 mL/min.
Detection : spectrophotometer at 230 nm.
04/2010:0543 Injection : 20 μL of test solution (a) and reference solution (a).
Run time : 8 times the retention time of carbamazepine.
CARBAMAZEPINE Relative retention with reference to carbamazepine
(retention time = about 10 min) : impurity A = about 0.9 ;
Carbamazepinum impurity E = about 3.5.
System suitability :
– resolution : minimum 1.7 between the peaks due to
impurity A and carbamazepine in the chromatogram
obtained with reference solution (a).
Limits :
– impurities A, E : for each impurity, not more than 1.5 times
the area of the corresponding peak in the chromatogram
C15H12N2O Mr 236.3 obtained with reference solution (a) (0.15 per cent) ;
[298-46-4] – unspecified impurities : not more than the area of the peak
DEFINITION due to carbamazepine in the chromatogram obtained with
reference solution (a) (0.10 per cent) ;
5H-Dibenzo[b,f]azepine-5-carboxamide.
– total : not more than 5 times the area of the peak due
Content : 98.0 per cent to 102.0 per cent (dried substance). to carbamazepine in the chromatogram obtained with
CHARACTERS reference solution (a) (0.5 per cent) ;
Appearance : white or almost white, crystalline powder. – disregard limit : 0.5 times the area of the peak due to
carbamazepine in the chromatogram obtained with
Solubility : very slightly soluble in water, freely soluble in reference solution (a) (0.05 per cent).
methylene chloride, sparingly soluble in acetone and in
ethanol (96 per cent). Chlorides (2.4.4) : maximum 140 ppm.
It shows polymorphism (5.9). The acceptable crystalline form Suspend 0.715 g in 20 mL of water R and boil for 10 min. Cool
corresponds to carbamazepine CRS. and dilute to 20 mL with water R. Filter through a membrane
filter (nominal pore size 0.8 μm). Dilute 10 mL of the filtrate
IDENTIFICATION to 15 mL with water R.
A. Melting point (2.2.14) : 189 °C to 193 °C. Heavy metals (2.4.8) : maximum 20 ppm.
B. Infrared absorption spectrophotometry (2.2.24). 1.0 g complies with test C. Prepare the reference solution using
Comparison : carbamazepine CRS. 2 mL of lead standard solution (10 ppm Pb) R.
Preparation : examine the substances as discs without prior Loss on drying (2.2.32) : maximum 0.5 per cent, determined
treatment. on 1.000 g by drying in an oven at 105 °C for 2 h.

General Notices (1) apply to all monographs and other texts 1761
Carbasalate calcium EUROPEAN PHARMACOPOEIA 8.0

Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on

1.0 g.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications.
Injection : test solution (b) and reference solution (b). F. 5H-dibenzo[b,f]azepine-5-carbonyl chloride
System suitability : (5-chlorocarbonyliminostilbene),
– repeatability : reference solution (b).
Calculate the percentage content of C15H12N2O from the
declared content of carbamazepine CRS.
STORAGE
In an airtight container.
IMPURITIES G. 10-bromo-5H-dibenzo[b,f]azepine-5-carboxamide
Specified impurities : A, E. (10-bromocarbamazepine).
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of 04/2010:1185
the tests in the monograph. They are limited by the general corrected 7.0
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities CARBASALATE CALCIUM
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : B, C, D, F, G. Carbasalatum calcicum

C19H18CaN2O9 Mr 458.4
A. 10,11-dihydro-5H-dibenzo[b,f]azepine-5-carboxamide [5749-67-7]
(10,11-dihydrocarbamazepine),
DEFINITION
Equimolecular compound of calcium di[2-(acetyloxy)ben-
zoate] and urea.
Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
B. 9-methylacridine, Solubility : freely soluble in water and in dimethylformamide,
practically insoluble in acetone and in anhydrous methanol.
Protect the substance from moisture during handling.
Examination in aqueous solutions has to be performed
immediately after preparation.
IDENTIFICATION
First identification : B, E.
Second identification : A, C, D, E.
C. (5H-dibenzo[b,f]azepin-5-ylcarbonyl)urea (N-carbamoyl-
carbamazepine), A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Dissolve 0.250 g in water R and dilute to
100.0 mL with the same solvent. To 1.0 mL of the solution
add 75 mL of water R and 5 mL of dilute hydrochloric
acid R, mix and dilute to 100.0 mL with water R. Examine
immediately.
Spectral range : 220-350 nm.
Absorption maxima : at 228 nm and 276 nm.
D. 5H-dibenzo[b,f]azepine (iminostilbene),
Specific absorbance at the absorption maxima :
– at 228 nm : 363 to 379,
– at 276 nm : 49 to 53.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: Ph. Eur. reference spectrum of carbasalate
calcium.
C. Dissolve 0.1 g in 10 mL of water R, boil for 2 min and cool.
E. 10,11-dihydro-5H-dibenzo[b,f]azepine (iminodibenzyl), The solution gives reaction (a) of salicylates (2.3.1).

1762 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Carbasalate calcium

D. Heat 0.2 g with 0.2 g of sodium hydroxide R ; a yellow or – disregard limit : 0.3 times the area of the principal peak in
yellowish-brown colour is produced and the vapour turns the chromatogram obtained with reference solution (b)
red litmus paper R blue. (0.03 per cent).
E. It gives reaction (a) of calcium (2.3.1). Sodium : maximum 0.1 per cent.
Atomic emission spectrometry (2.2.22, Method I).
TESTS
Test solution. Dissolve 1.0 g in 500.0 mL of water R.
Appearance of solution. The solution is not more opalescent
than reference suspension II (2.2.1) and is colourless Heavy metals (2.4.8) : maximum 10 ppm.
(2.2.2, Method II). Dissolve 2.0 g in 8 mL of water R with heating, cool and add
Dissolve 2.5 g in 50 mL of water R. 12 mL of acetone R. 12 mL of the solution complies with test B.
Prepare the reference solution using 10 mL of lead standard
Related substances. Liquid chromatography (2.2.29). Prepare solution (1 ppm Pb) R.
the solutions immediately before use.
Water (2.5.12) : maximum 0.1 per cent, determined on 1.000 g.
Solvent mixture : phosphoric acid R, methanol R, acetonitrile Use a mixture of 15 mL of anhydrous methanol R and 15 mL
for chromatography R (0.5:8:92 V/V/V). of dimethylformamide R as the solvent.
Test solution. Dissolve 0.100 g of the substance to be examined
in 5 mL of the solvent mixture, sonicate for 15 min and ASSAY
dilute to 10.0 mL with the solvent mixture. Filter the solution In a flask with a ground-glass stopper, dissolve 0.400 g in
through a membrane filter (nominal pore size 0.45 μm). 25 mL of water R. Add 25.0 mL of 0.1 M sodium hydroxide.
Close the flask and allow to stand for 2 h. Titrate with 0.1 M
Reference solution (a). Dissolve 10.0 mg of salicylic acid CRS
hydrochloric acid, using 0.2 mL of phenolphthalein solution R.
(impurity C) in the solvent mixture and dilute to 100.0 mL
Carry out a blank titration.
with the solvent mixture.
1 mL of 0.1 M sodium hydroxide is equivalent to 22.92 mg
Reference solution (b). Dilute 1.0 mL of reference solution (a) of C H CaN O .
19 18 2 9
to 10.0 mL with the solvent mixture.
Reference solution (c). Dissolve 2 mg of carbasalate STORAGE
impurity B CRS in 20.0 mL of the solvent mixture. In an airtight container.
Reference solution (d). Dilute 1.0 mL of the test solution to
10.0 mL with the solvent mixture. Mix 1.0 mL of this solution IMPURITIES
with 5.0 mL of reference solution (a), add 1.0 mL of reference Specified impurities : B, C.
solution (c) and dilute to 10.0 mL with the solvent mixture. Other detectable impurities (the following substances would,
Column : if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
– size : l = 0.25 m, Ø = 4.0 mm ; acceptance criterion for other/unspecified impurities and/or
– stationary phase : spherical end-capped octadecylsilyl silica by the general monograph Substances for pharmaceutical use
gel for chromatography R (5 μm) ; (2034). It is therefore not necessary to identify these impurities
– temperature : 40 °C. for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : A, D.
Mobile phase : phosphoric acid R, acetonitrile for
chromatography R, water R (0.5:40:60 V/V/V).
Flow rate : 1.8 mL/min.
Detection : spectrophotometer at 240 nm.
Injection : 10 μL of the test solution and reference solutions (b)
and (d).
Run time : 8 times the retention time of acetylsalicylic acid. A. 2-(acetyloxy)benzoic anhydride,
Identification of impurities : use the chromatogram obtained
with reference solution (d) to identify the peaks due to
impurities B and C.
Relative retention with reference to acetylsalicylic acid
(retention time = about 2 min) : impurity C = about 1.3 ;
impurity B = about 2.5.
System suitability : reference solution (d) :
B. 2-[[2-(acetyloxy)benzoyl]oxy]benzoic acid (acetylsalicyl-
– resolution : minimum 5.0 between the peaks due to salicylic acid),
acetylsalicylic acid and impurity C.
Limits :
– impurity C : not more than 5 times the area of the
corresponding peak in the chromatogram obtained with
reference solution (b) (0.5 per cent) ; C. 2-hydroxybenzenecarboxylic acid (salicylic acid),
– impurity B : not more than 1.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.15 per cent) ;
– unspecified impurities : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.05 per cent) ;
– total : not more than 7 times the area of the principal peak
in the chromatogram obtained with reference solution (b) D. 2-[(2-hydroxybenzoyl)oxy]benzoic acid (salicylsalicylic
(0.7 per cent) ; acid).

General Notices (1) apply to all monographs and other texts 1763
Carbidopa EUROPEAN PHARMACOPOEIA 8.0

01/2008:0755 from time to time during 30 min. Decant the liquid from both
corrected 6.0 flasks and repeat the process with further quantities, each of
150 mL, of carbon dioxide-free water R.
CARBIDOPA Take two 100 mL measuring cylinders 3.5-4.5 cm in internal
diameter and label these A and B. Into cylinder A, transfer
Carbidopum as completely as possible the resin from 1 conical flask using
60 mL of carbon dioxide-free water R ; into cylinder B, transfer
the 2nd quantity of resin, this time using 20 mL of carbon
dioxide-free water R.
Into each cylinder, insert a gas-inlet tube, the end of which has
an internal diameter of 2-3 mm and which reaches almost to
the bottom of the cylinder. Pass a rapid stream of nitrogen for
C10H14N2O4,H2O Mr 244.2 chromatography R through each mixture so that homogeneous
[38821-49-7] suspensions are formed. After 30 min, without interrupting
DEFINITION the gas flow, add 1.0 mL of test solution (a) to cylinder A ;
after 1 min stop the gas flow into cylinder A and transfer the
(2S)-3-(3,4-Dihydroxyphenyl)-2-hydrazino-2-methyl-
contents, through a moistened filter paper, into cylinder B.
propanoic acid monohydrate.
After 1 min, stop the gas flow to cylinder B and pour the
Content : 98.5 per cent to 101.0 per cent (dried substance). solution immediately through a moistened filter paper into
CHARACTERS a freshly prepared mixture of 1 mL of a 200 g/L solution
of salicylaldehyde R in methanol R and 20 mL of phosphate
Appearance : white or yellowish-white powder. buffer solution pH 5.5 R in a conical flask ; shake thoroughly
Solubility : slightly soluble in water, very slightly soluble in for 1 min and heat in a water-bath at 60 °C for 15 min. The
ethanol (96 per cent), practically insoluble in methylene liquid becomes clear. Allow to cool, add 2.0 mL of toluene R
chloride. It dissolves in dilute solutions of mineral acids. and shake vigorously for 2 min. Transfer the mixture into a
IDENTIFICATION centrifuge tube and centrifuge.
First identification : A, C. Separate the toluene layer in a 100 mL separating funnel and
shake vigorously with 2 quantities, each of 20 mL, of a 200 g/L
Second identification : A, B, D, E. solution of sodium metabisulfite R and finally with 2 quantities,
A. Specific optical rotation (see Tests). each of 50 mL, of water R. Separate the toluene layer.
B. Ultraviolet and visible absorption spectrophotometry Reference solution (a). Dissolve 10 mg of hydrazine sulfate R
(2.2.25). in dilute hydrochloric acid R and dilute to 50 mL with the
Test solution. Dissolve 50.0 mg in a 8.5 g/L solution of same acid. Dilute 1.0 mL of this solution to 10.0 mL with
hydrochloric acid R in methanol R and dilute to 100.0 mL dilute hydrochloric acid R.
with the same solution. Dilute 10.0 mL of this solution to Reference solution (b). Prepare the solution at the same time
100.0 mL with a 8.5 g/L solution of hydrochloric acid R in and in the same manner as described for test solution (b)
methanol R. using 1.0 mL of reference solution (a) instead of 1.0 mL of
Spectral range : 230-350 nm. test solution (a).
Absorption maximum : at 283 nm. Plate : TLC silanised silica gel plate R.
Specific absorbance at the absorption maximum : 135 to 150 Mobile phase : water R, methanol R (10:20 V/V).
(dried substance). Application : 10 μL of test solution (b) and reference
C. Infrared absorption spectrophotometry (2.2.24). solution (b).
Preparation : discs. Development : over a path of 10 cm.
Comparison : carbidopa CRS. Drying : in air.
D. Shake vigorously about 5 mg with 10 mL of water R for Detection : examine in ultraviolet light at 365 nm.
1 min and add 0.3 mL of ferric chloride solution R2. An Limit :
intense green colour is produced, which quickly turns to
reddish-brown. – hydrazine : any spot showing a yellow fluorescence is
not more intense than the corresponding spot in the
E. Suspend about 20 mg in 5 mL of water R and add 5 mL chromatogram obtained with reference solution (b)
of cupri-tartaric solution R. On heating, the colour of the (20 ppm).
solution changes to dark brown and a red precipitate is
formed. Methyldopa and methylcarbidopa. Liquid chromatography
(2.2.29).
TESTS Test solution. Dissolve 0.100 g of the substance to be examined
Appearance of solution. The solution is clear (2.2.1) and not in 0.1 M hydrochloric acid and dilute to 10.0 mL with the
more intensely coloured than reference solution BY6 or B6 same acid.
(2.2.2, Method II). Reference solution (a). Dissolve the contents of a vial of
Dissolve 0.25 g in 25 mL of 1 M hydrochloric acid. methylcarbidopa CRS in 0.1 M hydrochloric acid, add 1 mg of
Specific optical rotation (2.2.7) : − 22.5 to − 26.5 (dried methyldopa CRS and dilute to 20.0 mL with the same acid.
substance). Reference solution (b). Dissolve 5 mg of carbidopa CRS and
With the aid of an ultrasonic bath, dissolve completely 0.250 g 5 mg of methyldopa CRS in 0.1 M hydrochloric acid and dilute
in aluminium chloride solution R and dilute to 25.0 mL with to 10.0 mL with the same acid.
the same solution. Column :
Hydrazine. Thin-layer chromatography (2.2.27). – size : l = 0.25 m, Ø = 4.6 mm ;
Test solution (a). Dissolve 0.50 g in dilute hydrochloric acid R – stationary phase : octylsilyl silica gel for chromatography R
and dilute to 2.0 mL with the same acid. (5 μm).
Test solution (b). Place 25 g of strongly basic anion-exchange Mobile phase : methanol R, 14 g/L solution of potassium
resin R into each of 2 conical flasks with ground-glass stoppers. dihydrogen phosphate R (2:98 V/V).
To each, add 150 mL of carbon dioxide-free water R and shake Flow rate : 1 mL/min.

1764 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Carbimazole

Detection : spectrophotometer at 282 nm. Drying : in air for 30 min.

Injection : 20 μL. Detection : examine in ultraviolet light at 254 nm.
System suitability : reference solution (b) : Results : the principal spot in the chromatogram obtained
– resolution : minimum 4.0 between the peaks due to with the test solution is similar in position and size to the
methyldopa and carbidopa. principal spot in the chromatogram obtained with the
Limits : reference solution.
– methyldopa and methylcarbidopa : for each impurity, not D. Dissolve about 10 mg in a mixture of 0.05 mL of dilute
more than the area of the corresponding peak in the hydrochloric acid R and 50 mL of water R. Add 1 mL of
chromatogram obtained with reference solution (a) (0.5 per potassium iodobismuthate solution R. A red precipitate is
cent). formed.
Heavy metals (2.4.8) : maximum 20 ppm. TESTS
1.0 g complies with test C. Prepare the reference solution using Related substances. Liquid chromatography (2.2.29). Prepare
2 mL of lead standard solution (10 ppm Pb) R. the solutions immediately before use.
Loss on drying (2.2.32) : 6.9 per cent to 7.9 per cent, Solvent mixture : acetonitrile R, water R (20:80 V/V).
determined on 1.000 g by drying in an oven at 105 °C. Test solution. Dissolve 25.0 mg of the substance to be
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on examined in the solvent mixture and dilute to 50.0 mL with
1.0 g. the solvent mixture.
Reference solution (a). Dissolve 5 mg of thiamazole CRS
ASSAY (impurity A) in the solvent mixture and dilute to 100.0 mL
Dissolve 0.150 g with gentle heating in 75 mL of anhydrous with the solvent mixture. Mix 1 mL of the solution with 2 mL
acetic acid R. Titrate with 0.1 M perchloric acid, determining of the test solution and dilute to 10.0 mL with the solvent
the end-point potentiometrically (2.2.20). mixture.
1 mL of 0.1 M perchloric acid is equivalent to 22.62 mg Reference solution (b). Dissolve 5.0 mg of thiamazole CRS
of C10H14N2O4. (impurity A) in the solvent mixture and dilute to 100.0 mL
with the solvent mixture. Dilute 1.0 mL of the solution to
STORAGE 50.0 mL with the solvent mixture.
Protected from light. Reference solution (c). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
07/2012:0884 solution to 10.0 mL with the solvent mixture.
Reference solution (d). Dissolve 25.0 mg of carbimazole CRS
CARBIMAZOLE in the solvent mixture and dilute to 50.0 mL with the solvent
mixture.
Carbimazolum Column :
– size : l = 0.15 m, Ø = 3.9 mm;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : acetonitrile R, water R (10:90 V/V).
C7H10N2O2S Mr 186.2 Flow rate : 1 mL/min.
[22232-54-8] Detection : spectrophotometer at 254 nm.
DEFINITION Injection : 10 μL of the test solution and reference solutions (a),
Ethyl 3-methyl-2-thioxo-2,3-dihydro-1H-imidazole-1- (b) and (c).
carboxylate. Run time : 1.5 times the retention time of carbimazole.
Content : 98.0 per cent to 102.0 per cent (dried substance). Identification of impurities: use the chromatogram obtained
with reference solution (a) to identify the peak due to
CHARACTERS impurity A.
Appearance : white or yellowish-white, crystalline powder. Relative retention with reference to carbimazole (retention
Solubility : slightly soluble in water, soluble in acetone and in time = about 6 min) : impurity A = about 0.2.
ethanol (96 per cent). System suitability : reference solution (a) :
IDENTIFICATION – resolution : minimum 5.0 between the peaks due to
impurity A and carbimazole.
First identification : B.
Second identification : A, C, D. Limits :
A. Melting point (2.2.14) : 122 °C to 125 °C. – impurity A : not more than 0.75 times the area of the
principal peak in the chromatogram obtained with
B. Infrared absorption spectrophotometry (2.2.24). reference solution (b) (0.15 per cent) ;
Comparison : carbimazole CRS. – unspecified impurities : for each impurity, not more than the
C. Thin-layer chromatography (2.2.27). area of the principal peak in the chromatogram obtained
Test solution. Dissolve 10 mg of the substance to be with reference solution (c) (0.10 per cent) ;
examined in methylene chloride R and dilute to 10 mL with – total : maximum 0.2 per cent ;
the same solvent. – disregard limit : 0.5 times the area of the principal peak in
Reference solution. Dissolve 10 mg of carbimazole CRS in the chromatogram obtained with reference solution (c)
methylene chloride R and dilute to 10 mL with the same (0.05 per cent).
solvent.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Plate : TLC silica gel F254 plate R. on 1.000 g by drying in a desiccator over diphosphorus
Mobile phase : acetone R, methylene chloride R (20:80 V/V). pentoxide R at a pressure not exceeding 0.7 kPa for 24 h.
Application : 10 μL. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Development : over 3/4 of the plate. 1.0 g.

General Notices (1) apply to all monographs and other texts 1765
Carbocisteine EUROPEAN PHARMACOPOEIA 8.0

ASSAY Ninhydrin-positive substances. Examine by thin-layer

Liquid chromatography (2.2.29) as described in the test for chromatography (2.2.27), using a suitable silica gel as the
related substances with the following modification. coating substance.
Injection : test solution and reference solution (d). Test solution (a). Dissolve 0.10 g of the substance to be
Calculate the percentage content of C7H10N2O2S taking into examined in dilute ammonia R2 and dilute to 10 mL with the
account the assigned content of carbimazole CRS. same solvent.
Test solution (b). Dilute 1 mL of test solution (a) to 50 mL
IMPURITIES with water R.
Specified impurities : A. Reference solution (a). Dissolve 10 mg of carbocisteine CRS in
dilute ammonia R2 and dilute to 50 mL with the same solvent.
Reference solution (b). Dilute 5 mL of test solution (b) to
20 mL with water R.
Reference solution (c). Dissolve 10 mg of carbocisteine CRS
A. 1-methyl-1H-imidazole-2-thiol (thiamazole). and 10 mg of arginine hydrochloride CRS in 5 mL of dilute
ammonia R2 and dilute to 25 mL with water R.
01/2008:0885 Apply separately to the plate 5 μL of each solution. Allow
corrected 6.0 the plate to dry in air. Develop over a path of 15 cm using
a mixture of 20 volumes of glacial acetic acid R, 20 volumes
CARBOCISTEINE of water R and 60 volumes of butanol R. Dry the plate in a
current of warm air. Spray with ninhydrin solution R and heat
Carbocisteinum at 100 °C to 105 °C for 15 min. Any spot in the chromatogram
obtained with test solution (a), apart from the principal
spot, is not more intense than the spot in the chromatogram
obtained with reference solution (b) (0.5 per cent). The test
is not valid unless the chromatogram obtained with reference
C5H9NO4S Mr 179.2 solution (c) shows two clearly separated principal spots.
[638-23-3] Chlorides (2.4.4). Dissolve 33 mg in 5 mL of dilute nitric
DEFINITION acid R and dilute to 15 mL with water R. The solution, without
further addition of nitric acid, complies with the limit test for
Carbocisteine contains not less than 98.5 per cent
chlorides (0.15 per cent).
and not more than the equivalent of 101.0 per cent of
(2R)-2-amino-3-[(carboxymethyl)sulfanyl]propanoic acid, Sulfates (2.4.13). Dissolve 0.5 g in 5 mL of dilute hydrochloric
calculated with reference to the dried substance. acid R and dilute to 15 mL with distilled water R. The solution
complies with the limit test for sulfates (300 ppm).
CHARACTERS
Heavy metals (2.4.8). 2.0 g complies with test D for heavy
A white or almost white, crystalline powder, practically metals (10 ppm). Prepare the reference solution using 2 mL
insoluble in water and in alcohol. It dissolves in dilute mineral of lead standard solution (10 ppm Pb) R.
acids and in dilute solutions of alkali hydroxides.
Loss on drying (2.2.32). Not more than 0.5 per cent,
IDENTIFICATION determined on 1.000 g by drying in an oven at 105 °C for 2 h.
First identification : A, B. Sulfated ash (2.4.14). Not more than 0.3 per cent, determined
Second identification : A, C, D. on 1.0 g.
A. Specific optical rotation (see Tests). ASSAY
B. Examine by infrared absorption spectrophotometry
Dissolve 0.150 g in 10 mL of anhydrous formic acid R with
(2.2.24), comparing with the spectrum obtained with
slight heating and shake until dissolution is complete. Add
carbocisteine CRS. Examine the substances prepared as
50 mL of anhydrous acetic acid R. Titrate with 0.1 M perchloric
discs.
acid, determining the end-point potentiometrically (2.2.20).
C. Examine the chromatograms obtained in the test for
ninhydrin-positive substances. The principal spot in the 1 mL of 0.1 M perchloric acid is equivalent to 17.92 mg of
C5H9NO4S.
chromatogram obtained with test solution (b) is similar
in position, colour and size to the principal spot in the STORAGE
chromatogram obtained with reference solution (a). Store protected from light.
D. Dissolve 0.1 g in 4.5 mL of dilute sodium hydroxide
solution R. Heat on a water-bath for 10 min. Cool and add
1 mL of a 25 g/L solution of sodium nitroprusside R. A dark 04/2009:1299
red colour is produced, which changes to brown and then
to yellow within a few minutes. CARBOMERS
TESTS
Solution S. Disperse 5.00 g in 20 mL of water R and add Carbomera
dropwise with shaking 2.5 mL of strong sodium hydroxide DEFINITION
solution R. Adjust to pH 6.3 with 1 M sodium hydroxide and
dilute to 50.0 mL with water R. High-molecular-mass polymers of acrylic acid cross-linked
with alkenyl ethers of sugars or polyalcohols.
Appearance of solution. Solution S is clear (2.2.1) and
Content : 56.0 per cent to 68.0 per cent of carboxylic
colourless (2.2.2, Method II).
acid (-CO2H) groups (dried substance).
pH (2.2.3). Shake 0.2 g with 20 mL of carbon dioxide-free
water R. The pH of the suspension is 2.8 to 3.0. CHARACTERS
Specific optical rotation (2.2.7) : − 32.5 to − 35.5, determined Appearance : white or almost white, fluffy, hygroscopic powder.
on solution S and calculated with reference to the dried Solubility : swells in water and in other polar solvents after
substance. dispersion and neutralisation with sodium hydroxide solution.

1766 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Carbomers

IDENTIFICATION Close the vials with a tight rubber membrane stopper coated
First identification : A. with polytetrafluoroethylene and secure with an aluminium
Second identification : B, C, D. crimped cap. Shake to obtain a homogeneous dispersion.
A. Infrared absorption spectrophotometry (2.2.24). Static head-space conditions that may be used :
– equilibration temperature : 80 °C ;
Main bands : at 1710 ± 5 cm− 1, 1454 ± 5 cm− 1,
– equilibration time : 60 min ;
1414 ± 5 cm− 1, 1245 ± 5 cm− 1, 1172 ± 5 cm− 1, 1115 ± 5 cm− 1
– transfer-line temperature : 90 °C.
and 801 ± 5 cm− 1, with the strongest band at 1710 ± 5 cm− 1.
B. Adjust a 10 g/L dispersion to about pH 7.5 with 1 M sodium Injection : 1 mL of the gaseous phase of the test solution and
hydroxide. A highly viscous gel is formed. 1 mL of the gaseous phase of the reference solution ; repeat
C. Add 2 mL of a 100 g/L solution of calcium chloride R, with these injections twice more.
continuous stirring, to 10 mL of the gel from identificationSystem suitability :
test B. A white precipitate is immediately produced. – repeatability : maximum relative standard deviation of the
D. Add 0.5 mL of thymol blue solution R to 10 mL of a 10 g/L differences in area between the analyte peaks obtained
dispersion. An orange colour is produced. Add 0.5 mL of from the 3 replicate pair injections of the reference solution
cresol red solution R to 10 mL of a 10 g/L dispersion. A and the test solution is 15 per cent.
yellow colour is produced. Limit :
TESTS – benzene : the mean area of the peak due to benzene in
the chromatograms obtained with the test solution is not
Free acrylic acid. Liquid chromatography (2.2.29). greater than 0.5 times the mean area of the peak due to
Test solution. Mix 0.125 g of the substance to be examined benzene in the chromatograms obtained with the reference
with a 25 g/L solution of aluminium potassium sulfate R and solution (2 ppm).
dilute to 25.0 mL with the same solution. Heat the suspension Heavy metals (2.4.8) : maximum 20 ppm.
at 50 °C for 20 min with shaking, then shake the suspension
at room temperature for 60 min. Centrifuge and use the clear 1.0 g complies with test C. Prepare the reference solution using
supernatant solution as the test solution. 2 mL of lead standard solution (10 ppm Pb) R.
Reference solution. Dissolve 62.5 mg of acrylic acid R in a Loss on drying (2.2.32) : maximum 3.0 per cent, determined
25 g/L solution of aluminium potassium sulfate R and dilute on 1.000 g by drying in vacuo at 80 °C for 60 min.
to 100.0 mL with the same solution. Dilute 1.0 mL of this Sulfated ash (2.4.14) : maximum 4.0 per cent, determined on
solution to 50.0 mL with a 25 g/L solution of aluminium 1.0 g.
potassium sulfate R.
Column : ASSAY
– size : l = 0.12 m, Ø = 4.6 mm ; Slowly add 50 mL of water R to 0.120 g whilst stirring and
heating at 60 °C for 15 min. Stop heating, add 150 mL
– stationary phase : octadecylsilyl silica gel for of water R and continue stirring for 30 min. Add 2 g of
chromatography R (5 μm). potassium chloride R and titrate with 0.2 M sodium hydroxide,
Mobile phase : determining the end-point potentiometrically (2.2.20).
– mobile phase A : 1.361 g/L solution of potassium dihydrogen 1 mL of 0.2 M sodium hydroxide is equivalent to 9.0 mg of
phosphate R, adjusted to pH 2.5 using dilute phosphoric carboxylic acid (-CO2H) groups.
acid R ;
– mobile phase B : mixture of equal volumes of a 1.361 g/L STORAGE
solution of potassium dihydrogen phosphate R and In an airtight container.
acetonitrile for chromatography R ; FUNCTIONALITY-RELATED CHARACTERISTICS
Time Mobile phase A Mobile phase B This section provides information on characteristics that are
(min) (per cent V/V) (per cent V/V) recognised as being relevant control parameters for one or
0-8 100 0 more functions of the substance when used as an excipient (see
8-9 100 → 0 0 → 100 chapter 5.15). This section is a non-mandatory part of the
monograph and it is not necessary to verify the characteristics
9 - 20 0 100 to demonstrate compliance. Control of these characteristics can
however contribute to the quality of a medicinal product by
Flow rate : 1 mL/min. improving the consistency of the manufacturing process and
Detection : spectrophotometer at 205 nm. the performance of the medicinal product during use. Where
Injection : 20 μL. control methods are cited, they are recognised as being suitable
Retention time : acrylic acid = about 6.0 min. for the purpose, but other methods can also be used. Wherever
results for a particular characteristic are reported, the control
Limit :
method must be indicated.
– acrylic acid : not more than the area of the corresponding
The following characteristics may be relevant for carbomers
peak in the chromatogram obtained with the reference used as viscosity-increasing agents and gelling agents.
solution (0.25 per cent).
Apparent viscosity (2.2.10) : the nominal apparent viscosity
Benzene. Gas chromatography (2.4.24, System A). is typically between 300 mPa·s and 115 000 mPa·s. For a
Solution A. Dissolve 0.100 g of benzene R in dimethyl product with a nominal apparent viscosity of 20 000 mPa·s
sulfoxide R and dilute to 100.0 mL with the same solvent. or greater, the apparent viscosity is typically 70.0 per cent
Dilute 1.0 mL of the solution to 100.0 mL with water R. Dilute to 130.0 per cent of the nominal value ; for a product with
1.0 mL of this solution to 100.0 mL with water R. a nominal apparent viscosity of less than 20 000 mPa·s, the
Test solution. Weigh 50.0 mg of the substance to be examined apparent viscosity is typically 50.0 per cent to 150.0 per cent
into an injection vial and add 5.0 mL of water R and 1.0 mL of the nominal value.
of dimethyl sulfoxide R. Dry the substance to be examined in vacuo at 80 °C for 1 h.
Reference solution. Weigh 50.0 mg of the substance to be Carefully add 2.50 g of the previously dried substance to be
examined into an injection vial and add 4.0 mL of water R, examined to 500 mL of water R in a 1000 mL beaker while
1.0 mL of dimethyl sulfoxide R and 1.0 mL of solution A. stirring continuously at 1000 ± 50 r/min, with the stirrer

General Notices (1) apply to all monographs and other texts 1767
Carbon dioxide EUROPEAN PHARMACOPOEIA 8.0

shaft set at an angle of 60° to one side of the beaker. Add Limit :
the previously dried substance over a period of 45-90 s, at a – carbon monoxide : not more than the area of the
uniform rate, ensuring that loose agglomerates of powder corresponding peak in the chromatogram obtained with
are broken up, and continue stirring at 1000 ± 50 r/min for the reference gas (5 ppm V/V).
15 min. Remove the stirrer and place the beaker containing
the dispersion in a water-bath at 25 ± 1 °C for 30 min. Insert Nitrogen monoxide and nitrogen dioxide : maximum
2 ppm V/V in total, determined using a chemiluminescence
the stirrer to a depth necessary to ensure that air is not drawn
into the dispersion and, while stirring at 300 ± 25 r/min, analyser (2.5.26).
titrate with a glass-calomel electrode system to pH 7.3-7.8 Gas to be examined. The substance to be examined.
by adding a 180 g/L solution of sodium hydroxide R below Reference gas (a). Carbon dioxide R1.
the surface, determining the end-point potentiometrically Reference gas (b). A mixture containing 2 ppm V/V of nitrogen
(2.2.20). The total volume of the 180 g/L solution of sodium monoxide R in carbon dioxide R1 or in nitrogen R1.
hydroxide R used is about 6.2 mL. Allow 2-3 min before the Calibrate the apparatus and set the sensitivity using reference
final pH determination. If the final pH exceeds 7.8, discard gases (a) and (b). Measure the content of nitrogen monoxide
the preparation and prepare another using a smaller amount and nitrogen dioxide in the gas to be examined.
of sodium hydroxide for titration. Return the neutralised
preparation to the water-bath at 25 °C for 1 h, then perform If nitrogen is used instead of carbon dioxide in reference
the viscosity determination without delay to avoid slight gas (b), multiply the result obtained by the quenching
viscosity changes that occur 75 min after neutralisation. correction factor in order to correct the quenching effect on
Determine the viscosity using a rotating viscometer with a the analyser response caused by the carbon dioxide matrix
spindle rotating at 20 r/min, using a spindle suitable for the effect.
expected apparent viscosity. The quenching correction factor is determined by applying
a known reference mixture of nitrogen monoxide in carbon
Carboxylic acid groups : see Assay. dioxide and comparing the actual content with the content
indicated by the analyser which has been calibrated with a
01/2008:0375 NO/N reference mixture.
2

CARBON DIOXIDE =

Total sulfur : maximum 1 ppm V/V, determined using an

Carbonei dioxidum ultraviolet fluorescence analyser after oxidation of the sulfur
compounds by heating at 1000 °C (Figure 0375.-1).
CO2 Mr 44.01
[124-38-9] The apparatus consists of the following :
– a system generating ultraviolet radiation with a wavelength
DEFINITION of 210 nm, made up of an ultraviolet lamp, a collimator,
Content : minimum 99.5 per cent V/V of CO2 in the gaseous and a selective filter ; the beam is blocked periodically by a
phase. chopper rotating at high speed,
This monograph applies to carbon dioxide for medicinal use. – a reaction chamber through which flows the previously
filtered gas to be examined,
CHARACTERS
– a system that detects radiation emitted at a wavelength of
Appearance : colourless gas. 350 nm, made up of a selective filter, a photomultiplier
Solubility : at 20 °C and at a pressure of 101 kPa, 1 volume tube and an amplifier.
dissolves in about 1 volume of water. Gas to be examined. The substance to be examined.
PRODUCTION Reference gas (a). Carbon dioxide R1.
Examine the gaseous phase. Reference gas (b). A mixture containing between 0.5 ppm V/V
If the test is performed on a cylinder of gas, keep the cylinder of and 2 ppm V/V of hydrogen sulfide R1 in carbon dioxide R1.
the substance to be examined at room temperature for not less Calibrate the apparatus and set the sensitivity using reference
than 6 h before carrying out the tests. Keep the cylinder in the gases (a) and (b). Pass the gas to be examined through a quartz
vertical position with the outlet valve uppermost. oven heated to 1000 °C. Oxygen R is circulated in the oven at a
Carbon monoxide. Gas chromatography (2.2.28). tenth of the flow rate of the gas to be examined. Measure the
sulfur dioxide content in the gaseous mixture leaving the oven.
Gas to be examined. The substance to be examined.
Water : maximum 67 ppm V/V, determined using an
Reference gas. A mixture containing 5 ppm V/V of carbon
electrolytic hygrometer (2.5.28).
monoxide R in nitrogen R1.
Column : Assay. Infrared analyser (2.5.24).
– material : stainless steel, Gas to be examined. The substance to be examined. It must be
filtered to avoid stray light phenomena.
– size : l = 2 m, Ø = 4 mm,
Reference gas (a). Carbon dioxide R1.
– stationary phase : an appropriate molecular sieve for
chromatography (0.5 nm). Reference gas (b). A mixture containing 95.0 per cent V/V of
carbon dioxide R1 and 5.0 per cent V/V of nitrogen R1.
Carrier gas : helium for chromatography R.
Calibrate the apparatus and set the sensitivity using reference
Flow rate : 60 mL/min.
gases (a) and (b). Measure the content of carbon dioxide in
Temperature : the gas to be examined.
– column : 50 °C,
– injection port and detector : 130 °C. IDENTIFICATION
Detection : flame ionisation with methaniser. First identification : A.
Injection : loop injector. Second identification : B, C.
Adjust the injected volumes and the operating conditions so A. Infrared absorption spectrophotometry (2.2.24).
that the height of the peak due to carbon monoxide in the Comparison: Ph. Eur. reference spectrum of carbon dioxide.
chromatogram obtained with the reference gas is at least B. Place a glowing splinter of wood in an atmosphere of the
35 per cent of the full scale of the recorder. substance to be examined. It is extinguished.

1768 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Carbon monoxide

Figure 0375.-1.– UV Fluorescence Analyser

C. Pass a stream of the substance to be examined through DEFINITION
barium hydroxide solution R. A white precipitate is formed Gas obtained by steam reforming (catalytic oxidation) of
which dissolves with effervescence in dilute acetic acid R. hydrocarbons.
TESTS Content : minimum 99.5 per cent V/V of CO.
Examine the gaseous phase. This monograph applies to carbon monoxide for medicinal
If the test is performed on a cylinder of gas, keep the cylinder of use.
the substance to be examined at room temperature for not less CHARACTERS
than 6 h before carrying out the tests. Keep the cylinder in the
vertical position with the outlet valve uppermost. Appearance : colourless, flammable gas.
Solubility : at 20 °C and at a pressure of 101 kPa, 2.266 volumes
Carbon monoxide : maximum 5 ppm V/V, determined using of carbon monoxide dissolve in 100 volumes of water.
a carbon monoxide detector tube (2.1.6).
Hydrogen sulfide : maximum 1 ppm V/V, determined using a IDENTIFICATION
hydrogen sulfide detector tube (2.1.6). Carry out either test A or B.
Nitrogen monoxide and nitrogen dioxide : maximum A. Infrared absorption spectrophotometry (2.2.24).
2 ppm V/V in total, determined using a nitrogen monoxide Comparison: Ph. Eur. reference spectrum of carbon
and nitrogen dioxide detector tube (2.1.6). monoxide.
Sulfur dioxide : maximum 2 ppm V/V, determined using a B. It complies with the limits of the assay.
sulfur dioxide detector tube (2.1.6).
TESTS
Water vapour: maximum 67 ppm V/V, determined using a
water vapour detector tube (2.1.6). Carbon dioxide. Gas chromatography (2.2.28).
Gas to be examined. The substance to be examined.
STORAGE Reference gas. A mixture containing 300 ppm V/V of carbon
Store liquefied under pressure in suitable containers complying dioxide R1 in carbon monoxide R.
with the legal regulations. Column :
IMPURITIES – material : stainless steel ;
– size : l = 2 m, Ø = 2 mm ;
A. NO : nitrogen monoxide,
– stationary phase : an appropriate divinylbenzene porous
B. NO2 : nitrogen dioxide, polymer (149-177 μm).
C. CO : carbon monoxide, Carrier gas : helium for chromatography R.
Flow rate : 30 mL/min.
D. total sulfur, Temperature :
E. H2O : water. – column : 50 °C ;
– detector : 220 °C.
Detection : thermal conductivity.
01/2011:2408
corrected 7.2 Injection : 1 mL.
Run time : 3 min.
CARBON MONOXIDE Relative retention with reference to carbon monoxide
(retention time = about 0.4 min) : carbon dioxide = about 3.5.
Limit :
Carbonei monoxidum – carbon dioxide : not more than the area of the corresponding
peak in the chromatogram obtained with the reference gas
CO Mr 28.00 (300 ppm V/V).
[630-08-0]

General Notices (1) apply to all monographs and other texts 1769
Carboplatin EUROPEAN PHARMACOPOEIA 8.0

Methane. Gas chromatography (2.2.28). STORAGE

Gas to be examined. The substance to be examined. Under pressure in suitable containers complying with the
Reference gas. A mixture containing 100 ppm V/V of legal regulations.
methane R in carbon monoxide R. IMPURITIES
Column :
Specified impurities : A, B, C, D, E, F.
– material : stainless steel ;
A. CO2 : carbon dioxide,
– size : l = 2 m, Ø = 4 mm ;
B. CH4 : methane,
– stationary phase : ethylvinylbenzene-divinylbenzene
copolymer R (177-250 μm). C. H2 : hydrogen,
Carrier gas : nitrogen for chromatography R. D. Ni(CO)4 : nickel tetracarbonyl,
Flow rate : 10 mL/min. E. Fe(CO)5 : iron pentacarbonyl,
Temperature : F. H2O : water.
– column : 95 °C ;
– detector : 240 °C. 07/2009:1081
corrected 7.5
Detection : flame ionisation.
Injection : 1 mL. CARBOPLATIN
Run time : 3 min.
Retention time : methane = about 1.8 min. Carboplatinum
Limit :
– methane : not more than the area of the corresponding
peak in the chromatogram obtained with the reference gas
(100 ppm V/V).
Hydrogen. Gas chromatography.
Gas to be examined. The substance to be examined. C6H12N2O4Pt Mr 371.3
[41575-94-4]
Reference gas. A mixture containing 300 ppm V/V of hydrogen
for chromatography R in carbon monoxide R. DEFINITION
Column : (SP-4-2)-Diammine[cyclobutan-1,1-dicarboxylato(2-)-O,O′]-
– material : stainless steel ; platin.
– size : l = 2 m, Ø = 2 mm ; Content : 98.0 per cent to 102.0 per cent (dried substance).
– stationary phase : molecular sieve for chromatography CHARACTERS
(149-177 μm) with a nominal pore size of 0.5 nm. Appearance : colourless, crystalline powder.
Carrier gas : argon for chromatography R. Solubility : sparingly soluble in water, very slightly soluble in
Flow rate : 30 mL/min. acetone and in ethanol (96 per cent).
Temperature : mp : about 200 °C, with decomposition.
– column : 100 °C ; IDENTIFICATION
– detector : 160 °C. Infrared absorption spectrophotometry (2.2.24).
Detection : thermal conductivity. Comparison: Ph. Eur. reference spectrum of carboplatin.
Injection : 1 mL. TESTS
Run time : 4 min. Solution S. Dissolve 0.25 g in carbon dioxide-free water R and
Relative retention with reference to carbon monoxide dilute to 25 mL with the same solvent.
(retention time = about 2.3 min) : hydrogen = about 0.4. Appearance of solution. Solution S is clear (2.2.1) and
Limit : colourless (2.2.2, Method II).
– hydrogen : not more than the area of the corresponding Impurity B and acidity : maximum 0.5 per cent, calculated
peak in the chromatogram obtained with the reference gas as impurity B.
(300 ppm V/V). To 10 mL of solution S add 0.1 mL of phenolphthalein
Nickel tetracarbonyl and iron pentacarbonyl : not solution R1. The solution is colourless. Not more than 0.7 mL
detectable, using a detector tube having a limit of detection of 0.01 M sodium hydroxide is required to change the colour
of 0.1 ppm V/V (2.1.6). of the indicator to pink.
Water: maximum 10 ppm V/V, determined using an Related substances. Liquid chromatography (2.2.29).
electrolytic hygrometer (2.5.28). Test solution. Dissolve 20.0 mg of the substance to be
examined in a mixture of equal volumes of acetonitrile R
ASSAY and water R and dilute to 20.0 mL with the same mixture of
Infrared analyser (2.5.25). solvents.
Gas to be examined. The substance to be examined, previously Reference solution. Dilute 0.5 mL of the test solution to
filtered to avoid stray light phenomena. 200.0 mL with the mobile phase.
Reference gas (a). Carbon monoxide R. Column :
Reference gas (b). A mixture containing 95.0 per cent V/V of – size : l = 0.25 m, Ø = 4.6 mm ;
carbon monoxide R and 5.0 per cent V/V of nitrogen R1. – stationary phase : aminopropylsilyl silica gel for
Calibrate the apparatus and set the sensitivity using reference chromatography R (5 μm).
gases (a) and (b). Measure the content of carbon monoxide in Mobile phase : water R, acetonitrile R (13:87 V/V).
the gas to be examined. Flow rate : 2 mL/min.

1770 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Carboprost trometamol

Detection : spectrophotometer at 230 nm.

Injection : 10 μL.
Run time : 2.5 times the retention time of carboplatin. B. cyclobutane-1,1-dicarboxylic acid.
Relative retention with reference to carboplatin (retention
time = about 7 min) : impurity A = about 0.3.
01/2008:1712
System suitability : test solution :
– number of theoretical plates : minimum 5000 ; if necessary, CARBOPROST TROMETAMOL
adjust the concentration of acetonitrile in the mobile phase ;
– mass distribution ratio : minimum 4.0 ; if necessary, adjust Carboprostum trometamolum
the concentration of acetonitrile in the mobile phase ;
– symmetry factor : maximum 2.0 ; if necessary, adjust the
concentration of acetonitrile in the mobile phase.
Limits :
– impurity A : not more than the area of the principal peak
in the chromatogram obtained with the reference solution
(0.25 per cent) ; C25H47NO8 Mr 489.7
– total : not more than twice the area of the principal peak in [58551-69-2]
the chromatogram obtained with the reference solution
(0.5 per cent) ; DEFINITION
– disregard limit : 0.2 times the area of the principal peak in 2-Amino-2-(hydroxymethyl)propane-1,3-diol
the chromatogram obtained with the reference solution (5Z)-7-[(1R,2R,3R,5S)-3,5-dihydroxy-2-[(1E,3S)-3-
(0.05 per cent). hydroxy-3-methyloct-1-enyl]cyclopentyl]hept-5-enoate
((15S)-15-methyl-PGF2).
Chlorides (2.4.4): maximum 100 ppm.
Content : 94.0 per cent to 102.0 per cent (anhydrous substance).
Dissolve 0.5 g in water R, heating slightly if necessary, and
dilute to 20 mL with the same solvent. Filter if necessary. CHARACTERS
Dilute 10 mL of this solution to 15 mL with water R. Prepare Appearance : white or almost white powder.
the standard using 5 mL of chloride standard solution (5 ppm
Cl) R. Solubility : soluble in water.
Ammonium (2.4.1, Method B) : maximum 100 ppm, IDENTIFICATION
determined on 0.20 g. A. Specific optical rotation (see Tests).
Prepare the standard using 0.2 mL of ammonium standard B. Infrared absorption spectrophotometry (2.2.24).
solution (100 ppm NH4) R. Comparison: Ph. Eur. reference spectrum of carboprost
Silver : maximum 10 ppm. trometamol.
Inductively coupled plasma-atomic emission spectrometry
TESTS
(2.2.57).
Test solution. Dissolve 0.50 g in a 1 per cent V/V solution of Specific optical rotation (2.2.7) : + 18 to + 24 (anhydrous
nitric acid R and dilute to 50.0 mL with the same solution. substance).
Reference solutions. Prepare the reference solutions using Dissolve 0.100 g in ethanol (96 per cent) R and dilute to
silver standard solution (5 ppm Ag) R, diluting with a 1 per 10.0 mL with the same solvent.
cent V/V solution of nitric acid R. Related substances. Liquid chromatography (2.2.29).
Wavelength : 328.1 nm. Test solution. Dissolve 15.0 mg of the substance to be
Soluble barium : maximum 10 ppm. examined in a mixture of 23 volumes of acetonitrile R and
77 volumes of water for chromatography R and dilute to
Inductively coupled plasma-atomic emission spectrometry 10.0 mL with the same mixture of solvents.
(2.2.57).
Reference solution (a). Dissolve 15.0 mg of carboprost
Test solution. Use the solution described in the test for silver. trometamol CRS (containing impurity A) in a mixture of
Reference solutions. Prepare the reference solutions using 23 volumes of acetonitrile R and 77 volumes of water for
barium standard solution (50 ppm Ba) R, diluting with a 1 per chromatography R and dilute to 10.0 mL with the same
cent V/V solution of nitric acid R. mixture of solvents.
Wavelength : 455.4 nm. Reference solution (b). Dilute 1.0 mL of reference solution (a)
Loss on drying (2.2.32) : maximum 0.5 per cent, determined and 0.15 mL of (15R)-15-methylprostaglandin F2α R
on 1.000 g by drying in an oven at 105 °C. (impurity B) to 100.0 mL with a mixture of 23 volumes of
acetonitrile R and 77 volumes of water for chromatography R.
ASSAY Reference solution (c). Dilute 2.0 mL of the test solution to
Use the residue obtained in the test for loss on drying. Ignite 20.0 mL with a mixture of 23 volumes of acetonitrile R and
0.200 g of the residue to constant mass at 800 ± 50 °C. 77 volumes of water for chromatography R. Dilute 2.0 mL
1 mg of the residue is equivalent to 1.903 mg of C6H12N2O4Pt. of this solution to 20.0 mL with a mixture of 23 volumes of
acetonitrile R and 77 volumes of water for chromatography R.
STORAGE Column :
Protected from light. – size : l = 0.15 m, Ø = 4.6 mm,
IMPURITIES – stationary phase : octadecylsilyl silica gel for
Specified impurities : A, B. chromatography R1 (5 μm) with a pore size of
8-10 nm and a carbon loading of 12-19 per cent.
Mobile phase : mix 23 volumes of acetonitrile R1 and
77 volumes of a 2.44 g/L solution of sodium dihydrogen
phosphate R in water for chromatography R previously adjusted
A. cis-diamminedichloroplatinum(II) (cisplatin), to pH 2.5 with phosphoric acid R.

General Notices (1) apply to all monographs and other texts 1771
Carisoprodol EUROPEAN PHARMACOPOEIA 8.0

Flow rate : 1.0 mL/min. IMPURITIES

Specified impurities : A, B.
Detection : spectrophotometer at 200 nm.
Injection : 20 μL.
Run time : 1.3 times the retention time of carboprost.
Relative retention with reference to carboprost (retention
time = about 80 min): impurity B = about 0.85 ; A. (5E)-7-[(1R,2R,3R,5S)-3,5-dihydroxy-2-[(1E,3S)-3-
impurity A = about 0.9. hydroxy-3-methyloct-1-enyl]cyclopentyl]hept-5-enoic
Identification of impurities : use the chromatogram obtained acid,
with reference solution (a) and the chromatogram supplied
with carboprost trometamol CRS to identify the peak due to
impurity A.
System suitability :
– resolution : minimum 3.4 between the peaks due to B. (5Z)-7-[(1R,2R,3R,5S)-3,5-dihydroxy-2-[(1E,3R)-3-
impurity B and carboprost in the chromatogram obtained hydroxy-3-methyloct-1-enyl]cyclopentyl]hept-5-enoic
with reference solution (b) ; acid.
– peak-to-valley ratio : minimum 3.0, where Hp = height
above the baseline of the peak due to impurity A and
Hv = height above the baseline of the lowest point of the 01/2008:1689
curve separating this peak from the peak due to impurity B
in the chromatogram obtained with reference solution (a). CARISOPRODOL
Limits :
Carisoprodolum
– impurity A : not more than 3 times the area of the principal
peak in the chromatogram obtained with reference
solution (c) (3.0 per cent),
– impurity B : not more than the area of the principal peak
in the chromatogram obtained with reference solution (c)
(1.0 per cent), C12H24N2O4 Mr 260.3
– unspecified impurities : for each impurity, not more than [78-44-4]
0.1 times the area of the principal peak in the chromatogram DEFINITION
obtained with reference solution (c) (0.10 per cent),
(2RS)-2-[(Carbamoyloxy)methyl]-2-methylpentyl
– total : not more than 4 times the area of the principal peak (1-methylethyl)carbamate.
in the chromatogram obtained with reference solution (c) Content : 98.0 per cent to 102.0 per cent (dried substance).
(4.0 per cent),
CHARACTERS
– disregard limit : 0.05 times the area of the principal peak Appearance : white or almost white, fine powder.
in the chromatogram obtained with reference solution (c) Solubility : very slightly soluble in water, freely soluble in
(0.05 per cent). acetone, in alcohol and in methylene chloride.
Water (2.5.32) : maximum 0.5 per cent, determined on 50 mg.
IDENTIFICATION
First identification : A, B.
ASSAY Second identification : A, C, D.
A. Melting point (2.2.14) : 92 °C to 95 °C.
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications. B. Infrared absorption spectrophotometry (2.2.24).
Comparison: carisoprodol CRS.
Mobile phase : mix 27 volumes of acetonitrile R1 and C. Examine the chromatograms obtained in the test for
73 volumes of a 2.44 g/L solution of sodium dihydrogen related substances.
phosphate R in water for chromatography R previously adjusted
to pH 2.5 with phosphoric acid R. Results : the principal spot in the chromatogram obtained
with test solution (b) is similar in position, colour and size
Injection : test solution and reference solution (a). to the principal spot in the chromatogram obtained with
reference solution (d).
Run time : 1.2 times the retention time of carboprost. D. Dissolve 0.2 g in 15 mL of a 28 g/L solution of potassium
hydroxide R in alcohol R and boil under a reflux condenser
Retention time : carboprost = about 29 min. for 15 min. Add 0.5 mL of glacial acetic acid R and 1 mL
Calculate the percentage content of C25H47NO8 using the of a 50 g/L solution of cobalt nitrate R in ethanol R. An
declared content of carboprost trometamol CRS. intense blue colour develops.
TESTS
STORAGE Optical rotation (2.2.7) : − 0.10° to + 0.10°.
Dissolve 2.5 g in alcohol R and dilute to 25.0 mL with the
At a temperature below − 15 ° C. same solvent.

1772 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Carmellose

Related substances. Thin-layer chromatography (2.2.27). IMPURITIES

Test solution (a). Dissolve 0.20 g of the substance to be
examined in methylene chloride R and dilute to 10 mL with
the same solvent.
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
with methylene chloride R.
A. (2RS)-2-(hydroxymethyl)-2-methylpentyl (1-methylethyl)-
Reference solution (a). Dissolve 5.0 mg of meprobamate CRS carbamate,
in methylene chloride R and dilute to 50 mL with the same
solvent.
Reference solution (b). Dilute 1 mL of test solution (b) to
50 mL with methylene chloride R.
Reference solution (c). Dilute 5 mL of reference solution (b) to
10 mL with methylene chloride R.
Reference solution (d). Dissolve 20 mg of carisoprodol CRS
in methylene chloride R and dilute to 10 mL with the same B. 5-methyl-5-propyl-1,3-dioxan-2-one,
solvent.
Reference solution (e). Dissolve 10 mg of carisoprodol
impurity A CRS in 5 mL of reference solution (d) and dilute to
50 mL with methylene chloride R.
C. 2-methyl-2-propylpropane-1,3-diol,
Plate : TLC silica gel plate R.
Mobile phase : acetone R, methylene chloride R (20:80 V/V).
Application : 5 μL.
Development : over a path of 15 cm.
Drying : in air for 15 min.
Detection : spray with a solution prepared as follows : dissolve D. 2-methyl-2-propylpropane-1,3-diyl dicarbamate
5 g of phosphomolybdic acid R in a mixture of 50 mL of glacial (meprobamate).
acetic acid R and 10 mL of sulfuric acid R, and dilute to 100 mL
with glacial acetic acid R. Heat the plate at 100-105 °C for
30 min. 04/2013:2360
System suitability :
CARMELLOSE
– the chromatogram obtained with reference solution (c)
shows 1 clearly visible spot,
Carmellosum
– the chromatogram obtained with reference solution (e)
shows 2 clearly separated spots. [9000-11-7]
Limits : in the chromatogram obtained with test solution (a) :
DEFINITION
– impurity D : any spot due to impurity D is not more intense Carboxymethylether of cellulose.
than the spot in the chromatogram obtained with reference
solution (a) (0.5 per cent), Partly O-carboxymethylated cellulose.
– any other impurity : any spot, apart from the principal spot CHARACTERS
and any spot due to impurity D, is not more intense than Appearance : white or almost white powder, hygroscopic.
the spot in the chromatogram obtained with reference Solubility : practically insoluble in anhydrous ethanol. It swells
solution (b) (0.2 per cent). with water to form a suspension and becomes viscid in 1 M
Heavy metals (2.4.8) : maximum 10 ppm. sodium hydroxide.
2.0 g complies with test C. Prepare the reference solution using IDENTIFICATION
2 mL of lead standard solution (10 ppm Pb) R.
A. pH (2.2.3) : 3.5 to 5.0.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined Suspend 1.0 g in 100 mL of carbon dioxide-free water R.
on 1.000 g in vacuo at 60 °C for 3 h.
B. Infrared absorption spectrophotometry (2.2.24).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Comparison: carmellose CRS.
1.0 g.
TESTS
ASSAY
Chlorides : maximum 0.36 per cent.
Dissolve 0.100 g in 15 mL of a 25 per cent V/V solution of Shake 0.8 g with 50 mL of water R, dissolve in 10 mL of 1 M
sulfuric acid R and boil under a reflux condenser for 3 h. Cool, sodium hydroxide and dilute to 100 mL with water R. Heat on a
dissolve by cautiously adding 30 mL of water R, cool again and water-bath a mixture of 10 mL of dilute nitric acid R and 20 mL
place in a steam-distillation apparatus. Add 40 mL of strong of this solution until a flocculent precipitate is produced. Cool,
sodium hydroxide solution R and distil immediately by passing centrifuge and take out the supernatant. Wash the precipitate
steam through the mixture. Collect the distillate into 40 mL with 3 quantities, each of 10 mL, of water R, centrifuging each
of a 40 g/L solution of boric acid R until the total volume in time. Combine the supernatant and the washings and dilute
the receiver reaches about 200 mL. Add 0.25 mL of methyl red to 100 mL with water R. To 25 mL of this solution add 6 mL
mixed solution R. Titrate with 0.1 M hydrochloric acid, until of dilute nitric acid R and dilute to 50 mL with water R (test
the colour changes from green to violet. Carry out a blank solution). Prepare the reference solution in the same manner,
titration. using 0.40 mL of 0.01 M hydrochloric acid. Add 1 mL of
1 mL of 0.1 M hydrochloric acid is equivalent to 13.02 mg of silver nitrate solution R2 to the test solution and the reference
C12H24N2O4. solution. Allow to stand protected from light for 5 min. Any

General Notices (1) apply to all monographs and other texts 1773
Carmellose calcium EUROPEAN PHARMACOPOEIA 8.0

opalescence in the test solution is not more intense than that Solubility : practically insoluble in acetone, in alcohol and in
in the reference solution. toluene. It swells with water to form a suspension.
Sulfates : maximum 0.72 per cent. IDENTIFICATION
Shake 0.40 g with 25 mL of water R, dissolve in 5 mL of 1 M A. Shake 0.1 g thoroughly with 10 mL of water R. Add 2 mL
sodium hydroxide and add 20 mL of water R. Heat this solution of dilute sodium hydroxide solution R and allow to stand
with 2.5 mL of hydrochloric acid R in a water-bath until a for 10 min (solution A). Dilute 1 mL of solution A to 5 mL
flocculent precipitate is produced. Cool, centrifuge, and take with water R. To 0.05 mL add 0.5 mL of a 0.5 g/L solution
out the supernatant. Wash the precipitate with 3 quantities, of chromotropic acid, sodium salt R in a 75 per cent m/m
each of 10 mL, of water R, centrifuging each time. Combine solution of sulfuric acid R and heat on a water-bath for
the supernatant and the washings, and dilute to 100 mL with 10 min. A reddish-violet colour develops.
water R. Filter, and discard the first 5 mL of the filtrate. To
25 mL of the filtrate add 1 mL of dilute hydrochloric acid R B. Shake 5 mL of solution A obtained in identification test A
and dilute to 50 mL with water R (test solution). Prepare with 10 mL of acetone R. A white, flocculent precipitate
the reference solution in the same manner, using 1.5 mL is produced.
of 0.005 M sulfuric acid. Add 2 mL of a 120 g/L solution C. Shake 5 mL of solution A obtained in identification test A
of barium chloride R to the test solution and the reference with 1 mL of ferric chloride solution R1. A brown, flocculent
solution. Mix and allow to stand for 10 min. The white precipitate is formed.
turbidity produced in the test solution is not thicker than that D. Ignite 1 g and dissolve the residue in a mixture of 5 mL
in the reference solution. of acetic acid R and 10 mL of water R. Filter if necessary
Heavy metals : maximum 20 ppm. and boil the filtrate for a few minutes. Cool and neutralise
with dilute ammonia R1. The solution gives reaction (a) of
Place 1.0 g in a quartz or porcelain crucible. Cover loosely calcium (2.3.1).
with a lid and carbonise by gentle ignition. Cool and add 2 mL
of nitric acid R and 5 drops of sulfuric acid R. Heat cautiously
TESTS
until white fumes are no longer evolved and incinerate by
Solution S. Shake 1.0 g with 50 mL of distilled water R, add
ignition at 500-600 °C. Cool and add 2 mL of hydrochloric
5 mL of dilute sodium hydroxide solution R and dilute to
acid R. Evaporate to dryness on a water-bath. Moisten the
100 mL with distilled water R.
residue with 3 drops of hydrochloric acid R, add 10 mL of hot
water R and heat for 2 min. Add 1 drop of phenolphthalein Alkalinity. Shake 1.0 g thoroughly with 50 mL of carbon
solution R1, add dilute ammonia R1 dropwise until the dioxide-free water R and add 0.05 mL of phenolphthalein
solution develops a pale red colour. Add 2 mL of dilute acetic solution R. No red colour develops.
acid R, filter if necessary, and wash with 10 mL of water R. Chlorides (2.4.4) : maximum 0.36 per cent.
Transfer the filtrate and washings to a test-tube, and dilute Heat 28 mL of solution S with 10 mL of dilute nitric acid R on
to 50 mL with water R (test solution). Prepare the reference a water-bath until a flocculent precipitate is produced. Cool,
solution as follows : evaporate a mixture of 2 mL of nitric centrifuge and separate the supernatant. Wash the precipitate
acid R, 5 drops of sulfuric acid R and 2 mL of hydrochloric with 3 quantities, each of 10 mL, of water R, centrifuging each
acid R on a water-bath, then evaporate to dryness on a time. Combine the supernatant and the washings and dilute
sand-bath. Moisten the residue with 3 drops of hydrochloric to 100 mL with water R. To 25 mL add 6 mL of dilute nitric
acid R. Proceed as described for the test solution, then add acid R and dilute to 50 mL with water R. Dilute 10 mL of the
2.0 mL of lead standard solution (10 ppm Pb) R and dilute to solution to 15 mL with water R.
50 mL with water R.
Sulfates (2.4.13) : maximum 1 per cent.
Add 0.1 mL of sodium sulfide solution R1 to the test solution
and the reference solution and allow to stand for 5 min. The Heat 20 mL of solution S with 1 mL of hydrochloric acid R
on a water-bath until a flocculent precipitate is produced.
colour of the test solution is not more intense than that of the
reference solution. Cool, centrifuge and separate the supernatant. Wash the
precipitate with 3 quantities, each of 10 mL, of distilled
Loss on drying (2.2.32) : maximum 8.0 per cent, determined water R, centrifuging each time. Combine the supernatant
on 1.000 g by drying in an oven at 105 °C for 4 h. and the washings and dilute to 100 mL with distilled water R.
Sulfated ash (2.4.14) : maximum 1.5 per cent (dried To 25 mL add 1 mL of dilute hydrochloric acid R and dilute to
substance), determined on 1.0 g. 50 mL with distilled water R.
STORAGE Heavy metals (2.4.8) : maximum 20 ppm.
In an airtight container. 1.0 g complies with test D. Prepare the reference solution
using 2 mL of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 10.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 4 h.
01/2008:0886 Sulfated ash (2.4.14) : 10.0 per cent to 20.0 per cent,
corrected 6.0 determined on 1.0 g in a platinum crucible.

CARMELLOSE CALCIUM STORAGE

In an airtight container.
Carmellosum calcicum
01/2008:0472
[9050-04-8] corrected 8.0
DEFINITION
Calcium salt of a partly O-carboxymethylated cellulose. CARMELLOSE SODIUM
CHARACTERS Carmellosum natricum
Appearance : white or yellowish-white powder, hygroscopic
after drying. [9004-32-4]

1774 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Carmellose sodium, low-substituted

DEFINITION flat-bottomed tube. Examine the solutions viewing vertically.

Carmellose sodium (carboxymethylcellulose sodium) is the The test solution is not more intensely coloured than the
sodium salt of a partly O-carboxymethylated cellulose. It reference solution (0.4 per cent).
contains not less than 6.5 per cent and not more than 10.8 per Chlorides (2.4.4). Dilute 2 mL of solution S to 15 mL with
cent of sodium (Na), calculated with reference to the dried water R. The solution complies with the limit test for chlorides
substance. (0.25 per cent).
CHARACTERS Heavy metals (2.4.8). To the residue obtained in the
determination of the sulfated ash, add 1 mL of hydrochloric
A white or almost white, granular powder, hygroscopic acid R and evaporate on a water-bath. Take up the residue in
after drying, practically insoluble in acetone, in ethanol and 20 mL of water R. 12 mL of the solution complies with test A
in toluene. It is easily dispersed in water giving colloidal for heavy metals (20 ppm). Prepare the reference solution
solutions. using lead standard solution (2 ppm Pb) R.
IDENTIFICATION Loss on drying (2.2.32). Not more than 10.0 per cent,
A. To 10 mL of solution S (see Tests) add 1 mL of copper determined on 1.000 g by drying in an oven at 105 °C.
sulfate solution R. A blue, cotton-like precipitate is formed. Sulfated ash (2.4.14) : 20.0 per cent to 33.3 per cent,
B. Boil 5 mL of solution S for a few minutes. No precipitate determined on 1.0 g using a mixture of equal volumes of
is formed. sulfuric acid R and water R and calculated with reference to
the dried substance. These limits correspond to a content of
C. The solution prepared from the sulfated ash in the test for
6.5 per cent to 10.8 per cent of sodium (Na).
heavy metals gives the reactions of sodium (2.3.1).
LABELLING
TESTS
The label states the apparent viscosity in millipascal seconds
Solution S. Sprinkle a quantity of the substance to be for a 20 g/L solution ; for a product of low viscosity, the label
examined equivalent to 1.0 g of the dried substance onto states the concentration of the solution to be used and the
90 mL of carbon dioxide-free water R at 40 °C to 50 °C stirring apparent viscosity in millipascal seconds.
vigorously. Continue stirring until a colloidal solution is
obtained, cool and dilute to 100 mL with carbon dioxide-free
water R. 01/2008:1186
corrected 7.0
Appearance of solution. Solution S is not more opalescent
than reference suspension III (2.2.1) and not more intensely
coloured than reference solution Y6 (2.2.2, Method II).
CARMELLOSE SODIUM,
pH (2.2.3). The pH of solution S is 6.0 to 8.0.
LOW-SUBSTITUTED
Apparent viscosity. While stirring, introduce a quantity of Carmellosum natricum substitutum humile
the substance to be examined equivalent to 2.00 g of the dried
substance into 50 mL of water R heated to 90 °C. For a product [9050-32-4]
of low viscosity, use if necessary, the quantity required to give
the concentration indicated on the label. Allow to cool, dilute DEFINITION
to 100.0 mL with water R and stir until dissolution is complete. Low-substituted sodium carboxymethylcellulose. Sodium salt
Determine the viscosity (2.2.10) using a rotating viscometer at of a partly O-(carboxymethylated) cellulose.
20 °C and a shear rate of 10 s− 1. If it is impossible to obtain a
Content : 2.0 per cent to 4.5 per cent of sodium (Na) (dried
shear rate of exactly 10 s− 1, use a shear rate slightly higher and
substance).
a rate slightly lower and interpolate. The apparent viscosity is
not less than 75 per cent and not more than 140 per cent of CHARACTERS
the value stated on the label. Appearance : white or almost white powder or short fibres.
Sodium glycollate. Place a quantity of the substance to Solubility : practically insoluble in acetone, in anhydrous
be examined equivalent to 0.500 g of dried substance in a ethanol and in toluene. It swells in water to form a gel.
beaker. Add 5 mL of acetic acid R and 5 mL of water R. Stir
until dissolution is complete (about 30 min). Add 80 mL of IDENTIFICATION
acetone R and 2 g of sodium chloride R. Filter through a fast A. Shake 1 g with 100 mL of a 100 g/L solution of sodium
filter paper impregnated with acetone R into a volumetric hydroxide R. A suspension is produced.
flask, rinse the beaker and filter with acetone R and dilute the B. Shake 1 g with 50 mL of water R. Transfer 1 mL of the
filtrate to 100.0 mL with the same solvent. Allow to stand for mixture to a test tube, add 1 mL of water R and 0.05 mL
24 h without shaking. Use the clear supernatant to prepare of a freshly prepared 40 g/L solution of α-naphthol R in
the test solution. methanol R. Incline the test tube and add carefully 2 mL of
In a volumetric flask, dissolve 0.310 g of glycollic acid R, sulfuric acid R down the side so that it forms a lower layer.
previously dried in vacuo over diphosphorus pentoxide R, A reddish-purple colour develops at the interface.
in water R and dilute to 1000.0 mL with the same solvent. C. Sulfated ash (2.4.14) (see Tests).
Place 5.0 mL of this solution in a volumetric flask, add 5 mL
D. The solution prepared for the test for heavy metals gives
of acetic acid R and allow to stand for about 30 min. Add
reaction (a) of sodium (2.3.1).
80 mL of acetone R and 2 g of sodium chloride R and dilute
to 100.0 mL with acetone R. Use this solution to prepare the TESTS
reference solution.
pH (2.2.3) : 6.0 to 8.5.
Place 2.0 mL of each solution in a separate 25 mL volumetric Shake 1 g with 100 mL of carbon dioxide-free water R for
flask. Heat on a water-bath to eliminate acetone. Cool to room 5 min. Centrifuge.
temperature and add 5.0 mL of 2,7-dihydroxynaphthalene
solution R to each flask. Shake and add 15.0 mL of Sodium chloride and sodium glycollate : maximum 0.5 per
2,7-dihydroxynaphthalene solution R. Close the flasks with cent (dried substance) for the sum of the percentage contents.
aluminium foil and heat on a water-bath for 20 min. Cool Sodium chloride. Place 5.00 g in a 250 mL conical flask, add
under running water and dilute to 25.0 mL with sulfuric 50 mL of water R and 5 mL of strong hydrogen peroxide
acid R. Within 10 min, transfer 10.0 mL of each solution to a solution R and heat on a water bath for 20 min, stirring

General Notices (1) apply to all monographs and other texts 1775
Carmustine EUROPEAN PHARMACOPOEIA 8.0

occasionally to ensure total hydration. Cool, add 100 mL of Sulfated ash (2.4.14) : 6.5 per cent to 13.5 per cent (dried
water R and 10 mL of nitric acid R. Titrate with 0.05 M silver substance), corresponding to a content of 2.0 per cent to
nitrate determining the end-point potentiometrically (2.2.20) 4.5 per cent of Na.
using a silver-based indicator electrode and a double-junction Use 1.0 g with a mixture of equal volumes of sulfuric acid R
reference electrode containing a 100 g/L solution of potassium and water R.
nitrate R in the outer jacket and a standard filling solution in
the inner jacket. FUNCTIONALITY-RELATED CHARACTERISTICS
1 mL of 0.05 M silver nitrate is equivalent to 2.922 mg of NaCl. This section provides information on characteristics that are
recognised as being relevant control parameters for one or
Sodium glycollate. Place a quantity of the substance to be
more functions of the substance when used as an excipient (see
examined equivalent to 0.500 g of the dried substance in
chapter 5.15). This section is a non-mandatory part of the
a beaker. Add 5 mL of glacial acetic acid R and 5 mL of
monograph and it is not necessary to verify the characteristics
water R and stir to ensure total hydration (about 30 min).
to demonstrate compliance. Control of these characteristics can
Add 80 mL of acetone R and 2 g of sodium chloride R. Stir
however contribute to the quality of a medicinal product by
for several minutes to ensure complete precipitation of the
improving the consistency of the manufacturing process and
carboxymethylcellulose. Filter through a fast filter paper
the performance of the medicinal product during use. Where
impregnated with acetone R into a volumetric flask, rinse
control methods are cited, they are recognised as being suitable
the beaker and filter with acetone R and dilute the filtrate
for the purpose, but other methods can also be used. Wherever
to 100.0 mL with the same solvent. Allow to stand for 24 h
results for a particular characteristic are reported, the control
without shaking. Use the clear supernatant as the test solution.
method must be indicated.
Prepare the reference solutions as follows : in a 100 mL The following characteristic may be relevant for low-substituted
volumetric flask, dissolve 0.100 g of glycollic acid R, previously carmellose sodium used as disintegrant.
dried in vacuo over diphosphorus pentoxide R, in water R
and dilute to 100.0 mL with the same solvent. Transfer Settling volume : 15.0 mL to 35.0 mL.
0.5 mL, 1.0 mL, 1.5 mL and 2.0 mL of the solution to separate In a 100 mL graduated cylinder, place 20 mL of 2-propanol R,
volumetric flasks ; dilute the contents of each flask to 5.0 mL add 5.0 g of the substance to be examined and shake
with water R, add 5 mL of glacial acetic acid R, dilute to vigorously. Dilute to 30 mL with 2-propanol R then to 50 mL
100.0 mL with acetone R and mix. with water R and shake vigorously. Within 15 min, repeat
Transfer 2.0 mL of the test solution and 2.0 mL of each of the the shaking 3 times. Allow to stand for 4 h and determine
reference solutions to separate 25 mL volumetric flasks. Heat the volume of the settled mass.
the uncovered flasks in a water-bath to eliminate the acetone.
Allow to cool and add 5.0 mL of 2,7-dihydroxynaphthalene
solution R to each flask. Mix, add a further 15.0 mL of 01/2008:1187
2,7-dihydroxynaphthalene solution R and mix again. Close
the flasks with aluminium foil and heat in a water-bath for CARMUSTINE
20 min. Cool and dilute to 25.0 mL with sulfuric acid R.
Measure the absorbance (2.2.25) of each solution at 540 nm. Carmustinum
Prepare a blank using 2.0 mL of a solution containing 5 per
cent V/V each of glacial acetic acid R and water R in acetone R.
Prepare a standard curve using the absorbances obtained
with the reference solutions. From the standard curve and
the absorbance of the test solution, determine the mass a, in
milligrams, of glycollic acid in the substance to be examined C5H9Cl2N3O2 Mr 214.1
and calculate the content of sodium glycollate from the [154-93-8]
following expression :
DEFINITION
Carmustine contains not less than 98.0 per cent and
not more than the equivalent of 102.0 per cent of
1,3-bis(2-chloroethyl)-1-nitrosourea, calculated with reference
1.29 = the factor converting glycollic acid to sodium to the anhydrous substance.
glycollate,
b = the loss on drying as a percentage, CHARACTERS
m A yellowish, granular powder, very slightly soluble in water,
= the mass of the substance to be examined, in grams.
very soluble in methylene chloride, freely soluble in ethanol.
Water-soluble substances : maximum 70.0 per cent. It melts at about 31 °C with decomposition.
Disperse 5.00 g in 400.0 mL of water R and stir for 1 min every IDENTIFICATION
10 min during the first 30 min. Allow to stand for 1 h and
centrifuge, if necessary. Decant 100.0 mL of the supernatant Examine by infrared absorption spectrophotometry (2.2.24),
onto a fast filter paper in a vacuum filtration funnel, apply comparing with the Ph. Eur. reference spectrum of carmustine.
vacuum and collect 75.0 mL of the filtrate. Evaporate to Examine the melted substances prepared as films.
dryness and dry the residue at 100-105 °C for 4 h. TESTS
Heavy metals (2.4.8) : maximum 20 ppm. 1,3-Bis(2-chloroethyl)urea (impurity A). Examine by
To the residue obtained in the determination of the sulfated thin-layer chromatography (2.2.27), using a suitable silica gel
ash add 1 mL of hydrochloric acid R and evaporate on a as the coating substance.
water-bath. Take up the residue in 20 mL of water R (this Test solution. Dissolve 0.10 g of the substance to be examined
solution is used for identification test D). 12 mL of the solution in methylene chloride R and dilute to 5 mL with the same
complies with test A. Prepare the reference solution using lead solvent.
standard solution (1 ppm Pb) R. Reference solution (a). Dissolve 2 mg of carmustine
Loss on drying (2.2.32) : maximum 10.0 per cent, determined impurity A CRS in methylene chloride R and dilute to 10 mL
on 1.000 g by drying in an oven at 105 °C. with the same solvent.

1776 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Carnauba wax

Reference solution (b). Dilute 1 mL of the test solution to Application : 30 μL of the test solution and 10 μL of the
10 mL with methylene chloride R. To 5 mL of this solution, add reference solution as bands 20 mm by 3 mm.
5 mL of reference solution (a).
Development : over half of the plate.
Apply separately to the plate 2 μL of each solution. Develop
over a path of 10 cm using a mixture of 10 volumes of Drying : in air.
methanol R and 90 volumes of methylene chloride R. Allow Detection : spray with a freshly prepared 200 g/L solution of
the plate to dry in air. Spray with diethylamine R and heat phosphomolybdic acid R in alcohol R (about 10 mL for a 20 cm
at 125 °C for 10 min. Allow to cool and spray with silver plate). Heat at 100-105 °C for 10-15 min.
nitrate solution R2. Expose to ultraviolet light at 365 nm
until brown to black spots appear. Any spot corresponding Results : the chromatogram obtained with the reference
to carmustine impurity A in the chromatogram obtained solution shows in the lower part a dark blue zone (menthol),
with the test solution is not more intense than the spot in the above this zone a reddish zone (thymol) and in the upper
chromatogram obtained with reference solution (a) (1 per part a dark blue zone (menthyl acetate). The chromatogram
cent). The test is not valid unless the chromatogram obtained obtained with the test solution shows a large blue zone
with reference solution (b) shows two clearly separated spots. (triacontanol = melissyl alcohol) at a level between the thymol
and menthol zones in the chromatogram obtained with the
Water (2.5.12). Not more than 1.0 per cent, determined on reference solution. Further blue zones are visible in the upper
0.50 g by the semi-micro determination of water. part of the chromatogram obtained with the test solution,
at levels between those of the menthyl acetate and thymol
ASSAY zones in the chromatogram obtained with the reference
Dissolve 0.100 g in 30 mL of ethanol R and dilute to 100.0 mL solution ; above these zones further zones are visible in the
with water R. Dilute 3.0 mL of the solution to 100.0 mL with chromatogram obtained with the test solution ; the zone with
water R. Measure the absorbance (2.2.25) at the maximum the highest RF value is very pronounced. A number of faint
at 230 nm. zones are visible below the triacontanol zone and the point of
application is coloured blue.
Calculate the content of C5H9Cl2N3O2 taking the specific
absorbance to be 270.
TESTS
STORAGE Appearance of solution. The solution is clear (2.2.1) and not
Store in an airtight container, protected from light, at a more intensely coloured than a 50 mg/L solution of potassium
temperature of 2 °C to 8 °C. dichromate R (2.2.2, Method II).
Dissolve 0.10 g with heating in chloroform R and dilute to
IMPURITIES 10 mL with the same solvent.
Melting point (2.2.15) : 80 °C to 88 °C.
Melt the substance to be examined carefully on a water-bath
before introduction into the capillary tubes. Allow the tubes
to stand in the refrigerator for 24 h or at 0 °C for 2 h.
A. 1,3-bis(2-chloroethyl)urea. Acid value : 2 to 7.
To 2.000 g (m g) in a 250 mL conical flask fitted with a reflux
condenser add 40 mL of xylene R and a few glass beads. Heat
with stirring until the substance is completely dissolved. Add
01/2008:0597 20 mL of alcohol R and 1 mL of bromothymol blue solution R3
and titrate the hot solution with 0.5 M alcoholic potassium
hydroxide until a green colour persisting for at least 10 s is
CARNAUBA WAX obtained (n1 mL). Carry out a blank test (n2 mL). Calculate the
acid value from the expression :
Cera carnauba
DEFINITION
Purified wax obtained from the leaves of Copernicia cerifera Saponification value : 78 to 95.
Mart. To 2.000 g (m g) in a 250 mL conical flask fitted with a reflux
condenser add 40 mL of xylene R and a few glass beads. Heat
CHARACTERS with stirring until the substance is completely dissolved. Add
Appearance : pale yellow or yellow powder, flakes or hard 20 mL of alcohol R and 20.0 mL of 0.5 M alcoholic potassium
masses. hydroxide. Boil under a reflux condenser for 3 h. Add 1 mL
of phenolphthalein solution R1 and titrate the hot solution
Solubility : practically insoluble in water, soluble on heating in immediately with 0.5 M hydrochloric acid until the red colour
ethyl acetate and in xylene, practically insoluble in alcohol. disappears. Repeat the heating and titration until the colour
Relative density : about 0.97. no longer reappears on heating (n3 mL). Carry out a blank
test (n4 mL). Calculate the saponification value from the
IDENTIFICATION expression :
Thin-layer chromatography (2.2.27).
Test solution. Dissolve 0.10 g of the substance to be examined
with heating in 5 mL of chloroform R. Use the warm solution.
Total ash (2.4.16) : maximum 0.25 per cent, determined on
Reference solution. Dissolve 5 mg of menthol R, 5 μL of 2.0 g.
menthyl acetate R and 5 mg of thymol R in 10 mL of toluene R.
Plate : TLC silica gel plate R. STORAGE
Mobile phase : ethyl acetate R, chloroform R (2:98 V/V). Protected from light.

General Notices (1) apply to all monographs and other texts 1777
Carprofen for veterinary use EUROPEAN PHARMACOPOEIA 8.0

07/2008:2201 System suitability : reference solution (a) :

corrected 7.8 – resolution : minimum 1.5 between the peaks due to
impurity C and carprofen.
CARPROFEN FOR VETERINARY USE Limits :
– unspecified impurities : for each impurity, not more than
Carprofenum ad usum veterinarium twice the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.20 per cent) ;
– total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.5 per cent) ;
– disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.1 per
cent).
C15H12ClNO2 Mr 273.7 Heavy metals (2.4.8) : maximum 20 ppm.
[53716-49-7] Dissolve 1.0 g in ethanol (96 per cent) R and dilute to 20 mL
with the same solvent. 12 mL of the solution complies with
DEFINITION test B. Prepare the reference solution using lead standard
(2RS)-2-(6-Chloro-9H-carbazol-2-yl)propanoic acid. solution (1 ppm Pb) R.
Content : 98.5 per cent to 101.5 per cent (dried substance). Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 2 h.
CHARACTERS Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Appearance : white or almost white, crystalline powder. 1.0 g.
Solubility : practically insoluble in water, freely soluble in ASSAY
acetone, soluble in methanol, slightly soluble in 2-propanol.
Dissolve 0.200 g in 50 mL of ethanol (96 per cent) R. Add
It shows polymorphism (5.9). 1.0 mL of 0.1 M hydrochloric acid. Titrate with 0.1 M sodium
hydroxide, determining the end-point potentiometrically
IDENTIFICATION (2.2.20). Read the volume added between the 2 points of
Infrared absorption spectrophotometry (2.2.24). inflexion.
Comparison : carprofen CRS. 1 mL of 0.1 M sodium hydroxide is equivalent to 27.37 mg
If the spectra obtained in the solid state show differences, of C15H12ClNO2.
dissolve the substance to be examined and the reference STORAGE
substance separately in acetone R, evaporate to dryness and
record new spectra using the residues. Protected from light.
IMPURITIES
TESTS
Other detectable impurities (the following substances would,
Appearance of solution. The solution is clear (2.2.1) and not if present at a sufficient level, be detected by one or other of
more intensely coloured than reference solution BY3 (2.2.2, the tests in the monograph. They are limited by the general
Method II). acceptance criterion for other/unspecified impurities and/or
Dissolve 1.0 g in methanol R and dilute to 25 mL with the by the general monograph Substances for pharmaceutical
same solvent. use (2034). It is therefore not necessary to identify these
Related substances. Liquid chromatography (2.2.29). Carry impurities for demonstration of compliance. See also 5.10.
out the test protected from light. Control of impurities in substances for pharmaceutical use) :
A, B, C, D, E, F, G, H.
Test solution. Dissolve 50 mg of the substance to be examined
in the mobile phase and dilute to 100.0 mL with the mobile
phase.
Reference solution (a). Dissolve 2.5 mg of carprofen for system
suitability CRS (containing impurity C) in the mobile phase
and dilute to 10.0 mL with the mobile phase.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution A. 2-(6-chloro-9H-carbazol-2-yl)-2-methylpropanedioic acid,
to 10.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped polar-embedded octadecylsilyl
amorphous organosilica polymer R (5 μm).
Mobile phase : mix 30 volumes of a 1.36 g/L solution of B. (2RS)-2-(9H-carbazol-2-yl)propanoic acid,
potassium dihydrogen phosphate R adjusted to pH 3.0 with
phosphoric acid R and 70 volumes of methanol R2.
Flow rate : 1.3 mL/min.
Detection : spectrophotometer at 235 nm.
Injection : 20 μL.
Run time : 4 times the retention time of carprofen.
Retention time : carprofen = about 10 min. C. (1RS)-1-(6-chloro-9H-carbazol-2-yl)ethanol,

1778 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Carrageenan

To 10 mL of solution A, while still hot, add 4 drops of

a 100 g/L solution of potassium chloride R, mix and
allow to cool. A ‘brittle’ gel indicates a carrageenan
of a predominantly κ-type ; an ‘elastic’ gel indicates a
predominantly ι-type ; if the solution does not form a gel,
the carrageenan is of a predominantly λ-type.
D. 1-(6-chloro-9H-carbazol-2-yl)ethanone, B. Dilute 1 volume of solution A with about 4 volumes of
water R and add 2-3 drops of a 0.5 g/L solution of methylene
blue R in ethanol (96 per cent) R. A blue precipitate is
formed.
C. Infrared absorption spectrophotometry (2.2.24).
Preparation : prepare a 2 g/L solution of the substance to
be examined and cast films (5 μm thick when dry) on a
E. 3-chloro-9H-carbazole, suitable non-sticking surface.
Carrageenan has strong, broad absorption bands, typical
of all polysaccharides, in the 1000-1100 cm− 1 region.
Absorption maxima are 1065 cm− 1 and 1020 cm− 1
for gelling and non-gelling types, respectively. Other
characteristic absorption bands and their intensities relative
to the absorbance at 1050 cm− 1 are shown in Table 2138.-1.
Table 2138.-1. – Characteristic absorption bands for
F. diethyl 2-(6-chloro-9H-carbazol-2-yl)-2- carrageenan identification by infrared absorption
methylpropanedioate, spectrophotometry

Wave- Absorbance relative to the

number absorbance at 1050 cm− 1
Molecular structure
(cm− 1) κ ι λ

1220 - 1260 Ester sulfate 0.7 - 1.2 1.2 - 1.6 1.4 - 2.0

928 - 933 3,6-anhydro-D- 0.3 - 0.6 0.2 - 0.4 ≤ 0.2

galactose
G. ethyl (2RS)-2-(6-chloro-9H-carbazol-2-yl)propanoate,
840 - 850 Galactose-4-sulfate 0.3 - 0.5 0.2 - 0.4 -

825 - 830 Galactose-2-sulfate - - 0.2 - 0.4

810 - 820 Galactose-6-sulfate - - 0.1 - 0.3

800 - 805 3,6-anhydro-D- ≤ 0.2 0.2 - 0.4 -

galactose-2-sulfate
H. 6-chloro-2-ethyl-9H-carbazole.
TESTS
01/2011:2138 Apparent viscosity (2.2.10) : minimum 5 mPa·s. Heat a 15 g/L
dispersion (dried substance) at 80 °C for at least 15 min to
CARRAGEENAN dissolve. Compensate for any loss of water by evaporation,
allow to cool to 75 °C and carry out the test at this temperature.
Carrageenanum Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 2.0 g in 30 mL of water R and shake for 2 min. Allow
DEFINITION to stand and separate the aqueous layer. 12 mL of the solution
Carrageenans are polysaccharides extracted from different complies with test A. Prepare the reference solution using lead
Rhodophyceae with boiling water or aqueous alkali solutions. standard solution (1 ppm Pb) R.
Carrageenan is separated by alcohol precipitation, potassium Loss on drying (2.2.32) : maximum 12.0 per cent, determined
chloride precipitation, gel pressing, drum drying or freezing. on 1.000 g by drying in an oven at 105 °C.
The alcohol used during separation and purification is
generally 2-propanol. The main components are potassium, Total ash (2.4.16) : maximum 40.0 per cent.
sodium, calcium or magnesium salts of the sulfate esters of Ash insoluble in hydrochloric acid (2.8.1) : maximum 2.0 per
D-galactose and 3,6-anhydro-D-galactose copolymers. They cent.
exist in different proportions depending on the biological
origin of the polymer. LABELLING
The prevalent copolymers are designated as κ-, ι- and The label states the type of carrageenan.
λ-carrageenan.
FUNCTIONALITY-RELATED CHARACTERISTICS
CHARACTERS This section provides information on characteristics that are
Appearance : yellowish, brownish, or white or almost white recognised as being relevant control parameters for one or
powder. more functions of the substance when used as an excipient (see
Solubility : soluble in water giving a viscous or colloidal chapter 5.15). This section is a non-mandatory part of the
solution, insoluble in organic solvents. monograph and it is not necessary to verify the characteristics
to demonstrate compliance. Control of these characteristics can
IDENTIFICATION however contribute to the quality of a medicinal product by
A. Prepare a 20 g/L dispersion and heat in a water-bath at improving the consistency of the manufacturing process and
80 °C (Solution A). Allow to cool ; it becomes more viscous the performance of the medicinal product during use. Where
upon cooling and may form a gel. control methods are cited, they are recognised as being suitable

General Notices (1) apply to all monographs and other texts 1779
Carteolol hydrochloride EUROPEAN PHARMACOPOEIA 8.0

for the purpose, but other methods can also be used. Wherever – stationary phase : octadecylsilyl silica gel for
results for a particular characteristic are reported, the control chromatography R (5 μm).
method must be indicated. Mobile phase : mix 1 volume of methanol R2, 20 volumes of
The following characteristics may be relevant for carrageenan acetonitrile R and 79 volumes of a 2.82 g/L solution of sodium
used as viscosity-increasing agent. hexanesulfonate R.
Gel formation : see Identification A. Flow rate : 1 mL/min.
Apparent viscosity : see Tests. Detection : spectrophotometer at 252 nm.
Injection : 20 μL.
Identification of impurities : use the chromatogram supplied
with carteolol for system suitability CRS to identify the peak
01/2008:1972 due to impurity H.
corrected 6.0
System suitability :
CARTEOLOL HYDROCHLORIDE – the chromatogram obtained with reference solution (c) is
similar to the chromatogram provided with carteolol for
system suitability CRS ; the peaks due to impurity H and
Carteololi hydrochloridum carteolol show base-line separation ;
– signal-to-noise ratio : minimum 10 for the principal peak in
the chromatogram obtained with reference solution (d) ;
– number of theoretical plates : minimum 6000, calculated
for the principal peak in the chromatogram obtained with
reference solution (a).
Limits :
C16H25N2O3Cl Mr 328.8
[51781-21-6] – impurity H : not more than twice the area of the principal
peak in the chromatogram obtained with reference
DEFINITION solution (b) (0.2 per cent) ;
5-[(2RS)-3-[(1,1-Dimethylethyl)amino]-2-hydroxypropoxy]- – unspecified impurities : for each impurity, not more than the
3,4-dihydroquinolin-2(1H)-one hydrochloride. area of the principal peak in the chromatogram obtained
Content : 99.0 per cent to 101.0 per cent (dried substance). with reference solution (b) (0.10 per cent) ;
– total : not more than half the area of the principal peak in
CHARACTERS the chromatogram obtained with reference solution (a)
Appearance : white or almost white crystals or crystalline (0.5 per cent) ;
powder. – disregard limit : 0.2 times the area of the principal peak in
Solubility : soluble in water, sparingly soluble in methanol, the chromatogram obtained with reference solution (b)
slightly soluble in ethanol 96 per cent, practically insoluble (0.02 per cent).
in methylene chloride. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 3 h.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24). Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
Comparison : Ph. Eur. reference spectrum of carteolol
hydrochloride. ASSAY
B. It gives reaction (a) of chlorides (2.3.1). Dissolve 0.250 g in 60 mL of ethanol (96 per cent) R. Add
5.0 mL of 0.01 M hydrochloric acid. Carry out a potentiometric
TESTS titration (2.2.20), using 0.1 M sodium hydroxide. Read the
Appearance of solution. The solution is clear (2.2.1) and volume added between the 2 points of inflexion.
colourless (2.2.2, Method II). 1 mL of 0.1 M sodium hydroxide is equivalent to 32.88 mg
Dissolve 0.300 g in water R and dilute to 10 mL with the same of C16H25N2O3Cl.
solvent.
STORAGE
pH (2.2.3) : 5.0 to 6.0.
In an airtight container.
Dissolve 0.250 g in carbon dioxide-free water R and dilute to
25 mL with the same solvent. IMPURITIES
Related substances. Liquid chromatography (2.2.29). Specified impurities : H.
Test solution. Dissolve 20.0 mg of the substance to be Other detectable impurities (the following substances would,
examined in the mobile phase and dilute to 10.0 mL with the if present at a sufficient level, be detected by one or other of
mobile phase. the tests in the monograph. They are limited by the general
Reference solution (a). Dilute 1.0 mL of the test solution to acceptance criterion for other/unspecified impurities and/or
100.0 mL with the mobile phase. by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
Reference solution (b). Dilute 1.0 mL of reference solution (a) impurities for demonstration of compliance. See also 5.10.
to 10.0 mL with the mobile phase. Control of impurities in substances for pharmaceutical use) : A,
Reference solution (c). Dissolve 10 mg of carteolol for system B, C, D, E, F, G, I.
suitability CRS in the mobile phase and dilute to 5 mL with
the mobile phase.
Reference solution (d). Dilute 5.0 mL of reference solution (b)
to 10.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ; A. 4,6,7,8-tetrahydroquinoline-2,5(1H,3H)-dione,

1780 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Carvedilol

It shows polymorphism (5.9).

IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: carvedilol CRS.
B. 5-hydroxy-3,4-dihydroquinolin-2(1H)-one,
If the spectra obtained show differences, dissolve the substance
to be examined and the reference substance separately in
2-propanol R, evaporate to dryness and record new spectra
using the residues.
TESTS
C. 5-[[(2RS)-oxiran-2-yl]methoxy]-3,4-dihydroquinolin- Related substances. Liquid chromatography (2.2.29).
2(1H)-one, Test solution. Dissolve 25 mg of the substance to be examined
in the mobile phase and dilute to 25.0 mL with the mobile
phase.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10.0 mL with the mobile phase.
Reference solution (b). Dissolve 5 mg of carvedilol
D. R = Cl, R′ = H : 5-[(2RS)-3-chloro-2-hydroxypropoxy]-3,4- impurity C CRS in 5.0 mL of the mobile phase and dilute to
dihydroquinolin-2(1H)-one, 100.0 mL with the mobile phase. Dilute 4.0 mL of the solution
F. R = OCH3, R′ = H : 5-[(2RS)-2-hydroxy-3- to 100.0 mL with the mobile phase. Dilute 1.0 mL of this
methoxypropoxy]-3,4-dihydroquinolin-2(1H)-one, solution to 10.0 mL with the mobile phase.
Reference solution (c). Dissolve 5 mg of carvedilol for system
G. R = OH, R′ = H : 5-[(2RS)-2,3-dihydroxypropoxy]-3,4-
suitability CRS (containing impurities A and D) in the mobile
dihydroquinolin-2(1H)-one,
phase and dilute to 50.0 mL with the mobile phase.
I. R = NH-C(CH3)3, R′ = Br : 7-bromo-5-[(2RS)-3- Column :
[(1,1-(dimethylethyl)amino]-2-hydroxypropoxy]-3,4-
dihydroquinolin-2(1H)-one, – size : l = 0.150 m, Ø = 4.6 mm ;
– stationary phase : end-capped octylsilyl silica gel for
chromatography R (5 μm) ;
– temperature : 55 °C.
Mobile phase : dissolve 1.77 g of potassium dihydrogen
phosphate R in water R and dilute to 650 mL with the same
E. 5,5′-[(2-hydroxypropan-1,3-diyl)bis(oxy)]bis(3,4- solvent ; adjust to pH 2.0 with phosphoric acid R and add
dihydroquinolin-2(1H)-one), 350 mL of acetonitrile R.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 240 nm.
Injection : 20 μL.
Run time : 6 times the retention time of carvedilol.
Identification of impurities : use the chromatogram
H. 5-[(2RS)-3-[(1,1-dimethylethyl)amino]-2-
supplied with carvedilol for system suitability CRS and the
hydroxypropoxy]quinolin-2(1H)-one.
chromatogram obtained with reference solution (c) to identify
the peaks due to impurities A and D ; use the chromatogram
04/2012:1745 obtained with reference solution (b) to identify the peak due
corrected 8.0 to impurity C.
Relative retention with reference to carvedilol (retention
CARVEDILOL time = about 4 min) : impurity A = about 0.5 ;
impurity C = about 2.9 ; impurity D = about 3.8.
Carvedilolum System suitability :
– resolution : minimum 3.5 between the peaks due to
impurity A and carvedilol in the chromatogram obtained
with reference solution (c) ;
– signal-to-noise ratio : minimum 10 for the peak due to
impurity C in the chromatogram obtained with reference
solution (b).
C24H26N2O4 Mr 406.5 Limits :
[72956-09-3] – correction factor : for the calculation of content, multiply
DEFINITION the peak area of impurity A by 2.0 ;
(2RS)-1-(9H-Carbazol-4-yloxy)-3-[[2-(2-methoxyphenoxy)- – impurity A : not more than twice the area of the principal
ethyl]amino]propan-2-ol. peak in the chromatogram obtained with reference
Content : 99.0 per cent to 101.0 per cent (dried substance). solution (a) (0.2 per cent) ;
– impurity D : not more than 1.5 times the area of the
CHARACTERS principal peak in the chromatogram obtained with
Appearance : white or almost white, crystalline powder. reference solution (a) (0.15 per cent) ;
Solubility : practically insoluble in water, sparingly soluble in – impurity C : not more than the area of the corresponding
methylene chloride, slightly soluble in ethanol (96 per cent). peak in the chromatogram obtained with reference
It is practically insoluble in dilute acids. solution (b) (0.02 per cent);

General Notices (1) apply to all monographs and other texts 1781
Castor oil, hydrogenated EUROPEAN PHARMACOPOEIA 8.0

– unspecified impurities : for each impurity, not more than the

area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
– sum of impurities other than C : not more than 5 times the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.5 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a) C. (2RS)-1-[benzyl[2-(2-methoxyphenoxy)ethyl]amino]-3-
(0.05 per cent). (9H-carbazol-4-yloxy)propan-2-ol,
Heavy metals (2.4.8) : maximum 10 ppm.
Solvent : dimethyl sulfoxide R.
2.0 g complies with test H. Prepare the reference solution
using 2 mL of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
D. 1-(9H-carbazol-4-yloxy)-3-[4-[2-hydroxy-3-[[2-(2-
ASSAY methoxyphenoxy)ethyl]amino]propoxy]-9H-carbazol-9-
yl]propan-2-ol.
Dissolve 0.350 g in 60 mL of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point 01/2008:1497
potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 40.65 mg of CASTOR OIL, HYDROGENATED
C24H26N2O4.
Ricini oleum hydrogenatum
IMPURITIES DEFINITION
Specified impurities : A, C, D. Fatty oil obtained by hydrogenation of Virgin Castor oil (0051).
It consists mainly of the triglyceride of 12-hydroxystearic
Other detectable impurities (the following substances would, (12-hydroxyoctadecanoic) acid.
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general CHARACTERS
acceptance criterion for other/unspecified impurities and/or Appearance : fine, almost white or pale yellow powder or
by the general monograph Substances for pharmaceutical almost white or pale yellow masses or flakes.
use (2034). It is therefore not necessary to identify these Solubility : practically insoluble in water, slightly soluble in
impurities for demonstration of compliance. See also 5.10. methylene chloride, very slightly soluble in anhydrous ethanol,
Control of impurities in substances for pharmaceutical use) : B. practically insoluble in light petroleum.
IDENTIFICATION
A. Melting point (2.2.14) : 83 °C to 88 °C.
B. Hydroxyl value (see Tests).
C. Composition of fatty acids (see Tests).
TESTS
Acid value (2.5.1) : maximum 4.0, determined on 10.0 g
dissolved in 75 mL of hot ethanol (96 per cent) R.
Hydroxyl value (2.5.3, Method A) : 145 to 165, determined on
a warm solution.
A. 1-[[9-[2-hydroxy-3-[[2-(2-methoxyphenoxy)ethyl]amino]- Iodine value (2.5.4, Method A) : maximum 5.0.
propyl]-9H-carbazol-4-yl]oxy]-3-[[2-(2- Alkaline impurities. Dissolve 1.0 g by gentle heating in a
methoxyphenoxy)ethyl]amino]propan-2-ol, mixture of 1.5 mL of ethanol (96 per cent) R and 3 mL of
toluene R. Add 0.05 mL of a 0.4 g/L solution of bromophenol
blue R in ethanol (96 per cent) R. Not more than 0.2 mL of
0.01 M hydrochloric acid is required to change the colour of
the indicator to yellow.
Composition of fatty acids (2.4.22). Use the mixture of
calibrating substances in Table 2.4.22.-3.
Test solution. Introduce 75 mg of the substance to be examined
into a 10 mL centrifuge tube with a screw cap. Dissolve in
2 mL of 1,1-dimethylethyl methyl ether R1 by shaking and heat
gently (50-60 °C). Add, when still warm, 1 mL of a 12 g/L
solution of sodium R in anhydrous methanol R, prepared with
the necessary precautions, and mix vigorously for at least
5 min. Add 5 mL of distilled water R and mix vigorously for
B. 1,1′-[[2-(2-methoxyphenoxy)ethyl]nitrilo]bis[3-(9H- about 30 s. Centrifuge for 15 min at 1500 g. Use the upper
carbazol-4-yloxy)propan-2-ol], layer.

1782 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Castor oil, refined

Reference solution. Dissolve 50 mg of methyl 01/2013:2367

12-hydroxystearate CRS and 50 mg of methyl stearate CRS in
10.0 mL of 1,1-dimethylethyl methyl ether R1. CASTOR OIL, REFINED
Column :
– material : fused silica ; Ricini oleum raffinatum
– size : l = 30 m ; Ø = 0.25 mm ;
DEFINITION
– stationary phase : macrogol 20 000 R (film thickness
0.25 μm). Fatty oil obtained from the seeds of Ricinus communis L. by
Carrier gas : helium for chromatography R. cold expression. It is then refined. A suitable antioxidant may
be added.
Flow rate : 0.9 mL/min.
Split ratio : 1:100. PRODUCTION
Temperature : During the expression step, the temperature of the oil must
Time Temperature not exceed 50 °C.
(min) (°C) CHARACTERS
Column 0 - 55 215
Appearance : clear, almost colourless or slightly yellow, viscous,
Injection port 250 hygroscopic liquid.
Detector 250 Solubility : slightly soluble in light petroleum, miscible with
ethanol (96 per cent) and with glacial acetic acid.
Detection : flame ionisation. Relative density : about 0.958.
Injection : 1 μL. Refractive index : about 1.479.
Calculate the fraction of each fatty-acid using the following Viscosity : about 1000 mPa·s.
expression :
IDENTIFICATION
First identification : B, C.
Second identification : A, B.
Ax,s,c = corrected peak area of the fatty acid in the test A. A mixture of 2 mL of the substance to be examined and
solution : 8 mL of ethanol (96 per cent) R is clear (2.2.1).
B. Specific absorbance (see Tests).
C. Composition of fatty acids (see Tests).
Rc = relative correction factor for the peak due to methyl
TESTS
12-hydroxystearate :
Appearance. The substance to be examined is clear (2.2.1)
and not more intensely coloured (2.2.2, Method II) than 20 mL
of a mixture of 0.25 mL of blue primary solution, 0.25 mL
of red primary solution, 0.8 mL of yellow primary solution,
Rc = 1 for peaks corresponding to each of the other and 18.7 mL of a solution prepared by diluting 4.0 mL of
specified fatty acids or any unspecified fatty acid ; hydrochloric acid R1 to 100.0 mL with water R.
m1,r = mass of methyl 12-hydroxystearate in the Optical rotation (2.2.7) : + 3.5° to + 6.0°.
reference solution ; Specific absorbance (2.2.25) : greater than 0.7 and maximum
m2,r = mass of methyl stearate in the reference solution ; 1.5, determined at the absorption maximum at 270 nm.
A1,r = area of any peak due to methyl 12-hydroxystearate To 1.00 g add ethanol (96 per cent) R and dilute to 100.0 mL
in the chromatogram obtained with the reference with the same solvent.
solution ; Acid value (2.5.1) : maximum 0.8.
A2,r = area of any peak due to methyl stearate in the Dissolve 5.00 g in 25 mL of the prescribed mixture of solvents.
chromatogram obtained with the reference Hydroxyl value (2.5.3, Method A) : minimum 160.
solution ;
Peroxide value (2.5.5, Method A) : maximum 5.0.
Ax,s = area of the peaks due to any specified or
unspecified fatty acid methyl esters. Unsaponifiable matter (2.5.7) : maximum 0.8 per cent,
determined on 5.0 g.
Composition of the fatty acid fraction of the oil :
Oil obtained by extraction and adulteration. In a
– palmitic acid : not more than 2.0 per cent ; ground-glass-stoppered tube about 125 mm long and 18 mm
– stearic acid : 7.0 per cent to 14.0 per cent ; in internal diameter, thoroughly mix 3 mL of the substance to
– arachidic acid : not more than 1.0 per cent ; be examined with 3 mL of carbon disulfide R. Shake for 3 min
– 12-oxostearic acid : not more than 5.0 per cent ; with 1 mL of sulfuric acid R. The mixture is less intensely
– 12-hydroxystearic acid : 78.0 per cent to 91.0 per cent ; coloured than a freshly prepared mixture of 3.2 mL of ferric
chloride solution R1, 2.3 mL of water R and 0.5 mL of dilute
– any other fatty acid : not more than 3.0 per cent. ammonia R1.
Nickel (2.4.31) : maximum 1 ppm. Composition of fatty acids. Gas chromatography (2.4.22)
STORAGE with the following modifications.
In a well-filled container. Use the mixture of calibrating substances in Table 2.4.22.-3.
Test solution. Introduce 75 mg of the substance to be examined
IMPURITIES into a 10 mL centrifuge tube with a screw cap. Dissolve in
2 mL of 1,1-dimethylethyl methyl ether R1 with shaking and
heat gently (50-60 °C). To the still-warm solution, add 1 mL
of a 12 g/L solution of sodium R in anhydrous methanol R,
A. 12-oxostearic acid. prepared with the necessary precautions, and shake vigorously

General Notices (1) apply to all monographs and other texts 1783
Castor oil, virgin EUROPEAN PHARMACOPOEIA 8.0

for at least 5 min. Add 5 mL of distilled water R and shake 01/2013:0051

vigorously for about 30 s. Centrifuge for 15 min at 1500 g.
Use the upper layer. CASTOR OIL, VIRGIN
Reference solution. Dissolve 50 mg of methyl ricinoleate CRS
and 50 mg of methyl stearate CRS in 10.0 mL of Ricini oleum virginale
1,1-dimethylethyl methyl ether R1.
Column : DEFINITION
Fatty oil obtained by cold expression from the seeds of Ricinus
– material : fused silica ; communis L. A suitable antioxidant may be added.
– size : l = 30 m, Ø = 0.25 mm ;
PRODUCTION
– stationary phase : macrogol 20 000 R (film thickness
0.25 μm). During the expression step, the temperature of the oil must
not exceed 50 °C.
Carrier gas : helium for chromatography R.
CHARACTERS
Flow rate : 0.9 mL/min.
Appearance : clear at 40 °C, slightly yellow, viscous, hygroscopic
Split ratio : 1:100. liquid.
Temperature : Solubility : slightly soluble in light petroleum, miscible with
ethanol (96 per cent) and with glacial acetic acid.
Time Temperature
(min) (°C) Relative density : about 0.958.
Column 0 - 55 215 Refractive index : about 1.479.
Injection port 250 IDENTIFICATION
Detector 250 First identification : B, C.
Second identification : A, B.
Detection : flame ionisation. A. A mixture of 2 mL of the substance to be examined and
Injection : 1 μL. 8 mL of ethanol (96 per cent) R is clear (2.2.1).
Calculate the percentage content of each fatty acid by the B. Specific absorbance (see Tests).
normalisation procedure. C. Composition of fatty acids (see Tests).
Correct the area of the peak due to methyl ricinoleate, by TESTS
multiplying by a factor R calculated using the following
expression : Optical rotation (2.2.7) : + 3.5° to + 6.0°.
Specific absorbance (2.2.25) : maximum 0.7, determined at
the absorption maximum at 270 nm.
To 1.00 g add ethanol (96 per cent) R and dilute to 100.0 mL
m1 = mass of methyl ricinoleate in the reference solution ; with the same solvent.
Acid value (2.5.1) : maximum 1.5.
m2 = mass of methyl stearate in the reference solution ;
Dissolve 5.00 g in 25 mL of the prescribed mixture of solvents.
A1 = area of the peak due to methyl ricinoleate in Hydroxyl value (2.5.3, Method A) : minimum 160.
the chromatogram obtained with the reference
solution ; Peroxide value (2.5.5, Method A) : maximum 10.0.
A2 = area of the peak due to methyl stearate in the Unsaponifiable matter (2.5.7) : maximum 0.8 per cent,
chromatogram obtained with the reference determined on 5.0 g.
solution. Composition of fatty acids. Gas chromatography (2.4.22)
Composition of the fatty-acid fraction of the oil: with the following modifications.
Use the mixture of calibrating substances in Table 2.4.22.-3.
– palmitic acid : maximum 2.0 per cent ;
Test solution. Introduce 75 mg of the substance to be examined
– stearic acid : maximum 2.5 per cent ; into a 10 mL centrifuge tube with a screw cap. Dissolve in
– oleic acid and isomers : 2.5 per cent to 6.0 per cent ; 2 mL of 1,1-dimethylethyl methyl ether R1 with shaking and
heat gently (50-60 °C). Add, while still warm, 1 mL of a 12 g/L
– linoleic acid : 2.5 per cent to 7.0 per cent ; solution of sodium R in anhydrous methanol R, prepared with
– linolenic acid : maximum 1.0 per cent ; the necessary precautions, and mix vigorously for at least
5 min. Add 5 mL of distilled water R and mix vigorously for
– eicosenoic acid : maximum 1.0 per cent ; about 30 s. Centrifuge for 15 min at 1500 g. Use the upper
– ricinoleic acid : 85.0 per cent to 92.0 per cent ; layer.
– any other fatty acid : maximum 1.0 per cent. Reference solution. Dissolve 50 mg of methyl ricinoleate CRS
and 50 mg of methyl stearate CRS in 10.0 mL of
Water (2.5.32) : maximum 0.3 per cent, or maximum 0.2 per 1,1-dimethylethyl methyl ether R1.
cent if intended for use in the manufacture of parenteral
preparations, determined on 1.00 g. Column :
– material : fused silica ;
STORAGE – size : l = 30 m, Ø = 0.25 mm ;
In an airtight, well-filled container, protected from light. – stationary phase : macrogol 20 000 R (film thickness
0.25 μm).
LABELLING Carrier gas : helium for chromatography R.
The label states, where applicable, that the substance is suitable Flow rate : 0.9 mL/min.
for use in the manufacture of parenteral preparations. Split ratio : 1:100.

1784 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cefaclor

Temperature : CHARACTERS
Time Temperature Appearance : white or slightly yellow powder.
(min) (°C) Solubility : slightly soluble in water, practically insoluble in
Column 0 - 55 215 methanol and in methylene chloride.
Injection port 250 IDENTIFICATION
Detector 250 Infrared absorption spectrophotometry (2.2.24).
Detection : flame ionisation. Comparison: cefaclor CRS.
Injection : 1 μL. TESTS
Calculate the percentage content of each fatty acid by the pH (2.2.3) : 3.0 to 4.5.
normalisation procedure.
Suspend 0.250 g in carbon dioxide-free water R and dilute to
Correct the area of the peak due to methyl ricinoleate, by 10 mL with the same solvent.
multiplying by a factor R calculated using the following
expression : Specific optical rotation (2.2.7) : + 101 to + 111 (anhydrous
substance).
Dissolve 0.250 g in a 10 g/L solution of hydrochloric acid R
and dilute to 25.0 mL with the same solution.
Related substances. Liquid chromatography (2.2.29).
m1 = mass of methyl ricinoleate in the reference solution ;
Test solution. Dissolve 50.0 mg of the substance to be
m2 = mass of methyl stearate in the reference solution ; examined in 10.0 mL of a 2.7 g/L solution of sodium
dihydrogen phosphate R adjusted to pH 2.5 with phosphoric
A1 = area of the peak due to methyl ricinoleate in acid R.
the chromatogram obtained with the reference Reference solution (a). Dissolve 2.5 mg of cefaclor CRS and
solution ; 5.0 mg of delta-3-cefaclor CRS (impurity D) in 100.0 mL of a
2.7 g/L solution of sodium dihydrogen phosphate R adjusted to
A2 = area of the peak due to methyl stearate in the pH 2.5 with phosphoric acid R.
chromatogram obtained with the reference Reference solution (b). Dilute 1.0 mL of the test solution
solution. to 100.0 mL with a 2.7 g/L solution of sodium dihydrogen
phosphate R adjusted to pH 2.5 with phosphoric acid R.
Composition of the fatty-acid fraction of the oil :
Column :
– palmitic acid : maximum 2.0 per cent ;
– stearic acid : maximum 2.5 per cent ; – size : l = 0.25 m, Ø = 4.6 mm ;
– oleic acid and isomers : 2.5 per cent to 6.0 per cent ; – stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
– linoleic acid : 2.5 per cent to 7.0 per cent ;
Mobile phase :
– linolenic acid : maximum 1.0 per cent ;
– mobile phase A : 7.8 g/L solution of sodium dihydrogen
– eicosenoic acid : maximum 1.0 per cent ; phosphate R adjusted to pH 4.0 with phosphoric acid R ;
– ricinoleic acid : 85.0 per cent to 92.0 per cent ;
– mobile phase B : mix 450 mL of acetonitrile R with 550 mL
– any other fatty acid : maximum 1.0 per cent. of mobile phase A ;
Water (2.5.32) : maximum 0.3 per cent, determined on 1.00 g. Time Mobile phase A Mobile phase B
STORAGE (min) (per cent V/V) (per cent V/V)
0 - 30 95 → 75 5 → 25
In an airtight, well-filled container, protected from light.
30 - 45 75 → 0 25 → 100

01/2008:0986 45 - 55 0 100
corrected 6.5
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 220 nm.
CEFACLOR
Injection : 20 μL.
Cefaclorum System suitability : reference solution (a) :
– resolution : minimum 2 between the peaks due to cefaclor
and impurity D ; if necessary, adjust the acetonitrile content
in the mobile phase ;
– symmetry factor : maximum 1.2 for the peak due to cefaclor ;
if necessary, adjust the acetonitrile content in the mobile
phase.
C15H14ClN3O4S,H2O Mr 385.8 Limits :
[70356-03-5] – any impurity : for each impurity, not more than 0.5 times
the area of the principal peak in the chromatogram
DEFINITION obtained with reference solution (b) (0.5 per cent) ;
(6R,7R)-7-[[(2R)-2-Amino-2-phenylacetyl]amino]-3-chloro- – total : not more than twice the area of the principal peak
8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid in the chromatogram obtained with reference solution (b)
monohydrate. (2 per cent) ;
Semi-synthetic product derived from a fermentation product. – disregard limit : 0.1 times the area of the principal peak in
Content : 96.0 per cent to 102.0 per cent of C15H14ClN3O4S the chromatogram obtained with reference solution (b)
(anhydrous substance). (0.1 per cent).

General Notices (1) apply to all monographs and other texts 1785
Cefadroxil monohydrate EUROPEAN PHARMACOPOEIA 8.0

Heavy metals (2.4.8) : maximum 30 ppm.

1.0 g complies with test C. Prepare the reference solution using
3 mL of lead standard solution (10 ppm Pb) R.
Water (2.5.12) : 3.0 per cent to 6.5 per cent, determined on
0.200 g.
D. (2R,6R,7R)- and (2S,6R,7R)-7-[[(2R)-2-amino-
ASSAY 2-phenylacetyl]amino]-3-chloro-8-oxo-5-thia-
Liquid chromatography (2.2.29). 1-azabicyclo[4.2.0]oct-3-ene-2-carboxylic acid
(delta-3-cefaclor),
Test solution. Dissolve 15.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with the
mobile phase.
Reference solution (a). Dissolve 15.0 mg of cefaclor CRS in the
mobile phase and dilute to 50.0 mL with the mobile phase.
Reference solution (b). Dissolve 3.0 mg of cefaclor CRS and
3.0 mg of delta-3-cefaclor CRS (impurity D) in the mobile E. 2-[[(2R)-2-amino-2-phenylacetyl]amino]-2-(5-chloro-4-
phase and dilute to 10.0 mL with the mobile phase. oxo-3,4-dihydro-2H-1,3-thiazin-2-yl)acetic acid,
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : add 220 mL of methanol R to a mixture of F. 3-phenylpyrazin-2-ol,
780 mL of water R, 10 mL of triethylamine R and 1 g of sodium
pentanesulfonate R, then adjust to pH 2.5 with phosphoric
acid R.
Flow rate : 1.5 mL/min.
Detection : spectrophotometer at 265 nm.
Injection : 20 μL. G. (2R,6R,7R)- and (2S,6R,7R)-7-[[(2R)-2-amino-2-
System suitability : phenylacetyl]amino]-3-methylene-8-oxo-5-thia-1-
azabicyclo[4.2.0]octane-2-carboxylic acid (isocefalexine),
– resolution : minimum 2.5 between the peaks due to cefaclor
and impurity D in the chromatogram obtained with
reference solution (b) ; if necessary, adjust the concentration
of methanol in the mobile phase ;
– symmetry factor : maximum 1.5 for the peak due to cefaclor
in the chromatogram obtained with reference solution (b) ;
– repeatability : maximum relative standard deviation of
1.0 per cent after 6 injections of reference solution (a). H. (6R,7R)-7-[[(2R)-2-[[(2R)-2-amino-2-phenylacetyl]ami-
no]-2-phenylacetyl]amino]-3-chloro-8-oxo-5-thia-1-aza-
IMPURITIES bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (N-phenylglycyl
cefaclor).

04/2008:0813
corrected 7.0

CEFADROXIL MONOHYDRATE
A. (2R)-2-amino-2-phenylacetic acid (phenylglycine),
Cefadroxilum monohydricum

B. (6R,7R)-7-amino-3-chloro-8-oxo-5-thia-1-azabicyclo-
[4.2.0]oct-2-ene-2-carboxylic acid, C16H17N3O5S,H2O Mr 381.4
[66592-87-8]
DEFINITION
(6R,7R)-7-[[(2R)-2-Amino-2-(4-hydroxyphenyl)ace-
tyl]amino]-3-methyl-8-oxo-5-thia-1-azabicy-
clo[4.2.0]oct-2-ene-2-carboxylic acid monohydrate.
Semi-synthetic product derived from a fermentation product.
Content : 95.0 per cent to 102.0 per cent (anhydrous substance).
C. (6R,7R)-7-[[(2S)-2-amino-2-phenylacetyl]amino]-3-
chloro-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2- CHARACTERS
carboxylic acid, Appearance : white or almost white powder.

1786 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cefadroxil monohydrate

Solubility : slightly soluble in water, very slightly soluble in – any other impurity : for each impurity, not more than the
ethanol (96 per cent). area of the 2nd peak in the chromatogram obtained with
reference solution (c) (1.0 per cent),
IDENTIFICATION – total : not more than 3 times the area of the 2nd peak in
Infrared absorption spectrophotometry (2.2.24). the chromatogram obtained with reference solution (c)
Comparison : cefadroxil CRS. (3.0 per cent),
– disregard limit : 0.05 times the area of the 2nd peak
TESTS in the chromatogram obtained with reference
pH (2.2.3) : 4.0 to 6.0. solution (c) (0.05 per cent) ; disregard the peaks due to
Suspend 1.0 g in carbon dioxide-free water R and dilute to dimethylformamide and dimethylacetamide.
20 mL with the same solvent. N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm.
Specific optical rotation (2.2.7) : + 165 to + 178 (anhydrous Water (2.5.12) : 4.0 per cent to 6.0 per cent, determined on
substance). 0.200 g.
Dissolve 0.500 g in water R and dilute to 50.0 mL with the Sulfated ash (2.4.14) : maximum 0.5 per cent, determined on
same solvent. 1.0 g.
Related substances. Liquid chromatography (2.2.29). ASSAY
Test solution. Dissolve 50.0 mg of the substance to be Liquid chromatography (2.2.29).
examined in mobile phase A and dilute to 50.0 mL with
mobile phase A. Test solution. Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 100.0 mL with
Reference solution (a). Dissolve 10.0 mg of D-α-(4- the mobile phase.
hydroxyphenyl)glycine CRS (impurity A) in mobile phase A
and dilute to 10.0 mL with mobile phase A. Reference solution (a). Dissolve 50.0 mg of cefadroxil CRS in
the mobile phase and dilute to 100.0 mL with the mobile phase.
Reference solution (b). Dissolve 10.0 mg of 7-aminodesacet-
Reference solution (b). Dissolve 5 mg of cefadroxil CRS and
oxycephalosporanic acid CRS (impurity B) in phosphate buffer
50 mg of amoxicillin trihydrate CRS in the mobile phase and
solution pH 7.0 R5 and dilute to 10.0 mL with the same buffer
dilute to 100 mL with the mobile phase.
solution.
Column :
Reference solution (c). Dilute 1.0 mL of reference solution (a)
and 1.0 mL of reference solution (b) to 100.0 mL with mobile – size : l = 0.25 m, Ø = 4.6 mm,
phase A. – stationary phase : octadecylsilyl silica gel for
Reference solution (d). Dissolve 10 mg of dimethylformamide R chromatography R (5 μm).
and 10 mg of dimethylacetamide R in mobile phase A and Mobile phase : acetonitrile R, a 2.72 g/L solution of potassium
dilute to 10.0 mL with mobile phase A. Dilute 1.0 mL of this dihydrogen phosphate R (4:96 V/V).
solution to 100.0 mL with mobile phase A. Flow rate : 1 mL/min.
Reference solution (e). Dilute 1.0 mL of reference solution (c) Detection : spectrophotometer at 254 nm.
to 25.0 mL with mobile phase A. Injection : 20 μL.
Column : System suitability : reference solution (b) :
– size : l = 0.10 m, Ø = 4.6 mm, – resolution : minimum 5.0 between the peaks due to
– stationary phase : spherical octadecylsilyl silica gel for cefadroxil and to amoxicillin.
chromatography R (5 μm). Calculate the percentage content of cefadroxil.
Mobile phase :
STORAGE
– mobile phase A : phosphate buffer solution pH 5.0 R,
Protected from light.
– mobile phase B : methanol R2,
Time
IMPURITIES
Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-1 98 2
1 - 20 98 → 70 2 → 30

Flow rate : 1.5 mL/min. A. (2R)-2-amino-2-(4-hydroxyphenyl)acetic acid,

Detection : spectrophotometer at 220 nm.
Injection : 20 μL of the test solution and reference solutions (c),
(d) and (e).
Relative retention with reference to cefadroxil (retention
time = about 6 min) : dimethylformamide = about 0.4 ;
dimethylacetamide = about 0.75. B. (6R,7R)-7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo-
System suitability : [4.2.0]oct-2-ene-2-carboxylic acid (7-ADCA),
– resolution : minimum 5.0 between the peaks due to
impurities A and B in the chromatogram obtained with
reference solution (c),
– signal-to-noise ratio : minimum 10 for the 2nd peak in the
chromatogram obtained with reference solution (e).
Limits :
– impurity A : not more than the area of the 1st peak in the C. (2R,5RS)-2-[(R)-[[(2R)-2-amino-2-(4-hydroxyphenyl)-
chromatogram obtained with reference solution (c) (1.0 per acetyl]amino]carboxymethyl]-5-methyl-5,6-dihydro-2H-
cent), 1,3-thiazine-4-carboxylic acid,

General Notices (1) apply to all monographs and other texts 1787
Cefalexin monohydrate EUROPEAN PHARMACOPOEIA 8.0

IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: cefalexin monohydrate CRS.
TESTS
pH (2.2.3) : 4.0 to 5.5.
D. (6R,7R)-7-[[(2S)-2-amino-2-(4-hydroxyphenyl)- Dissolve 50 mg in carbon dioxide-free water R and dilute to
acetyl]amino]-3-methyl-8-oxo-5-thia-1-azabicyclo-
10 mL with the same solvent.
[4.2.0]oct-2-ene-2-carboxylic acid (L-cefadroxil),
Specific optical rotation (2.2.7) : + 149 to + 158 (anhydrous
substance).
Dissolve 0.125 g in phthalate buffer solution pH 4.4 R and
dilute to 25.0 mL with the same solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50.0 mg of the substance to be
E. (6RS)-3-(aminomethylene)-6-(4-hydroxyphenyl)pipera- examined in mobile phase A and dilute to 50.0 mL with
zine-2,5-dione, mobile phase A.
Reference solution (a). Dissolve 10.0 mg of D-phenylglycine R
in mobile phase A and dilute to 10.0 mL with mobile phase A.
Reference solution (b). Dissolve 10.0 mg of 7-aminodesacet-
oxycephalosporanic acid CRS in phosphate buffer solution
pH 7.0 R5 and dilute to 10.0 mL with mobile phase A.
Reference solution (c). Dilute 1.0 mL of reference solution (a)
and 1.0 mL of reference solution (b) to 100.0 mL with mobile
F. (6R,7R)-7-[[(2R)-2-[[(2RS)-2-amino-2-(4-hydroxyphenyl)- phase A.
acetyl]amino]-2-(4-hydroxyphenyl)acetyl]amino]-3-
methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2- Reference solution (d). Dissolve 10 mg of dimethylformamide R
carboxylic acid, and 10 mg of dimethylacetamide R in mobile phase A and
dilute to 10.0 mL with mobile phase A. Dilute 1.0 mL of this
solution to 100.0 mL with mobile phase A.
Reference solution (e). Dilute 1.0 mL of reference solution (c)
to 20.0 mL with mobile phase A.
Reference solution (f). Dissolve 10 mg of cefotaxime
G. 3-hydroxy-4-methylthiophen-2(5H)-one, sodium CRS in mobile phase A and dilute to 10.0 mL with
mobile phase A. To 1.0 mL of this solution add 1.0 mL of the
test solution and dilute to 100 mL with mobile phase A.
Column :
– size : l = 0.10 m, Ø = 4.6 mm ;
– stationary phase : spherical octadecylsilyl silica gel for
H. (6R,7R)-7-[(2,2-dimethylpropanoyl)amino]-3-methyl-8- chromatography R (5 μm).
oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid Mobile phase :
(7-ADCA pivalamide). – mobile phase A : phosphate buffer solution pH 5.0 R ;
– mobile phase B : methanol R2 ;
04/2008:0708
corrected 7.0 Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
CEFALEXIN MONOHYDRATE 0-1 98 2
1 - 20 98 → 70 2 → 30
Cefalexinum monohydricum
Flow rate : 1.5 mL/min.
Detection : spectrophotometer at 220 nm.
Injection : 20 μL of the test solution and reference solutions (c),
(d), (e) and (f).
System suitability :
– resolution : minimum 2.0 between the peaks due to
impurities A and B in the chromatogram obtained with
C16H17N3O4S,H2O Mr 365.4 reference solution (c) and minimum 1.5 between the peaks
[23325-78-2] due to cefalexin and cefotaxime in the chromatogram
DEFINITION obtained with reference solution (f).
(6R,7R)-7-[[(2R)-2-Amino-2-phenylacetyl]amino]-3-methyl- Limits :
8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid – impurity B : not more than the area of the 2nd peak in the
monohydrate. chromatogram obtained with reference solution (c) (1.0 per
Semi-synthetic product derived from a fermentation product. cent) ;
Content : 95.0 per cent to 102.0 per cent (anhydrous substance). – any other impurity : not more than the area of the 1st peak
in the chromatogram obtained with reference solution (c)
CHARACTERS (1.0 per cent) ;
Appearance : white or almost white, crystalline powder. – total : not more than 3 times the area of the 1st peak in the
Solubility : sparingly soluble in water, practically insoluble in chromatogram obtained with reference solution (c) (3.0 per
ethanol (96 per cent). cent) ;

1788 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cefalotin sodium

– disregard limit : the area of the 2nd peak in the chromatogram

obtained with reference solution (e) (0.05 per cent) ;
disregard any peaks due to dimethylformamide or
dimethylacetamide.
N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm. D. 3-hydroxy-4-methylthiophen-2(5H)-one,
Water (2.5.12) : 4.0 per cent to 8.0 per cent, determined on
0.300 g.
Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
1.0 g.
ASSAY
E. (6R,7R)-7-[(2,2-dimethylpropanoyl)amino]-3-methyl-8-
Liquid chromatography (2.2.29). oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
Test solution. Dissolve 50.0 mg of the substance to be (7-ADCA pivalamide),
examined in water R and dilute to 100.0 mL with the same
solvent.
Reference solution (a). Dissolve 50.0 mg of cefalexin
monohydrate CRS in water R and dilute to 100.0 mL with the
same solvent.
Reference solution (b). Dissolve 10 mg of cefradine CRS in
20 mL of reference solution (a) and dilute to 100 mL with F. (2RS,6R,7R)-7-[[(2R)-2-amino-2-phenylacetyl]amino]-
water R. 3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-3-ene-2-
Column : carboxylic acid (delta-2-cefalexin).
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm). 04/2013:0987
Mobile phase : methanol R, acetonitrile R, 13.6 g/L
solution of potassium dihydrogen phosphate R, water R CEFALOTIN SODIUM
(2:5:10:83 V/V/V/V).
Flow rate : 1.5 mL/min. Cefalotinum natricum
Detection : spectrophotometer at 254 nm.
Injection : 20 μL.
System suitability : reference solution (b) :
– resolution : minimum 4.0 between the peaks due to cefalexin
and cefradine.
Calculate the percentage content of cefalexin monohydrate.
C16H15N2NaO6S2 Mr 418.4
STORAGE [58-71-9]
Protected from light. DEFINITION
IMPURITIES Sodium (6R,7R)-3-[(acetyloxy)methyl]-8-oxo-7-[(thiophen-
2-ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
carboxylate.
Semi-synthetic product derived from a fermentation product.
Content : 96.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
A. (2R)-2-amino-2-phenylacetic acid (D-phenylglycine),
Appearance : white or almost white powder.
Solubility : freely soluble in water, slightly soluble in anhydrous
ethanol.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
B. (6R,7R)-7-amino-3-methyl-8-oxo-5-thia-1-aza- Comparison: cefalotin sodium CRS.
bicyclo[4.2.0]oct-2-ene-2-carboxylic acid B. It gives reaction (a) of sodium (2.3.1).
(7-aminodesacetoxycephalosporanic acid, 7-ADCA),
TESTS
Solution S. Dissolve 2.50 g in carbon dioxide-free water R and
dilute to 25.0 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and its
absorbance (2.2.25) at 450 nm is not greater than 0.20.
pH (2.2.3) : 4.5 to 7.0 for solution S.
Specific optical rotation (2.2.7) : + 124 to + 134 (anhydrous
C. (6R,7R)-7-[[(2R)-2-[[(2R)-2-amino-2-phenylacetyl]ami- substance).
no]-2-phenylacetyl]amino]-3-methyl-8-oxo-5-thia-1-aza- Dissolve 1.25 g in water R and dilute to 25.0 mL with the same
bicyclo[4.2.0]oct-2-ene-2-carboxylic acid, solvent.

General Notices (1) apply to all monographs and other texts 1789
Cefalotin sodium EUROPEAN PHARMACOPOEIA 8.0

Related substances. Liquid chromatography (2.2.29). Prepare 2-Ethylhexanoic acid (2.4.28) : maximum 0.5 per cent.
the solutions immediately before use. Water (2.5.12) : maximum 1.5 per cent, determined on 0.500 g.
Test solution (a). Dissolve 75.0 mg of the substance to be
Bacterial endotoxins (2.6.14) : less than 0.13 IU/mg, if
examined in water R and dilute to 25.0 mL with the same intended for use in the manufacture of parenteral preparations
solvent. without a further appropriate procedure for the removal of
Test solution (b). Dilute 5.0 mL of test solution (a) to 50.0 mL bacterial endotoxins.
with water R.
Reference solution (a). Dissolve 75.0 mg of cefalotin ASSAY
sodium CRS in water R and dilute to 25.0 mL with the same Liquid chromatography (2.2.29) as described in the test for
solvent. Dilute 5.0 mL of the solution to 50.0 mL with water R. related substances with the following modifications.
Reference solution (b). Dilute 1.0 mL of test solution (a) to Mobile phase : mix 14 volumes of acetonitrile R and 86 volumes
100.0 mL with water R. of a 6.967 g/L solution of dipotassium hydrogen phosphate R
Reference solution (c). Mix 1 mL of test solution (a), 1 mL of previously adjusted to pH 6.0 with phosphoric acid R.
hydrochloric acid R1 and 8 mL of water R. Heat at 60 °C for Detection : spectrophotometer at 260 nm.
12 min and cool to room temperature in iced water. Inject Injection : 5 μL of test solution (b) and reference solution (a).
immediately. Run time : 1.5 times the retention time of cefalotin (retention
Reference solution (d). Dissolve 5 mg of cefalotin for impurity B time = about 10 min).
identification CRS in water R and dilute to 5 mL with the same Calculate the percentage content of C16H15N2NaO6S2 using the
solvent. chromatogram obtained with reference solution (a) and taking
Column : into account the assigned content of cefalotin sodium CRS.
– size : l = 0.25 m, Ø = 4.6 mm ;
STORAGE
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm) ; Protected from light. If the substance is sterile, store in a
sterile, airtight, tamper-proof container.
– temperature : 40 °C.
Mobile phase : IMPURITIES
– mobile phase A : mix 3 volumes of acetonitrile R1 and Specified impurities : B, D.
97 volumes of a 1.742 g/L solution of dipotassium hydrogen Other detectable impurities (the following substances would,
phosphate R previously adjusted to pH 2.5 with phosphoric if present at a sufficient level, be detected by one or other of
acid R ; the tests in the monograph. They are limited by the general
– mobile phase B : mix 40 volumes of acetonitrile R1 and acceptance criterion for other/unspecified impurities and/or
60 volumes of a 1.742 g/L solution of dipotassium hydrogen by the general monograph Substances for pharmaceutical use
phosphate R previously adjusted to pH 2.5 with phosphoric (2034). It is therefore not necessary to identify these impurities
acid R ; for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : A, C.
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 30 100 → 0 0 → 100
30 - 35 0 100

Flow rate : 1.0 mL/min.

Detection : spectrophotometer at 220 nm. A. (6R,7R)-3-methyl-8-oxo-7-[(thiophen-2-ylacetyl)amino]-
Injection : 20 μL of test solution (a) and reference solutions (b), 5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
(c) and (d). (deacetoxycefalotin),
Relative retention with reference to cefalotin (retention
time = about 26 min) : impurity C = about 0.2 ;
impurity B = about 0.7 ; impurity D = about 0.88 ;
impurity A = about 0.96.
System suitability: reference solution (c) :
– resolution : minimum 7.0 between the peaks due to
impurity D and cefalotin. B. (6R,7R)-3-(hydroxymethyl)-8-oxo-7-[(thiophen-2-
ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
Limits : carboxylic acid (deacetylcefalotin),
– impurity B : not more than the area of the principal peak
in the chromatogram obtained with reference solution (b)
(1.0 per cent) ;
– impurity D : not more than 0.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent) ;
– any other impurity : for each impurity, not more C. (6R,7R)-3-[(acetyloxy)methyl]-7-amino-8-oxo-5-thia-1-
than 0.25 times the area of the principal peak in the azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (7-ACA),
chromatogram obtained with reference solution (b)
(0.25 per cent) ;
– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(3.0 per cent) ;
– disregard limit : 0.05 times the area of the principal peak
in the chromatogram obtained with reference solution (b) D. (5aR,6R)-6-[(thiophen-2-ylacetyl)amino]-5a,6-dihydro-
(0.05 per cent). 3H,7H-azeto[2,1-b]furo[3,4-d][1,3]thiazine-1,7(4H)-dione
N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm. (cefalotin lactone).

1790 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cefamandole nafate

01/2008:1402 Reference solution (a). Dilute 1 mL of the test solution to

corrected 7.0 10 mL with the solvent mixture, then heat at 60 °C for 30 min.
Reference solution (b). Dilute 1.0 mL of the test solution to
CEFAMANDOLE NAFATE 100.0 mL with the solvent mixture.
Column :
Cefamandoli nafas – size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase :
– triethylamine phosphate solution : dissolve 2.0 g of sodium
pentanesulfonate R in 350 mL of water R, add 40 mL of
triethylamine R, adjust to pH 2.5 with phosphoric acid R
and dilute to 700 mL with water R ;
– mobile phase A : mix 1 volume of the triethylamine
phosphate solution and 2 volumes of water R ;
– mobile phase B : mix equal volumes of the triethylamine
Cefamandole nafate : [42540-40-9] phosphate solution, methanol R and acetonitrile R ;
Cefamandole sodium : [30034-03-8] Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
DEFINITION 0-1 100 0
Mixture of sodium (6R,7R)-7-[[(2R)-2-(formyloxy)-
2-phenylacetyl]amino]-3-[[(1-methyl-1H-tetrazol-5- 1 - 35 100 → 0 0 → 100
yl)sulfanyl]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-
2-ene-2-carboxylate and sodium (6R,7R)-7-[[(2R)-2- Flow rate : 1.5 mL/min.
hydroxy-2-phenylacetyl]amino]-3-[[(1-methyl-1H-tetrazol- Detection : spectrophotometer at 254 nm.
5-yl)sulfanyl]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct- Injection : 20 μL loop injector.
2-ene-2-carboxylate (cefamandole sodium), with sodium Relative retention with reference to cefamandole nafate
carbonate. (retention time = about 24 min) : cefamandole = about 0.8.
Semi-synthetic product derived from a fermentation product. System suitability : reference solution (a) :
Content : – resolution : minimum 5.0 between the peaks due to
– cefamandole nafate (C19H17N6NaO6S2) : 93.0 per cent to cefamandole and cefamandole nafate.
102.0 per cent (anhydrous and sodium carbonate-free Limits :
substance), for the sum of the content of cefamandole
nafate and cefamandole sodium expressed as cefamandole – any impurity : for each impurity, not more than the area
nafate ; of the principal peak in the chromatogram obtained with
reference solution (b) (1.0 per cent) ;
– cefamandole sodium (C18H17N6NaO5S2) : maximum 10.0 per
cent (anhydrous and sodium carbonate-free substance) ; – total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
– sodium carbonate (Na2CO3) : 4.8 per cent to 6.4 per cent. (5.0 per cent) ;
CHARACTERS – disregard limit : 0.1 times the area of the principal peak in
Appearance : white or almost white powder. the chromatogram obtained with reference solution (b)
(0.1 per cent).
Solubility : freely soluble in water, sparingly soluble in
methanol. 2-Ethylhexanoic acid (2.4.28) : maximum 0.3 per cent m/m.
Heavy metals (2.4.8) : maximum 20 ppm.
IDENTIFICATION
1.0 g complies with test C. Prepare the reference solution using
A. Infrared absorption spectrophotometry (2.2.24). 2 mL of lead standard solution (10 ppm Pb) R.
Comparison : cefamandole nafate CRS.
Water (2.5.12) : maximum 2.0 per cent, determined on 0.500 g.
B. It gives reaction (a) of sodium (2.3.1).
Bacterial endotoxins (2.6.14) : less than 0.15 IU/mg, if
TESTS intended for use in the manufacture of parenteral preparations
Solution S. Dissolve 2.5 g in carbon dioxide-free water R and without a further appropriate procedure for the removal of
dilute to 25 mL with the same solvent. bacterial endotoxins.
Appearance of solution. Solution S is clear (2.2.1) and its ASSAY
absorbance (2.2.25) at 475 nm is not greater than 0.03. Cefamandole nafate. Liquid chromatography (2.2.29).
pH : 6.0 to 8.0 for solution S, measured after 30 min. Prepare the solutions immediately before use.
Specific optical rotation (2.2.7) : − 35.0 to − 45.0 (anhydrous Test solution. Dissolve 50.0 mg of the substance to be
and sodium carbonate-free substance). examined in the mobile phase and dilute to 100.0 mL with
Dissolve 1.00 g in acetate buffer solution pH 4.7 R1 and dilute the mobile phase.
to 10.0 mL with the same solvent. Reference solution (a). Dissolve 50.0 mg of cefamandole
nafate CRS in the mobile phase and dilute to 100.0 mL with
Related substances. Liquid chromatography (2.2.29). Prepare
the mobile phase.
the solutions immediately before use.
Reference solution (b). Dilute 1 mL of the test solution to
Solvent mixture. Mix 18 volumes of acetonitrile R and
10 mL with the mobile phase, then heat at 60 °C for 30 min.
75 volumes of a 10 per cent V/V solution of triethylamine R
previously adjusted to pH 2.5 with phosphoric acid R. Column :
Test solution. Dissolve 0.100 g of the substance to be examined – size : l = 0.25 m, Ø = 4.6 mm ;
in the solvent mixture and dilute to 10.0 mL with the solvent – stationary phase : octadecylsilyl silica gel for
mixture. chromatography R (5 μm).

General Notices (1) apply to all monographs and other texts 1791
Cefapirin sodium EUROPEAN PHARMACOPOEIA 8.0

Mobile phase : mix 25 volumes of acetonitrile R and 75 volumes 01/2008:1650

of a 10 per cent V/V solution of triethylamine R previously corrected 6.0
adjusted to pH 2.5 with phosphoric acid R.
Flow rate : 1.0 mL/min. CEFAPIRIN SODIUM
Detection : spectrophotometer at 254 nm.
Injection : 20 μL loop injector. Cefapirinum natricum
System suitability :
– resolution : minimum 7.0 between the 2 principal peaks in
the chromatogram obtained with reference solution (b) ;
– repeatability : maximum relative standard deviation of
0.8 per cent after a series of single injections of not less
than 3 freshly prepared reference solutions (a).
Calculate the percentage content of cefamandole nafate C17H16N3NaO6S2 Mr 445.5
(C19H17N6NaO6S2) from the sum of the contents of cefamandole [24356-60-3]
nafate and cefamandole sodium expressed as cefamandole
DEFINITION
nafate, using the declared content of cefamandole nafate CRS.
Sodium (6R,7R)-3-[(acetyloxy)methyl]-8-oxo-7-[[[(pyridin-
1 mg of cefamandole sodium is equivalent to 1.0578 mg of 4-yl)sulfanyl]acetyl]amino]-5-thia-1-azabicyclo[4.2.0]oct-2-
cefamandole nafate. ene-2-carboxylate.
Sodium carbonate. Dissolve 0.500 g in 50 mL of water R. Semi-synthetic product derived from a fermentation product.
Titrate with 0.1 M hydrochloric acid, determining the
Content : 96.0 per cent to 102.0 per cent (anhydrous substance).
end-point potentiometrically (2.2.20).
1 mL of 0.1 M hydrochloric acid is equivalent to 5.3 mg of CHARACTERS
Na2CO3. Appearance : white or pale yellow powder.
STORAGE Solubility : soluble in water, practically insoluble in methylene
chloride.
In an airtight container, protected from light. If the substance
is sterile, store in a sterile, airtight, tamper-proof container.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
LABELLING
Comparison: cefapirin sodium CRS.
The label states that the substance contains sodium carbonate.
B. It gives reaction (a) of sodium (2.3.1).
IMPURITIES
TESTS
Appearance of solution. Dissolve 2.0 g in water R and dilute
to 10.0 mL with the same solvent. The solution is clear (2.2.1).
Dilute 5.0 mL to 10.0 mL with water R. The absorbance
(2.2.25) of this solution at 450 nm is not greater than 0.25.
pH (2.2.3) : 6.5 to 8.5.
Dissolve 0.100 g in carbon dioxide-free water R and dilute to
A. (6R,7R)-7-[[(2R)-2-(formyloxy)-2-phenylacetyl]amino]-
10.0 mL with the same solvent.
3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
carboxylic acid (formylmandeloyl-7-amino-desacetoxy- Specific optical rotation (2.2.7) : + 150 to + 165 (anhydrous
cephalosporanic acid), substance).
Dissolve 0.500 g in water R and dilute to 25.0 mL with the
same solvent.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
Test solution. Dissolve 42 mg of the substance to be examined
in the mobile phase and dilute to 200.0 mL with the mobile
phase.
C. (6R,7R)-7-[[(2R)-2-(acetyloxy)-2-phenylacetyl]amino]-
3-[[(1-methyl-1H-tetrazol-5-yl)sulfanyl]methyl]-8-oxo- Reference solution (a). Dissolve 42 mg of cefapirin sodium CRS
5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid in the mobile phase and dilute to 200.0 mL with the mobile
(O-acetylcefamandole), phase.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase.
Reference solution (c). Dilute 1.0 mL of reference solution (b)
to 20.0 mL with the mobile phase.
Reference solution (d). Mix 1 mL of the test solution, 8 mL of
D. 1-methyl-1H-tetrazole-5-thiol, the mobile phase and 1 mL of hydrochloric acid R1. Heat at
60 °C for 10 min.
Column :
– size : l = 0.30 m, Ø = 4 mm,
– stationary phase : octadecylsilyl silica gel for
chromatography R (10 μm).
Mobile phase : mix 80 mL of dimethylformamide R, 4.0 mL of
E. (6R,7R)-7-[[(2R)-2-(formyloxy)-2-phenylacetyl]amino]-3- glacial acetic acid R and 20 mL of a 4.5 per cent m/m solution
[(acetyloxy)methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2- of potassium hydroxide R. Dilute to 2 L with water R.
ene-2-carboxylic acid (formylmandeloyl-7-ACA). Flow rate : 2.0 mL/min.

1792 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cefatrizine propylene glycol

Detection : spectrophotometer at 254 nm. 01/2008:1403

Injection : 20 μL of the test solution and reference solutions (b),
(c) and (d). CEFATRIZINE PROPYLENE GLYCOL
Run time : twice the retention time of cefapirin.
Relative retention with reference to cefapirin (retention Cefatrizinum propylen glycolum
time = about 13 min) : impurity B = about 0.3 ;
impurity C = about 0.5 ; impurity A = about 0.75.
System suitability : reference solution (d) :
– resolution : minimum 2.0 between the peaks due to cefapirin
and impurity A.
Limits :
– any impurity : for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
reference solution (b) (1.0 per cent), and not more than
1 such peak has an area greater than 0.3 times the area C18H18N6O5S2,(C3H8O2)n Mr 462.5 (base)
of the principal peak in the chromatogram obtained with
reference solution (b) (0.3 per cent), DEFINITION
– total : not more than twice the area of the principal peak Mixture of (6R,7R)-7-[[(2R)-2-amino-2-(4-hydroxyphenyl)-
in the chromatogram obtained with reference solution (b) acetyl]amino]-8-oxo-3-[[(1H-1,2,3-triazol-4-yl)sulfanyl]-
(2.0 per cent), methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
– disregard limit : area of the principal peak in the and propane-1,2-diol in molecular proportions of about 1:1.
chromatogram obtained with reference solution (c) Content : 95.0 per cent to 102.0 per cent of C18H18N6O5S2
(0.05 per cent). (anhydrous substance).
N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm. CHARACTERS
2-Ethylhexanoic acid (2.4.28) : maximum 0.5 per cent. Appearance : white or almost white powder.
Water (2.5.12) : maximum 2.0 per cent, determined on 0.300 g. Solubility : slightly soluble in water, practically insoluble in
Bacterial endotoxins (2.6.14) : less than 0.17 IU/mg, if ethanol (96 per cent) and in methylene chloride.
intended for use in the manufacture of parenteral preparations IDENTIFICATION
without a further appropriate procedure for the removal of
bacterial endotoxins. A. Infrared absorption spectrophotometry (2.2.24).
Comparison: cefatrizine propylene glycol CRS.
ASSAY B. Examine the chromatograms obtained in the test for
Liquid chromatography (2.2.29) as described in the test for propylene glycol.
related substances with the following modification. Results : the principal peak in the chromatogram obtained
Injection : test solution and reference solution (a). with the test solution is similar in retention time and size
Calculate the percentage content of C17H16N3NaO6S2. to the principal peak in the chromatogram obtained with
reference solution (b).
STORAGE
TESTS
Protected from light. If the substance is sterile, store in a
sterile, tamper-proof container. Specific optical rotation (2.2.7) : + 63 to + 69 (anhydrous
substance).
IMPURITIES Dissolve 0.400 g in 1 M hydrochloric acid and dilute to 20.0 mL
Specified impurities : A, B, C. with the same acid.
Propylene glycol. Gas chromatography (2.2.28).
Solvent mixture : acetone R, water R (20:80 V/V).
Internal standard solution. Dissolve 1.0 g of
dimethylacetamide R in the solvent mixture and
dilute to 50.0 mL with the solvent mixture.
Test solution. Introduce 0.40 g of the substance to be examined
into a ground-glass-stoppered test-tube. Add 3.0 mL of the
A. (5aR,6R)-6-[[[(pyridin-4-yl)sulfanyl]acetyl]amino]-5a,6- internal standard solution, 1.0 mL of the solvent mixture and
dihydro-3H,7H-azeto[2,1-b]furo[3,4-d][1,3]thiazine- 2.0 mL of hydrochloric acid R. Seal the test-tube and shake.
1,7(4H)-dione (deacetylcefapirin lactone), Reference solution (a). Dissolve 2.0 g of propylene glycol R in
the solvent mixture and dilute to 100.0 mL with the solvent
mixture.
Reference solution (b). Introduce into a ground-glass-stoppered
test-tube 1.0 mL of reference solution (a) and 1.0 mL of the
internal standard solution.
Column :
B. R = OH : (6R,7R)-3-(hydroxymethyl)-8-oxo-7-[[[(pyridin- – material : stainless steel ;
4-yl)sulfanyl]acetyl]amino]-5-thia-1-azabicyclo[4.2.0]oct- – size : l = 2 m, Ø = 2 mm ;
2-ene-2-carboxylic acid (deacetylcefapirin), – stationary phase : ethylvinylbenzene-divinylbenzene
copolymer R (150-180 μm).
C. R = H : (6R,7R)-3-methyl-8-oxo-7-[[[(pyridin-4-
yl)sulfanyl]acetyl]amino]-5-thia-1-azabicyclo[4.2.0]oct-2- Carrier gas : nitrogen for chromatography R.
ene-2-carboxylic acid (deacetoxycefapirin). Flow rate : 30 mL/min.

General Notices (1) apply to all monographs and other texts 1793
Cefazolin sodium EUROPEAN PHARMACOPOEIA 8.0

Temperature : IMPURITIES
– column : 200 °C ; Specified impurities : A.
– injection port and detector : 250 °C.
Detection : flame ionisation.
Injection : 1 μL of the test solution and reference solution (b).
Limit :
– propylene glycol : 13.0 per cent to 18.0 per cent.
Related substances. Liquid chromatography (2.2.29). A. (6R,7R)-7-amino-8-oxo-3-[[(1H-1,2,3-triazol-4-
Test solution. Dissolve 60.0 mg of the substance to be yl)sulfanyl]methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
examined in the mobile phase and dilute to 100.0 mL with carboxylic acid (7-ACA triazole).
the mobile phase.
Reference solution (a). Dissolve 60.0 mg of cefatrizine
propylene glycol CRS in the mobile phase and dilute to 04/2013:0988
100.0 mL with the mobile phase.
Reference solution (b). Dissolve 30.0 mg of cefatrizine CEFAZOLIN SODIUM
impurity A CRS in buffer solution pH 7.0 R and dilute to
100.0 mL with the same buffer solution. Cefazolinum natricum
Reference solution (c). Dilute 0.6 mL of reference solution (a)
to 100.0 mL with the mobile phase.
Reference solution (d). Dilute 1.0 mL of reference solution (b)
to 100.0 mL with buffer solution pH 7.0 R.
Reference solution (e). To 1.0 mL of reference solution (a) add
1.0 mL of reference solution (b) and dilute to 10.0 mL with
the mobile phase.
Column : C14H13N8NaO4S3 Mr 476.5
– size : l = 0.25 m, Ø = 4 mm ; [27164-46-1]
– stationary phase : octadecylsilyl silica gel for DEFINITION
chromatography R (5 μm).
Sodium (6R,7R)-3-[[(5-methyl-1,3,4-thiadiazol-2-
Mobile phase : mix 5 volumes of acetonitrile R and 95 volumes yl)sulfanyl]methyl]-8-oxo-7-[(1H-tetrazol-1-ylacetyl)amino]-
of a 2.72 g/L solution of potassium dihydrogen phosphate R 5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate.
in water R.
Semi-synthetic product derived from a fermentation product.
Flow rate : 2 mL/min.
Content : 95.0 per cent to 102.0 per cent (anhydrous substance).
Detection : spectrophotometer at 272 nm.
Injection : 20 μL of the test solution and reference solutions (c), CHARACTERS
(d) and (e). Appearance : white or almost white powder, very hygroscopic.
Run time : at least twice the retention time of cefatrizine. Solubility : freely soluble in water, very slightly soluble in
System suitability: reference solution (e) : ethanol (96 per cent).
– resolution : minimum 5.0 between the peaks due to It shows polymorphism (5.9).
cefatrizine and impurity A.
IDENTIFICATION
Limits :
– impurity A : not more than the area of the corresponding A. Infrared absorption spectrophotometry (2.2.24).
peak in the chromatogram obtained with reference Preparation : dissolve 0.150 g in 5 mL of water R, add
solution (d) (0.5 per cent) ; 0.5 mL of dilute acetic acid R, swirl and allow to stand for
– any other impurity : for each impurity, not more than the 10 min in iced water. Filter the precipitate and rinse with
area of the principal peak in the chromatogram obtained 1-2 mL of water R. Dissolve in a mixture of 1 volume of
with reference solution (c) (0.6 per cent) ; water R and 9 volumes of acetone R. Evaporate the solvent
almost to dryness, then dry in an oven at 60 °C for 30 min.
– sum of impurities other than A : not more than 3.5 times the
area of the principal peak in the chromatogram obtained Comparison: cefazolin CRS.
with reference solution (c) (2.1 per cent) ; B. It gives reaction (a) of sodium (2.3.1).
– disregard limit : 0.05 times the area of the principal peak TESTS
in the chromatogram obtained with reference solution (c)
(0.03 per cent). Solution S. Dissolve 2.50 g in carbon dioxide-free water R and
dilute to 25.0 mL with the same solvent.
Water (2.5.12) : maximum 1.5 per cent, determined on 0.500 g.
Appearance of solution. Solution S is clear (2.2.1) and its
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on absorbance (2.2.25) at 430 nm is not greater than 0.15.
1.0 g.
pH (2.2.3) : 4.0 to 6.0 for solution S.
ASSAY Specific optical rotation (2.2.7) : − 24 to − 15 (anhydrous
Liquid chromatography (2.2.29) as described in the test for substance).
related substances with the following modifications. Dissolve 1.25 g in water R and dilute to 25.0 mL with the same
Injection : test solution and reference solution (a). solvent.
System suitability: reference solution (a) : Absorbance (2.2.25). Dissolve 0.100 g in water R and dilute to
– repeatability : maximum relative standard deviation of 100.0 mL with the same solvent. Dilute 2.0 mL of the solution
1.0 per cent after 6 injections. to 100.0 mL with sodium hydrogen carbonate solution R.
Calculate the percentage content of C18H18N6O5S2 from the Examined between 220 nm and 350 nm, the solution shows
declared content of C18H18N6O5S2 in cefatrizine propylene an absorption maximum at 272 nm. The specific absorbance
glycol CRS. at the maximum is 260 to 300 (anhydrous substance).

1794 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cefazolin sodium

Related substances. Liquid chromatography (2.2.29). Limits :

Test solution. Dissolve 50.0 mg of the substance to be – any impurity : for each impurity, not more than the area
examined in mobile phase A and dilute to 20.0 mL with of the principal peak in the chromatogram obtained with
mobile phase A. reference solution (a) (1.0 per cent) ;
Reference solution (a). Dilute 1.0 mL of the test solution to – total : not more than 3.5 times the area of the principal peak
100.0 mL with mobile phase A. in the chromatogram obtained with reference solution (a)
Reference solution (b). Dissolve 20 mg of the substance to be (3.5 per cent) ;
examined in 10 mL of a 2 g/L solution of sodium hydroxide R. – disregard limit : 0.05 times the area of the principal peak
Allow to stand for 15-30 min. Dilute 1.0 mL of the solution to in the chromatogram obtained with reference solution (a)
20 mL with mobile phase A. (0.05 per cent).
Column :
N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm.
– size : l = 0.125 m, Ø = 4.0 mm ;
Water (2.5.12) : maximum 6.0 per cent, determined on 0.300 g.
– stationary phase : octadecylsilyl silica gel for
chromatography R (3 μm) ; Bacterial endotoxins (2.6.14) : less than 0.15 IU/mg, if
– temperature : 45 °C. intended for use in the manufacture of parenteral preparations
without a further appropriate procedure for the removal of
Mobile phase : bacterial endotoxins.
– mobile phase A : solution containing 14.54 g/L of disodium
hydrogen phosphate R and 3.53 g/L of potassium dihydrogen ASSAY
phosphate R ; Liquid chromatography (2.2.29).
– mobile phase B : acetonitrile for chromatography R ; Test solution. Dissolve 50.0 mg of the substance to be
Time Mobile phase A Mobile phase B examined in the mobile phase and dilute to 50.0 mL with the
(min) (per cent V/V) (per cent V/V) mobile phase.
0-2 98 2 Reference solution (a). Dissolve 50.0 mg of cefazolin CRS in
2-4 98 → 85 2 → 15 the mobile phase and dilute to 50.0 mL with the mobile phase.
4 - 10 85 → 60 15 → 40
Reference solution (b). Dissolve 5.0 mg of cefuroxime
sodium CRS in 10.0 mL of reference solution (a) and dilute to
10 - 11.5 60 → 35 40 → 65 100.0 mL with the mobile phase.
11.5 - 12 35 65 Column :
12 - 15 35 → 98 65 → 2 – size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
15 - 21 98 2
chromatography R (5 μm).
Flow rate : 1.2 mL/min. Mobile phase : mix 10 volumes of acetonitrile R and 90 volumes
Detection : spectrophotometer at 254 nm. of a solution containing 2.77 g/L of disodium hydrogen
phosphate R and 1.86 g/L of citric acid R.
Injection : 5 μL.
System suitability : reference solution (b) : Flow rate : 1.0 mL/min.
– resolution : minimum 2.0 between the peaks due to cefazolin Detection : spectrophotometer at 270 nm.
and impurity L (see Figure 0988.-1). Injection : 20 μL.

1. unknown structure 2. impurity E 3. unknown structure 4. cefazolin 5. impurity L

Figure 0988.-1. – Chromatogram for the test for related substances of cefazolin sodium : reference solution (b) (in situ degradation)

General Notices (1) apply to all monographs and other texts 1795
Cefepime dihydrochloride monohydrate EUROPEAN PHARMACOPOEIA 8.0

System suitability : reference solution (b) :

– resolution : minimum 2.0 between the peaks due to cefazolin
and cefuroxime.
Calculate the percentage content of cefazolin sodium by
multiplying the percentage content of cefazolin by 1.048.

STORAGE G. (5aR,6R)-6-[(1H-tetrazol-1-ylacetyl)amino]-5a,6-dihydro-
In an airtight container, protected from light. If the substance 3H,7H-azeto[2,1-b]furo[3,4-d][1,3]thiazine-1,7(4H)-dione,
is sterile, store in a sterile, airtight, tamper-proof container.

IMPURITIES
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or H. (6R,7R)-3-[(acetyloxy)methyl]-7-amino-8-oxo-5-thia-1-
by the general monograph Substances for pharmaceutical azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (7-ACA),
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use): A,
B, C, D, E, G, H, I, K, L.

I. 2-[carboxy[(1H-tetrazol-1-ylacetyl)amino]methyl]-5-[[(5-
methyl-1,3,4-thiadiazol-2-yl)sulfanyl]methyl]-5,6-dihydro-
2H-1,3-thiazine-4-carboxylic acid (cefazoloic acid),

A. (6R,7R)-7-amino-3-[[(5-methyl-1,3,4-thiadiazol-2-
yl)sulfanyl]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-
ene-2-carboxylic acid,

K. (6R,7R)-3-[[(5-methyl-1,3,4-thiadiazol-2-
yl)sulfanyl]methyl]-8-oxo-7-[(1H-tetrazol-1-
ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
carboxamide (cefazolinamide),
B. (6R,7R)-7-[(2,2-dimethylpropanoyl)amino]-3-[[(5-methyl-
1,3,4-thiadiazol-2-yl)sulfanyl]methyl]-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid,

L. (6R,7S)-3-[[(5-methyl-1,3,4-thiadiazol-2-
yl)sulfanyl]methyl]-8-oxo-7-[(1H-tetrazol-1-
C. (6R,7R)-3-methyl-8-oxo-7-[(1H-tetrazol-1- ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2- carboxylic acid.
ene-2-carboxylic acid,

07/2011:2126

CEFEPIME DIHYDROCHLORIDE
MONOHYDRATE
D. (6R,7R)-3-[(acetyloxy)methyl]-8-oxo-7-[(1H-tetrazol-1- Cefepimi dihydrochloridum monohydricum
ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
carboxylic acid,

C19H26Cl2N6O5S2,H2O Mr 571.5
E. 5-methyl-1,3,4-thiadiazol-2-thiol (MMTD), [123171-59-5]

1796 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cefepime dihydrochloride monohydrate

DEFINITION Related substances. Liquid chromatography (2.2.29). Prepare

(6R,7R)-7-[[(2Z)-(2-Aminothiazol-4-yl)(methoxy- the solutions immediately before use or keep refrigerated at
imino)acetyl]amino]-3-[(1-methylpyrrolidinio)methyl]- 4-8 °C for not more than 12 h.
8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate Test solution. Dissolve 70.0 mg of the substance to be
dihydrochloride monohydrate. Semi-synthetic product examined in mobile phase A and dilute to 50.0 mL with
derived from a fermentation product. mobile phase A. Sonicate for 30 s and stir for about 5 min.
Content : 97.0 per cent to 102.0 per cent (anhydrous substance). Reference solution (a). Dissolve 70.0 mg of cefepime
dihydrochloride monohydrate CRS in mobile phase A and
CHARACTERS dilute to 50.0 mL with mobile phase A. Sonicate for 30 s and
Appearance : white or almost white, crystalline powder. stir for about 5 min.
Solubility : freely soluble in water and in methanol, practically Reference solution (b). Dilute 1.0 mL of the test solution to
insoluble in methylene chloride. 10.0 mL with mobile phase A. Dilute 2.0 mL of this solution
to 100.0 mL with mobile phase A.
IDENTIFICATION Reference solution (c). Dissolve 7 mg of cefepime
A. Infrared absorption spectrophotometry (2.2.24). dihydrochloride monohydrate for system suitability CRS
Comparison : cefepime dihydrochloride monohydrate CRS. (containing impurities A, B and F) in mobile phase A and
dilute to 5 mL with mobile phase A.
B. It gives reaction (a) of chlorides (2.3.1).
Reference solution (d). Dissolve 2 mg of cefepime
TESTS impurity E CRS in mobile phase A and dilute to 25.0 mL with
mobile phase A. Dilute 1.0 mL of the solution to 10.0 mL with
Appearance of solution. The solution is clear (2.2.1) and not mobile phase A.
more intensely coloured than reference solution Y3 (2.2.2,
Method II). Column :
Dissolve 2.0 g in water R and dilute to 20 mL with the same – size : l = 0.25 m, Ø = 4.6 mm ;
solvent. – stationary phase : end-capped octadecylsilyl silica gel for
Specific optical rotation (2.2.7) : + 40 to + 45 (anhydrous chromatography R (5 μm).
substance). Mobile phase :
Dissolve 0.250 g in water R and dilute to 25.0 mL with the – mobile phase A : mix 10 volumes of acetonitrile R and
same solvent. 90 volumes of a 0.68 g/L solution of potassium dihydrogen
Impurity G. Liquid chromatography (2.2.29). Prepare the phosphate R previously adjusted to pH 5.0 with 0.5 M
solutions immediately before use. potassium hydroxide;
Test solution. Dissolve 0.100 g of the substance to be examined – mobile phase B : mix equal volumes of acetonitrile R and
in 0.01 M nitric acid and dilute to 10.0 mL with the same acid. a 0.68 g/L solution of potassium dihydrogen phosphate R
previously adjusted to pH 5.0 with 0.5 M potassium
Reference solution (a). Dilute 0.250 g of N-methylpyrrolidine R hydroxide ;
(impurity G) to 100.0 mL with water R. Dilute 2.0 mL of this
solution to 100.0 mL with 0.01 M nitric acid. Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
Reference solution (b). Dilute 0.250 g of pyrrolidine R to
0 - 10 100 0
100 mL with 0.01 M nitric acid. Dilute 2 mL of the solution
to 100 mL with 0.01 M nitric acid. Mix 5 mL of this solution 10 - 30 100 → 50 0 → 50
with 5 mL of reference solution (a).
30 - 35 50 50
Column :
35 - 36 50 → 100 50 → 0
– size : l = 0.05 m, Ø = 4.6 mm ;
– stationary phase : strong cation-exchange resin R (5 μm). 36 - 45 100 0

Mobile phase : mix 1 volume of acetonitrile R and 100 volumes Flow rate : 1 mL/min.
of 0.01 M nitric acid ; filter through a 0.2 μm filter.
Detection : spectrophotometer at 254 nm.
Flow rate : 1 mL/min.
Injection : 10 μL of the test solution and reference solutions (b),
Detection : conductivity detector. (c) and (d).
Injection : 100 μL. Identification of impurities : use the chromatogram supplied
Run time : 1.1 times the retention time of cefepime. with cefepime dihydrochloride monohydrate for system
Retention time : cefepime = about 50 min, eluting as a suitability CRS and the chromatogram obtained with reference
broadened peak. solution (c) to identify the peaks due to impurities A, B and
F ; use the chromatogram obtained with reference solution (d)
System suitability : to identify the peak due to impurity E.
– symmetry factor : maximum 2.5 for the peak due to Relative retention with reference to cefepime (retention
impurity G in the chromatogram obtained with reference time = about 7 min) : impurity E = about 0.4 ;
solution (a); impurity F = about 0.8 ; impurity A = about 2.5 ;
– repeatability : maximum relative standard deviation of impurity B = about 4.1.
5.0 per cent after 6 injections of reference solution (a) ; System suitability : reference solution (c) :
– peak-to-valley ratio : minimum 3 between the peaks due to – resolution : minimum 1.5 between the peaks due to
pyrrolidine and impurity G in the chromatogram obtained impurity F and cefepime.
with reference solution (b).
Limits :
Calculate the percentage content of impurity G in the test
solution using reference solution (a). – correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
Limit : corresponding correction factor : impurity A = 1.4 ;
– impurity G : maximum 0.5 per cent. impurity B = 1.4 ; impurity E = 1.8 ;

General Notices (1) apply to all monographs and other texts 1797
Cefepime dihydrochloride monohydrate EUROPEAN PHARMACOPOEIA 8.0

– impurity A : not more than 1.5 times the area of the

principal peak in the chromatogram obtained with
reference solution (b) (0.3 per cent) ;
– impurities B, F : for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
reference solution (b) (0.2 per cent) ;
– impurity E : not more than 0.5 times the area of the
principal peak in the chromatogram obtained with
B. (6R,7R)-7-[[(2Z)-[2-[[(2Z)-(2-aminothiazol-4-
reference solution (b) (0.1 per cent) ;
yl)(methoxyimino)acetyl]amino]thiazol-4-yl](methoxy-
– unspecified impurities : for each impurity, not more than imino)acetyl]amino]-3-[(1-methylpyrrolidinio)methyl]-8-
0.5 times the area of the principal peak in the chromatogram oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate,
obtained with reference solution (b) (0.10 per cent) ;
– total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(1.0 per cent) ;
– disregard limit : 0.25 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent).
Water (2.5.12) : 3.0 per cent to 4.5 per cent, determined on
0.400 g. C. (2Z)-2-(2-aminothiazol-4-yl)-N-(formylmethyl)-2-
(methoxyimino)acetamide,
Bacterial endotoxins (2.6.14) : less than 0.04 IU/mg, if
intended for use in the manufacture of parenteral preparations
without a further appropriate procedure for the removal of
bacterial endotoxins.

ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications.
Mobile phase : mobile phase A. D. (2Z)-(2-aminothiazol-4-yl)(methoxyimino)acetic acid,
Injection : test solution and reference solution (a).
Run time : 1.4 times the retention time of cefepime.
Calculate the percentage content of C19H26Cl2N6O5S2
from the declared content of cefepime dihydrochloride
monohydrate CRS.

STORAGE E. (6R,7R)-7-amino-3-[(1-methylpyrrolidinio)methyl]-8-oxo-
Protected from light. If the substance is sterile, store in a 5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate,
sterile, airtight, tamper-proof container.

IMPURITIES
Specified impurities : A, B, E, F, G.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : C, D.
F. (6R,7R)-7-[[[(6R,7R)-7-[[(2Z)-(2-aminothiazol-4-yl)-
(methoxyimino)acetyl]amino]-3-[(1-methylpyrrolidinio)-
methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-yl]-
carbonyl]amino]-3-[(1-methylpyrrolidinio)methyl]-8-oxo-
5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate,

A. (6R,7R)-7-[[(2E)-(2-aminothiazol-4-yl)(methoxy-
imino)acetyl]amino]-3-[(1-methylpyrrolidinio)methyl]-
8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate
(anti-cefepime), G. 1-methylpyrrolidine (N-methylpyrrolidine).

1798 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cefixime

01/2008:1188 Run time : 3 times the retention time of cefixime.

corrected 6.0 System suitability : reference solution (c) :
– resolution : minimum 2.0 between the peaks due to cefixime
CEFIXIME and impurity D ; if necessary, adjust the concentration of
acetonitrile in the mobile phase.
Cefiximum Limits :
– any impurity : for each impurity, not more than 0.5 times
the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.5 per cent) ;
– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(3 per cent) ;
C16H15N5O7S2,3H2O Mr 507.5 – disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
DEFINITION (0.1 per cent).
(6R,7R)-7-[[(Z)-2-(2-Aminothiazol-4-yl)-2- Ethanol (2.4.24). Head-space gas chromatography (2.2.28) :
[(carboxymethoxy)imino]acetyl]amino]-3-ethen- use the standard additions method.
yl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic Sample solution. Dissolve 0.250 g of the substance to be
acid trihydrate. examined in a mixture of 1 volume of dimethylacetamide R
Semi-synthetic product derived from a fermentation product. and 4 volumes of water R and dilute to 25.0 mL with the same
Content : 95.0 per cent to 102.0 per cent (anhydrous substance). mixture of solvents.
Limit:
CHARACTERS – ethanol : maximum 1.0 per cent m/m.
Appearance : white or almost white, slightly hygroscopic
powder. Water (2.5.12) : 9.0 per cent to 12.0 per cent, determined on
0.200 g.
Solubility : slightly soluble in water, soluble in methanol,
sparingly soluble in anhydrous ethanol, practically insoluble Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
in ethyl acetate. 1.0 g.

IDENTIFICATION ASSAY
Infrared absorption spectrophotometry (2.2.24). Liquid chromatography (2.2.29) as described in the test for
Comparison : cefixime CRS. related substances with the following modifications.
If the spectra obtained show differences, dissolve the substance Injection : the test solution and reference solution (a).
to be examined and the reference substance separately in System suitability : reference solution (a) :
methanol R, evaporate to dryness and record new spectra – repeatability : maximum relative standard deviation of
using the residues. 1.0 per cent after 6 injections.
TESTS Calculate the percentage content of C16H15N5O7S2 from the
declared content of cefixime CRS.
pH (2.2.3) : 2.6 to 4.1.
Suspend 0.5 g in carbon dioxide-free water R and dilute to STORAGE
10 mL with the same solvent. In an airtight container, protected from light.
Related substances. Liquid chromatography (2.2.29). IMPURITIES
Test solution. Dissolve 25.0 mg of the substance to be
examined in the mobile phase and dilute to 25.0 mL with the
mobile phase.
Reference solution (a). Dissolve 25.0 mg of cefixime CRS in the
mobile phase and dilute to 25.0 mL with the mobile phase.
Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 100.0 mL with the mobile phase.
Reference solution (c). Dissolve 10 mg of cefixime CRS in A. R = CO2H : 2-[[(Z)-2-(2-aminothiazol-4-yl)-2-
10 mL of water R. Heat on a water-bath for 45 min and cool [(carboxymethoxy)imino]acetyl]amino]-2-[(2R)-5-methyl-
(in situ preparation of impurity D). Inject immediately. 7-oxo-1,2,5,7-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-
Column : yl]acetic acid,
– size : l = 0.125 m, Ø = 4 mm ; B. R = H : 2-[[[(Z)-1-(2-aminothiazol-4-yl)-2-
– stationary phase : octadecylsilyl silica gel for [[[(2R,5RS)-5-methyl-7-oxo-1,2,5,7-tetrahydro-
chromatography R (5 μm) ; 4H-furo[3,4-d][1,3]thiazin-2-yl]methyl]amino]-2-
oxoethylidene]amino]oxy]acetic acid,
– temperature : 40 °C.
– Mobile phase : mix 250 volumes of acetonitrile R and
750 volumes of a tetrabutylammonium hydroxide solution
prepared as follows : dissolve 8.2 g of tetrabutylammonium
hydroxide R in water R and dilute to 800 mL with the same
solvent ; adjust to pH 6.5 with dilute phosphoric acid R and
dilute to 1000 mL with water R.
Flow rate : 1.0 mL/min. C. (6R,7S)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2-[(carboxy-
Detection : spectrophotometer at 254 nm. methoxy)imino]acetyl]amino]-3-ethenyl-8-oxo-
Injection : 10 μL of the test solution and reference solutions (b) 5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
and (c). (cefixime 7-epimer),

General Notices (1) apply to all monographs and other texts 1799
Cefoperazone sodium EUROPEAN PHARMACOPOEIA 8.0

Results : the principal peak in the chromatogram obtained

with test solution (a) is similar in retention time and size
to the principal peak in the chromatogram obtained with
reference solution (a).
C. It gives reaction (a) of sodium (2.3.1).
TESTS
D. (6R,7R)-7-[[(E)-2-(2-aminothiazol-4-yl)-2-[(carboxy-
methoxy)imino]acetyl]amino]-3-ethenyl-8-oxo- Appearance of solution. The solution is clear (2.2.1) and its
5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid absorbance (2.2.25) at 430 nm is not greater than 0.15.
(cefixime (E)-isomer), Dissolve 2.5 g in water R and dilute to 25.0 mL with the same
solvent.
pH (2.2.3) : 4.5 to 6.5.
Dissolve 2.5 g in carbon dioxide-free water R and dilute to
10 mL with the same solvent.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
Test solution (a). Dissolve 25.0 mg of the substance to be
examined in the mobile phase and dilute to 250.0 mL with
E. R = H, R′ = CH3 : (6R,7R)-7-[[(Z)-2-(2-aminothiazol-4-
the mobile phase.
yl)-2-[(carboxymethoxy)imino]acetyl]amino]-3-methyl-8-
oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, Test solution (b). Dissolve 25.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with the
F. R = C2H5, R′ = CH=CH2 : (6R,7R)-7-[[(Z)-2-(2-ami- mobile phase.
nothiazol-4-yl)-2-[(2-ethoxy-2-oxoethoxy)imi- Reference solution (a). Dissolve 25.0 mg of cefoperazone
no]acetyl]amino]-3-ethenyl-8-oxo-5-thia-1-azabicy- dihydrate CRS in the mobile phase and dilute to 250.0 mL
clo[4.2.0]oct-2-ene-2-carboxylic acid. with the mobile phase.
Reference solution (b). Dilute 5.0 mL of reference solution (a)
to 100.0 mL with the mobile phase.
01/2008:1404 Column :
corrected 6.4
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
CEFOPERAZONE SODIUM chromatography R (5 μm).
Mobile phase : mix 884 volumes of water R, 110 volumes of
Cefoperazonum natricum acetonitrile R, 3.5 volumes of a 60 g/L solution of acetic acid R
and 2.5 volumes of a triethylammonium acetate solution
prepared as follows : dilute 14 mL of triethylamine R and
5.7 mL of glacial acetic acid R to 100 mL with water R.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 254 nm.
Injection : 20 μL of test solution (b) and reference solutions (a)
and (b).
Run time : 2.5 times the retention time of cefoperazone.
Retention time : cefoperazone = about 15 min.
C25H26N9NaO8S2 Mr 668 System suitability : reference solution (a) :
[62893-20-3] – number of theoretical plates : minimum 5000, calculated
for the principal peak ; if necessary, adjust the content of
DEFINITION acetonitrile R in the mobile phase ;
Sodium (6R,7R)-7-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin- – symmetry factor : maximum 1.6 for the principal peak ; if
1-yl)carbonyl]amino]-2-(4-hydroxyphenyl)acetyl]amino]-3- necessary, adjust the content of acetonitrile R in the mobile
[[(1-methyl-1H-tetrazol-5-yl)sulfanyl]methyl]-8-oxo-5-thia- phase.
1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate. Limits :
Semi-synthetic product derived from a fermentation product. – any impurity : for each impurity, not more than 1.5 times
Content : 95.0 per cent to 102.0 per cent (anhydrous substance). the area of the principal peak in the chromatogram
obtained with reference solution (b) (1.5 per cent) ;
CHARACTERS
– total : not more than 4.5 times the area of the principal peak
Appearance : white or slightly yellow, hygroscopic powder. in the chromatogram obtained with reference solution (b)
Solubility : freely soluble in water, soluble in methanol, slightly (4.5 per cent) ;
soluble in ethanol (96 per cent). – disregard limit : 0.1 times the area of the principal peak in
If crystalline, it shows polymorphism (5.9). the chromatogram obtained with reference solution (b)
(0.1 per cent).
IDENTIFICATION
Acetone (2.4.24, System B) : maximum 2.0 per cent.
A. Infrared absorption spectrophotometry (2.2.24).
Sample solution. Dissolve 0.500 g of the substance to be
Preparation : dissolve the substance to be examined in examined in water R and dilute to 10.0 mL with the same
methanol R and evaporate to dryness ; examine the residue. solvent.
Comparison : Ph. Eur. reference spectrum of cefoperazone Solvent solution. Dissolve 0.350 g of acetone R in water R and
sodium. dilute to 100.0 mL with the same solvent. Dilute 10.0 mL of
B. Examine the chromatograms obtained in the assay. this solution to 100.0 mL with water R.

1800 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cefotaxime sodium

Prepare each of 4 injection vials as shown in the table below :

Vial No. Sample solution Solvent solution Water R
(mL) (mL) (mL)
1 1.0 0 4.0
C. 1-methyl-1H-tetrazole-5-thiol,
2 1.0 1.0 3.0
3 1.0 2.0 2.0
4 1.0 3.0 1.0

Static head-space conditions that may be used :

– equilibration time : 15 min ; D. (6R,7R)-7-amino-8-oxo-3-[(1H-1,2,3-triazol-4-yl-
– transfer-line temperature : 110 °C. sulfanyl)methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
carboxylic acid (7-TACA),
Temperature :
– Column : 40 °C for 10 min.
Heavy metals (2.4.8) : maximum 5 ppm.
2.0 g complies with test C. Prepare the reference solution using
1 mL of lead standard solution (10 ppm Pb) R.
Water (2.5.12) : maximum 5.0 per cent, determined on 0.200 g. E. (6R,7R)-3-[(acetyloxy)methyl]-7-amino-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (7-ACA),
Bacterial endotoxins (2.6.14) : less than 0.20 IU/mg, if
intended for use in the manufacture of parenteral preparations
without a further appropriate procedure for the removal of
bacterial endotoxins.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications.
Injection : test solution (a) and reference solution (a).
System suitability: reference solution (a) :
F. (6R,7S)-7-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazine-1-yl)-
– repeatability : maximum relative standard deviation of carbonyl]amino]-2-(4-hydroxyphenyl)acetyl]amino]-3-
1.0 per cent after 6 injections. [[(1-methyl-1H-tetrazol-5-yl)sulfanyl]methyl]-8-oxo-5-
Calculate the percentage content of cefoperazone sodium by thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid.
multiplying the percentage content of cefoperazone by 1.034.
01/2008:0989
STORAGE
In an airtight container, protected from light, at a temperature CEFOTAXIME SODIUM
of 2 °C to 8 °C. If the substance is sterile, store in a sterile,
airtight, tamper-proof container. Cefotaximum natricum
IMPURITIES

C16H16N5NaO7S2 Mr 477.4
[64485-93-4]
DEFINITION
Sodium (6R,7R)-3-[(acetyloxy)methyl]-7-[[(2Z)-2-(2-
A. (5aR,6R)-6-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1-yl)- aminothiazol-4-yl)-2-(methoxyimino)acetyl]amino]-8-oxo-5-
carbonyl]amino]-2-(4-hydroxyphenyl)acetyl]amino]- thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate.
5a,6-dihydro-3H,7H-azeto[2,1-b]furo[3,4-d][1,3]thiazine- Semi-synthetic product derived from a fermentation product.
1,7(4H)-dione, Content : 96.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
Appearance : white or slightly yellow powder, hygroscopic.
Solubility : freely soluble in water, sparingly soluble in
methanol.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: cefotaxime sodium CRS.
B. It gives reaction (a) of sodium (2.3.1).
B. (6R,7R)-7-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1-yl)-
carbonyl]amino]-2-(4-hydroxyphenyl)acetyl]amino]-3-[(4- TESTS
methyl-5-thioxo-4,5-dihydro-1H-tetrazol-1-yl)methyl]-8- Solution S. Dissolve 2.5 g in carbon dioxide-free water R and
oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, dilute to 25.0 mL with the same solvent.

General Notices (1) apply to all monographs and other texts 1801
Cefotaxime sodium EUROPEAN PHARMACOPOEIA 8.0

Appearance of solution. Solution S is clear (2.2.1). Add 1 mL System suitability : reference solution (c) :
of glacial acetic acid R to 10 mL of solution S. The solution, – resolution : minimum 3.5 between the peaks due to
examined immediately, is clear. impurity E and cefotaxime ;
pH (2.2.3): 4.5 to 6.5 for solution S. – symmetry factor : maximum 2.0 for the peak due to
Specific optical rotation (2.2.7) : + 58.0 to + 64.0 (anhydrous cefotaxime.
substance). Limits :
Dissolve 0.100 g in water R and dilute to 10.0 mL with the – impurities A, B, C, D, E, F : for each impurity, not more
same solvent. than the area of the principal peak in the chromatogram
Absorbance (2.2.25) : maximum 0.40 at 430 nm for solution S. obtained with reference solution (b) (1.0 per cent) ;
– any other impurity : for each impurity, not more than
Specific absorbance (2.2.25) : 360 to 390, determined at the
0.2 times the area of the principal peak in the chromatogram
absorption maximum at 235 nm (anhydrous substance).
obtained with reference solution (b) (0.2 per cent) ;
Dissolve 20.0 mg in water R and dilute to 100.0 mL with the
– total : not more than 3 times the area of the principal peak
same solvent. Dilute 10.0 mL of the solution to 100.0 mL with
in the chromatogram obtained with reference solution (b)
water R.
(3.0 per cent) ;
Related substances. Liquid chromatography (2.2.29). Prepare – disregard limit : 0.05 times the area of the principal peak
the solutions immediately before use. in the chromatogram obtained with reference solution (b)
Solution A : mobile phase B, mobile phase A (14:86 V/V). (0.05 per cent).
Test solution. Dissolve 40.0 mg of the substance to be Ethanol (2.4.24, System A) : maximum 1.0 per cent.
examined in solution A and dilute to 50.0 mL with the same
solution. N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm.
Reference solution (a). Dissolve 8.0 mg of cefotaxime acid CRS 2-Ethylhexanoic acid (2.4.28) : maximum 0.5 per cent m/m.
in solution A and dilute to 10.0 mL with the same solution. Water (2.5.12) : maximum 3.0 per cent, determined on 0.300 g.
Reference solution (b). Dilute 1.0 mL of the test solution to Bacterial endotoxins (2.6.14) : less than 0.05 IU/mg, if
100.0 mL with solution A. intended for use in the manufacture of parenteral preparations
Reference solution (c). Add 1.0 mL of dilute hydrochloric acid R without a further appropriate procedure for the removal of
to 4.0 mL of the test solution. Heat the solution at 40 °C for bacterial endotoxins.
2 h. Add 5.0 mL of buffer solution pH 6.6 R and 1.0 mL of
dilute sodium hydroxide solution R. ASSAY
Reference solution (d). Dissolve 4 mg of cefotaxime for peak Liquid chromatography (2.2.29) as described in the test for
identification CRS (containing impurities A, B, C, E and F) related substances with the following modification.
in 5 mL of solution A. Injection : test solution and reference solution (a).
Column : Calculate the percentage content of C16H16N5NaO7S2 by
– size : l = 0.15 m, Ø = 3.9 mm, multiplying the percentage content of cefotaxime by 1.048.
– stationary phase : octadecylsilyl silica gel for STORAGE
chromatography R (5 μm), In an airtight container, protected from light. If the substance
– temperature : 30 °C. is sterile, store in a sterile, airtight, tamper-proof container.
Mobile phase :
IMPURITIES
– mobile phase A : 7.1 g/L solution of disodium hydrogen
Specified impurities : A, B, C, D, E, F.
phosphate R adjusted to pH 6.25 using phosphoric acid R ;
Other detectable impurities (the following substances would,
– mobile phase B : methanol R ;
if present at a sufficient level, be detected by one or other of
Time Mobile phase A Mobile phase B the tests in the monograph. They are limited by the general
(min) (per cent V/V) (per cent V/V) acceptance criterion for other/unspecified impurities and/or
0-7 86 14 by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
7-9 86 → 82 14 → 18
impurities for demonstration of compliance. See also 5.10.
9 - 16 82 18 Control of impurities in substances for pharmaceutical use) : G.
16 - 45 82 → 60 18 → 40
45 - 50 60 40
50 - 55 60 → 86 40 → 14
55 - 60 86 14

Flow rate : 1.0 mL/min. A. R = R′ = H : (6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-

Detection : spectrophotometer at 235 nm. yl)-2-(methoxyimino)acetyl]amino]-3-methyl-8-oxo-
5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
Injection : 10 μL of the test solution and reference solutions (b), (deacetoxycefotaxime),
(c) and (d).
Identification of impurities : use the chromatogram supplied B. R = OH, R′ = H : (6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-
with cefotaxime for peak identification CRS and the yl)-2-(methoxyimino)acetyl]amino]-3-(hydroxymethyl)-8-
chromatogram obtained with reference solution (d) to identify oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
the peaks due to impurities A, B, C, E and F. (deacetylcefotaxime),
Relative retention with reference to cefotaxime (retention C. R = O-CO-CH3, R′ = CHO : (6R,7R)-3-[(acetyloxy)methyl]-
time = about 13 min) : impurity B = about 0.3 ; 7-[[(2Z)-2-[2-(formylamino)thiazol-4-yl]-2-
impurity A = about 0.5 ; impurity E = about 0.6 ; (methoxyimino)acetyl]amino]-8-oxo-5-thia-
impurity C = about 1.9 ; impurity D = about 2.3 ; 1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
impurity F = about 2.4 ; impurity G = about 3.1. (N-formylcefotaxime),

1802 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cefoxitin sodium

DEFINITION
Sodium (6R,7S)-3-[(carbamoyloxy)methyl]-7-methoxy-
8-oxo-7-[[(thiophen-2-yl)acetyl]amino]-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate.
Semi-synthetic product derived from a fermentation product.
D. (6R,7R)-3-[(acetyloxy)methyl]-7-[[(2E)-2-(2- Content : 95.0 per cent to 102.0 per cent (anhydrous substance).
aminothiazol-4-yl)-2-(methoxyimino)acetyl]amino]-8- CHARACTERS
oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
(E-cefotaxime), Appearance : white or almost white, very hygroscopic powder.
Solubility : very soluble in water, sparingly soluble in ethanol
(96 per cent).
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: cefoxitin sodium CRS.
B. It gives reaction (a) of sodium (2.3.1).
E. (5aR,6R)-6-[[(2Z)-2-(2-aminothiazol-4-yl)-2- TESTS
(methoxyimino)acetyl]amino]-5a,6-dihydro-3H,7H-
azeto[2,1-b]furo[3,4-d][1,3]thiazine-1,7(4H)-dione Solution S. Dissolve 2.50 g in carbon dioxide-free water R and
(deacetylcefotaxime lactone), dilute to 25 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than intensity 5 of the range of
reference solutions of the most appropriate colour (2.2.2,
Method II).
pH (2.2.3) : 4.2 to 7.0.
Dilute 2 mL of solution S to 20 mL with carbon dioxide-free
water R.
Specific optical rotation (2.2.7) : + 206 to + 214 (anhydrous
substance).
Dissolve 0.250 g in methanol R and dilute to 25.0 mL with
F. (6R,7R)-3-[(acetyloxy)methyl]-7-[[(2Z)-2-[2- the same solvent.
[[[(6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-
(methoxyimino)acetyl]amino]-2-carboxy-8-oxo-5-thia- Related substances. Liquid chromatography (2.2.29). Prepare
1-azabicyclo[4.2.0]oct-2-en-2-yl]methyl]amino]thiazol- the solutions immediately before use.
4-yl]-2-(methoxyimino)acetyl]amino]-8-oxo-5-thia-1- Solution A. Dissolve 1.0 g of potassium dihydrogen phosphate R
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (cefotaxime and 1.8 g of anhydrous disodium hydrogen phosphate R in
dimer), 1000 mL of water R. To 100 mL of the solution add 800 mL of
water R, adjust to pH 7.0 with phosphoric acid R or a 40 g/L
solution of sodium hydroxide R and dilute to 1000 mL with
water R.
Test solution. Dissolve 50 mg of the substance to be examined
in solution A and dilute to 50.0 mL with solution A.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with solution A.
Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 20.0 mL with solution A.
Reference solution (c). Dissolve 5 mg of cefoxitin for peak
identification CRS (containing impurities A, B, E, H, I and J)
G. (6R,7R)-3-[(acetyloxy)methyl]-7-[[(2Z)-2-[2-[[(2Z)- in solution A and dilute to 5 mL with solution A.
2-(2-aminothiazol-4-yl)-2-(methoxyimino)ace-
tyl]amino]thiazol-4-yl]-2-(methoxyimino)acetyl]ami- Column :
no]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carbox- – size : l = 0.25 m, Ø = 4.6 mm ;
ylic acid (ATA cefotaxime). – stationary phase : phenylsilyl silica gel for chromatography R
(3.0 μm) ;
– temperature : 35 °C.
01/2013:0990 Mobile phase :
– mobile phase A : 1.0 g/L solution of ammonium formate R
CEFOXITIN SODIUM adjusted to pH 2.7 with anhydrous formic acid R ;
– mobile phase B : acetonitrile R ;
Cefoxitinum natricum Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)

0-5 92 8
5 - 50 92 → 74 8 → 26
50 - 85 74 26

C16H16N3NaO7S2 Mr 449.4 Flow rate : 1.0 mL/min.

[33564-30-6] Detection : spectrophotometer at 254 nm.

General Notices (1) apply to all monographs and other texts 1803
Cefoxitin sodium EUROPEAN PHARMACOPOEIA 8.0

Injection : 20 μL. Run time : 12 min.

Identification of impurities : use the chromatogram System suitability : reference solution (c) :
supplied with cefoxitin for peak identification CRS and the – resolution : minimum 3.5 between the 2 principal peaks.
chromatogram obtained with reference solution (c) to identify Calculate the percentage content of C16H16N3NaO7S2 taking
the peaks due to impurities A, B, E, H, I and J. into account the assigned content of cefoxitin sodium CRS.
Relative retention with reference to cefoxitin (retention
time = about 30 min) : impurity A = about 0.83 ; STORAGE
impurity I = about 0.98 ; impurity H = about 1.06 ; In an airtight container. If the substance is sterile, store in a
impurity E = about 1.11 ; impurity B = about 1.18 ; sterile, airtight, tamper-proof container.
impurity J = about 1.66.
IMPURITIES
System suitability: reference solution (c) :
Specified impurities : A, B, E, H, I, J.
– resolution : minimum 2.0 between the peaks due to
impurities H and E ; Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
– peak-to-valley ratio : minimum 2.0, where Hp = height the tests in the monograph. They are limited by the general
above the baseline of the peak due to impurity I and acceptance criterion for other/unspecified impurities and/or
Hv = height above the baseline of the lowest point of the by the general monograph Substances for pharmaceutical use
curve separating this peak from the peak due to cefoxitin. (2034). It is therefore not necessary to identify these impurities
Limits : for demonstration of compliance. See also 5.10. Control of
– impurity I : not more than the area of the principal peak impurities in substances for pharmaceutical use) : C, D, F, G.
in the chromatogram obtained with reference solution (a)
(1.0 per cent) ;
– impurities E, H : not more than 0.5 times the area of
the principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent) ;
– impurity J : not more than 0.3 times the area of the principal
peak in the chromatogram obtained with reference A. (6R,7S)-3-(hydroxymethyl)-7-methoxy-8-oxo-
solution (a) (0.3 per cent) ; 7-[[(thiophen-2-yl)acetyl]amino]-5-thia-1-
– impurities A, B : for each impurity, not more than 0.2 times azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
the area of the principal peak in the chromatogram (decarbamoylcefoxitin),
obtained with reference solution (a) (0.2 per cent) ;
– unspecified impurities : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.10 per cent) ;
– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(3.0 per cent) ; B. (2RS,6R,7S)-3-[(carbamoyloxy)methyl]-7-methoxy-
– disregard limit : the area of the principal peak in the 8-oxo-7-[[(thiophen-2-yl)acetyl]amino]-5-thia-
chromatogram obtained with reference solution (b) 1-azabicyclo[4.2.0]oct-3-ene-2-carboxylic acid
(0.05 per cent). (delta-3-cefoxitin),
Water (2.5.12) : maximum 1.0 per cent, determined on 0.500 g.
Bacterial endotoxins (2.6.14) : less than 0.13 IU/mg, if
intended for use in the manufacture of parenteral preparations
without a further appropriate procedure for the removal of
bacterial endotoxins.
ASSAY
C. (5aR,6R)-6-[[(thiophen-2-yl)acetyl]amino]-5a,6-dihydro-
Liquid chromatography (2.2.29). 3H,7H-azeto[2,1-b]furo[3,4-d][1,3]thiazine-1,7(4H)-dione
Test solution. Dissolve 25.0 mg of the substance to be (cefalotin lactone),
examined in water R and dilute to 25.0 mL with the same
solvent.
Reference solution (a). Dissolve 25.0 mg of cefoxitin
sodium CRS in water R and dilute to 25.0 mL with the same
solvent.
Reference solution (b). Dissolve 20.0 mg of 2-(2-thienyl)acetic
acid R in water R and dilute to 25.0 mL with the same solvent.
Reference solution (c). Mix 1.0 mL of reference solution (a) D. (5aR,6S)-6-methoxy-6-[[(thiophen-2-yl)acetyl]amino]-
and 5.0 mL of reference solution (b). 5a,6-dihydro-3H,7H-azeto[2,1-b]furo[3,4-d][1,3]thiazine-
Column : 1,7(4H)-dione (cefoxitin lactone),
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : acetic acid R, acetonitrile R, water R
(1:19:81 V/V/V).
Flow rate : 1 mL/min. E. (6R,7S)-3-[(carbamoyloxy)methyl]-7-methoxy-7-[[(2R)-2-
Detection : spectrophotometer at 254 nm. methoxy-2-(thiophen-2-yl)acetyl]amino]-8-oxo-5-thia-1-
Injection : 20 μL of the test solution and reference solutions (a) azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid ((R)-methoxy
and (c). cefoxitin),

1804 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cefpodoxime proxetil

Comparison: cefpodoxime proxetil CRS.

TESTS
Diastereoisomer ratio. Liquid chromatography (2.2.29) as
described under Assay. Use the normalisation procedure.
Limit : test solution :
F. (6R,7S)-3-[(carbamoyloxy)methyl]-7-methoxy-7-[[(2S)-2- – the ratio of the area of the peak due to cefpodoxime proxetil
methoxy-2-(thiophen-2-yl)acetyl]amino]-8-oxo-5-thia-1- diastereoisomer II to the sum of the areas of the peaks
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid ((S)-methoxy due to cefpodoxime proxetil diastereoisomers I and II is
cefoxitin), between 0.5 and 0.6.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use or store them at 2-8 °C.
Solvent mixture: glacial acetic acid R, acetonitrile R, water R
(2:99:99 V/V/V).
Test solution. Dissolve 50 mg of the substance to be examined
in the solvent mixture and dilute to 50.0 mL with the solvent
mixture.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture.
Reference solution (b). Dissolve 5 mg of cefpodoxime proxetil
G. (6R,7S)-3-[[[[[[(6R,7S)-2-carboxy-7-methoxy-8- for peak identification CRS (containing impurities B, C and D)
oxo-7-[[2-(thiophen-2-yl)acetyl]amino]-5-thia-1- in 5.0 mL of the solvent mixture.
azabicyclo[4.2.0]oct-2-en-3-yl]methyl]oxy]carbamoyl]- Reference solution (c). Dissolve 5 mg of cefpodoxime proxetil
oxy]methyl]-7-methoxy-8-oxo-7-[[2-(thiophen-2- for impurity H identification CRS in 5.0 mL of the solvent
yl)acetyl]amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2- mixture.
carboxylic acid (cefoxitin dimer), Column :
H. unknown structure, – size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
I. unknown structure, chromatography R (5 μm) ;
J. unknown structure. – temperature : maintain at a constant temperature of 20 °C.
Mobile phase :
– mobile phase A : anhydrous formic acid R, methanol R,
01/2011:2341 water R (1:400:600 V/V/V) ;
– mobile phase B : anhydrous formic acid R, water R,
methanol R (1:50:950 V/V/V) ;
CEFPODOXIME PROXETIL
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
Cefpodoximum proxetili 0 - 65 95 5
65 - 145 95 → 15 5 → 85
145 - 155 15 85

Flow rate : 0.6 mL/min.

Detection : spectrophotometer at 254 nm.
Injection : 20 μL.
Identification of impurities : use the chromatogram supplied
with cefpodoxime proxetil for peak identification CRS and
C21H27N5O9S2 Mr 557.6 the chromatogram obtained with reference solution (b)
[87239-81-4] to identify the peaks due to impurities B, C and D ; use
the chromatogram supplied with cefpodoxime proxetil for
DEFINITION impurity H identification CRS and the chromatogram obtained
(1RS)-1-[[(1-Methylethoxy)carbonyl]oxy]ethyl with reference solution (c) to identify the peaks due to
(6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-(methoxyimi- impurity H.
no)acetyl]amino]-3-(methoxymethyl)-8-oxo-5-thia-1-azabi- Relative retention with reference to cefpodoxime
cyclo[4.2.0]oct-2-ene-2-carboxylate. proxetil diastereoisomer II (retention time = about 58 min) :
Semi-synthetic product derived from a fermentation product. diastereoisomer I of impurity B = about 0.68 ; diastereoisomer I
Content : 94.0 per cent to 102.0 per cent (anhydrous substance). of cefpodoxime proxetil = about 0.74 ; impurity C = about 0.82 ;
diastereoisomer II of impurity B = about 0.85 ; impurity D
CHARACTERS (2 peaks) = about 0.88 and 1.13 ; peaks due to diastereoisomers
Appearance : white or pale yellow or light brown, amorphous of impurity H : between about 1.9 and 2.3.
powder. System suitability :
Solubility : very slightly soluble or practically insoluble in – the chromatogram obtained with reference solution (b) is
water, very soluble in acetonitrile and in methanol, freely similar to the chromatogram supplied with cefpodoxime
soluble in anhydrous ethanol. proxetil for peak identification CRS ;
– resolution : minimum 6.0 between the peaks due to
IDENTIFICATION cefpodoxime proxetil diastereoisomers I and II in the
Infrared absorption spectrophotometry (2.2.24). chromatogram obtained with reference solution (a) ;

General Notices (1) apply to all monographs and other texts 1805
Cefpodoxime proxetil EUROPEAN PHARMACOPOEIA 8.0

– peak-to-valley ratio : minimum 1.1, where Hp = height STORAGE

above the baseline of the peak due to diastereoisomer II Protected from light.
of impurity B and Hv = height above the baseline of the
lowest point of the curve separating this peak from the IMPURITIES
peak due to impurity C in the chromatogram obtained with
reference solution (b). Specified impurities : B, C, D, H.
Limits : Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
– impurity C : not more than twice the sum of the areas of the tests in the monograph. They are limited by the general
the 2 principal peaks in the chromatogram obtained with acceptance criterion for other/unspecified impurities and/or
reference solution (a) (2.0 per cent) ; by the general monograph Substances for pharmaceutical use
– impurity D (sum of the 2 diastereoisomers) : not more (2034). It is therefore not necessary to identify these impurities
than the sum of the areas of the 2 principal peaks in the for demonstration of compliance. See also 5.10. Control of
chromatogram obtained with reference solution (a) (1.0 per impurities in substances for pharmaceutical use) : A, E, F, G.
cent) ;
– impurity H (sum of the diastereoisomers) : not more
than the sum of the areas of the 2 principal peaks in the
chromatogram obtained with reference solution (a) (1.0 per
cent) ;
– impurity B (sum of the 2 diastereoisomers) : not more than
0.5 times the sum of the areas of the 2 principal peaks in
the chromatogram obtained with reference solution (a) A. (6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-
(0.5 per cent) ; (methoxyimino)acetyl]amino]-3-(methoxymethyl)-8-
– any other impurity : for each impurity, not more than oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
0.2 times the sum of the areas of the 2 principal peaks in (cefpodoxime),
the chromatogram obtained with reference solution (a)
(0.2 per cent) ;
– total : not more than 4 times the sum of the areas of the
2 principal peaks in the chromatogram obtained with
reference solution (a) (4.0 per cent) ;
– disregard limit : 0.05 times the sum of the areas of the
2 principal peaks in the chromatogram obtained with
reference solution (a) (0.05 per cent).
Water (2.5.12) : maximum 2.5 per cent, determined on 0.500 g.
B. (1RS)-1-[[(1-methylethoxy)carbonyl]oxy]ethyl
ASSAY (6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-
(methoxyimino)acetyl]amino]-3-methyl-8-oxo-
Liquid chromatography (2.2.29). 5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate
Solution A : 20 mg/L solution of anhydrous citric acid R in (ADCA-analogue of cefpodoxime proxetil),
acetonitrile R.
Test solution. Dissolve 30.0 mg of the substance to be
examined in solution A and dilute to 50.0 mL with solution A.
Reference solution. Dissolve 30.0 mg of cefpodoxime
proxetil CRS in solution A and dilute to 50.0 mL with
solution A.
Column:
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm) ; C. (1RS)-1-[[(1-methylethoxy)carbonyl]oxy]ethyl
(6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-
– temperature : 40 °C. (methoxyimino)acetyl]amino]-3-(methoxymethyl)-
Mobile phase : methanol R, water R (9:11 V/V). 8-oxo-5-thia-1-azabicyclo[4.2.0]oct-3-ene-2-carboxylate
(delta-2-cefpodoxime proxetil),
Flow rate : 0.8 mL/min.
Detection : spectrophotometer at 240 nm.
Injection : 10 μL.
Run time : 1.2 times the retention time of cefpodoxime proxetil
diastereoisomer II.
Retention time : cefpodoxime proxetil diastereoisom-
er II = about 30 min.
System suitability: reference solution :
– resolution : minimum 4.0 between the peaks due to D. (1RS)-1-[[(1-methylethoxy)carbonyl]oxy]ethyl
cefpodoxime proxetil diastereoisomers I and II. (6R,7R)-7-[[(2E)-2-(2-aminothiazol-4-yl)-2-
Calculate the percentage content of C21H27N5O9S2 from the (methoxyimino)acetyl]amino]-3-(methoxymethyl)-
sum of the areas of the 2 peaks due to the diastereoisomers 8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate
and using the declared content of cefpodoxime proxetil CRS. (anti-cefpodoxime proxetil),

1806 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cefprozil monohydrate

01/2013:2342

CEFPROZIL MONOHYDRATE
Cefprozilum monohydricum

E. (1RS)-1-[[(1-methylethoxy)carbonyl]oxy]ethyl
(6R,7R)-3-(acetoxymethyl)-7-[[(2Z)-2-(2-aminothiazol-
4-yl)-2-(methoxyimino)acetyl]amino]-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate (ACA-analogue
of cefpodoxime proxetil),
C18H19N3O5S, H2O Mr 407.4
[121123-17-9]
DEFINITION
Mixture of the 2 diastereoisomers of (6R,7R)-7-[[(2R)-2-
amino-2-(4-hydroxyphenyl)acetyl]amino]-8-oxo-3-[(1EZ)-
prop-1-enyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic
acid monohydrate.
Semi-synthetic product derived from a fermentation product.
F. (1RS)-1-[[(1-methylethoxy)carbonyl]oxy]ethyl Content : 96.0 per cent to 102.0 per cent (anhydrous substance).
(6R,7R)-7-[[(2Z)-2-[(2-formylamino)thiazol-4-yl)-2- CHARACTERS
(methoxyimino)acetyl]amino]-3-(methoxymethyl)-8-
oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate Appearance : white or yellow, crystalline powder, slightly
(N-formyl cefpodoxime proxetil), hygroscopic.
Solubility : slightly soluble in water and in methanol, practically
insoluble in acetone.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: cefprozil CRS.
TESTS
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
G. (1RS)-1-[[(1-methylethoxy)carbonyl]oxy]ethyl Test solution (a). Dissolve 0.125 g of the substance to be
(6R,7R)-7-[[(2Z)-[2-(2-acetylamino)thiazol-4-yl]-2- examined in 1 mL of a 103 g/L solution of hydrochloric acid R
(methoxyimino)acetyl]amino]-3-(methoxymethyl)-8- and dilute to 25.0 mL with mobile phase A.
oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate
(N-acetyl-cefpodoxime proxetil), Test solution (b). Dissolve 30.0 mg of the substance to be
examined in water R and dilute to 100.0 mL with the same
solvent.
Reference solution (a). Dilute 1.0 mL of test solution (a) to
100.0 mL with mobile phase A.
Reference solution (b). Dissolve 5 mg of cefprozil for peak
identification CRS (containing impurities B, H and M) in
0.05 mL of a 103 g/L solution of hydrochloric acid R and add
1 mL of mobile phase A.
Reference solution (c). Dissolve 3 mg of cefprozil CRS and 6 mg
of cefprozil impurity mixture CRS (containing impurities D
and F) in 2 mL of a 103 g/L solution of hydrochloric acid R
and dilute to 50 mL with mobile phase A.
Reference solution (d). Dissolve 30.0 mg of cefprozil CRS in
water R and dilute to 100.0 mL with the same solvent.
Reference solution (e). Dissolve 10.0 mg of cefadroxil CRS
(impurity B) in water R and dilute to 20.0 mL with the same
solvent. Dilute 1.0 mL of the solution to 20.0 mL with water R.
Reference solution (f). Dissolve 10.0 mg of cefprozil
H. mixture of the diastereoisomers of 1-[[(1-methyl- impurity A CRS in water R and dilute to 100.0 mL with the
ethoxy)carbonyl]oxy]ethyl (6R,7R)-7-[[(2Z)-2-[2- same solvent. Dilute 1.0 mL of the solution to 10.0 mL with
[[(2R)-2-[[(2Z)-2-(2-aminothiazol-4-yl)-2-(methox- water R.
yimino)acetyl]amino]-2-[(2R)-5-(methoxymeth- Column :
yl)-4-[[1-[[(1-methylethoxy)carbonyl]oxy]ethoxy]carbon- – size : l = 0.25 m, Ø = 4.6 mm ;
yl]-3,6-dihydro-2H-1,3-thiazin-2-yl]acetyl]amino]thia-
zol-4-yl]-2-(methoxyimino)acetyl]amino]-3-(methoxy- – stationary phase : end-capped octadecylsilyl silica gel for
methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-car- chromatography R (5 μm) ;
boxylate (cefpodoxime proxetil dimer). – temperature : 40 °C.

General Notices (1) apply to all monographs and other texts 1807
Cefprozil monohydrate EUROPEAN PHARMACOPOEIA 8.0

Mobile phase : Water (2.5.12) : 3.5 per cent to 6.5 per cent, determined on
– mobile phase A : dissolve 11.5 g of ammonium dihydrogen 0.500 g.
phosphate R in water R, adjust to pH 4.4 with phosphoric Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
acid R and dilute to 1000 mL with water R ; 1.0 g.
– mobile phase B : acetonitrile R, mobile phase A (50:50 V/V) ;
ASSAY
Time Mobile phase A Mobile phase B Liquid chromatography (2.2.29) as described in the test for
(min) (per cent V/V) (per cent V/V) related substances with the following modifications.
0-8 81 19 Mobile phase : mobile phase B, mobile phase A (18:82 V/V).
8 - 20 81 → 36 19 → 64 Detection : spectrophotometer at 280 nm.
20 - 25 36 64 Injection : 10 μL of test solution (b) and reference solution (d).
Run time : twice the retention time of cefprozil (Z)-isomer.
Flow rate : 1.0 mL/min.
Elution order : (Z)-isomer, (E)-isomer.
Detection : spectrophotometer at 230 nm. Retention time : cefprozil (Z)-isomer = about 8 min.
Injection : 10 μL of test solution (a) and reference solutions (a), System suitability : reference solution (d) :
(b), (c), (e) and (f).
– resolution : minimum 2.5 between the peaks due to cefprozil
Identification of impurities : use the chromatogram (Z)-isomer and the (E)-isomer.
supplied with cefprozil for peak identification CRS and the
chromatogram obtained with reference solution (b) to Calculate the percentage content of the sum of both isomers
identify the peaks due to impurities B, H and M ; use the of cefprozil (C18H19N3O5S) taking into account the assigned
chromatogram supplied with cefprozil impurity mixture CRS contents of both (E)-isomer and (Z)-isomer of cefprozil CRS.
and the chromatogram obtained with reference solution (c) STORAGE
to identify the peaks due to impurities D and F ; impurities G
and I are identified by their relative retention. In an airtight container.
Relative retention with reference to cefprozil (Z)-isomer IMPURITIES
(retention time = about 7 min) : impurity A = about 0.4 ; Specified impurities : A, B, D, G, H, I, M.
impurity B = about 0.5 ; impurity D = about 0.7 ;
impurity F = about 0.9 ; cefprozil (E)-isomer = about 1.4 ; Other detectable impurities (the following substances would,
impurity G = about 1.7 ; impurity H = about 2.0 ; if present at a sufficient level, be detected by one or other of
impurity I = about 2.1 ; impurity M = about 2.9. the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
System suitability: reference solution (c) : by the general monograph Substances for pharmaceutical
– resolution : minimum 1.4 between the peaks due to use (2034). It is therefore not necessary to identify these
impurity F and cefprozil (Z)-isomer. impurities for demonstration of compliance. See also 5.10.
Limits : Control of impurities in substances for pharmaceutical use) :
C, E, F, J, K, L, N.
– correction factor : for the calculation of content, multiply
the peak area of impurity D by 2.3 ;
– impurity B : not more than the area of the principal peak
in the chromatogram obtained with reference solution (e)
(0.5 per cent) ;
– impurities D, G, H, I, M : for each impurity, not more than A. (2R)-2-amino-2-(4-hydroxyphenyl)acetic acid
0.3 times the sum of the areas of the 2 principal peaks in (p-hydroxyphenylglycine),
the chromatogram obtained with reference solution (a)
(0.3 per cent) ;
– impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (f)
(0.2 per cent) ;
– any other impurity : for each impurity, not more than
0.2 times the sum of the areas of the 2 principal peaks in
the chromatogram obtained with reference solution (a) B. (6R,7R)-7-[[(2R)-2-amino-2-(4-hydroxyphenyl)ace-
(0.2 per cent) ; tyl]amino]-3-methyl-8-oxo-5-thia-1-azabicy-
clo[4.2.0]oct-2-ene-2-carboxylic acid (cefadroxil),
– total : maximum 2.0 per cent ;
– disregard limit : 0.05 times the sum of the areas of the
2 principal peaks in the chromatogram obtained with
reference solution (a) (0.05 per cent).
(E)-isomer ratio. Liquid chromatography (2.2.29) as
described under Assay.
Determine the area of the peak due to the (E)-isomer in the C. (6RS)-3-(aminomethylene)-6-(4-hydroxyphenyl)piper-
chromatogram obtained with test solution (b) and reference azine-2,5-dione,
solution (d). Calculate the ratio of the (E)-isomer to the sum
of both cefprozil isomers, as determined under Assay.
Limit :
– (E)-isomer ratio : 0.06 to 0.11.
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test C. Prepare the reference solution using D. (6R,7R)-7-amino-8-oxo-3-[(1Z)-prop-1-enyl]-5-thia-1-
2 mL of lead standard solution (10 ppm Pb) R. azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid,

1808 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cefradine

E. (6R,7R)-7-[[(2R)-2-amino-2-[4-[[(2R)-2-amino-2-(4- K. mixture of 4 diastereoisomers of (3RS,6RS)-3-[(2R,5R)-5-

hydroxyphenyl)acetyl]oxy]phenyl]acetyl]amino]-8-oxo-3- ethyl-7-oxo-1,2,5,7-tetrahydro-4H-furo[3,4-d][1,3]thiazin-
[(1Z)-prop-1-enyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2- 2-yl]-6-(4-hydroxyphenyl)piperazine-2,5-dione,
carboxylic acid,

L. 2-hydroxyethyl (2R)-2-amino-2-(4-hydroxyphenyl)acetate,

F. (6R,7R)-7-amino-8-oxo-3-[(1E)-prop-1-enyl]-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid,

M. (6R,7R)-7-[[(2R)-2-amino-2-[4-[(ethoxycarbonyl)-
oxy]phenyl]acetyl]amino]-8-oxo-3-[(1Z)-prop-1-enyl]-5-
thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid,

G. (2R)-2-[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino-
2-[(2R)-4-carboxy-5-[(1Z)-prop-1-enyl]-3,6-dihydro-2H-
1,3-thiazin-2-yl]-acetic acid,

N. (6R,7R)-7-[[(2R)-2-amino-2-[4-(ethoxycarbonyl)-
oxy]phenyl]acetyl]amino]-8-oxo-3-[(1E)-prop-1-enyl]-5-
thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid.

01/2014:0814

CEFRADINE
H. (6R,7R)-7-[[(2R)-2-[[(2R)-2-amino-2-(4-hydroxy- Cefradinum
phenyl)acetyl]amino]-2-(4-hydroxyphenyl)acetyl]-
amino]-8-oxo-3-[(1Z)-prop-1-enyl]-5-thia-1-aza-
bicyclo[4.2.0]oct-2-ene-2-carboxylic acid,

I. (2R)-2-[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino-
2-[(2R)-4-carboxy-5-[(1E)-prop-1-enyl]-3,6-dihydro-2H-
1,3-thiazin-2-yl]-acetic acid,

Cefradine : [38821-53-3]
DEFINITION
Main component : (6R,7R)-7-[[(2R)-amino(cyclohexa-
1,4-dienyl)acetyl]amino]-3-methyl-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (cefradine).
Semi-synthetic product derived from a fermentation product.
J. (6R,7R)-7-[[(2R)-2-[[(2R)-2-amino-2-(4-hydroxy-
phenyl)acetyl]amino]-2-(4-hydroxyphenyl)acetyl]- Content :
amino]-8-oxo-3-[(1E)-prop-1-enyl]-5-thia-1- – cefradine : minimum 90.0 per cent (anhydrous substance);
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, – cefalexin : maximum 5.0 per cent (anhydrous substance) ;

General Notices (1) apply to all monographs and other texts 1809
Cefradine EUROPEAN PHARMACOPOEIA 8.0

– 4′,5′-dihydrocefradine : maximum 2.0 per cent (anhydrous Time Mobile phase A Mobile phase B
substance) ; (min) (per cent V/V) (per cent V/V)
0 - 2.5 99.5 → 97 0.5 → 3
– sum of the percentage contents of cefradine, cefalexin and
4′,5′-dihydrocefradine : 96.0 per cent to 102.0 per cent 2.5 - 11 97 → 75 3 → 25
(anhydrous substance).
11 - 13 75 → 60 25 → 40

CHARACTERS 13 - 16 60 40

Appearance : white or slightly yellow, hygroscopic powder. 16 - 19 60 → 20 40 → 80

Solubility : sparingly soluble in water, practically insoluble in 19 - 19.1 20 → 99.5 80 → 0.5
ethanol (96 per cent) and in hexane.
19.1 - 25 99.5 0.5

IDENTIFICATION Flow rate : 1.0 mL/min.

Infrared absorption spectrophotometry (2.2.24). Detection : spectrophotometer at 220 nm.
Comparison : cefradine CRS. Injection : 25 μL.
Identification of impurities : use the chromatogram
If the spectra obtained in the solid state show differences, supplied with cefradine for peak identification CRS and the
dissolve 30 mg of the substance to be examined and 30 mg of chromatogram obtained with reference solution (d) to identify
the reference substance separately in 10 mL of methanol R, the peaks due to impurities C, D and E ; use the chromatogram
evaporate to dryness at 40 °C at a pressure less than 2 kPa and obtained with reference solution (a) to identify the peak due
record new spectra using the residues. to impurity B ; use the chromatogram supplied with cefradine
impurity mixture CRS and the chromatogram obtained with
TESTS reference solution (e) to identify the peaks due to impurities
Solution S. Dissolve 2.50 g in sodium carbonate solution R A and G.
and dilute to 25.0 mL with the same solvent. Relative retention with reference to cefradine (retention
Appearance of solution. Solution S is not more opalescent time = about 15 min) : impurity A = about 0.27 ;
than reference suspension II (2.2.1). Allow solution S to stand impurity B = about 0.32 ; impurity C = about 0.53 ;
for 5 min. The absorbance (2.2.25) of solution S measured at impurity D = about 0.63 ; impurity E = about 0.80 ;
450 nm is not greater than 0.60. impurity F = about 0.92 ; cefalexin = about 0.95 ;
4′,5′-dihydrocefradine = about 1.06 ; impurity G = about 1.32.
pH (2.2.3) : 3.5 to 6.0. System suitability : reference solution (b) :
Dissolve 0.100 g in carbon dioxide-free water R and dilute to – resolution : minimum 4.0 between the peaks due to cefalexin
10 mL with the same solvent. and cefradine.
Specific optical rotation (2.2.7) : + 80.0 to + 90.0 (anhydrous Limits :
substance). – correction factor : for the calculation of content, multiply
Dissolve 0.250 g in acetate buffer solution pH 4.6 R and dilute the peak area of impurity B by 3.4 ;
to 25.0 mL with the same solution. – impurities A, B, C, D, E, F, G : for each impurity, not
Related substances. Liquid chromatography (2.2.29). more than 0.25 times the area of the principal peak in
the chromatogram obtained with reference solution (c)
Test solution. Dissolve 0.300 g of the substance to be examined (0.25 per cent);
in mobile phase A and dilute to 50.0 mL with mobile phase A. – any other impurity : for each impurity, not more
Reference solution (a). Dissolve 3.0 mg of cyclohexa-1,4- than 0.25 times the area of the principal peak in the
dienylglycine CRS (impurity B) in mobile phase A and dilute chromatogram obtained with reference solution (c)
to 100.0 mL with mobile phase A. (0.25 per cent);
Reference solution (b). Dissolve 3 mg of the substance to be – total : not more than twice the area of the principal peak
examined and 3 mg of cefalexin monohydrate CRS in mobile in the chromatogram obtained with reference solution (c)
phase A and dilute to 25 mL with mobile phase A. (2.0 per cent) ;
– disregard limit : 0.05 times the area of the principal peak
Reference solution (c). Dilute 1.0 mL of the test solution to in the chromatogram obtained with reference solution (c)
100.0 mL with mobile phase A. (0.05 per cent) ; disregard the peaks due to cefalexin and
Reference solution (d). Dissolve 6 mg of cefradine for peak 4′,5′-dihydrocefradine.
identification CRS (containing impurities C, D and E) in N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm.
1.0 mL of mobile phase A.
Water (2.5.12) : maximum 6.0 per cent, determined on 0.300 g.
Reference solution (e). Dissolve the contents of a vial of
cefradine impurity mixture CRS (impurities A and G) in Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
1.0 mL of mobile phase A. 1.0 g.
Column : ASSAY
– size : l = 0.15 m, Ø = 4.6 mm ; Liquid chromatography (2.2.29).
Test solution. Dissolve 50.0 mg of the substance to be
– stationary phase : octadecylsilyl silica gel for examined in phosphate buffer solution pH 5.0 R and dilute to
chromatography R (5 μm) ; 100.0 mL with the same solution.
– temperature : 30 °C. Reference solution (a). Dissolve 50.0 mg of cefradine CRS
Mobile phase : (containing 4′,5′-dihydrocefradine) in phosphate buffer
solution pH 5.0 R and dilute to 100.0 mL with the same
– mobile phase A : 2.72 g/L solution of potassium dihydrogen solution.
phosphate R adjusted to pH 3.0 with dilute phosphoric Reference solution (b). Dissolve 5.0 mg of cefalexin
acid R ; monohydrate CRS in phosphate buffer solution pH 5.0 R and
– mobile phase B : methanol R2 ; dilute to 100.0 mL with the same solution.

1810 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ceftazidime pentahydrate

Reference solution (c). Dilute 1 mL of reference solution (a) to

10 mL with phosphate buffer solution pH 5.0 R. Mix 5 mL of
this solution and 5 mL of reference solution (b).
Column :
– size : l = 0.10 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for E. (6R,7R)-7-[[(2R)-amino(2-hydroxyphenyl)acetyl]amino]-
chromatography R (5 μm). 3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
Mobile phase : methanol R, phosphate buffer solution pH 5.0 R carboxylic acid,
(25:75 V/V).
Flow rate : 1.5 mL/min.
Detection : spectrophotometer at 254 nm.
Injection : 5 μL.
F. 3-hydroxy-4-methylthiophen-2(5H)-one,
Run time : twice the retention time of cefradine.
Relative retention with reference to cefradine
(retention time = about 3 min): cefalexin = about 0.7 ;
4′,5′-dihydrocefradine = about 1.5.
System suitability: reference solution (c) :
– resolution : minimum 4.0 between the peaks due to cefalexin
and cefradine. G. (6R,7R)-7-[(2,2-dimethylpropanoyl)amino]-3-methyl-8-
Calculate the percentage content of cefradine using the oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
chromatogram obtained with reference solution (a) and taking (7-ADCA pivalamide).
into account the assigned content of cefradine CRS. Calculate
the percentage content of cefalexin using the chromatogram 01/2013:1405
obtained with reference solution (b) and taking into account
the assigned content of cefalexin monohydrate CRS. Calculate
the percentage content of 4′,5′-dihydrocefradine using CEFTAZIDIME PENTAHYDRATE
the chromatogram obtained with reference solution (b),
taking into account the assigned content of cefalexin Ceftazidimum pentahydricum
monohydrate CRS and multiplying the area of the peak due to
4′,5′-dihydrocefradine by a correction factor of 1.6.
STORAGE
In an airtight container, protected from light, at a temperature
of 2 °C to 8 °C.
IMPURITIES
Specified impurities : A, B, C, D, E, F, G. C22H22N6O7S2,5H2O Mr 637
[78439-06-2]
DEFINITION
(6R,7R)-7-[[(2Z)-2-(2-Aminothiazol-4-yl)-2-[(1-carboxy-
1-methylethoxy)imino]acetyl]amino]-8-oxo-3-[(pyridin-
1-ium-1-yl)methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
carboxylate pentahydrate.
A. (6R,7R)-7-amino-3-methyl-8-oxo-5-thia-1- Semi-synthetic product derived from a fermentation product.
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
(7-aminodeacetoxycephalosporanic acid, 7-ADCA), Content : 95.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : slightly soluble in water and in methanol, practically
insoluble in acetone and in ethanol (96 per cent). It dissolves
in acid and alkali solutions.
B. (2R)-amino(cyclohexa-1,4-dienyl)acetic acid
(D-dihydrophenylglycine, cyclohexa-1,4-dienylglycine), IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: ceftazidime CRS.
TESTS
Solution S. Dissolve 0.25 g in carbon dioxide-free water R and
dilute to 50 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
C. (6R,7R)-7-[[(2R)-amino(cyclohexa-1,4-dienyl)ace- colourless (2.2.2, Method II).
tyl]amino]-3-methyl-8-oxo-5-thia-1-azabicy- pH (2.2.3) : 3.0 to 4.0 for solution S.
clo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (isomer 1),
Related substances. Liquid chromatography (2.2.29).
D. (6R,7R)-7-[[(2R)-amino(cyclohexa-1,4-dienyl)ace- Test solution. Suspend 0.150 g of the substance to be examined
tyl]amino]-3-methyl-8-oxo-5-thia-1-azabicy- in 5 mL of acetonitrile R, dissolve by adding water R and dilute
clo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide (isomer 2), to 100 mL with water R.

General Notices (1) apply to all monographs and other texts 1811
Ceftazidime pentahydrate EUROPEAN PHARMACOPOEIA 8.0

Reference solution (a). To 1.0 mL of the test solution add Impurity F. Liquid chromatography (2.2.29). Prepare the
5.0 mL of acetonitrile R and dilute to 100.0 mL with water R. solutions immediately before use.
Dilute 1.0 mL of this solution to 5.0 mL with water R. Phosphate buffer solution. Prepare a 10 per cent V/V solution
Reference solution (b). In order to prepare impurity B in situ, of phosphate buffer solution pH 7.0 R4.
expose 5 mL of the test solution to ultraviolet light at 254 nm Test solution. Dissolve 0.500 g of the substance to be examined
for about 24 h. in phosphate buffer solution and dilute to 100.0 mL with the
Reference solution (c). Suspend 3 mg of ceftazidime for peak same solution.
identification CRS (containing impurities A and G) in 0.5 mL Reference solution (a). Dissolve 1.00 g of pyridine R in water R
of acetonitrile R, dissolve by adding water R and dilute to 2 mL and dilute to 100.0 mL with the same solvent. Dilute 5.0 mL
with water R. of the solution to 200.0 mL with water R. Dilute 1.0 mL of this
Column : solution to 100.0 mL with phosphate buffer solution.
– size : l = 0.25 m, Ø = 4.6 mm ; Reference solution (b). Dilute 1 mL of the test solution to
200 mL with phosphate buffer solution. To 1 mL of this
– stationary phase : octadecylsilyl silica gel for solution add 20 mL of reference solution (a) and dilute to
chromatography R (5 μm) ; 200 mL with phosphate buffer solution.
– temperature : 40 °C. Column :
Mobile phase : – size : l = 0.25 m, Ø = 4.6 mm ;
– mobile phase A : solution containing 3.6 g/L of disodium – stationary phase : octadecylsilyl silica gel for
hydrogen phosphate R and 1.4 g/L of potassium dihydrogen chromatography R (5 μm).
phosphate R, adjusted to pH 3.4 with a 10 per cent V/V Mobile phase : mix 8 volumes of a 28.8 g/L solution of
solution of phosphoric acid R ; ammonium dihydrogen phosphate R previously adjusted to
– mobile phase B : acetonitrile for chromatography R ; pH 7.0 with ammonia R, 24 volumes of acetonitrile R and
68 volumes of water R.
Time Mobile phase A Mobile phase B
Flow rate : 1.0 mL/min.
(min) (per cent V/V) (per cent V/V)
0-4 96 → 89 4 → 11 Detection : spectrophotometer at 255 nm.
Injection : 20 μL.
4-5 89 11
Run time : 10 min.
5-8 89 → 84 11 → 16
System suitability : reference solution (b) :
8 - 11 84 → 80 16 → 20 – resolution : minimum 7.0 between the peaks due to
11 - 15 80 → 50 20 → 50 ceftazidime and impurity F.
Limit :
15 - 18 50 → 20 50 → 80
– impurity F : not more than the area of the principal peak
18 - 22 20 80 in the chromatogram obtained with reference solution (a)
(500 ppm).
Flow rate : 1.3 mL/min.
Heavy metals (2.4.8) : maximum 20 ppm.
Detection : spectrophotometer at 254 nm. 1.0 g complies with test F. Prepare the reference solution using
Injection : 10 μL. 2.0 mL of lead standard solution (10 ppm Pb) R.
Relative retention with reference to ceftazidime Water (2.5.12) : 13.0 per cent to 15.0 per cent, determined on
(retention time = about 8 min) : impurity F = about 0.4 ; 0.100 g.
impurity G = about 0.8 ; impurity A = about 0.9 ; Bacterial endotoxins (2.6.14) : less than 0.10 IU/mg, if
impurity B = about 1.4. intended for use in the manufacture of parenteral preparations
Identification of impurities : use the chromatogram supplied without a further appropriate procedure for the removal of
with ceftazidime for peak identification CRS and the bacterial endotoxins.
chromatogram obtained with reference solution (c) to identify
the peaks due to impurities A and G ; use the chromatogram ASSAY
obtained with reference solution (b) to identify the peak due Liquid chromatography (2.2.29).
to impurity B. Test solution. Dissolve 25.0 mg of the substance to be
System suitability: reference solution (c) : examined in the mobile phase and dilute to 25.0 mL with the
– resolution : minimum 4.0 between the peaks due to mobile phase.
impurity A and ceftazidime. Reference solution (a). Dissolve 25.0 mg of ceftazidime CRS in
the mobile phase and dilute to 25.0 mL with the mobile phase.
Limits :
Reference solution (b). Dissolve 5.0 mg of ceftazidime for peak
– correction factor : for the calculation of content, multiply identification CRS (containing impurities A and G) in the
the peak area of impurity G by 3.0 ; mobile phase and dilute to 5.0 mL with the mobile phase.
– impurities A, B, G : for each impurity, not more than the Column :
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.2 per cent) ; – size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : hexylsilyl silica gel for chromatography R
– unspecified impurities : for each impurity, not more than (5 μm).
0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent) ; Mobile phase : dissolve 4.3 g of disodium hydrogen phosphate R
and 2.7 g of potassium dihydrogen phosphate R in 980 mL of
– total : not more than 5 times the area of the principal peak water R, then add 20 mL of acetonitrile R.
in the chromatogram obtained with reference solution (a)
(1.0 per cent) ; Flow rate : 2 mL/min.
Detection : spectrophotometer at 245 nm.
– disregard limit : 0.25 times the area of the principal peak
in the chromatogram obtained with reference solution (a) Injection : 20 μL.
(0.05 per cent) ; disregard the peak due to impurity F. Run time : 6 min.

1812 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ceftazidime pentahydrate with sodium carbonate for injection

Relative retention with reference to ceftazidime (retention

time = about 4.5 min) : impurity A = about 0.7.
System suitability : reference solution (b) :
F. pyridine,
– resolution : minimum 1.5 between the peaks due to
impurity A and ceftazidime.
Calculate the content of ceftazidime (C22H22N6O7S2) taking
into account the assigned content of C22H22N6O7S2 in
ceftazidime CRS.
STORAGE
In an airtight container. If the substance is sterile, store in a
sterile, airtight, tamper-proof container. G. 2-[[[(1Z)-1-(2-aminothiazol-4-yl)-2-[(oxoethyl)amino]-2-
oxoethylidene]amino]oxy]-2-methylpropanoic acid,
IMPURITIES
Specified impurities : A, B, F, G.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of H. (6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-[(2-methoxy-
impurities in substances for pharmaceutical use) : C, E, H. 1,1-dimethyl-2-oxoethoxy)imino]acetyl]amino]-
8-oxo-3-[(pyridin-1-ium-1-yl)methyl]-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate.

01/2013:2344

CEFTAZIDIME PENTAHYDRATE
WITH SODIUM CARBONATE FOR
A. (2RS,6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-
[(1-carboxy-1-methylethoxy)imino]acetyl]amino]-
INJECTION
8-oxo-3-[(pyridin-1-ium-1-yl)methyl]-5-thia-
1-azabicyclo[4.2.0]oct-3-ene-2-carboxylate Ceftazidimum pentahydricum et natrii
(Δ-2-ceftazidime), carbonas ad iniectabile
DEFINITION
Sterile mixture of Ceftazidime pentahydrate (1405) and
Anhydrous sodium carbonate (0773).
Semi-synthetic product derived from a fermentation product.
Content :
– ceftazidime : 93.0 per cent to 105.0 per cent (dried and
B. (6R,7R)-7-[[(2E)-2-(2-aminothiazol-4-yl)-2-[(1-carboxy- carbonate-free substance) ;
1-methylethoxy)imino]acetyl]amino]-8-oxo-3-[(pyridin- – sodium carbonate : 8.0 per cent to 10.0 per cent.
1-ium-1-yl)methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
carboxylate, CHARACTERS
Appearance : white or pale yellow powder.
Solubility : freely soluble in water and in methanol, practically
insoluble in acetone.
IDENTIFICATION
A. Examine the chromatograms obtained in the assay.
C. (6R,7R)-7-amino-8-oxo-3-[(pyridin-1-ium-1-yl)methyl]-5- Results : the principal peak in the chromatogram obtained
thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate, with the test solution is similar in retention time to
the principal peak in the chromatogram obtained with
reference solution (a).
B. It gives the reaction of carbonates (2.3.1).
TESTS
Solution S. Dissolve 2.60 g in carbon dioxide-free water R and
dilute to 20.0 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and its
absorbance (2.2.25) at 425 nm is not greater than 0.50.
pH (2.2.3) : 5.0 to 7.5 for solution S.
E. (6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-
2-[[2-(1,1-dimethylethoxy)-1,1-dimethyl-2- Related substances. Liquid chromatography (2.2.29).
oxoethoxy]imino]acetyl]amino]-8-oxo-3-[(pyridin- Test solution. Suspend 0.150 g of the substance to be examined
1-ium-1-yl)methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2- in 5 mL of acetonitrile R, dissolve by adding water R and dilute
carboxylate, to 100 mL with water R.

General Notices (1) apply to all monographs and other texts 1813
Ceftazidime pentahydrate with sodium carbonate for injection EUROPEAN PHARMACOPOEIA 8.0

Reference solution (a). To 1.0 mL of the test solution add Impurity F. Liquid chromatography (2.2.29). Prepare the
5.0 mL of acetonitrile R and dilute to 100.0 mL with water R. solutions immediately before use.
Dilute 1.0 mL of this solution to 5.0 mL with water R. Phosphate buffer solution. Prepare a 10 per cent V/V solution
Reference solution (b). In order to prepare impurity B in situ, of phosphate buffer solution pH 7.0 R4.
expose 5 mL of the test solution to ultraviolet light at 254 nm Test solution. Dissolve 0.500 g of the substance to be examined
for about 24 h. in phosphate buffer solution and dilute to 100.0 mL with the
Reference solution (c). Suspend 3 mg of ceftazidime for peak same solution.
identification CRS (containing impurities A and G) in 0.5 mL Reference solution (a). Dissolve 1.00 g of pyridine R in water R
of acetonitrile R, dissolve by adding water R and dilute to 2 mL and dilute to 100.0 mL with the same solvent. Dilute 5.0 mL
with water R. of the solution to 200.0 mL with water R. Dilute 1.0 mL of this
Column : solution to 100.0 mL with phosphate buffer solution.
Reference solution (b). Dilute 1.0 mL of the test solution to
– size : l = 0.25 m, Ø = 4.6 mm ;
200.0 mL with phosphate buffer solution. To 1.0 mL of this
– stationary phase : octadecylsilyl silica gel for solution add 20.0 mL of reference solution (a) and dilute to
chromatography R (5 μm) ; 200.0 mL with phosphate buffer solution.
– temperature : 40 °C. Column :
Mobile phase : – size : l = 0.25 m, Ø = 4.6 mm ;
– mobile phase A : solution containing 3.6 g/L of disodium – stationary phase : octadecylsilyl silica gel for
hydrogen phosphate R and 1.4 g/L of potassium dihydrogen chromatography R (5 μm).
phosphate R, adjusted to pH 3.4 with a 10 per cent V/V Mobile phase : mix 8 volumes of a 28.8 g/L solution of
solution of phosphoric acid R ; ammonium dihydrogen phosphate R previously adjusted to
– mobile phase B : acetonitrile for chromatography R ; pH 7.0 with ammonia R, 24 volumes of acetonitrile R and
68 volumes of water R.
Time Mobile phase A Mobile phase B Flow rate : 1.0 mL/min.
(min) (per cent V/V) (per cent V/V)
Detection : spectrophotometer at 255 nm.
0-4 96 → 89 4 → 11
Injection : 20 μL.
4-5 89 11 Run time : 10 min.
5-8 89 → 84 11 → 16 System suitability : reference solution (b) :
8 - 11 84 → 80 16 → 20 – resolution : minimum 7.0 between the peaks due to
ceftazidime and impurity F.
11 - 15 80 → 50 20 → 50
Limit :
15 - 18 50 → 20 50 → 80 – impurity F : not more than 6 times the area of the principal
18 - 22 20 80 peak in the chromatogram obtained with reference
solution (a) (0.3 per cent).
Flow rate : 1.3 mL/min. Loss on drying (2.2.32) : maximum 13.5 per cent, determined
Detection : spectrophotometer at 254 nm. on 0.300 g. Dry at 25 °C at a pressure not exceeding 0.67 kPa
for 4 h then heat the residue at 100 °C at a pressure not
Injection : 10 μL. exceeding 0.67 kPa for 3 h.
Relative retention with reference to ceftazidime Bacterial endotoxins (2.6.14) : less than 0.10 IU/mg, if
(retention time = about 8 min) : impurity F = about 0.4 ; intended for use in the manufacture of parenteral preparations
impurity G = about 0.8 ; impurity A = about 0.9 ; without a further appropriate procedure for the removal of
impurity B = about 1.4. bacterial endotoxins.
Identification of impurities : use the chromatogram supplied
with ceftazidime for peak identification CRS and the ASSAY
chromatogram obtained with reference solution (c) to identify Ceftazidime. Liquid chromatography (2.2.29).
the peaks due to impurities A and G ; use the chromatogram Test solution. Dissolve 25.0 mg of the substance to be
obtained with reference solution (b) to identify the peak due examined in the mobile phase and dilute to 25.0 mL with the
to impurity B. mobile phase.
System suitability: reference solution (c) : Reference solution (a). Dissolve 25.0 mg of ceftazidime CRS in
– resolution : minimum 4.0 between the peaks due to the mobile phase and dilute to 25.0 mL with the mobile phase.
impurity A and ceftazidime. Reference solution (b). Dissolve 5.0 mg of ceftazidime for peak
Limits : identification CRS (containing impurities A and G) in the
mobile phase and dilute to 5.0 mL with the mobile phase.
– correction factor : for the calculation of content, multiply
the peak area of impurity G by 3.0 ; Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– impurities A, B, G : for each impurity, not more than the
area of the principal peak in the chromatogram obtained – stationary phase : hexylsilyl silica gel for chromatography R
with reference solution (a) (0.2 per cent) ; (5 μm).
– unspecified impurities : for each impurity, not more than Mobile phase : dissolve 4.3 g of disodium hydrogen phosphate R
0.5 times the area of the principal peak in the chromatogram and 2.7 g of potassium dihydrogen phosphate R in 980 mL of
obtained with reference solution (a) (0.10 per cent) ; water R, then add 20 mL of acetonitrile R.
Flow rate : 2 mL/min.
– total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a) Detection : spectrophotometer at 245 nm.
(1.0 per cent) ; Injection : 20 μL.
– disregard limit : 0.25 times the area of the principal peak Run time : 6 min.
in the chromatogram obtained with reference solution (a) Relative retention with reference to ceftazidime (retention
(0.05 per cent) ; disregard the peak due to impurity F. time = about 4.5 min) : impurity A = about 0.7.

1814 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ceftriaxone sodium

System suitability : reference solution (b) :

– resolution : minimum 1.5 between the peaks due to
impurity A and ceftazidime.
Calculate the content of ceftazidime (C22H22N6O7S2) taking
into account the assigned content of C22H22N6O7S2 in
ceftazidime CRS. C. (6R,7R)-7-amino-8-oxo-3-[(pyridin-1-ium-1-yl)methyl]-5-
Sodium carbonate. Atomic absorption spectrometry (2.2.23, thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate,
Method I).
Caesium chloride buffer solution. To 12.7 g of caesium
chloride R add 500 mL of water R and 86 mL of hydrochloric
acid R and dilute to 1000.0 mL with water R.
Sodium standard solution (1000 mg/L). Dissolve 3.70 g of
sodium nitrate R in water R and dilute to 500 mL with the
same solvent, add 48.5 g of nitric acid R and dilute to 1000 mL
with water R.
Test solution. Dissolve 650.0 mg of the substance to be
examined in water R and dilute to 100.0 mL with the same E. (6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-[[2-(1,1-
solvent. To 10.0 mL of this solution add 5.0 mL of caesium dimethylethoxy)-1,1-dimethyl-2-oxoethoxy]imino]acetyl]-
chloride buffer solution and dilute to 50.0 mL with water R. amino]-8-oxo-3-[(pyridin-1-ium-1-yl)methyl]-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate,
Reference solution. Into 4 identical flasks, each containing
20.0 mL of caesium chloride buffer solution, introduce
respectively 0 mL, 5.00 mL, 10.00 mL and 15.00 mL of sodium
standard solution (1000 mg/L) and dilute to 200.0 mL with
water R. F. pyridine,
Source : sodium hollow-cathode lamp.
Wavelength : 330.2 nm to 330.3 nm.
Atomisation device : air-acetylene flame.
Calculate the percentage content of sodium carbonate.
STORAGE
In a sterile, airtight, tamper-proof container, protected from
light and humidity. G. 2-[[[(1Z)-1-(2-aminothiazol-4-yl)-2-[(oxoethyl)amino]-2-
oxoethylidene]amino]oxy]-2-methylpropanoic acid,
LABELLING
The label states the percentage content m/m of ceftazidime.
IMPURITIES
Specified impurities : A, B, F, G.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or H. (6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-[(2-methoxy-
by the general monograph Substances for pharmaceutical use 1,1-dimethyl-2-oxoethoxy)imino]acetyl]amino]-
(2034). It is therefore not necessary to identify these impurities 8-oxo-3-[(pyridin-1-ium-1-yl)methyl]-5-thia-1-
for demonstration of compliance. See also 5.10. Control of azabicyclo[4.2.0]oct-2-ene-2-carboxylate.
impurities in substances for pharmaceutical use) : C, E, H.
01/2008:0991

CEFTRIAXONE SODIUM
Ceftriaxonum natricum

A. (2RS,6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-
[(1-carboxy-1-methylethoxy)imino]acetyl]amino]-
8-oxo-3-[(pyridin-1-ium-1-yl)methyl]-5-thia-
1-azabicyclo[4.2.0]oct-3-ene-2-carboxylate
(Δ-2-ceftazidime),

C18H16N8Na2O7S3,3½H2O Mr 662
[104376-79-6]
DEFINITION
Disodium (6R,7R)-7-[[(2Z)-(2-aminothiazol-4-
yl)(methoxyimino)acetyl]amino]-3-[[(2-methyl-6-oxido-5-
B. (6R,7R)-7-[[(2E)-2-(2-aminothiazol-4-yl)-2-[(1-carboxy- oxo-2,5-dihydro-1,2,4-triazin-3-yl)sulfanyl]methyl]-8-oxo-5-
1-methylethoxy)imino]acetyl]amino]-8-oxo-3-[(pyridin- thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate 3.5 hydrate.
1-ium-1-yl)methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2- Semi-synthetic product derived from a fermentation product.
carboxylate, Content : 96.0 per cent to 102.0 per cent (anhydrous substance).

General Notices (1) apply to all monographs and other texts 1815
Ceftriaxone sodium EUROPEAN PHARMACOPOEIA 8.0

CHARACTERS 2-Ethylhexanoic acid (2.4.28) : maximum 0.8 per cent m/m.

Appearance : almost white or yellowish, slightly hygroscopic, Water (2.5.12) : 8.0 per cent to 11.0 per cent, determined on
crystalline powder. 0.100 g.
Solubility : freely soluble in water, sparingly soluble in Bacterial endotoxins (2.6.14) : less than 0.08 IU/mg, if
methanol, very slightly soluble in anhydrous ethanol. intended for use in the manufacture of parenteral preparations
IDENTIFICATION without a further appropriate procedure for the removal of
bacterial endotoxins.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : ceftriaxone sodium CRS. ASSAY
B. It gives reaction (a) of sodium (2.3.1). Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
TESTS Injection : test solution and reference solution (a).
Solution S. Dissolve 2.40 g in carbon dioxide-free water R and Calculate the percentage content of C18H16N8Na2O7S3 from the
dilute to 20.0 mL with the same solvent. declared content of ceftriaxone sodium CRS.
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution Y5 or BY5 STORAGE
(2.2.2). In an airtight container protected from light. If the substance
Dilute 2 mL of solution S to 20 mL with water R. is sterile, store in a sterile, airtight, tamper-proof container.
pH (2.2.3): 6.0 to 8.0 for solution S. IMPURITIES
Specific optical rotation (2.2.7) : − 155 to − 170 (anhydrous
substance).
Dissolve 0.250 g in water R and dilute to 25.0 mL with the
same solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 30.0 mg of the substance to be
examined in the mobile phase and dilute to 100.0 mL with
the mobile phase. A. (6R,7R)-7-[[(2E)-(2-aminothiazol-4-yl)(methoxy-
Reference solution (a). Dissolve 30.0 mg of ceftriaxone imino)acetyl]amino]-3-[[(2-methyl-5,6-dioxo-1,2,5,6-
sodium CRS in the mobile phase and dilute to 100.0 mL with tetrahydro-1,2,4-triazin-3-yl)sulfanyl]methyl]-8-oxo-5-
the mobile phase. thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
Reference solution (b). Dissolve 5.0 mg of ceftriaxone ((E)-isomer),
sodium CRS and 5.0 mg of ceftriaxone impurity A CRS in the
mobile phase and dilute to 100.0 mL with the mobile phase.
Reference solution (c). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm). B. (5aR,6R)-6-[[(2Z)-(2-aminothiazol-4-yl)(methox-
yimino)acetyl]amino]-5a,6-dihydro-3H,7H-azeto-
Mobile phase : dissolve 2.0 g of tetradecylammonium bromide R [2,1-b]furo[3,4-d][1,3]thiazine-1,7(4H)-dione,
and 2.0 g of tetraheptylammonium bromide R in a mixture
of 440 mL of water R, 55 mL of 0.067 M phosphate buffer
solution pH 7.0 R, 5.0 mL of citrate buffer solution pH 5.0
prepared by dissolving 20.17 g of citric acid R in 800 mL of
water R, adjusting to pH 5.0 with strong sodium hydroxide
solution R and diluting to 1000.0 mL with water R, and 500 mL
of acetonitrile R.
Flow rate : 1.5 mL/min. C. 2-methyl-3-sulfanyl-1,2-dihydro-1,2,4-triazine-5,6-dione,
Detection : spectrophotometer at 254 nm.
Injection : 20 μL of the test solution and reference solutions (b)
and (c).
Run time : twice the retention time of ceftriaxone.
System suitability : reference solution (b) :
– resolution : minimum 3.0 between the peaks due to
ceftriaxone and impurity A.
D. S-benzothiazol-2-yl (2Z)-(2-aminothiazol-4-
Limits : yl)(methoxyimino)thioacetate,
– any impurity : not more than the area of the principal peak
in the chromatogram obtained with reference solution (c)
(1.0 per cent) ;
– total : not more than 4 times the area of the principal peak
in the chromatogram obtained with reference solution (c)
(4.0 per cent) ;
– disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (c) E. (6R,7R)-7-amino-3-[[(2-methyl-5,6-dioxo-1,2,5,6-
(0.1 per cent). tetrahydro-1,2,4-triazin-3-yl)sulfanyl]methyl]-8-oxo-5-
N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm. thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid.

1816 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cefuroxime axetil

01/2008:1300 Flow rate : 1.0 mL/min.

corrected 6.0 Detection : spectrophotometer at 278 nm.
Injection : 20 μL of the test solution and reference solutions (a),
CEFUROXIME AXETIL (b) and (c).
Identification of impurities: use the chromatogram obtained
Cefuroximum axetili with reference solution (b) to identify the pair of peaks due to
impurity A and use the chromatogram obtained with reference
solution (c) to identify the pair of peaks due to impurity B.
Relative retention with reference to cefuroxime axetil
diastereoisomer A : cefuroxime axetil diastereoisomer B = about
0.9, impurity A = about 1.2 ; impurity B = 1.7 and 2.1.
System suitability : reference solution (b) :
– resolution : minimum 1.5 between the peaks due to
cefuroxime axetil diastereoisomer A and impurity A.
C20H22N4O10S Mr 510.5 Limits :
[64544-07-6] – impurity A : maximum 1.5 per cent for the sum of the pair
of peaks ;
DEFINITION
– impurity B : maximum 1.0 per cent for the sum of the pair
Mixture of the 2 diastereoisomers of (1RS)-1-(acetyloxy)ethyl
of peaks ;
(6R,7R)-3-[(carbamoyloxy)methyl]-7-[[(Z)-2-(furan-2-yl)-2-
(methoxyimino)acetyl]amino]-8-oxo-5-thia-1-azabicy- – impurity E : maximum 0.5 per cent ;
clo[4.2.0]oct-2-ene-2-carboxylate. – any other impurity : for each impurity, maximum 0.5 per
Semi-synthetic product derived from a fermentation product. cent ;
Content : 96.0 per cent to 102.0 per cent (anhydrous substance). – total : maximum 3.0 per cent ;
– disregard limit : 0.05 times the area of the 2 principal peaks
CHARACTERS in the chromatogram obtained with reference solution (a)
Appearance : white or almost white powder. (0.05 per cent).
Solubility : slightly soluble in water, soluble in acetone, in ethyl Diastereoisomer ratio. Liquid chromatography (2.2.29) as
acetate and in methanol, slightly soluble in ethanol (96 per described in the test for related substances.
cent). Limit : test solution :
IDENTIFICATION – the ratio of the area of the peak due to cefuroxime axetil
diastereoisomer A to the sum of the areas of the peaks due
A. Infrared absorption spectrophotometry (2.2.24). to cefuroxime axetil diastereoisomers A and B is between
Comparison : cefuroxime axetil CRS. 0.48 and 0.55.
B. Examine the chromatograms obtained in the assay. Acetone (2.4.24) : maximum 1.1 per cent.
Results : the principal peaks in the chromatogram obtained Water (2.5.12) : maximum 1.5 per cent, determined on 0.400 g.
with the test solution are similar in retention time and size
to the peaks due to cefuroxime axetil diastereoisomers ASSAY
A and B in the chromatogram obtained with reference
solution (d). Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications.
TESTS Injection : test solution and reference solution (d).
Related substances. Liquid chromatography (2.2.29) : use System suitability : reference solution (d) :
the normalisation procedure. Prepare the test solution and – resolution : minimum 1.5 between the peaks due to
reference solution (d) immediately before use. cefuroxime axetil diastereoisomers A and B ;
Test solution. Dissolve 10.0 mg of the substance to be – repeatability : maximum relative standard deviation of
examined in the mobile phase and dilute to 50.0 mL with the 2.0 per cent for the sum of the peaks due to cefuroxime
mobile phase. axetil diastereoisomers A and B after 6 injections.
Reference solution (a). Dilute 1.0 mL of the test solution to Calculate the percentage content of C20H22N4O10S from the
100.0 mL with the mobile phase. sum of the areas of the 2 diastereoisomer peaks and the
Reference solution (b). In order to prepare in situ impurity A, declared content of C20H22N4O10S in cefuroxime axetil CRS.
heat 5 mL of the test solution at 60 °C for 1 h.
Reference solution (c). In order to prepare in situ impurity B, STORAGE
expose 5 mL of the test solution to ultraviolet light at 254 nm In an airtight container, protected from light.
for 24 h.
Reference solution (d). Dissolve 10.0 mg of cefuroxime IMPURITIES
axetil CRS in the mobile phase and dilute to 50.0 mL with the Specified impurities : A, B, E.
mobile phase. Other detectable impurities (the following substances would,
Column : if present at a sufficient level, be detected by one or other of
– size : l = 0.25 m, Ø = 4.6 mm ; the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
– stationary phase : trimethylsilyl silica gel for by the general monograph Substances for pharmaceutical use
chromatography R (5 μm). (2034). It is therefore not necessary to identify these impurities
Mobile phase : methanol R, 23 g/L solution of ammonium for demonstration of compliance. See also 5.10. Control of
dihydrogen phosphate R (38:62 V/V). impurities in substances for pharmaceutical use) : C, D.

General Notices (1) apply to all monographs and other texts 1817
Cefuroxime sodium EUROPEAN PHARMACOPOEIA 8.0

Content : 96.0 per cent to 102.0 per cent (anhydrous substance).

CHARACTERS
Appearance : white or almost white, slightly hygroscopic
powder.
Solubility : freely soluble in water, very slightly soluble in
ethanol (96 per cent).

A. 1-(acetyloxy)ethyl (6R,7R)-3-[(carbamoyloxy)methyl]- IDENTIFICATION

7-[[(Z)-2-(furan-2-yl)-2-(methoxyimino)acetyl]amino]- A. Infrared absorption spectrophotometry (2.2.24).
8-oxo-5-thia-1-azabicyclo[4.2.0]oct-3-ene-2-carboxylate Comparison: cefuroxime sodium CRS.
(Δ3-isomers),
B. It gives reaction (a) of sodium (2.3.1).
TESTS
Solution S. Dissolve 2.0 g in carbon dioxide-free water R and
dilute to 20.0 mL with the same solvent.
Appearance of solution. Solution S is not more opalescent
than reference suspension II (2.2.1). The absorbance (2.2.25)
of solution S measured at 450 nm is not greater than 0.25.
pH (2.2.3) : 5.5 to 8.5.
B. (1RS)-1-(acetyloxy)ethyl (6R,7R)-3-[(carbamoyloxy)-
Dilute 2 mL of solution S to 20 mL with carbon dioxide-free
methyl]-7-[[(E)-2-(furan-2-yl)-2-(methoxyimino)acetyl]-
water R.
amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
carboxylate ((E)-isomers), Specific optical rotation (2.2.7) : + 59 to + 66 (anhydrous
substance).
Dissolve 0.500 g in acetate buffer solution pH 4.6 R and dilute
to 25.0 mL with the same buffer solution.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use or keep at 2-8 °C.
Test solution (a). Dissolve 25.0 mg of the substance to be
C. R = CO-CCl3 : (6R,7R)-7-[[(Z)-2-(furan-2-yl)- examined in water R and dilute to 25.0 mL with the same
2-(methoxyimino)acetyl]amino]-8-oxo-3- solvent.
[[[(trichloroacetyl)carbamoyl]oxy]methyl]-5-thia- Test solution (b). Dilute 5.0 mL of test solution (a) to 50.0 mL
1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, with water R.
D. R = H : cefuroxime. Reference solution (a). Dissolve 25.0 mg of cefuroxime
sodium CRS in water R and dilute to 25.0 mL with the same
solvent. Dilute 5.0 mL to 50.0 mL with water R.
Reference solution (b). Place 20 mL of reference solution (a) in
a water-bath at 80 °C for 15 min. Cool and inject immediately.
Reference solution (c). Dilute 1.0 mL of test solution (a) to
100.0 mL with water R.
Column :
E. (5aR,6R)-6-[[(2Z)-2-(furan-2-yl)-2-(methoxy- – size : l = 0.125 m, Ø = 4.6 mm ;
imino)acetyl]amino]-5a,6-dihydro-3H,7H-
azeto[2,1-b]furo[3,4-d][1,3]thiazine-1,7(4H)-dione – stationary phase : hexylsilyl silica gel for chromatography R
(descarbamoylcefuroxime lactone). (5 μm).
Mobile phase : mix 1 volume of acetonitrile R and 99 volumes
of an acetate buffer solution pH 3.4, prepared by dissolving
6.01 g of glacial acetic acid R and 0.68 g of sodium acetate R in
01/2008:0992
water R and diluting to 1000 mL with the same solvent.
corrected 6.0
Flow rate : 1.5 mL/min.
CEFUROXIME SODIUM Detection : spectrophotometer at 273 nm.
Injection : 20 μL loop injector ; inject test solution (a) and
reference solutions (b) and (c).
Cefuroximum natricum Run time : 4 times the retention time of cefuroxime.
System suitability : reference solution (b) :
– resolution : minimum 2.0 between the peaks due to
cefuroxime and impurity A.
Limits :
– impurity A : not more than the area of the principal peak
C16H15N4NaO8S Mr 446.4 in the chromatogram obtained with reference solution (c)
[56238-63-2] (1.0 per cent) ;
– any other impurity : not more than the area of the principal
DEFINITION peak in the chromatogram obtained with reference
Sodium (6R,7R)-3-[(carbamoyloxy)methyl]-7-[[(Z)- solution (c) (1.0 per cent) ;
(furan-2-yl)(methoxyimino)acetyl]amino]-8-oxo-5-thia-1- – total : not more than 3 times the area of the principal peak
azabicyclo[4.2.0]oct-2-ene-2-carboxylate. in the chromatogram obtained with reference solution (c)
Semi-synthetic product derived from a fermentation product. (3.0 per cent) ;

1818 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Celecoxib

– disregard limit : 0.05 times the area of the principal peak

in the chromatogram obtained with reference solution (c)
(0.05 per cent).
N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm.
2-Ethylhexanoic acid (2.4.28) : maximum 0.5 per cent m/m. I. (Z)-(furan-2-yl)(methoxyimino)acetic acid.
Water (2.5.12) : maximum 3.5 per cent, determined on 0.400 g.
Bacterial endotoxins (2.6.14) : less than 0.10 IU/mg, if
intended for use in the manufacture of parenteral preparations 07/2012:2591
without a further appropriate procedure for the removal of
bacterial endotoxins. CELECOXIB
ASSAY
Liquid chromatography (2.2.29) as described in the test for Celecoxibum
related substances with the following modification.
Injection : test solution (b) and reference solution (a).
Calculate the percentage content of cefuroxime sodium.
STORAGE
In an airtight container. If the substance is sterile, store in a
sterile, airtight, tamper-proof container.
IMPURITIES
C17H14F3N3O2S Mr 381.4
[169590-42-5]
DEFINITION
4-[5-(4-Methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzenesulfonamide.
A. R = OH : (6R,7R)-7-[[(Z)-(furan-2-yl)(methoxyimino)ace-
tyl]amino]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicy- Content : 98.0 per cent to 102.0 per cent (anhydrous substance).
clo[4.2.0]oct-2-ene-2-carboxylic acid (descarbamoyl- CHARACTERS
cefuroxime),
Appearance : white or almost white, crystalline or amorphous
B. R = O-CO-CH3 : (6R,7R)-3-[(acetyloxy)methyl]-7-[[(Z)- powder.
(furan-2-yl)(methoxyimino)acetyl]amino]-8-oxo-5-thia-1- Solubility : practically insoluble in water, freely soluble to
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, soluble in anhydrous ethanol, soluble in methylene chloride.
C. R = H : (6R,7R)-7-[[(Z)-(furan-2-yl)(methoxyimi- It shows polymorphism (5.9).
no)acetyl]amino]-3-methyl-8-oxo-5-thia-1-azabi-
cyclo[4.2.0]oct-2-ene-2-carboxylic acid, IDENTIFICATION
D. R = O-CO-NH-CO-CCl3 : (6R,7R)-7-[[(Z)-(furan- Infrared absorption spectrophotometry (2.2.24).
2-yl)(methoxyimino)acetyl]amino]-8-oxo-3- Comparison: celecoxib CRS.
[[[(trichloroacetyl)carbamoyl]oxy]methyl]-5-thia-1- If the spectra obtained show differences, dissolve the substance
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, to be examined and the reference substance separately in
2-propanol R, evaporate to dryness and record new spectra
using the residues.
TESTS
Related substances. Liquid chromatography (2.2.29).
Solvent mixture : water R, methanol R2 (25:75 V/V).
E. R = O-CO-NH2 : (6R,7R)-3-[(carbamoyloxy)methyl]-7- Test solution. Dissolve 50.0 mg of the substance to be
[[(E)-(furan-2-yl)(methoxyimino)acetyl]amino]-8-oxo- examined in the solvent mixture and dilute to 100.0 mL with
5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid the solvent mixture.
(trans-cefuroxime),
Reference solution (a). Dissolve 50.0 mg of celecoxib CRS in
F. R = OH : (6R,7R)-7-[[(E)-(furan-2-yl)(methoxyimino)ace- the solvent mixture and dilute to 100.0 mL with the solvent
tyl]amino]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabi- mixture.
cyclo[4.2.0]oct-2-ene-2-carboxylic acid, Reference solution (b). Dissolve 3 mg of celecoxib
G. R = O-CO-CH3 : (6R,7R)-3-[(acetyloxy)methyl]-7-[[(E)- impurity A CRS and 3 mg of celecoxib impurity B CRS in
(furan-2-yl)(methoxyimino)acetyl]amino]-8-oxo-5-thia-1- the solvent mixture and dilute to 50.0 mL with the solvent
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, mixture. Dilute 1.0 mL of the solution to 25.0 mL with
reference solution (a).
Reference solution (c). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
H. (5aR,6R)-6-[[(Z)-(furan-2-yl)(methoxyimino)acetyl]- – stationary phase : end-capped phenylsilyl silica gel for
amino]-5a,6-dihydro-3H,7H-azeto[2,1-b]furo[3,4- chromatography R (5 μm) ;
d][1,3]thiazine-1,7(4H)-dione, – temperature : 60 °C.

General Notices (1) apply to all monographs and other texts 1819
Celiprolol hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Mobile phase : mix 10 volumes of acetonitrile R1, 30 volumes of

methanol R2 and 60 volumes of a 2.7 g/L solution of potassium
dihydrogen phosphate R previously adjusted to pH 3.0 with
phosphoric acid R.
Flow rate : 1.5 mL/min.
Detection : spectrophotometer at 215 nm.
Injection : 25 μL of the test solution and reference solutions (b)
and (c).
Run time : 1.5 times the retention time of celecoxib.
Identification of impurities : use the chromatogram obtained
with reference solution (b) to identify the peaks due to B. 4-[3-(4-methylphenyl)-5-(trifluoromethyl)-1H-pyrazol-1-
impurities A and B. yl]benzenesulfonamide.
Relative retention with reference to celecoxib (retention
time = about 27 min) : impurity A = about 0.9 ;
impurity B = about 1.1. 01/2008:1632
corrected 6.0
System suitability :
– resolution : minimum 1.8 between the peaks due to CELIPROLOL HYDROCHLORIDE
impurity A and celecoxib and minimum 1.8 between the
peaks due to celecoxib and impurity B in the chromatogram
obtained with reference solution (b). Celiprololi hydrochloridum
Calculation of percentage contents :
– for all impurities, use the concentration of celecoxib in
reference solution (c).
Limits :
– impurity A : maximum 0.4 per cent ;
– unspecified impurities : for each impurity, maximum
0.10 per cent ;
– total : maximum 0.5 per cent ;
C20H34ClN3O4 Mr 416.0
– reporting threshold : 0.05 per cent. [57470-78-7]
Heavy metals (2.4.8) : maximum 20 ppm.
DEFINITION
Solvent mixture : water R, acetone R (15:85 V/V).
3-[3-Acetyl-4-[(2RS)-3-[(1,1-dimethylethyl)amino]-2-
0.50 g complies with test H. Prepare the reference solution hydroxypropoxy]phenyl]-1,1-diethylurea hydrochloride.
using 1 mL of lead standard solution (10 ppm Pb) R. Content : 99.0 per cent to 101.0 per cent (dried substance).
Water (2.5.12) : maximum 0.5 per cent, determined on 0.400 g.
CHARACTERS
Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
1.0 g in a platinum crucible. Appearance : white or very slightly yellow, crystalline powder.
Solubility : freely soluble in water and in methanol, soluble
ASSAY in ethanol (96 per cent), very slightly soluble in methylene
Liquid chromatography (2.2.29) as described in the test for chloride.
related substances with the following modifications. It shows polymorphism (5.9).
Injection : test solution and reference solution (a). IDENTIFICATION
Calculate the percentage content of C17H14F3N3O2S taking into A. Infrared absorption spectrophotometry (2.2.24).
account the assigned content of celecoxib CRS. Comparison: celiprolol hydrochloride CRS.
IMPURITIES If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
Specified impurities : A. substance separately in methanol R, evaporate to dryness
Other detectable impurities (the following substances would, and record new spectra using the residues.
if present at a sufficient level, be detected by one or other of B. It gives reaction (a) of chlorides (2.3.1).
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or TESTS
by the general monograph Substances for pharmaceutical Optical rotation (2.2.7) : − 0.10° to + 0.10°.
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10. Dissolve 1.0 g in water R and dilute to 10.0 mL with the same
Control of impurities in substances for pharmaceutical use) : B. solvent.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
Test solution. Dissolve 0.100 g of the substance to be examined
in mobile phase A and dilute to 20.0 mL with mobile phase A.
Reference solution (a). Dissolve 2 mg of the substance to be
examined and 2 mg of acebutolol hydrochloride R in mobile
phase A and dilute to 50.0 mL with mobile phase A.
Reference solution (b). Dissolve 10 mg of the substance to be
A. 4-[5-(3-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1- examined in 2 mL of mobile phase A and allow to stand for
yl]benzenesulfonamide, 24 h (for identification of impurity A).

1820 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Celiprolol hydrochloride

Reference solution (c). Dilute 1.0 mL of the test solution to – disregard limit : 0.5 times the area of the principal peak in
100.0 mL with mobile phase A. Dilute 1.0 mL of this solution the chromatogram obtained with reference solution (c)
to 10.0 mL with mobile phase A. (0.05 per cent).
Reference solution (d). Dissolve 10 mg of celiprolol for peak Loss on drying (2.2.32) : maximum 0.5 per cent, determined
identification CRS in mobile phase A and dilute to 2 mL with on 1.000 g by drying in an oven at 105 °C for 3 h.
mobile phase A.
ASSAY
Reference solution (e). This solution is only prepared if required
(see below) and is used to determine the identity of impurity I Dissolve 0.350 g under an atmosphere of nitrogen in 50 mL of
which co-elutes with impurity H (the 2 impurities originate ethanol (96 per cent) R and add 1.0 mL of 0.1 M hydrochloric
from different routes of synthesis). Dissolve the contents of a acid. Carry out a potentiometric titration (2.2.20), using
vial of celiprolol impurity I CRS in mobile phase A and dilute 0.1 M sodium hydroxide. Read the volume added between the
to 2.0 mL with mobile phase A. 2 points of inflexion.
Column : 1 mL of 0.1 M sodium hydroxide is equivalent to 41.60 mg
of C20H34ClN3O4.
– size : l = 0.15 m, Ø = 4.6 mm,
– stationary phase : octylsilyl silica gel for chromatography R STORAGE
(5 μm), Protected from light.
– temperature : 30 °C.
IMPURITIES
Mobile phase :
Specified impurities : A, B, C, D, E, F, G, H, I.
– mobile phase A : mix 91 mL of tetrahydrofuran R, 63 mL
of acetonitrile R1, 0.6 mL of pentafluoropropanoic acid R
and 0.2 mL of trifluoroacetic acid R ; dilute to 1000 mL with
water R ;
– mobile phase B : acetonitrile R1 ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V) A. R1 = H, R2 = NH-C(CH3)3 : 1-[5-amino-
0 - 50 100 → 80 0 → 20 2-[(2RS)-3-[(1,1-dimethylethyl)amino]-2-
hydroxypropoxy]phenyl]ethanone,
50 - 51 80 → 100 20 → 0
C. R1 = CO-NH-C(CH3)3, R2 = NH-C(CH3)3 :
51 - 65 100 0 1-[3-acetyl-4-[(2RS)-3-[(1,1-dimethylethyl)amino]-
2-hydroxypropoxy]phenyl]-3-(1,1-dimethylethyl)urea,
Flow rate : 1.4 mL/min.
D. R1 = CO-N(C2H5)2, R2 = N(C2H5)2 : 3-[3-acetyl-4-
Detection : spectrophotometer at 232 nm. [(2RS)-3-(diethylamino)-2-hydroxypropoxy]phenyl]-1,1-
Injection : 10 μL. diethylurea,
Identification of impurities : use the chromatogram H. R1 = CO-N(C2H5)2, R2 = Br : 3-[3-acetyl-4-[(2RS)-
supplied with celiprolol for peak identification CRS and the 3-bromo-2-hydroxypropoxy]phenyl]-1,1-diethylurea
chromatogram obtained with reference solution (d) to identify (bromhydrin compound),
the peaks due to impurities B, E and F.
Relative retention with reference to celiprolol (retention
time = about 10 min) : impurity A = about 0.3 ;
impurity D = about 0.7 ; impurity G = about 1.2 ;
impurity B = about 1.4 ; impurity F = about 1.6 ;
impurity C = about 2.2 ; impurity H or I = about 2.5 ;
impurity E = about 3.9.
System suitability: reference solution (a) : B. 1,3-bis[3-acetyl-4-[3-[(1,1-dimethylethyl)amino]-2-
hydroxypropoxy]phenyl]urea,
– resolution : minimum 4.0 between the peaks due to
celiprolol and acebutolol.
Limits :
– correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
corresponding correction factor : impurity A = 4.0 ;
impurity B = 1.5 ; impurity E = 2.3 ; impurity F = 0.5 ;
impurity I = 1.7 ;
– any impurity : for each impurity, not more than twice the
area of the principal peak in the chromatogram obtained
with reference solution (c) (0.2 per cent), and not more E. 1,1′-[[(1,1-dimethylethyl)imino]bis[(2-hydroxypropane-
than 1 such peak has an area greater than the area of 1,3-diyl)oxy(3-acetyl-1,4-phenylene)]]bis(3,3-diethylurea),
the principal peak in the chromatogram obtained with
reference solution (c) (0.1 per cent) ;
– total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (c)
(0.5 per cent) ;
– if any of the above limits are exceeded and if a peak occurs
with a relative retention of about 2.5 (impurity H or I),
the identity of this peak has to be clarified by use of a UV F. R1 = R3 = H, R2 = CO-CH3 : 3-(3-acetyl-4-hydroxyphenyl)-
spectrum recorded with a diode array detector ; if this 1,1-diethylurea,
spectrum is different from the one obtained with reference I. R1 = CO-CH3, R2 = H, R3 = C2H5 : 1-acetyl-1-(4-
solution (e), no correction factor is applied ; ethoxyphenyl)-3,3-diethylurea,

General Notices (1) apply to all monographs and other texts 1821
Cellulose acetate EUROPEAN PHARMACOPOEIA 8.0

chapter 5.15). This section is a non-mandatory part of the

monograph and it is not necessary to verify the characteristics
to demonstrate compliance. Control of these characteristics can
however contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process and
the performance of the medicinal product during use. Where
control methods are cited, they are recognised as being suitable
G. 3-[3-acetyl-4-[[(RS)-oxiranyl]methoxy]phenyl]-1,1- for the purpose, but other methods can also be used. Wherever
diethylurea. results for a particular characteristic are reported, the control
method must be indicated.
The following characteristics may be relevant for cellulose
01/2009:0887 acetate used as film former.
Apparent viscosity. Dissolve 10 g in a mixture of 50 mL of
CELLULOSE ACETATE methanol R and 50 mL of methylene chloride R by shaking.
Determine the viscosity of this solution at 20 ± 0.1 °C using a
Cellulosi acetas rotating viscometer (2.2.10).
DEFINITION Acetyl groups (C2H3O) : typically 29.0 per cent to 44.8 per
cent of acetyl groups (dried substance) and typically 90.0 per
Partly or completely O-acetylated cellulose. cent to 110.0 per cent of the nominal acetyl content (dried
CHARACTERS substance).
Appearance : white, yellowish-white or greyish-white, A. Cellulose acetate containing not more than 42.0 per cent
hygroscopic powder or granules. of acetyl groups
Solubility : practically insoluble in water, soluble in acetone, in To 2.000 g in a 500 mL conical flask, add 100 mL of
formic acid and in a mixture of equal volumes of methanol acetone R and 10 mL of water R. Close the flask and stir
and methylene chloride, practically insoluble in ethanol with a magnetic stirrer until dissolution is complete. Add
(96 per cent). 30.0 mL of 1 M sodium hydroxide with constant stirring. A
finely divided precipitate of regenerated cellulose, free from
IDENTIFICATION lumps, is obtained. Close the flask and stir with a magnetic
Infrared absorption spectrophotometry (2.2.24). stirrer for 30 min. Add 100 mL of water R at 80 °C, washing
Comparison : cellulose acetate CRS. down the sides of the flask, stir for 2 min and cool to room
temperature. Titrate with 0.5 M sulfuric acid, using 0.1 mL
Preparation : prepare a 100 g/L solution of cellulose acetate,
of phenolphthalein solution R as indicator. Carry out a
previously dried, in dioxan R, and spread 1 drop of the solution
blank titration.
between 2 sodium chloride plates ; separate the plates, heat
them both at 105 °C for 1 h, and reassemble the dried plates. Calculate the percentage content of acetyl groups using the
following expression :
TESTS
Free acid : maximum 0.1 per cent, calculated as acetic acid
(dried substance).
To 5.00 g in a 250 mL conical flask, add 150 mL of carbon
dioxide-free water R, insert the stopper, swirl the suspension d = loss on drying as a percentage ;
gently and allow to stand for 3 h. Filter, then wash the flask m = mass of the substance to be examined, in grams ;
and the filter with carbon dioxide-free water R, adding these
washings to the filtrate. Add 0.1 mL of phenolphthalein n 1 = number of millilitres of 0.5 M sulfuric acid used
solution R1 and titrate the combined filtrate and washings with in the test ;
0.01 M sodium hydroxide until a pale pink colour is obtained. n2 = number of millilitres of 0.5 M sulfuric acid used
1 mL of 0.01 M sodium hydroxide is equivalent to 0.6005 mg in the blank titration.
of free acid, calculated as acetic acid. B. Cellulose acetate containing more than 42.0 per cent of
Heavy metals (2.4.8) : maximum 10 ppm. acetyl groups
2.0 g complies with test D. Prepare the reference solution To 2.000 g in a 500 mL conical flask, add 30 mL of dimethyl
using 2 mL of lead standard solution (10 ppm Pb) R. sulfoxide R and 100 mL of acetone R. Close the flask and
Loss on drying (2.2.32) : maximum 5.0 per cent, determined stir with a magnetic stirrer for 16 h. Add 30.0 mL of
on 1.000 g by drying in an oven at 105 °C for 3 h. 1 M sodium hydroxide with constant stirring. Close the
flask and stir with a magnetic stirrer for 6 min. Allow to
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
stand without stirring for 60 min. Resume stirring and
1.0 g.
add 100 mL of water R at 80 °C, washing down the sides
Microbial contamination of the flask, stir for 2 min and cool to room temperature.
TAMC : acceptance criterion 103 CFU/g (2.6.12). Titrate with 0.5 M hydrochloric acid, using 0.1 mL of
TYMC : acceptance criterion 102 CFU/g (2.6.12). phenolphthalein solution R as indicator. Add 0.5 mL of
0.5 M hydrochloric acid in excess, stir for 5 min and allow
Absence of Escherichia coli (2.6.13). to stand for 30 min. Titrate with 0.5 M sodium hydroxide,
Absence of Salmonella (2.6.13). until a persistent pink colour is obtained, stirring with a
magnetic stirrer. Calculate the net number of millimoles
STORAGE of 0.5 M sodium hydroxide consumed, taking the mean of
In an airtight container. 2 blank titrations into consideration.
FUNCTIONALITY-RELATED CHARACTERISTICS Calculate the percentage content of acetyl groups using the
following expression :
This section provides information on characteristics that are
recognised as being relevant control parameters for one or
more functions of the substance when used as an excipient (see

1822 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cellulose acetate phthalate

d = loss on drying as a percentage ; a magnetic stirrer for 30 min. Add 100 mL of hot water R at
m 80 °C, washing down the sides of the flask and stir for 2 min.
= mass of the substance to be examined, in grams ; Cool, centrifuge or filter the suspension and wash the residue
n = net number of millimoles of 0.5 M sodium with water R. Combine the filtrate and washings, adjust to
hydroxide consumed. pH 3 with dilute phosphoric acid R and dilute to 500.0 mL
with water R.
The following characteristics may be relevant for cellulose
acetate used as matrix former in prolonged-release tablets. Reference solution. Dissolve 0.200 g of glacial acetic acid R and
0.400 g of butyric acid R in water R, adjust to pH 3 with dilute
Apparent viscosity : see test above. phosphoric acid R and dilute to 500.0 mL with water R.
Acetyl groups : see test above. Column :
Molecular mass distribution (2.2.30). – size : l = 0.25 m, Ø = 4.6 mm ;
Particle-size distribution (2.9.31). – stationary phase : octadecylsilyl silica gel for
Powder flow (2.9.36). chromatography R (5 μm).
Mobile phase :
– mobile phase A : methanol R ;
07/2013:1406 – mobile phase B : phosphate buffer solution pH 3.0 R1 ;

CELLULOSE ACETATE BUTYRATE Time

(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V)
0 - 30 5 95
Cellulosi acetas butyras
30 - 35 5 → 20 95 → 80
DEFINITION
35 - 60 20 80
Partly or completely O-acetylated and O-butyrated cellulose.
60 - 61 5 95
Content :
– acetyl groups (C2H3O) : 2.0 per cent to 30.0 per cent (dried Flow rate : 1.2 mL/min.
substance) ; 90.0 per cent to 110.0 per cent of that stated on Detection : spectrophotometer at 210 nm.
the label (dried substance) ;
Injection : 20 μL.
– butyryl groups (C4H7O) : 16.0 per cent to 53.0 per cent
(dried substance) ; 90.0 per cent to 110.0 per cent of that Calculate the percentage content of acetic acid and butyric
stated on the label (dried substance). acid using the chromatograms obtained with the 2 solutions.
To calculate the percentage content of acetyl (C2H3O) and of
CHARACTERS butyryl (C4H7O) groups, multiply the percentage content of
Appearance : white, yellowish-white or greyish-white powder acetic acid and butyric acid by 0.717 and 0.807, respectively.
or granules, slightly hygroscopic. STORAGE
Solubility : practically insoluble in water, soluble in acetone, in In an airtight container.
formic acid and in a mixture of equal volumes of methanol
and methylene chloride, practically insoluble in ethanol LABELLING
(96 per cent). The label states the nominal percentage content of acetyl and
IDENTIFICATION butyryl groups.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : cellulose acetate butyrate CRS. 01/2012:0314
The intensity of the bands may vary according to the degree
of substitution. CELLULOSE ACETATE PHTHALATE
B. It complies with the limits of the assay.
TESTS Cellulosi acetas phthalas
Acidity. To 5.00 g in a 250 mL conical flask, add 150 mL [9004-38-0]
of carbon dioxide-free water R, insert the stopper, swirl the
suspension gently and allow to stand for 3 h. Filter, wash the DEFINITION
flask and the filter with carbon dioxide-free water R. Combine Partly O-acetylated and O-phthalylated cellulose.
the filtrate and washings. Add 0.1 mL of phenolphthalein
solution R1. Not more than 3.0 mL of 0.01 M sodium hydroxide Content :
is required to change the colour of the indicator. – phthaloyl groups (C8H5O3 ; Mr 149.1) : 30.0 per cent to
Heavy metals (2.4.8) : maximum 20 ppm. 36.0 per cent (anhydrous and acid-free substance) ;
1.0 g complies with test F. Prepare the reference solution using – acetyl groups (C2H3O ; Mr 43.04) : 21.5 per cent to 26.0 per
2 mL of lead standard solution (10 ppm Pb) R. cent (anhydrous and acid-free substance).
Loss on drying (2.2.32) : maximum 2.0 per cent, determined CHARACTERS
on 1.000 g by drying in an oven at 105 °C for 3 h. Appearance : white or almost white, free-flowing powder or
Total ash (2.4.16) : maximum 0.1 per cent. colourless flakes, hygroscopic.
Solubility : practically insoluble in water, freely soluble in
ASSAY acetone, soluble in diethylene glycol, practically insoluble in
Liquid chromatography (2.2.29). ethanol (96 per cent) and in methylene chloride. It dissolves
Test solution. To 1.000 g of the substance to be examined in in dilute solutions of alkali hydroxides.
a 500 mL conical flask, add 100 mL of acetone R and 10 mL
of water R. Close the flask and stir with a magnetic stirrer IDENTIFICATION
until dissolution is complete. Add 30.0 mL of 1 M sodium Infrared absorption spectrophotometry (2.2.24).
hydroxide with constant stirring. Close the flask and stir with Comparison: cellulose acetate phthalate CRS.

General Notices (1) apply to all monographs and other texts 1823
Cellulose, microcrystalline EUROPEAN PHARMACOPOEIA 8.0

TESTS FUNCTIONALITY-RELATED CHARACTERISTICS

Viscosity (2.2.9) : 45 mPa·s to 90 mPa·s, determined at This section provides information on characteristics that are
25 ± 0.2 °C. recognised as being relevant control parameters for one or
Dissolve 15 g, calculated with reference to the anhydrous more functions of the substance when used as an excipient (see
substance, in 85 g of a mixture of 1 part by weight of water R chapter 5.15). This section is a non-mandatory part of the
and 249 parts by weight of acetone R. monograph and it is not necessary to verify the characteristics
to demonstrate compliance. Control of these characteristics can
Free acid : maximum 3.0 per cent, calculated as phthalic acid however contribute to the quality of a medicinal product by
(anhydrous substance). improving the consistency of the manufacturing process and
Shake 3.0 g for 2 h with 100 mL of a 50 per cent V/V solution the performance of the medicinal product during use. Where
of methanol R and filter. Wash the flask and the filter with control methods are cited, they are recognised as being suitable
2 quantities, each of 10 mL, of a 50 per cent V/V solution for the purpose, but other methods can also be used. Wherever
of methanol R. Combine the filtrate and washings, add results for a particular characteristic are reported, the control
phenolphthalein solution R and titrate with 0.1 M sodium method must be indicated.
hydroxide until a faint pink colour is obtained. Carry out a The following characteristics may be relevant for cellulose
blank titration using 120 mL of a 50 per cent V/V solution acetate phthalate used as film former in gastro-resistant tablets
of methanol R. and capsules.
1 mL of 0.1 M sodium hydroxide is equivalent to 8.3 mg of free
acid, calculated as phthalic acid. Viscosity : see Tests.
Heavy metals (2.4.8) : maximum 10 ppm. Solubility of a film. Dissolve about 0.15 g in 1 mL of acetone R
and pour onto a clear glass plate. A film is formed. Take
2.0 g complies with test C. Prepare the reference solution using a piece of the film and place it in a flask containing 0.1 M
2 mL of lead standard solution (10 ppm Pb) R. hydrochloric acid. It does not dissolve. Then place the piece of
Water (2.5.12) : maximum 5.0 per cent, determined on 0.500 g. film in a flask containing phosphate buffer solution pH 6.8 R. It
Carry out the test using a mixture of 2 volumes of methylene dissolves.
chloride R and 3 volumes of anhydrous ethanol R. Phthaloyl groups : see Assay.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Acetyl groups : see Assay.
1.0 g.
ASSAY
Phthaloyl groups. Dissolve 1.000 g in 50 mL of a mixture 01/2009:0316
of 2 volumes of acetone R and 3 volumes of ethanol (96 per corrected 7.0
cent) R. Add about 0.1 mL of phenolphthalein solution R1
and titrate with 0.1 M sodium hydroxide. Carry out a blank CELLULOSE, MICROCRYSTALLINE
titration.
Calculate the percentage content of phthaloyl groups (P) using Cellulosum microcristallinum
the following expression :

a = percentage content of water (see Tests) ;

m = mass of the substance to be examined, in grams ;
n = volume of 0.1 M sodium hydroxide used, in
millilitres ;
S = percentage content of free acid (see Tests). C6nH10n+2O5n+1
Acetyl groups. To 0.100 g add 25.0 mL of 0.1 M sodium DEFINITION
hydroxide and heat on a water-bath under a reflux condenser
for 30 min. Cool, add about 0.1 mL of phenolphthalein Purified, partly depolymerised cellulose prepared by treating
solution R1 and titrate with 0.1 M hydrochloric acid. Carry out alpha-cellulose, obtained as a pulp from fibrous plant material,
a blank titration. with mineral acids.
Calculate the percentage content of acetyl groups using the CHARACTERS
following expression : Appearance : white or almost white, fine or granular powder.
Solubility : practically insoluble in water, in acetone, in
anhydrous ethanol, in toluene, in dilute acids and in a 50 g/L
solution of sodium hydroxide.
a = percentage content of water (see Tests) ;
IDENTIFICATION
m = mass of the substance to be examined, in grams ; A. Place about 10 mg on a watch-glass and disperse in 2 mL of
n1 = volume of 0.1 M hydrochloric acid used in the test, iodinated zinc chloride solution R. The substance becomes
in millilitres ; violet-blue.
n2 = volume of 0.1 M hydrochloric acid used in the B. The degree of polymerisation is not more than 350.
blank titration, in millilitres ; Transfer 1.300 g to a 125 mL conical flask. Add 25.0 mL of
P = percentage content of phthaloyl groups ; water R and 25.0 mL of cupriethylenediamine hydroxide
solution R. Immediately purge the solution with nitrogen R,
S = percentage content of free acid (see Tests). insert the stopper and shake until completely dissolved.
Transfer an appropriate volume of the solution to a suitable
STORAGE capillary viscometer (2.2.9). Equilibrate the solution at
In an airtight container. 25 ± 0.1 °C for at least 5 min. Record the flow time (t1) in

1824 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cellulose, microcrystalline

seconds between the 2 marks on the viscometer. Calculate TESTS

the kinematic viscosity (ν1) of the solution using the Solubility. Dissolve 50 mg in 10 mL of ammoniacal solution
following expression : of copper tetrammine R. It dissolves completely, leaving no
residue.
pH (2.2.3) : 5.0 to 7.5 for the supernatant.
where k1 is the viscometer constant. Shake 5 g with 40 mL of carbon dioxide-free water R for 20 min
and centrifuge.
Dilute a suitable volume of cupriethylenediamine hydroxide
solution R with an equal volume of water R and measure Conductivity (2.2.38). The conductivity of the test solution
the flow time (t2) using a suitable capillary viscometer. does not exceed the conductivity of the water by more than
Calculate the kinematic viscosity (ν2) of the solvent using 75 μS·cm− 1.
the following expression : Use as test solution the supernatant obtained in the test for
pH. Measure the conductivity of the supernatant after a stable
reading has been obtained and measure the conductivity of
the water used to prepare the test solution.
where k2 is the viscometer constant. Ether-soluble substances : maximum 0.05 per cent (5 mg)
Determine the relative viscosity (ηrel) of the substance to be for the difference between the weight of the residue and the
examined using the following expression : weight obtained from a blank determination.
Place 10.0 g in a chromatography column about 20 mm in
internal diameter and pass 50 mL of peroxide-free ether R
through the column. Evaporate the eluate to dryness. Dry the
Determine the intrinsic viscosity ([η]c) by interpolation, residue at 105 °C for 30 min, allow to cool in a desiccator and
using the intrinsic viscosity table (Table 0316.-1). weigh. Carry out a blank determination.
Calculate the degree of polymerisation (P) using the Water-soluble substances : maximum 0.25 per cent (12.5 mg)
following expression : for the difference between the mass of the residue and the
mass obtained from a blank determination.
Shake 5.0 g with 80 mL of water R for 10 min. Filter through
a filter paper with the aid of vacuum into a tared flask.
Evaporate to dryness on a water-bath avoiding charring. Dry
where m is the mass in grams of the substance to be at 105 °C for 1 h, allow to stand in a desiccator and weigh.
examined and b is the loss on drying as a percentage. Carry out a blank determination.
Table 0316.-1. – Intrinsic viscosity table
Intrinsic viscosity [η]c at different values of relative viscosity ηrel
[η]c
ηrel 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09

1.1 0.098 0.106 0.115 0.125 0.134 0.143 0.152 0.161 0.170 0.180
1.2 0.189 0.198 0.207 0.216 0.225 0.233 0.242 0.250 0.259 0.268
1.3 0.276 0.285 0.293 0.302 0.310 0.318 0.326 0.334 0.342 0.350
1.4 0.358 0.367 0.375 0.383 0.391 0.399 0.407 0.414 0.422 0.430
1.5 0.437 0.445 0.453 0.460 0.468 0.476 0.484 0.491 0.499 0.507
1.6 0.515 0.522 0.529 0.536 0.544 0.551 0.558 0.566 0.573 0.580
1.7 0.587 0.595 0.602 0.608 0.615 0.622 0.629 0.636 0.642 0.649
1.8 0.656 0.663 0.670 0.677 0.683 0.690 0.697 0.704 0.710 0.717
1.9 0.723 0.730 0.736 0.743 0.749 0.756 0.762 0.769 0.775 0.782

2.0 0.788 0.795 0.802 0.809 0.815 0.821 0.827 0.833 0.840 0.846
2.1 0.852 0.858 0.864 0.870 0.876 0.882 0.888 0.894 0.900 0.906
2.2 0.912 0.918 0.924 0.929 0.935 0.941 0.948 0.953 0.959 0.965
2.3 0.971 0.976 0.983 0.988 0.994 1.000 1.006 1.011 1.017 1.022
2.4 1.028 1.033 1.039 1.044 1.050 1.056 1.061 1.067 1.072 1.078
2.5 1.083 1.089 1.094 1.100 1.105 1.111 1.116 1.121 1.126 1.131
2.6 1.137 1.142 1.147 1.153 1.158 1.163 1.169 1.174 1.179 1.184
2.7 1.190 1.195 1.200 1.205 1.210 1.215 1.220 1.225 1.230 1.235
2.8 1.240 1.245 1.250 1.255 1.260 1.265 1.270 1.275 1.280 1.285
2.9 1.290 1.295 1.300 1.305 1.310 1.314 1.319 1.324 1.329 1.333

General Notices (1) apply to all monographs and other texts 1825
Cellulose, microcrystalline EUROPEAN PHARMACOPOEIA 8.0

Intrinsic viscosity [η]c at different values of relative viscosity ηrel

[η]c
ηrel 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
3.0 1.338 1.343 1.348 1.352 1.357 1.362 1.367 1.371 1.376 1.381
3.1 1.386 1.390 1.395 1.400 1.405 1.409 1.414 1.418 1.423 1.427
3.2 1.432 1.436 1.441 1.446 1.450 1.455 1.459 1.464 1.468 1.473
3.3 1.477 1.482 1.486 1.491 1.496 1.500 1.504 1.508 1.513 1.517
3.4 1.521 1.525 1.529 1.533 1.537 1.542 1.546 1.550 1.554 1.558
3.5 1.562 1.566 1.570 1.575 1.579 1.583 1.587 1.591 1.595 1.600
3.6 1.604 1.608 1.612 1.617 1.621 1.625 1.629 1.633 1.637 1.642
3.7 1.646 1.650 1.654 1.658 1.662 1.666 1.671 1.675 1.679 1.683
3.8 1.687 1.691 1.695 1.700 1.704 1.708 1.712 1.715 1.719 1.723
3.9 1.727 1.731 1.735 1.739 1.742 1.746 1.750 1.754 1.758 1.762

4.0 1.765 1.769 1.773 1.777 1.781 1.785 1.789 1.792 1.796 1.800
4.1 1.804 1.808 1.811 1.815 1.819 1.822 1.826 1.830 1.833 1.837
4.2 1.841 1.845 1.848 1.852 1.856 1.859 1.863 1.867 1.870 1.874
4.3 1.878 1.882 1.885 1.889 1.893 1.896 1.900 1.904 1.907 1.911
4.4 1.914 1.918 1.921 1.925 1.929 1.932 1.936 1.939 1.943 1.946
4.5 1.950 1.954 1.957 1.961 1.964 1.968 1.971 1.975 1.979 1.982
4.6 1.986 1.989 1.993 1.996 2.000 2.003 2.007 2.010 2.013 2.017
4.7 2.020 2.023 2.027 2.030 2.033 2.037 2.040 2.043 2.047 2.050
4.8 2.053 2.057 2.060 2.063 2.067 2.070 2.073 2.077 2.080 2.083
4.9 2.087 2.090 2.093 2.097 2.100 2.103 2.107 2.110 2.113 2.116

5.0 2.119 2.122 2.125 2.129 2.132 2.135 2.139 2.142 2.145 2.148
5.1 2.151 2.154 2.158 2.160 2.164 2.167 2.170 2.173 2.176 2.180
5.2 2.183 2.186 2.190 2.192 2.195 2.197 2.200 2.203 2.206 2.209
5.3 2.212 2.215 2.218 2.221 2.224 2.227 2.230 2.233 2.236 2.240
5.4 2.243 2.246 2.249 2.252 2.255 2.258 2.261 2.264 2.267 2.270
5.5 2.273 2.276 2.279 2.282 2.285 2.288 2.291 2.294 2.297 2.300
5.6 2.303 2.306 2.309 2.312 2.315 2.318 2.320 2.324 2.326 2.329
5.7 2.332 2.335 2.338 2.341 2.344 2.347 2.350 2.353 2.355 2.358
5.8 2.361 2.364 2.367 2.370 2.373 2.376 2.379 2.382 2.384 2.387
5.9 2.390 2.393 2.396 2.400 2.403 2.405 2.408 2.411 2.414 2.417

6.0 2.419 2.422 2.425 2.428 2.431 2.433 2.436 2.439 2.442 2.444
6.1 2.447 2.450 2.453 2.456 2.458 2.461 2.464 2.467 2.470 2.472
6.2 2.475 2.478 2.481 2.483 2.486 2.489 2.492 2.494 2.497 2.500
6.3 2.503 2.505 2.508 2.511 2.513 2.516 2.518 2.521 2.524 2.526
6.4 2.529 2.532 2.534 2.537 2.540 2.542 2.545 2.547 2.550 2.553
6.5 2.555 2.558 2.561 2.563 2.566 2.568 2.571 2.574 2.576 2.579
6.6 2.581 2.584 2.587 2.590 2.592 2.595 2.597 2.600 2.603 2.605
6.7 2.608 2.610 2.613 2.615 2.618 2.620 2.623 2.625 2.627 2.630
6.8 2.633 2.635 2.637 2.640 2.643 2.645 2.648 2.650 2.653 2.655
6.9 2.658 2.660 2.663 2.665 2.668 2.670 2.673 2.675 2.678 2.680

1826 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cellulose, microcrystalline

Intrinsic viscosity [η]c at different values of relative viscosity ηrel

[η]c
ηrel 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
7.0 2.683 2.685 2.687 2.690 2.693 2.695 2.698 2.700 2.702 2.705
7.1 2.707 2.710 2.712 2.714 2.717 2.719 2.721 2.724 2.726 2.729
7.2 2.731 2.733 2.736 2.738 2.740 2.743 2.745 2.748 2.750 2.752
7.3 2.755 2.757 2.760 2.762 2.764 2.767 2.769 2.771 2.774 2.776
7.4 2.779 2.781 2.783 2.786 2.788 2.790 2.793 2.795 2.798 2.800
7.5 2.802 2.805 2.807 2.809 2.812 2.814 2.816 2.819 2.821 2.823
7.6 2.826 2.828 2.830 2.833 2.835 2.837 2.840 2.842 2.844 2.847
7.7 2.849 2.851 2.854 2.856 2.858 2.860 2.863 2.865 2.868 2.870
7.8 2.873 2.875 2.877 2.879 2.881 2.884 2.887 2.889 2.891 2.893
7.9 2.895 2.898 2.900 2.902 2.905 2.907 2.909 2.911 2.913 2.915

8.0 2.918 2.920 2.922 2.924 2.926 2.928 2.931 2.933 2.935 2.937
8.1 2.939 2.942 2.944 2.946 2.948 2.950 2.952 2.955 2.957 2.959
8.2 2.961 2.963 2.966 2.968 2.970 2.972 2.974 2.976 2.979 2.981
8.3 2.983 2.985 2.987 2.990 2.992 2.994 2.996 2.998 3.000 3.002
8.4 3.004 3.006 3.008 3.010 3.012 3.015 3.017 3.019 3.021 3.023
8.5 3.025 3.027 3.029 3.031 3.033 3.035 3.037 3.040 3.042 3.044
8.6 3.046 3.048 3.050 3.052 3.054 3.056 3.058 3.060 3.062 3.064
8.7 3.067 3.069 3.071 3.073 3.075 3.077 3.079 3.081 3.083 3.085
8.8 3.087 3.089 3.092 3.094 3.096 3.098 3.100 3.102 3.104 3.106
8.9 3.108 3.110 3.112 3.114 3.116 3.118 3.120 3.122 3.124 3.126

9.0 3.128 3.130 3.132 3.134 3.136 3.138 3.140 3.142 3.144 3.146
9.1 3.148 3.150 3.152 3.154 3.156 3.158 3.160 3.162 3.164 3.166
9.2 3.168 3.170 3.172 3.174 3.176 3.178 3.180 3.182 3.184 3.186
9.3 3.188 3.190 3.192 3.194 3.196 3.198 3.200 3.202 3.204 3.206
9.4 3.208 3.210 3.212 3.214 3.215 3.217 3.219 3.221 3.223 3.225
9.5 3.227 3.229 3.231 3.233 3.235 3.237 3.239 3.241 3.242 3.244
9.6 3.246 3.248 3.250 3.252 3.254 3.256 3.258 3.260 3.262 3.264
9.7 3.266 3.268 3.269 3.271 3.273 3.275 3.277 3.279 3.281 3.283
9.8 3.285 3.287 3.289 3.291 3.293 3.295 3.297 3.298 3.300 3.302
9.9 3.304 3.305 3.307 3.309 3.311 3.313 3.316 3.318 3.320 3.321

Intrinsic viscosity [η]c at different values of relative viscosity ηrel

[η]c
ηrel 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
10 3.32 3.34 3.36 3.37 3.39 3.41 3.43 3.45 3.46 3.48
11 3.50 3.52 3.53 3.55 3.56 3.58 3.60 3.61 3.63 3.64
12 3.66 3.68 3.69 3.71 3.72 3.74 3.76 3.77 3.79 3.80
13 3.80 3.83 3.85 3.86 3.88 3.89 3.90 3.92 3.93 3.95
14 3.96 3.97 3.99 4.00 4.02 4.03 4.04 4.06 4.07 4.09
15 4.10 4.11 4.13 4.14 4.15 4.17 4.18 4.19 4.20 4.22
16 4.23 4.24 4.25 4.27 4.28 4.29 4.30 4.31 4.33 4.34
17 4.35 4.36 4.37 4.38 4.39 4.41 4.42 4.43 4.44 4.45
18 4.46 4.47 4.48 4.49 4.50 4.52 4.53 4.54 4.55 4.56
19 4.57 4.58 4.59 4.60 4.61 4.62 4.63 4.64 4.65 4.66

General Notices (1) apply to all monographs and other texts 1827
Cellulose, powdered EUROPEAN PHARMACOPOEIA 8.0

IDENTIFICATION
Heavy metals (2.4.8) : maximum 10 ppm. A. Place about 10 mg on a watch-glass and disperse in 2 mL of
2.0 g complies with test C. Prepare the reference solution using iodinated zinc chloride solution R. The substance becomes
2 mL of lead standard solution (10 ppm Pb) R. violet-blue.
B. The degree of polymerisation is greater than 440.
Loss on drying (2.2.32) : maximum 7.0 per cent, determined Transfer 0.250 g to a 125 mL conical flask. Add 25.0 mL of
on 1.000 g by drying in an oven at 105 °C for 3 h. water R and 25.0 mL of cupriethylenediamine hydroxide
solution R. Immediately purge the solution with nitrogen R,
insert the stopper and shake until completely dissolved.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Transfer an appropriate volume of the solution to a suitable
1.0 g. capillary viscometer (2.2.9). Equilibrate the solution at
25 ± 0.1 °C for at least 5 min. Record the flow time (t1) in
seconds between the 2 marks on the viscometer. Calculate
Microbial contamination
the kinematic viscosity (ν1) of the solution using the
TAMC : acceptance criterion 103 CFU/g (2.6.12). following expression :
2
TYMC : acceptance criterion 10 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Pseudomonas aeruginosa (2.6.13). where k1 is the viscometer constant.
Absence of Staphylococcus aureus (2.6.13). Dilute a suitable volume of cupriethylenediamine hydroxide
Absence of Salmonella (2.6.13). solution R with an equal volume of water R and measure
the flow time (t2) using a suitable capillary viscometer.
Calculate the kinematic viscosity (ν2) of the solvent using
FUNCTIONALITY-RELATED CHARACTERISTICS the following expression :
This section provides information on characteristics that are
recognised as being relevant control parameters for one or
more functions of the substance when used as an excipient (see
chapter 5.15). This section is a non-mandatory part of the where k2 is the viscometer constant.
monograph and it is not necessary to verify the characteristics Determine the relative viscosity (ηrel) of the substance to be
to demonstrate compliance. Control of these characteristics can examined using the following expression :
however contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process and
the performance of the medicinal product during use. Where Determine the intrinsic viscosity ([η]c) by interpolation,
control methods are cited, they are recognised as being suitable using the intrinsic viscosity table (Table 0315.-1).
for the purpose, but other methods can also be used. Wherever
results for a particular characteristic are reported, the control Calculate the degree of polymerisation (P) using the
method must be indicated. following expression :
The following characteristics may be relevant for
microcrystalline cellulose used as binder, diluent or disintegrant.
where m is the mass in grams of the substance to be
Particle-size distribution (2.9.31 or 2.9.38). examined and b is the loss on drying as a percentage.
Powder flow (2.9.36).
TESTS
01/2009:0315 Solubility. Dissolve 50 mg in 10 mL of ammoniacal solution
of copper tetrammine R. It dissolves completely, leaving no
CELLULOSE, POWDERED residue.
pH (2.2.3) : 5.0 to 7.5 for the supernatant.
Cellulosi pulvis Mix 10 g with 90 mL of carbon dioxide-free water R and allow
to stand with occasional stirring for 1 h.
Ether-soluble substances : maximum 0.15 per cent (15 mg)
for the difference between the mass of the residue and the
mass obtained from a blank determination.
Place 10.0 g in a chromatography column about 20 mm in
internal diameter and pass 50 mL of peroxide-free ether R
through the column. Evaporate the eluate to dryness in a
previously dried and tared evaporating dish, with the aid of
a current of air in a fume cupboard. After all the ether has
evaporated, dry the residue at 105 °C for 30 min, allow to cool
C6nH10n+2O5n+1 in a desiccator and weigh. Carry out a blank determination.
DEFINITION Water-soluble substances : maximum 1.5 per cent (15.0 mg)
Purified, mechanically disintegrated cellulose prepared by for the difference between the mass of the residue and the
processing alpha-cellulose obtained as a pulp from fibrous mass obtained from a blank determination.
plant material. Shake 6.0 g with 90 mL of carbon dioxide-free water R for
10 min. Filter with the aid of vacuum into a tared flask.
CHARACTERS Discard the first 10 mL of the filtrate and pass the filtrate
Appearance : white or almost white, fine or granular powder. through the same filter a second time, if necessary, to obtain
Solubility : practically insoluble in water, slightly soluble in a a clear filtrate. Evaporate a 15.0 mL portion of the filtrate to
50 g/L solution of sodium hydroxide, practically insoluble in dryness in a tared evaporating dish without charring. Dry at
acetone, in anhydrous ethanol, in toluene, in dilute acids and 105 °C for 1 h, allow to cool in a desiccator and weigh. Carry
in most organic solvents. out a blank determination.

1828 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cellulose, powdered

Heavy metals (2.4.8) : maximum 10 ppm. FUNCTIONALITY-RELATED CHARACTERISTICS

2.0 g complies with test C. Prepare the reference solution using This section provides information on characteristics that are
2 mL of lead standard solution (10 ppm Pb) R. recognised as being relevant control parameters for one or
more functions of the substance when used as an excipient (see
Loss on drying (2.2.32) : maximum 6.5 per cent, determined chapter 5.15). This section is a non-mandatory part of the
on 1.000 g by drying in an oven at 105 °C for 3 h. monograph and it is not necessary to verify the characteristics
Sulfated ash (2.4.14) : maximum 0.3 per cent (dried to demonstrate compliance. Control of these characteristics can
substance), determined on 1.0 g. however contribute to the quality of a medicinal product by
improving the consistency of the manufacturing process and
Microbial contamination the performance of the medicinal product during use. Where
TAMC : acceptance criterion 103 CFU/g (2.6.12). control methods are cited they are recognised as being suitable
2
for the purpose but other methods can also be used. Wherever
TYMC : acceptance criterion 10 CFU/g (2.6.12). results for a particular characteristic are reported, the control
Absence of Escherichia coli (2.6.13). method must be indicated.
The following characteristics may be relevant for powdered
Absence of Pseudomonas aeruginosa (2.6.13). cellulose used as diluent or disintegrant.
Absence of Staphylococcus aureus (2.6.13). Particle-size distribution (2.9.31 or 2.9.38).
Absence of Salmonella (2.6.13). Powder flow (2.9.36).
Table 0315.-1. – Intrinsic viscosity table
Intrinsic viscosity [η]c at different values of relative viscosity ηrel
[η]c
ηrel 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09

1.1 0.098 0.106 0.115 0.125 0.134 0.143 0.152 0.161 0.170 0.180
1.2 0.189 0.198 0.207 0.216 0.225 0.233 0.242 0.250 0.259 0.268
1.3 0.276 0.285 0.293 0.302 0.310 0.318 0.326 0.334 0.342 0.350
1.4 0.358 0.367 0.375 0.383 0.391 0.399 0.407 0.414 0.422 0.430
1.5 0.437 0.445 0.453 0.460 0.468 0.476 0.484 0.491 0.499 0.507
1.6 0.515 0.522 0.529 0.536 0.544 0.551 0.558 0.566 0.573 0.580
1.7 0.587 0.595 0.602 0.608 0.615 0.622 0.629 0.636 0.642 0.649
1.8 0.656 0.663 0.670 0.677 0.683 0.690 0.697 0.704 0.710 0.717
1.9 0.723 0.730 0.736 0.743 0.749 0.756 0.762 0.769 0.775 0.782

2.0 0.788 0.795 0.802 0.809 0.815 0.821 0.827 0.833 0.840 0.846
2.1 0.852 0.858 0.864 0.870 0.876 0.882 0.888 0.894 0.900 0.906
2.2 0.912 0.918 0.924 0.929 0.935 0.941 0.948 0.953 0.959 0.965
2.3 0.971 0.976 0.983 0.988 0.994 1.000 1.006 1.011 1.017 1.022
2.4 1.028 1.033 1.039 1.044 1.050 1.056 1.061 1.067 1.072 1.078
2.5 1.083 1.089 1.094 1.100 1.105 1.111 1.116 1.121 1.126 1.131
2.6 1.137 1.142 1.147 1.153 1.158 1.163 1.169 1.174 1.179 1.184
2.7 1.190 1.195 1.200 1.205 1.210 1.215 1.220 1.225 1.230 1.235
2.8 1.240 1.245 1.250 1.255 1.260 1.265 1.270 1.275 1.280 1.285
2.9 1.290 1.295 1.300 1.305 1.310 1.314 1.319 1.324 1.329 1.333

3.0 1.338 1.343 1.348 1.352 1.357 1.362 1.367 1.371 1.376 1.381
3.1 1.386 1.390 1.395 1.400 1.405 1.409 1.414 1.418 1.423 1.427
3.2 1.432 1.436 1.441 1.446 1.450 1.455 1.459 1.464 1.468 1.473
3.3 1.477 1.482 1.486 1.491 1.496 1.500 1.504 1.508 1.513 1.517
3.4 1.521 1.525 1.529 1.533 1.537 1.542 1.546 1.550 1.554 1.558
3.5 1.562 1.566 1.570 1.575 1.579 1.583 1.587 1.591 1.595 1.600
3.6 1.604 1.608 1.612 1.617 1.621 1.625 1.629 1.633 1.637 1.642
3.7 1.646 1.650 1.654 1.658 1.662 1.666 1.671 1.675 1.679 1.683
3.8 1.687 1.691 1.695 1.700 1.704 1.708 1.712 1.715 1.719 1.723
3.9 1.727 1.731 1.735 1.739 1.742 1.746 1.750 1.754 1.758 1.762

General Notices (1) apply to all monographs and other texts 1829
Cellulose, powdered EUROPEAN PHARMACOPOEIA 8.0

Intrinsic viscosity [η]c at different values of relative viscosity ηrel

[η]c
ηrel 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09

4.0 1.765 1.769 1.773 1.777 1.781 1.785 1.789 1.792 1.796 1.800
4.1 1.804 1.808 1.811 1.815 1.819 1.822 1.826 1.830 1.833 1.837
4.2 1.841 1.845 1.848 1.852 1.856 1.859 1.863 1.867 1.870 1.874
4.3 1.878 1.882 1.885 1.889 1.893 1.896 1.900 1.904 1.907 1.911
4.4 1.914 1.918 1.921 1.925 1.929 1.932 1.936 1.939 1.943 1.946
4.5 1.950 1.954 1.957 1.961 1.964 1.968 1.971 1.975 1.979 1.982
4.6 1.986 1.989 1.993 1.996 2.000 2.003 2.007 2.010 2.013 2.017
4.7 2.020 2.023 2.027 2.030 2.033 2.037 2.040 2.043 2.047 2.050
4.8 2.053 2.057 2.060 2.063 2.067 2.070 2.073 2.077 2.080 2.083
4.9 2.087 2.090 2.093 2.097 2.100 2.103 2.107 2.110 2.113 2.116

5.0 2.119 2.122 2.125 2.129 2.132 2.135 2.139 2.142 2.145 2.148
5.1 2.151 2.154 2.158 2.160 2.164 2.167 2.170 2.173 2.176 2.180
5.2 2.183 2.186 2.190 2.192 2.195 2.197 2.200 2.203 2.206 2.209
5.3 2.212 2.215 2.218 2.221 2.224 2.227 2.230 2.233 2.236 2.240
5.4 2.243 2.246 2.249 2.252 2.255 2.258 2.261 2.264 2.267 2.270
5.5 2.273 2.276 2.279 2.282 2.285 2.288 2.291 2.294 2.297 2.300
5.6 2.303 2.306 2.309 2.312 2.315 2.318 2.320 2.324 2.326 2.329
5.7 2.332 2.335 2.338 2.341 2.344 2.347 2.350 2.353 2.355 2.358
5.8 2.361 2.364 2.367 2.370 2.373 2.376 2.379 2.382 2.384 2.387
5.9 2.390 2.393 2.396 2.400 2.403 2.405 2.408 2.411 2.414 2.417

6.0 2.419 2.422 2.425 2.428 2.431 2.433 2.436 2.439 2.442 2.444
6.1 2.447 2.450 2.453 2.456 2.458 2.461 2.464 2.467 2.470 2.472
6.2 2.475 2.478 2.481 2.483 2.486 2.489 2.492 2.494 2.497 2.500
6.3 2.503 2.505 2.508 2.511 2.513 2.516 2.518 2.521 2.524 2.526
6.4 2.529 2.532 2.534 2.537 2.540 2.542 2.545 2.547 2.550 2.553
6.5 2.555 2.558 2.561 2.563 2.566 2.568 2.571 2.574 2.576 2.579
6.6 2.581 2.584 2.587 2.590 2.592 2.595 2.597 2.600 2.603 2.605
6.7 2.608 2.610 2.613 2.615 2.618 2.620 2.623 2.625 2.627 2.630
6.8 2.633 2.635 2.637 2.640 2.643 2.645 2.648 2.650 2.653 2.655
6.9 2.658 2.660 2.663 2.665 2.668 2.670 2.673 2.675 2.678 2.680

7.0 2.683 2.685 2.687 2.690 2.693 2.695 2.698 2.700 2.702 2.705
7.1 2.707 2.710 2.712 2.714 2.717 2.719 2.721 2.724 2.726 2.729
7.2 2.731 2.733 2.736 2.738 2.740 2.743 2.745 2.748 2.750 2.752
7.3 2.755 2.757 2.760 2.762 2.764 2.767 2.769 2.771 2.774 2.776
7.4 2.779 2.781 2.783 2.786 2.788 2.790 2.793 2.795 2.798 2.800
7.5 2.802 2.805 2.807 2.809 2.812 2.814 2.816 2.819 2.821 2.823
7.6 2.826 2.828 2.830 2.833 2.835 2.837 2.840 2.842 2.844 2.847
7.7 2.849 2.851 2.854 2.856 2.858 2.860 2.863 2.865 2.868 2.870
7.8 2.873 2.875 2.877 2.879 2.881 2.884 2.887 2.889 2.891 2.893
7.9 2.895 2.898 2.900 2.902 2.905 2.907 2.909 2.911 2.913 2.915

1830 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cetirizine dihydrochloride

Intrinsic viscosity [η]c at different values of relative viscosity ηrel

[η]c
ηrel 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
8.0 2.918 2.920 2.922 2.924 2.926 2.928 2.931 2.933 2.935 2.937
8.1 2.939 2.942 2.944 2.946 2.948 2.950 2.952 2.955 2.957 2.959
8.2 2.961 2.963 2.966 2.968 2.970 2.972 2.974 2.976 2.979 2.981
8.3 2.983 2.985 2.987 2.990 2.992 2.994 2.996 2.998 3.000 3.002
8.4 3.004 3.006 3.008 3.010 3.012 3.015 3.017 3.019 3.021 3.023
8.5 3.025 3.027 3.029 3.031 3.033 3.035 3.037 3.040 3.042 3.044
8.6 3.046 3.048 3.050 3.052 3.054 3.056 3.058 3.060 3.062 3.064
8.7 3.067 3.069 3.071 3.073 3.075 3.077 3.079 3.081 3.083 3.085
8.8 3.087 3.089 3.092 3.094 3.096 3.098 3.100 3.102 3.104 3.106
8.9 3.108 3.110 3.112 3.114 3.116 3.118 3.120 3.122 3.124 3.126

9.0 3.128 3.130 3.132 3.134 3.136 3.138 3.140 3.142 3.144 3.146
9.1 3.148 3.150 3.152 3.154 3.156 3.158 3.160 3.162 3.164 3.166
9.2 3.168 3.170 3.172 3.174 3.176 3.178 3.180 3.182 3.184 3.186
9.3 3.188 3.190 3.192 3.194 3.196 3.198 3.200 3.202 3.204 3.206
9.4 3.208 3.210 3.212 3.214 3.215 3.217 3.219 3.221 3.223 3.225
9.5 3.227 3.229 3.231 3.233 3.235 3.237 3.239 3.241 3.242 3.244
9.6 3.246 3.248 3.250 3.252 3.254 3.256 3.258 3.260 3.262 3.264
9.7 3.266 3.268 3.269 3.271 3.273 3.275 3.277 3.279 3.281 3.283
9.8 3.285 3.287 3.289 3.291 3.293 3.295 3.297 3.298 3.300 3.302
9.9 3.304 3.305 3.307 3.309 3.311 3.313 3.316 3.318 3.320 3.321

Intrinsic viscosity [η]c at different values of relative viscosity ηrel

[η]c
ηrel 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
10 3.32 3.34 3.36 3.37 3.39 3.41 3.43 3.45 3.46 3.48
11 3.50 3.52 3.53 3.55 3.56 3.58 3.60 3.61 3.63 3.64
12 3.66 3.68 3.69 3.71 3.72 3.74 3.76 3.77 3.79 3.80
13 3.80 3.83 3.85 3.86 3.88 3.89 3.90 3.92 3.93 3.95
14 3.96 3.97 3.99 4.00 4.02 4.03 4.04 4.06 4.07 4.09
15 4.10 4.11 4.13 4.14 4.15 4.17 4.18 4.19 4.20 4.22
16 4.23 4.24 4.25 4.27 4.28 4.29 4.30 4.31 4.33 4.34
17 4.35 4.36 4.37 4.38 4.39 4.41 4.42 4.43 4.44 4.45
18 4.46 4.47 4.48 4.49 4.50 4.52 4.53 4.54 4.55 4.56
19 4.57 4.58 4.59 4.60 4.61 4.62 4.63 4.64 4.65 4.66

04/2013:1084 DEFINITION
(RS)-2-[2-[4-[(4-Chlorophenyl)phenylmethyl]piperazin-1-
CETIRIZINE DIHYDROCHLORIDE yl]ethoxy]acetic acid dihydrochloride.
Content : 99.0 per cent to 101.0 per cent (dried substance).
Cetirizini dihydrochloridum CHARACTERS
Appearance : white or almost white powder.
Solubility : freely soluble in water, practically insoluble in
acetone and in methylene chloride.
IDENTIFICATION
First identification : B, D.
Second identification : A, C, D.
C21H27Cl3N2O3 Mr 461.8 A. Ultraviolet and visible absorption spectrophotometry
[83881-52-1] (2.2.25).

General Notices (1) apply to all monographs and other texts 1831
Cetirizine dihydrochloride EUROPEAN PHARMACOPOEIA 8.0

Test solution. Dissolve 20.0 mg in 50 mL of a 10.3 g/L Identification of impurities : use the chromatogram
solution of hydrochloric acid R and dilute to 100.0 mL with supplied with cetirizine for peak identification CRS and
the same acid. Dilute 10.0 mL of this solution to 100.0 mL the chromatogram obtained with reference solution (c) to
with a 10.3 g/L solution of hydrochloric acid R. identify the peaks due to impurities B, C, D, E and F ; use the
Spectral range : 210-350 nm. chromatogram obtained with reference solution (a) to identify
Absorption maximum : at 231 nm. the peak due to impurity A.
Specific absorbance at the absorption maximum : 359 to 381. Relative retention with reference to cetirizine (retention
time = about 9 min) : impurity D = about 0.6 ;
B. Infrared absorption spectrophotometry (2.2.24). impurity B = about 0.8 ; impurity C = about 0.9 ;
Comparison : cetirizine dihydrochloride CRS. impurity E = about 1.2 ; impurity F = about 1.37 ;
C. Thin-layer chromatography (2.2.27). impurity A = about 1.42.
Test solution. Dissolve 10 mg of the substance to be System suitability : reference solution (c) :
examined in water R and dilute to 5 mL with the same – peak-to-valley ratio : minimum 5, where Hp = height
solvent. above the baseline of the peak due to impurity C and
Reference solution (a). Dissolve 10 mg of cetirizine Hv = height above the baseline of the lowest point of the
dihydrochloride CRS in water R and dilute to 5 mL with curve separating this peak from the peak due to cetirizine.
the same solvent. Limits :
Reference solution (b). Dissolve 10 mg of chlorphenamine – correction factors : for the calculation of content, multiply
maleate CRS in water R and dilute to 5 mL with the same the peak areas of the following impurities by the
solvent. Mix 1 mL of the solution and 1 mL of reference corresponding correction factor : impurity A = 0.7 ;
solution (a). impurity C = 1.9 ; impurity D = 0.6 ; impurity E = 1.3 ;
Plate : TLC silica gel GF254 plate R. impurity F = 1.9 ;
Mobile phase : ammonia R, methanol R, methylene – impurities A, B, C, D, E, F : for each impurity, not more
chloride R (1:10:90 V/V/V). than 1.5 times the area of the principal peak in the
Application : 5 μL. chromatogram obtained with reference solution (b)
Development : over 2/3 of the plate. (0.15 per cent);
Drying : in a current of cold air. – unspecified impurities : for each impurity, not more than the
Detection : examine in ultraviolet light at 254 nm. area of the principal peak in the chromatogram obtained
with reference solution (b) (0.10 per cent) ;
System suitability: reference solution (b) :
– the chromatogram shows 2 clearly separated spots. – total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
Results : the principal spot in the chromatogram obtained (0.3 per cent) ;
with the test solution is similar in position and size to
the principal spot in the chromatogram obtained with – disregard limit : 0.5 times the area of the principal peak in
reference solution (a). the chromatogram obtained with reference solution (b)
(0.05 per cent).
D. It gives reaction (a) of chlorides (2.3.1).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
TESTS on 1.000 g by drying in an oven at 105 °C.
Solution S. Dissolve 1.0 g in carbon dioxide-free water R and Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
dilute to 20 mL with the same solvent. 1.0 g.
Appearance of solution. Solution S is clear (2.2.1) and ASSAY
not more intensely coloured than reference solution BY7
(2.2.2, Method II). Dissolve 0.100 g in 70 mL of a mixture of 30 volumes of
water R and 70 volumes of acetone R. Titrate with 0.1 M
pH (2.2.3): 1.2 to 1.8 for solution S. sodium hydroxide to the 2nd point of inflexion. Determine
Related substances. Liquid chromatography (2.2.29). the end-point potentiometrically (2.2.20). Carry out a blank
Test solution. Dissolve 20 mg of the substance to be examined titration.
in the mobile phase and dilute to 100.0 mL with the mobile 1 mL of 0.1 M sodium hydroxide is equivalent to 15.39 mg
phase. of C21H27Cl3N2O3.
Reference solution (a). Dissolve 2 mg of cetirizine
dihydrochloride CRS and 2 mg of cetirizine impurity A CRS in STORAGE
the mobile phase and dilute to 50.0 mL with the mobile phase. Protected from light.
Dilute 1.0 mL of the solution to 100.0 mL with the mobile
IMPURITIES
phase.
Specified impurities : A, B, C, D, E, F.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution Other detectable impurities (the following substances would,
to 10.0 mL with the mobile phase. if present at a sufficient level, be detected by one or other of
Reference solution (c). Dissolve the contents of a vial of the tests in the monograph. They are limited by the general
cetirizine for peak identification CRS (containing impurities B, acceptance criterion for other/unspecified impurities and/or
C, D, E and F) in 5.0 mL of the mobile phase. by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
Column : impurities for demonstration of compliance. See also 5.10.
– size : l = 0.25 m, Ø = 4.6 mm ; Control of impurities in substances for pharmaceutical use) : G.
– stationary phase : silica gel for chromatography R (5 μm).
Mobile phase : dilute sulfuric acid R, water R, acetonitrile R
(0.4:6.6:93 V/V/V).
Flow rate : 1 mL/min.
Detection : spectrophotometer at 230 nm.
Injection : 20 μL.
Run time : 3 times the retention time of cetirizine. A. (RS)-1-[(4-chlorophenyl)phenylmethyl]piperazine,

1832 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cetostearyl alcohol

Content :
– stearyl alcohol : minimum 40.0 per cent,
– sum of the contents of stearyl alcohol and cetyl alcohol :
minimum 90.0 per cent.
CHARACTERS
B. (RS)-2-[4-[(4-chlorophenyl)phenylmethyl]piperazin-1- Appearance : white or pale yellow, wax-like mass, plates, flakes
yl]acetic acid, or granules.
Solubility : practically insoluble in water, soluble in ethanol
(96 per cent) and in light petroleum. When melted, it is
miscible with fatty oils, with liquid paraffin and with melted
wool fat.
IDENTIFICATION
Examine the chromatograms obtained in the assay.
C. (RS)-2-[2-[4-[(2-chlorophenyl)phenylmethyl]piperazin-1- Results : the 2 principal peaks in the chromatogram obtained
yl]ethoxy]acetic acid, with the test solution are similar in retention time to the
principal peaks in the chromatogram obtained with the
reference solution.
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution B6 (2.2.2,
Method II).
Dissolve 0.50 g in 20 mL of boiling ethanol (96 per cent) R.
Allow to cool.
Melting point (2.2.14) : 49 °C to 56 °C.
D. 1,4-bis[(4-chlorophenyl)phenylmethyl]piperazine, Acid value (2.5.1) : maximum 1.0.
Hydroxyl value (2.5.3, Method A) : 208 to 228.
Iodine value (2.5.4, Method A) : maximum 2.0.
Dissolve 2.00 g in methylene chloride R and dilute to 25 mL
with the same solvent.
Saponification value (2.5.6) : maximum 2.0.
ASSAY
E. (RS)-2-[2-[2-[4-[(4-chlorophenyl)phenylmethyl]piperazin-
Gas chromatography (2.2.28) : use the normalisation
1-yl]ethoxy]ethoxy]acetic acid (ethoxycetirizine),
procedure.
Test solution. Dissolve 0.100 g of the substance to be examined
in ethanol (96 per cent) R and dilute to 10.0 mL with the same
solvent.
Reference solution. Dissolve 60 mg of cetyl alcohol CRS and
40 mg of stearyl alcohol CRS in ethanol (96 per cent) R and
dilute to 10 mL with the same solvent. Dilute 1 mL of this
solution to 10 mL with ethanol (96 per cent) R.
F. 2-[2-[4-(diphenylmethyl)piperazin-1-yl]ethoxy]acetic acid, Column :
– size : l = 30 m, Ø = 0.32 mm,
– stationary phase : poly(dimethyl)siloxane R (1 μm).
Carrier gas : helium for chromatography R.
Flow rate : 1 mL/min.
Split ratio : 1:100.
Temperature :
G. (RS)-2-[4-[(4-chlorophenyl)phenylmethyl]piperazin-1-
yl]ethan-1-ol. Time Temperature
(min) (°C)
Column 0 - 20 150 → 250
20 - 40 250
01/2008:0702
Injection port 250

CETOSTEARYL ALCOHOL Detector 250

Detection : flame ionisation.

Alcohol cetylicus et stearylicus Injection : 1 μL.
DEFINITION System suitability : reference solution :
Mixture of solid aliphatic alcohols, mainly octadecan-1-ol – resolution : minimum 5.0 between the peaks due to cetyl
(stearyl alcohol ; C18H38O ; Mr 270.5) and hexadecan-1-ol (cetyl alcohol and stearyl alcohol.
alcohol ; C16H34O ; Mr 242.4), of animal or vegetable origin. Calculate the percentage contents of C16H34O and C18H38O.

General Notices (1) apply to all monographs and other texts 1833
Cetostearyl alcohol (type A), emulsifying EUROPEAN PHARMACOPOEIA 8.0

04/2011:0801 B. Examine the chromatograms obtained in the assay of

cetostearyl alcohol.
CETOSTEARYL ALCOHOL (TYPE A), Results : the 2 principal peaks in the chromatogram
obtained with the test solution are similar in retention time
EMULSIFYING to the 2 principal peaks in the chromatogram obtained
with the reference solution.
Alcohol cetylicus et stearylicus C. It gives a yellow colour to a non-luminous flame.
emulsificans A D. To 0.3 g add 20 mL of anhydrous ethanol R and heat to
boiling on a water-bath with shaking. Filter the mixture
DEFINITION immediately, evaporate to dryness and take up the residue
in 7 mL of water R. To 1 mL of the solution add 0.1 mL of a
Mixture of cetostearyl alcohol and sodium cetostearyl sulfate. 1 g/L solution of methylene blue R, 2 mL of dilute sulfuric
A suitable buffer may be added. acid R and 2 mL of methylene chloride R and shake. A blue
Content : colour develops in the lower layer.
– cetostearyl alcohol : minimum 80.0 per cent (anhydrous
substance) ; TESTS
– sodium cetostearyl sulfate : minimum 7.0 per cent Acid value (2.5.1) : maximum 2.0.
(anhydrous substance). Iodine value (2.5.4, Method A) : maximum 3.0.
Dissolve 2.00 g in 25 mL of methylene chloride R.
CHARACTERS
Saponification value (2.5.6) : maximum 2.0.
Appearance : white or pale yellow, waxy mass, plates, flakes or
granules. Water (2.5.12) : maximum 3.0 per cent, determined on 2.50 g.
Solubility : soluble in hot water giving an opalescent solution,
practically insoluble in cold water, slightly soluble in ethanol ASSAY
(96 per cent). Cetostearyl alcohol. Gas chromatography (2.2.28).
Internal standard solution. Dissolve 0.200 g of
IDENTIFICATION 1-nonadecanol CRS in anhydrous ethanol R and dilute to
First identification : B, C, D. 100.0 mL with the same solvent.
Second identification : A, C. Test solution. Dissolve 0.200 g of the substance to be examined
A. Thin-layer chromatography (2.2.27). in 25.0 mL of the internal standard solution. Add 25 mL
of water R and shake with 4 quantities, each of 25 mL, of
Test solution (a). Dissolve 0.1 g of the substance to pentane R, adding sodium chloride R, if necessary, to facilitate
be examined in 10 mL of trimethylpentane R, heating the separation of the layers. Combine the upper layers,
on a water-bath. Shake with 2 mL of ethanol (70 per wash with 2 quantities, each of 30 mL, of water R, dry over
cent V/V) R and allow to separate. Use the lower layer as anhydrous sodium sulfate R and filter.
test solution (b). Dilute 1 mL of the upper layer to 8 mL
with trimethylpentane R. Reference solution. Dissolve 0.100 g of cetyl alcohol CRS and
0.100 g of stearyl alcohol CRS in 25.0 mL of the internal
Test solution (b). Use the lower layer obtained in the standard solution. Add 25 mL of water R and shake with
preparation of test solution (a). 4 quantities, each of 25 mL, of pentane R, adding sodium
Reference solution (a). Dissolve 24 mg of cetyl chloride R, if necessary, to facilitate the separation of the
alcohol CRS and 16 mg of stearyl alcohol CRS in 10 mL of layers. Combine the upper layers, wash with 2 quantities, each
trimethylpentane R. of 30 mL, of water R, dry over anhydrous sodium sulfate R
and filter.
Reference solution (b). Dissolve 20 mg of sodium cetostearyl
sulfate R in 10 mL of ethanol (70 per cent V/V) R, heating Column :
on a water-bath. – material : fused silica ;
Plate : TLC silanised silica gel plate R. – size : l = 25 m, Ø = 0.25 mm ;
Mobile phase : water R, acetone R, methanol R – stationary phase : poly(dimethyl)siloxane R (film thickness
(20:40:40 V/V/V). 0.25 μm).
Application : 2 μL. Carrier gas : helium for chromatography R.
Development : over 2/3 of the plate. Flow rate : 1 mL/min.
Drying : in air. Split ratio : 1:100.
Detection : spray with a 50 g/L solution of phosphomolybdic Temperature :
acid R in ethanol (96 per cent) R ; heat at 120 °C until spots
appear (about 3 h). Time Temperature
Results : (min) (°C)
Column 0 - 20 150 → 250
– the 2 principal spots in the chromatogram obtained with
test solution (a) are similar in position and colour to Injection port 250
the principal spots in the chromatogram obtained with Detector 250
reference solution (a) ;
– 2 of the spots in the chromatogram obtained with test Detection : flame ionisation.
solution (b) are similar in position and colour to the
Injection : 1 μL.
principal spots in the chromatogram obtained with
reference solution (b). Elution order : cetyl alcohol, stearyl alcohol, 1-nonadecanol.

1834 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cetostearyl alcohol (type B), emulsifying

Calculate the percentage content of cetyl alcohol and of stearyl A. Thin-layer chromatography (2.2.27).
alcohol in the substance to be examined using the following Test solution (a). Dissolve 0.1 g of the substance to
expression and taking into account the declared content of the be examined in 10 mL of trimethylpentane R, heating
chemical reference substances : on a water-bath. Shake with 2 mL of ethanol (70 per
cent V/V) R and allow to separate. Use the lower layer as
test solution (b). Dilute 1 mL of the upper layer to 8 mL
with trimethylpentane R.
Ax = area of the peak due to cetyl alcohol or stearyl Test solution (b). Use the lower layer obtained in the
alcohol in the chromatogram obtained with the preparation of test solution (a).
test solution ; Reference solution (a). Dissolve 24 mg of cetyl
Ax,y = area of the peak due to cetyl alcohol CRS or stearyl alcohol CRS and 16 mg of stearyl alcohol CRS in 10 mL of
alcohol CRS in the chromatogram obtained with trimethylpentane R.
the reference solution ; Reference solution (b). Dissolve 20 mg of sodium
A1 = area of the peak due to the internal standard in the laurilsulfate CRS in 10 mL of ethanol (70 per cent V/V) R,
chromatogram obtained with the test solution ; heating on a water-bath.
Plate : TLC silanised silica gel plate R.
A2 = area of the peak due to the internal standard in
the chromatogram obtained with the reference Mobile phase : water R, acetone R, methanol R
solution ; (20:40:40 V/V/V).
m = mass of the substance to be examined in the test Application : 2 μL.
solution, in milligrams ; Development : over 2/3 of the plate.
mx,y = mass of cetyl alcohol CRS or stearyl alcohol CRS in Drying : in air.
the reference solution, in milligrams. Detection : spray with a 50 g/L solution of phosphomolybdic
The percentage content of cetostearyl alcohol corresponds to acid R in ethanol (96 per cent) R ; heat at 120 °C until spots
the sum of the percentage contents of cetyl alcohol and stearyl appear (about 3 h).
alcohol. Results :
Sodium cetostearyl sulfate. Disperse 0.300 g in 25 mL of – the 2 principal spots in the chromatogram obtained with
methylene chloride R. Add 50 mL of water R and 10 mL of test solution (a) are similar in position and colour to
dimidium bromide-sulfan blue mixed solution R. Titrate with the principal spots in the chromatogram obtained with
0.004 M benzethonium chloride, using sonication, heating, and reference solution (a) ;
allowing the layers to separate before each addition, until the – 1 of the spots in the chromatogram obtained with test
colour of the lower layer changes from pink to grey. solution (b) is similar in position and colour to the
1 mL of 0.004 M benzethonium chloride is equivalent to principal spot in the chromatogram obtained with
1.434 mg of sodium cetostearyl sulfate. reference solution (b).
B. Examine the chromatograms obtained in the assay of
LABELLING cetostearyl alcohol.
The label states, where applicable, the name and concentration Results : the 2 principal peaks in the chromatogram
of any added buffer. obtained with the test solution are similar in retention time
to the 2 principal peaks in the chromatogram obtained
with the reference solution.
04/2011:0802 C. It gives a yellow colour to a non-luminous flame.
D. To 0.3 g add 20 mL of anhydrous ethanol R and heat to
CETOSTEARYL ALCOHOL (TYPE B), boiling on a water-bath with shaking. Filter the mixture
immediately, evaporate to dryness and take up the residue
EMULSIFYING in 7 mL of water R. To 1 mL of the solution add 0.1 mL of a
1 g/L solution of methylene blue R, 2 mL of dilute sulfuric
Alcohol cetylicus et stearylicus acid R and 2 mL of methylene chloride R and shake. A blue
colour develops in the lower layer.
emulsificans B
TESTS
DEFINITION
Acid value (2.5.1) : maximum 2.0.
Mixture of cetostearyl alcohol and sodium laurilsulfate. A
suitable buffer may be added. Iodine value (2.5.4, Method A) : maximum 3.0.
Content : Dissolve 2.00 g in 25 mL of methylene chloride R.
– cetostearyl alcohol : minimum 80.0 per cent (anhydrous Saponification value (2.5.6) : maximum 2.0.
substance) ; Water (2.5.12) : maximum 3.0 per cent, determined on 2.50 g.
– sodium laurilsulfate : minimum 7.0 per cent (anhydrous
substance). ASSAY
Cetostearyl alcohol. Gas chromatography (2.2.28).
CHARACTERS
Internal standard solution. Dissolve 0.200 g of
Appearance : white or pale yellow, waxy mass, plates, flakes or 1-nonadecanol CRS in anhydrous ethanol R and dilute to
granules. 100.0 mL with the same solvent.
Solubility : soluble in hot water giving an opalescent solution, Test solution. Dissolve 0.200 g of the substance to be examined
practically insoluble in cold water, slightly soluble in ethanol in 25.0 mL of the internal standard solution. Add 25 mL
(96 per cent). of water R and shake with 4 quantities, each of 25 mL, of
IDENTIFICATION pentane R, adding sodium chloride R, if necessary, to facilitate
the separation of the layers. Combine the upper layers,
First identification : B, C, D. wash with 2 quantities, each of 30 mL, of water R, dry over
Second identification : A, C. anhydrous sodium sulfate R and filter.

General Notices (1) apply to all monographs and other texts 1835
Cetostearyl isononanoate EUROPEAN PHARMACOPOEIA 8.0

Reference solution. Dissolve 0.100 g of cetyl alcohol CRS and 01/2008:1085

0.100 g of stearyl alcohol CRS in 25.0 mL of the internal
standard solution. Add 25 mL of water R and shake with CETOSTEARYL ISONONANOATE
4 quantities, each of 25 mL, of pentane R, adding sodium
chloride R, if necessary, to facilitate the separation of the Cetostearylis isononanoas
layers. Combine the upper layers, wash with 2 quantities, each
of 30 mL, of water R, dry over anhydrous sodium sulfate R DEFINITION
and filter. Mixture of esters of cetostearyl alcohol with isononanoic acid,
Column : mainly 3,5,5-trimethylhexanoic acid.
– material : fused silica ; CHARACTERS
– size : l = 25 m, Ø = 0.25 mm ; Appearance : clear, colourless or slightly yellowish liquid.
– stationary phase : poly(dimethyl)siloxane R (film thickness Solubility : practically insoluble in water, soluble in ethanol
0.25 μm). (96 per cent) and in light petroleum, miscible with fatty oils
Carrier gas : helium for chromatography R. and with liquid paraffins.
Viscosity : 15 mPa·s to 30 mPa·s.
Flow rate : 1 mL/min.
Relative density : 0.85 to 0.86.
Split ratio : 1:100.
Refractive index : 1.44 to 1.45.
Temperature :
IDENTIFICATION
Time Temperature
A. On cooling, turbidity occurs below 15 °C.
(min) (°C)
Column 0 - 20 150 → 250
B. Saponification value (see Tests).
C. Infrared absorption spectrophotometry (2.2.24).
Injection port 250
Comparison: Ph. Eur. reference spectrum of cetostearyl
Detector 250 isononanoate.

Detection : flame ionisation. TESTS

Injection : 1 μL. Appearance. The substance to be examined is clear (2.2.1)
and not more intensely coloured than reference solution Y6
Elution order : cetyl alcohol, stearyl alcohol, 1-nonadecanol. (2.2.2, Method I).
Calculate the percentage content of cetyl alcohol and of stearyl Acid value (2.5.1) : maximum 1.0, determined on 5.0 g.
alcohol in the substance to be examined using the following
expression and taking into account the declared content of the Hydroxyl value (2.5.3, Method A) : maximum 5.0.
chemical reference substances : Iodine value (2.5.4, Method A) : maximum 1.0.
Saponification value (2.5.6) : 135 to 148, determined on 1.0 g.
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test D. Prepare the reference solution
Ax = area of the peak due to cetyl alcohol or stearyl using 2 mL of lead standard solution (10 ppm Pb) R.
alcohol in the chromatogram obtained with the Water (2.5.12) : maximum 0.2 per cent, determined on 10.0 g.
test solution ;
Total ash (2.4.16) : maximum 0.2 per cent, determined on
Ax,y = area of the peak due to cetyl alcohol CRS or stearyl
2.0 g.
alcohol CRS in the chromatogram obtained with
the reference solution ; 01/2008:0378
A1 = area of the peak due to the internal standard in the corrected 6.0
chromatogram obtained with the test solution ;
A2 = area of the peak due to the internal standard in CETRIMIDE
the chromatogram obtained with the reference
solution ; Cetrimidum
m = mass of the substance to be examined in the test
solution, in milligrams ;
mx,y = mass of cetyl alcohol CRS or stearyl alcohol CRS in
the reference solution, in milligrams.
DEFINITION
The percentage content of cetostearyl alcohol corresponds to Cetrimide consists of trimethyltetradecylammonium
the sum of the percentage contents of cetyl alcohol and stearyl bromide and may contain smaller amounts of dodecyl- and
alcohol. hexadecyl-trimethylammonium bromides.
Sodium laurilsulfate. Disperse 0.300 g in 25 mL of methylene Content : 96.0 per cent to 101.0 per cent of alkyltrimethyl-
chloride R. Add 50 mL of water R and 10 mL of dimidium ammonium bromides, calculated as C17H38BrN (Mr 336.4)
bromide-sulfan blue mixed solution R. Titrate with 0.004 M (dried substance).
benzethonium chloride, using sonication, heating, and allowing CHARACTERS
the layers to separate before each addition, until the colour of
the lower layer changes from pink to grey. Appearance : white or almost white, voluminous, free-flowing
powder.
1 mL of 0.004 M benzethonium chloride is equivalent to Solubility : freely soluble in water and in alcohol.
1.154 mg of sodium laurilsulfate.
IDENTIFICATION
LABELLING A. Dissolve 0.25 g in alcohol R and dilute to 25.0 mL with the
The label states, where applicable, the name and concentration same solvent. At wavelengths from 260 nm to 280 nm, the
of any added buffer. absorbance (2.2.25) of the solution has a maximum of 0.05.

1836 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cetyl alcohol

B. Dissolve about 5 mg in 5 mL of buffer solution pH 8.0 R. 1 mL of 0.05 M potassium iodate is equivalent to 33.64 mg
Add about 10 mg of potassium ferricyanide R. A yellow of C17H38BrN.
precipitate is formed. Prepare a blank in the same manner
but omitting the substance to be examined : a yellow
solution is observed but no precipitate is formed.
01/2008:0540
C. Solution S (see Tests) froths copiously when shaken.
D. Thin-layer chromatography (2.2.27). CETYL ALCOHOL
Test solution. Dissolve 0.10 g of the substance to be
examined in water R and dilute to 5 mL with the same Alcohol cetylicus
solvent.
Reference solution. Dissolve 0.10 g of trimethyltetradecyl- DEFINITION
ammonium bromide CRS in water R and dilute to 5 mL Mixture of solid alcohols, mainly hexadecan-1-ol (C16H34O ;
with the same solvent. Mr 242.4), of animal or vegetable origin.
Plate : TLC silanised silica gel F254 plate R. Content : minimum 95.0 per cent of C16H34O.
Mobile phase : acetone R, 270 g/L solution of sodium CHARACTERS
acetate R, methanol R (20:35:45 V/V/V).
Appearance : white or almost white, unctuous mass, powder,
Application : 1 μL. flakes or granules.
Development : over a path of 12 cm. Solubility : practically insoluble in water, freely soluble or
Drying : in a current of hot air. sparingly soluble in ethanol (96 per cent). When melted, it is
miscible with vegetable and animal oils, with liquid paraffin
Detection : allow to cool ; expose the plate to iodine vapour and with melted wool fat.
and examine in daylight.
IDENTIFICATION
Result : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size Examine the chromatograms obtained in the assay.
to the principal spot in the chromatogram obtained with Results : the principal peak in the chromatogram obtained
the reference solution. with the test solution is similar in retention time to the
E. It gives reaction (a) of bromides (2.3.1). principal peak in the chromatogram obtained with reference
solution (a).
TESTS TESTS
Solution S. Dissolve 2.0 g in carbon dioxide-free water R and Appearance of solution. The solution is clear (2.2.1) and not
dilute to 100 mL with the same solvent. more intensely coloured than reference solution B6 (2.2.2,
Appearance of solution. Solution S is clear (2.2.1) and Method II).
colourless (2.2.2, Method II). Dissolve 0.50 g in 20 mL of boiling ethanol (96 per cent) R.
Acidity or alkalinity. To 50 mL of solution S add 0.1 mL of Allow to cool.
bromocresol purple solution R. Not more than 0.1 mL of 0.1 M Melting point (2.2.14) : 46 °C to 52 °C.
hydrochloric acid or 0.1 M sodium hydroxide is required to
Acid value (2.5.1) : maximum 1.0.
change the colour of the indicator.
Hydroxyl value (2.5.3, Method A) : 218 to 238.
Amines and amine salts. Dissolve 5.0 g in 30 mL of a mixture
of 1 volume of 1 M hydrochloric acid and 99 volumes of Iodine value (2.5.4, Method A) : maximum 2.0.
methanol R and add 100 mL of 2-propanol R. Pass a stream Dissolve 2.00 g in methylene chloride R and dilute to 25 mL
of nitrogen R slowly through the solution. Gradually add with the same solvent.
15.0 mL of 0.1 M tetrabutylammonium hydroxide and record
Saponification value (2.5.6) : maximum 2.0.
the potentiometric titration curve (2.2.20). If the curve shows
2 points of inflexion, the volume of titrant added between the ASSAY
2 points is not greater than 2.0 mL.
Gas chromatography (2.2.28) : use the normalisation
Loss on drying (2.2.32) : maximum 2.0 per cent, determined procedure.
on 1.000 g by drying in an oven at 105 °C for 2 h. Test solution. Dissolve 0.100 g of the substance to be examined
Sulfated ash (2.4.14) : maximum 0.5 per cent, determined on in ethanol (96 per cent) R and dilute to 10.0 mL with the same
1.0 g. solvent.
Reference solution (a). Dissolve 50 mg of cetyl alcohol CRS
ASSAY in ethanol (96 per cent) R and dilute to 5 mL with the same
Dissolve 2.000 g in water R and dilute to 100.0 mL with the solvent.
same solvent. Transfer 25.0 mL of the solution to a separating Reference solution (b). Dissolve 50 mg of stearyl alcohol R in
funnel, add 25 mL of chloroform R, 10 mL of 0.1 M sodium ethanol (96 per cent) R and dilute to 10 mL with the same
hydroxide and 10.0 mL of a freshly prepared 50 g/L solution of solvent.
potassium iodide R. Shake, allow to separate and discard the Reference solution (c). Mix 1 mL of reference solution (a)
chloroform layer. Shake the aqueous layer with 3 quantities, and 1 mL of reference solution (b) and dilute to 10 mL with
each of 10 mL, of chloroform R and discard the chloroform ethanol (96 per cent) R.
layers. Add 40 mL of hydrochloric acid R, allow to cool and
titrate with 0.05 M potassium iodate until the deep brown Column :
colour is almost discharged. Add 2 mL of chloroform R and – size : l = 30 m, Ø = 0.32 mm,
continue the titration, shaking vigorously, until the colour of – stationary phase : poly(dimethyl)siloxane R (1 μm).
the chloroform layer no longer changes. Carry out a blank Carrier gas : helium for chromatography R.
titration on a mixture of 10.0 mL of the freshly prepared
50 g/L solution of potassium iodide R, 20 mL of water R and Flow rate : 1 mL/min.
40 mL of hydrochloric acid R. Split ratio : 1:100.

General Notices (1) apply to all monographs and other texts 1837
Cetyl palmitate EUROPEAN PHARMACOPOEIA 8.0

Temperature : Nickel (2.4.31) : maximum 1 ppm.

Time Temperature Water (2.5.12) : maximum 0.3 per cent, determined on 1.0 g
(min) (°C) using a mixture of equal volumes of anhydrous methanol R
Column 0 - 20 150 → 250 and methylene chloride R as solvent.
20 - 40 250 Total ash (2.4.16) : maximum 0.2 per cent, determined on
1.0 g.
Injection port 250
ASSAY
Detector 250
Gas chromatography (2.2.28) : use the normalisation
Detection : flame ionisation. procedure.
Injection : 1 μL of the test solution and reference solutions (a) Test solution. Dissolve 20.0 mg of the substance to be
and (c). examined in hexane R and dilute to 20.0 mL with the same
System suitability: reference solution (c) : solvent.
– resolution : minimum 5.0 between the peaks due to cetyl Reference solution (a). Dissolve 20.0 mg of cetyl palmi-
alcohol and stearyl alcohol. tate 95 CRS in hexane R and dilute to 20.0 mL with the same
solvent.
Calculate the percentage content of C16H34O.
Reference solution (b). Dissolve 20.0 mg of cetyl palmi-
tate 15 CRS in hexane R and dilute to 20.0 mL with the same
solvent.
01/2008:1906
Column :
– material : stainless steel ;
CETYL PALMITATE
– size : l = 10 m, Ø = 0.53 mm ;
Cetylis palmitas – stationary phase : poly(dimethyl)siloxane R (film thickness
2.65 μm).
DEFINITION Carrier gas : helium for chromatography R.
Mixture of C14-C18 esters of lauric (dodecanoic), myristic Flow rate : 6.5 mL/min.
(tetradecanoic), palmitic (hexadecanoic) and stearic Split ratio : 1:10.
(octadecanoic) acids (‘Cetyl esters wax’).
Temperature :
Content (expressed as hexadecyl hexadecanoate) : 10.0 per
cent to 20.0 per cent for Cetyl palmitate 15, 60.0 per cent to Time Temperature
70.0 per cent for Cetyl palmitate 65 and minimum 90.0 per (min) (°C)
cent for Cetyl palmitate 95. Column 0 - 10 100 → 300

CHARACTERS 10 - 15 300

Appearance : white or almost white, waxy plates, flakes or Injection port 350
powder. Detector 350
Solubility : practically insoluble in water, soluble in boiling
anhydrous ethanol and in methylene chloride, slightly soluble Detection : flame ionisation.
in light petroleum, practically insoluble in anhydrous ethanol. Injection : 1 μL.
mp : about 45 °C for Cetyl palmitate 15 and Cetyl palmitate 65 Relative retention with reference to cetyl palmitate (retention
and about 52 °C for Cetyl palmitate 95. time = about 9 min) : cetyl alcohol = about 0.3 ; palmitic
acid = about 0.4 ; lauric ester = about 0.8 ; myristic
IDENTIFICATION ester = about 0.9 ; stearic ester = about 1.1.
A. It complies with the limits of the assay and the System suitability : reference solution (b) :
chromatogram obtained with the test solution shows the
typical main peak(s). – resolution : minimum of 1.5 between the peaks due to cetyl
palmitate and cetyl stearate.
B. Saponification value (see Tests).
STORAGE
TESTS
At a temperature not exceeding 25 °C.
Appearance of solution. The solution is not more intensely
coloured than reference solution Y6 (2.2.2, Method II). LABELLING
Dissolve 4.0 g in methylene chloride R and dilute to 20 mL The label states the type of cetyl palmitate.
with the same solvent.
Acid value (2.5.1) : maximum 4.0.
Dissolve 10.0 g in 50 mL of the solvent mixture described by 01/2008:0379
heating under reflux on a water-bath for 5 min. corrected 6.0
Hydroxyl value (2.5.3, Method A) : maximum 20.0.
Iodine value (2.5.4, Method A) : maximum 2.0. CETYLPYRIDINIUM CHLORIDE
Saponification value (2.5.6) : 105 to 120.
Heat under reflux for 2 h. Cetylpyridinii chloridum
Alkaline impurities. Dissolve 2.0 g ‘with gentle heating’ in
a mixture of 1.5 mL of ethanol (96 per cent) R and 3 mL of
toluene R. Add 0.05 mL of a 0.4 g/L solution of bromophenol
blue R in ethanol (96 per cent) R. Not more than 0.4 mL of
0.01 M hydrochloric acid is required to change the colour of C21H38ClN,H2O Mr 358.0
the solution to yellow. [6004-24-6]

1838 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Charcoal, activated

DEFINITION hydrochloric acid R, allow to cool and titrate with 0.05 M

Cetylpyridinium chloride contains not less than 96.0 per potassium iodate until the deep-brown colour is almost
cent and not more than the equivalent of 101.0 per cent of discharged. Add 2 mL of chloroform R and continue the
1-hexadecylpyridinium chloride, calculated with reference titration, shaking vigorously, until the chloroform layer no
to the anhydrous substance. longer changes colour. Carry out a blank titration on a mixture
of 10.0 mL of the freshly prepared 50 g/L solution of potassium
CHARACTERS iodide R, 20 mL of water R and 40 mL of hydrochloric acid R.
A white or almost white powder, slightly soapy to the touch, 1 mL of 0.05 M potassium iodate is equivalent to 34.0 mg of
soluble in water and in alcohol. An aqueous solution froths C21H38ClN.
copiously when shaken.
01/2009:0313
IDENTIFICATION
corrected 7.0
First identification : B, D.
Second identification : A, C, D. CHARCOAL, ACTIVATED
A. Dissolve 0.10 g in water R and dilute to 100.0 mL with the
same solvent. Dilute 5.0 mL of this solution to 100.0 mL Carbo activatus
with water R. Examined between 240 nm and 300 nm
(2.2.25), the solution shows an absorption maximum at DEFINITION
259 nm and 2 shoulders at about 254 nm and at about Obtained from vegetable matter by suitable carbonisation
265 nm. The specific absorbance at the maximum is 126 to processes intended to confer a high adsorption power.
134, calculated with reference to the anhydrous substance.
B. Examine by infrared absorption spectrophotometry CHARACTERS
(2.2.24), comparing with the spectrum obtained with Appearance : black, light powder free from grittiness.
cetylpyridinium chloride CRS. Examine the substances in Solubility : practically insoluble in all usual solvents.
the solid state.
IDENTIFICATION
C. To 5 mL of dilute sodium hydroxide solution R add 0.1 mL
of bromophenol blue solution R1 and 5 mL of chloroform R A. When heated to redness it burns slowly without a flame.
and shake. The chloroform layer is colourless. Add 0.1 mL B. Adsorption power (see Tests).
of solution S (see Tests) and shake. The chloroform layer
becomes blue. TESTS
D. Solution S gives reaction (a) of chlorides (2.3.1). Solution S. To 2.0 g in a conical flask with a ground-glass
neck add 50 mL of dilute hydrochloric acid R. Boil gently
TESTS under a reflux condenser for 1 h, filter and wash the filter with
Solution S. Dissolve 1.0 g in carbon dioxide-free water R and dilute hydrochloric acid R. Evaporate the combined filtrate and
dilute to 100 mL with the same solvent. washings to dryness on a water-bath, dissolve the residue in
0.1 M hydrochloric acid and dilute to 50.0 mL with the same
Appearance of solution. Solution S is not more opalescent acid.
than reference suspension II (2.2.1) and is colourless (2.2.2,
Method II). Acidity or alkalinity. To 2.0 g add 40 mL of water R and
boil for 5 min. Cool, restore to the original mass with carbon
Acidity. To 50 mL of solution S add 0.1 mL of phenolphthalein dioxide-free water R and filter. Reject the first 20 mL of the
solution R. Not more than 2.5 mL of 0.02 M sodium hydroxide filtrate. To 10 mL of the filtrate add 0.25 mL of bromothymol
is required to change the colour of the indicator. blue solution R1 and 0.25 mL of 0.02 M sodium hydroxide. The
Amines and amine salts. Dissolve 5.0 g with heating in solution is blue. Not more than 0.75 mL of 0.02 M hydrochloric
20 mL of a mixture of 3 volumes of 1 M hydrochloric acid and acid is required to change the colour of the indicator to yellow.
97 volumes of methanol R and add 100 mL of 2-propanol R. Acid-soluble substances : maximum 3 per cent.
Pass a stream of nitrogen R slowly through the solution. To 1.0 g add 25 mL of dilute nitric acid R and boil for 5 min.
Gradually add 12.0 mL of 0.1 M tetrabutylammonium Filter whilst hot through a sintered-glass filter (10) (2.1.2) and
hydroxide and record the potentiometric titration curve wash with 10 mL of hot water R. Evaporate the combined
(2.2.20). If the curve shows 2 points of inflexion, the volume filtrate and washings to dryness on a water-bath, add to the
of titrant added between the two points is not greater than residue 1 mL of hydrochloric acid R, evaporate to dryness
5.0 mL. If the curve shows no point of inflexion, the substance again and dry the residue to constant mass at 100-105 °C. The
to be examined does not comply with the test. If the curve residue weighs a maximum of 30 mg.
shows one point of inflexion, repeat the test but add 3.0 mL of
a 25.0 g/L solution of dimethyldecylamine R in 2-propanol R Alkali-soluble coloured substances. To 0.25 g add 10 mL of
before the titration. If the titration curve after the addition of dilute sodium hydroxide solution R and boil for 1 min. Cool,
12.0 mL of the titrant shows only one point of inflexion, the filter and dilute the filtrate to 10 mL with water R. The solution
substance to be examined does not comply with the test. is not more intensely coloured than reference solution GY4
(2.2.2, Method II).
Water (2.5.12) : 4.5 per cent to 5.5 per cent, determined on
0.300 g by the semi-micro determination of water. Ethanol (96 per cent) soluble substances : maximum 0.5 per
cent.
Sulfated ash (2.4.14). Not more than 0.2 per cent, determined
on 1.0 g. To 2.0 g add 50 mL of ethanol (96 per cent) R and boil under
a reflux condenser for 10 min. Filter immediately, cool, and
ASSAY dilute to 50 mL with ethanol (96 per cent) R. The filtrate is
Dissolve 2.00 g in water R and dilute to 100.0 mL with the not more intensely coloured than reference solution Y6 or BY6
same solvent. Transfer 25.0 mL of the solution to a separating (2.2.2, Method II). Evaporate 40 mL of the filtrate to dryness
funnel, add 25 mL of chloroform R, 10 mL of 0.1 M sodium and dry to constant mass at 100-105 °C. The residue weighs a
hydroxide and 10.0 mL of a freshly prepared 50 g/L solution maximum of 8 mg.
of potassium iodide R. Shake well, allow to separate and Fluorescent substances. In an intermittent-extraction
discard the chloroform layer. Shake the aqueous layer with apparatus, treat 10.0 g with 100 mL of cyclohexane R1 for 2 h.
three quantities, each of 10 mL, of chloroform R and discard Collect the liquid and dilute to 100 mL with cyclohexane R1.
the chloroform layers. To the aqueous layer add 40 mL of Examine in ultraviolet light at 365 nm. The fluorescence of the

General Notices (1) apply to all monographs and other texts 1839
Chenodeoxycholic acid EUROPEAN PHARMACOPOEIA 8.0

solution is not more intense than that of a solution of 83 μg STORAGE

of quinine R in 1000 mL of 0.005 M sulfuric acid examined In an airtight container.
under the same conditions.
Sulfides. To 1.0 g in a conical flask add 5 mL of hydrochloric 01/2008:1189
acid R1 and 20 mL of water R. Heat to boiling. The fumes corrected 6.0
released do not turn lead acetate paper R brown.
Copper : maximum 25 ppm. CHENODEOXYCHOLIC ACID
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Use solution S. Acidum chenodeoxycholicum
Reference solutions. Prepare the reference solutions using
copper standard solution (0.1 per cent Cu) R and diluting with
0.1 M hydrochloric acid.
Source : copper hollow-cathode lamp.
Wavelength : 325.0 nm.
Atomisation device : air-acetylene flame.
Lead : maximum 10 ppm.
Atomic absorption spectrometry (2.2.23, Method I). C24H40O4 Mr 392.6
Test solution. Use solution S. [474-25-9]
Reference solutions. Prepare the reference solutions using lead DEFINITION
standard solution (100 ppm Pb) R and diluting with 0.1 M Chenodeoxycholic acid contains not less than 99.0 per
hydrochloric acid. cent and not more than the equivalent of 101.0 per cent
Source : lead hollow-cathode lamp. of 3α,7α-dihydroxy-5β-cholan-24-oic acid, calculated with
Wavelength : 283.3 nm ; 217.0 nm may be used depending on reference to the dried substance.
the apparatus.
CHARACTERS
Atomisation device : air-acetylene flame.
A white or almost white powder, very slightly soluble in water,
Zinc : maximum 25 ppm. freely soluble in alcohol, soluble in acetone, slightly soluble
Atomic absorption spectrometry (2.2.23, Method I). in methylene chloride.
Test solution. Use solution S.
IDENTIFICATION
Reference solutions. Prepare the reference solutions using zinc
First identification : A.
standard solution (100 ppm Zn) R and diluting with 0.1 M
hydrochloric acid. Second identification : B, C.
Source : zinc hollow-cathode lamp. A. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
Wavelength : 214.0 nm.
chenodeoxycholic acid CRS. Examine the substances
Atomisation device : air-acetylene flame. prepared as discs using potassium bromide R.
Loss on drying (2.2.32) : maximum 15 per cent, determined B. Examine the chromatograms obtained in the test for related
on 1.00 g by drying in an oven at 120 °C for 4 h. substances. The principal spot in the chromatogram
Sulfated ash (2.4.14) : maximum 5.0 per cent, determined on obtained with test solution (b) is similar in position,
1.0 g. colour and size to the principal spot in the chromatogram
obtained with reference solution (a).
Adsorption power. To 0.300 g in a 100 mL
ground-glass-stoppered conical flask add 25.0 mL of C. Dissolve about 10 mg in 1 mL of sulfuric acid R. Add
a freshly prepared solution of 0.5 g of phenazone R in 50 mL 0.1 mL of formaldehyde solution R and allow to stand for
of water R. Shake thoroughly for 15 min. Filter and reject 5 min. Add 5 mL of water R. The suspension obtained is
the first 5 mL of filtrate. To 10.0 mL of the filtrate add 1.0 g greenish-blue.
of potassium bromide R and 20 mL of dilute hydrochloric TESTS
acid R. Using 0.1 mL of methyl red solution R as indicator,
titrate with 0.0167 M potassium bromate until the red colour Specific optical rotation (2.2.7). Dissolve 0.500 g in
is discharged. Titrate slowly (1 drop every 15 s) towards themethanol R and dilute to 25.0 mL with the same solvent. The
specific optical rotation is + 11.0 to + 13.0, calculated with
end of the titration. Carry out a blank titration using 10.0 mL
of the phenazone solution. reference to the dried substance.
Calculate the quantity of phenazone adsorbed per 100 g of Related substances. Examine by thin-layer chromatography
activated charcoal from the following expression : (2.2.27), using a suitable silica gel as the coating substance.
Test solution (a). Dissolve 0.40 g of the substance to be
examined in a mixture of 1 volume of water R and 9 volumes
of acetone R and dilute to 10 mL with the same mixture of
solvents.
a = number of millilitres of 0.0167 M potassium
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL with
bromate used for the blank ;
a mixture of 1 volume of water R and 9 volumes of acetone R.
b = number of millilitres of 0.0167 M potassium Reference solution (a). Dissolve 40 mg of chenodeoxycholic
bromate used for the test ; acid CRS in a mixture of 1 volume of water R and 9 volumes
m = mass in grams of the substance to be examined. of acetone R and dilute to 10 mL with the same mixture of
Minimum 40 g of phenazone is adsorbed per 100 g of activated solvents.
charcoal, calculated with reference to the dried substance. Reference solution (b). Dissolve 20 mg of lithocholic acid CRS
in a mixture of 1 volume of water R and 9 volumes of acetone R
Microbial contamination and dilute to 10 mL with the same mixture of solvents. Dilute
TAMC : acceptance criterion 103 CFU/g (2.6.12). 2 mL of the solution to 100 mL with a mixture of 1 volume of
TYMC : acceptance criterion 102 CFU/g (2.6.12). water R and 9 volumes of acetone R.

1840 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Chitosan hydrochloride

Reference solution (c). Dissolve 20 mg of ursodeoxycholic E. R = H, R1 = H, R2 = H, R3 = OH : 3α,12α-dihydroxy-5β-

acid CRS in a mixture of 1 volume of water R and 9 volumes cholan-24-oic acid (deoxycholic acid),
of acetone R and dilute to 50 mL with the same mixture of
solvents. F. R = H, R1+R2 = = O, R3 = H : 3α-hydroxy-7-oxo-5β-
cholan-24-oic acid,
Reference solution (d). Dissolve 20 mg of cholic acid CRS in a
mixture of 1 volume of water R and 9 volumes of acetone R G. R = CH3, R1 = OH, R2 = H, R3 = H : methyl
and dilute to 100 mL with the same mixture of solvents. 3α,7β-dihydroxy-5β-cholan-24-oate.
Reference solution (e). Dilute 0.5 mL of test solution (a) to
20 mL with a mixture of 1 volume of water R and 9 volumes of
acetone R. Dilute 1 mL of the solution to 10 mL with a mixture 01/2008:1774
of 1 volume of water R and 9 volumes of acetone R. corrected 6.5
Reference solution (f). Dissolve 10 mg of chenodeoxycholic
acid CRS in reference solution (c) and dilute to 25 mL with CHITOSAN HYDROCHLORIDE
the same solution.
Apply separately to the plate 5 μL of each solution. Develop in Chitosani hydrochloridum
an unsaturated tank over a path of 15 cm using a mixture of
1 volume of glacial acetic acid R, 30 volumes of acetone R and DEFINITION
60 volumes of methylene chloride R. Dry the plate at 120 °C for
Chitosan hydrochloride is the chloride salt of an unbranched
10 min. Spray the plate immediately with a 47.6 g/L solution
binary heteropolysaccharide consisting of the two units
of phosphomolybdic acid R in a mixture of 1 volume of sulfuric
N-acetyl-D-glucosamine and D-glucosamine, obtained by
acid R and 20 volumes of glacial acetic acid R and heat again
partial deacetylation of chitin normally leading to a degree
at 120 °C until blue spots appear on a lighter background.
of deacetylation of 70.0 per cent to 95.0 per cent. Chitin is
In the chromatogram obtained with test solution (a): any
extracted from the shells of shrimp and crab.
spot corresponding to lithocholic acid is not more intense
than the principal spot in the chromatogram obtained with PRODUCTION
reference solution (b) (0.1 per cent) ; any spot corresponding
to ursodeoxycholic acid is not more intense than the principal The animals from which chitosan hydrochloride is derived
spot in the chromatogram obtained with reference solution (c) must fulfil the requirements for the health of animals suitable
(1 per cent); any spot corresponding to cholic acid is not more for human consumption to the satisfaction of the competent
intense than the principal spot in the chromatogram obtained authority. It must have been shown to what extent the
with reference solution (d) (0.5 per cent) ; any spot apart from method of production allows inactivation or removal of any
the principal spot and any spots corresponding to lithocholic contamination by viruses or other infectious agents.
acid, ursodeoxycholic acid and cholic acid, is not more intense CHARACTERS
than the principal spot in the chromatogram obtained with
reference solution (e) (0.25 per cent). The test is not valid Appearance : white or almost white, fine powder.
unless the chromatogram obtained with reference solution (f) Solubility : sparingly soluble in water, practically insoluble in
shows two clearly separated principal spots. anhydrous ethanol.
Heavy metals (2.4.8). 1.0 g complies with test C for heavy IDENTIFICATION
metals (20 ppm). Prepare the reference solution using 2 mL
of lead standard solution (10 ppm Pb) R. A. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs.
Loss on drying (2.2.32). Not more than 1.5 per cent,
determined on 1.000 g by drying in an oven at 105 °C. Comparison: chitosan hydrochloride CRS.
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined B. It gives reaction (a) of chlorides (2.3.1).
on 1.0 g. C. Dilute 50 mL of solution S (see Tests) to 250 mL with a
25 per cent V/V solution of ammonia R. A voluminous
ASSAY gelatinous mass is formed.
Dissolve 0.350 g in 50 mL of alcohol R, previously neutralised D. To 10 mL of solution S add 90 mL of acetone R. A
to 0.2 mL of phenolphthalein solution R. Add 50 mL of water R voluminous gelatinous mass is formed.
and titrate with 0.1 M sodium hydroxide until a pink colour is
obtained. TESTS
1 mL of 0.1 M sodium hydroxide is equivalent to 39.26 mg of Solution S. Dissolve 1.0 g in 100 mL of water R and stir
C24H40O4. vigorously for 20 min with a mechanical stirrer.
IMPURITIES Appearance of solution. Solution S is not more opalescent
than reference suspension II (2.2.1) and not more intensely
coloured than reference solution BY5 (2.2.2, Method II).
Matter insoluble in water : maximum 0.5 per cent.
Add 2.00 g to 400.0 mL of water R while stirring until no
further dissolution takes place. Transfer the solution to a 2 L
beaker, and add 200 mL of water R. Boil the solution gently for
2 h, covering the beaker during the operation. Filter through
a sintered-glass filter (40) (2.1.2), wash the residue with water
A. R = H, R1 = OH, R2 = H, R3 = H : 3α-7β-dihydroxy-5β- and dry to constant weight in an oven at 100-105 °C. The
cholan-24-oic acid (ursodeoxycholic acid), residue weighs a maximum of 10 mg.
B. R = H, R1 = H, R2 = OH, R3 = OH : 3α,7α,12α-trihydroxy- pH (2.2.3) : 4.0 to 6.0 for solution S.
5β-cholan-24-oic acid (cholic acid),
Viscosity (2.2.10) : 80 per cent to 120 per cent of the value
C. R = H, R1 = H, R2 = H, R3 = H : 3α-hydroxy-5β-cholan- stated on the label, determined on solution S.
24-oic acid (lithocholic acid), Determine the viscosity using a rotating viscometer at 20 °C
D. R = H, R1 = OH, R2 = H, R3 = OH : 3α,7β,12α-trihydroxy- with a spindle rotating at 20 r/min, using a suitable spindle for
5β-cholan-24-oic acid (ursocholic acid), the range of the expected viscosity.

General Notices (1) apply to all monographs and other texts 1841
Chloral hydrate EUROPEAN PHARMACOPOEIA 8.0

Degree of deacetylation LABELLING

Test solution. Dissolve 0.250 g in water R and dilute to 50.0 mL The label states the nominal viscosity in millipascal seconds
with the same solvent, stirring vigorously. Dilute 1.0 mL of this for a 10 g/L solution in water R.
solution to 100.0 mL with water R. Measure the absorbance
(2.2.25) from 200 nm to 205 nm as the first derivative of the 01/2008:0265
absorbance curve. Determine the pH of the solution.
Reference solutions. Prepare solutions of 1.0 μg/mL, 5.0 μg/mL, CHLORAL HYDRATE
15.0 μg/mL and 35.0 μg/mL of N-acetylglucosamine R in
water R. Measure the absorbance (2.2.25) from 200 nm
to 205 nm of each solution as the first derivative of the Chlorali hydras
absorption curve. Make a standard curve by plotting the first
derivative at 202 nm as a function of the concentration of
N-acetylglucosamine, and calculate the slope of the curve
by least squares linear regression. Use the standard curve to
determine the equivalent amount of N-acetylglucosamine for C2H3Cl3O2 Mr 165.4
the substance to be examined. [302-17-0]
Calculate the degree of deacetylation (molar) using the DEFINITION
following expression : 2,2,2-Trichloroethane-1,1-diol.
Content : 98.5 per cent to 101.0 per cent.
CHARACTERS
Appearance : colourless, transparent crystals.
C1 = concentration of chitosan hydrochloride in the test Solubility : very soluble in water, freely soluble in ethanol
solution in micrograms per millilitre ; (96 per cent).
C2 = concentration of N-acetylglucosamine in the
test solution, as determined from the standard IDENTIFICATION
curve prepared using the reference solution in A. To 10 mL of solution S (see Tests) add 2 mL of dilute
micrograms per millilitre ; sodium hydroxide solution R. The mixture becomes cloudy
M1 = 203 (relative molecular mass of N-acetylglucos- and, when heated, gives off an odour of chloroform.
amine unit (C8H13NO5) in polymer) ; B. To 1 mL of solution S add 2 mL of sodium sulfide
M3 solution R. A yellow colour develops which quickly
= relative molecular mass of chitosan hydrochloride.
becomes reddish-brown. On standing for a short time, a
M3 is calculated from the pH in solution, assuming a pKa red precipitate may be formed.
value of 6.8, using the following equations : TESTS
Solution S. Dissolve 3.0 g in carbon dioxide-free water R and
dilute to 30 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
pH (2.2.3) : 3.5 to 5.5 for solution S.
Chloral alcoholate. Warm 1.0 g with 10 mL of dilute sodium
hydroxide solution R, filter the supernatant solution and add
M2 = 161 (relative molecular mass of deacetylated unit 0.05 M iodine dropwise until a yellow colour is obtained.
(glucosamine) (C6H11NO4) in polymer). Allow to stand for 1 h. No precipitate is formed.
Chlorides : 10.0 per cent to 20.0 per cent. Chlorides (2.4.4) : maximum 100 ppm.
Introduce 0.200 g into a 250 mL borosilicate flask fitted with a Dilute 5 mL of solution S to 15 mL with water R.
reflux condenser. Add 40 mL of a mixture of 1 volume of nitric Heavy metals (2.4.8) : maximum 20 ppm.
acid R and 2 volumes of water R. Boil gently under a reflux 10 mL of solution S diluted to 20 mL with water R complies
condenser for 5 min. Cool and add 25 mL of water R through with test A. Prepare the reference solution using lead standard
the condenser. Add 16.0 mL of 0.1 M silver nitrate, shake solution (1 ppm Pb) R.
vigorously and titrate with 0.1 M ammonium thiocyanate,
Non-volatile residue : maximum 0.1 per cent.
using 1 mL of ferric ammonium sulfate solution R2 as indicator,
and shaking vigorously towards the end-point. Carry out a Evaporate 2.000 g on a water-bath. The residue weighs a
blank titration. maximum of 2 mg.
1 mL of 0.1 M silver nitrate is equivalent to 3.55 mg of Cl. ASSAY
Heavy metals (2.4.8) : maximum 40 ppm. Dissolve 4.000 g in 10 mL of water R and add 40.0 mL of 1 M
sodium hydroxide. Allow to stand for exactly 2 min and titrate
1.0 g complies with test F. Prepare the reference solution using with 0.5 M sulfuric acid, using 0.1 mL of phenolphthalein
4 mL of lead standard solution (10 ppm Pb) R. solution R as indicator. Titrate the neutralised solution with
Loss on drying (2.2.32) : maximum 10 per cent, determined 0.1 M silver nitrate, using 0.2 mL of potassium chromate
on 1.000 g by drying in an oven at 105 °C. solution R as indicator. Calculate the number of millilitres of
1 M sodium hydroxide used by deducting from the volume of
Sulfated ash (2.4.14) : maximum 1.0 per cent, determined on 1 M sodium hydroxide, added at the beginning of the titration,
1.0 g. the volume of 0.5 M sulfuric acid used in the 1st titration and
two-fifteenths of the volume of 0.1 M silver nitrate used in
STORAGE the 2nd titration.
At a temperature of 2 °C to 8 °C, protected from moisture 1 mL of 1 M sodium hydroxide is equivalent to 0.1654 g
and light. of C2H3Cl3O2.

1842 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Chlorambucil

STORAGE Test solution. Dissolve 25 mg of the substance to be examined

In an airtight container. in the solvent mixture and dilute to 100.0 mL with the solvent
mixture.
Reference solution (a). Dilute 1.0 mL of the test solution to
04/2011:0137 100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
CHLORAMBUCIL Reference solution (b). Dissolve 5 mg of chlorambucil for
system suitability CRS (containing impurities B and E) in the
Chlorambucilum solvent mixture and dilute to 20.0 mL with the solvent mixture.
Column :
– size : l = 0.25 m, Ø = 3.0 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase :
– mobile phase A : 1.9 g/L solution of ammonium acetate R
C14H19Cl2NO2 Mr 304.2 adjusted to pH 3.9 with acetic acid R ;
[305-03-3] – mobile phase B : acetonitrile for chromatography R ;
DEFINITION Time Mobile phase A Mobile phase B
4-[4-[Bis(2-chloroethyl)amino]phenyl]butanoic acid. (min) (per cent V/V) (per cent V/V)
Content : 98.5 per cent to 101.0 per cent (anhydrous substance). 0-5 60 40
5 - 15 60 → 10 40 → 90
CHARACTERS
Appearance : white or almost white, crystalline powder. 15 - 25 10 90
Solubility : practically insoluble in water, freely soluble in Flow rate : 0.8 mL/min.
acetone and in ethanol (96 per cent).
Detection : spectrophotometer at 260 nm.
IDENTIFICATION Injection : 10 μL.
Infrared absorption spectrophotometry (2.2.24). Identification of impurities : use the chromatogram supplied
Comparison : chlorambucil CRS. with chlorambucil for system suitability CRS and the
chromatogram obtained with reference solution (b) to identify
TESTS the peaks due to impurities B and E.
Impurity G. Liquid chromatography (2.2.29). The solutions Relative retention with reference to chlorambucil
are stable for 8 h at room temperature or for 24 h at 4-8 °C ; (retention time = about 12 min) : impurity B = about 0.5 ;
protect them from light. impurity E = about 1.4.
Test solution. Dissolve 10 mg of the substance to be examined System suitability : reference solution (b) :
in methanol R and dilute to 20.0 mL with the same solvent.
– resolution : minimum 5.0 between the peaks due to
Reference solution (a). Dilute 1.0 mL of the test solution to impurity B and chlorambucil.
50.0 mL with the mobile phase. Dilute 2.0 mL of this solution
to 10.0 mL with the mobile phase. Limits :
Reference solution (b). Dissolve 5 mg of chlorambucil with – impurity E : not more than 6 times the area of the principal
impurity G CRS in methanol R and dilute to 10.0 mL with the peak in the chromatogram obtained with reference
same solvent. solution (a) (0.6 per cent) ;
Column : – impurity B : not more than 4 times the area of the principal
peak in the chromatogram obtained with reference
– size : l = 0.15 m, Ø = 3.9 mm ; solution (a) (0.4 per cent) ;
– stationary phase : phenylsilyl silica gel for chromatography R – unspecified impurities : for each impurity, not more than the
(5 μm). area of the principal peak in the chromatogram obtained
Mobile phase : methanol R, 1 per cent V/V solution of with reference solution (a) (0.10 per cent) ;
trifluoroacetic acid R (50:50 V/V). – total : not more than 10 times the area of the principal peak
Flow rate : 1.8 mL/min. in the chromatogram obtained with reference solution (a)
Detection : spectrophotometer at 260 nm. (1.0 per cent) ;
Injection : 20 μL. – disregard limit : 0.5 times the area of the principal peak in
Run time : twice the retention time of chlorambucil. the chromatogram obtained with reference solution (a)
(0.05 per cent).
Relative retention with reference to chlorambucil (retention
time = about 11 min) : impurity G = about 1.2. Water (2.5.12) : maximum 0.5 per cent, determined on 1.00 g.
System suitability : reference solution (b) : Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
– resolution : minimum 1.5 between the peaks due to 1.0 g.
chlorambucil and impurity G.
ASSAY
Limit :
Dissolve 0.200 g in 10 mL of acetone R and add 10 mL of
– impurity G : not more than the area of the principal peak water R. Titrate with 0.1 M sodium hydroxide, using 0.1 mL of
in the chromatogram obtained with reference solution (a) phenolphthalein solution R as indicator.
(0.4 per cent).
1 mL of 0.1 M sodium hydroxide is equivalent to 30.42 mg
Related substances. Liquid chromatography (2.2.29). Prepare of C14H19Cl2NO2.
the solutions immediately before use and protect from light.
Solvent mixture : 10.3 g/L solution of hydrochloric acid R, STORAGE
acetonitrile for chromatography R (10:90 V/V). Protected from light.

General Notices (1) apply to all monographs and other texts 1843
Chloramphenicol EUROPEAN PHARMACOPOEIA 8.0

IMPURITIES
Specified impurities : B, E, G.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use G. 4-[2-[bis(2-chloroethyl)amino]phenyl]butanoic acid or
(2034). It is therefore not necessary to identify these impurities 4-[3-[bis(2-chloroethyl)amino]phenyl]butanoic acid (meta
for demonstration of compliance. See also 5.10. Control of or ortho chlorambucil).
impurities in substances for pharmaceutical use) : A, C, D, F.

01/2008:0071
corrected 6.0

CHLORAMPHENICOL
A. 4-[4-[(2-chloroethyl)(2-hydroxyethyl)amino]phenyl]buta-
noic acid,
Chloramphenicolum

B. 4-[4-[(2-chloroethyl)amino]phenyl]butanoic acid,

C11H12Cl2N2O5 Mr 323.1
[56-75-7]
DEFINITION
Chloramphenicol is 2,2-dichloro-N-[(1R,2R)-2-hydroxy-1-
C. 4-[4-[[2-[[bis(2-chloroethoxy)phosphoryl]oxy]ethyl](2- (hydroxymethyl)-2-(4-nitrophenyl)ethyl]acetamide, produced
chloroethyl)amino]phenyl]butanoic acid, by the growth of certain strains of Streptomyces venezuelae in
a suitable medium. It is normally prepared by synthesis. It
contains not less than 98.0 per cent and not more than the
equivalent of 102.0 per cent of C11H12Cl2N2O5, calculated with
reference to the dried substance.
CHARACTERS
A white, greyish-white or yellowish-white, fine, crystalline
D. 2-chloroethyl 4-[4-[bis(2-chloroethyl)amino]phenyl]- powder or fine crystals, needles or elongated plates, slightly
butanoate, soluble in water, freely soluble in alcohol and in propylene
glycol.
A solution in ethanol is dextrorotatory and a solution in ethyl
acetate is laevorotatory.
IDENTIFICATION
First identification : A, B.
Second identification : A, C, D, E.
A. Melting point (2.2.14) : 149 °C to 153 °C.
E. 4-[4-[[2-[[4-[4-[bis(2-chloroethyl)amino]phenyl]- B. Examine by infrared absorption spectrophotometry
butanoyl]oxy]ethyl](2-chloroethyl)amino]phenyl]butanoic (2.2.24), comparing with the spectrum obtained with
acid, chloramphenicol CRS.
C. Examine the chromatograms obtained in the test for
related substances. The principal spot in the chromatogram
obtained with 1 μL of the test solution is similar in position
and size to the principal spot in the chromatogram obtained
with reference solution (a).
D. Dissolve about 10 mg in 1 mL of alcohol (50 per cent V/V) R,
add 3 mL of a 10 g/L solution of calcium chloride R and
50 mg of zinc powder R and heat on a water-bath for 10 min.
Filter the hot solution and allow to cool. Add 0.1 mL of
benzoyl chloride R and shake for 1 min. Add 0.5 mL of ferric
chloride solution R1 and 2 mL of chloroform R and shake.
The aqueous layer is coloured light violet-red to purple.
E. To 50 mg in a porcelain crucible add 0.5 g of anhydrous
F. 4-[4-[[2-[[4-[4-[[2-[[4-[4-[bis(2-chloroethyl)amino]- sodium carbonate R. Heat over an open flame for 10 min.
phenyl]butanoyl]oxy]ethyl](2-chloroethyl)amino]- Allow to cool. Take up the residue with 5 mL of dilute
phenyl]butanoyl]oxy]ethyl](2-chloroethyl)amino]- nitric acid R and filter. To 1 mL of the filtrate add 1 mL of
phenyl]butanoic acid, water R. The solution gives reaction (a) of chlorides (2.3.1).

1844 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Chloramphenicol palmitate

TESTS 01/2008:0473
corrected 6.0
Acidity or alkalinity. To 0.1 g add 20 mL of carbon
dioxide-free water R, shake and add 0.1 mL of bromothymol
blue solution R1. Not more than 0.1 mL of 0.02 M hydrochloric CHLORAMPHENICOL PALMITATE
acid or 0.02 M sodium hydroxide is required to change the
colour of the indicator. Chloramphenicoli palmitas
Specific optical rotation (2.2.7). Dissolve 1.50 g in ethanol R
and dilute to 25.0 mL with the same solvent. The specific
optical rotation is + 18.5 to + 20.5.
Related substances. Examine by thin-layer chromatography
(2.2.27), using silica gel GF254 R as the coating substance.

Test solution. Dissolve 0.10 g of the substance to be examined C27H42Cl2N2O6 Mr 561.6

in acetone R and dilute to 10 mL with the same solvent. [530-43-8]

Reference solution (a). Dissolve 0.10 g of chloramphenicol CRS DEFINITION

in acetone R and dilute to 10 mL with the same solvent. Chloramphenicol palmitate contains not less than 98.0 per
cent and not more than the equivalent of 102.0 per cent
Reference solution (b). Dilute 0.5 mL of reference solution (a) of (2R,3R)-2-[(dichloroacetyl)amino]-3-hydroxy-3-(4-
to 100 mL with acetone R. nitrophenyl)propyl hexadecanoate, calculated with reference
to the dried substance.
Apply separately to the plate 1 μL and 20 μL of the test Semi-synthetic product derived from a fermentation product.
solution, 1 μL of reference solution (a) and 20 μL of reference
solution (b). Develop over a path of 15 cm using a mixture CHARACTERS
of 1 volume of water R, 10 volumes of methanol R and A white or almost white, fine, unctuous powder, practically
90 volumes of chloroform R. Allow the plate to dry in air insoluble in water, freely soluble in acetone, sparingly soluble
and examine in ultraviolet light at 254 nm. Any spot in the in ethanol (96 per cent), very slightly soluble in hexane.
chromatogram obtained with 20 μL of the test solution, apart It melts at 87 °C to 95 °C.
from the principal spot, is not more intense than the spot It shows polymorphism (5.9). The thermodynamically stable
in the chromatogram obtained with reference solution (b) form has low bioavailability following oral administration.
(0.5 per cent).
IDENTIFICATION
Chlorides (2.4.4). To 1.00 g add 20 mL of water R and
A. Examine by thin-layer chromatography (2.2.27), using TLC
10 mL of nitric acid R and shake for 5 min. Filter through a
silanised silica gel plate R.
filter paper previously washed by filtering 5 mL portions of
water R until 5 mL of filtrate no longer becomes opalescent on Test solution. Dissolve 50 mg of the substance to be
addition of 0.1 mL of nitric acid R and 0.1 mL of silver nitrate examined in a mixture of 1 mL of 1 M sodium hydroxide
solution R1. 15 mL of the filtrate complies with the limit test and 5 mL of acetone R and allow to stand for 30 min. Add
for chlorides (100 ppm). 1.1 mL of 1 M hydrochloric acid and 3 mL of acetone R.
Reference solution (a). Dissolve 10 mg of
Loss on drying (2.2.32). Not more than 0.5 per cent, chloramphenicol CRS in acetone R and dilute to
determined on 1.000 g by drying in an oven at 105 °C. 5 mL with the same solvent.
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined Reference solution (b). Dissolve 10 mg of palmitic acid R in
on 2.0 g. acetone R and dilute to 5 mL with the same solvent.
Pyrogens (2.6.8). If intended for use in the manufacture Reference solution (c). Dissolve 10 mg of the substance to
of parenteral preparations without a further appropriate be examined in acetone R and dilute to 5 mL with the same
procedure for the removal of pyrogens, it complies with the solvent.
test for pyrogens. Inject per kilogram of the rabbit’s mass Apply to the plate 4 μL of each solution. Develop over a
2.5 mL of a solution containing per millilitre 2 mg of the path of 15 cm using a mixture of 30 volumes of a 100 g/L
substance to be examined. solution of ammonium acetate R and 70 volumes of ethanol
(96 per cent) R. Allow the plate to dry in air and spray
with a solution containing 0.2 g/L of dichlorofluorescein R
and 0.1 g/L of rhodamine B R in ethanol (96 per cent) R.
ASSAY Allow the plate to dry in air and examine in ultraviolet
light at 254 nm. The chromatogram obtained with the
Dissolve 0.100 g in water R and dilute to 500.0 mL with the test solution shows 3 spots corresponding in position to
same solvent. Dilute 10.0 mL of this solution to 100.0 mL with the principal spots in the chromatograms obtained with
water R. Measure the absorbance (2.2.25) at the maximum reference solutions (a), (b) and (c).
at 278 nm. B. Dissolve 0.2 g in 2 mL of pyridine R, add 2 mL of a 100 g/L
solution of potassium hydroxide R and heat on a water-bath.
Calculate the content of C11H12Cl2N2O5 taking the specific A red colour is produced.
absorbance to be 297. C. Dissolve about 10 mg in 5 mL of ethanol (96 per cent) R
and add 4.5 mL of dilute sulfuric acid R and 50 mg of zinc
powder R. Allow to stand for 10 min and if necessary decant
the supernatant or filter. Cool the solution in iced water
STORAGE and add 0.5 mL of sodium nitrite solution R. Allow to stand
for 2 min and add 1 g of urea R, 2 mL of strong sodium
Store protected from light. If the substance is sterile, store in a hydroxide solution R and 1 mL of β-naphthol solution R. A
sterile, airtight, tamper-proof container. red colour develops.

General Notices (1) apply to all monographs and other texts 1845
Chloramphenicol sodium succinate EUROPEAN PHARMACOPOEIA 8.0

TESTS IMPURITIES
Acidity. Dissolve 1.0 g in 5 mL of a mixture of equal volumes
of ethanol (96 per cent) R and ether R, warming to 35 °C. Add
0.2 mL of phenolphthalein solution R. Not more than 0.4 mL of
0.1 M sodium hydroxide is required to produce a pink colour
persisting for 30 s.
Specific optical rotation (2.2.7). Dissolve 1.25 g in anhydrous
ethanol R and dilute to 25.0 mL with the same solvent. The A. (1R,2R)-2-[(dichloroacetyl)amino]-3-hydroxy-1-(4-
specific optical rotation is + 22.5 to + 25.5. nitrophenyl)propyl hexadecanoate (chloramphenicol
Free chloramphenicol : maximum 450 ppm. Dissolve 1.0 g, palmitate isomer),
with gentle heating, in 80 mL of xylene R. Cool and shake with
3 quantities, each of 15 mL, of water R. Dilute the combined
aqueous extracts to 50 mL with water R and shake with
10 mL of toluene R. Allow to separate and discard the toluene
layer. Centrifuge a portion of the aqueous layer and measure
the absorbance (A) (2.2.25) at the maximum at 278 nm
using as the compensation liquid a blank solution having an
absorbance not greater than 0.05.
Calculate the content of free chloramphenicol in parts per B. (1R,2R)-2-[(dichloroacetyl)amino]-1-(4-nitrophen-
million from the expression : yl)propane-1,3-diyl bishexadecanoate (chloramphenicol
dipalmitate).

01/2008:0709
Related substances. Examine by thin-layer chromatography corrected 6.0
(2.2.27), using silica gel GF254 R as the coating substance.
Test solution. Dissolve 0.1 g of the substance to be examined CHLORAMPHENICOL SODIUM
in acetone R and dilute to 10 mL with the same solvent. SUCCINATE
Reference solution (a). Dissolve 20 mg of chloramphenicol
palmitate isomer CRS in acetone R and dilute to 10 mL with Chloramphenicoli natrii succinas
the same solvent. Dilute 1 mL of this solution to 10 mL with
acetone R.
Reference solution (b). Dissolve 20 mg of chloramphenicol
dipalmitate CRS in acetone R and dilute to 10 mL with the
same solvent. Dilute 1 mL of this solution to 10 mL with
acetone R.
Reference solution (c). Dissolve 5 mg of chloramphenicol CRS
in acetone R and dilute to 10 mL with the same solvent. Dilute
1 mL of this solution to 10 mL with acetone R. C15H15Cl2N2NaO8 Mr 445.2
Apply to the plate 10 μL of each solution. Develop over a DEFINITION
path of 15 cm using a mixture of 10 volumes of methanol R, Mixture in variable proportions of sodium (2R,3R)-2-
40 volumes of chloroform R and 50 volumes of cyclohexane R. [(dichloroacetyl)amino]-3-hydroxy-3-(4-nitrophenyl)propyl
Allow the plate to dry in air and examine in ultraviolet light butanedioate (3 isomer) and of sodium (1R,2R)-2-
at 254 nm. In the chromatogram obtained with the test [(dichloroacetyl)amino]-3-hydroxy-1-(4-nitrophenyl)propyl
solution, any spots due to chloramphenicol palmitate isomer butanedioate (1 isomer).
and chloramphenicol dipalmitate are not more intense than
Semi-synthetic product derived from a fermentation product.
the corresponding spots in the chromatograms obtained with
reference solutions (a) and (b) respectively (2.0 per cent) and Content : 98.0 per cent to 102.0 per cent (anhydrous substance).
any spot, apart from the principal spot and the spots due CHARACTERS
to chloramphenicol palmitate isomer and chloramphenicol
dipalmitate, is not more intense than the principal spot in the Appearance : white or yellowish-white powder, hygroscopic.
chromatogram obtained with reference solution (c) (0.5 per Solubility : very soluble in water, freely soluble in ethanol
cent). (96 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined IDENTIFICATION
on 1.000 g by heating at 80 °C over diphosphorus pentoxide R A. Thin-layer chromatography (2.2.27).
at a pressure not exceeding 0.1 kPa for 3 h.
Test solution. Dissolve 20 mg of the substance to be
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on examined in 2 mL of acetone R.
1.0 g. Reference solution (a). Dissolve 20 mg of chloramphenicol
sodium succinate CRS in 2 mL of acetone R.
ASSAY
Reference solution (b). Dissolve 20 mg of
Dissolve 90.0 mg in ethanol (96 per cent) R and dilute to chloramphenicol CRS in 2 mL of acetone R.
100.0 mL with the same solvent. Dilute 10.0 mL of this
Plate : TLC silica gel GF254 plate R.
solution to 250.0 mL with ethanol (96 per cent) R. Measure the
absorbance (2.2.25) of the solution at the maximum at 271 nm. Mobile phase : dilute acetic acid R, methanol R, chloroform R
(1:14:85 V/V/V).
Calculate the content of C27H42Cl2N2O6taking the specific
absorbance to be 178. Application : 2 μL.
Development : over a path of 15 cm.
STORAGE Drying : in air.
Protected from light. Detection : examine in ultraviolet light at 254 nm.

1846 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Chlorcyclizine hydrochloride

Results : the 2 principal spots in the chromatogram obtained Pyrogens (2.6.8). If intended for use in the manufacture
with the test solution are similar in position and size to of parenteral preparations without a further appropriate
the 2 principal spots in the chromatogram obtained with procedure for removal of pyrogens, it complies with the test
reference solution (a) ; their positions are different from for pyrogens. Inject per kilogram of the rabbit’s mass 2.5 mL
that of the principal spot in the chromatogram obtained of a solution in water for injections R containing 2 mg of the
with reference solution (b). substance to be examined per millilitre.
B. Dissolve about 10 mg in 1 mL of ethanol (50 per cent V/V) R,
ASSAY
add 3 mL of a 10 g/L solution of calcium chloride R and
50 mg of zinc powder R and heat on a water-bath for 10 min. Dissolve 0.200 g in water R and dilute to 500.0 mL with the
Filter the hot solution and allow to cool. Add 0.1 mL of same solvent. Dilute 5.0 mL of this solution to 100.0 mL with
benzoyl chloride R and shake for 1 min. Add 0.5 mL of water R. Measure the absorbance (2.2.25) at the absorption
ferric chloride solution R1 and 2 mL of chloroform R and maximum at 276 nm.
shake. The upper layer is light violet-red or purple. Calculate the content of C15H15Cl2N2NaO8, taking the specific
C. Dissolve 50 mg in 1 mL of pyridine R. Add 0.5 mL of dilute absorbance to be 220.
sodium hydroxide solution R and 1.5 mL of water R. Heat in STORAGE
a water-bath for 3 min. A red colour develops. Add 2 mL
of nitric acid R and cool under running water. Add 1 mL of In an airtight container, protected from light. If the substance
is sterile, store in a sterile, airtight, tamper-proof container,
0.1 M silver nitrate. A white precipitate is formed slowly.
protected from light.
D. It gives reaction (a) of sodium (2.3.1).
TESTS 01/2008:1086
corrected 7.0
pH (2.2.3) : 6.4 to 7.0.
Dissolve 2.50 g in carbon dioxide-free water R and dilute to
10 mL with the same solvent.
CHLORCYCLIZINE HYDROCHLORIDE
Specific optical rotation (2.2.7) : + 5.0 to + 8.0 (anhydrous Chlorcyclizini hydrochloridum
substance).
Dissolve 0.50 g in water R and dilute to 10.0 mL with the same
solvent.
Chloramphenicol and chloramphenicol disodium
disuccinate. Liquid chromatography (2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be
examined in the mobile phase and dilute to 100.0 mL with
the mobile phase. C18H22Cl2N2 Mr 337.3
[14362-31-3]
Reference solution (a). Dissolve 10.0 mg of chlorampheni-
col CRS in the mobile phase and dilute to 100.0 mL with the DEFINITION
mobile phase (solution A). Dilute 5.0 mL of this solution to Chlorcyclizine hydrochloride contains not less than 99.0 per
100.0 mL with the mobile phase. cent and not more than the equivalent of 101.0 per cent of
Reference solution (b). Dissolve 10.0 mg of chloramphenicol (RS)-1-[(4-chlorophenyl)phenylmethyl]-4-methylpiperazine
disodium disuccinate CRS in the mobile phase and dilute to hydrochloride, calculated with reference to the dried
100.0 mL with the mobile phase (solution B). Dilute 5.0 mL of substance.
this solution to 100.0 mL with the mobile phase.
Reference solution (c). Dissolve 25 mg of the substance to be CHARACTERS
examined in the mobile phase, add 5 mL of solution A and A white or almost white, crystalline powder, freely soluble in
5 mL of solution B and dilute to 100 mL with the mobile phase. water and in methylene chloride, soluble in alcohol.
Column : IDENTIFICATION
– size : l = 0.25 m, Ø = 4.6 mm ; First identification : B, D.
– stationary phase : octadecylsilyl silica gel for Second identification : A, C, D.
chromatography R (5 μm).
A. Dissolve 10.0 mg in a 5 g/L solution of sulfuric acid R and
Mobile phase : 20 g/L solution of phosphoric acid R, methanol R, dilute to 100.0 mL with the same acid. Dilute 10.0 mL of
water R (5:40:55 V/V/V). the solution to 100.0 mL with a 5 g/L solution of sulfuric
Flow rate : 1.0 mL/min. acid R. Examined between 215 nm and 300 nm (2.2.25),
Detection : spectrophotometer at 275 nm. the solution shows an absorption maximum at 231 nm.
Injection : 20 μL. The specific absorbance at the maximum is 475 to 525,
calculated with reference to the dried substance.
System suitability: reference solution (c) :
B. Examine by infrared absorption spectrophotometry
– the 2 peaks corresponding to those in the chromatograms
(2.2.24), comparing with the spectrum obtained with
obtained with reference solutions (a) and (b) are clearly
chlorcyclizine hydrochloride CRS. Examine the substances
separated from the peaks corresponding to the 2 principal
prepared as discs.
peaks in the chromatogram obtained with the test solution ;
if necessary, adjust the methanol content of the mobile C. Examine the chromatograms obtained in the test for
phase. related substances (see Tests). The principal spot in the
chromatogram obtained with test solution (b) is similar in
Limits :
position and size to the principal spot in the chromatogram
– chloramphenicol : not more than the area of the principal obtained with reference solution (a).
peak in the chromatogram obtained with reference
D. It gives reaction (a) of chlorides (2.3.1).
solution (a) (2.0 per cent) ;
– chloramphenicol disodium disuccinate : not more than the TESTS
area of the principal peak in the chromatogram obtained Appearance of solution. Dissolve 0.5 g in water R and dilute
with reference solution (b) (2.0 per cent). to 10 mL with the same solvent. The solution is clear (2.2.1)
Water (2.5.12) : maximum 2.0 per cent, determined on 0.500 g. and colourless (2.2.2, Method II).

General Notices (1) apply to all monographs and other texts 1847
Chlordiazepoxide EUROPEAN PHARMACOPOEIA 8.0

pH (2.2.3). Dissolve 0.10 g in carbon dioxide-free water R and 01/2008:0656

dilute to 10 mL with the same solvent. The pH of the solution corrected 6.0
is 5.0 to 6.0.
Related substances. Examine by thin-layer chromatography CHLORDIAZEPOXIDE
(2.2.27), using a plate coated with a suitable silica gel.
Test solution (a). Dissolve 0.20 g of the substance to be Chlordiazepoxidum
examined in methanol R and dilute to 10 mL with the same
solvent.
Test solution (b). Dilute 5 mL of test solution (a) to 100 mL
with methanol R.
Reference solution (a). Dissolve 10 mg of chlorcyclizine
hydrochloride CRS in methanol R and dilute to 10 mL with
the same solvent.
Reference solution (b). Dissolve 5 mg of methylpiperazine R in C16H14ClN3O Mr 299.8
methanol R and dilute to 50 mL with the same solvent. [58-25-3]

Reference solution (c). Dilute 1 mL of test solution (b) to DEFINITION

25 mL with methanol R. 7-Chloro-N-methyl-5-phenyl-3H-1,4-benzodiazepin-2-amine
4-oxide.
Reference solution (d). Dissolve 10 mg of hydroxyzine
Content : 99.0 per cent to 101.0 per cent (dried substance).
hydrochloride CRS and 10 mg of chlorcyclizine
hydrochloride CRS in methanol R and dilute to 10 mL with CHARACTERS
the same solvent.
Appearance : almost white or light yellow, crystalline powder.
Apply separately to the plate 10 μL of each solution and Solubility : practically insoluble in water, sparingly soluble in
develop over a path of 15 cm using a mixture of 2 volumes ethanol (96 per cent).
of concentrated ammonia R, 13 volumes of methanol R and It shows polymorphism (5.9).
85 volumes of methylene chloride R. Allow the plate to dry
in air and expose it to iodine vapour for 10 min. In the IDENTIFICATION
chromatogram obtained with test solution (a) : any spot Infrared absorption spectrophotometry (2.2.24).
corresponding to methylpiperazine is not more intense
than the spot in the chromatogram obtained with reference Comparison: chlordiazepoxide CRS.
solution (b) (0.5 per cent) ; any spot, apart from the principal If the spectra obtained in the solid state show differences,
spot and any spot corresponding to methylpiperazine, is not dissolve the substance to be examined and the reference
more intense than the spot in the chromatogram obtained substance separately in methylene chloride R, evaporate to
with reference solution (c) (0.2 per cent). The test is not valid dryness and record new spectra using the residues.
unless the chromatogram obtained with reference solution (d)
shows two clearly separated spots. TESTS
Related substances. Liquid chromatography (2.2.29). Carry
Loss on drying (2.2.32). Not more than 1.0 per cent, out the test protected from bright light and prepare the solutions
determined on 1.000 g by drying in an oven at 130 °C. immediately before use.
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined Test solution. Dissolve 20.0 mg of the substance to be
on 1.0 g. examined in the mobile phase and dilute to 100.0 mL with
the mobile phase.
ASSAY Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 2.0 mL of this solution
Dissolve 0.200 g in a mixture of 1 mL of 0.1 M hydrochloric to 10.0 mL with the mobile phase.
acid and 50 mL of methanol R. Carry out a potentiometric Reference solution (b). Dissolve 5 mg of chlordiazepoxide
titration (2.2.20), using 0.1 M sodium hydroxide. Read impurity A CRS in the mobile phase, add 25.0 mL of the test
the volume added between the two points of inflexion. solution and dilute to 100.0 mL with the mobile phase. Dilute
2.0 mL of this solution to 50.0 mL with the mobile phase.
1 mL of 0.1 M sodium hydroxide is equivalent to 33.73 mg of
C18H22Cl2N2. Reference solution (c). Dissolve 4.0 mg of aminochlorobenzo-
phenone R in the mobile phase and dilute to 100.0 mL with
the mobile phase. Dilute 1.0 mL of this solution to 100.0 mL
STORAGE with the mobile phase.
Column :
Store protected from light. – size : l = 0.15 m, Ø = 4.6 mm,
– stationary phase : octadecylsilyl silica gel for
IMPURITIES chromatography R (5 μm).
Mobile phase : acetonitrile R, water R (50:50 V/V).
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 254 nm.
Injection : 10 μL.
Run time : 6 times the retention time of chlordiazepoxide.
Relative retention with reference to chlordiazepoxide
(retention time = about 3.6 min) : impurity A = about 0.7 ;
A. N-methylpiperazine. impurity B = about 2.3 ; impurity C = about 3.9.

1848 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Chlordiazepoxide hydrochloride

System suitability : reference solution (b) : 01/2008:0474

– resolution : minimum 5.0 between the peaks due to
impurity A and chlordiazepoxide. CHLORDIAZEPOXIDE
Limits : HYDROCHLORIDE
– impurities A, B : for each impurity, not more than the area
of the principal peak in the chromatogram obtained with Chlordiazepoxidi hydrochloridum
reference solution (a) (0.2 per cent),
– impurity C : not more than the area of the principal peak
in the chromatogram obtained with reference solution (c)
(0.2 per cent),
– unspecified impurities : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent),
– total : not more than 2.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a) C16H15Cl2N3O Mr 336.2
(0.5 per cent), [438-41-5]
– disregard limit : 0.25 times the area of the principal peak DEFINITION
in the chromatogram obtained with reference solution (a) 7-Chloro-N-methyl-5-phenyl-3H-1,4-benzodiazepin-2-amine
(0.05 per cent). 4-oxide hydrochloride.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined Content : 99.0 per cent to 101.0 per cent (dried substance).
on 1.000 g by drying in an oven at 105 °C.
CHARACTERS
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. Appearance : white or slightly yellow, crystalline powder.
Solubility : soluble in water, sparingly soluble in ethanol
ASSAY (96 per cent).
Dissolve 0.250 g, with heating if necessary, in 80 mL of It shows polymorphism (5.9).
anhydrous acetic acid R. Titrate with 0.1 M perchloric acid IDENTIFICATION
determining the end-point potentiometrically (2.2.20).
A. Infrared absorption spectrophotometry (2.2.24).
1 mL of 0.1 M perchloric acid is equivalent to 29.98 mg Comparison: chlordiazepoxide hydrochloride CRS.
of C16H14ClN3O.
If the spectra obtained in the solid state show differences,
STORAGE dissolve 100 mg in 9 mL of water R and add 1 mL of
dilute sodium hydroxide solution R. Extract with 10 mL of
Protected from light. methylene chloride R in a separating funnel. Evaporate the
organic layer and dry the residue obtained at 100-105 °C.
IMPURITIES Proceed in the same way with the reference substance.
Specified impurities : A, B, C. Record new spectra using the residues.
B. Dissolve 50 mg in 5 mL of water R, add 1 mL of dilute
ammonia R1, mix, allow to stand for 5 min and filter.
Acidify the filtrate with dilute nitric acid R. The solution
gives reaction (a) of chlorides (2.3.1).
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution GY6 (2.2.2,
Method II).
A. 7-chloro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2- Dissolve 2.5 g in water R and dilute to 25 mL with the same
one 4-oxide, solvent.
Related substances. Liquid chromatography (2.2.29). Carry
out the following operations protected from bright light and
prepare the solutions immediately before use.
Test solution. Dissolve 20.0 mg of the substance to be
examined in the mobile phase and dilute to 100.0 mL with
the mobile phase.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 2.0 mL of this solution
B. 6-chloro-2-(chloromethyl)-4-phenylquinazoline 3-oxide, to 10.0 mL with the mobile phase.
Reference solution (b). Dissolve 5 mg of chlordiazepoxide
impurity A CRS in the mobile phase, add 25.0 mL of the test
solution and dilute to 100.0 mL with the mobile phase. Dilute
2.0 mL of this solution to 50.0 mL with the mobile phase.
Reference solution (c). Dissolve 4.0 mg of aminochlorobenzo-
phenone R in the mobile phase and dilute to 100.0 mL with
the mobile phase. Dilute 1.0 mL of this solution to 100.0 mL
with the mobile phase.
C. (2-amino-5-chlorophenyl)phenylmethanone Column :
(aminochlorobenzophenone). – size : l = 0.15 m, Ø = 4.6 mm,

General Notices (1) apply to all monographs and other texts 1849
Chlorhexidine diacetate EUROPEAN PHARMACOPOEIA 8.0

– stationary phase : octadecylsilyl silica gel for

chromatography R (5 μm).
Mobile phase : acetonitrile R, water R (50:50 V/V).
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 254 nm.
Injection : 10 μL. C. (2-amino-5-chlorophenyl)phenylmethanone
Run time : 6 times the retention time of chlordiazepoxide. (aminochlorobenzophenone).
Relative retention with reference to chlordiazepoxide 01/2008:0657
(retention time = about 3.6 min) : impurity A = about 0.7 ; corrected 7.0
impurity B = about 2.3 ; impurity C = about 3.9.
System suitability : reference solution (b) : CHLORHEXIDINE DIACETATE
– resolution : minimum 5.0 between the peaks due to
impurity A and chlordiazepoxide. Chlorhexidini diacetas
Limits :
– impurities A, B : for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent),
– impurity C : not more than the area of the principal peak
in the chromatogram obtained with reference solution (c)
(0.2 per cent),
– unspecified impurities : for each impurity, not more than C26H38Cl2N10O4 Mr 625.6
0.5 times the area of the principal peak in the chromatogram [56-95-1]
obtained with reference solution (a) (0.10 per cent),
– total : not more than 2.5 times the area of the principal peak DEFINITION
in the chromatogram obtained with reference solution (a) 1,1′-(Hexane-1,6-diyl)bis[5-(4-chlorophenyl)biguanide]
(0.5 per cent), diacetate.
– disregard limit : 0.25 times the area of the principal peak Content : 98.0 per cent to 101.0 per cent (dried substance).
in the chromatogram obtained with reference solution (a) CHARACTERS
(0.05 per cent). Appearance : white or almost white, microcrystalline powder.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined Solubility : sparingly soluble in water, soluble in ethanol (96 per
on 1.000 g by drying in vacuo at 60 °C for 4 h. cent), slightly soluble in glycerol and in propylene glycol.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. IDENTIFICATION
First identification : A.
ASSAY Second identification : B, C, D.
Dissolve 0.250 g in 50 mL of water R. Titrate with 0.1 M silver A. Infrared absorption spectrophotometry (2.2.24).
nitrate, determining the end-point potentiometrically (2.2.20). Comparison: chlorhexidine diacetate CRS.
1 mL of 0.1 M silver nitrate is equivalent to 33.62 mg B. Dissolve about 5 mg in 5 mL of a warm 10 g/L solution
of C16H15Cl2N3O. of cetrimide R and add 1 mL of strong sodium hydroxide
solution R and 1 mL of bromine water R. A deep red colour
STORAGE is produced.
Protected from light. C. Dissolve 0.3 g in 10 mL of a mixture of equal volumes of
hydrochloric acid R and water R. Add 40 mL of water R,
IMPURITIES filter if necessary and cool in iced water. Make alkaline to
titan yellow paper R by adding dropwise, and with stirring,
Specified impurities : A, B, C. strong sodium hydroxide solution R and add 1 mL in excess.
Filter, wash the precipitate with water R until the washings
are free from alkali and recrystallise from ethanol (70 per
cent V/V) R. Dry at 100-105 °C. The residue melts (2.2.14)
at 132 °C to 136 °C.
D. It gives reaction (a) of acetates (2.3.1).
TESTS
Chloroaniline : maximum 500 ppm.
Dissolve 0.20 g in 25 mL of water R with shaking if necessary.
A. 7-chloro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2- Add 1 mL of hydrochloric acid R and dilute to 30 mL with
one 4-oxide, water R. Add rapidly and with thorough mixing after each
addition : 2.5 mL of dilute hydrochloric acid R, 0.35 mL
of sodium nitrite solution R, 2 mL of a 50 g/L solution
of ammonium sulfamate R, 5 mL of a 1.0 g/L solution of
naphthylethylenediamine dihydrochloride R and 1 mL of
ethanol (96 per cent) R, dilute to 50.0 mL with water R and
allow to stand for 30 min. Any reddish-blue colour in the
solution is not more intense than that in a standard prepared
at the same time and in the same manner, using a mixture of
B. 6-chloro-2-(chloromethyl)-4-phenylquinazoline 3-oxide, 10.0 mL of a 0.010 g/L solution of chloroaniline R in dilute

1850 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Chlorhexidine digluconate solution

hydrochloric acid R and 20 mL of dilute hydrochloric acid R

instead of the solution of the substance to be examined.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.200 g of the substance to be examined
in the mobile phase and dilute to 100 mL with the mobile
phase.
Reference solution (a). Dissolve 15 mg of chlorhexidine for
performance test CRS in the mobile phase and dilute to B. [[6-[[[(4-chlorophenyl)carbamimidoyl]carbamimidoyl]-
10.0 mL with the mobile phase. amino]hexyl]carbamimidoyl]urea,
Reference solution (b). Dilute 2.5 mL of the test solution to
100 mL with the mobile phase.
Reference solution (c). Dilute 2.0 mL of reference solution (b)
to 10 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10 mL with the mobile phase.
Column :
– size : l = 0.2 m, Ø = 4 mm ;
– stationary phase : octadecylsilyl silica gel for C. 1,1′-[hexane-1,6-diylbis(iminocarbonimidoyl)]bis[3-(4-
chromatography R (5 μm). chlorophenyl)urea],
Mobile phase : solution of 2.0 g of sodium octanesulfonate R in
a mixture of 120 mL of glacial acetic acid R, 270 mL of water R
and 730 mL of methanol R.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 254 nm.
Equilibration : with the mobile phase for at least 1 h.
Injection : 10 μL.
Run time : 6 times the retention time of chlorhexidine.
System suitability: reference solution (a) : D. 1,1′-[[[[(4-chlorophenyl)carbamimidoyl]imino]-
– the chromatogram obtained is similar to the chromatogram methylene]bis[imino(hexane-1,6-diyl)]]bis[5-(4-
supplied with chlorhexidine for performance test CRS chlorophenyl)biguanide].
in that the peaks due to impurity A and impurity B
precede that due to chlorhexidine ; if necessary, adjust the 07/2013:0658
concentration of acetic acid in the mobile phase (increasing
the concentration decreases the retention times). CHLORHEXIDINE DIGLUCONATE
Limits : SOLUTION
– total : not more than the area of the principal peak in the
chromatogram obtained with reference solution (b) (2.5 per Chlorhexidini digluconatis solutio
cent) ;
– disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (c)
(0.05 per cent) ; disregard any peak with a relative retention
time with reference to chlorhexidine of 0.25 or less.
Loss on drying (2.2.32) : maximum 3.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulfated ash (2.4.14) : maximum 0.15 per cent, determined
on 1.0 g. C34H54Cl2N10O14 Mr 898
[18472-51-0]
ASSAY
Dissolve 0.140 g in 100 mL of anhydrous acetic acid R and DEFINITION
titrate with 0.1 M perchloric acid. Determine the end-point Aqueous solution of 1,1′-(hexane-1,6-diyl)bis[5-(4-
potentiometrically (2.2.20). chlorophenyl)biguanide] di-D-gluconate.
1 mL of 0.1 M perchloric acid is equivalent to 15.64 mg Content : 190 g/L to 210 g/L.
of C26H38Cl2N10O4. CHARACTERS
IMPURITIES Appearance : almost colourless or pale-yellowish liquid.
Solubility : miscible with water, with not more than 3 parts of
acetone and with not more than 5 parts of ethanol (96 per
cent).
IDENTIFICATION
First identification : A, B.
Second identification : B, C, D.
A. Infrared absorption spectrophotometry (2.2.24).
Preparation : to 1 mL add 40 mL of water R, cool in iced
A. 1-(4-chlorophenyl)-5-[6-[(cyanocarbamimidoyl)amino]- water, make alkaline to titan yellow paper R by adding
hexyl]biguanide, dropwise, and with stirring, strong sodium hydroxide

General Notices (1) apply to all monographs and other texts 1851
Chlorhexidine digluconate solution EUROPEAN PHARMACOPOEIA 8.0

solution R and add 1 mL in excess. Filter, wash the Related substances. Liquid chromatography (2.2.29). Store
precipitate with water R until the washings are free from the solutions at a temperature not exceeding 12 °C.
alkali and recrystallise from ethanol (70 per cent V/V) R. Test solution. Dilute 1.0 mL of the preparation to be examined
Dry at 100-105 °C. Examine the residue. to 100.0 mL with mobile phase A.
Comparison : chlorhexidine CRS. Reference solution (a). Dissolve the contents of a vial
B. Thin-layer chromatography (2.2.27). of chlorhexidine for system suitability CRS (containing
Test solution. Dilute 10.0 mL of the preparation to be impurities A, B, F, G, H, I, J, K, L, N and O) in 1.0 mL of
examined to 50 mL with water R. mobile phase A.
Reference solution. Dissolve 25 mg of calcium gluconate CRS Reference solution (b). Dilute 1.0 mL of the test solution to
in 1 mL of water R. 100.0 mL with mobile phase A.
Plate : TLC silica gel plate R. Column :
Mobile phase : concentrated ammonia R, ethyl acetate R, – size : l = 0.25 m, Ø = 4.6 mm ;
water R, ethanol (96 per cent) R (10:10:30:50 V/V/V/V). – stationary phase : end-capped octadecylsilyl silica gel for
Application : 5 μL. chromatography R (5 μm) ;
Development : over 1/2 of the plate. – temperature : 30 °C.
Drying : at 100 °C for 20 min and allow to cool. Mobile phase :
Detection : spray with a solution containing 25 g/L of – mobile phase A : mix 20 volumes of a solution containing
ammonium molybdate R and 10 g/L of cerium sulfate R in 0.1 per cent V/V of trifluoroacetic acid R in acetonitrile R
dilute sulfuric acid R, and heat at 110 °C for about 10 min. and 80 volumes of a solution containing 0.1 per cent V/V
Results : the principal spot in the chromatogram obtained of trifluoroacetic acid R in water R ;
with the test solution is similar in position, colour and size – mobile phase B : mix 10 volumes of a solution containing
to the principal spot in the chromatogram obtained with 0.1 per cent V/V of trifluoroacetic acid R in water R and
the reference solution. 90 volumes of a solution containing 0.1 per cent V/V of
C. To 1 mL add 40 mL of water R, cool in iced water, make trifluoroacetic acid R in acetonitrile R ;
alkaline to titan yellow paper R by adding dropwise, and
with stirring, strong sodium hydroxide solution R and add Time Mobile phase A Mobile phase B
1 mL in excess. Filter, wash the precipitate with water R (min) (per cent V/V) (per cent V/V)
until the washings are free from alkali and recrystallise 0-2 100 0
from ethanol (70 per cent V/V) R. Dry at 100-105 °C. The 2 - 32 100 → 80 0 → 20
residue melts (2.2.14) at 132 °C to 136 °C.
D. To 0.05 mL add 5 mL of a 10 g/L solution of cetrimide R, 32 - 37 80 20
1 mL of strong sodium hydroxide solution R and 1 mL of 37 - 47 80 → 70 20 → 30
bromine water R ; a deep red colour is produced.
47 - 54 70 30
TESTS
Flow rate : 1.0 mL/min.
Relative density (2.2.5) : 1.06 to 1.07.
Detection : spectrophotometer at 254 nm.
pH (2.2.3) : 5.5 to 7.0.
Dilute 5.0 mL to 100 mL with carbon dioxide-free water R. Injection : 10 μL.
Identification of impurities : use the chromatogram supplied
Impurity P (Chloroaniline) : maximum 500 ppm, calculated
with chlorhexidine for system suitability CRS and the
with reference to chlorhexidine digluconate solution.
chromatogram obtained with reference solution (a) to identify
Test solution. Dilute 0.20 g of the preparation to be examined the peaks due to impurities A, B, F, G, H, I, J, K, L, N and O.
to 30 mL with water R. Add rapidly and with thorough mixing
after each addition : 5 mL of a 103 g/L solution of hydrochloric Relative retention with reference to chlorhexidine
acid R, 0.35 mL of sodium nitrite solution R, 2 mL of a 50 g/L (retention time = about 35 min) : impurity L = about 0.23 ;
solution of ammonium sulfamate R, 5 mL of a 1 g/L solution impurity Q = about 0.24 ; impurity G = about 0.25 ;
of naphthylethylenediamine dihydrochloride R and 1 mL of impurity N = about 0.35 ; impurity B = about 0.36 ;
ethanol (96 per cent) R ; transfer quantitatively to a volumetric impurity F = about 0.5 ; impurity A = about 0.6 ;
flask, dilute to 50.0 mL with water R and allow to stand for impurity H = about 0.85 ; impurity O = about 0.90 ;
30 min. impurity I = about 0.91 ; impurity J = about 0.96 ;
impurity K = about 1.4.
Reference solutions. Prepare reference solutions containing
respectively 50 ppm, 100 ppm, 200 ppm, 500 ppm and System suitability : reference solution (a) :
600 ppm of chloroaniline R (impurity P) as follows : dilute – resolution : minimum 3.0 between the peaks due to
1.0 mL, 2.0 mL, 4.0 mL, 10.0 mL and 12.0 mL of a solution impurities L and G ;
containing 0.010 g/L of chloroaniline R (impurity P) in dilute – peak-to-valley ratio : minimum 2.0, where Hp = height
hydrochloric acid R to 20 mL with water R. Then, add 10 mL above the baseline of the peak due to impurity B and
of water R. Add rapidly and with thorough mixing after Hv = height above the baseline of the lowest point of the
each addition : 5 mL of a 103 g/L solution of hydrochloric curve separating this peak from the peak due to impurity N.
acid R, 0.35 mL of sodium nitrite solution R, 2 mL of a 50 g/L
solution of ammonium sulfamate R, 5 mL of a 1 g/L solution Limits :
of naphthylethylenediamine dihydrochloride R and 1 mL of – impurity N : not more than the area of the principal peak
ethanol (96 per cent) R ; transfer each solution quantitatively to in the chromatogram obtained with reference solution (b)
a volumetric flask, dilute to 50.0 mL with water R and allow to (1.0 per cent) ;
stand for 30 min. – impurity H : not more than 0.5 times the area of the
Measure the absorbance (2.2.25) of each reference solution principal peak in the chromatogram obtained with
and plot a calibration curve. reference solution (b) (0.5 per cent) ;
Measure the absorbance (2.2.25) of the test solution at – impurities A, J ,K : for each impurity, not more than
556 nm. Determine the concentration of chloroaniline from 0.4 times the area of the principal peak in the chromatogram
the calibration curve. obtained with reference solution (b) (0.4 per cent) ;

1852 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Chlorhexidine digluconate solution

– sum of impurities I and O : not more than 0.4 times the area
of the principal peak in the chromatogram obtained with
reference solution (b) (0.4 per cent) ;
– impurity G : not more than 0.3 times the area of the
principal peak in the chromatogram obtained with F. N-(4-chlorophenyl)urea,
reference solution (b) (0.3 per cent) ;
– impurities B, F, L, Q : for each impurity, not more than
0.2 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.2 per cent) ;
– unspecified impurities : for each impurity, not more than
0.1 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.10 per cent) ;
G. 1-(6-aminohexyl)-5-(4-chlorophenyl)biguanide,
– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(3.0 per cent) ;
– disregard limit : 0.05 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent).
ASSAY
Determine the density (2.2.5) of the preparation to be
examined. Transfer 1.00 g to a 250 mL beaker and add 50 mL
of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, H. 1,1′-[iminobis(carbonimidoyliminohexane-6,1-diyl)]bis[5-
determining the end-point potentiometrically (2.2.20). (4-chlorophenyl)biguanide],
1 mL of 0.1 M perchloric acid is equivalent to 22.44 mg I. unknown structure,
of C34H54Cl2N10O14.
STORAGE
Protected from light.
IMPURITIES
Specified impurities : A, B, F, G, H, I, J, K, L, N, O, P, Q.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use J. 1-(4-chlorophenyl)-5-[6-[[4-[(4-chlorophenyl)amino]-
(2034). It is therefore not necessary to identify these impurities 6-[(1S,2R,3R,4R)-1,2,3,4,5-pentahydroxypentyl]-1,3,5-
for demonstration of compliance. See also 5.10. Control of triazin-2-yl]amino]hexyl]biguanide,
impurities in substances for pharmaceutical use) : E, M.

K. N-(4-chlorophenyl)-N′-[[6-[[[(4-chlorophenyl)carbamimi-
doyl]carbamimidoyl]amino]hexyl]carbamimidoyl]urea,
A. 1-(4-chlorophenyl)-5-[6-[(cyanocarbamimidoyl)amino]-
hexyl]biguanide,

L. (5R,6S)-2-[(4-chlorophenyl)amino]-5-hydroxy-6-[(1R,2R)-
1,2,3-trihydroxypropyl]-5,6-dihydro-4H-1,3-oxazin-4-one,

B. N-[[6-[[[(4-chlorophenyl)carbamimidoyl]carbamimidoyl]-
amino]hexyl]carbamimidoyl]urea,

M. 1-(4-chlorophenyl)-5-[6-[[(phenylcarbamimidoyl)carba-
E. N-(4-chlorophenyl)guanidine, mimidoyl]amino]hexyl]biguanide,

General Notices (1) apply to all monographs and other texts 1853
Chlorhexidine dihydrochloride EUROPEAN PHARMACOPOEIA 8.0

titan yellow paper R by adding dropwise, and with stirring,

strong sodium hydroxide solution R and add 1 mL in excess.
Filter, wash the precipitate with water R until the washings
are free from alkali and recrystallise from ethanol (70 per
cent V/V) R. Dry at 100-105 °C. The residue melts (2.2.14)
at 132 °C to 136 °C.
D. It gives reaction (a) of chlorides (2.3.1).
N. 1-[6-(carbamimidoylamino)hexyl]-5-(4-chlorophenyl)- TESTS
biguanide, Chloroaniline : maximum 500 ppm.
To 0.20 g add 1 mL of hydrochloric acid R, shake for about
30 s, dilute to 30 mL with water R and shake until a clear
solution is obtained. Add rapidly and with thorough mixing
after each addition : 2.5 mL of dilute hydrochloric acid R,
0.35 mL of sodium nitrite solution R, 2 mL of a 50 g/L solution
of ammonium sulfamate R, 5 mL of a 1.0 g/L solution of
naphthylethylenediamine dihydrochloride R and 1 mL of
ethanol (96 per cent) R ; dilute to 50.0 mL with water R and
O. 1-(2-chlorophenyl)-5-[6-[[[(4-chlorophenyl)carbamimido- allow to stand for 30 min. Any reddish-blue colour in the
yl]carbamimidoyl]amino]hexyl]biguanide, solution is not more intense than that in a standard prepared
at the same time and in the same manner using a mixture of
10.0 mL of a 0.010 g/L solution of chloroaniline R in dilute
hydrochloric acid R and 20 mL of dilute hydrochloric acid R
instead of the solution of the substance to be examined.
P. 4-chloroaniline, Related substances. Liquid chromatography (2.2.29).
Q. unknown structure. Test solution. Dissolve 0.200 g of the substance to be examined
in the mobile phase and dilute to 100 mL with the mobile
phase.
01/2008:0659 Reference solution (a). Dissolve 15 mg of chlorhexidine for
corrected 7.0 performance test CRS in the mobile phase and dilute to
10.0 mL with the mobile phase.
CHLORHEXIDINE Reference solution (b). Dilute 2.5 mL of the test solution to
DIHYDROCHLORIDE 100 mL with the mobile phase.
Reference solution (c). Dilute 2.0 mL of reference solution (b)
Chlorhexidini dihydrochloridum to 10 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10 mL with the mobile phase.
Column :
– size : l = 0.2 m, Ø = 4 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : solution of 2.0 g of sodium octanesulfonate R in
a mixture of 120 mL of glacial acetic acid R, 270 mL of water R
and 730 mL of methanol R.
C22H32Cl4N10 Mr 578.4 Flow rate : 1.0 mL/min.
[3697-42-5] Detection : spectrophotometer at 254 nm.
Equilibration : with the mobile phase for at least 1 h.
DEFINITION
Injection : 10 μL.
1,1′-(Hexane-1,6-diyl)bis[5-(4-chlorophenyl)biguanide]
dihydrochloride. Run time : 6 times the retention time of chlorhexidine.
Content : 98.0 per cent to 101.0 per cent (dried substance). System suitability : reference solution (a) :
– the chromatogram obtained is similar to the chromatogram
CHARACTERS supplied with chlorhexidine for performance test CRS
Appearance : white or almost white, crystalline powder. in that the peaks due to impurity A and impurity B
Solubility : sparingly soluble in water and in propylene glycol, precede that due to chlorhexidine ; if necessary, adjust the
very slightly soluble in ethanol (96 per cent). concentration of acetic acid in the mobile phase (increasing
the concentration decreases the retention times).
IDENTIFICATION Limits :
First identification : A, D. – total : not more than the area of the principal peak in the
Second identification : B, C, D. chromatogram obtained with reference solution (b) (2.5 per
A. Infrared absorption spectrophotometry (2.2.24). cent) ;
Comparison : chlorhexidine dihydrochloride CRS. – disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (c)
B. Dissolve about 5 mg in 5 mL of a warm 10 g/L solution
(0.05 per cent) ; disregard any peak with a relative retention
of cetrimide R and add 1 mL of strong sodium hydroxide
time with reference to chlorhexidine of 0.25 or less.
solution R and 1 mL of bromine water R. A dark red colour
is produced. Loss on drying (2.2.32) : maximum 1.0 per cent, determined
C. Dissolve 0.3 g in 10 mL of a mixture of equal volumes of on 1.000 g by drying in an oven at 105 °C.
hydrochloric acid R and water R. Add 40 mL of water R, Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
filter if necessary and cool in iced water. Make alkaline to 1.0 g.

1854 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Chlorobutanol hemihydrate

ASSAY CHARACTERS
Dissolve 100.0 mg in 5 mL of anhydrous formic acid R and Appearance : white or almost white, crystalline powder or
add 70 mL of acetic anhydride R. Titrate with 0.1 M perchloric colourless crystals, sublimes readily.
acid, determining the end-point potentiometrically (2.2.20). Solubility : slightly soluble in water, very soluble in ethanol
1 mL of 0.1 M perchloric acid is equivalent to 14.46 mg (96 per cent), soluble in glycerol (85 per cent).
of C22H32Cl4N10. mp : about 95 °C (without previous drying).
IMPURITIES IDENTIFICATION
A. Add about 20 mg to a mixture of 1 mL of pyridine R and
2 mL of strong sodium hydroxide solution R. Heat in a
water-bath and shake. Allow to stand. The pyridine layer
becomes red.
B. Add about 20 mg to 5 mL of ammoniacal silver nitrate
solution R and warm slightly. A black precipitate is formed.
C. To about 20 mg add 3 mL of 1 M sodium hydroxide and
shake to dissolve. Add 5 mL of water R and then, slowly,
A. 1-(4-chlorophenyl)-5-[6-[(cyanocarbamimidoyl)amino]- 2 mL of iodinated potassium iodide solution R. A yellowish
hexyl]biguanide, precipitate is formed.
D. Water (see Tests).
TESTS
Solution S. Dissolve 5 g in ethanol (96 per cent) R and dilute
to 10 mL with the same solvent.
Appearance of solution. Solution S is not more opalescent
than reference suspension II (2.2.1) and not more intensely
coloured than reference solution BY5 (2.2.2, Method II).
B. [[6-[[[(4-chlorophenyl)carbamimidoyl]carbamimidoyl]-
amino]hexyl]carbamimidoyl]urea, Acidity. To 4 mL of solution S add 15 mL of ethanol (96 per
cent) R and 0.1 mL of bromothymol blue solution R1. Not
more than 1.0 mL of 0.01 M sodium hydroxide is required to
change the colour of the indicator to blue.
Chlorides (2.4.4) : maximum 300 ppm.
Dissolve 0.17 g in 5 mL of ethanol (96 per cent) R and dilute
to 15 mL with water R. When preparing the standard, replace
the 5 mL of water R by 5 mL of ethanol (96 per cent) R.
Water (2.5.12) : maximum 1.0 per cent, determined on 2.00 g.
C. 1,1′-[hexane-1,6-diylbis(iminocarbonimidoyl)]bis[3-(4- Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
chlorophenyl)urea], 1.0 g.
ASSAY
Dissolve 0.100 g in 20 mL of ethanol (96 per cent) R. Add 10 mL
of dilute sodium hydroxide solution R, heat in a water-bath for
5 min and cool. Add 20 mL of dilute nitric acid R, 25.0 mL of
0.1 M silver nitrate and 2 mL of dibutyl phthalate R and shake
vigorously. Add 2 mL of ferric ammonium sulfate solution R2
and titrate with 0.1 M ammonium thiocyanate until an orange
colour is obtained.
1 mL of 0.1 M silver nitrate is equivalent to 5.92 mg
of C4H7Cl3O.
D. 1,1′-[[[[(4-chlorophenyl)carbamimidoyl]imino]-
methylene]bis[imino(hexane-1,6-diyl)]]bis[5-(4- STORAGE
chlorophenyl)biguanide]. In an airtight container.

01/2008:0382 01/2008:0383
corrected 6.0 corrected 6.0

CHLOROBUTANOL, ANHYDROUS CHLOROBUTANOL HEMIHYDRATE

Chlorobutanolum anhydricum Chlorobutanolum hemihydricum

C4H7Cl3O Mr 177.5 C4H7Cl3O,½H2O Mr 186.5

[57-15-8] [6001-64-5]
DEFINITION DEFINITION
1,1,1-Trichloro-2-methylpropan-2-ol. 1,1,1-Trichloro-2-methylpropan-2-ol hemihydrate.
Content : 98.0 per cent to 101.0 per cent (anhydrous substance). Content : 98.0 per cent to 101.0 per cent (anhydrous substance).

General Notices (1) apply to all monographs and other texts 1855
Chlorocresol EUROPEAN PHARMACOPOEIA 8.0

CHARACTERS Content : 98.0 per cent to 101.0 per cent.

Appearance : white or almost white, crystalline powder or CHARACTERS
colourless crystals, sublimes readily.
Appearance : white or almost white, crystalline powder or
Solubility : slightly soluble in water, very soluble in ethanol compacted crystalline masses supplied as pellets or colourless
(96 per cent), soluble in glycerol (85 per cent). or white crystals.
mp : about 78 °C (without previous drying).
Solubility : slightly soluble in water, very soluble in ethanol
IDENTIFICATION (96 per cent), freely soluble in fatty oils. It dissolves in
A. Add about 20 mg to a mixture of 1 mL of pyridine R and solutions of alkali hydroxides.
2 mL of strong sodium hydroxide solution R. Heat in a IDENTIFICATION
water-bath and shake. Allow to stand. The pyridine layer
becomes red. A. Melting point (2.2.14) : 64 °C to 67 °C.
B. Add about 20 mg to 5 mL of ammoniacal silver nitrate B. To 0.1 g add 0.2 mL of benzoyl chloride R and 0.5 mL of
solution R and warm slightly. A black precipitate is formed. dilute sodium hydroxide solution R. Shake vigorously until
a white, crystalline precipitate is formed. Add 5 mL of
C. To about 20 mg add 3 mL of 1 M sodium hydroxide and water R and filter. The precipitate, recrystallised from 5 mL
shake to dissolve. Add 5 mL of water R and then, slowly, of methanol R and dried at 70 °C, melts (2.2.14) at 85 °C
2 mL of iodinated potassium iodide solution R. A yellowish to 88 °C.
precipitate is formed.
C. To 5 mL of solution S (see Tests) add 0.1 mL of ferric
D. Water (see Tests). chloride solution R1. A bluish colour is produced.
TESTS
TESTS
Solution S. Dissolve 5 g in ethanol (96 per cent) R and dilute
Solution S. To 3.0 g, finely powdered, add 60 mL of carbon
to 10 mL with the same solvent.
dioxide-free water R, shake for 2 min and filter.
Appearance of solution. Solution S is not more opalescent
than reference suspension II (2.2.1) and not more intensely Appearance of solution. The solution is clear (2.2.1) and not
coloured than reference solution BY5 (2.2.2, Method II). more intensely coloured than reference solution BY6 (2.2.2,
Method II).
Acidity. To 4 mL of solution S add 15 mL of ethanol (96 per Dissolve 1.25 g in ethanol (96 per cent) R and dilute to 25 mL
cent) R and 0.1 mL of bromothymol blue solution R1. Not with the same solvent.
more than 1.0 mL of 0.01 M sodium hydroxide is required to
change the colour of the indicator to blue. Acidity. To 10 mL of solution S add 0.1 mL of methyl red
solution R. The solution is orange or red. Not more than
Chlorides (2.4.4) : maximum 100 ppm.
0.2 mL of 0.01 M sodium hydroxide is required to produce a
To 1 mL of solution S add 4 mL of ethanol (96 per cent) R and pure yellow colour.
dilute to 15 mL with water R. When preparing the standard,
replace the 5 mL of water R by 5 mL of ethanol (96 per cent) R. Related substances. Gas chromatography (2.2.28) : use the
normalisation procedure.
Water (2.5.12) : 4.5 per cent to 5.5 per cent, determined on
0.300 g. Test solution. Dissolve 1.0 g of the substance to be examined
in acetone R and dilute to 100 mL with the same solvent.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Reference solution. Dilute 1.0 mL of the test solution to
1.0 g.
100.0 mL with acetone R. Dilute 5.0 mL of this solution to
ASSAY 100.0 mL with acetone R.
Dissolve 0.100 g in 20 mL of ethanol (96 per cent) R. Add 10 mL Column :
of dilute sodium hydroxide solution R, heat in a water-bath for – material : glass ;
5 min and cool. Add 20 mL of dilute nitric acid R, 25.0 mL of – size : l = 1.80 m, Ø = 3-4 mm ;
0.1 M silver nitrate and 2 mL of dibutyl phthalate R and shake
vigorously. Add 2 mL of ferric ammonium sulfate solution R2 – stationary phase : silanised diatomaceous earth for gas
and titrate with 0.1 M ammonium thiocyanate until an orange chromatography R impregnated with 3-5 per cent m/m of
colour is obtained. polymethylphenylsiloxane R.
1 mL of 0.1 M silver nitrate is equivalent to 5.92 mg Carrier gas : nitrogen for chromatography R.
of C4H7Cl3O. Flow rate : 30 mL/min.
Temperature :
STORAGE
In an airtight container. – column : 125 °C ;
– injection port : 210 °C ;
– detector : 230 °C.
01/2011:0384
Detection : flame ionisation.
CHLOROCRESOL Run time : 3 times the retention time of chlorocresol.
Retention time : chlorocresol = about 8 min.
Chlorocresolum Limits :
– unspecified impurities : for each impurity, maximum
0.10 per cent ;
– total : maximum 1 per cent ;
– disregard limit : the area of the principal peak in the
chromatogram obtained with the reference solution
C7H7ClO Mr 142.6 (0.05 per cent).
[59-50-7]
Non-volatile matter : maximum 0.1 per cent.
DEFINITION Evaporate 2.0 g to dryness on a water-bath and dry the residue
4-Chloro-3-methylphenol. at 100-105 °C. The residue weighs not more than 2 mg.

1856 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Chloroquine sulfate

ASSAY C. Dissolve 25 mg in 20 mL of water R and add 8 mL of picric

In a ground-glass-stoppered flask, dissolve 70.0 mg in 30 mL acid solution R1. The precipitate, washed with water R,
of glacial acetic acid R. Add 25.0 mL of 0.0167 M potassium with alcohol R and finally with methylene chloride R, melts
bromate, 20 mL of a 150 g/L solution of potassium bromide R (2.2.14) at 206-209 °C.
and 10 mL of hydrochloric acid R. Allow to stand protected D. Dissolve 0.1 g in 10 mL of water R, add 2 mL of dilute
from light for 15 min. Add 1 g of potassium iodide R and sodium hydroxide solution R and shake with 2 quantities,
100 mL of water R. Titrate with 0.1 M sodium thiosulfate, each of 20 mL, of methylene chloride R. The aqueous layer,
shaking vigorously and using 1 mL of starch solution R, added acidified by the addition of nitric acid R, gives reaction (b)
towards the end of the titration, as indicator. Carry out a of phosphates (2.3.1).
blank titration.
TESTS
1 mL of 0.0167 M potassium bromate is equivalent to 3.565 mg
of C7H7ClO. Solution S. Dissolve 2.5 g in carbon dioxide-free water R and
dilute to 25 mL with the same solvent.
STORAGE Appearance of solution. Solution S is clear (2.2.1) and not
Protected from light. more intensely coloured than reference solution BY5 or GY5
(2.2.2, Method II).
pH (2.2.3). The pH of solution S is 3.8 to 4.3.
01/2008:0544 Related substances. Examine by thin-layer chromatography
corrected 6.0 (2.2.27), using silica gel GF254 R as the coating substance.
Test solution. Dissolve 0.50 g of the substance to be examined
CHLOROQUINE PHOSPHATE in water R and dilute to 10 mL with the same solvent.
Reference solution (a). Dilute 1 mL of the test solution to
100 mL with water R.
Chloroquini phosphas Reference solution (b). Dilute 5 mL of reference solution (a)
to 10 mL with water R.
Apply to the plate 2 μL of each solution. Develop over a path
of 12 cm using a mixture of 10 volumes of diethylamine R,
40 volumes of cyclohexane R and 50 volumes of chloroform R.
Allow the plate to dry in air. Examine in ultraviolet light at
254 nm. Any spot in the chromatogram obtained with the test
solution, apart from the principal spot, is not more intense
than the spot in the chromatogram obtained with reference
C18H32ClN3O8P2 Mr 515.9 solution (a) (1.0 per cent) and not more than one such spot
[50-63-5] is more intense than the spot in the chromatogram obtained
with reference solution (b) (0.5 per cent).
DEFINITION
Heavy metals (2.4.8). Dissolve 2.0 gin 10 mL of water R. Add
Chloroquine phosphate contains not less than 98.5 per
5 mL of concentrated ammonia R and shake with 40 mL of
cent and not more than the equivalent of 101.0 per cent of
methylene chloride R. Filter the aqueous layer and neutralise
N -(7-chloroquinolin-4-yl)-N ,N -diethylpentane-1,4-diamine
4 1 1
the filtrate with glacial acetic acid R. Heat on a water-bath
bis(dihydrogen phosphate), calculated with reference to the
to eliminate methylene chloride, allow to cool and dilute
dried substance.
to 20.0 mL with water R. 12 mL of this solution complies
CHARACTERS with test A for heavy metals (20 ppm). Prepare the reference
solution using lead standard solution (2 ppm Pb) R.
A white or almost white, crystalline powder, hygroscopic,
freely soluble in water, very slightly soluble in alcohol and in Loss on drying (2.2.32) : maximum 2.0 per cent, determined
methanol. on 1.000 g by drying in an oven at 105 °C.
It exists in 2 forms, one of which melts at about 195 °C and the ASSAY
other at about 218 °C. Dissolve 0.200 g in 50 mL of anhydrous acetic acid R.
IDENTIFICATION Titrate with 0.1 M perchloric acid determining the end-point
potentiometrically (2.2.20).
First identification : B, D.
1 mL of 0.1 M perchloric acid is equivalent to 25.79 mg of
Second identification : A, C, D. C18H32ClN3O8P2.
A. Dissolve 0.100 g in water R and dilute to 100.0 mL with the
same solvent. Dilute 1.0 mL of this solution to 100.0 mL STORAGE
with water R. Examined between 210 nm and 370 nm In an airtight container, protected from light.
(2.2.25), the solution shows absorption maxima at 220 nm,
235 nm, 256 nm, 329 nm and 342 nm. The specific 01/2008:0545
absorbances at the maxima are respectively 600 to 660, 350
to 390, 300 to 330, 325 to 355 and 360 to 390. CHLOROQUINE SULFATE
B. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with Chloroquini sulfas
the base isolated from chloroquine sulfate CRS. Record
the spectra using solutions prepared as follows : dissolve
separately 0.1 g of the substance to be examined and 80 mg
of the reference substance in 10 mL of water R, add 2 mL
of dilute sodium hydroxide solution R and shake with 2
quantities, each of 20 mL, of methylene chloride R ; combine
the organic layers, wash with water R, dry over anhydrous
sodium sulfate R, evaporate to dryness and dissolve the
residues separately, each in 2 mL of methylene chloride R. C18H28ClN3O4S,H2O Mr 436.0

General Notices (1) apply to all monographs and other texts 1857
Chlorphenamine maleate EUROPEAN PHARMACOPOEIA 8.0

DEFINITION allow to cool and dilute to 20.0 mL with water R. 12 mL of this

Chloroquine sulfate contains not less than 98.5 per cent solution complies with test A (20 ppm). Prepare the reference
and not more than the equivalent of 101.0 per cent of solution using lead standard solution (2 ppm Pb) R.
N4-(7-chloroquinolin-4-yl)-N1,N1-diethylpentane-1,4-diamine Water (2.5.12) : 3.0 per cent to 5.0 per cent, determined on
sulfate, calculated with reference to the anhydrous substance. 0.500 g.
CHARACTERS Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
on 1.0 g.
A white or almost white, crystalline powder, freely soluble in
water and in methanol, very slightly soluble in ethanol (96 per ASSAY
cent). Dissolve 0.400 g in 50 mL of anhydrous acetic acid R.
It melts at about 208 °C (instantaneous method). Titrate with 0.1 M perchloric acid determining the end-point
potentiometrically (2.2.20).
IDENTIFICATION
1 mL of 0.1 M perchloric acid is equivalent to 41.8 mg of
First identification : B, D. C18H28ClN3O4S.
Second identification : A, C, D.
A. Dissolve 0.100 g in water R and dilute to 100.0 mL with the STORAGE
same solvent. Dilute 1.0 mL of this solution to 100.0 mL Store in an airtight container, protected from light.
with water R. Examined between 210 nm and 370 nm
(2.2.25), the solution shows absorption maxima at 220 nm, 04/2008:0386
235 nm, 256 nm, 329 nm and 342 nm. The specific
absorbances at the maxima are respectively 730 to 810, 430 CHLORPHENAMINE MALEATE
to 470, 370 to 410, 400 to 440 and 430 to 470.
B. Examine by infrared absorption spectrophotometry Chlorphenamini maleas
(2.2.24), comparing with the spectrum obtained with
the base isolated from chloroquine sulfate CRS. Record
the spectra using solutions prepared as follows : dissolve
separately 0.1 g of the substance to be examined and of the
reference substance in 10 mL of water R, add 2 mL of dilute
sodium hydroxide solution R and shake with 2 quantities,
each of 20 mL, of methylene chloride R ; combine the
organic layers, wash with water R, dry over anhydrous C20H23ClN2O4 Mr 390.9
sodium sulfate R, evaporate to dryness and dissolve the [113-92-8]
residues separately each in 2 mL of methylene chloride R.
C. Dissolve 25 mg in 20 mL of water R and add 8 mL of picric DEFINITION
acid solution R1. The precipitate, washed with water R, (3RS)-3-(4-Chlorophenyl)-N,N-dimethyl-3-(pyridin-2-
with ethanol (96 per cent) R and finally with ether R, melts yl)propan-1-amine hydrogen (Z)-butenedioate.
(2.2.14) at 206 °C to 209 °C. Content : 98.0 per cent to 101.0 per cent (dried substance).
D. It gives reaction (a) of sulfates (2.3.1).
CHARACTERS
TESTS Appearance : white or almost white, crystalline powder.
Solution S. Dissolve 2.0 g in carbon dioxide-free water R and Solubility : freely soluble in water, soluble in ethanol (96 per
dilute to 25 mL with the same solvent. cent).
Appearance of solution. Solution S is clear (2.2.1) and not IDENTIFICATION
more intensely coloured than reference solution BY5 or GY5
(2.2.2, Method II). A. Melting point (2.2.14) : 130 °C to 135 °C.
B. Infrared absorption spectrophotometry (2.2.24).
pH (2.2.3). The pH of solution S is 4.0 to 5.0.
Comparison: chlorphenamine maleate CRS.
Related substances. Examine by thin-layer chromatography
C. Optical rotation (see Tests).
(2.2.27), using silica gel GF254 R as the coating substance.
Test solution. Dissolve 0.50 g of the substance to be examined TESTS
in water R and dilute to 10 mL with the same solvent. Solution S. Dissolve 2.0 g in water R and dilute to 20.0 mL
Reference solution (a). Dilute 1 mL of the test solution to with the same solvent.
100 mL with water R. Appearance of solution. Solution S is clear (2.2.1) and not
Reference solution (b). Dilute 5 mL of reference solution (a) more intensely coloured than reference solution BY6 (2.2.2,
to 10 mL with water R. Method II).
Apply separately to the plate 2 μL of each solution. Develop Optical rotation (2.2.7): − 0.10° to + 0.10°, determined on
over a path of 12 cm using a mixture of 10 volumes of solution S.
diethylamine R, 40 volumes of cyclohexane R and 50 volumes
of methylene chloride R. Allow the plate to dry in air. Examine Related substances. Liquid chromatography (2.2.29).
in ultraviolet light at 254 nm. Any spot in the chromatogram Test solution. Dissolve 0.100 g of the substance to be examined
obtained with the test solution, apart from the principal in the mobile phase and dilute to 100.0 mL with the mobile
spot, is not more intense than the spot in the chromatogram phase.
obtained with reference solution (a) (1.0 per cent) and not Reference solution (a). Dilute 0.5 mL of the test solution to
more than one such spot is more intense than the spot in the 100.0 mL with the mobile phase.
chromatogram obtained with reference solution (b) (0.5 per Reference solution (b). Dilute 1.0 mL of reference solution (a)
cent). to 10.0 mL with the mobile phase.
Heavy metals (2.4.8). Dissolve 2.0 g in 10 mL of water R. Add Reference solution (c). Dissolve 5 mg of chlorphenamine
5 mL of concentrated ammonia R and shake with 40 mL of impurity C CRS in 5 mL of the test solution and dilute to
ether R. Filter the aqueous layer and neutralise the filtrate with 50.0 mL with the mobile phase. Dilute 2 mL of this solution to
glacial acetic acid R. Heat on a water-bath to eliminate ether, 20 mL with the mobile phase.

1858 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Chlorpromazine hydrochloride

Reference solution (d). Dissolve 5 mg of 2,2′-dipyridylamine R

(impurity B) in the mobile phase and dilute to 100 mL with
the mobile phase.
Reference solution (e). Dissolve the contents of a vial of
chlorphenamine impurity A CRS in 2 mL of the test solution.
Sonicate for 5 min.
Column : A. 2-(4-chlorophenyl)-4-(dimethylamino)-2-[2-
– size : l = 0.30 m, Ø = 3.9 mm ; (dimethylamino)ethyl]butanenitrile,
– stationary phase : octadecylsilyl silica gel for
chromatography R (10 μm).
Mobile phase : mix 20 volumes of acetonitrile R and 80 volumes
of a 8.57 g/L solution of ammonium dihydrogen phosphate R
previously adjusted to pH 3.0 with phosphoric acid R. B. N-(pyridin-2-yl)pyridin-2-amine (2,2′-dipyridylamine),
Flow rate : 1.2 mL/min.
Detection : spectrophotometer at 225 nm.
Injection : 20 μL.
Run time : 3.5 times the retention time of chlorphenamine.
Relative retention with reference to chlorphenamine
(retention time = about 11 min) : maleic acid = about 0.2 ;
impurity A = about 0.3 ; impurity B = about 0.4 ; C. (3RS)-3-(4-chlorophenyl)-N-methyl-3-(pyridin-2-
impurity C = about 0.9 ; impurity D = about 3.0. yl)propan-1-amine,
System suitability: reference solution (c) :
– resolution : minimum 1.5 between the peaks due to
impurity C and chlorphenamine.
Limits :
– correction factors : for the calculation of contents,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity A = 1.5 ; D. (2RS)-2-(4-chlorophenyl)-4-(dimethylamino)-2-(pyridin-
impurity B = 1.4 ; 2-yl)butanenitrile.
– impurity A : not more than 0.4 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent) ; 07/2012:0475
– impurities B, C, D : for each impurity, not more than
0.2 times the area of the principal peak in the chromatogram CHLORPROMAZINE
obtained with reference solution (a) (0.1 per cent) ; HYDROCHLORIDE
– unspecified impurities : for each impurity, not more than
0.2 times the area of the principal peak in the chromatogram Chlorpromazini hydrochloridum
obtained with reference solution (a) (0.10 per cent) ;
– total : not more than the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.5 per
cent) ;
– disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.05 per cent) ; disregard the peaks due to the blank and
maleic acid.
C17H20Cl2N2S Mr 355.3
Heavy metals (2.4.8) : maximum 20 ppm. [69-09-0]
1.0 g complies with test C. Prepare the reference solution using
2 mL of lead standard solution (10 ppm Pb) R. DEFINITION
Loss on drying (2.2.32) : maximum 0.5 per cent, determined 3-(2-Chloro-10H-phenothiazin-10-yl)-N,N-dimethylpropan-
on 1.000 g by drying in an oven at 105 °C for 4 h. 1-amine hydrochloride.
Content : 99.0 per cent to 101.0 per cent (dried substance).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. CHARACTERS
ASSAY Appearance : white or almost white, crystalline powder.
Dissolve 0.150 g in 25 mL of anhydrous acetic acid R. Titrate Solubility : very soluble in water, freely soluble in ethanol
with 0.1 M perchloric acid, determining the end-point (96 per cent).
potentiometrically (2.2.20). It decomposes on exposure to air and light.
1 mL of 0.1 M perchloric acid is equivalent to 19.54 mg It shows polymorphism (5.9).
of C20H23ClN2O4.
IDENTIFICATION
STORAGE First identification : B, D.
Protected from light. Second identification : A, C, D.
A. Ultraviolet and visible absorption spectrophotometry
IMPURITIES (2.2.25). Prepare the solutions protected from bright light
Specified impurities : A, B, C, D. and measure the absorbances immediately.

General Notices (1) apply to all monographs and other texts 1859
Chlorpromazine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Test solution. Dissolve 50.0 mg in a 10.3 g/L solution of Reference solution (c). Dissolve 4.0 mg of chlorpromazine
hydrochloric acid R and dilute to 500.0 mL with the same impurity A CRS in the mobile phase and dilute to 100.0 mL
solution. Dilute 5.0 mL of the solution to 100.0 mL with a with the mobile phase. Dilute 1.0 mL of the solution to
10.3 g/L solution of hydrochloric acid R. 100.0 mL with the mobile phase.
Spectral range : 230-340 nm. Reference solution (d). Dissolve 4 mg of promazine
Absorption maxima : at 254 nm and 306 nm. hydrochloride CRS (impurity C) and 4.0 mg of chlorpromazine
impurity E CRS in the mobile phase and dilute to 100.0 mL
Specific absorbance at the absorption maximum at 254 nm :
with the mobile phase. Dilute 1.0 mL of the solution to
890 to 960.
100.0 mL with the mobile phase.
B. Infrared absorption spectrophotometry (2.2.24).
Column :
Preparation : 60 g/L solutions in methylene chloride R using
– size : l = 0.25 m, Ø = 4.0 mm ;
a 0.1 mm cell.
Comparison : chlorpromazine hydrochloride CRS. – stationary phase : base-deactivated octylsilyl silica gel for
chromatography R (5 μm).
C. Identification of phenothiazines by thin-layer
chromatography (2.3.3) : use chlorpromazine Mobile phase : mix 0.2 volumes of thiodiethylene glycol R with
hydrochloride CRS to prepare the reference solution. 50 volumes of acetonitrile R and 50 volumes of a 0.5 per
cent V/V solution of trifluoroacetic acid R previously adjusted
D. It gives reaction (b) of chlorides (2.3.1). to pH 5.3 with tetramethylethylenediamine R.
TESTS Flow rate : 1.0 mL/min.
pH (2.2.3) : 3.5 to 4.5. Carry out the test protected from light Detection : spectrophotometer at 254 nm.
and use freshly prepared solutions. Injection : 10 μL.
Dissolve 1.0 g in carbon dioxide-free water R and dilute to Run time : 4 times the retention time of chlorpromazine.
10 mL with the same solvent. Identification of impurities: use the chromatogram obtained
Impurity F. Thin-layer chromatography (2.2.27). Prepare the with reference solution (c) to identify the peak due to
solutions immediately before use and protect from light. impurity A ; use the chromatogram obtained with reference
Solvent mixture : diethylamine R, methanol R (5:95 V/V). solution (d) to identify the peaks due to impurities C and E ;
use the chromatogram obtained with reference solution (a) to
Test solution. Dissolve 0.100 g of the substance to be examined identify the peak due to impurity D.
in the solvent mixture and dilute to 5.0 mL with the solvent
mixture. Relative retention with reference to chlorpromazine
(retention time = about 8 min) : impurity A = about 0.4 ;
Reference solution (a). Dissolve the contents of a vial of impurity B = about 0.5 ; impurity C = about 0.7 ;
chlorpromazine impurity F CRS in 2.0 mL of the solvent impurity D = about 0.9 ; impurity E = about 3.4.
mixture.
System suitability : reference solution (a) :
Reference solution (b). Dilute 300 μL of reference solution (a)
to 10.0 mL with the solvent mixture. – resolution : minimum 2.0 between the peaks due to
impurity D and chlorpromazine.
Reference solution (c). Dissolve 0.10 g of the substance to be
examined in the solvent mixture, add 1.0 mL of reference Limits :
solution (a) and dilute to 5.0 mL with the solvent mixture. – impurities B, C, D : for each impurity, not more than
Plate : TLC silica gel F254 plate R. 0.6 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.3 per cent) ;
Mobile phase : acetone R, diethylamine R, cyclohexane R
(10:10:80 V/V/V). – impurity A : not more than 1.5 times the area of the
corresponding peak in the chromatogram obtained with
Application : 10 μL of the test solution and reference reference solution (c) (0.15 per cent) ;
solutions (b) and (c).
– impurity E : not more than 1.5 times the area of the
Development : over 3/4 of the plate. corresponding peak in the chromatogram obtained with
Drying : in air. reference solution (d) (0.15 per cent) ;
Detection : examine in ultraviolet light at 254 nm. – unspecified impurities : for each impurity, not more than
Retardation factors : impurity F = about 0.5 ; 0.2 times the area of the principal peak in the chromatogram
chlorpromazine = about 0.6. obtained with reference solution (b) (0.10 per cent) ;
System suitability: reference solution (c) : – total : maximum 1.0 per cent ;
– the chromatogram shows 2 clearly separated spots due to – disregard limit : 0.1 times the area of the principal peak in
impurity F and chlorpromazine. the chromatogram obtained with reference solution (b)
Limit : (0.05 per cent).
– impurity F : any spot due to impurity F is not more intense Heavy metals (2.4.8) : maximum 10 ppm.
than the spot in the chromatogram obtained with reference Solvent : water R.
solution (b) (0.15 per cent). 0.25 g complies with test H. Prepare the reference solution
Related substances. Liquid chromatography (2.2.29). Prepare using 0.25 mL of lead standard solution (10 ppm Pb) R.
the solutions immediately before use and protect from light. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Test solution. Dissolve 40.0 mg of the substance to be on 1.000 g by drying in an oven at 105 °C.
examined in the mobile phase and dilute to 100.0 mL with
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
the mobile phase.
1.0 g.
Reference solution (a). Dissolve 4 mg of chlorpromazine
impurity D CRS in the mobile phase and dilute to 10.0 mL ASSAY
with the mobile phase. To 1 mL of the solution add 1 mL of Dissolve 0.250 g in a mixture of 5.0 mL of 0.1 M hydrochloric
the test solution and dilute to 100.0 mL with the mobile phase. acid and 50 mL of ethanol (96 per cent) R. Carry out a
Reference solution (b). Dilute 1.0 mL of the test solution to potentiometric titration (2.2.20), using 0.1 M sodium
20.0 mL with the mobile phase. Dilute 1.0 mL of this solution hydroxide. Read the volume added between the 2 points of
to 10.0 mL with the mobile phase. inflexion.

1860 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Chlorpropamide

1 mL of 0.1 M sodium hydroxide is equivalent to 35.53 mg 01/2008:1087

of C17H20Cl2N2S. corrected 6.0

STORAGE CHLORPROPAMIDE
In an airtight container, protected from light.
Chlorpropamidum
IMPURITIES
Specified impurities : A, B, C, D, E, F.

C10H13ClN2O3S Mr 276.7
[94-20-2]
DEFINITION
Chlorpropamide contains not less than 99.0 per cent
and not more than the equivalent of 101.0 per cent of
A. 3-(2-chloro-10H-phenothiazin-10-yl)-N,N- 1-[(4-chlorophenyl)sulfonyl]-3-propylurea, calculated with
dimethylpropan-1-amine S-oxide (chlorpromazine reference to the dried substance.
sulfoxide),
CHARACTERS
A white or almost white, crystalline powder, practically
insoluble in water, freely soluble in acetone and in methylene
chloride, soluble in alcohol. It dissolves in dilute solutions of
alkali hydroxides.
It shows polymorphism (5.9).
IDENTIFICATION
B. N-[3-(2-chloro-10H-phenothiazin-10-yl)propyl]-N,N′,N′- First identification : C, D.
trimethylpropane-1,3-diamine, Second identification : A, B, D.
A. Melting point (2.2.14) : 126 °C to 130 °C.
B. Dissolve 0.10 g in methanol R and dilute to 50.0 mL
with the same solvent. Dilute 5.0 mL of the solution to
100.0 mL with 0.01 M hydrochloric acid. Dilute 10.0 mL
of the solution to 100.0 mL with 0.01 M hydrochloric
acid. Examined between 220 nm and 350 nm (2.2.25), the
solution shows an absorption maximum at 232 nm. The
specific absorption at the maximum is 570 to 630.
C. 3-(10H-phenothiazin-10-yl)-N,N-dimethylpropan-1- C. Examine by infrared absorption spectrophotometry
amine (promazine), (2.2.24), comparing with the spectrum obtained with
chlorpropamide CRS. Examine the substances prepared as
discs. If the spectra obtained show differences, dissolve the
substance to be examined and the reference substance in
methylene chloride R, evaporate to dryness and record the
new spectra using the residues.
D. Heat 0.1 g with 2 g of anhydrous sodium carbonate R until
a dull red colour appears for 10 min. Allow to cool, extract
the residue with about 5 mL of water R, dilute to 10 mL
with water R and filter. The solution gives the reaction (a)
D. 3-(2-chloro-10H-phenothiazin-10-yl)-N-methylpropan-1- of chloride (2.3.1).
amine (desmethylchlorpromazine),
TESTS
Related substances. Examine by thin-layer chromatography
(2.2.27), using a suitable silica gel as the coating substance.
Test solution. Dissolve 0.50 g of the substance to be examined
in acetone R and dilute to 10 mL with the same solvent.
Reference solution (a). Dissolve 15 mg of 4-chlorobenzenesul-
fonamide R (chlorpropamide impurity A) in acetone R and
dilute to 100 mL with the same solvent.
E. 2-chloro-10H-phenothiazine, Reference solution (b). Dissolve 15 mg of chlorpropamide
impurity B CRS in acetone R and dilute to 100 mL with the
same solvent.
Reference solution (c). Dilute 0.3 mL of the test solution to
100 mL with acetone R.
Reference solution (d). Dilute 5 mL of reference solution (c) to
15 mL with acetone R.
Reference solution (e). Dissolve 0.10 g of the substance to be
examined, 5 mg of 4-chlorobenzenesulfonamide R and 5 mg
F. 3-(4-chloro-10H-phenothiazin-10-yl)-N,N- of chlorpropamide impurity B CRS in acetone R and dilute to
dimethylpropan-1-amine. 10 mL with the same solvent.

General Notices (1) apply to all monographs and other texts 1861
Chlorprothixene hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Apply to the plate 5 μL of each solution. Develop over a path 01/2008:0815

of 15 cm using a mixture of 11.5 volumes of concentrated
ammonia R, 30 volumes of cyclohexane R, 50 volumes of
methanol R and 100 volumes of methylene chloride R. Allow CHLORPROTHIXENE
the plate to dry in a current of cold air, heat at 110 °C for HYDROCHLORIDE
10 min. At the bottom of a chromatographic tank, place
an evaporating dish containing a mixture of 1 volume of
hydrochloric acid R, 1 volume of water R and 2 volumes of a Chlorprothixeni hydrochloridum
50 g/L solution of potassium permanganate R, close the tank
and allow to stand for 15 min. Place the dried hot plate in the
tank and close the tank. Leave the plate in contact with the
chlorine vapour for 2 min. Withdraw the plate and place it in
a current of cold air until the excess of chlorine is removed
and an area of coating below the points of application does not
give a blue colour with a drop of potassium iodide and starch
solution R. Spray with potassium iodide and starch solution R.
In the chromatogram obtained with the test solution : any spot C18H19Cl2NS Mr 352.3
corresponding to impurity A is not more intense than the spot [6469-93-8]
in the chromatogram obtained with reference solution (a)
(0.3 per cent) ; any spot corresponding to impurity B is not DEFINITION
more intense than the spot in the chromatogram obtained (Z)-3-(2-Chloro-9H-thioxanthen-9-ylidene)-N,N-
with reference solution (b) (0.3 per cent) ; any spot, apart from dimethylpropan-1-amine hydrochloride.
the principal spot and any spot corresponding to impurity A
and B, is not more intense than the spot in the chromatogram Content : 99.0 per cent to 101.0 per cent (dried substance).
obtained with reference solution (c) (0.3 per cent) ; not more
than two such spots are more intense than the spot in the CHARACTERS
chromatogram obtained with reference solution (d) (0.1 per Appearance : white or almost white, crystalline powder.
cent). The test is not valid unless the chromatogram obtained Solubility : soluble in water and in alcohol, slightly soluble in
with reference solution (e) shows three clearly separated spots methylene chloride.
with approximate RF values of 0.4, 0.6 and 0.9 corresponding
to chlorpropamide, impurity A and impurity B respectively. mp : about 220 °C.
Heavy metals (2.4.8). Dissolve 2.0 g in a mixture of IDENTIFICATION
15 volumes of water R and 85 volumes of acetone R and dilute
to 20 mL with the same mixture of solvents. 12 mL of the First identification : A, E.
solution complies with test B for heavy metals (20 ppm). Second identification : B, C, D, E.
Prepare the reference solution using lead standard solution A. Infrared absorption spectrophotometry (2.2.24).
(2 ppm Pb) prepared by diluting lead standard solution
(100 ppm Pb) R with a mixture of 15 volumes of water R and Preparation : dissolve 0.25 g in 10 mL of water R. Add 1 mL
85 volumes of acetone R. of dilute sodium hydroxide solution R. Shake with 20 mL
of methylene chloride R. Separate the organic layer and
Loss on drying (2.2.32). Not more than 0.5 per cent, wash with 5 mL of water R. Evaporate the organic layer
determined on 1.000 g by drying in an oven at 100 °C to to dryness and dry the residue at 40-50 °C. Examine the
105 °C. residues prepared as discs.
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined Comparison: chlorprothixene hydrochloride CRS.
on 1.0 g.
B. Dissolve 0.2 g in a mixture of 5 mL of dioxan R and 5 mL
ASSAY of a 1.5 g/L solution of sodium nitrite R. Add 0.8 mL of
nitric acid R. After 10 min add the solution to 20 mL of
Dissolve 0.250 g in 50 mL of alcohol R previously neutralised
water R. 1 h later filter the precipate formed. The filtrate
using phenolphthalein solution R1 as indicator and add 25 mL
is used immediately for identification test C. Dissolve the
of water R. Titrate with 0.1 M sodium hydroxide until a pink
precipitate by warming in about 15 mL of alcohol R and
colour is obtained.
add the solution to 10 mL of water R. Filter and dry the
1 mL of 0.1 M sodium hydroxide is equivalent to 27.67 mg precipitate at 100-105 °C for 2 h. The melting point (2.2.14)
of C10H13ClN2O3S. is 152 °C to 154 °C.
STORAGE C. To 1 mL of the filtrate obtained in identification test B,
add 0.2 mL of a suspension of 50 mg of fast red B salt R in
Store protected from light. 1 mL of alcohol R. Add 1 mL of 0.5 M alcoholic potassium
hydroxide. A dark red colour is produced. Carry out a
IMPURITIES
blank test.
D. Dissolve about 20 mg in 2 mL of nitric acid R. A red
colour is produced. Add 5 mL of water R and examine
in ultraviolet light at 365 nm. The solution shows green
fluorescence.
A. R = H : 4-chlorobenzenesulfonamide, E. It gives reaction (a) of chlorides (2.3.1).

C. R = CO-NH2 : [(4-chlorophenyl)sulfonyl]urea. TESTS

Solution S. Dissolve 0.25 g in carbon dioxide-free water R and
dilute to 25 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
B. 1,3-dipropylurea, pH (2.2.3) : 4.4 to 5.2 for solution S.

1862 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Chlortalidone

Related substances. Liquid chromatography (2.2.29). Carry STORAGE

out the test protected from bright light. Protected from light.
Test solution. Dissolve 20.0 mg of the substance to be
examined in the mobile phase and dilute to 20.0 mL with the IMPURITIES
mobile phase. Specified impurities : A, B, C, D, E, F.
Reference solution (a). Dissolve 20.0 mg of chlorprothixene
hydrochloride CRS (with a defined content of (E)-isomer) in
the mobile phase and dilute to 20.0 mL with the mobile phase.
Reference solution (b). Dilute 2.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 3.0 mL of this solution
to 20.0 mL with the mobile phase.
Column :
– size : l = 0.12 m, Ø = 4.0 mm, A. (RS)-2-chloro-9-[3-(dimethylamino)propyl]-9H-
thioxanthen-9-ol,
– stationary phase : base-deactivated octadecylsilyl silica gel
for chromatography R (3 μm or 5 μm).
Mobile phase : solution containing 6.0 g/L of potassium
dihydrogen phosphate R, 2.9 g/L of sodium laurilsulfate R
and 9 g/L of tetrabutylammonium bromide R in a mixture of
50 volumes of methanol R, 400 volumes of acetonitrile R and
550 volumes of distilled water R.
Flow rate : 1.5 mL/min.
B. R1 = H, R2 = CH-CH2-CH2-N(CH3)2, R3 = H : N,N-
Detection : spectrophotometer at 254 nm. dimethyl-3-(9H-thioxanthen-9-ylidene)propan-1-amine,
Equilibration : for about 30 min with the mobile phase.
C. R1 = Cl, R2 = CH-CH2-CH2-NH-CH3, R3 = H :
Injection : 20 μL. (Z)-3-(2-chloro-9H-thioxanthen-9-ylidene)-N-
Run time : twice the retention time of chlorprothixene. methylpropan-1-amine,
Relative retention with reference to chlorprothixene :
impurity E = about 1.55. D. R1 = H, R2 = CH-CH2-CH2-N(CH3)2, R3 = Cl :
(Z)-3-(4-chloro-9H-thioxanthen-9-ylidene)-N,N-
System suitability: reference solution (a) : dimethylpropan-1-amine,
– retention time : chlorprothixene = about 10 min,
E. R1 = Cl, R2 = O, R3 = H : 2-chloro-9H-thioxanthen-9-one,
– relative retention with reference to chlorprothixene :
(E)-isomer = about 1.35.
Limits :
– (E)-isomer : not more than 2.0 per cent, calculated from
the area of the corresponding peak in the chromatogram
obtained with reference solution (a) and taking into account
the assigned content of this isomer in chlorprothixene
hydrochloride CRS,
F. (E)-3-(2-chloro-9H-thioxanthen-9-ylidene)-N,N-
– impurity E : not more than 3 times the area of the principal dimethylpropan-1-amine ((E)-isomer).
peak in the chromatogram obtained with reference
solution (b) (0.3 per cent taking into account a response
factor of 3),
– any other impurity : not more than the area of the principal 01/2008:0546
peak in the chromatogram obtained with reference
solution (b) (0.3 per cent), CHLORTALIDONE
– total of any other impurity : not more than 2.33 times the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.7 per cent), Chlortalidonum
– disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.03 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test F. Prepare the reference solution using C14H11ClN2O4S Mr 338.8
2 mL of lead standard solution (10 ppm Pb) R. [77-36-1]
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in vacuo at 60 °C for 3 h. DEFINITION
2-Chloro-5-[(1RS)-1-hydroxy-3-oxo-2,3-dihydro-1H-
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on isoindol-1-yl]benzenesulfonamide.
1.0 g.
Content : 97.0 per cent to 102.0 per cent (dried substance).
ASSAY
CHARACTERS
Dissolve 0.300 g in a mixture of 5.0 mL of 0.01 M hydrochloric
acid and 50 mL of alcohol R. Carry out a potentiometric Appearance : white or yellowish-white powder.
titration (2.2.20), using 0.1 M sodium hydroxide. Read Solubility : very slightly soluble in water, soluble in acetone
the volume added between the 2 points of inflexion. and in methanol, practically insoluble in methylene chloride.
1 mL of 0.1 M sodium hydroxide is equivalent to 35.23 mg of It dissolves in dilute solutions of alkali hydroxides.
C18H19Cl2NS. It shows polymorphism (5.9).

General Notices (1) apply to all monographs and other texts 1863
Chlortalidone EUROPEAN PHARMACOPOEIA 8.0

IDENTIFICATION System suitability : reference solution (b) :

Infrared absorption spectrophotometry (2.2.24). – resolution : minimum 1.5 between the peaks due to
impurity J and chlortalidone.
Comparison : chlortalidone CRS.
Limits :
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference – impurity B : not more than 7 times the area of the principal
substance separately in methanol R, evaporate to dryness and peak in the chromatogram obtained with reference
record new spectra using the residues. solution (a) (0.7 per cent) ;
– impurity J : not more than 3 times the area of the principal
TESTS peak in the chromatogram obtained with reference
solution (a) (0.3 per cent) ;
Acidity. Dissolve 1.0 g with heating in a mixture of 25 mL
of acetone R and 25 mL of carbon dioxide-free water R. – impurity G : not more than 2 times the area of the principal
Cool. Titrate with 0.1 M sodium hydroxide, determining the peak in the chromatogram obtained with reference
end-point potentiometrically (2.2.20). Not more than 0.75 mL solution (a) (0.2 per cent) ;
of 0.1 M sodium hydroxide is required. – unspecified impurities : for each impurity, not more than the
Related substances. Liquid chromatography (2.2.29). area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
Solvent mixture. Mix 2 volumes of a 2 g/L solution of sodium
hydroxide R, 48 volumes of mobile phase B and 50 volumes of – total : not more than 12 times the area of the principal peak
mobile phase A. in the chromatogram obtained with reference solution (a)
(1.2 per cent) ;
Test solution (a). Dissolve 50.0 mg of the substance to be
examined in the solvent mixture and dilute to 50.0 mL with – disregard limit : 0.5 times the area of the principal peak in
the solvent mixture. the chromatogram obtained with reference solution (a)
(0.05 per cent).
Test solution (b). Dilute 10.0 mL of test solution (a) to
100.0 mL with the solvent mixture. Chlorides (2.4.4) : maximum 350 ppm.
Triturate 0.3 g finely, add 30 mL of water R, shake for 5 min
Reference solution (a). Dilute 1.0 mL of test solution (a) to and filter. 15 mL of the filtrate complies with the test. Prepare
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
the standard using 10 mL of chloride standard solution (5 ppm
solution to 10.0 mL with the solvent mixture. Cl) R.
Reference solution (b). Dissolve the contents of a vial Loss on drying (2.2.32) : maximum 0.5 per cent, determined
of chlortalidone for peak identification CRS (containing on 1.000 g by drying in an oven at 105 °C.
impurities B, G and J) in 1 mL of the solvent mixture.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Reference solution (c). Dissolve 50.0 mg of chlortalidone CRS 1.0 g.
in the solvent mixture and dilute to 50.0 mL with the solvent
mixture. Dilute 10.0 mL of this solution to 100.0 mL with the ASSAY
solvent mixture.
Liquid chromatography (2.2.29) as described in the test for
Column : related substances with the following modification.
– size : l = 0.25 m, Ø = 4.6 mm ; Injection : 20 μL of test solution (b) and reference solution (c).
– stationary phase : octylsilyl silica gel for chromatography R Calculate the percentage content of C14H11ClN2O4S from the
(5 μm) ; declared content of chlortalidone CRS.
– temperature : 40 °C.
IMPURITIES
Mobile phase : Specified impurities : B, G, J.
– mobile phase A : dissolve 1.32 g of ammonium phosphate R Other detectable impurities (the following substances would,
in about 900 mL of water R and adjust to pH 5.5 with dilute if present at a sufficient level, be detected by one or other of
phosphoric acid R ; dilute to 1000 mL with water R ; the tests in the monograph. They are limited by the general
– mobile phase B : methanol R2 ; acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
Time Mobile phase A Mobile phase B use (2034). It is therefore not necessary to identify these
(min) (per cent V/V) (per cent V/V) impurities for demonstration of compliance. See also 5.10.
0 - 16 65 35 Control of impurities in substances for pharmaceutical use):
16 - 21 65 → 50 35 → 50
A, C, D, E, F, H, I.

21 - 35 50 50
35 - 45 50 → 65 50 → 35

Flow rate : 1.4 mL/min.

Detection : spectrophotometer at 220 nm.
A. R = H, R′ = OH : 2-(4-chloro-3-sulfobenzoyl)benzoic acid,
Injection : 20 μL of test solution (a) and reference solutions (a)
and (b).
B. R = H, R′ = NH2 : 2-(4-chloro-3-sulfamoylbenzoyl)benzoic
Identification of impurities : use the chromatogram obtained acid,
with reference solution (b) and the chromatogram supplied
with chlortalidone for peak identification CRS to identify the C. R = C2H5, R′ = NH2 : ethyl 2-(4-chloro-3-sulfamoylbenzoyl)-
peaks due to impurities B, G and J. benzoate,
Relative retention with reference to chlortalidone
(retention time = about 7 min): impurity B = about 0.7 ; I. R = CH(CH3)2, R′ = NH2 : 1-methylethyl
impurity J = about 0.9 ; impurity G = about 6. 2-(4-chloro-3-sulfamoylbenzoyl)benzoate,

1864 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Chlortetracycline hydrochloride

IDENTIFICATION
First identification : C, D.
Second identification : A, B, C.
A. Thin-layer chromatography (2.2.27).
D. R = OC2H5, R′ = SO2-NH2 : 2-chloro-5-[(1RS)-1-ethoxy-3- Test solution. Dissolve 5 mg of the substance to be
oxo-2,3-dihydro-1H-isoindol-1-yl]benzenesulfonamide, examined in methanol R and dilute to 10 mL with the same
solvent.
E. R = H, R′ = SO2-NH2 : 2-chloro-5-[(1RS)-3-oxo-2,3-
dihydro-1H-isoindol-1-yl]benzenesulfonamide, Reference solution (a). Dissolve 5 mg of chlortetracycline
hydrochloride CRS in methanol R and dilute to 10 mL with
G. R = OH, R′ = Cl : (3RS)-3-(3,4-dichlorophenyl)-3-hydroxy- the same solvent.
2,3-dihydro-1H-isoindol-1-one,
Reference solution (b). Dissolve 5 mg of chlortetracycline
H. R = OCH(CH3)2, R′ = SO2-NH2 : 2-chloro-5-[(1RS)- hydrochloride CRS, 5 mg of demeclocycline hydrochloride R
1-(1-methylethoxy)-3-oxo-2,3-dihydro-1H-isoindol-1- and 5 mg of doxycycline R in methanol R and dilute to
yl]benzenesulfonamide, 10 mL with the same solvent.
Plate : TLC octadecylsilyl silica gel F254 plate R.
Mobile phase : mix 20 volumes of acetonitrile R, 20 volumes
of methanol R and 60 volumes of a 63 g/L solution of
oxalic acid R previously adjusted to pH 2 with concentrated
ammonia R.
F. bis[2-chloro-5-(1-hydroxy-3-oxo-2,3-dihydro-1H-isoindol- Application : 1 μL.
1-yl)benzenesulfonyl]amine, Development : over 3/4 of the plate.
J. impurity of unknown structure with a relative retention Drying : in air.
of about 0.9. Detection : examine in ultraviolet light at 254 nm.
07/2012:0173 System suitability : the chromatogram obtained with
corrected 7.8 reference solution (b) shows 3 clearly separated spots.
Results : the principal spot in the chromatogram obtained
CHLORTETRACYCLINE with the test solution is similar in position and size to
the principal spot in the chromatogram obtained with
HYDROCHLORIDE reference solution (a).
B. To about 2 mg add 5 mL of sulfuric acid R. A deep
Chlortetracyclini hydrochloridum blue colour develops and becomes bluish-green. Add
the solution to 2.5 mL of water R. The colour becomes
brownish.
C. It gives reaction (a) of chlorides (2.3.1).
D. Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : test solution and reference solution (a).
Compound R Molecular formula Mr
Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time and size
Chlortetracycline hydrochloride Cl C22H24Cl2N2O8 515.3 to the principal peak in the chromatogram obtained with
Tetracycline hydrochloride H C22H25ClN2O8 480.9
reference solution (a).

Chlortetracycline hydrochloride : [64-72-2] TESTS

Tetracycline hydrochloride : [64-75-5] pH (2.2.3) : 2.3 to 3.3.
DEFINITION Dissolve 0.1 g in 10 mL of carbon dioxide-free water R, heating
slightly.
Mixture of antibiotics, the main component being
the hydrochloride of (4S,4aS,5aS,6S,12aS)-7-chloro-4- Specific optical rotation (2.2.7) : − 250 to − 235 (anhydrous
(dimethylamino)-3,6,10,12,12a-pentahydroxy-6-methyl-1,11- substance).
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dissolve 0.125 g in water R and dilute to 50.0 mL with the
(chlortetracycline hydrochloride), a substance produced by same solvent.
the growth of certain strains of Streptomyces aureofaciens or Absorbance (2.2.25) : maximum 0.40 at 460 nm.
obtained by any other means.
Dissolve 0.125 g in water R and dilute to 25.0 mL with the
Content : same solvent.
– chlortetracycline hydrochloride (C22H24Cl2N2O8) : minimum
89.5 per cent (anhydrous substance) ; Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
– tetracycline hydrochloride (C22H25ClN2O8) : maximum
6.0 per cent (anhydrous substance) ; Test solution. Dissolve 25.0 mg of the substance to be
examined in mobile phase B and dilute to 25.0 mL with mobile
– sum of the contents of chlortetracycline hydrochloride and phase B.
tetracycline hydrochloride: 94.5 per cent to 102.0 per cent
(anhydrous substance). Reference solution (a). Dissolve 25.0 mg of chlortetracycline
hydrochloride CRS in mobile phase B and dilute to 25.0 mL
CHARACTERS with mobile phase B.
Appearance : yellow powder. Reference solution (b). Dilute 1.0 mL of the test solution to
Solubility : slightly soluble in water and in ethanol (96 per 100.0 mL with mobile phase B.
cent). It dissolves in solutions of alkali hydroxides and Reference solution (c). Dilute 1.0 mL of reference solution (b)
carbonates. to 10.0 mL with mobile phase B.

General Notices (1) apply to all monographs and other texts 1865
Chlortetracycline hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Reference solution (d). Dissolve 5 mg of chlortetracycline for – unspecified impurities : for each impurity, not more than the
system suitability CRS (containing impurities A, B, D, E, G, H, area of the principal peak in the chromatogram obtained
J, K and L) in mobile phase B and dilute to 5 mL with mobile with reference solution (c) (0.10 per cent) ;
phase B. – sum of impurities other than A : not more than twice the
Reference solution (e). Dissolve 25.0 mg of tetracycline area of the principal peak in the chromatogram obtained
hydrochloride CRS in mobile phase B and dilute to 25.0 mL with reference solution (b) (2.0 per cent) ;
with mobile phase B. Dilute 5.0 mL of this solution to – disregard limit : 0.5 times the area of the principal peak in
100.0 mL with mobile phase B. the chromatogram obtained with reference solution (c)
Column : (0.05 per cent).
– size : l = 0.075 m, Ø = 4.6 mm ; Heavy metals (2.4.8) : maximum 50 ppm.
– stationary phase : end-capped octylsilyl silica gel for 0.5 g complies with test C. Prepare the reference solution using
chromatography with polar incorporated groups R (3.5 μm) ; 2.5 mL of lead standard solution (10 ppm Pb) R.
– temperature : 45 °C. Water (2.5.12) : maximum 2.0 per cent, determined on 0.300 g.
Mobile phase : Sulfated ash (2.4.14) : maximum 0.5 per cent, determined on
1.0 g.
– mobile phase A : to 725 mL of water R add 50 mL of
perchloric acid solution R, shake and add 225 mL of Bacterial endotoxins (2.6.14) : less than 1 IU/mg, if intended
dimethyl sulfoxide R ; for use in the manufacture of parenteral preparations without
a further appropriate procedure for the removal of bacterial
– mobile phase B : to 250 mL of water R add 50 mL of endotoxins.
perchloric acid solution R, shake and add 700 mL of
dimethyl sulfoxide R ; ASSAY
Time Mobile phase A Mobile phase B Liquid chromatography (2.2.29) as described in the test for
(min) (per cent V/V) (per cent V/V) related substances with the following modification.
0 - 46 100 → 0 0 → 100 Injection : 10 μL of the test solution and reference solutions (a)
and (e).
Flow rate : 0.4 mL/min. Calculate the percentage content of C22H24Cl2N2O8 using
Detection : spectrophotometer at 280 nm. the chromatogram obtained with reference solution (a) and
taking into account the assigned content of chlortetracycline
Injection : 20 μL of the test solution and reference solutions (b),
hydrochloride CRS. Calculate the percentage content of
(c) and (d).
C22H25ClN2O8 using the chromatogram obtained with
Identification of impurities : use the chromatogram supplied reference solution (e) and taking into account the assigned
with chlortetracycline for system suitability CRS and the content of tetracycline hydrochloride CRS.
chromatogram obtained with reference solution (d) to identify
the peaks due to impurities A, B, D, E, G, H, J, K and L. STORAGE
Relative retention with reference to chlortetracycline Protected from light. If the substance is sterile, store in a
(retention time = about 26 min): impurity D = about 0.5 ; sterile, airtight, tamper-proof container.
tetracycline = about 0.6 ; impurity E = about 0.7 ;
impurity B = about 0.8, impurity A = about 0.86 ; IMPURITIES
impurity G = about 0.9 ; impurity H = about 1.1 ; Specified impurities : A, B, D, E, G, H, J, K, L.
impurity J = about 1.4, impurity K = about 1.67 ; Other detectable impurities (the following substances would,
impurity L = about 1.71. if present at a sufficient level, be detected by one or other of
System suitability : reference solution (d) : the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
– resolution : minimum 1.5 between the peaks due to by the general monograph Substances for pharmaceutical use
tetracycline and impurity E ; minimum 1.5 between the (2034). It is therefore not necessary to identify these impurities
peaks due to impurities A and G ; minimum 1.5 between for demonstration of compliance. See also 5.10. Control of
the peaks due to impurities K and L ; if necessary, adjust the impurities in substances for pharmaceutical use) : C, F, I.
concentration of dimethyl sulfoxide in mobile phase A.
Limits :
– correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
corresponding correction factor : impurity G = 1.4 ;
impurity J = 0.3 ; impurity K = 0.4 ; impurity L = 0.4 ;
– impurity A : not more than 4 times the area of the principal
peak in the chromatogram obtained with reference A. (4R,4aS,5aS,6S,12aS)-7-chloro-4-(dimethylamino)-
solution (b) (4.0 per cent) ; 3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide
– impurities B, E : for each impurity, not more than the area
(4-epichlortetracycline),
of the principal peak in the chromatogram obtained with
reference solution (b) (1.0 per cent) ;
– impurity J : not more than 3 times the area of the principal
peak in the chromatogram obtained with reference
solution (c) (0.3 per cent) ;
– impurities D, G, H, L : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.2 per cent) ; B. (4S,4aS,5aS,6S,12aS)-7-chloro-4-(dimethyl-
– impurity K : not more than 1.5 times the area of the amino)-3,6,10,12,12a-pentahydroxy-1,11-di-
principal peak in the chromatogram obtained with oxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
reference solution (c) (0.15 per cent) ; mide (demeclocycline),

1866 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cholecalciferol

C. (4R,4aS,5aS,6S,12aS)-4-(dimethylamino)-3,6,10,12,12a- I. (4R,4aS,12aS)-4-(dimethylamino)-3,10,12,12a-tetrahy-
pentahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrote- droxy-6-methyl-1,11-dioxo-1,4,4a,5,11,12a-hexahydrote-
tracene-2-carboxamide (4-epidemethyltetracycline), tracene-2-carboxamide (4-epianhydrotetracycline),

J. (4S,4aS,12aS)-4-(dimethylamino)-3,10,12,12a-
D. (4R,4aS,5aS,6S,12aS)-4-(dimethylamino)-3,6,10,12,12a- tetrahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,11,12a-
pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a- hexahydrotetracene-2-carboxamide (anhydrotetracycline),
octahydrotetracene-2-carboxamide (4-epitetracycline),

K. (4R,4aS,12aS)-7-chloro-4-(dimethylamino)-
3,10,12,12a-tetrahydroxy-6-methyl-1,11-dioxo-
E. (4R,4aS,5aS,6S,12aS)-7-chloro-4-(dimethyl- 1,4,4a,5,11,12a-hexahydrotetracene-2-carboxamide
amino)-3,6,10,12,12a-pentahydroxy-1,11-di- (4-epianhydrochlortetracycline),
oxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide (4-epidemethylchlortetracycline),

L. (4S,4aS,12aS)-7-chloro-4-(dimethylamino)-
3,10,12,12a-tetrahydroxy-6-methyl-1,11-dioxo-
1,4,4a,5,11,12a-hexahydrotetracene-2-carboxamide
F. (4R,4aS,6S,8aS)-6-[(1R)-7-chloro-4-hydroxy-1- (anhydrochlortetracycline).
methyl-3-oxo-1,3-dihydro-2-benzofuran-1-yl]-
4-(dimethylamino)-3,8a-dihydroxy-1,8-dioxo- 01/2013:0072
1,4,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide
(4-epiisochlortetracycline), CHOLECALCIFEROL
Cholecalciferolum

G. (4S,4aS,6S,8aS)-6-[(1R)-7-chloro-4-hydroxy-1-
methyl-3-oxo-1,3-dihydro-2-benzofuran-1-yl]-
4-(dimethylamino)-3,8a-dihydroxy-1,8-dioxo-
1,4,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide
(isochlortetracycline),
C27H44O Mr 384.6
[67-97-0]
DEFINITION
(5Z,7E)-9,10-Secocholesta-5,7,10(19)-trien-3β-ol.
Content : 97.0 per cent to 102.0 per cent.
A reversible isomerisation to pre-cholecalciferol takes place
H. (4S,4aS,5aS,6S,12aS)-2-acetyl-7-chloro-4- in solution, depending on temperature and time. The activity
(dimethylamino)-3,6,10,12,12a-pentahydroxy-6-methyl- is due to both compounds (see Assay).
4a,5a,6,12a-tetrahydrotetracene-1,11(4H,5H)-dione 1 mg of cholecalciferol is equivalent to 40 000 IU of antirachitic
(2-acetyl-2-decarboxamidochlortetracycline), activity (vitamin D) in rats.

General Notices (1) apply to all monographs and other texts 1867
Cholecalciferol EUROPEAN PHARMACOPOEIA 8.0

CHARACTERS ASSAY
Appearance : white or almost white crystals. Liquid chromatography (2.2.29) as described in the test for
Solubility : practically insoluble in water, freely soluble in related substances with the following modification.
ethanol (96 per cent), soluble in trimethylpentane and in fatty Injection : test solution and reference solution (a).
oils. Calculate the percentage content of C27H44O taking into
It is sensitive to air, heat and light. Solutions in solvents account the assigned content of cholecalciferol CRS and, if
without an antioxidant are unstable and are to be used necessary, the peak due to pre-cholecalciferol.
immediately.
STORAGE
IDENTIFICATION Under nitrogen, in an airtight container, protected from light,
Infrared absorption spectrophotometry (2.2.24). at a temperature of 2 °C to 8 °C.
The contents of an opened container are to be used
Comparison : cholecalciferol CRS.
immediately.
TESTS IMPURITIES
Specific optical rotation (2.2.7) : + 105 to + 112, determined Specified impurities : A.
within 30 min of preparing the solution.
Other detectable impurities (the following substances would,
Dissolve 0.200 g rapidly in aldehyde-free alcohol R without if present at a sufficient level, be detected by one or other of
heating and dilute to 25.0 mL with the same solvent. the tests in the monograph. They are limited by the general
Related substances. Liquid chromatography (2.2.29). Prepare acceptance criterion for other/unspecified impurities and/or
the solutions immediately before use, avoiding exposure to by the general monograph Substances for pharmaceutical use
actinic light and air. (2034). It is therefore not necessary to identify these impurities
Test solution. Dissolve 10.0 mg of the substance to be for demonstration of compliance. See also 5.10. Control of
examined in trimethylpentane R without heating and dilute to impurities in substances for pharmaceutical use) : B, C, D, E.
10.0 mL with the same solvent.
Reference solution (a). Dissolve 10.0 mg of cholecalciferol CRS
in trimethylpentane R without heating and dilute to 10.0 mL
with the same solvent.
Reference solution (b). Dilute 1.0 mL of cholecalciferol for
system suitability CRS (containing impurity A) to 5.0 mL
with the mobile phase. Heat in a water-bath at 90 °C under
a reflux condenser for 45 min and cool (formation of
pre-cholecalciferol).
Reference solution (c). Dilute 10.0 mL of reference solution (a)
to 100.0 mL with the mobile phase. Dilute 1.0 mL of this
A. (5E,7E)-9,10-secocholesta-5,7,10(19)-trien-3β-ol
solution to 100.0 mL with the mobile phase.
(trans-cholecalciferol, trans-vitamin D3),
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : silica gel for chromatography R (5 μm).
Mobile phase : pentanol R, hexane R (0.3:99.7 V/V).
Flow rate : 2 mL/min.
Detection : spectrophotometer at 265 nm.
Injection : 5 μL of the test solution and reference solutions (b)
and (c). B. cholesta-5,7-dien-3β-ol (7,8-didehydrocholesterol,
provitamin D3),
Run time : twice the retention time of cholecalciferol.
Relative retention with reference to cholecalciferol (retention
time = about 19 min) : pre-cholecalciferol = about 0.5 ;
impurity A = about 0.6.
System suitability : reference solution (b) :
– resolution : minimum 1.5 between the peaks due to
pre-cholecalciferol and impurity A.
Limits : C. 9β,10α-cholesta-5,7-dien-3β-ol (lumisterol3),
– impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (c)
(0.1 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (c) (0.10 per cent) ;
– total : not more than 10 times the area of the principal peak
in the chromatogram obtained with reference solution (c)
(1.0 per cent) ;
– disregard limit : 0.5 times the area of the principal
peak in the chromatogram obtained with reference
solution (c) (0.05 per cent) ; disregard the peak due to D. (6E)-9,10-secocholesta-5(10),6,8(14)-trien-3β-ol
pre-cholecalciferol. (iso-tachysterol3),

1868 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cholecalciferol concentrate (oily form)

obtained with reference solution (b) shows immediately at

the same level an orange principal spot which gradually
becomes reddish-brown and remains so for 10 min.
B. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Prepare a solution in cyclohexane R
containing the equivalent of about 400 IU/mL.
Spectral range : 250-300 nm.
Absorption maximum : at 267 nm.
C. Examine the chromatograms obtained in the assay.
E. (6E)-9,10-secocholesta-5(10),6,8-trien-3β-ol (tachysterol3). Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time to
the principal peak in the chromatogram obtained with
01/2008:0575 reference solution (a).
corrected 6.5
TESTS
CHOLECALCIFEROL CONCENTRATE Acid value (2.5.1) : maximum 2.0.
(OILY FORM) Dissolve 5.0 g in 25 mL of the prescribed mixture of solvents.
Peroxide value (2.5.5, Method A) : maximum 20.
Cholecalciferolum densatum oleosum Related substances
The thresholds indicated under Related substances
DEFINITION
(Table 2034.-1) in the general monograph Substances for
Solution of Cholecalciferol (0072) in a suitable vegetable fatty pharmaceutical use (2034) do not apply.
oil, authorised by the competent authority.
Content : 90.0 per cent to 110.0 per cent of the cholecalciferol ASSAY
content stated on the label, which is not less than 500 000 IU/g. Carry out the assay as rapidly as possible, avoiding exposure
It may contain suitable stabilisers such as antioxidants. to actinic light and air.
Liquid chromatography (2.2.29).
CHARACTERS Test solution. Dissolve a quantity of the preparation to
Appearance : clear, yellow liquid. be examined, weighed with an accuracy of 0.1 per cent,
Solubility : practically insoluble in water, slightly soluble in equivalent to about 400 000 IU, in 10.0 mL of toluene R and
anhydrous ethanol, miscible with solvents of fats. dilute to 100.0 mL with the mobile phase.
Partial solidification may occur, depending on the temperature. Reference solution (a). Dissolve 10.0 mg of cholecalciferol CRS
without heating in 10.0 mL of toluene R and dilute to 100.0 mL
IDENTIFICATION with the mobile phase.
First identification : A, C. Reference solution (b). Dilute 1.0 mL of cholecalciferol for
Second identification : A, B. system suitability CRS to 5.0 mL with the mobile phase. Heat
A. Thin-layer chromatography (2.2.27). Prepare the solutions in a water-bath at 90 °C under a reflux condenser for 45 min
immediately before use. and cool.
Test solution. Dissolve an amount of the preparation to Reference solution (c). Dissolve 0.10 g of cholecalciferol CRS
be examined corresponding to 400 000 IU in ethylene without heating in toluene R and dilute to 100.0 mL with the
chloride R containing 10 g/L of squalane R and 0.1 g/L of same solvent.
butylhydroxytoluene R and dilute to 4 mL with the same Reference solution (d). Dilute 5.0 mL of reference solution (c)
solution. to 50.0 mL with the mobile phase. Keep the solution in iced
Reference solution (a). Dissolve 10 mg of cholecalciferol CRS water.
in ethylene chloride R containing 10 g/L of squalane R and Reference solution (e). Place 5.0 mL of reference solution (c) in
0.1 g/L of butylhydroxytoluene R and dilute to 4 mL with a volumetric flask, add about 10 mg of butylhydroxytoluene R
the same solution. and displace air from the flask with nitrogen R. Heat in a
Reference solution (b). Dissolve 10 mg of ergocalciferol CRS water-bath at 90 °C under a reflux condenser protected from
in ethylene chloride R containing 10 g/L of squalane R and light and under nitrogen R for 45 min. Cool and dilute to
0.1 g/L of butylhydroxytoluene R and dilute to 4 mL with 50.0 mL with the mobile phase.
the same solution. Column :
Plate : TLC silica gel G plate R. – size : l = 0.25 m, Ø = 4.6 mm ;
Mobile phase : a 0.1 g/L solution of butylhydroxytoluene R – stationary phase : silica gel for chromatography R (5 μm).
in a mixture of equal volumes of cyclohexane R and Mobile phase : pentanol R, hexane R (3:997 V/V).
peroxide-free ether R. Flow rate : 2 mL/min.
Application : 20 μL. Detection : spectrophotometer at 254 nm.
Development : immediately, protected from light, over a Injection : the chosen volume of each solution (the same
path of 15 cm. volume for reference solution (a) and for the test solution) ;
Drying : in air. automatic injection device or sample loop recommended.
Detection : spray with sulfuric acid R. Relative retention with reference to cholecalciferol : pre-
Results : the chromatogram obtained with the test cholecalciferol = about 0.4 ; trans-cholecalciferol = about 0.5.
solution shows immediately a bright yellow principal spot System suitability : reference solution (b) :
which rapidly becomes orange-brown, then gradually – resolution : minimum 1.0 between the peaks due to
greenish-grey, remaining so for 10 min. This spot is similar pre-cholecalciferol and trans-cholecalciferol ; if necessary
in position, colour and size to the spot in the chromatogram adjust the proportions of the constituents and the flow rate
obtained with reference solution (a). The chromatogram of the mobile phase to obtain this resolution ;

General Notices (1) apply to all monographs and other texts 1869
Cholecalciferol concentrate (powder form) EUROPEAN PHARMACOPOEIA 8.0

– repeatability : maximum relative standard deviation of Content : 90.0 per cent to 110.0 per cent of the cholecalciferol
1.0 per cent for the peak due to cholecalciferol after content stated on the label, which is not less than 100 000 IU/g.
6 injections. It may contain suitable stabilisers such as antioxidants.
Calculate the conversion factor (f) using the following
expression : CHARACTERS
Appearance : white or yellowish-white, small particles.
Solubility : practically insoluble, swells, or forms a dispersion
in water, depending on the formulation.
K = area (or height) of the peak due to cholecalciferol IDENTIFICATION
in the chromatogram obtained with reference
solution (d) ; First identification : A, C.
L Second identification : A, B.
= area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with reference A. Thin-layer chromatography (2.2.27). Prepare the solutions
solution (e) ; immediately before use.
M = area (or height) of the peak due to Test solution. Place 10.0 mL of the test solution prepared
pre-cholecalciferol in the chromatogram for the assay in a suitable flask and evaporate to dryness
obtained with reference solution (e). under reduced pressure by swirling in a water-bath at
40 °C. Cool under running water and restore atmospheric
The value of f determined in duplicate on different days may pressure with nitrogen R. Dissolve the residue immediately
be used during the entire procedure. in 0.4 mL of ethylene chloride R containing 10 g/L of
Calculate the content of cholecalciferol in International Units squalane R and 0.1 g/L of butylhydroxytoluene R.
per gram using the following expression : Reference solution (a). Dissolve 10 mg of cholecalciferol CRS
in ethylene chloride R containing 10 g/L of squalane R and
0.1 g/L of butylhydroxytoluene R and dilute to 4 mL with
the same solution.
m = mass of the preparation to be examined in the test Reference solution (b). Dissolve 10 mg of ergocalciferol CRS
solution, in milligrams ; in ethylene chloride R containing 10 g/L of squalane R and
0.1 g/L of butylhydroxytoluene R and dilute to 4 mL with
m′ = mass of cholecalciferol CRS in reference solution (a), the same solution.
in milligrams ;
Plate : TLC silica gel G plate R.
V = volume of the test solution (100 mL) ; Mobile phase : a 0.1 g/L solution of butylhydroxytoluene R
V′ = volume of reference solution (a) (100 mL) ; in a mixture of equal volumes of cyclohexane R and
peroxide-free ether R.
SD = area (or height) of the peak due to cholecalciferol in
the chromatogram obtained with the test solution ; Application : 20 μL.
S′D = area (or height) of the peak due to cholecalciferol Development : immediately, protected from light, over a
path of 15 cm.
in the chromatogram obtained with reference
solution (a); Drying : in air.
Sp = area (or height) of the peak due to Detection : spray with sulfuric acid R.
pre-cholecalciferol in the chromatogram Results : the chromatogram obtained with the test solution
obtained with the test solution ; shows immediately a bright yellow principal spot,
f = conversion factor. which rapidly becomes orange-brown, then gradually
greenish-grey, remaining so for 10 min. This spot is similar
STORAGE in position, colour and size to the spot in the chromatogram
obtained with reference solution (a). The chromatogram
In an airtight, well-filled container, protected from light. The obtained with reference solution (b) shows immediately at
contents of an opened container are to be used as soon as the same level an orange principal spot, which gradually
possible ; any unused part is to be protected by an atmosphere becomes reddish-brown and remains so for 10 min.
of nitrogen.
B. Ultraviolet and visible absorption spectrophotometry
LABELLING (2.2.25).
The label states : Test solution. Place 5.0 mL of the test solution prepared for
– the number of International Units per gram ; the assay in a suitable flask and evaporate to dryness under
reduced pressure by swirling in a water-bath at 40 °C. Cool
– the method of restoring the solution if partial solidification under running water and restore atmospheric pressure with
occurs. nitrogen R. Dissolve the residue immediately in 50.0 mL
of cyclohexane R.
01/2008:0574 Spectral range : 250-300 nm.
corrected 6.5 Absorption maximum : at 265 nm.
C. Examine the chromatograms obtained in the assay.
CHOLECALCIFEROL CONCENTRATE Results : the principal peak in the chromatogram obtained
(POWDER FORM) with the test solution is similar in retention time to
the principal peak in the chromatogram obtained with
Cholecalciferoli pulvis reference solution (a).

DEFINITION TESTS
Powder concentrate obtained by dispersing an oily solution Related substances
of Cholecalciferol (0072) in an appropriate matrix, which is The thresholds indicated under Related substances
usually based on a combination of gelatin and carbohydrates (Table 2034.-1) in the general monograph Substances for
of suitable quality, authorised by the competent authority. pharmaceutical use (2034) do not apply.

1870 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cholecalciferol concentrate (powder form)

ASSAY System suitability : reference solution (b) :

Carry out the assay as rapidly as possible, avoiding exposure
to actinic light and air. – resolution : minimum 1.0 between the peaks due to
pre-cholecalciferol and trans-cholecalciferol ; if necessary,
Liquid chromatography (2.2.29). adjust the proportions of the constituents and the flow rate
Test solution. Introduce into a saponification flask a quantity of the mobile phase to obtain this resolution ;
of the preparation to be examined, weighed with an accuracy
of 0.1 per cent, equivalent to about 100 000 IU. Add 5 mL – repeatability : maximum relative standard deviation of
of water R, 20 mL of anhydrous ethanol R, 1 mL of sodium 1.0 per cent for the peak due to cholecalciferol after
ascorbate solution R and 3 mL of a freshly prepared 50 per 6 injections.
cent m/m solution of potassium hydroxide R. Heat in a
water-bath under a reflux condenser for 30 min. Cool rapidly Calculate the conversion factor (f) using the following
under running water. Transfer the liquid to a separating expression :
funnel with the aid of 2 quantities, each of 15 mL, of
water R, 1 quantity of 10 mL of ethanol (96 per cent) R and
2 quantities, each of 50 mL, of pentane R. Shake vigorously
for 30 s. Allow to stand until the 2 layers are clear. Transfer
the lower aqueous-alcoholic layer to a 2nd separating funnel
and shake with a mixture of 10 mL of ethanol (96 per K = area (or height) of the peak due to cholecalciferol
cent) R and 50 mL of pentane R. After separation, transfer in the chromatogram obtained with reference
the aqueous-alcoholic layer to a 3rd separating funnel and solution (d);
the pentane layer to the 1st separating funnel, washing the L = area (or height) of the peak due to cholecalciferol
2nd separating funnel with 2 quantities, each of 10 mL, of in the chromatogram obtained with reference
pentane R and adding the washings to the 1st separating solution (e) ;
funnel. Shake the aqueous-alcoholic layer with 50 mL of M = area (or height) of the peak due to
pentane R and add the pentane layer to the 1st funnel. Wash pre-cholecalciferol in the chromatogram
the pentane layer with 2 quantities, each of 50 mL, of a obtained with reference solution (e).
freshly prepared 30 g/L solution of potassium hydroxide R in
ethanol (10 per cent V/V) R, shaking vigorously, then wash
with successive quantities, each of 50 mL, of water R until the The value of f determined in duplicate on different days may
washings are neutral to phenolphthalein. Transfer the washed be used during the entire procedure.
pentane extract to a ground-glass-stoppered flask. Evaporate
the contents of the flask to dryness under reduced pressure by Calculate the content of cholecalciferol in International Units
swirling in a water-bath at 40 °C. Cool under running water per gram using the following expression :
and restore atmospheric pressure with nitrogen R. Dissolve the
residue immediately in 5.0 mL of toluene R and add 20.0 mL
of the mobile phase to obtain a solution containing about
4000 IU/mL.
Reference solution (a). Dissolve 10.0 mg of cholecalciferol CRS, m = mass of the preparation to be examined in the
without heating, in 10.0 mL of toluene R and dilute to test solution, in milligrams ;
100.0 mL with the mobile phase. m′ = mass of cholecalciferol CRS in reference
Reference solution (b). Dilute 1.0 mL of cholecalciferol for solution (a), in milligrams ;
system suitability CRS to 5.0 mL with the mobile phase. Heat V = volume of the test solution (25 mL) ;
in a water-bath at 90 °C under a reflux condenser for 45 min
and cool. V′ = volume of reference solution (a) (100 mL) ;
Reference solution (c). Dissolve 0.10 g of cholecalciferol CRS, SD = area (or height) of the peak due to cholecalciferol
without heating, in toluene R and dilute to 100.0 mL with the in the chromatogram obtained with the test
same solvent. solution ;
Reference solution (d). Dilute 5.0 mL of reference solution (c) S′D = area (or height) of the peak due to cholecalciferol
to 50.0 mL with the mobile phase. Keep the solution in iced in the chromatogram obtained with reference
water. solution (a) ;
Reference solution (e). Place 5.0 mL of reference solution (c) in Sp = area (or height) of the peak due to
a volumetric flask, add about 10 mg of butylhydroxytoluene R pre-cholecalciferol in the chromatogram
and displace the air from the flask with nitrogen R. Heat in a obtained with the test solution ;
water-bath at 90 °C under a reflux condenser, protected from f = conversion factor.
light and under nitrogen R, for 45 min. Cool and dilute to
50.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ; STORAGE
– stationary phase : silica gel for chromatography R (5 μm).
In an airtight, well-filled container, protected from light. The
Mobile phase : pentanol R, hexane R (3:997 V/V). contents of an opened container are to be used as soon as
Flow rate : 2 mL/min. possible ; any unused part is to be protected by an atmosphere
of nitrogen.
Detection : spectrophotometer at 254 nm.
Injection : the chosen volume of each solution (the same
volume for reference solution (a) and for the test solution) ;
automatic injection device or sample loop recommended. LABELLING
Relative retention with reference to cholecalciferol : pre-
cholecalciferol = about 0.4 ; trans-cholecalciferol = about 0.5. The label states the number of International Units per gram.

General Notices (1) apply to all monographs and other texts 1871
Cholecalciferol concentrate (water-dispersible form) EUROPEAN PHARMACOPOEIA 8.0

01/2008:0598 Spectral range : 250-300 nm.

corrected 6.5 Absorption maximum : at 265 nm.
CHOLECALCIFEROL CONCENTRATE C. Examine the chromatograms obtained in the assay.
Results : the principal peak in the chromatogram obtained
(WATER-DISPERSIBLE FORM) with the test solution is similar in retention time to
the principal peak in the chromatogram obtained with
Cholecalciferolum in aqua dispergibile reference solution (a).
DEFINITION D. Mix about 1 g with 10 mL of water R previously warmed
Solution of Cholecalciferol (0072) in a suitable vegetable fatty to 50 °C, and cool to 20 °C. Immediately after cooling, a
oil, authorised by the competent authority, to which suitable uniform, slightly opalescent and slightly yellow dispersion
solubilisers have been added. is obtained.
Content : 90.0 per cent to 115.0 per cent of the cholecalciferol
content stated on the label, which is not less than 100 000 IU/g. TESTS
It may contain suitable stabilisers such as antioxidants. Related substances
The thresholds indicated under Related substances
CHARACTERS
(Table 2034.-1) in the general monograph Substances for
Appearance : slightly yellowish liquid of variable opalescence pharmaceutical use (2034) do not apply.
and viscosity.
Highly concentrated solutions may become cloudy at low ASSAY
temperatures or form a gel at room temperature. Carry out the assay as rapidly as possible, avoiding exposure
IDENTIFICATION to actinic light and air.
First identification : A, C, D. Liquid chromatography (2.2.29).
Second identification : A, B, D. Test solution. Introduce into a saponification flask a quantity
A. Thin-layer chromatography (2.2.27). Prepare the solutions of the preparation to be examined, weighed with an accuracy
immediately before use. of 0.1 per cent, equivalent to about 100 000 IU. Add 5 mL
Test solution. Place 10.0 mL of the test solution prepared of water R, 20 mL of anhydrous ethanol R, 1 mL of sodium
for the assay in a suitable flask and evaporate to dryness ascorbate solution R and 3 mL of a freshly prepared 50 per
under reduced pressure by swirling in a water-bath at cent m/m solution of potassium hydroxide R. Heat in a
40 °C. Cool under running water and restore atmospheric water-bath under a reflux condenser for 30 min. Cool rapidly
pressure with nitrogen R. Dissolve the residue immediately under running water. Transfer the liquid to a separating
in 0.4 mL of ethylene chloride R containing 10 g/L of funnel with the aid of 2 quantities, each of 15 mL, of
squalane R and 0.1 g/L of butylhydroxytoluene R. water R, 1 quantity of 10 mL of ethanol (96 per cent) R and
2 quantities, each of 50 mL, of pentane R. Shake vigorously
Reference solution (a). Dissolve 10 mg of cholecalciferol CRS
for 30 s. Allow to stand until the 2 layers are clear. Transfer
in ethylene chloride R containing 10 g/L of squalane R and
the aqueous-alcoholic layer to a 2nd separating funnel and
0.1 g/L of butylhydroxytoluene R and dilute to 4 mL with
the same solution. shake with a mixture of 10 mL of ethanol (96 per cent) R
and 50 mL of pentane R. After separation, transfer the
Reference solution (b). Dissolve 10 mg of ergocalciferol CRS aqueous-alcoholic layer to a 3rd separating funnel and the
in ethylene chloride R containing 10 g/L of squalane R and pentane layer to the 1st separating funnel, washing the
0.1 g/L of butylhydroxytoluene R and dilute to 4 mL with 2nd separating funnel with 2 quantities, each of 10 mL, of
the same solution. pentane R and adding the washings to the 1st separating
Plate : TLC silica gel G plate R. funnel. Shake the aqueous-alcoholic layer with 50 mL of
Mobile phase : a 0.1 g/L solution of butylhydroxytoluene R pentane R and add the pentane layer to the 1st funnel. Wash
in a mixture of equal volumes of cyclohexane R and the pentane layer with 2 quantities, each of 50 mL, of a freshly
peroxide-free ether R. prepared 30 g/L solution of potassium hydroxide R in ethanol
Application : 20 μL. (10 per cent V/V) R, shaking vigorously, and then wash with
successive quantities, each of 50 mL, of water R until the
Development : immediately, protected from light, over a
washings are neutral to phenolphthalein. Transfer the washed
path of 15 cm.
pentane extract to a ground-glass-stoppered flask. Evaporate
Drying : in air. the contents of the flask to dryness under reduced pressure by
Detection : spray with sulfuric acid R. swirling in a water-bath at 40 °C. Cool under running water
Results : the chromatogram obtained with the test solution and restore atmospheric pressure with nitrogen R. Dissolve the
shows immediately a bright yellow principal spot, residue immediately in 5.0 mL of toluene R and add 20.0 mL
which rapidly becomes orange-brown, then gradually of the mobile phase to obtain a solution containing about
greenish-grey, remaining so for 10 min. This spot is similar 4000 IU/mL.
in position, colour and size to the principal spot in the Reference solution (a). Dissolve 10.0 mg of cholecalciferol CRS,
chromatogram obtained with reference solution (a). The without heating, in 10.0 mL of toluene R and dilute to
chromatogram obtained with reference solution (b) shows 100.0 mL with the mobile phase.
immediately at the same level an orange principal spot,
which gradually becomes reddish-brown and remains so Reference solution (b). Dilute 1.0 mL of cholecalciferol for
for 10 min. system suitability CRS to 5.0 mL with the mobile phase. Heat
in a water-bath at 90 °C under a reflux condenser for 45 min
B. Ultraviolet and visible absorption spectrophotometry and cool.
(2.2.25).
Test solution. Place 5.0 mL of the test solution prepared for Reference solution (c). Dissolve 0.10 g of cholecalciferol CRS,
the assay in a suitable flask and evaporate to dryness under without heating, in toluene R and dilute to 100.0 mL with the
reduced pressure by swirling in a water-bath at 40 °C. Cool same solvent.
under running water and restore atmospheric pressure with Reference solution (d). Dilute 5.0 mL of reference solution (c)
nitrogen R. Dissolve the residue immediately in 50.0 mL to 50.0 mL with the mobile phase. Keep the solution in iced
of cyclohexane R. water.

1872 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cholesterol

Reference solution (e). Place 5.0 mL of reference solution (c) in The contents of an opened container are to be used as soon as
a volumetric flask, add about 10 mg of butylhydroxytoluene R possible ; any unused part is to be protected by an atmosphere
and displace the air from the flask with nitrogen R. Heat in a of inert gas.
water-bath at 90 °C under a reflux condenser, protected from
light and under nitrogen R, for 45 min. Cool and dilute to LABELLING
50.0 mL with the mobile phase. The label states :
Column : – the number of International Units per gram ;
– size : l = 0.25 m, Ø = 4.6 mm ; – the storage temperature.
– stationary phase : silica gel for chromatography R (5 μm).
Mobile phase : pentanol R, hexane R (3:997 V/V). 01/2008:0993
Flow rate : 2 mL/min.
Detection : spectrophotometer at 254 nm. CHOLESTEROL
Injection : the chosen volume of each solution (the same
volume for reference solution (a) and for the test solution) ; Cholesterolum
automatic injection device or sample loop recommended.
Relative retention with reference to cholecalciferol : pre-
cholecalciferol = about 0.4 ; trans-cholecalciferol = about 0.5.
System suitability : reference solution (b) :
– resolution : minimum 1.0 between the peaks due to
pre-cholecalciferol and trans-cholecalciferol ; if necessary,
adjust the proportions of the constituents and the flow rate
of the mobile phase to obtain this resolution ;
C27H46O Mr 386.7
– repeatability : maximum relative standard deviation of [57-88-5]
1.0 per cent for the peak due to cholecalciferol after
6 injections. DEFINITION
Calculate the conversion factor (f) using the following Cholest-5-en-3β-ol.
expression : Content :
– cholesterol : minimum 95.0 per cent (dried substance) ;
– total sterols : 97.0 per cent to 103.0 per cent (dried
substance).
K = area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with reference CHARACTERS
solution (d) ; Appearance : white or almost white, crystalline powder.
L = area (or height) of the peak due to cholecalciferol Solubility : practically insoluble in water, sparingly soluble in
in the chromatogram obtained with reference acetone and in ethanol (96 per cent).
solution (e) ; It is sensitive to light.
M = area (or height) of the peak due to
pre-cholecalciferol in the chromatogram IDENTIFICATION
obtained with reference solution (e). A. Melting point (2.2.14) : 147 °C to 150 °C.
The value of f determined in duplicate on different days may B. Thin-layer chromatography (2.2.27). Prepare the solutions
be used during the entire procedure. immediately before use.
Calculate the content of cholecalciferol in International Units Test solution. Dissolve 10 mg of the substance to be
per gram using the following expression : examined in ethylene chloride R and dilute to 5 mL with
the same solvent.
Reference solution. Dissolve 10 mg of cholesterol CRS in
ethylene chloride R and dilute to 5 mL with the same solvent.
Plate : TLC silica gel G plate R.
m = mass of the preparation to be examined in the test
Mobile phase : ethyl acetate R, toluene R (33:66 V/V).
solution, in milligrams ;
Application : 20 μL.
m′ = mass of cholecalciferol CRS in reference solution (a),
in milligrams ; Development : immediately, protected from light, over a
path of 15 cm.
V = volume of the test solution (25 mL) ;
Drying : in air.
V′ = volume of reference solution (a) (100 mL) ; Detection : spray 3 times with antimony trichloride
SD = area (or height) of the peak due to cholecalciferol in solution R ; examine within 3-4 min.
the chromatogram obtained with the test solution ; Results : the principal spot in the chromatogram obtained
S′D = area (or height) of the peak due to cholecalciferol with the test solution is similar in position, colour and size
in the chromatogram obtained with reference to the principal spot in the chromatogram obtained with
solution (a); the reference solution.
Sp C. Dissolve about 5 mg in 2 mL of methylene chloride R. Add
= area (or height) of the peak due to
1 mL of acetic anhydride R, 0.01 mL of sulfuric acid R and
pre-cholecalciferol in the chromatogram
shake. A pink colour is produced which rapidly changes to
obtained with the test solution ;
red, then to blue and finally to brilliant green.
f = conversion factor.
TESTS
STORAGE Solubility in ethanol (96 per cent). In a stoppered flask,
In an airtight, well-filled container, protected from light, at the dissolve 0.5 g in 50 mL of ethanol (96 per cent) R at 50 °C.
temperature stated on the label. Allow to stand for 2 h. No deposit or turbidity is formed.

General Notices (1) apply to all monographs and other texts 1873
Cholesterol for parenteral use EUROPEAN PHARMACOPOEIA 8.0

Acidity. Dissolve 1.0 g in 10 mL of ether R, add 10.0 mL of

0.1 M sodium hydroxide and shake for about 1 min. Heat
gently to eliminate ether and then boil for 5 min. Cool, add
10 mL of water R and 0.1 mL of phenolphthalein solution R as
indicator and titrate with 0.1 M hydrochloric acid until the
pink colour just disappears, stirring the solution vigorously
throughout the titration. Carry out a blank titration. The
difference between the volumes of 0.1 M hydrochloric acid
B. cholesta-5,24-dien-3β-ol (desmosterol),
required to change the colour of the indicator in the blank
and in the test is not more than 0.3 mL.
Loss on drying (2.2.32) : maximum 0.3 per cent, determined
on 1.000 g by drying in vacuo at 60 °C for 4 h.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY
Gas chromatography (2.2.28). C. 5α-cholesta-7,24-dien-3β-ol.
Internal standard solution. Dissolve 0.100 g of pregnenolone
isobutyrate CRS in heptane R and dilute to 100.0 mL with the 01/2012:2397
same solvent.
Test solution. Dissolve 25.0 mg of the substance to be CHOLESTEROL FOR PARENTERAL
examined in the internal standard solution and dilute to
25.0 mL with the same solution. USE
Reference solution. Dissolve 25.0 mg of cholesterol CRS in
the internal standard solution and dilute to 25.0 mL with the Cholesterolum ad usum parenteralem
same solution.
Column :
– material : fused silica ;
– size : l = 30 m, Ø = 0.25 mm ;
– stationary phase : poly(dimethyl)siloxane R (film thickness
0.25 μm).
Carrier gas : helium for chromatography R.
Flow rate : 2 mL/min. C27H46O Mr 386.7
Split ratio : 1:25. [57-88-5]
Temperature : DEFINITION
– column : 275 °C ; Cholest-5-en-3β-ol obtained from Wool fat (0134).
– injection port : 285 °C ; Content :
– detector : 300 °C. – cholesterol : 99.0 per cent to 101.0 per cent (dried substance).
Detection : flame ionisation.
Injection : 1.0 μL. CHARACTERS
System suitability: reference solution : Appearance : white or almost white, crystalline powder.
– resolution : minimum 10.0 between the peaks due to Solubility : practically insoluble in water, sparingly soluble in
pregnenolone isobutyrate and cholesterol. acetone and in ethanol (96 per cent).
Calculate the percentage content of cholesterol from the It is sensitive to light.
declared content in cholesterol CRS. Calculate the percentage IDENTIFICATION
content of total sterols by adding together the contents of
cholesterol and other substances with a retention time less A. Melting point (2.2.14) : 147 °C to 150 °C.
than or equal to 1.5 times the retention time of cholesterol. B. Examine the chromatograms obtained in the assay.
Disregard the peaks due to the internal standard and the Results : the principal peak in the chromatogram obtained
solvent. with the test solution is similar in retention time and size
to the principal peak in the chromatogram obtained with
STORAGE the reference solution.
Protected from light. C. Dissolve about 5 mg in 2 mL of methylene chloride R.
LABELLING Add 1 mL of acetic anhydride R and 0.01 mL of sulfuric
acid R and shake. A pink colour is produced which rapidly
The label states the source material for the production of changes to red, then to blue and finally to bright green.
cholesterol (for example bovine brain and spinal cord, wool
fat or chicken eggs). TESTS
IMPURITIES Solubility in ethanol (96 per cent). In a stoppered flask,
dissolve 0.5 g in 50 mL of ethanol (96 per cent) R at 50 °C.
Allow to stand for 2 h. The solution is clear.
Acidity. Dissolve 1.0 g in 10 mL of ether R, add 10.0 mL of
0.1 M sodium hydroxide and shake for about 1 min. Heat
gently to eliminate the ether and then boil for 5 min. Cool,
add 10 mL of water R and 0.1 mL of phenolphthalein solution R
as indicator and titrate with 0.1 M hydrochloric acid until the
pink colour just disappears, stirring the solution vigorously
A. 5α-cholest-7-en-3β-ol (lathosterol), throughout the titration. Carry out a blank titration. The

1874 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cholesterol for parenteral use

difference between the volumes of 0.1 M hydrochloric acid acetonitrile R then 0.5 mL of water R to the residue. Suspend
required to change the colour of the indicator in the blank with the aid of ultrasound for about 5 min. Centrifuge the
titration and in the test is not more than 0.1 mL. suspension for 5 min and use the supernatant.
Peroxide value (2.5.5, Method A) : maximum 10. Column :
Other sterols. Gas chromatography (2.2.28) : use the – size : l = 0.25 m, Ø = 3 mm ;
normalisation procedure. – stationary phase : octadecylsilyl silica gel for
Internal standard solution. Dissolve 0.100 g of pregnenolone chromatography R (5 μm) ;
isobutyrate CRS in heptane R and dilute to 100.0 mL with the – temperature : 40 °C.
same solvent. Mobile phase :
Test solution. Dissolve 25.0 mg of the substance to be – mobile phase A : acetonitrile R, water R (50:50 V/V) ;
examined in the internal standard solution and dilute to – mobile phase B : acetonitrile R ;
25.0 mL with the same solution.
Time Mobile phase A Mobile phase B
Reference solution. Dissolve 25.0 mg of cholesterol CRS in (min) (per cent V/V) (per cent V/V)
the internal standard solution and dilute to 25.0 mL with the
same solution. 0 - 20 100 0
Column : 20 - 20.5 100 → 0 0 → 100
– material : fused silica ;
20.5 - 30 0 100
– size : l = 30 m, Ø = 0.25 mm ;
– stationary phase : poly(dimethyl)siloxane R (film thickness After elution of the components, a gradient is applied to
0.25 μm). prevent a strong drifting baseline due to cholesterol during
Carrier gas : helium for chromatography R. the following run.
Flow rate : 2 mL/min. Flow rate : 1 mL/min.
Detection : spectrophotometer at 254 nm.
Split ratio : 1:25.
Injection : 100 μL of the test solution and reference solutions (b)
Temperature : and (c).
– column : 275 °C ; Identification of impurities: use the chromatogram obtained
– injection port : 285 °C ; with reference solution (b) to identify the peaks due to
– detector : 300 °C. impurities A and B.
Detection : flame ionisation. Retention time : impurity A = about 10 min ;
Injection : 1.0 μL. impurity B = about 18 min.
Limits :
Relative retention with reference to cholesterol (retention
time = about 8.5 min) : pregnenolone isobutyrate = about 0.8. – impurity A : not more than 0.5 times the area of the
corresponding peak in the chromatogram obtained with
System suitability: reference solution : reference solution (c) (0.05 ppm) ;
– resolution : minimum 10.0 between the peaks due to – impurity B : not more than 0.5 times the area of the
pregnenolone isobutyrate and cholesterol. corresponding peak in the chromatogram obtained with
Limits : reference solution (c) (0.05 ppm).
– total of other substances with a retention time less than Heavy metals (2.4.8) : maximum 10 ppm.
or equal to 1.5 times the retention time of cholesterol : 2.0 g complies with test C. Prepare the reference solution using
maximum 0.5 per cent ; 2.0 mL of lead standard solution (10 ppm Pb) R.
– disregard limit : 0.05 per cent ; disregard the peak due to
Loss on drying (2.2.32) : maximum 0.1 per cent, determined
the internal standard.
on 1.000 g by drying in vacuo at 60 °C for 4 h.
Benzoyl ureas. Liquid chromatography (2.2.29). Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Test solution. Dissolve 1.0 g of the substance to be examined in 1.0 g.
200 mL of heptane R using a magnetic stirrer and add 10 mL
of acetonitrile R. Shake and allow the layers to separate. Isolate Microbial contamination
the lower layer (acetonitrile) and add 10 mL of acetonitrile R TAMC : acceptance criterion 102 CFU/g (2.6.12).
to the heptane layer and extract again. Combine the lower Bacterial endotoxins (2.6.14) : less than 0.1 IU/mg.
layers and evaporate to dryness using a rotary evaporator (for
example, at 40 °C and 17 kPa). Add 0.5 mL of acetonitrile R ASSAY
then 0.5 mL of water R to the residue. Suspend with the aid Gas chromatography (2.2.28) as described in the test for other
of ultrasound for about 5 min. Centrifuge the suspension for sterols.
5 min and use the supernatant. Calculate the percentage content of C27H46O from the declared
Reference solution (a). Dissolve 10.0 mg of diflubenzuron R content of cholesterol CRS.
(impurity A) and 10.0 mg of triflumuron R (impurity B) in
acetonitrile R and dilute to 100.0 mL with the same solvent. STORAGE
Dilute 0.1 mL of the solution to 100.0 mL with acetonitrile R. Protected from light.
Reference solution (b). Mix 0.5 mL of reference solution (a) IMPURITIES
and 0.5 mL of water R. Specified impurities : A, B.
Reference solution (c). Dissolve 1.0 g of the substance to
be examined in 200 mL of heptane R using a magnetic
stirrer. Add 0.5 mL of reference solution (a) and 9.5 mL of
acetonitrile R. Shake and allow the layers to separate. Isolate
the lower layer (acetonitrile) and add 10 mL of acetonitrile R
to the heptane layer and extract again. Combine the lower
layers and evaporate to dryness using a rotary evaporator A. 1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea
(for example, at e.g. 40 °C and 17 kPa). Add 0.5 mL of (diflubenzuron),

General Notices (1) apply to all monographs and other texts 1875
Chondroitin sulfate sodium EUROPEAN PHARMACOPOEIA 8.0

pH (2.2.3) : 5.5 to 7.5 for solution S1.

Specific optical rotation (2.2.7) : − 20 to − 30 (terrestrial
origin) or − 12 to − 19 (marine origin) (dried substance),
determined on solution S1.
Intrinsic viscosity : 0.01 m3/kg to 0.15 m3/kg.
B. 1-(2-chlorobenzoyl)-3-[(4-trifluoromethoxy)phenyl]urea
(triflumuron). Test solution (a). Weigh 5.000 g (m0p) of the substance to be
examined and add about 80 mL of an 11.7 g/L solution of
sodium chloride R at room temperature. Dissolve by shaking
at room temperature for 30 min. Dilute to 100.0 mL with
01/2009:2064 an 11.7 g/L solution of sodium chloride R. Filter through a
membrane filter (nominal pore size 0.45 μm) and discard the
CHONDROITIN SULFATE SODIUM first 10 mL. The concentration of test solution (a) is only
indicative and must be adjusted after an initial measurement
of the viscosity of test solution (a).
Chondroitini natrii sulfas Test solution (b). To 15.0 mL of test solution (a) add 5.0 mL of
an 11.7 g/L solution of sodium chloride R.
Test solution (c). To 10.0 mL of test solution (a) add 10.0 mL
of an 11.7 g/L solution of sodium chloride R.
Test solution (d). To 5.0 mL of test solution (a) add 15.0 mL of
an 11.7 g/L solution of sodium chloride R.
Determine the flow-time (2.2.9) for an 11.7 g/L solution
of sodium chloride R (t0) and the flow times for the 4 test
solutions (t1, t2, t3 and t4), at 25.00 ± 0.03 °C. Use an
H2O(C14H19NNa2O14S)x appropriate suspended level viscometer (specifications :
viscometer constant = about 0.005 mm2/s2, kinematic
DEFINITION viscosity range = 1-5 mm2/s, internal diameter of
Natural copolymer based mainly on the 2 disaccharides : tube R = 0.53 mm, volume of bulb C = 5.6 mL, internal
[4)-(β-D-glucopyranosyluronic acid)-(1→3)-[2-(acetyl- diameter of tube N = 2.8-3.2 mm) with a funnel-shaped lower
amino)-2-deoxy-β-D-galactopyranosyl 4-sulfate]-(1→] and capillary end. Use the same viscometer for all measurements ;
[4)-(β-D-glucopyranosyluronic acid)-(1→3)-[2-(acetylamino)- measure all outflow times in triplicate. The test is not valid
2-deoxy-β-D-galactopyranosyl 6-sulfate]-(1→], sodium unless the results do not differ by more than 0.35 per cent
salt. On complete hydrolysis it liberates D-galactosamine, from the mean and if the flow time t1 is not less than 1.6 × t0
D-glucuronic acid, acetic acid and sulfuric acid. It is obtained and not more than 1.8 × t0. If this is not the case, adjust the
from cartilage of both terrestrial and marine origins. concentration of test solution (a) and repeat the procedure.
Depending on the animal species of origin, it shows different Calculation of the relative viscosities
proportions of 4-sulfate and 6-sulfate groups. Since the densities of the chondroitin sulfate solutions and of
Content : 95 per cent to 105 per cent (dried substance). the solvent are almost equal, the relative viscosities ηri (being
ηr1, ηr2, ηr3 and ηr4) can be calculated from the ratio of the flow
PRODUCTION times for the respective solutions ti (being t1, t2, t3 and t4) to the
The animals from which chondroitin sulfate sodium is derived flow time of the solvent t0, but taking into account the kinetic
must fulfil the requirements for the health of animals suitable energy correction factor for the capillary (B = 30 800 s3), as
for human consumption. shown below :
CHARACTERS
Appearance : white or almost white, hygroscopic powder.
Solubility : freely soluble in water, practically insoluble in
acetone and in ethanol (96 per cent).
IDENTIFICATION Calculation of the concentrations
A. Infrared absorption spectrophotometry (2.2.24). Calculate the concentration c1 (expressed in kg/m3) of
chondroitin sulfate sodium in test solution (a) using the
Preparation : discs of potassium bromide R. following expression :
Comparison : for chondroitin sulfate sodium of terrestrial
origin use chondroitin sulfate sodium CRS and for
chondroitin sulfate sodium of marine origine use
chondroitin sulfate sodium (marine) CRS.
x = percentage content of chondroitin sulfate sodium
B. Solution S1 (see Tests) gives reaction (b) of sodium (2.3.1).
as determined in the assay ;
C. Examine the electropherograms obtained in the test for
related substances. h = loss on drying as a percentage.
Results : the principal band in the electropherogram Calculate the concentration c2 (expressed in kg/m3) of
obtained with the test solution is similar in position to chondroitin sulfate sodium in test solution (b) using the
the principal band in the electropherogram obtained with following expression :
reference solution (a).
TESTS
Solution S1. Dissolve 2.500 g in 50.0 mL of carbon dioxide-free Calculate the concentration c3 (expressed in kg/m ) of
3

water R. chondroitin sulfate sodium in test solution (c) using the

following expression :
Solution S2. Dilute 1.0 mL of solution S1 to 10.0 mL with
water R.

1876 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Chondroitin sulfate sodium

Calculate the concentration c4 (expressed in kg/m3) of tap water followed by 10-15 min with water R until the band
chondroitin sulfate sodium in test solution (d) using the in the electropherogram obtained with reference solution (c)
following expression : is visible. Allow the gel to dry.
System suitability :
– the electropherogram obtained with reference solution (c)
Calculation of the intrinsic viscosity shows a visible band ;
The specific viscosity ηsi of the test solution (being ηs1, ηs2, ηs3 – the band in the electropherogram obtained with reference
and ηs4) is calculated from the relative viscosities ηri (being ηr1, solution (b) is clearly visible and similar in position to the
ηr2, ηr3 and ηr4) according to the following expression : band in the electropherogram obtained with reference
solution (a).
Results : any secondary band in the electropherogram obtained
with the test solution is not more intense than the band in
The intrinsic viscosity [η], defined as the electropherogram obtained with reference solution (b)
(2 per cent).
Protein (2.5.33, Method 2) : maximum 3.0 per cent (dried
substance).
is calculated by linear least-squares regression analysis using
the following equation : Test solution. Dilute 1.0 mL of solution S1 to 50.0 mL with
0.1 M sodium hydroxide.
Reference solutions. Dissolve about 0.100 g of bovine
albumin R, accurately weighed, in 0.1 M sodium hydroxide
and dilute to 50.0 mL with the same solvent. Carry out all
ci = concentration of the substance to be examined additional dilutions using 0.1 M sodium hydroxide.
3
expressed in kg/m ;
Chlorides (2.4.4) : maximum 0.5 per cent.
kH = Huggins’ constant.
Dilute 1 mL of solution S2 to 15 mL with water R. Do not
Related substances. Electrophoresis (2.2.31). add diluted nitric acid. Prepare the standard using 5 mL of
chloride standard solution (5 ppm Cl) R and 10 mL of water R.
Buffer solution A (0.1 M barium acetate pH 5.0). Dissolve
25.54 g of barium acetate R in 900 mL of water R. Adjust to Heavy metals (2.4.8) : maximum 20 ppm.
pH 5.0 with glacial acetic acid R and dilute to 1000.0 mL with 1.0 g complies with test C. Prepare the reference solution using
water R. 2 mL of lead standard solution (10 ppm Pb) R.
Buffer solution B (1 M barium acetate pH 5.0). Dissolve Loss on drying (2.2.32) : maximum 12.0 per cent, determined
255.43 g of barium acetate R in 900 mL of water R. Adjust on 1.000 g by drying in an oven at 105 °C for 4 h.
to pH 5.0 with glacial acetic acid R and dilute to 1000.0 mL Microbial contamination
with water R.
TAMC : acceptance criterion 103 CFU/g (2.6.12).
Staining solution. Dissolve 1.0 g of toluidine blue R and 2.0 g 2
of sodium chloride R in 1000 mL of 0.01 M hydrochloric acid. TYMC : acceptance criterion 10 CFU/g (2.6.12).
Filter. Absence of Staphylococcus aureus (2.6.13).
Test solution. Prepare a 30 mg/mL solution of the substance to Absence of Pseudomonas aeruginosa (2.6.13).
be examined in water R. Absence of Escherichia coli (2.6.13).
Reference solution (a). Prepare a 30 mg/mL solution of Absence of Salmonella (2.6.13).
chondroitin sulfate sodium CRS in water R. Absence of bile-tolerant gram-negative bacteria (2.6.13).
Reference solution (b). Dilute 2.0 mL of reference solution (a)
to 100.0 mL with water R. ASSAY
Test solution (a). Weigh 0.100 g (m1) of the substance to be
Reference solution (c). Mix equal volumes of reference examined, dissolve in water R and dilute to 100.0 mL with
solution (b) and water R. the same solvent.
Procedure. Allow the electrophoresis support to cool the plate Test solution (b). Dilute 5.0 mL of test solution (a) to 50.0 mL
to 10 °C. Pre-equilibrate the agarose gel for 1 min in buffer with water R.
solution A. Remove excess liquid by careful decanting. Dry the
gel for approximately 5 min. Place 400 mL of buffer solution B Reference solution (a). Weigh 0.100 g (m0) of chondroitin
into each of the containers of the electrophoresis equipment. sulfate sodium CRS, previously dried as described in the test
Transfer 1 μL of each solution to the slots of the agarose for loss on drying, dissolve in water R and dilute to 100.0 mL
gel. Pipette a few millilitres of a 50 per cent V/V solution with the same solvent.
of glycerol R onto the cooled plate of the electrophoresis Reference solution (b). Dilute 5.0 mL of reference solution (a)
equipment and place the gel in the middle of the ceramic to 50.0 mL with water R.
plate. Place a wick, saturated with buffer solution B, at the Titrant solution (a). Weigh 4.000 g of cetylpyridinium chloride
positive and negative sides of the agarose gel. Ensure that monohydrate R and dilute to 1000 mL with water R.
there is good contact between the electrophoresis buffer Titrant solution (b). Weigh 1.000 g of cetylpyridinium chloride
and the agarose gel. Perform the electrophoresis under monohydrate R and dilute to 1000 mL with water R.
the following conditions : 75 mA/gel, resulting in a voltage
of 100-150 V (maximum 300-400 V) for a gel of about Perform either visual or photometric titration as follows :
12 cm × 10 cm. Carry out the electrophoresis for 12 min. Place Visual titration. Titrate 40.0 mL of reference solution (a) and
the gel in a mixture consisting of 10 volumes of anhydrous 40.0 mL of test solution (a) with titrant solution (a). The
ethanol R and 90 volumes of buffer solution A for 2 min. solution becomes turbid. At the end point, the liquid appears
Carry out the electrophoresis for 20 min. Place the gel in a clear, with an almost-white precipitate in suspension. The
mixture consisting of 30 volumes of anhydrous ethanol R precipitate is more apparent if 0.1 mL of a 1 per cent solution
and 70 volumes of buffer solution A for 2 min. Carry out of methylene blue R is added before starting the titration.
the electrophoresis for 20 min. Stain the gel in the staining The precipitated particles are more apparent against the blue
solution for 10 min. Destain the gel for 15 min under running background.

General Notices (1) apply to all monographs and other texts 1877
Chymotrypsin EUROPEAN PHARMACOPOEIA 8.0

Photometric titration. Titrate 50.0 mL of reference solution (b) Substrate solution. To 24.0 mg of acetyltyrosine ethyl ester R
and 50.0 mL of test solution (b) with titrant solution (b). To add 0.2 mL of ethanol (96 per cent) R and swirl to dissolve.
determine the end point, use a suitable autotitrator equipped Add 2.0 mL of 0.067 M phosphate buffer solution pH 7.0 R
with a phototrode at a suitable wavelength (none is critical) in and 1 mL of methyl red mixed solution R and dilute to
the visible range. 10.0 mL with water R.
Calculate the percentage content of chondroitin sulfate B. Dilute 0.5 mL of solution S to 5 mL with
sodium using the following expression : water R. Add 0.10 mL of a 20 g/L solution of
tosylphenylalanylchloromethane R in ethanol (96 per
cent) R. Adjust to pH 7.0 and shake for 2 h. In a depression
in a white spot-plate, mix 0.05 mL of this solution with
0.2 mL of the substrate solution (see Identification test A).
v0 = volume of appropriate titrant solution when No colour develops within 3 min of mixing.
titrating the appropriate reference solution, in
millilitres ; TESTS
v1 = volume of appropriate titrant solution when Solution S. Dissolve 0.10 g in carbon dioxide-free water R and
titrating the appropriate test solution, in millilitres ; dilute to 10.0 mL with the same solvent.
h = loss on drying of the substance to be examined, as Appearance of solution. Solution S is not more opalescent
a percentage ; than reference suspension II (2.2.1).
Z = percentage content of H2O(C14H19NNa2O14S)x in pH (2.2.3) : 3.0 to 5.0 for solution S.
chondroitin sulfate sodium CRS.
Specific absorbance (2.2.25): 18.5 to 22.5, determined at the
STORAGE absorption maximum at 281 nm ; maximum 8, determined at
the absorption minimum at 250 nm.
In an airtight container, protected from light.
Dissolve 30.0 mg in 0.001 M hydrochloric acid and dilute to
LABELLING 100.0 mL with the same acid.
The label states the origin of the substance (marine or Trypsin.
terrestrial). Substrate solution. To 98.5 mg of tosylarginine methyl ester
hydrochloride R, suitable for assaying trypsin, add 5 mL of
tris(hydroxymethyl)aminomethane buffer solution pH 8.1 R
and swirl to dissolve. Add 2.5 mL of methyl red mixed
01/2011:0476 solution R and dilute to 25.0 mL with water R.
Test solution. Transfer to a depression in a white spot-plate
CHYMOTRYPSIN 0.01 mL of tris(hydroxymethyl)aminomethane buffer solution
pH 8.1 R and 0.1 mL of solution S. Add 0.2 mL of the substrate
Chymotrypsinum solution.
Reference solution. At the same time and in the same manner
as for the test solution, prepare a solution using the substance
[9004-07-3] to be examined to which not more than 1 per cent m/m of
DEFINITION trypsin BRP has been added.
Chymotrypsin is a proteolytic enzyme obtained by the Start a timer. No colour appears in the test solution within
activation of chrymotrypsinogen extracted from the pancreas 3-5 min after the addition of the substrate solution. A purple
of beef (Bos taurus L.). It has an activity of not less than 5.0 colour is produced in the control solution.
microkatals per milligram. In solution it has maximal enzymic Loss on drying (2.2.32) : not more than 5.0 per cent,
activity at about pH 8 ; the activity is reversibly inhibited at determined on 0.100 g by drying at 60 °C at a pressure not
pH 3, the pH at which it is most stable. exceeding 0.7 kPa for 2 h.
PRODUCTION ASSAY
The animals from which chymotrypsin is derived must The activity of chymotrypsin is determined by comparing the
fulfil the requirements for the health of animals suitable for rate at which it hydrolyses acetyltyrosine ethyl ester R with the
human consumption. Furthermore, the tissues used shall not rate at which chymotrypsin BRP hydrolyses the same substrate
include any specified risk material as defined by any relevant under the same conditions.
international or, where appropriate, national legislation. Apparatus. Use a reaction vessel of about 30 mL capacity
The method of manufacture is validated to demonstrate that provided with :
the product, if tested, would comply with the following test. – a device that will maintain a temperature of 25.0 ± 0.1 °C ;
Histamine (2.6.10) : not more than 1 μg (calculated as – a stirring device, for example a magnetic stirrer ;
histamine base) per 5 microkatals of chymotrypsin activity.
Before carrying out the test, heat the solution of the substance– a lid with holes for the insertion of electrodes, the tip of
to be examined on a water-bath for 30 min. a burette, a tube for the admission of nitrogen and the
introduction of reagents.
CHARACTERS An automatic or manual titration apparatus may be used.
Appearance : white or almost white, crystalline or amorphous For the latter, the burette is graduated in 0.005 mL and the
powder, hygroscopic if amorphous. pH meter is provided with a wide scale and glass-calomel or
glass-silver-silver chloride electrodes.
Solubility : sparingly soluble in water.
Test solution. Dissolve 25.0 mg of the substance to be
IDENTIFICATION examined in 0.001 M hydrochloric acid and dilute to 250.0 mL
A. Dilute 1 mL of solution S (see Tests) to 10 mL with water R. with the same acid.
In a depression in a white spot-plate, mix 0.05 mL of this Reference solution. Dissolve 25.0 mg of chymotrypsin BRP in
solution with 0.2 mL of the substrate solution. A purple 0.001 M hydrochloric acid and dilute to 250.0 mL with the
colour develops. same acid.

1878 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ciclesonide

Store the solutions at 0-5 °C. Warm 1 mL of each solution IDENTIFICATION

to about 25 °C over 15 min and use 50 μL of each solution A. Infrared absorption spectrophotometry (2.2.24).
(corresponding to about 25 nanokatals) for each titration. Comparison: ciclesonide CRS.
Carry out the titration in an atmosphere of nitrogen. Transfer
10.0 mL of 0.01 M calcium chloride solution R to the reaction B. Examine the chromatograms obtained in the assay.
vessel and, while stirring, add 0.35 mL of 0.2 M acetyltyrosine Results : the principal peak in the chromatogram obtained
ethyl ester R. When the temperature is steady at 25.0 ± 0.1 °C with the test solution is similar in retention time and size
(after about 5 min), adjust to pH 8.0 exactly with 0.02 M to the principal peak in the chromatogram obtained with
sodium hydroxide. Add 50 μL of the test solution (equivalent reference solution (a).
to about 5 μg of the substance to be examined) and start a
timer. Maintain at pH 8.0 by the addition of 0.02 M sodium TESTS
hydroxide, noting the volume added every 30 s. Calculate Related substances. Liquid chromatography (2.2.29).
the volume of 0.02 M sodium hydroxide used per second Test solution. Dissolve 50.0 mg of the substance to be
between 30 s and 210 s. Carry out a titration in the same examined in anhydrous ethanol R and dilute to 50.0 mL with
manner using the reference solution and calculate the volume the same solvent.
of 0.02 M sodium hydroxide used per second. Reference solution (a). Dissolve 50.0 mg of ciclesonide CRS
Calculate the activity in microkatals per milligram using the in anhydrous ethanol R and dilute to 50.0 mL with the same
following expression : solvent.
Reference solution (b). Dissolve 3 mg of ciclesonide
impurity B CRS, 3 mg of ciclesonide impurity C CRS and
5 mg of ciclesonide containing impurity A CRS in anhydrous
m = mass of the substance to be examined, in ethanol R and dilute to 10.0 mL with the same solvent.
milligrams ; Reference solution (c). Dissolve 50 mg of the substance to be
m′ = mass of chymotrypsin BRP, in milligrams ; examined in anhydrous ethanol R, add 1.0 mL of reference
solution (b) and dilute to 50.0 mL with anhydrous ethanol R.
V = volume of 0.02 M sodium hydroxide used per Reference solution (d). Dilute 1.0 mL of the test solution to
second by the test solution ; 100.0 mL with anhydrous ethanol R. Dilute 1.0 mL of this
V′ = volume of 0.02 M sodium hydroxide used per solution to 10.0 mL with anhydrous ethanol R.
second by the reference solution ; Column :
A = activity of chymotrypsin BRP, in microkatals per – size : l = 0.25 m, Ø = 4.6 mm ;
milligram.
– stationary phase : phenylsilyl silica gel for chromatography R
STORAGE (5 μm) ;
In an airtight container at 2 °C to 8 °C, protected from light. – temperature : 60 °C.
Mobile phase : water R, anhydrous ethanol R (38:62 V/V).
LABELLING Flow rate : 1.0 mL/min.
The label states : Detection : spectrophotometer at 243 nm.
– the quantity of chymotrypsin and the total activity in Injection : 20 μL of the test solution and reference solutions (c)
microkatals per container ; and (d).
– for the amorphous substance, that it is hygroscopic. Run time : 2.2 times the retention time of ciclesonide.
Identification of impurities: use the chromatogram obtained
04/2013:2703 with reference solution (c) to identify the peaks due to
impurities A, B and C.
Relative retention with reference to ciclesonide (retention
CICLESONIDE time = about 16 min) : impurity B = about 0.4 ;
impurity C = about 0.9 ; impurity A = about 1.4.
Ciclesonidum System suitability : reference solution (c) :
– resolution : minimum 1.5 between the peaks due to
impurity C and ciclesonide.
Calculation of percentage contents :
– for each impurity, use the concentration of ciclesonide in
reference solution (d).
Limits :
– impurity A : maximum 1.0 per cent ;
– impurities B, C : for each impurity, maximum 0.15 per cent ;
– unspecified impurities : for each impurity, maximum
C32H44O7 Mr 540.7 0.10 per cent ;
[126544-47-6]
– total of unspecified impurities : maximum 0.2 per cent ;
DEFINITION – total : maximum 1.2 per cent ;
(2′R)-2′-Cyclohexyl-11β-hydroxy-3,20-dioxo-16βH- – reporting threshold : 0.05 per cent.
[1,3]dioxolo[4′,5′:16,17]pregna-1,4-dien-21-yl Heavy metals (2.4.8) : maximum 20 ppm.
2-methylpropanoate.
Solvent mixture : water R, ethanol (96 per cent) R (15:85 V/V).
Content : 98.0 per cent to 102.0 per cent (anhydrous substance).
0.250 g complies with test H. Prepare the reference solution
CHARACTERS using 0.5 mL of lead standard solution (10 ppm Pb) R.
Appearance : white or yellowish-white, crystalline powder. Water (2.5.12) : maximum 0.5 per cent, determined on 0.500 g.
Solubility : practically insoluble in water, freely soluble to Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
soluble in acetone and in anhydrous ethanol. 1.0 g.

General Notices (1) apply to all monographs and other texts 1879
Ciclopirox EUROPEAN PHARMACOPOEIA 8.0

ASSAY CHARACTERS
Liquid chromatography (2.2.29) as described in the test for Appearance : white or yellowish-white, crystalline powder.
related substances with the following modifications. Solubility : slightly soluble in water, freely soluble in anhydrous
Injection : test solution and reference solution (a). ethanol and in methylene chloride.
Run time : 1.6 times the retention time of ciclesonide. IDENTIFICATION
System suitability: reference solution (a) : First identification : B.
– symmetry factor : maximum 2.2 for the peak due to Second identification : A, C.
ciclesonide.
A. Melting point (2.2.14) : 140 °C to 145 °C.
Calculate the percentage content of C32H44O7 taking into
account the assigned content of ciclesonide CRS. B. Infrared absorption spectrophotometry (2.2.24).
Comparison: ciclopirox CRS.
IMPURITIES C. Thin-layer chromatography (2.2.27).
Specified impurities : A, B, C. Test solution. Dissolve 20 mg of the substance to be
examined in methanol R and dilute to 10 mL with the same
solvent.
Reference solution. Dissolve 20 mg of ciclopirox CRS in
methanol R and dilute to 10 mL with the same solvent.
Plate : TLC silica gel F254 plate R.
Pretreatment : before use, predevelop with the mobile phase
until the solvent front has migrated to the top of the plate.
Allow to dry in air for 5 min.
Mobile phase : concentrated ammonia R, water R, ethanol
A. (2′S)-2′-cyclohexyl-11β-hydroxy-3,20-dioxo-16βH- (96 per cent) R (10:15:75 V/V/V).
[1,3]dioxolo[4′,5′:16,17]pregna-1,4-dien-21-yl Application : 10 μL.
2-methylpropanoate (S-epimer of ciclesonide),
Development : over 2/3 of the plate.
Drying : in air for 10 min.
Detection A : examine in ultraviolet light at 254 nm.
Results A : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the
reference solution.
Detection B : spray with a 20 g/L solution of ferric chloride R
B. (2′R)-2′-cyclohexyl-11β,21-dihydroxy-16βH- in anhydrous ethanol R.
[1,3]dioxolo[4′,5′:16,17]pregna-1,4-diene-3,20-dione,
Results B : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
the reference solution.
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution Y5 (2.2.2,
Method II).
Dissolve 2.0 g in methanol R and dilute to 10 mL with the
C. (2′R)-2′-[(1RS)-cyclohex-3-enyl]-11β-hydroxy-3,20-dioxo- same solvent.
16βH-[1,3]dioxolo[4′,5′:16,17]pregna-1,4-dien-21-yl Related substances. Liquid chromatography (2.2.29). Carry
2-methylpropanoate. out the test avoiding exposure to actinic light. All materials in
direct contact with the substance to be examined like column
materials, reagents, solvents, etc. should contain only very low
07/2010:1407 amounts of extractable metal cations.
corrected 7.5
Solvent mixture : acetonitrile R, mobile phase (10:90 V/V).
Test solution. Dissolve 30.0 mg of the substance to be
CICLOPIROX examined in 15 mL of the solvent mixture, using an ultrasonic
bath if necessary, and dilute to 20.0 mL with the solvent
Ciclopiroxum mixture.
Reference solution (a). Dissolve 15.0 mg of ciclopirox
impurity A CRS and 15.0 mg of ciclopirox impurity B CRS in
the solvent mixture and dilute to 10.0 mL with the solvent
mixture.
Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 200.0 mL with the solvent mixture.
Reference solution (c). Dilute 2.0 mL of reference solution (b)
C12H17NO2 Mr 207.3 to 10.0 mL with the solvent mixture.
[29342-05-0]
Reference solution (d). Mix 5.0 mL of reference solution (a)
DEFINITION and 5.0 mL of the test solution.
6-Cyclohexyl-1-hydroxy-4-methylpyridin-2(1H)-one. Column :
Content : 98.0 per cent to 101.0 per cent (dried substance). – size : l = 0.08 m, Ø = 4 mm ;

1880 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ciclopirox olamine

– stationary phase : nitrile silica gel for chromatography R2

(5 μm).
In order to ensure desorption of interfering metal ions, every
new column is to be rinsed with the rinsing solution over a
period of not less than 15 h and then with the mobile phase
for not less than 5 h at a flow rate of 0.2 mL/min.
A. [(5RS)-3-cyclohexyl-5-methyl-4,5-dihydro-1,2-oxazol-5-
Rinsing solution : glacial acetic acid R, acetylacetone R, yl]acetic acid,
acetonitrile R, water R (0.1:0.1:50:50 V/V/V/V).
Mobile phase : glacial acetic acid R, acetonitrile R, 0.96 g/L
solution of sodium edetate R (0.01:23:77 V/V/V).
Flow rate : 0.7 mL/min.
Detection : spectrophotometer at 220 nm and at 298 nm.
Injection : 10 μL of the test solution and reference solutions (b),
(c) and (d) ; inject the solvent mixture as a blank. B. 6-cyclohexyl-4-methyl-2H-pyran-2-one,
Run time : 2.5 times the retention time of ciclopirox.
Retention time : ciclopirox = 8 min to 11 min ; if necessary
adjust the ratio of the 0.96 g/L solution of sodium edetate to
acetonitrile in the mobile phase.
Relative retention with reference to ciclopirox :
impurity A = about 0.5 ; impurity C = about 0.9 ;
impurity B = about 1.3. C. 6-cyclohexyl-4-methylpyridin-2(1H)-one.
System suitability : at 298 nm :
07/2010:1302
– resolution : minimum 2.0 between the peaks due to corrected 7.5
ciclopirox and impurity B in the chromatogram obtained
with reference solution (d) ;
– symmetry factor : 0.8 to 2.0 for the principal peak in the
CICLOPIROX OLAMINE
chromatogram obtained with the test solution.
Ciclopirox olaminum
Limits :
– impurity A at 220 nm : not more than the area of the
corresponding peak in the chromatogram obtained with
reference solution (b) (0.5 per cent) ;
– impurities B, C at 298 nm : for each impurity, not more than
the area of the peak due to impurity B in the chromatogram
obtained with reference solution (b) (0.5 per cent) ;
– unspecified impurities at 298 nm : for each impurity, not C14H24N2O3 Mr 268.4
more than the area of the peak due to impurity B in [41621-49-2]
the chromatogram obtained with reference solution (c) DEFINITION
(0.10 per cent) ;
6-Cyclohexyl-1-hydroxy-4-methylpyridin-2(1H)-one and
– sum of impurities other than B at 298 nm : not more than 2-aminoethanol.
the area of the peak due to impurity B in the chromatogram Content :
obtained with reference solution (b) (0.5 per cent) ;
– ciclopirox (C12H17NO2 ; Mr 207.3) : 76.0 per cent to 78.5 per
– disregard limit at 298 nm : 0.5 times the area of the peak due cent (dried substance) ;
to impurity B in the chromatogram obtained with reference
solution (c) (0.05 per cent). – 2-aminoethanol (C2H7NO ; Mr 61.1): 22.2 per cent to
23.3 per cent (dried substance).
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test C. Prepare the reference solution using CHARACTERS
2 mL of lead standard solution (10 ppm Pb) R. Appearance : white or pale yellow, crystalline powder.
Loss on drying (2.2.32) : maximum 1.5 per cent, determined Solubility : sparingly soluble in water, very soluble in ethanol
on 1.000 g by drying in vacuo at 60 °C over diphosphorus (96 per cent) and in methylene chloride, slightly soluble in
pentoxide R. ethyl acetate, practically insoluble in cyclohexane.
It shows polymorphism (5.9).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. IDENTIFICATION
First identification : A.
ASSAY
Second identification : B.
Dissolve 0.150 g in 20 mL of methanol R. Add 20 mL of
water R and titrate with 0.1 M sodium hydroxide, determining A. Infrared absorption spectrophotometry (2.2.24).
the end-point potentiometrically (2.2.20). Carry out a blank Comparison: ciclopirox olamine CRS.
titration. If the spectra obtained in the solid state show differences,
1 mL of 0.1 M sodium hydroxide is equivalent to 20.73 mg dissolve the substance to be examined and the reference
of C12H17NO2. substance separately in the minimum volume of ethyl
acetate R, evaporate to dryness on a water-bath and record
STORAGE new spectra using the residues.
Protected from light. B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 25 mg of the substance to be
IMPURITIES examined in methanol R and dilute to 10 mL with the same
Specified impurities : A, B, C. solvent.

General Notices (1) apply to all monographs and other texts 1881
Ciclopirox olamine EUROPEAN PHARMACOPOEIA 8.0

Reference solution. Dissolve 25 mg of ciclopirox In order to ensure desorption of interfering metal ions, every
olamine CRS in methanol R and dilute to 10 mL with the new column is to be rinsed with the rinsing solution over a
same solvent. period of not less than 15 h and then with the mobile phase
Plate : TLC silica gel F254 plate R. for not less than 5 h at a flow rate of 0.2 mL/min.
Pretreatment : before use, predevelop 2 plates with the Rinsing solution : acetylacetone R, anhydrous acetic acid R,
mobile phase until the solvent front has migrated to the top acetonitrile R, water R (0.1:0.1:50:50 V/V/V/V).
of the plates. Allow to dry in air for 5 min. Mobile phase : anhydrous acetic acid R, acetonitrile R, 0.96 g/L
Mobile phase : concentrated ammonia R, water R, anhydrous solution of sodium edetate R (0.01:23:77 V/V/V).
ethanol R (10:15:75 V/V/V). Flow rate : 0.7 mL/min.
Application : 10 μL. Detection : spectrophotometer at 220 nm and at 298 nm.
Development : over 2/3 of the plate. Injection : 10 μL of the test solution and reference solutions (b),
(c) and (d).
Drying : in air for 10 min.
Run time : 2.5 times the retention time of ciclopirox.
Detection A : examine in ultraviolet light at 254 nm.
Retention time : ciclopirox = 8 min to 11 min ; if necessary
Results A : the principal spot in the chromatogram obtained adjust the ratio of the 0.96 g/L solution of sodium edetate to
with the test solution is similar in position and size to the acetonitrile in the mobile phase.
principal spot in the chromatogram obtained with the
reference solution. Relative retention with reference to ciclopirox :
impurity A = about 0.5 ; impurity C = about 0.9 ;
Detection B : spray 1 plate with ferric chloride solution R3. impurity B = about 1.3.
Results B : the principal spot in the chromatogram obtained System suitability : at 298 nm :
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with – resolution : minimum of 2.0 between the peaks due to
the reference solution. impurity B and ciclopirox in the chromatogram obtained
with reference solution (d) ;
Detection C : spray the 2nd second plate with ninhydrin
– symmetry factor : 0.8 to 2.0 for the principal peak in the
solution R. Heat at 110 °C until the spots appear.
chromatogram obtained with the test solution.
Results C : the principal spot in the chromatogram obtained Limits :
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with – impurity A at 220 nm : not more than the area of the
the reference solution. corresponding peak in the chromatogram obtained with
reference solution (b) (0.5 per cent) ;
TESTS – impurities B, C at 298 nm : for each impurity, not more than
Appearance of solution. The solution is clear (2.2.1) and not the area of the peak due to impurity B in the chromatogram
more intensely coloured than reference solution BY7 (2.2.2, obtained with reference solution (b) (0.5 per cent) ;
Method II). – unspecified impurities at 298 nm : for each impurity, not
Dissolve 2.0 g in methanol R and dilute to 20 mL with the more than the area of the peak due to impurity B in
same solvent. the chromatogram obtained with reference solution (c)
(0.10 per cent);
pH (2.2.3) : 8.0 to 9.0.
– sum of impurities other than B at 298 nm : not more than
Dissolve 1.0 g in carbon dioxide-free water R and dilute to the area of the peak due to impurity B in the chromatogram
100 mL with the same solvent. obtained with reference solution (b) (0.5 per cent) ;
Related substances. Liquid chromatography (2.2.29). Carry – disregard limit at 298 nm : 0.5 times the area of the peak due
out the test avoiding exposure to actinic light. All materials to impurity B in the chromatogram obtained with reference
in direct contact with the substance to be examined, such as solution (c) (0.05 per cent).
column materials, reagents, solvents, etc. should contain only
small amounts of extractable metal cations. Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test C. Prepare the reference solution using
Solvent mixture : acetonitrile R, mobile phase (10:90 V/V).
2 mL of lead standard solution (10 ppm Pb) R.
Test solution. Dissolve 40.0 mg of the substance to be
examined (corresponding to about 30 mg of ciclopirox) Loss on drying (2.2.32) : maximum 1.5 per cent, determined
in a mixture of 20 μL of anhydrous acetic acid R, 2 mL of on 1.000 g by drying under high vacuum.
acetonitrile R, and 15 mL of the mobile phase, using an Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
ultrasonic bath if necessary. Dilute the solution to 20.0 mL 1.0 g.
with the mobile phase.
ASSAY
Reference solution (a). Dissolve 15.0 mg of ciclopirox
impurity A CRS and 15.0 mg of ciclopirox impurity B CRS in 2-Aminoethanol. Dissolve 0.250 g in 25 mL of anhydrous
a mixture of 1 mL of acetonitrile R and 7 mL of the mobile acetic acid R. Titrate with 0.1 M perchloric acid, determining
phase, and dilute to 10.0 mL with the mobile phase. the end-point potentiometrically (2.2.20).
Reference solution (b). Dilute 1.0 mL of reference solution (a) 1 mL of 0.1 M perchloric acid is equivalent to 6.108 mg of
to 200.0 mL with the solvent mixture. C2H7NO.
Reference solution (c). Dilute 2.0 mL of reference solution (b) Ciclopirox. Dissolve 0.200 g in 2 mL of methanol R.
to 10.0 mL with the solvent mixture. Add 38 mL of water R, swirl and titrate immediately
with 0.1 M sodium hydroxide, determining the end-point
Reference solution (d). Mix 5.0 mL of reference solution (a) potentiometrically (2.2.20). Carry out a blank titration.
and 5.0 mL of the test solution.
Use 0.1 M sodium hydroxide, the titre of which has been
Column : determined under the conditions prescribed above using
– size : l = 80 mm, Ø = 4 mm ; 0.100 g of benzoic acid RV.
– stationary phase : nitrile silica gel for chromatography R 1 mL of 0.1 M sodium hydroxide is equivalent to 20.73 mg
(5 μm). of C12H17NO2.

1882 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ciclosporin

STORAGE TESTS
Protected from light. Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution Y5, BY5 or R7
IMPURITIES (2.2.2, Method II).
Specified impurities : A, B, C. Dissolve 1.5 g in anhydrous ethanol R and dilute to 15 mL
with the same solvent.
Specific optical rotation (2.2.7) : − 193 to − 185 (dried
substance).
Dissolve 0.125 g in methanol R and dilute to 25.0 mL with
the same solvent.
A. [(5RS)-3-cyclohexyl-5-methyl-4,5-dihydro-1,2-oxazol-5- Related substances. Liquid chromatography (2.2.29).
yl]acetic acid,
Solvent mixture : acetonitrile R, water R (50:50 V/V).
Test solution. Dissolve 30.0 mg of the substance to be
examined in the solvent mixture and dilute to 25.0 mL with
the solvent mixture.
Reference solution (a). Dissolve 30.0 mg of ciclosporin CRS in
the solvent mixture and dilute to 25.0 mL with the solvent
mixture.
B. 6-cyclohexyl-4-methyl-2H-pyran-2-one, Reference solution (b). Dilute 2.0 mL of reference solution (a)
to 200.0 mL with the solvent mixture.
Reference solution (c). Dissolve the contents of a vial of
ciclosporin for system suitability CRS in 5.0 mL of the mobile
phase.
Column :
– size : l = 0.25 m, Ø = 4 mm ;
– stationary phase : octadecylsilyl silica gel for
C. 6-cyclohexyl-4-methylpyridin-2(1H)-one. chromatography R (3-5 μm) ;
– temperature : 80 °C.
The column is connected to the injection port by a steel
07/2012:0994 capillary tube about 1 m long, having an internal diameter of
0.25 mm and maintained at 80 °C.
CICLOSPORIN Mobile phase : phosphoric acid R, 1,1-dimethylethyl methyl
ether R, acetonitrile R, water R (0.1:5:43:52 V/V/V/V).
Ciclosporinum Flow rate : 1.5 mL/min.
Detection : spectrophotometer at 210 nm.
Injection : 20 μL of the test solution and reference solutions (b)
and (c).
Run time : 1.7 times the retention time of ciclosporin.
System suitability : reference solution (c) :
– retention time : ciclosporin = 25 min to 30 min ; if necessary,
adjust the ratio of acetonitrile to water in the mobile phase ;
C62H111N11O12 Mr 1203 – peak-to-valley ratio : minimum 1.4, where Hp = height
[59865-13-3] above the baseline of the peak due to ciclosporin U and
Hv = height above the baseline of the lowest point of the
DEFINITION curve separating this peak from the peak due to ciclosporin ;
Cyclo[[(2S,3R,4R,6E)-3-hydroxy-4-methyl-2-(methylamino)- if necessary, adjust the ratio of 1,1-dimethylethyl methyl
oct-6-enoyl]-L-2-aminobutanoyl-N-methylglycyl-N-methyl-L- ether to acetonitrile in the mobile phase.
leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl- Limits :
L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl] (ciclosporin A). – any impurity : for each impurity, not more than 0.7 times
Substance produced by Beauveria nivea (Tolypocladium the area of the principal peak in the chromatogram
inflatum Gams) or obtained by any other means. obtained with reference solution (b) (0.7 per cent) ;
Content : 97.0 per cent to 102.0 per cent (dried substance). – total : not more than 1.5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
CHARACTERS (1.5 per cent) ;
Appearance : white or almost white powder. – disregard limit : 0.05 times the area of the principal peak
Solubility : practically insoluble in water, freely soluble in in the chromatogram obtained with reference solution (b)
anhydrous ethanol and in methylene chloride. (0.05 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
IDENTIFICATION The residue obtained in the test for loss on drying complies
A. Infrared absorption spectrophotometry (2.2.24). with test C. Prepare the reference solution using 2 mL of lead
Comparison : ciclosporin CRS. standard solution (10 ppm Pb) R.
B. Examine the chromatograms obtained in the assay. Loss on drying (2.2.32) : maximum 2.0 per cent, determined
Results : the principal peak in the chromatogram obtained on 1.000 g at 60 °C at a pressure not exceeding 15 Pa for 3 h.
with the test solution is similar in retention time to Bacterial endotoxins (2.6.14) : less than 0.84 IU/mg, if
the principal peak in the chromatogram obtained with intended for use in the manufacture of parenteral preparations
reference solution (a). without a further appropriate procedure for the removal of

General Notices (1) apply to all monographs and other texts 1883
Cilastatin sodium EUROPEAN PHARMACOPOEIA 8.0

bacterial endotoxins. Dissolve 50 mg of the substance to be Solubility : very soluble in water and in methanol, slightly
examined in a mixture of 280 mg of ethanol (96 per cent) R soluble in anhydrous ethanol, very slightly soluble in dimethyl
and 650 mg of polyoxyethylated castor oil R and dilute to the sulfoxide, practically insoluble in acetone and in methylene
required concentration using water for BET. chloride.
ASSAY IDENTIFICATION
Liquid chromatography (2.2.29) as described in the test for A. Specific optical rotation (see Tests).
related substances with the following modifications. B. Infrared absorption spectrophotometry (2.2.24).
Injection : test solution and reference solution (a). Comparison: cilastatin sodium CRS.
System suitability: reference solution (a) : C. It gives reaction (a) of sodium (2.3.1).
– repeatability : maximum relative standard deviation of
1.0 per cent after 6 injections. TESTS
Calculate the percentage content of C62H111N11O12 taking into Solution S. Dissolve 1.0 g in carbon dioxide-free water R and
account the assigned content of ciclosporin CRS. dilute to 100 mL with the same solvent.
STORAGE Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y6 (2.2.2,
In an airtight container, protected from light. If the substance
Method II).
is sterile, store in a sterile, airtight, tamper-proof container.
pH (2.2.3) : 6.5 to 7.5 for solution S.
IMPURITIES
Specific optical rotation (2.2.7) : + 41.5 to + 44.5 (anhydrous
substance).
Dissolve 0.250 g in a mixture of 1 volume of hydrochloric
acid R and 120 volumes of methanol R, then dilute to 25.0 mL
with the same mixture of solvents.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 32.0 mg of the substance to be
A. different ciclosporins [difference from ciclosporin examined in water R and dilute to 20.0 mL with the same
(R = CH3 : ciclosporin A)] : ciclosporin B solvent.
[7-L-Ala] ; ciclosporin C [7-L-Thr] ; ciclosporin D Reference solution (a). Dilute 2.0 mL of the test solution to
[7-L-Val] ; ciclosporin E [5-L-Val] ; ciclosporin G 100.0 mL with water R. Dilute 5.0 mL of this solution to
[7-(L-2-aminopentanoyl)] ; ciclosporin H [5-D-MeVal] ; 100.0 mL with water R.
ciclosporin L [R = H] ; ciclosporin T [4-L-Leu] ;
ciclosporin U [11-L-Leu] ; ciclosporin V [1-L-Abu], Reference solution (b). Dilute 5.0 mL of the test solution to
100.0 mL with water R. Dilute 2.0 mL of this solution to
20.0 mL with water R.
Reference solution (c). Dissolve 16 mg of the substance to be
examined in dilute hydrogen peroxide solution R and dilute to
10.0 mL with the same solution. Allow to stand for 30 min.
Dilute 1 mL of this solution to 100 mL with water R.
Reference solution (d). Dissolve 32 mg of mesityl oxide R
B. [6-[(2S,3R,4R)-3-hydroxy-4-methyl-2-(methylamino)- (impurity D) in 100 mL of water R. Dilute 1 mL of this
octanoic acid]]ciclosporin A, solution to 50 mL with water R.
Column :
C. isociclosporin A.
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
01/2008:1408 chromatography R (5 μm) ;
corrected 6.1 – temperature : 50 °C.
Mobile phase :
CILASTATIN SODIUM – mobile phase A : mix 300 volumes of acetonitrile R1 and
700 volumes of a 0.1 per cent V/V solution of phosphoric
Cilastatinum natricum acid R in water R ;
– mobile phase B : 0.1 per cent V/V solution of phosphoric
acid R in water R ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 30 15 → 100 85 → 0
C16H25N2NaO5S Mr 380.4
[81129-83-1] 30 - 46 100 0
46 - 56 100 → 15 0 → 85
DEFINITION
Sodium (Z)-7-[[(R)-2-amino-2-carboxyethyl]sulfanyl]-2- Flow rate : 2.0 mL/min.
[[[(1S)-2,2-dimethylcyclopropyl]carbonyl]amino]hept-2- Detection : spectrophotometer at 210 nm.
enoate.
Injection : 20 μL.
Content : 98.0 per cent to 101.5 per cent (anhydrous substance).
System suitability :
CHARACTERS – the chromatogram obtained with reference solution (c)
Appearance : white or light yellow amorphous, hygroscopic shows 3 principal peaks : the first 2 peaks (impurity A) may
powder. elute without being completely resolved ;

1884 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cilazapril

– mass distribution ratio : minimum 10 for the peak due to Limits :

cilastatin (3rd peak) in the chromatogram obtained with – acetone : maximum 1.0 per cent m/m ;
reference solution (c) ; – methanol : maximum 0.5 per cent m/m ;
– signal-to-noise ratio : minimum 5.0 for the principal peak in – impurity D : maximum 0.4 per cent m/m.
the chromatogram obtained with reference solution (a).
Heavy metals (2.4.8) : maximum 20 ppm.
Limits :
1.0 g complies with test C. Prepare the reference solution using
– impurities A, B, C : for each impurity, not more than the 2.0 mL of lead standard solution (10 ppm Pb) R.
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.5 per cent) ; Water (2.5.12) : maximum 2.0 per cent, determined on 0.50 g.
– total : not more than twice the area of the principal peak Bacterial endotoxins (2.6.14) : less than 0.17 IU/mg, if
in the chromatogram obtained with reference solution (b) intended for use in the manufacture of parenteral preparations
(1 per cent) ; without a further appropriate procedure for the removal of
bacterial endotoxins.
– disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.1 per ASSAY
cent) ; disregard any peak corresponding to the peak due to Dissolve 0.300 g in 30 mL of methanol R and add 5 mL of
impurity D in the chromatogram obtained with reference water R. Add 0.1 M hydrochloric acid to a pH of about 3.0.
solution (d). Carry out a potentiometric titration (2.2.20), using 0.1 M
Impurity D, acetone and methanol. Gas chromatography sodium hydroxide. 3 jumps of potential are observed. Titrate
(2.2.28). to the 3rd equivalence point.
Internal standard solution. Dissolve 0.5 mL of propanol R in 1 mL of 0.1 M sodium hydroxide is equivalent to 19.02 mg
water R and dilute to 1000 mL with the same solvent. of C16H25N2NaO5S.
Test solution. Dissolve 0.200 g of the substance to be examined STORAGE
in water R, add 2.0 mL of the internal standard solution and
In an airtight container, at a temperature not exceeding 8 °C. If
dilute to 10.0 mL with water R.
the substance is sterile, store in a sterile, airtight, tamper-proof
Reference solution. Dissolve 2.0 mL of acetone R, 0.5 mL of container.
methanol R and 0.5 mL of mesityl oxide R (impurity D) in
water R and dilute to 1000 mL with the same solvent. To IMPURITIES
2.0 mL of this solution add 2.0 mL of the internal standard Specified impurities : A, B, C, D.
solution and dilute to 10.0 mL with water R. This solution
contains 316 μg of acetone, 79 μg of methanol and 86 μg of
impurity D per millilitre.
Column :
– material : fused silica ;
A. (Z)-7-[(RS)-[(R)-2-amino-2-carboxyethyl]sulfinyl]-2-
– size : l = 30 m, Ø = 0.53 mm ; [[[(1S)-2,2-dimethylcyclopropyl]carbonyl]amino]hept-2-
– stationary phase : macrogol 20 000 R (film thickness 1.0 μm). enoic acid,
Carrier gas : helium for chromatography R.
Flow rate : 9 mL/min.
Temperature :
Time Temperature
(min) (°C)
Column 0 - 2.5 50 B. R = H : (Z)-7-[[(R)-2-[[(1RS)-1-methyl-3-oxobutyl]amino]-
2-carboxyethyl]sulfanyl]-2-[[[(1S)-2,2-dimethyl-
2.5 - 5 50 → 70
cyclopropyl]carbonyl]amino]hept-2-enoic acid,
5 - 5.5 70
C. R = CH3 : (Z)-7-[[(R)-2-[(1,1-dimethyl-3-oxobutyl)amino]-
Injection port 160 2-carboxyethyl]sulfanyl]-2-[[[(1S)-2,2-dimethyl-
cyclopropyl]carbonyl]amino]hept-2-enoic acid,
Detector 220

Detection : flame ionisation.

Injection : 1 μL.
Calculate the percentage contents of acetone, methanol and
impurity D using the following expression : D. 4-methylpent-3-en-2-one (mesityl oxide).

01/2008:1499

C = concentration of the solvent in the reference CILAZAPRIL

solution, in μg/mL ;
Cilazaprilum
W = quantity of cilastatin sodium in the test solution,
in milligrams ;
Ru = ratio of the area of the solvent peak to the area of
the propanol peak in the chromatogram obtained
with the test solution ;
Rs = ratio of the area of the solvent peak to the area of
the propanol peak in the chromatogram obtained C22H31N3O5,H2O Mr 435.5
with the reference solution. [92077-78-6]

General Notices (1) apply to all monographs and other texts 1885
Cilazapril EUROPEAN PHARMACOPOEIA 8.0

DEFINITION Flow rate : 1.0 mL/min.

(1S,9S)-9-[[(1S)-1-(Ethoxycarbonyl)-3-phenylpropyl]amino]- Detection : spectrophotometer at 214 nm.
10-oxooctahydro-6H-pyridazino[1,2-a][1,2]diazepine-1- Injection : 20 μL.
carboxylic acid monohydrate.
Run time : twice the retention time of cilazapril ; when
Content : 98.5 per cent to 101.5 per cent (anhydrous substance). impurity A is present, it may be necessary to continue the
chromatography until it is eluted.
CHARACTERS
Relative retention with reference to cilazapril :
Appearance : white or almost white, crystalline powder. impurity B = about 0.6 ; impurity D = about 0.9 ;
Solubility : slightly soluble in water, freely soluble in methanol impurity C = about 1.6 ; impurity A = 4 to 5.
and in methylene chloride. System suitability : reference solution (b) :
IDENTIFICATION – resolution : minimum 2.5 between the peaks due to
A. Infrared absorption spectrophotometry (2.2.24). impurity D and cilazapril ;
Comparison : cilazapril CRS. – symmetry factor : maximum 3.0 for the peak due to
cilazapril.
B. Specific optical rotation (see Tests).
Limits :
TESTS – impurity B : not more than the area of the principal peak
Specific optical rotation (2.2.7) : − 383 to − 399 (anhydrous in the chromatogram obtained with reference solution (a)
substance). (0.5 per cent) ;
Dissolve 0.200 g in 0.067 M phosphate buffer solution pH 7.0 R, – impurity D : not more than 0.4 times the area of the
with the aid of ultrasound if necessary, and dilute to 50.0 mL principal peak in the chromatogram obtained with
with the same buffer solution. Carry out the determination reference solution (a) (0.2 per cent) ;
at 365 nm. – impurity C : not more than 0.2 times the area of the
Impurity A. Thin-layer chromatography (2.2.27). principal peak in the chromatogram obtained with
reference solution (a) (0.1 per cent) ;
Test solution. Dissolve 0.20 g of the substance to be examined
in methanol R and dilute to 5.0 mL with the same solvent. – unspecified impurities : for each impurity, not more than
0.2 times the area of the principal peak in the chromatogram
Reference solution (a). Dissolve 2 mg of cilazapril obtained with reference solution (a) (0.10 per cent) ;
impurity A CRS in methanol R and dilute to 50.0 mL with the
same solvent. – total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (a)
Reference solution (b). Dissolve 5 mg of cilazapril (1 per cent) ;
impurity A CRS and 5 mg of the substance to be examined in
– disregard limit : 0.1 times the area of the principal peak in
methanol R and dilute to 10.0 mL with the same solvent.
the chromatogram obtained with reference solution (a)
Plate : TLC silica gel plate R. (0.05 per cent) ; disregard any peak due to impurity A.
Mobile phase : glacial acetic acid R, water R, hexane R, Water (2.5.12) : 3.5 per cent to 5.0 per cent, determined on
methanol R, ethyl acetate R (5:5:15:15:60 V/V/V/V/V). 0.300 g.
Application : 5 μL. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Development : over a path of 10 cm. 1.0 g.
Drying : in a current of cold air for 10 min.
ASSAY
Detection : spray with a freshly prepared mixture of 1 volume
of potassium iodobismuthate solution R and 10 volumes of Dissolve 0.300 g in 10 mL of anhydrous ethanol R and add
dilute acetic acid R and then with dilute hydrogen peroxide 50 mL of water R. Titrate with 0.1 M sodium hydroxide,
solution R. determining the end-point potentiometrically (2.2.20). Carry
out a blank titration.
System suitability : reference solution (b) :
1 mL of 0.1 M sodium hydroxide is equivalent to 41.75 mg
– the chromatogram shows 2 clearly separated spots. of C22H31N3O5.
Limit :
STORAGE
– impurity A : any spot due to impurity A is not more intense
than the corresponding spot in the chromatogram obtained Protected from light.
with reference solution (a) (0.1 per cent). IMPURITIES
Related substances. Liquid chromatography (2.2.29). Specified impurities : A, B, C, D.
Test solution. Dissolve 25.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with the
mobile phase.
Reference solution (a). Dilute 1.0 mL of the test solution to
50.0 mL with the mobile phase. Dilute 5.0 mL of this solution
to 20.0 mL with the mobile phase.
Reference solution (b). Dissolve 5.0 mg of cilazapril A. R = C(CH3)3, R′ = C2H5 : 1,1-dimethylethyl (1S,9S)-9-
impurity D CRS in the test solution and dilute to 10.0 mL with [[(S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]-10-
the test solution. oxooctahydro-6H-pyridazino[1,2-a][1,2]diazepine-1-
Column : carboxylate,
– size : l = 0.25 m, Ø = 4.6 mm ; B. R = R′ = H : (1S,9S)-9-[[(S)-1-carboxy-3-phenylpropyl]-
– stationary phase : octadecylsilyl silica gel for amino]-10-oxooctahydro-6H-pyridazino[1,2-
chromatography R (5 μm). a][1,2]diazepine-1-carboxylic acid,
Mobile phase : mix 10 volumes of triethylamine R and C. R = R′ = C2H5 : ethyl (1S,9S)-9-[[(S)-1-(ethoxycarbonyl)-
750 volumes of water R, adjust to pH 2.30 with phosphoric 3-phenylpropyl]amino]-10-oxooctahydro-6H-
acid R, and add 200 volumes of tetrahydrofuran R. pyridazino[1,2-a][1,2]diazepine-1-carboxylate,

1886 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cimetidine

TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution Y5 (2.2.2,
Method II).
Dissolve 3.0 g in 12 mL of 1 M hydrochloric acid and dilute
D. (1S,9S)-9-[[(R)-1-(ethoxycarbonyl)-3-phenyl- to 20 mL with water R.
propyl]amino]-10-oxooctahydro-6H-pyridazi-
no-[1,2-a][1,2]diazepine-1-carboxylic acid. Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 20 mg of the substance to be examined
01/2010:0756 in mobile phase A and dilute to 50.0 mL with mobile phase A.
corrected 6.8 Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with mobile phase A. Dilute 2.0 mL of this solution
CIMETIDINE to 10.0 mL with mobile phase A.
Reference solution (b). Dissolve the contents of a vial of
Cimetidinum cimetidine for system suitability CRS (containing impurities B,
C, D, E, G and H) in 1.0 mL of mobile phase A.
Reference solution (c). Dissolve 4 mg of cimetidine for peak
identification CRS (containing impurity F) in mobile phase A
and dilute to 10.0 mL with mobile phase A.
Column :
C10H16N6S Mr 252.3 – size : l = 0.25 m, Ø = 4.6 mm ;
[51481-61-9] – stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
DEFINITION Mobile phase A : mix 0.4 volumes of diethylamine R and
2-Cyano-1-methyl-3-[-2-[[(5-methyl-1H-imidazol-4- 780 volumes of a 1.1 g/L solution of sodium hexanesulfonate R ;
yl)methyl]sulfanyl]ethyl]guanidine. adjust to pH 2.8 with phosphoric acid R ; add 250 volumes of
Content : 98.5 per cent to 101.5 per cent (dried substance). methanol R2 ;
Mobile phase B : methanol R2 ;
CHARACTERS
Time Mobile phase A Mobile phase B
Appearance : white or almost white powder.
(min) (per cent V/V) (per cent V/V)
Solubility : slightly soluble in water, soluble in ethanol (96 per
0 - 60 100 0
cent), practically insoluble in methylene chloride. It dissolves
in dilute mineral acids. 60 - 65 100 → 90 0 → 10
It shows polymorphism (5.9). 65 - 120 90 10
IDENTIFICATION
Flow rate : 1.1 mL/min.
First identification : B.
Detection : spectrophotometer at 220 nm.
Second identification : A, C.
Injection : 50 μl.
A. Melting point (2.2.14) : 139 °C to 144 °C.
Identification of impurities : use the chromatogram
If necessary, dissolve the substance to be examined in
supplied with cimetidine for system suitability CRS and
2-propanol R, evaporate to dryness and determine the
the chromatogram obtained with reference solution (b) to
melting point again.
identify the peaks due to impurities B, C, D, E, G and H ;
B. Infrared absorption spectrophotometry (2.2.24). use the chromatogram supplied with cimetidine for peak
Comparison : cimetidine CRS. identification CRS and the chromatogram obtained with
If the spectra obtained in the solid state show differences, reference solution (c) to identify the peak due to impurity F.
dissolve the substance to be examined and the reference Relative retention with reference to cimetidine
substance separately in 2-propanol R, evaporate to dryness (retention time = about 18 min) : impurity G = about
and record new spectra using the residues. 0.2 ; impurity E = about 0.4 ; impurity D = about
C. Thin-layer chromatography (2.2.27). 1.5 ; impurity C = about 1.6 ; impurity B = about 2.0 ;
Test solution. Dissolve 10 mg of the substance to be impurity H = about 2.3 ; impurity F = about 4.6.
examined in methanol R and dilute to 10 mL with the same System suitability : reference solution (b) :
solvent. – resolution : minimum 1.5 between the peaks due to
Reference solution. Dissolve 10 mg of cimetidine CRS in impurities D and C.
methanol R and dilute to 10 mL with the same solvent. Limits :
Plate : TLC silica gel GF254 plate R. – correction factors : for the calculation of content, multiply
Mobile phase : concentrated ammonia R, methanol R, ethyl the peak areas of the following impurities by the
acetate R (15:20:65 V/V/V). corresponding correction factor : impurity C = 2.5 ;
Application : 5 μL. impurity D = 3.3 ; impurity E = 0.7 ; impurity G = 0.6.
Development : over 3/4 of the plate. – impurities B, C, D, E, F, G, H : for each impurity, not more
Drying : in a current of cold air. than the area of the principal peak in the chromatogram
Detection : expose to iodine vapour until maximum obtained with reference solution (a) (0.2 per cent) ;
contrast has been obtained and examine in ultraviolet light – unspecified impurities : for each impurity, not more than
at 254 nm. 0.5 times the area of the principal peak in the chromatogram
Results : the principal spot in the chromatogram obtained obtained with reference solution (a) (0.10 per cent) ;
with the test solution is similar in position and size to the – total : not more than 5 times the area of the principal peak
principal spot in the chromatogram obtained with the in the chromatogram obtained with reference solution (a)
reference solution. (1.0 per cent) ;

General Notices (1) apply to all monographs and other texts 1887
Cimetidine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

– disregard limit : 0.25 times the area of the principal peak

in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test C. Prepare the reference solution using
2 mL of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined H. 1,1′-(disulfanediyldiethylene)bis(2-cyano-3-
on 1.000 g by drying in an oven at 105 °C. methylguanidine),
Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
1.0 g.
ASSAY
Dissolve 0.200 g in 60 mL of anhydrous acetic acid R. I. R1 = OH, R2 = C2H5 : (5-ethyl-1H-imidazol-4-yl)methanol,
Titrate with 0.1 M perchloric acid determining the end point
potentiometrically (2.2.20). J. R1 = S-CH2-CH2-NH2, R2 = CH3 : 2-[[(5-methyl-1H-
imidazol-4-yl)methyl]sulfanyl]ethanamine.
1 mL of 0.1 M perchloric acid is equivalent to 25.23 mg of
C10H16N6S
STORAGE 01/2010:1500
Protected from light.
CIMETIDINE HYDROCHLORIDE
IMPURITIES
Specified impurities : B, C, D, E, F, G, H. Cimetidini hydrochloridum
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
C10H17ClN6S Mr 288.8
impurities in substances for pharmaceutical use) : A, I, J.
[70059-30-2]
DEFINITION
2-Cyano-1-methyl-3-[-2-[[(5-methyl-1H-imidazol-4-
yl)methyl]sulfanyl]ethyl]guanidine hydrochloride.
Content : 98.5 per cent to 101.5 per cent (dried substance).
A. methyl 3-cyano-1-[2-[[(5-methyl-1H-imidazol-4-
yl)methyl]sulfanyl]ethyl]carbamimidothioate, CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : freely soluble in water, sparingly soluble in
anhydrous ethanol.
IDENTIFICATION
First identification : B, D.
B. R1 = CN, R2 = O-CH3, X = S : methyl 3-cyano-1-[2- Second identification : A, C, D.
[[(5-methyl-1H-imidazol-4-yl)methyl]sulfanyl]ethyl]-
carbamimidate, A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
C. R1 = CO-NH2, R2 = NH-CH3, X = S : 1-[(methylamino)- Test solution. Dissolve 70 mg in 0.2 M sulfuric acid and
[[2-[[(5-methyl-1H-imidazol-4-yl)methyl]sulfanyl]- dilute to 100.0 mL with the same acid. Dilute 2.0 mL of this
ethyl]amino]methylidene]urea, solution to 100.0 mL with 0.2 M sulfuric acid.
D. R1 = H, R2 = NH-CH3, X = S : 1-methyl-3-[2-[[(5-methyl- Specific absorbance at the absorption maximum at 218 nm :
1H-imidazol-4-yl)methyl]sulfanyl]ethyl]guanidine, 650 to 705.
E. R1 = CN, R2 = NH-CH3, X = SO : 2-cyano-1-methyl-3- B. Infrared absorption spectrophotometry (2.2.24).
[2-[[(5-methyl-1H-imidazol-4-yl)methyl]sulfinyl]- Comparison: cimetidine hydrochloride CRS.
ethyl]guanidine, C. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 10 mg of the substance to be
examined in methanol R and dilute to 10 mL with the same
solvent.
Reference solution. Dissolve 10 mg of cimetidine
hydrochloride CRS in methanol R and dilute to 10 mL with
the same solvent.
F. 2-cyano-1,3-bis[2-[[(5-methyl-1H-imidazol-4-yl)methyl]-
sulfanyl]ethyl]guanidine, Plate : TLC silica gel GF254 plate R.
Mobile phase : concentrated ammonia R, methanol R, ethyl
acetate R (15:20:65 V/V/V).
Application : 5 μL.
Development : over 3/4 of the plate.
G. 2-cyano-1,3-dimethylguanidine, Drying : in a current of cold air

1888 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cimetidine hydrochloride

Detection : expose to iodine vapour until maximum System suitability : reference solution (b) :
contrast has been obtained and examine in ultraviolet light
at 254 nm. – resolution : minimum 1.5 between the peaks due to
impurities D and C.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to the Limits :
principal spot in the chromatogram obtained with the – correction factors : for the calculation of content, multiply
reference solution. the peak areas of the following impurities by the
D. It gives reaction (a) of chlorides (2.3.1). corresponding correction factor : impurity C = 2.5 ;
impurity D = 3.3 ; impurity E = 0.7 ; impurity G = 0.6 ;
TESTS – impurities B, C, D, E, F, G, H : for each impurity, not more
Appearance of solution. The solution is clear (2.2.1) and not than the area of the principal peak in the chromatogram
more intensely coloured than reference solution Y5 (2.2.2, obtained with reference solution (a) (0.2 per cent) ;
Method II). – unspecified impurities : for each impurity, not more than
Dissolve 3.0 g in 12 mL of 1 M hydrochloric acid and dilute 0.5 times the area of the principal peak in the chromatogram
to 20 mL with water R. obtained with reference solution (a) (0.10 per cent) ;
pH (2.2.3) : 4.0 to 5.0. – total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
Dissolve 100 mg in carbon dioxide-free water R and dilute to (1.0 per cent) ;
10.0 mL with the same solvent.
Related substances. Liquid chromatography (2.2.29). – disregard limit : 0.25 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
Test solution. Dissolve 20 mg of the substance to be examined (0.05 per cent).
in mobile phase A and dilute to 50.0 mL with mobile phase A.
Heavy metals (2.4.8) : maximum 20 ppm.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with mobile phase A. Dilute 2.0 mL of this solution 1.0 g complies with test C. Prepare the reference solution using
to 10.0 mL with mobile phase A. 2 mL of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 1.0 per cent, determined
Reference solution (b). Dissolve the contents of a vial of
on 1.000 g by drying in an oven at 105 °C.
cimetidine for system suitability CRS (containing impurities B,
C, D, E, G and H) in 1.0 mL of mobile phase A. Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
1.0 g.
Reference solution (c). Dissolve 4 mg of cimetidine for peak
identification CRS (containing impurity F) in mobile phase A
and dilute to 10.0 mL with mobile phase A. ASSAY
Column : Dissolve 0.200 g in a mixture of 5 mL of 0.01 M hydrochloric
– size : l = 0.25 m, Ø = 4.6 mm ; acid and 50 mL of ethanol (96 per cent) R. Carry out a
potentiometric titration (2.2.20), using 0.1 M sodium
– stationary phase : end-capped octadecylsilyl silica gel for hydroxide. Read the volume added between the 2 points of
chromatography R (5 μm). inflexion.
Mobile phase A : mix 0.4 volumes of diethylamine R and 1 mL of 0.1 M sodium hydroxide is equivalent to 28.88 mg of
780 volumes of a 1.1 g/L solution of sodium hexanesulfonate R. C H ClN S.
10 17 6
Adjust to pH 2.8 with phosphoric acid R and add 250 volumes
of methanol R2 ;
STORAGE
Mobile phase B : methanol R2 ;
Protected from light.
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 60 100 0 IMPURITIES
60 - 65 100 → 90 0 → 10 Specified impurities : B, C, D, E, F, G, H.
65 - 120 90 10 Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
Flow rate : 1.1 mL/min. the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
Detection : spectrophotometer at 220 nm. by the general monograph Substances for pharmaceutical use
Injection : 50 μL. (2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
Identification of impurities : use the chromatogram impurities in substances for pharmaceutical use) : A, I, J.
supplied with cimetidine for system suitability CRS and
the chromatogram obtained with reference solution (b) to
identify the peaks due to the impurities B, C, D, E, G and
H ; use the chromatogram supplied with cimetidine for peak
identification CRS and the chromatogram obtained with
reference solution (c) to identify the peak due to impurity F.
Relative retention with reference to cimetidine (retention
time = about 18 min) : impurity G = about 0.2 ;
impurity E = about 0.4 ; impurity D = about 1.5 ;
impurity C = about 1.6 ; impurity B = about 2.0 ; A. methyl 3-cyano-1-[2-[[(5-methyl-1H-imidazol-4-
impurity H = about 2.3 ; impurity F = about 4.6. yl)methyl]sulfanyl]ethyl]carbamimidothioate,

General Notices (1) apply to all monographs and other texts 1889
Cinchocaine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

CHARACTERS
A white or almost white, crystalline powder or colourless
crystals, hygroscopic, very soluble in water, freely soluble in
acetone, in alcohol and in methylene chloride. It agglomerates
very easily.
B. R1 = CN, R2 = O-CH3, X = S : methyl 3-cyano-1-[2-
[[(5-methyl-1H-imidazol-4-yl)methyl]sulfanyl]ethyl]- IDENTIFICATION
carbamimidate, First identification : B, E.
C. R1 = CO-NH2, R2 = NH-CH3, X = S : 1-[(methylamino)- Second identification : A, C, D, E.
[[2-[[(5-methyl-1H-imidazol-4-yl)methyl]sulfanyl]- A. Dissolve 60.0 mg in 1 M hydrochloric acid and dilute to
ethyl]amino]methylidene]urea, 100 mL with the same acid. Dilute 2 mL of the solution to
100 mL with 1 M hydrochloric acid. Examined between
D. R1 = H, R2 = NH-CH3, X = S : 1-methyl-3-[2-[[(5-methyl- 220 nm and 350 nm (2.2.25), the solution shows two
1H-imidazol-4-yl)methyl]sulfanyl]ethyl]guanidine, absorption maxima, at 246 nm and 319 nm. The ratio of
E. R1 = CN, R2 = NH-CH3, X = SO : 2-cyano-1-methyl-3- the absorbance measured at 246 nm to that measured at
[2-[[(5-methyl-1H-imidazol-4-yl)methyl]sulfinyl]- 319 nm is 2.7 to 3.0.
ethyl]guanidine, B. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
cinchocaine hydrochloride CRS. Examine the substances
prepared as discs using potassium chloride R.
C. Examine the chromatograms obtained in the test for
related substances. The principal spot in the chromatogram
F. 2-cyano-1,3-bis[2-[[(5-methyl-1H-imidazol-4-yl)methyl]- obtained with test solution (b) is similar in position and
sulfanyl]ethyl]guanidine. size to the principal spot in the chromatogram obtained
with reference solution (a).
D. Dissolve 0.5 g in 5 mL of water R. Add 1 mL of dilute
ammonia R2. A white precipitate is formed. Filter, wash the
precipitate with five quantities, each of 10 mL, of water R
and dry in a desiccator. It melts at 64 °C to 66 °C (2.2.14).
G. 2-cyano-1,3-dimethylguanidine, E. It gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S. Dissolve 5.0 g in carbon dioxide-free water R
prepared from distilled water R, and dilute to 50 mL with the
same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
H. 1,1’-(disulfanediyldiethylene)bis(2-cyano-3- more intensely coloured than reference solution Y6 (2.2.2,
methylguanidine), Method II).
pH (2.2.3). Dilute 10 mL of solution S to 50 mL with carbon
dioxide-free water R. The pH of the solution is 5.0 to 6.0.
Related substances. Examine by thin-layer chromatography
(2.2.27), using as the coating substance a suitable silica gel with
a fluorescent indicator having an optimal intensity at 254 nm.
I. R1 = OH, R2 = C2H5 : (5-ethyl-1H-imidazol-4-yl)methanol,
Test solution (a). Dissolve 0.20 g of the substance to be
J. R1 = S-CH2-CH2-NH2, R2 = CH3 : 2-[[(5-methyl-1H- examined in methanol R and dilute to 5 mL with the same
imidazol-4-yl)methyl]sulfanyl]ethanamine. solvent.
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
with methanol R.
01/2008:1088
Reference solution (a). Dissolve 20 mg of cinchocaine
hydrochloride CRS in methanol R and dilute to 5 mL with the
CINCHOCAINE HYDROCHLORIDE same solvent.
Reference solution (b). Dilute 1 mL of test solution (b) to
Cinchocaini hydrochloridum 20 mL with methanol R.
Reference solution (c). Dilute 1 mL of test solution (b) to
50 mL with methanol R.
Reference solution (d). Dissolve 20 mg of benzocaine CRS in
methanol R and dilute to 5 mL with the same solvent. Dilute
1 mL of the solution and 1 mL of reference solution (a) to
20 mL with methanol R.
Apply separately to the plate 5 μL of each solution. Develop
C20H30ClN3O2 Mr 379.9 over a path of 15 cm using a mixture of 1 volume of
[61-12-1] ammonia R, 5 volumes of methanol R, 30 volumes of acetone R
and 50 volumes of toluene R. Dry the plate in a current of
DEFINITION warm air for 15 min. Examine in ultraviolet light at 254 nm.
Cinchocaine hydrochloride contains not less than 98.5 per Any spot in the chromatogram obtained with test solution (a),
cent and not more than the equivalent of 101.0 per cent of apart from the principal spot, is not more intense than the
2-butoxy-N-[2-(diethylamino)ethyl]quinoline-4-carboxamide principal spot in the chromatogram obtained with reference
hydrochloride, calculated with reference to the dried solution (b) (0.5 per cent) and at most one such spot is more
substance. intense than the spot in the chromatogram obtained with

1890 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cineole

reference solution (c) (0.2 per cent). The test is not valid Test solution. Dilute 1 mL of solution S (see Tests) to 25 mL
unless the chromatogram obtained with reference solution (d) with alcohol R.
shows two clearly separated spots. Reference solution. Mix 80 mg of cineole CRS with alcohol R
Heavy metals (2.4.8). 12 mL of solution S complies with test A and dilute to 10 mL with the same solvent.
for heavy metals (20 ppm). Prepare the reference solution Plate : TLC silica gel plate R.
using lead standard solution (2 ppm Pb) R.
Mobile phase : ethyl acetate R, toluene R (10:90 V/V).
Loss on drying (2.2.32). Not more than 2.0 per cent, Application : 2 μL.
determined on 0.500 g by drying in vacuo at 60 °C.
Development : over 2/3 of the plate.
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
on 1.0 g. Drying : in a current of cold air.
Detection : spray with anisaldehyde solution R, heat at
ASSAY 100-105 °C for 5 min.
Dissolve 0.300 g in a mixture of 15.0 mL of 0.01 M hydrochloric Results : the principal spot in the chromatogram obtained
acid and 50 mL of alcohol R. Carry out a potentiometric with the test solution is similar in position, colour and size
titration (2.2.20), using 0.1 M sodium hydroxide. Read to the principal spot in the chromatogram obtained with
the volume added between the two points of inflexion. the reference solution.
1 mL of 0.1 M sodium hydroxide is equivalent to 37.99 mg C. To 0.1 mL add 4 mL of sulfuric acid R. An orange-red
of C20H30ClN3O2. colour develops. Add 0.2 mL of formaldehyde solution R.
The colour changes to deep brown.
STORAGE
Store in an airtight container, protected from light. TESTS
Solution S. Dilute 2.00 g to 10.0 mL with alcohol R.
IMPURITIES
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method I).
Chiral impurities. The optical rotation (2.2.7) of solution S is
− 0.10° to + 0.10°.
Refractive index (2.2.6) : 1.456 to 1.460.
Related substances. Gas chromatography (2.2.28).
A. R1 = Cl, R2 = NH-[CH2]2-N(C2H5)2 : 2-chloro-N-[2- Internal standard solution. Dissolve 1.0 g of camphor R in
(diethylamino)ethyl]quinoline-4-carboxamide, heptane R and dilute to 200 mL with the same solvent.
Test solution (a). Dissolve 2.5 g of the substance to be
B. R1 = R2 = OH : 2-hydroxyquinoline-4-carboxylic acid, examined in heptane R and dilute to 25.0 mL with the same
solvent.
C. R1 = OH, R2 = NH-[CH2]2-N(C2H5)2 : N-[2-
(diethylamino)ethyl]-2-hydroxyquinoline-4-carboxamide, Test solution (b). Dissolve 2.5 g of the substance to be
examined in heptane R, add 5.0 mL of the internal standard
D. R1 = O-[CH2]3-CH3, R2 = OH : 2-butoxyquinoline-4- solution and dilute to 25.0 mL with heptane R.
carboxylic acid. Reference solution (a). To 2.0 mL of test solution (a) add
20.0 mL of the internal standard solution and dilute to
100.0 mL with heptane R.
Reference solution (b). Dissolve 50 mg of 1,4-cineole R and
01/2008:1973
50 mg of the substance to be examined in heptane R and dilute
to 50.0 mL with the same solvent.
CINEOLE Column :
– size : l = 30 m, Ø = 0.25 mm,
Cineolum – stationary phase : macrogol 20 000 R (film thickness
0.25 μm).
Carrier gas : helium for chromatography R.
Linear velocity : 45 cm/s.
Split-ratio : 1:70.
Temperature :
C10H18O Mr 154.3
[470-82-6] Time Temperature
(min) (°C)
DEFINITION Column 0 - 10 50
1,3,3-Trimethyl-2-oxabicyclo[2.2.2]octane. 10 - 35 50 → 100
35 - 45 100 → 200
CHARACTERS 45 - 55 200
Appearance : clear colourless liquid. Injection port 220

Solubility : practically insoluble in water, miscible with alcohol Detector 250

and with methylene chloride.
Detection : flame ionisation.
It solidifies at about 0.5 °C.
Injection : 1 μL.
IDENTIFICATION System suitability : reference solution (b) :
A. Refractive index (see Tests). – resolution : minimum 10 between the peaks due to
B. Thin-layer chromatography (2.2.27). impurity A and to cineole.

General Notices (1) apply to all monographs and other texts 1891
Cinnarizine EUROPEAN PHARMACOPOEIA 8.0

Limits : Reference solution (b). Dissolve 10 mg of cinnarizine CRS

– total : calculate the ratio (R) of the area of the peak due and 10 mg of flunarizine dihydrochloride CRS in methanol R
to cineole to the area of the peak due to the internal and dilute to 20 mL with the same solvent.
standard from the chromatogram obtained with reference Plate : TLC octadecylsilyl silica gel F254 plate R.
solution (a) ; from the chromatogram obtained with test Mobile phase : 58.4 g/L solution of sodium chloride R,
solution (b), calculate the ratio of the sum of the areas of methanol R, acetone R (20:30:50 V/V/V).
any peaks, apart from the principal peak and the peak due Application : 5 μL.
to the internal standard, to the area of the peak due to
internal standard : this ratio is not greater than R (2 per Development : in an unsaturated tank, over 3/4 of the plate.
cent), Drying : in air.
– disregard limit : 0.025 times the area of the principal peak Detection : examine in ultraviolet light at 254 nm.
in the chromatogram obtained with reference solution (a) System suitability : reference solution (b) :
(0.05 per cent). – the chromatogram shows 2 clearly separated spots.
Residue on evaporation : maximum 0.1 per cent. Results : the principal spot in the chromatogram obtained
To 2.0 g add 5 mL of water R, evaporate to dryness on a with the test solution is similar in position and size to
water-bath and dry at 100-105 °C for 1 h. The residue weighs the principal spot in the chromatogram obtained with
a maximum of 2 mg. reference solution (a).
D. Dissolve 0.2 g of anhydrous citric acid R in 10 mL of acetic
STORAGE anhydride R in a water-bath at 80 °C and maintain the
In an airtight container, protected from light. temperature of the water-bath at 80 °C for 10 min. Add
about 20 mg of the substance to be examined. A purple
IMPURITIES colour develops.
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution BY7 (2.2.2,
Method II).
Dissolve 0.5 g in methylene chloride R and dilute to 20 mL
A. 1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane with the same solvent.
(1,4-cineole). Acidity or alkalinity. Suspend 0.5 g in 15 mL of water R. Boil
for 2 min. Cool and filter. Dilute the filtrate to 20 mL with
carbon dioxide-free water R. To 10 mL of this solution add
07/2011:0816 0.1 mL of phenolphthalein solution R and 0.25 mL of 0.01 M
sodium hydroxide. The solution is pink. To 10 mL of the
CINNARIZINE solution add 0.1 mL of methyl red solution R and 0.25 mL of
0.01 M hydrochloric acid. The solution is red.
Cinnarizinum Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be
examined in methanol R and dilute to 10.0 mL with the same
solvent.
Reference solution (a). Dissolve 12.5 mg of cinnarizine CRS
and 15.0 mg of flunarizine dihydrochloride CRS in methanol R
and dilute to 100.0 mL with the same solvent. Dilute 1.0 mL
of this solution to 20.0 mL with methanol R.
C26H28N2 Mr 368.5 Reference solution (b). Dilute 1.0 mL of the test solution to
[298-57-7] 100.0 mL with methanol R. Dilute 5.0 mL of this solution to
20.0 mL with methanol R.
DEFINITION
Column :
(E)-1-(Diphenylmethyl)-4-(3-phenylprop-2-enyl)piperazine.
– size : l = 0.1 m, Ø = 4.0 mm ;
Content : 99.0 per cent to 101.0 per cent (dried substance).
– stationary phase : base-deactivated octadecylsilyl silica gel for
CHARACTERS chromatography R (3 μm).
Appearance : white or almost white powder. Mobile phase :
Solubility : practically insoluble in water, freely soluble in – mobile phase A : 10 g/L solution of ammonium acetate R ;
methylene chloride, soluble in acetone, slightly soluble in – mobile phase B : 0.2 per cent V/V solution of glacial acetic
ethanol (96 per cent) and in methanol. acid R in acetonitrile R1 ;
IDENTIFICATION Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
First identification : A, B.
0 - 20 75 → 10 25 → 90
Second identification : A, C, D.
A. Melting point (2.2.14) : 118 °C to 122 °C. 20 - 25 10 90
B. Infrared absorption spectrophotometry (2.2.24). Flow rate : 1.5 mL/min.
Comparison : cinnarizine CRS. Detection : spectrophotometer at 230 nm.
C. Thin-layer chromatography (2.2.27). Injection : 10 μL.
Test solution. Dissolve 10 mg of the substance to be Relative retention with reference to cinnarizine (retention
examined in methanol R and dilute to 20 mL with the same time = about 11 min) : impurity A = about 0.4 ;
solvent. flunarizine = about 1.05 ; impurity B = about 1.1 ;
Reference solution (a). Dissolve 10 mg of cinnarizine CRS impurity C = about 1.2 ; impurity D = about 1.6 ;
in methanol R and dilute to 20 mL with the same solvent. impurity E = about 1.8.

1892 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ciprofibrate

System suitability: reference solution (a) :

– resolution : minimum 5.0 between the peaks due to
cinnarizine and flunarizine.
Limits :
– impurities A, B, C, D, E : for each impurity, not more
than the area of the principal peak in the chromatogram D. 1-(diphenylmethyl)-4-[(1RS,3E)-4-phenyl-1-[(E)-2-
obtained with reference solution (b) (0.25 per cent) ; phenylethenyl]but-3-enyl]piperazine,
– unspecified impurities : for each impurity, not more than
0.4 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.10 per cent) ;
– total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.5 per cent) ;
– disregard limit : 0.2 times the area of the principal peak in E. 1,4-bis(diphenylmethyl)piperazine.
the chromatogram obtained with reference solution (b)
(0.05 per cent). 01/2008:2013
Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 1.0 g in a mixture of 15 volumes of water R and CIPROFIBRATE
85 volumes of acetone R. Add dilute hydrochloric acid R until
dissolution is complete. Dilute to 20 mL with a mixture of Ciprofibratum
15 volumes of water R and 85 volumes of acetone R. 12 mL
of the solution complies with test B. Prepare the reference
solution using 10 mL of lead standard solution (1 ppm Pb)
obtained by diluting lead standard solution (100 ppm Pb) R
with a mixture of 15 volumes of water R and 85 volumes of
acetone R.
C13H14Cl2O3 Mr 289.2
Loss on drying (2.2.32) : maximum 0.5 per cent, determined [52214-84-3]
on 1.000 g by drying in an oven in vacuo at 60 °C for 4 h.
DEFINITION
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. 2-[4-[(1RS)-2,2-Dichlorocyclopropyl]phenoxy]-2-
methylpropanoic acid.
ASSAY Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
Dissolve 0.150 g in 50 mL of a mixture of 1 volume of
CHARACTERS
anhydrous acetic acid R and 7 volumes of methyl ethyl
ketone R. Titrate with 0.1 M perchloric acid, using 0.2 mL of Appearance : white or slightly yellow, crystalline powder.
naphtholbenzein solution R as indicator. Solubility : practically insoluble in water, freely soluble in
1 mL of 0.1 M perchloric acid is equivalent to 18.43 mg anhydrous ethanol, soluble in toluene.
of C26H28N2. mp : about 115 °C.

STORAGE IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Protected from light.
Comparison: ciprofibrate CRS.
IMPURITIES
TESTS
Specified impurities : A, B, C, D, E.
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution BY4 (2.2.2,
Method II).
Dissolve 1.0 g in anhydrous ethanol R and dilute to 10.0 mL
with the same solvent.
Related substances. Liquid chromatography (2.2.29).
A. 1-(diphenylmethyl)piperazine, Test solution. Dissolve 0.125 g of the substance to be examined
in a mixture of equal volumes of acetonitrile R and water R
and dilute to 50 mL with the same mixture of solvents.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with a mixture of equal volumes of acetonitrile R
and water R. Dilute 1.0 mL of this solution to 10.0 mL with a
mixture of equal volumes of acetonitrile R and water R.
Reference solution (b). Dissolve the contents of a vial of
B. (Z)-1-(diphenylmethyl)-4-(3-phenylprop-2- ciprofibrate for system suitability CRS in 2.0 mL of a mixture
enyl)piperazine, of equal volumes of acetonitrile R and water R.
Column :
– size : l = 0.15 m, Ø = 4.6 mm,
– stationary phase : octylsilyl silica gel for chromatography R
(5 μm).
Mobile phase :
C. 4-(diphenylmethyl)-1,1-bis[(E)-3-phenylprop-2- – mobile phase A : 1.36 g/L solution of potassium dihydrogen
enyl]piperazinium chloride, phosphate R adjusted to pH 2.2 with phosphoric acid R,

General Notices (1) apply to all monographs and other texts 1893
Ciprofloxacin EUROPEAN PHARMACOPOEIA 8.0

– mobile phase B : acetonitrile R,

Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 30 75 → 30 25 → 70
B. 4-[(1RS)-2,2-dichlorocyclopropyl]phenol,
30 - 40 30 70
40 - 42 30 → 75 70 → 25

Flow rate : 1.5 mL/min.

Detection : spectrophotometer at 230 nm.
Injection : 10 μL. C. R = CH2OH : 2-[4-[(1RS)-2,2-dichlorocyclopropyl]phe-
noxy]-2-methylpropan-1-ol,
Identification of impurities : use the chromatogram supplied
with ciprofibrate for system suitability CRS to identify the D. R = CO-OCH3 : methyl 2-[4-[(1RS)-2,2-dichlorocycloprop-
peaks due to impurities A, B, C, D and E. yl]phenoxy]-2-methylpropanoate,
Relative retention with reference to ciprofibrate (retention E. R = CO-OC2H5 : ethyl 2-[4-[(1RS)-2,2-dichlorocycloprop-
time = about 18 min) : impurity A = about 0.7 ; yl]phenoxy]-2-methylpropanoate.
impurity B = about 0.8 ; impurity C = about 0.95 ;
impurity D = about 1.3 ; impurity E = about 1.5.
01/2008:1089
System suitability : reference solution (b) :
– resolution : baseline separation between the peaks due to CIPROFLOXACIN
impurity C and ciprofibrate.
Limits : Ciprofloxacinum
– correction factor : for the calculation of content, multiply
the peak area of impurity A by 2.3,
– impurities A, C, D : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent),
– impurity B : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.2 per cent), C17H18FN3O3 Mr 331.4
– impurity E : not more than 8 times the area of the principal [85721-33-1]
peak in the chromatogram obtained with reference
DEFINITION
solution (a) (0.8 per cent),
– any other impurity : for each impurity, not more than the 1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-
area of the principal peak in the chromatogram obtained dihydroquinoline-3-carboxylic acid.
with reference solution (a) (0.1 per cent), Content : 99.0 per cent to 101.0 per cent (dried substance).
– total of other impurities: not more than 5 times the area CHARACTERS
of the principal peak in the chromatogram obtained with
Appearance : almost white or pale yellow, crystalline powder,
reference solution (a) (0.5 per cent),
slightly hygroscopic.
– disregard limit : 0.5 times the area of the principal peak in Solubility : practically insoluble in water, very slightly soluble
the chromatogram obtained with reference solution (a) in anhydrous ethanol and in methylene chloride.
(0.05 per cent).
Chlorides (2.4.4): maximum 350 ppm. IDENTIFICATION
To 0.190 g add 20 mL of water R and treat in an ultrasonic bath Infrared absorption spectrophotometry (2.2.24).
for 8 min. Filter. 15 mL of the filtrate complies with the test. Comparison: ciprofloxacin CRS.
Water (2.5.12) : maximum 0.5 per cent, determined on 1.000 g. TESTS
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Appearance of solution. The solution is clear (2.2.1) and not
1.0 g. more intensely coloured than reference solution GY5 (2.2.2,
ASSAY Method II).
Dissolve 0.25 g in 0.1 M hydrochloric acid and dilute to 20 mL
Dissolve 0.250 g in a mixture of 20 mL of water R and 40 mL with the same solvent.
of anhydrous ethanol R. Titrate with 0.1 M sodium hydroxide,
determining the end-point potentiometrically (2.2.20). Impurity A. Thin-layer chromatography (2.2.27).
1 mL of 0.1 M sodium hydroxide is equivalent to 28.92 mg Test solution. Dissolve 50 mg of the substance to be examined
of C13H14Cl2O3. in dilute ammonia R1 and dilute to 5 mL with the same solvent.
Reference solution. Dissolve 10 mg of ciprofloxacin
STORAGE impurity A CRS in a mixture of 0.1 mL of dilute ammonia R1
In an airtight container, protected from light. and 90 mL of water R and dilute to 100 mL with water R.
Dilute 2 mL of the solution to 10 mL with water R.
IMPURITIES Plate : TLC silica gel F254 plate R.
Specified impurities : A, B, C, D, E. Application : 5 μL.
At the bottom of a chromatographic tank, place an evaporating
dish containing 50 mL of concentrated ammonia R. Expose
the plate to the ammonia vapour for 15 min in the closed
tank. Withdraw the plate, transfer to a 2nd chromatographic
A. 2-(4-ethenylphenoxy)-2-methylpropanoic acid, tank and proceed with development.

1894 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ciprofloxacin

Mobile phase : acetonitrile R, concentrated ammonia R, buffer solution pH 3.5 R. The filtrate complies with test E.
methanol R, methylene chloride R (10:20:40:40 V/V/V/V). Prepare the reference solution using 10 mL of lead standard
Development : over 3/4 of the plate. solution (1 ppm Pb) R.
Drying : in air. Loss on drying (2.2.32) : maximum 1.0 per cent, determined
on 1.000 g by drying under vacuum at 120 °C.
Detection : examine in ultraviolet light at 254 nm.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Limit : 1.0 g in a platinum crucible.
– impurity A : any spot corresponding to impurity A is not
more intense than the principal spot in the chromatogram ASSAY
obtained with the reference solution (0.2 per cent). Dissolve 0.300 g in 80 mL of glacial acetic acid R. Titrate
Related substances. Liquid chromatography (2.2.29). with 0.1 M perchloric acid, determining the end-point
Test solution. To 25.0 mg of the substance to be examined add potentiometrically (2.2.20).
0.2 mL of dilute phosphoric acid R and dilute to 50.0 mL with 1 mL of 0.1 M perchloric acid is equivalent to 33.14 mg
the mobile phase and treat in an ultrasonic bath until a clear of C17H18FN3O3.
solution is obtained.
Reference solution (a). Dilute 1.0 mL of the test solution to STORAGE
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution In an airtight container, protected from light.
to 5.0 mL with the mobile phase.
IMPURITIES
Reference solution (b). Dissolve 5 mg of ciprofloxacin
hydrochloride for peak identification CRS in the mobile phase Specified impurities : A, B, C, D, E.
and dilute to 10.0 mL with the mobile phase. Other detectable impurities (the following substances would,
Column : if present at a sufficient level, be detected by one or other of
– size : l = 0.25 m, Ø = 4.6 mm ; the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
– stationary phase : base-deactivated octadecylsilyl silica gel for by the general monograph Substances for pharmaceutical
chromatography R (5 μm) ; use (2034). It is therefore not necessary to identify these
– temperature : 40 °C. impurities for demonstration of compliance. See also 5.10.
Mobile phase : mix 13 volumes of acetonitrile R and 87 volumes Control of impurities in substances for pharmaceutical use) : F.
of a 2.45 g/L solution of phosphoric acid R, previously adjusted
to pH 3.0 with triethylamine R.
Flow rate : 1.5 mL/min.
Detection : spectrophotometer at 278 nm.
Injection : 50 μL.
Run time : twice the retention time of ciprofloxacin.
Identification of impurities : use the chromatogram supplied A. R = Cl : 7-chloro-1-cyclopropyl-6-fluoro-4-oxo-1,4-
with ciprofloxacin hydrochloride for peak identification CRS dihydroquinoline-3-carboxylic acid (fluoroquinolonic
and the chromatogram obtained with reference solution (b) to acid),
identify the peaks due to impurities B, C, D and E.
Relative retention with reference to ciprofloxacin C. R = NH-[CH2]2-NH2 : 7-[(2-aminoethyl)amino]-1-
(retention time = about 9 min) : impurity E = about 0.4 ; cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
impurity F = about 0.5 ; impurity B = about 0.6 ; carboxylic acid (ethylenediamine compound),
impurity C = about 0.7 ; impurity D = about 1.2.
System suitability : reference solution (b) :
– resolution : minimum 1.3 between the peaks due to
impurity B and impurity C.
Limits :
– correction factors : for the calculation of contents,
multiply the peak areas of the following impurities by B. R = CO2H, R′ = H : 1-cyclopropyl-4-oxo-7-(piperazin-
the corresponding correction factor : impurity B = 0.7 ; 1-yl)-1,4-dihydroquinoline-3-carboxylic acid (desfluoro
impurity C = 0.6 ; impurity D = 1.4 ; impurity E = 6.7 ; compound),
– impurities B, C, D, E : for each impurity, not more than the
area of the principal peak in the chromatogram obtained E. R = H, R′ = F : 1-cyclopropyl-6-fluoro-7-(piperazin-1-
with reference solution (a) (0.2 per cent) ; yl)quinolin-4(1H)-one (decarboxylated compound),
– unspecified impurities : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram F. R = CO2H, R′ = OH : 1-cyclopropyl-6-hydroxy-4-oxo-7-
obtained with reference solution (a) (0.10 per cent) ; (piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid,
– total : not more than 2.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
– disregard limit : 0.25 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.05 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 0.5 g in dilute acetic acid R and dilute to 30 mL with D. 7-chloro-1-cyclopropyl-4-oxo-6-(piperazin-1-yl)-1,4-
the same solvent. Add 2 mL of water R instead of 2 mL of dihydroquinoline-3-carboxylic acid.

General Notices (1) apply to all monographs and other texts 1895
Ciprofloxacin hydrochloride EUROPEAN PHARMACOPOEIA 8.0

04/2011:0888 Related substances. Liquid chromatography (2.2.29).

corrected 7.4 Test solution. Dissolve 25.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with the
CIPROFLOXACIN HYDROCHLORIDE mobile phase.
Reference solution (a). Dissolve 25.0 mg of ciprofloxacin
hydrochloride CRS in the mobile phase and dilute to 50.0 mL
Ciprofloxacini hydrochloridum with the mobile phase.
Reference solution (b). Dissolve 5 mg of ciprofloxacin
hydrochloride for peak identification CRS (containing
impurities B, C, D and E) in the mobile phase and dilute to
10.0 mL with the mobile phase.
Reference solution (c). Dilute 1.0 mL of the test solution to
50.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10.0 mL with the mobile phase.
C17H19ClFN3O3,xH2O Mr 367.8 (anhydrous) Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
DEFINITION
– stationary phase : base-deactivated octadecylsilyl silica gel for
1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4- chromatography R (5 μm) ;
dihydroquinoline-3-carboxylic acid hydrochloride. It contains
– temperature : 40 °C.
a variable quantity of water.
Mobile phase : mix 13 volumes of acetonitrile R and 87 volumes
Content : 98.0 per cent to 102.0 per cent (anhydrous substance). of a 2.45 g/L solution of phosphoric acid R previously adjusted
CHARACTERS to pH 3.0 with triethylamine R.
Flow rate : 1.5 mL/min.
Appearance : pale yellow, crystalline, slightly hygroscopic
powder. Detection : spectrophotometer at 278 nm.
Solubility : soluble in water, slightly soluble in methanol, very Injection : 50 μL of the test solution and reference solutions (b)
slightly soluble in anhydrous ethanol, practically insoluble in and (c).
acetone, in ethyl acetate and in methylene chloride. Run time : 2.3 times the retention time of ciprofloxacin.
Identification of impurities : use the chromatogram supplied
IDENTIFICATION with ciprofloxacin hydrochloride for peak identification CRS
A. Infrared absorption spectrophotometry (2.2.24). and the chromatogram obtained with reference solution (b) to
Comparison : ciprofloxacin hydrochloride CRS. identify the peaks due to impurities B, C, D and E.
Relative retention with reference to ciprofloxacin
B. 0.1 g gives reaction (b) of chlorides (2.3.1).
(retention time = about 9 min) : impurity E = about 0.4 ;
TESTS impurity B = about 0.6 ; impurity C = about 0.7 ;
impurity D = about 1.2.
Solution S. Dissolve 0.5 g in carbon dioxide-free water R and
System suitability : reference solution (b) :
dilute to 20 mL with the same solvent.
– resolution : minimum 1.3 between the peaks due to
Appearance of solution. The solution is clear (2.2.1) and not impurities B and C.
more intensely coloured than reference solution GY5 (2.2.2,
Method II). Limits :
– correction factors : for the calculation of content, multiply
Dilute 10 mL of solution S to 20 mL with carbon dioxide-free
the peak areas of the following impurities by the
water R.
corresponding correction factor : impurity B = 0.7 ;
pH (2.2.3): 3.5 to 4.5 for solution S. impurity C = 0.6 ; impurity D = 1.4 ; impurity E = 6.7 ;
Impurity A. Thin-layer chromatography (2.2.27). – impurity E : not more than 1.5 times the area of the
Test solution. Dissolve 50 mg of the substance to be examined principal peak in the chromatogram obtained with
in water R and dilute to 5 mL with the same solvent. reference solution (c) (0.3 per cent) ;
Reference solution. Dissolve 10 mg of ciprofloxacin – impurities B, C, D : for each impurity, not more than the
impurity A CRS in a mixture of 0.1 mL of dilute ammonia R1 area of the principal peak in the chromatogram obtained
and 90 mL of water R and dilute to 100 mL with water R. with reference solution (c) (0.2 per cent) ;
Dilute 2 mL of the solution to 10 mL with water R. – unspecified impurities : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram
Plate : TLC silica gel F254 plate R. obtained with reference solution (c) (0.10 per cent) ;
Mobile phase : acetonitrile R, concentrated ammonia R, – total : not more than 2.5 times the area of the principal peak
methanol R, methylene chloride R (10:20:40:40 V/V/V/V). in the chromatogram obtained with reference solution (c)
Application : 5 μL. (0.5 per cent) ;
Development : at the bottom of a chromatographic tank, – disregard limit : 0.25 times the area of the principal peak
place an evaporating dish containing 50 mL of concentrated in the chromatogram obtained with reference solution (c)
ammonia R. Expose the plate to the ammonia vapour for (0.05 per cent).
15 min in the closed tank. Withdraw the plate, transfer to a Heavy metals (2.4.8) : maximum 20 ppm.
2nd chromatographic tank and develop over 3/4 of the plate.
Dissolve 0.25 g in water R and dilute to 30 mL with the same
Drying : in air. solvent. Carry out the prefiltration. The filtrate complies
Detection : examine in ultraviolet light at 254 nm. with test E. Prepare the reference solution using 5 mL of lead
Limit : standard solution (1 ppm Pb) R.
– impurity A : any spot corresponding to impurity A is not Water (2.5.12) : maximum 6.7 per cent, determined on 0.200 g.
more intense than the principal spot in the chromatogram Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
obtained with the reference solution (0.2 per cent). 1.0 g in a platinum crucible.

1896 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cisplatin

ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : 10 μL of the test solution and reference solution (a).
Calculate the percentage content of C17H19ClFN3O3.
STORAGE F. 1-cyclopropyl-6-hydroxy-4-oxo-7-(piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylic acid.
In an airtight container, protected from light.
IMPURITIES
01/2009:0599
Specified impurities : A, B, C, D, E. corrected 7.0
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of CISPLATIN
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical Cisplatinum
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use) : F.

PtCl2(NH3)2 Mr 300.0
[15663-27-1]
DEFINITION
cis-Diamminedichloroplatinum(II).
Content : 97.0 per cent to 102.0 per cent.
A. 7-chloro-1-cyclopropyl-6-fluoro-4-oxo-1,4- CHARACTERS
dihydroquinoline-3-carboxylic acid (fluoroquinolonic Appearance : yellow powder, or yellow or orange-yellow
acid),
crystals.
Solubility : slightly soluble in water, sparingly soluble in
dimethylformamide, practically insoluble in ethanol (96 per
cent).
Carry out identification test B, the tests (except that for silver)
and the assay protected from light.
IDENTIFICATION
B. 1-cyclopropyl-4-oxo-7-(piperazin-1-yl)-1,4- First identification : A, B.
dihydroquinoline-3-carboxylic acid (desfluoro Second identification : B, C.
compound),
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: cisplatin CRS.
B. Thin-layer chromatography (2.2.27).
Test solution. Dilute 1 mL of solution S2 (see Tests) to
10 mL with dimethylformamide R.
Reference solution. Dissolve 10 mg of cisplatin CRS in 5 mL
of dimethylformamide R.
C. 7-[(2-aminoethyl)amino]-1-cyclopropyl-6-fluoro-4-oxo- Plate : cellulose for chromatography R1 as the coating
1,4-dihydroquinoline-3-carboxylic acid (ethylenediamine substance.
compound), Pretreatment : activate the plate by heating at 150 °C for 1 h.
Mobile phase : acetone R, dimethylformamide R (10:90 V/V).
Application : 2 μL.
Development : over 2/3 of the plate.
Drying : in air.
Detection : spray with a 50 g/L solution of stannous
chloride R in a mixture of equal volumes of dilute
hydrochloric acid R and water R. Examine after 1 h.
D. 7-chloro-1-cyclopropyl-4-oxo-6-(piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylic acid, Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
the reference solution.
C. Add 50 mg to 2 mL of dilute sodium hydroxide solution R
in a glass dish. Evaporate to dryness. Dissolve the residue
in a mixture of 0.5 mL of nitric acid R and 1.5 mL of
hydrochloric acid R. Evaporate to dryness. The residue
is orange. Dissolve the residue in 0.5 mL of water R and
E. 1-cyclopropyl-6-fluoro-7-(piperazin-1-yl)quinolin-4(1H)- add 0.5 mL of ammonium chloride solution R. A yellow,
one (decarboxylated compound), crystalline precipitate is formed.

General Notices (1) apply to all monographs and other texts 1897
Cisplatin EUROPEAN PHARMACOPOEIA 8.0

TESTS Limits :
Solution S1. Dissolve 25 mg in a 9 g/L solution of sodium – impurity A : not more than the area of the corresponding
chloride R in carbon dioxide-free water R and dilute to 25 mL peak in the chromatogram obtained with reference
with the same solvent. solution (d) (2.0 per cent) ;
Solution S2. Dissolve 0.20 g in dimethylformamide R and – impurity B : not more than the area of the corresponding
dilute to 10 mL with the same solvent. peak in the chromatogram obtained with reference
solution (d) (1.0 per cent) ;
Appearance of solution S1. Solution S1 is clear (2.2.1) and
not more intensely coloured than reference solution GY5 – unspecified impurities : for each impurity, not more than
(2.2.2, Method II). 0.5 times the area of the peak due to cisplatin in the
chromatogram obtained with reference solution (d)
Appearance of solution S2. Solution S2 is clear (2.2.1). (0.10 per cent);
pH (2.2.3) : 4.5 to 6.0 for solution S1, measured immediately – sum of impurities other than A and B : not more than
after preparation. 2.5 times the area of the peak due to cisplatin in the
Related substances. Liquid chromatography (2.2.29). Carry chromatogram obtained with reference solution (d) (0.5 per
out the test protected from light. Do not heat or sonicate any cent) ;
platinum-containing solution. All solutions are to be used – disregard limit : the area of the peak due to cisplatin in
within 4 h. the chromatogram obtained with reference solution (e)
Test solution. Dissolve 25.0 mg of the substance to be (0.05 per cent). Disregard any peak due to the cisplatin
examined in a 9.0 g/L solution of sodium chloride R and dilute aquo complex.
to 25.0 mL with the same solution. Silver: maximum 250 ppm.
Reference solution (a). Dissolve 25.0 mg of cisplatin CRS in a Atomic absorption spectrometry (2.2.23, Method I).
9.0 g/L solution of sodium chloride R and dilute to 25.0 mL Test solution. Dissolve 0.100 g in 15 mL of nitric acid R,
with the same solution. heating to 80 °C. Cool and dilute to 25.0 mL with water R.
Reference solution (b). Dissolve 5.0 mg of cisplatin Reference solutions. To suitable volumes (10 mL to 30 mL)
impurity A CRS in a 9.0 g/L solution of sodium chloride R and of silver standard solution (5 ppm Ag) R add 50 mL of nitric
dilute to 50.0 mL with the same solution. acid R and dilute to 100.0 mL with water R.
Reference solution (c). Dissolve 5.6 mg of cisplatin Source : silver hollow-cathode lamp, preferably using a
impurity B CRS in a 9.0 g/L solution of sodium chloride R and transmission band of 0.5 nm.
dilute to 100.0 mL with the same solution.
Wavelenth : 328 nm.
Reference solution (d). Mix 0.05 mL of the test solution with
5.0 mL of reference solution (b) and 5.0 mL of reference Atomisation device : fuel-lean air-acetylene flame.
solution (c) and dilute to 25.0 mL with a 9.0 g/L solution of Carry out a blank determination.
sodium chloride R.
ASSAY
Reference solution (e). Dilute 5.0 mL of reference solution (d) Liquid chromatography (2.2.29) as described in the test for
to 20.0 mL with a 9.0 g/L solution of sodium chloride R. related substances with the following modification.
Blank solution : 9.0 g/L solution of sodium chloride R. Injection : 10 μL of the test solution and reference solution (a).
Column : Calculate the percentage content of PtCl2(NH3)2 from the sum
– size : l = 0.25 m, Ø = 4.0 mm ; of the areas of the peaks due to cisplatin and cisplatin aquo
– stationary phase : base-deactivated octylsilyl silica gel for complex and from the declared content of cisplatin CRS.
chromatography R (4 μm) ;
STORAGE
– temperature : 30 °C.
In an airtight container, protected from light.
Mobile phase : dissolve 1.08 g of sodium octanesulfonate R,
1.70 g of tetrabutylammonium hydrogen sulfate R and IMPURITIES
2.72 g of potassium dihydrogen phosphate R in water for Specified impurities : A, B.
chromatography R and dilute to 950 mL with the same solvent.
Adjust to pH 5.9 with 1 M sodium hydroxide and dilute to Other detectable impurities (the following substances would,
1000 mL with water for chromatography R. if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
Flow rate : 1.0 mL/min. acceptance criterion for other/unspecified impurities and/or
Detection : spectrophotometer at 210 nm. by the general monograph Substances for pharmaceutical
Injection : 20 μL of the test solution, reference solutions (d) use (2034). It is therefore not necessary to identify these
and (e), and the blank solution. impurities for demonstration of compliance. See also 5.10.
Run time : 7 times the retention time of cisplatin. Control of impurities in substances for pharmaceutical use) : C.
The displacement peak is the latest eluting peak of the group
of injection peaks in the chromatogram obtained with the
blank solution.
Identification of cisplatin aquo complex : use the chromatogram A. trans-diamminedichloroplatinum(II) (transplatin),
supplied with cisplatin CRS and the chromatogram obtained
with reference solution (a) to identify the peak due to cisplatin
aquo complex.
Relative retention with reference to cisplatin (retention
time = about 3.8 min) : displacement peak = about 0.5 ;
impurity A = about 0.6 ; impurity B = about 0.7 ; cisplatin aquo B. amminetrichloroplatinate(–),
complex = about 1.2.
System suitability : reference solution (d) :
– resolution : minimum 2.5 between the peaks due to
impurities A and B, the displacement peak and the peak
due to impurity A are well separated. C. tetrachloroplatinate(2–).

1898 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Citalopram hydrobromide

04/2011:2288 Detection : spectrophotometer at 230 nm and, for impurity G,

at 254 nm.
CITALOPRAM HYDROBROMIDE Injection : 40 μL.
Identification of impurities : use the chromatogram
Citaloprami hydrobromidum supplied with citalopram for system suitability CRS and the
chromatogram obtained with reference solution (b) to identify
the peaks due to impurities B, D and G.
Relative retention with reference to citalopram (retention
time = about 19 min) : impurity G = about 0.5 ;
impurity B = about 0.7 ; impurity D = about 0.9.
System suitability : reference solution (b) :
– resolution : minimum 1.5 between the peaks due to
impurity D and citalopram at 230 nm.
C20H22BrFN2O Mr 405.3 Limits :
[59729-32-7]
– correction factor : for the calculation of content, multiply
DEFINITION the peak area of impurity G by 0.6 ;
(1RS)-1-[3-(Dimethylamino)propyl]-1-(4-fluorophenyl)-1,3- – impurity D : not more than twice the area of the principal
dihydroisobenzofuran-5-carbonitrile hydrobromide. peak in the chromatogram obtained with reference
Content : 99.0 per cent to 101.5 per cent (dried substance). solution (a) (0.2 per cent) ;
– impurity B : not more than 1.5 times the area of the
CHARACTERS principal peak in the chromatogram obtained with
Appearance : white or almost white, crystalline powder. reference solution (a) (0.15 per cent) ;
Solubility : sparingly soluble in water and in anhydrous ethanol. – impurity G at 254 nm : not more than 1.5 times the area
of the principal peak in the chromatogram obtained with
IDENTIFICATION reference solution (a) (0.15 per cent) ;
A. Optical rotation (see Tests). – unspecified impurities : for each impurity, not more than the
B. Infrared absorption spectrophotometry (2.2.24). area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
Comparison : citalopram hydrobromide CRS.
– sum of impurities other than G : not more than 5 times the
C. It gives reaction (a) of bromides (2.3.1). area of the principal peak in the chromatogram obtained
TESTS with reference solution (a) (0.5 per cent) ;
Optical rotation (2.2.7) : − 0.10° to + 0.10°. – disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
Dissolve 1.0 g in methanol R and dilute to 20 mL with the (0.05 per cent).
same solvent.
Heavy metals (2.4.8) : maximum 20 ppm.
Related substances. Liquid chromatography (2.2.29).
Dissolve 0.5 g in ethanol (96 per cent) R and dilute to 20 mL
Test solution. Dissolve 50 mg of the substance to be examined with the same solvent. 12 mL of the solution complies with
in mobile phase A and dilute to 100.0 mL with mobile phase A. test B. Prepare the reference solution using lead standard
Reference solution (a). Dilute 1.0 mL of the test solution to solution (0.5 ppm Pb) obtained by diluting lead standard
100.0 mL with mobile phase A (solution A). Dilute 1.0 mL of solution (100 ppm Pb) R with ethanol (96 per cent) R. Filter
solution A to 10.0 mL with mobile phase A. the solutions through a membrane filter (nominal pore size
Reference solution (b). Dissolve the contents of a vial of 0.45 μm).
citalopram for system suitability CRS (containing impurities B, Loss on drying (2.2.32) : maximum 0.5 per cent, determined
D and G) in 1.0 mL of solution A. on 1.000 g by drying in an oven at 105 °C for 4 h.
Column : Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
– size : l = 0.25 m, Ø = 4.6 mm ; 1.0 g in a platinum crucible.
– stationary phase : end-capped octadecylsilyl silica gel for ASSAY
chromatography R (4 μm) ;
Dissolve 0.300 g in 50 mL of ethanol (96 per cent) R and add
– temperature : 40 °C.
0.5 mL of 0.1 M hydrochloric acid. Carry out a potentiometric
Mobile phase : titration (2.2.20), using 0.1 M sodium hydroxide. Read the
– mobile phase A : dissolve 1.58 g of ammonium formate R volume added between the 2 points of inflexion.
in 500 mL of a mixture of 4 volumes of acetonitrile R, 1 mL of 0.1 M sodium hydroxide is equivalent to 40.53 mg
32 volumes of methanol R and 64 volumes of water R ; of C20H22BrFN2O.
– mobile phase B : dissolve 1.58 g of ammonium formate R
in 500 mL of a mixture of 32 volumes of water R and IMPURITIES
68 volumes of acetonitrile R ; Specified impurities : B, D, G.
Time Mobile phase A Mobile phase B Other detectable impurities (the following substances would,
(min) (per cent V/V) (per cent V/V) if present at a sufficient level, be detected by one or other of
0-2 100 0 the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
2 - 25 100 → 40 0 → 60
by the general monograph Substances for pharmaceutical use
25 - 30 40 60 (2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
Flow rate : 1.0 mL/min. impurities in substances for pharmaceutical use) : A, C, E, F.

General Notices (1) apply to all monographs and other texts 1899
Citalopram hydrochloride EUROPEAN PHARMACOPOEIA 8.0

IDENTIFICATION
A. Optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: citalopram hydrochloride CRS.
C. It gives reaction (a) of chlorides (2.3.1).
TESTS
A. R = CO-NH2, X = H2 : (1RS)-1-[3-(dimethylamino)propyl]- Solution S. Dissolve 1.0 g in methanol R and dilute to 20 mL
1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5- with the same solvent.
carboxamide,
Appearance of solution. Solution S, examined immediately
C. R = CN, X = O : (3RS)-6-cyano-3-[3-(dimethylamino)prop- after preparation, is clear (2.2.1) and not more intensely
yl]-3-(4-fluorophenyl)isobenzofuran-1(3H)-one, coloured than reference solution Y6 (2.2.2, Method II).
E. R = Cl, X = H2 : 3-[(1RS)-5-chloro-1-(4-fluorophenyl)- Optical rotation (2.2.7): − 0.10° to + 0.10°, determined on
1,3-dihydroisobenzofuran-1-yl]-N,N-dimethylpropan-1- solution S.
amine, Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50 mg of the substance to be examined
in mobile phase A and dilute to 100.0 mL with mobile phase A.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with mobile phase A (solution A). Dilute 1.0 mL of
solution A to 10.0 mL with mobile phase A.
Reference solution (b). Dissolve the contents of a vial of
citalopram for system suitability CRS (impurities B and D) in
B. 1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-3- 1.0 mL of solution A.
hydroxy-1,3dihydroisobenzofuran-5-carbonitrile, Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (4 μm) ;
– temperature : 40 °C.
Mobile phase :
– mobile phase A : dissolve 1.58 g of ammonium formate R
in 500 mL of a mixture of 4 volumes of acetonitrile R,
D. R1 = CN, R2 = H : (1RS)-1-(4-fluorophenyl)-1-[3-
32 volumes of methanol R and 64 volumes of water R ;
(methylamino)propyl]-1,3-dihydroisobenzofuran-5-
carbonitrile, – mobile phase B : dissolve 1.58 g of ammonium formate R
in 500 mL of a mixture of 32 volumes of water R and
F. R1 = Br, R2 = CH3 : 3-[(1RS)-5-bromo-1-(4-fluorophenyl)- 68 volumes of acetonitrile R ;
1,3-dihydroisobenzofuran-1-yl]-N,N-dimethylpropan-1-
amine, Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
G. R1 = CO-[CH2]3-N(CH3)2, R2 = CH3 : 4-(dimethylamino)-
0-2 100 0
1-[(1RS)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-
1,3-dihydroisobenzofuran-5-yl]butan-1-one. 2 - 25 100 → 40 0 → 60
25 - 30 40 60
01/2009:2203
corrected 6.4 Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 230 nm.
CITALOPRAM HYDROCHLORIDE Injection : 40 μL.
Identification of impurities : use the chromatogram
Citaloprami hydrochloridum supplied with citalopram for system suitability CRS and the
chromatogram obtained with reference solution (b) to identify
the peaks due to impurities B and D.
Relative retention with reference to citalopram (retention
time = about 19 min) : impurity B = about 0.7 ;
impurity D = about 0.9.
System suitability : reference solution (b) :
– resolution : minimum 1.5 between the peaks due to
C20H22ClFN2O Mr 360.9 impurity D and citalopram.
[85118-27-0] Limits :
DEFINITION – impurity B : not more than 1.5 times the area of the
(1RS)-1-[3-(Dimethylamino)propyl]-1-(4-fluorophenyl)-1,3- principal peak in the chromatogram obtained with
dihydroisobenzofuran-5-carbonitrile hydrochloride. reference solution (a) (0.15 per cent) ;
Content : 99.0 per cent to 101.5 per cent (dried substance). – unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
CHARACTERS with reference solution (a) (0.10 per cent) ;
Appearance : white or almost white, crystalline powder. – total : not more than twice the area of the principal peak
Solubility : very soluble in water, freely soluble in anhydrous in the chromatogram obtained with reference solution (a)
ethanol. (0.2 per cent) ;

1900 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Citric acid, anhydrous

– disregard limit : 0.5 times the area of the principal peak in 01/2008:0455
the chromatogram obtained with reference solution (a) corrected 6.0
(0.05 per cent).
Heavy metals (2.4.8) : maximum 20 ppm. CITRIC ACID, ANHYDROUS
Dissolve 1.0 g in 20 mL of water R. 12 mL of the solution
complies with test A. Prepare the reference solution using lead Acidum citricum anhydricum
standard solution (1 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 4 h.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on C6H8O7 Mr 192.1
1.0 g in a platinum crucible. [77-92-9]
DEFINITION
ASSAY
2-Hydroxypropane-1,2,3-tricarboxylic acid.
Dissolve 0.250 g in 50 mL of ethanol (96 per cent) R and add
0.5 mL of 0.1 M hydrochloric acid. Carry out a potentiometric Content : 99.5 per cent to 100.5 per cent (anhydrous substance).
titration (2.2.20), using 0.1 M sodium hydroxide. Read the CHARACTERS
volume added between the 2 points of inflexion.
Appearance : white or almost white, crystalline powder,
1 mL of 0.1 M sodium hydroxide is equivalent to 36.09 mg colourless crystals or granules.
of C20H22ClFN2O.
Solubility : very soluble in water, freely soluble in ethanol
IMPURITIES (96 per cent).
Specified impurities : B. mp : about 153 °C, with decomposition.
Other detectable impurities (the following substances would, IDENTIFICATION
if present at a sufficient level, be detected by one or other of First identification : B, E.
the tests in the monograph. They are limited by the general Second identification : A, C, D, E.
acceptance criterion for other/unspecified impurities and/or
A. Dissolve 1 g in 10 mL of water R. The solution is strongly
by the general monograph Substances for pharmaceutical use
acidic (2.2.4).
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of B. Infrared absorption spectrophotometry (2.2.24).
impurities in substances for pharmaceutical use) : A, C, D, E, F. Preparation : dry the substance to be examined and the
reference substance at 100-105 °C for 2 h.
Comparison: anhydrous citric acid CRS.
C. Add about 5 mg to a mixture of 1 mL of acetic anhydride R
and 3 mL of pyridine R. A red colour develops.
D. Dissolve 0.5 g in 5 mL of water R, neutralise using 1 M
sodium hydroxide (about 7 mL), add 10 mL of calcium
chloride solution R and heat to boiling. A white precipitate
is formed.
E. Water (see Tests).
A. R1 = CO-NH2, R2 = CH3, X = H2 : (1RS)-1-[3-
(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3- TESTS
dihydroisobenzofuran-5-carboxamide, Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution Y7, BY7 or
C. R1 = CN, R2 = CH3, X = O : (3RS)-6-cyano-3-[3- GY7 (2.2.2, Method II).
(dimethylamino)propyl]-3-(4-fluorophenyl)isobenzofuran- Dissolve 2.0 g in water R and dilute to 10 mL with the same
1(3H)-one, solvent.
D. R1 = CN, R2 = H, X = H2 : (1RS)-1-(4-fluorophenyl)-1- Readily carbonisable substances. To 1.0 g in a cleaned test
[3-(methylamino)propyl]-1,3-dihydroisobenzofuran-5- tube add 10 mL of sulfuric acid R and immediately heat the
carbonitrile, mixture in a water-bath at 90 ± 1 °C for 60 min. Cool rapidly
immediately afterwards. The solution is not more intensely
E. R1 = Cl, R2 = CH3, X = H2 : 3-[(1RS)-5-chloro-1-(4- coloured than a mixture of 1 mL of red primary solution and
fluorophenyl)-1,3-dihydroisobenzofuran-1-yl]-N,N- 9 mL of yellow primary solution (2.2.2, Method I).
dimethylpropan-1-amine, Oxalic acid : maximum 360 ppm, calculated as anhydrous
oxalic acid.
F. R1 = Br, R2 = CH3, X = H2 : 3-[(1RS)-5-bromo-1-(4- Dissolve 0.80 g in 4 mL of water R. Add 3 mL of hydrochloric
fluorophenyl)-1,3-dihydroisobenzofuran-1-yl]-N,N- acid R and 1 g of zinc R in granules. Boil for 1 min. Allow
dimethylpropan-1-amine, to stand for 2 min. Transfer the supernatant to a test-tube
containing 0.25 mL of a 10 g/L solution of phenylhydrazine
hydrochloride R and heat to boiling. Cool rapidly, transfer to a
graduated cylinder and add an equal volume of hydrochloric
acid R and 0.25 mL of a 50 g/L solution of potassium
ferricyanide R. Shake and allow to stand for 30 min. Any
pink colour in the solution is not more intense than that in a
standard prepared at the same time in the same manner using
4 mL of a 0.1 g/L solution of oxalic acid R.
Sulfates (2.4.13) : maximum 150 ppm.
B. 1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-3- Dissolve 2.0 g in distilled water R and dilute to 30 mL with
hydroxy-1,3-dihydroisobenzofuran-5-carbonitrile. the same solvent.

General Notices (1) apply to all monographs and other texts 1901
Citric acid monohydrate EUROPEAN PHARMACOPOEIA 8.0

Aluminium (2.4.17) : maximum 0.2 ppm, if intended for use C. Add about 5 mg to a mixture of 1 mL of acetic anhydride R
in the manufacture of dialysis solutions. and 3 mL of pyridine R. A red colour develops.
Prescribed solution. Dissolve 20 g in 100 mL of water R and D. Dissolve 0.5 g in 5 mL of water R, neutralise using 1 M
add 10 mL of acetate buffer solution pH 6.0 R. sodium hydroxide (about 7 mL), add 10 mL of calcium
Reference solution. Mix 2 mL of aluminium standard solution chloride solution R and heat to boiling. A white precipitate
(2 ppm Al) R, 10 mL of acetate buffer solution pH 6.0 R and is formed.
98 mL of water R. E. Water (see Tests).
Blank solution. Mix 10 mL of acetate buffer solution pH 6.0 R TESTS
and 100 mL of water R.
Appearance of solution. The solution is clear (2.2.1) and not
Heavy metals (2.4.8) : maximum 10 ppm. more intensely coloured than reference solution Y7, BY7 or
Dissolve 5.0 g in several portions in 39 mL of dilute sodium GY7 (2.2.2, Method II).
hydroxide solution R and dilute to 50 mL with distilled water R. Dissolve 2.0 g in water R and dilute to 10 mL with the same
12 mL of the solution complies with test A. Prepare the solvent.
reference solution using lead standard solution (1 ppm Pb) R.
Readily carbonisable substances. To 1.0 g in a cleaned test
Water (2.5.12) : maximum 1.0 per cent, determined on 2.000 g. tube add 10 mL of sulfuric acid R and immediately heat the
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on mixture in a water-bath at 90 ± 1 °C for 60 min. Cool rapidly
1.0 g. immediately afterwards. The solution is not more intensely
coloured than a mixture of 1 mL of red primary solution and
Bacterial endotoxins (2.6.14) : less than 0.5 IU/mg, if intended 9 mL of yellow primary solution (2.2.2, Method I).
for use in the manufacture of parenteral preparations without
a further appropriate procedure for the removal of bacterial Oxalic acid : maximum 360 ppm, calculated as anhydrous
endotoxins. oxalic acid.
Dissolve 0.80 g in 4 mL of water R. Add 3 mL of hydrochloric
ASSAY acid R and 1 g of zinc R in granules. Boil for 1 min. Allow
Dissolve 0.550 g in 50 mL of water R. Titrate with 1 M sodium to stand for 2 min. Transfer the supernatant to a test-tube
hydroxide, using 0.5 mL of phenolphthalein solution R as containing 0.25 mL of a 10 g/L solution of phenylhydrazine
indicator. hydrochloride R and heat to boiling. Cool rapidly, transfer to a
1 mL of 1 M sodium hydroxide is equivalent to 64.03 mg graduated cylinder and add an equal volume of hydrochloric
of C6H8O7. acid R and 0.25 mL of a 50 g/L solution of potassium
ferricyanide R. Shake and allow to stand for 30 min. Any
LABELLING pink colour in the solution is not more intense than that in a
The label states, where applicable, that the substance is standard prepared at the same time in the same manner using
intended for use in the manufacture of dialysis solutions. 4 mL of a 0.1 g/L solution of oxalic acid R.
Sulfates (2.4.13) : maximum 150 ppm.
Dissolve 2.0 g in distilled water R and dilute to 30 mL with
the same solvent.
01/2008:0456
corrected 6.0 Aluminium (2.4.17) : maximum 0.2 ppm, if intended for use
in the manufacture of dialysis solutions.
Prescribed solution. Dissolve 20 g in 100 mL of water R and
CITRIC ACID MONOHYDRATE add 10 mL of acetate buffer solution pH 6.0 R.
Reference solution. Mix 2 mL of aluminium standard solution
Acidum citricum monohydricum (2 ppm Al) R, 10 mL of acetate buffer solution pH 6.0 R and
98 mL of water R.
Blank solution. Mix 10 mL of acetate buffer solution pH 6.0 R
and 100 mL of water R.
C6H8O7,H2O Mr 210.1 Heavy metals (2.4.8) : maximum 10 ppm.
[5949-29-1] Dissolve 5.0 g in several portions in 39 mL of dilute sodium
hydroxide solution R and dilute to 50 mL with distilled water R.
DEFINITION 12 mL of the solution complies with test A. Prepare the
2-Hydroxypropane-1,2,3-tricarboxylic acid monohydrate. reference solution using lead standard solution (1 ppm Pb) R.
Content : 99.5 per cent to 100.5 per cent (anhydrous substance). Water (2.5.12) : 7.5 per cent to 9.0 per cent, determined on
0.500 g.
CHARACTERS
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Appearance : white or almost white, crystalline powder, 1.0 g.
colourless crystals or granules, efflorescent.
Bacterial endotoxins (2.6.14) : less than 0.5 IU/mg, if intended
Solubility : very soluble in water, freely soluble in ethanol for use in the manufacture of parenteral preparations without
(96 per cent). a further appropriate procedure for the removal of bacterial
IDENTIFICATION endotoxins.
First identification : B, E. ASSAY
Second identification : A, C, D, E. Dissolve 0.550 g in 50 mL of water R. Titrate with 1 M sodium
A. Dissolve 1 g in 10 mL of water R. The solution is strongly hydroxide, using 0.5 mL of phenolphthalein solution R as
acidic (2.2.4). indicator.
B. Infrared absorption spectrophotometry (2.2.24). 1 mL of 1 M sodium hydroxide is equivalent to 64.03 mg
of C6H8O7.
Preparation : dry the substance to be examined and the
reference substance at 100-105 °C for 2 h. STORAGE
Comparison : citric acid monohydrate CRS. In an airtight container.

1902 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cladribine

LABELLING Mobile phase : concentrated ammonia R, ethanol (96 per

The label states, where applicable, that the substance is cent) R, ethyl acetate R (20:40:40 V/V/V).
intended for use in the manufacture of dialysis solutions. Application : 5 μL as bands of 10 mm ; thoroughly dry the
points of application in a current of warm air.
Development : over 2/3 of the plate.
01/2011:2174 Drying : in air, then heat at 45 °C for 10 min.
Detection : spray with a solution containing 0.5 g of thymol R
CLADRIBINE in a mixture of 5 mL of sulfuric acid R and 95 mL of ethanol
(96 per cent) R ; heat at 110 °C for 20 min or until the spots
appear.
Cladribinum System suitability : reference solution (b) :
– the chromatogram shows 2 clearly separated spots.
Limit :
– impurity E : any spot due to impurity E is not more intense
than the spot in the chromatogram obtained with reference
solution (a) (0.3 per cent).
Related substances. Liquid chromatography (2.2.29).
Solvent mixture : acetonitrile R, water R (10:90 V/V).
Test solution (a). Dissolve 25.0 mg of the substance to be
examined in the solvent mixture and dilute to 5.0 mL with the
C10H12ClN5O3 Mr 285.7 solvent mixture.
[4291-63-8]
Test solution (b). Dissolve 20.0 mg of the substance to be
DEFINITION examined in the solvent mixture and dilute to 100.0 mL with
the solvent mixture.
2-Chloro-9-(2-deoxy-β-D-erythro-pentofuranosyl)-9H-purin-
6-amine. Reference solution (a). Dissolve 20.0 mg of cladribine CRS in
the solvent mixture and dilute to 100.0 mL with the solvent
Content : 97.0 per cent to 102.0 per cent (anhydrous substance). mixture.
CHARACTERS Reference solution (b). Dilute 1.0 mL of test solution (a) to
Appearance : white or almost white, crystalline powder. 100.0 mL with the solvent mixture.
Reference solution (c). Dilute 1.0 mL of reference solution (b)
Solubility : slightly soluble in water, soluble in dimethyl
sulfoxide, slightly soluble in methanol, practically insoluble to 10.0 mL with the solvent mixture.
in acetonitrile. Reference solution (d). Dissolve 1.0 mg of cladribine
impurity C CRS in reference solution (b) and dilute to 25.0 mL
It shows polymorphism (5.9). with the same solution.
IDENTIFICATION Reference solution (e). Dilute 5.0 mL of reference solution (c)
A. Specific optical rotation (see Tests). to 10.0 mL with the solvent mixture.
B. Infrared absorption spectrophotometry (2.2.24). Reference solution (f). Dissolve 3 mg of cladribine for peak
identification CRS (containing impurities A, B, C and D) in
Comparison : cladribine CRS. 2 mL of the solvent mixture.
If the spectra obtained in the solid state show differences, Column :
dissolve the substance to be examined in the minimum – size : l = 0.25 m, Ø = 4.6 mm ;
volume of methanol R and evaporate to dryness. Dry the
precipitate at 100 °C for 2 h and record a new spectrum – stationary phase : base-deactivated octylsilyl silica gel for
using the residue. chromatography R (5 μm).
Mobile phase :
TESTS – mobile phase A : water for chromatography R ;
Appearance of solution. The solution is clear (2.2.1) and – mobile phase B : acetonitrile for chromatography R ;
colourless (2.2.2, Method II). – mobile phase C : 50 g/L solution of phosphoric acid R in
Disperse 0.15 g in water R, dilute to 50 mL with the same water for chromatography R ;
solvent and sonicate until dissolution is complete. Time Mobile phase A Mobile phase B Mobile phase C
Specific optical rotation (2.2.7) : − 21.0 to − 27.0 (anhydrous (min) (per cent V/V) (per cent V/V) (per cent V/V)
substance). 0 - 10 80 → 70 10 → 20 10
Dissolve 0.25 g in dimethyl sulfoxide R and dilute to 25.0 mL 10 - 25 70 → 20 20 → 70 10
with the same solvent.
25 - 30 20 70 10
Impurity E. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 40.0 mg of the substance to be Flow rate : 0.8 mL/min.
examined in dimethylformamide R and dilute to 2.0 mL with Detection : spectrophotometer at 252 nm.
the same solvent. Injection : 20 μL of test solution (a) and reference
Reference solution (a). Dissolve 5.0 mg of 2-deoxy-D-ribose R solutions (c), (d), (e) and (f).
(impurity E) in dimethylformamide R and dilute to 25.0 mL Identification of impurities : use the chromatogram supplied
with the same solvent. Dilute 3.0 mL of this solution to with cladribine for peak identification CRS and the
10.0 mL with dimethylformamide R. chromatogram obtained with reference solution (f) to identify
Reference solution (b). Dissolve 10.0 mg of 2-deoxy-D-ribose R the peaks due to impurities A, B, C and D.
(impurity E) in dimethylformamide R and dilute to 5.0 mL Relative retention with reference to cladribine (retention
with the same solvent. Mix 9 volumes of this solution with time = about 10 min) : impurity A = about 0.33 ;
1 volume of the test solution. impurity B = about 0.44 ; impurity C = about 0.73 ;
Plate : TLC silica gel F254 plate R. impurity D = about 0.92.

General Notices (1) apply to all monographs and other texts 1903
Clarithromycin EUROPEAN PHARMACOPOEIA 8.0

System suitability : reference solution (d) :

– resolution : minimum 4.5 between the peaks due to
impurity C and cladribine.
Limits :
– correction factors : for the calculation of content, multiply C. 2-chloro-7H-purin-6-amine (2-chloroadenine),
the peak areas of the following impurities by the
corresponding correction factor : impurity B = 1.7 ;
impurity C = 0.8 ;
– impurities A, C : for each impurity, not more than 3 times
the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.3 per cent) ;
– impurities B, D : for each impurity, not more than twice the
area of the principal peak in the chromatogram obtained
D. 2-chloro-9-(2-deoxy-α-D-erythro-pentofuranosyl)-9H-
with reference solution (c) (0.2 per cent) ;
purin-6-amine,
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (c) (0.10 per cent) ;
– total : not more than 10 times the area of the principal peak
in the chromatogram obtained with reference solution (c)
(1.0 per cent) ;
– disregard limit : the area of the principal peak in the E. 2-deoxy-D-erythro-pentofuranose (2-deoxy-D-ribose),
chromatogram obtained with reference solution (e)
(0.05 per cent).
Water (2.5.32) : maximum 0.5 per cent, determined on 0.100 g.
Bacterial endotoxins (2.6.14): less than 3 IU/mg, if intended
for use in the manufacture of parenteral preparations without F. R = NH2 : 4-methylbenzamide,
a further appropriate procedure for the removal of bacterial
endotoxins. G. R = OCH3 : methyl 4-methylbenzoate.
ASSAY
Liquid chromatography (2.2.29) as described in the test for 01/2008:1651
related substances with the following modification. corrected 7.0
Injection : test solution (b) and reference solution (a).
Calculate the percentage content of C10H12ClN5O3 from the
CLARITHROMYCIN
declared content of cladribine CRS.
Clarithromycinum
STORAGE
Protected from light, at a temperature of 2 °C to 8 °C. If the
substance is sterile, store in a sterile, airtight, tamper-proof
container.
LABELLING
The label states, where applicable, that the substance is suitable
for use in the manufacture of parenteral preparations.
IMPURITIES
Specified impurities : A, B, C, D, E.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of C38H69NO13 Mr 748
the tests in the monograph. They are limited by the general [81103-11-9]
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use DEFINITION
(2034). It is therefore not necessary to identify these impurities (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-4-[(2,6-Dideoxy-
for demonstration of compliance. See also 5.10. Control of 3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-
impurities in substances for pharmaceutical use) : F, G. 14-ethyl-12,13-dihydroxy-7-methoxy-3,5,7,9,11,13-
hexamethyl-6-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-
xylo-hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione
(6-O-methylerythromycin A).
Semi-synthetic product derived from a fermentation product.
Content : 96.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, soluble in acetone
and in methylene chloride, slightly soluble in methanol.
A. R = NH2 : 9-(2-deoxy-β-D-erythro-pentofuranosyl)-9H-
purin-2,6-diamine, IDENTIFICATION
B. R = OCH3 : 9-(2-deoxy-β-D-erythro-pentofuranosyl)-2- Infrared absorption spectrophotometry (2.2.24).
methoxy-9H-purin-6-amine, Comparison: clarithromycin CRS.

1904 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Clarithromycin

TESTS – peak-to-valley ratio : minimum 3.0, where Hp = height above

Solution S. Dissolve 0.500 g in methylene chloride R and dilute the baseline of the peak due to impurity D and Hv = height
to 50.0 mL with the same solvent. above the baseline of the lowest point of the curve
separating this peak from the peak due to clarithromycin
Appearance of solution. Solution S is clear or not more in the chromatogram obtained with reference solution (d).
opalescent than reference suspension II (2.2.1) and not
more intensely coloured than reference solution Y7 (2.2.2, Limits :
Method II). – correction factors : for the calculation of contents,
Specific optical rotation (2.2.7) : − 94 to − 102 (anhydrous multiply the peak areas of the following impurities by
substance), determined on solution S. the corresponding correction factor : impurity G = 0.27 ;
impurity H = 0.15 ; use the chromatogram supplied with
Related substances. Liquid chromatography (2.2.29). clarithromycin for peak identification CRS to identify the
Test solution. Dissolve 75.0 mg of the substance to be peaks ;
examined in 25 mL of acetonitrile R1 and dilute to 50.0 mL – any impurity : not more than twice the area of the principal
with water R. peak in the chromatogram obtained with reference
solution (c) (1.0 per cent), and not more than 4 such peaks
Reference solution (a). Dissolve 75.0 mg of clarithromycin CRS
have an area greater than 0.8 times the area of the principal
in 25 mL of acetonitrile R1 and dilute to 50.0 mL with water R.
peak in the chromatogram obtained with reference
Reference solution (b). Dilute 5.0 mL of reference solution (a) solution (c) (0.4 per cent) ;
to 100.0 mL with a mixture of equal volumes of acetonitrile R1 – total : not more than 7 times the area of the principal peak
and water R. in the chromatogram obtained with reference solution (c)
Reference solution (c). Dilute 1.0 mL of reference solution (b) (3.5 per cent) ;
to 10.0 mL with a mixture of equal volumes of acetonitrile R1 – disregard limit : 0.2 times the area of the principal peak in
and water R. the chromatogram obtained with reference solution (c)
Reference solution (d). Dissolve 15.0 mg of clarithromycin for (0.1 per cent) ; disregard the peaks eluting before impurity I
peak identification CRS in 5.0 mL of acetonitrile R1 and dilute and after impurity H.
to 10.0 mL with water R. Heavy metals (2.4.8) : maximum 20 ppm.
Blank solution. Dilute 25.0 mL of acetonitrile R1 to 50.0 mL Dissolve 1.0 g in a mixture of 15 volumes of water R and
with water R and mix. 85 volumes of dioxan R and dilute to 20 mL with the same
mixture of solvents. 12 mL of the solution complies with
Column :
test B. Prepare the reference solution using lead standard
– size : l = 0.10 m, Ø = 4.6 mm, solution (1 ppm Pb) obtained by diluting lead standard
solution (100 ppm Pb) R with a mixture of 15 volumes of
– stationary phase : octadecylsilyl silica gel for water R and 85 volumes of dioxan R.
chromatography R (3.5 μm),
Water (2.5.12) : maximum 2.0 per cent, determined on 0.500 g.
– temperature : 40 °C.
Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
Mobile phase : 0.5 g.
– mobile phase A : a 4.76 g/L solution of potassium dihydrogen
phosphate R adjusted to pH 4.4 with dilute phosphoric ASSAY
acid R or a 45 g/L solution of potassium hydroxide R, Liquid chromatography (2.2.29) as described in the test for
filtered through a C18 filtration kit, related substances with the following modifications.
– mobile phase B : acetonitrile R1, Injection : test solution and reference solution (a).
Time Mobile phase A Mobile phase B Calculate the percentage content of C38H69NO13.
(min) (per cent V/V) (per cent V/V)
0 - 32 75 → 40 25 → 60 IMPURITIES
32 - 34 40 60
Specified impurities : A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P.

Flow rate : 1.1 mL/min.

Detection : spectrophotometer at 205 nm.
Injection : 10 μL of the blank solution, the test solution and
reference solutions (b), (c) and (d).
Relative retention r (not rG) with reference to clarithromycin
(retention time = about 11 min) : impurity I = about 0.38 ;
impurity A = about 0.42 ; impurity J = about 0.63 ;
impurity L = about 0.74 ; impurity B = about 0.79 ;
impurity M = about 0.81 ; impurity C = about 0.89 ;
impurity D = about 0.96 ; impurity N = about 1.15 ;
impurity E = about 1.27 ; impurity F = about 1.33 ;
A. R1 = CH3, R2 = OH, R3 = H : 2-demethyl-2-
impurity P = about 1.35 ; impurity O = about 1.41 ;
(hydroxymethyl)-6-O-methylerythromycin A
impurity K = about 1.59 ; impurity G = about 1.72 ;
(clarithromycin F),
impurity H = about 1.82.
System suitability :
B. R1 = R2 = R3 = H : 6-O-methyl-15-norerythromycin A,
– symmetry factor : maximum 1.7 for the peak due to
clarithromycin in the chromatogram obtained with
reference solution (b), P. R1 = R3 = CH3, R2 = H : 4′,6-di-O-methylerythromycin A,

General Notices (1) apply to all monographs and other texts 1905
Clazuril for veterinary use EUROPEAN PHARMACOPOEIA 8.0

C. R1 = R2 = CH3, R3 = H : 6-O-methylerythromycin A L. R = H : 6-O-methylerythromycin A (Z)-9-oxime,

(E)-9-oxime, O. R = CH3 : 6-O-methylerythromycin A (Z)-9-(O-
methyloxime),
G. R1 = R2 = R3 = CH3 : 6-O-methylerythromycin A
(E)-9-(O-methyloxime),

J. R1 = CH3, R2 = R3 = H : erythromycin A (E)-9-oxime,

M. R1 = R3 = H, R2 = CH3 : 3″-N-demethyl-6-O-
methylerythromycin A (E)-9-oxime,

N. (10E)-10,11-didehydro-11-deoxy-6-O-methylerythro-
mycin A.

07/2010:1714

CLAZURIL FOR VETERINARY USE

D. R1 = R2 = R3 = H : 3″-N-demethyl-6-O-methylerythro- Clazurilum ad usum veterinarium
mycin A,

E. R1 = R2 = CH3, R3 = H : 6,11-di-O-methylerythromycin A,

F. R1 = R3 = CH3, R2 = H : 6,12-di-O-methylerythromycin A,

H. R1 = CHO, R2 = R3 = H : 3″-N-demethyl-3′-N-formyl-6- C17H10Cl2N4O2 Mr 373.2

O-methylerythromycin A, [101831-36-1]
DEFINITION
(2RS)-[2-Chloro-4-(3,5-dioxo-4,5-dihydro-1,2,4-triazin-
2(3H)-yl)phenyl](4-chlorophenyl)acetonitrile.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or light yellow powder.
Solubility : practically insoluble in water, freely soluble in
dimethylformamide, slightly soluble in ethanol (96 per cent)
I. 3-O-decladinosyl-6-O-methylerythromycin A, and in methylene chloride.
IDENTIFICATION
A. Melting point (2.2.14) : 199 °C to 203 °C.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: clazuril CRS.
TESTS
Related substances. Liquid chromatography (2.2.29).
Solvent mixture : tetrahydrofuran R, water R (50:50 V/V).
Test solution. Dissolve 20.0 mg of the substance to be
K. (1S,2R,5R,6S,7S,8R,9R,11Z)-2-ethyl-6-hydroxy-9- examined in the solvent mixture and dilute to 20.0 mL with
methoxy-1,5,7,9,11,13-hexamethyl-8-[[3,4,6-trideoxy- the solvent mixture.
3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]- Reference solution (a). Dissolve 5 mg of clazuril for system
3,15-dioxabicyclo[10.2.1]pentadeca-11,13-dien-4-one suitability CRS (containing impurities A, B, C, D, E, F, G, H
(3-O-decladinosyl-8,9:10,11-dianhydro-6-O- and I) in the solvent mixture and dilute to 5.0 mL with the
methylerythromycin A-9,12-hemiketal), solvent mixture.

1906 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Clazuril for veterinary use

Reference solution (b). Dilute 1.0 mL of the test solution to ASSAY

100.0 mL with the solvent mixture. Dilute 2.0 mL of this Dissolve about 0.260 g in 35 mL of tetrahydrofuran R and
solution to 10.0 mL with the solvent mixture. add 35 mL of water R. Titrate with 0.1 M sodium hydroxide,
Column : determining the end-point potentiometrically (2.2.20). Carry
– size : l = 0.10 m, Ø = 4.6 mm ; out a blank titration.
– stationary phase : octadecylsilyl silica gel for 1 mL of 0.1 M sodium hydroxide is equivalent to 37.32 mg
chromatography R (3 μm) ; of C17H10Cl2N4O2.
– temperature : 35 °C. STORAGE
Mobile phase : Protected from light.
– mobile phase A : mix 100 volumes of a 7.7 g/L solution of IMPURITIES
ammonium acetate R adjusted to pH 6.2 with a 10 per
cent V/V solution of anhydrous formic acid R, 150 volumes Specified impurities : A, B, C, D, E, F, G, H, I.
of acetonitrile R and 750 volumes of water R ;
– mobile phase B : mix 50 volumes of water R, 100 volumes
of a 7.7 g/L solution of ammonium acetate R adjusted to
pH 6.2 with a 10 per cent V/V solution of anhydrous formic
acid R and 850 volumes of acetonitrile R ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 20 100 → 0 0 → 100 A. (2RS)-[2-chloro-4-(3,5-dioxo-4,5-dihydro-1,2,4-triazin-
20 - 25 0 100
2(3H)-yl)phenyl](4-chlorophenyl)acetic acid,

Flow rate : 1.0 mL/min.

Detection : spectrophotometer at 230 nm.
Injection : 5 μL.
Identification of impurities : use the chromatogram supplied
with clazuril for system suitability CRS and the chromatogram
obtained with reference solution (a) to identify the peaks due
to impurities A, B, C, D, E, F, G, H and I.
Relative retention with reference to clazuril (retention B. 2-[3-chloro-4-[(RS)-(4-chlorophenyl)cyanometh-
time = about 16 min) : impurity A = about 0.6 ; yl]phenyl]-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-tria-
impurity B = about 0.78 ; impurity C = about 0.80 ; zine-6-carboxamide,
impurity D = about 0.86 ; impurity E = about 0.9 ;
impurity F = about 0.95 ; impurity G = about 0.98 ;
impurity H = about 1.1 ; impurity I = about 1.2.
System suitability: reference solution (a) :
– peak-to-valley ratio : minimum 1.5, where Hp = height above
the baseline of the peak due to impurity G and Hv = height
above the baseline of the lowest point of the curve
separating this peak from the peak due to clazuril, C. (2RS)-2-[2-chloro-4-(3,5-dioxo-4,5-dihydro-1,2,4-triazin-
– the chromatogram obtained is similar to the chromatogram 2(3H)-yl)phenyl]-2-(4-chlorophenyl)acetamide,
supplied with clazuril for system suitability CRS.
Limits :
– correction factors : for the calculation of contents,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity G = 1.4 ;
impurity H = 0.8 ;
– impurities A, B, C, D, E, F, G, H, I : for each impurity,
not more than the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.2 per
cent) ; D. 2-[3-chloro-4-[(RS)-(4-chlorophenyl)cyanometh-
– unspecified impurities : for each impurity, not more than the yl]phenyl]-N,N-dimethyl-3,5-dioxo-2,3,4,5-tetra-
area of the principal peak in the chromatogram obtained hydro-1,2,4-triazine-6-carboxamide,
with reference solution (b) (0.20 per cent) ;
– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.6 per cent) ;
– disregard limit : 0.25 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent) ; disregard the peaks due to the solvents.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 4 h. E. methyl 2-[3-chloro-4-[(RS)-(4-chlorophenyl)cyano-
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on methyl]phenyl]-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-tria-
1.0 g. zine-6-carboxylate,

General Notices (1) apply to all monographs and other texts 1907
Clebopride malate EUROPEAN PHARMACOPOEIA 8.0

IDENTIFICATION
First identification : B, C.
Second identification : A, C, D.
A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Dissolve 20.0 mg in water R and dilute to
100.0 mL with the same solvent. Dilute 10.0 mL of the
F. ethyl 2-[3-chloro-4-[(RS)-(4-chlorophenyl)cyano- solution to 100.0 mL with water R.
methyl]phenyl]-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4- Spectral range : 230-350 nm.
triazine-6-carboxylate, Absorption maxima : at 270 nm and 307 nm.
Specific absorbance at the absorption maxima :
– at 270 nm : 252 to 278 ;
– at 307 nm : 204 to 226.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: clebopride malate CRS.
C. Dissolve 20 mg in 1 mL of sulfuric acid R, add 1 mL of
G. 2-[3-chloro-4-(4-chlorobenzoyl)phenyl]-1,2,4-triazine- β-naphthol solution R1 and mix. The solution examined in
3,5(2H,4H)-dione, daylight is yellow with blue fluorescence.
D. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 5 mg of the substance to be
examined in anhydrous ethanol R and dilute to 10 mL with
the same solvent.
Reference solution (a). Dissolve 5 mg of clebopride
malate CRS in anhydrous ethanol R and dilute to 10 mL
with the same solvent.
Reference solution (b). Dissolve 5 mg of clebopride
malate CRS and 5 mg of metoclopramide hydrochloride CRS
H. [2-chloro-4-(3,5-dioxo-4,5-dihydro-1,2,4-triazin-2(3H)- in anhydrous ethanol R and dilute to 10 mL with the same
yl)phenyl][4-[[2-chloro-4-(3,5-dioxo-4,5-dihydro- solvent.
1,2,4-triazin-2(3H)-yl)phenyl]cyanomethyl]phenyl](4- Plate : TLC silica gel F254 plate R.
chlorophenyl)acetonitrile, Mobile phase : concentrated ammonia R, acetone R,
methanol R, toluene R (2:14:14:70 V/V/V/V).
Application : 5 μL as bands of 10 mm by 3 mm.
Development : over 3/4 of the plate.
Drying : in air.
Detection : examine in ultraviolet light at 254 nm.
System suitability : reference solution (b) :
I. (Z)-2-[[3-chloro-4-[(RS)-(4-chlorophenyl)cyanometh- – the chromatogram shows 2 clearly separated zones.
yl]phenyl]diazanylidene]acetamide. Results : the principal zone in the chromatogram obtained
with the test solution is similar in position and size to
the principal zone in the chromatogram obtained with
01/2011:1303 reference solution (a).
TESTS
CLEBOPRIDE MALATE Solution S. Dissolve 1.0 g in carbon dioxide-free water R and
dilute to 100 mL with the same solvent.
Clebopridi malas Appearance of solution. Solution S, examined immediately
after preparation, is clear (2.2.1) and colourless (2.2.2,
Method I).
pH (2.2.3) : 3.8 to 4.2 for solution S.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.100 g of the substance to be examined
C24H30ClN3O7 Mr 508.0 in the mobile phase and dilute to 100.0 mL with the mobile
[57645-91-7] phase.
Reference solution (a). Dilute 1.0 mL of the test solution to
DEFINITION 100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
4-Amino-N-(1-benzylpiperidin-4-yl)-5-chloro-2-methoxy- to 10.0 mL with the mobile phase.
benzamide acid (RS)-2-hydroxybutanedioate. Reference solution (b). Dissolve 10 mg of the substance to be
Content : 98.5 per cent to 101.0 per cent (dried substance). examined and 10 mg of metoclopramide hydrochloride CRS
in the mobile phase and dilute to 100.0 mL with the mobile
CHARACTERS phase. Dilute 1.0 mL of the solution to 10.0 mL with the
Appearance : white or almost white, crystalline powder. mobile phase.
Solubility : sparingly soluble in water and in methanol, Column :
slightly soluble in anhydrous ethanol, practically insoluble – size : l = 0.12 m, Ø = 4.0 mm ;
in methylene chloride. – stationary phase : octadecylsilyl silica gel for
mp : about 164 °C, with decomposition. chromatography R (5 μm).

1908 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Clemastine fumarate

Mobile phase : mix 20 volumes of acetonitrile R1 and STORAGE

80 volumes of a 1 g/L solution of sodium heptanesulfonate R Protected from light.
adjusted to pH 2.5 with phosphoric acid R.
Flow rate : 1 mL/min. IMPURITIES
Detection : spectrophotometer at 215 nm. Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
Injection : 20 μL. the tests in the monograph. They are limited by the general
Run time : twice the retention time of clebopride. acceptance criterion for other/unspecified impurities and/or
Relative retention with reference to clebopride (retention by the general monograph Substances for pharmaceutical use
time = about 15 min) : metoclopramide = about 0.45. (2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
System suitability : reference solution (b) :
impurities in substances for pharmaceutical use) : A, B, C.
– resolution : minimum 5.0 between the peaks due to
metoclopramide and clebopride.
Limits :
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained A. 4-amino-5-chloro-2-methoxybenzoic acid,
with reference solution (a) (0.10 per cent) ;
– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.3 per cent) ;
B. 1-benzylpiperidin-4-amine,
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent) ; disregard the 2 peaks eluting within the
first 2 min.
Chlorides : maximum 100 ppm.
Prepare the solutions at the same time. C. 4-amino-N-(1-benzylpiperidin-4-yl)-2-methoxybenza-
Test solution. Dissolve 0.530 g in 20.0 mL of anhydrous acetic mide.
acid R, add 6 mL of dilute nitric acid R and dilute to 50.0 mL
with water R. 01/2008:1190
Reference solution. To 1.5 mL of 0.001 M hydrochloric acid corrected 6.1
add 20.0 mL of anhydrous acetic acid R and 6 mL of dilute
nitric acid R and dilute to 50.0 mL with water R. CLEMASTINE FUMARATE
Transfer both recently prepared solutions to separate
test-tubes. Add to each tube 1 mL of silver nitrate solution R2. Clemastini fumaras
Allow to stand for 5 min protected from light. Examine the
tubes laterally against a black background. Any opalescence in
the test solution is not more intense than that in the reference
solution.
Sulfates : maximum 100 ppm.
Prepare the solutions at the same time.
Test solution. Dissolve 3.00 g in 20.0 mL of glacial acetic C25H30ClNO5 Mr 460.0
acid R, heating gently if necessary. Allow to cool and dilute to [14976-57-9]
50.0 mL with water R.
DEFINITION
Reference solution. To 9 mL of sulfate standard solution
(2R)-2-[2-[(R)-1-(4-Chlorophenyl)-1-phenylethoxy]ethyl]-1-
(10 ppm SO4) R1 add 6 mL of glacial acetic acid R.
methylpyrrolidine (E)-butenedioate.
Into 2 test-tubes introduce 1.5 mL of sulfate standard solution Content : 98.5 per cent to 101.0 per cent (dried substance).
(10 ppm SO4) R1 and add 1 mL of a 250 g/L solution of barium
chloride R. Shake and allow to stand for 1 min. To one of the CHARACTERS
tubes add 15 mL of the test solution and to the other add Appearance : white or almost white, crystalline powder.
15 mL of the reference solution. After 5 min, any opalescence
Solubility : very slightly soluble in water, sparingly soluble in
in the tube containing the test solution is not more intense
ethanol (70 per cent V/V), slightly soluble in ethanol (50 per
than that in the tube containing the reference solution.
cent V/V) and in methanol.
Heavy metals (2.4.8) : maximum 20 ppm.
IDENTIFICATION
1.0 g complies with test D. Prepare the reference solution
using 2 mL of lead standard solution (10 ppm Pb) R. First identification : A, B.
Second identification : A, C, D.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C. A. Specific optical rotation (see Tests).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on B. Infrared absorption spectrophotometry (2.2.24).
1.0 g. Comparison: clemastine fumarate CRS.
C. Examine the chromatograms obtained in the test for
ASSAY related substances.
Dissolve 0.400 g in 50 mL of anhydrous acetic acid R. Titrate Results : the principal spot in the chromatogram obtained
with 0.1 M perchloric acid, determining the end-point with test solution (b) is similar in position, colour and size
potentiometrically (2.2.20). to the principal spot in the chromatogram obtained with
1 mL of 0.1 M perchloric acid is equivalent to 50.80 mg reference solution (a).
of C24H30ClN3O7. D. Thin-layer chromatography (2.2.27).

General Notices (1) apply to all monographs and other texts 1909
Clemastine fumarate EUROPEAN PHARMACOPOEIA 8.0

Test solution. Dissolve 40 mg of the substance to be – disregard limit : disregard any spot remaining at the point
examined in methanol R and dilute to 2 mL with the same of application (fumaric acid).
solvent. Impurity C. Liquid chromatography (2.2.29).
Reference solution. Dissolve 50 mg of fumaric acid CRS in Solvent mixture : acetonitrile R1, 10 g/L solution of ammonium
ethanol (96 per cent) R and dilute to 10 mL with the same dihydrogen phosphate R (25:75 V/V).
solvent.
Test solution. Dissolve 20 mg of the substance to be examined
Plate : TLC silica gel G plate R.
in the solvent mixture and dilute to 100 mL with the solvent
Mobile phase : water R, anhydrous formic acid R, di-isopropyl mixture.
ether R (5:25:70 V/V/V).
Reference solution (a). Dissolve 6 mg of 1-(4-chlorophenyl)-1-
Application : 5 μL. phenylethanol CRS (impurity C) in the solvent mixture and
Development : over a path of 15 cm. dilute to 100 mL with the solvent mixture.
Drying : at 100-105 °C for 30 min and allow to cool. Reference solution (b). Dilute 1 mL of reference solution (a) to
Detection : spray with a 16 g/L solution of potassium 100 mL with the solvent mixture.
permanganate R and examine in daylight. Reference solution (c). Dissolve 10 mg of the substance to be
Results : the spot with the highest RF value in the examined in the solvent mixture and dilute to 100 mL with the
chromatogram obtained with the test solution is similar solvent mixture. To 1 mL of this solution add 1 mL of reference
in position, colour and size to the principal spot in the solution (a) and dilute to 100 mL with the solvent mixture.
chromatogram obtained with the reference solution. Column :
TESTS – size : l = 0.1 m, Ø = 4.6 mm ;
Solution S. Dissolve 0.500 g in methanol R and dilute to – stationary phase : octadecylsilyl silica gel for
50.0 mL with the same solvent. chromatography R (5 μm).
Appearance of solution. Solution S is clear (2.2.1) and not Mobile phase : phosphoric acid R, acetonitrile R1, 10 g/L solution
more intensely coloured than reference solution BY7 (2.2.2, of ammonium dihydrogen phosphate R (0.1:45:55 V/V/V).
Method II). Flow rate : 1 mL/min.
pH (2.2.3) : 3.2 to 4.2. Detection : spectrophotometer at 220 nm.
Suspend 1.0 g in 10 mL of carbon dioxide-free water R. Injection : 100 μL.
Specific optical rotation (2.2.7) : + 15.0 to + 18.0 (dried System suitability : reference solution (c) :
substance), determined on solution S. – resolution : minimum 2.2 between the peaks due to
Related substances. Thin-layer chromatography (2.2.27). clemastine and impurity C.
Test solution (a). Dissolve 0.100 g of the substance to be Limit :
examined in methanol R and dilute to 5.0 mL with the same – impurity C : not more than the area of the principal peak
solvent. in the chromatogram obtained with reference solution (b)
Test solution (b). Dilute 1.0 mL of test solution (a) to 10.0 mL (0.3 per cent).
with methanol R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Reference solution (a). Dissolve 20.0 mg of clemastine on 1.000 g by drying in an oven at 105 °C for 6 h.
fumarate CRS in methanol R and dilute to 10.0 mL with the
same solvent. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
Reference solution (b). Dilute 1.5 mL of test solution (b) to
50.0 mL with methanol R. ASSAY
Reference solution (c). Dilute 0.5 mL of test solution (b) to Dissolve 0.350 g in 60 mL of anhydrous acetic acid R. Titrate
50.0 mL with methanol R. with 0.1 M perchloric acid, determining the end-point
Reference solution (d). Dissolve 10.0 mg of diphenhydramine potentiometrically (2.2.20).
hydrochloride CRS in 5.0 mL of reference solution (a). 1 mL of 0.1 M perchloric acid is equivalent to 46.00 mg
Plate : TLC silica gel G plate R. of C25H30ClNO5.
Mobile phase : concentrated ammonia R, methanol R,
tetrahydrofuran R (1:20:80 V/V/V). IMPURITIES
Application : 5 μL. Specified impurities : A, B, C.
Development : over a path of 15 cm. Other detectable impurities (the following substances would,
Drying : in a current of cold air for 5 min. if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
Detection : spray with a freshly prepared mixture of 1 volume acceptance criterion for other/unspecified impurities and/or
of potassium iodobismuthate solution R and 10 volumes of by the general monograph Substances for pharmaceutical
dilute acetic acid R and then with dilute hydrogen peroxide use (2034). It is therefore not necessary to identify these
solution R ; cover the plate immediately with a glass plate of impurities for demonstration of compliance. See also 5.10.
the same size and examine the chromatograms after 2 min. Control of impurities in substances for pharmaceutical use) : D.
System suitability : reference solution (d) :
– the chromatogram shows 2 clearly separated spots.
Limits : test solution (a) :
– any impurity : any spot, apart from the principal spot, is not
more intense than the principal spot in the chromatogram
obtained with reference solution (b) (0.3 per cent) and at
most 4 such spots are more intense than the principal spot
in the chromatogram obtained with reference solution (c) A. (1RS,2R)-2-[2-[(R)-1-(4-chlorophenyl)-1-phenylethoxy]-
(0.1 per cent) ; ethyl]-1-methylpyrrolidine 1-oxide,

1910 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Clenbuterol hydrochloride

Results : the principal spot in the chromatogram obtained

with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
the reference solution.
C. It gives reaction (a) of chlorides (2.3.1).

B. 4-[1-(4-chlorophenyl)-1-phenylethoxy]-1-methylazepane, TESTS
Solution S. Dissolve 0.5 g in 10 mL of carbon dioxide-free
water R.
Appearance of solution. Solution S is not more opalescent
than reference suspension II (2.2.1) and not more intensely
coloured than reference solution Y6 (2.2.2, Method II).
pH (2.2.3) : 5.0 to 7.0 for solution S.
C. (RS)-1-(4-chlorophenyl)-1-phenylethanol, Optical rotation (2.2.7) : − 0.10° to + 0.10°.
Dissolve 0.30 g in water R and dilute to 10.0 mL with the same
solvent. Filter if necessary.
Related substances. Liquid chromatography (2.2.29).
Test solution. Disperse 100.0 mg of the substance to be
D. 2-[(2RS)-1-methylpyrrolidin-2-yl]ethanol. examined in the mobile phase and dilute to 50.0 mL with the
mobile phase.
Reference solution (a). Dilute 0.1 mL of the test solution to
01/2008:1409 100.0 mL with water R.
Reference solution (b). Dissolve 5 mg of clenbuterol
CLENBUTEROL HYDROCHLORIDE impurity B CRS in 10 mL of the mobile phase, add 2.5 mL of
the test solution and dilute to 25.0 mL with the mobile phase.
Clenbuteroli hydrochloridum Column :
– size : l = 0.125 m, Ø = 4 mm,
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm),
– temperature : 40 °C.
Mobile phase : mix 200 volumes of acetonitrile R, 200 volumes
of methanol R and 600 volumes of a solution prepared as
C12H19Cl3N2O Mr 313.7 follows : dissolve 3.0 g of sodium decanesulfonate R and 5.0 g
[21898-19-1] of potassium dihydrogen phosphate R in 900 mL of water R,
adjust to pH 3.0 with dilute phosphoric acid R and dilute to
DEFINITION 1000 mL with water R.
(1RS)-1-(4-Amino-3,5-dichlorophenyl)-2-[(1,1- Flow rate : 0.5 mL/min.
dimethylethyl)amino]ethanol hydrochloride. Detection : spectrophotometer at 215 nm.
Content : 99.0 per cent to 101.0 per cent (anhydrous substance). Injection : 5 μL.
CHARACTERS Run time : 1.5 times the retention time of clenbuterol.
Retention time : clenbuterol = about 29 min.
Appearance : white or almost white, crystalline powder.
System suitability : reference solution (b) :
Solubility : soluble in water and in ethanol (96 per cent),
slightly soluble in acetone. – resolution : minimum 4.0 between the peaks due to
impurity B and clenbuterol.
mp : about 173 °C, with decomposition.
Limits :
IDENTIFICATION – impurities A, B, C, D, E, F : for each impurity, not more
First identification : A, C. than the area of the principal peak in the chromatogram
Second identification : B, C. obtained with reference solution (a) (0.1 per cent),
– any other impurity : for each impurity, not more than the
A. Infrared absorption spectrophotometry (2.2.24).
area of the principal peak in the chromatogram obtained
Comparison : clenbuterol hydrochloride CRS. with reference solution (a) (0.1 per cent),
B. Thin-layer chromatography (2.2.27). – total : not more than twice the area of the principal peak
Test solution. Dissolve 10 mg of the substance to be in the chromatogram obtained with reference solution (a)
examined in 10 mL of methanol R. (0.2 per cent),
Reference solution. Dissolve 10 mg of clenbuterol – disregard limit : 0.5 times the area of the principal peak in
hydrochloride CRS in 10 mL of methanol R. the chromatogram obtained with reference solution (a)
Plate : TLC silica gel F254 plate R. (0.05 per cent).
Mobile phase : ammonia R, anhydrous ethanol R, toluene R Water (2.5.12) : maximum 1.0 per cent, determined on 0.500 g.
(0.15:10:15 V/V/V). Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Application : 10 μL. 1.0 g.
Development : over a path of 10 cm. ASSAY
Drying : in air. Dissolve 0.250 g in 50 mL of ethanol (96 per cent) R and add
Detection : spray with a 10 g/L solution of sodium nitrite R 5.0 mL of 0.01 M hydrochloric acid. Titrate with 0.1 M sodium
in 1 M hydrochloric acid and dip after 10 min in a 4 g/L hydroxide, determining the end-point potentiometrically
solution of naphthylethylenediamine dihydrochloride R in (2.2.20). Read the volume added between the 2 points of
methanol R. Allow to dry in air. inflexion.

General Notices (1) apply to all monographs and other texts 1911
Clindamycin hydrochloride EUROPEAN PHARMACOPOEIA 8.0

1 mL of 0.1 M sodium hydroxide is equivalent to 31.37 mg Reference solution (a). Dissolve 10 mg of clindamycin
of C12H19Cl3N2O. hydrochloride CRS in methanol R and dilute to 10 mL with
the same solvent.
IMPURITIES
Reference solution (b). Dissolve 10 mg of clindamycin
Specified impurities : A, B, C, D, E, F. hydrochloride CRS and 10 mg of lincomycin
hydrochloride CRS in methanol R and dilute to
10 mL with the same solvent.
Plate : TLC silica gel G plate R.
Mobile phase : mix 19 volumes of 2-propanol R, 38 volumes
of a 150 g/L solution of ammonium acetate R adjusted to
pH 9.6 with ammonia R, and 43 volumes of ethyl acetate R.
A. R1 = H, R2 = Cl : 4-amino-3,5-dichlorobenzaldehyde, Application : 5 μL.
B. R1 = CH2-NH-C(CH3)3, R2 = Cl : 1-(4-amino-3,5- Development : over a path of 15 cm using the upper layer
dichlorophenyl)-2-[(1,1-dimethylethyl)amino]ethanone, of the mobile phase.
C. R1 = CH3, R2 = Cl : 1-(4-amino-3,5-dichlorophenyl)ethan- Drying : in air.
one, Detection : spray with a 1 g/L solution of potassium
permanganate R.
D. R1 = CH3, R2 = H : 1-(4-aminophenyl)ethanone,
System suitability : the chromatogram obtained with
E. R1 = CH2Br, R2 = Cl : 1-(4-amino-3,5-dichlorophenyl)-2- reference solution (b) shows 2 clearly separated spots.
bromoethanone, Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
reference solution (a).
C. Dissolve about 10 mg in 2 mL of dilute hydrochloric acid R
and heat on a water-bath for 3 min. Add 3 mL of sodium
carbonate solution R and 1 mL of a 20 g/L solution of
F. (1RS)-1-(4-amino-3-bromo-5-chlorophenyl)-2-[(1,1- sodium nitroprusside R. A violet-red colour develops.
dimethylethyl)amino]ethanol. D. Dissolve 0.1 g in water R and dilute to 10 mL with the same
solvent. The solution gives reaction (a) of chlorides (2.3.1).
01/2008:0582
corrected 6.0 TESTS
pH (2.2.3) : 3.0 to 5.0.
CLINDAMYCIN HYDROCHLORIDE Dissolve 1.0 g in carbon dioxide-free water R and dilute to
10 mL with the same solvent.
Clindamycini hydrochloridum Specific optical rotation (2.2.7) : + 135 to + 150 (anhydrous
substance).
Dissolve 1.000 g in water R and dilute to 25.0 mL with the
same solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with the
mobile phase.
Reference solution (a). Dissolve 50.0 mg of clindamycin
C18H34Cl2N2O5S Mr 461.5 hydrochloride CRS in the mobile phase and dilute to 50.0 mL
[21462-39-5] with the mobile phase.
DEFINITION Reference solution (b). Dilute 2.0 mL of the test solution to
100.0 mL with the mobile phase.
Methyl 7-chloro-6,7,8-trideoxy-6-[[[(2S,4R)-1-methyl-4-
propylpyrrolidin-2-yl]carbonyl]amino]-1-thio-L-threo-α-D- Column :
galacto-octopyranoside hydrochloride. It contains a variable – size : l = 0.25 m, Ø = 4.6 mm,
quantity of water. – stationary phase : octadecylsilyl silica gel for
Semi-synthetic product derived from a fermentation product. chromatography R (5 μm).
Content : 91.0 per cent to 102.0 per cent (anhydrous substance). Mobile phase : mix 45 volumes of acetonitrile R and 55 volumes
of a 6.8 g/L solution of potassium dihydrogen phosphate R
CHARACTERS adjusted to pH 7.5 with a 250 g/L solution of potassium
Appearance : white or almost white, crystalline powder. hydroxide R.
Solubility : very soluble in water, slightly soluble in ethanol Flow rate : 1 mL/min.
(96 per cent). Detection : spectrophotometer at 210 nm.
Injection : 20 μL.
IDENTIFICATION
Run time : twice the retention time of clindamycin.
First identification : A, D.
System suitability : reference solution (a) :
Second identification : B, C, D. – relative retention with reference to clindamycin
A. Infrared absorption spectrophotometry (2.2.24). (retention time = about 10 min) : impurity A = about 0.4 ;
Comparison : clindamycin hydrochloride CRS. impurity B = about 0.65 ; impurity C = about 0.8.
B. Thin-layer chromatography (2.2.27). Limits :
Test solution. Dissolve 10 mg of the substance to be – impurity B : not more than the area of the principal peak
examined in methanol R and dilute to 10 mL with the same in the chromatogram obtained with reference solution (b)
solvent. (2.0 per cent),

1912 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Clindamycin phosphate

– impurity C : not more than twice the area of the principal DEFINITION
peak in the chromatogram obtained with reference Methyl 7-chloro-6,7,8-trideoxy-6-[[[(2S,4R)-1-methyl-4-
solution (b) (4.0 per cent), propylpyrrolidin-2-yl]carbonyl]amino]-1-thio-L-threo-α-D-
– any other impurity : not more than 0.5 times the area of galacto-octopyranoside 2-(dihydrogen phosphate).
the principal peak in the chromatogram obtained with Semi-synthetic product derived from a fermentation product.
reference solution (b) (1.0 per cent), Content : 95.0 per cent to 102.0 per cent (anhydrous substance).
– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b) CHARACTERS
(6.0 per cent), Appearance : white or almost white, slightly hygroscopic
– disregard limit : 0.025 times the area of the principal peak powder.
in the chromatogram obtained with reference solution (b) Solubility : freely soluble in water, very slightly soluble in
(0.05 per cent). ethanol (96 per cent), practically insoluble in methylene
Water (2.5.12) : 3.0 per cent to 6.0 per cent, determined on chloride.
0.500 g. It shows polymorphism (5.9).
Sulfated ash (2.4.14) : maximum 0.5 per cent, determined on IDENTIFICATION
1.0 g. First identification : A, D.
ASSAY Second identification : B, C, D.
Liquid chromatography (2.2.29) as described in the test for A. Infrared absorption spectrophotometry (2.2.24).
related substances with the following modifications. Preparation : discs of potassium bromide R.
Injection : 20 μL of the test solution and reference solution (a). In 2 separate tubes place 50 mg of the substance to be
System suitability : examined and 50 mg of clindamycin phosphate CRS. Add
0.2 mL of water R and heat until completely dissolved.
– repeatability : maximum relative standard deviation of Evaporate to dryness under reduced pressure and dry the
0.85 per cent after 6 injections of reference solution (a). residues at 100-105 °C for 2 h.
STORAGE Comparison: clindamycin phosphate CRS.
In an airtight container. B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 20 mg of the substance to be
IMPURITIES examined in methanol R and dilute to 10 mL with the same
solvent.
Reference solution (a). Dissolve 20 mg of clindamycin
phosphate CRS in methanol R and dilute to 10 mL with the
same solvent.
Reference solution (b). Dissolve 10 mg of lincomycin
hydrochloride CRS in 5 mL of reference solution (a).
Plate : TLC silica gel plate R.
A. R1 = CH2-CH2-CH3, R2 = OH, R3 = H : methyl Mobile phase : glacial acetic acid R, water R, butanol R
6,8-dideoxy-6-[[[(2S,4R)-1-methyl-4-propylpyrrolidin- (20:20:60 V/V/V).
2-yl]carbonyl]amino]-1-thio-D-erythro-α-D-galacto- Application : 5 μL.
octopyranoside (lincomycin), Development : over a path of 12 cm.
B. R1 = C2H5, R2 = H, R3 = Cl : methyl 7-chloro-6,7,8- Drying : at 100-105 °C for 30 min.
trideoxy-6-[[[(2S,4R)-4-ethyl-1-methylpyrrolidin- Detection : spray with a 1 g/L solution of potassium
2-yl]carbonyl]amino]-1-thio-L-threo-α-D-galacto- permanganate R.
octopyranoside (clindamycin B), System suitability : reference solution (b) :
C. R1 = CH2-CH2-CH3, R2 = Cl, R3 = H : methyl – the chromatogram shows 2 principal spots.
7-chloro-6,7,8-trideoxy-6-[[[(2S,4R)-1-methyl-4- Results : the principal spot in the chromatogram obtained
propylpyrrolidin-2-yl]carbonyl]amino]-1-thio-D-erythro- with the test solution is similar in position, colour and size
α-D-galacto-octopyranoside (7-epiclindamycin). to the principal spot in the chromatogram obtained with
reference solution (a).
C. Dissolve about 10 mg in 2 mL of dilute hydrochloric acid R
01/2008:0996 and heat in a water-bath for 3 min. Add 4 mL of sodium
corrected 6.0 carbonate solution R and 1 mL of a 20 g/L solution of
sodium nitroprusside R. Prepare a standard in the same
CLINDAMYCIN PHOSPHATE manner using clindamycin phosphate CRS. The colour of
the test solution corresponds to that of the standard.
Clindamycini phosphas D. Boil 0.1 g under a reflux condenser with a mixture of 5 mL
of strong sodium hydroxide solution R and 5 mL of water R
for 90 min. Cool and add 5 mL of nitric acid R. Extract with
3 quantities, each of 15 mL, of methylene chloride R and
discard the extracts. Filter the upper layer through a paper
filter. The filtrate gives reaction (b) of phosphates (2.3.1).
TESTS
Solution S. Dissolve 1.00 g in carbon dioxide-free water R.
Heat gently if necessary. Cool and dilute to 25.0 mL with
carbon dioxide-free water R.
C18H34ClN2O8PS Mr 505.0 Appearance of the solution. Solution S is clear (2.2.1) and
[24729-96-2] colourless (2.2.2, Method II).

General Notices (1) apply to all monographs and other texts 1913
Clioquinol EUROPEAN PHARMACOPOEIA 8.0

pH (2.2.3) : 3.5 to 4.5. Calculate the percentage content of C18H34ClN2O8PS from the
Dilute 5.0 mL of solution S to 20 mL with carbon dioxide-free declared content of clindamycin phosphate CRS.
water R. STORAGE
Specific optical rotation (2.2.7) : + 115 to + 130 (anhydrous In an airtight container, at a temperature not exceeding
substance). 30 °C. If the substance is sterile, store in a sterile, airtight,
Dissolve 0.250 g in water R and dilute to 25.0 mL with the tamper-proof container.
same solvent.
IMPURITIES
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 75.0 mg of the substance to be
examined in the mobile phase and dilute to 25.0 mL with the
mobile phase.
Reference solution (a). Dissolve 75.0 mg of clindamycin
phosphate CRS in the mobile phase and dilute to 25.0 mL with
the mobile phase.
Reference solution (b). Dissolve 5.0 mg of lincomycin
hydrochloride CRS (impurity A) and 15.0 mg of clindamycin A. methyl 6,8-dideoxy-6-[[[(2S,4R)-1-methyl-4-
hydrochloride CRS (impurity E) in 5.0 mL of reference propylpyrrolidin-2-yl]carbonyl]amino]-1-thio-D-erythro-
solution (a), then dilute to 100.0 mL with the mobile phase. α-D-galacto-octopyranoside (lincomycin),
Reference solution (c). Dilute 1.0 mL of reference solution (a)
to 100.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octylsilyl silica gel for chromatography R
(5-10 μm).
Mobile phase : mix 200 mL of acetonitrile R1 and 800 mL
B. R1 = PO3H2, R2 = R3 = H, R4 = C2H5 : clindamycin B
of a 13.6 g/L solution of potassium dihydrogen phosphate R
2-(dihydrogen phosphate),
previously adjusted to pH 2.5 with phosphoric acid R.
Flow rate : 1.0 mL/min. C. R1 = R3 = H, R2 = PO3H2, R4 = C3H7 : clindamycin
3-(dihydrogen phosphate),
Detection : spectrophotometer at 210 nm.
Injection : 20 μL of the test solution and reference solutions (b) D. R1 = R2 = H, R3 = PO3H2, R4 = C3H7 : clindamycin
and (c). 4-(dihydrogen phosphate),
Run time : the retention time of impurity E. E. R1 = R2 = R3 = H, R4 = C3H7 : clindamycin.
System suitability : reference solution (b) :
– resolution : minimum 6.0 between the peaks due to 01/2008:2111
clindamycin phosphate (2nd peak) and impurity E
(3rd peak) ; if necessary, adjust the concentration of CLIOQUINOL
acetonitrile in the mobile phase ;
– symmetry factor : maximum 1.5 for the peak due to Clioquinolum
clindamycin phosphate ;
– the peak due to impurity A (1st peak) is clearly separated
from the peak due to the solvent.
Limits :
– any impurity : for each impurity, not more than 2.5 times
the area of the peak due to clindamycin phosphate in the
chromatogram obtained with reference solution (c) (2.5 per C9H5ClINO Mr 305.5
cent) ; [130-26-7]
– total : not more than 4 times the area of the peak due to
DEFINITION
clindamycin phosphate in the chromatogram obtained with
reference solution (c) (4.0 per cent) ; 5-Chloro-7-iodoquinolin-8-ol.
– disregard limit : 0.1 times the area of the principal peak in Content : 98.0 per cent to 102.0 per cent (dried substance).
the chromatogram obtained with reference solution (c)
CHARACTERS
(0.1 per cent).
Appearance : almost white, light yellow, brownish-yellow or
Water (2.5.12) : maximum 6.0 per cent, determined on 0.250 g. yellowish-grey powder.
Bacterial endotoxins (2.6.14) : less than 0.6 IU/mg, if intended Solubility : practically insoluble in water, sparingly soluble in
for use in the manufacture of parenteral preparations without methylene chloride, very slightly soluble or slightly soluble in
a further appropriate procedure for removal of bacterial ethanol (96 per cent).
endotoxins.
IDENTIFICATION
ASSAY
First identification : B.
Liquid chromatography (2.2.29) as described in the test for
Second identification : A, C, D.
related substances with the following modifications.
A. Dissolve 40.0 mg in methanol R and dilute to 100.0 mL
Injection : the test solution and reference solution (a). with the same solvent. Dilute 10.0 mL to 100.0 mL with
System suitability: reference solution (a) : methanol R (solution A). Examined between 280 nm and
– repeatability : maximum relative standard deviation of 350 nm (2.2.25), solution A shows an absorption maximum
1.0 per cent after 6 injections ; if necessary, adjust the at 321 nm. Dilute 10.0 mL of solution A to 100.0 mL
integrator parameters. with methanol R (solution B). Examined between 230 nm

1914 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Clobazam

and 280 nm, solution B shows an absorption maximum – disregard limit : the area of the principal peak in the
at 255 nm. The specific absorbance at this absorption chromatogram obtained with reference solution (c)
maximum is 1530 to 1660. (0.05 per cent).
B. Infrared absorption spectrophotometry (2.2.24). Halides : maximum 140 ppm, expressed as chlorides.
Preparation : discs of potassium bromide R. Shake 0.5 g with 25 mL of water R for 1 min and filter. To the
Comparison : clioquinol CRS. filtrate add 0.5 mL of dilute nitric acid R and 0.5 mL of silver
nitrate solution R2. Allow to stand for 5 min. Any opalescence
C. When heated, violet fumes are produced.
is not more intense than that in a standard prepared at the
D. Dissolve about 1 mg in 5 mL of ethanol (96 per cent) R. same time by adding 0.5 mL of silver nitrate solution R2 to
Add 0.05 mL of ferric chloride solution R1. A dark green 25 mL of water R containing 0.2 mL of 0.01 M hydrochloric
colour develops. acid and 0.5 mL of dilute nitric acid R.
TESTS Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying over diphosphorus pentoxide R at a
Acidity or alkalinity. Shake 0.5 g with 10 mL of carbon
pressure not exceeding 0.7 kPa for 24 h.
dioxide-free water R and filter. To the filtrate add 0.2 mL of
phenolphthalein solution R. The solution is colourless. Not Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
more than 0.5 mL of 0.01 M sodium hydroxide is required to 1.0 g.
change the colour of the indicator to pink.
ASSAY
Related substances. Liquid chromatography (2.2.29).
Dissolve 0.200 g in 20 mL of acetic anhydride R and add 30 mL
Test solution. Dissolve 50.0 mg of the substance to be of glacial acetic acid R. Titrate with 0.1 M perchloric acid,
examined in methanol R and dilute to 50.0 mL with the same determining the end-point potentiometrically (2.2.20).
solvent, heating gently if necessary. Dilute 10.0 mL of the 1 mL of 0.1 M perchloric acid is equivalent to 30.55 mg of total
solution to 25.0 mL with the mobile phase. quinolines, calculated as clioquinol.
Reference solution (a). Dissolve 20.0 mg of 5-chloroquinolin-
8-ol R, 10.0 mg of 5,7-dichloroquinolin-8-ol R, 5 mg STORAGE
of the substance to be examined and 10.0 mg of Protected from light.
5,7-diiodoquinolin-8-ol R in methanol R, heating gently if
necessary and dilute to 20.0 mL with the same solvent. Dilute IMPURITIES
4.0 mL of the solution to 50.0 mL with the mobile phase. Specified impurities : A, B, C.
Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 10.0 mL with the mobile phase.
Reference solution (c). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 20.0 mL with the mobile phase.
Column :
A. R1 = Cl, R2 = H : 5-chloroquinolin-8-ol,
– size : l = 0.15 m, Ø = 3.9 mm,
B. R1 = R2 = Cl : 5,7-dichloroquinolin-8-ol,
– stationary phase : octylsilyl silica gel for chromatography R
(5 μm). C. R1 = R2 = I : 5,7-diiodoquinolin-8-ol.
Mobile phase : dissolve 0.50 g of sodium edetate R in 350 mL
of water R, add 4.0 mL of hexylamine R and mix. Adjust to 01/2008:1974
pH 3.0 with phosphoric acid R. Add 600 mL of methanol R and corrected 6.0
dilute to 1000 mL with water R.
Flow rate : 1.3 mL/min. CLOBAZAM
Detection : spectrophotometer at 254 nm.
Injection : 20 μL.
Clobazamum
Run time : 4 times the retention time of clioquinol.
Relative retention with reference to clioquinol (retention
time = about 10 min) : impurity A = about 0.4 ;
impurity B = about 0.7 ; impurity C = about 1.3.
System suitability: reference solution (a) :
– resolution : minimum 3.0 between the peaks due to
clioquinol and impurity C.
Limits : C16H13ClN2O2 Mr 300.7
– impurity A : not more than the area of the corresponding [22316-47-8]
peak in the chromatogram obtained with reference DEFINITION
solution (b) (2.0 per cent),
7-Chloro-1-methyl-5-phenyl-1,5-dihydro-3H-1,5-
– impurity B : not more than the area of the corresponding benzodiazepine-2,4-dione.
peak in the chromatogram obtained with reference
solution (b) (1.0 per cent), Content : 97.0 per cent to 103.0 per cent (dried substance).
– impurity C : not more than the area of the corresponding CHARACTERS
peak in the chromatogram obtained with reference Appearance : white or almost white, crystalline powder.
solution (b) (1.0 per cent), Solubility : slightly soluble in water, freely soluble in methylene
– unspecified impurities : for each impurity, not more than chloride, sparingly soluble in alcohol.
twice the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.10 per cent), IDENTIFICATION
– total of the nominal contents of impurities A, B, C and Infrared absorption spectrophotometry (2.2.24).
unspecified impurities : maximum 3.0 per cent, Comparison: Ph. Eur. reference spectrum of clobazam.

General Notices (1) apply to all monographs and other texts 1915
Clobetasol propionate EUROPEAN PHARMACOPOEIA 8.0

TESTS C. R1 = R3 = CH3, R2 = Cl, R4 = H : (3RS)-7-chloro-1,3-

Related substances. Liquid chromatography (2.2.29). dimethyl-5-phenyl-1,5-dihydro-3H-1,5-benzodiazepine-
2,4-dione,
Test solution. Dissolve 10.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with the D. R1 = R3 = R4 = CH3, R2 = Cl : 7-chloro-1,3,3-trimethyl-5-
mobile phase. phenyl-1,5-dihydro-3H-1,5-benzodiazepine-2,4-dione,
Reference solution (a). Dissolve 5.0 mg of clobazam
impurity A CRS in the mobile phase and dilute to 50.0 mL with
the mobile phase. Dilute 1.0 mL of the solution to 100.0 mL
with the mobile phase.
Reference solution (b). Dissolve 5 mg of chlordiazepoxide CRS
and 5 mg of clonazepam CRS in the mobile phase and dilute
to 50 mL with the mobile phase. Dilute 1 mL of the solution
to 100 mL with the mobile phase.
Reference solution (c). Dilute 1.0 mL of the test solution to E. N-[4-chloro-2-(phenylamino)phenyl]-N-methylacetamide,
200.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm,
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : acetonitrile R, water R (40:60 V/V).
Flow rate : 1 mL/min.
Detection : spectrophotometer at 230 nm. F. methyl 3-[[4-chloro-2-(phenylamino)phenyl]methylami-
Injection : 20 μL. no]-3-oxopropanoate.
Run time : 5 times the retention time of clobazam.
Retention time : clobazam = about 15 min. 01/2008:2127
System suitability : reference solution (b) : corrected 6.0
– resolution : minimum 1.3 between the peaks due to
chlordiazepoxide and clonazepam. CLOBETASOL PROPIONATE
Limits :
– impurity A : not more than the area of the principal peak Clobetasoli propionas
in the chromatogram obtained with reference solution (a)
(0.5 per cent),
– any other impurity : not more than 0.4 times the area of
the principal peak in the chromatogram obtained with
reference solution (c) (0.2 per cent),
– total of other impurities: not more than twice the area of
the principal peak in the chromatogram obtained with
reference solution (c) (1.0 per cent),
– disregard limit : 0.1 times the area of the principal peak in C25H32ClFO5 Mr 467.0
the chromatogram obtained with reference solution (c) [25122-46-7]
(0.05 per cent). DEFINITION
Loss on drying (2.2.32) : maximum 0.5 per cent, determined 21-Chloro-9-fluoro-11β-hydroxy-16β-methyl-3,20-
on 1.000 g by drying in an oven at 105 °C. dioxopregna-1,4-dien-17-yl propanoate.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Content : 97.0 per cent to 102.0 per cent (dried substance).
the residue obtained in the test for loss on drying.
CHARACTERS
ASSAY
Appearance : white or almost white, crystalline powder.
Dissolve 50.0 mg in alcohol R and dilute to 100.0 mL with the
same solvent. Dilute 2.0 mL of the solution to 250.0 mL with Solubility : practically insoluble in water, freely soluble in
alcohol R. Measure the absorbance (2.2.25) at the maximum acetone, sparingly soluble in ethanol (96 per cent).
at 232 nm. IDENTIFICATION
Calculate the content of C16H13ClN2O2 taking the specific Infrared absorption spectrophotometry (2.2.24).
absorbance to be 1380.
Comparison: clobetasol propionate CRS.
IMPURITIES
TESTS
Specific optical rotation (2.2.7) : + 112 to + 118 (dried
substance).
Dissolve 0.500 g in acetone R and dilute to 50.0 mL with the
same solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution (a). Dissolve 20.0 mg of the substance to be
examined in the mobile phase and dilute to 20.0 mL with the
A. R1 = R3 = R4 = H, R2 = Cl : 7-chloro-5-phenyl-1,5- mobile phase.
dihydro-3H-1,5-benzodiazepine-2,4-dione, Test solution (b). Dissolve 20.0 mg of the substance to be
B. R1 = CH3, R2 = R3 = R4 = H : 1-methyl-5-phenyl-1,5- examined in the mobile phase and dilute to 100.0 mL with
dihydro-3H-1,5-benzodiazepine-2,4-dione, the mobile phase.

1916 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Clobetasol propionate

Reference solution (a). Dissolve 20.0 mg of clobetasol – disregard limit : 0.1 times the area of the principal peak in
propionate CRS in the mobile phase and dilute to 100.0 mL the chromatogram obtained with reference solution (d)
with the mobile phase. (0.05 per cent).
Reference solution (b). Dissolve the contents of a vial of Loss on drying (2.2.32) : maximum 0.5 per cent, determined
clobetasol impurity J CRS in 2.0 mL of the mobile phase. To on 1.000 g by drying in an oven at 105 °C for 3 h.
0.5 mL of this solution add 0.5 mL of test solution (b) and Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
dilute to 20.0 mL with the mobile phase. 1.0 g.
Reference solution (c). Dissolve the contents of a vial of
clobetasol for peak identification CRS (containing impurities ASSAY
A, B, C, D, E, L and M) in 2 mL of the mobile phase. Liquid chromatography (2.2.29) as described in the test for
Reference solution (d). Dilute 1.0 mL of test solution (a) to related substances with the following modification.
50.0 mL with the mobile phase. Dilute 5.0 mL of this solution Injection : test solution (b) and reference solution (a).
to 20.0 mL with the mobile phase. Calculate the percentage content of C25H32ClFO5 using the
Column : chromatogram obtained with reference solution (a) and the
– size : l = 0.15 m, Ø = 4.6 mm; declared content of clobetasol propionate CRS.
– stationary phase : spherical octadecylsilyl silica gel for STORAGE
chromatography R (5 μm);
– temperature : 30 °C. Protected from light.
Mobile phase : mix 10 volumes of methanol R, 42.5 volumes IMPURITIES
of a 7.85 g/L solution of sodium dihydrogen phosphate Specified impurities : A, B, C, D, E, L, M.
monohydrate R adjusted to pH 5.5 with a 100 g/L solution of
sodium hydroxide R and 47.5 volumes of acetonitrile R. Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
Flow rate : 1.0 mL/min. the tests in the monograph. They are limited by the general
Detection : spectrophotometer at 240 nm. acceptance criterion for other/unspecified impurities and/or
Injection : 10 μL of test solution (a) and reference solutions (b), by the general monograph Substances for pharmaceutical use
(c) and (d). (2034). It is therefore not necessary to identify these impurities
Run time : 3 times the retention time of clobetasol propionate. for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use): F, G, H, I, J, K.
Identification of impurities : use the chromatogram
supplied with clobetasol for peak identification CRS and the
chromatogram obtained with reference solution (c) to identify
the peaks due to impurities A, B, C, D, E, L and M.
Relative retention with reference to clobetasol propionate
(retention time = about 10 min) : impurity A = about 0.4 ;
impurity B = about 0.6 ; impurity C = about 0.9 ;
impurity J = about 1.1 ; impurity D = about 1.2 ;
impurity L = about 1.3 ; impurity M = about 1.6 ; A. R1 = CO-C2H5, R2 = OH : 9-fluoro-11β,21-dihydroxy-
impurity E = about 2.1. 16β-methyl-3,20-dioxopregna-1,4-dien-17-yl propanoate
System suitability : (betamethasone 17-propionate),
– resolution : minimum 2.0 between the peaks due to G. R1 = H, R2 = Cl : 21-chloro-9-fluoro-11β,17-dihydroxy-
clobetasol propionate and impurity J in the chromatogram 16β-methylpregna-1,4-diene-3,20-dione (clobetasol),
obtained with reference solution (b) ;
H. R1 = CO-C2H5, R2 = H : 9-fluoro-11β-hydroxy-16β-methyl-
– the chromatogram obtained with reference solution (c) is 3,20-dioxopregna-1,4-dien-17-yl propanoate,
similar to the chromatogram supplied with clobetasol for
peak identification CRS. I. R1 = CO-C2H5, R2 = O-SO2-CH3 : 9-fluoro-11β-hydroxy-
Limits : 16β-methyl-21-[(methylsulfonyl)oxy]-3,20-dioxopregna-
1,4-dien-17-yl propanoate,
– correction factors : for the calculation of content,
multiply the peak areas of the following impurities by K. R1 = H, R2 = O-CO-C2H5 : 9-fluoro-11β,17-dihydroxy-
the corresponding correction factor : impurity B = 0.6; 16β-methyl-3,20-dioxopregna-1,4-dien-21-yl propanoate
impurity C = 1.5 ; (betamethasone 21-propionate),
– impurity E : not more than 1.4 times the area of the
principal peak in the chromatogram obtained with
reference solution (d) (0.7 per cent) ;
– impurity D : not more than the area of the principal peak
in the chromatogram obtained with reference solution (d)
(0.5 per cent) ;
– impurities B, C : for each impurity, not more than 0.6 times
the area of the principal peak in the chromatogram B. 21-chloro-9-fluoro-11β-hydroxy-16-methylpregna-1,4,16-
obtained with reference solution (d) (0.3 per cent) ; triene-3,20-dione,
– impurities A, L, M : for each impurity, not more than
0.4 times the area of the principal peak in the chromatogram
obtained with reference solution (d) (0.2 per cent) ;
– unspecified impurities : for each impurity, not more than
0.2 times the area of the principal peak in the chromatogram
obtained with reference solution (d) (0.10 per cent) ;
– total : not more than 4 times the area of the principal peak
in the chromatogram obtained with reference solution (d) C. 21-chloro-9-fluoro-11β-hydroxy-16α-methyl-3,20-
(2.0 per cent) ; dioxopregna-1,4-dien-17-yl propanoate,

General Notices (1) apply to all monographs and other texts 1917
Clobetasone butyrate EUROPEAN PHARMACOPOEIA 8.0

mp : about 178 °C.

IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: clobetasone butyrate CRS.
TESTS
D. 21-chloro-9-fluoro-11β-hydroxy-16β-methyl-3,20- Specific optical rotation (2.2.7) : + 131 to + 138 (dried
dioxopregn-4-en-17-yl propanoate (1,2-dihydroclobetasol substance).
17-propionate), Dissolve 0.250 g in ethanol R1 and dilute to 25.0 mL with the
same solvent.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
Solvent mixture : anhydrous formic acid R, acetonitrile R,
water R (0.1:43:57 V/V/V).
Test solution. Dissolve 65 mg of the substance to be examined
in 5.0 mL of acetonitrile R and dilute to 25.0 mL with the
E. 21-chloro-16β-methyl-3,20-dioxopregna-1,4-dien-17-yl solvent mixture.
propanoate, Reference solution (a). Dissolve 13 mg of clobetasone butyrate
for system suitability CRS (containing impurity F) in 1 mL of
acetonitrile R and dilute to 5.0 mL with the solvent mixture.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Column :
F. 9-fluoro-11β-hydroxy-16β-methyl-3-oxopregna-1,4,17(20)- – size : l = 0.15 m, Ø = 4.6 mm ;
trien-21-oic acid, – stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (3.5 μm) ;
– temperature : 40 °C.
Mobile phase :
– mobile phase A : anhydrous formic acid R, water R
(0.1:99.9 V/V) ;
– mobile phase B : anhydrous formic acid R, acetonitrile R
(0.1:99.9 V/V) ;
J. (17R)-4′-chloro-5′-ethyl-9-fluoro-11β-hydroxy-16β- Time Mobile phase A Mobile phase B
methylspiro[androsta-1,4-diene-17,2′(3′H)-furan]-3,3′- (min) (per cent V/V) (per cent V/V)
dione (17α-spiro compound), 0-3 57 43
L. unknown structure, 3 - 26 57 → 43 43 → 57
M. unknown structure.
Flow rate : 1.5 mL/min.
01/2010:1090 Detection : spectrophotometer at 241 nm.
corrected 6.7 Injection : 10 μL.
Identification of impurities : use the chromatogram supplied
CLOBETASONE BUTYRATE with clobetasone butyrate for system suitability CRS and the
chromatogram obtained with reference solution (a) to identify
the peak due to impurity F.
Clobetasoni butyras
Relative retention with reference to clobetasone butyrate
(retention time = about 14 min) : impurity F = about 0.9.
System suitability :
– resolution : minimum 3.5 between the peaks due to
impurity F and clobetasone butyrate in the chromatogram
obtained with reference solution (a) ;
– signal-to-noise ratio : minimum 10 for the principal peak in
the chromatogram obtained with reference solution (b).
C26H32ClFO5 Mr 479.0 Limits :
[25122-57-0] – unspecified impurities : for each impurity, not more than the
DEFINITION area of the principal peak in the chromatogram obtained
with reference solution (b) (0.10 per cent) ;
21-Chloro-9-fluoro-16β-methyl-3,11,20-trioxopregna-1,4-
dien-17-yl butanoate. – total : not more than 5 times the area of the principal peak
Content : 97.0 per cent to 102.0 per cent (dried substance). in the chromatogram obtained with reference solution (b)
(0.5 per cent) ;
CHARACTERS – disregard limit : 0.5 times the area of the principal peak in
Appearance : white or almost white powder. the chromatogram obtained with reference solution (b)
Solubility : practically insoluble in water, freely soluble in (0.05 per cent).
acetone and in methylene chloride, slightly soluble in ethanol Loss on drying (2.2.32) : maximum 0.5 per cent, determined
(96 per cent). on 1.000 g by drying in an oven at 105 °C.

1918 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Clodronate disodium tetrahydrate

ASSAY
Dissolve 20.0 mg in ethanol (96 per cent) R and dilute
to 100.0 mL with the same solvent. Dilute 5.0 mL of the
solution to 50.0 mL with ethanol (96 per cent) R. Measure the
absorbance (2.2.25) at the absorption maximum at 235 nm.
Calculate the content of C26H32ClFO5, taking the specific
absorbance to be 327.
F. 21-chloro-9-fluoro-16α-methyl-3,11,20-trioxopregna-1,4-
STORAGE dien-17-yl butanoate (16α-methyl clobetasone butyrate).
Protected from light.
07/2008:1777
IMPURITIES
Other detectable impurities (the following substances would,
CLODRONATE DISODIUM
if present at a sufficient level, be detected by one or other of TETRAHYDRATE
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or Dinatrii clodronas tetrahydricus
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use): A,
C, D, E, F, G, H, I.
CH2Cl2Na2O6P2,4H2O Mr 360.9
DEFINITION
Disodium (dichloromethylene)bis(hydrogen phosphonate)
tetrahydrate.
Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
A. R1 = H, R2 = Cl : 21-chloro-9-fluoro-17-hydroxy-16β- Solubility : freely soluble in water, practically insoluble in
methylpregna-1,4-diene-3,11,20-trione (clobetasone), ethanol (96 per cent), slightly soluble in methanol.
G. R1 = CO-CH2-CH2-CH3, R2 = O-CO-CH2-CH3 : 9-fluoro- IDENTIFICATION
16β-methyl-3,11,20-trioxo-21-(propanoyloxy)pregna-1,4- A. Infrared absorption spectrophotometry (2.2.24).
dien-17-yl butanoate, Comparison: clodronate disodium tetrahydrate CRS.
B. Dissolve 0.5 g in 10 mL of water R. The solution gives
H. R1 = CO-CH2-CH3, R2 = Cl : 21-chloro-9-fluoro-16β- reaction (a) of sodium (2.3.1).
methyl-3,11,20-trioxopregna-1,4-dien-17-yl propanoate
(17-O-propionyl clobetasone), TESTS
Solution S. Dissolve 1.0 g in carbon dioxide-free water R and
I. R1 = CO-CH(CH3)2, R2 = Cl : 21-chloro-9-fluoro- dilute to 20 mL with the same solvent.
16β-methyl-3,11,20-trioxopregna-1,4-dien-17-yl Appearance of solution. Solution S is clear (2.2.1) and
2-methylpropanoate (17-O-isobutyryl clobetasone),
colourless (2.2.2, Method II).
pH (2.2.3) : 3.0 to 4.5, for solution S.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.125 g of the substance to be examined
in 30 mL of water R, sonicate for 10 min and dilute to 50.0 mL
with water R (test stock solution). Dilute 10.0 mL of the test
stock solution to 20.0 mL of water R.
Reference solution (a). Dilute 1.0 mL of the test solution
to 10.0 mL with water R. Dilute 1.0 mL of this solution to
C. 21-chloro-9-fluoro-16β-methyl-3,11,20-trioxopregn-1-en- 50.0 mL with water R.
17-yl butanoate (4,5-dihydroclobetasone butyrate), Reference solution (b). Dissolve 1 mg of clodronate
impurity D CRS in 10 mL of water R, sonicate for 10 min and
dilute to 20.0 mL with water R. Mix 2.0 mL of this solution
with 10.0 mL of the test stock solution and dilute to 20.0 mL
with water R.
Reference solution (c). Dilute 1.0 mL of a 0.3 g/L solution of
phosphoric acid R (impurity B) to 100.0 mL with water R.
Precolumn :
– size : l = 0.05 m, Ø = 4 mm ;
D. R = Br : 2α-bromo-21-chloro-9-fluoro-16β-methyl-3,11,20- – stationary phase : anion-exchange resin R ;
trioxopregn-1-en-17-yl butanoate (2-bromoclobetasone – particle size : 9 μm.
butyrate),
Column :
E. R = H : 21-chloro-9-fluoro-16β-methyl-3,11,20- – size : l = 0.25 m, Ø = 4 mm ;
trioxopregn-4-en-17-yl butanoate (1,2-dihydroclobetasone – stationary phase : anion-exchange resin R ;
butyrate), – particle size : 9 μm.

General Notices (1) apply to all monographs and other texts 1919
Clofazimine EUROPEAN PHARMACOPOEIA 8.0

Mobile phase : use (2034). It is therefore not necessary to identify these

– mobile phase A : 0.21 g/L solution of sodium hydroxide R in impurities for demonstration of compliance. See also 5.10.
carbon dioxide-free water R ; close immediately, mix and Control of impurities in substances for pharmaceutical use) : D.
use under helium pressure ;
– mobile phase B : 4.2 g/L solution of sodium hydroxide R in
carbon dioxide-free water R ; close immediately, mix and
use under helium pressure ;
Time
A. R = CH(CH3)2, R′ = Cl : [dichloro[hydroxy(1-
Mobile phase A Mobile phase B
methylethoxy)phosphinoyl]methyl]phosphonic acid,
(min) (per cent V/V) (per cent V/V)
0 - 10 90 → 60 10 → 40 D. R = R′ = H : (chloromethylene)bis(phosphonic acid),
10 - 22 60 → 50 40 → 50 B. H3PO4 : phosphoric acid.
22 - 23 50 → 20 50 → 80
23 - 25 20 80 01/2008:2054

Flow rate : 1 mL/min. CLOFAZIMINE

Detection : conductivity detector. Use a self-regenerating anion
suppressor. Clofaziminum
Injection : 20 μL.
Identification of impurities : use the chromatogram obtained
with reference solution (c) to identify the peak due to
impurity B.
Relative retention with reference to clodronate (retention
time = about 13 min) : impurities A and B = about 0.7 ;
impurity D = about 1.1.
System suitability : reference solution (b) :
– peak-to-valley ratio : minimum 3, where Hp = height
above the baseline of the peak due to impurity D and C27H22Cl2N4 Mr 473.4
Hv = height above the baseline of the lowest point of the [2030-63-9]
curve separating this peak from the peak due to clodronate.
Limits : DEFINITION
– sum of impurities A and B : not more than the area of N,5-Bis(4-chlorophenyl)-3-[(1-methylethyl)imino]-3,5-
the principal peak in the chromatogram obtained with dihydrophenazin-2-amine.
reference solution (a) (0.2 per cent) ; Content : 99.0 per cent to 101.0 per cent (dried substance).
– unspecified impurities : for each impurity, not more than CHARACTERS
0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent) ; Appearance : reddish-brown, fine powder.
– total : not more than 1.5 times the area of the principal peak Solubility : practically insoluble in water, soluble in methylene
in the chromatogram obtained with reference solution (a) chloride, very slightly soluble in ethanol (96 per cent).
(0.3 per cent) ; It shows polymorphism (5.9).
– disregard limit : 0.25 times the area of the principal peak IDENTIFICATION
in the chromatogram obtained with reference solution (a) First identification : A.
(0.05 per cent).
Second identification : B, C.
Heavy metals (2.4.8) : maximum 20 ppm.
A. Infrared absorption spectrophotometry (2.2.24).
0.5 g complies with test G. Prepare the reference solution Comparison: clofazimine CRS.
using 1 mL of lead standard solution (10 ppm Pb) R.
If the spectra obtained in the solid state show differences,
Water (2.5.12) : 18.5 per cent to 21.0 per cent, determined on dissolve the substance to be examined and the reference
0.100 g. substance separately in methylene chloride R, evaporate to
dryness and record new spectra using the residues.
ASSAY
B. Thin-layer chromatography (2.2.27).
Dissolve 0.140 g in 10 mL of water R. Add 10 mL of strong
sodium hydroxide solution R and some glass beads. Boil until Test solution. Dissolve 10 mg of the substance to be
the solution is completely decolourised (about 10 min). Cool examined in methylene chloride R and dilute to 10 mL with
in an ice-bath and add 30 mL of water R and 10 mL of nitric the same solvent.
acid R. Titrate with 0.1 M silver nitrate, determining the Reference solution. Dissolve 10 mg of clofazimine CRS in
end-point potentiometrically (2.2.20). methylene chloride R and dilute to 10 mL with the same
1 mL of 0.1 M silver nitrate is equivalent to 14.44 mg of solvent.
CH2Cl2Na2O6P2. Plate : TLC silica gel GF254 plate R.
Mobile phase : propanol R, methylene chloride R (6:85 V/V).
IMPURITIES Application : 5 μL.
Specified impurities : A, B. First development : over 2/3 of the plate.
Other detectable impurities (the following substances would, Drying : horizontally in air for 5 min.
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general Second development : over 2/3 of the plate.
acceptance criterion for other/unspecified impurities and/or Drying : in air for 5 min.
by the general monograph Substances for pharmaceutical Detection : examine in ultraviolet light at 254 nm.

1920 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Clofibrate

Results : the principal spot in the chromatogram obtained ASSAY

with the test solution is similar in position and size to the Dissolve 0.400 g in 5 mL of methylene chloride R and add
principal spot in the chromatogram obtained with the 20 mL of acetone R and 5 mL of anhydrous acetic acid R.
reference solution. Titrate with 0.1 M perchloric acid, determining the end-point
C. Dissolve 2 mg in 3 mL of acetone R and add 0.1 mL of potentiometrically (2.2.20).
hydrochloric acid R. An intense violet colour is produced. 1 mL of 0.1 M perchloric acid is equivalent to 47.34 mg
Add 0.5 mL of a 200 g/L solution of sodium hydroxide R ; of C27H22Cl2N4.
the colour changes to orange-red.
IMPURITIES
TESTS
Specified impurities : A, B.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
Test solution. Dissolve 50 mg of the substance to be examined
in the mobile phase and dilute to 100 mL with the mobile
phase.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10.0 mL with the mobile phase.
Reference solution (b). Dissolve 5.0 mg of clofazimine for
system suitability CRS in the mobile phase and dilute to A. R1 = Cl, R2 = H : N,5-bis(4-chlorophenyl)-3-imino-3,5-
10.0 mL with the mobile phase. dihydrophenazin-2-amine,
Column : B. R1 = H, R2 = CH(CH3)2 : 5-(4-chlorophenyl)-3-[(1-
– size : l = 0.25 m, Ø = 4.6 mm, methylethyl)imino]-N-phenyl-3,5-dihydrophenazin-2-
– stationary phase : octylsilyl silica gel for chromatography R amine.
(5 μm).
Mobile phase : dissolve 2.25 g of sodium laurilsulfate R, 0.85 g 01/2008:0318
of tetrabutylammonium hydrogen sulfate R and 0.885 g of
disodium hydrogen phosphate R in water R. Adjust to pH 3.0 CLOFIBRATE
with dilute phosphoric acid R and dilute to 500 mL with
water R. Mix 35 volumes of this solution and 65 volumes of
acetonitrile R. Clofibratum
Flow rate : 1 mL/min.
Detection : spectrophotometer at 280 nm.
Injection : 20 μL.
Run time : 3 times the retention time of clofazimine.
Identification of impurities : use the chromatogram supplied C12H15ClO3 Mr 242.7
with clofazimine for system suitability CRS to identify the peak [637-07-0]
due to impurity B.
DEFINITION
Relative retention with reference to clofazimine (retention
Ethyl 2-(4-chlorophenoxy)-2-methylpropionate.
time = about 15 min) : impurity A = about 0.7 ;
impurity B = about 0.8. CHARACTERS
System suitability : reference solution (b) : Appearance : clear, almost colourless liquid.
– resolution : baseline separation between the peaks due to Solubility : very slightly soluble in water, miscible with ethanol
impurity B and clofazimine. (96 per cent).
Limits :
IDENTIFICATION
– impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (a) A. Infrared absorption spectrophotometry (2.2.24).
(0.1 per cent), Comparison: clofibrate CRS.
– impurity B : not more than 3 times the area of the principal B. Ultraviolet and visible absorption spectrophotometry
peak in the chromatogram obtained with reference (2.2.25).
solution (a) (0.3 per cent), Test solution (a). Dissolve 0.10 g in methanol R and dilute
– any other impurity : for each impurity, not more than the to 100.0 mL with the same solvent. Dilute 10.0 mL of this
area of the principal peak in the chromatogram obtained solution to 100.0 mL with methanol R.
with reference solution (a) (0.1 per cent), Test solution (b). Dilute 10.0 mL of test solution (a) to
– total : not more than 5 times the area of the principal peak 100.0 mL with methanol R.
in the chromatogram obtained with reference solution (a) Spectral range : 250-350 nm for test solution (a) ; 220-250 nm
(0.5 per cent), for test solution (b).
– disregard limit : 0.5 times the area of the principal peak in Absorption maxima : at 280 nm and 288 nm for test
the chromatogram obtained with reference solution (a) solution (a) ; at 226 nm for test solution (b).
(0.05 per cent). Specific absorbances at the absorption maxima :
Heavy metals (2.4.8) : maximum 10 ppm. – at 226 nm : about 460 for test solution (b) ;
2.0 g complies with test C. Prepare the reference solution using – at 280 nm : about 44 for test solution (a);
2 mL of lead standard solution (10 ppm Pb) R. – at 288 nm : about 31 for test solution (a).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C. TESTS
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Relative density (2.2.5) : 1.138 to 1.147.
1.0 g. Refractive index (2.2.6) : 1.500 to 1.505.

General Notices (1) apply to all monographs and other texts 1921
Clomifene citrate EUROPEAN PHARMACOPOEIA 8.0

Acidity. To 1.0 g add 10 mL of anhydrous ethanol R and 01/2008:0997

0.1 mL of phenol red solution R. Not more than 1.0 mL of
0.01 M sodium hydroxide is required to change the colour of CLOMIFENE CITRATE
the indicator.
Volatile related substances. Gas chromatography (2.2.28). Clomifeni citras
Test solution. To 10.0 g of the substance to be examined add
a mixture of 10 mL of dilute sodium hydroxide solution R
and 10 mL of water R. Shake, separate the lower (organic)
layer, wash with 5 mL of water R and add the washings to the
aqueous layer. Dry the organic layer with anhydrous sodium
sulfate R and use as the test solution. Reserve the aqueous
layer for the test for 4-chlorophenol.
Reference solution (a). Dissolve 0.12 g of the substance to be
examined in chloroform R and dilute to 100.0 mL with the C32H36ClNO8 Mr 598.1
same solvent. Dilute 1.0 mL of this solution to 10.0 mL with [50-41-9]
chloroform R.
DEFINITION
Reference solution (b). Dissolve 0.12 g of methyl
2-(4-chlorophenoxy)-2-methylpropionate CRS in the substance Mixture of the (E)- and (Z)-isomers of 2-[4-(2-chloro-1,2-
to be examined and dilute to 10.0 mL with the same solvent. diphenylethenyl)phenoxy]-N,N-diethylethanamine
Dilute 1.0 mL of the solution to 10.0 mL with the substance to dihydrogen citrate.
be examined. Dilute 1.0 mL of this solution to 10.0 mL with Content : 98.0 per cent to 101.0 per cent (anhydrous substance).
the substance to be examined.
CHARACTERS
Column : Appearance : white or pale yellow, crystalline powder.
– size : l = 1.5 m, Ø = 4 mm ; Solubility : slightly soluble in water, sparingly soluble in
ethanol (96 per cent).
– stationary phase : silanised diatomaceous earth for
gas chromatography R (250-420 μm) impregnated IDENTIFICATION
with 30 per cent m/m of poly(dimethyl)siloxane R ; or A. Infrared absorption spectrophotometry (2.2.24).
silanised diatomaceous earth for gas chromatography R
Preparation : discs of potassium bromide R.
(150-180 μm) impregnated with 10 per cent m/m of
poly(dimethyl)siloxane R ; Comparison: clomifene citrate CRS.
B. Dissolve about 5 mg in 5 mL of a mixture of 1 volume of
– temperature : 185 °C. acetic anhydride R and 5 volumes of pyridine R, then heat
Carrier gas : nitrogen for chromatography R. in a water-bath. A deep red colour is produced.
Detection : flame ionisation. TESTS
Prepare the solutions protected from light in brown-glass
Injection : 2 μL.
vessels. Ensure minimum exposure of the solutions to daylight
System suitability : reference solution (b) : until they are required for chromatography.
– peak-to-valley ratio : minimum 4, where Hp = height Related substances. Liquid chromatography (2.2.29).
above the baseline of the peak due to methyl Test solution. Dissolve 12.5 mg of the substance to be
2-(4-chlorophenoxy)-2-methylpropionate and Hv = height examined in the mobile phase and dilute to 10.0 mL with the
above the baseline of the lowest point of the curve mobile phase.
separating this peak from the peak due to clofibrate. Reference solution (a). Dissolve 12.5 mg of clomifene citrate
for performance test CRS in the mobile phase and dilute to
Limit :
10.0 mL with the mobile phase.
– total : not more than 10 times the area of the peak due to Reference solution (b). Dilute 1.0 mL of the test solution to
clofibrate in the chromatogram obtained with reference 50.0 mL with the mobile phase.
solution (a) (0.1 per cent). Column :
4-Chlorophenol. Gas chromatography (2.2.28) as described – size : l = 0.25 m, Ø = 4.6 mm ;
in the test for volatile related substances with the following – stationary phase : butylsilyl silica gel for chromatography R
modifications. (5 μm).
Test solution. Shake the aqueous layer reserved in the test Mobile phase : mix 400 mL of acetonitrile R with 600 mL of
for volatile related substances with 2 quantities, each of water R and add 8.0 mL of diethylamine R ; adjust to pH 6.2
5 mL, of chloroform R and discard the organic layers. Acidify with about 1-2 mL of phosphoric acid R, taking care to reduce
the aqueous layer by the dropwise addition of hydrochloric progressively the volume of each addition as the required pH
acid R. Shake with 3 quantities, each of 3 mL, of chloroform R. is approached.
Combine the organic layers and dilute to 10.0 mL with Flow rate : 1.2 mL/min.
chloroform R.
Detection : spectrophotometer at 233 nm.
Reference solution. Dissolve 0.25 g of chlorophenol R in Equilibration : with the mobile phase for about 1 h.
chloroform R and dilute to 100.0 mL with the same solvent.
Injection : 10 μL.
Dilute 1.0 mL of this solution to 100.0 mL with chloroform R.
Run time : 4 times the retention time of clomifene.
Limit : System suitability : reference solution (a) :
– 4-chlorophenol : not more than the area of the peak due to – peak-to-valley ratio : minimum 15, where Hp = height
4-chlorophenol in the chromatogram obtained with the above the baseline of the peak due to impurity A and
reference solution (25 ppm). Hv = height above the baseline of the lowest point of the

1922 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Clomifene citrate

curve separating this peak from the peak due to clomifene ; ASSAY
if necessary, adjust the concentration of acetonitrile in the Dissolve 0.500 g in 50 mL of anhydrous acetic acid R. Titrate
mobile phase ; with 0.1 M perchloric acid, determining the end-point
– the chromatogram obtained is similar to the chromatogram potentiometrically (2.2.20).
supplied with clomifene citrate for performance test CRS. 1 mL of 0.1 M perchloric acid is equivalent to 59.81 mg
Limits : of C32H36ClNO8.
– impurity A : not more than the area of the principal peak STORAGE
in the chromatogram obtained with reference solution (b)
(2.0 per cent) ; Protected from light.

– impurities B, C, D, E, F, G, H : for each impurity, not IMPURITIES

more than 0.5 times the area of the principal peak in the Specified impurities : A, B, C, D, E, F, G, H.
chromatogram obtained with reference solution (b) (1.0 per
cent) ;
– total : not more than 1.25 times the area of the principal
peak in the chromatogram obtained with reference
solution (b) (2.5 per cent) ;
– disregard limit : 0.025 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent) ; disregard any peak with a retention time
relative to the clomifene peak of 0.2 or less.
(Z)-isomer. Liquid chromatography (2.2.29). A. 2-[4-(1,2-diphenylethenyl)phenoxy]-N,N-diethyl-
Test solution. Dissolve 25 mg of the substance to be examined ethanamine,
in 25 mL of 0.1 M hydrochloric acid, add 5 mL of 1 M sodium
hydroxide and shake with 3 quantities, each of 25 mL, of
ethanol-free chloroform R. Wash the combined extracts with
10 mL of water R, dry over anhydrous sodium sulfate R and
dilute to 100 mL with ethanol-free chloroform R. To 20 mL B. [4-[2-(diethylamino)ethoxy]phenyl]phenylmethanone,
of this solution add 0.1 mL of triethylamine R and dilute to
100 mL with hexane R.
Reference solution. Dissolve 25 mg of clomifene citrate CRS in
25 mL of 0.1 M hydrochloric acid, add 5 mL of 1 M sodium
hydroxide and shake with 3 quantities, each of 25 mL, of
ethanol-free chloroform R. Wash the combined extracts with C. (2RS)-2-[4-[2-(diethylamino)ethoxy]phenyl]-1,2-
10 mL of water R, dry over anhydrous sodium sulfate R and diphenylethanone,
dilute to 100 mL with ethanol-free chloroform R. To 20 mL
of this solution add 0.1 mL of triethylamine R and dilute to
100 mL with hexane R.
Column :
– size : l = 0.3 m, Ø = 4 mm ;
D. 2,2-bis[4-[2-(diethylamino)ethoxy]phenyl]-1,2-
– stationary phase : silica gel for chromatography R (10 μm). diphenylethanone,
Mobile phase : triethylamine R, ethanol-free chloroform R,
hexane R (1:200:800 V/V/V).
Flow rate : 2 mL/min.
Detection : spectrophotometer at 302 nm.
Equilibration : with the mobile phase for about 2 h.
Injection : 50 μL.
E. 2-[4-[1,2-bis(4-chlorophenyl)ethenyl]phenoxy]-N,N-
Identification of peaks : the chromatogram obtained with the diethylethanamine,
reference solution shows a peak due to the (E)-isomer just
before a peak due to the (Z)-isomer.
System suitability: reference solution :
– resolution : minimum 1.0 between the peaks due to the
(E)- and (Z)-isomers ; if necessary, adjust the relative
proportions of ethanol-free chloroform and hexane in the
mobile phase. F. 2-[4-[2-chloro-2-(4-chlorophenyl)-1-phenylethenyl]-
phenoxy]-N,N-diethylethanamine,
Measure the area of the peak due to the (Z)-isomer in the
chromatograms obtained with the test solution and the
reference solution. Calculate the content of the (Z)-isomer, as
a percentage of the total clomifene citrate present, from the
declared content of clomifene citrate CRS.
Limit :
GH. 2-[2-chloro-4-(2-chloro-1,2-diphenylethenyl)phenoxy]-
– (Z)-isomer : 30.0 per cent to 50.0 per cent. N,N-diethylethanamine (G. higher-melting-point isomer ;
Water (2.5.12) : maximum 1.0 per cent, determined on 1.000 g. H. lower-melting-point isomer).

General Notices (1) apply to all monographs and other texts 1923
Clomipramine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

01/2008:0889 TESTS
corrected 6.0 Solution S. Dissolve 2.0 g in carbon dioxide-free water R and
dilute to 20 mL with the same solvent.
CLOMIPRAMINE HYDROCHLORIDE Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y5 (2.2.2,
Clomipramini hydrochloridum Method I).
pH (2.2.3) : 3.5 to 5.0 for solution S.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use and protected from light.
Test solution. Dissolve 20.0 mg of the substance to be
examined in a mixture of 25 volumes of mobile phase B and
75 volumes of mobile phase A and dilute to 10.0 mL with the
same mixture of mobile phases.
Reference solution (a). Dissolve 22.6 mg of imipramine
C19H24Cl2N2 Mr 351.3 hydrochloride CRS, 4.0 mg of clomipramine impurity C CRS,
[17321-77-6] 4.0 mg of clomipramine impurity D CRS and 2.0 mg of
clomipramine impurity F CRS in a mixture of 25 volumes of
DEFINITION mobile phase B and 75 volumes of mobile phase A and dilute
3-(3-Chloro-10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N,N- to 100.0 mL with the same mixture of mobile phases. Dilute
dimethylpropan-1-amine hydrochloride. 1.0 mL of this solution to 10.0 mL with a mixture of 25 volumes
of mobile phase B and 75 volumes of mobile phase A.
Content : 99.0 per cent to 101.0 per cent (dried substance).
Reference solution (b). Dilute 1.0 mL of the test solution to
CHARACTERS 100.0 mL with a mixture of 25 volumes of mobile phase B and
75 volumes of mobile phase A.
Appearance : white or slightly yellow, crystalline powder,
slightly hygroscopic. Reference solution (c). Dissolve 10.0 mg of clomipramine
hydrochloride CRS and 3.0 mg of clomipramine impurity C CRS
Solubility : freely soluble in water and in methylene chloride, in a mixture of 25 volumes of mobile phase B and 75 volumes
soluble in alcohol. of mobile phase A and dilute to 20.0 mL with the same
It shows polymorphism (5.9). mixture of mobile phases. Dilute 1.0 mL of this solution to
10.0 mL with a mixture of 25 volumes of mobile phase B and
IDENTIFICATION 75 volumes of mobile phase A.
First identification : B, E. Column :
Second identification : A, C, D, E. – size : l = 0.25 m, Ø = 4.6 mm,
A. Melting point (2.2.14) : 191 °C to 195 °C. – stationary phase : cyanopropylsilyl silica gel for
B. Infrared absorption spectrophotometry (2.2.24). chromatography R (5 μm),
Preparation : discs of potassium bromide R. The – temperature : 30 °C.
transmittance at about 2000 cm− 1 (5 μm) is at least 65 per Mobile phase :
cent without compensation. – mobile phase A : dissolve 1.2 g of sodium dihydrogen
Comparison : clomipramine hydrochloride CRS. phosphate R in water R, add 1.1 mL of nonylamine R, adjust
C. Thin-layer chromatography (2.2.27). Prepare the solutions to pH 3.0 with phosphoric acid R and dilute to 1000 mL
immediately before use and protected from light. with water R,
Test solution. Dissolve 20 mg of the substance to be – mobile phase B : acetonitrile R.
examined in methanol R and dilute to 10 mL with the same Time Mobile phase A Mobile phase B
solvent. (min) (per cent V/V) (per cent V/V)
Reference solution. Dissolve 20 mg of clomipramine 0 - 10 75 25
hydrochloride CRS in methanol R and dilute to 10 mL with 10 - 20 75 → 65 25 → 35
the same solvent.
Plate : TLC silica gel G plate R. 20 - 32 65 35

Mobile phase : concentrated ammonia R, acetone R, ethyl 32 - 34 65 → 75 35 → 25

acetate R (5:25:75 V/V/V). 34 - 44 75 25
Application : 5 μL.
Development : over a path of 15 cm. Flow rate : 1.5 mL/min.
Drying : in air. Detection : spectrophotometer at 254 nm.
Detection : spray with a 5 g/L solution of potassium Injection : 20 μL.
dichromate R in a 20 per cent V/V solution of sulfuric Relative retentions with reference to clomipramine
acid R. Examine immediately. (retention time = about 8 min) : impurity A = about 0.5 ;
Results : the principal spot in the chromatogram obtained impurity B = about 0.7 ; impurity C = about 0.9 ;
with the test solution is similar in position, colour and size impurity D = about 1.7 ; impurity E = about 2.5 ;
to the principal spot in the chromatogram obtained with impurity F = about 3.4 ; impurity G = about 4.3.
the reference solution. System suitability : reference solution (c) :
D. Dissolve about 5 mg in 2 mL of nitric acid R. An intense – resolution : minimum 3.0 between the peaks due to
blue colour develops. clomipramine and to impurity C.
E. Dissolve about 50 mg in 5 mL of water R and add 1 mL of Limits :
dilute ammonia R1. Mix, allow to stand for 5 min and filter. – impurity B : not more than the area of the corresponding
Acidify the filtrate with dilute nitric acid R. The solution peak in the chromatogram obtained with reference
gives reaction (a) of chlorides (2.3.1). solution (a) (1.0 per cent),

1924 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Clonazepam

– impurity C, D : for each impurity, not more than the area

of the corresponding peak in the chromatogram obtained
with reference solution (a) (0.2 per cent),
– impurity F : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (a) (0.1 per cent),
– any other impurity : not more than 0.1 times the area of
the principal peak in the chromatogram obtained with D. R1 = R3 = Cl, R2 = CH2-CH2-CH2-N(CH3)2 :
reference solution (b) (0.1 per cent), 3-(3,7-dichloro-10,11-dihydro-5H-dibenzo[b,f]azepin-5-
– total of other impurities : not more than 0.2 times the area yl)-N,N-dimethylpropan-1-amine,
of the principal peak in the chromatogram obtained with E. R1 = R2 = R3 = H : 10,11-dihydro-5H-dibenzo[b,f]azepine
reference solution (b) (0.2 per cent), (iminodibenzyl),
– total : not more than the area of the principal peak in the
chromatogram obtained with reference solution (b) (1.0 per F. R1 = Cl, R2 = R3 = H : 3-chloro-10,11-dihydro-5H-
cent), dibenzo[b,f]azepine,
– disregard limit : 0.01 times the area of the principal peak G. R1 = Cl, R2 = CH2-CH=CH2, R3 = H : 3-chloro-5-(prop-2-
in the chromatogram obtained with reference solution (b) enyl)-10,11-dihydro-5H-dibenzo[b,f]azepine.
(0.01 per cent).
Heavy metals (2.4.8) : maximum 20 ppm. 01/2008:0890
corrected 6.0
2.0 g complies with test C. Prepare the reference solution using
4 mL of lead standard solution (10 ppm Pb) R.
CLONAZEPAM
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C. Clonazepamum
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY
Dissolve 0.250 g in 50 mL of alcohol R and add 5.0 mL of
0.01 M hydrochloric acid. Carry out a potentiometric titration
(2.2.20), using 0.1 M sodium hydroxide. Read the volume
added between the 2 points of inflexion.
1 mL of 0.1 M sodium hydroxide is equivalent to 35.13 mg of C15H10ClN3O3 Mr 315.7
C19H24Cl2N2. [1622-61-3]
STORAGE DEFINITION
In an airtight container, protected from light. 5-(2-Chlorophenyl)-7-nitro-1,3-dihydro-2H-1,4-
benzodiazepin-2-one.
IMPURITIES Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : slightly yellowish, crystalline powder.
Solubility : practically insoluble in water, slightly soluble in
alcohol and in methanol.
mp : about 239 °C.
IDENTIFICATION
A. N-[3-(3-chloro-10,11-dihydro-5H-dibenzo[b,f]azepin-5- Infrared absorption spectrophotometry (2.2.24).
yl)propyl]-N,N′,N′-trimethylpropane-1,3-diamine, Comparison: Ph. Eur. reference spectrum of clonazepam.
TESTS
Related substances. Liquid chromatography (2.2.29). Carry
out the test protected from light and prepare the solutions
immediately before use.
Solvent mixture : tetrahydrofuran R, methanol R, water R
(10:42:48 V/V/V).
Test solution. Dissolve 0.100 g of the substance to be examined
B. 3-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N,N- in methanol R and dilute to 20.0 mL with the same solvent.
dimethylpropan-1-amine (imipramine), Dilute 1.0 mL to 10.0 mL with the solvent mixture.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of the
solution to 10.0 mL with the solvent mixture.
Reference solution (b). Dissolve 5 mg of the substance to be
examined and 5 mg of flunitrazepam R in the solvent mixture
and dilute to 100.0 mL with the solvent mixture.
Reference solution (c). Dissolve 1.0 mg of clonazepam
impurity B CRS in the solvent mixture and dilute to 20.0 mL
C. 3-(3-chloro-5H-dibenzo[b,f]azepin-5-yl)-N,N- with the solvent mixture. Dilute 1.0 mL of the solution to
dimethylpropan-1-amine, 100.0 mL with the solvent mixture.

General Notices (1) apply to all monographs and other texts 1925
Clonidine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Column : 01/2008:0477
– size : l = 0.15 m, Ø = 4.6 mm, corrected 6.3
– stationary phase : end-capped octylsilyl silica gel for
chromatography R (5 μm). CLONIDINE HYDROCHLORIDE
Mobile phase : mix 10 volumes of tetrahydrofuran R,
42 volumes of methanol R and 48 volumes of a 6.6 g/L solution Clonidini hydrochloridum
of ammonium phosphate R previously adjusted to pH 8.0 with
a 40 g/L solution of sodium hydroxide R or dilute phosphoric
acid R.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 254 nm.
Injection : 10 μL.
C9H10Cl3N3 Mr 266.6
Run time : 3 times the retention time of clonazepam. [4205-91-8]
Relative retention with reference to clonazepam
(retention time = about 7 min): impurity B = about 2.1 ; DEFINITION
impurity A = about 2.4. 2,6-Dichloro-N-(imidazolidin-2-ylidene)aniline
System suitability : reference solution (b) : hydrochloride.
– resolution : minimum 1.8 between the peaks due to Content : 98.5 per cent to 101.0 per cent (dried substance).
flunitrazepam and to clonazepam.
CHARACTERS
Limits :
Appearance : white or almost white, crystalline powder.
– impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (a) Solubility : soluble in water and in anhydrous ethanol.
(0.1 per cent),
IDENTIFICATION
– impurity B : not more than the area of the principal peak
in the chromatogram obtained with reference solution (c) First identification : B, D.
(0.1 per cent) Second identification : A, C, D.
– any other impurity : for each impurity, not more than the A. Ultraviolet and visible absorption spectrophotometry
area of the principal peak in the chromatogram obtained (2.2.25).
with reference solution (a) (0.1 per cent), Test solution. Dissolve 30.0 mg in 0.01 M hydrochloric acid
– total : not more than twice the area of the principal peak and dilute to 100.0 mL with the same acid.
in the chromatogram obtained with reference solution (a) Spectral range : 245-350 nm.
(0.2 per cent), Absorption maxima : at 272 nm and 279 nm.
– disregard limit : 0.5 times the area of the principal peak in
Point of inflexion : at 265 nm.
the chromatogram obtained with reference solution (a)
(0.05 per cent). Specific absorbance at the absorption maxima :
Loss on drying (2.2.32) : maximum 0.5 per cent, determined – at 272 nm : about 18 ;
on 1.000 g by drying in an oven at 105 °C for 4 h. – at 279 nm : about 16.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on B. Infrared absorption spectrophotometry (2.2.24).
1.0 g. Comparison: clonidine hydrochloride CRS.
ASSAY C. Thin-layer chromatography (2.2.27).
Dissolve 0.275 g in 50 mL of acetic anhydride R. Titrate Test solution. Dissolve 5 mg of the substance to be
with 0.1 M perchloric acid, determining the end-point examined in methanol R and dilute to 5 mL with the same
potentiometrically (2.2.20). solvent.
1 mL of 0.1 M perchloric acid is equivalent to 31.57 mg Reference solution. Dissolve 5 mg of clonidine
of C15H10ClN3O3. hydrochloride CRS in methanol R and dilute to 5 mL with
the same solvent.
STORAGE Plate : TLC silica gel G plate R.
Protected from light. Mobile phase : glacial acetic acid R, butanol R, water R
IMPURITIES (10:40:50 V/V/V) ; allow to separate, filter the upper layer
and use the filtrate.
Specified impurities : A, B.
Application : 10 μL.
Development : over 2/3 of the plate.
Drying : in air.
Detection : spray with potassium iodobismuthate solution R2.
Allow to dry in air for 1 h. Spray again with potassium
iodobismuthate solution R2 and then immediately spray
with a 50 g/L solution of sodium nitrite R.
A. (2-amino-5-nitrophenyl)(2-chlorophenyl)methanone,
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
the reference solution.
D. It gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S. Dissolve 1.25 g in carbon dioxide-free water R and
B. 3-amino-4-(2-chlorophenyl)-6-nitroquinolin-2(1H)-one. dilute to 25 mL with the same solvent.

1926 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Clopamide

Appearance of solution. Solution S is clear (2.2.1) and not (2034). It is therefore not necessary to identify these impurities
more intensely coloured than reference solution Y7 (2.2.2, for demonstration of compliance. See also 5.10. Control of
Method II). impurities in substances for pharmaceutical use): A, B, C.
pH (2.2.3): 4.0 to 5.0 for solution S.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50 mg of the substance to be examined
in mobile phase A and dilute to 50 mL with mobile phase A.
Reference solution (a). Dilute 1.0 mL of the test solution to A. 1-acetylimidazolidin-2-one,
100.0 mL with mobile phase A. Dilute 1.0 mL of this solution
to 10.0 mL with mobile phase A.
Reference solution (b). Dissolve 5 mg of clonidine
impurity B CRS in 2 mL of acetonitrile R and dilute to 5 mL
with mobile phase A. To 1 mL of this solution, add 1 mL of
the test solution and dilute to 10 mL with mobile phase A.
B. 1-acetyl-2-[(2,6-dichlorophenyl)amino]-4,5-dihydro-1H-
Column : imidazole,
– size : l = 0.15 m, Ø = 3.0 mm ;
– stationary phase : propylsilyl silica gel for chromatography R
(5 μm) ;
– temperature : 40 °C.
Mobile phase : C. 2,6-dichloroaniline.
– mobile phase A : dissolve 4 g of potassium dihydrogen
phosphate R in 1000 mL of water R, and adjust to pH 4.0 04/2008:1747
with phosphoric acid R ; corrected 7.0
– mobile phase B : mobile phase A, acetonitrile R1 (25:75 V/V) ;
Time Mobile phase A Mobile phase B CLOPAMIDE
(min) (per cent V/V) (per cent V/V)
0 90 10 Clopamidum
0 - 15 90 → 30 10 → 70
15 - 15.1 30 → 90 70 → 10
15.1 - 20 90 10

Flow rate : 1.5 mL/min.

Detection : spectrophotometer at 210 nm. C14H20ClN3O3S Mr 345.8
Injection : 5 μL. [636-54-4]
System suitability : reference solution (b) : DEFINITION
– resolution : minimum 5 between the peaks due to clonidine 4-Chloro-N-[(2RS,6SR)-2,6-dimethylpiperidin-1-yl]-3-
and impurity B. sulfamoylbenzamide.
Limits : Content : 99.0 per cent to 101.0 per cent (dried substance).
– unspecified impurities : for each impurity, not more than the PRODUCTION
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ; The production method is evaluated to determine the
potential for formation of an N-nitroso compound
– total : not more than twice the area of the principal peak (cis-2,6-dimethyl-1-nitrosopiperidine). Where necessary,
in the chromatogram obtained with reference solution (a) the production method is validated to demonstrate that the
(0.2 per cent) ; N-nitroso compound is absent in the final product.
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a) CHARACTERS
(0.05 per cent). Appearance : white or almost white, hygroscopic, crystalline
Loss on drying (2.2.32) : maximum 0.5 per cent, determined powder.
on 1.000 g by drying in an oven at 105 °C. Solubility : slightly soluble in water and in anhydrous ethanol,
sparingly soluble in methanol.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. It shows polymorphism (5.9).

ASSAY IDENTIFICATION
Dissolve 0.200 g in 70 mL of ethanol (96 per cent) R. Titrate Infrared absorption spectrophotometry (2.2.24).
with 0.1 M ethanolic sodium hydroxide determining the Comparison: clopamide CRS.
end-point potentiometrically (2.2.20). If the spectra obtained in the solid state show differences,
1 mL of 0.1 M sodium hydroxide is equivalent to 26.66 mg dissolve the substance to be examined and the reference
of C9H10Cl3N3. substance separately in the minimum volume of methanol R,
evaporate to dryness on a water-bath and record new spectra
IMPURITIES using the residues.
Other detectable impurities (the following substances would, TESTS
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general Related substances. Liquid chromatography (2.2.29).
acceptance criterion for other/unspecified impurities and/or Test solution. Dissolve 100 mg of the substance to be examined
by the general monograph Substances for pharmaceutical use in methanol R and dilute to 10.0 mL with the same solvent.

General Notices (1) apply to all monographs and other texts 1927
Clopidogrel hydrogen sulfate EUROPEAN PHARMACOPOEIA 8.0

Reference solution (a). Dissolve 10 mg of clopamide for system Filter the solutions through a membrane filter (nominal pore
suitability CRS (containing impurities B, C and H) in 1.0 mL size 0.45 μm) to evaluate the result.
of methanol R. Loss on drying (2.2.32) : maximum 2.5 per cent, determined
Reference solution (b). Dilute 2.0 mL of the test solution to on 1.000 g by drying in an oven at 105 °C.
100.0 mL with methanol R. Dilute 2.0 mL of this solution to Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
40.0 mL with methanol R. 1.0 g.
Column :
– size : l = 0.15 m, Ø = 4.6 mm ; ASSAY
– stationary phase : end-capped octylsilyl silica gel for Dissolve 0.280 g in 70 mL of anhydrous acetic acid R. Titrate
chromatography R (5 μm). with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
Mobile phase :
1 mL of 0.1 M perchloric acid is equivalent to 34.58 mg
– mobile phase A : dissolve 1.0 g of ammonium acetate R in of C14H20ClN3O3S.
950 mL of water R, adjust to pH 2.0 with phosphoric acid R
and dilute to 1000 mL with water R ; STORAGE
– mobile phase B : acetonitrile R ; In an airtight container, protected from light.
– mobile phase C : water R, tetrahydrofuran for IMPURITIES
chromatography R (20:80 V/V) ; this mobile phase allows
adequate rinsing of the system ; Specified impurities : B, C, H.
Other detectable impurities (the following substances would,
Time Mobile phase A Mobile phase B Mobile phase C if present at a sufficient level, be detected by one or other of
(min) (per cent V/V) (per cent V/V) (per cent V/V) the tests in the monograph. They are limited by the general
0 - 35 95 → 75 5 → 25 0 acceptance criterion for other/unspecified impurities and/or
35 - 45 75 → 35 25 → 65 0 by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
45 - 50 35 → 30 65 → 0 0 → 70 for demonstration of compliance. See also 5.10. Control of
50 - 60 30 0 70 impurities in substances for pharmaceutical use) : A, G.

Flow rate : 0.4 mL/min.

Detection : spectrophotometer at 235 nm.
Injection : 10 μL.
Identification of impurities : use the chromatogram
supplied with clopamide for system suitability CRS and the A. R = CH3 : 4-chloro-N-[(2RS,6RS)-2,6-dimethylpiperidin-1-
chromatogram obtained with reference solution (a) to identify yl]-3-sulfamoylbenzamide (trans-clopamide),
the peaks due to impurities B, C and H.
G. R = H : 4-chloro-N-[(2RS)-2-methylpiperidin-1-yl]-3-
Relative retention with reference to clopamide (retention sulfamoylbenzamide,
time = about 33 min) : impurity C = about 0.8 ;
impurity H = about 1.2 ; impurity B = about 1.4.
System suitability: reference solution (a) :
– resolution : minimum 3 between the peaks due to impurity C
and clopamide. B. R = H : 4-chlorobenzoic acid,
Limits : C. R = SO2-NH2 : 4-chloro-3-sulfamoylbenzoic acid,
– correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
corresponding correction factor : impurity B = 0.5 ;
impurity H = 0.4 ;
– impurities B, C, H : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.2 per cent) ; H. 4-chloro-3-[(E)-[(dimethylamino)methylene]sulfamoyl]-
N-[(2RS,6SR)-2,6-dimethylpiperidin-1-yl]benzamide.
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.10 per cent) ; 04/2011:2531
– total : not more than 10 times the area of the principal peak
in the chromatogram obtained with reference solution (b) CLOPIDOGREL HYDROGEN SULFATE
(1.0 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in Clopidogreli hydrogenosulfas
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 0.25 g in a mixture of 20 volumes of acetone R and
85 volumes of methanol R and dilute to 20 mL with the same
mixture of solvents. 20 mL of the solution complies with
modified test B. Prepare the reference solution by diluting C16H18ClNO6S2 Mr 419.9
0.5 mL of lead standard solution (10 ppm Pb) R to 20 mL [120202-66-6]
with a mixture of 20 volumes of acetone R and 85 volumes
of methanol R. Prepare the blank solution by using 20 mL DEFINITION
of a mixture of 20 volumes of acetone R and 85 volumes of Methyl (2S)-(2-chlorophenyl)[6,7-dihydrothieno[3,2-
methanol R. c]pyridin-5(4H)-yl]acetate sulfate.

1928 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Clopidogrel hydrogen sulfate

Content : 99.0 per cent to 101.0 per cent (anhydrous substance). Reference solution (a). Dissolve 5 mg of clopidogrel
impurity A CRS in the solvent mixture and dilute to 25.0 mL
CHARACTERS with the solvent mixture.
Appearance : white or almost white powder. Reference solution (b). Dissolve 32 mg of clopidogrel for system
Solubility : freely soluble in water and in methanol, practically suitability CRS (containing impurities B and C) in the solvent
insoluble in cyclohexane. mixture, add 0.5 mL of reference solution (a) and dilute to
It shows polymorphism (5.9). 5.0 mL with the solvent mixture.
Reference solution (c). Dilute 1.0 mL of the test solution to
IDENTIFICATION 100.0 mL with the solvent mixture. Dilute 1.0 mL of this
Carry out either tests A, B, D or tests B, C, D. solution to 10.0 mL with the solvent mixture.
A. Specific optical rotation (2.2.7): + 54.0 to + 58.0 (anhydrous Column :
substance). – size : l = 0.15 m, Ø = 3.9 mm ;
Dissolve 0.250 g in methanol R and dilute to 25.0 mL with – stationary phase : end-capped octadecylsilyl silica gel for
the same solvent. chromatography R (5 μm) ;
B. Infrared absorption spectrophotometry (2.2.24). – temperature : 30 °C.
Comparison : clopidogrel hydrogen sulfate CRS. Mobile phase :
If the spectra obtained show differences, dissolve the – mobile phase A : mix 5 volumes of methanol R2
substance to be examined and the reference substance and 95 volumes of a 0.96 g/L solution of sodium
separately in anhydrous ethanol R, evaporate to dryness pentanesulfonate monohydrate R adjusted to pH 2.5 with
and record new spectra using the residues (the substance phosphoric acid R ;
may stick to the surface of the recipient used). – mobile phase B : methanol R2, acetonitrile R1 (5:95 V/V) ;
C. Enantiomeric purity (see Tests).
Time Mobile phase A Mobile phase B
D. It gives reaction (a) of sulfates (2.3.1). (min) (per cent V/V) (per cent V/V)
TESTS 0-3 89.5 10.5

Appearance of solution. The solution is clear (2.2.1) and not 3 - 48 89.5 → 31.5 10.5 → 68.5
more intensely coloured than reference solution Y6 (2.2.2, 48 - 68 31.5 68.5
Method I).
Dissolve 1.0 g in methanol R and dilute to 20.0 mL with the Flow rate : 1.0 mL/min.
same solvent. Detection : spectrophotometer at 220 nm.
Enantiomeric purity. Liquid chromatography (2.2.29) : use Injection : 10 μL of the test solution and reference solutions (b)
the normalisation procedure. and (c).
Test solution. Dissolve 0.1 g of the substance to be examined Identification of impurities : use the chromatogram
in 25.0 mL of anhydrous ethanol R and dilute to 50.0 mL with supplied with clopidogrel for system suitability CRS and the
heptane R. chromatogram obtained with reference solution (b) to identify
Reference solution. Dissolve 10 mg of clopidogrel for system the peaks due to impurities A and B.
suitability CRS (containing impurities B and C) in 2.5 mL of Relative retention with reference to clopidogrel (retention
anhydrous ethanol R and dilute to 5.0 mL with heptane R. time = about 25 min) : impurity A = about 0.4 ;
Column : impurity B = about 1.1.
– size : l = 0.25 m, Ø = 4.6 mm ; System suitability : reference solution (b) :
– stationary phase : silica gel OJ for chiral separations R – peak-to-valley ratio : minimum 10, where Hp = height above
(10 μm). the baseline of the peak due to impurity B and Hv = height
Mobile phase : anhydrous ethanol R, heptane R (15:85 V/V). above the baseline of the lowest point of the curve
separating this peak from the peak due to clopidogrel.
Flow rate : 0.8 mL/min.
Limits :
Detection : spectrophotometer at 220 nm.
– impurity B : not more than 3 times the area of the principal
Injection : 10 μL. peak in the chromatogram obtained with reference
Run time : 1.25 times the retention time of clopidogrel. solution (c) (0.3 per cent) ;
Identification of impurities : use the chromatogram – impurity A : not more than twice the area of the principal
supplied with clopidogrel for system suitability CRS and the peak in the chromatogram obtained with reference
chromatogram obtained with the reference solution to identify solution (c) (0.2 per cent) ;
the peaks due to impurities B and C. – unspecified impurities : for each impurity, not more than the
Relative retention with reference to clopidogrel (retention area of the principal peak in the chromatogram obtained
time = about 18 min) : impurity C = about 0.6 ; with reference solution (c) (0.10 per cent) ;
impurity B = about 0.7. – total : not more than 5 times the area of the principal peak
System suitability: reference solution : in the chromatogram obtained with reference solution (c)
– resolution : minimum 2.0 between the peaks due to (0.5 per cent) ;
impurities C and B ; – disregard limit : 0.5 times the area of the principal peak in
– signal-to-noise ratio : minimum 20 for the peak due to the chromatogram obtained with reference solution (c)
impurity C. (0.05 per cent).
Limit : Heavy metals (2.4.8) : maximum 20 ppm.
– impurity C : maximum 0.5 per cent. 1.0 g complies with test C. Prepare the reference solution using
Related substances. Liquid chromatography (2.2.29). 2 mL of lead standard solution (10 ppm Pb) R.
Solvent mixture : mobile phase A, acetonitrile R1 (40:60 V/V). Water (2.5.12) : maximum 0.5 per cent, determined on 1.00 g.
Test solution. Dissolve 65 mg of the substance to be examined Replace the solvent after each titration.
in the solvent mixture and dilute to 10.0 mL with the solvent Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
mixture. 1.0 g.

General Notices (1) apply to all monographs and other texts 1929
Closantel sodium dihydrate for veterinary use EUROPEAN PHARMACOPOEIA 8.0

ASSAY 01/2008:1716
corrected 7.0
Dissolve 0.160 g in a mixture of 10 mL of acetone R, 10 mL of
methanol R and 30 mL of water R. Titrate with 0.1 M sodium
hydroxide, determining the end-point potentiometrically CLOSANTEL SODIUM DIHYDRATE
(2.2.20). A precipitate may be formed during the titration. FOR VETERINARY USE
1 mL of 0.1 M sodium hydroxide is equivalent to 20.99 mg
of C16H18ClNO6S2. Closantelum natricum dihydricum
ad usum veterinarium
STORAGE
Protected from light.

IMPURITIES
Specified impurities : A, B, C.
Other detectable impurities (the following substances would, C22H13Cl2I2N2NaO2,2H2O Mr 721
if present at a sufficient level, be detected by one or other of [61438-64-0]
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or DEFINITION
by the general monograph Substances for pharmaceutical N-[5-Chloro-4-[(RS)-(4-chlorophenyl)cyanomethyl]-2-
use (2034). It is therefore not necessary to identify these methylphenyl]-2-hydroxy-3,5-diiodobenzamide sodium salt
impurities for demonstration of compliance. See also 5.10. dihydrate.
Control of impurities in substances for pharmaceutical use): D.
Content : 98.5 per cent to 101.5 per cent (anhydrous substance).
CHARACTERS
Appearance : yellow powder, slightly hygroscopic.
Solubility : very slightly soluble in water, freely soluble in
ethanol (96 per cent), soluble in methanol.
It shows polymorphism (5.9).
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
A. (2S)-(2-chlorophenyl)[6,7-dihydrothieno[3,2-c]pyridin- Preparation : discs without recrystallisation.
5(4H)-yl]acetic acid,
Comparison: closantel sodium dihydrate CRS.
B. Dissolve 0.1 g in 2 mL of ethanol (96 per cent) R. The
solution gives reaction (a) of sodium (2.3.1).
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution GY4 (2.2.2,
Method II).
Dissolve 0.50 g in ethanol (96 per cent) R and dilute to 50 mL
with the same solvent.
B. methyl (2S)-(2-chlorophenyl)[4,7-dihydrothieno[2,3-
c]pyridin-6(5H)-yl]acetate, Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use and protect from light.
Test solution. Dissolve 0.100 g of the substance to be examined
in methanol R and dilute to 10.0 mL with the same solvent.
Reference solution (a). Dissolve 10 mg of closantel for system
suitability CRS (containing impurities A to J) in methanol R
and dilute to 1.0 mL with the same solvent.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with methanol R. Dilute 5.0 mL of this solution to
25.0 mL with methanol R.
C. methyl (2R)-(2-chlorophenyl)[6,7-dihydrothieno[3,2- Column :
c]pyridin-5(4H)-yl]acetate, – size : l = 0.10 m, Ø = 4.6 mm,
– stationary phase : base-deactivated octadecylsilyl silica gel for
chromatography R (3 μm),
– temperature : 35 °C.
Mobile phase :
– mobile phase A : to 100 mL of a 7.7 g/L solution of
ammonium acetate R previously adjusted to pH 4.3 with
acetic acid R, add 50 mL of acetonitrile R and 850 mL of
water R;
– mobile phase B : to 100 mL of a 7.7 g/L solution of
ammonium acetate R previously adjusted to pH 4.3
D. methyl (2R)-(2-chlorophenyl)[(2S)-(2-chlorophenyl)[6,7- with acetic acid R, add 50 mL of water R and 850 mL of
dihydrothieno[3,2-c]pyridin-5(4H)-yl]acetyloxy]acetate. acetonitrile R;

1930 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Clotrimazole

Time Mobile phase A Mobile phase B

(min) (per cent V/V) (per cent V/V)
0-2 50 50
2 - 22 50 → 20 50 → 80
B. (2RS)-(4-amino-2-chloro-5-methylphenyl)(4-
22 - 27 20 80
chlorophenyl)ethanenitrile,
Flow rate : 1.5 mL/min.
Detection : spectrophotometer at 240 nm.
Injection : 10 μL.
Relative retention with reference to closantel (retention
time = about 16 min) : impurity A = about 0.07 ;
impurity B = about 0.48 ; impurity C = about 0.62 ;
impurity D = about 0.65 ; impurity E = about 0.82 ;
impurity F = about 0.89 ; impurity G = about 0.93 ; C. R1 = H, R2 = CO2H, R3 = I : (2RS)-[2-chloro-4-[(2-
impurity H = about 1.13 ; impurity I = about 1.16 ; hydroxy-3,5-diiodobenzoyl)amino]-5-methylphenyl](4-
impurity J = about 1.55. chlorophenyl)acetic acid,
System suitability: reference solution (a) : D. R1 = H, R2 = CONH2, R3 = I : N-[4-[(1RS)-2-amino-1-(4-
– resolution : baseline separation between the peaks due to chlorophenyl)-2-oxoethyl]-5-chloro-2-methylphenyl]-2-
impurity G and closantel, hydroxy-3,5-diiodobenzamide,
– the chromatogram obtained is similar to the chromatogram E. R1 = H, R2 = CN, R3 = Cl : 3-chloro-N-[5-chloro-4-
supplied with closantel for system suitability CRS. [(RS)-(4-chlorophenyl)cyanomethyl]-2-methylphenyl]-2-
Limits : hydroxy-5-iodobenzamide,
– correction factors : for the calculation of contents, F. R1 + R2 = O, R3 = I : N-[5-chloro-4-(4-chlorobenzoyl)-2-
multiply the peak areas of the following impurities by methylphenyl]-2-hydroxy-3,5-diiodobenzamide,
the corresponding correction factor : impurity A = 1.5 ; G. R1 = H, R2 = C(=NH)OCH3, R3 = I : methyl (2RS)-2-
impurity B = 1.3 ; [2-chloro-4-[(2-hydroxy-3,5-diiodobenzoyl)amino]-5-
– impurity G : not more than 2.5 times the area of the methylphenyl]-2-(4-chlorophenyl)acetimidate,
principal peak in the chromatogram obtained with H. R1 = H, R2 = CO-OCH3, R3 = I : methyl
reference solution (b) (0.5 per cent) ; (2RS)-[2-chloro-4-[(2-hydroxy-3,5-diiodobenzoyl)amino]-
– impurities F, H, I : for each impurity, not more than 1.5 times 5-methylphenyl](4-chlorophenyl)acetate,
the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.3 per cent) ; I. R1 = R3 = H, R2 = CN : N-[5-chloro-4-[(RS)-(4-
chlorophenyl)cyanomethyl]-2-methylphenyl]-2-hydroxy-
– impurities A, B, C, D, E, J : for each impurity, not more 5-iodobenzamide,
than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.2 per cent) ;
– any other impurity : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.2 per cent) ;
– total : not more than 7.5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(1.5 per cent) ;
– disregard limit : 0.25 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent).
Water (2.5.12) : 4.8 per cent to 5.8 per cent, determined on
0.250 g. J. N-[5-chloro-4-[[4-[[2-chloro-4-[(2-hydroxy-3,5-
Use a mixture of 1 volume of dimethylformamide R and diiodobenzoyl)amino]-5-methylphenyl]cyanometh-
4 volumes of methanol R as the solvent. yl]phenyl](4-chlorophenyl)cyanomethyl]-2-methylphen-
yl]-2-hydroxy-3,5-diiodobenzamide.
ASSAY
Dissolve 0.500 g in 50 mL of a mixture of 1 volume of 04/2008:0757
anhydrous acetic acid R and 7 volumes of methyl ethyl
ketone R. Titrate with 0.1 M perchloric acid, determining the CLOTRIMAZOLE
end-point potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 68.5 mg Clotrimazolum
of C22H13Cl2I2N2NaO2.
STORAGE
In an airtight container, protected from light.
IMPURITIES
Specified impurities : A, B, C, D, E, F, G, H, I, J.

C22H17ClN2 Mr 344.8
[23593-75-1]
DEFINITION
A. 2-hydroxy-3,5-diiodobenzoic acid, 1-[(2-Chlorophenyl)diphenylmethyl]-1H-imidazole.

General Notices (1) apply to all monographs and other texts 1931
Clotrimazole EUROPEAN PHARMACOPOEIA 8.0

Content : 98.5 per cent to 100.5 per cent (dried substance). Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 210 nm.
CHARACTERS
Injection : 10 μL.
Appearance : white or pale yellow, crystalline powder.
Relative retention with reference to clotrimazole
Solubility : practically insoluble in water, soluble in ethanol (retention time = about 12 min) : impurity D = about 0.1 ;
(96 per cent) and in methylene chloride. impurity F = about 0.9 ; impurity B = about 1.1 ;
IDENTIFICATION impurity E = about 1.5 ; impurity A = about 1.8.
First identification : B. System suitability : reference solution (b) :
Second identification : A, C. – resolution : minimum 1.5 between the peaks due to
impurity F and clotrimazole ;
A. Melting point (2.2.14) : 141 °C to 145 °C.
– the chromatogram obtained is similar to the chromatogram
B. Infrared absorption spectrophotometry (2.2.24). supplied with clotrimazole for peak identification CRS.
Comparison : clotrimazole CRS. Limits :
C. Thin-layer chromatography (2.2.27). – impurities A, B : for each impurity, not more than twice the
Test solution. Dissolve 50 mg of the substance to be area of the principal peak in the chromatogram obtained
examined in ethanol (96 per cent) R and dilute to 5 mL with reference solution (a) (0.2 per cent) ;
with the same solvent. – impurities D, E : for each impurity, not more than the area
Reference solution. Dissolve 50 mg of clotrimazole CRS in of the corresponding peak in the chromatogram obtained
ethanol (96 per cent) R and dilute to 5 mL with the same with reference solution (c) (0.2 per cent) ;
solvent. – impurity F : not more than the area of the principal peak
Plate : TLC silica gel F254 plate R. in the chromatogram obtained with reference solution (a)
(0.1 per cent) ;
Mobile phase : concentrated ammonia R1, propanol R,
toluene R (0.5:10:90 V/V/V). – unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
Application : 10 μL.
with reference solution (a) (0.10 per cent) ;
Development : over 2/3 of the plate. – total : not more than 5 times the area of the principal peak
Drying : in air. in the chromatogram obtained with reference solution (a)
Detection : examine in ultraviolet light at 254 nm. (0.5 per cent) ;
Results : the principal spot in the chromatogram obtained – disregard limit : 0.5 times the area of the principal peak in
with the test solution is similar in position and size to the the chromatogram obtained with reference solution (a)
principal spot in the chromatogram obtained with the (0.05 per cent).
reference solution. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
TESTS
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Related substances. Liquid chromatography (2.2.29). 1.0 g.
Test solution. Dissolve 50.0 mg of the substance to be
examined in acetonitrile R1 and dilute to 50.0 mL with the ASSAY
same solvent. Dissolve 0.300 g in 80 mL of anhydrous acetic acid R. Using
Reference solution (a). Dilute 1.0 mL of the test solution to 0.3 mL of naphtholbenzein solution R as indicator, titrate
100.0 mL with acetonitrile R1. Dilute 1.0 mL of this solution with 0.1 M perchloric acid until the colour changes from
to 10.0 mL with acetonitrile R1. brownish-yellow to green.
Reference solution (b). Dissolve the contents of a vial of 1 mL of 0.1 M perchloric acid is equivalent to 34.48 mg
clotrimazole for peak identification CRS (containing impurities of C22H17ClN2.
A, B and F) in 1.0 mL of acetonitrile R1.
STORAGE
Reference solution (c). Dissolve 5.0 mg of imidazole CRS
Protected from light.
(impurity D) and 5.0 mg of clotrimazole impurity E CRS in
acetonitrile R1 and dilute to 100.0 mL with the same solvent. IMPURITIES
Dilute 1.0 mL of this solution to 25.0 mL with acetonitrile R1. Specified impurities : A, B, D, E, F.
Column : Other detectable impurities (the following substances would,
– size : l = 0.15 m, Ø = 4.6 mm ; if present at a sufficient level, be detected by one or other of
– stationary phase : spherical end-capped octylsilyl silica gel for the tests in the monograph. They are limited by the general
chromatography R (5 μm) ; acceptance criterion for other/unspecified impurities and/or
– temperature : 40 °C. by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
Mobile phase : impurities for demonstration of compliance. See also 5.10.
– mobile phase A : dissolve 1.0 g of potassium dihydrogen Control of impurities in substances for pharmaceutical use) : C.
phosphate R and 0.5 g of tetrabutylammonium hydrogen
sulfate R1 in water R and dilute to 1000 mL with the same
solvent ;
– mobile phase B : acetonitrile R1 ;
Time Mobile phase A Mobile phase B
A. R = OH, R′ = C6H5 : (2-chlorophenyl)diphenylmethanol,
(min) (per cent V/V) (per cent V/V)
0-3 75 25 C. R = Cl, R′ = C6H5 : 1-chloro-2-(chlorodiphenylmethyl)-
benzene,
3 - 25 75 → 20 25 → 80
25 - 30 20 80 E. R + R′ = O : (2-chlorophenyl)phenylmethanone
(2-chlorobenzophenone),

1932 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cloxacillin sodium

System suitability : reference solution (b) :

– the chromatogram shows 3 clearly separated spots.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
reference solution (a).
B. R = Cl : 1-[(4-chlorophenyl)diphenylmethyl]-1H-imidazole, C. Place about 2 mg in a test-tube about 150 mm long and
15 mm in diameter. Moisten with 0.05 mL of water R and
F. R = H : 1-(triphenylmethyl)-1H-imidazole (deschloro- add 2 mL of sulfuric acid-formaldehyde reagent R. Mix the
clotrimazole), contents of the tube by swirling ; the solution is slightly
greenish-yellow. Place the test-tube in a water-bath for
1 min ; the solution becomes yellow.
D. It gives reaction (a) of sodium (2.3.1).
D. imidazole. TESTS
Solution S. Dissolve 2.50 g in carbon dioxide-free water R and
01/2008:0661 dilute to 25.0 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and its
CLOXACILLIN SODIUM absorbance (2.2.25) at 430 nm is not greater than 0.04.
pH (2.2.3) : 5.0 to 7.0 for solution S.
Cloxacillinum natricum Specific optical rotation (2.2.7) : + 160 to + 169 (anhydrous
substance).
Dissolve 0.250 g in water R and dilute to 25.0 mL with the
same solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution (a). Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with the
mobile phase.
C19H17ClN3NaO5S,H2O Mr 475.9 Test solution (b). Dilute 5.0 mL of test solution (a) to 50.0 mL
[7081-44-9] with the mobile phase.
DEFINITION Reference solution (a). Dissolve 50.0 mg of cloxacillin
Sodium (2S,5R,6R)-6-[[[3-(2-chlorophenyl)-5- sodium CRS in the mobile phase and dilute to 50.0 mL with
methylisoxazol-4-yl]carbonyl]amino]-3,3-dimethyl-7-oxo-4- the mobile phase. Dilute 5.0 mL of this solution to 50.0 mL
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate monohydrate. with the mobile phase.
Semi-synthetic product derived from a fermentation product. Reference solution (b). Dilute 5.0 mL of test solution (b) to
50.0 mL with the mobile phase.
Content : 95.0 per cent to 102.0 per cent (anhydrous substance).
Reference solution (c). Dissolve 5 mg of flucloxacillin
CHARACTERS sodium CRS and 5 mg of cloxacillin sodium CRS in the mobile
Appearance : white or almost white, hygroscopic, crystalline phase and dilute to 50.0 mL with the mobile phase.
powder. Column :
Solubility : freely soluble in water and in methanol, soluble in – size : l = 0.25 m, Ø = 4 mm ;
ethanol (96 per cent). – stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
IDENTIFICATION
Mobile phase : mix 25 volumes of acetonitrile R and 75 volumes
First identification : A, D. of a 2.7 g/L solution of potassium dihydrogen phosphate R
Second identification : B, C, D. adjusted to pH 5.0 with dilute sodium hydroxide solution R.
A. Infrared absorption spectrophotometry (2.2.24). Flow rate : 1.0 mL/min.
Preparation : discs. Detection : spectrophotometer at 225 nm.
Comparison : cloxacillin sodium CRS. Injection : 20 μL of test solution (a) and reference solutions (b)
B. Thin-layer chromatography (2.2.27). and (c).
Test solution. Dissolve 25 mg of the substance to be Run time : 5 times the retention time of cloxacillin.
examined in 5 mL of water R. System suitability : reference solution (c) :
Reference solution (a). Dissolve 25 mg of cloxacillin – resolution : minimum 2.5 between the peaks due to
sodium CRS in 5 mL of water R. cloxacillin (1st peak) and flucloxacillin (2nd peak).
Reference solution (b). Dissolve 25 mg of cloxacillin Limits :
sodium CRS, 25 mg of dicloxacillin sodium CRS and 25 mg – any impurity : not more than the area of the principal peak
of flucloxacillin sodium CRS in 5 mL of water R. in the chromatogram obtained with reference solution (b)
Plate : TLC silanised silica gel plate R. (1.0 per cent) ;
Mobile phase : mix 30 volumes of acetone R and 70 volumes – total : not more than 5 times the area of the principal peak
of a 154 g/L solution of ammonium acetate R, then adjust in the chromatogram obtained with reference solution (b)
to pH 5.0 with glacial acetic acid R. (5.0 per cent) ;
Application : 1 μL. – disregard limit : 0.05 times the area of the principal peak
Development : over a path of 15 cm. in the chromatogram obtained with reference solution (b)
Drying : in air. (0.05 per cent).
Detection : expose to iodine vapour until the spots appear ; N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm.
examine in daylight. 2-Ethylhexanoic acid (2.4.28) : maximum 0.8 per cent m/m.

General Notices (1) apply to all monographs and other texts 1933
Clozapine EUROPEAN PHARMACOPOEIA 8.0

Water (2.5.12) : 3.0 per cent to 4.5 per cent, determined on 01/2008:1191
0.300 g.
Bacterial endotoxins (2.6.14) : less than 0.20 IU/mg, if CLOZAPINE
intended for use in the manufacture of parenteral preparations
without a further appropriate procedure for the removal of Clozapinum
bacterial endotoxins.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications.
Injection : test solution (b) and reference solution (a).
System suitability :
– repeatability : maximum relative standard deviation of C18H19ClN4 Mr 326.8
1.0 per cent after 6 injections of reference solution (a). [5786-21-0]
Calculate the percentage content of C19H17ClN3NaO5S from DEFINITION
the declared content of cloxacillin sodium CRS.
8-Chloro-11-(4-methylpiperazin-1-yl)-5H-dibenzo[b,e][1,4]-
STORAGE diazepine.
In an airtight container, at a temperature not exceeding Content : 99.0 per cent to 101.0 per cent (dried substance).
25 °C. If the substance is sterile, store in a sterile, airtight,
CHARACTERS
tamper-proof container.
Appearance : yellow, crystalline powder.
IMPURITIES Solubility : practically insoluble in water, freely soluble in
methylene chloride, soluble in ethanol (96 per cent). It
dissolves in dilute acetic acid.
IDENTIFICATION
A. Melting point (2.2.14) : 182 °C to 186 °C.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: clozapine CRS.
A. R = CO2H : (4S)-2-[carboxy[[[3-(2-chlorophenyl)-
TESTS
5-methylisoxazol-4-yl]carbonyl]amino]methyl]-5,5-
dimethylthiazolidine-4-carboxylic acid (penicilloic acid of Related substances. Liquid chromatography (2.2.29).
cloxacillin), Solvent mixture : water R, methanol R2 (20:80 V/V).
B. R = H : (2RS,4S)-2-[[[[3-(2-chlorophenyl)-5- Solution A. Dissolve 2.04 g of potassium dihydrogen
methylisoxazol-4-yl]carbonyl]amino]methyl]-5,5- phosphate R in 1000 mL of water R and adjust to pH 2.4 ± 0.05
dimethylthiazolidine-4-carboxylic acid (penilloic acid of with dilute phosphoric acid R.
cloxacillin), Test solution. Dissolve 75 mg of the substance to be examined
in 80 mL of methanol R2 and dilute to 100 mL with water R.
Reference solution (a). Dilute 1.0 mL of the test solution
to 10.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 100.0 mL with the solvent mixture.
Reference solution (b). Dissolve the contents of a vial of
clozapine for peak identification CRS (containing impurities A,
C. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia- B, C and D) in 1.0 mL of the solvent mixture.
1-azabicyclo[3.2.0]heptane-2-carboxylic acid
(6-aminopenicillanic acid), Column :
– size : l = 0.125 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase :
– mobile phase A : acetonitrile for chromatography R,
methanol R2, solution A (1:1:8 V/V/V) ;
– mobile phase B : acetonitrile for chromatography R,
D. 3-(2-chlorophenyl)-5-methylisoxazole-4-carboxylic acid, methanol R2, solution A (4:4:2 V/V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-4 100 0
4 - 24 100 → 0 0 → 100
24 - 29 0 100

Flow rate : 1.2 mL/min.

Detection : spectrophotometer at 257 nm.
E. (2S,5R,6R)-6-[[[(2S,5R,6R)-6-[[[3-(2-chlorophenyl)-5- Injection : 20 μL.
methylisoxazol-4-yl]carbonyl]amino]-3,3-dimethyl-7- Identification of impurities : use the chromatogram
oxo-4-thia-1-azabicyclo[3.2.0]hept-2-yl]carbonyl]amino]- supplied with clozapine for peak identification CRS and the
3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2- chromatogram obtained with reference solution (b) to identify
carboxylic acid (6-APA cloxacillin amide). the peaks due to impurities A, B, C and D.

1934 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cocaine hydrochloride

Relative retention with reference to clozapine (retention

time = about 11 min) : impurity C = about 0.9 ;
impurity D = about 1.1 ; impurity A = about 1.6 ;
impurity B = about 1.7.
System suitability : reference solution (b) :
– resolution : minimum 2.5 between the peaks due to
impurity C and clozapine ; C. 8-chloro-11-(piperazin-1-yl)-5H-diben-
– the chromatogram obtained with reference solution (b) is zo[b,e][1,4]diazepine,
similar to the chromatogram supplied with clozapine for
peak identification CRS.
Limits :
– correction factor : for the calculation of content, multiply
the peak area of impurity D by 2.7 ;
– impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.1 per cent) ; D. 1-[2-[(2-amino-4-chlorophenyl)amino]benzoyl]-4-
– impurities B, D : for each impurity, not more than twice the methylpiperazine.
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.2 per cent) ; 01/2008:0073
– impurity C : not more than 3 times the area of the principal corrected 6.0
peak in the chromatogram obtained with reference
solution (a) (0.3 per cent) ; COCAINE HYDROCHLORIDE
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained Cocaini hydrochloridum
with reference solution (a) (0.10 per cent) ;
– total : not more than 6 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.6 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent).
C17H22ClNO4 Mr 339.8
Heavy metals (2.4.8) : maximum 20 ppm. [53-21-4]
1.0 g complies with test C. Prepare the reference solution using
2 mL of lead standard solution (10 ppm Pb) R. DEFINITION
Loss on drying (2.2.32) : maximum 0.5 per cent, determined Methyl (1R,2R,3S,5S)-3-(benzoyloxy)-8-methyl-8-
on 1.000 g by drying in an oven at 105 °C. azabicyclo[3.2.1]octane-2-carboxylate hydrochloride.
Content : 98.5 per cent to 101.0 per cent (dried substance).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. CHARACTERS
ASSAY Appearance : white or almost white, crystalline powder or
colourless crystals.
Dissolve 0.100 g in 50 mL of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point Solubility : very soluble in water, freely soluble in alcohol,
potentiometrically (2.2.20). slightly soluble in methylene chloride.
1 mL of 0.1 M perchloric acid is equivalent to 16.34 mg mp : about 197 °C, with decomposition.
of C18H19ClN4. IDENTIFICATION
IMPURITIES First identification : B, D.
Specified impurities : A, B, C, D. Second identification : A, C, D, E.
A. Dissolve 20.0 mg in 0.01 M hydrochloric acid and dilute to
100.0 mL with the same acid. Dilute 5.0 mL of the solution
to 50.0 mL with 0.01 M hydrochloric acid. Examined
between 220 nm and 350 nm (2.2.25), the solution shows
2 absorption maxima, at 233 nm and 273 nm. The specific
absorbance at 233 nm is 378 to 402.
B. Infrared absorption spectrophotometry (2.2.24).
A. 8-chloro-5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-
one, Comparison: Ph. Eur. reference spectrum of cocaine
hydrochloride.
C. Dissolve 0.1 g in 5 mL of water R and add 1 mL of dilute
ammonia R2. A white precipitate is formed. Initiate
crystallisation by scratching the wall of the tube with a
glass rod. The crystals, washed with water R and dried in
vacuo, melt (2.2.14) at 96 °C to 99 °C.
D. It gives reaction (a) of chlorides (2.3.1).
E. It gives the reaction of alkaloids (2.3.1).
TESTS
B. 11,11′-(piperazine-1,4-diyl)bis(8-chloro-5H- Solution S. Dissolve 0.5 g in water R and dilute to 25 mL with
dibenzo[b,e][1,4]diazepine), the same solvent.

General Notices (1) apply to all monographs and other texts 1935
Coconut oil, refined EUROPEAN PHARMACOPOEIA 8.0

Appearance of solution. Solution S is clear (2.2.1) and IMPURITIES

colourless (2.2.2, Method II).
Acidity. To 10 mL of solution S add 0.05 mL of methyl red
solution R. Not more than 0.2 mL of 0.02 M sodium hydroxide
is required to change the colour of the indicator.
Specific optical rotation (2.2.7) : − 70 to − 73 (dried
substance).
Dissolve 0.50 g in water R and dilute to 20.0 mL with the same
solvent.
Readily carbonisable substances. To 0.2 g add 2 mL of A. methyl (1R,2R,3S,5S)-8-methyl-3-[[(E)-3-
sulfuric acid R. After 15 min, the solution is not more intensely phenylpropenoyl]oxy]-8-azabicyclo[3.2.1]octane-2-
coloured than reference solution BY5 (2.2.2, Method I). carboxylate (cinnamoylcocaine),
Related substances. Examine by liquid chromatography
(2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with the
mobile phase.
Reference solution (a). Dilute 1.0 mL of the test solution to
50.0 mL with the mobile phase. Dilute 5.0 mL of this solution B. bis[(1R,2R,3S,5S)-2-(methoxycarbonyl)-8-methyl-
to 100.0 mL with the mobile phase. 8-azabicyclo[3.2.1]oct-3-yl] (1r,2c,3t,4t)-2,4-
Reference solution (b). Dissolve 25 mg of the substance to be diphenylcyclobutane-1,3-dicarboxylate (α-truxilline),
examined in 0.01 M sodium hydroxide and dilute to 10.0 mL
with the same solvent. Dilute 1.0 mL of the solution to
10.0 mL with 0.01 M sodium hydroxide. Allow the solution to
stand for 15 min.
Column :
– size : l = 0.15 m, Ø = 4.6 mm,
C. bis[(1R,2R,3S,5S)-2-(methoxycarbonyl)-8-methyl-
– stationary phase : end-capped octadecylsilyl silica gel for 8-azabicyclo[3.2.1]oct-3-yl] (1r,2c,3t,4t)-3,4-
chromatography R (5 μm) with a specific surface area of diphenylcyclobutane-1,2-dicarboxylate (β-truxilline).
335 m2/g, a pore size of 10 nm and a carbon loading of
19.1 per cent,
01/2010:1410
– temperature : 35 °C.
Mobile phase : triethylamine R, tetrahydrofuran R, COCONUT OIL, REFINED
acetonitrile R, water R (0.5:100:430:479.5 V/V/V/V).
Flow rate : 1 mL/min. Cocois oleum raffinatum
Detection : spectrophotometer at 216 nm.
Injection : 20 μL. [8001-31-8]
Relative retention with reference to cocaine (retention DEFINITION
time = about 7.4 min) : degradation product = about 0.7.
Fatty oil obtained from the dried, solid part of the endosperm
System suitability : reference solution (b) : of Cocos nucifera L., then refined.
– resolution : minimum of 5 between the peaks due to cocaine
and to the degradation product. CHARACTERS
Limits : Appearance : white or almost white, unctuous mass.
– any impurity eluting after the principal peak : not more Solubility : practically insoluble in water, freely soluble in
than the area of the principal peak in the chromatogram methylene chloride and in light petroleum (bp : 65-70 °C),
obtained with reference solution (a) (0.1 per cent), very slightly soluble in ethanol (96 per cent).
– total : not more than 5 times the area of the principal peak Refractive index : about 1.449, determined at 40 °C.
in the chromatogram obtained with reference solution (a)
(0.5 per cent), IDENTIFICATION
– disregard limit: 0.5 times the area of the principal peak in A. Melting point (see Tests).
the chromatogram obtained with reference solution (a) B. Composition of fatty acids (see Tests).
(0.05 per cent). TESTS
Loss on drying (2.2.32) : maximum 0.5 per cent, determined Melting point (2.2.14) : 23 °C to 26 °C.
on 1.000 g by drying in an oven at 105 °C.
Acid value (2.5.1) : maximum 0.5, determined on 20.0 g.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
the residue from the test for loss on drying. Peroxide value (2.5.5, Method A) : maximum 5.0.
Unsaponifiable matter (2.5.7) : maximum 1.0 per cent,
ASSAY determined on 5.0 g.
Dissolve 0.250 g in a mixture of 5.0 mL of 0.01 M hydrochloric
acid and 50 mL of alcohol R. Carry out a potentiometric Alkaline impurities (2.4.19). It complies with the test.
titration (2.2.20), using 0.1 M sodium hydroxide. Read Composition of fatty acids (2.4.22, Method B). Refined
the volume added between the 2 points of inflexion. coconut oil is melted under gentle heating to a homogeneous
1 mL of 0.1 M sodium hydroxide is equivalent to 33.98 mg liquid prior to sampling.
of C17H22ClNO4. Reference solution. Dissolve 15.0 mg of tricaproin CRS,
80.0 mg of tristearin CRS, 0.150 g of tricaprin CRS, 0.200 g
STORAGE of tricaprylin CRS, 0.450 g of trimyristin CRS and 1.25 g of
Protected from light. trilaurin CRS in a mixture of 2 volumes of methylene chloride R

1936 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cocoyl caprylocaprate

and 8 volumes of heptane R, then dilute to 50 mL with the 01/2008:1411

same mixture of solvents heating at 45-50 °C. Transfer 2 mL of
this mixture to a 10 mL centrifuge tube with a screw cap and COCOYL CAPRYLOCAPRATE
evaporate the solvent in a current of nitrogen R. Dissolve with
1 mL of heptane R and 1 mL of dimethyl carbonate R and mix
vigorously under gentle heating (50-60 °C). Add, while still
Cocoylis caprylocapras
warm, 1 mL of a 12 g/L solution of sodium R in anhydrous DEFINITION
methanol R, prepared with the necessary precautions, and mix Mixture of esters of saturated C12 - C18 alcohols with caprylic
vigorously for about 5 min. Add 3 mL of distilled water R and (octanoic) and capric (decanoic) acids obtained by the reaction
mix vigorously for about 30 s. Centrifuge for 15 min at 1500 g. of these acids with vegetable saturated fatty alcohols.
Inject 1 μL of the organic phase.
CHARACTERS
Calculate the percentage content of each fatty acid using the
following expression : Appearance : slightly yellowish liquid.
Solubility : practically insoluble in water, miscible with ethanol
(96 per cent) and with liquid paraffin.
Relative density : about 0.86.
Ax,s,c is the corrected peak area of each fatty acid in the test Refractive index : about 1.445.
solution : Viscosity : about 11 mPa·s.
IDENTIFICATION
A. Freezing point (2.2.18) : maximum 15 °C.
Rc is the relative correction factor : B. Infrared absorption spectrophotometry (2.2.24).
Comparison: cocoyl caprylocaprate CRS.
C. Composition of fatty acids and fatty alcohols (see Tests).
TESTS
for the peaks due to caproic, caprylic, capric, lauric and Appearance. The substance to be examined is not more
myristic acid methyl esters. intensely coloured than reference solution Y5 (2.2.2, Method I).
mx,r = mass of tricaproin, tricaprylin, tricaprin, trilaurin Acid value (2.5.1) : maximum 0.5, determined on 5.00 g.
or trimyristin in the reference solution, in
milligrams ; Hydroxyl value (2.5.3, Method A) : maximum 5.0.
m1,r = mass of tristearin in the reference solution, in Iodine value (2.5.4, Method A) : maximum 1.0.
milligrams : Saponification value (2.5.6) : 160 to 173.
Ax,r = area of the peaks due to caproic, caprylic, capric, Composition of fatty acids and fatty alcohols (2.4.22,
lauric and myristic acid methyl esters in the Method C). Use the chromatogram obtained with the following
reference solution ; reference solution for identification of the peaks due to the
A1,r = area of the peak due to stearic acid methyl ester in fatty alcohols.
the reference solution ; Reference solution. Dissolve the amounts of the substances
Ax,s = area of the peaks due to any specified or unspecified listed in the following table in 10 mL of heptane R.
fatty acid methyl esters ; Substance Amount (mg)
Rc = 1 for the peaks due to each of the remaining Methyl caproate R 10
specified fatty acid methyl esters or any unspecified
fatty acid methyl ester. Methyl caprylate R 90

Methyl decanoate R 50
Composition of the fatty-acid fraction of the oil:
Methyl laurate R 20
– caproic acid (RRt 0.11) : maximum 1.5 per cent,
Methyl myristate R 10
– caprylic acid (RRt 0.23) : 5.0 per cent to 11.0 per cent,
Methyl palmitate R 10
– capric acid (RRt 0.56) : 4.0 per cent to 9.0 per cent,
Methyl stearate R 10
– lauric acid (RRt 0.75) : 40.0 per cent to 50.0 per cent,
Decanol R 10
– myristic acid (RRt 0.85) : 15.0 per cent to 20.0 per cent,
Lauryl alcohol R 100
– palmitic acid (RRt 0.93) : 7.0 per cent to 12.0 per cent,
Myristyl alcohol R 40
– stearic acid (RRt 1.00) : 1.5 per cent to 5.0 per cent,
Cetyl alcohol CRS 30
– oleic acid and isomers (RRt 1.01) : 4.0 per cent to 10.0 per
Stearyl alcohol CRS 20
cent,
– linoleic acid (RRt 1.03) : 1.0 per cent to 3.0 per cent, Consider the sum of the areas of the peaks due to the fatty
acids listed below to be equal to 100 and the sum of the areas
– linolenic acid (RRt 1.06) : maximum 0.2 per cent, of the peaks due to the fatty alcohols listed below to be equal
– arachidic acid (RRt 1.10) : maximum 0.2 per cent, to 100.
Composition of the fatty acid fraction of the substance :
– eicosenoic acid (RRt 1.11) : maximum 0.2 per cent.
– caproic acid : maximum 2.0 per cent,
Water (2.5.32) : maximum 0.1 per cent, determined on 1.00 g. – caprylic acid : 50.0 per cent to 80.0 per cent,
– capric acid : 20.0 per cent to 50.0 per cent,
STORAGE – lauric acid : maximum 3.0 per cent,
In a well-filled container, protected from light. – myristic acid : maximum 2.0 per cent.

General Notices (1) apply to all monographs and other texts 1937
Codeine EUROPEAN PHARMACOPOEIA 8.0

Composition of the fatty alcohol fraction of the substance : Appearance of solution. Solution S is clear (2.2.1) and
– capric alcohol : maximum 3.0 per cent, colourless (2.2.2, Method II).
– lauryl alcohol : 48.0 per cent to 63.0 per cent, Specific optical rotation (2.2.7) : − 142 to − 146 (dried
substance).
– myristyl alcohol : 18.0 per cent to 27.0 per cent,
Dissolve 0.50 g in ethanol (96 per cent) R and dilute to 25.0 mL
– cetyl alcohol : 6.0 per cent to 13.0 per cent, with the same solvent.
– stearyl alcohol : 9.0 per cent to 16.0 per cent.
Related substances. Liquid chromatography (2.2.29).
Water (2.5.12) : maximum 0.1 per cent, determined on 5.00 g. Test solution. Dissolve 0.100 g of the substance to be examined
Total ash (2.4.16) : maximum 0.1 per cent, determined on and 0.100 g of sodium octanesulfonate R in the mobile phase
1.0 g. and dilute to 10.0 mL with the mobile phase.
Reference solution (a). Dissolve 5.0 mg of codeine
impurity A CRS in the mobile phase and dilute to 5.0 mL with
the mobile phase.
04/2008:0076
corrected 7.0 Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 20.0 mL with the mobile phase.
Reference solution (c). Dilute 1.0 mL of the test solution to
CODEINE 50.0 mL with the mobile phase. Dilute 5.0 mL of this solution
to 100.0 mL with the mobile phase.
Codeinum Reference solution (d). To 0.25 mL of the test solution, add
2.5 mL of reference solution (a).
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : dissolve 1.08 g of sodium octanesulfonate R
in a mixture of 20 mL of glacial acetic acid R and 250 mL of
C18H21NO3,H2O Mr 317.4 acetonitrile R and dilute to 1000 mL with water R.
[6059-47-8] Flow rate : 2 mL/min.
Detection : spectrophotometer at 245 nm.
DEFINITION
Injection : 10 μL.
7,8-Didehydro-4,5α-epoxy-3-methoxy-17-methylmorphinan- Run time : 10 times the retention time of codeine.
6α-ol monohydrate.
Relative retention with reference to codeine (retention
Content : 99.0 per cent to 101.0 per cent (dried substance). time = about 6 min) : impurity B = about 0.6 ;
CHARACTERS impurity E = about 0.7 ; impurity A = about 2.0 ;
impurity C = about 2.3 ; impurity D = about 3.6.
Appearance : white or almost white, crystalline powder or
System suitability : reference solution (d) :
colourless crystals.
– resolution : minimum 3 between the peaks due to codeine
Solubility : soluble in boiling water, freely soluble in ethanol and impurity A.
(96 per cent).
Limits :
IDENTIFICATION – correction factor : for the calculation of content, multiply
First identification : A, C. the peak area of impurity C by 0.25 ;
Second identification : A, B, D, E. – impurity A : not more than twice the area of the principal
peak in the chromatogram obtained with reference
A. Melting point (2.2.14) : 155 °C to 159 °C. solution (b) (1.0 per cent) ;
B. Ultraviolet and visible absorption spectrophotometry – impurities B, C, D, E : for each impurity, not more than
(2.2.25). twice the area of the principal peak in the chromatogram
Test solution. To 2.0 mL of solution S (see Tests) add 50 mL obtained with reference solution (c) (0.2 per cent) ;
of water R then 10 mL of 1 M sodium hydroxide and dilute – unspecified impurities : for each impurity, not more than the
to 100.0 mL with water R. area of the principal peak in the chromatogram obtained
Spectral range : 250-350 nm. with reference solution (c) (0.10 per cent) ;
Absorption maximum : at 284 nm. – sum of impurities other than A : not more than 10 times the
Specific absorbance at the absorption maximum : about 50 area of the principal peak in the chromatogram obtained
(dried substance). with reference solution (c) (1.0 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
C. Infrared absorption spectrophotometry (2.2.24).
the chromatogram obtained with reference solution (c)
Preparation : dried substance prepared as a disc of (0.05 per cent).
potassium bromide R.
Loss on drying (2.2.32) : 4.0 per cent to 6.0 per cent,
Comparison : codeine CRS. determined on 1.000 g by drying in an oven at 105 °C.
D. To about 10 mg add 1 mL of sulfuric acid R and 0.05 mL Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
of ferric chloride solution R2 and heat on a water-bath. A 1.0 g.
blue colour develops. Add 0.05 mL of nitric acid R. The
colour changes to red. ASSAY
E. It gives the reaction of alkaloids (2.3.1). Dissolve 0.250 g in 10 mL of anhydrous acetic acid R. Add
20 mL of dioxan R. Titrate with 0.1 M perchloric acid, using
TESTS 0.05 mL of crystal violet solution R as indicator.
Solution S. Dissolve 50 mg in carbon dioxide-free water R and 1 mL of 0.1 M perchloric acid is equivalent to 29.94 mg
dilute to 10.0 mL with the same solvent. of C18H21NO3.

1938 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Codeine hydrochloride dihydrate

STORAGE
Protected from light.

IMPURITIES
Specified impurities : A, B, C, D, E.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of F. 7,8-didehydro-4,5α-epoxy-3-methoxy-17-
the tests in the monograph. They are limited by the general methylmorphinan-6α,14-diol,
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : F, G.

G. 6,7,8,14-tetradehydro-4,5α-epoxy-3,6-dimethoxy-17-
methylmorphinan (thebaine).

01/2008:1412

A. 7,8-didehydro-4,5α-epoxy-3,6α-dimethoxy-17-
CODEINE HYDROCHLORIDE
methylmorphinan (methylcodeine), DIHYDRATE
Codeini hydrochloridum dihydricum

B. 7,8-didehydro-4,5α-epoxy-17-methylmorphinan-3,6α-diol
(morphine), C18H22ClNO3,2H2O Mr 371.9
DEFINITION
7,8-Didehydro-4,5α-epoxy-3-methoxy-17-methylmorphinan-
6α-ol hydrochloride dihydrate.
Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
small, colourless crystals.
C. 7,7′,8,8′-tetradehydro-4,5α:4′,5′α-diepoxy-3,3′-dimethoxy- Solubility : soluble in water, slightly soluble in ethanol (96 per
17,17′-dimethyl-2,2′-bimorphinanyl-6α,6′α-diol (codeine cent), practically insoluble in cyclohexane.
dimer),
IDENTIFICATION
First identification : A, D.
Second identification : B, C, D, E.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: Ph. Eur. reference spectrum of codeine
hydrochloride dihydrate.
B. To 5 mL of solution S (see Tests) add 1 mL of a mixture
of equal volumes of strong sodium hydroxide solution R
and water R and initiate crystallisation, if necessary, by
scratching the wall of the tube with a glass rod and cooling
D. 7,8-didehydro-2-[(7,8-didehydro-4,5α-epoxy-6α-hydroxy- in iced water. Wash the precipitate with water R and dry at
17-methylmorphinan-3-yl)oxy]-4,5α-epoxy-3-methoxy- 100-105 °C. It melts (2.2.15) at 155 °C to 159 °C.
17-methylmorphinan-6α-ol (3-O-(codein-2-yl)morphine), C. To about 10 mg add 1 mL of sulfuric acid R and 0.05 mL
of ferric chloride solution R2 and heat on a water-bath. A
blue colour develops. Add 0.05 mL of nitric acid R. The
colour changes to red.
D. Solution S gives reaction (a) of chlorides (2.3.1).
E. It gives the reaction of alkaloids (2.3.1).
TESTS
Solution S. Dissolve 2.00 g in carbon dioxide-free water R
E. 7,8-didehydro-4,5α-epoxy-3-methoxy-17- prepared from distilled water R and dilute to 50.0 mL with
methylmorphinan-6α,10-diol, the same solvent.

General Notices (1) apply to all monographs and other texts 1939
Codeine hydrochloride dihydrate EUROPEAN PHARMACOPOEIA 8.0

Appearance of solution. Solution S is clear (2.2.1) and not Water (2.5.12) : 8.0 per cent to 10.5 per cent, determined on
more intensely coloured than reference solution Y6 (2.2.2, 0.250 g.
Method II).
ASSAY
Acidity or alkalinity. To 5 mL of solution S add 5 mL of
carbon dioxide-free water R. Add 0.05 mL of methyl red Dissolve 0.300 g in a mixture of 5 mL of 0.01 M hydrochloric
solution R and 0.2 mL of 0.02 M hydrochloric acid ; the solution acid and 30 mL of ethanol (96 per cent) R. Carry out a
is red. Add 0.4 mL of 0.02 M sodium hydroxide ; the solution potentiometric titration (2.2.20), using 0.1 M sodium
becomes yellow. hydroxide. Read the volume added between the 2 points of
inflexion.
Specific optical rotation (2.2.7) : − 117 to − 121 (anhydrous
substance). 1 mL of 0.1 M sodium hydroxide is equivalent to 33.59 mg
of C18H22ClNO3.
Dilute 5.0 mL of solution S to 10.0 mL with water R.
Related substances. Liquid chromatography (2.2.29). STORAGE
Test solution. Dissolve 0.100 g of the substance to be examined Protected from light.
and 0.100 g of sodium octanesulfonate R in the mobile phase
and dilute to 10.0 mL with the mobile phase. IMPURITIES
Reference solution (a). Dissolve 5.0 mg of codeine Specified impurities : A, B, C, D, E.
impurity A CRS in the mobile phase and dilute to 5.0 mL with Other detectable impurities (the following substances would,
the mobile phase. if present at a sufficient level, be detected by one or other of
Reference solution (b). Dilute 1.0 mL of reference solution (a) the tests in the monograph. They are limited by the general
to 20.0 mL with the mobile phase. acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
Reference solution (c). Dilute 1.0 mL of the test solution to (2034). It is therefore not necessary to identify these impurities
50.0 mL with the mobile phase. Dilute 5.0 mL of this solution for demonstration of compliance. See also 5.10. Control of
to 100.0 mL with the mobile phase. impurities in substances for pharmaceutical use) : F, G.
Reference solution (d). To 0.25 mL of the test solution add
2.5 mL of reference solution (a).
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : dissolve 1.08 g of sodium octanesulfonate R
in a mixture of 20 mL of glacial acetic acid R and 250 mL of A. 7,8-didehydro-4,5α-epoxy-3,6α-dimethoxy-17-
acetonitrile R and dilute to 1000 mL with water R. methylmorphinan (methylcodeine),
Flow rate : 2 mL/min.
Detection : spectrophotometer at 245 nm.
Injection : 10 μL.
Run time : 10 times the retention time of codeine.
Relative retention with reference to codeine (retention
time = about 6 min) : impurity B = about 0.6 ;
impurity E = about 0.7 ; impurity A = about 2.0 ;
impurity C = about 2.3 ; impurity D = about 3.6. B. 7,8-didehydro-4,5α-epoxy-17-methylmorphinan-3,6α-diol
System suitability : reference solution (d) : (morphine),
– resolution : minimum 3 between the peaks due to codeine
and impurity A.
Limits :
– correction factor : for the calculation of content, multiply
the peak area of impurity C by 0.25 ;
– impurity A : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (b) (1.0 per cent) ;
C. 7,7′,8,8′-tetradehydro-4,5α:4′,5′α-diepoxy-3,3′-dimethoxy-
– impurities B, C, D, E : for each impurity, not more than 17,17′-dimethyl-2,2′-bimorphinanyl-6α,6′α-diol (codeine
twice the area of the principal peak in the chromatogram dimer),
obtained with reference solution (c) (0.2 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (c) (0.10 per cent) ;
– sum of impurities other than A : not more than 10 times the
area of the principal peak in the chromatogram obtained
with reference solution (c) (1.0 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (c)
(0.05 per cent).
D. 7,8-didehydro-2-[(7,8-didehydro-4,5α-epoxy-6α-hydroxy-
Sulfates (2.4.13) : maximum 0.1 per cent. 17-methylmorphinan-3-yl)oxy]-4,5α-epoxy-3-methoxy-
Dilute 5 mL of solution S to 20 mL with distilled water R. 17-methylmorphinan-6α-ol (3-O-(codein-2-yl)morphine),

1940 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Codeine phosphate hemihydrate

B. Infrared absorption spectrophotometry (2.2.24).

Preparation : dissolve 0.20 g in 4 mL of water R. Add 1 mL
of a mixture of equal volumes of strong sodium hydroxide
solution R and water R and initiate crystallisation, if
necessary, by scratching the wall of the tube with a glass
rod and cooling in iced water. Wash the precipitate
with water R and dry at 100-105 °C. Examine the dried
E. 7,8-didehydro-4,5α-epoxy-3-methoxy-17- precipitate prepared as discs using potassium bromide R.
methylmorphinan-6α,10-diol, Comparison: Ph. Eur. reference spectrum of codeine.
C. Dissolve 0.20 g in 4 mL of water R. Add 1 mL of a mixture
of equal volumes of strong sodium hydroxide solution R
and water R and initiate crystallisation, if necessary, by
scratching the wall of the tube with a glass rod and cooling
in iced water. The precipitate, washed with water R and
dried at 100-105 °C, melts (2.2.14) at 155 °C to 159 °C.
D. To about 10 mg add 1 mL of sulfuric acid R and 0.05 mL
F. 7,8-didehydro-4,5α-epoxy-3-methoxy-17- of ferric chloride solution R2 and heat on a water-bath. A
methylmorphinan-6α,14-diol, blue colour develops. Add 0.05 mL of nitric acid R. The
colour changes to red.
E. Loss on drying (see Tests).
F. Solution S gives reaction (a) of phosphates (2.3.1).
G. It gives the reaction of alkaloids (2.3.1).
TESTS
Solution S. Dissolve 1.00 g in carbon dioxide-free water R
G. 6,7,8,14-tetradehydro-4,5α-epoxy-3,6-dimethoxy-17- prepared from distilled water R and dilute to 25.0 mL with
methylmorphinan (thebaine). the same solvent.
pH (2.2.3) : 4.0 to 5.0 for solution S.
01/2011:0074
Specific optical rotation (2.2.7) : − 98 to − 102 (dried
substance).
CODEINE PHOSPHATE Dilute 5.0 mL of solution S to 10.0 mL with water R.
HEMIHYDRATE Related substances. Liquid chromatography (2.2.29).
Codeini phosphas hemihydricus Test solution. Dissolve 0.100 g of the substance to be examined
and 0.100 g of sodium octanesulfonate R in the mobile phase
and dilute to 10.0 mL with the mobile phase.
Reference solution (a). Dissolve 5.0 mg of codeine
impurity A CRS in the mobile phase and dilute to 5.0 mL with
the mobile phase.
Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 20.0 mL with the mobile phase.
Reference solution (c). Dilute 1.0 mL of the test solution to
C18H24NO7P,½H2O Mr 406.4 50.0 mL with the mobile phase. Dilute 5.0 mL of this solution
[41444-62-6] to 100.0 mL with the mobile phase.
DEFINITION Reference solution (d). To 0.25 mL of the test solution add
2.5 mL of reference solution (a).
7,8-Didehydro-4,5α-epoxy-3-methoxy-17-methylmorphinan-
6α-ol phosphate hemihydrate. Column :
Content : 98.5 per cent to 101.0 per cent (dried substance). – size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octylsilyl silica gel for
CHARACTERS chromatography R (5 μm).
Appearance : white or almost white, crystalline powder or Mobile phase : dissolve 1.08 g of sodium octanesulfonate R
small, colourless crystals. in a mixture of 20 mL of glacial acetic acid R and 250 mL of
Solubility : freely soluble in water, slightly soluble or very acetonitrile R and dilute to 1000 mL with water R.
slightly soluble in ethanol (96 per cent). Flow rate : 2 mL/min.
IDENTIFICATION Detection : spectrophotometer at 245 nm.
First identification : B, E, F. Injection : 10 μL.
Second identification : A, C, D, E, F, G. Run time : 10 times the retention time of codeine.
A. Ultraviolet and visible absorption spectrophotometry Relative retention with reference to codeine (retention
(2.2.25). time = about 6 min) : impurities B and E = about 0.7 ;
Test solution. Dilute 1.0 mL of solution S (see Tests) to impurity A = about 2.0 ; impurity C = about 2.3 ;
100.0 mL with water R. To 25.0 mL of this solution add impurity D = about 3.6.
25 mL of water R then 10 mL of 1 M sodium hydroxide and System suitability : reference solution (d) :
dilute to 100.0 mL with water R. – resolution : minimum 3 between the peaks due to codeine
Spectral range : 250-350 nm. and impurity A.
Absorption maximum : at 284 nm. Limits :
Specific absorbance at the absorption maximum : about 38 – correction factor : for the calculation of content, multiply
(dried substance). the peak area of impurity C by 0.25 ;

General Notices (1) apply to all monographs and other texts 1941
Codeine phosphate sesquihydrate EUROPEAN PHARMACOPOEIA 8.0

– impurity A : not more than twice the area of the principal

peak in the chromatogram obtained with reference
solution (b) (1.0 per cent) ;
– sum of impurities B and E : not more than 4 times the area
of the principal peak in the chromatogram obtained with
reference solution (c) (0.4 per cent) ;
– impurities C, D : for each impurity, not more than twice the
area of the principal peak in the chromatogram obtained C. 7,7′,8,8′-tetradehydro-4,5α:4′,5′α-diepoxy-3,3′-dimethoxy-
with reference solution (c) (0.2 per cent) ; 17,17′-dimethyl-2,2′-bimorphinanyl-6α,6′α-diol (codeine
– unspecified impurities : for each impurity, not more than the dimer),
area of the principal peak in the chromatogram obtained
with reference solution (c) (0.10 per cent) ;
– sum of impurities other than A : not more than 10 times the
area of the principal peak in the chromatogram obtained
with reference solution (c) (1.0 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (c)
(0.05 per cent).
Sulfates (2.4.13) : maximum 0.1 per cent. D. 7,8-didehydro-2-[(7,8-didehydro-4,5α-epoxy-6α-hydroxy-
Dilute 5 mL of solution S to 20 mL with distilled water R. 17-methylmorphinan-3-yl)oxy]-4,5α-epoxy-3-methoxy-
17-methylmorphinan-6α-ol (3-O-(codein-2-yl)morphine),
Loss on drying (2.2.32) : 1.5 per cent to 3.0 per cent,
determined on 1.000 g by drying in an oven at 105 °C.

ASSAY
Dissolve 0.350 g in a mixture of 10 mL of anhydrous acetic
acid R and 20 mL of dioxan R. Titrate with 0.1 M perchloric
acid using 0.05 mL of crystal violet solution R as indicator.
1 mL of 0.1 M perchloric acid is equivalent to 39.74 mg E. 7,8-didehydro-4,5α-epoxy-3-methoxy-17-
of C18H24NO7P. methylmorphinan-6α,10-diol,

STORAGE
Protected from light.

IMPURITIES
Specified impurities : A, B, C, D, E.
Other detectable impurities (the following substances would, F. 7,8-didehydro-4,5α-epoxy-3-methoxy-17-
if present at a sufficient level, be detected by one or other of methylmorphinan-6α,14-diol,
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : F, G.

G. 6,7,8,14-tetradehydro-4,5α-epoxy-3,6-dimethoxy-17-
methylmorphinan (thebaine).

01/2008:0075
corrected 6.0

CODEINE PHOSPHATE
A. 7,8-didehydro-4,5α-epoxy-3,6α-dimethoxy-17- SESQUIHYDRATE
methylmorphinan (methylcodeine),
Codeini phosphas sesquihydricus

B. 7,8-didehydro-4,5α-epoxy-17-methylmorphinan-3,6α-diol C18H24NO7P,1½H2O Mr 424.4

(morphine), [5913-76-8]

1942 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Codeine phosphate sesquihydrate

DEFINITION Reference solution (c). Dilute 1.0 mL of the test solution to

7,8-Didehydro-4,5α-epoxy-3-methoxy-17-methylmorphinan- 50.0 mL with the mobile phase. Dilute 5.0 mL of this solution
6α-ol phosphate sesquihydrate. to 100.0 mL with the mobile phase.
Content : 98.5 per cent to 101.0 per cent (dried substance). Reference solution (d). To 0.25 mL of the test solution add
2.5 mL of reference solution (a).
CHARACTERS Column :
Appearance : white or almost white, crystalline powder or – size : l = 0.25 m, Ø = 4.6 mm ;
small, colourless crystals. – stationary phase : end-capped octylsilyl silica gel for
Solubility : freely soluble in water, slightly soluble in ethanol chromatography R (5 μm).
(96 per cent). Mobile phase : dissolve 1.08 g of sodium octanesulfonate R
in a mixture of 20 mL of glacial acetic acid R and 250 mL of
IDENTIFICATION acetonitrile R and dilute to 1000 mL with water R.
First identification : B, E, F. Flow rate : 2 mL/min.
Second identification : A, C, D, E, F, G. Detection : spectrophotometer at 245 nm.
A. Ultraviolet and visible absorption spectrophotometry Injection : 10 μL.
(2.2.25). Run time : 10 times the retention time of codeine.
Test solution. Dilute 1.0 mL of solution S (see Tests) to Relative retention with reference to codeine (retention
100.0 mL with water R. To 25.0 mL of this solution add time = about 6 min) : impurity B = about 0.6 ;
25 mL of water R then 10 mL of 1 M sodium hydroxide and impurity E = about 0.7 ; impurity A = about 2.0 ;
dilute to 100.0 mL with water R. impurity C = about 2.3 ; impurity D = about 3.6.
Spectral range : 250-350 nm. System suitability : reference solution (d) :
Absorption maximum : at 284 nm. – resolution : minimum 3 between the peaks due to codeine
Specific absorbance at the absorption maximum : about 38 and impurity A.
(dried substance). Limits :
B. Infrared absorption spectrophotometry (2.2.24). – correction factor : for the calculation of content, multiply
Preparation : dissolve 0.20 g in 4 mL of water R. Add 1 mL the peak area of impurity C by 0.25 ;
of a mixture of equal volumes of strong sodium hydroxide – impurity A : not more than twice the area of the principal
solution R and water R and initiate crystallisation, if peak in the chromatogram obtained with reference
necessary, by scratching the wall of the tube with a glass solution (b) (1.0 per cent) ;
rod and cooling in iced water. Wash the precipitate – impurities B, C, D, E : for each impurity, not more than
with water R and dry at 100-105 °C. Examine the dried twice the area of the principal peak in the chromatogram
precipitate prepared as discs using potassium bromide R. obtained with reference solution (c) (0.2 per cent) ;
Comparison : Ph. Eur. reference spectrum of codeine. – unspecified impurities : for each impurity, not more than the
C. Dissolve 0.20 g in 4 mL of water R. Add 1 mL of a mixture area of the principal peak in the chromatogram obtained
of equal volumes of strong sodium hydroxide solution R with reference solution (c) (0.10 per cent) ;
and water R and initiate crystallisation, if necessary, by – sum of impurities other than A : not more than 10 times the
scratching the wall of the tube with a glass rod and cooling area of the principal peak in the chromatogram obtained
in iced water. The precipitate, washed with water R and with reference solution (c) (1.0 per cent) ;
dried at 100-105 °C, melts (2.2.14) at 155 °C to 159 °C. – disregard limit : 0.5 times the area of the principal peak in
D. To about 10 mg add 1 mL of sulfuric acid R and 0.05 mL the chromatogram obtained with reference solution (c)
of ferric chloride solution R2 and heat on a water-bath. A (0.05 per cent).
blue colour develops. Add 0.05 mL of nitric acid R. The
Sulfates (2.4.13) : maximum 0.1 per cent.
colour changes to red.
Dilute 5 mL of solution S to 20 mL with distilled water R.
E. Loss on drying (see Tests).
Loss on drying (2.2.32) : 5.0 per cent to 7.5 per cent,
F. Solution S gives reaction (a) of phosphates (2.3.1).
determined on 0.500 g by drying in an oven at 105 °C.
G. It gives the reaction of alkaloids (2.3.1).
ASSAY
TESTS Dissolve 0.350 g in a mixture of 10 mL of anhydrous acetic
Solution S. Dissolve 1.00 g in carbon dioxide-free water R acid R and 20 mL of dioxan R. Titrate with 0.1 M perchloric
prepared from distilled water R and dilute to 25.0 mL with acid using 0.05 mL of crystal violet solution R as indicator.
the same solvent. 1 mL of 0.1 M perchloric acid is equivalent to 39.74 mg
pH (2.2.3): 4.0 to 5.0 for solution S. of C18H24NO7P.
Specific optical rotation (2.2.7) : − 98 to − 102 (dried STORAGE
substance). Protected from light.
Dilute 5.0 mL of solution S to 10.0 mL with water R.
IMPURITIES
Related substances. Liquid chromatography (2.2.29).
Specified impurities : A, B, C, D, E.
Test solution. Dissolve 0.100 g of the substance to be examined
and 0.100 g of sodium octanesulfonate R in the mobile phase Other detectable impurities (the following substances would,
and dilute to 10.0 mL with the mobile phase. if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
Reference solution (a). Dissolve 5.0 mg of codeine acceptance criterion for other/unspecified impurities and/or
impurity A CRS in the mobile phase and dilute to 5.0 mL with by the general monograph Substances for pharmaceutical use
the mobile phase. (2034). It is therefore not necessary to identify these impurities
Reference solution (b). Dilute 1.0 mL of reference solution (a) for demonstration of compliance. See also 5.10. Control of
to 20.0 mL with the mobile phase. impurities in substances for pharmaceutical use) : F, G.

General Notices (1) apply to all monographs and other texts 1943
Codergocrine mesilate EUROPEAN PHARMACOPOEIA 8.0

07/2013:2060

CODERGOCRINE MESILATE
Codergocrini mesilas
A. 7,8-didehydro-4,5α-epoxy-3,6α-dimethoxy-17-
methylmorphinan (methylcodeine),

B. 7,8-didehydro-4,5α-epoxy-17-methylmorphinan-3,6α-diol
(morphine),

C. 7,7′,8,8′-tetradehydro-4,5α:4′,5′α-diepoxy-3,3′-dimethoxy-
17,17′-dimethyl-2,2′-bimorphinanyl-6α,6′α-diol (codeine
dimer),
[8067-24-1]
DEFINITION
A mixture of :
– (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2,5-
bis(1-methylethyl)-3,6-dioxooctahydro-8H-oxazolo[3,2-
a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10,10a-
octahydroindolo[4,3-fg]quinoline-9-carboxamide
methanesulfonate (dihydroergocornine mesilate) ;
D. 7,8-didehydro-2-[(7,8-didehydro-4,5α-epoxy-6α-hydroxy- – (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-
17-methylmorphinan-3-yl)oxy]-4,5α-epoxy-3-methoxy- hydroxy-2-(1-methylethyl)-3,6-dioxooctahydro-8H-
17-methylmorphinan-6α-ol (3-O-(codein-2-yl)morphine), oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-
9-carboxamide methanesulfonate (dihydroergocristine
mesilate) ;
– (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-(1-
methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro-
8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9-
carboxamide methanesulfonate (α-dihydroergocryptine
E. 7,8-didehydro-4,5α-epoxy-3-methoxy-17- mesilate) ;
methylmorphinan-6α,10-diol, – (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-
2-(1-methylethyl)-5-[(1RS)-1-methylpropyl]-3,6-
dioxooctahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-
2-yl]-7-methyl-4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-
fg]quinoline-9-carboxamide methanesulfonate
(β-dihydroergocryptine mesilate or epicriptine mesilate).
Content : 98.0 per cent to 102.0 per cent (dried substance).

F. 7,8-didehydro-4,5α-epoxy-3-methoxy-17- PRODUCTION
methylmorphinan-6α,14-diol, It is considered that alkylsulfonate esters are genotoxic
and are potential impurities in codergocrine mesilate. The
manufacturing process should be developed taking into
consideration the principles of quality risk management,
together with considerations of the quality of starting
materials, process capability and validation. The general
methods 2.5.37. Methyl, ethyl and isopropyl methanesulfonate
in methanesulfonic acid, 2.5.38. Methyl, ethyl and
isopropyl methanesulfonate in active substances and
G. 6,7,8,14-tetradehydro-4,5α-epoxy-3,6-dimethoxy-17- 2.5.39. Methanesulfonyl chloride in methanesulfonic acid are
methylmorphinan (thebaine). available to assist manufacturers.

1944 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Codergocrine mesilate

CHARACTERS System suitability : test solution :

Appearance : white or yellowish powder. – resolution : minimum 3 between any 2 consecutive principal
Solubility : sparingly soluble in water, sparingly soluble to peaks.
soluble in ethanol (96 per cent), slightly soluble in methylene Composition :
chloride.
– dihydroergocornine : 30.0 per cent to 35.0 per cent ;
IDENTIFICATION – α-dihydroergocryptine : 20.0 per cent to 25.0 per cent ;
A. Thin-layer chromatography (2.2.27). – dihydroergocristine : 30.0 per cent to 35.0 per cent ;
Test solution. Dissolve 0.20 g of the substance to be – β-dihydroergocryptine : 10.0 per cent to 13.0 per cent ;
examined in a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R and dilute to 5 mL with – disregard limit : 1.0 per cent.
the same mixture of solvents. Related substances. Thin-layer chromatography (2.2.27).
Reference solution. Dissolve 0.20 g of methanesulfonic Perform the test as rapidly as possible and protected from direct
acid R in a mixture of 1 volume of methanol R and light. Prepare the test solution last and immediately before
9 volumes of methylene chloride R and dilute to 5 mL with application on the plate.
the same mixture of solvents. Test solution. Dissolve 0.40 g of the substance to be examined
Plate : TLC silica gel plate R. in a mixture of 1 volume of methanol R and 9 volumes of
Mobile phase : water R, concentrated ammonia R, butanol R, methylene chloride R and dilute to 5.0 mL with the same
acetone R (5:10:20:65 V/V/V/V). mixture of solvents.
Application : 10 μL. Reference solution (a). Dissolve 40 mg of dihydroergocristine
mesilate CRS in a mixture of 1 volume of methanol R and
Development : over 2/3 of the plate. 9 volumes of methylene chloride R and dilute to 10.0 mL with
Drying : in a current of cold air for not more than 1 min. the same mixture of solvents. Dilute 3.0 mL of the solution
Detection : spray with a 1 g/L solution of bromocresol to 50.0 mL with a mixture of 1 volume of methanol R and
purple R in methanol R, adjusted to a violet-red colour with 9 volumes of methylene chloride R.
0.05 mL of dilute ammonia R1. Reference solution (b). To 2.0 mL of reference solution (a), add
Drying : in a current of hot air at 100 °C. 1.0 mL of a mixture of 1 volume of methanol R and 9 volumes
of methylene chloride R.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position and colour to Reference solution (c). To 1.0 mL of reference solution (a), add
the principal spot in the chromatogram obtained with the 2.0 mL of a mixture of 1 volume of methanol R and 9 volumes
reference solution. of methylene chloride R.
B. Examine the chromatograms obtained in the test for Reference solution (d). To 1.0 mL of reference solution (a), add
composition. 5.0 mL of a mixture of 1 volume of methanol R and 9 volumes
Results : the 4 principal peaks in the chromatogram of methylene chloride R.
obtained with the test solution are similar in retention time Plate : TLC silica gel plate R.
to the 4 principal peaks in the chromatogram obtained
with the reference solution. Mobile phase : concentrated ammonia R, methanol R, ethyl
acetate R, methylene chloride R (1:3:50:50 V/V/V/V).
TESTS Application : 10 μL.
pH (2.2.3) : 4.2 to 5.2. Drying : in the dark for 2 min after the application of the last
Dissolve 0.10 g in carbon dioxide-free water R and dilute to solution.
20 mL with the same solvent. First development : in an unsaturated tank, over 2/3 of the plate.
Composition. Liquid chromatography (2.2.29) : use the Drying : in a current of cold air for not more than 1 min.
normalisation procedure.
Second development : in an unsaturated tank, over 2/3 of the
Test solution. Dissolve 20 mg of the substance to be examined plate ; use freshly prepared mobile phase.
in a mixture of 1 volume of anhydrous ethanol R and 2 volumes
of a 10 g/L solution of tartaric acid R and dilute to 10 mL with Drying : in a current of cold air for not more than 1 min.
the same mixture of solvents. Detection : spray thoroughly with dimethylaminobenzaldehyde
Reference solution. Dissolve 20 mg of codergocrine solution R7 and dry in a current of hot air until the spot in the
mesilate CRS in a mixture of 1 volume of anhydrous ethanol R chromatogram obtained with reference solution (d) is clearly
and 2 volumes of a 10 g/L solution of tartaric acid R and dilute visible.
to 10 mL with the same mixture of solvents. System suitability : test solution :
Column :
– the chromatogram shows at least 3 separated secondary
– size : l = 0.15 m, Ø = 4.6 mm ; spots.
– stationary phase : octadecylsilyl silica gel for Limits :
chromatography R (5 μm).
– any impurity : any spots, apart from the principal spot,
Mobile phase : triethylamine R, acetonitrile R, water R are not more intense than the spot in the chromatogram
(2.5:25:75 V/V/V). obtained with reference solution (a) (0.3 per cent) ; not
Flow rate : 1.5 mL/min. more than 4 such spots are more intense than the spot in
Detection : spectrophotometer at 280 nm. the chromatogram obtained with reference solution (c)
(0.1 per cent) and 2 of these may be more intense than
Injection : 20 μL. the spot in the chromatogram obtained with reference
Run time : 20 min. solution (b) (0.2 per cent).
Elution order : dihydroergocornine, α-dihydroergocryptine, Loss on drying (2.2.32) : maximum 5.0 per cent, determined
dihydroergocristine, β-dihydroergocryptine. on 0.500 g by drying at 120 °C under high vacuum.

General Notices (1) apply to all monographs and other texts 1945
Cod-liver oil, farmed EUROPEAN PHARMACOPOEIA 8.0

ASSAY Positional distribution (β(2)-acyl) of fatty acids. Nuclear

Dissolve 0.500 g in 60 mL of pyridine R. Pass a stream of magnetic resonance spectrometry (2.2.33).
nitrogen R over the surface of the solution and titrate with Test solution. Dissolve 190-210 mg of the substance to be
0.1 M tetrabutylammonium hydroxide, determining the examined in 500 μL of deuterated chloroform R. Prepare at
end-point potentiometrically (2.2.20). least 3 samples and examine within 3 days.
1 mL of 0.1 M tetrabutylammonium hydroxide is equivalent to Apparatus : high-resolution FT-NMR spectrometer operating
68.04 mg of codergocrine mesilate (average Mr = 680). at minimum 300 MHz.
Acquisition of 13C NMR spectra. The following parameters
STORAGE may be used :
Protected from light. – sweep width : 200 ppm (− 5 ppm to 195 ppm) ;
– irradiation frequency offset : 95 ppm ;
– time domain : 64 K ;
07/2012:2398 – pulse delay : 2 s ;
– pulse program : zgig 30 (inverse gated, 30° excitation pulse) ;
COD-LIVER OIL, FARMED – dummy scans : 4 ;
– number of scans : 4096.
Iecoris aselli domestici oleum Processing and plotting. The following parameters may be
used :
DEFINITION – size : 64 K (zero-filling) ;
Purified fatty oil obtained from the fresh livers of farmed cod, – window multiplication: exponential ;
Gadus morhua L., solid substances being removed by cooling – Lorentzian broadening factor: 0.2 Hz.
and filtering.
Use the CDCl3 signal for shift referencing. The shift of the
Content : central peak of the 1:1:1 triplet is set to 77.16 ppm.
– sum of the contents of EPA and DHA (expressed as Plot the spectral region δ 171.5-173.5 ppm. Compare the
triglycerides) : 10.0 per cent to 28.0 per cent ; spectrum with the spectrum shown in Figure 2398.-1. The
– vitamin A : 50 IU (15 μg) to 500 IU (150 μg) per gram ; shift values lie within the ranges given in Table 2398.-1.
– vitamin D3 : maximum 50 IU (1.3 μg) per gram. Table 2398.-1. – Shift values
A suitable antioxidant may be added. Signal Shift range (ppm)
PRODUCTION β DHA 172.05 - 172.09
The fish shall only be given feed with a composition that is α DHA 172.43 - 172.47
in accordance with the relevant European Union or other
applicable regulations. β EPA 172.52 - 172.56

The content of dioxins and dioxin-like PCBs (polychlorinated α EPA 172.90 - 172.94
biphenyls) is controlled using methods and limits in 172.56 - 172.60
β C18:4
accordance with the requirements set in the European Union
or other applicable regulations. α C18:4 172.95 - 172.99

CHARACTERS System suitability :

Appearance : clear, pale yellowish liquid. – signal-to-noise ratio : minimum 5 for the smallest
Solubility : practically insoluble in water, miscible with light relevant peak corresponding to α C18:4 signal (in the
petroleum, slightly soluble in ethanol (96 per cent). range δ 172.95-172.99 ppm) ;
– peak width at half-height : maximum 0.02 ppm for the
IDENTIFICATION central CDCl3 signal (at δ 77.16 ppm).
A. Examine the 13C NMR spectra obtained in the test for Calculation of positional distribution (β(2)-acyl) : use the
positional distribution (β(2)-acyl) of fatty acids (see Tests). following expression :
The spectra contain peaks between 172 ppm and 173 ppm
with shifts similar to those in the spectrum shown in
Figure 2398.-1.
The positional distribution (β(2)-acyl) for cervonic α = peak area of the corresponding α-carbonyl peak ;
(docosahexaenoic) acid (C22:6 n-3 ; DHA), timnodonic
(eicosapentaenoic) acid (C20:5 n-3 ; EPA) and moroctic β = peak area of β-carbonyl peak from C22:6 n-3,
acid (C18:4 n-3) complies with the limits. C20:5 n-3 or C18:4 n-3, respectively.
B. Linoleic acid (see Tests). Limits :
TESTS – positional distribution (β(2)-acyl) :
– cervonic (docosahexaenoic) acid (C22:6 n-3 ; DHA) :
Acid value (2.5.1) : maximum 2.0.
71 per cent to 81 per cent ;
Anisidine value (2.5.36) : maximum 10.0. – timnodonic (eicosapentaenoic) acid (C20:5 n-3 EPA) :
Peroxide value (2.5.5, Method B) : maximum 5.0. 32 per cent to 40 per cent ;
Unsaponifiable matter (2.5.7) : maximum 1.5 per cent, – moroctic acid (C18:4 n-3) : 28 per cent to 38 per cent.
determined on 2.0 g, and extracting with 3 quantities, each of Composition of fatty acids (2.4.29). For identification of the
50 mL, of peroxide-free ether R. peaks, see the chromatogram shown in Figure 2398.-2.
Stearin. Heat at least 10 mL to 60-90 °C then allow to cool for The 24 largest peaks of the methyl esters account for more
3 h in a bath of iced water or a thermostatically controlled than 90 per cent of the total area (these correspond to, in
bath at 0 ± 0.5 °C. If necessary, to eliminate insoluble matter, common elution order : 14:0, 15:0, 16:0, 16:1 n-7, 16:4 n-1,
filter the sample after heating. The sample remains clear. 18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6, 18:3 n-3, 18:4 n-3, 20:1 n-11,

1946 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cod-liver oil, farmed

1. α C18:4 2. α EPA 3. β C18:4 4. β EPA 5. α DHA 6. β DHA

Figure 2398.-1. – 13C NMR spectrum : carbonyl region of farmed cod-liver oil

20:1 n-9, 20:1 n-7, 20:2 n-6, 20:4 n-6, 20:3 n-3, 20:4 n-3, each of 50 mL, of water R, and evaporate to dryness under a
20:5 n-3, 22:1 n-11, 22:1 n-9, 21:5 n-3, 22:5 n-3, 22:6 n-3). gentle current of nitrogen R at a temperature not exceeding
30 °C or in a rotary evaporator at a temperature not exceeding
Linoleic acid (2.4.29) : 3.0 per cent to 11.0 per cent.
30 °C under reduced pressure (water ejector). Dissolve
the residue in sufficient 2-propanol R1 to give an expected
concentration of vitamin A equivalent to 10-15 IU/mL.
ASSAY
EPA and DHA (2.4.29). See the chromatogram shown in Measure the absorbances of the solution at 300 nm, 310 nm,
Figure 2398.-2. 325 nm and 334 nm and at the wavelength of maximum
absorption with a suitable spectrophotometer in specially
Vitamin A. Carry out the test as rapidly as possible, avoiding matched 1 cm cells, using 2-propanol R1 as the compensation
exposure to actinic light and air, oxidising agents, oxidation liquid.
catalysts (for example, copper and iron) and acids.
Calculate the content of vitamin A, as all-trans-retinol, in
Use method A. If method A is found not to be valid, use International Units per gram, using the following expression :
method B.
METHOD A
Ultraviolet absorption spectrophotometry (2.2.25).

Test solution. To 1.00 g in a round-bottomed flask, add 3 mL A325 = absorbance at 325 nm ;

of a freshly prepared 50 per cent m/m solution of potassium m = mass of the substance to be examined, in grams ;
hydroxide R and 30 mL of anhydrous ethanol R. Boil under
reflux in a current of nitrogen R for 30 min. Cool rapidly and V = total volume of solution containing 10-15 IU of
add 30 mL of water R. Extract with 50 mL of ether R. Repeat the vitamin A per millilitre ;
extraction 3 times and discard the lower layer after complete 1821 = conversion factor for the specific absorbance of
separation. Wash the combined upper layers with 4 quantities, all-trans-retinol, in International Units.

General Notices (1) apply to all monographs and other texts 1947
Cod-liver oil, farmed EUROPEAN PHARMACOPOEIA 8.0

1. C14:0 5. C16:4 n-1 9. C18:2 n-6 13. C20:1 n-9 17. C20:3 n-3 21. C22:1 n-9
2. C15:0 6. C18:0 10 C18:3 n-3 14. C20:1 n-7 18. C20:4 n-3 22. C21:5 n-3
3. C16:0 7. C18:1 n-9 11. C18:4 n-3 15. C20:2 n-6 19. C20:5 n-3 23. C22:5 n-3
4. C16:1 n-7 8. C18:1 n-7 12. C20:1 n-11 16. C20:4 n-6 20. C22:1 n-11 24. C22:6 n-3

Figure 2398.-2. – Chromatogram for the test for composition of fatty acids of farmed cod-liver oil

The above expression can be used only if A325 has a value 10 g/L solution of sodium chloride R and then with 150 mL of
not greater than A325,corr /0.970, where A325,corr is the corrected a mixture of equal volumes of ether R and light petroleum R1.
absorbance at 325 nm and is given by the following equation : Shake for 1 min. When the layers have separated completely,
discard the lower layer and wash the upper layer, first with
50 mL of a 30 g/L solution of potassium hydroxide R in a
10 per cent V/V solution of anhydrous ethanol R and then
A designates the absorbance at the wavelength indicated by with 3 quantities, each of 50 mL, of a 10 g/L solution of
the subscript. sodium chloride R. Filter the upper layer through 5 g of
If A325 has a value greater than A325,corr /0.970, calculate the anhydrous sodium sulfate R on a fast filter paper into a 250 mL
content of vitamin A using the following expression : flask suitable for a rotary evaporator. Wash the funnel with
10 mL of fresh extraction mixture, filter and combine the
upper layers. Distil them at a temperature not exceeding
30 °C under reduced pressure (water ejector) and fill with
nitrogen R when evaporation is completed. Alternatively,
The assay is not valid unless : evaporate the solvent under a gentle current of nitrogen R at
– the wavelength of maximum absorption lies between a temperature not exceeding 30 °C. Dissolve the residue in
323 nm and 327 nm ; 2-propanol R, transfer to a 25 mL volumetric flask and dilute
to 25 mL with 2-propanol R. Gentle heating in an ultrasonic
– the absorbance at 300 nm relative to that at 325 nm is at bath may be required. A large fraction of the white residue is
most 0.73. cholesterol, constituting approximately 50 per cent m/m of the
METHOD B unsaponifiable matter of cod-liver oil.
Liquid chromatography (2.2.29). Reference solution (a). Prepare a solution of retinol acetate CRS
Test solution. Prepare duplicates. To 2.00 g in a in 2-propanol R1 so that 1 mL contains about 1000 IU of
round-bottomed flask, add 5 mL of a freshly prepared 100 g/L all-trans-retinol.
solution of ascorbic acid R, 10 mL of a freshly prepared 800 g/L The exact concentration of reference solution (a) is assessed
solution of potassium hydroxide R and 100 mL of anhydrous by ultraviolet absorption spectrophotometry (2.2.25). Dilute
ethanol R. Boil under a reflux condenser on a water-bath for reference solution (a) with 2-propanol R1 to a presumed
15 min. Add 100 mL of a 10 g/L solution of sodium chloride R concentration of 10-15 IU/mL and measure the absorbance
and cool. Transfer the solution to a 500 mL separating funnel, at 326 nm in matched 1 cm cells using 2-propanol R1 as the
rinsing the round-bottomed flask with about 75 mL of a compensation liquid.

1948 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cod-liver oil, farmed

Calculate the content of vitamin A in International Units A1 = area of the peak due to all-trans-retinol in the
per millilitre of reference solution (a) using the following chromatogram obtained with the test solution ;
expression, taking into account the assigned content of retinol A2 = area of the peak due to all-trans-retinol in
acetate CRS : the chromatogram obtained with reference
solution (b);
C = concentration of retinol acetate CRS in reference
solution (a) as assessed prior to the saponification,
in International Units per millilitre (= 1000 IU/mL) ;
A326 = absorbance at 326 nm ; V = volume of reference solution (a) treated (2.00 mL) ;
V1 = volume of reference solution (a) used ; m = mass of the substance to be examined in the test
V2 = volume of the diluted solution ; solution (2.00 g).
1900 = conversion factor for the specific absorbance of Vitamin D3. Liquid chromatography (2.2.29). Carry out the
retinol acetate CRS, in International Units. assay as rapidly as possible, avoiding exposure to actinic light
Reference solution (b). Proceed as described for the test and air.
solution but using 2.00 mL of reference solution (a) in place of Internal standard solution. Dissolve 0.50 mg of
the substance to be examined. ergocalciferol CRS in 100 mL of anhydrous ethanol R.
Test solution (a). To 4.00 g in a round-bottomed flask, add
The exact concentration of reference solution (b) is assessed
5 mL of a freshly prepared 100 g/L solution of ascorbic acid R,
by ultraviolet absorption spectrophotometry (2.2.25). Dilute
10 mL of a freshly prepared 800 g/L solution of potassium
reference solution (b) with 2-propanol R1 to a presumed
hydroxide R and 100 mL of anhydrous ethanol R. Boil under a
all-trans-retinol concentration of 10-15 IU/mL and measure
reflux condenser on a water-bath for 30 min. Add 100 mL of
the absorbance at 325 nm in matched 1 cm cells using
a 10 g/L solution of sodium chloride R and cool the solution
2-propanol R1 as the compensation liquid.
to room temperature. Transfer the solution to a 500 mL
separating funnel, rinsing the round-bottomed flask with
about 75 mL of a 10 g/L solution of sodium chloride R and
Calculate the content of all-trans-retinol in International Units then with 150 mL of a mixture of equal volumes of ether R and
per millilitre of reference solution (b), using the following light petroleum R1. Shake for 1 min. When the layers have
expression : separated completely, discard the lower layer and wash the
upper layer, first with 50 mL of a 30 g/L solution of potassium
hydroxide R in a 10 per cent V/V solution of anhydrous
ethanol R, and then with 3 quantities, each of 50 mL, of a 10 g/L
solution of sodium chloride R. Filter the upper layer through
5 g of anhydrous sodium sulfate R on a fast filter paper into a
A325 = absorbance at 325 nm ; 250 mL flask suitable for a rotary evaporator. Wash the funnel
with 10 mL of fresh extraction mixture, filter and combine the
V3 = volume of the diluted solution ; upper layers. Distil them at a temperature not exceeding 30 °C
V4 = volume of reference solution (b) used ; under reduced pressure (water ejector) and fill with nitrogen R
when evaporation is completed. Alternatively, evaporate the
1821 = conversion factor for the specific absorbance of solvent under a gentle current of nitrogen R at a temperature
all-trans-retinol, in International Units. not exceeding 30 °C. Dissolve the residue in 1.5 mL of the
Column : mobile phase described under Purification. Gentle heating
in an ultrasonic bath may be required. A large fraction of the
– size : l = 0.25 m, Ø = 4.6 mm ; white residue is cholesterol, constituting approximately 50 per
cent m/m of the unsaponifiable matter of cod-liver oil.
– stationary phase : octadecylsilyl silica gel for
chromatography R (5-10 μm). Test solution (b). Prepare duplicates. To 4.00 g add 2.0 mL of
the internal standard solution and proceed as described for
Mobile phase : water R, methanol R (3:97 V/V). test solution (a).
Flow rate : 1 mL/min. Reference solution (a). Dissolve 0.50 mg of cholecalciferol CRS
in 100.0 mL of anhydrous ethanol R.
Detection : spectrophotometer at 325 nm.
Reference solution (b). In a round-bottomed flask, add 2.0 mL
Injection : 10 μL ; inject in triplicate the test solution and of reference solution (a) and 2.0 mL of the internal standard
reference solution (b). solution and proceed as described for test solution (a).
Retention time : all-trans-retinol = 5 ± 1 min. PURIFICATION
Column :
System suitability : – size : l = 0.25 m, Ø = 4.6 mm ;
– the chromatogram obtained with the test solution shows a – stationary phase : nitrile silica gel for chromatography R
peak corresponding to the peak due to all-trans-retinol in (10 μm).
the chromatogram obtained with reference solution (b) ; Mobile phase : isoamyl alcohol R, hexane R (1.6:98.4 V/V).
– the results obtained with the duplicate test solutions do not Flow rate : 1.1 mL/min.
differ by more than 5 per cent ; Detection : spectrophotometer at 265 nm.
– the recovery of all-trans-retinol in reference solution (b) as Injection : 350 μL of reference solution (b) and test
assessed by direct absorption spectrophotometry is greater solutions (a) and (b). Collect each eluate from 2 min before
than 95 per cent. until 2 min after the retention time of cholecalciferol, in a
ground-glass-stoppered tube containing 1 mL of a 1 g/L
Calculate the content of vitamin A using the following solution of butylhydroxytoluene R in hexane R. Evaporate
expression : separately to dryness at a temperature not exceeding 30 °C
under a gentle current of nitrogen R. Dissolve each residue in
1.5 mL of acetonitrile R.

General Notices (1) apply to all monographs and other texts 1949
Cod-liver oil (type A) EUROPEAN PHARMACOPOEIA 8.0

DETERMINATION 07/2012:1192
Column :
– size : l = 0.15 m, Ø = 4.6 mm ; COD-LIVER OIL (TYPE A)
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm). Iecoris aselli oleum A
Mobile phase : phosphoric acid R, 96 per cent V/V solution of DEFINITION
acetonitrile R (0.2:99.8 V/V).
Purified fatty oil obtained from the fresh livers of wild
Flow rate : 1.0 mL/min. cod, Gadus morhua L. and other species of Gadidae, solid
substances being removed by cooling and filtering. A suitable
Detection : spectrophotometer at 265 nm. antioxidant may be added.
Injection : 2 quantities not exceeding 200 μL of each of the Content :
3 solutions obtained under Purification.
– vitamin A : 600 IU (180 μg) to 2500 IU (750 μg) per gram ;
System suitability : – vitamin D3 : 60 IU (1.5 μg) to 250 IU (6.25 μg) per gram.
– resolution : minimum 1.4 between the peaks due to
ergocalciferol and cholecalciferol in the chromatogram PRODUCTION
obtained with reference solution (b) ; The content of dioxins and dioxin-like PCBs (polychlorinated
biphenyls) is controlled using methods and limits in
– the results obtained with the test solution (b) duplicates do
accordance with the requirements set in the European Union
not differ by more than 5 per cent.
or other applicable regulations.
Calculate the content of vitamin D3 in International Units per
gram using the following expression, taking into account the CHARACTERS
assigned content of cholecalciferol CRS : Appearance : clear, yellowish liquid.
Solubility : practically insoluble in water, miscible with light
petroleum, slightly soluble in ethanol (96 per cent).

IDENTIFICATION
m1 = mass of the sample in test solution (b), in grams ; First identification : A, B, C.
m2 = total mass of cholecalciferol CRS used for Second identification : C, D.
the preparation of reference solution (a), in A. In the assay for vitamin A using method A, the test solution
micrograms (500 μg) ; shows an absorption maximum (2.2.25) at 325 ± 2 nm. In
A1 = area (or height) of the peak due to cholecalciferol in the assay for vitamin A using method B, the chromatogram
the chromatogram obtained with test solution (a) ; obtained with the test solution shows a peak corresponding
A2 = area (or height) of the peak due to cholecalciferol in to the peak due to all-trans-retinol in the chromatogram
the chromatogram obtained with test solution (b) ; obtained with the reference solution.
A3 = area (or height) of the peak due to ergocalciferol B. In the assay for vitamin D3, the chromatogram obtained
in the chromatogram obtained with reference with test solution (a) shows a peak corresponding to the
solution (b) ; peak due to cholecalciferol in the chromatogram obtained
A4 = area (or height) of the peak due to ergocalciferol in with reference solution (b).
the chromatogram obtained with test solution (b) ; C. Composition of fatty acids (see Tests).
A5 = area (or height) of a possible peak in the D. To 0.1 g add 0.5 mL of methylene chloride R and 1 mL of
chromatogram obtained with test solution (a) with antimony trichloride solution R. Mix. A deep blue colour
the same retention time as the peak co-eluting with develops in about 10 s.
ergocalciferol in test solution (b) ;
A6 = area (or height) of the peak due to cholecalciferol TESTS
in the chromatogram obtained with reference
solution (b) ; Appearance. The substance to be examined is not more
V1 = total volume of reference solution (a) (100 mL) ; intensely coloured than a reference solution prepared as
follows : to 3.0 mL of red primary solution add 25.0 mL of
V2 = volume of reference solution (a) used for preparing yellow primary solution and dilute to 50.0 mL with a 10 g/L
reference solution (b) (2.0 mL). solution of hydrochloric acid R (2.2.2, Method II).
Relative density (2.2.5) : 0.917 to 0.930.
STORAGE Refractive index (2.2.6) : 1.477 to 1.484.
In an airtight and well-filled container, protected from light. If Acid value (2.5.1) : maximum 2.0.
no antioxidant is added, store under an inert gas. Anisidine value (2.5.36) : maximum 30.0.
Once the container has been opened, its contents are used as Iodine value (2.5.4, Method B) : 150 to 180.
soon as possible and any part of the contents not used at once
is protected by an atmosphere of inert gas. Use starch solution R2.
Peroxide value (2.5.5, Method B) : maximum 10.0.
LABELLING Unsaponifiable matter (2.5.7) : maximum 1.5 per cent,
determined on 2.0 g, and extracting with 3 quantities, each of
The label states : 50 mL, of peroxide-free ether R.
– the concentration of EPA and DHA as a sum ; Stearin. Heat at least 10 mL to 60-90 °C then allow to cool for
– the number of International Units of vitamin A per gram ; 3 h in a bath of iced water or a thermostatically controlled
bath at 0 ± 0.5 °C. If necessary, to eliminate insoluble matter,
– the number of International Units of vitamin D3 per gram. filter the sample after heating. The sample remains clear.

1950 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cod-liver oil (type A)

Composition of fatty acids. Gas chromatography (2.2.28). Column :

Trivial name of Nomenclature Lower limit Upper limit – material : fused silica ;
fatty acid area area – size : l = 30 m, Ø = 0.25 mm ;
(per cent) (per cent)
Saturated fatty acids : – stationary phase : macrogol 20 000 R (film thickness
0.25 μm).
Myristic acid 14:0 2.0 6.0
Carrier gas : hydrogen for chromatography R or helium for
Palmitic acid 16:0 7.0 14.0
chromatography R, where oxygen scrubber is applied.
Stearic acid 18:0 1.0 4.0
Mono-unsaturated fatty acids :
Split ratio : 1:200.
Temperature :
Palmitoleic acid 16:1 n-7 4.5 11.5
cis-Vaccenic acid 18:1 n-7 2.0 7.0 Time Temperature
Oleic acid 18:1 n-9 12.0 21.0 (min) (°C)
Gadoleic acid 20:1 n-11 1.0 5.5 Column 0 - 55 170 → 225
Gondoic acid 20:1 n-9 5.0 17.0 55 - 75 225
Erucic acid 22:1 n-9 0 1.5
Cetoleic acid 22:1 n-11+13 5.0 12.0 Injection port 250
(22:1 n-11) Detector 280
Poly-unsaturated fatty acids :

Linoleic acid 18:2 n-6 0.5 Detection : flame ionisation.

3.0
α-Linolenic acid 18:3 n-3 0 Injection : 1 μL, twice.
2.0
Moroctic acid 18:4 n-3 0.5 System suitability :
4.5
Timnodonic 20:5 n-3 7.0 16.0
(eicosapentaenoic)
– the 15 fatty acids to be tested are satisfactorily identified
acid (EPA) from the chromatogram shown in Figure 1192.-1 ;
Cervonic 22:6 n-3 6.0 18.0 – injection of a mixture of equal amounts of methyl
(docosahexaenoic) palmitate R, methyl stearate R, methyl arachidate R and
acid (DHA)
methyl behenate R gives area percentages of 24.4, 24.8, 25.2
Test solution. Introduce about 0.45 g of the substance to be and 25.6 (± 0.5 per cent), respectively ;
examined into a 10 mL volumetric flask, dissolve in hexane R – resolution : minimum 1.3 between the peaks due to methyl
containing 50 mg of butylhydroxytoluene R per litre and dilute oleate and methyl cis-vaccenate ; the resolution between
to 10.0 mL with the same solvent. Transfer 2.0 mL of this the pair due to methyl gadoleate and methyl gondoate
solution into a quartz tube and evaporate the solvent with a is sufficient for purposes of identification and area
gentle current of nitrogen R. Add 1.5 mL of a 20 g/L solution measurement.
of sodium hydroxide R in methanol R, cover with nitrogen R,
cap tightly with a polytetrafluoroethylene-lined cap, mix and Calculate the area per cent for each fatty acid methyl ester
heat on a water-bath for 7 min. Cool, add 2 mL of boron using the following expression :
trichloride-methanol solution R, cover with nitrogen R, cap
tightly, mix and heat on a water-bath for 30 min. Cool to
40-50 °C, add 1 mL of trimethylpentane R, cap and vortex or
shake vigorously for at least 30 s. Immediately add 5 mL of Ax = peak area of fatty acid x ;
saturated sodium chloride solution R, cover with nitrogen R,
cap and vortex or shake vigorously for at least 15 s. Allow A t = sum of the peak areas (up to C22:6 n-3).
the upper layer to become clear and transfer it to a separate The calculation is not valid unless :
tube. Shake the methanol layer once more with 1 mL of
trimethylpentane R and combine the trimethylpentane – the total area is based only on peaks due solely to fatty acid
extracts. Wash the combined extracts with 2 quantities, each methyl esters ;
of 1 mL, of water R and dry over anhydrous sodium sulfate R. – the number of fatty acid methyl ester peaks exceeding
Prepare 2 solutions for each sample. 0.05 per cent of the total area is at least 24 ;

Figure 1192.-1. – Chromatogram for the test for composition of fatty acids of cod-liver oil (type A)

General Notices (1) apply to all monographs and other texts 1951
Cod-liver oil (type A) EUROPEAN PHARMACOPOEIA 8.0

– the 24 largest peaks of the methyl esters account for more solution of potassium hydroxide R and 100 mL of anhydrous
than 90 per cent of the total area (these correspond to, in ethanol R. Boil under a reflux condenser on a water-bath for
common elution order : 14:0, 15:0, 16:0, 16:1 n-7, 16:4 n-1, 15 min. Add 100 mL of a 10 g/L solution of sodium chloride R
18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6, 18:3 n-3, 18:4 n-3, and cool. Transfer the solution to a 500 mL separating funnel,
20:1 n-11, 20:1 n-9, 20:1 n-7, 20:2 n-6, 20:4 n-6, 20:3 n-3, rinsing the round-bottomed flask with about 75 mL of a
20:4 n-3, 20:5 n-3, 22:1 n-11, 22:1 n-9, 21:5 n-3, 22:5 n-3, 10 g/L solution of sodium chloride R and then with 150 mL of
22:6 n-3). a mixture of equal volumes of ether R and light petroleum R1.
Shake for 1 min. When the layers have separated completely,
ASSAY discard the lower layer and wash the upper layer, first with
Vitamin A. Carry out the test as rapidly as possible, avoiding 50 mL of a 30 g/L solution of potassium hydroxide R in a
exposure to actinic light and air, oxidising agents, oxidation 10 per cent V/V solution of anhydrous ethanol R and then
catalysts (for example, copper and iron) and acids. with 3 quantities, each of 50 mL, of a 10 g/L solution of
Use method A. If method A is found not to be valid, use sodium chloride R. Filter the upper layer through 5 g of
method B. anhydrous sodium sulfate R on a fast filter paper into a 250 mL
flask suitable for a rotary evaporator. Wash the funnel with
METHOD A 10 mL of fresh extraction mixture, filter and combine the
Ultraviolet absorption spectrophotometry (2.2.25). upper layers. Distil them at a temperature not exceeding
Test solution. To 1.00 g in a round-bottomed flask, add 3 mL 30 °C under reduced pressure (water ejector) and fill with
of a freshly prepared 50 per cent m/m solution of potassium nitrogen R when evaporation is completed. Alternatively,
hydroxide R and 30 mL of anhydrous ethanol R. Boil under evaporate the solvent under a gentle current of nitrogen R at
reflux in a current of nitrogen R for 30 min. Cool rapidly and a temperature not exceeding 30 °C. Dissolve the residue in
add 30 mL of water R. Extract with 50 mL of ether R. Repeat the 2-propanol R, transfer to a 25 mL volumetric flask and dilute
extraction 3 times and discard the lower layer after complete to 25 mL with 2-propanol R. Gentle heating in an ultrasonic
separation. Wash the combined upper layers with 4 quantities, bath may be required. A large fraction of the white residue is
each of 50 mL, of water R, and evaporate to dryness under a cholesterol, constituting approximately 50 per cent m/m of the
gentle current of nitrogen R at a temperature not exceeding unsaponifiable matter of cod-liver oil.
30 °C or in a rotary evaporator at a temperature not exceeding Reference solution (a). Prepare a solution of retinol acetate CRS
30 °C under reduced pressure (water ejector). Dissolve in 2-propanol R1 so that 1 mL contains about 1000 IU of
the residue in sufficient 2-propanol R1 to give an expected all-trans-retinol.
concentration of vitamin A equivalent to 10-15 IU/mL. The exact concentration of reference solution (a) is assessed
Measure the absorbances of the solution at 300 nm, 310 nm, by ultraviolet absorption spectrophotometry (2.2.25). Dilute
325 nm and 334 nm and at the wavelength of maximum reference solution (a) with 2-propanol R1 to a presumed
absorption with a suitable spectrophotometer in specially concentration of 10-15 IU/mL and measure the absorbance
matched 1 cm cells, using 2-propanol R1 as the compensation at 326 nm in matched 1 cm cells using 2-propanol R1 as the
liquid. compensation liquid.
Calculate the content of vitamin A, as all-trans-retinol, in Calculate the content of vitamin A in International Units
International Units per gram, using the following expression : per millilitre of reference solution (a) using the following
expression, taking into account the assigned content of retinol
acetate CRS :

A325 = absorbance at 325 nm ;

m = mass of the substance to be examined, in grams ; A326 = absorbance at 326 nm ;
V = total volume of solution containing 10-15 IU of V1 = volume of reference solution (a) used ;
vitamin A per millilitre ;
1821 = conversion factor for the specific absorbance of V2 = volume of the diluted solution ;
all-trans-retinol, in International Units. 1900 = conversion factor for the specific absorbance of
The above expression can be used only if A325 has a value retinol acetate CRS, in International Units.
not greater than A325,corr/0.970, where A325,corr is the corrected Reference solution (b). Proceed as described for the test
absorbance at 325 nm and is given by the following equation : solution but using 2.00 mL of reference solution (a) in place of
the substance to be examined.
The exact concentration of reference solution (b) is assessed
A designates the absorbance at the wavelength indicated by by ultraviolet absorption spectrophotometry (2.2.25). Dilute
the subscript. reference solution (b) with 2-propanol R1 to a presumed
If A325 has a value greater than A325,corr/0.970, calculate the all-trans-retinol concentration of 10-15 IU/mL and measure
content of vitamin A using the following expression : the absorbance at 325 nm in matched 1 cm cells using
2-propanol R1 as the compensation liquid.
Calculate the content of all-trans-retinol in International Units
per millilitre of reference solution (b), using the following
The assay is not valid unless : expression :
– the wavelength of the maximum absorption lies between
323 nm and 327 nm ;
– the absorbance at 300 nm relative to that at 325 nm is at
most 0.73. A325 = absorbance at 325 nm ;
METHOD B V3 = volume of the diluted solution ;
Liquid chromatography (2.2.29). V4 = volume of reference solution (b) used ;
Test solution. Prepare duplicates. To 2.00 g in a
round-bottomed flask, add 5 mL of a freshly prepared 100 g/L 1821 = conversion factor for the specific absorbance of
solution of ascorbic acid R, 10 mL of a freshly prepared 800 g/L all-trans-retinol, in International Units.

1952 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cod-liver oil (type A)

Column : Test solution (b). Prepare duplicates. To 4.00 g add 2.0 mL of

– size : l = 0.25 m, Ø = 4.6 mm ; the internal standard solution and proceed as described for
– stationary phase : octadecylsilyl silica gel for test solution (a).
chromatography R (5-10 μm). Reference solution (a). Dissolve 0.50 mg of cholecalciferol CRS
Mobile phase : water R, methanol R (3:97 V/V). in 100.0 mL of anhydrous ethanol R.
Flow rate : 1 mL/min. Reference solution (b). Into a round-bottomed flask,
add 2.0 mL of reference solution (a) and 2.0 mL of the
Detection : spectrophotometer at 325 nm. internal standard solution and proceed as described for test
Injection : 10 μL ; inject in triplicate the test solution and solution (a).
reference solution (b).
PURIFICATION
Retention time : all-trans-retinol = 5 ± 1 min. Column :
System suitability : – size : l = 0.25 m, Ø = 4.6 mm ;
– the chromatogram obtained with the test solution shows a – stationary phase : nitrile silica gel for chromatography R
peak corresponding to the peak due to all-trans-retinol in (10 μm).
the chromatogram obtained with reference solution (b) ;
Mobile phase : isoamyl alcohol R, hexane R (1.6:98.4 V/V).
– the results obtained with the duplicate test solutions do not
differ by more than 5 per cent ; Flow rate : 1.1 mL/min.
– the recovery of all-trans-retinol in reference solution (b) as Detection : spectrophotometer at 265 nm.
assessed by direct absorption spectrophotometry is greater Injection : 350 μL of reference solution (b) and test
than 95 per cent. solutions (a) and (b). Collect each eluate from 2 min before
Calculate the content of vitamin A using the following until 2 min after the retention time of cholecalciferol, in a
expression : ground-glass-stoppered tube containing 1 mL of a 1 g/L
solution of butylhydroxytoluene R in hexane R. Evaporate
separately to dryness at a temperature not exceeding 30 °C
under a gentle current of nitrogen R. Dissolve each residue in
1.5 mL of acetonitrile R.
A1 = area of the peak due to all-trans-retinol in the
chromatogram obtained with the test solution ; DETERMINATION
A2 = area of the peak due to all-trans-retinol in Column :
the chromatogram obtained with reference – size : l = 0.15 m, Ø = 4.6 mm ;
solution (b) ; – stationary phase : octadecylsilyl silica gel for
C = concentration of retinol acetate CRS in reference chromatography R (5 μm).
solution (a) as assessed prior to the saponification, Mobile phase : phosphoric acid R, 96 per cent V/V solution of
in International Units per millilitre (= 1000 IU/mL) ; acetonitrile R (0.2:99.8 V/V).
V = volume of reference solution (a) treated (2.00 mL) ; Flow rate : 1.0 mL/min.
m = mass of the substance to be examined in the test Detection : spectrophotometer at 265 nm.
solution (2.00 g). Injection : 2 quantities not exceeding 200 μL of each of the
3 solutions obtained under Purification.
Vitamin D3. Liquid chromatography (2.2.29). Carry out the
assay as rapidly as possible, avoiding exposure to actinic light System suitability :
and air. – resolution : minimum 1.4 between the peaks due to
Internal standard solution. Dissolve 0.50 mg of ergocalciferol and cholecalciferol in the chromatogram
ergocalciferol CRS in 100 mL of anhydrous ethanol R. obtained with reference solution (b) ;
Test solution (a). To 4.00 g in a round-bottomed flask, add – the results obtained with test solution (b) duplicates do not
5 mL of a freshly prepared 100 g/L solution of ascorbic acid R, differ by more than 5 per cent.
10 mL of a freshly prepared 800 g/L solution of potassium Calculate the content of vitamin D3 in International Units per
hydroxide R and 100 mL of anhydrous ethanol R. Boil under a gram using the following expression, taking into account the
reflux condenser on a water-bath for 30 min. Add 100 mL of assigned content of cholecalciferol CRS :
a 10 g/L solution of sodium chloride R and cool the solution
to room temperature. Transfer the solution to a 500 mL
separating funnel, rinsing the round-bottomed flask with
about 75 mL of a 10 g/L solution of sodium chloride R and
then with 150 mL of a mixture of equal volumes of ether R and
light petroleum R1. Shake for 1 min. When the layers have m1 = mass of the sample in test solution (b), in grams ;
separated completely, discard the lower layer and wash the m2 = total mass of cholecalciferol CRS used for
upper layer, first with 50 mL of a 30 g/L solution of potassium the preparation of reference solution (a), in
hydroxide R in a 10 per cent V/V solution of anhydrous micrograms (500 μg) ;
ethanol R, and then with 3 quantities, each of 50 mL, of a 10 g/L A1 = area (or height) of the peak due to cholecalciferol in
solution of sodium chloride R. Filter the upper layer through the chromatogram obtained with test solution (a) ;
5 g of anhydrous sodium sulfate R on a fast filter paper into a A2 = area (or height) of the peak due to cholecalciferol in
250 mL flask suitable for a rotary evaporator. Wash the funnel
the chromatogram obtained with test solution (b) ;
with 10 mL of fresh extraction mixture, filter and combine the
upper layers. Distil them at a temperature not exceeding 30 °C A3 = area (or height) of the peak due to ergocalciferol
under reduced pressure (water ejector) and fill with nitrogen R in the chromatogram obtained with reference
when evaporation is completed. Alternatively, evaporate the solution (b);
solvent under a gentle current of nitrogen R at a temperature A4 = area (or height) of the peak due to ergocalciferol in
not exceeding 30 °C. Dissolve the residue in 1.5 mL of the the chromatogram obtained with test solution (b) ;
mobile phase described under Purification. Gentle heating A5 = area (or height) of a possible peak in the
in an ultrasonic bath may be required. A large fraction of the chromatogram obtained with test solution (a) with
white residue is cholesterol, constituting approximately 50 per the same retention time as the peak co-eluting with
cent m/m of the unsaponifiable matter of cod-liver oil. ergocalciferol in test solution (b) ;

General Notices (1) apply to all monographs and other texts 1953
Cod-liver oil (type B) EUROPEAN PHARMACOPOEIA 8.0

A6 = area (or height) of the peak due to cholecalciferol Refractive index (2.2.6) : 1.477 to 1.484.
in the chromatogram obtained with reference Acid value (2.5.1) : maximum 2.0.
solution (b) ;
V1 = total volume of reference solution (a) (100 mL) ; Iodine value (2.5.4, Method B) : 150 to 180.
Use starch solution R2.
V2 = volume of reference solution (a) used for preparing
reference solution (b) (2.0 mL). Peroxide value (2.5.5, Method B) : maximum 10.0.
Unsaponifiable matter (2.5.7) : maximum 1.5 per cent,
STORAGE determined on 2.0 g and extracting with 3 quantities, each of
In an airtight and well-filled container, protected from light. If 50 mL, of peroxide-free ether R.
no antioxidant is added, store under an inert gas. Stearin. Heat at least 10 mL to 60-90 °C then allow to cool for
Once the container has been opened, its contents are used as 3 h in a bath of iced water or a thermostatically controlled
soon as possible and any part of the contents not used at once bath at 0 ± 0.5 °C. If necessary, to eliminate insoluble matter,
is protected by an atmosphere of inert gas. filter the sample after heating. The sample remains clear.
LABELLING Composition of fatty acids. Gas chromatography (2.2.28).
The label states : Trivial name Nomenclature Lower limit Upper limit
– the number of International Units of vitamin A per gram ; of fatty acid area area
(per cent) (per cent)
– the number of International Units of vitamin D3 per gram.
Saturated fatty acids :

07/2012:1193 Myristic acid 14:0 2.0 6.0

Palmitic acid 16:0 7.0 14.0
COD-LIVER OIL (TYPE B) Stearic acid 18:0 1.0 4.0
Mono-unsaturated fatty acids :
Iecoris aselli oleum B Palmitoleic acid 16:1 n-7 4.5 11.5
cis-Vaccenic acid 18:1 n-7 2.0 7.0
DEFINITION 12.0
Oleic acid 18:1 n-9 21.0
Purified fatty oil obtained from the fresh livers of wild Gadoleic acid 20:1 n-11 1.0 5.5
cod, Gadus morhua L. and other species of Gadidae, solid Gondoic acid 20:1 n-9 5.0 17.0
substances being removed by cooling and filtering. A suitable Erucic acid 22:1 n-9 0 1.5
antioxidant may be added. 22:1 n-11+13 5.0 12.0
Cetoleic acid
Content : (22:1 n-11)
– vitamin A : 600 IU (180 μg) to 2500 IU (750 μg) per gram ; Poly-unsaturated fatty acids :
– vitamin D3 : 60 IU (1.5 μg) to 250 IU (6.25 μg) per gram. Linoleic acid 18:2 n-6 0.5 3.0
α-Linolenic acid 18:3 n-3 0 2.0
PRODUCTION
Moroctic acid 18:4 n-3 0.5 4.5
The content of dioxins and dioxin-like PCBs (polychlorinated Timnodonic 20:5 n-3 7.0 16.0
biphenyls) is controlled using methods and limits in (eicosapentaenoic)
accordance with the requirements set in the European Union acid (EPA)
or other applicable regulations. Cervonic 22:6 n-3 6.0 18.0
(docosahexaenoic)
CHARACTERS acid (DHA)
Appearance : clear, yellowish liquid. Test solution. Introduce about 0.45 g of the substance to be
Solubility : practically insoluble in water, miscible with lightexamined into a 10 mL volumetric flask, dissolve in hexane R
petroleum, slightly soluble in ethanol (96 per cent). containing 50 mg of butylhydroxytoluene R per litre and dilute
to 10.0 mL with the same solvent. Transfer 2.0 mL of the
IDENTIFICATION solution into a quartz tube and evaporate the solvent with a
First identification : A, B, C. gentle current of nitrogen R. Add 1.5 mL of a 20 g/L solution
Second identification : C, D. of sodium hydroxide R in methanol R, cover with nitrogen R,
A. In the assay for vitamin A using method A, the test solution cap tightly with a polytetrafluoroethylene-lined cap, mix and
shows an absorption maximum (2.2.25) at 325 ± 2 nm. In heat on a water-bath for 7 min. Cool, add 2 mL of boron
the assay for vitamin A using method B, the chromatogram trichloride-methanol solution R, cover with nitrogen R, cap
obtained with the test solution shows a peak corresponding tightly, mix and heat on a water-bath for 30 min. Cool to
to the peak due to all-trans-retinol in the chromatogram 40-50 °C, add 1 mL of trimethylpentane R, cap and vortex or
obtained with the reference solution. shake vigorously for at least 30 s. Immediately add 5 mL of
saturated sodium chloride solution R, cover with nitrogen R,
B. In the assay for vitamin D3, the chromatogram obtained
cap and vortex or shake thoroughly for at least 15 s. Allow
with test solution (a) shows a peak corresponding to the
the upper layer to become clear and transfer to a separate
peak due to cholecalciferol in the chromatogram obtained
tube. Shake the methanol layer once more with 1 mL of
with reference solution (b).
trimethylpentane R and combine the trimethylpentane
C. Composition of fatty acids (see Tests). extracts. Wash the combined extracts with 2 quantities, each
D. To 0.1 g add 0.5 mL of methylene chloride R and 1 mL of of 1 mL, of water R and dry over anhydrous sodium sulfate R.
antimony trichloride solution R. Mix. A deep blue colour Prepare 2 solutions for each sample.
develops in about 10 s. Column :
TESTS – material : fused silica ;
Appearance. The substance to be examined is not more – size : l = 30 m, Ø = 0.25 mm ;
intensely coloured than a reference solution prepared as – stationary phase : macrogol 20 000 R (film thickness
follows : to 3.0 mL of red primary solution add 25.0 mL of 0.25 μm).
yellow primary solution and dilute to 50.0 mL with a 10 g/L Carrier gas : hydrogen for chromatography R or helium for
solution of hydrochloric acid R (2.2.2, Method II). chromatography R, where oxygen scrubber is applied.
Relative density (2.2.5) : 0.917 to 0.930. Split ratio : 1:200.

1954 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cod-liver oil (type B)

Temperature : ASSAY
Time Temperature Vitamin A. Carry out the test as rapidly as possible, avoiding
(min) (°C) exposure to actinic light and air, oxidising agents, oxidation
Column 0 - 55 170 → 225 catalysts (for example, copper and iron) and acids.
Use method A. If method A is found not to be valid, use
55 - 75 225
method B.
Injection port 250 METHOD A
Detector 280 Ultraviolet absorption spectrophotometry (2.2.25).
Test solution. To 1.00 g in a round-bottomed flask, add 3 mL
Detection : flame ionisation. of a freshly prepared 50 per cent m/m solution of potassium
Injection : 1 μL, twice. hydroxide R and 30 mL of anhydrous ethanol R. Boil under
reflux in a current of nitrogen R for 30 min. Cool rapidly and
System suitability : add 30 mL of water R. Extract with 50 mL of ether R. Repeat the
– the 15 fatty acids to be tested are satisfactorily identified extraction 3 times and discard the lower layer after complete
from the chromatogram shown in Figure 1193.-1 ; separation. Wash the combined upper layers with 4 quantities,
– injection of a mixture of equal amounts of methyl each of 50 mL, of water R and evaporate to dryness under a
palmitate R, methyl stearate R, methyl arachidate R, and gentle current of nitrogen R at a temperature not exceeding
methyl behenate R give area percentages of 24.4, 24.8, 25.2 30 °C or in a rotary evaporator at a temperature not exceeding
and 25.6 (± 0.5 per cent), respectively ; 30 °C under reduced pressure (water ejector). Dissolve
the residue in sufficient 2-propanol R1 to give an expected
– resolution : minimum of 1.3 between the peaks due to concentration of vitamin A equivalent to 10-15 IU/mL.
methyl oleate and methyl cis-vaccenate ; the resolution
between the pair due to methyl gadoleate and methyl Measure the absorbances of the solution at 300 nm, 310 nm,
gondoate is sufficient for purposes of identification and 325 nm and 334 nm and at the wavelength of maximum
area measurement. absorption with a suitable spectrophotometer in specially
matched 1 cm cells, using 2-propanol R1 as the compensation
Calculate the area per cent for each fatty acid methyl ester liquid.
using the following expression :
Calculate the content of vitamin A, as all-trans-retinol, in
International Units per gram using the following expression :

Ax = peak area of fatty acid x ;

At = sum of the peak areas (up to C22:6 n-3). A325 = absorbance at 325 nm ;
m = mass of the substance to be examined, in grams ;
The calculation is not valid unless :
– the total area is based only on peaks due to solely fatty acids V = total volume of solution containing 10-15 IU of
methyl esters ; vitamin A per millilitre ;
1821 = conversion factor for the specific absorbance of
– the number of fatty acid methyl ester peaks exceeding all-trans-retinol, in International Units.
0.05 per cent of the total area is at least 24 ;
The above expression can be used only if A325 has a value
– the 24 largest peaks of the methyl esters account for more not greater than A325,corr/0.970 where A325,corr is the corrected
than 90 per cent of the total area (these correspond to, in absorbance at 325 nm and is given by the equation :
common elution order : 14:0, 15:0, 16:0, 16:1 n-7, 16:4 n-1,
18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6, 18:3 n-3, 18:4 n-3,
20:1 n-11, 20:1 n-9, 20:1 n-7, 20:2 n-6, 20:4 n-6, 20:3 n-3,
20:4 n-3, 20:5 n-3, 22:1 n-11, 22:1 n-9, 21:5 n-3, 22:5 n-3, A designates the absorbance at the wavelength indicated by
22:6 n-3). the subscript.

Figure 1193.-1. – Chromatogram for the test for composition of fatty acids of cod-liver oil (type B)

General Notices (1) apply to all monographs and other texts 1955
Cod-liver oil (type B) EUROPEAN PHARMACOPOEIA 8.0

If A325 has a value greater than A325,corr/0.970, calculate the concentration of 10-15 IU/mL of all-trans-retinol and measure
content of vitamin A using the following expression : the absorbance at 325 nm in matched 1 cm cells using
2-propanol R1 as the compensation liquid.
Calculate the content of all-trans-retinol in International Units
per millilitre of reference solution (b) from the expression :
The assay is not valid unless :
– the wavelength of maximum absorption lies between
323 nm and 327 nm ;
– the absorbance at 300 nm relative to that at 325 nm is at A325 = absorbance at 325 nm ;
most 0.73.
V3 = volume of the diluted solution ;
METHOD B
V4 = volume of reference solution (b) used ;
Liquid chromatography (2.2.29).
Test solution. Prepare duplicates. To 2.00 g in a 1821 = conversion factor for the specific absorbance of
round-bottomed flask, add 5 mL of a freshly prepared 100 g/L all-trans-retinol, in International Units.
solution of ascorbic acid R and 10 mL of a freshly prepared Column :
800 g/L solution of potassium hydroxide R and 100 mL of – size : l = 0.25 m, Ø = 4.6 mm ;
anhydrous ethanol R. Boil under a reflux condenser on a
water-bath for 15 min. Add 100 mL of a 10 g/L solution of – stationary phase : octadecylsilyl silica gel for
sodium chloride R and cool. Transfer the solution to a 500 mL chromatography R (5-10 μm).
separating funnel, rinsing the round-bottomed flask with Mobile phase : water R, methanol R (3:97 V/V).
about 75 mL of a 10 g/L solution of sodium chloride R and Flow rate : 1 mL/min.
then with 150 mL of a mixture of equal volumes of ether R and Detection : spectrophotometer at 325 nm.
light petroleum R1. Shake for 1 min. When the layers have
separated completely, discard the lower layer and wash the Injection : 10 μL ; inject in triplicate the test solution and
upper layer, first with 50 mL of a 30 g/L solution of potassium reference solution (b).
hydroxide R in a 10 per cent V/V solution of anhydrous Retention time : all-trans-retinol = 5 ± 1 min.
ethanol R and then with 3 quantities, each of 50 mL, of a 10 g/L System suitability :
solution of sodium chloride R. Filter the upper layer through – the chromatogram obtained with the test solution shows a
5 g of anhydrous sodium sulfate R on a fast filter paper into a peak corresponding to the peak due to all-trans-retinol in
250 mL flask suitable for a rotary evaporator. Wash the funnel the chromatogram obtained with reference solution (b);
with 10 mL of fresh extraction mixture, filter and combine
the upper layers. Distil them at a temperature not exceeding – the results obtained with the duplicate test solutions do not
differ by more than 5 per cent ;
30 °C under reduced pressure (water ejector) and fill with
nitrogen R when evaporation is completed. Alternatively – the recovery of all-trans-retinol in reference solution (b) as
evaporate the solvent under a gentle current of nitrogen R at assessed by direct absorption spectrophotometry is greater
a temperature not exceeding 30 °C. Dissolve the residue in than 95 per cent.
2-propanol R, transfer to a 25 mL volumetric flask and dilute Calculate the content of vitamin A using the following
to 25 mL with 2-propanol R. Gentle heating in an ultrasonic expression :
bath may be required. A large fraction of the white residue is
cholesterol, constituting approximately 50 per cent m/m of the
unsaponifiable matter of cod-liver oil.
Reference solution (a). Prepare a solution of retinol acetate CRS A1 = area of the peak due to all-trans-retinol in the
in 2-propanol R1 so that 1 mL contains about 1000 IU of chromatogram obtained with the test solution ;
all-trans-retinol. A2 = area of the peak due to all-trans-retinol in
The exact concentration of reference solution (a) is assessed the chromatogram obtained with reference
by ultraviolet absorption spectrophotometry (2.2.25). Dilute solution (b);
reference solution (a) with 2-propanol R1 to a presumed C = concentration of retinol acetate CRS in reference
concentration of 10-15 IU/mL and measure the absorbance solution (a) as assessed prior to the saponification,
at 326 nm in matched 1 cm cells using 2-propanol R1 as the in International Units per millilitre (= 1000 IU/mL) ;
compensation liquid. V = volume of reference solution (a) treated (2.00 mL) ;
Calculate the content of vitamin A in International Units m = mass of the substance to be examined in the test
per millilitre of reference solution (a) using the following solution (2.00 g).
expression, taking into account the assigned content of retinol
acetate CRS : Vitamin D3. Liquid chromatography (2.2.29). Carry out the
assay as rapidly as possible, avoiding exposure to actinic light
and air.
Internal standard solution. Dissolve 0.50 mg of
A326 = absorbance at 326 nm ; ergocalciferol CRS in 100 mL of anhydrous ethanol R.
V1 = volume of reference solution (a) used ; Test solution (a). To 4.00 g in a round-bottomed flask, add
5 mL of a freshly prepared 100 g/L solution of ascorbic acid R,
V2 = volume of the diluted solution ; 10 mL of a freshly prepared 800 g/L solution of potassium
1900 = conversion factor for the specific absorbance of hydroxide R and 100 mL of anhydrous ethanol R. Boil under a
retinol acetate CRS, in International Units. reflux condenser on a water-bath for 30 min. Add 100 mL of
a 10 g/L solution of sodium chloride R and cool the solution
Reference solution (b). Proceed as described for the test to room temperature. Transfer the solution to a 500 mL
solution but using 2.00 mL of reference solution (a) in place of separating funnel, rinsing the round-bottomed flask with
the substance to be examined. about 75 mL of a 10 g/L solution of sodium chloride R and
The exact concentration of reference solution (b) is assessed then with 150 mL of a mixture of equal volumes of ether R and
by ultraviolet absorption spectrophotometry (2.2.25). Dilute light petroleum R1. Shake for 1 min. When the layers have
reference solution (b) with 2-propanol R1 to a presumed separated completely, discard the lower layer and wash the

1956 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Colchicine

upper layer, first with 50 mL of a 30 g/L solution of potassium m1 = mass of the sample in test solution (b), in grams ;
hydroxide R in a 10 per cent V/V solution of anhydrous m2 = total mass of cholecalciferol CRS used for
ethanol R, and then with 3 quantities, each of 50 mL, of a 10 g/L the preparation of reference solution (a), in
solution of sodium chloride R. Filter the upper layer through micrograms (500 μg) ;
5 g of anhydrous sodium sulfate R on a fast filter paper into a A1 = area (or height) of the peak due to cholecalciferol in
250 mL flask suitable for a rotary evaporator. Wash the funnel
the chromatogram obtained with test solution (a) ;
with 10 mL of fresh extraction mixture, filter and combine the
upper layers. Distil them at a temperature not exceeding 30 °C A2 = area (or height) of the peak due to cholecalciferol in
under reduced pressure (water ejector) and fill with nitrogen R the chromatogram obtained with test solution (b) ;
when evaporation is completed. Alternatively evaporate the A3 = area (or height) of the peak due to ergocalciferol
solvent under a gentle current of nitrogen R at a temperature in the chromatogram obtained with reference
not exceeding 30 °C. Dissolve the residue in 1.5 mL of the solution (b);
mobile phase described under Purification. Gentle heating A4 = area (or height) of the peak due to ergocalciferol in
in an ultrasonic bath may be required. A large fraction of the the chromatogram obtained with test solution (b) ;
white residue is cholesterol, constituting approximately 50 per A5 = area (or height) of a possible peak in the
cent m/m of the unsaponifiable matter of cod-liver oil. chromatogram obtained with test solution (a) with
the same retention time as the peak co-eluting with
Test solution (b). Prepare duplicates. To 4.00 g add 2.0 mL of ergocalciferol in test solution (b) ;
the internal standard solution and proceed as described for
A6 = area (or height) of the peak due to cholecalciferol
test solution (a).
in the chromatogram obtained with reference
Reference solution (a). Dissolve 0.50 mg of cholecalciferol CRS solution (b);
in 100.0 mL of anhydrous ethanol R. V1 = total volume of reference solution (a) (100 mL) ;
Reference solution (b). In a round-bottomed flask, add 2.0 mL V2 = volume of reference solution (a) used for preparing
of reference solution (a) and 2.0 mL of the internal standard reference solution (b) (2.0 mL).
solution and proceed as described for test solution (a).
PURIFICATION STORAGE
Column : In an airtight and well-filled container, protected from light. If
no antioxidant is added, store under an inert gas.
– size : l = 0.25 m, Ø = 4.6 mm ;
Once the container has been opened, its contents are used as
– stationary phase : nitrile silica gel for chromatography R soon as possible and any part of the contents not used at once
(10 μm). is protected by an atmosphere of inert gas.
Mobile phase : isoamyl alcohol R, hexane R (1.6:98.4 V/V). LABELLING
Flow rate : 1.1 mL/min. The label states :
Detection : spectrophotometer at 265 nm. – the number of International Units of vitamin A per gram ;
Injection : 350 μL of reference solution (b) and test – the number of International Units of vitamin D3 per gram.
solutions (a) and (b). Collect each eluate from 2 min before
until 2 min after the retention time of cholecalciferol, in a 01/2008:0758
ground-glass-stoppered tube containing 1 mL of a 1 g/L corrected 7.2
solution of butylhydroxytoluene R in hexane R. Evaporate
separately to dryness at a temperature not exceeding 30 °C COLCHICINE
under a gentle current of nitrogen R. Dissolve each residue in
1.5 mL of acetonitrile R. Colchicinum
DETERMINATION
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : phosphoric acid R, 96 per cent V/V solution of
acetonitrile R (0.2:99.8 V/V). C22H25NO6 Mr 399.4
Flow rate : 1.0 mL/min. [64-86-8]
Detection : spectrophotometer at 265 nm. DEFINITION
Injection : 2 quantities not exceeding 200 μL of each of the (-)-N-[(7S,12aRa)-1,2,3,10-Tetramethoxy-9-oxo-5,6,7,9-
3 solutions obtained under Purification. tetrahydrobenzo[a]heptalen-7-yl]acetamide.
System suitability : Content : 97.0 per cent to 102.0 per cent (anhydrous substance).
– resolution : minimum 1.4 between the peaks due to CHARACTERS
ergocalciferol and cholecalciferol in the chromatogram Appearance : yellowish-white, amorphous or crystalline
obtained with reference solution (b) ; powder.
– the results obtained with the test solution (b) duplicates do Solubility : very soluble in water, rapidly recrystallising from
not differ by more than 5 per cent. concentrated solutions as the sesquihydrate, freely soluble in
ethanol (96 per cent), practically insoluble in cyclohexane.
Calculate the content of vitamin D3 in International Units per
gram using the following expression, taking into account the IDENTIFICATION
assigned content of cholecalciferol CRS : First identification : B.
Second identification : A, C, D.
A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).

General Notices (1) apply to all monographs and other texts 1957
Colchicine EUROPEAN PHARMACOPOEIA 8.0

Test solution. Dissolve 5 mg in ethanol (96 per cent) R and System suitability : reference solution (a) :
dilute to 100.0 mL with the same solvent. Dilute 5.0 mL of Peak-to-valley ratio : minimum 2, where Hp = height above the
this solution to 25.0 mL with ethanol (96 per cent) R. baseline of the peak due to impurity A and Hv = height above
Spectral range : 230-400 nm. the baseline of the lowest point of the curve separating this
Absorption maxima : at 243 nm and 350 nm. peak from the peak due to colchicine.
Absorbance ratio : A243/A350 = 1.7 to 1.9. Limits :
B. Infrared absorption spectrophotometry (2.2.24). – impurity A : not more than 3.5 times the area of the
principal peak in the chromatogram obtained with
Preparation : discs of potassium bromide R. reference solution (b) (3.5 per cent) ;
Comparison : colchicine CRS. – any other impurity : not more than the area of the principal
C. To 0.5 mL of solution S (see Tests) add 0.5 mL of peak in the chromatogram obtained with reference
dilute hydrochloric acid R and 0.15 mL of ferric chloride solution (b) (1 per cent);
solution R1. The solution is yellow and becomes dark green – total : not more than 5 times the area of the principal peak
on boiling for 30 s. Cool, add 2 mL of methylene chloride R in the chromatogram obtained with reference solution (b)
and shake. The organic layer is greenish-yellow. (5 per cent) ;
D. Dissolve about 30 mg in 1 mL of ethanol (96 per cent) R and – disregard limit : the area of the principal peak in the
add 0.15 mL of ferric chloride solution R1. A brownish-red chromatogram obtained with reference solution (c)
colour develops. (0.05 per cent).
TESTS Colchiceine : maximum 0.2 per cent.
Solution S. Dissolve 0.10 g in water R and dilute to 20 mL Dissolve 50 mg in water R and dilute to 5 mL with the same
with the same solvent. solvent. Add 0.1 mL of ferric chloride solution R1. The solution
is not more intensely coloured than a mixture of 1 mL of red
Appearance of solution. Solution S is clear (2.2.1) and not primary solution, 2 mL of yellow primary solution and 2 mL
more intensely coloured than reference solution GY3 (2.2.2, of blue primary solution (2.2.2, Method II).
Method II).
Chloroform (2.4.24) : maximum 500 ppm.
Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of
bromothymol blue solution R1. Either the solution does not Ethyl acetate (2.4.24) : maximum 6.0 per cent m/m.
change colour or it becomes green. Not more than 0.1 mL Water (2.5.12) : maximum 2.0 per cent, determined on 0.500 g.
of 0.01 M sodium hydroxide is required to change the colour
of the indicator to blue. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
0.5 g.
Specific optical rotation (2.2.7) : − 235 to − 250 (anhydrous
substance). ASSAY
Dissolve 50.0 mg in ethanol (96 per cent) R and dilute to Dissolve 0.250 g with gentle heating in a mixture of 10 mL of
10.0 mL with the same solvent. acetic anhydride R and 20 mL of toluene R. Titrate with 0.1 M
Related substances. Liquid chromatography (2.2.29). perchloric acid, determining the end-point potentiometrically
(2.2.20).
Solvent mixture : methanol R, water R (50:50 V/V).
1 mL of 0.1 M perchloric acid is equivalent to 39.94 mg of
Test solution. Dissolve 20.0 mg of the substance to be
C22H25NO6.
examined in the solvent mixture and dilute to 20.0 mL with
the solvent mixture.
STORAGE
Reference solution (a). Dissolve 5 mg of colchicine for system
suitability CRS in the solvent mixture and dilute to 5.0 mL Protected from light.
with the solvent mixture.
IMPURITIES
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture.
Reference solution (c). Dilute 1 mL of reference solution (b) to
20.0 mL with the solvent mixture.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octylsilyl silica gel for chromatography R1
(5 μm).
Mobile phase : mix 450 volumes of a 6.8 g/L solution of A. N-[(7S,12aRa)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-
potassium dihydrogen phosphate R and 530 volumes of tetrahydrobenzo[a]heptalen-7-yl]formamide
methanol R. After cooling to room temperature, adjust the (N-deacetyl-N-formylcolchicine),
volume to 1000 mL with methanol R. Adjust the apparent pH
to 5.5 with dilute phosphoric acid R.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 254 nm.
Injection : 20 μL.
Run time : 3 times the retention time of colchicine.
Relative retention with reference to colchicine (retention
time = about 7 min) : impurity D = about 0.4 ; B. (-)-N-[(7S,12aSa)-1,2,3,10-tetramethoxy-9-oxo-
impurity E = about 0.7 ; impurity B = about 0.8 ; 5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide
impurity A = about 0.94 ; impurity C = about 1.2. (conformational isomer),

1958 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Colestyramine

TESTS
pH (2.2.3) : 4.0 to 6.0.
Suspend 0.100 g in 10 mL of water R and allow to stand for
10 min.
Dialysable quaternary amines : maximum 500 ppm,
expressed as benzyltrimethylammonium chloride.
C. N-[(7S,7bR,10aS)-1,2,3,9-tetramethoxy-8-oxo- Test solution. Place a 25 cm piece of cellulose dialysis tubing
5,6,7,7b,8,10a-hexahydrobenzo[a]cyclopenta[3,4]- having a molecular weight cut-off of 12 000-14 000 and an
cyclobuta[1,2-c]cyclohepten-7-yl]acetamide inflated diameter of 3-6 cm (flat width of 5-9 cm) in water R to
(β-lumicolchicine), hydrate until pliable, appropriately sealing one end. Introduce
2.0 g of the substance to be examined into the tube and add
10 mL of water R. Seal the tube and completely immerse it in
100 mL of water R in a suitable vessel and stir the liquid for
16 h to effect dialysis. Use the dialysate as test solution.
Reference solution. Prepare the reference solution in a similar
manner but using 10 mL of a freshly prepared 0.1 g/L solution
of benzyltrimethylammonium chloride R instead of the
substance to be examined.
Transfer 5.0 mL of the test solution to a separating funnel and
add 5 mL of a 3.8 g/L solution of disodium tetraborate R, 1 mL
D. N-[(7S,12aRa)-3-(β-D-glucopyranosyloxy)-1,2,10- of a solution containing 1.5 g/L of bromothymol blue R and
trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7- 4.05 g/L of sodium carbonate R and 10 mL of chloroform R.
yl]acetamide (colchicoside), Shake the mixture vigourously for 1 min, allow the phases
to separate and transfer the clear organic layer to a 25 mL
volumetric flask. Repeat the extraction with a further 10 mL
of chloroform R, combine the organic layers and dilute to
25 mL with chloroform R. Measure the absorbance (2.2.25) of
the solution at the absorption maximum at 420 nm, using as
compensation liquid a solution prepared in the same manner
but using 5.0 mL of water R instead of the test solution.
Repeat the operation using 5.0 mL of the reference solution.
E. N-[(7S,12aRa)-3-hydroxy-1,2,10-trimethoxy-9-oxo-
5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide The absorbance obtained with the test solution is not greater
than that obtained with the reference solution.
(3-O-demethylcolchicine),
Impurity A. Liquid chromatography (2.2.29).
Test solution. Shake 5.0 g with 10 mL of acetone R for 30 min.
Centrifuge and use the supernatant.
Reference solution (a). Dissolve 5 mg of styrene R in acetone R
and dilute to 100.0 mL with the same solvent. Dilute 1.0 mL
of the solution to 100.0 mL with acetone R.
Reference solution (b). Dissolve 0.35 mL of styrene R in
F. N-[(7S,12aRa)-10-hydroxy-1,2,3-trimethoxy-9-oxo-5,6,7,9- acetone R and dilute to 100.0 mL with the same solvent. Dilute
tetrahydrobenzo[a]heptalen-7-yl]acetamide (colchiceine). 1.0 mL of the solution to 100.0 mL with acetone R.
Reference solution (c). Dissolve 0.35 mL of toluene R in
acetone R and dilute to 100.0 mL with the same solvent.
01/2008:1775 Reference solution (d). Mix 1.0 mL of reference solution (b)
and 1.0 mL of reference solution (c) with acetone R and dilute
COLESTYRAMINE to 100.0 mL with the same solvent.
Column :
Colestyraminum – size : l = 0.30 m, Ø = 3.9 mm,
– stationary phase : octadecylsilyl silica gel for
[11041-12-6] chromatography R (10 μm) with a specific surface
area of 330 m2/g and a pore size of 12.5 nm.
DEFINITION Mobile phase : acetonitrile R, water R (50:50 V/V).
Strongly basic anion-exchange resin in chloride form, Flow rate : 2.0 mL/min.
consisting of styrene-divinylbenzene copolymer with Detection : spectrophotometer at 254 nm.
quaternary ammonium groups.
Injection : 20 μL of test solution and reference solutions (a)
Nominal exchange capacity : 1.8 g to 2.2 g of sodium and (d).
glycocholate per gram (dried substance).
System suitability : reference solution (d) :
CHARACTERS – resolution : minimum 1.5 between the peaks due to
Appearance : white or almost white, fine powder, hygroscopic. impurity A and toluene.
Solubility : insoluble in water, in methylene chloride and in Limit :
ethanol (96 per cent). – impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (a)
IDENTIFICATION (1 ppm).
A. Infrared absorption spectrophotometry (2.2.24). Chloride : 13.0 per cent to 17.0 per cent (dried substance).
Comparison : colestyramine CRS. To 0.2 g add 100 mL of water R and 50 mg of potassium
B. Chloride (see Tests). nitrate R. Add, with stirring, 2 mL of nitric acid R and

General Notices (1) apply to all monographs and other texts 1959
Colistimethate sodium EUROPEAN PHARMACOPOEIA 8.0

titrate with 0.1 M silver nitrate, determining the end-point IMPURITIES

potentiometrically (2.2.20). Specified impurities : A.
1 mL of 0.1 M silver nitrate is equivalent to 3.55 mg of Cl. A. styrene.
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test F. Prepare the reference solution using 01/2008:0319
2 mL of lead standard solution (10 ppm Pb) R. corrected 6.0
Loss on drying (2.2.32) : maximum 12 per cent, determined
on 1.000 g by drying in an oven at 70 °C over diphosphorus COLISTIMETHATE SODIUM
pentoxide R at a pressure not exceeding 7 kPa for 16 h.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Colistimethatum natricum
1.0 g.
[8068-28-8]
ASSAY
Exchange capacity. Liquid chromatography (2.2.29). DEFINITION
Solution A. Dissolve 1.500 g of sodium glycocholate R in a Colistimethate sodium is prepared from colistin by the action
solution containing 4 g/L of potassium dihydrogen phosphate R of formaldehyde and sodium hydrogen sulfite.
and 12 g/L of dipotassium hydrogen phosphate R and dilute to Semi-synthetic product derived from a fermentation product.
100.0 mL with the same solution. Content : minimum 11 500 IU/mg (dried substance).
Test solution. Add 20.0 mL of solution A to a quantity of the
substance to be examined equivalent to about 0.100 g of the CHARACTERS
dried substance. Shake mechanically for 2 h and centrifuge Appearance : white or almost white, hygroscopic powder.
for 15 min. Dilute 5.0 mL of the supernatant to 50.0 mL with Solubility : very soluble in water, slightly soluble in ethanol
water R. (96 per cent), practically insoluble in acetone.
Reference solution (a). Dilute 4.0 mL of solution A to 100.0 mL IDENTIFICATION
with water R.
A. Thin-layer chromatography (2.2.27).
Reference solution (b). Dissolve 60 mg of sodium glycocholate R
and 30 mg of sodium taurodeoxycholate R in water R and Test solution. Dissolve 5 mg of the substance to be
dilute to 100 mL with the same solvent. Dilute 1 mL of the examined in 1 mL of a mixture of equal volumes of
solution to 10 mL with water R. hydrochloric acid R and water R. Heat at 135 °C in a
sealed tube for 5 h. Evaporate to dryness on a water-bath
Column : and continue the heating until the hydrochloric acid has
– size : l = 0.25 m, Ø = 4.6 mm, evaporated. Dissolve the residue in 0.5 mL of water R.
– stationary phase : octadecylsilyl silica gel for Reference solution (a). Dissolve 20 mg of leucine R in
chromatography R (5 μm). water R and dilute to 10 mL with the same solvent.
Mobile phase : mix 35 volumes of acetonitrile R and 65 volumes Reference solution (b). Dissolve 20 mg of threonine R in
of a 10.9 g/L solution of potassium dihydrogen phosphate R water R and dilute to 10 mL with the same solvent.
adjusted to pH 3.0 with phosphoric acid R. Reference solution (c). Dissolve 20 mg of phenylalanine R
Flow rate : 1.5 mL/min. in water R and dilute to 10 mL with the same solvent.
Detection : spectrophotometer at 214 nm. Reference solution (d). Dissolve 20 mg of serine R in water R
and dilute to 10 mL with the same solvent.
Injection : 50 μL.
Plate : TLC silica gel G plate R.
Run time : twice the retention time of glycocholate. Carry out the following procedures protected from light.
System suitability : reference solution (b) : Mobile phase : water R, phenol R (25:75 V/V).
– resolution : minimum 1.5 between the peaks due to Application : 5 μL as bands of 10 mm, then place the plate
glycocholate and taurodeoxycholate. in the chromatographic tank so that it is not in contact with
Calculate the nominal exchange capacity using the following the mobile phase, and allow it to become impregnated with
expression : the vapour of the mobile phase for at least 12 h.
Development : over a path of 12 cm using the same mobile
phase.
Drying : at 100-105 °C.
A1 = area of the peak due to glycocholate in the Detection : spray with ninhydrin solution R1 and heat at
chromatogram obtained with reference 110 °C for 5 min.
solution (a), Results : the chromatogram obtained with the test solution
A2 = area of the peak due to glycocholate in the shows zones corresponding to those in the chromatograms
chromatogram obtained with the test solution, obtained with reference solutions (a) and (b), but shows
m1 = mass, in milligrams, of sodium glycocholate R used no zones corresponding to those in the chromatograms
obtained with reference solutions (c) and (d) ; the
in the preparation of solution A, chromatogram obtained with the test solution also shows a
m2 = mass, in milligrams, of the dried substance to zone with a very low RF value (2,4-diaminobutyric acid).
be examined used in the preparation of the test B. Dissolve about 5 mg in 3 mL of water R. Add 3 mL of
solution, dilute sodium hydroxide solution R. Shake and add 0.5 mL
1.2 = correction factor to convert the true exchange of a 10 g/L solution of copper sulfate R. A violet colour is
capacity to the conventionally used nominal produced.
exchange capacity. C. Dissolve about 50 mg in 1 mL of 1 M hydrochloric acid and
add 0.5 mL of 0.01 M iodine. The solution is decolourised
STORAGE and gives reaction (a) of sulfates (2.3.1).
In an airtight container. D. It gives reaction (b) of sodium (2.3.1).

1960 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Colistin sulfate

TESTS Solubility : freely soluble in water, practically insoluble in

Appearance of solution. The solution is clear (2.2.1). acetone and in ethanol (96 per cent).
Dissolve 0.16 g in 10 mL of water R. IDENTIFICATION
pH (2.2.3) : 6.5 to 8.5. First identification : B, E.
Dissolve 0.1 g in carbon dioxide-free water R and dilute to Second identification : A, C, D, E.
10 mL with the same solvent. Measure after 30 min. A. Thin-layer chromatography (2.2.27).
Specific optical rotation (2.2.7) : − 46 to − 51 (dried Test solution. Dissolve 5 mg of the substance to be examined
substance). in 1 mL of a mixture of equal volumes of hydrochloric
Dissolve 1.25 g in water R and dilute to 25.0 mL with the same acid R and water R. Heat at 135 °C in a sealed tube for 5 h.
solvent. Evaporate to dryness on a water-bath and continue the
Free colistin. Dissolve 80 mg in 3 mL of water R. Add 0.1 mL heating until moistened blue litmus paper R does not turn
of a 100 g/L solution of silicotungstic acid R ; 10-20 s after red. Dissolve the residue in 0.5 mL of water R.
addition of the reagent, the solution is not more opalescent Reference solution (a). Dissolve 20 mg of leucine R in
than reference suspension II (2.2.1). water R and dilute to 10 mL with the same solvent.
Total sulfite. Work in a fume cupboard. Dissolve 0.100 g in Reference solution (b). Dissolve 20 mg of threonine R in
50 mL of water R and add 5 mL of a 100 g/L solution of sodium water R and dilute to 10 mL with the same solvent.
hydroxide R and 0.3 g of potassium cyanide R. Boil gently Reference solution (c). Dissolve 20 mg of phenylalanine R
for 3 min and then cool. Neutralise with 0.5 M sulfuric acid in water R and dilute to 10 mL with the same solvent.
using 0.2 mL of methyl orange solution R as indicator. Add an
excess of 0.5 mL of the acid and 0.2 g of potassium iodide R. Reference solution (d). Dissolve 20 mg of serine R in water R
Titrate with 0.05 M iodine using 1 mL of starch solution R as and dilute to 10 mL with the same solvent.
indicator. The volume of 0.05 M iodine used in the titration is Plate : TLC silica gel G plate R.
5.5 mL to 7.0 mL. Carry out the following procedures protected from light.
Loss on drying (2.2.32) : maximum 5.0 per cent, determined Mobile phase : water R, phenol R (25:75 V/V).
on 1.000 g by drying at 60 °C over diphosphorus pentoxide R at Application : 5 μL as bands of 10 mm, then place the plate
a pressure not exceeding 670 Pa for 3 h. in the chromatographic tank so that it is not in contact with
Sulfated ash (2.4.14): 16 per cent to 21 per cent, determined the mobile phase, and allow it to become impregnated with
on 0.50 g. the vapour of the mobile phase for at least 12 h.
Pyrogens (2.6.8). If intended for use in the manufacture Development : over half of the plate.
of parenteral preparations without a further appropriate Drying : at 105 °C.
procedure for removal of pyrogens, it complies with the test. Detection : spray with ninhydrin solution R1 and heat at
Inject, per kilogram of the rabbit’s mass, 1 mL of a solution in 110 °C for 5 min.
water for injections R containing 2.5 mg of the substance to be
examined per millilitre. Results : the chromatogram obtained with the test solution
shows zones corresponding to those in the chromatograms
ASSAY obtained with reference solutions (a) and (b), but shows
Carry out the microbiological assay of antibiotics (2.7.2). no zones corresponding to those in the chromatograms
obtained with reference solutions (c) and (d) ; the
STORAGE chromatogram obtained with the test solution also shows a
In an airtight container, protected from light. If the substance zone with a very low RF value (2,4-diaminobutyric acid).
is sterile, store in a sterile, airtight, tamper-proof container. B. Examine the chromatograms obtained in the test for
composition.
01/2013:0320 Results : the peaks due to polymyxin E1 and polymyxin E2
in the chromatogram obtained with the test solution are
COLISTIN SULFATE similar in retention time to the corresponding peaks in the
chromatogram obtained with reference solution (a).
Colistini sulfas C. Dissolve about 5 mg in 3 mL of water R. Add 3 mL of
dilute sodium hydroxide solution R. Shake and add 0.5 mL
of a 10 g/L solution of copper sulfate R. A violet colour is
produced.
D. Dissolve about 50 mg in 1 mL of 1 M hydrochloric acid
and add 0.5 mL of 0.01 M iodine. The solution remains
coloured.
E. It gives reaction (a) of sulfates (2.3.1).
TESTS
pH (2.2.3) : 4.0 to 6.0.
Dissolve 0.1 g in carbon dioxide-free water R and dilute to
10 mL with the same solvent.
Specific optical rotation (2.2.7) : − 63 to − 73 (dried
DEFINITION substance).
A mixture of the sulfates of polypeptides produced by certain Dissolve 1.25 g in water R and dilute to 25.0 mL with the same
strains of Bacillus polymyxa var. colistinus or obtained by any solvent.
other means.
Content : minimum 19 000 IU/mg (dried substance). Composition. Liquid chromatography (2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be
CHARACTERS examined in 40 mL of water R and dilute to 50.0 mL with
Appearance : white or almost white, hygroscopic powder. acetonitrile R1.

General Notices (1) apply to all monographs and other texts 1961
Copovidone EUROPEAN PHARMACOPOEIA 8.0

Reference solution (a). Dissolve 25.0 mg of colistin sulfate CRS Related substances. Liquid chromatography (2.2.29) as
in 40 mL of water R and dilute to 50.0 mL with acetonitrile R1. described in the test for composition with the following
Reference solution (b). Dilute 1.0 mL of reference solution (a) modifications. Use the normalisation procedure.
to 100.0 mL with a mixture of 20 volumes of acetonitrile R1 Injection : test solution and reference solution (b).
and 80 volumes of water R. Limits :
Column : – any impurity : maximum 4.0 per cent ;
– size : l = 0.15 m, Ø = 4.6 mm ; – total : maximum 23.0 per cent ;
– stationary phase : end-capped octadecylsilyl silica gel for – disregard limit : the area of the peak due to polymyxin E1
chromatography R (3.5 μm) ; in the chromatogram obtained with reference solution (b) ;
disregard the peaks due to polymyxins E2, E3, E1-I, E1
– temperature : 30 °C. and E1-7MOA.
Mobile phase : mix 22 volumes of acetonitrile R1 and Sulfate : 16.0 per cent to 18.0 per cent (dried substance).
78 volumes of a solution prepared as follows : dissolve 4.46 g
of anhydrous sodium sulfate R in 900 mL of water R, adjust to Dissolve 0.250 g in 100 mL of water R and adjust to pH 11
pH 2.4 with dilute phosphoric acid R and dilute to 1000 mL with concentrated ammonia R. Add 10.0 mL of 0.1 M barium
with water R. chloride and about 0.5 mg of phthalein purple R. Titrate
with 0.1 M sodium edetate, adding 50 mL of ethanol (96 per
Flow rate : 1.0 mL/min. cent) R when the colour of the solution begins to change and
Detection : spectrophotometer at 215 nm. continuing the titration until the violet-blue colour disappears.
Injection : 20 μL of the test solution and reference solution (a). 1 mL of 0.1 M barium chloride is equivalent to 9.606 mg of SO4.
Run time : 1.5 times the retention time of polymyxin E1. Loss on drying (2.2.32) : maximum 3.5 per cent, determined
on 1.000 g by drying at 60 °C over diphosphorus pentoxide R at
Identification of peaks : use the chromatogram supplied with
a pressure not exceeding 0.67 kPa for 3 h.
colistin sulfate CRS to identify the peaks due to polymyxins
E1, E2, E3, E1-I and E1-7MOA. Sulfated ash (2.4.14) : maximum 1.0 per cent, determined on
Relative retention with reference to polymyxin E1 (retention 1.0 g.
time = about 16 min) : polymyxin E2 = about 0.45 ; ASSAY
polymyxin E3 = about 0.5 ; polymyxin E1-I = about 0.8 ; Carry out the microbiological assay of antibiotics (2.7.2).
polymyxin E1-7MOA = about 1.1.
System suitability: reference solution (a) : STORAGE
– resolution : minimum 8.0 between the peaks due to In an airtight container, protected from light.
polymyxin E2 and polymyxin E1 ; minimum 6.0 between
the peaks due to polymyxin E2 and polymyxin E1-I ; 07/2011:0891
minimum 2.5 between the peaks due to polymyxin E1-I
and polymyxin E1 ; minimum 1.5 between the peaks due to
polymyxin E1 and polymyxin E1-7MOA.
COPOVIDONE
Calculate the percentage content of polymyxin E3, of Copovidonum
polymyxin E1-I, of polymyxin E1-7MOA, and of the sum
of polymyxins E1, E2, E3, E1-I and E1-7MOA, using the
following expression :

CEi = percentage content of polymyxin Ei ;

(C6H9NO)n, (C4H6O2)m Mr (111.1)n + (86.1)m
AEi = area of the peak due to polymyxin Ei in the [25086-89-9]
chromatogram obtained with the test solution ;
DEFINITION
m1 = mass of the substance to be examined (dried Copovidone is a copolymer of 1-ethenylpyrrolidin-2-one and
substance) used to prepare the test solution, in ethenyl acetate in the mass proportion 3:2.
milligrams ;
Content :
BEi = area of the peak due to polymyxin Ei in – nitrogen (N ; Ar 14.01) : 7.0 per cent to 8.0 per cent (dried
the chromatogram obtained with reference substance),
solution (a) ; – ethenyl acetate C4H6O2 ; Mr 86.10) : 35.3 per cent to 42.0 per
cent (dried substance).
m2 = mass of colistin sulfate CRS used to prepare K-value : 90.0 per cent to 110.0 per cent of the value stated
reference solution (a), in milligrams ; on the label.
DEi = assigned percentage content of polymyxin Ei in CHARACTERS
colistin sulfate CRS. Aspect : white or yellowish-white hygroscopic powder or flakes.
Limits : Solubility : freely soluble in water, in ethanol (96 per cent) and
in methylene chloride.
– polymyxin E3 : maximum 10.0 per cent (dried substance) ;
– polymyxin E1-I : maximum 10.0 per cent (dried substance) ; IDENTIFICATION
– polymyxin E1-7MOA : maximum 10.0 per cent (dried First identification : A.
substance) ; Second identification : B, C.
– sum of polymyxins E1, E2, E3, E1-I and E1-7MOA : A. Infrared absorption spectrophotometry (2.2.24).
minimum 77.0 per cent (dried substance). Comparison: Ph. Eur. reference spectrum of copovidone.

1962 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Copovidone

B. To 1 mL of solution S (see Tests) add 5 mL of water R and Ab2 = absorbance of the blank after the addition of
0.2 mL of 0.05 M iodine. A red colour appears. aldehyde dehydrogenase ;
C. Dissolve 0.7 g of hydroxylamine hydrochloride R in 10 mL m = mass of povidone, in grams, calculated with
of methanol R, add 20 mL of a 40 g/L solution of sodium reference to the dried substance ;
hydroxide R and filter if necessary. To 5 mL of the solution C = concentration (mg/ml), of acetaldehyde in the
add 0.1 g of the substance to be examined and boil for reference solution, calculated from the weight of
2 min. Transfer 50 μL to a filter paper and add 0.1 mL of a the acetaldehyde ammonia trimer trihydrate with
mixture of equal volumes of ferric chloride solution R1 and the factor 0.72.
hydrochloric acid R. A violet colour appears.
Peroxides : maximum 400 ppm, expressed as H2O2.
TESTS Dilute 10 mL of solution S to 25 mL with water R. Add 2 mL of
Solution S. Dissolve 10.0 g in water R and dilute to 100.0 mL titanium trichloride-sulfuric acid reagent R and allow to stand
with the same solvent. Add the substance to be examined to for 30 min. The absorbance (2.2.25) of the solution, measured
the water R in small portions with constant stirring. at 405 nm using a mixture of 25 mL of a 40 g/L solution of
Appearance of solution. Solution S is not more opalescent the substance to be examined and 2 mL of a 13 per cent V/V
than reference suspension III (2.2.1) and not more intensely solution of sulfuric acid R as the compensation liquid, is not
coloured than reference solution B5, R5 or BY5 (2.2.2, greater than 0.35.
Method II). Hydrazine. Thin-layer chromatography (2.2.27). Use freshly
Viscosity, expressed as K-value. Dilute 5.0 mL of solution S prepared solutions.
to 50.0 mL with water R. Allow to stand for 1 h and determine Test solution. To 25 mL of solution S add 0.5 mL of a 50 g/L
solution of salicylaldehyde R in methanol R, mix and heat in
the viscosity (2.2.9) of the solution at 25 ± 0.1 °C, using a size
n° 1 viscometer with a minimum flow time of 100 s. Calculate a water-bath at 60 °C for 15 min. Allow to cool, add 2.0 mL
the K-value using the following expression : of xylene R, shake for 2 min and centrifuge. Use the clear
supernatant layer.
Reference solution. Dissolve 9 mg of salicylaldehyde azine R in
xylene R and dilute to 100 mL with the same solvent. Dilute
1 mL of the solution to 10 mL with xylene R.
Plate : TLC silanised silica gel plate R.
c = percentage concentration (g/100 mL) of the Mobile phase : water R, methanol R (20:80 V/V).
substance to be examined, calculated with Application : 10 μL.
reference to the dried substance ; Development : over 3/4 of the plate.
= viscosity of the solution relative to that of water. Drying : in air.
Aldehydes : maximum 500 ppm, expressed as acetaldehyde. Detection : examine in ultraviolet light at 365 nm.
Test solution. Dissolve 1.0 g of the substance to be examined Limit :
in phosphate buffer solution pH 9.0 R and dilute to 100.0 mL – hydrazine : any spot due to salicylaldehyde azine is not
with the same solvent. Stopper the flask and heat at 60 °C for more intense than the spot in the chromatogram obtained
1 h. Allow to cool. with the reference solution (1 ppm).
Reference solution. Dissolve 0.140 g of acetaldehyde ammonia Monomers : maximum 0.1 per cent.
trimer trihydrate R in water R and dilute to 200.0 mL with the Dissolve 10.0 g in 30 mL of methanol R and add slowly 20.0 mL
same solvent. Dilute 1.0 mL of the solution to 100.0 mL with of iodine bromide solution R. Allow to stand for 30 min
phosphate buffer solution pH 9.0 R. protected from light with repeated shaking. Add 10 mL of a
Into 3 identical spectrophotometric cells with a path length of 100 g/L solution of potassium iodide R and titrate with 0.1 M
1 cm, introduce separately 0.5 mL of the test solution, 0.5 mL sodium thiosulfate until a yellow colour is obtained. Continue
of the reference solution and 0.5 mL of water R (blank). To titration dropwise until the solution becomes colourless.
each cell add 2.5 mL of phosphate buffer solution pH 9.0 R and Carry out a blank titration. Not more than 1.8 mL of 0.1 M
0.2 mL of nicotinamide-adenine dinucleotide solution R. Mix sodium thiosulfate is used.
and stopper tightly. Allow to stand at 22 ± 2 °C for 2-3 min Impurity A. Liquid chromatography (2.2.29).
and measure the absorbance (2.2.25) of each solution at Test solution. Dissolve 0.100 g of the substance to be examined
340 nm, using water R as the compensation liquid. To each in water R and dilute to 50.0 mL with the same solvent.
cell, add 0.05 mL of aldehyde dehydrogenase solution R, mix
and stopper tightly. Allow to stand at 22 ± 2 °C for 5 min. Reference solution. Dissolve 0.100 g of 2-pyrrolidone R
Measure the absorbance of each solution at 340 nm using (impurity A) in water R and dilute to 100 mL with the same
water R as compensation liquid. Determine the content of solvent. Dilute 1.0 mL to 100.0 mL with water R.
aldehydes using the following expression : Precolumn :
– size : l = 0.025 m, Ø = 4 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
Column :
At1 = absorbance of the test solution before the addition
of aldehyde dehydrogenase ; – size : l = 0.25 m, Ø = 4 mm ;
At2 = absorbance of the test solution after the addition of – stationary phase : spherical aminohexadecylsilyl silica gel for
chromatography R (5 μm) ;
aldehyde dehydrogenase ;
– temperature : 30 °C.
As1 = absorbance of the reference solution before the
addition of aldehyde dehydrogenase ; Mobile phase : water R adjusted to pH 2.4 with phosphoric
acid R.
As2 = absorbance of the reference solution after the Flow rate : 1 mL/min.
addition of aldehyde dehydrogenase ;
Detection : spectrophotometer at 205 nm. A detector is placed
Ab1 = absorbance of the blank before the addition of between the precolumn and the analytical column. A second
aldehyde dehydrogenase ; detector is placed after the analytical column.

General Notices (1) apply to all monographs and other texts 1963
Copper sulfate, anhydrous EUROPEAN PHARMACOPOEIA 8.0

Injection : 10 μL. When impurity A has left the precolumn Viscosity (2.2.9) : see above.
(after about 1.2 min) switch the flow directly from the pump
to the analytical column. Before the next chromatogram is
run, wash the precolumn by reversed flow.
Limit : 01/2008:0893
corrected 7.0
– impurity A : not more than the area of the principal peak
in the chromatogram obtained with the reference solution
(0.5 per cent). COPPER SULFATE, ANHYDROUS
Heavy metals (2.4.8) : maximum 20 ppm.
12 mL of solution S complies with test A. Prepare the reference Cupri sulfas anhydricus
solution using lead standard solution (2 ppm Pb) R.
Loss on drying (2.2.32) : maximum 5.0 per cent, determined CuSO4 Mr 159.6
on 0.500 g by drying in an oven at 105 °C. [7758-98-7]
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on DEFINITION
1.0 g.
Content : 99.0 per cent to 101.0 per cent (dried substance).
ASSAY
CHARACTERS
Ethenyl acetate. Determine the saponification value (2.5.6)
Appearance : greenish-grey powder, very hygroscopic.
on 2.00 g of the substance to be examined. Multiply the result
obtained by 0.1534 to obtain the percentage content of the Solubility : freely soluble in water, slightly soluble in methanol,
ethenyl acetate component. practically insoluble in ethanol (96 per cent).
Nitrogen. Carry out the determination of nitrogen (2.5.9) IDENTIFICATION
using 30.0 mg of the substance to be examined and 1 g of
a mixture of 3 parts of copper sulfate R and 997 parts of A. Add several drops of dilute ammonia R2 to 1 mL of
dipotassium sulfate R, heating until a clear, light green solution solution S (see Tests). A blue precipitate is formed. On
is obtained and then for a further 45 min. further addition of dilute ammonia R2 the precipitate
dissolves and a dark blue colour is produced.
STORAGE B. Loss on drying (see Tests).
In an airtight container. C. Dilute 1 mL of solution S to 5 mL with water R. The
solution gives reaction (a) of sulfates (2.3.1).
LABELLING
The label states the K-value. TESTS
Solution S. Dissolve 1.6 g in water R and dilute to 50 mL with
IMPURITIES the same solvent.
Appearance of solution. Solution S is clear (2.2.1).
Chlorides (2.4.4) : maximum 150 ppm.
Dilute 10 mL of solution S to 15 mL with water R.
A. pyrrolidin-2-one (2-pyrrolidone). Iron : maximum 150 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
FUNCTIONALITY-RELATED CHARACTERISTICS
Test solution. Dissolve 0.32 g in 10 mL of water R, add 2.5 mL
This section provides information on characteristics that are of lead-free nitric acid R and dilute to 25.0 mL with water R.
recognised as being relevant control parameters for one or
more functions of the substance when used as an excipient Reference solutions. Prepare the reference solutions using iron
(see chapter 5.15). Some of the characteristics described in standard solution (20 ppm Fe) R, adding 2.5 mL of lead-free
the Functionality-related characteristics section may also be nitric acid R and diluting to 25.0 mL with water R.
present in the mandatory part of the monograph since they Source : iron hollow-cathode lamp.
also represent mandatory quality criteria. In such cases, a Wavelength : 248.3 nm.
cross-reference to the tests described in the mandatory part is
Atomisation device : air-acetylene flame.
included in the Functionality-related characteristics section.
Control of the characteristics can contribute to the quality Copper may form explosive acetylides with acetylene. Therefore,
of a medicinal product by improving the consistency of the clean the burner thoroughly before any residues become dry.
manufacturing process and the performance of the medicinal Lead : maximum 80 ppm.
product during use. Where control methods are cited, they are
recognised as being suitable for the purpose, but other methods Atomic absorption spectrometry (2.2.23, Method I).
can also be used. Wherever results for a particular characteristic Test solution. Dissolve 1.6 g in 10 mL of water R, add 2.5 mL
are reported, the control method must be indicated. of lead-free nitric acid R and dilute to 25.0 mL with water R.
The following characteristics may be relevant for copovidone Reference solutions. Prepare the reference solutions using lead
used as binder in tablets and granules. standard solution (100 ppm Pb) R, adding 2.5 mL of lead-free
nitric acid R and diluting to 25.0 mL with water R.
Viscosity (2.2.9) : determine the dynamic viscosity using a
capillary viscometer on a 10 per cent solution (dried substance) Source : lead hollow-cathode lamp.
or on a 20 per cent solution (dried substance) at 25 °C. It is Wavelength : 217.0 nm.
typically about 8 mPa·s or about 23 mPa·s, respectively. Atomisation device : air-acetylene flame.
Particle-size distribution (2.9.31 or 2.9.38). Copper may form explosive acetylides with acetylene. Therefore,
Bulk and tapped density (2.9.34). clean the burner thoroughly before any residues become dry.
The following characteristic may be relevant for copovidone Loss on drying (2.2.32) : maximum 1.0 per cent, determined
used as film former in coated dosage forms and in aerosols. on 0.500 g by drying in an oven at 250 ± 10 °C.

1964 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cortisone acetate

ASSAY Copper may form explosive acetylides with acetylene. Therefore,

Dissolve 0.125 g in 50 mL of water R. Add 2 mL of sulfuric clean the burner thoroughly before any residues become dry.
acid R and 3 g of potassium iodide R. Titrate with 0.1 M sodium Loss on drying (2.2.32) : 35.0 per cent to 36.5 per cent,
thiosulfate, using 1 mL of starch solution R, added towards determined on 0.500 g by drying in an oven at 250 ± 10 °C.
the end of the titration.
ASSAY
1 mL of 0.1 M sodium thiosulfate is equivalent to 15.96 mg
of CuSO4. Dissolve 0.200 g in 50 mL of water R. Add 2 mL of sulfuric
acid R and 3 g of potassium iodide R. Titrate with 0.1 M
STORAGE sodium thiosulfate, adding 1 mL of starch solution R towards
In an airtight container. the end of the titration.
1 mL 0.1 M sodium thiosulfate is equivalent to 24.97 mg of
01/2008:0894 CuSO4,5H2O.
corrected 7.0
01/2008:0321
COPPER SULFATE PENTAHYDRATE corrected 6.0

Cupri sulfas pentahydricus CORTISONE ACETATE

CuSO4,5H2O Mr 249.7 Cortisoni acetas
[7758-99-8]
DEFINITION
Content : 99.0 per cent to 101.0 per cent.
CHARACTERS
Appearance : blue, crystalline powder or transparent, blue
crystals.
C23H30O6 Mr 402.5
Solubility : freely soluble in water, soluble in methanol, [50-04-4]
practically insoluble in ethanol (96 per cent).
DEFINITION
IDENTIFICATION
17-Hydroxy-3,11,20-trioxopregn-4-en-21-yl acetate.
A. Add several drops of dilute ammonia R2 to 1 mL of
solution S (see Tests). A blue precipitate is formed. On Content : 97.0 per cent to 103.0 per cent (dried substance).
further addition of dilute ammonia R2 the precipitate CHARACTERS
dissolves and a dark blue colour is produced.
Appearance : white or almost white, crystalline powder.
B. Loss on drying (see Tests).
Solubility : practically insoluble in water, freely soluble in
C. Dilute 1 mL of solution S to 5 mL with water R. The methylene chloride, soluble in dioxan, sparingly soluble
solution gives reaction (a) of sulfates (2.3.1). in acetone, slightly soluble in ethanol (96 per cent) and in
TESTS methanol.
It shows polymorphism (5.9).
Solution S. Dissolve 5 g in water R and dilute to 100 mL with
the same solvent. IDENTIFICATION
Appearance of solution. Solution S is clear (2.2.1). First identification : A, B.
Chlorides (2.4.4) : maximum 100 ppm. Second identification : C, D, E.
Dilute 10 mL of solution S to 15 mL with water R. A. Infrared absorption spectrophotometry (2.2.24).
Iron : maximum 100 ppm. Comparison: cortisone acetate CRS.
Atomic absorption spectrometry (2.2.23, Method I). If the spectra obtained in the solid state show differences,
Test solution. Dissolve 0.5 g in 10 mL of water R, add 2.5 mL record new spectra using 50 g/L solutions in methylene
of lead-free nitric acid R and dilute to 25.0 mL with water R. chloride R in a 0.2 mm cell.
Reference solutions. Prepare the reference solutions using iron B. Thin-layer chromatography (2.2.27).
standard solution (20 ppm Fe) R, adding 2.5 mL of lead-free Solvent mixture : methanol R, methylene chloride R
nitric acid R and diluting to 25.0 mL with water R. (1:9 V/V).
Source : iron hollow-cathode lamp. Test solution. Dissolve 10 mg of the substance to be
Wavelength : 248.3 nm. examined in the solvent mixture and dilute to 10 mL with
the solvent mixture.
Atomisation device : air-acetylene flame.
Reference solution (a). Dissolve 20 mg of cortisone
Copper may form explosive acetylides with acetylene. Therefore, acetate CRS in the solvent mixture and dilute to 20 mL
clean the burner thoroughly before any residues become dry. with the solvent mixture.
Lead : maximum 50 ppm. Reference solution (b). Dissolve 10 mg of hydrocortisone
Atomic absorption spectrometry (2.2.23, Method I). acetate R in reference solution (a) and dilute to 10 mL with
Test solution. Dissolve 2.5 g in 10 mL of water R, add 2.5 mL reference solution (a).
of lead-free nitric acid R and dilute to 25.0 mL with water R. Plate : TLC silica gel F254 plate R.
Reference solutions. Prepare the reference solutions using lead Mobile phase : add a mixture of 1.2 volumes of water R and
standard solution (100 ppm Pb) R, adding 2.5 mL of lead-free 8 volumes of methanol R to a mixture of 15 volumes of
nitric acid R and diluting to 25.0 mL with water R. ether R and 77 volumes of methylene chloride R.
Source : lead hollow-cathode lamp. Application : 5 μL.
Wavelength : 217.0 nm. Development : over a path of 15 cm.
Atomisation device : air-acetylene flame. Drying : in air.

General Notices (1) apply to all monographs and other texts 1965
Cortisone acetate EUROPEAN PHARMACOPOEIA 8.0

Detection A : examine in ultraviolet light at 254 nm. E. About 10 mg gives the reaction of acetyl (2.3.1).
Results A : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to TESTS
the principal spot in the chromatogram obtained with Specific optical rotation (2.2.7) : + 211 to + 220 (dried
reference solution (a). substance).
Detection B : spray with alcoholic solution of sulfuric acid R. Dissolve 0.250 g in dioxan R and dilute to 25.0 mL with the
Heat at 120 °C for 10 min or until the spots appear. Allow to same solvent.
cool. Examine in daylight and in ultraviolet light at 365 nm.
Related substances. Liquid chromatography (2.2.29). Prepare
Results B : the principal spot in the chromatogram obtained
the solutions immediately before use.
with the test solution is similar in position, colour in
daylight, fluorescence in ultraviolet light at 365 nm and Test solution. Dissolve 25.0 mg of the substance to be
size to the principal spot in the chromatogram obtained examined in acetonitrile R and dilute to 10.0 mL with the
with reference solution (a). same solvent.
System suitability: reference solution (b) : Reference solution (a). Dissolve 2 mg of cortisone acetate CRS
– the chromatogram shows 2 clearly separated spots. and 2 mg of hydrocortisone acetate CRS (impurity A) in
acetonitrile R and dilute to 100.0 mL with the same solvent.
C. Thin-layer chromatography (2.2.27).
Test solution (a). Dissolve 25 mg of the substance to be Reference solution (b). Dilute 1.0 mL of the test solution to
examined in methanol R with gentle heating and dilute to 100.0 mL with acetonitrile R.
5 mL with the same solvent (solution A). Dilute 2 mL of Column :
this solution to 10 mL with methylene chloride R. – size : l = 0.25 m, Ø = 4.6 mm ;
Test solution (b). Transfer 2 mL of solution A to
a 15 mL glass tube with a ground-glass stopper or – stationary phase : octadecylsilyl silica gel for
a polytetrafluoroethylene cap. Add 10 mL of saturated chromatography R (5 μm).
methanolic potassium hydrogen carbonate solution R and Mobile phase : in a 1000 mL volumetric flask mix 400 mL of
immediately pass a stream of nitrogen R briskly through the acetonitrile R with 550 mL of water R and allow to equilibrate ;
solution for 5 min. Stopper the tube. Heat in a water-bath dilute to 1000 mL with water R and mix again.
at 45 °C protected from light for 2.5 h. Allow to cool. Flow rate : 1 mL/min.
Reference solution (a). Dissolve 25 mg of cortisone
Detection : spectrophotometer at 254 nm.
acetate CRS in methanol R with gentle heating and dilute
to 5 mL with the same solvent (solution B). Dilute 2 mL of Equilibration : with the mobile phase for about 30 min.
this solution to 10 mL with methylene chloride R. Injection : 20 μL ; inject acetonitrile R as a blank.
Reference solution (b). Transfer 2 mL of solution B
Run time : twice the retention time of cortisone acetate.
to a 15 mL glass tube with a ground-glass stopper or
a polytetrafluoroethylene cap. Add 10 mL of saturated Retention time : impurity A = about 10 min ; cortisone
methanolic potassium hydrogen carbonate solution R and acetate = about 12 min.
immediately pass a stream of nitrogen R briskly through the System suitability : reference solution (a) :
solution for 5 min. Stopper the tube. Heat in a water-bath
at 45 °C protected from light for 2.5 h. Allow to cool. – resolution : minimum 4.2 between the peaks due to
impurity A and cortisone acetate ; if necessary, adjust the
Plate : TLC silica gel F254 plate R.
concentration of acetonitrile in the mobile phase.
Mobile phase : add a mixture of 1.2 volumes of water R and
8 volumes of methanol R to a mixture of 15 volumes of Limits :
ether R and 77 volumes of methylene chloride R. – impurity A : not more than 0.5 times the area of the
Application : 5 μL. principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent) ;
Development : over a path of 15 cm.
Drying : in air. – total : not more than 1.5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
Detection A : examine in ultraviolet light at 254 nm. (1.5 per cent) ;
Results A : the principal spot in each of the chromatograms – disregard limit : 0.05 times the area of the principal peak
obtained with the test solutions is similar in position and in the chromatogram obtained with reference solution (b)
size to the principal spot in the chromatogram obtained (0.05 per cent).
with the corresponding reference solution.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Detection B : spray with alcoholic solution of sulfuric acid R
on 0.500 g by drying in an oven at 105 °C.
and heat at 120 °C for 10 min or until the spots appear.
Allow to cool. Examine in daylight and in ultraviolet light
at 365 nm. ASSAY
Results B : the principal spot in each of the chromatograms Dissolve 0.100 g in ethanol (96 per cent) R and dilute to
obtained with the test solutions is similar in position, colour 100.0 mL with the same solvent. Dilute 2.0 mL of this
in daylight, fluorescence in ultraviolet light at 365 nm and solution to 100.0 mL with ethanol (96 per cent) R. Measure the
size to the principal spot in the chromatogram obtained absorbance (2.2.25) at the absorption maximum at 237 nm.
with the corresponding reference solution. The principal Calculate the content of C23H30O6 taking the specific
spots in the chromatograms obtained with test solution (b) absorbance to be 395.
and reference solution (b) have an RF value distinctly lower
than that of the principal spots in the chromatograms STORAGE
obtained with test solution (a) and reference solution (a).
Protected from light.
D. Add about 2 mg to 2 mL of sulfuric acid R and shake to
dissolve. Within 5 min, a faint yellow colour develops. Add
this solution to 10 mL of water R and mix. The colour is IMPURITIES
discharged and a clear solution remains. Specified impurities : A.

1966 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cotton, absorbent

Absorbency
Apparatus. A dry cylindrical copper wire basket 8.0 cm high
and 5.0 cm in diameter. The wire of which the basket is
constructed is about 0.4 mm in diameter, the mesh is 1.5 cm
to 2.0 cm wide and the mass of the basket is 2.7 ± 0.3 g.
Sinking time. Not more than 10 s. Weigh the basket to the
A. 11β,17-dihydroxy-3,20-dioxopregn-4-en-21-yl acetate nearest centigram (m1). Take a total of 5.00 g in approximately
(hydrocortisone acetate). equal quantities from 5 different places in the product to be
examined, place loosely in the basket and weigh the filled
basket to the nearest centigram (m2). Fill a beaker 11 cm to
12 cm in diameter to a depth of 10 cm with water at about
01/2008:0036 20 °C. Hold the basket horizontally and drop it from a height
corrected 7.0 of about 10 mm into the water. Measure with a stopwatch
the time taken for the basket to sink below the surface of the
water. Calculate the result as the average of 3 tests.
COTTON, ABSORBENT Water-holding capacity. Not less than 23.0 g of water per
gram. After the sinking time has been measured, remove
Lanugo gossypii absorbens the basket from the water, allow it to drain for exactly 30 s
suspended in a horizontal position over the beaker, transfer it
DEFINITION to a tared beaker (m3) and weigh to the nearest centigram (m4).
Calculate the water-holding capacity per gram of absorbent
Absorbent cotton consists of new fibres or good quality
cotton using the following expression :
combers obtained from the seed-coat of various species of the
genus Gossypium L., cleaned, purified, bleached and carefully
carded. It may not contain any compensatory colouring
matter.
Calculate the result as the average of 3 tests.
CHARACTERS
Ether-soluble substances. Not more than 0.50 per cent. In an
It is white or almost white and is composed of fibres of average extraction apparatus, extract 5.00 g with ether R for 4 h at a rate
length not less than 10 mm, determined by a suitable method, of at least 4 extractions per hour. Evaporate the ether extract
and contains not more than traces of leaf residue, pericarp, and dry the residue to constant mass at 100 °C to 105 °C.
seed-coat or other impurities. It offers appreciable resistance
when pulled. It does not shed any appreciable quantity of dust Extractable colouring matter. In a narrow percolator, slowly
when gently shaken. extract 10.0 g with alcohol R until 50 mL of extract is obtained.
The liquid obtained is not more intensely coloured (2.2.2,
IDENTIFICATION Method II) than reference solution Y5, GY6 or a reference
solution prepared as follows : to 3.0 mL of blue primary
A. Examined under a microscope, each fibre is seen to consist solution add 7.0 mL of hydrochloric acid (10 g/L HCl). Dilute
of a single cell, up to about 4 cm long and up to 40 μm wide, 0.5 mL of this solution to 10.0 mL with hydrochloric acid
in the form of a flattened tube with thick and rounded walls (10 g/L HCl).
and often twisted.
Surface-active substances. Introduce the 10 mL portion of
B. When treated with iodinated zinc chloride solution R, the solution S reserved before filtration into a 25 mL graduated
fibres become violet. ground-glass-stoppered cylinder with an external diameter
C. To 0.1 g add 10 mL of zinc chloride-formic acid solution R. of 20 mm and a wall thickness of not greater than 1.5 mm,
Heat to 40 °C and allow to stand for 2 h 30 min, shaking previously rinsed 3 times with sulfuric acid R and then with
occasionally. It does not dissolve. water R. Shake vigorously 30 times in 10 s, allow to stand for
1 min and repeat the shaking. After 5 min, any foam present
TESTS must not cover the entire surface of the liquid.
Solution S. Place 15.0 g in a suitable vessel, add 150 mL Water-soluble substances. Not more than 0.50 per cent. Boil
of water R, close the vessel and allow to macerate for 2 h. 5.000 g in 500 mL of water R for 30 min, stirring frequently.
Decant the solution, squeeze the residual liquid carefully from Replace the water lost by evaporation. Decant the liquid,
the sample with a glass rod and mix. Reserve 10 mL of the squeeze the residual liquid carefully from the sample with
solution for the test for surface-active substances and filter a glass rod and mix. Filter the liquid whilst hot. Evaporate
the remainder. 400 mL of the filtrate (corresponding to 4/5 of the mass of the
Acidity or alkalinity. To 25 mL of solution S add 0.1 mL of sample taken) and dry the residue to constant mass at 100 °C
phenolphthalein solution R and to another 25 mL add 0.05 mL to 105 °C.
of methyl orange solution R. Neither solution is pink. Loss on drying (2.2.32). Not more than 8.0 per cent,
Foreign fibres. Examined under a microscope, it is seen determined on 5.000 g by drying in an oven at 105 °C.
to consist exclusively of typical cotton fibres, except that Sulfated ash (2.4.14). Not more than 0.40 per cent. Introduce
occasionally a few isolated foreign fibres may be present. 5.00 g into a previously heated and cooled, tared crucible.
Fluorescence. Examine a layer about 5 mm in thickness Heat cautiously over a naked flame and then carefully to
under ultraviolet light at 365 nm. It displays only a slight dull redness at 600 °C. Allow to cool, add a few drops of
brownish-violet fluorescence and a few yellow particles. It dilute sulfuric acid R, then heat and incinerate until all the
shows no intense blue fluorescence, apart from that which black particles have disappeared. Allow to cool. Add a few
may be shown by a few isolated fibres. drops of ammonium carbonate solution R. Evaporate and
incinerate carefully, allow to cool and weigh again. Repeat the
Neps. Spread about 1 g evenly between 2 colourless incineration for periods of 5 min to constant mass.
transparent plates each 10 cm square. Examine for neps by
transmitted light and compare with Cotton wool standard for
neps CRS. The product to be examined is not more neppy STORAGE
than the standard. Store in a dust-proof package in a dry place.

General Notices (1) apply to all monographs and other texts 1967
Cottonseed oil, hydrogenated EUROPEAN PHARMACOPOEIA 8.0

01/2008:1305 – behenic acid : maximum 1.0 per cent ;

corrected 7.0 – lignoceric acid : maximum 0.5 per cent.
Nickel : maximum 1 ppm.
COTTONSEED OIL, HYDROGENATED Atomic absorption spectrometry (2.2.23, Method II).
Test solution. Introduce 5.0 g into a platinum or silica
Gossypii oleum hydrogenatum crucible tared after ignition. Cautiously heat and introduce
DEFINITION into the substance a wick formed from twisted ashless filter
paper. Ignite the wick. When the substance ignites, stop
Product obtained by refining and hydrogenation of oil
heating. After combustion, ignite in a muffle furnace at about
obtained from seeds of cultivated plants of various varieties
600 ± 50 °C. Continue the incineration until white ash is
of Gossypium hirsutum L. or of other species of Gossypium. obtained. After cooling, take up the residue with 2 quantities,
The product consists mainly of triglycerides of palmitic and
each of 2 mL, of dilute hydrochloric acid R and transfer into a
stearic acids.
25 mL graduated flask. Add 0.3 mL of nitric acid R and dilute
CHARACTERS to 25.0 mL with distilled water R.
Appearance : white or almost white mass or powder which Reference solutions. Prepare 3 reference solutions by adding
melts to a clear, pale yellow liquid when heated. 1.0 mL, 2.0 mL and 4.0 mL of nickel standard solution (0.2 ppm
Ni) R to 2.0 mL portions of the test solution, diluting to
Solubility : practically insoluble in water, freely soluble in 10.0 mL with distilled water R.
methylene chloride and in toluene, very slightly soluble in
ethanol (96 per cent). Source : nickel hollow-cathode lamp.
Wavelength : 232 nm.
IDENTIFICATION Atomisation device : graphite furnace.
A. Melting point (see Tests).
Carrier gas : argon R.
B. Composition of fatty acids (see Tests).
STORAGE
TESTS
Protected from light.
Melting point (2.2.14) : 57 °C to 70 °C.
Acid value (2.5.1) : maximum 0.5.
Dissolve 10.0 g in 50 mL of a hot mixture of equal volumes 01/2008:1628
of ethanol (96 per cent) R and toluene R, previously
neutralised with 0.1 M potassium hydroxide using 0.5 mL of CRESOL, CRUDE
phenolphthalein solution R1 as indicator. Titrate the solution
immediately while still hot.
Peroxide value (2.5.5, Method A) : maximum 5.0.
Cresolum crudum
Unsaponifiable matter (2.5.7) : maximum 1.0 per cent,
determined on 5.0 g.
Alkaline impurities. Dissolve by gentle heating 2.0 g in a
mixture of 1.5 mL of ethanol (96 per cent) R and 3 mL of C 7H 8O Mr 108.1
toluene R. Add 0.05 mL of a 0.4 g/L solution of bromophenol
blue R in ethanol (96 per cent) R. Not more than 0.4 mL of DEFINITION
0.01 M hydrochloric acid is required to change the colour to Mixture of 2-, 3- and 4-methylphenol.
yellow.
CHARACTERS
Composition of fatty acids (2.4.22, Method A). Use the
mixture of calibrating substances in Table 2.4.22.-3. Appearance : colourless or pale brown liquid.
Column : Solubility : sparingly soluble in water, miscible with alcohol
and with methylene chloride.
– material : fused silica ;
– size : l = 25 m, Ø = 0.25 mm ; IDENTIFICATION
– stationary phase : poly(cyanopropyl)siloxane R (film A. To 0.5 mL add 300 mL of water R, mix and filter. To 10 mL
thickness 0.2 μm). of the filtrate add 1 mL of ferric chloride solution R1. A
Carrier gas : helium for chromatography R. blue colour is produced.
Flow rate : 0.65 mL/min. B. To 10 mL of the filtrate obtained in identification test A,
add 1 mL of bromine water R. A pale yellow flocculent
Split ratio : 1:100.
precipitate is produced.
Temperature :
C. Relative density (see Tests).
– column : 180 °C for 35 min ;
– injection port and detector : 250 °C. TESTS
Detection : flame ionisation. Solution S. To 2.5 g of the substance to be examined add
Composition of the fatty-acid fraction of the oil: 50 mL of water R, shake for 1 min and filter through a
moistened filter.
– saturated fatty acids of chain length less than C14 : maximum
0.2 per cent ; Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of
methyl red solution R and 0.2 mL of 0.01 M sodium hydroxide.
– myristic acid : maximum 1.0 per cent ;
The solution is yellow. Add 0.3 mL of 0.01 M hydrochloric
– palmitic acid : 19.0 per cent to 26.0 per cent ; acid. The solution is red.
– stearic acid : 68.0 per cent to 80.0 per cent ; Relative density (2.2.5) : 1.029 to 1.044.
– oleic acid and isomers : maximum 4.0 per cent ;
Distillation range (2.2.11) : a maximum of 2.0 per cent V/V
– linoleic acid and isomers : maximum 1.0 per cent ; distils below 188 °C and a minimum of 80 per cent V/V distils
– arachidic acid : maximum 1.0 per cent ; between 195 °C and 205 °C.

1968 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Croscarmellose sodium

Sulfur compounds. Place 20 mL in a small conical flask. Over filtrate to 100.0 mL with the same solvent. Allow to stand for
the mouth of the flask fix a piece of filter paper moistened with 24 h without shaking. Use the clear supernatant to prepare
lead acetate solution R. Heat on a water-bath for 5 min. Not the test solution.
more than a light yellow colour is produced on the filter paper. Prepare the reference solutions as follows : in a 100 mL
Residue on evaporation : maximum 0.1 per cent. volumetric flask, dissolve 0.100 g of glycollic acid R, previously
Evaporate 2.0 g to dryness on a water-bath and dry at dried in vacuo over diphosphorus pentoxide R at room
100-105 °C for 1 h. The residue weighs not more than 2 mg. temperature overnight, in water R and dilute to 100.0 mL with
the same solvent ; use the solution within 30 days ; transfer
STORAGE 1.0 mL, 2.0 mL, 3.0 mL and 4.0 mL of the solution to separate
Protected from light. volumetric flasks, dilute the contents of each flask to 5.0 mL
with water R, add 5 mL of glacial acetic acid R, dilute to
100.0 mL with acetone R and mix.
01/2009:0985 Transfer 2.0 mL of the test solution and 2.0 mL of each
corrected 6.5 of the reference solutions to separate 25 mL volumetric
flasks. Heat the uncovered flasks for 20 min on a water-bath
to eliminate acetone. Allow to cool and add 5.0 mL of
CROSCARMELLOSE SODIUM 2,7-dihydroxynaphthalene solution R to each flask. Mix, add a
further 15.0 mL of 2,7-dihydroxynaphthalene solution R and
Carmellosum natricum conexum mix again. Close the flasks with aluminium foil and heat on
a water-bath for 20 min. Cool and dilute to 25.0 mL with
DEFINITION sulfuric acid R.
Cross-linked sodium carboxymethylcellulose. Measure the absorbance (2.2.25) of each solution at 540 nm.
Sodium salt of a cross-linked, partly O-carboxymethylated Prepare a blank using 2.0 mL of a solution containing 5 per
cellulose. cent V/V each of glacial acetic acid R and water R in acetone R.
Prepare a standard curve using the absorbances obtained
CHARACTERS with the reference solutions. From the standard curve and
Appeareance : white or greyish-white powder. the absorbance of the test solution, determine the mass (a) of
Solubility : practically insoluble in acetone, in anhydrous glycollic acid in the substance to be examined, in milligrams,
ethanol and in toluene. and calculate the content of sodium glycollate using the
following expression :
IDENTIFICATION
A. Mix 1 g with 100 mL of a solution containing 4 ppm of
methylene blue R, stir the mixture and allow it to settle. The
substance to be examined absorbs the methylene blue and
1.29 = the factor converting glycollic acid to sodium
settles as a blue, fibrous mass.
glycollate ;
B. Mix 1 g with 50 mL of water R. Transfer 1 mL of the mixture
to a small test-tube and add 1 mL of water R and 0.05 mL b = loss on drying as a percentage ;
of a freshly prepared 40 g/L solution of α-naphthol R in m = mass of the substance to be examined, in grams.
methanol R. Incline the test-tube and carefully add 2 mL of
sulfuric acid R down the side so that it forms a lower layer. Water-soluble substances : maximum 10.0 per cent.
A reddish-violet colour develops at the interface. Disperse 10.00 g in 800.0 mL of water R and stir for 1 min
C. The solution prepared from the sulfated ash in the test for every 10 min during the first 30 min. Allow to stand for
heavy metals (see Tests) gives reaction (a) of sodium (2.3.1). 1 h and centrifuge if necessary. Decant 200.0 mL of the
supernatant onto a fast filter paper in a vacuum filtration
TESTS funnel, apply vacuum and collect 150.0 mL of the filtrate.
pH (2.2.3) : 5.0 to 7.0 for the suspension. Evaporate to dryness and dry the residue at 100-105 °C for 4 h.
Shake 1 g with 100 mL of carbon dioxide-free water R for 5 min. Heavy metals (2.4.8) : maximum 20 ppm.
Sodium chloride and sodium glycollate : maximum 0.5 per To the residue obtained in the determination of the sulfated
cent (dried substance) for the sum of the percentage contents ash add 1 mL of hydrochloric acid R and evaporate on a
of sodium chloride and sodium glycollate. water-bath. Take up the residue in 20 mL of water R. 12 mL
Sodium chloride. Place 5.00 g in a 250 mL conical flask, add of the solution complies with test A. Prepare the reference
50 mL of water R and 5 mL of strong hydrogen peroxide solution using lead standard solution (1 ppm Pb) R.
solution R and heat on a water-bath for 20 min, stirring Loss on drying (2.2.32) : maximum 10.0 per cent, determined
occasionally to ensure total hydration. Cool, add 100 mL of on 1.000 g by drying in an oven at 105 °C for 6 h.
water R and 10 mL of nitric acid R. Titrate with 0.05 M silver Sulfated ash(2.4.14) : 14.0 per cent to 28.0 per cent (dried
nitrate, determining the end-point potentiometrically (2.2.20) substance), determined on 1.0 g, using a mixture of
using a silver indicator electrode and a double-junction equal volumes of sulfuric acid R and water R.
reference electrode containing a 100 g/L solution of potassium
nitrate R in the outer jacket and a standard filling solution in Microbial contamination
the inner jacket, and stirring constantly. TAMC : acceptance criterion 103 CFU/g (2.6.12).
1 mL of 0.05 M silver nitrate is equivalent to 2.922 mg of NaCl. TYMC : acceptance criterion 102 CFU/g (2.6.12).
Sodium glycollate. Place a quantity of the substance to be Absence of Escherichia coli (2.6.13).
examined equivalent to 0.500 g of the dried substance in a
100 mL beaker. Add 5 mL of glacial acetic acid R and 5 mL FUNCTIONALITY-RELATED CHARACTERISTICS
of water R and stir to ensure total hydration (about 15 min). This section provides information on characteristics that are
Add 50 mL of acetone R and 1 g of sodium chloride R. Stir recognised as being relevant control parameters for one or
for several minutes to ensure complete precipitation of the more functions of the substance when used as an excipient (see
carboxymethylcellulose. Filter through a fast filter paper chapter 5.15). This section is a non-mandatory part of the
impregnated with acetone R into a volumetric flask, rinse the monograph and it is not necessary to verify the characteristics
beaker and the filter with 30 mL of acetone R and dilute the to demonstrate compliance. Control of these characteristics can

General Notices (1) apply to all monographs and other texts 1969
Crospovidone EUROPEAN PHARMACOPOEIA 8.0

however contribute to the quality of a medicinal product by Solubility : practically insoluble in water, in ethanol 96 per cent
improving the consistency of the manufacturing process and and in methylene chloride.
the performance of the medicinal product during use. Where
control methods are cited, they are recognised as being suitable IDENTIFICATION
for the purpose, but other methods can also be used. Wherever A. Infrared absorption spectrophotometry (2.2.24).
results for a particular characteristic are reported, the control
method must be indicated. Comparison: crospovidone CRS.
The following characteristics may be relevant for croscarmellose B. Suspend 1 g in 10 mL of water R, add 0.1 mL of 0.05 M
sodium used as disintegrant. iodine and shake for 30 s. Add 1 mL of starch solution R
and shake. No blue colour develops within 30 s.
Settling volume. Place 75 mL of water R in a 100 mL
graduated cylinder and add 1.5 g of the substance to be C. To 10 mL of water R, add 0.1 g and shake. A suspension is
examined in 0.5 g portions, shaking vigorously after each formed and no clear solution is obtained within 15 min.
addition. Dilute to 100.0 mL with water R and shake again D. The analytical sieves must be clean and dry. For this
until the substance is homogeneously distributed. Allow to purpose the sieves are washed in hot water and allowed to
stand for 4 h. The settling volume is between 10.0 mL and dry overnight in a drying cabinet at 105 °C.
30.0 mL. Place 20 g (dried substance) in a 1000 mL conical flask, add
Degree of substitution : 0.60 to 0.85 (dried substance). 500 mL of water R and shake the suspension for 30 min.
Place 1.000 g in a 500 mL conical flask, add 300 mL of a Pour the suspension through a 63 μm analytical sieve,
100 g/L solution of sodium chloride R and 25.0 mL of 0.1 M previously tared, and rinse the sieve with water R until the
sodium hydroxide, stopper the flask and allow to stand for filtrate is clear. Dry the sieve and sample residue at 105 °C
5 min, shaking occasionally. Add 0.05 mL of m-cresol purple for 5 h in a drying cabinet without circulating air. Cool in a
solution R and about 15 mL of 0.1 M hydrochloric acid from desiccator for 30 min and weigh.
a burette. Insert the stopper and shake. If the solution is Calculate the percentage sieving residue (fraction of sample
violet, add 0.1 M hydrochloric acid in 1 mL portions until the particles having a diameter of more than 63 μm), using the
solution becomes yellow, shaking after each addition. Titrate following expression :
with 0.1 M sodium hydroxide until the colour turns to violet.
Calculate the number of milliequivalents (M) of base required
to neutralise the equivalent of 1 g of dried substance.
Calculate the degree of acid carboxymethyl substitution (A) m1 = mass of the sieve and sample residue, after
using the following expression : drying for 5 h, in grams ;
m2 = initial mass of the sample, in grams ;
m3 = mass of the sieve, in grams.
C = sulfated ash as a percentage. If the sieving residue fraction is more than 15 per cent,
Calculate the degree of sodium carboxymethyl substitution (S) the substance is classified as type A ; if the sieving residue
using the following expression : fraction is less than or equal to 15 per cent, the substance
is classified as type B.

TESTS
The degree of substitution is the sum of A and S. Peroxides. Type A : maximum 400 ppm expressed as H2O2 ;
type B : maximum 1000 ppm expressed as H2O2.
Particle size distribution (2.9.31 or 2.9.38).
Suspend 2.0 g in 50 mL of water R. To 25 mL of this suspension
Hausner ratio (2.9.36). add 2 mL of titanium trichloride-sulfuric acid reagent R. Allow
to stand for 30 min and filter. The absorbance (2.2.25) of the
04/2012:0892 filtrate, measured at 405 nm using a mixture of 25 mL of a
filtered 40 g/L suspension of the substance to be examined
and 2 mL of a 13 per cent V/V solution of sulfuric acid R as
CROSPOVIDONE the compensation liquid, has a maximum of 0.35.
For type B use 10 mL of the suspension and dilute to 25 mL
Crospovidonum with water R for the test.
Water-soluble substances : maximum 1.5 per cent.
Place 25.0 g in a 400 mL beaker, add 200 mL of water R and
stir for 1 h using a magnetic stirrer. Transfer the suspension to
a 250.0 mL volumetric flask, rinsing with water R, and dilute
to volume with the same solvent. Allow the bulk of the solids
(C6H9NO)n Mr (111.1)n to settle. Filter about 100 mL of the almost clear supernatant
[9003-39-8] through a membrane filter (nominal pore size 0.45 μm),
protected by superimposing a membrane filter (nominal pore
DEFINITION size 3 μm). While filtering, stir the liquid above the membrane
Cross-linked homopolymer of 1-ethenylpyrrolidin-2-one. filter manually or by means of a mechanical stirrer, taking care
not to damage the membrane filter. Transfer 50.0 mL of the
Content : 11.0 per cent to 12.8 per cent of N (Ar 14.01) (dried clear filtrate to a tared 100 mL beaker, evaporate to dryness
substance). and dry at 105-110 °C for 3 h. The residue weighs a maximum
2 types of crospovidone are available, depending on the of 75 mg.
particle size : type A and type B.
Impurity A. Liquid chromatography (2.2.29).
CHARACTERS Test solution. Suspend 1.250 g in 50.0 mL of methanol R and
Appearance : hygroscopic, white or yellowish-white powder shake for 60 min. Leave the bulk to settle and filter through a
or flakes. membrane filter (nominal pore size 0.2 μm).

1970 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Crotamiton

Reference solution (a). Dissolve 50 mg of 1-vinylpyrrolidin-2- changes from green through pale greyish-blue to pale greyish
one R (impurity A) in methanol R and dilute to 100.0 mL with red-purple. Carry out a blank determination and make any
the same solvent. Dilute 1.0 mL of the solution to 100.0 mL necessary correction.
with methanol R. Dilute 5.0 mL of this solution to 100.0 mL 1 mL of 0.025 M sulfuric acid is equivalent to 0.700 mg of N.
with the mobile phase.
Reference solution (b). Dissolve 10 mg of 1-vinylpyrrolidin-2- STORAGE
one R (impurity A) and 0.50 g of vinyl acetate R in methanol R In an airtight container.
and dilute to 100 mL with the same solvent. Dilute 1.0 mL of
the solution to 100.0 mL with the mobile phase. LABELLING
Precolumn : The label states the type of crospovidone (type A or type B).
– size : l = 0.025 m, Ø = 4 mm ; IMPURITIES
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Column :
– size : l = 0.25 m, Ø = 4 mm ;
– stationary phase : octadecylsilyl silica gel for A. 1-ethenylpyrrolidin-2-one (1-vinylpyrrolidin-2-one).
chromatography R (5 μm) ;
FUNCTIONALITY-RELATED CHARACTERISTICS
– temperature : 40 °C.
This section provides information on characteristics that are
Mobile phase : acetonitrile R, water R (10:90 V/V). recognised as being relevant control parameters for one or
Flow rate : 1 mL/min. more functions of the substance when used as an excipient
Detection : spectrophotometer at 235 nm. (see chapter 5.15). Some of the characteristics described in
Injection : 50 μL. After each injection of the test solution, washthe Functionality-related characteristics section may also be
the precolumn by passing the mobile phase backwards, at the present in the mandatory part of the monograph since they
same flow rate as applied in the test, for 30 min. also represent mandatory quality criteria. In such cases, a
cross-reference to the tests described in the mandatory part is
System suitability : included in the Functionality-related characteristics section.
– resolution : minimum 2.0 between the peaks due to Control of the characteristics can contribute to the quality
impurity A and vinyl acetate in the chromatogram obtained of a medicinal product by improving the consistency of the
with reference solution (b) ; manufacturing process and the performance of the medicinal
– repeatability : maximum relative standard deviation of product during use. Where control methods are cited, they are
2.0 per cent after 6 injections of reference solution (a). recognised as being suitable for the purpose, but other methods
Calculation of percentage content : can also be used. Wherever results for a particular characteristic
are reported, the control method must be indicated.
– for impurity A, use the concentration of impurity A in
reference solution (a). The following characteristics may be relevant for crospovidone
used as disintegrant.
Limit :
Hydration capacity. Introduce 2.0 g into a 100 mL centrifuge
– impurity A : maximum 10 ppm. tube and add 40 mL of water R. Shake vigorously until a
Heavy metals (2.4.8) : maximum 10 ppm. suspension is obtained. Shake again 5 min and 10 min later,
2.0 g complies with test D. Prepare the reference solution then centrifuge for 15 min at 750 g. Decant the supernatant
using 2 mL of lead standard solution (10 ppm Pb) R. and weigh the residue. The hydration capacity is the ratio of
the mass of the residue to the initial mass of the sample. It
Loss on drying (2.2.32) : maximum 5.0 per cent, determined
is typically 3 to 9.
on 0.500 g by drying in an oven at 105 °C.
Particle-size distribution (2.9.31).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. Powder flow (2.9.36).
The following characteristic may be relevant for crospovidone
ASSAY used as suspension stabiliser.
Place 0.100 g of the substance to be examined (m mg) in a Settling volume. Introduce 10 g into a 100 mL graduated
combustion flask and add 5 g of a mixture of 1 g of copper cylinder and add 90 mL of water R. Shake vigorously. Dilute
sulfate R, 1 g of titanium dioxide R and 33 g of dipotassium to 100 mL with water R, washing the powder residues from
sulfate R, and 3 glass beads. Wash any adhering particles from the walls of the cylinder. Allow to stand for 24 h, then read the
the neck into the flask with a small quantity of water R. Add volume of the sediment. It is typically greater than 60 mL.
7 mL of sulfuric acid R, allowing it to run down the inside
wall of the flask. Gradually heat the flask until the solution
has a clear, yellowish-green colour, and the inside wall of 07/2010:1194
the flask is free from carbonised material, and then heat for
a further 45 min. After cooling, cautiously add 20 mL of CROTAMITON
water R, and connect the flask to the distillation apparatus,
which has been previously washed by passing steam through Crotamitonum
it. To the absorption flask add 30 mL of a 40 g/L solution
of boric acid R, 0.15 mL of bromocresol green-methyl red
solution R and sufficient water R to immerse the lower end of
the condenser tube. Add 30 mL of strong sodium hydroxide
solution R through a funnel, cautiously rinse the funnel with
10 mL of water R, immediately close the clamp attached
to the rubber tube, then start the distillation with steam to C13H17NO Mr 203.3
obtain 80-100 mL of distillate. Remove the absorption flask [483-63-6]
from the lower end of the condenser tube, rinsing the end
part with a small quantity of water R, and titrate the distillate DEFINITION
with 0.025 M sulfuric acid until the colour of the solution N-Ethyl-N-(2-methylphenyl)but-2-enamide.

General Notices (1) apply to all monographs and other texts 1971
Crotamiton EUROPEAN PHARMACOPOEIA 8.0

Content : diluting 5 mL of a 200 g/L solution of sodium hydroxide R to

– sum of the (E)- and (Z)-isomers : 96.0 per cent to 102.0 per 20 mL with water R and adding 1.5 mL of 0.01 M hydrochloric
cent ; acid, 5 mL of nitric acid R and diluting to 50 mL with water R.
– (Z)-isomer : maximum 15.0 per cent. Related substances. Liquid chromatography (2.2.29).
Test solution (a). Dissolve 50.0 mg of the substance to be
CHARACTERS examined in the mobile phase and dilute to 100.0 mL with
Appearance : colourless or pale yellow, oily liquid. the mobile phase.
Solubility : slightly soluble in water, miscible with ethanol Test solution (b). Dilute 1.0 mL of test solution (a) to 20.0 mL
(96 per cent). with the mobile phase.
At low temperatures it may partly or completely solidify. Reference solution (a). Dissolve 50.0 mg of crotamiton CRS
in the mobile phase and dilute to 100.0 mL with the mobile
IDENTIFICATION
phase. Dilute 1.0 mL of this solution to 20.0 mL with the
First identification : B. mobile phase.
Second identification : A, C, D. Reference solution (b). Dissolve 15.0 mg of crotamiton
A. Ultraviolet and visible absorption spectrophotometry impurity A CRS in the mobile phase and dilute to 20.0 mL with
(2.2.25). the mobile phase. Dilute 1.0 mL of this solution to 50.0 mL
Test solution. Dissolve 25.0 mg in cyclohexane R and dilute with the mobile phase.
to 100.0 mL with the same solvent. Dilute 1.0 mL of this Reference solution (c). Dilute 1.0 mL of test solution (a) to
solution to 10.0 mL with cyclohexane R. 100.0 mL with the mobile phase.
Spectral range : 220-300 nm. Reference solution (d). Dissolve 15 mg of crotamiton
Absorption maximum : at 242 nm. impurity A CRS in the mobile phase and dilute to 100.0 mL
Specific absorbance at the absorption maximum : 300 to 330. with the mobile phase. Dilute 1.0 mL of this solution to
10.0 mL with test solution (a).
B. Infrared absorption spectrophotometry (2.2.24).
Column :
Comparison : crotamiton CRS.
– size : l = 0.25 m, Ø = 4 mm ;
C. Thin-layer chromatography (2.2.27).
– stationary phase : silica gel for chromatography R (5 μm).
Test solution. Dissolve 25 mg of the substance to be
examined in anhydrous ethanol R and dilute to 10 mL with Mobile phase : tetrahydrofuran R, cyclohexane R (8:92 V/V).
the same solvent. Flow rate : 1.0 mL/min.
Reference solution. Dissolve 25 mg of crotamiton CRS in Detection : spectrophotometer at 242 nm.
anhydrous ethanol R and dilute to 10 mL with the same Injection : 20 μL of test solution (a) and reference solutions (b),
solvent. (c) and (d).
Plate : TLC silica gel F254 plate R. Run time : 2.5 times the retention time of the (E)-isomer.
Mobile phase : shake 98 volumes of methylene chloride R Relative retention with reference to the (E)-isomer :
with 2 volumes of concentrated ammonia R, dry over (Z)-isomer = about 0.5 ; impurity A = about 0.8.
anhydrous sodium sulfate R, filter and mix 97 volumes of System suitability : reference solution (d) :
the filtrate with 3 volumes of 2-propanol R. – resolution : minimum 4.5 between the peaks due to
Application : 5 μL. impurity A and the (E)-isomer.
Development : over a 2/3 of the plate. Limits :
Drying : in air. – impurity A : not more than the area of the corresponding
Detection : examine in ultraviolet light at 254 nm. peak in the chromatogram obtained with reference
Results : the principal spot in the chromatogram obtained solution (b) (3.0 per cent) ;
with the test solution is similar in position and size to the – unspecified impurities : for each impurity, not more than
principal spot in the chromatogram obtained with the 0.1 times the sum of the areas of the peaks due to the
reference solution. (Z)- and (E)- isomers in the chromatogram obtained with
D. To 10 mL of a saturated solution add a few drops of a 3 g/L reference solution (c) (0.10 per cent) ;
solution of potassium permanganate R. A brown colour is – sum of impurities other than A : not more than the sum of
produced and a brown precipitate is formed on standing. the areas of the peaks due to the (Z)- and (E)-isomers in
the chromatogram obtained with reference solution (c)
TESTS (1.0 per cent) ;
Relative density (2.2.5) : 1.006 to 1.011. – disregard limit : 0.02 times the sum of the areas of the peaks
Refractive index (2.2.6) : 1.540 to 1.542. due to the (Z)- and (E)-isomers in the chromatogram
obtained with reference solution (c) (0.02 per cent).
Free amines : maximum 500 ppm, expressed as
ethylaminotoluene. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
Dissolve 5.00 g in 16 mL of methylene chloride R and add
4.0 mL of glacial acetic acid R. Add 0.1 mL of metanil yellow ASSAY
solution R and 1.0 mL of 0.02 M perchloric acid. The solution Liquid chromatography (2.2.29) as described in the test for
is red-violet. related substances with the following modification.
Chlorides : maximum 100 ppm. Injection : test solution (b) and reference solution (a).
Boil 5.0 g under a reflux condenser for 1 h with 25 mL of Calculate the percentage content of C13H17NO from the sum
ethanol (96 per cent) R and 5 mL of a 200 g/L solution of of the areas of the peaks due to the (Z)- and (E)-isomers in
sodium hydroxide R. Cool, add 5 mL of water R and shake the chromatograms obtained. Calculate the content of the
with 25 mL of ether R. Dilute the lower layer to 20 mL with (Z)-isomer, as a percentage of the total content of the (E)-
water R ; add 5 mL of nitric acid R, dilute to 50 mL with and (Z)-isomers, from the chromatogram obtained with test
water R and add 1 mL of a freshly prepared 50 g/L solution of solution (b).
silver nitrate R. Any opalescence in the solution is not more
intense than that in a mixture of 1 mL of a freshly prepared STORAGE
50 g/L solution of silver nitrate R and a solution prepared by Protected from light.

1972 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cyanocobalamin

IMPURITIES Test solution. Dissolve 2 mg of the substance to be

Specified impurities : A. examined in 1 mL of a mixture of equal volumes of ethanol
(96 per cent) R and water R.
Reference solution. Dissolve 2 mg of cyanocobalamin CRS
in 1 mL of a mixture of equal volumes of ethanol (96 per
cent) R and water R.
Plate : TLC silica gel G plate R.
Mobile phase : dilute ammonia R1, methanol R, methylene
A. N-ethyl-N-(2-methylphenyl)but-3-enamide. chloride R (9:30:45 V/V/V).
Application : 10 μL.
Development : in an unsaturated tank, over a path of 12 cm.
01/2008:0547 Drying : in air.
corrected 6.0 Detection : examine in daylight.
Results : the principal spot in the chromatogram obtained
CYANOCOBALAMIN with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
the reference solution.
Cyanocobalaminum
TESTS
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 10.0 mg of the substance to be
examined in the mobile phase and dilute to 10.0 mL with the
mobile phase. Use within 1 h.
Reference solution (a). Dilute 3.0 mL of the test solution to
100.0 mL with the mobile phase. Use within 1 h.
Reference solution (b). Dilute 5.0 mL of the test solution to
50.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 100.0 mL with the mobile phase. Use within 1 h.
Reference solution (c). Dissolve 25 mg of the substance to be
examined in 10 mL of water R, warming if necessary. Allow
to cool and add 5 mL of a 1.0 g/L solution of chloramine R
and 0.5 mL of 0.05 M hydrochloric acid, then dilute to 25 mL
with water R. Shake and allow to stand for 5 min. Dilute 1 mL
of this solution to 10 mL with the mobile phase and inject
immediately.
Column :
C63H88CoN14O14P Mr 1355
[68-19-9] – size : l = 0.25 m, Ø = 4 mm ;
– stationary phase : octylsilyl silica gel for chromatography R
DEFINITION (5 μm).
α-(5,6-Dimethylbenzimidazol-1-yl)cobamide cyanide. Mobile phase : mix 26.5 volumes of methanol R and
Content : 96.0 per cent to 102.0 per cent (dried substance). 73.5 volumes of a 10 g/L solution of disodium hydrogen
This monograph applies to cyanocobalamin produced by phosphate R adjusted to pH 3.5 with phosphoric acid R and
fermentation. use within 2 days.
Flow rate : 0.8 mL/min.
CHARACTERS Detection : spectrophotometer at 361 nm.
Appearance : dark red, crystalline powder or dark red crystals. Injection : 20 μL.
Solubility : sparingly soluble in water and in ethanol (96 per Run time : 3 times the retention time of cyanocobalamin.
cent), practically insoluble in acetone.
System suitability :
The anhydrous substance is very hygroscopic.
– the chromatogram obtained with reference solution (c)
IDENTIFICATION shows 2 principal peaks ;
A. Ultraviolet and visible absorption spectrophotometry – resolution : minimum 2.5 between the 2 principal peaks in
(2.2.25). the chromatogram obtained with reference solution (c) ;
Test solution. Dissolve 2.5 mg in water R and dilute to – signal-to-noise ratio : minimum 5 for the principal peak in
100.0 mL with the same solvent. the chromatogram obtained with reference solution (b).
Spectral range : 260-610 nm. Limits :
Absorption maxima : at 278 nm, 361 nm and from 547 nm – total : not more than the area of the principal peak in the
to 559 nm. chromatogram obtained with reference solution (a) (3 per
cent) ;
Absorbance ratio :
– disregard limit : the area of the principal peak in the
– A361 / A547-559 = 3.15 to 3.45 ; chromatogram obtained with reference solution (b) (0.1 per
– A361 / A278 = 1.70 to 1.90. cent).
B. Thin-layer chromatography (2.2.27). Carry out the test Loss on drying (2.2.32) : maximum 12.0 per cent, determined
protected from light. on 20.00 mg by drying in vacuo at 105 °C for 2 h.

General Notices (1) apply to all monographs and other texts 1973
Cyclizine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

ASSAY Drying : in air for 30 min.

Dissolve 25.00 mg in water R and dilute to 1000.0 mL with Detection : expose to iodine vapour for 10 min.
the same solvent. Measure the absorbance (2.2.25) at the Results : the principal spot in the chromatogram obtained
absorption maximum at 361 nm. with the test solution is similar in position, colour and size
Calculate the content of C63H88CoN14O14P taking the specific to the principal spot in the chromatogram obtained with
absorbance to be 207. the reference solution.
STORAGE D. Dissolve 0.5 g in 10 mL of ethanol (60 per cent V/V) R,
heating if necessary. Cool in iced water. Add 1 mL of dilute
In an airtight container, protected from light. sodium hydroxide solution R and 10 mL of water R. Filter,
wash the precipitate with water R and dry at 60 °C at a
07/2008:1092 pressure not exceeding 0.7 kPa for 2 h. The melting point
(2.2.14) is 105 °C to 108 °C.
CYCLIZINE HYDROCHLORIDE E. It gives reaction (a) of chlorides (2.3.1).
TESTS
Cyclizini hydrochloridum pH (2.2.3) : 4.5 to 5.5.
Dissolve 0.5 g in a mixture of 40 volumes of ethanol (96 per
cent) R and 60 volumes of carbon dioxide-free water R and
dilute to 25 mL with the same mixture of solvents.
Related substances. Gas chromatography (2.2.28). Prepare
the solutions immediately before use.
Test solution. Dissolve 0.250 g of the substance to be examined
in 4.0 mL of methanol R and dilute to 5.0 mL with 1 M sodium
C18H23ClN2 Mr 302.8 hydroxide.
[305-25-3] Reference solution (a). Dissolve 25 mg of the substance to be
DEFINITION examined in 10.0 mL of methanol R. Dilute 1.0 mL of this
solution to 50.0 mL with methanol R.
1-(Diphenylmethyl)-4-methylpiperazine hydrochloride.
Reference solution (b). Dissolve 5 mg of the substance to be
Content : 98.5 per cent to 101.0 per cent (dried substance). examined, 5.0 mg of cyclizine impurity A CRS and 5.0 mg of
CHARACTERS cyclizine impurity B CRS in methanol R and dilute to 20.0 mL
with the same solvent.
Appearance : white or almost white, crystalline powder.
Column :
Solubility : slightly soluble in water and in ethanol (96 per
cent). – material : fused silica ;
– size : l = 25 m, Ø = 0.33 mm ;
IDENTIFICATION – stationary phase : poly(dimethyl)(diphenyl)siloxane R (film
First identification : B, E. thickness 0.50 μm).
Second identification : A, C, D, E. Carrier gas : helium for chromatography R.
A. Ultraviolet and visible absorption spectrophotometry Flow rate : 1.0 mL/min.
(2.2.25). Split ratio : 1:25.
Test solution (a). Dissolve 20.0 mg in a 5 g/L solution of Temperature :
sulfuric acid R and dilute to 100.0 mL with the same acid
solution. Time Temperature
Test solution (b). Dilute 10.0 mL of test solution (a) to (min) (°C)
100.0 mL with a 5 g/L solution of sulfuric acid R. Column 0 - 14 100 → 240
Spectral range : 240-350 nm for test solution (a) ; 210-240 nm 14 - 16 240 → 270
for test solution (b).
16 - 30 270
Resolution (2.2.25) : minimum 1.7.
Injection port 250
Absorption maxima : at 258 nm and 262 nm for test
solution (a) ; at 225 nm for test solution (b). Detector 290
Absorbance ratio : A262/A258 = 1.0 to 1.1.
Detection : flame ionisation.
Specific absorbance at the absorption maximum at 225 nm :
370 to 410 for test solution (b). Injection : 1 μL.
B. Infrared absorption spectrophotometry (2.2.24). Relative retention with reference to cyclizine (retention
time = about 15 min) : impurity A = about 0.2 ;
Comparison : cyclizine hydrochloride CRS. impurity B = about 0.7.
C. Thin-layer chromatography (2.2.27). System suitability : reference solution (b) :
Test solution. Dissolve 10 mg of the substance to be – peak-to-valley ratio : minimum 50, where Hp = height
examined in methanol R and dilute to 10 mL with the same above the baseline of the peak due to impurity A and
solvent. Hv = height above the baseline of the lowest point of the
Reference solution. Dissolve 10 mg of cyclizine curve separating this peak from the peak due to methanol.
hydrochloride CRS in methanol R and dilute to 10 mL with Limits :
the same solvent.
– impurities A, B : for each impurity, not more than the area
Plate : TLC silica gel GF254 plate R. of the corresponding peak in the chromatogram obtained
Mobile phase : concentrated ammonia R, methanol R, with reference solution (b) (0.5 per cent) ;
methylene chloride R (2:13:85 V/V/V). – unspecified impurities : for each impurity, not more than the
Application : 20 μL. area of the principal peak in the chromatogram obtained
Development : over 2/3 of the plate. with reference solution (a) (0.10 per cent) ;

1974 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cyclopentolate hydrochloride

– total : not more than 10 times the area of the principal peak A. Melting point (2.2.14) : 135 °C to 141 °C.
in the chromatogram obtained with reference solution (a) B. Infrared absorption spectrophotometry (2.2.24).
(1.0 per cent) ; Preparation : discs of potassium chloride R.
– disregard limit : 0.5 times the area of the principal peak in Comparison: cyclopentolate hydrochloride CRS.
the chromatogram obtained with reference solution (a)
(0.05 per cent). If the spectra obtained show differences, dissolve the
substance to be examined and the reference substance
Loss on drying (2.2.32) : maximum 1.0 per cent, determined separately in ethanol (96 per cent) R, evaporate to dryness
on 1.000 g by drying in an oven at 130 °C. and record new spectra using the residues.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on C. Thin-layer chromatography (2.2.27).
1.0 g. Test solution. Dissolve 10 mg of the substance to be
ASSAY examined in 5 mL of ethanol (96 per cent) R.
In order to avoid overheating in the reaction medium, mix Reference solution. Dissolve 10 mg of cyclopentolate
thoroughly throughout and stop the titration immediately after hydrochloride CRS in ethanol (96 per cent) R and dilute to
the end-point has been reached. 5 mL with the same solvent.
Dissolve 0.120 g in 15 mL of anhydrous formic acid R and add Plate : TLC silica gel plate R.
40 mL of acetic anhydride R. Titrate with 0.1 M perchloric Mobile phase : concentrated ammonia R, water R, butyl
acid, determining the end-point potentiometrically (2.2.20). acetate R, 2-propanol R (5:15:30:50 V/V/V/V).
1 mL of 0.1 M perchloric acid is equivalent to 15.14 mg Application : 10 μL.
of C18H23ClN2. Development : over 2/3 of the plate.
STORAGE Drying : in air.
Protected from light. Detection : spray with alcoholic solution of sulfuric acid R
and heat at 120 °C for 30 min ; examine in ultraviolet light
IMPURITIES at 365 nm.
Specified impurities : A, B. Result : the principal spot in the chromatogram obtained
with the test solution is similar in position, fluorescence
and size to the principal spot in the chromatogram obtained
with the reference solution.
D. It gives reaction (a) of chlorides (2.3.1).
A. 1-methylpiperazine,
TESTS
pH (2.2.3) : 4.5 to 5.5.
Dissolve 0.2 g in carbon dioxide-free water R and dilute to
20 mL with the same solvent.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
B. diphenylmethanol (benzhydrol). Test solution. Dissolve 20 mg of the substance to be examined
in water R and dilute to 20.0 mL with the same solvent.
04/2009:1093 Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with water R. Dilute 5.0 mL of this solution to
10.0 mL with water R.
CYCLOPENTOLATE
Reference solution (b). Dissolve 10 mg of cyclopentolate for
HYDROCHLORIDE system suitability CRS (containing impurity C) in water R and
dilute to 10.0 mL with the same solvent.
Cyclopentolati hydrochloridum Column :
– size : l = 0.125 m, Ø = 4.0 mm ;
– stationary phase : spherical end-capped hexylsilyl silica gel
for chromatography R (5 μm).
Mobile phase : dissolve 0.66 g of ammonium phosphate R in
water R, adjust to pH 3.0 with phosphoric acid R and dilute to
1000 mL with water R ; mix and filter ; mix 55 volumes of this
C17H26ClNO3 Mr 327.8 solution and 45 volumes of acetonitrile R1.
[5870-29-1] Flow rate : 1.0 mL/min.
DEFINITION Detection : spectrophotometer at 220 nm.
2-(Dimethylamino)ethyl (2RS)-(1-hydroxycyclopentyl)- Injection : 20 μl.
(phenyl)acetate hydrochloride. Run time : 2.5 times the retention time of cyclopentolate.
Content : 98.5 per cent to 101.5 per cent (dried substance). Identification of impurities : use the chromatogram supplied
with cyclopentolate for system suitability CRS and the
CHARACTERS chromatogram obtained with reference solution (b) to identify
Appearance : white or almost white, crystalline powder. the peak due to impurity C.
Solubility : very soluble in water, freely soluble in ethanol Relative retention with reference to cyclopentolate (retention
(96 per cent). time = about 4 min) : impurity C = about 0.9.
It shows polymorphism (5.9). System suitability : reference solution (b) :
– peak-to-valley ratio : minimum 6, where Hp = height above
IDENTIFICATION the baseline of the peak due to impurity C and Hv = height
First identification : B, D. above the baseline of the lowest point of the curve
Second identification : A, C, D. separating this peak from the peak due to cyclopentolate.

General Notices (1) apply to all monographs and other texts 1975
Cyclophosphamide EUROPEAN PHARMACOPOEIA 8.0

Limits : DEFINITION
– correction factor : for the calculation of content, multiply Cyclophosphamide contains not less than 98.0 per cent and not
the peak area of impurity C by 2.0 ; more than the equivalent of 102.0 per cent of (2RS)-N,N-bis(2-
– impurity C : not more than the area of the principal peak chloroethyl)tetrahydro-2H-1,3,2-oxazaphosphorin-2-amine
in the chromatogram obtained with reference solution (a) 2-oxide, calculated with reference to the anhydrous substance.
(0.5 per cent) ;
CHARACTERS
– unspecified impurities : for each impurity, not more than
0.2 times the area of the principal peak in the chromatogram A white or almost white, crystalline powder, soluble in water,
obtained with reference solution (a) (0.10 per cent) ; freely soluble in alcohol.
– total : not more than twice the area of the principal peak IDENTIFICATION
in the chromatogram obtained with reference solution (a) First identification : B.
(1.0 per cent) ;
Second identification : A, C, D.
– disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (a) A. Determine the melting point (2.2.14) of the substance to be
(0.05 per cent). examined. Mix equal parts of the substance to be examined
and cyclophosphamide CRS and determine the melting
Loss on drying (2.2.32) : maximum 0.5 per cent, determined point of the mixture. The difference between the melting
on 1.000 g by drying in an oven at 105 °C for 4 h. points (which are about 51 °C) is not greater than 2 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on B. Examine by infrared absorption spectrophotometry
1.0 g. (2.2.24), comparing with the spectrum obtained with
ASSAY cyclophosphamide CRS.
Dissolve 0.250 g in a mixture of 1.0 mL of 0.1 M hydrochloric C. Examine the chromatograms obtained in the test for
acid and 50 mL of ethanol (96 per cent) R. Carry out a related substances. The principal spot in the chromatogram
potentiometric titration (2.2.20), using 0.1 M sodium obtained with test solution (b) is similar in position,
hydroxide. Read the volume added between the 2 points of colour and size to the principal spot in the chromatogram
inflexion. obtained with reference solution (a).
1 mL of 0.1 M sodium hydroxide is equivalent to 32.79 mg D. Dissolve 0.1 g in 10 mL of water R and add 5 mL of silver
of C17H26ClNO3. nitrate solution R1 ; the solution remains clear. Boil, a
white precipitate is formed which dissolves in concentrated
IMPURITIES ammonia R and is reprecipitated on the addition of dilute
Specified impurities : C. nitric acid R.
Other detectable impurities (the following substances would, TESTS
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general Solution S. Dissolve 0.50 g in carbon dioxide-free water R and
acceptance criterion for other/unspecified impurities and/or dilute to 25.0 mL with the same solvent.
by the general monograph Substances for pharmaceutical use Appearance of solution. Solution S is clear (2.2.1) and not
(2034). It is therefore not necessary to identify these impurities
more intensely coloured than reference solution Y6 (2.2.2,
for demonstration of compliance. See also 5.10. Control of Method II).
impurities in substances for pharmaceutical use) : A, B. pH (2.2.3). The pH of solution S is 4.0 to 6.0, determined
immediately after preparation of the solution.
Related substances. Examine by thin-layer chromatography
(2.2.27), using silica gel G R as the coating substance.
Test solution (a). Dissolve 0.10 g of the substance to be
examined in alcohol R and dilute to 5 mL with the same
solvent.
A. (2RS)-(1-hydroxycyclopentyl)(phenyl)acetic acid,
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
with alcohol R.
Reference solution (a). Dissolve 10 mg of cyclophosphamide CRS
in alcohol R and dilute to 5 mL with the same solvent.
B. phenylacetic acid,
Reference solution (b). Dilute 0.1 mL of test solution (a) to
10 mL with alcohol R.
Apply separately to the plate 10 μL of each solution. Develop
over a path of 15 cm using a mixture of 2 volumes of anhydrous
C. 2-(dimethylamino)ethyl phenylacetate. formic acid R, 4 volumes of acetone R, 12 volumes of water R
and 80 volumes of methyl ethyl ketone R. Dry the plate in a
01/2008:0711 current of warm air and heat at 110 °C for 10 min. At the
bottom of a chromatographic tank, place an evaporating dish
containing a 50 g/L solution of potassium permanganate R
CYCLOPHOSPHAMIDE and add an equal volume of hydrochloric acid R. Place the
plate whilst still hot in the tank and close the tank. Leave the
Cyclophosphamidum plate in contact with the chlorine gas for 2 min. Withdraw
the plate and place it in a current of cold air until the excess
of chlorine is removed and an area of coating below the
points of application gives at most a very faint blue colour
with a drop of potassium iodide and starch solution R. Avoid
prolonged exposure to cold air. Spray with potassium iodide
and starch solution R and allow to stand for 5 min. Any spot
C7H15Cl2N2O2P,H2O Mr 279.1 in the chromatogram obtained with test solution (a), apart
[6055-19-2] from the principal spot, is not more intense than the spot

1976 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cyproheptadine hydrochloride

in the chromatogram obtained with reference solution (b) Reference solution (b). Dissolve 2.0 mg of dibenzocyclo-
(1.0 per cent). Disregard any spot remaining at the point of heptene CRS (impurity A), 2.0 mg of dibenzosuberone CRS
application. (impurity B) and 2.0 mg of cyproheptadine impurity C CRS in
Chlorides (2.4.4). Dissolve 0.15 g in water R and dilute to mobile phase A, add 1.0 mL of the test solution and dilute to
15 mL with the same solvent. The freshly prepared solution 100.0 mL with mobile phase A.
complies with the limit test for chlorides (330 ppm). Reference solution (c). Dilute 1.0 mL of reference solution (b)
Phosphates (2.4.11). Dissolve 0.10 g in water R and dilute to to 10.0 mL with mobile phase A.
100 mL with the same solvent. The solution complies with the Column :
limit test for phosphates (100 ppm). – size : l = 0.25 m, Ø = 4.6 mm ;
Heavy metals (2.4.8). 1.0 g complies with test C for heavy – stationary phase : octylsilyl silica gel for chromatography R
metals (20 ppm). Prepare the reference solution using 2 mL (5 μm).
of lead standard solution (10 ppm Pb) R. Mobile phase :
Water (2.5.12) : 6.0 per cent to 7.0 per cent, determined on – mobile phase A : dissolve 6.12 g of potassium dihydrogen
0.300 g by the semi-micro determination of water. phosphate R in 900 mL of water R, adjust to pH 4.5 with
phosphoric acid R and dilute to 1000 mL with water R ; mix
ASSAY 60 volumes of this solution and 40 volumes of acetonitrile
Dissolve 0.100 g in 50 mL of a 1 g/L solution of sodium for chromatography R ;
hydroxide R in ethylene glycol R and boil under a reflux – mobile phase B : dissolve 6.12 g of potassium dihydrogen
condenser for 30 min. Allow to cool and rinse the condenser phosphate R in 900 mL of water R, adjust to pH 4.5 with
with 25 mL of water R. Add 75 mL of 2-propanol R, 15 mL of phosphoric acid R and dilute to 1000 mL with water R ; mix
dilute nitric acid R, 10.0 mL of 0.1 M silver nitrate and 2.0 mL 40 volumes of this solution and 60 volumes of acetonitrile
of ferric ammonium sulfate solution R2 and titrate with 0.1 M for chromatography R ;
ammonium thiocyanate.
Time Mobile phase A Mobile phase B
1 mL of 0.1 M silver nitrate is equivalent to 13.05 mg of
(min) (per cent V/V) (per cent V/V)
C7H15Cl2N2O2P.
0 - 10.0 100 0
07/2009:0817 10.0 - 10.1 100 → 0 0 → 100

CYPROHEPTADINE 10.1 - 35 0 100

HYDROCHLORIDE Flow rate : 1.0 mL/min.

Detection : spectrophotometer at 230 nm.
Cyproheptadini hydrochloridum Injection : 10 μL.
Relative retention with reference to cyproheptadine
(retention time = about 8 min) : impurity C = about 0.7 ;
impurity B = about 2.6 ; impurity A = about 3.9.
System suitability : reference solution (b) :
– resolution : minimum 7.0 between the peaks due to
impurity C and cyproheptadine.
Limits :
C21H22ClN,1½H2O Mr 350.9
[41354-29-4] – impurities A, B, C : for each impurity, not more than
1.5 times the area of the corresponding peak in the
DEFINITION chromatogram obtained with reference solution (c)
4-(5H-Dibenzo[a,d][7]annulen-5-ylidene)-1-methyl- (0.15 per cent);
piperidine hydrochloride sesquihydrate. – unspecified impurities : for each impurity, not more than the
Content : 98.5 per cent to 101.0 per cent (anhydrous substance). area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
CHARACTERS – total : not more than 5 times the area of the principal peak
Appearance : white or slightly yellow, crystalline powder. in the chromatogram obtained with reference solution (a)
Solubility : slightly soluble in water, freely soluble in methanol, (0.5 per cent) ;
sparingly soluble in ethanol (96 per cent). – disregard limit : 0.5 times the area of the principal peak in
IDENTIFICATION the chromatogram obtained with reference solution (a)
(0.05 per cent).
A. Infrared absorption spectrophotometry (2.2.24).
Water (2.5.12) : 7.0 per cent to 9.0 per cent, determined on
Comparison : cyproheptadine hydrochloride CRS.
0.200 g.
B. A saturated solution gives reaction (b) of chlorides (2.3.1).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
TESTS 1.0 g.
Acidity. Dissolve 0.10 g in water R and dilute to 25 mL with ASSAY
the same solvent. Add 0.1 mL of methyl red solution R. Not
more than 0.15 mL of 0.01 M sodium hydroxide is required to Dissolve 0.250 g in a mixture of 5.0 mL of 0.01 M hydrochloric
change the colour of the indicator. acid and 50 mL of ethanol (96 per cent) R. Carry out a
potentiometric titration (2.2.20), using 0.1 M sodium
Related substances. Liquid chromatography (2.2.29). hydroxide. Read the volume added between the 2 points of
Test solution. Dissolve 40.0 mg of the substance to be inflexion.
examined in mobile phase A and dilute to 20.0 mL with 1 mL of 0.1 M sodium hydroxide is equivalent to 32.39 mg
mobile phase A. of C21H22ClN.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with mobile phase A. Dilute 1.0 mL of this solution STORAGE
to 10.0 mL with mobile phase A. Protected from light.

General Notices (1) apply to all monographs and other texts 1977
Cyproterone acetate EUROPEAN PHARMACOPOEIA 8.0

IMPURITIES Plate : TLC silica gel F254 plate R.

Specified impurities : A, B, C. Mobile phase : cyclohexane R, ethyl acetate R (50:50 V/V).
Application : 5 μL.
Development : twice over 3/4 of the plate ; dry in air between
the 2 developments.
Drying : in air.
Detection : examine in ultraviolet light at 254 nm.
Results : the principal spot in the chromatogram obtained
A. 5H-dibenzo[a,d][7]annulene (dibenzocycloheptene), with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the
reference solution.
C. To about 1 mg add 2 mL of sulfuric acid R and heat on a
water-bath for 2 min. A red colour develops. Cool. Add
this solution cautiously to 4 mL of water R and shake. The
solution becomes violet.
D. Incinerate about 30 mg with 0.3 g of anhydrous sodium
B. 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-one carbonate R over a naked flame for about 10 min. Cool and
(dibenzosuberone), dissolve the residue in 5 mL of dilute nitric acid R. Filter.
To 1 mL of the filtrate add 1 mL of water R. The solution
gives reaction (a) of chlorides (2.3.1).
E. It gives the reaction of acetyl (2.3.1).
TESTS
Specific optical rotation (2.2.7) : + 152 to + 157 (dried
substance).
C. 5-(1-methylpiperidin-4-yl)-5H-dibenzo[a,d][7]annulen- Dissolve 0.25 g in acetone R and dilute to 25.0 mL with the
5-ol. same solvent.
Related substances. Liquid chromatography (2.2.29).
04/2012:1094
Test solution. Dissolve 10 mg of the substance to be examined
in acetonitrile R and dilute to 10.0 mL with the same solvent.
CYPROTERONE ACETATE Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with acetonitrile R.
Cyproteroni acetas Reference solution (b). Dissolve the contents of a vial of
cyproterone impurity mixture CRS (impurities F and I) in
1.0 mL of the test solution.
Reference solution (c). Dissolve 2 mg of cyproterone acetate for
peak identification CRS (containing impurities B, C, E and G)
in 2.0 mL of acetonitrile R.
Column :
– size : l = 0.125 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
C24H29ClO4 Mr 416.9 chromatography R (3 μm).
[427-51-0] Mobile phase : acetonitrile R, water R (40:60 V/V).
DEFINITION Flow rate : 1.5 mL/min.
6-Chloro-3,20-dioxo-1β,2β-dihydro-3′H-cyclopropa- Detection : spectrophotometer at 254 nm.
[1,2]pregna-1,4,6-trien-17-yl acetate. Injection : 20 μL.
Content : 97.0 per cent to 103.0 per cent (dried substance). Run time : twice the retention time of cyproterone acetate.
CHARACTERS Identification of impurities : use the chromatogram supplied
with cyproterone impurity mixture CRS and the chromatogram
Appearance : white or almost white, crystalline powder.
obtained with reference solution (b) to identify the peaks
Solubility : practically insoluble in water, very soluble in due to impurities F and I ; use the chromatogram supplied
methylene chloride, freely soluble in acetone, soluble in with cyproterone acetate for peak identification CRS and the
methanol, sparingly soluble in anhydrous ethanol. chromatogram obtained with reference solution (c) to identify
mp : about 210 °C. the peaks due to impurities B, C, E and G.
IDENTIFICATION Relative retention with reference to cyproterone acetate
(retention time = about 22 min) : impurity E = about 0.27 ;
First identification : A. impurity G = about 0.3 ; impurity F = about 0.5 ;
Second identification : B, C, D, E. impurity B = about 0.7 ; impurity I = about 0.9 ;
A. Infrared absorption spectrophotometry (2.2.24). impurity C = about 1.5.
Comparison : cyproterone acetate CRS. System suitability : reference solution (b) :
B. Thin-layer chromatography (2.2.27). – resolution : minimum 1.5 between the peaks due to
Test solution. Dissolve 20 mg of the substance to be impurity I and cyproterone acetate.
examined in methylene chloride R and dilute to 10 mL with Limits :
the same solvent. – correction factors : for the calculation of content, multiply
Reference solution. Dissolve 10 mg of cyproterone the peak areas of the following impurities by the
acetate CRS in methylene chloride R and dilute to 5 mL corresponding correction factor : impurity C = 1.8 ;
with the same solvent. impurity E = 0.7 ;

1978 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cyproterone acetate

– impurity F : not more than 0.4 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.4 per cent) ;
– impurity E : not more than 0.2 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent) ;
– impurities B, C, G : for each impurity, not more
than 0.15 times the area of the principal peak in the C. 6-chloro-1α-(chloromethyl)-3,20-dioxopregna-4,6-dien-
chromatogram obtained with reference solution (a) 17-yl acetate,
(0.15 per cent) ;
– unspecified impurities : for each impurity, not more than
0.1 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent) ;
– total : not more than 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
– disregard limit : 0.05 times the area of the principal peak
in the chromatogram obtained with reference solution (a) D. 1α-(chloromethyl)-3,6,20-trioxopregn-4-en-17-yl acetate,
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying at 80 °C at a pressure not exceeding
0.7 kPa.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.

ASSAY
Dissolve 50.0 mg in methanol R and dilute to 50.0 mL with the E. 3,6,20-trioxo-1β,2β-dihydro-3′H-cyclopropa[1,2]pregna-
same solvent. Dilute 1.0 mL of the solution to 100.0 mL with 1,4-dien-17-yl acetate,
methanol R. Measure the absorbance (2.2.25) at the absorption
maximum at 282 nm.
Calculate the content of C24H29ClO4 taking the specific
absorbance to be 414.

STORAGE
Protected from light.

IMPURITIES F. 6-chloro-17-hydroxy-1β,2β-dihydro-3′H-cyclopropa[1,2]-
Specified impurities : B, C, E, F, G. pregna-1,4,6-triene-3,20-dione,
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : A, D, H, I, J.
G. 6β-chloro-7α-hydroxy-3,20-dioxo-1β,2β-dihydro-3′H-
cyclopropa[1,2]pregna-1,4-dien-17-yl acetate,

A. 3,20-dioxo-1β,2β-dihydro-3′H-cyclopropa-
[1,2]pregna-1,4,6-trien-17-yl acetate,
H. 3,20-dioxopregna-1,4-dien-17-yl acetate,

B. 6-methoxy-3,20-dioxo-1β,2β-dihydro-3′H- I. 6-chloro-3,20-dioxopregna-1,4,6-trien-17-yl acetate

cyclopropa[1,2]pregna-1,4,6-trien-17-yl acetate, (delmadinone acetate),

General Notices (1) apply to all monographs and other texts 1979
Cysteine hydrochloride monohydrate EUROPEAN PHARMACOPOEIA 8.0

D. Dissolve about 5 mg in 1 mL of dilute sodium hydroxide

solution R. Add 1 mL of a 30 g/L solution of sodium
nitroprusside R. An intense violet colour develops which
becomes brownish-red and then orange. Add 1 mL of
hydrochloric acid R. The solution becomes green.
E. Dissolve about 50 mg in 5 mL of water R. Heat to about
60 °C on a water-bath and carefully add, dropwise, 5 mL of
strong hydrogen peroxide solution R. Heat the water-bath
J. 6α,7α-epoxy-3,20-dioxo-1β,2β-dihydro-3′H-
to boiling and maintain the sample on the water-bath for
cyclopropa[1,2]pregna-1,4-dien-17-yl acetate. 1 h. After cooling to room temperature reconstitute the
sample to 10 mL with water R. 2 mL of the solution gives
01/2014:0895 reaction (a) of chlorides (2.3.1).
TESTS
CYSTEINE HYDROCHLORIDE Solution S. Dissolve 2.5 g in distilled water R and dilute to
MONOHYDRATE 50 mL with the same solvent.
Appearance of solution. The solution is clear (2.2.1) and not
Cysteini hydrochloridum monohydricum more intensely coloured than reference solution BY6 (2.2.2,
Method II).
Dilute 10 mL of solution S to 20 mL with water R.
Specific optical rotation (2.2.7) : + 5.5 to + 7.0 (dried
C3H8ClNO2S,H2O Mr 175.6 substance).
[7048-04-6] Dissolve 2.00 g in hydrochloric acid R1 and dilute to 25.0 mL
DEFINITION with the same acid.
(2R)-2-Amino-3-sulfanylpropanoic acid hydrochloride Ninhydrin-positive substances. Amino acid analysis
monohydrate. (2.2.56). For analysis, use Method 1. Prepare the solutions
immediately before use.
Fermentation product, extract or hydrolysate of protein.
Content : 98.5 per cent to 101.0 per cent (dried substance). The concentrations of the test solution and the reference
solutions may be adapted according to the sensitivity of the
CHARACTERS equipment used. The concentrations of all solutions are
Appearance : white or almost white, crystalline powder or adjusted so that the system suitability requirements described
colourless crystals. in general chapter 2.2.46 are fulfilled, keeping the ratios of
concentrations between all solutions as described.
Solubility : freely soluble in water, slightly soluble in ethanol
(96 per cent). Solution A : dilute hydrochloric acid R1 or a sample preparation
buffer suitable for the apparatus used.
IDENTIFICATION Test solution. Dissolve 30.0 mg of the substance to be
First identification : A, B, E. examined in solution A and dilute to 50.0 mL with solution A.
Second identification : A, C, D, E. Reference solution (a). Dilute 1.0 mL of the test solution to
A. Specific optical rotation (see Tests). 100.0 mL with solution A. Dilute 2.0 mL of this solution to
B. Infrared absorption spectrophotometry (2.2.24). 10.0 mL with solution A.
Comparison : cysteine hydrochloride monohydrate CRS. Reference solution (b). Dissolve 30.0 mg of L-cystine R
(impurity A) in solution A and dilute to 100.0 mL with
C. Thin-layer chromatography (2.2.27). solution A. Dilute 1.0 mL of the solution to 250.0 mL with
Test solution. Dissolve 20 mg of the substance to be solution A.
examined in water R and dilute to 10 mL with the Reference solution (c). Dissolve 30.0 mg of proline R in
same solvent. Add 10 mL of a 40 g/L solution of solution A and dilute to 100.0 mL with solution A. Dilute
N-ethylmaleimide R in ethanol (96 per cent) R. Allow to 1.0 mL of the solution to 250.0 mL with solution A.
stand for 5 min. Dilute 2 mL of the solution to 10 mL with Reference solution (d). Dilute 6.0 mL of ammonium standard
water R. solution (100 ppm NH4) R to 50.0 mL with solution A. Dilute
Reference solution. Dissolve 20 mg of cysteine hydrochloride 1.0 mL of this solution to 100.0 mL with solution A.
monohydrate CRS in water R and dilute to 10 mL with Reference solution (e). Dissolve 30 mg of isoleucine R and
the same solvent. Add 10 mL of a 40 g/L solution of 30 mg of leucine R in solution A and dilute to 50.0 mL with
N-ethylmaleimide R in ethanol (96 per cent) R. Allow to solution A. Dilute 1.0 mL of the solution to 200.0 mL with
stand for 5 min. Dilute 2 mL of the solution to 10 mL with solution A.
water R.
Blank solution : solution A.
Plate : TLC silica gel plate R.
Mobile phase : glacial acetic acid R, water R, butanol R Inject suitable, equal amounts of the test, blank and reference
(20:20:60 V/V/V). solutions into the amino acid analyser. Run a program suitable
for the determination of physiological amino acids.
Application : 5 μL.
System suitability : reference solution (e):
Development : over 2/3 of the plate.
– resolution : minimum 1.5 between the peaks due to
Drying : at 80 °C for 30 min.
isoleucine and leucine.
Detection : spray with ninhydrin solution R and heat at
105 °C for 15 min. Calculation of percentage contents:
Results : the principal spot in the chromatogram obtained – for impurity A, use the concentration of impurity A in
with the test solution is similar in position, colour and size reference solution (b) ;
to the principal spot in the chromatogram obtained with – for any ninhydrin-positive substance detected at 570 nm,
the reference solution. use the concentration of cysteine in reference solution (a) ;

1980 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cystine

– for any ninhydrin-positive substance detected at 440 nm,

use the concentration of proline in reference solution (c) ; if
a peak is above the reporting threshold at both wavelengths,
use the result obtained at 570 nm for quantification.
A. (2R,2′R)-3,3′-disulfanediylbis(2-aminopropanoic acid)
Limits: (cystine),
– impurity A at 570 nm : maximum 0.5 per cent ;
– any ninhydrin-positive substance : for each impurity,
maximum 0.2 per cent ;
– total : maximum 1.0 per cent ; B. (2S)-2-amino-3-hydroxypropanoic acid (serine).
– reporting threshold : 0.05 per cent.
01/2008:0998
The thresholds indicated under Related substances corrected 6.0
(Table 2034.-1) in the general monograph Substances for
pharmaceutical use (2034) do not apply.
CYSTINE
Sulfates (2.4.13) : maximum 300 ppm.
Dilute 10 mL of solution S to 15 mL with distilled water R. Cystinum
Ammonium. Amino acid analysis (2.2.56) as described in
the test for ninhydrin-positive substances with the following
modifications.
Injection : test solution, reference solution (d) and blank
solution. C6H12N2O4S2 Mr 240.3
Limit : [56-89-3]
– ammonium at 570 nm : not more than the area of the DEFINITION
corresponding peak in the chromatogram obtained with Cystine contains not less than 98.5 per cent and
reference solution (d) (0.02 per cent), taking into account not more than the equivalent of 101.0 per cent of
the peak due to ammonium in the chromatogram obtained 3,3′-disulfanediylbis[(2R)-2-aminopropanoic acid], calculated
with the blank solution. with reference to the dried substance.
Iron (2.4.9) : maximum 20 ppm.
CHARACTERS
In a separating funnel, dissolve 0.50 g in 10 mL of dilute
A white or almost white, crystalline powder, practically
hydrochloric acid R. Shake with 3 quantities, each of 10 mL, of
insoluble in water and in alcohol. It dissolves in dilute
methyl isobutyl ketone R1, shaking for 3 min each time. To the
solutions of alkali hydroxides.
combined organic layers add 10 mL of water R and shake for
3 min. Use the aqueous layer. IDENTIFICATION
Heavy metals (2.4.8) : maximum 10 ppm. First identification : A, B.
0.5 g complies with test G. Prepare the reference solution Second identification : A, C, D.
using 0.5 mL of lead standard solution (10 ppm Pb) R. A. Specific optical rotation (see Tests).
Loss on drying (2.2.32) : 8.0 per cent to 12.0 per cent, B. Examine by infrared absorption spectrophotometry
determined on 1.000 g by drying at a pressure not exceeding (2.2.24), comparing with the spectrum obtained with
0.7 kPa for 24 h. cystine CRS. Examine the substances prepared as discs.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on C. Examine the chromatograms obtained in the test for
1.0 g. ninhydrin-positive substances. The principal spot in the
chromatogram obtained with test solution (b) is similar
ASSAY in position, colour and size to the principal spot in the
chromatogram obtained with reference solution (a).
In a ground-glass stoppered flask dissolve 0.300 g of the D. To 0.1 g carefully add 1 mL of strong hydrogen peroxide
substance to be examined and 4 g of potassium iodide R in solution R and 0.1 mL of ferric chloride solution R1. Allow
20 mL of water R. Cool the solution in iced water and add to cool. Add 1 mL of dilute hydrochloric acid R and 5 mL
3 mL of hydrochloric acid R1 and 25.0 mL of 0.05 M iodine. of water R. Add 1 mL of barium chloride solution R1.
Stopper the flask and allow to stand in the dark for 20 min. Turbidity or a white precipitate develops within 3 min.
Titrate with 0.1 M sodium thiosulfate using 3 mL of starch
solution R, added towards the end of the titration, as indicator. TESTS
Carry out a blank titration.
Appearance of solution. Dissolve 1.0 g in dilute hydrochloric
1 mL of 0.05 M iodine is equivalent to 15.76 mg of C3H8ClNO2S. acid R and dilute to 10 mL with the same acid. The solution is
clear (2.2.1) and not more intensely coloured than reference
STORAGE solution Y7 (2.2.2, Method II).
Protected from light. Specific optical rotation (2.2.7). Dissolve 0.50 g in 1 M
hydrochloric acid and dilute to 25.0 mL with the same acid.
IMPURITIES The specific optical rotation is − 218 to − 224, calculated with
Specified impurities : A. reference to the dried substance.
Other detectable impurities (the following substances would, Ninhydrin-positive substances. Examine by thin-layer
if present at a sufficient level, be detected by one or other of chromatography (2.2.27), using a TLC silica gel plate R.
the tests in the monograph. They are limited by the general Test solution (a). Dissolve 0.10 g of the substance to be
acceptance criterion for other/unspecified impurities. It examined in 1 M hydrochloric acid and dilute to 10 mL with
is therefore not necessary to identify these impurities for the same acid.
demonstration of compliance. See also 5.10. Control of Test solution (b). Dilute 1 mL of test solution (a) to 50 mL
impurities in substances for pharmaceutical use) : B. with water R.

General Notices (1) apply to all monographs and other texts 1981
Cytarabine EUROPEAN PHARMACOPOEIA 8.0

Reference solution (a). Dissolve 10 mg of cystine CRS in 1 mL 01/2008:0760

of 1 M hydrochloric acid and dilute to 50 mL with water R.
CYTARABINE
Reference solution (b). Dilute 2 mL of test solution (b) to
20 mL with water R. Cytarabinum
Reference solution (c). Dissolve 10 mg of cystine CRS and 10 mg
of arginine hydrochloride CRS in 1 mL of 1 M hydrochloric
acid and dilute to 25 mL with water R.

Apply separately to the plate 5 μL of each solution. Develop

over a path of 15 cm using a mixture of 30 volumes of
concentrated ammonia R and 70 volumes of 2-propanol R.
Allow the plate to dry in air. Spray with ninhydrin solution R
and heat at 100 °C to 105 °C for 15 min. Any spot in the
chromatogram obtained with test solution (a), apart from C9H13N3O5 Mr 243.2
the principal spot, is not more intense than the spot in the [147-94-4]
chromatogram obtained with reference solution (b) (0.2 per
cent). The test is not valid unless the chromatogram obtained DEFINITION
with reference solution (c) shows two clearly separated spots. Cytarabine contains not less than 99.0 per cent and
not more than the equivalent of 100.5 per cent of
Chlorides (2.4.4). Dissolve 0.25 g in 5 mL of dilute nitric 4-amino-1-β-D-arabinofuranosylpyrimidin-2(1H)-one,
acid R and dilute to 15 mL with water R. The solution, without calculated with reference to the dried substance.
further addition of nitric acid, complies with the limit test for
chlorides (200 ppm). CHARACTERS
Sulfates (2.4.13). Dissolve 0.5 g in 5 mL of dilute hydrochloric A white or almost white, crystalline powder, freely soluble
acid R and dilute to 15 mL with distilled water R. The solution in water, very slightly soluble in alcohol and in methylene
complies with the limit test for sulfates (300 ppm). chloride.
It melts at about 215 °C.
Ammonium (2.4.1). 0.10 g complies with limit test B for
ammonium (200 ppm). Prepare the standard using 0.2 mL of IDENTIFICATION
ammonium standard solution (100 ppm NH4) R. A. Dissolve 20.0 mg in 0.1 M hydrochloric acid and dilute to
Iron (2.4.9). In a separating funnel, dissolve 1.0 g in 10 mL of 100.0 mL with the same acid. Dilute 5.0 mL of the solution
dilute hydrochloric acid R. Shake with three quantities, each of to 100.0 mL with 0.1 M hydrochloric acid. Examined
10 mL, of methyl isobutyl ketone R1, shaking for 3 min each between 230 nm and 350 nm (2.2.25), the solution shows an
time. To the combined organic layers add 10 mL of water R absorption maximum at 281 nm. The specific absorbance
and shake for 3 min. The aqueous layer complies with the at the maximum is 540 to 570.
limit test for iron (10 ppm). B. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
Heavy metals (2.4.8). 2.0 g complies with test D for heavy cytarabine CRS. Examine the substances prepared as discs.
metals (10 ppm). Prepare the reference solution using 2 mL C. Examine the chromatograms obtained in the test for related
of lead standard solution (10 ppm Pb) R. substances in ultraviolet light at 254 nm. The principal
Loss on drying (2.2.32). Not more than 0.5 per cent, spot in the chromatogram obtained with test solution (b)
determined on 1.000 g by drying in an oven at 105 °C. is similar in position and size to the principal spot in the
chromatogram obtained with reference solution (a).
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
on 1.0 g. TESTS
Appearance of solution. Dissolve 1.0 g in water R and dilute
to 10 mL with the same solvent. The solution is clear (2.2.1)
and not more intensely coloured than reference solution Y5
ASSAY
(2.2.2, Method II).
In a flask with a ground-glass stopper, dissolve 0.100 g in a Specific optical rotation (2.2.7). Dissolve 0.250 g in water R
mixture of 2 mL of dilute sodium hydroxide solution R and and dilute to 25.0 mL with the same solvent. The specific
10 mL of water R. Add 10 mL of a 200 g/L solution of potassium optical rotation is + 154 to + 160, calculated with reference
bromide R, 50.0 mL of 0.0167 M potassium bromate and 15 mL to the dried substance.
of dilute hydrochloric acid R. Stopper the flask and cool in iced Related substances. Examine by thin-layer chromatography
water. Allow to stand in the dark for 10 min. Add 1.5 g of (2.2.27), using silica gel GF254 R as the coating substance.
potassium iodide R. After 1 min, titrate with 0.1 M sodium Test solution (a). Dissolve 0.25 g of the substance to be
thiosulfate, using 2 mL of starch solution R, added towards the examined in water R and dilute to 5 mL with the same solvent.
end-point, as indicator. Carry out a blank titration.
Test solution (b). Dilute 2 mL of test solution (a) to 50 mL
with water R.
1 mL of 0.0167 M potassium bromate is equivalent to 2.403 mg
of C6H12N2O4S2. Reference solution (a). Dissolve 10 mg of cytarabine CRS in
water R and dilute to 5 mL with the same solvent.
Reference solution (b). Dilute 0.5 mL of test solution (a) to
100 mL with water R.
STORAGE Reference solution (c). Dissolve 20 mg of uridine R and 20 mg
of uracil arabinoside CRS in methanol R and dilute to 10 mL
Store protected from light. with the same solvent.

1982 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Cytarabine

Apply separately to the plate 5 μL of each solution. Develop 1 mL of 0.1 M perchloric acid is equivalent to 24.32 mg of
over a path of 15 cm using a mixture of 15 volumes of C9H13N3O5.
water R, 20 volumes of acetone R and 65 volumes of methyl
ethyl ketone R. Allow the plate to dry in air and examine in STORAGE
ultraviolet light at 254 nm. Any spot in the chromatogram Store in an airtight container, protected from light.
obtained with test solution (a), apart from the principal
spot, is not more intense than the spot in the chromatogram IMPURITIES
obtained with reference solution (b) (0.5 per cent). The test
is not valid unless the chromatogram obtained with reference
solution (c) shows two clearly separated spots.
Loss on drying (2.2.32). Not more than 1.0 per cent,
determined on 0.250 g by drying over diphosphorus
pentoxide R at 60 °C at a pressure of 0.2 kPa to 0.7 kPa for 3 h.
Sulfated ash (2.4.14). Not more than 0.5 per cent, determined
on 1.0 g.
ASSAY A. R = OH, R′ = H : 1-β-D-arabinofuranosylpyrimidine-
2,4(1H,3H)-dione (uracil arabonoside),
Dissolve 0.200 g in 60 mL of anhydrous acetic acid R, warming
if necessary. Titrate with 0.1 M perchloric acid determining B. R = H, R′ = OH : 1-β-D-ribofuranosylpyrimidine-
the end-point potentiometrically (2.2.20). 2,4(1H,3H)-dione (uridine).

General Notices (1) apply to all monographs and other texts 1983
EUROPEAN PHARMACOPOEIA 8.0 Dacarbazine

01/2008:1691 Test solution. Dissolve 50.0 mg of the substance to be

corrected 8.0 examined and 75 mg of citric acid R in distilled water R and
dilute to 5.0 mL with the same solvent.
DACARBAZINE Reference solution (a). Dissolve 5.0 mg of dacarbazine
impurity A CRS in distilled water R and dilute to 50.0 mL
Dacarbazinum with the same solvent. Dilute 5.0 mL of this solution to
25.0 mL with distilled water R.
Reference solution (b). Dissolve 5.0 mg of dacarbazine
impurity B CRS in distilled water R, add 0.5 mL of the test
solution and dilute to 10.0 mL with distilled water R. Dilute
1.0 mL of this solution to 50.0 mL with distilled water R.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
C6H10N6O Mr 182.2
[4342-03-4] – stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
DEFINITION Mobile phase : 15.63 g/L solution of glacial acetic acid R
5-[(1E)-3,3-Dimethyltriaz-1-enyl]-1H-imidazole-4- containing 2.33 g/L of sodium dioctyl sulfosuccinate R. As
carboxamide. the mobile phase contains sodium dioctyl sulfosuccinate, it
Content : 98.5 per cent to 101.0 per cent (anhydrous substance). must be freshly prepared every day, and the column must
be flushed with a mixture of equal volumes of methanol R
CHARACTERS and water R, after all tests have been completed or at the
Appearance : white or slightly yellowish, crystalline powder. end of the day, for at least 2 h.
Solubility : slightly soluble in water and in anhydrous ethanol, Flow rate : 1.2 mL/min.
practically insoluble in methylene chloride. Detection : spectrophotometer at 254 nm.
IDENTIFICATION Injection : 25 μL of the test solution and reference
solution (a).
First identification : B.
Run time : 3 times the retention time of impurity A.
Second identification : A, C.
Retention time : impurity A = about 3 min.
A. Ultraviolet and visible absorption spectrophotometry
(2.2.25). Limits :
Test solution. Dissolve 15.0 mg in 100.0 mL of 0.1 M – impurity A : not more than the area of the corresponding
hydrochloric acid. Dilute 5.0 mL of this solution to peak in the chromatogram obtained with reference
100.0 mL with 0.1 M hydrochloric acid. solution (a) (0.2 per cent) ;
Spectral range : 200-400 nm. – unspecified impurities eluting after impurity A : for
each impurity, not more than 0.5 times the area of the
Absorption maximum : at 323 nm. principal peak in the chromatogram obtained with
Shoulder : at 275 nm. reference solution (a) (0.10 per cent).
Specific absorbance at the absorption maximum : 1024 to B. Liquid chromatography (2.2.29) as described in test A for
1131. related substances with the following modifications.
B. Infrared absorption spectrophotometry (2.2.24). Mobile phase : mix 45 volumes of a 15.63 g/L solution of
Comparison : dacarbazine CRS. glacial acetic acid R containing 2.33 g/L of sodium dioctyl
C. Thin-layer chromatography (2.2.27). sulfosuccinate R with 55 volumes of methanol R.
Test solution. Dissolve 2.0 mg of the substance to be Injection : 10 μL of the test solution and reference
examined in methanol R and dilute to 5.0 mL with the solution (b).
same solvent. Run time : twice the retention time of dacarbazine.
Reference solution. Dissolve 2.0 mg of dacarbazine CRS in Relative retention with reference to dacarbazine (retention
methanol R and dilute to 5.0 mL with the same solvent. time = about 12 min) : impurity B = about 0.7.
Plate : TLC silica gel F254 plate R. System suitability : reference solution (b) :
Mobile phase : glacial acetic acid R, water R, butanol R – resolution : minimum 1.5 between the peaks due to
(1:2:5 V/V/V). impurity B and dacarbazine.
Application : 10 μL. Limits :
Development : over 2/3 of the plate. – impurity B : not more than the area of the corresponding
Drying : in air. peak in the chromatogram obtained with reference
Detection : examine in ultraviolet light at 254 nm. solution (b) (0.1 per cent) ;
Results : the principal spot in the chromatogram obtained – unspecified impurities : for each impurity, not more
with the test solution is similar in position and size to the than the area of the peak due to dacarbazine in the
principal spot in the chromatogram obtained with the chromatogram obtained with reference solution (b)
reference solution. (0.10 per cent);
– total : not more than 5 times the area of the peak due
TESTS to dacarbazine in the chromatogram obtained with
Appearance of solution. The solution is clear (2.2.1) and not reference solution (b) (0.5 per cent) ;
more intensely coloured than reference solution BY6 (2.2.2, – disregard limit : 0.5 times the area of the peak due
Method II). to dacarbazine in the chromatogram obtained with
Dissolve 0.25 g in a 210 g/L solution of citric acid R and dilute reference solution (b) (0.05 per cent).
to 25.0 mL with the same solution. Impurity D. Head-space gas chromatography (2.2.28).
Related substances Test solution. Introduce 0.200 g of the substance to be
A. Liquid chromatography (2.2.29). Use freshly prepared examined into a 20 mL vial and firmly attach the septum and
solutions and protect them from light. cap. Using a 10 μL syringe, inject 5 μL of water R into the vial.

General Notices (1) apply to all monographs and other texts 1987
Dalteparin sodium EUROPEAN PHARMACOPOEIA 8.0

Reference solution (a). Dilute 2.5 mL of dimethylamine

solution R (impurity D) to 100.0 mL with water R (solution A).
Firmly attach the septum and cap to a 20 mL vial. Using a
10 μL syringe, inject 10 μL of solution A into the vial.
Reference solution (b). Firmly attach the septum and cap to a A. 3,7-dihydro-4H-imidazo[4,5-d]-1,2,3-triazin-4-one
20 mL vial. Using a 10 μL syringe, inject 10 μL of solution A (2-azahypoxanthine),
and 10 μL of a 10 g/L solution of triethylamine R into the vial.
Column :
– material : fused silica ;
– size : l = 30.0 m, Ø = 0.53 mm ;
B. 5-amino-1H-imidazole-4-carboxamide,
– stationary phase : base-deactivated polyethyleneglycol R (film
thickness 1.0 μm).
Carrier gas : helium for chromatography R.
Flow rate : 13 mL/min.
Split ratio : 1:1. C. 5-diazenyl-1H-imidazole-4-carboxamide,
Static head-space conditions that may be used :
– equilibration temperature : 60 °C ;
– equilibration time : 10 min ; D. N-methylmethanamine.
– transfer-line temperature : 90 °C ;
01/2008:1195
– pressurisation time : 30 s.
Temperature : DALTEPARIN SODIUM
Time Temperature
(min) (°C) Dalteparinum natricum
Column 0-3 35

3 - 11 35 → 165

Injection port 180

Detector 220

Detection : flame ionisation.

Injection : 1 mL.
System suitability : reference solution (b) :
– resolution : minimum 2.5 between the peaks due to
impurity D and triethylamine.
Limit :
– impurity D : not more than the area of the corresponding DEFINITION
peak in the chromatogram obtained with reference Dalteparin sodium is the sodium salt of a low-molecular-mass
solution (a) (0.05 per cent). heparin that is obtained by nitrous acid depolymerisation of
heparin from porcine intestinal mucosa. The majority of the
Water (2.5.12) : maximum 0.5 per cent, determined on 1.00 g. components have a 2-O-sulfo-α-L-idopyranosuronic
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on acid structure at the non-reducing end and a
1.0 g. 6-O-sulfo-2,5-anhydro-D-mannitol structure at the
reducing end of their chain.
ASSAY Dalteparin sodium complies with the monograph
Dissolve 0.150 g in 30 mL of anhydrous acetic acid R. Titrate Low-molecular-mass heparins (0828) with the modifications
with 0.1 M perchloric acid, determining the end-point and additional requirements below.
potentiometrically (2.2.20). The mass-average relative molecular mass ranges between
1 mL of 0.1 M perchloric acid is equivalent to 18.22 mg 5600 and 6400, with a characteristic value of about 6000.
of C6H10N6O. The degree of sulfatation is 2.0 to 2.5 per disaccharide unit.
The potency is not less than 110 IU and not more than 210 IU
STORAGE of anti-factor Xa activity per milligram, calculated with
At a temperature of 2 °C to 8 °C, protected from light. reference to the dried substance. The anti-factor IIa activity
is not less than 35 IU/mg and not more than 100 IU/mg,
IMPURITIES calculated with reference to the dried substance. The ratio of
anti-factor Xa activity to anti-factor IIa activity is between
Specified impurities : A, B, D. 1.9 and 3.2.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of PRODUCTION
the tests in the monograph. They are limited by the general Dalteparin sodium is produced by a validated manufacturing
acceptance criterion for other/unspecified impurities and/or and purification procedure under conditions designed to
by the general monograph Substances for pharmaceutical minimise the presence of N-NO groups.
use (2034). It is therefore not necessary to identify these The manufacturing procedure must have been shown to
impurities for demonstration of compliance. See also 5.10. reduce any contamination by N-NO groups to approved limits
Control of impurities in substances for pharmaceutical use) : C. using an appropriate, validated quantification method.

1988 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dalteparin sodium

IDENTIFICATION Inject 100 μL of reference solution (d). When the

chromatograms are recorded in the prescribed conditions,
Carry out identification test A as described in the monograph the retention time for nitrite is 3.3 to 4.0 min. The test is not
Low-molecular-mass heparins (0828) using dalteparin valid unless :
sodium CRS.
– the number of theoretical plates calculated for the nitrite
Carry out identification test C as described in the monograph peak is at least 7000 per metre per column (dalteparin
Low-molecular-mass heparins (0828). The following sodium will block the binding sites of the stationary
requirements apply. phase, which will cause shorter retention times and
lower separation efficiency for the analyte ; the initial
The mass-average relative molecular mass ranges between 5600 performance of the column may be partially restored using
and 6400. The mass percentage of chains lower than 3000 is a 58 g/L solution of sodium chloride R at a flow rate of
not more than 13.0 per cent. The mass percentage of chains 1.0 mL/min for 1 h ; after regeneration the column is rinsed
higher than 8000 ranges between 15.0 per cent and 25.0 per with 200 mL to 400 mL of water R) ;
cent. – the symmetry factor for the nitrite peak is less than 3 ;
– the relative standard deviation of the peak area for nitrite
obtained from 6 injections is less than 3.0 per cent.
TESTS Inject 100 μL each of reference solutions (c) and (e). The test
Appearance of solution. Dissolve 1 g in 10 mL of water R. is not valid unless :
The solution is clear (2.2.1) and not more intensely coloured – the correlation factor for a linear relationship between
than intensity 5 of the range of reference solutions of the most concentration and response for reference solutions (c), (d)
appropriate colour (2.2.2, Method II). and (e) is at least 0.995 ;
Nitrite. Not more than 5 ppm. Examine by liquid – the signal-to-noise ratio for reference solution (c) is not less
chromatography (2.2.29). Rinse all volumetric flasks at least than 5 (if the noise level is too high, electrode recalibration
three times with water R before the preparation of the solutions. is recommended) ;
– a blank injection of water R does not give rise to spurious
Test solution. Dissolve 80.0 mg of the substance to be
peaks.
examined in water R and dilute to 10.0 mL with the same
solvent. Allow to stand for at least 30 min. Inject 100 μL of the test solution. Calculate the content of
nitrite from the peak areas in the chromatogram obtained
Reference solution (a). Dissolve 60.0 mg of sodium nitrite R in with reference solutions (c), (d) and (e).
water R and dilute to 1000.0 mL with the same solvent. Boron. Not more than 1 ppm, determined by inductively
coupled plasma atomic emission spectroscopy.
For the preparation of reference solution (b), use a pipette
previously rinsed with reference solution (a). Boron is determined by measurement of the emission from
an inductively coupled plasma (ICP) at a wavelength specific
to boron. The emission line at 249.733 nm is used. Use an
Reference solution (b). Dilute 1.00 mL of reference solution (a) appropriate apparatus, whose settings have been optimised as
to 50.0 mL with water R. directed by the manufacturer.
Before preparing reference solutions (c), (d) and (e), rinse all Test solution. Dissolve 0.2500 g of the substance to be
pipettes with reference solution (b). examined in about 2 mL of water for chromatography R, add
100 μL of nitric acid R and dilute to 10.00 mL with the same
Reference solution (c). Dilute 1.00 mL of reference solution (b) solvent.
to 100.0 mL with water R (corresponding to 1 ppm of nitrite Reference solution (a). Prepare a 1 per cent V/V solution of
in the test sample). nitric acid R in water for chromatography R (blank).

Reference solution (d). Dilute 3.00 mL of reference solution (b) Reference solution (b). Prepare a 11.4 μg/mL solution of boric
to 100.0 mL with water R (corresponding to 3 ppm of nitrite acid R in a 1 per cent V/V solution of nitric acid R in water for
in the test sample). chromatography R (STDcal).
Reference solution (c). Dissolve 0.2500 g of a reference
Reference solution (e). Dilute 5.00 mL of reference solution (b) dalteparin sodium with no detectable boron in about 2 mL of
to 100.0 mL with water R (corresponding to 5 ppm of nitrite water for chromatography R, add 100 μL of nitric acid R and
in the test sample). dilute to 10.00 mL with the same solvent (STD0).
Reference solution (d). Dissolve 0.2500 g of a reference
The chromatographic procedure may be carried out using : dalteparin sodium with no boron detected in about 2 mL
of a 1 per cent V/V solution of nitric acid R in water for
– a column 0.125 m long and 4.3 mm in internal diameter chromatography R, add 10 μL of a 5.7 mg/mL solution of boric
packed with a strong anion-exchange resin ; acid R and dilute to 10.00 mL with the same solvent (STD1).
This solution contains 1 μg/mL of boron.
– as mobile phase at a flow rate of 1.0 mL/min a solution Calculate the content of boron in the substance to be
consisting of 13.61 g of sodium acetate R dissolved in examined, using the following correction factor :
water R, adjusted to pH 4.3 with phosphoric acid R and
diluted to 1000 mL with water R ;

– as detector an appropriate electrochemical device with the

following characteristics and settings : a suitable working Loss on drying (2.2.32). Not more than 5.0 per cent,
electrode, a detector potential of + 1.00 V versus Ag/AgCl determined on 1.000 g by drying in an oven at 60 °C over
reference electrode and a detector sensitivity of 0.1 μA full diphosphorus pentoxide R at a pressure not exceeding 670 Pa
scale. for 3 h.

General Notices (1) apply to all monographs and other texts 1989
Danaparoid sodium EUROPEAN PHARMACOPOEIA 8.0

01/2011:2090 Reference solutions. Prepare 2 independent series of dilutions

in geometric progression of danaparoid sodium CRS in
DANAPAROID SODIUM phosphate buffer solution pH 6.5 R and in the concentration
range of 0.0005 to 0.005 units of anti-factor IIa activity per
millilitre.
Danaparoidum natricum Transfer 50 μL of each solution into the wells of a 96-well
microtitre plate. To each well add 50 μL of antithrombin III
solution R3 and 50 μL of human thrombin solution R1. Shake
the microtitre plate but do not allow bubbles to form. Incubate
for 75 min. To each well add 50 μL of chromogenic substrate R4.
Shake the microtitre plate. Measure the absorbances at 405 nm
(2.2.25) using a suitable reading device, exactly 4 min after the
addition of the chromogenic substrate. The reaction may be
stopped using 75 μL of a 20 per cent V/V solution of glacial
acetic acid R. Determine the blank amidolytic activity in a
similar manner, using phosphate buffer solution pH 6.5 R as
the blank solution (minimum 10 blanks per microtitre plate).
Calculate the activity of the substance to be examined in units
of anti-factor IIa activity per milligram using a suitable
statistical method, for example the parallel-line assay.
Chondroitin sulfate and dermatan sulfate. Chondroitin
sulfate : maximum 8.5 per cent (dried substance) ; dermatan
sulfate : 8.0 per cent to 16.0 per cent (dried substance).
Determine by selective enzymatic degradation.
Test solutions. Dry the substance to be examined at 60 °C over
DEFINITION diphosphorus pentoxide R at a pressure of about 670 Pa for 3 h.
Preparation containing the sodium salts of a mixture of Dissolve 0.200 g of the dried substance in 10.0 mL of water R.
sulfated glycosaminoglycans present in porcine tissues. Dilute this solution as necessary to obtain 3 test solutions
Danaparoid sodium is prepared from the intestinal mucosa of containing 20 mg/mL, 10 mg/mL and 5 mg/mL of the dried
pigs. Its major constituents are heparan sulfate and dermatan substance to be examined in water R.
sulfate. On complete hydrolysis it liberates D-glucosamine, Chondroitin sulfate reference solutions. Dry chondroitin
D-galactosamine, D-glucuronic acid, L-iduronic acid, acetic sulfate CRS over diphosphorus pentoxide R at room
acid and sulfuric acid. It has the characteristic property of temperature at a pressure of about 670 Pa for 16 h. Prepare
enhancing the inactivation of activated factor X (factor Xa) by solutions containing 1 mg/mL, 2 mg/mL and 3 mg/mL of
antithrombin. It has a negligible effect on the inactivation rate dried chondroitin sulfate CRS in water R.
of thrombin by antithrombin. Dermatan sulfate reference solutions. Dry dermatan sulfate CRS
Potency : 11.0 to 17.0 anti-factor Xa units per milligram (dried over diphosphorus pentoxide R at room temperature at a
substance). pressure of about 670 Pa for 16 h. Prepare solutions containing
1 mg/mL, 2 mg/mL and 3 mg/mL of dried dermatan
PRODUCTION sulfate CRS in water R.
The animals from which danaparoid sodium is derived must Chondroitinase ABC solution. Dissolve chondroitinase ABC R
fulfil the requirements for the health of animals suitable in tris-sodium acetate-sodium chloride buffer solution pH 8.0 R
for human consumption. It is prepared using a process to obtain an activity of 0.5-1.0 units per millilitre.
that ensures that the relative proportion of active sulfated
glycosaminoglycans is consistent. It is produced by methods of Chondroitinase AC solution. Dissolve chondroitinase AC R in
manufacturing designed to minimise or eliminate endotoxins tris-sodium acetate-sodium chloride buffer solution pH 7.4 R to
and hypotensive substances. obtain an activity of 1.0-2.0 units per millilitre.
Procedure :
CHARACTERS – Degradation with chondroitinase ABC : label 2 sets of
Appearance : white or almost white, hygroscopic powder. 10 tubes in triplicate : T1, T2 and T3 for the test solutions ;
Solubility : freely soluble in water. SD1, SD2 and SD3 for the dermatan sulfate reference
solutions ; SC1, SC2 and SC3 for the chondroitin sulfate
IDENTIFICATION reference solutions ; and B for the blank (water R). To each
A. The ratio of anti-factor Xa activity to anti-factor IIa tube add 1.25 mL of tris-sodium acetate buffer solution
activity, determined as described under Assay and Tests pH 8.0 R and 150 μL of the test solutions, dermatan sulfate
respectively, is not less than 22. reference solutions, chondroitin sulfate reference solutions
B. Molecular mass distribution (see Tests) : the mass-average or water R. To each tube in 1 set of tubes add 75 μL of
relative molecular mass ranges between 4000 and 7000. chondroitinase ABC solution. To determine the blank
response level, add 75 μL of tris-sodium acetate-sodium
TESTS chloride buffer solution pH 8.0 R to each tube in the other
pH (2.2.3) : 5.5 to 7.0. set of tubes. Mix the contents of the tubes using a vortex
mixer, cover with appropriate stoppers and incubate at
Dissolve 0.5 g of the dried substance to be examined in carbon 37 °C for at least 24 h.
dioxide-free water R and dilute to 50 mL with the same solvent.
– Degradation with chondroitinase AC : label 7 tubes in
Anti-factor IIa activity : maximum 0.5 units per milligram triplicate : T1, T2 and T3 for the test solutions ; SC1, SC2
(dried substance). and SC3 for the chondroitin sulfate reference solutions ;
Test solutions. Prepare 2 independent series of dilutions in and B for the blank (water R). To each tube add 1.25 mL of
geometric progression of the substance to be examined in tris-sodium acetate buffer solution pH 7.4 R and 150 μL of
phosphate buffer solution pH 6.5 R and in the concentration the test solutions, chondroitin sulfate reference solutions or
range of 0.0005 to 0.005 units of anti-factor IIa activity per water R. Add 75 μL of chondroitinase AC solution to each
millilitre. tube. Mix the contents of the tubes using a vortex mixer,

1990 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Danaparoid sodium

cover with appropriate stoppers and incubate at 37 °C for Flow rate : 0.9 mL/min ± 2 per cent.
at least 24 h. After the incubation period mix the contents Detection : spectrophotometer at 210 nm.
of the tubes using a vortex mixer and dilute to 12 times
with water R. Measure the absorbances (2.2.25) of the Injection : 100 μL.
diluted solutions at 234 nm against water R using a suitable Run time : for a period of time ensuring complete elution of
spectrophotometer. sample and solvent peaks (about 40 min).
Calculation : calculate the mean blank absorbance of each System suitability : inject the reference solution twice. The
reference solution, i.e. the mean of the absorbances of the difference between the retention times corresponding to the
reference solutions to which no chondroitinase ABC has maxima of the peaks is not more than 5 s.
been added. Subtract the mean blank absorbance value Calibration : calibration is achieved by taking the relevant part
from the individual absorbance of each reference solution. of the chromatogram obtained with the reference solution, i.e.
Calculate linear regression curves for the 2 chondroitin sulfate excluding the sharp peak at the end of the chromatogram, and
reference and the dermatan sulfate reference by plotting the matching the chromatogram obtained with the test solution
blank-corrected absorbances against the concentrations. with the calibration table obtained with the reference solution.
Calculate the average percentage content of dermatan sulfate From the calibration curve obtained, determine the molecular
in the test solutions of all tested concentrations using the mass distribution of the sample. A calibration table is supplied
following expression : with danaparoid sodium CRS.
Limits :
– chains with a relative molecular mass less than 2000 :
maximum 13 per cent ;
– chains with a relative molecular mass less than 4000 :
A1 = blank absorbance of the test solution ; maximum 39 per cent ;
– chains with a relative molecular mass between 4000 and
A2 = absorbance of the test solution with
8000 : minimum 50 per cent ;
chondroitinase ABC ;
– chains with a relative molecular mass higher than 8000 :
A3 = absorbance of the test solution with
maximum 19 per cent ;
chondroitinase AC ;
B1 = gradient of the curve obtained with the chondroitin – chains with a relative molecular mass higher than 10 000 :
maximum 11 per cent.
sulfate reference solutions with chondroitinase AC ;
B2 = gradient of the curve obtained with the Nitrogen (2.5.9) : 2.4 per cent to 3.0 per cent (dried substance).
chondroitin sulfate reference solutions with Nucleic acids : maximum 0.5 per cent (dried substance).
chondroitinase ABC ; Test solution. Weigh about 50 mg of the dried substance to
B3 = gradient of the curve obtained with the be examined into a centrifuge tube and dissolve in 200 μL of
dermatan sulfate reference solutions with water R.
chondroitinase ABC ; Reference solution. Dissolve about 50 mg of ribonucleic
C = concentration of the test solution, in milligrams acid CRS in 5 mL of 0.1 M sodium hydroxide and dilute to
per millilitre ; 20.0 mL with water R. Transfer 200 μL of the solution into
I1 = y-intercept of the curve obtained with the a centrifuge tube.
chondroitin sulfate reference solutions with Add 4.0 mL of a 50 g/L solution of trichloroacetic acid R to
chondroitinase AC ; each tube and mix. Place all tubes in boiling water for 30 min.
I2 = y-intercept of the curve obtained with the Allow to cool to room temperature. Add again 4.0 mL of a
chondroitin sulfate reference solutions with 50 g/L solution of trichloroacetic acid R to each tube and mix.
chondroitinase ABC ; If any of the test solutions is not clear, sonicate all the tubes
in an ultrasonic bath for 10 min and centrifuge at 1500 g for
I3 = y-intercept of the curve obtained with the
15 min. Dilute 1.0 mL of the clear supernatant to 4.0 mL with
dermatan sulfate reference solutions with water R. Measure the absorbances of the diluted reference
chondroitinase ABC. and test solutions at 265 nm (2.2.25) against a blank solution
Calculate the average percentage content of chondroitin prepared in the same manner, and calculate the percentage
sulfate in the test solutions for all tested concentrations using nucleic acid content of the sample.
the following expression : Total protein (2.5.33, Method 2) : maximum 0.5 per cent.
Dissolve the substance to be examined in water R. Use bovine
albumin R as the reference substance.
Sodium : 9.0 per cent to 11.0 per cent (dried substance).
Molecular mass distribution. Size-exclusion chromatography
(2.2.30). Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Dissolve 10 mg of the substance to be examined Test solution. Dissolve 0.125 g of the substance to be examined
in 2 mL of the mobile phase. in 100.0 mL of a 1.27 mg/mL solution of caesium chloride R in
0.1 M hydrochloric acid.
Reference solution. Dissolve 10 mg of danaparoid sodium CRS
in 2 mL of the mobile phase. Reference solutions. Prepare reference solutions containing
50 ppm, 100 ppm and 150 ppm of Na by diluting sodium
Column : standard solution (1000 ppm Na) R with a 1.27 mg/mL solution
– size : l = 0.60 m, Ø = 7.5 mm ; of caesium chloride R in 0.1 M hydrochloric acid.
– stationary phase : hydrophilic silica gel for chromatography R Source : sodium hollow-cathode lamp.
(10 μm) with a fractionation range for proteins with a Wavelength : 330.3 nm.
relative molecular mass of approximately 5000-100 000 ; Atomisation device : air-acetylene flame.
– temperature : 30 °C. Loss on drying (2.2.32) : maximum 5.0 per cent, determined
Mobile phase : 28.4 g/L solution of anhydrous sodium sulfate R on 0.500 g by drying in an oven at 60 °C over diphosphorus
adjusted to pH 5.0 with dilute sulfuric acid R. pentoxide R at a pressure of 670 Pa for 3 h.

General Notices (1) apply to all monographs and other texts 1991
Dapsone EUROPEAN PHARMACOPOEIA 8.0

Bacterial endotoxins (2.6.14) : less than 0.02 IU per unit of CHARACTERS

anti-factor Xa activity, if intended for use in the manufacture
of parenteral preparations without a further appropriate A white or slightly yellowish-white, crystalline powder, very
procedure for the removal of bacterial endotoxins. slightly soluble in water, freely soluble in acetone, sparingly
soluble in alcohol. It dissolves freely in dilute mineral acids.
ASSAY
IDENTIFICATION
The anticoagulant activity of danaparoid sodium is determined
in vitro by an assay which determines its ability to accelerate A. Melting point (2.2.14) : 175 °C to 181 °C.
the inhibition of factor Xa by antithrombin III (anti-factor
Xa assay). B. Dissolve 50.0 mg in methanol R and dilute to 100.0 mL
with the same solvent. Dilute 1.0 mL of this solution to
Test solutions. Prepare 2 independent series of dilutions in 100.0 mL with methanol R. Examined between 230 nm and
geometric progression of the substance to be examined in 350 nm (2.2.25), the solution shows 2 absorption maxima,
tris(hydroxymethyl)aminomethane EDTA buffer solution at 260 nm and 295 nm. The specific absorbances at these
pH 8.4 R and in the concentration range of 0.1 to 0.32 units maxima are 700 to 760 and 1150 to 1250, respectively.
of anti-factor Xa activity per millilitre.
C. Examine the chromatograms obtained in the test for
Reference solutions. Prepare 2 independent series of dilutions related substances. The principal spot in the chromatogram
in geometric progression of danaparoid sodium CRS in obtained with test solution (b) is similar in position,
tris(hydroxymethyl)aminomethane EDTA buffer solution colour and size to the principal spot in the chromatogram
pH 8.4 R and in the concentration range of 0.08 to 0.35 units obtained with reference solution (a).
of anti-factor Xa activity per millilitre.
Transfer 40 μL of each solution into the wells of a 96-well TESTS
microtitre plate. Add 40 μL of antithrombin III solution R4
to each well and shake the microtitre plate but do not allow Related substances. Examine by thin-layer chromatography
bubbles to form. Add 40 μL of bovine factor Xa solution R1 to (2.2.27), using silica gel G R as the coating substance.
each well. Exactly 2 min after the addition of the factor Xa Test solution (a). Dissolve 0.10 g of the substance to be
solution, add 80 μL of chromogenic substrate R5. Measure examined in methanol R and dilute to 10 mL with the same
the absorbance at 405 nm (2.2.25) using a suitable reading solvent.
device, exactly 4 min after the addition of the factor Xa
solution. The reaction may be stopped using 75 μL of a Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
20 per cent V/V solution of glacial acetic acid R. Determine with methanol R.
the blank amidolytic activity in the same manner, using
Reference solution (a). Dissolve 10 mg of dapsone CRS in
tris(hydroxymethyl)aminomethane EDTA buffer solution
methanol R and dilute to 10 mL with the same solvent.
pH 8.4 R as the blank (minimum 8 blanks per microtitre
plate). Calculate the potency of the substance to be examined Reference solution (b). Dilute 1 mL of test solution (b) to
in units of anti-factor Xa activity per milligram using a 10 mL with methanol R.
suitable statistical method, for example the parallel-line assay.
Reference solution (c). Dilute 2 mL of reference solution (b) to
10 mL with methanol R.
STORAGE
Apply separately to the plate 1 μL of test solution (b), 1 μL
In an airtight container. If the substance is sterile, store in a
sterile, airtight, tamper-proof container. of reference solution (a), 10 μL of test solution (a), 10 μL of
reference solution (b) and 10 μL of reference solution (c).
Develop in an unsaturated tank over a path of 15 cm using a
LABELLING mixture of 1 volume of concentrated ammonia R, 6 volumes of
The label states the number of units of anti-factor Xa activity methanol R, 20 volumes of ethyl acetate R and 20 volumes of
per milligram. heptane R. Allow the plate to dry in air. Spray the plate with
a 1 g/L solution of 4-dimethylaminocinnamaldehyde R in a
mixture of 1 volume of hydrochloric acid R and 99 volumes of
alcohol R. Examine in daylight. Any spot in the chromatogram
obtained with test solution (a), apart from the principal spot, is
01/2008:0077 not more intense than the spot in the chromatogram obtained
corrected 6.0 with reference solution (b) (1.0 per cent) and not more than 2
such spots are more intense than the spot in the chromatogram
obtained with reference solution (c) (0.2 per cent).
DAPSONE Loss on drying (2.2.32). Not more than 1.5 per cent,
determined on 1.000 g by drying in an oven at 105 °C.
Dapsonum Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
on 1.0 g.

ASSAY
Dissolve 0.100 g in 50 mL of dilute hydrochloric acid R. Carry
out the determination of primary aromatic amino-nitrogen
C12H12N2O2S Mr 248.3 (2.5.8).
[80-08-0]
1 mL of 0.1 M sodium nitrite is equivalent to 12.42 mg of
C12H12N2O2S.
DEFINITION
Dapsone contains not less than 99.0 per cent and not more STORAGE
than the equivalent of 101.0 per cent of 4,4′-sulfonyldianiline,
calculated with reference to the dried substance. Store protected from light.

1992 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Daunorubicin hydrochloride

01/2008:0662 Column :
– size : l = 0.25 m, Ø = 4.0 mm,
DAUNORUBICIN HYDROCHLORIDE – stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
Daunorubicini hydrochloridum Mobile phase : mixture of equal volumes of acetonitrile R and
a solution containing 2.88 g/L of sodium laurilsulfate R and
2.25 g/L of phosphoric acid R.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 254 nm.
Injection : 5 μL ; inject the test solution and reference
solutions (b), (c) and (d).
Run time : twice the retention time of daunorubicin.
Relative retention with reference to daunorubicin
(retention time = about 15 min) : impurity A = about
0.4 ; impurity D = about 0.5 ; epirubicin = about 0.6 ;
C27H30ClNO10 Mr 564.0 impurity B = about 0.7.
[23541-50-6] System suitability : reference solution (b) :
– resolution : minimum of 2.0 between the peaks due to
DEFINITION
impurity D and epirubicin.
(8S,10S)-8-Acetyl-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo- Limits :
hexopyranosyl)oxy]-6,8,11-trihydroxy-1-methoxy-7,8,9,10-
– impurity A : not more than the area of the corresponding
tetrahydrotetracene-5,12-dione hydrochloride.
peak in the chromatogram obtained with reference
Substance produced by certain strains of Streptomyces solution (c) (0.5 per cent),
coeruleorubidus or of Streptomyces peucetius or obtained by – impurity B : not more than 3 times the area of the principal
any other means. peak in the chromatogram obtained with reference
Content : 95.0 per cent to 102.0 per cent (anhydrous substance). solution (d) (1.5 per cent),
– impurity D : not more than the area of the corresponding
PRODUCTION
peak in the chromatogram obtained with reference
It is produced by methods of manufacture designed to solution (c) (0.5 per cent),
eliminate or minimise the presence of histamine. – any other impurity : not more than the area of the principal
CHARACTERS peak in the chromatogram obtained with reference
solution (d) (0.5 per cent),
Appearance : crystalline, orange-red powder, hygroscopic. – total of other impurities: not more than 5 times the area
Solubility : freely soluble in water and in methanol, slightly of the principal peak in the chromatogram obtained with
soluble in alcohol, practically insoluble in acetone. reference solution (d) (2.5 per cent),
– disregard limit : 0.1 times the area of the principal peak in
IDENTIFICATION
the chromatogram obtained with reference solution (d)
A. Infrared absorption spectrophotometry (2.2.24). (0.05 per cent).
Comparison : daunorubicin hydrochloride CRS. Butanol (2.4.24, System B) : maximum 1.0 per cent.
B. Dissolve about 10 mg in 0.5 mL of nitric acid R, add 0.5 mL Water (2.5.12) : maximum 3.0 per cent, determined on 0.100 g.
of water R and heat over a flame for 2 min. Allow to
cool and add 0.5 mL of silver nitrate solution R1. A white Bacterial endotoxins (2.6.14) : less than 4.3 IU/mg, if intended
precipitate is formed. for use in the manufacture of parenteral preparations without
a further appropriate procedure for the removal of bacterial
TESTS endotoxins.
pH (2.2.3) : 4.5 to 6.5. ASSAY
Dissolve 50 mg in carbon dioxide-free water R and dilute to Liquid chromatography (2.2.29) as described in the test for
10 mL with the same solvent. related substances.
Related substances. Liquid chromatography (2.2.29). Prepare Injection : test solution and reference solution (a).
the solutions immediately before use. Calculate the percentage content of C27H30ClNO10.
Test solution. Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with the STORAGE
mobile phase. In an airtight container, protected from light. If the substance
Reference solution (a). Dissolve 50.0 mg of daunorubicin is sterile, store in a sterile, airtight, tamper-proof container.
hydrochloride CRS in the mobile phase and dilute to 50.0 mL IMPURITIES
with the mobile phase.
Reference solution (b). Dissolve 10 mg of doxorubicin
hydrochloride CRS and 10 mg of epirubicin hydrochloride CRS
in the mobile phase and dilute to 100.0 mL with the mobile
phase. Dilute 1.0 mL of the solution to 10.0 mL with the
mobile phase.
Reference solution (c). Dissolve 5.0 mg of daunorubicinone CRS A. R = CO-CH3 : (8S,10S)-8-acetyl-6,8,10,11-tetrahydroxy-
and 5.0 mg of doxorubicin hydrochloride CRS in the mobile 1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione
phase and dilute to 100.0 mL with the mobile phase. Dilute (daunorubicin aglycone, daunorubicinone),
1.0 mL of the solution to 10.0 mL with the mobile phase. E. R = CHOH-CH3 : (8S,10S)-6,8,10,11-tetrahydroxy-8-[(1RS)-
Reference solution (d). Dilute 1.0 mL of reference solution (a) 1-hydroxyethyl]-1-methoxy-7,8,9,10-tetrahydrotetracene-
to 200.0 mL with the mobile phase. 5,12-dione (13-dihydrodaunorubicinone),

General Notices (1) apply to all monographs and other texts 1993
Decyl oleate EUROPEAN PHARMACOPOEIA 8.0

07/2013:0896

DEFEROXAMINE MESILATE
Deferoxamini mesilas

B. R = CHOH-CH3 : (8S,10S)-10-[(3-amino-2,3,6-trideoxy-
α-L-lyxo-hexopyranosyl)oxy]-6,8,11-trihydroxy-8-[(1RS)-
1-hydroxyethyl]-1-methoxy-7,8,9,10-tetrahydrotetracene-
5,12-dione (daunorubicinol),
C26H52N6O11S Mr 657
C. R = CH2-CO-CH3 : (8S,10S)-10-[(3-amino-2,3,6-trideoxy- [138-14-7]
α-L-lyxo-hexopyranosyl)oxy]-6,8,11-trihydroxy-1- DEFINITION
methoxy-8-(2-oxopropyl)-7,8,9,10-tetrahydrotetracene-
5,12-dione (feudomycin B), N′-[5-[[4-[[5-(Acetylhydroxyamino)pentyl]amino]-4-
oxobutanoyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N-
hydroxybutanediamide methanesulfonate.
D. R = CO-CH2-OH : doxorubicin,
Content : 98.0 per cent to 102.0 per cent (anhydrous substance).
F. R = CO-CH2-CH3 : (8S,10S)-10-[(3-amino-2,3,6-trideoxy-α- PRODUCTION
L-lyxo-hexopyranosyl)oxy]-6,8,11-trihydroxy-1-methoxy- It is considered that alkylsulfonate esters are genotoxic and
8-propanoyl-7,8,9,10-tetrahydrotetracene-5,12-dione are potential impurities in deferoxamine mesilate. The
(8-ethyldaunorubicin). manufacturing process should be developed taking into
consideration the principles of quality risk management,
together with considerations of the quality of starting
materials, process capability and validation. The general
01/2008:1307 methods 2.5.37. Methyl, ethyl and isopropyl methanesulfonate
in methanesulfonic acid, 2.5.38. Methyl, ethyl and
isopropyl methanesulfonate in active substances and 2.5.39.
DECYL OLEATE Methanesulfonyl chloride in methanesulfonic acid are available
to assist manufacturers.
Decylis oleas CHARACTERS
Appearance : white or almost white powder.
DEFINITION
Solubility : freely soluble in water, slightly soluble in methanol,
Mixture consisting of decyl esters of fatty acids, mainly oleic very slightly soluble in ethanol (96 per cent).
(cis-9-octadecenoic) acid.
IDENTIFICATION
A suitable antioxidant may be added.
First identification : A, D.
CHARACTERS Second identification : B, C, D.
Appearance : clear, pale yellow or colourless liquid. A. Infrared absorption spectrophotometry (2.2.24).
Solubility : practically insoluble in water, miscible with Preparation : discs.
ethanol (96 per cent), with methylene chloride and with light Comparison: deferoxamine mesilate CRS.
petroleum (bp : 40-60 °C). If the spectra obtained show differences, dissolve the
substance to be examined and the reference substance
IDENTIFICATION separately in ethanol (96 per cent) R, evaporate to dryness
A. Relative density (see Tests). and record new spectra using the residues.
B. Dissolve about 5 mg in 5 mL of water R. Add 2 mL of a 5 g/L
B. Saponification value (see Tests). solution of trisodium phosphate dodecahydrate R and 0.5 mL
C. Oleic acid (see Tests). of a 25 g/L solution of sodium naphthoquinonesulfonate R.
A brownish-black colour develops.
TESTS C. Solution A obtained in the assay is brownish-red. To
Relative density (2.2.5) : 0.860 to 0.870. 10 mL of solution A add 3 mL of ether R and shake. The
organic layer is colourless. To 10 mL of solution A add
Acid value (2.5.1) : maximum 1.0, determined on 10.0 g.
3 mL of benzyl alcohol R and shake. The organic layer is
Iodine value (2.5.4, Method A) : 55 to 70. brownish-red.
Peroxide value (2.5.5, Method A) : maximum 10.0. D. Dissolve 0.1 g in 5 mL of dilute hydrochloric acid R. Add
1 mL of barium chloride solution R2. The solution is clear.
Saponification value (2.5.6) : 130 to 140, determined on 2.0 g.
In a porcelain crucible, mix 0.1 g with 1 g of anhydrous
Oleic acid (2.4.22, Method A) : minimum 60.0 per cent in the sodium carbonate R, heat and ignite over a naked flame.
fatty acid fraction of the substance. Allow to cool. Dissolve the residue in 10 mL of water R,
Water (2.5.12) : maximum 1.0 per cent, determined on 1.00 g. heating if necessary, and filter. The filtrate gives reaction (a)
of sulfates (2.3.1).
Total ash (2.4.16) : maximum 0.1 per cent, determined on
2.0 g. TESTS
Solution S. Dissolve 2.5 g in carbon dioxide-free water R
STORAGE prepared from distilled water R and dilute to 25 mL with the
Protected from light. same solvent.

1994 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dembrexine hydrochloride monohydrate for veterinary use

Appearance of solution. Solution S is clear (2.2.1) and not STORAGE

more intensely coloured than reference solution Y5 (2.2.2, Protected from light. If the substance is sterile, store in a
Method II). sterile, airtight, tamper-proof container.
pH (2.2.3): 3.7 to 5.5 for freshly prepared solution S.
IMPURITIES
Related substances. Liquid chromatography (2.2.29). Prepare
Specified impurities : A.
the solutions immediately before use, protected from light.
Other detectable impurities (the following substances would,
Test solution. Dissolve 50.0 mg of the substance to be if present at a sufficient level, be detected by one or other of
examined in the mobile phase and dilute to 50.0 mL with the the tests in the monograph. They are limited by the general
mobile phase. acceptance criterion for other/unspecified impurities and/or
Reference solution (a). Dissolve 10.0 mg of deferoxamine by the general monograph Substances for pharmaceutical
mesilate CRS in the mobile phase and dilute to 10.0 mL with use (2034). It is therefore not necessary to identify these
the mobile phase. impurities for demonstration of compliance. See also 5.10.
Reference solution (b). Dilute 1.0 mL of the test solution to Control of impurities in substances for pharmaceutical use) : B.
25.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (10 μm).
Mobile phase : dissolve 1.32 g of ammonium phosphate R and
0.37 g of sodium edetate R in 950 mL of water R ; adjust to
pH 2.8 with phosphoric acid R (about 3-4 mL) and add 55 mL A. N′-[5-[[4-[[4-(acetylhydroxyamino)butyl]amino]-4-
of tetrahydrofuran R. oxobutanoyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-
Flow rate : 2 mL/min. N-hydroxybutanediamide (desferrioxamine A1),
Detection : spectrophotometer at 220 nm. B. other desferrioxamines.
Injection : 20 μL.
Run time : 3 times the retention time of deferoxamine. 01/2008:2169
System suitability: reference solution (a) :
DEMBREXINE HYDROCHLORIDE
– resolution : minimum 1.0 between the peak with a relative
retention time of about 0.8 and the principal peak. MONOHYDRATE FOR VETERINARY
Limits : USE
– impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (b) Dembrexini hydrochloridum
(4.0 per cent) ; monohydricum ad usum veterinarium
– total : not more than 1.75 times the area of the principal
peak in the chromatogram obtained with reference
solution (b) (7.0 per cent) ;
– disregard limit : 0.02 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.08 per cent).
Chlorides (2.4.4): maximum 330 ppm. C13H18Br2ClNO2,H2O Mr 433.6
Dilute 2 mL of solution S to 20 mL with water R. [52702-51-9]
Sulfates (2.4.13) : maximum 400 ppm. DEFINITION
Dilute 5 mL of solution S to 20 mL with distilled water R. trans-4-[(3,5-Dibromo-2-hydroxybenzyl)amino]cyclohexanol
Heavy metals (2.4.8) : maximum 10 ppm. hydrochloride monohydrate.
2.0 g complies with test C. Prepare the reference solution using Content : 98.0 per cent to 101.0 per cent (anhydrous substance).
2 mL of lead standard solution (10 ppm Pb) R. CHARACTERS
Water (2.5.12) : maximum 2.0 per cent, determined on 1.000 g. Appearance : white or almost white, crystalline powder.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Solubility : slightly soluble in water, freely soluble in methanol,
1.0 g. slightly soluble in anhydrous ethanol.
Bacterial endotoxins (2.6.14) : less than 0.025 IU/mg, if IDENTIFICATION
intended for use in the manufacture of parenteral preparations A. Infrared absorption spectrophotometry (2.2.24).
without a further appropriate procedure for the removal of
bacterial endotoxins. Comparison: dembrexine hydrochloride monohydrate CRS.
B. It gives reaction (a) of chlorides (2.3.1).
ASSAY
TESTS
Dissolve 0.500 g in 25 mL of water R. Add 4 mL of
0.05 M sulfuric acid. Titrate with 0.1 M ferric ammonium Related substances. Liquid chromatography (2.2.29). Prepare
sulfate. Towards the end of the titration, titrate uniformly the solutions immediately before use.
and at a rate of about 0.2 mL/min. Determine the end-point Test solution. Dissolve 25.0 mg of the substance to be
potentiometrically (2.2.20) using a platinum indicator examined in methanol R and dilute to 10.0 mL with the same
electrode and a calomel reference electrode. Retain the titrated solvent.
solution (solution A) for identification test C. Reference solution (a). Dilute 1.0 mL of the test solution to
1 mL of 0.1 M ferric ammonium sulfate is equivalent to 50.0 mL with methanol R. Dilute 1.0 mL of this solution to
65.68 mg of C26H52N6O11S. 10.0 mL with methanol R.

General Notices (1) apply to all monographs and other texts 1995
Demeclocycline hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Reference solution (b). Dissolve 2.5 mg of tribromophenol R IMPURITIES

(impurity E) in methanol R and dilute to 50.0 mL with the Specified impurities : A, B.
same solvent. To 1.0 mL of this solution add 1.0 mL of the test Other detectable impurities (the following substances would,
solution and dilute to 10.0 mL with methanol R. if present at a sufficient level, be detected by one or other of
Blank solution. Methanol R. the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
Column :
by the general monograph Substances for pharmaceutical use
– size : l = 0.15 m, Ø = 4.0 mm ; (2034). It is therefore not necessary to identify these impurities
– stationary phase : end-capped octadecylsilyl silica gel for for demonstration of compliance. See also 5.10. Control of
chromatography R (5 μm) ; impurities in substances for pharmaceutical use) : C, D, E.
– temperature : 40 °C.
Mobile phase :
– mobile phase A : dissolve 1.0 g of potassium dihydrogen
phosphate R in 900 mL of water R, adjust to pH 7.4 with
0.5 M potassium hydroxide and dilute to 1000 mL with
water R ; mix 80 volumes of this solution with 20 volumes A. trans-4-[(3,5-dibromo-2-hydroxybenzylidene)amino]-
of methanol R ; cyclohexanol,
– mobile phase B : methanol R, acetonitrile R (20:80 V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-7 75 25

7 - 15 75 → 50 25 → 50
B. cis-4-[(3,5-dibromo-2-hydroxybenzyl)amino]cyclohexanol,
15 - 20 50 50

20 - 25 50 → 75 50 → 25

25 - 30 75 25

Flow rate : 1.0 mL/min. C. R1 = CHO, R2 = R3 = Br : 3,5-dibromo-2-

Detection : spectrophotometer at 250 nm. hydroxybenzaldehyde,
Injection : 10 μL. D. R1 = CHO, R2 = R3 = H : 2-hydroxybenzaldehyde
(salicylaldehyde),
Relative retention with reference to dembrexine
(retention time = about 6 min) : impurity A = about 2.3 ; E. R1 = R2 = R3 = Br : 2,4,6-tribromophenol.
impurity B = about 1.3.
System suitability : reference solution (b) : 01/2008:0176
– resolution : minimum 2 between the peaks due to DEMECLOCYCLINE
dembrexine and impurity E.
Limits :
HYDROCHLORIDE
– impurities A, B : for each impurity, not more than the area Demeclocyclini hydrochloridum
of the principal peak in the chromatogram obtained with
reference solution (a) (0.2 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.2 per cent) ;
– total : not more than 2.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ; C21H22Cl2N2O8 Mr 501.3
– disregard limit : 0.5 times the area of the principal peak in [64-73-3]
the chromatogram obtained with reference solution (a) DEFINITION
(0.1 per cent) ; disregard any peak due to the blank.
(4S,4aS,5aS,6S,12aS)-7-Chloro-4-(dimethylamino)-
Water (2.5.12) : 3.5 per cent to 5.0 per cent, determined on 3,6,10,12,12a-pentahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-
0.500 g. octahydrotetracene-2-carboxamide hydrochloride.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Substance produced by certain strains of Streptomyces
1.0 g. aureofaciens or obtained by any other means.
Content : 89.5 per cent to 102.0 per cent (anhydrous substance).
ASSAY
CHARACTERS
Dissolve 0.350 g in 40 mL of methanol R. Add 40 mL of Appearance : yellow powder.
acetone R and 1 mL of 0.1 M hydrochloric acid. Carry out
a potentiometric titration (2.2.20) using 0.1 M sodium Solubility : soluble or sparingly soluble in water, slightly
hydroxide. Read the volume added between the 2 points of soluble in alcohol, very slightly soluble in acetone. It dissolves
inflexion. in solutions of alkali hydroxides and carbonates.
1 mL of 0.1 M sodium hydroxide is equivalent to 41.56 mg IDENTIFICATION
of C13H18Br2ClNO2. A. Thin-layer chromatography (2.2.27).

1996 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Demeclocycline hydrochloride

Test solution. Dissolve 5 mg of the substance to be – stationary phase : styrene-divinylbenzene copolymer R

examined in methanol R and dilute to 10 mL with the same (8 μm),
solvent. – temperature : 60 °C,
Reference solution (a). Dissolve 5 mg of demeclocycline Mobile phase : weigh 80.0 g of 2-methyl-2-propanol R and
hydrochloride CRS in methanol R and dilute to 10 mL with transfer to a 1000 mL volumetric flask with the aid of 200 mL
the same solvent. of water R ; add 100 mL of a 35 g/L solution of dipotassium
Reference solution (b). Dissolve 5 mg of demeclocycline hydrogen phosphate R adjusted to pH 9.0 with dilute phosphoric
hydrochloride CRS, 5 mg of chlortetracycline hydrochloride R acid R, 150 mL of a 10 g/L solution of tetrabutylammonium
and 5 mg of tetracycline hydrochloride R in methanol R and hydrogen sulfate R adjusted to pH 9.0 with dilute sodium
dilute to 10 mL with the same solvent. hydroxide solution R and 10 mL of a 40 g/L solution of sodium
Plate : TLC octadecylsilyl silica gel F254 plate R. edetate R adjusted to pH 9.0 with dilute sodium hydroxide
solution R ; dilute to 1000 mL with water R.
Mobile phase : mix 20 volumes of acetonitrile R, 20 volumes
of methanol R and 60 volumes of a 63 g/L solution of Flow rate : 1 mL/min.
oxalic acid R previously adjusted to pH 2 with concentrated Detection : spectrophotometer at 254 nm.
ammonia R. Injection : 20 μL ; inject the test solution and reference
Application : 1 μL. solutions (c) and (d).
Development : over 3/4 of the plate. System suitability : reference solution (c) :
Drying : in air. – resolution : minimum of 2.8 between the peaks due to
impurity B (1st peak) and demeclocycline (2nd peak) ; if
Detection : examine in ultraviolet light at 254 nm.
necessary, adjust the 2-methyl-2-propanol content of the
System suitability : the chromatogram obtained with mobile phase or lower the pH of the mobile phase,
reference solution (b) shows 3 clearly separated spots. – symmetry factor : maximum 1.25 for the peak due to
Results : the principal spot in the chromatogram obtained demeclocycline.
with the test solution is similar in position and size to Limits :
the principal spot in the chromatogram obtained with
reference solution (a). – any impurity : not more than the area of the principal peak
in the chromatogram obtained with reference solution (d)
B. To about 2 mg add 5 mL of sulfuric acid R. A violet colour (5.0 per cent), and not more than 1 such peak has an area
develops. Add the solution to 2.5 mL of water R. The greater than 0.8 times the area of the principal peak in
colour becomes yellow. the chromatogram obtained with reference solution (d)
C. It gives reaction (a) of chlorides (2.3.1). (4.0 per cent),
– total : not more than twice the area of the principal peak
TESTS in the chromatogram obtained with reference solution (d)
pH (2.2.3) : 2.0 to 3.0. (10.0 per cent),
Dissolve 0.1 g in carbon dioxide-free water R and dilute to – disregard limit : 0.02 times the area of the principal peak
10 mL with the same solvent. in the chromatogram obtained with reference solution (d)
(0.1 per cent).
Specific optical rotation (2.2.7) : − 248 to − 263 (anhydrous
substance). Heavy metals (2.4.8) : maximum 50 ppm.
Dissolve 0.250 g in 0.1 M hydrochloric acid and dilute to 0.5 g complies with test C. Prepare the reference solution using
25.0 mL with the same acid. 2.5 mL of lead standard solution (10 ppm Pb) R.
Specific absorbance (2.2.25) : 340 to 370 determined at the Water (2.5.12) : maximum 3.0 per cent, determined on 1.000 g.
maximum at 385 nm (anhydrous substance). Sulfated ash (2.4.14) : maximum 0.5 per cent, determined on
Dissolve 10.0 mg in 0.01 M hydrochloric acid and dilute to 1.0 g.
100.0 mL with the same acid. To 10.0 mL of the solution add
12 mL of dilute sodium hydroxide solution R and dilute to ASSAY
100.0 mL with water R. Liquid chromatography (2.2.29) as described in the test for
Related substances. Liquid chromatography (2.2.29). Prepare related substances with the following modification.
the solutions immediately before use. Injection : test solution and reference solution (a).
Test solution. Dissolve 25.0 mg of the substance to be Calculate the percentage content of C21H22Cl2N2O8.
examined in 0.01 M hydrochloric acid and dilute to 25.0 mL
with the same acid. STORAGE
Reference solution (a). Dissolve 25.0 mg of demeclocycline Protected from light.
hydrochloride CRS in 0.01 M hydrochloric acid and dilute to
25.0 mL with the same acid.
IMPURITIES
Reference solution (b). Dissolve 5.0 mg of 4-epidemeclocycline
hydrochloride CRS in 0.01 M hydrochloric acid and dilute to
25.0 mL with the same acid.
Reference solution (c). Mix 1.0 mL of reference solution (a)
and 5.0 mL of reference solution (b) and dilute to 25.0 mL
with 0.01 M hydrochloric acid.
Reference solution (d). Dilute 5.0 mL of reference solution (a)
to 100.0 mL with 0.01 M hydrochloric acid.
A. (4S,4aS,5aS,6S,12aS)-4-(dimethylamino)-3,6,10,12,12a-
Column : pentahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-
– size : l = 0.25 m, Ø = 4.6 mm, octahydrotetracene-2-carboxamide (demethyltetracycline),

General Notices (1) apply to all monographs and other texts 1997
Deptropine citrate EUROPEAN PHARMACOPOEIA 8.0

Related substances. Examine by thin-layer chromatography

(2.2.27), using as the coating substance a suitable silica gel with
a fluorescent indicator having an optimal intensity at 254 nm.
Test solution (a). Dissolve 0.10 g of the substance to be
examined in methanol R and dilute to 10 mL with the same
solvent.
B. (4R,4aS,5aS,6S,12aS)-7-chloro-4-(dimethyl- Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
amino)-3,6,10,12,12a-pentahydroxy-1,11-di- with methanol R.
oxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa- Reference solution (a). Dilute 1.0 mL of test solution (a) to
mide (4-epidemeclocycline).
100.0 mL with methanol R.
Reference solution (b). Dissolve 20 mg of deptropine citrate CRS
in methanol R and dilute to 2 mL with the same solvent. Dilute
01/2008:1308 1 mL of the solution to 10 mL with methanol R.
corrected 6.0 Reference solution (c). Dissolve 5 mg of tropine CRS in
methanol R and dilute to 100.0 mL with the same solvent.
DEPTROPINE CITRATE Reference solution (d). Dissolve 10 mg of deptropine
citrate CRS and 10 mg of tropine CRS in methanol R and dilute
to 25 mL with the same solvent.
Deptropini citras
Apply to the plate 40 μL of each solution. Develop over a
path of 10 cm using a mixture of 8 volumes of concentrated
ammonia R and 92 volumes of butanol R. Dry the plate
at 100 °C to 105 °C until the ammonia has completely
evaporated. Examine in ultraviolet light at 254 nm. Any spot
in the chromatogram obtained with test solution (a), apart
from the principal spot, is not more intense than the spot in
the chromatogram obtained with reference solution (a) (1 per
cent). Spray with dilute potassium iodobismuthate solution R
C29H35NO8 Mr 525.6 and then with a 10 g/L solution of sodium nitrite R. Expose the
[2169-75-7] plate to iodine vapours. Examine in daylight and in ultraviolet
DEFINITION light at 254 nm. In the chromatogram obtained with test
solution (a) : any spot corresponding to tropine is not more
Deptropine citrate contains not less than 98.0 per cent intense than the spot in the chromatogram obtained with
and not more than the equivalent of 101.0 per cent of reference solution (c) (0.5 per cent) ; any spot, apart from the
(1R,3r,5S)-3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5- principal spot and any spot corresponding to tropine, is not
yloxy)-8-methyl-8-azabicyclo[3.2.1]octane dihydrogen citrate, more intense than the spot in the chromatogram obtained
calculated with reference to the dried substance. with reference solution (a) (1 per cent). The test is not valid
unless the chromatogram obtained with reference solution (d)
CHARACTERS shows two clearly separated spots.
A white or almost white, microcrystalline powder, very Heavy metals (2.4.8). 1.0 g complies with test C for heavy
slightly soluble in water and in ethanol, practically insoluble metals (20 ppm). Prepare the reference solution using 2 mL
in methylene chloride. of lead standard solution (10 ppm Pb) R.
It melts at about 170 °C, with decomposition. Loss on drying (2.2.32). Not more than 2.0 per cent,
IDENTIFICATION determined on 1.000 g by drying in an oven at 105 °C for 4 h.
First identification : A. Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
on 1.0 g.
Second identification : B, C, D, E.
A. Examine by infrared absorption spectrophotometry ASSAY
(2.2.24), comparing with the spectrum obtained with
deptropine citrate CRS. Dissolve 0.400 g in 50 mL of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point
B. Examine the chromatograms obtained in the test for related potentiometrically (2.2.20).
substances. The principal spot in the chromatogram
obtained with test solution (b) is similar in position, 1 mL of 0.1 M perchloric acid is equivalent to 52.56 mg of
colour and size to the principal spot in the chromatogram C29H35NO8.
obtained with reference solution (b).
C. To about 1 mg add 0.5 mL of sulfuric acid R. A stable STORAGE
red-orange colour develops. Store protected from light.
D. Dissolve about 1 mg in 0.25 mL of perchloric acid R and
warm gently until the solution becomes turbid. Add 5 mL IMPURITIES
of glacial acetic acid R ; a pink colour with an intense green
fluorescence appears.
E. To about 5 mg add 1 mL of acetic anhydride R and 5 mL of
pyridine R. A purple colour develops.

TESTS
pH (2.2.3). Suspend 0.25 g in carbon dioxide-free water R,
dilute to 25 mL with the same solvent and filter. The pH of A. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-ol
the solution is 3.7 to 4.5. (tropine),

1998 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dequalinium chloride

Absorbance ratios:
– A240/A326 = 1.56 to 1.80 ;
– A326/A336 = 1.12 to 1.30.
B. Infrared absorption spectrophotometry (2.2.24).
Spectral range : 600-2000 cm− 1.
Comparison: dequalinium chloride CRS.
B. (1R,3s,5S)-3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen- C. To 5 mL of solution S (see Tests) add 5 mL of potassium
5-yloxy)-8-methyl-8-azabicyclo[3.2.1]octane ferricyanide solution R. A yellow precipitate is formed.
(pseudodeptropine),
D. To 10 mL of solution S add 1 mL of dilute nitric acid R. A
white precipitate is formed. Filter and reserve the filtrate
for identification test E.
E. The filtrate from identification test D gives reaction (a) of
chlorides (2.3.1).
TESTS
Solution S. Dissolve 0.2 g in 90 mL of carbon dioxide-free
C. 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ol water R, heating if necessary, and dilute to 100 mL with the
(dibenzocycloheptadienol), same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Acidity or alkalinity. To 5 mL of solution S add 0.1 mL
of bromothymol blue solution R1. Not more than 0.2 mL
of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is
required to change the colour of the indicator.
D. (1R,3r,5S)-3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen- Related substances. Liquid chromatography (2.2.29).
5-yloxy)-8-azabicyclo[3.2.1]octane (demethyldeptropine). Test solution. Dissolve 10.0 mg of the substance to be
examined in the mobile phase and dilute to 10.0 mL with the
mobile phase.
01/2008:1413 Reference solution (a). Dissolve 10.0 mg of dequalinium
corrected 6.0 chloride for performance test CRS in the mobile phase and
dilute to 10.0 mL with the mobile phase.
DEQUALINIUM CHLORIDE Reference solution (b). Dissolve 10.0 mg of dequalinium
chloride CRS in the mobile phase and dilute to 10.0 mL with
Dequalinii chloridum the mobile phase. Dilute 1.0 mL of the solution to 50.0 mL
with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R.
Mobile phase : dissolve 2 g of sodium hexanesulfonate R in
300 mL of water R ; adjust to pH 4.0 with acetic acid R and add
700 mL of methanol R.
Flow rate : 1.5 mL/min.
C30H40Cl2N4 Mr 527.6
[522-51-0] Detection : spectrophotometer at 240 nm.
Injection : 10 μL.
DEFINITION
Run time : 5 times the retention time of dequalinium chloride.
1,1′-(Decane-1,10-diyl)bis(4-amino-2-methylquinolinium) System suitability : reference solution (a) :
dichloride (dried substance).
– peak-to-valley ratio : minimum 2.0, where Hp = height above
Content : 95.0 per cent to 101.0 per cent. the baseline of the peak due to impurity B and Hv = height
CHARACTERS above the baseline of the lowest point of the curve
separating this peak from the peak due to dequalinium
Appearance : white or yellowish-white powder, hygroscopic. chloride. If necessary, adjust the concentration of methanol
Solubility : slightly soluble in water and in ethanol (96 per in the mobile phase.
cent). Limits :
IDENTIFICATION – impurity A : not more than 0.5 times the area of the
First identification : B, E. principal peak in the chromatogram obtained with
reference solution (b) (1 per cent) ;
Second identification : A, C, D, E.
– total of impurities other than A : not more than 5 times the
A. Ultraviolet and visible absorption spectrophotometry area of the principal peak in the chromatogram obtained
(2.2.25). with reference solution (b) (10 per cent) ;
Test solution. Dissolve about 10 mg in water R and dilute to – disregard limit : 0.025 times the area of the principal peak
100 mL with the same solvent. Dilute 10 mL of the solution in the chromatogram obtained with reference solution (b)
to 100 mL with water R. (0.05 per cent).
Spectral range : 230-350 nm. Readily carbonisable substances. Dissolve 20 mg in 2 mL of
Absorption maxima : at 240 nm and 326 nm. sulfuric acid R. After 5 min the solution is not more intensely
Shoulder : at 336 nm. coloured than reference solution BY4 (2.2.2, Method I).

General Notices (1) apply to all monographs and other texts 1999
3-O-Desacyl-4′-monophosphoryl lipid A EUROPEAN PHARMACOPOEIA 8.0

Loss on drying (2.2.32) : maximum 7.0 per cent, determined activities of the parent LPS. It consists of a mixture of
on 1.000 g by drying at 105 °C at a pressure not exceeding congeners, all containing a backbone of β1′→6-linked
0.7 kPa. disaccharide of 2-deoxy-2-aminoglucose phosphorylated at
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on the 4′-position, but differing in the fatty acid substitutions at
1.0 g. the 2, 2′ and 3′ positions. The immunostimulatory activities
of 3-O-desacyl-4′-monophosphoryl lipid A combined
ASSAY with the vaccine include up-regulation of co-stimulatory
In order to avoid overheating in the reaction medium, mix molecules on antigen-presenting cells and secretion of
thoroughly throughout and stop the titration immediately after pro-inflammatory cytokines, resulting in an enhanced
the end-point has been reached. immune response of the Th1-type against the antigens.
3-O-desacyl-4′-monophosphoryl lipid A is a lyophilised
Dissolve 0.200 g in 5 mL of anhydrous formic acid R and add powder or a sterile liquid.
50 mL of acetic anhydride R. Titrate with 0.1 M perchloric
acid, determining the end-point potentiometrically (2.2.20). Requirements given in the sections up to and including the
section Triethylamine salt of 3-O-desacyl-4′-monophosphoryl
1 mL of 0.1 M perchloric acid is equivalent to 26.38 mg of lipid A also apply to formulations that do not proceed to the
C30H40Cl2N4. 3-O-desacyl-4′-monophosphoryl lipid A liquid bulk.
STORAGE
PRODUCTION
In an airtight container.
GENERAL PROVISIONS
IMPURITIES The production method shall have been shown to yield
Specified impurities : A. consistently a 3-O-desacyl-4′-monophosphoryl lipid A
comparable in structure and function with a preparation of
Other detectable impurities (the following substances would,
3-O-desacyl-4′-monophosphoryl lipid A used as adjuvant in
if present at a sufficient level, be detected by one or other of
the particular vaccine of proven clinical efficacy and safety
the tests in the monograph. They are limited by the general
in man.
acceptance criterion for other/unspecified impurities and/or
During development studies, and wherever revalidation is
by the general monograph Substances for pharmaceutical use
necessary, a test for residual endotoxin activity is carried out
(2034). It is therefore not necessary to identify these impurities
by injecting intravenously 12-day-old embryonated hens’
for demonstration of compliance. See also 5.10. Control of
eggs with 0.1 mL of dilutions of the test sample (8 eggs
impurities in substances for pharmaceutical use) : B, C.
per dilution) of 3-O-desacyl-4′-monophosphoryl lipid A.
Eggs are candled and read for mortality at 18-24 hours
post-inoculation and the chick embryo 50 per cent lethal dose
(CELD50) is calculated. The residual endotoxin activity of the
3-O-desacyl-4′-monophosphoryl lipid A is acceptable if the
CELD50 is more than 100 μg.
An endotoxin standard of Salmonella typhimurium is prepared
A. 2-methylquinolin-4-amine, and selected dilutions are injected into each group of 8 eggs.
For a test to be valid, the CELD50 of the endotoxin standard
must not be more than 0.05 μg.
Reference preparation : a batch of 3-O-desacyl-4′-
monophosphoryl lipid A shown to be comparable
in structure and function with a preparation of
3-O-desacyl-4′-monophosphoryl lipid A used as adjuvant in
the particular vaccine of proven clinical efficacy and safety in
B. 4-amino-1-[10-[(2-methylquinolin-4-yl)amino]decyl]-2- man or a batch representative thereof.
methylquinolinium chloride,
BACTERIAL SEED LOTS
The bacterial strain used for master seed lots shall be identified
by historical records that include information on its origin and
the tests used to characterise the strain, in particular genotypic
and phenotypic information. Only a working seed lot that
complies with the following requirements may be used.
Identification. The working seed lot is identified by suitable
methods such as Gram staining and fatty acid profiling (5.1.6).
Microbial Purity. Each seed lot complies with the
C. 1-[10-(4-amino-2-methylquinolinio)decyl]-4-[[10- requirements for absence of contaminating organisms.
(4-amino-2-methylquinolinio)decyl]amino]-2- Purity of bacterial cultures is verified by methods of suitable
methylquinolinium trichloride. sensitivity and specificity.
07/2011:2537 PROPAGATION AND HARVEST
The bacteria is grown using a suitable liquid medium. At the
3-O-DESACYL-4′-MONOPHOSPHORYL end of cultivation, the culture is tested for purity and yield.
The culture medium is separated from the bacterial mass by a
LIPID A suitable method, for example filtration. Only a harvest that is
consistent with respect to the profiles for growth rate, pH, and
Adeps A 3-O-desacyl-4′- O2-consumption may be used for the extraction of LPS.
monophosphorylatus TRIETHYLAMINE SALT OF 3-O-DESACYL-4′-
MONOPHOSPHORYL LIPID A
DEFINITION LPS is extracted from the bacterial cells by successive
3-O-Desacyl-4′-monophosphoryl lipid A is a detoxified alcohol and chloroform-methanol extractions and is then
derivative of the lipopolysaccharide (LPS) of Salmonella converted to 3-O-desacyl-4′-monophosphoryl lipid A by
minnesota, strain R595, which retains the immunostimulatory hydrolysis, then purified and salified by triethanolamine

2000 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 3-O-Desacyl-4′-monophosphoryl lipid A

before freeze-drying. The freeze-dried triethylamine salt of The liquid bulk is sterilised by filtration through a
3-O-desacyl-4′-monophosphoryl lipid A must comply with bacteria-retentive filter.
the following requirements.
Only a 3-O-desacyl-4′-monophosphoryl lipid A liquid bulk
Appearance. A visual description of the particular that complies with the requirements given below under
preparation after freeze-drying is established and approved Identification, Tests and Assay and that is within the limits
by the competent authority ; each batch of freeze-dried approved for the particular product may be used for the
triethylamine salt of 3-O-desacyl-4′-monophosphoryl lipid A preparation of 3-O-desacyl-4′-monophosphoryl lipid A in the
must comply with this description. final lots.
Protein : less than 0.5 per cent m/m, determined using a
suitable method, for example a reversed-phase HPLC method CHARACTERS
for amino acid analysis (2.2.56). The total amino acid content When dispersed in an aqueous solution : slightly turbid
in micrograms is calculated by comparison to amino acid suspension.
standards and is equal to the protein concentration.
Nucleic acid : maximum 0.3 per cent m/m, determined using When dissolved in an organic solvent : a description of its
a suitable method. For example, a fluorimetric method may be appearance is established and approved by the competent
used where nucleic acids are extracted from the freeze-dried authority ; the 3-O-desacyl-4′-monophosphoryl lipid A liquid
triethylamine salt of 3-O-desacyl-4′-monophosphoryl lipid A, bulk complies with this description.
using a solution containing NH4OH and a suitable non-ionic
detergent, and stained by a suitable fluorescent dye. The IDENTIFICATION
nucleic acid content in the test sample is interpolated from a Congener distribution (see Tests).
calibration curve.
Hexosamine (2.5.20) : 1000 nmol/mg to 1450 nmol/mg. TESTS
Phosphorus (2.5.18) : 0.5 μmol/mg to 0.8 μmol/mg. Particle size. Where applicable, the particle size in the
Congener distribution. The relative amount of tetraacyl, microfluidised liquid bulk is determined by a suitable method,
pentaacyl, hexaacyl and heptaacyl congener groups are for example dynamic light scattering. The particle size for
determined by a suitable method, for example reversed-phase each batch of liquid bulk is within the limits approved for the
HPLC analysis (2.2.29). particular product.
The relative amount of each congener group in the Sterility (2.6.1). It complies with the test, carried out using
triethylamine salt of 3-O-desacyl-4′-monophosphoryl lipid A 10 mL for each medium.
is : Congener distribution. The relative amount of tetraacyl,
– tetraacyl : 15 per cent to 35 per cent ; pentaacyl, hexaacyl and heptaacyl congener groups are
– pentaacyl : 35 per cent to 60 per cent ; determined by a suitable method, for example reversed-phase
– hexaacyl : 20 per cent to 40 per cent ; HPLC analysis (2.2.29).
– heptaacyl : less than 0.5 per cent. The relative amount of each congener group in the
3-O-desacyl-4′-monophosphoryl lipid A liquid bulk is :
Triethylamine : 4.2 to 5.8 per cent m/m, determined by a
suitable method, for example gas chromatography (2.2.28). – tetraacyl : 15 per cent to 35 per cent ;
Water (2.5.12) : maximum 6.7 per cent m/m. – pentaacyl : 35 per cent to 60 per cent ;
Free fatty acids : maximum 2.6 per cent m/m, determined – hexaacyl : 20 per cent to 40 per cent ;
by a suitable method, for example reversed-phase HPLC
analysis (2.2.29). – heptaacyl : less than 0.5 per cent.
2-Keto-3-deoxyoctonate : less than 0.5 per cent m/m,
ASSAY
determined by a suitable method. For example, a colorimetric
method may be used where 2-keto-3-deoxyoctonate is The 3-O-desacyl-4′-monophosphoryl lipid A content
released by hydrolysis (0.2 N H2SO4 at 100 °C for 30 min), is determined by a suitable method, for example gas
oxidised by periodic acid, and reacted with sodium arsenite chromatographic quantification (2.2.28) of trifluoroacetic
to yield β-formylpyruvic acid, which subsequently is coupled anhydride derivatised fatty acid methyl esters of the
to thiobarbituric acid to give a red coloured chromophore 3-O-desacyl-4′-monophosphoryl lipid A fatty acids
with absorption maximum at 550 nm. The amount of dodecanoic acid (C12:0), tetradecanoic acid (C14:0),
2-keto-3-deoxyoctonate is interpolated from a calibration 3-hydroxy tetradecanoic acid (3-OH-C14:0) and
curve. hexadecanoic acid (C16:0) obtained by hydrolysis
Identity. The test for congener distribution also serves to of 3-O-desacyl-4′-monophosphoryl lipid A in an
identify the product. aqueous/methanol (50:50 V/V) solution, containing 5 per cent
of sodium hydroxide. To the test sample, a reference sample
Microbial contamination and the dilutions of the calibration curve, pentadecanoic acid
TAMC : acceptance criterion 101 CFU/10 mg (2.6.12). (C15:0) is added as an internal standard. The temperature
gradient applied must allow the separation of the fatty acid
Pyrogens (2.6.8). The triethylamine salt of 3-O-desacyl-4′-
methyl esters in about 40 min.
monophosphoryl lipid A complies with the test for pyrogens.
Inject into each rabbit per kilogram of body mass 3 mL of a The sum of the ratios between the area for each individual
solution containing 2.5 μg of 3-O-desacyl-4′-monophosphoryl fatty acid methyl ester (C12:0, C14:0, 3-OH-C14:0 and C16:0)
lipid A. and the area of the internal standard (ratio = area Cx / area
3-O-DESACYL-4′-MONOPHOSPHORYL LIPID A LIQUID C15:0) is calculated. The 3-O-desacyl-4′-monophosphoryl
BULK lipid A quantity corresponding to the sum ratio value on
the calibration curve, established with the dilutions of the
The triethylamine salt of 3-O-desacyl-4′-monophosphoryl
3-O-desacyl-4′-monophosphoryl lipid A standard, is reported.
lipid A is dispersed in a liquid suitable for the subsequent
processing steps at a defined target concentration. If the salt The content of 3-O-desacyl-4′-monophosphoryl lipid A is not
is not soluble in water a microfluidisation step is necessary to less than 80 per cent and not greater than 120 per cent of the
prepare a stable aqueous suspension. estimated content.

General Notices (1) apply to all monographs and other texts 2001
Desflurane EUROPEAN PHARMACOPOEIA 8.0

04/2008:1666 Detection : flame ionisation.

corrected 7.0 Injection : 2.0 μL.
Run time : 35 min.
DESFLURANE Relative retention with reference to desflurane (retention
time = about 11.5 min) : impurity C = about 1.06 ;
Desfluranum impurity D = about 1.09 ; impurity A = about 1.14 ;
impurity G = about 1.39 ; impurity E = about 1.5 ;
impurity B = about 1.7 ; impurity F = about 2.2 ;
impurity H = about 2.6.
System suitability : reference solution (a) :
C 3H 2F 6O Mr 168.0
– number of theoretical plates : minimum 20 000, calculated
[57041-67-5]
for the peak due to impurity A ;
DEFINITION – symmetry factor : maximum 2.0 for the peak due to
(2RS)-2-(Difluoromethoxy)-1,1,1,2-tetrafluoroethane. impurity B.
Limits :
CHARACTERS – impurity B : not more than the difference between the
Appearance : clear, colourless, mobile, heavy liquid. area of the corresponding peak in the chromatogram
Solubility : practically insoluble in water, miscible with obtained with reference solution (a) and the area of the
anhydrous ethanol. corresponding peak in the chromatogram obtained with
Relative density : 1.47, determined at 15 °C. the test solution (0.2 per cent V/V) ;
bp : about 22 °C. – impurity A : not more than the difference between the
area of the corresponding peak in the chromatogram
IDENTIFICATION obtained with reference solution (a) and the area of the
Infrared absorption spectrophotometry (2.2.24). corresponding peak in the chromatogram obtained with
Preparation : examine the substance in the gaseous state. the test solution (0.1 per cent V/V) ;
– impurities C, D, G : for each impurity, not more than the
Comparison : Ph. Eur. reference spectrum of desflurane.
difference between the area of the peak due to impurity A in
TESTS the chromatogram obtained with reference solution (b) and
the area of the peak due to impurity A in the chromatogram
The substance to be examined must be cooled to a temperature obtained with the test solution (0.01 per cent V/V) ;
below 10 °C and the tests must be carried out at a temperature
below 20 °C. – impurities E, H : for each impurity, not more than the
difference between the area of the corresponding peak in
Acidity or alkalinity. To 20 mL add 20 mL of carbon the chromatogram obtained with reference solution (a) and
dioxide-free water R, shake for 3 min and allow to stand. the area of the corresponding peak in the chromatogram
Collect the upper layer and add 0.2 mL of bromocresol purple obtained with the test solution (0.01 per cent V/V) ;
solution R. Not more than 0.1 mL of 0.01 M sodium hydroxide
– impurity F : not more than the difference between the
or 0.6 mL of 0.01 M hydrochloric acid is required to change
area of the corresponding peak in the chromatogram
the colour of the indicator.
obtained with reference solution (a) and the area of the
Related substances. Gas chromatography (2.2.28). corresponding peak in the chromatogram obtained with
Test solution. The substance to be examined. the test solution (0.002 per cent V/V) ;
Reference solution (a). Introduce 25 mL of the substance – unspecified impurities : for each impurity, not more than
to be examined into a 50 mL flask fitted with a septum, 0.5 times the difference between the area of the peak due to
and add 0.50 mL of desflurane impurity A CRS and 1.0 mL impurity A in the chromatogram obtained with reference
of isoflurane CRS (impurity B). Add 50 μL of acetone R solution (b) and the area of the peak due to impurity A
(impurity H), 10 μL of chloroform R (impurity F) and 50 μL in the chromatogram obtained with the test solution
of methylene chloride R (impurity E) to the solution, using an (0.005 per cent V/V) ;
airtight syringe, and dilute to 50.0 mL with the substance to be – sum of impurities other than A, B, C, D, E, F, G and H : not
examined. Dilute 5.0 mL of this solution to 50.0 mL with the more than the difference between the area of the peak due
substance to be examined. Store at a temperature below 10 °C. to impurity A in the chromatogram obtained with reference
Reference solution (b). Dilute 5.0 mL of reference solution (a) solution (b) and the area of the peak due to impurity A in
to 50.0 mL with the substance to be examined. Store at a the chromatogram obtained with the test solution (0.01 per
temperature below 10 °C. cent V/V) ;
Reference solution (c). Dilute 5.0 mL of reference solution (b) – disregard limit : the difference between the area of the
to 25.0 mL with the substance to be examined. Store at a peak due to impurity A in the chromatogram obtained
temperature below 10 °C. with reference solution (c) and the area of the peak due to
Column : impurity A in the chromatogram obtained with the test
solution (0.002 per cent V/V).
– material : fused silica ;
Fluorides : maximum 10 ppm.
– size : l = 105 m, Ø = 0.32 mm ;
Potentiometry (2.2.36, Method I).
– stationary phase : poly[methyl(trifluoropropylmethyl)silox-
ane] R (film thickness 1.5 μm). Test solution. To 10.0 mL in a separating funnel, add 10 mL
of a mixture of 30.0 mL of dilute ammonia R2 and 70.0 mL
Carrier gas : helium for chromatography R. of distilled water R. Shake for 1 min and collect the upper
Flow rate : 2.0 mL/min. layer. Repeat this extraction procedure twice, collecting the
Split ratio : 1:25. upper layer each time. Adjust the combined upper layers to
Temperature : pH 5.2 with dilute hydrochloric acid R. Add 5.0 mL of fluoride
standard solution (1 ppm F) R and dilute to 50.0 mL with
– column : 30 °C ;
distilled water R. To 20.0 mL of this solution add 20.0 mL of
– injection port : 150 °C ; total-ionic-strength-adjustment buffer R and dilute to 50.0 mL
– detector : 200 °C. with distilled water R.

2002 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Desipramine hydrochloride

Reference solutions. To each of 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL 01/2008:0481
and 5.0 mL of fluoride standard solution (10 ppm F) R add corrected 6.0
20.0 mL of total-ionic-strength-adjustment buffer R and dilute
to 50.0 mL with distilled water R. DESIPRAMINE HYDROCHLORIDE
Indicator electrode : fluoride selective.
Reference electrode : silver-silver chloride. Desipramini hydrochloridum
Carry out the measurements on 20 mL of each solution.
Calculate the concentration of fluorides using the calibration
curve, taking into account the addition of fluoride to the test
solution.
Antimony : maximum 3 ppm.
Atomic absorption spectrometry (2.2.23, Method I).
Solvent mixture : hydrochloric acid R, nitric acid R (50:50 V/V). C18H23ClN2 Mr 302.8
Test solution. Transfer 10 g, cooled to below 10 °C, to a tared [58-28-6]
flask containing 20 mL of water R cooled to below 5 °C. Add
1 mL of the solvent mixture and leave at room temperature DEFINITION
until the desflurane has evaporated completely. Subsequently, Desipramine hydrochloride contains not less than 99.0 per
reduce the volume to about 8 mL on a hot plate. Cool to room cent and not more than the equivalent of 101.0 per cent
temperature and transfer to a volumetric flask. Add 1 mL of of 3-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N-
the solvent mixture and adjust to 10.0 mL with water R. methylpropan-1-amine hydrochloride, calculated with
reference to the dried substance.
Reference solutions. To each of 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL
and 5.0 mL of antimony standard solution (100 ppm Sb) R add CHARACTERS
20 mL of the solvent mixture and dilute to 100.0 mL with A white or almost white, crystalline powder, soluble in water
water R. and in alcohol.
Source : antimony hollow-cathode lamp using a transmission It melts at about 214 °C.
band of 0.2 nm and a 75 per cent lamp current.
Wavelength : 217.6 nm. IDENTIFICATION
Atomisation device : air-acetylene flame. First identification : B, E.
Second identification : A, C, D, E.
Non-volatile matter : maximum 100 mg/L.
A. Dissolve 40.0 mg in 0.01 M hydrochloric acid and dilute to
Evaporate 20.0 mL to dryness with the aid of a stream of 100.0 mL with the same acid. Dilute 5.0 mL of the solution
nitrogen R. The residue weighs not more than 2.0 mg. to 100.0 mL with 0.01 M hydrochloric acid. Examined
STORAGE between 230 nm and 350 nm (2.2.25), the solution shows an
absorption maximum at 251 nm and a shoulder at 270 nm.
In a glass bottle fitted with a polyethylene-lined cap. Before The specific absorbance at the maximum is 255 to 285.
opening the bottle, cool the contents to below 10 °C.
B. Examine by infrared absorption spectrophotometry
IMPURITIES (2.2.24), comparing with the spectrum obtained with
Specified impurities : A, B, C, D, E, F, G, H. desipramine hydrochloride CRS.
C. Examine the chromatograms obtained in the test for
related substances. The principal spot in the chromatogram
obtained with test solution (b) is similar in position,
colour and size to the principal spot in the chromatogram
A. 1,1′-oxybis(1,2,2,2-tetrafluoroethane), obtained with reference solution (a).
D. Dissolve about 50 mg in 3 mL of water R and add 0.05 mL
of a 25 g/L solution of quinhydrone R in methanol R. An
intense pink colour develops within about 15 min.
E. To 0.5 mL of solution S (see Tests) add 1.5 mL of water R.
B. (2RS)-2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane The solution gives reaction (a) of chlorides (2.3.1).
(isofluorane),
TESTS
Solution S. Dissolve 1.25 g in carbon dioxide-free water R,
warming to not more than 30 °C if necessary, and dilute to
25 mL with the same solvent.
C. R = H, R′ = F : dichlorofluoromethane,
Appearance of solution. Solution S, examined immediately
D. R = Cl, R′ = F : trichlorofluoromethane, after preparation, is not more intensely coloured than
reference solution BY6 (2.2.2, Method II).
E. R = R′ = H : dichloromethane (methylene chloride),
Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of
F. R = H, R′ = Cl : trichloromethane (chloroform), methyl red solution R and 0.3 mL of 0.01 M sodium hydroxide.
The solution is yellow. Not more than 0.5 mL of 0.01 M
hydrochloric acid is required to change the colour of the
indicator to red.
Related substances. Carry out the test protected from bright
G. 1,1,2-trichloro-1,2,2-trifluoroethane, light. Examine by thin-layer chromatography (2.2.27), using a
TLC silica gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be
examined in a mixture of equal volumes of ethanol R and
methylene chloride R and dilute to 10 mL with the same
H. propanone (acetone). mixture of solvents. Prepare immediately before use.

General Notices (1) apply to all monographs and other texts 2003
Deslanoside EUROPEAN PHARMACOPOEIA 8.0

Test solution (b). Dilute 1 mL of test solution (a) to 10 mL DEFINITION

with a mixture of equal volumes of ethanol R and methylene Deslanoside contains not less than 95.0 per cent and
chloride R. not more than the equivalent of 105.0 per cent of
Reference solution (a). Dissolve 25 mg of desipramine 3β-[(O-β-D-glucopyranosyl-(1→4)-O-2,6-dideoxy-β-D-ribo-
hydrochloride CRS in a mixture of equal volumes of ethanol R hexopyranosyl-(1→4)-O-2,6-dideoxy-β-D-ribo-hexopyranosyl-
and methylene chloride R and dilute to 25 mL with the same (1→4)-2,6-dideoxy-β-D-ribo-hexopyranosyl)oxy]-12β,14-
mixture of solvents. Prepare immediately before use. dihydroxy-5β,14β-card-20(22)-enolide, calculated with
Reference solution (b). Dilute 1 mL of reference solution (a) reference to the dried substance.
to 50 mL with a mixture of equal volumes of ethanol R and CHARACTERS
methylene chloride R.
A white or almost white, crystalline or finely crystalline
Apply to the plate 5 μL of each solution. Develop over a path powder, hygroscopic, practically insoluble in water, very
of 7 cm using a mixture of 1 volume of water R, 10 volumes of slightly soluble in alcohol. In an atmosphere of low relative
anhydrous acetic acid R and 10 volumes of toluene R. Dry the humidity, it loses water.
plate in a current of air for 10 min, spray with a 5 g/L solution
of potassium dichromate R in a mixture of 1 volume of sulfuricIDENTIFICATION
acid R and 4 volumes of water R and examine immediately. First identification : A.
Any spot in the chromatogram obtained with test solution (a),
apart from the principal spot, is not more intense than the Second identification : B, C, D.
spot in the chromatogram obtained with reference solution (b) A. Examine by infrared absorption spectrophotometry
(0.2 per cent). (2.2.24), comparing with the spectrum obtained with
deslanoside CRS. When comparing the spectra, special
Heavy metals (2.4.8). 2.0 g complies with test C for heavy attention is given to the absence of a distinct absorption
metals (20 ppm). Prepare the reference solution using 4 mL maximum at about 1260 cm–1 and to the intensity of
of lead standard solution (10 ppm Pb) R. the absorption maximum at about 1740 cm–1. Examine
Loss on drying (2.2.32). Not more than 0.5 per cent, the substances in discs prepared by dissolving 1 mg of
determined on 1.000 g by drying in an oven at 105 °C. the substance to be examined or 1 mg of the reference
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined substance in 0.3 mL of methanol R and triturating with
on 1.0 g. about 0.4 g of dry, finely powdered potassium bromide R
until the mixture is uniform and completely dry.
ASSAY B. Examine the chromatograms obtained in the test for related
Dissolve 0.2500 g in a mixture of 5 mL of 0.01 M hydrochloric substances. The principal zone in the chromatogram
acid and 50 mL of alcohol R. Carry out a potentiometric obtained with test solution (b) is similar in position,
titration (2.2.20), using 0.1 M sodium hydroxide. Read colour and size to the principal zone in the chromatogram
the volume added between the two points of inflexion. obtained with reference solution (a).
1 mL of 0.1 M sodium hydroxide is equivalent to 30.28 mg C. Suspend about 0.5 mg in 0.2 mL of alcohol (60 per
of C18H23ClN2. cent V/V) R. Add 0.1 mL of dinitrobenzoic acid solution R
and 0.1 mL of dilute sodium hydroxide solution R. A violet
STORAGE colour develops.
Store protected from light. D. Dissolve about 5 mg in 5 mL of glacial acetic acid R and
add 0.05 mL of ferric chloride solution R1. Cautiously add
2 mL of sulfuric acid R, avoiding mixing the two liquids.
Allow to stand ; a brown but not reddish ring develops
01/2008:0482 at the interface and a greenish-yellow, then bluish-green
corrected 6.0 colour diffuses from it to the upper layer.
TESTS
DESLANOSIDE Solution S. Dissolve 0.20 g in a mixture of equal volumes of
chloroform R and methanol R and dilute to 10 mL with the
Deslanosidum same mixture of solvents.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Specific optical rotation (2.2.7). Dissolve 0.200 g in
anhydrous pyridine R and dilute to 10.0 mL with the same
solvent. The specific optical rotation is + 6.5 to + 8.5,
calculated with reference to the dried substance.
Related substances. Examine by thin-layer chromatography
(2.2.27), using silica gel G R as the coating substance.
Test solution (a). Use solution S.
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL with
a mixture of equal volumes of chloroform R and methanol R.
Reference solution (a). Dissolve 20 mg of deslanoside CRS in a
mixture of equal volumes of chloroform R and methanol R and
dilute to 10 mL with the same mixture of solvents.
Reference solution (b). Dilute 2.5 mL of reference solution (a)
to 10 mL with a mixture of equal volumes of chloroform R
and methanol R.
Reference solution (c). Dilute 1 mL of reference solution (a)
C47H74O19 Mr 943 to 10 mL with a mixture of equal volumes of chloroform R
[17598-65-1] and methanol R.

2004 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Desloratadine

Apply separately to the plate as 10 mm bands 5 μL of each If the spectra obtained in the solid state show differences,
solution. Develop immediately over a path of 15 cm using a dissolve the substance to be examined and the reference
mixture of 3 volumes of water R, 36 volumes of methanol R substance separately in methyl isobutyl ketone R, evaporate to
and 130 volumes of methylene chloride R. Dry the plate in dryness and record new spectra using the residues.
a current of warm air, spray with a mixture of 5 volumes
of sulfuric acid R and 95 volumes of alcohol R and heat at TESTS
140 °C for 15 min. Examine in daylight. In the chromatogram Related substances. Liquid chromatography (2.2.29).
obtained with test solution (a), any zone, apart from the Test solution. Dissolve 20.0 mg of the substance to be
principal zone, is not more intense than the zone in the examined in the mobile phase and dilute to 25.0 mL with the
chromatogram obtained with reference solution (b) (2.5 per mobile phase. Dilute 5.0 mL of the solution to 50.0 mL with
cent) and at most two such zones are more intense than the the mobile phase.
zone in the chromatogram obtained with reference solution (c) Reference solution (a). Dissolve 20.0 mg of desloratadine CRS
(1.0 per cent). in the mobile phase and dilute to 25.0 mL with the mobile
Loss on drying (2.2.32). Not more than 5.0 per cent, phase. Dilute 5.0 mL of the solution to 50.0 mL with the
determined on 0.500 g by drying in vacuo at 105 °C. mobile phase.
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined Reference solution (b). Dilute 1.0 mL of the test solution to
on the residue obtained in the test for loss on drying. 100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10.0 mL with the mobile phase.
ASSAY Reference solution (c). Dissolve 4 mg of desloratadine for
Dissolve 50.0 mg in alcohol R and dilute to 50.0 mL with the system suitability CRS (containing impurities A and B) in the
same solvent. Dilute 5.0 mL of this solution to 100.0 mL with mobile phase and dilute to 5.0 mL with the mobile phase.
alcohol R. Prepare a reference solution in the same manner, Dilute 1.0 mL of the solution to 10.0 mL with the mobile phase.
using 50.0 mg of deslanoside CRS (undried). To 5.0 mL of Column :
each solution add 3.0 mL of alkaline sodium picrate solution R
and allow to stand protected from bright light in a water-bath – size : l = 0.25 m, Ø = 4.6 mm ;
at 20 ± 1 °C for 40 min. Measure the absorbance (2.2.25) – stationary phase : end-capped octadecylsilyl silica gel for
of each solution at the maximum at 484 nm, using as the chromatography R (4 μm) ;
compensation liquid a mixture of 3.0 mL of alkaline sodium – temperature : 35 °C.
picrate solution R and 5.0 mL of alcohol R prepared at the Mobile phase : dissolve 0.865 g of sodium dodecyl sulfate R in
same time. water R, add 0.5 mL of trifluoroacetic acid R and dilute to
Calculate the content of C47H74O19 from the absorbances 1000 mL with water R; mix 57 volumes of this solution and
measured and the concentrations of the solutions. 43 volumes of acetonitrile R.
Flow rate : 1.0 mL/min.
STORAGE
Detection : spectrophotometer at 280 nm.
Store in an airtight, glass container, protected from light, at a
Injection : 100 μL of the test solution and reference solutions (b)
temperature below 10 °C.
and (c).
Run time : 2.5 times the retention time of desloratadine.
Identification of impurities : use the chromatogram supplied
01/2014:2570 with desloratadine for system suitability CRS and the
chromatogram obtained with reference solution (c) to identify
DESLORATADINE the peaks due to impurities A and B.
Relative retention with reference to desloratadine
Desloratadinum (retention time = about 21 min) : impurity A = about 0.8 ;
impurity B = about 0.9.
System suitability : reference solution (c) :
– resolution : minimum 2.0 between the peaks due to
impurity B and desloratadine.
Calculation of percentage contents :
– correction factors : multiply the peak areas of the following
impurities by the corresponding correction factor :
impurity A = 1.6 ; impurity B = 1.6 ;
C19H19ClN2 Mr 310.8
[100643-71-8] – for each impurity, use the concentration of desloratadine in
reference solution (b).
DEFINITION Limits :
8-Chloro-11-(piperidin-4-ylidene)-6,11-dihydro-5H- – impurity B : maximum 0.3 per cent ;
benzo[5,6]cyclohepta[1,2-b]pyridine. – impurity A : maximum 0.2 per cent ;
Content : 98.0 per cent to 102.0 per cent (anhydrous substance). – unspecified impurities : for each impurity, maximum
0.10 per cent ;
CHARACTERS – total : maximum 0.4 per cent ;
Appearance : white or almost white powder. – reporting threshold : 0.05 per cent.
Solubility : very slightly soluble or pratically insoluble in water, Heavy metals (2.4.8) : maximum 20 ppm.
freely soluble in ethanol (96 per cent), slightly soluble or very
slightly soluble in heptane. Solvent : methanol R.
It shows polymorphism (5.9). 0.250 g complies with test H. Prepare the reference solution
using 0.5 mL of lead standard solution (10 ppm Pb) R.
IDENTIFICATION Water (2.5.32) : maximum 0.5 per cent, determined on 0.250 g.
Infrared absorption spectrophotometry (2.2.24). Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
Comparison : desloratadine CRS. 0.5 g.

General Notices (1) apply to all monographs and other texts 2005
Desmopressin EUROPEAN PHARMACOPOEIA 8.0

ASSAY CHARACTERS
Liquid chromatography (2.2.29) as described in the test for Appearance : white or almost white, fluffy powder.
related substances with the following modifications. Solubility : soluble in water, in ethanol (96 per cent) and in
Injection : test solution and reference solution (a). glacial acetic acid.
System suitability: reference solution (a) : IDENTIFICATION
– symmetry factor : 0.5 to 1.5 for the peak due to desloratadine. A. Examine the chromatograms obtained in the assay.
Calculate the percentage content of C19H19ClN2 taking into Results : the retention time and size of the principal peak
account the assigned content of desloratadine CRS. in the chromatogram obtained with the test solution are
IMPURITIES approximately the same as those of the principal peak in
the chromatogram obtained with the reference solution.
Specified impurities : A, B.
B. Amino acid analysis (2.2.56). For hydrolysis use Method 1
Other detectable impurities (the following substances would, and for analysis use Method 1.
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general Express the content of each amino acid in moles. Calculate
acceptance criterion for other/unspecified impurities and/or the relative proportions of the amino acids, taking 1/6 of
by the general monograph Substances for pharmaceutical the sum of the number of moles of aspartic acid, glutamic
use (2034). It is therefore not necessary to identify these acid, proline, glycine, arginine and phenylalanine as
impurities for demonstration of compliance. See also 5.10. equal to 1. The values fall within the following limits :
Control of impurities in substances for pharmaceutical use): C. aspartic acid : 0.90 to 1.10 ; glutamic acid : 0.90 to 1.10 ;
proline : 0.90 to 1.10 ; glycine : 0.90 to 1.10 ; arginine : 0.90
to 1.10 ; phenylalanine : 0.90 to 1.10 ; tyrosine : 0.70 to 1.05 ;
half-cystine : 0.30 to 1.05. Lysine, isoleucine and leucine
are absent ; not more than traces of other amino acids are
present.
TESTS
Specific optical rotation (2.2.7) : − 72 to − 82 (anhydrous and
A. (11RS)-8-chloro-11-fluoro-11-(piperidin-4-yl)-6,11- acetic acid-free substance).
dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridine, Dissolve 10.0 mg in a 1 per cent V/V solution of glacial acetic
acid R and dilute to 5.0 mL with the same acid.
Related substances. Liquid chromatography (2.2.29) : use the
normalisation procedure.
Test solution. Dissolve 1.0 mg of the substance to be examined
in 2.0 mL of water R.
Resolution solution. Dissolve the contents of a vial of
oxytocin/desmopressin validation mixture CRS in 500 μL of
B. (11RS)-8-chloro-11-(1,2,3,6-tetrahydropyridin-4-yl)-6,11- water R.
dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridine, Column :
– size : l = 0.12 m, Ø = 4.0 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase :
– mobile phase A : 0.067 M phosphate buffer solution pH 7.0 R ;
filter and degas ;
– mobile phase B : acetonitrile for chromatography R, mobile
C. ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta- phase A (50:50 V/V) ; filter and degas.
[1,2-b]pyridin-11-ylidene)piperidine-1-carboxylate
(loratadine). Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-4 76 24
07/2009:0712
4 - 18 76 → 58 24 → 42
DESMOPRESSIN 18 - 35 58 → 48 42 → 52

Desmopressinum 35 - 40 48 → 76 52 → 24

40 - 50 76 24

Flow rate : 1.5 mL/min.

Detection : spectrophotometer at 220 nm.
Injection : 50 μL.
C46H64N14O12S2 Mr 1069 Retention time : desmopressin = about 16 min ; oxytocin = about
[16679-58-6] 17 min.
DEFINITION System suitability : resolution solution :
(3-Sulfanylpropanoyl)-L-tyrosyl-L-phenylalanyl-L-glutaminyl- – resolution : minimum 1.5 between the peaks due to
L-asparaginyl-L-cysteinyl-L-prolyl-D-arginylglycinamide cyclic desmopressin and oxytoxin.
(1→6)-disulfide. Limits :
Synthetic cyclic nonapeptide, available as an acetate. – unspecified impurities : for each impurity, maximum 0.5 per
Content : 95.0 per cent to 105.0 per cent (anhydrous and acetic cent ;
acid-free substance). – total : maximum 1.5 per cent ;

2006 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Desogestrel

– disregard limit : 0.05 per cent. 01/2008:1717

Acetic acid (2.5.34) : 3.0 per cent to 8.0 per cent.
Test solution. Dissolve 20.0 mg of the substance to be DESOGESTREL
examined in a mixture of 5 volumes of mobile phase B and
95 volumes of mobile phase A and dilute to 10.0 mL with the Desogestrelum
same mixture of mobile phases.
Water (2.5.32) : maximum 6.0 per cent, determined on
20.0 mg.
Bacterial endotoxins (2.6.14) : less than 500 IU/mg, if
intended for use in the manufacture of parenteral preparations
without a further appropriate procedure for the removal of
bacterial endotoxins.
ASSAY C22H30O Mr 310.5
Liquid chromatography (2.2.29) as described in the test for [54024-22-5]
related substances with the following modifications. DEFINITION
Reference solution. Dissolve the contents of a vial of 13-Ethyl-11-methylidene-18,19-dinor-17α-pregn-4-en-20-
desmopressin CRS in water R to obtain a concentration of yn-17-ol.
0.5 mg/mL.
Content : 98.0 per cent to 102.0 per cent (dried substance).
Mobile phase : mobile phase B, mobile phase A (40:60 V/V).
Flow rate : 2.0 mL/min. CHARACTERS
Retention time : desmopressin = about 5 min. Appearance : white or almost white, crystalline powder.
Calculate the content of desmopressin (C46H64N14O12S2) from Solubility : practically insoluble in water, very soluble in
the declared content of C46H64N14O12S2 in desmopressin CRS. methanol, freely soluble in anhydrous ethanol and in
methylene chloride.
STORAGE
IDENTIFICATION
In an airtight container, protected from light, at a temperature
of 2 °C to 8 °C. If the substance is sterile, store in a sterile, A. Infrared absorption spectrophotometry (2.2.24).
airtight, tamper-proof container. Comparison: desogestrel CRS.
B. Specific optical rotation (see Tests).
LABELLING
The label states : TESTS
– the mass of peptide per container ; Specific optical rotation (2.2.7) : + 53 to + 57 (dried
– where applicable, that the substance is suitable for use in substance).
the manufacture of parenteral preparations. Dissolve 0.250 g in anhydrous ethanol R and dilute to 25.0 mL
with the same solvent.
IMPURITIES
Related substances. Liquid chromatography (2.2.29).
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of Test solution. Dissolve 20.0 mg of the substance to be
the tests in the monograph. They are limited by the general examined in 25 mL of acetonitrile R1 and dilute to 50.0 mL
acceptance criterion for other/unspecified impurities and/or with water R.
by the general monograph Substances for pharmaceutical Reference solution (a). Dissolve 4 mg of desogestrel for system
use (2034). It is therefore not necessary to identify these suitability CRS (containing impurities A, B, C and D) in 5 mL
impurities for demonstration of compliance. See also 5.10. of acetonitrile R1 and dilute to 10.0 mL with water R.
Control of impurities in substances for pharmaceutical use) : Reference solution (b). Dilute 1.0 mL of the test solution to
A, B, C, D, E, F, G. 100.0 mL with a mixture of equal volumes of acetonitrile R1
and water R.
Reference solution (c). Dilute 1.0 mL of reference solution (b)
to 10.0 mL with a mixture of equal volumes of acetonitrile R1
and water R.
A. X = Gln, Y = Asp, Z = D-Arg : [5-L-aspartic acid]- Reference solution (d). Dissolve 20.0 mg of desogestrel CRS in
desmopressin, 25 mL of acetonitrile R1 and dilute to 50.0 mL with water R.
B. X = Glu, Y = Asn, Z = D-Arg : [4-L-glutamic acid]- Column :
desmopressin, – size : l = 0.25 m, Ø = 4.6 mm,
– stationary phase : sterically protected octadecylsilyl silica gel
D. X = Gln, Y = Asn, Z = L-Arg : [8-L-arginine]desmopressin, for chromatography R (5 μm),
– temperature : 50 °C.
Mobile phase : water R, acetonitrile R1 (27:73 V/V).
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 205 nm.
C. R = OH, R4 = R5 = H : [9-glycine]desmopressin, Injection : 15 μL of the test solution and reference solutions (a),
E. R = NH2, R4 = CH2-NH-CO-CH3, R5 = H : (b) and (c).
N5.4-[(acetylamino)methyl]desmopressin, Run time : 2.5 times the retention time of desogestrel.
F. R = NH2, R4 = H, R5 = CH2-NH-CO-CH3 : Identification of impurities : use the chromatogram
N4.5-[(acetylamino)methyl]desmopressin, supplied with desogestrel for system suitability CRS and the
chromatogram obtained with reference solution (a) to identify
G. R = N(CH3)2, R4 = R5 = H : N1.9,N1.9-dimethyldesmopressin. the peaks due to impurities A, B, C and D.

General Notices (1) apply to all monographs and other texts 2007
Desoxycortone acetate EUROPEAN PHARMACOPOEIA 8.0

Relative retention with reference to desogestrel (retention

time = about 22 min) : impurity E = about 0.2 ;
impurity D = about 0.25 ; impurity B = about 0.7 ;
impurity A = about 0.95 ; impurity C = about 1.05.
System suitability: reference solution (a) :
– peak-to-valley ratio : minimum 2.0, where Hp = height B. R1 = CH3, R2 = OH, R3 = C≡CH, R4 = R5 = H :
above the baseline of the peak due to impurity C and 11-methylidene-19-nor-17α-pregn-4-en-20-yn-17-ol,
Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to desogestrel. C. R1 = C2H5, R2 + R3 = O, R4 = R5 = H :
Limits : 13-ethyl-11-methylidenegon-4-en-17-one,

– correction factors : for the calculation of content, D. R1 = C2H5, R2 = OH, R3 = C≡CH, R4 + R5 = O :

multiply the peak area of the following impurities by 13-ethyl-17-hydroxy-11-methylidene-18,19-dinor-17α-
the corresponding correction factor : impurity A = 1.8, pregn-4-en-20-yn-3-one,
impurity D = 1.5 ;
– impurities A, B, C : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.2 per cent) ;
– impurity D : not more than the area of the principal peak
in the chromatogram obtained with reference solution (c)
(0.1 per cent) ;
E. 13-ethyl-11-methylidene-18,19-dinor-17α-pregn-4-en-20-
– unspecified impurities : for each impurity, not more than the
yne-3β,17-diol.
area of the principal peak in the chromatogram obtained
with reference solution (c) (0.10 per cent) ;
– total : not more than 0.5 times the area of the principal peak 04/2010:0322
in the chromatogram obtained with reference solution (b)
(0.5 per cent) ; DESOXYCORTONE ACETATE
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (c) Desoxycortoni acetas
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in vacuo at a pressure not exceeding
2 kPa.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.

ASSAY C23H32O4 Mr 372.5

[56-47-3]
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification. DEFINITION
Injection : test solution and reference solution (d). 3,20-Dioxopregn-4-en-21-yl acetate.
Calculate the percentage content of C22H30O from the areas of Content : 97.0 per cent to 103.0 per cent (dried substance).
the peaks and the declared content of desogestrel CRS.
CHARACTERS
IMPURITIES Appearance : white or almost white, crystalline powder or
colourless crystals.
Specified impurities : A, B, C, D. Solubility : practically insoluble in water, freely soluble in
Other detectable impurities (the following substances would, methylene chloride, soluble in acetone, sparingly soluble in
if present at a sufficient level, be detected by one or other of ethanol (96 per cent), slightly soluble in propylene glycol and
the tests in the monograph. They are limited by the general in fatty oils.
acceptance criterion for other/unspecified impurities and/or
IDENTIFICATION
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these First identification : B, C.
impurities for demonstration of compliance. See also 5.10. Second identification : A, C, D, E.
Control of impurities in substances for pharmaceutical use) : E. A. Melting point (2.2.14) : 157 °C to 161 °C.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: desoxycortone acetate CRS.
C. Thin-layer chromatography (2.2.27).
Solvent mixture : methanol R, methylene chloride R
(1:9 V/V).
Test solution. Dissolve 10 mg of the substance to be
examined in the solvent mixture and dilute to 10 mL with
the solvent mixture.
Reference solution (a). Dissolve 20 mg of desoxycortone
A. 13-ethyl-11-methylidene-18,19-dinor-5α,17α-pregn-3-en- acetate CRS in the solvent mixture and dilute to 20 mL
20-yn-17-ol (desogestrel Δ3-isomer), with the solvent mixture.

2008 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Detomidine hydrochloride for veterinary use

Reference solution (b). Dissolve 10 mg of cortisone acetate R Limits :

in reference solution (a) and dilute to 10 mL with reference – unspecified impurities : for each impurity, not more than
solution (a). 0.2 times the area of the principal peak in the chromatogram
Plate : TLC silica gel F254 plate R. obtained with reference solution (b) (0.10 per cent) ;
Mobile phase : add a mixture of 1.2 volumes of water R and – total : not more than the area of the principal peak in the
8 volumes of methanol R to a mixture of 15 volumes of chromatogram obtained with reference solution (b) (0.5 per
ether R and 77 volumes of methylene chloride R. cent) ;
Application : 5 μL. – disregard limit : 0.1 times the area of the principal peak in
Development : over 2/3 of the plate. the chromatogram obtained with reference solution (b)
Drying : in air. (0.05 per cent).
Detection A : examine in ultraviolet light at 254 nm. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Results A : the principal spot in the chromatogram obtained on 0.500 g by drying in an oven at 105 °C.
with the test solution is similar in position and size to
ASSAY
the principal spot in the chromatogram obtained with
reference solution (a). Dissolve 0.100 g in ethanol (96 per cent) R and dilute to
Detection B : spray with alcoholic solution of sulfuric acid R, 100.0 mL with the same solvent. Dilute 2.0 mL of this
heat at 120 °C for 10 min or until the spots appear, and solution to 100.0 mL with ethanol (96 per cent) R. Measure the
allow to cool ; examine in daylight and in ultraviolet light absorbance (2.2.25) at the absorption maximum at 240 nm.
at 365 nm. Calculate the content of C23H32O4 taking the specific
Results B : the principal spot in the chromatogram obtained absorbance to be 450.
with the test solution is similar in position, colour in STORAGE
daylight, fluorescence in ultraviolet light at 365 nm and
size to the principal spot in the chromatogram obtained Protected from light.
with reference solution (a).
System suitability: reference solution (b) :
– the chromatogram shows 2 clearly separated spots. 01/2008:1414
corrected 6.0
D. Add about 2 mg to 2 mL of sulfuric acid R and shake to
dissolve. Within 5 min, a yellow colour develops. Add
this solution to 2 mL of water R and mix. The resulting DETOMIDINE HYDROCHLORIDE FOR
solution is dichroic, showing an intense blue colour by VETERINARY USE
transparency, and red fluorescence that is particularly
intense in ultraviolet light at 365 nm.
E. About 10 mg gives the reaction of acetyl (2.3.1).
Detomidini hydrochloridum ad usum
veterinarium
TESTS
Specific optical rotation (2.2.7) : + 171 to + 179 (dried
substance).
Dissolve 0.250 g in dioxan R and dilute to 25.0 mL with the
same solvent.
Related substances. Liquid chromatography (2.2.29). C12H15ClN2 Mr 222.7
Test solution. Dissolve 25.0 mg of the substance to be [90038-01-0]
examined in the mobile phase and dilute to 10.0 mL with the DEFINITION
mobile phase.
4-(2,3-Dimethylbenzyl)-1H-imidazole hydrochloride.
Reference solution (a). Dissolve 2 mg of desoxycortone
acetate CRS and 2 mg of betamethasone 17-valerate CRS in the Content : 98.5 per cent to 101.5 per cent (dried substance).
mobile phase and dilute to 200.0 mL with the mobile phase. CHARACTERS
Reference solution (b). Dilute 1.0 mL of the test solution to Appearance : white or almost white, hygroscopic, crystalline
200.0 mL with the mobile phase. powder.
Column :
Solubility : soluble in water, freely soluble in ethanol (96 per
– size : l =0.25 m, Ø = 4.6 mm ; cent), very slightly soluble in methylene chloride, practically
– stationary phase : octadecylsilyl silica gel for insoluble in acetone.
chromatography R (5 μm). mp : about 160 °C.
Mobile phase : in a 1000 mL volumetric flask mix 350 mL of
water R with 600 mL of acetonitrile R and allow to equilibrate ; IDENTIFICATION
dilute to 1000 mL with water R and mix again. A. Infrared absorption spectrophotometry (2.2.24).
Flow rate : 1 mL/min. Preparation : discs.
Detection : spectrophotometer at 254 nm. Comparison: detomidine hydrochloride CRS.
Equilibration : with the mobile phase for about 30 min. If the spectra obtained show differences, dry the substance
Injection : 20 μL. to be examined and the reference substance separately in
Run time : 3 times the retention time of desoxycortone acetate. an oven at 100-105 °C and record new spectra.
Retention time : betamethasone 17-valerate = about 7.5 min ; B. It gives reaction (a) of chlorides (2.3.1).
desoxycortone acetate = about 9.5 min.
System suitability: reference solution (a) : TESTS
– resolution : minimum 4.5 between the peaks due to Appearance of solution. The solution is clear (2.2.1) and
betamethasone 17-valerate and desoxycortone acetate ; if colourless (2.2.2, Method II).
necessary, adjust the concentration of acetonitrile in the Dissolve 0.25 g in water R and dilute to 25 mL with the same
mobile phase. solvent.

General Notices (1) apply to all monographs and other texts 2009
Dexamethasone EUROPEAN PHARMACOPOEIA 8.0

Related substances. Liquid chromatography (2.2.29). (2034). It is therefore not necessary to identify these impurities
Test solution. Dissolve 25.0 mg of the substance to be for demonstration of compliance. See also 5.10. Control of
examined in 20 mL of the mobile phase and dilute to 50.0 mL impurities in substances for pharmaceutical use) : A, B.
with the mobile phase.
Reference solution (a). Dilute 0.20 mL of the test solution to
100.0 mL with the mobile phase.
Reference solution (b). Dissolve 1 mg of detomidine
impurity B CRS in the mobile phase and dilute to 100 mL with A. (RS)-(2,3-dimethylphenyl)(1H-imidazol-4-yl)methanol,
the mobile phase. Dilute 1 mL of this solution to 10 mL with
reference solution (a).
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : octylsilyl silica gel for chromatography R
(5 μm).
Mobile phase : acetonitrile R, 2.64 g/L solution of ammonium B. (RS)-(1-benzyl-1H-imidazol-5-yl)(2,3-dimethylphenyl)-
phosphate R (35:65 V/V). methanol,
Flow rate : 1 mL/min.
Detection : spectrophotometer at 220 nm.
Injection : 20 μL.
Run time : 4 times the retention time of detomidine. C. 4-[(2,3-dimethylcyclohexyl)methyl]-1H-imidazole.
Relative retention with reference to detomidine
(retention time = about 7 min) : impurity A = about 0.4 ; 01/2014:0388
impurity B = about 2.0 ; impurity C = about 3.0.
System suitability : reference solution (b) : DEXAMETHASONE
– resolution : minimum 5 between the peaks due to
detomidine and impurity B. Dexamethasonum
Limits :
– correction factor : multiply by 2.7 the area of any peak due
to impurity C and its diastereoisomer eluting with a relative
retention time of about 3 ;
– impurity C : for the sum of the areas of the peaks due to
impurity C and its diastereoisomer, not more than 2.5 times
the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.5 per cent) ; C22H29FO5 Mr 392.5
[50-02-2]
– any other impurity : for each impurity, not more than the
area of the principal peak in the chromatogram obtained DEFINITION
with reference solution (a) (0.2 per cent) ; 9-Fluoro-11β,17,21-trihydroxy-16α-methylpregna-1,4-diene-
– total : not more than 5 times the area of the principal peak 3,20-dione.
in the chromatogram obtained with reference solution (a) Content : 97.0 per cent to 103.0 per cent (dried substance).
(1 per cent) ;
– disregard limit : 0.25 times the area of the principal peak CHARACTERS
in the chromatogram obtained with reference solution (a) Appearance : white or almost white, crystalline powder.
(0.05 per cent). Solubility : practically insoluble in water, sparingly soluble in
Loss on drying (2.2.32) : maximum 0.5 per cent, determined anhydrous ethanol, slightly soluble in methylene chloride.
on 1.000 g by drying in oven at 105 °C. IDENTIFICATION
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on First identification : B, C.
1.0 g. Second identification : A, C, D, E.
ASSAY A. Dissolve 10.0 mg in anhydrous ethanol R and dilute
to 100.0 mL with the same solvent. Place 2.0 mL of
Dissolve 0.170 g in 50 mL of ethanol (96 per cent) R. Add this solution in a stoppered test tube, add 10.0 mL of
5.0 mL of 0.01 M hydrochloric acid. Carry out a potentiometric phenylhydrazine-sulfuric acid solution R, mix and heat in
titration (2.2.20), using 0.1 M sodium hydroxide. Read a water-bath at 60 °C for 20 min. Cool immediately. The
the volume added between the 2 points of inflexion.
absorbance (2.2.25) measured at the absorption maximum
1 mL of 0.1 M sodium hydroxide is equivalent to 22.27 mg at 419 nm is not less than 0.4.
of C12H15ClN2. B. Infrared absorption spectrophotometry (2.2.24).
STORAGE Comparison: dexamethasone CRS.
In an airtight container. C. Thin-layer chromatography (2.2.27).
Solvent mixture : methanol R, methylene chloride R
IMPURITIES (1:9 V/V).
Specified impurities : C. Test solution. Dissolve 10 mg of the substance to be
Other detectable impurities (the following substances would, examined in the solvent mixture and dilute to 10 mL with
if present at a sufficient level, be detected by one or other of the solvent mixture.
the tests in the monograph. They are limited by the general Reference solution (a). Dissolve 20 mg of
acceptance criterion for other/unspecified impurities and/or dexamethasone CRS in the solvent mixture and
by the general monograph Substances for pharmaceutical use dilute to 20 mL with the solvent mixture.

2010 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dexamethasone

Reference solution (b). Dissolve 10 mg of betamethasone CRS – stationary phase : end-capped octadecylsilyl silica gel for
in reference solution (a) and dilute to 10 mL with reference chromatography R (5 μm) ;
solution (a). – temperature : 45 °C.
Plate : TLC silica gel F254 plate R. Mobile phase :
Mobile phase : butanol R saturated with water R, toluene R, – mobile phase A : mix 250 mL of acetonitrile R with 700 mL
ether R (5:10:85 V/V/V). of water R and allow to equilibrate ; dilute to 1000.0 mL
Application : 5 μL. with water R and mix again ;
Development : over 2/3 of the plate. – mobile phase B : acetonitrile R ;
Drying : in air. Time Mobile phase A Mobile phase B
Detection A : examine in ultraviolet light at 254 nm. (min) (per cent V/V) (per cent V/V)
0 - 15 100 0
Results A : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to 15 - 40 100 → 0 0 → 100
the principal spot in the chromatogram obtained with
reference solution (a). Flow rate : 1.2 mL/min.
Detection : spectrophotometer at 254 nm.
Detection B : spray with alcoholic solution of sulfuric acid R.
Injection : 20 μL ; inject mobile phase A as a blank.
Heat at 120 °C for 10 min or until the spots appear. Allow to
Identification of impurities : use the chromatogram supplied
cool. Examine in daylight and in ultraviolet light at 365 nm.
Results B : the principal spot in the chromatogram obtainedwith dexamethasone for system suitability CRS and the
with the test solution is similar in position, colour in chromatogram obtained with reference solution (a) to identify
daylight, fluorescence in ultraviolet light at 365 nm and the peaks due to impurities B, F and G ; use the chromatogram
size to the principal spot in the chromatogram obtained supplied with dexamethasone for peak identification CRS and
with reference solution (a). the chromatogram obtained with reference solution (c) to
System suitability: reference solution (b) : identify the peaks due to impurities J and K.
– the chromatogram shows 2 spots which may, however, Relative retention with reference to dexamethasone
not be completely separated. (retention time = about 15 min) : impurity J = about 0.90 ;
impurity B = about 0.94 ; impurity K = about 1.3 ;
D. Add about 2 mg to 2 mL of sulfuric acid R and shake to impurity F = about 1.5 ; impurity G = about 1.7.
dissolve. Within 5 min, a faint reddish-brown colour
develops. Add this solution to 10 mL of water R and mix ; System suitability : reference solution (a) :
the colour is discharged. – peak-to-valley ratio : minimum 2.0, where Hp = height above
the baseline of the peak due to impurity B and Hv = height
E. Mix about 5 mg with 45 mg of heavy magnesium oxide R
above the baseline of the lowest point of the curve
and ignite in a crucible until an almost white residue is separating this peak from the peak due to dexamethasone.
obtained (usually less than 5 min). Allow to cool, add
1 mL of water R, 0.05 mL of phenolphthalein solution R1 Limits :
and about 1 mL of dilute hydrochloric acid R to render the – impurity G : not more than 3 times the area of the principal
solution colourless. Filter. To a freshly prepared mixture peak in the chromatogram obtained with reference
of 0.1 mL of alizarin S solution R and 0.1 mL of zirconyl solution (b) (0.3 per cent) ;
nitrate solution R, add 1.0 mL of the filtrate. Mix, allow – impurities B, F, J, K : for each impurity, not more than
to stand for 5 min and compare the colour of the solution 1.5 times the area of the principal peak in the chromatogram
with that of a blank prepared in the same manner. The test obtained with reference solution (b) (0.15 per cent) ;
solution is yellow and the blank solution is red. – unspecified impurities : for each impurity, not more than the
TESTS area of the principal peak in the chromatogram obtained
with reference solution (b) (0.10 per cent) ;
Specific optical rotation (2.2.7) : + 86 to + 92 (dried – total : not more than 5 times the area of the principal peak
substance). in the chromatogram obtained with reference solution (b)
Dissolve 0.250 g in anhydrous ethanol R and dilute to 25.0 mL (0.5 per cent) ;
with the same solvent. – disregard limit : 0.5 times the area of the principal peak in
Related substances. Liquid chromatography (2.2.29). Carry the chromatogram obtained with reference solution (b)
out the test protected from light. (0.05 per cent).
Test solution. Dissolve 25 mg of the substance to be examined Loss on drying (2.2.32) : maximum 0.5 per cent, determined
in 1.5 mL of acetonitrile R and add 5 mL of mobile phase A. on 0.500 g by drying in an oven at 105 °C.
Sonicate until dissolution is complete and dilute to 10.0 mL
with mobile phase A. ASSAY
Reference solution (a). Dissolve 5 mg of dexamethasone for Dissolve 0.100 g in ethanol (96 per cent) R and dilute to
system suitability CRS (containing impurities B, F and G) in 100.0 mL with the same solvent. Dilute 2.0 mL of this
0.5 mL of acetonitrile R and add 1 mL of mobile phase A. solution to 100.0 mL with ethanol (96 per cent) R. Measure the
Sonicate until dissolution is complete and dilute to 2.0 mL absorbance (2.2.25) at the absorption maximum at 238.5 nm.
with mobile phase A. Calculate the content of C22H29FO5 taking the specific
Reference solution (b). Dilute 1.0 mL of the test solution to absorbance to be 394.
100.0 mL with mobile phase A. Dilute 1.0 mL of this solution STORAGE
to 10.0 mL with mobile phase A.
Protected from light.
Reference solution (c). Dissolve 5 mg of dexamethasone for
peak identification CRS (containing impurities J and K) in IMPURITIES
0.5 mL of acetonitrile R and add 1 mL of mobile phase A. Specified impurities : B, F, G, J, K.
Sonicate until dissolution is complete and dilute to 2.0 mL
with mobile phase A. Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
Column : the tests in the monograph. They are limited by the general
– size : l = 0.15 m, Ø = 4.6 mm ; acceptance criterion for other/unspecified impurities and/or

General Notices (1) apply to all monographs and other texts 2011
Dexamethasone acetate EUROPEAN PHARMACOPOEIA 8.0

by the general monograph Substances for pharmaceutical

use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use): A,
C, D, E, H, I.

H. 17-hydroxy-16α-methyl-3,20-dioxopregna-1,4,9(11)-trien-
21-yl acetate,

A. 14-fluoro-11β,17,21-trihydroxy-16α-methylpregna-1,4-
diene-3,20-dione,

I. 9α,11α-epoxy-17,21-dihydroxy-16α-methylpregna-1,4-
diene-3,20-dione,

B. 9-fluoro-11β,17,21-trihydroxy-16β-methylpregna-1,4-
diene-3,20-dione (betamethasone),

J. 17,21-dihydroxy-16α-methylpregna-1,4-diene-3,11,20-
trione,

C. 9-fluoro-11β,17,21-trihydroxy-16α-methylpregn-4-ene-
3,20-dione,
K. 17,21-dihydroxy-16α-methylpregna-1,4,7,9(11)-tetraene-
3,20-dione.

04/2010:0548

DEXAMETHASONE ACETATE
D. 9β,11β-epoxy-17,21-dihydroxy-16α-methylpregna-1,4-
diene-3,20-dione, Dexamethasoni acetas

E. 17,21-dihydroxy-16α-methylpregna-1,4,9(11)-triene-3,20- C24H31FO6 Mr 434.5

dione, [1177-87-3]
DEFINITION
9-Fluoro-11β,17-dihydroxy-16α-methyl-3,20-dioxopregna-
1,4-dien-21-yl acetate.
Content : 97.0 per cent to 103.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
F. 9-fluoro-11β,21-dihydroxy-16α-methylpregna-1,4-diene- Solubility : practically insoluble in water, freely soluble in
3,20-dione, ethanol (96 per cent), slightly soluble in methylene chloride.
It shows polymorphism (5.9).
IDENTIFICATION
First identification : B, C.
Second identification : A, C, D, E, F.
A. Dissolve 10.0 mg in anhydrous ethanol R and dilute to
100.0 mL with the same solvent. Place 2.0 mL of this
G. 9-fluoro-11β,17-dihydroxy-16α-methyl-3,20-dioxopregna- solution in a ground-glass-stoppered tube, add 10.0 mL of
1,4-dien-21-yl acetate (dexamethasone acetate), phenylhydrazine-sulfuric acid solution R, mix and heat in

2012 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dexamethasone acetate

a water-bath at 60 °C for 20 min. Cool immediately. The Related substances. Liquid chromatography (2.2.29). Carry
absorbance (2.2.25) measured at the absorption maximum out the test protected from light.
at 419 nm is not less than 0.35. Test solution. Dissolve 25 mg of the substance to be examined
B. Infrared absorption spectrophotometry (2.2.24). in about 4 mL of acetonitrile R and dilute to 10.0 mL with
Comparison : dexamethasone acetate CRS. water R.
If the spectra obtained in the solid state show differences, Reference solution (a). Dissolve 2 mg of dexamethasone CRS
dissolve the substance to be examined and the reference (impurity A) and 2 mg of betamethasone acetate CRS
substance separately in methylene chloride R, evaporate to (impurity D) in 100.0 mL of the mobile phase and sonicate for
dryness and record new spectra using the residues. about 10 min (solution A). Mix 6.0 mL of the test solution and
1.0 mL of solution A and dilute to 10.0 mL with the mobile
C. Thin-layer chromatography (2.2.27). phase.
Solvent mixture : methanol R, methylene chloride R Reference solution (b). Dilute 1.0 mL of the test solution to
(1:9 V/V). 100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
Test solution. Dissolve 10 mg of the substance to be to 10.0 mL with the mobile phase.
examined in the solvent mixture and dilute to 10 mL with Reference solution (c). Dissolve the contents of a vial of
the solvent mixture. dexamethasone acetate impurity E CRS in 1.0 mL of the mobile
Reference solution (a). Dissolve 20 mg of dexamethasone phase.
acetate CRS in the solvent mixture and dilute to 20 mL Column :
with the solvent mixture. – size : l = 0.25 m, Ø = 4.6 mm ;
Reference solution (b). Dissolve 10 mg of cortisone acetate R – stationary phase : octadecylsilyl silica gel for
in reference solution (a) and dilute to 10 mL with reference chromatography R (5 μm).
solution (a). Mobile phase : mix 380 mL of acetonitrile R with 550 mL of
Plate : TLC silica gel F254 plate R. water R and allow to equilibrate ; dilute to 1000.0 mL with
Mobile phase : add a mixture of 1.2 volumes of water R and water R and mix again.
8 volumes of methanol R to a mixture of 15 volumes of Flow rate : 1 mL/min.
ether R and 77 volumes of methylene chloride R. Detection : spectrophotometer at 254 nm.
Application : 5 μL. Injection : 20 μL.
Development : over 3/4 of the plate. Run time : 2.5 times the retention time of dexamethasone
Drying : in air. acetate.
Detection A : examine in ultraviolet light at 254 nm. Identification of impurities: use the chromatogram obtained
with reference solution (a) to identify the peaks due to
Results A : the principal spot in the chromatogram obtained impurities A and D ; use the chromatogram obtained with
with the test solution is similar in position and size to reference solution (c) to identify the peak due to impurity E.
the principal spot in the chromatogram obtained with
Relative retention with reference to dexamethasone acetate
reference solution (a).
(retention time = about 22 min) : impurity A = about 0.4 ;
Detection B : spray with alcoholic solution of sulfuric acid R, impurity D = about 0.9 ; impurity E = about 1.2.
heat at 120 °C for 10 min or until the spots appear, and System suitability : reference solution (a) :
allow to cool; examine in daylight and in ultraviolet light
at 365 nm. – resolution : minimum 3.3 between the peaks due to
impurity D and dexamethasone acetate.
Results B : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour in Limits :
daylight, fluorescence in ultraviolet light at 365 nm and – impurity D : not more than 3 times the area of the principal
size to the principal spot in the chromatogram obtained peak in the chromatogram obtained with reference
with reference solution (a). solution (b) (0.3 per cent) ;
System suitability: reference solution (b) : – impurities A, E : for each impurity, not more than twice the
area of the principal peak in the chromatogram obtained
– the chromatogram shows 2 clearly separated spots. with reference solution (b) (0.2 per cent) ;
D. Add about 2 mg to 2 mL of sulfuric acid R and shake to – unspecified impurities : for each impurity, not more than the
dissolve. Within 5 min, a faint reddish-brown colour area of the principal peak in the chromatogram obtained
develops. Add this solution to 10 mL of water R and mix. with reference solution (b) (0.10 per cent) ;
The colour is discharged and a clear solution remains.
– total : not more than 5 times the area of the principal peak
E. Mix about 5 mg with 45 mg of heavy magnesium oxide R in the chromatogram obtained with reference solution (b)
and ignite in a crucible until an almost white residue is (0.5 per cent) ;
obtained (usually less than 5 min). Allow to cool, add – disregard limit : 0.5 times the area of the principal peak in
1 mL of water R, 0.05 mL of phenolphthalein solution R1 the chromatogram obtained with reference solution (b)
and about 1 mL of dilute hydrochloric acid R to render the (0.05 per cent).
solution colourless. Filter. To a freshly prepared mixture
of 0.1 mL of alizarin S solution R and 0.1 mL of zirconyl Loss on drying (2.2.32) : maximum 0.5 per cent, determined
nitrate solution R, add 1.0 mL of the filtrate. Mix, allow on 0.500 g by drying in vacuo in an oven at 105 °C.
to stand for 5 min and compare the colour of the solution ASSAY
with that of a blank prepared in the same manner. The test
solution is yellow and the blank is red. Dissolve 0.100 g in ethanol (96 per cent) R and dilute to
100.0 mL with the same solvent. Dilute 2.0 mL of this
F. About 10 mg gives the reaction of acetyl (2.3.1). solution to 100.0 mL with ethanol (96 per cent) R. Measure the
TESTS absorbance (2.2.25) at the absorption maximum at 238.5 nm.
Calculate the content of C24H31FO6 taking the specific
Specific optical rotation (2.2.7) : + 94 to + 99 (dried absorbance to be 357.
substance).
Dissolve 0.250 g in anhydrous ethanol R and dilute to 25.0 mL STORAGE
with the same solvent. Protected from light.

General Notices (1) apply to all monographs and other texts 2013
Dexamethasone isonicotinate EUROPEAN PHARMACOPOEIA 8.0

IMPURITIES
Specified impurities : A, D, E.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use G. 9-fluoro-11β-hydroxy-16α-methyl-3,20-dioxopregna-1,4-
(2034). It is therefore not necessary to identify these impurities dien-21-yl acetate,
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : B, C, F, G, H.

H. 17-hydroxy-16α-methyl-3,20-dioxopregna-1,4,9(11)-trien-
21-yl acetate.
A. 9-fluoro-11β,17,21-trihydroxy-16α-methylpregna-1,4- 01/2008:2237
diene-3,20-dione (dexamethasone),
DEXAMETHASONE ISONICOTINATE
Dexamethasoni isonicotinas

B. 14-fluoro-11β,17-dihydroxy-16α-methyl-3,20-
dioxopregna-1,4-dien-21-yl acetate,
C28H32FNO6 Mr 497.6
[2265-64-7]
DEFINITION
9-Fluoro-11β,17-dihydroxy-16α-methyl-3,20-dioxopregna-
1,4-dien-21-yl pyridine-4-carboxylate.
Content : 99.0 per cent to 101.0 per cent (dried substance).
C. 9-fluoro-11β,17β-dihydroxy-16α-methyl-3,20- CHARACTERS
dioxopregna-1,4-dien-21-yl acetate,
Appearance : white or almost white crystalline powder.
Solubility : practically insoluble in water, slightly soluble in
anhydrous ethanol and in acetone.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: dexamethasone isonicotinate CRS.
TESTS
D. 9-fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregna-
Specific optical rotation (2.2.7) : + 142 to + 146 (dried
1,4-dien-21-yl acetate (betamethasone acetate),
substance).
Suspend 0.200 g in 4.0 mL of ethyl acetate R and dilute to
20.0 mL with ethanol (96 per cent) R. Treat in an ultrasonic
bath until a clear solution is obtained.
Related substances. Liquid chromatography (2.2.29). Prepare
solutions immediately before use.
Test solution. Suspend 50.0 mg in 7 mL of acetonitrile R and
dilute to 10.0 mL with water R. Treat in an ultrasonic bath
E. 9-fluoro-11β,17-dihydroxy-16α-methyl-3,20-dioxopregn-4- until a clear solution is obtained.
en-21-yl acetate, Reference solution (a). Suspend 5.0 mg of dexamethasone CRS
and 5.0 mg of dexamethasone acetate CRS in 70 mL of
acetonitrile R, add 1.0 mL of the test solution and dilute to
100.0 mL with water R. Treat in an ultrasonic bath until a
clear solution is obtained.
Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 10.0 mL with water R.
Reference solution (c). Suspend 5 mg of dexamethasone
isonicotinate for impurity C identification CRS in 0.7 mL of
F. 17-hydroxy-16α-methyl-3,20-dioxo-9β,11β-epoxypregna- acetonitrile R and dilute to 1 mL with water R. Treat in an
1,4-dien-21-yl acetate, ultrasonic bath until a clear solution is obtained.

2014 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dexamethasone sodium phosphate

Column :
– size : l = 0.125 m, Ø = 4.0 mm,
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase :
– mobile phase A : water R, A. 9-fluoro-11β,17,21-trihydroxy-16α-methylpregna-1,4-
– mobile phase B : acetonitrile R, diene-3,20-dione (dexamethasone),
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-2 68 32

2 - 20 68 → 50 32 → 50

20 - 25 50 → 68 50 → 32

25 - 35 68 32 B. 9-fluoro-11β,17-dihydroxy-16α-methyl-3,20-dioxopregna-
1,4-dien-21-yl acetate (dexamethasone acetate),
Flow rate : 1.2 mL/min.
Detection : spectrophotometer at 240 nm.
Injection : 10 μL.
Identification of impurities : use the chromatogram
supplied with dexamethasone isonicotinate for impurity C
identification CRS and the chromatogram obtained with
reference solution (c) to identify the peak due to impurity C.
C. 9-fluoro-11β,17-dihydroxy-16α-methylpregna-1,4-diene-
Relative retention with reference to dexamethasone 3,20-dione (21-deoxydexamethasone).
isonicotinate (retention time = about 12 min) :
impurity A = about 0.4 ; impurity C = about 0.6 ;
impurity B = about 0.8. 07/2012:0549
System suitability: reference solution (a) :
– resolution : minimum 5.0 between the peaks due to DEXAMETHASONE SODIUM
impurity B and dexamethasone isonicotinate. PHOSPHATE
Limits :
– impurity A : not more than 5 times the area of the Dexamethasoni natrii phosphas
corresponding peak in the chromatogram obtained with
reference solution (b) (0.5 per cent),
– impurity B : not more than 3 times the area of the
corresponding peak in the chromatogram obtained with
reference solution (b) (0.3 per cent),
– impurity C : not more than 3 times the area of the peak
due to dexamethasone isonicotinate in the chromatogram
obtained with reference solution (b) (0.3 per cent), C22H28FNa2O8P Mr 516.4
– unspecified impurities : for each impurity, not more than [2392-39-4]
the area of the peak due to dexamethasone isonicotinate
in the chromatogram obtained with reference solution (b) DEFINITION
(0.1 per cent), 9-Fluoro-11β,17-dihydroxy-16α-methyl-3,20-dioxopregna-
1,4-dien-21-yl disodium phosphate.
– total : not more than 8 times the area of the peak due
to dexamethasone isonicotinate in the chromatogram Content : 97.0 per cent to 102.0 per cent (anhydrous substance).
obtained with reference solution (b) (0.8 per cent), CHARACTERS
– disregard limit : 0.5 times the area of the peak due to Appearance : white or almost white, very hygroscopic powder.
dexamethasone isonicotinate in the chromatogram
Solubility : freely soluble in water, slightly soluble in ethanol
obtained with reference solution (b) (0.05 per cent).
(96 per cent), practically insoluble in methylene chloride.
Loss on drying (2.2.32) : maximum 1.0 per cent, determined It shows polymorphism (5.9).
on 1.000 g by drying in an oven at 102 °C under high vacuum
for 4 h. IDENTIFICATION
First identification : B, G.
ASSAY
Second identification : A, C, D, E, F.
Dissolve 0.400 g in a mixture of 5 mL of anhydrous formic A. Dissolve 10.0 mg in 5 mL of water R and dilute to
acid R and 50 mL of glacial acetic acid R. Titrate with 0.1 M 100.0 mL with anhydrous ethanol R. Place 2.0 mL of this
perchloric acid, determining the end-point potentiometrically solution in a ground-glass-stoppered tube, add 10.0 mL of
(2.2.20). phenylhydrazine-sulfuric acid solution R, mix and heat in
1 mL of 0.1 M perchloric acid is equivalent to 49.76 mg a water-bath at 60 °C for 20 min. Cool immediately. The
of C28H32FNO6. absorbance (2.2.25) measured at the absorption maximum
at 419 nm is at least 0.20.
IMPURITIES B. Infrared absorption spectrophotometry (2.2.24).
Specified impurities : A, B, C. Comparison: dexamethasone sodium phosphate CRS.

General Notices (1) apply to all monographs and other texts 2015
Dexamethasone sodium phosphate EUROPEAN PHARMACOPOEIA 8.0

If the spectra obtained in the solid state show differences, TESTS

dissolve the substance to be examined and the reference Solution S. Dissolve 1.0 g in carbon dioxide-free water R and
substance separately in the minimum volume of ethanol dilute to 20 mL with the same solvent.
(96 per cent) R, evaporate to dryness on a water-bath and
record new spectra using the residues. Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution B7 (2.2.2,
C. Thin-layer chromatography (2.2.27). Method II).
Test solution. Dissolve 10 mg of the substance to be pH (2.2.3) : 7.5 to 9.5.
examined in methanol R and dilute to 10 mL with the same
solvent. Dilute 1 mL of solution S to 5 mL with carbon dioxide-free
water R.
Reference solution (a). Dissolve 20 mg of dexamethasone
sodium phosphate CRS in methanol R and dilute to 20 mL Specific optical rotation (2.2.7) : + 75 to + 83 (anhydrous
with the same solvent. substance).
Dissolve 0.250 g in water R and dilute to 25.0 mL with the
Reference solution (b). Dissolve 10 mg of prednisolone same solvent.
sodium phosphate CRS in reference solution (a) and dilute
to 10 mL with reference solution (a). Related substances. Liquid chromatography (2.2.29).
Plate : TLC silica gel F254 plate R. Solution A. Dissolve 7.0 g of ammonium acetate R in 1000 mL
of water R.
Mobile phase : glacial acetic acid R, water R, butanol R
Test solution. Dissolve 10 mg of the substance to be examined
(20:20:60 V/V/V).
in mobile phase A and dilute to 10.0 mL with mobile phase A.
Application : 5 μL. Reference solution (a). Dissolve 2 mg of betamethasone sodium
Development : over 3/4 of the plate. phosphate CRS (impurity B) and 2 mg of dexamethasone
Drying : in air. sodium phosphate CRS in mobile phase A, then dilute to
100.0 mL with mobile phase A.
Detection A : examine in ultraviolet light at 254 nm.
Reference solution (b). Dissolve 2 mg of dexamethasone
Results A : the principal spot in the chromatogram obtained sodium phosphate for peak identification CRS (containing
with the test solution is similar in position and size to impurities A, C, D, E, F and G) in mobile phase A and dilute
the principal spot in the chromatogram obtained with to 2.0 mL with mobile phase A.
reference solution (a). Reference solution (c). Dilute 1.0 mL of the test solution to
Detection B : spray with alcoholic solution of sulfuric acid R, 100.0 mL with mobile phase A. Dilute 1.0 mL of this solution
heat at 120 °C for 10 min or until the spots appear, and to 10.0 mL with mobile phase A.
allow to cool ; examine in daylight and in ultraviolet light Column :
at 365 nm.
– size : l = 0.125 m, Ø = 4.6 mm ;
Results B : the principal spot in the chromatogram obtained – stationary phase : end-capped octylsilyl silica gel for
with the test solution is similar in position, colour in chromatography R (5 μm) ;
daylight, fluorescence in ultraviolet light at 365 nm and
size to the principal spot in the chromatogram obtained – temperature : 30 °C.
with reference solution (a). Mobile phase :
System suitability: reference solution (b) : – mobile phase A : mix 300 mL of solution A and 350 mL
of water R, adjust to pH 3.8 with acetic acid R, then add
– the chromatogram shows 2 spots which may, however, 350 mL of methanol R ;
not be completely separated.
– mobile phase B : adjust 300 mL of solution A to pH 4.0 with
D. Add about 2 mg to 2 mL of sulfuric acid R and shake to acetic acid R, then add 700 mL of methanol R ;
dissolve. Within 5 min, a faint yellowish-brown colour
Time Mobile phase A Mobile phase B
develops. Add this solution to 10 mL of water R and mix.
The colour fades and a clear solution remains. (min) (per cent V/V) (per cent V/V)
0 - 3.5 90 10
E. Mix about 5 mg with 45 mg of heavy magnesium oxide R
and ignite in a crucible until an almost white residue is 3.5 - 23.5 90 → 60 10 → 40
obtained (usually less than 5 min). Allow to cool, add 23.5 - 34.5 60 → 5 40 → 95
1 mL of water R, 0.05 mL of phenolphthalein solution R1
and about 1 mL of dilute hydrochloric acid R to render the 34.5 - 50 5 95
solution colourless. Filter. To a freshly prepared mixture
of 0.1 mL of alizarin S solution R and 0.1 mL of zirconyl Flow rate : 1.0 mL/min.
nitrate solution R, add 1.0 mL of the filtrate. Mix, allow Detection : spectrophotometer at 254 nm.
to stand for 5 min and compare the colour of the solution Injection : 20 μL.
with that of a blank prepared in the same manner. The test Identification of impurities : use the chromatogram
solution is yellow and the blank is red. supplied with dexamethasone sodium phosphate for peak
F. To 40 mg add 2 mL of sulfuric acid R and heat gently identification CRS and the chromatogram obtained with
until white fumes are evolved, add nitric acid R dropwise, reference solution (b) to identify the peaks due to impurities
continue the heating until the solution is almost colourless A, C, D, E, F and G ; use the chromatogram obtained with
and cool. Add 2 mL of water R, heat until white fumes are reference solution (a) to identify the peak due to impurity B.
again evolved, cool, add 10 mL of water R and neutralise to Relative retention with reference to dexamethasone
red litmus paper R with dilute ammonia R1. The solution sodium phosphate (retention time = about 22 min) :
gives reaction (a) of sodium (2.3.1) and reaction (b) of impurity C = about 0.5 ; impurity D = about 0.6 ;
phosphates (2.3.1). impurity E = about 0.8 ; impurity F = about 0.92 ;
G. Examine the chromatograms obtained in the assay. impurity B = about 0.95 ; impurity A = about 1.37 ;
Results : the principal peak in the chromatogram obtained impurity G = about 1.41.
with the test solution is similar in retention time and size System suitability : reference solution (a) :
to the principal peak in the chromatogram obtained with – resolution : minimum 2.0 between the peaks due to
reference solution (b). impurity B and dexamethasone sodium phosphate.

2016 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dexamethasone sodium phosphate

Limits : Reference solution (a). Dissolve 2 mg of dexamethasone CRS

– correction factor : for the calculation of content, multiply (impurity A) and 2 mg of dexamethasone sodium
the peak area of impurity A by 0.75 ; phosphate CRS in 2 mL of tetrahydrofuran R, then dilute to
100.0 mL with the mobile phase. Dilute 5.0 mL of this solution
– impurity A : not more than 5 times the area of the principal to 50.0 mL with the mobile phase.
peak in the chromatogram obtained with reference
solution (c) (0.5 per cent) ; Reference solution (b). Dissolve 30.0 mg of dexamethasone
sodium phosphate CRS in the mobile phase and dilute to
– impurity G : not more than 3 times the area of the principal 50.0 mL with the mobile phase. Dilute 5.0 mL of the solution
peak in the chromatogram obtained with reference to 50.0 mL with the mobile phase.
solution (c) (0.3 per cent) ;
Column :
– impurities B, C, D, E, F : for each impurity, not more than
twice the area of the principal peak in the chromatogram – size : l = 0.15 m, Ø = 4.6 mm ;
obtained with reference solution (c) (0.2 per cent) ; – stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (7 μm).
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained Mobile phase : mix 520 mL of water R with 2 mL of phosphoric
with reference solution (c) (0.10 per cent) ; acid R. Adjust the temperature to 20 °C, then adjust to pH 2.6
with sodium hydroxide R. Mix this solution with 36 mL of
– total : not more than 10 times the area of the principal peak tetrahydrofuran R and 364 mL of methanol R.
in the chromatogram obtained with reference solution (c)
(1.0 per cent) ; Flow rate : 1.5 mL/min.
– disregard limit : 0.5 times the area of the principal peak in Detection : spectrophotometer at 254 nm.
the chromatogram obtained with reference solution (c) Injection : 20 μL.
(0.05 per cent). Run time : 3 times the retention time of dexamethasone
Inorganic phosphates : maximum 1 per cent. sodium phosphate.
Dissolve 50 mg in water R and dilute to 100 mL with the same Identification of impurities: use the chromatogram obtained
solvent. To 10 mL of this solution add 5 mL of molybdovanadic with reference solution (a) to identify the peak due to
reagent R, mix and allow to stand for 5 min. Any yellow colour impurity A.
in the solution is not more intense than that in a standard Relative retention with reference to dexamethasone
prepared at the same time in the same manner using 10 mL of sodium phosphate (retention time = about 8 min) :
phosphate standard solution (5 ppm PO4) R. impurity A = about 2.0.
Ethanol. Gas chromatography (2.2.28). System suitability : reference solution (a) :
Internal standard solution. Dilute 1.0 mL of propanol R to – resolution : minimum 6.0 between the peaks due to
100.0 mL with water R. dexamethasone sodium phosphate and impurity A.
Test solution. Dissolve 0.50 g of the substance to be examined Calculate the percentage content of C22H28FNa2O8P using
in 5.0 mL of the internal standard solution and dilute to the chromatogram obtained with reference solution (b) and
10.0 mL with water R. taking into account the assigned content of dexamethasone
sodium phosphate CRS.
Reference solution. Dilute 1.0 g of anhydrous ethanol R to
100.0 mL with water R. To 2.0 mL of this solution add 5.0 mL STORAGE
of the internal standard solution and dilute to 10.0 mL with In an airtight container, protected from light.
water R.
Column : IMPURITIES
– size : l = 1 m, Ø = 3.2 mm ; Specified impurities : A, B, C, D, E, F, G.
– stationary phase : ethylvinylbenzene-divinylbenzene Other detectable impurities (the following substances would,
copolymer R1 (150-180 μm). if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
Carrier gas : nitrogen for chromatography R. acceptance criterion for other/unspecified impurities and/or
Flow rate : 30 mL/min. by the general monograph Substances for pharmaceutical
Temperature : use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
– column : 150 °C ;
Control of impurities in substances for pharmaceutical use) : H.
– injection port : 250 °C ;
– detector : 280 °C.
Detection : flame ionisation.
Injection : 2 μL.
Limit :
– ethanol : maximum 3.0 per cent m/m.
Ethanol and water : maximum 13.0 per cent m/m for the sum A. 9-fluoro-11β,17,21-trihydroxy-16α-methylpregna-1,4-
of the percentage contents. diene-3,20-dione (dexamethasone),
Determine the water content using 0.200 g (2.5.12). Add the
percentage content of water and the percentage content of
ethanol obtained in the test for ethanol.
ASSAY
Liquid chromatography (2.2.29).
Test solution. Dissolve 30.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with the B. 9-fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregna-
mobile phase. Dilute 5.0 mL of the solution to 50.0 mL with 1,4-dien-21-yl dihydrogen phosphate (betamethasone
the mobile phase. phosphate),

General Notices (1) apply to all monographs and other texts 2017
Dexchlorpheniramine maleate EUROPEAN PHARMACOPOEIA 8.0

A. Specific optical rotation (see Tests).

B. Melting point (2.2.14) : 110 °C to 115 °C.
C. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs of potassium bromide R.
Comparison: dexchlorpheniramine maleate CRS.
D. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 0.10 g of the substance to be
examined in methanol R and dilute to 5.0 mL with the
same solvent.
Reference solution. Dissolve 56 mg of maleic acid R in
methanol R and dilute to 10 mL with the same solvent.
C, D, E, F. for each impurity, one or more diastereoisomer(s) Plate : TLC silica gel F254 plate R.
of (9-fluoro-11β,17a-dihydroxy-16-methyl-3,17-dioxo- Mobile phase : water R, anhydrous formic acid R, methanol R,
D-homo-androsta-1,4-dien-17a-yl)methyl dihydrogen di-isopropyl ether R (3:7:20:70 V/V/V/V).
phosphate (undefined stereochemistry at C-16 and C-17a), Application : 5 μL.
or (9-fluoro-11β,17-dihydroxy-16α-methyl-3,17a-dioxo- Development : over a path of 12 cm.
D-homo-androsta-1,4-dien-17-yl)methyl dihydrogen
phosphate (undefined stereochemistry at C-17), Drying : in a current of air for a few minutes.
Detection : examine in ultraviolet light at 254 nm.
Results : the chromatogram obtained with the test solution
shows 2 clearly separated spots. The upper spot is similar in
position and size to the spot in the chromatogram obtained
with the reference solution.
E. To 0.15 g in a porcelain crucible add 0.5 g of anhydrous
sodium carbonate R. Heat over an open flame for 10 min.
G. 9-fluoro-11β,17-dihydroxy-16α-methyl-3-oxoandrosta-
Allow to cool. Take up the residue with 10 mL of dilute
1,4-diene-17β-carboxylic acid,
nitric acid R and filter. To 1 mL of the filtrate add 1 mL of
water R. The solution gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S. Dissolve 2.0 g in water R and dilute to 20.0 mL
with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution BY6 (2.2.2,
H. 9-fluoro-11β,17-dihydroxy-16α-methyl-3,20-dioxopregn- Method II).
4-en-21-yl dihydrogen phosphate.
pH (2.2.3) : 4.5 to 5.5.
Dissolve 0.20 g in 20 mL of water R.
01/2008:1196 Specific optical rotation (2.2.7) : + 22 to + 23 (dried
corrected 6.8 substance), determined on solution S.
Related substances. Gas chromatography (2.2.28).
DEXCHLORPHENIRAMINE MALEATE Test solution. Dissolve 10.0 mg of the substance to be
examined in 1.0 mL of methylene chloride R.
Dexchlorpheniramini maleas Reference solution. Dissolve 5.0 mg of brompheniramine
maleate CRS in 0.5 mL of methylene chloride R and add 0.5 mL
of the test solution. Dilute 0.5 mL of this solution to 50.0 mL
with methylene chloride R.
Column :
– material : glass ;
– size : l = 2.3 m, Ø = 2 mm ;
– stationary phase : acid- and base-washed silanised
C20H23ClN2O4 Mr 390.9 diatomaceous earth for gas chromatography R (135-175 μm)
[2438-32-6] impregnated with 3 per cent m/m of a mixture of 50 per
cent of poly(dimethyl)siloxane and 50 per cent of
DEFINITION poly(diphenyl)siloxane.
(3S)-3-(4-Chlorophenyl)-N,N-dimethyl-3-(pyridin-2- Carrier gas : nitrogen for chromatography R.
yl)propan-1-amine (Z)-butenedioate. Flow rate : 20 mL/min.
Content : 98.0 per cent to 100.5 per cent (dried substance). Temperature :
– column : 205 °C ;
CHARACTERS
– injection port and detector : 250 °C.
Appearance : white or almost white, crystalline powder.
Detection : flame ionisation.
Solubility : very soluble in water, freely soluble in ethanol
Injection : 1 μL.
(96 per cent), in methanol and in methylene chloride.
Run time : 2.5 times the retention time of dexchlorpheniramine.
IDENTIFICATION System suitability : reference solution :
First identification : A, C, E. – resolution : minimum 1.5 between the peaks due to
Second identification : A, B, D, E. dexchlorpheniramine and brompheniramine.

2018 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dexpanthenol

Limits : ASSAY
– impurity A : not more than 0.8 times the area of the peak Dissolve 0.150 g in 25 mL of anhydrous acetic acid R. Titrate
due to dexchlorpheniramine in the chromatogram obtained with 0.1 M perchloric acid, determining the end-point
with the reference solution (0.4 per cent) ; potentiometrically (2.2.20).
– total : not more than twice the area of the peak due to 1 mL of 0.1 M perchloric acid is equivalent to 19.54 mg
dexchlorpheniramine in the chromatogram obtained with of C20H23ClN2O4.
the reference solution (1 per cent). STORAGE
Enantiomeric purity. Liquid chromatography (2.2.29). Protected from light.
Test solution. Dissolve 10.0 mg of the substance to be IMPURITIES
examined in 3 mL of water R. Add a few drops of concentrated
ammonia R until an alkaline reaction is produced. Shake with Specified impurities : A, B.
5 mL of methylene chloride R. Separate the layers. Evaporate
the lower, methylene chloride layer to an oily residue on a
water-bath. Dissolve the oily residue in 2-propanol R and
dilute to 10.0 mL with the same solvent.
Reference solution (a). Dissolve 10.0 mg of dexchlorphenir-
amine maleate CRS in 3 mL of water R. Add a few drops
of concentrated ammonia R until an alkaline reaction is A. (3RS)-N,N-dimethyl-3-phenyl-3-(pyridin-2-yl)propan-1-
produced. Shake with 5 mL of methylene chloride R. Separate amine,
the layers. Evaporate the lower, methylene chloride layer to
an oily residue on a water-bath. Dissolve the oily residue in
2-propanol R and dilute to 10.0 mL with the same solvent.
Reference solution (b). Dissolve 10.0 mg of chlorphenamine
maleate CRS in 3 mL of water R. Add a few drops of
concentrated ammonia R until an alkaline reaction is
produced. Shake with 5 mL of methylene chloride R. Separate
the layers. Evaporate the lower, methylene chloride layer to B. (3R)-3-(4-chlorophenyl)-N,N-dimethyl-3-(pyridin-2-
an oily residue on a water-bath. Dissolve the oily residue in yl)propan-1-amine ((R)-enantiomer).
2-propanol R and dilute to 10.0 mL with the same solvent.
Reference solution (c). Dilute 1.0 mL of the test solution to 01/2008:0761
50 mL with 2-propanol R.
Column : DEXPANTHENOL
– size : l = 0.25 m, Ø = 4.6 mm ;
Dexpanthenolum
– stationary phase : amylose derivative of silica gel for
chromatography R.
Mobile phase : diethylamine R, 2-propanol R, hexane R
(3:20:980 V/V/V).
Flow rate : 1 mL/min. C9H19NO4 Mr 205.3
Detection : spectrophotometer at 254 nm. [81-13-0]
Injection : 10 μL. DEFINITION
Under these conditions the peak due to the (S)-isomer appears Dexpanthenol contains not less than 98.0 per cent and not
first. more than the equivalent of 101.0 per cent of (2R)-2,4-
System suitability : dihydroxy-N-(3-hydroxypropyl)-3,3-dimethylbutanamide,
calculated with reference to the anhydrous substance.
– resolution : minimum 1.5 between the peaks due to the
(R)-enantiomer (impurity B) and the (S)-enantiomer in the CHARACTERS
chromatogram obtained with reference solution (b) ; A colourless or slightly yellowish, viscous hygroscopic liquid,
– the retention times of the principal peaks in the or a white or almost white, crystalline powder, very soluble in
chromatograms obtained with the test solution and water, freely soluble in ethanol (96 per cent).
reference solution (a) are identical ((S)-enantiomer).
IDENTIFICATION
Limits :
First identification : A, B.
– (R)-enantiomer (impurity B) : not more than the area of Second identification : A, C, D.
the principal peak in the chromatogram obtained with
A. Specific optical rotation (see Tests).
reference solution (c) (2 per cent) ;
B. Examine by infrared absorption spectrophotometry
– unspecified impurities : for each impurity, not more (2.2.24), comparing with the spectrum obtained with
than 0.25 times the area of the principal peak in the dexpanthenol CRS. Examine the substances using discs
chromatogram obtained with reference solution (c) (0.5 per prepared as follows : dissolve the substance to be examined
cent). and the reference substance separately in 1.0 mL of
Heavy metals (2.4.8) : maximum 20 ppm. anhydrous ethanol R to obtain a concentration of 5 mg/mL.
1.0 g complies with test C. Prepare the reference solution using Place dropwise 0.5 mL of this solution on a disc of
2 mL of lead standard solution (10 ppm Pb) R. potassium bromide R. Dry the disc at 100-105 °C for 15 min.
C. Examine the chromatograms obtained in the test for
Loss on drying (2.2.32) : maximum 0.5 per cent, determined 3-aminopropanol. The principal spot in the chromatogram
on 1.000 g by drying in an oven at 65 °C for 4 h. obtained with test solution (b) is similar in position,
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on colour and size to the principal spot in the chromatogram
1.0 g. obtained with reference solution (a).

General Notices (1) apply to all monographs and other texts 2019
Dextran 1 for injection EUROPEAN PHARMACOPOEIA 8.0

D. To 1 mL of solution S (see Tests) add 1 mL of dilute sodium Average relative molecular mass : about 1000.
hydroxide solution R and 0.1 mL of copper sulfate solution R.
A blue colour develops. PRODUCTION
It is obtained by hydrolysis and fractionation of dextrans
TESTS produced by fermentation of sucrose using Leuconostoc
Solution S. Dissolve 2.500 g in carbon dioxide-free water R mesenteroides strain NRRL B-512 = CIP 78.59 or substrains
and dilute to 50.0 mL with the same solvent. thereof (for example L. mesenteroides B-512 F = NCTC 10817).
Appearance of solution. Solution S is clear (2.2.1) and not It is prepared in conditions designed to minimise the risk of
more intensely coloured than reference solution B6 (2.2.2, microbial contamination.
Method II). CHARACTERS
pH (2.2.3). The pH of solution S is not greater than 10.5. Appearance : white or almost white hygroscopic powder.
Specific optical rotation (2.2.7). The specific optical rotation Solubility : very soluble in water, very slightly soluble in
is + 29.0 to + 32.0, determined on solution S and calculated ethanol (96 per cent).
with reference to the anhydrous substance.
IDENTIFICATION
3-Aminopropanol. Examine by thin-layer chromatography
(2.2.27), using silica gel G R as the coating substance. A. Dissolve 3.000 g in water R, heat on a water-bath and dilute
to 100.0 mL with the same solvent. The specific optical
Test solution (a). Dissolve 0.25 g of the substance to be
rotation (2.2.7) is + 148 to + 164, calculated with reference
examined in anhydrous ethanol R and dilute to 5 mL with the
to the dried substance. Dry an aliquot of the solution first
same solvent.
on a water-bath and then to constant weight in vacuo at
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL 70 °C. Calculate the dextran content after correction for the
with anhydrous ethanol R. content of sodium chloride.
Reference solution (a). Dissolve the contents of a vial of B. Infrared absorption spectrophotometry (2.2.24).
dexpanthenol CRS in 1.0 mL of anhydrous ethanol R to obtain
Preparation : to 1-2 mg add 1 or a few drops of water R.
a concentration of 5 mg/mL.
Grind in an agate mortar for 1-2 min. Add about 300 mg of
Reference solution (b). Dissolve 25 mg of 3-aminopropanol R potassium bromide R and mix to a slurry but do not grind.
in anhydrous ethanol R and dilute to 100 mL with the same Dry in vacuo at 40 °C for 15 min. Crush the residue. If it
solvent. is not dry, dry for another 15 min. Prepare a disc using
Apply separately to the plate 10 μL of each solution. Develop potassium bromide R.
over a path of 15 cm using a mixture of 20 volumes of Comparison: repeat the operations using dextran 1 CRS.
concentrated ammonia R, 25 volumes of methanol R and Blank : run the infrared spectrum with a blank disc using
55 volumes of butanol R. Allow the plate to dry in air, spray
potassium bromide R in the reference beam.
with a 100 g/L solution of trichloroacetic acid R in methanol R
and heat at 150 °C for 10 min. Spray with a 1 g/L solution C. Molecular-mass distribution (see Tests).
of ninhydrin R in methanol R and heat at 120 °C until a TESTS
colour appears. Any spot due to 3-aminopropanol in the
chromatogram obtained with test solution (a) is not more Solution S. Dissolve 7.5 g in carbon dioxide-free water R, heat
intense than the spot in the chromatogram obtained with on a water-bath and dilute to 50 mL with the same solvent.
reference solution (b) (0.5 per cent). Absorbance (2.2.25) : maximum 0.12, determined at 375 nm
Heavy metals (2.4.8). 12 mL of solution S complies with on solution S.
limit test A for heavy metals (20 ppm). Prepare the reference Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of
solution using lead standard solution (1 ppm Pb) R. phenolphthalein solution R. The solution is colourless. Add
Water (2.5.12). Not more than 1.0 per cent, determined on 0.2 mL of 0.01 M sodium hydroxide. The solution is pink. Add
1.000 g. 0.4 mL of 0.01 M hydrochloric acid. The solution is colourless.
Add 0.1 mL of methyl red solution R. The solution is red or
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined orange.
on 1.0 g.
Nitrogen-containing substances : maximum 110 ppm of N.
ASSAY Carry out the determination of nitrogen by sulfuric acid
To 0.400 g add 50.0 mL of 0.1 M perchloric acid. Boil under digestion (2.5.9), using 0.200 g and heating for 2 h. Collect
a reflux condenser for 5 h protected from humidity. Allow the distillate in a mixture of 0.5 mL of bromocresol green
to cool. Add 50 mL of dioxan R by rinsing the condenser, solution R, 0.5 mL of methyl red solution R and 20 mL of
protected from humidity. Add 0.2 mL of naphtholbenzein water R. Titrate with 0.01 M hydrochloric acid. Not more than
solution R and titrate with 0.1 M potassium hydrogen phthalate 0.15 mL of 0.01 M hydrochloric acid is required to change the
until the colour changes from green to yellow. Carry out a colour of the indicator.
blank titration. Sodium chloride : maximum 1.5 per cent.
1 mL of 0.1 M perchloric acid is equivalent to 20.53 mg Accurately weigh 3-5 g and dissolve in 100 mL of water R.
of C9H19NO4. Add 0.3 mL of potassium chromate solution R and titrate with
STORAGE 0.1 M silver nitrate until the yellowish-white colour changes
to reddish-brown.
In an airtight container.
1 mL of 0.1 M silver nitrate is equivalent to 5.844 mg of NaCl.
01/2009:1506 Molecular-mass distribution. Size-exclusion
chromatography (2.2.30).
DEXTRAN 1 FOR INJECTION Test solution. Dissolve 6.0-6.5 mg of the substance to be
examined in 1.0 mL of the mobile phase.
Dextranum 1 ad iniectabile Reference solution (a). Dissolve 6.0-6.5 mg of dextran 1 CRS
in 1.0 mL of the mobile phase.
DEFINITION Reference solution (b). Dissolve the content of an ampoule
Low-molecular-weight fraction of dextran, consisting of a of isomaltooligosaccharide CRS in 1 mL of the mobile
mixture of isomaltooligosaccharides. phase, and mix. This corresponds to approximately 45 μg

2020 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dextran 40 for injection

of isomaltotriose (3 glucose units), approximately 45 μg of Heavy metals (2.4.8) : maximum 10 ppm.

isomaltononaose (9 glucose units), and approximately 60 μg Dilute 20 mL of solution S to 30 mL with water R. 12 mL of
of sodium chloride per 100 μL. solution complies with test A. Prepare the reference solution
Column : 2 columns coupled in series : using lead standard solution (1 ppm Pb) R.
– size : l = 0.30 m, Ø = 10 mm ; Loss on drying (2.2.32) : maximum 5.0 per cent, determined
– stationary phase : dextran covalently bound to highly on 5.000 g by drying in an oven at 105 °C for 5 h.
cross-linked porous agarose beads, allowing resolution of Bacterial endotoxins (2.6.14) : less than 25 IU/g.
oligosaccharides in the molecular mass range of 180 to
Microbial contamination
3000 ;
TAMC : acceptance criterion 102 CFU/g (2.6.12).
– temperature : 20-25 °C.
Mobile phase : 2.92 g/L solution of sodium chloride R. 01/2009:0999
Flow rate : 0.07-0.08 mL/min maintained constant to ± 1 per
cent. DEXTRAN 40 FOR INJECTION
Detection : differential refractometer.
Injection : 100 μL. Dextranum 40 ad iniectabile
Identification of peaks : use the chromatogram obtained
with reference solution (b) to identify the peaks due to DEFINITION
isomaltotriose, isomaltononaose and sodium chloride. Mixture of polysaccharides, principally of the α-1,6-glucan
Determine the peak areas. Disregard any peak due to sodium type.
chloride. Calculate the average relative molecular mass Mw Average relative molecular mass : about 40 000.
and the amount of the fraction with less than 3 and more than PRODUCTION
9 glucose units, of dextran 1 CRS and of the substance to be
examined, using the following expression : It is obtained by hydrolysis and fractionation of dextrans
produced by fermentation of sucrose using Leuconostoc
mesenteroides strain NRRL B-512 = CIP 78.59 or substrains
thereof (for example L. mesenteroides B-512F = NCTC 10817).
Mw = average molecular mass of the dextran ; It is prepared in conditions designed to minimise the risk of
microbial contamination.
mi = molecular mass of oligosaccharide i ;
CHARACTERS
wi = weight proportion of oligosaccharide i.
Appearance : white or almost white powder.
Use the following mi values for the calculation : Solubility : very soluble in water, very slightly soluble in
Oligosaccharide i mi ethanol (96 per cent).
glucose 180 IDENTIFICATION
isomaltose 342
A. Specific optical rotation (2.2.7) : + 195 to + 201 (dried
substance).
isomaltotriose 504 Dissolve 1.0 g in water R, heating on a water-bath, and
isomaltotetraose 666 dilute to 50.0 mL with the same solvent.
B. Infrared absorption spectrophotometry (2.2.24).
isomaltopentaose 828
Comparison: dextran CRS.
isomaltohexaose 990
C. Molecular-mass distribution (see Tests).
isomaltoheptaose 1152
TESTS
1314
isomaltooctaose Solution S. Dissolve 5.0 g in distilled water R, heating on a
isomaltononaose 1476 water-bath, and dilute to 50 mL with the same solvent.
isomaltodecaose 1638 Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
isomaltoundecaose 1800
Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of
isomaltododecaose 1962 phenolphthalein solution R. The solution remains colourless.
2124
Add 0.2 mL of 0.01 M sodium hydroxide. The solution is
isomaltotridecaose
red. Add 0.4 mL of 0.01 M hydrochloric acid. The solution is
isomaltotetradecaose 2286 colourless. Add 0.1 mL of methyl red solution R. The solution
2448
is red or orange.
isomaltopentadecaose
Nitrogen-containing substances : maximum 110 ppm N.
isomaltohexadecaose 2610
Carry out the determination of nitrogen by sulfuric acid
isomaltoheptadecaose 2772 digestion (2.5.9), using 0.200 g and heating for 2 h. Collect
2934
the distillate in a mixture of 0.5 mL of bromocresol green
isomaltooctadecaose
solution R, 0.5 mL of methyl red solution R and 20 mL of
isomaltononadecaose 3096 water R. Titrate with 0.01 M hydrochloric acid. Not more than
0.15 mL of 0.01 M hydrochloric acid is required to change the
System suitability : the values obtained for dextran 1 CRS are colour of the indicator.
within the values stated on the label.
Residual solvents. Gas chromatography (2.2.28).
Limits :
Internal standard : propanol R.
– average molecular mass (Mw) : 850 to 1150 ;
Test solution. Dissolve 5 g of the substance to be examined in
– fraction with less than 3 glucose units : less than 15 per cent ; 100 mL of water R and distil. Collect the first 45 mL of the
– fraction with more than 9 glucose units : less than 20 per distillate, add 1 mL of a 25 g/L solution of propanol R and
cent. dilute to 50 mL with water R.

General Notices (1) apply to all monographs and other texts 2021
Dextran 60 for injection EUROPEAN PHARMACOPOEIA 8.0

Reference solution. Mix 0.5 mL of a 25 g/L solution of IDENTIFICATION

anhydrous ethanol R, 0.5 mL of a 25 g/L solution of propanol R A. Specific optical rotation (2.2.7) : + 195 to + 201 (dried
and 0.5 mL of a 2.5 g/L solution of methanol R and dilute to substance).
25.0 mL with water R. Dissolve 1.0 g in water R, heating on a water-bath, and
Column : dilute to 50.0 mL with the same solvent.
– material : stainless steel ; B. Infrared absorption spectrophotometry (2.2.24).
– size : l = 1.8 m, Ø = 2 mm ; Comparison: dextran CRS.
– stationary phase : ethylvinylbenzene-divinylbenzene C. Molecular-mass distribution (see Tests).
copolymer R (125-150 μm).
Carrier gas : nitrogen for chromatography R. TESTS
Flow rate : 25 mL/min. Solution S. Dissolve 5.0 g in distilled water R, heating on a
Temperature : water-bath, and dilute to 50 mL with the same solvent.
– column : 190 °C ; Appearance of solution. Solution S is clear (2.2.1) and
– injection port : 240 °C ; colourless (2.2.2, Method II).
– detector : 210 °C. Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of
phenolphthalein solution R. The solution remains colourless.
Detection : flame ionisation. Add 0.2 mL of 0.01 M sodium hydroxide. The solution is
Injection : the chosen volume of each solution. red. Add 0.4 mL of 0.01 M hydrochloric acid. The solution is
Limits : colourless. Add 0.1 mL of methyl red solution R. The solution
– ethanol : not more than the area of the corresponding peak is red or orange.
in the chromatogram obtained with the reference solution Nitrogen-containing substances : maximum 110 ppm of N.
(0.5 per cent) ; Carry out the determination of nitrogen by sulfuric acid
– methanol : not more than the area of the corresponding digestion (2.5.9), using 0.200 g and heating for 2 h. Collect
peak in the chromatogram obtained with the reference the distillate in a mixture of 0.5 mL of bromocresol green
solution (0.05 per cent); solution R, 0.5 mL of methyl red solution R and 20 mL of
– sum of solvents other than ethanol, methanol and propanol : water R. Titrate with 0.01 M hydrochloric acid. Not more than
not more than the area of the peak due to the internal 0.15 mL of 0.01 M hydrochloric acid is required to change the
standard (0.5 per cent, calculated as propanol). colour of the indicator.
Molecular-mass distribution (2.2.39). The average molecular Residual solvents. Gas chromatography (2.2.28).
mass (Mw) is 35 000 to 45 000. The average molecular mass Internal standard : propanol R.
of the 10 per cent high fraction is not greater than 110 000. Test solution. Dissolve 5 g of the substance to be examined in
The average molecular mass of the 10 per cent low fraction is 100 mL of water R and distil. Collect the first 45 mL of the
not less than 7000. distillate, add 1 mL of a 25 g/L solution of propanol R and
Heavy metals (2.4.8) : maximum 10 ppm. dilute to 50 mL with water R.
12 mL of solution S complies with test A. Prepare the reference Reference solution. Mix 0.5 mL of a 25 g/L solution of
solution using lead standard solution (1 ppm Pb) R. anhydrous ethanol R, 0.5 mL of a 25 g/L solution of propanol R
Loss on drying (2.2.32) : maximum 7.0 per cent, determined and 0.5 mL of a 2.5 g/L solution of methanol R and dilute to
on 0.200 g by heating in an oven at 105 ± 2 °C for 5 h. 25.0 mL with water R.
Sulfated ash (2.4.14) : maximum 0.3 per cent, determined on Column :
0.50 g. – material : stainless steel ;
Bacterial endotoxins (2.6.14) : less than 10 IU/g. – size : l = 1.8 m, Ø = 2 mm ;
– stationary phase : ethylvinylbenzene-divinylbenzene
Microbial contamination
copolymer R (125-150 μm).
TAMC : acceptance criterion 102 CFU/g (2.6.12).
Carrier gas : nitrogen for chromatography R.
Flow rate : 25 mL/min.
01/2009:1000
Temperature :
DEXTRAN 60 FOR INJECTION – column : 190 °C ;
– injection port : 240 °C ;
Dextranum 60 ad iniectabile – detector : 210 °C.
Detection : flame ionisation.
DEFINITION Injection : the chosen volume of each solution.
Mixture of polysaccharides, principally of the α-1,6-glucan Limits :
type.
– ethanol : not more than the area of the corresponding peak
Average relative molecular mass : about 60 000. in the chromatogram obtained with the reference solution
PRODUCTION (0.5 per cent) ;
It is obtained by hydrolysis and fractionation of dextrans – methanol : not more than the area of the corresponding
produced by fermentation of sucrose using Leuconostoc peak in the chromatogram obtained with the reference
mesenteroides strain NRRL B-512 = CIP 78.59 or substrains solution (0.05 per cent);
thereof (for example L. mesenteroides B-512F = NCTC 10817). – sum of solvents other than ethanol, methanol and propanol :
It is prepared in conditions designed to minimise the risk of not more than the area of the peak due to the internal
microbial contamination. standard (0.5 per cent, calculated as propanol).
Molecular-mass distribution (2.2.39). The average molecular
CHARACTERS mass (Mw) is 54 000 to 66 000. The average molecular mass of
Appearance : white or almost white powder. the 10 per cent high fraction is not greater than 180 000. The
Solubility : very soluble in water, very slightly soluble in average molecular mass of the 10 per cent low fraction is not
ethanol (96 per cent). less than 14 000.

2022 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dextranomer

Heavy metals (2.4.8) : maximum 10 ppm. Test solution. Dissolve 5 g of the substance to be examined in
12 mL of solution S complies with test A. Prepare the reference 100 mL of water R and distil. Collect the first 45 mL of the
solution using lead standard solution (1 ppm Pb) R. distillate, add 1 mL of a 25 g/L solution of propanol R and
dilute to 50 mL with water R.
Loss on drying (2.2.32) : maximum 7.0 per cent, determined
on 0.200 g by heating in an oven at 105 ± 2 °C for 5 h. Reference solution. Mix 0.5 mL of a 25 g/L solution of
anhydrous ethanol R, 0.5 mL of a 25 g/L solution of propanol R
Sulfated ash (2.4.14) : maximum 0.3 per cent, determined on and 0.5 mL of a 2.5 g/L solution of methanol R and dilute to
0.50 g. 25.0 mL with water R.
Bacterial endotoxins (2.6.14) : less than 16 IU/g. Column :
Microbial contamination – material : stainless steel ;
TAMC : acceptance criterion 102 CFU/g (2.6.12). – size : l = 1.8 m, Ø = 2 mm ;
– stationary phase : ethylvinylbenzene-divinylbenzene
copolymer R (125-150 μm).
01/2009:1001 Carrier gas : nitrogen for chromatography R.
Flow rate : 25 mL/min.
DEXTRAN 70 FOR INJECTION Temperature :
– column : 190 °C ;
Dextranum 70 ad iniectabile – injection port : 240 °C ;
DEFINITION – detector : 210 °C.
Mixture of polysaccharides, principally of the α-1,6-glucan Detection : flame ionisation.
type. Injection : the chosen volume of each solution.
Average relative molecular mass : about 70 000. Limits :
– ethanol : not more than the area of the corresponding peak
PRODUCTION in the chromatogram obtained with the reference solution
It is obtained by hydrolysis and fractionation of dextrans (0.5 per cent) ;
produced by fermentation of sucrose using Leuconostoc – methanol : not more than the area of the corresponding
mesenteroides strain NRRL B-512 = CIP 78.59 or substrains peak in the chromatogram obtained with the reference
thereof (for example L. mesenteroides B-512F = NCTC 10817). solution (0.05 per cent);
It is prepared in conditions designed to minimise the risk of – sum of solvents other than ethanol, methanol and propanol :
microbial contamination. not more than the area of the peak due to the internal
CHARACTERS standard (0.5 per cent, calculated as propanol).
Molecular-mass distribution (2.2.39). The average molecular
Appearance : white or almost white powder.
mass (Mw) is 64 000 to 76 000. The average molecular mass of
Solubility : very soluble in water, very slightly soluble in the 10 per cent high fraction is not greater than 185 000. The
ethanol (96 per cent). average molecular mass of the 10 per cent low fraction is not
IDENTIFICATION less than 15 000.
A. Specific optical rotation (2.2.7) : + 195 to + 201 (dried Heavy metals (2.4.8) : maximum 10 ppm.
substance). 12 mL of solution S complies with test A. Prepare the reference
Dissolve 1.0 g in water R, heating on a water-bath, and solution using lead standard solution (1 ppm Pb) R.
dilute to 50.0 mL with the same solvent. Loss on drying (2.2.32) : maximum 7.0 per cent, determined
B. Infrared absorption spectrophotometry (2.2.24). on 0.200 g by heating in an oven at 105 ± 2 °C for 5 h.
Comparison : dextran CRS. Sulfated ash (2.4.14) : maximum 0.3 per cent, determined on
0.50 g.
C. Molecular-mass distribution (see Tests).
Bacterial endotoxins (2.6.14) : less than 16 IU/g.
TESTS
Microbial contamination
Solution S. Dissolve 5.0 g in distilled water R, heating on a TAMC : acceptance criterion 102 CFU/g (2.6.12).
water-bath, and dilute to 50 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II). 01/2014:2238
Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of
phenolphthalein solution R. The solution remains colourless. DEXTRANOMER
Add 0.2 mL of 0.01 M sodium hydroxide. The solution is
red. Add 0.4 mL of 0.01 M hydrochloric acid. The solution is Dextranomerum
colourless. Add 0.1 mL of methyl red solution R. The solution
is red or orange. [56087-11-7]
Nitrogen-containing substances : maximum 110 ppm of N. DEFINITION
Carry out the determination of nitrogen by sulfuric acid Three-dimensional network made of dextran chains
digestion (2.5.9), using 0.200 g and heating for 2 h. Collect O,O′-cross-linked with 2-hydroxypropane-1,3-diyl
the distillate in a mixture of 0.5 mL of bromocresol green bridges and O-substituted with 2,3-dihydroxypropyl and
solution R, 0.5 mL of methyl red solution R and 20 mL of 2-hydroxy-1-(hydroxymethyl)ethyl groups.
water R. Titrate with 0.01 M hydrochloric acid. Not more than
0.15 mL of 0.01 M hydrochloric acid is required to change the CHARACTERS
colour of the indicator. Appearance : white or almost white, spherical beads.
Residual solvents. Gas chromatography (2.2.28). Solubility : practically insoluble in water. It swells in water and
Internal standard : propanol R. in electrolyte solutions.

General Notices (1) apply to all monographs and other texts 2023
Dextrin EUROPEAN PHARMACOPOEIA 8.0

PRODUCTION IDENTIFICATION
The absorption capacity is determined using a 9.0 g/L solution A. Suspend 1 g in 50 mL of water R, boil for 1 min and cool.
of sodium chloride R containing 20 μL/L of polysorbate 20 R or To 1 mL of the solution add 0.05 mL of iodine solution R1.
another suitable solution, with a suitable, validated method. A dark blue or reddish-brown colour is produced, which
The particle size is controlled to a minimum of 80 per cent of disappears on heating.
the number of dry beads within 100-300 μm and a maximum B. Centrifuge 5 mL of the mucilage obtained in identification
of 7 per cent of their number below 100 μm using a suitable, test A. To the upper layer add 2 mL of dilute sodium
validated method. hydroxide solution R and, dropwise with shaking, 0.5 mL
of copper sulfate solution R and boil. A red precipitate is
IDENTIFICATION produced.
A. The substance to be examined is practically insoluble in C. It is very soluble in boiling water R, forming a mucilaginous
water R. It swells in water R. solution.
B. Infrared absorption spectrophotometry (2.2.24).
TESTS
Preparation : grind the substance to be examined in
acetone R. Evaporate the solvent at room temperature and pH (2.2.3) : 2.0 to 8.0.
use the residue. Disperse 5.0 g in 100 mL of carbon dioxide-free water R.
Comparison : dextranomer CRS. Chlorides : maximum 0.2 per cent.
Dissolve 2.5 g in 50 mL of boiling water R, dilute to 100 mL
TESTS
with water R and filter. Dilute 1 mL of the filtrate to 15 mL,
pH (2.2.3) : 5.3 to 7.5. add 1 mL of dilute nitric acid R, pour the mixture as a
Introduce 0.50 g to 30 mL of a freshly prepared 74.6 g/L single addition into 1 mL of silver nitrate solution R2 and
solution of potassium chloride R. Allow to stand for 2 min. allow to stand for 5 min protected from light. When viewed
Determine the pH on the mucilage obtained. transversely against a black background any opalescence
Boron : maximum 30 ppm. produced is not more intense than that obtained by treating a
mixture of 10 mL of chloride standard solution (5 ppm Cl) R
Inductively coupled plasma-atomic emission spectrometry and 5 mL of water R, prepared in the same manner.
(ICP-AES) (2.2.57).
Reducing sugars : maximum 10 per cent, calculated as glucose
Test solution. Introduce 3.0 g into a platinum dish and moisten C H O .
with 5 mL of a 32.1 g/L solution of magnesium nitrate R in 6 12 6

a mixture of equal volumes of ethanol (96 per cent) R and To a quantity of dextrin equivalent to 2.0 g (dried substance)
distilled water R. Evaporate to dryness on a water-bath. Ignite add 100 mL of water R, shake for 30 min, dilute to 200.0 mL
at 550 °C for 5 h. Take up the residue with 5 mL of 6 M with water R and filter. To 10.0 mL of alkaline cupri-tartaric
hydrochloric acid R and transfer to a 50 mL volumetric flask. solution R add 20.0 mL of the filtrate, mix, and heat on a hot
Add about 20 mL of distilled water R and allow to digest for plate adjusted to bring the solution to boil within 3 min. Boil
1 h on a water-bath. Allow to cool and dilute to 50.0 mL with for 2 min, and cool immediately. Add 5 mL of a 300 g/L
distilled water R. solution of potassium iodide R and 10 mL of 1 M sulfuric acid,
mix, and titrate immediately with 0.1 M sodium thiosulfate,
Reference solutions. Prepare the reference solutions using a using starch solution R, added towards the end of the titration,
solution of boric acid R containing 10 ppm of boron. Proceed as indicator. Repeat the procedure beginning with “To
as described for the test solution. 10.0 mL of...”, using, in place of the filtrate, 20.0 mL of a 1 g/L
Wavelength : 249.773 nm. solution of glucose R, accurately prepared. Perform a blank
Heavy metals (2.4.8) : maximum 30 ppm. titration. (VB − VU) is not greater than (VB − VS), in which
VB, VU and VS are the number of millilitres of 0.1 M sodium
1.0 g complies with test F. Prepare the reference solution using
thiosulfate consumed in the titrations of the blank, the dextrin
3 mL of lead standard solution (10 ppm Pb) R.
and the glucose, respectively.
Loss on drying (2.2.32) : maximum 10.0 per cent, determined
Heavy metals (2.4.8) : maximum 20 ppm.
on 1.000 g by drying in an oven at 105 °C for 15 h.
1.0 g complies with test C. Prepare the reference solution using
Sulfated ash (2.4.14) : maximum 0.4 per cent, determined on 2 mL of lead standard solution (10 ppm Pb) R.
1.0 g.
Loss on drying (2.2.32) : maximum 13.0 per cent, determined
Microbial contamination on 1.000 g by drying at 130-135 °C for 90 min.
2
TAMC : acceptance criterion 10 CFU/g (2.6.12), determined
Sulfated ash (2.4.14) : maximum 0.5 per cent, determined on
using the pour-plate method. 1.0 g.
FUNCTIONALITY-RELATED CHARACTERISTICS
04/2009:1507 This section provides information on characteristics that are
recognised as being relevant control parameters for one or
DEXTRIN more functions of the substance when used as an excipient (see
chapter 5.15). This section is a non-mandatory part of the
Dextrinum monograph and it is not necessary to verify the characteristics
to demonstrate compliance. Control of these characteristics can
DEFINITION however contribute to the quality of a medicinal product by
Maize, potato or cassava starch partly hydrolysed and modified improving the consistency of the manufacturing process and
by heating with or without the presence of acids, alkalis or the performance of the medicinal product during use. Where
pH-control agents. control methods are cited, they are recognised as being suitable
for the purpose, but other methods can also be used. Wherever
CHARACTERS results for a particular characteristic are reported, the control
Appearance : white or almost white, free-flowing powder. method must be indicated.
Solubility : very soluble in boiling water forming a The following characteristics may be relevant for dextrin used
mucilaginous solution, slowly soluble in cold water, practically as filler and binder, in tablets and capsules.
insoluble in ethanol (96 per cent). Particle-size distribution (2.9.31 or 2.9.38).

2024 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dextromethorphan hydrobromide

Powder flow (2.9.36). Results : the principal spot in the chromatogram obtained
The following characteristic may be relevant for dextrin used with the test solution is similar in position and size to the
as viscosity-increasing agent. principal spot in the chromatogram obtained with the
reference solution.
Apparent viscosity (2.2.10) : typically 100 mPa·s to 350 mPa·s
(dried substance), depending on the grade of dextrin. D. It gives reaction (a) of bromides (2.3.1).
In a beaker, prepare a 10-50 per cent slurry so that the viscosity TESTS
value ranges from 100 mPa·s to 350 mPa·s. The total mass of Solution S. Dissolve 1.0 g in ethanol (96 per cent) R and dilute
the sample plus water must be 600 g. Mix with a plastic rod to to 20 mL with the same solvent.
obtain a homogeneous slurry. Place the beaker in a water-bath
at 100 ± 1 °C. Introduce the paddle of a stirrer into the beaker Appearance of solution. Solution S is clear (2.2.1) and
and close the beaker with a lid. Start agitation at 250 r/min as colourless (2.2.2, Method II).
rapidly as possible and carry on for exactly 30 min. Transfer Acidity or alkalinity. Dissolve 0.4 g in carbon dioxide-free
the paste immediately to the beaker to be used for viscosity water R with gentle heating, cool and dilute to 20 mL with the
measurement, placed in a water-bath at 40 ± 1 °C. Stir until same solvent. Add 0.1 mL of methyl red solution R and 0.2 mL
the temperature in the beaker is 40 ± 1 °C then measure the of 0.01 M sodium hydroxide. The solution is yellow. Not more
apparent viscosity using spindle no. 2 and a rotation speed than 0.4 mL of 0.01 M hydrochloric acid is required to change
of 100 r/min. the colour of the indicator to red.
Specific optical rotation (2.2.7) : + 28 to + 30 (anhydrous
07/2010:0020 substance).
Dissolve 0.200 g in 0.1 M hydrochloric acid and dilute to
DEXTROMETHORPHAN 10.0 mL with the same acid.
HYDROBROMIDE Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 10.0 mg of the substance to be
Dextromethorphani hydrobromidum examined in the mobile phase and dilute to 10.0 mL with the
mobile phase.
Reference solution (a). Dissolve 2 mg of dextromethorphan
impurity A CRS in 2 mL of the test solution and dilute to
25.0 mL with the mobile phase.
Reference solution (b). Dilute 1.0 mL of the test solution to
200.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
C18H26BrNO,H2O Mr 370.3 – stationary phase : octadecylsilyl silica gel for
[6700-34-1] chromatography R (5 μm).
DEFINITION Mobile phase : dissolve 3.11 g of docusate sodium R in a
ent-3-Methoxy-17-methylmorphinan hydrobromide mixture of 400 mL of water R and 600 mL of acetonitrile R,
monohydrate. add 0.56 g of ammonium nitrate R and adjust to apparent
pH 2.0 with glacial acetic acid R.
Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
Flow rate : 1.0 mL/min.
CHARACTERS Detection : spectrophotometer at 280 nm.
Appearance : almost white, crystalline powder. Injection : 20 μL.
Solubility : sparingly soluble in water, freely soluble in ethanol Run time : twice the retention time of dextromethorphan.
(96 per cent). Relative retention with reference to dextromethorphan
mp : about 125 °C, with decomposition. (retention time = about 22 min) : impurity B = about 0.4 ;
impurity C = about 0.8 ; impurity D = about 0.9 ;
IDENTIFICATION impurity A = about 1.1.
First identification : A, B, D. System suitability : reference solution (a) :
Second identification : A, C, D. – resolution : minimum 1.5 between the peaks due to
A. Specific optical rotation (see Tests). dextromethorphan and impurity A.
B. Infrared absorption spectrophotometry (2.2.24). Limits :
Comparison : dextromethorphan hydrobromide CRS. – correction factor : for the calculation of content, multiply
C. Thin-layer chromatography (2.2.27). the peak area of impurity C by 0.2 ;
Test solution. Dissolve 25 mg of the substance to be – impurities A, B, C, D : for each impurity, not more than the
examined in methanol R and dilute to 10 mL with the same area of the principal peak in the chromatogram obtained
solvent. with reference solution (b) (0.5 per cent), and not more
Reference solution. Dissolve 25 mg of dextromethorphan than 1 such peak has an area greater than 0.5 times the area
hydrobromide CRS in methanol R and dilute to 10 mL with of the principal peak in the chromatogram obtained with
the same solvent. reference solution (b) (0.25 per cent) ;
Plate : TLC silica gel G plate R. – unspecified impurities : for each impurity, not more than
0.2 times the area of the principal peak in the chromatogram
Mobile phase : concentrated ammonia R, methylene obtained with reference solution (b) (0.10 per cent) ;
chloride R, methanol R, ethyl acetate R, toluene R
(2:10:13:20:55 V/V/V/V/V). – total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (b)
Application : 5 μL. (1.0 per cent) ;
Development : over 2/3 of the plate. – disregard limit : 0.1 times the area of the principal peak in
Drying : in air. the chromatogram obtained with reference solution (b)
Detection : spray with potassium iodobismuthate solution R2. (0.05 per cent).

General Notices (1) apply to all monographs and other texts 2025
Dextromoramide tartrate EUROPEAN PHARMACOPOEIA 8.0

N,N-Dimethylaniline : maximum 10 ppm. 01/2008:0021

corrected 6.0
Dissolve 0.5 g with heating in 20 mL of water R. Allow to
cool, add 2 mL of dilute acetic acid R and 1 mL of a 10 g/L
solution of sodium nitrite R and dilute to 25 mL with water R. DEXTROMORAMIDE TARTRATE
The solution is not more intensely coloured than a reference
solution prepared at the same time and in the same manner Dextromoramidi tartras
using 20 mL of a 0.25 mg/L solution of N,N-dimethylaniline R.
Water (2.5.12) : 4.0 per cent to 5.5 per cent, determined on
0.200 g.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.

ASSAY
Dissolve 0.300 g in a mixture of 5.0 mL of 0.01 M hydrochloric
acid and 20 mL of ethanol (96 per cent) R. Titrate with
0.1 M sodium hydroxide, determining the end-point
C29H38N2O8 Mr 542.6
potentiometrically (2.2.20). Read the volume added between
[2922-44-3]
the 2 points of inflexion.
1 mL of 0.1 M sodium hydroxide is equivalent to 35.23 mg DEFINITION
of C18H26BrNO. Dextromoramide tartrate contains not less than 98.0 per cent
and not more than the equivalent of 101.0 per cent of 1-[(3S)-3-
STORAGE methyl-4-(morpholin-4-yl)-2,2-diphenylbutanoyl]pyrrolidine
hydrogen (2R,3R)-2,3-dihydroxybutanedioate, calculated with
Protected from light. reference to the dried substance.

IMPURITIES CHARACTERS
Specified impurities : A, B, C, D. A white or almost white, amorphous or crystalline powder,
soluble in water, sparingly soluble in alcohol.
It melts at about 190 °C, with slight decomposition.
IDENTIFICATION
A. Dissolve 75 mg in 1 M hydrochloric acid and dilute to
100.0 mL with the same acid. Examined between 230 nm
and 350 nm (2.2.25), the solution shows 3 absorption
maxima, at 254 nm, 259 nm and 264 nm. The specific
absorbances at the maxima are about 6.9, 7.7 and 6.5,
A. ent-3-methoxymorphinan, respectively.
B. Dissolve about 50 mg in water R and dilute to 10 mL
with the same solvent. To 2 mL of the solution add 3 mL
of ammoniacal silver nitrate solution R and heat on a
water-bath. A grey or black precipitate is formed.
C. It gives reaction (b) of tartrates (2.3.1).
TESTS
pH (2.2.3). Dissolve 0.2 g in carbon dioxide-free water R and
dilute to 20 mL with the same solvent. The pH of the solution
is 3.0 to 4.0.
B. ent-17-methylmorphinan-3-ol,
Specific optical rotation (2.2.7). Dissolve 0.50 g in 0.1 M
hydrochloric acid and dilute to 10.0 mL with the same acid.
The specific optical rotation is + 21 to + 23.
Related substances. Examine by thin-layer chromatography
(2.2.27), using silica gel G R as the coating substance.
Test solution. Dissolve 0.2 g of the substance to be examined
in methanol R and dilute to 10 mL with the same solvent.
Reference solution. Dilute 1 mL of the test solution to 100 mL
with methanol R.
C. ent-3-methoxy-17-methylmorphinan-10-one, Apply separately to the plate 10 μL of each solution. Develop
over a path of 15 cm using methanol R. Allow the plate to
dry in air and spray with dilute potassium iodobismuthate
solution R. Any spot in the chromatogram obtained with
the test solution, apart from the principal spot, is not more
intense than the spot in the chromatogram obtained with the
reference solution (1.0 per cent).
Loss on drying (2.2.32). Not more than 0.5 per cent,
determined on 1.00 g by drying in an oven at 105 °C.
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
D. ent-(14S)-3-methoxy-17-methylmorphinan. on 1.0 g.

2026 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dextropropoxyphene hydrochloride

ASSAY Reference solution (c). Dilute 1.0 mL of toluene R to 50.0 mL

with the solvent mixture. Dilute 1.0 mL of this solution to
Dissolve 0.250 g in 30 mL of anhydrous acetic acid R. Titrate
10.0 mL with the solvent mixture.
with 0.05 M perchloric acid using 0.15 mL of naphtholbenzein
solution R as indicator. Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
1 mL of 0.05 M perchloric acid is equivalent to 27.13 mg of
C29H38N2O8. – stationary phase : octylsilyl silica gel for chromatography R
(5 μm).
Mobile phase :
01/2010:0713
– mobile phase A : dissolve 2.5 g of ammonium phosphate R
in water R, adjust to pH 5.6 with dilute phosphoric acid R
DEXTROPROPOXYPHENE and dilute to 1000 mL with the same solvent ;
HYDROCHLORIDE – mobile phase B : acetonitrile R1.
Time Mobile phase A Mobile phase B
Dextropropoxypheni hydrochloridum (min) (per cent V/V) (per cent V/V)
0-2 85 15

2-7 85 → 75 15 → 25

7 - 24 75 → 50 25 → 50

24 - 32 50 → 40 50 → 60

Flow rate : 1.5 mL/min.

Detection : spectrophotometer at 214 nm.
C22H30ClNO2 Mr 375.9 Injection : 10 μL.
[1639-60-7] Identification of impurities : use the chromatogram supplied
with dextropropoxyphene for system suitability CRS and
DEFINITION the chromatogram obtained with reference solution (b) to
(1S,2R)-1-Benzyl-3-(dimethylamino)-2-methyl-1- identify the peaks due to impurities A, B, C and D. Use the
phenylpropyl propanoate hydrochloride. chromatogram obtained with reference solution (c) to identify
Content : 98.5 per cent to 101.5 per cent (dried substance). the peak due to toluene.
Relative retention with reference to dextropropoxyphene
CHARACTERS (retention time = about 18 min) : impurity A = about 0.8 ;
Appearance : white or almost white, crystalline powder. impurity B = about 0.9 ; impurity D = about 1.1 ;
Solubility : very soluble in water, freely soluble in ethanol impurity C = about 1.2.
(96 per cent). System suitability : reference solution (b) :
mp : about 165 °C. – peak-to-valley ratio : minimum 5, where Hp = height
above the baseline of the peak due to impurity D and
IDENTIFICATION Hv = height above the baseline of the lowest point of
A. Specific optical rotation (see Tests). the curve separating this peak from the peak due to
B. Infrared absorption spectrophotometry (2.2.24). dextropropoxyphene.
Comparison : dextropropoxyphene hydrochloride CRS. Limits :
C. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1). – impurities A, B : for each impurity, not more than 5 times
the area of the principal peak in the chromatogram
TESTS obtained with reference solution (a) (0.5 per cent) ;
Solution S. Dissolve 1.5 g in carbon dioxide-free water R and – impurities C, D : for each impurity, not more than twice the
dilute to 30 mL with the same solvent. area of the principal peak in the chromatogram obtained
Appearance of solution. Solution S is clear (2.2.1) and with reference solution (a) (0.2 per cent) ;
colourless (2.2.2, Method II). – unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
Acidity or alkalinity. Dilute 10 mL of solution S to 25 mL with reference solution (a) (0.10 per cent) ;
with carbon dioxide-free water R. To 10 mL of this solution
add 0.1 mL of methyl red solution R and 0.2 mL of 0.01 M – total : not more than 10 times the area of the principal peak
sodium hydroxide. The solution is yellow. Add 0.4 mL of in the chromatogram obtained with reference solution (a)
0.01 M hydrochloric acid. The solution is red. (1.0 per cent) ;
Specific optical rotation (2.2.7) : + 52 to + 57. – disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
Dissolve 0.100 g in water R and dilute to 10.0 mL with the (0.05 per cent) ; disregard any peak due to toluene (relative
same solvent. retention = about 1.24).
Related substances. Liquid chromatography (2.2.29). Loss on drying (2.2.32) : maximum 1.0 per cent, determined
Solvent mixture : acetonitrile R, methanol R (50:50 V/V). on 1.000 g by drying in an oven at 105 °C for 4 h.
Test solution. Dissolve 0.100 g of the substance to be examined Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
in the solvent mixture and dilute to 50.0 mL with the solvent 1.0 g.
mixture.
Reference solution (a). Dilute 1.0 mL of the test solution ASSAY
to 50.0 mL with the solvent mixture. Dilute 1.0 mL of this Dissolve 0.270 g in 60 mL of acetic anhydride R. Titrate
solution to 20.0 mL with the solvent mixture. with 0.1 M perchloric acid, determining the end-point
Reference solution (b). Dissolve 2 mg of dextropropoxyphene potentiometrically (2.2.20).
for system suitability CRS (containing impurities A, B, C 1 mL of 0.1 M perchloric acid is equivalent to 37.59 mg of
and D) in 1.0 mL of the solvent mixture. C22H30ClNO2.

General Notices (1) apply to all monographs and other texts 2027
Diacerein EUROPEAN PHARMACOPOEIA 8.0

STORAGE CHARACTERS
Protected from light. Appearance : yellow, crystalline powder.
Solubility : practically insoluble in water, soluble in
IMPURITIES
dimethylacetamide, slightly soluble in tetrahydrofuran,
Specified impurities : A, B, C, D. practically insoluble in anhydrous ethanol.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of IDENTIFICATION
the tests in the monograph. They are limited by the general Infrared absorption spectrophotometry (2.2.24).
acceptance criterion for other/unspecified impurities and/or Comparison: diacerein CRS.
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these TESTS
impurities for demonstration of compliance. See also 5.10. Impurities B and H. Liquid chromatography (2.2.29).
Control of impurities in substances for pharmaceutical use) : F.Carry out the test protected from light.
Solution A. Dissolve 10 g of sodium hydroxide R in 500 mL
of water R.
Solution B. Dissolve 14.7 g of sodium chloride R and 18.8 g of
glycine R in 500 mL of water R.
Solution C. Mix 25.3 volumes of solution A and 74.6 volumes
of solution B. If necessary, adjust to pH 9.5 using dilute sodium
hydroxide solution R or dilute sulfuric acid R.
Solution D. Dilute 5 mL of dilute sulfuric acid R to 500 mL
A. R = H : (2S,3R)-4-(dimethylamino)-1,2-diphenyl-3-methyl- with water R.
butan-2-ol (oxyphene), Test solution. Dissolve 0.100 g of the substance to be examined
B. R = CO-CH3 : (1S,2R)-1-benzyl-3-(dimethylamino)-2- in 30 mL of solution A, mix for 10 min. Add 70 mL of
methyl-1-phenylpropyl acetate (acetoxyphene), solution B and adjust to pH 9.5 with dilute sodium hydroxide
solution R or dilute sulfuric acid R, if necessary. Extract with
C. R = CO-CH2-CH2-CH3 : (1S,2R)-1-benzyl-3- 3 quantities, each of 25 mL, of methylene chloride R. Combine
(dimethylamino)-2-methyl-1-phenylpropyl butanoate the methylene chloride extracts and wash with 2 quantities,
(butyroxyphene), each of 8 mL, of solution C and then once with 10 mL of
solution D. Evaporate the organic layer to dryness at 33 °C,
completing the drying procedure using compressed air.
Dissolve the residue in 2.0 mL of the mobile phase.
Reference solution (a). Dissolve 7.5 mg of diacerein
impurity B CRS in tetrahydrofuran R and dilute to 25.0 mL
with the same solvent. Sonicate for not more than 30 s. Dilute
1.0 mL of the solution to 100.0 mL with solution A. Dilute
5.0 mL of this solution to 50.0 mL with solution A. Mix 5.0 mL
of this solution with 25 mL of solution A for 10 min. Add
D. (1S,2S)-1-benzyl-3-(dimethylamino)-2-methyl-1- 70 mL of solution B and adjust to pH 9.5 with dilute sodium
phenylpropyl propanoate (isopropoxyphene), hydroxide solution R or dilute sulfuric acid R, if necessary.
Perform the extraction as described for the test solution. Care
should be taken that the time between dissolution of diacerein
impurity B in tetrahydrofuran and extraction does not exceed
30 min.
Reference solution (b). Dilute 1.0 mL of reference solution (a)
F. (2RS)-3-(dimethylamino)-2-methyl-1-phenylpropan-1- to 5.0 mL with the mobile phase.
one. Column :
– size : l = 0.125 m, Ø = 4.6 mm ;
01/2014:2409 – stationary phase : irregular octadecylsilyl silica gel for
chromatography R (5 μm) ;
– temperature : 16 ± 1 °C.
DIACEREIN Mobile phase : tetrahydrofuran R, acetonitrile R, 4 g/L solution
of citric acid R (8:27.5:64.5 V/V/V).
Diacereinum Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 254 nm.
Injection : 100 μL.
Run time : 2.5 times the retention time of impurity B.
Retention time : impurity B = about 11 min.
System suitability : reference solution (b) :
– signal-to-noise ratio : minimum 10 for the principal peak.
C19H12O8 Mr 368.3 Limit :
[13739-02-1] – sum of impurities B and H : not more than the area of the
peak due to impurity B in the chromatogram obtained with
DEFINITION reference solution (a) (15 ppm).
4,5-Diacetoxy-9,10-dioxo-9,10-dihydroanthracene-2- Related substances. Liquid chromatography (2.2.29). Carry
carboxylic acid. out the test protected from light.
Content : 98.0 per cent to 102.0 per cent (dried substance). Solvent mixture : mobile phase A, mobile phase B (50:50 V/V).

2028 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Diacerein

Test solution. Dissolve 0.100 g of the substance to be examined – total : not more than 20 times the area of the principal peak
in 50 mL of tetrahydrofuran R and dilute to 100.0 mL with the in the chromatogram obtained with reference solution (a)
solvent mixture. (2.0 per cent) ;
Reference solution (a). Dilute 1.0 mL of the test solution – disregard limit : 0.5 times the area of the principal peak in
to 100.0 mL with tetrahydrofuran R. Dilute 1.0 mL of this the chromatogram obtained with reference solution (a)
solution to 10.0 mL with the solvent mixture. (0.05 per cent).
Reference solution (b). In order to prepare impurities D and E Chromium : maximum 10 ppm.
in situ, add 10.0 mL of 0.01 M sodium hydroxide to 0.100 g of Atomic absorption spectrometry (2.2.23, Method I).
the substance to be examined. Add 40 mL of tetrahydrofuran R
and dilute to 100.0 mL with the solvent mixture. Test solution. In a digestion bomb, dissolve 0.25 g of the
substance to be examined in a mixture of 2 mL of strong
Reference solution (c). Dissolve the contents of a vial of hydrogen peroxide solution R and 6 mL of nitric acid R.
diacerein impurity mixture CRS (impurities C and F) in a Mineralise using a microwave oven with a power-incrementing
mixture of 0.5 mL of tetrahydrofuran R and 0.5 mL of the system. Transfer quantitatively to a volumetric flask with
solvent mixture. water R and dilute to 50.0 mL with water R. Centrifuge. Dilute
Column : 5.0 mL of the clear supernatant to 50.0 mL with water R.
– size : l = 0.10 m, Ø = 4.6 mm ; Blank solution. Prepare as described for the test solution,
– stationary phase : end-capped polar-embedded octadecylsilyl omitting the substance to be examined.
amorphous organosilica polymer R (5 μm) ; Stock solution. Dilute 5.0 mL of chromium standard solution
– temperature : 30 °C. (100 ppm Cr) R to 50.0 mL with water R. Dilute 5.0 mL of
Mobile phase : this solution to 100.0 mL with water R. Dilute 2.0 mL of this
solution to 100.0 mL with a 0.12 per cent V/V solution of
– mobile phase A : to 353 mL of water R add 147 mL of dilute nitric acid R.
phosphoric acid R and mix ; dilute 2 mL of the solution to
1000 mL with water R ; Reference solutions. Prepare the reference solutions using the
stock solution, diluting with the blank solution.
– mobile phase B : acetonitrile R ;
Source : chromium hollow-cathode lamp using a transmission
Time Mobile phase A Mobile phase B band preferably of 0.2 nm.
(min) (per cent V/V) (per cent V/V) Wavelength : 357.9 nm.
0-3 80 20
Atomisation device : graphite furnace.
3 - 13 80 → 60 20 → 40
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
13 - 20 60 40 on 1.000 g by drying in an oven at 105 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Flow rate : 1.2 mL/min. 1.0 g.
Detection : spectrophotometer at 254 nm.
Injection : 20 μL. ASSAY
Identification of impurities : use the chromatogram supplied Liquid chromatography (2.2.29) as described in the test for
with diacerein impurity mixture CRS and the chromatogram related substances with the following modifications.
obtained with reference solution (c) to identify the peaks due Test solution. Dissolve 60.0 mg of the substance to be
to impurities C and F ; use the chromatogram obtained with examined in tetrahydrofuran R and dilute to 50.0 mL with the
reference solution (b) to identify the peaks due to impurities D same solvent. Dilute 2.0 mL of the solution to 25.0 mL with
and E. the solvent mixture.
Relative retention with reference to diacerein (retention Reference solution. Dissolve 60.0 mg of diacerein CRS in
time = about 13.5 min) : impurity D = about 1.1 ; tetrahydrofuran R and dilute to 50.0 mL with the same solvent.
impurity E = about 1.15 ; impurity C = about 1.2 ; Dilute 2.0 mL of the solution to 25.0 mL with the solvent
impurity F = about 1.3. mixture.
System suitability : Calculate the percentage content of C19H12O8 taking into
– resolution : minimum 1.5 between the peaks due to account the assigned content of diacerein CRS.
impurities D and E in the chromatogram obtained with STORAGE
reference solution (b);
– signal-to-noise ratio : minimum 100 for the principal peak In an airtight container, protected from light.
in the chromatogram obtained with reference solution (a). IMPURITIES
Limits : Specified impurities : B, C, D, E, F, H.
– correction factors : for the calculation of content, multiply Other detectable impurities (the following substances would,
the peak areas of the following impurities by the if present at a sufficient level, be detected by one or other of
corresponding correction factor : impurity C = 1.4 ; the tests in the monograph. They are limited by the general
impurity D = 1.3 ; impurity E = 1.3 ; impurity F = 9.5 ; acceptance criterion for other/unspecified impurities and/or
– impurities D, E : for each impurity, not more than 5 times by the general monograph Substances for pharmaceutical
the area of the principal peak in the chromatogram use (2034). It is therefore not necessary to identify these
obtained with reference solution (a) (0.5 per cent) ; impurities for demonstration of compliance. See also 5.10.
– impurity C : not more than twice the area of the principal Control of impurities in substances for pharmaceutical use) : G.
peak in the chromatogram obtained with reference
solution (a) (0.2 per cent) ;
– impurity F : not more than 1.5 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.15 per cent);
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained B. 1,8-dihydroxy-3-(hydroxymethyl)-anthracene-9,10-dione
with reference solution (a) (0.10 per cent) ; (aloe-emodin),

General Notices (1) apply to all monographs and other texts 2029
Diazepam EUROPEAN PHARMACOPOEIA 8.0

01/2008:0022

DIAZEPAM
Diazepamum
C. 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-
carboxylic acid (rhein),

C16H13ClN2O Mr 284.7
D. 5-acetoxy-4-hydroxy-9,10-dioxo-9,10-dihydroanthracene- [439-14-5]
2-carboxylic acid (monoacetyl rhein isomer A), DEFINITION
7-Chloro-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-
benzodiazepin-2-one.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : very slightly soluble in water, soluble in ethanol
E. 4-acetoxy-5-hydroxy-9,10-dioxo-9,10-dihydroanthracene- (96 per cent).
2-carboxylic acid (monoacetyl rhein isomer B), IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : diazepam CRS.
TESTS
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions protected from bright light.
Test solution. Dissolve 25.0 mg of the substance to be
examined in 0.5 mL of acetonitrile R and dilute to 50.0 mL
with the mobile phase.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10.0 mL with the mobile phase.
Reference solution (b). Dissolve the contents of a vial of
F. (10S)-3-(acetoxymethyl)-10-(2,3,4,6-tetra-O-acetyl-β-D-
diazepam for system suitability CRS (containing impurities A,
glucopyranosyl)-9-oxo-9,10-dihydroanthracene-1,8-diyl
B and E) in 1.0 mL of the mobile phase.
diacetate (heptaacetyl aloin, heptaacetyl barbaloin),
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : spherical end-capped octylsilyl silica gel for
chromatography R (5 μm) ;
– temperature : 30 °C.
Mobile phase : mix 22 volumes of acetonitrile R, 34 volumes of
methanol R and 44 volumes of a 3.4 g/L solution of potassium
dihydrogen phosphate R previously adjusted to pH 5.0 with
dilute sodium hydroxide solution R.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 254 nm.
Injection : 20 μL.
G. 3-(acetoxymethyl)-10-(2,3,4,6-tetra-O-acetyl-β-D- Run time : about 4 times the retention time of diazepam.
glucopyranosyl)anthracene-1,8,9-triyl triacetate, Identification of impurities : use the chromatogram
supplied with diazepam for system suitability CRS and the
chromatogram obtained with reference solution (b) to identify
the peaks due to impurities A, B and E.
Relative retention with reference to diazepam (retention
time = about 9 min) : impurity E = about 0.7 ; impurity A = about
0.8 ; impurity B = about 1.3.
System suitability : reference solution (b) :
– resolution : minimum 2.5 between the peaks due to
H. 3-(acetoxymethyl)-9,10-dioxo-9,10-dihydroanthracene- impurities E and A and minimum 6.0 between the peaks
1,8-diyl diacetate (triacetyl aloe-emodin). due to impurity A and diazepam.

2030 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Diazoxide

Limits :
– correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
corresponding correction factor : impurity B = 1.3 ;
impurity E = 1.3 ;
– impurities A, B, E : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent) ; C. 3-amino-6-chloro-1-methyl-4-phenylquinolin-2(1H)-one,
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
– total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.2 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent). E. 6-chloro-1-methyl-4-phenylquinazolin-2(1H)-one,
Heavy metals (2.4.8) : maximum 20 ppm.
2.0 g complies with test C. Prepare the reference solution using
4 mL of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in vacuo at 60 °C for 4 h.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY F. 7-chloro-2-methoxy-5-phenyl-3H-1,4-benzodiazepine.
Dissolve 0.200 g in 50 mL of acetic anhydride R. Titrate
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20). 01/2008:0550
corrected 6.0
1 mL of 0.1 M perchloric acid is equivalent to 28.47 mg
of C16H13ClN2O.
DIAZOXIDE
STORAGE
Protected from light. Diazoxidum
IMPURITIES
Specified impurities : A, B, E.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
C8H7ClN2O2S Mr 230.7
acceptance criterion for other/unspecified impurities and/or
[364-98-7]
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities DEFINITION
for demonstration of compliance. See also 5.10. Control of
Diazoxide contains not less than 98.0 per cent and
impurities in substances for pharmaceutical use) : C, D, F.
not more than the equivalent of 101.0 per cent of
7-chloro-3-methyl-2H-1,2,4-benzothiadiazine 1,1-dioxide,
calculated with reference to the dried substance.
CHARACTERS
A white or almost white, fine or crystalline powder, practically
insoluble in water, freely soluble in dimethylformamide,
slightly soluble in alcohol. It is very soluble in dilute solutions
of the alkali hydroxides.
A. 7-chloro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-
one (nordazepam), IDENTIFICATION
First identification : B.
Second identification : A, C, D.
A. Dissolve 50.0 mg in 5 mL of 1 M sodium hydroxide
and dilute to 50.0 mL with water R. Dilute 1.0 mL of
this solution to 100.0 mL with 0.1 M sodium hydroxide.
Examined between 230 nm and 350 nm (2.2.25), the
solution shows an absorption maximum at 280 nm and
a shoulder at 304 nm. The specific absorbance at the
maximum is 570 to 610.
B. R = CO-CH2-Cl : 2-chloro-N-(4-chloro-2-benzoylphenyl)-
N-methylacetamide, B. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
D. R = H : [5-chloro-2-(methylamino)phenyl]phenylmethan- diazoxide CRS. Examine the substances prepared as discs
one, using potassium bromide R.

General Notices (1) apply to all monographs and other texts 2031
Dibrompropamidine diisetionate EUROPEAN PHARMACOPOEIA 8.0

C. Examine the chromatograms obtained in the test for related 01/2008:2300

substances in ultraviolet light at 254 nm. The principal corrected 6.0
spot in the chromatogram obtained with test solution (b)
is similar in position and size to the principal spot in the
chromatogram obtained with reference solution (b).
DIBROMPROPAMIDINE
DIISETIONATE
D. Dissolve about 20 mg in a mixture of 5 mL of hydrochloric
acid R and 10 mL of water R. Add 0.1 g of zinc powder R. Dibrompropamidini diisetionas
Boil for 5 min, cool and filter. To the filtrate add 2 mL
of a 1 g/L solution of sodium nitrite R and mix. Allow
to stand for 1 min and add 1 mL of a 5 g/L solution of
naphthylethylenediamine dihydrochloride R. A red or
violet-red colour develops.

TESTS
Appearance of solution. Dissolve 0.4 g in 2 mL of 1 M sodium C21H30Br2N4O10S2 Mr 722
hydroxide and dilute to 20 mL with water R. The solution is [614-87-9]
clear (2.2.1) and not more intensely coloured than reference
solution Y7 (2.2.2, Method II). DEFINITION
Acidity or alkalinity. To 0.5 g of the powdered substance 3,3′-Dibromo-4,4′-(propane-1,3-diylbisoxy)dibenzimidamide
to be examined add 30 mL of carbon dioxide-free water R, bis(2-hydroxyethanesulfonate).
shake for 2 min and filter. To 10 mL of the filtrate add 0.2 mL Content : 99.0 per cent to 101.0 per cent (dried substance).
of 0.01 M sodium hydroxide and 0.15 mL of methyl red
solution R. The solution is yellow. Not more than 0.4 mL of PRODUCTION
0.01 M hydrochloric acid is required to change the colour of The production method must be evaluated to determine the
the indicator to red. potential for formation of alkyl 2-hydroxyethanesulfonates,
which is particularly likely to occur if the reaction
Related substances. Examine by thin-layer chromatography medium contains lower alcohols. Where necessary, the
(2.2.27), using silica gel GF254 R as the coating substance. production method is validated to demonstrate that alkyl
2-hydroxyethanesulfonates are not detectable in the final
Test solution (a). Dissolve 0.1 g of the substance to be
product.
examined in a mixture of 0.5 mL of 1 M sodium hydroxide and
1 mL of methanol R and dilute to 5 mL with methanol R. CHARACTERS
Test solution (b). Dilute 1 mL of test solution (a) to 5 mL with Appearance : white or almost white, crystalline powder.
a mixture of 1 volume of 1 M sodium hydroxide and 9 volumes Solubility : freely soluble or soluble in water, slightly soluble
of methanol R. in ethanol (96 per cent), practically insoluble in methylene
chloride.
Reference solution (a). Dilute 0.5 mL of test solution (a) to
100 mL with a mixture of 1 volume of 1 M sodium hydroxide IDENTIFICATION
and 9 volumes of methanol R. A. Infrared absorption spectrophotometry (2.2.24).
Comparison: dibrompropamidine diisetionate CRS.
Reference solution (b). Dissolve 20 mg of diazoxide CRS in
a mixture of 0.5 mL of 1 M sodium hydroxide and 1 mL of B. Mix 0.1 g with 0.5 g of anhydrous sodium carbonate R,
methanol R and dilute to 5 mL with methanol R. ignite and take up the residue with 20 mL of water R. Filter
and neutralise the filtrate to blue litmus paper R with nitric
Apply separately to the plate 5 μL of each solution. Develop acid R. The filtrate gives reaction (a) of bromides (2.3.1).
over a path of 15 cm using a mixture of 7 volumes of
TESTS
concentrated ammonia R, 25 volumes of methanol R and
68 volumes of chloroform R. Allow the plate to dry in air pH (2.2.3) : 5.0 to 6.0.
and examine in ultraviolet light at 254 nm. Any spot in the Dissolve 0.50 g in carbon dioxide-free water R and dilute to
chromatogram obtained with test solution (a), apart from 10 mL with the same solvent.
the principal spot, is not more intense than the spot in the
Related substances. Liquid chromatography (2.2.29).
chromatogram obtained with reference solution (a) (0.5 per
cent). Solvent mixture: anhydrous formic acid R, methanol R, ethyl
acetate R (0.01:8:12 V/V/V).
Loss on drying (2.2.32). Not more than 0.5 per cent,
Test solution. To 8 mL of methanol R add 20.0 mg of the
determined on 1.000 g by drying in an oven at 105 °C for 2 h.
substance to be examined and dissolve with the aid of an
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined ultrasonic bath. Add 11 mL of ethyl acetate R then 10 μL of
on 1.0 g. anhydrous formic acid R and mix. Dilute to 20.0 mL with ethyl
acetate R.
Reference solution (a). Dilute 1.0 mL of the test solution to
ASSAY 100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Dissolve 0.200 g with gentle heating in 50 mL of a mixture of Reference solution (b). Dissolve 10 mg of dibrompropamidine
1 volume of water R and 2 volumes of dimethylformamide R. for system suitability CRS (containing impurities A and B) in
Titrate with 0.1 M sodium hydroxide, determining the 4 mL of methanol R using an ultrasonic bath. Add 5 mL of
end-point potentiometrically (2.2.20). Carry out a blank ethyl acetate R then 5 μL of anhydrous formic acid R and mix.
titration. Dilute to 10.0 mL with ethyl acetate R.
1 mL of 0.1 M sodium hydroxide is equivalent to 23.07 mg Column :
of C8H7ClN2O2S. – size : l = 0.25 m, Ø = 4.6 mm,

2032 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dibutyl phthalate

– stationary phase : strong cation-exchange silica gel for 01/2008:0762

chromatography R (5 μm).
Mobile phase : mix 4 volumes of a 25 g/L solution of ammonium DIBUTYL PHTHALATE
formate R in methanol R and 6 volumes of ethyl acetate R.
Flow rate : 1 mL/min. Dibutylis phthalas
Detection : spectrophotometer at 254 nm.
Injection : 40 μL.
Run time : 1.5 times the retention time of dibrompropamidine.
Identification of impurities : use the chromatogram supplied
with dibrompropamidine for system suitability CRS and the
chromatogram obtained with reference solution (b) to identify C16H22O4 Mr 278.3
the peaks due to impurities A and B. [84-74-2]
Relative retention with reference to dibrompropamidine
(retention time = about 20 min) : impurity A = about 0.4 ; DEFINITION
impurity B = about 1.1. Dibutyl benzene-1,2-dicarboxylate.
System suitability : reference solution (b) : Content : 99.0 per cent m/m to 101.0 per cent m/m.
– peak-to-valley ratio : minimum 1.5, where Hp = height CHARACTERS
above the baseline of the peak due to impurity B and Appearance : clear, oily liquid, colourless or very slightly
Hv = height above the baseline of the lowest point of yellow.
the curve separating this peak from the peak due to
dibrompropamidine. Solubility : practically insoluble in water, miscible with ethanol
(96 per cent).
Limits :
IDENTIFICATION
– impurity A : not more than 3 times the area of the principal
peak in the chromatogram obtained with reference First identification : B, C.
solution (a) (0.3 per cent) ; Second identification : A, D, E.
– impurity B : not more than 5 times the area of the principal
A. Relative density (2.2.5) : 1.043 to 1.048.
peak in the chromatogram obtained with reference B. Refractive index (2.2.6) : 1.490 to 1.495.
solution (a) (0.5 per cent) ; C. Infrared absorption spectrophotometry (2.2.24).
– unspecified impurities : for each impurity, not more than theComparison: dibutyl phthalate CRS.
area of the principal peak in the chromatogram obtained
D. Thin-layer chromatography (2.2.27).
with reference solution (a) (0.1 per cent) ;
Test solution. Dissolve 50 mg of the substance to be
– total : not more than 10 times the area of the principal peakexamined in ether R and dilute to 10 mL with the same
in the chromatogram obtained with reference solution (a) solvent.
(1.0 per cent) ;
Reference solution. Dissolve 50 mg of dibutyl phthalate CRS
– disregard limit : 0.5 times the area of the principal peak inin ether R and dilute to 10 mL with the same solvent.
the chromatogram obtained with reference solution (a) Plate : TLC silica gel GF254 plate R.
(0.05 per cent).
Mobile phase : heptane R, ether R (30:70 V/V).
Loss on drying (2.2.32) : maximum 2.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C. Application : 10 μL.
Development : over a path of 15 cm.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. Drying : in air.
Detection : examine in ultraviolet light at 254 nm.
ASSAY Results : the principal spot in the chromatogram obtained
Dissolve 0.250 g in 50 mL of dimethylformamide R. with the test solution is similar in position and size to the
Titrate with 0.1 M tetrabutylammonium hydroxide under principal spot in the chromatogram obtained with the
a current of nitrogen R, determining the end-point reference solution.
potentiometrically (2.2.20). E. To about 0.1 mL add 0.25 mL of sulfuric acid R and 50 mg
1 mL of 0.1 M tetrabutylammonium hydroxide is equivalent to of resorcinol R. Heat in a water-bath for 5 min. Allow to
36.12 mg of C21H30Br2N4O10S2. cool. Add 10 mL of water R and 1 mL of strong sodium
hydroxide solution R. The solution becomes yellow or
IMPURITIES brownish-yellow and shows a green fluorescence.
Specified impurities : A, B. TESTS
Appearance. The substance to be examined is clear (2.2.1)
and not more intensely coloured than reference solution Y6
(2.2.2, Method II).
Acidity. Dissolve 20.0 g in 50 mL of ethanol (96 per cent) R
previously neutralised to phenolphthalein solution R1. Add
0.2 mL of phenolphthalein solution R1. Not more than 0.50 mL
of 0.1 M sodium hydroxide is required to change the colour of
A. R = Br, X = O : 3-bromo-4-[3-(2-bromo-4- the indicator.
carbamimidoylphenoxy)propoxy]benzamide, Related substances. Gas chromatography (2.2.28).
Internal standard solution. Dissolve 60 mg of bibenzyl R
B. R = H, X = NH : 3-bromo-4-[3-(4-carbamimidoylphe- in methylene chloride R and dilute to 20 mL with the same
noxy)propoxy]benzimidamide. solvent.

General Notices (1) apply to all monographs and other texts 2033
Diclazuril for veterinary use EUROPEAN PHARMACOPOEIA 8.0

Test solution (a). Dissolve 1.0 g of the substance to be 01/2008:1718

examined in methylene chloride R and dilute to 20.0 mL with corrected 7.0
the same solvent.
Test solution (b). Dissolve 1.0 g of the substance to be DICLAZURIL FOR VETERINARY USE
examined in methylene chloride R, add 2.0 mL of the internal
standard solution and dilute to 20.0 mL with methylene Diclazurilum ad usum veterinarium
chloride R.
Reference solution. To 1.0 mL of test solution (a) add 10.0 mL
of the internal standard solution and dilute to 100.0 mL with
methylene chloride R.
Column :
– material : glass ;
C17H9Cl3N4O2 Mr 407.6
– size : l = 1.5 m, Ø = 4 mm ; [101831-37-2]
– stationary phase : silanised diatomaceous earth for gas DEFINITION
chromatography R (150-180 μm) impregnated with 3 per
(RS)-(4-Chlorophenyl)[2,6-dichloro-4-(3,5-dioxo-4,5-
cent m/m of polymethylphenylsiloxane R.
dihydro-1,2,4-triazin-2(3H)-yl)phenyl]acetonitrile.
Carrier gas : nitrogen for chromatography R. Content : 99.0 per cent to 101.0 per cent (dried substance).
Flow rate : 30 mL/min.
CHARACTERS
Temperature : Appearance : white or light yellow powder.
– column : 190 °C ; Solubility : practically insoluble in water, sparingly soluble
in dimethylformamide, practically insoluble in alcohol and
– injection port and detector : 225 °C.
methylene chloride.
Detection : flame ionisation.
IDENTIFICATION
Injection : 1 μL.
Infrared absorption spectrophotometry (2.2.24).
Run time : 3 times the retention time of dibutyl phthalate. Comparison: Ph. Eur. reference spectrum of diclazuril.
Elution order : bibenzyl, dibutyl phthalate. TESTS
Retention time : dibutyl phthalate = about 12 min. Related substances. Liquid chromatography (2.2.29).
System suitability : Test solution. Dissolve 20.0 mg of the substance to be
– resolution : minimum 12 between the peaks due to bibenzyl examined in dimethylformamide R and dilute to 20.0 mL with
and dibutyl phthalate in the chromatogram obtained with the same solvent.
the reference solution ; Reference solution (a). Dissolve 5 mg of diclazuril for system
suitability CRS in dimethylformamide R and dilute to 5.0 mL
– in the chromatogram obtained with test solution (a), there with the same solvent.
is no peak with the same retention time as the internal
standard. Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with dimethylformamide R. Dilute 5.0 mL of the
Limit : solution to 20.0 mL with dimethylformamide R.
– total : calculate the ratio (R) of the area of the peak due Column :
to dibutyl phthalate to the area of the peak due to the – size : l = 0.10 m, Ø = 4.6 mm,
internal standard from the chromatogram obtained with – stationary phase : base-deactivated octadecylsilyl silica gel for
the reference solution ; from the chromatogram obtained chromatography R (3 μm),
with test solution (b), calculate the ratio of the sum of the – temperature : 35 °C.
areas of any peaks, apart from the principal peak and the
peak due to the internal standard, to the area of the peak Mobile phase :
due to the internal standard : this ratio is not greater than – mobile phase A : mix 10 volumes of a 6.3 g/L solution of
R (1.0 per cent). ammonium formate R adjusted to pH 4.0 with anhydrous
formic acid R, 15 volumes of acetonitrile R and 75 volumes
Water (2.5.12) : maximum 0.2 per cent, determined on 10.00 g. of water R,
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on – mobile phase B : mix 10 volumes of a 6.3 g/L solution of
1.0 g. ammonium formate R adjusted to pH 4.0 with anhydrous
formic acid R, 85 volumes of acetonitrile R and 5 volumes
ASSAY of water R,
Introduce 0.750 g into a 250 mL borosilicate glass flask. Add Time Mobile phase A Mobile phase B
25.0 mL of 0.5 M alcoholic potassium hydroxide and a few (min) (per cent V/V) (per cent V/V)
glass beads. Heat in a water-bath under a reflux condenser 0 - 20 100 → 0 0 → 100
for 1 h. Add 1 mL of phenolphthalein solution R1 and titrate
20 - 25 0 100
immediately with 0.5 M hydrochloric acid until the colour
changes from red to colourless. Carry out a blank titration. Flow rate : 1.0 mL/min.
Calculate the volume of potassium hydroxide used in the
Detection : spectrophotometer at 230 nm.
saponification.
Injection : 5 μL.
1 mL of 0.5 M alcoholic potassium hydroxide is equivalent to System suitability : reference solution (a) :
69.59 mg of C16H22O4.
– peak-to-valley ratio : minimum of 1.5, where Hp = height
above the baseline of the peak due to impurity D and
STORAGE Hv = height above the baseline of the lowest point of the
In an airtight container. curve separating this peak from the peak due to diclazuril.

2034 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Diclofenac potassium

Limits :
– correction factors : for the calculation of contents,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity D = 1.9 ;
impurity H = 1.4, E. R = NH2 : (RS)-(4-amino-2,6-dichlorophenyl)(4-
– impurity D : not more than 0.4 times the area of the chlorophenyl)acetonitrile,
principal peak in the chromatogram obtained with H. R = H : (RS)-(4-chlorophenyl)(2,6-dichlorophenyl)-
reference solution (b) (0.1 per cent), acetonitrile,
– any other impurity : not more than the area of the principal
peak in the chromatogram obtained with reference
solution (b) (0.25 per cent),
– total : not more than 4 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(1.0 per cent),
– disregard limit : 0.2 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined I. N,2-bis[3,5-dichloro-4-[(4-chlorophenyl)cyanomethyl]-
on 1.000 g by drying in an oven at 105 °C for 4 h. phenyl]-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-
carboxamide.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
01/2008:1508
ASSAY
Dissolve 0.150 g in 75 mL of dimethylformamide R. DICLOFENAC POTASSIUM
Carry out a potentiometric titration (2.2.20), using 0.1 M
tetrabutylammonium hydroxide. Read the volume added at Diclofenacum kalicum
the second inflexion point. Carry out a blank titration.
1 mL of 0.1 M tetrabutylammonium hydroxide is equivalent
to 20.38 mg of C17H9Cl3N4O2.
STORAGE
Protected from light.
IMPURITIES
Specified impurities : A, B, C, D, E, F, G, H, I. C14H10Cl2KNO2 Mr 334.2
[15307-81-0]
DEFINITION
Potassium [2-[(2,6-dichlorophenyl)amino]phenyl]acetate.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or slightly yellowish, slightly hygroscopic,
crystalline powder.
A. R = Cl, R′ = CO2H : 2-[3,5-dichloro-4-[(RS)-(4- Solubility : sparingly soluble in water, freely soluble in
chlorophenyl)cyanomethyl]phenyl]-3,5-dioxo-2,3,4,5- methanol, soluble in ethanol (96 per cent), slightly soluble
tetrahydro-1,2,4-triazine-6-carboxylic acid, in acetone.
B. R = OH, R′ = H : (RS)-[2,6-dichloro-4-(3,5-dioxo- IDENTIFICATION
4,5-dihydro-1,2,4-triazin-2(3H)-yl)phenyl](4-
hydroxyphenyl)acetonitrile, First identification : A, D.
Second identification : B, C, D.
C. R = Cl, R′ = CONH2 : 2-[3,5-dichloro-4-[(RS)-(4- A. Infrared absorption spectrophotometry (2.2.24).
chlorophenyl)cyanomethyl]phenyl]-3,5-dioxo-2,3,4,5- Preparation : discs.
tetrahydro-1,2,4-triazine-6-carboxamide,
Comparison: diclofenac potassium CRS.
G. R = Cl, R′ = CO-O-[CH2]3-CH3 : butyl 2-[3,5-dichloro-4- B. Thin-layer chromatography (2.2.27).
[(RS)-(4-chlorophenyl)cyanomethyl]phenyl]-3,5-dioxo- Test solution. Dissolve 25 mg of the substance to be
2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxylate, examined in methanol R and dilute to 5 mL with the same
solvent.
Reference solution (a). Dissolve 25 mg of diclofenac
potassium CRS in methanol R and dilute to 5 mL with the
same solvent.
Reference solution (b). Dissolve 10 mg of indometacin R
in reference solution (a) and dilute to 2 mL with the same
solution.
D. X = O : 2-[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]-1,2,4- Plate : TLC silica gel GF254 plate R.
triazine-3,5(2H,4H)-dione,
Mobile phase : concentrated ammonia R, methanol R, ethyl
F. X = H2 : 2-[3,5-dichloro-4-(4-chlorobenzyl)phenyl]-1,2,4- acetate R (10:10:80 V/V/V).
triazine-3,5(2H,4H)-dione, Application : 5 μL.

General Notices (1) apply to all monographs and other texts 2035
Diclofenac sodium EUROPEAN PHARMACOPOEIA 8.0

Development : over a path of 10 cm. Heavy metals (2.4.8) : maximum 10 ppm.

Drying : in air. 2.0 g complies with test C. Use a quartz crucible. Prepare
Detection : examine in ultraviolet light at 254 nm. the reference solution using 2 mL of lead standard solution
(10 ppm Pb) R.
System suitability : reference solution (b):
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
– the chromatogram shows 2 clearly separated spots. on 1.000 g by drying in an oven at 105 °C for 3 h.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to ASSAY
the principal spot in the chromatogram obtained with Dissolve 0.250 g in 30 mL of anhydrous acetic acid R. Titrate
reference solution (a). with 0.1 M perchloric acid, determining the end-point
C. Dissolve about 10 mg in 10 mL of ethanol (96 per cent) R. potentiometrically (2.2.20).
To 1 mL of this solution add 0.2 mL of a mixture, prepared 1 mL of 0.1 M perchloric acid is equivalent to 33.42 mg
immediately before use, of equal volumes of a 6 g/L solution of C14H10Cl2KNO2.
of potassium ferricyanide R and a 9 g/L solution of ferric
chloride R. Allow to stand protected from light for 5 min. STORAGE
Add 3 mL of a 10 g/L solution of hydrochloric acid R. Allow In an airtight container, protected from light.
to stand protected from light for 15 min. A blue colour
develops and a precipitate is formed. IMPURITIES
D. Suspend 0.5 g in 10 mL of water R. Stir and add water R Specified impurities : A, B, C, D, E.
until the substance is dissolved. Add 2 mL of hydrochloric
acid R1, stir for 1 h and filter with the aid of vacuum.
Neutralise with sodium hydroxide solution R. The solution
gives reaction (b) of potassium (2.3.1).
TESTS
Appearance of solution. The solution is clear (2.2.1) and its
absorbance (2.2.25) at 440 nm is not greater than 0.05. A. 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-indol-2-one,
Dissolve 1.25 g in methanol R and dilute to 25.0 mL with the
same solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50.0 mg of the substance to be
examined in methanol R and dilute to 50.0 mL with the same
solvent.
Reference solution (a). Dilute 2.0 mL of the test solution to
100.0 mL with methanol R. Dilute 1.0 mL of this solution to B. R1 = CHO, R2 = Cl : 2-[(2,6-dichlorophenyl)amino]-
10.0 mL with methanol R. benzaldehyde,
Reference solution (b). Dilute 1.0 mL of the test solution to C. R1 = CH2OH, R2 = Cl : [2-[(2,6-dichlorophenyl)amino]-
200.0 mL with methanol R. In 1.0 mL of this solution dissolve phenyl]methanol,
the contents of a vial of diclofenac impurity A CRS. D. R1 = CH2-CO2H, R2 = Br : 2-[2-[(2-bromo-6-
Column : chlorophenyl)amino]phenyl]acetic acid,
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : mix 34 volumes of a solution containing E. 1,3-dihydro-2H-indol-2-one.
0.5 g/L of phosphoric acid R and 0.8 g/L of sodium dihydrogen
phosphate R, adjusted to pH 2.5 with phosphoric acid R, and
66 volumes of methanol R. 01/2008:1002
Flow rate : 1 mL/min.
DICLOFENAC SODIUM
Detection : spectrophotometer at 254 nm.
Injection : 20 μL. Diclofenacum natricum
Run time : 1.5 times the retention time of diclofenac.
Retention time : impurity A = about 12 min ; diclofenac = about
25 min.
System suitability: reference solution (b):
– resolution : minimum 6.5 between the peaks due to
impurity A and diclofenac.
Limits:
C14H10Cl2NNaO2 Mr 318.1
– impurities A, B, C, D, E : for each impurity, not more [15307-79-6]
than the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent) ; DEFINITION
– total : not more than 2.5 times the area of the principal peak Sodium 2-[(2,6-dichlorophenyl)amino]phenyl]acetate.
in the chromatogram obtained with reference solution (a) Content : 99.0 per cent to 101.0 per cent (dried substance).
(0.5 per cent) ;
– disregard limit : 0.25 times the area of the principal peak CHARACTERS
in the chromatogram obtained with reference solution (a) Appearance : white or slightly yellowish, slightly hygroscopic,
(0.05 per cent). crystalline powder.

2036 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Diclofenac sodium

Solubility : sparingly soluble in water, freely soluble in Mobile phase : mix 34 volumes of a solution containing
methanol, soluble in ethanol (96 per cent), slightly soluble 0.5 g/L of phosphoric acid R and 0.8 g/L of sodium dihydrogen
in acetone. phosphate R, adjusted to pH 2.5 with phosphoric acid R, and
mp : about 280 °C, with decomposition. 66 volumes of methanol R.
Flow rate : 1 mL/min.
IDENTIFICATION
Detection : spectrophotometer at 254 nm.
First identification : A, D. Injection : 20 μL.
Second identification : B, C, D. Run time : 1.5 times the retention time of diclofenac.
A. Infrared absorption spectrophotometry (2.2.24). Retention times : impurity A = about 12 min ; diclofenac = about
Preparation : discs. 25 min.
Comparison : diclofenac sodium CRS. System suitability : reference solution (b):
B. Thin-layer chromatography (2.2.27). – resolution : minimum 6.5 between the peaks due to
Test solution. Dissolve 25 mg of the substance to be impurity A and diclofenac.
examined in methanol R and dilute to 5 mL with the same Limits:
solvent. – impurities A, B, C, D, E : for each impurity, not more
Reference solution (a). Dissolve 25 mg of diclofenac than the area of the principal peak in the chromatogram
sodium CRS in methanol R and dilute to 5 mL with the obtained with reference solution (a) (0.2 per cent) ;
same solvent. – total : not more than 2.5 times the area of the principal peak
Reference solution (b). Dissolve 10 mg of indometacin R in the chromatogram obtained with reference solution (a)
in reference solution (a) and dilute to 2 mL with the same (0.5 per cent) ;
solution. – disregard limit : 0.25 times the area of the principal peak
Plate : TLC silica gel GF254 plate R. in the chromatogram obtained with reference solution (a)
Mobile phase : concentrated ammonia R, methanol R, ethyl (0.05 per cent).
acetate R (10:10:80 V/V/V). Heavy metals (2.4.8) : maximum 10 ppm.
Application : 5 μL. 2.0 g complies with test C. Use a quartz crucible. Prepare
Development : over a path of 10 cm. the reference solution using 2 mL of lead standard solution
Drying : in air. (10 ppm Pb) R.
Detection : examine in ultraviolet light at 254 nm. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 3 h.
System suitability: reference solution (b) :
– the chromatogram shows 2 clearly separated spots. ASSAY
Results : the principal spot in the chromatogram obtained Dissolve 0.250 g in 30 mL of anhydrous acetic acid R. Titrate
with the test solution is similar in position and size to with 0.1 M perchloric acid, determining the end-point
the principal spot in the chromatogram obtained with potentiometrically (2.2.20).
reference solution (a). 1 mL of 0.1 M perchloric acid is equivalent to 31.81 mg
C. Dissolve about 10 mg in 10 mL of ethanol (96 per cent) R. of C14H10Cl2NNaO2.
To 1 mL of this solution add 0.2 mL of a mixture, prepared
immediately before use, of equal volumes of a 6 g/L solution STORAGE
of potassium ferricyanide R and a 9 g/L solution of ferric In an airtight container, protected from light.
chloride R. Allow to stand protected from light for 5 min.
Add 3 mL of a 10 g/L solution of hydrochloric acid R. Allow IMPURITIES
to stand, protected from light, for 15 min. A blue colour Specified impurities : A, B, C, D, E.
develops and a precipitate is formed.
D. Dissolve 60 mg in 0.5 mL of methanol R and add 0.5 mL of
water R. The solution gives reaction (b) of sodium (2.3.1).
TESTS
Appearance of solution. The solution is clear (2.2.1) and its
absorbance (2.2.25) at 440 nm is not greater than 0.05.
Dissolve 1.25 g in methanol R and dilute to 25.0 mL with the
same solvent. A. 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-indol-2-one,
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50.0 mg of the substance to be
examined in methanol R and dilute to 50.0 mL with the same
solvent.
Reference solution (a). Dilute 2.0 mL of the test solution to
100.0 mL with methanol R. Dilute 1.0 mL of this solution to
10.0 mL with methanol R.
Reference solution (b). Dilute 1.0 mL of the test solution to B. R1 = CHO, R2 = Cl : 2-[(2,6-dichlorophenyl)amino]-
200.0 mL with methanol R. In 1.0 mL of this solution dissolve benzaldehyde,
the contents of a vial of diclofenac impurity A CRS.
Column : C. R1 = CH2OH, R2 = Cl : [2-[(2,6-dichlorophenyl)amino]-
phenyl]methanol,
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octylsilyl silica gel for D. R1 = CH2-CO2H, R2 = Br : 2-[2-[(2-bromo-6-
chromatography R (5 μm). chlorophenyl)amino]phenyl]acetic acid,

General Notices (1) apply to all monographs and other texts 2037
Dicloxacillin sodium EUROPEAN PHARMACOPOEIA 8.0

the contents of the tube by swirling ; the solution is slightly

greenish-yellow. Place the test-tube in a water-bath for
1 min ; a yellow colour develops.
D. It gives reaction (a) of sodium (2.3.1).
E. 1,3-dihydro-2H-indol-2-one.
TESTS
01/2008:0663 Solution S. Dissolve 2.50 g in carbon dioxide-free water R and
corrected 6.0 dilute to 25.0 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and its
DICLOXACILLIN SODIUM absorbance (2.2.25) at 430 nm is not greater than 0.04.
pH (2.2.3) : 5.0 to 7.0 for solution S.
Dicloxacillinum natricum Specific optical rotation (2.2.7) : + 128 to + 143 (anhydrous
substance).
Dissolve 0.250 g in water R and dilute to 25.0 mL with the
same solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution (a). Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with the
mobile phase.
C19H16Cl2N3NaO5S,H2O Mr 510.3
[13412-64-1] Test solution (b). Dilute 5.0 mL of test solution (a) to 50.0 mL
with the mobile phase.
DEFINITION Reference solution (a). Dissolve 50.0 mg of dicloxacillin
Sodium (2S,5R,6R)-6-[[[3-(2,6-dichlorophenyl)-5- sodium CRS in the mobile phase and dilute to 50.0 mL with
methylisoxazol-4-yl]carbonyl]amino]-3,3-dimethyl-7-oxo-4- the mobile phase. Dilute 5.0 mL of this solution to 50.0 mL
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate monohydrate. with the mobile phase.
Semi-synthetic product derived from a fermentation product. Reference solution (b). Dilute 5.0 mL of test solution (b) to
Content : 95.0 per cent to 102.0 per cent (anhydrous substance). 50.0 mL with the mobile phase.
Reference solution (c). Dissolve 5 mg of flucloxacillin
CHARACTERS sodium CRS and 5 mg of dicloxacillin sodium CRS in the
Appearance : white or almost white, hygroscopic, crystalline mobile phase, then dilute to 50.0 mL with the mobile phase.
powder. Column :
Solubility : freely soluble in water, soluble in ethanol (96 per – size : l = 0.25 m, Ø = 4 mm ;
cent) and in methanol. – stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
IDENTIFICATION
Mobile phase : mix 25 volumes of acetonitrile R and 75 volumes
First identification : A, D. of a 2.7 g/L solution of potassium dihydrogen phosphate R
Second identification : B, C, D. adjusted to pH 5.0 with dilute sodium hydroxide solution R.
A. Infrared absorption spectrophotometry (2.2.24). Flow rate : 1.0 mL/min.
Preparation : discs. Detection : spectrophotometer at 225 nm.
Comparison : dicloxacillin sodium CRS. Injection : 20 μL of test solution (a) and reference solutions (b)
B. Thin-layer chromatography (2.2.27). and (c).
Test solution. Dissolve 25 mg of the substance to be Run time : 5 times the retention time of dicloxacillin.
examined in 5 mL of water R. Retention time : dicloxacillin = about 10 min.
Reference solution (a). Dissolve 25 mg of dicloxacillin System suitability : reference solution (c) :
sodium CRS in 5 mL of water R. – resolution : minimum 2.5 between the peaks due to
Reference solution (b). Dissolve 25 mg of cloxacillin flucloxacillin (1st peak) and dicloxacillin (2nd peak).
sodium CRS, 25 mg of dicloxacillin sodium CRS and 25 mg Limits :
of flucloxacillin sodium CRS in 5 mL of water R. – any impurity : for each impurity, not more than the area
Plate : TLC silanised silica gel plate R. of the principal peak in the chromatogram obtained with
Mobile phase : mix 30 volumes of acetone R and 70 volumes reference solution (b) (1 per cent) ;
of a 154 g/L solution of ammonium acetate R adjusted to – total : not more than 5 times the area of the principal peak
pH 5.0 with glacial acetic acid R. in the chromatogram obtained with reference solution (b)
Application : 1 μL. (5 per cent) ;
Development : over a path of 15 cm. – disregard limit : 0.05 times the area of the principal peak
Drying : in air. in the chromatogram obtained with reference solution (b)
(0.05 per cent).
Detection : expose to iodine vapour until the spots appear
and examine in daylight. N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm.
System suitability: reference solution (b) : 2-Ethylhexanoic acid (2.4.28) : maximum 0.8 per cent m/m.
– the chromatogram shows 3 clearly separated spots. Water (2.5.12) : 3.0 per cent to 4.5 per cent, determined on
Results : the principal spot in the chromatogram obtained 0.300 g.
with the test solution is similar in position, colour and size Pyrogens (2.6.8). If intended for use in the manufacture
to the principal spot in the chromatogram obtained with of parenteral preparations without a further appropriate
reference solution (a). procedure for the removal of pyrogens, it complies with the
C. Place about 2 mg in a test-tube about 150 mm long and test for pyrogens. Inject per kilogram of the rabbit’s mass 1 mL
about 15 mm in diameter. Moisten with 0.05 mL of water R of a solution in water for injections R containing 20 mg of the
and add 2 mL of sulfuric acid-formaldehyde reagent R. Mix substance to be examined per millilitre.

2038 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dicycloverine hydrochloride

ASSAY CHARACTERS
Liquid chromatography (2.2.29) as described in the test for A white or almost white, crystalline powder, soluble in water,
related substances with the following modifications. freely soluble in alcohol and in methylene chloride.
Injection : test solution (b) and reference solution (a). It shows polymorphism (5.9).
System suitability: reference solution (a) :
IDENTIFICATION
– repeatability : maximum relative standard deviation of
1.0 per cent after 6 injections. First identification : A, D.
Second identification : B, C, D.
STORAGE
A. Examine by infrared absorption spectrophotometry
In an airtight container, at a temperature not exceeding (2.2.24), comparing with the spectrum obtained with
25 °C. If the substance is sterile, store in a sterile, airtight, dicycloverine hydrochloride CRS. Examine the substances
tamper-proof container. prepared as discs using potassium chloride R. If the spectra
IMPURITIES obtained show differences, dissolve the substance to
be examined and the reference substance separately in
acetone R, evaporate to dryness and record new spectra
using the residues.
B. Examine the chromatograms obtained in the test for related
substances. The principal spot in the chromatogram
obtained with test solution (b) is similar in position,
colour and size to the principal spot in the chromatogram
obtained with reference solution (b).
A. R = CO2H : (4S)-2-[carboxy[[[3-(2,6-dichlorophenyl)-
C. To 3 mL of a 1.0 g/L solution of sodium laurilsulfate R add
5-methylisoxazol-4-yl]carbonyl]amino]methyl]-5,5-
5 mL of methylene chloride R and 0.05 mL of a 2.5 g/L
dimethylthiazolidine-4-carboxylic acid (penicilloic acids
solution of methylene blue R, mix gently and allow to stand ;
of dicloxacillin),
the lower layer is blue. Add 2 mL of a 20 g/L solution of the
B. R = H : (2RS,4S)-2-[[[[3-(2,6-dichlorophenyl)-5- substance to be examined, mix gently and allow to stand ;
methylisoxazol-4-yl]carbonyl]amino]methyl]-5,5- the upper layer is blue and the lower layer is colourless.
dimethylthiazolidine-4-carboxylic acid (penilloic acids of D. It gives reaction (a) of chlorides (2.3.1).
dicloxacillin),
TESTS
pH (2.2.3). Dissolve 0.5 g in water R and dilute to 50 mL with
the same solvent. The pH of the solution is 5.0 to 5.5.
Related substances. Examine by thin-layer chromatography
(2.2.27), using a suitable silica gel as the coating substance.
C. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-
Test solution (a). Dissolve 0.25 g to the substance to be
1-azabicyclo[3.2.0]heptane-2-carboxylic acid
examined in methanol R and dilute to 5 mL with the same
(6-aminopenicillanic acid),
solvent.
Test solution (b). Dilute 1 mL of test solution (a) to 50 mL
with methanol R.
Reference solution (a). Dilute 1 mL of test solution (b) to
10 mL with methanol R.
Reference solution (b). Dissolve 10 mg of dicycloverine
hydrochloride CRS in methanol R and dilute to 10 mL with
the same solvent.
D. 3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carboxylic
acid. Reference solution (c). Dissolve 5 mg of tropicamide CRS
in reference solution (b) and dilute to 5 mL with the same
solution.
01/2008:1197 Apply separately to the plate 10 μL of each solution. Develop
corrected 6.0 over a path of 15 cm using a mixture of 5 volumes of
concentrated ammonia R, 10 volumes of ethyl acetate R,
DICYCLOVERINE HYDROCHLORIDE 10 volumes of water R and 75 volumes of propanol R. Dry the
plate in a current of warm air. Spray with dilute potassium
iodobismuthate solution R. Any spot in the chromatogram
Dicycloverini hydrochloridum obtained with test solution (a), apart from the principal
spot, is not more intense than the spot in the chromatogram
obtained with reference solution (a) (0.2 per cent). The test
is not valid unless the chromatogram obtained with reference
solution (c) shows two clearly separated spots.
Loss on drying (2.2.32). Not more than 1.0 per cent,
determined on 1.000 g by drying in an oven at 105 °C.
C19H36ClNO2 Mr 346.0 Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
on 1.0 g.
DEFINITION
Dicycloverine hydrochloride contains not less than 99.0 per ASSAY
cent and not more than the equivalent of 101.0 per cent Dissolve 0.300 g in a mixture of 5.0 mL of 0.01 M hydrochloric
of 2-(diethylamino)ethyl bicyclohexyl-1-carboxylate acid and 50 mL of alcohol R. Carry out a potentiometric
hydrochloride, calculated with reference to the dried titration (2.2.20), using 0.1 M sodium hydroxide. Read
substance. the volume added between the two points of inflexion.

General Notices (1) apply to all monographs and other texts 2039
Didanosine EUROPEAN PHARMACOPOEIA 8.0

1 mL of 0.1 M sodium hydroxide is equivalent to 34.60 mg – stationary phase : base-deactivated octadecylsilyl silica gel for
of C19H36ClNO2. chromatography R (5 μm).
IMPURITIES Mobile phase :
– mobile phase A : mix 8 volumes of methanol R and
92 volumes of a 3.86 g/L solution of ammonium acetate R
adjusted to pH 8.0 with concentrated ammonia R ;
– mobile phase B : mix 30 volumes of methanol R and
70 volumes of a 3.86 g/L solution of ammonium acetate R
A. bicyclohexyl-1-carboxylic acid. adjusted to pH 8.0 with concentrated ammonia R ;
Time Mobile phase A Mobile phase B
01/2008:2200 (min) (per cent V/V) (per cent V/V)
corrected 7.0 0 - 18 100 0

DIDANOSINE
18 - 25 100 → 0 0 → 100

25 - 45 0 100
Didanosinum 45 - 50 0 → 100 100 → 0

50 - 60 100 0

Flow rate : 1.0 mL/min.

Detection : spectrophotometer at 254 nm.
Injection : 20 μL.
Identification of impurities : use the chromatogram
supplied with didanosine for system suitability CRS and the
C10H12N4O3 Mr 236.2 chromatogram obtained with reference solution (c) to identify
[69655-05-6] the peaks due to impurities A to F and use the chromatogram
obtained with reference solution (d) to identify the peak due
DEFINITION
to impurity G.
9-(2,3-Dideoxy-β-D-glycero-pentofuranosyl)-1,9-dihydro-6H-
purin-6-one (2′,3′-dideoxyinosine). Relative retention with reference to didanosine (retention
time = about 13-15 min) : impurity A = about 0.3 ;
Content : 98.5 per cent to 101.0 per cent (anhydrous substance). impurity B = about 0.4 ; impurity C = about 0.44 ;
CHARACTERS impurity D = about 0.48 ; impurity E = about 0.5 ;
impurity F = about 0.8 ; impurity G = about 1.6.
Appearance : white or almost white, crystalline powder.
System suitability : reference solution (c) :
Solubility : sparingly soluble in water, freely soluble in dimethyl
sulfoxide, slightly soluble in methanol and in ethanol (96 per – resolution : minimum 2.5 between the peaks due to
cent). impurity C and impurity D.
Limits :
IDENTIFICATION – impurity A : not more than the area of the principal peak
A. Infrared absorption spectrophotometry (2.2.24). in the chromatogram obtained with reference solution (b)
Comparison : didanosine CRS. (0.5 per cent) ;
B. Specific optical rotation (2.2.7) : − 28.2 to − 24.2 (anhydrous – impurities B, C, D, E, F, G : for each impurity, not more than
substance). twice the area of the principal peak in the chromatogram
Dissolve 0.100 g in water R and dilute to 10.0 mL with the obtained with reference solution (a) (0.2 per cent) ;
same solvent. – unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
TESTS with reference solution (a) (0.10 per cent) ;
Related substances. Liquid chromatography (2.2.29). Prepare – total : not more than 10 times the area of the principal peak
the solutions immediately before use. in the chromatogram obtained with reference solution (a)
Solvent mixture. Mix 8 volumes of mobile phase B and (1.0 per cent) ;
92 volumes of mobile phase A. – disregard limit : 0.5 times the area of the principal peak in
Test solution. Dissolve 25.0 mg of the substance to be the chromatogram obtained with reference solution (a)
examined in 50.0 mL of the solvent mixture. (0.05 per cent).
Reference solution (a). Dilute 1.0 mL of the test solution to Heavy metals (2.4.8) : maximum 20 ppm.
100.0 mL with the solvent mixture. Dilute 1.0 mL of this 1.0 g complies with test F. Prepare the reference solution using
solution to 10.0 mL with the solvent mixture. 2 mL of lead standard solution (10 ppm Pb) R.
Reference solution (b). Dissolve 5.0 mg of didanosine Water (2.5.12) : maximum 2.0 per cent, determined on 0.500 g.
impurity A CRS in the solvent mixture and dilute to 100.0 mL
with the solvent mixture. Dilute 1.0 mL to 20.0 mL with the Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
solvent mixture. 1.0 g.
Reference solution (c). Dissolve 5 mg of didanosine for system ASSAY
suitability CRS (containing impurities A to F) in the solvent
Dissolve 0.200 g in 50 mL of glacial acetic acid R. Titrate
mixture and dilute to 10 mL with the solvent mixture.
with 0.1 M perchloric acid, determining the end-point
Reference solution (d). Dissolve 5 mg of didanosine potentiometrically (2.2.20).
impurity G CRS in the solvent mixture and dilute to 100 mL
1 mL of 0.1 M perchloric acid is equivalent to 23.62 mg
with the solvent mixture. Dilute 1 mL to 20 mL with the
of C10H12N4O3.
solvent mixture.
Column : IMPURITIES
– size : l = 0.25 m, Ø = 4.6 mm ; Specified impurities : A, B, C, D, E, F, G.

2040 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Diethyl phthalate

Other detectable impurities (the following substances would, 04/2008:0897

if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general DIETHYL PHTHALATE
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
Diethylis phthalas
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : H, I.

C12H14O4 Mr 222.2
[84-66-2]
A. 1,7-dihydro-6H-purin-6-one (hypoxanthine),
DEFINITION
Diethyl benzene-1,2-dicarboxylate.
Content : 99.0 per cent m/m to 101.0 per cent m/m.
CHARACTERS
Appearance : clear, colourless or very slightly yellow, oily
liquid.
Solubility : practically insoluble in water, miscible with ethanol
(96 per cent).
B. R = R′ = OH : 9-β-D-ribofuranosyl-1,9-dihydro-6H-purin- IDENTIFICATION
6-one (inosine), First identification : B, C.
Second identification : A, D, E.
C. R = H, R′ = OH : 9-(2-deoxy-β-D-erythro-pentofuranosyl)-
A. Relative density (2.2.5) : 1.117 to 1.121.
1,9-dihydro-6H-purin-6-one (2′-deoxyinosine),
B. Refractive index (2.2.6) : 1.500 to 1.505.
D. R = OH, R′ = H : 9-(3-deoxy-β-D-erythro-pentofuranosyl)- C. Infrared absorption spectrophotometry (2.2.24).
1,9-dihydro-6H-purin-6-one (3′-deoxyinosine), Preparation : thin films.
Comparison: diethyl phthalate CRS.
E. R + R′ = O : 9-(2,3-anhydro-β-D-ribofuranosyl)-1,9- D. Thin-layer chromatography (2.2.27).
dihydro-6H-purin-6-one (2′,3′-anhydroinosine), Test solution. Dissolve 50 mg of the substance to be
examined in ether R and dilute to 10 mL with the same
solvent.
Reference solution. Dissolve 50 mg of diethyl phthalate CRS
in ether R and dilute to 10 mL with the same solvent.
Plate : TLC silica gel GF254 plate R.
Mobile phase : heptane R, ether R (30:70 V/V).
Application : 10 μL.
F. 9-(2,3-dideoxy-β-D-glycero-pent-2-enofuranosyl)- Development : over 2/3 of the plate.
1,9-dihydro-6H-purin-6-one (2′,3′-dideoxy-2′,3′- Drying : in air.
didehydroinosine), Detection : examine in ultraviolet light at 254 nm.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the
reference solution.
E. To about 0.1 mL add 0.25 mL of sulfuric acid R and 50 mg
of resorcinol R. Heat on a water-bath for 5 min. Allow to
cool. Add 10 mL of water R and 1 mL of strong sodium
hydroxide solution R. The solution becomes yellow or
G. R = OH : 9-(2,3-dideoxy-β-D-glycero-pentofuranosyl)-9H- brownish-yellow and shows green fluorescence.
purin-6-amine (2′,3′-dideoxyadenosine),
TESTS
H. R = H : 9-(2,3,5-trideoxy-β-D-glycero-pentofuranosyl)-9H- Appearance. The substance to be examined is clear (2.2.1)
purin-6-amine (2′,3′,5′-trideoxyadenosine), and not more intensely coloured than reference solution Y6
(2.2.2, Method II).
Acidity. Dissolve 20.0 g in 50 mL of ethanol (96 per cent) R
previously neutralised to phenolphthalein solution R1. Add
0.2 mL of phenolphthalein solution R1. Not more than 0.1 mL
of 0.1 M sodium hydroxide is required to change the colour of
the indicator to pink.
Related substances. Gas chromatography (2.2.28).
Internal standard solution. Dissolve 60 mg of naphthalene R
I. 9-(2,3-dideoxy-β-D-glycero-pent-2-enofuranosyl)-9H- in methylene chloride R and dilute to 20 mL with the same
purin-6-amine (2′,3′-dideoxy-2′,3′-didehydroadenosine). solvent.

General Notices (1) apply to all monographs and other texts 2041
Diethylcarbamazine citrate EUROPEAN PHARMACOPOEIA 8.0

Test solution (a). Dissolve 1.0 g of the substance to be 01/2008:0271

examined in methylene chloride R and dilute to 20.0 mL with
the same solvent. DIETHYLCARBAMAZINE CITRATE
Test solution (b). Dissolve 1.0 g of the substance to be
examined in methylene chloride R, add 2.0 mL of the internal Diethylcarbamazini citras
standard solution and dilute to 20.0 mL with methylene
chloride R.
Reference solution. To 1.0 mL of test solution (a) add 10.0 mL
of the internal standard solution and dilute to 100.0 mL with
methylene chloride R.
Column : C16H29N3O8 Mr 391.4
[1642-54-2]
– material : glass ;
– size : l = 2 m, Ø = 2 mm ; DEFINITION
N,N-Diethyl-4-methylpiperazine-1-carboxamide dihydrogen
– stationary phase : silanised diatomaceous earth for gas 2-hydroxypropane-1,2,3-tricarboxylate.
chromatography R (150-180 μm) impregnated with 3 per
cent m/m of polymethylphenylsiloxane R. Content : 98.0 per cent to 102.0 per cent (dried substance).

Carrier gas : nitrogen for chromatography R. CHARACTERS

Appearance : white or almost white, crystalline powder, slightly
Flow rate : 30 mL/min.
hygroscopic.
Temperature : Solubility : very soluble in water, soluble in ethanol (96 per
– column : 150 °C ; cent), practically insoluble in acetone.
mp : about 138 °C, with decomposition.
– injection port and detector : 225 °C.
IDENTIFICATION
Detection : flame ionisation.
First identification : A, C.
Injection : 1 μL. Second identification : B, C.
Run time : 3 times the retention time of diethyl phthalate. A. Infrared absorption spectrophotometry (2.2.24).
Elution order : naphthalene, diethyl phtalate. Comparison: diethylcarbamazine citrate CRS.
System suitability : B. Examine the chromatograms obtained in the test for
impurities A and B.
– resolution : minimum 10 between the peaks due to Results : the principal spot in the chromatogram obtained
naphthalene and diethyl phthalate in the chromatogram with the test solution is similar in position, colour and size
obtained with the reference solution ; to the principal spot in the chromatogram obtained with
– in the chromatogram obtained with test solution (a), there reference solution (a).
is no peak with the same retention time as the internal C. Dissolve 0.1 g in 5 mL of water R. The solution gives the
standard. reaction of citrates (2.3.1).
Limit : TESTS
– total : calculate the ratio (R) of the area of the peak due Solution S. Shake 2.5 g with water R until dissolved and dilute
to diethyl phthalate to the area of the peak due to the to 25 mL with the same solvent.
internal standard from the chromatogram obtained with Appearance of solution. Solution S is not more opalescent
the reference solution ; from the chromatogram obtained than reference suspension II (2.2.1) and not more intensely
with test solution (b), calculate the ratio of the sum of the coloured than reference solution BY6 (2.2.2, Method II).
areas of any peaks, apart from the principal peak and the
peak due to the internal standard, to the area of the peak Impurities A and B. Thin-layer chromatography (2.2.27).
due to the internal standard : this ratio is not greater than Test solution. Dissolve 0.5 g of the substance to be examined
R (1.0 per cent). in methanol R and dilute to 10 mL with the same solvent.
Water (2.5.12) : maximum 0.2 per cent, determined on 10.0 g. Reference solution (a). Dissolve 0.1 g of diethylcarbamazine
citrate CRS in methanol R and dilute to 2.0 mL with the same
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on solvent.
1.0 g.
Reference solution (b). Dissolve 10 mg of methylpiperazine R
(impurity A) in methanol R and dilute to 100 mL with the
ASSAY same solvent.
Introduce 0.750 g into a 250 mL borosilicate glass flask. Add Reference solution (c). Dissolve 10 mg of dimethylpiperazine R
25.0 mL of 0.5 M alcoholic potassium hydroxide and a few (impurity B) in methanol R and dilute to 100 mL with the
glass beads. Boil in a water-bath under a reflux condenser same solvent.
for 1 h. Add 1 mL of phenolphthalein solution R1 and titrate Plate : TLC silica gel plate R.
immediately with 0.5 M hydrochloric acid. Carry out a blank Mobile phase : concentrated ammonia R, methyl ethyl ketone R,
titration. Calculate the volume of 0.5 M alcoholic potassium methanol R (5:30:65 V/V/V).
hydroxide used in the saponification.
Application : 10 μL.
1 mL of 0.5 M alcoholic potassium hydroxide is equivalent to Development : over 2/3 of the plate.
55.56 mg of C12H14O4.
Drying : at 100-105 °C.
Detection : expose to iodine vapour for 30 min.
STORAGE
Retardation factors : impurity A = about 0.2 ; impurity B = about
In an airtight container. 0.5.

2042 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Diethylene glycol monoethyl ether

Limits : ASSAY
– impurity A : any spot due to impurity A is not more intense Liquid chromatography (2.2.29) as described in the test for
than the corresponding spot in the chromatogram obtained related substances with the following modification.
with reference solution (b) (0.2 per cent) ; Injection : 20 μL of test solution (b) and reference solution (d).
– impurity B : any spot due to impurity B is not more intense Calculate the percentage content of C16H29N3O8 from the
than the corresponding spot in the chromatogram obtained declared content of diethylcarbamazine citrate CRS.
with reference solution (c) (0.2 per cent).
STORAGE
Related substances. Liquid chromatography (2.2.29).
In an airtight container.
Solution A. Dissolve 31.2 g of potassium dihydrogen
phosphate R in water R and dilute to 1000 mL with the same IMPURITIES
solvent. Specified impurities : A, B.
Test solution (a). Suspend 0.30 g of the substance to be
examined in solution A and dilute to 100 mL with solution A.
Filter or centrifuge and use the clear filtrate or supernatant.
Test solution (b). Dissolve 10.0 mg of the substance to be
examined in solution A and dilute to 100.0 mL with solution A. A. R = H : 1-methylpiperazine,
Reference solution (a). Dilute 1.0 mL of test solution (a) to B. R = CH3 : 1,4-dimethylpiperazine.
100.0 mL with solution A. Dilute 1.0 mL of this solution to
10.0 mL with solution A.
Reference solution (b). Dissolve 10 mg of citric acid R in 01/2008:1198
solution A and dilute to 10 mL with solution A.
Reference solution (c). To 3 mL of test solution (a) add 0.5 mL DIETHYLENE GLYCOL MONOETHYL
of strong hydrogen peroxide solution R and maintain at 80 °C ETHER
for 3 h. Dilute to 100 mL with solution A.
Reference solution (d). Dissolve 5.0 mg of diethylcarbamazine
citrate CRS in solution A and dilute to 50.0 mL with solution A.
Diethylenglycoli aether monoethylicus
Column :
– size : l = 0.15 m, Ø = 3.9 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for C6H14O3 Mr 134.2
chromatography R (5 μm). [111-90-0]
Mobile phase : mix 100 volumes of methanol R2 and DEFINITION
900 volumes of a 10 g/L solution of potassium dihydrogen 2-(2-Ethoxyethoxy)ethanol, produced by condensation of
phosphate R. ethylene oxide and alcohol, followed by distillation.
Flow rate : 0.8 mL/min.
CHARACTERS
Detection : spectrophotometer at 220 nm.
Appearance : clear, colourless, hygroscopic liquid.
Injection : 20 μL of test solution (a) and reference solutions (a),
(b) and (c). Solubility : miscible with water, with acetone and with alcohol,
miscible in certain proportions with vegetable oils, not
Run time : twice the retention time of diethylcarbamazine. miscible with mineral oils.
Identification of impurities : use the chromatogram obtained Relative density : about 0.991.
with reference solution (b) to identify the peak due to the
citrate. IDENTIFICATION
Relative retention with reference to diethylcarbamazine A. Refractive index (2.2.6) : 1.426 to 1.428.
(retention time = about 7 min) : citrate = about 0.2 ; B. Infrared absorption spectrophotometry (2.2.24).
degradation product = about 1.6. Comparison: Ph. Eur. reference spectrum of diethylene
System suitability: reference solution (c) : glycol monoethyl ether.
– resolution : minimum 5 between the peaks due to
diethylcarbamazine and the degradation product. TESTS
Limits : Acid value (2.5.1) : maximum 0.1.
– unspecified impurities : for each impurity, not more than the Mix 30.0 mL with 30 mL of alcohol R previously neutralised
area of the principal peak in the chromatogram obtained with 0.1 M potassium hydroxide using phenolphthalein
with reference solution (a) (0.10 per cent) ; solution R as indicator. Titrate with 0.01 M alcoholic potassium
hydroxide.
– total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a) Peroxide value (2.5.5) : maximum 8.0, determined on 2.00 g.
(0.5 per cent) ; Related substances. Gas chromatography (2.2.28).
– disregard limit : 0.5 times the area of the principal peak in Internal standard solution. Dilute 1.00 g of decane R to
the chromatogram obtained with reference solution (a) 100.0 mL with methanol R.
(0.05 per cent) ; disregard the peak due to the citrate. Test solution. To 5.00 g of the substance to be examined, add
Heavy metals (2.4.8) : maximum 20 ppm. 0.1 mL of the internal standard solution and dilute to 10.0 mL
with methanol R.
12 mL of solution S complies with test A. Prepare the reference
solution using 10 mL of lead standard solution (2 ppm Pb) R. Reference solution (a). Dilute 25.0 mg of ethylene glycol
monomethyl ether R, 80.0 mg of ethylene glycol monoethyl
Loss on drying (2.2.32) : maximum 0.5 per cent, determined ether R, 0.310 g of ethylene glycol R and 0.125 g of diethylene
on 1.000 g by drying in vacuo at 60 °C for 4 h. glycol R to 100.0 mL with methanol R. To 1.0 mL of this
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on solution add 0.1 mL of the internal standard solution and
1.0 g. dilute to 10.0 mL with methanol R.

General Notices (1) apply to all monographs and other texts 2043
Diethylene glycol palmitostearate EUROPEAN PHARMACOPOEIA 8.0

Reference solution (b). Dilute 25.0 mg of ethylene glycol Column :

monoethyl ether R and 25.0 mg of ethylene glycol R to 100.0 mL – material : fused silica,
with methanol R. Dilute 1.0 mL of this solution to 5.0 mL – size : l = 30 m, Ø = 0.32 mm,
with methanol R.
– stationary phase : poly(cyanoprop-
Reference solution (c). Dilute 1.00 g of the substance to be yl)(7)(phenyl)(7)(methyl)(86)siloxane R (film thickness
examined to 100.0 mL with methanol R. To 1.0 mL of this 1 μm).
solution add 0.1 mL of the internal standard solution and
dilute to 10.0 mL with methanol R. Carrier gas : helium for chromatography R.
Column : Flow rate : 1.1 mL/min.
Static head-space conditions which may be used :
– material : fused silica,
– equilibration temperature : 80 °C,
– size : l = 30 m, Ø = 0.32 mm,
– equilibration time : 45 min,
– stationary phase : poly(cyanoprop-
yl)(7)(phenyl)(7)(methyl)(86)siloxane R (film thickness – transfer line temperature : 110 °C,
1 μm). – pressurisation time : 2 min,
Carrier gas : nitrogen for chromatography R or helium for – injection time : 12 s.
chromatography R. Temperature :
Flow rate : 2.0 mL/min. Time Temperature
Split ratio : 1:80. (min) (°C)
Temperature : Column 0-5 40

Time Temperature 5 - 18 40 → 200

(min) (°C) Injection port 150
Column 0-1 120
Detector 250
1 - 10 120 → 225
Detection : flame ionisation.
10 - 12 225
Injection : 1.0 mL.
Injection port 275
The peak due to ethylene oxide is identified by injecting
Detector 250 solutions of ethylene oxide of increasing concentration.
Determine the content of ethylene oxide (ppm) in the
Detection : flame ionisation. substance to be examined using the following expression :
Injection : 0.5 μL.
Relative retentions with reference to diethylene glycol
monoethyl ether (retention time = about 4 min) : ethylene
glycol monomethyl ether = about 0.4 ; ethylene glycol ST = area of the peak due to ethylene oxide in the
monoethyl ether = about 0.5 ; ethylene glycol = about 0.55 ; chromatogram obtained with the test solution,
diethylene glycol = about 1.1.
SS = area of the peak due to ethylene oxide in the
System suitability :
chromatogram obtained with the reference
– resolution : minimum 3.0 between the peaks due to ethylene solution,
glycol monoethyl ether and to ethylene glycol in the MT = mass of the substance to be examined in the test
chromatogram obtained with reference solution (b),
solution, in grams,
– signal-to-noise ratio : minimum 3.0 for the peak due to
MS = mass of the substance to be examined in the
ethylene glycol monomethyl ether in the chromatogram
obtained with reference solution (a), reference solution, in grams,
Limits (take into account the impurity/internal standard peak C = mass of added ethylene oxide in the reference
area ratio) : solution, in micrograms.
– ethylene glycol monomethyl ether : not more than the area Limit :
of the corresponding peak in the chromatogram obtained – ethylene oxide : maximum 1 ppm.
with reference solution (a) (50 ppm), Water (2.5.12) : maximum 0.1 per cent, determined on 10.0 g.
– ethylene glycol monoethyl ether : not more than the area of
the corresponding peak in the chromatogram obtained STORAGE
with reference solution (a) (160 ppm), Under an inert gas, in an airtight container.
– ethylene glycol : not more than the area of the corresponding
peak in the chromatogram obtained with reference LABELLING
solution (a) (620 ppm), The label states that the substance is stored under an inert gas.
– diethylene glycol : not more than the area of the
corresponding peak in the chromatogram obtained with 01/2008:1415
reference solution (a) (250 ppm), corrected 6.0
– total : not more than the area of the principal peak in the
chromatogram obtained with reference solution (c) (0.2 per DIETHYLENE GLYCOL
cent).
Ethylene oxide. Head-space gas chromatography (2.2.28).
PALMITOSTEARATE
Test solution. To 1.00 g of the substance to be examined in Diethylenglycoli palmitostearas
a vial, add 50 μL of water R.
Reference solution. To 1.00 g of the substance to be examined DEFINITION
in a vial, add 50 μL of ethylene oxide solution R4 and close Mixture of diethylene glycol mono- and diesters of stearic
tightly. (octadecanoic) and palmitic (hexadecanoic) acids.

2044 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Diethylstilbestrol

It is produced by esterification of diethylene glycol and stearic A = area of the peak due to the monoesters,
acid 50 (see Stearic acid (1474)) of vegetable or animal origin.
B = area of the peak due to the diesters,
Content :
– monoesters : 45.0 per cent to 60.0 per cent ; D = percentage content of free diethylene glycol
+ percentage content of free fatty acids.
– diesters : 35.0 per cent to 55.0 per cent.
Calculate the percentage content of free fatty acids using the
CHARACTERS following expression :
Appearance : white or almost white, waxy solid.
Solubility : practically insoluble in water, soluble in acetone
and in hot ethanol (96 per cent).
IDENTIFICATION IA = acid value.
A. Melting point (see Tests). – diesters : calculate the percentage content of diesters using
B. Composition of fatty acids (see Tests). the following expression :
C. It complies with the limit of the assay (monoesters content).
TESTS
Melting point (2.2.15) : 43 °C to 50 °C. STORAGE
Acid value (2.5.1) : maximum 4.0. Protected from light.
Iodine value (2.5.4, Method A) : maximum 3.0.
Saponification value (2.5.6) : 155 to 180, determined on 2.0 g.
Composition of fatty acids (2.4.22, Method A). Use the 01/2008:0484
mixture of calibrating substances in Table 2.4.22.-1. corrected 6.0
Composition of the fatty acid fraction of the substance :
– stearic acid : 40.0 per cent to 60.0 per cent ; DIETHYLSTILBESTROL
– sum of contents of palmitic acid and stearic acid : minimum
90.0 per cent. Diethylstilbestrolum
Free diethylene glycol : maximum 2.5 per cent, determined
as described in the assay.
Total ash (2.4.16) : maximum 0.1 per cent.
ASSAY
Size-exclusion chromatography (2.2.30).
Test solution. Into a 15 mL flask, weigh 0.200 g (m). Add C18H20O2 Mr 268.4
5.0 mL of tetrahydrofuran R and shake to dissolve. Heat [56-53-1]
gently, if necessary. Reweigh the flask and calculate the total
mass of solvent and substance (M). DEFINITION
Reference solutions. Into four 15 mL flasks, weigh, 2.5 mg, Diethylstilbestrol contains not less than 97.0 per cent
5.0 mg, 10.0 mg and 20.0 mg respectively of diethylene glycol R. and not more than the equivalent of 101.0 per cent of
Add 5.0 mL of tetrahydrofuran R. Weigh the flasks again and (E)-4,4′-(1,2-diethylethene-1,2-diyl)diphenol, calculated with
calculate the concentration of diethylene glycol in milligrams reference to the dried substance.
per gram for each reference solution.
CHARACTERS
Column :
A white or almost white, crystalline powder, practically
– size : l = 0.6 m, Ø = 7 mm,
insoluble in water, freely soluble in alcohol. It dissolves in
– stationary phase : styrene-divinylbenzene copolymer R solutions of the alkali hydroxides.
(5 μm) with a pore size of 10 nm.
It melts at about 172 °C.
Mobile phase : tetrahydrofuran R.
Flow rate : 1 mL/min. IDENTIFICATION
Detection : differential refractometer. First identification : B, D.
Injection : 40 μL. Second identification : A, C, D.
Relative retention with reference to diethylene glycol : A. Examined between 230 nm and 450 nm (2.2.25), the
diesters = about 0.78 ; monoesters = about 0.84. irradiated solution of the substance to be examined
Calculations : prepared as prescribed in the assay shows two absorption
– free diethylene glycol : from the calibration curve maxima, at 292 nm and 418 nm.
obtained with the reference solutions, determine the B. Examine by infrared absorption spectrophotometry
concentration (C) of diethylene glycol in milligrams per (2.2.24), comparing with the spectrum obtained with
gram in the test solution and calculate the percentage diethylstilbestrol CRS. Examine the substances prepared as
content of free diethylene glycol in the substance to be discs.
examined using the following expression : C. Examine the chromatograms obtained in the test for
mono-and dimethyl ethers. The principal spot in the
chromatogram obtained with test solution (b) is similar
in position, colour and size to the principal spot in the
– monoesters : calculate the percentage content of monoesters chromatogram obtained with reference solution (a).
using the following expression : D. Dissolve about 0.5 mg in 0.2 mL of glacial acetic acid R,
add 1 mL of phosphoric acid R and heat on a water-bath for
3 min. A deep-yellow colour develops.

General Notices (1) apply to all monographs and other texts 2045
Difloxacin hydrochloride trihydrate for veterinary use EUROPEAN PHARMACOPOEIA 8.0

TESTS 04/2013:2239
4,4′-Dihydroxystilbene and related ethers. Dissolve 0.100 g
in ethanol R and dilute to 10.0 mL with the same solvent. The DIFLOXACIN HYDROCHLORIDE
absorbance (2.2.25) of the solution measured at 325 nm is not TRIHYDRATE FOR VETERINARY USE
greater than 0.50.
Mono- and dimethyl ethers. Examine by thin-layer Difloxacini hydrochloridum trihydricum ad
chromatography (2.2.27), using silica gel G R as the coating usum veterinarium
substance.
Test solution (a). Dissolve 0.2 g of the substance to be
examined in 2 mL of alcohol R.
Test solution (b). Dilute 1 mL of test solution (a) to 20 mL
with alcohol R.
Reference solution (a). Dissolve 10 mg of diethylstilbestrol CRS
in 2 mL of alcohol R.
Reference solution (b). Dissolve 5 mg of diethylstilbestrol
monomethyl ether CRS in alcohol R and dilute to 10 mL with C21H20ClF2N3O3,3H2O Mr 490.0
the same solvent. Anhydrous difloxacin hydrochloride : [91296-86-5]
Reference solution (c). Dissolve 5 mg of diethylstilbestrol DEFINITION
dimethyl ether CRS in alcohol R and dilute to 10 mL with the 6-Fluoro-1-(4-fluorophenyl)-7-(4-methylpiperazin-
same solvent. 1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
Reference solution (d). Dissolve 10 mg of dienestrol CRS in hydrochloride trihydrate.
2 mL of alcohol R. To 1 mL of this solution add 1 mL of Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
reference solution (a).
CHARACTERS
Apply to the plate 1 μL of each solution. Develop over a path Appearance : white or light yellow, crystalline powder.
of 15 cm using a mixture of 10 volumes of diethylamine R and Solubility : slightly soluble in water and in methanol, very
90 volumes of toluene R. Allow the plate to dry in air, spray slightly soluble in methylene chloride.
with alcoholic solution of sulfuric acid R and heat at 120 °C for
10 min. In the chromatogram obtained with test solution (a), It shows polymorphism (5.9).
any spots corresponding to diethylstilbestrol monomethyl IDENTIFICATION
ether and diethylstilbestrol dimethyl ether are not more
A. Infrared absorption spectrophotometry (2.2.24).
intense than the spots in the chromatograms obtained with
reference solutions (b) and (c) respectively (0.5 per cent). Comparison: difloxacin hydrochloride CRS.
Diethylstilbestrol gives one or sometimes two spots. The test If the spectra obtained in the solid state show differences,
is not valid unless the chromatogram obtained with reference dissolve the substance to be examined and the reference
solution (d) shows at least two clearly separated spots having substance separately in methanol R, evaporate to dryness
approximately the same intensity. and record new spectra using the residues.
Loss on drying (2.2.32). Not more than 0.5 per cent, B. Suspend 30 mg in 2 mL of water R, acidify with dilute nitric
determined on 1.000 g by drying in an oven at 105 °C. acid R and filter. The clear filtrate gives reaction (a) of
chlorides (2.3.1).
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined C. Water (see Tests).
on 1.0 g.
TESTS
ASSAY Related substances. Liquid chromatography (2.2.29).
Dissolve 20.0 mg in ethanol R and dilute to 100.0 mL with the Solvent mixture : acetonitrile R, water R (50:50 V/V).
same solvent. Dilute 10.0 mL of the solution to 100.0 mL with Solution A. Dissolve 2.72 g of potassium dihydrogen
ethanol R. To 25.0 mL of the resulting solution add 25.0 mL phosphate R in 900 mL of water R and adjust to pH 2.5 with
of a solution of 1 g of dipotassium hydrogen phosphate R in phosphoric acid R ; dilute to 1000 mL with water R.
55 mL of water R. Prepare in the same manner a reference Test solution. Dissolve 30.0 mg of the substance to be
solution using 20.0 mg of diethylstilbestrol CRS. Transfer an examined in 50.0 mL of the solvent mixture and dilute to
equal volume of each solution to separate 1 cm quartz cells 100.0 mL with mobile phase A.
and close the cells ; place the two cells about 5 cm from a Reference solution (a). Dissolve 6.0 mg of difloxacin
low-pressure, short-wave 2 W to 20 W mercury lamp and impurity G CRS in acetonitrile R and dilute to 20.0 mL with
irradiate for about 5 min. Measure the absorbance (2.2.25) the same solvent.
of the irradiated solutions at the maximum at 418 nm, using Reference solution (b). Mix 0.5 mL of reference solution (a),
water R as the compensation liquid. Continue the irradiation 1.0 mL of the test solution and 50 mL of the solvent mixture
for successive periods of 3 min to 15 min, depending on and dilute to 100.0 mL with mobile phase A.
the power of the lamp, and repeat the measurement of the
absorbances at 418 nm until the maximum absorbance (about Reference solution (c). Dissolve 3 mg of sarafloxacin
0.7) is obtained. If necessary, adjust the geometry of the hydrochloride R (impurity B) in 100.0 mL of solution A. Dilute
irradiation apparatus to obtain a maximum, reproducible 1.0 mL of the solution to 50.0 mL with the test solution.
absorbance at 418 nm. Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
Calculate the content of C18H20O2 from the measured
absorbances and the concentrations of the solutions. – stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase :
STORAGE
– mobile phase A : acetonitrile R, tetrahydrofuran R, solution A
Store protected from light. (5:5:90 V/V/V) ;

2046 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Difloxacin hydrochloride trihydrate for veterinary use

– mobile phase B : acetonitrile R, solution A, tetrahydrofuran R by the general monograph Substances for pharmaceutical
(5:35:60 V/V/V) ; use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Time Mobile phase A Mobile phase B
Control of impurities in substances for pharmaceutical use) : A,
(min) (per cent V/V) (per cent V/V)
B, C, D, E, F.
0 - 15 100 0

15 - 50 100 → 0 0 → 100

50 - 60 0 100

Flow rate : 1.0 mL/min.

Detection : spectrophotometer at 325 nm.
Injection : 30 μL of the test solution and reference solutions (b)
and (c).
Identification of impurities : use the chromatogram obtained
with reference solution (c) to identify the peak due to
impurity B ; use the chromatogram obtained with reference
solution (b) to identify the peak due to impurity G.
Relative retention with reference to difloxacin (retention A. 6-fluoro-7-(4-methylpiperazin-1-yl)-1-[4-(4-
time = about 10 min) : impurity B = about 1.2 ; methylpiperazin-1-yl)phenyl]-4-oxo-1,4-
impurity G = about 4.0. dihydroquinoline-3-carboxylic acid,
System suitability: reference solution (c) :
– peak-to-valley ratio : minimum 2.0, where Hp = height
above the baseline of the peak due to impurity B and
Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to difloxacin.
Limits :
– impurity G : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (b) (0.5 per cent) ;
– unspecified impurities : for each impurity, not more than B. 6-fluoro-1-(4-fluorophenyl)-4-oxo-7-piperazin-1-yl-1,4-
0.2 times the area of the peak due to difloxacin in the dihydroquinoline-3-carboxylic acid (sarafloxacin),
chromatogram obtained with reference solution (b)
(0.20 per cent) ;
– total : maximum 1.0 per cent ;
– disregard limit : 0.1 times the area of the peak due to
difloxacin in the chromatogram obtained with reference
solution (b) (0.10 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
Solvent mixture. Dissolve 30 g of propylene glycol R in 30 mL
of methanol R, add 4 g of arginine R and dilute to 100 mL
with water R. C. 1-(4-chlorophenyl)-6-fluoro-7-(4-methylpiperazin-1-yl)-4-
0.25 g complies with test H. Prepare the reference solution oxo-1,4-dihydroquinoline-3-carboxylic acid,
using 0.5 mL of lead standard solution (10 ppm Pb) R.
The substance precipitates after addition of buffer solution
pH 3.5 R. Dilute to 20 mL with methanol R ; the substance
re-dissolves completely.
Water (2.5.12) : 8.0 per cent to 12.0 per cent, determined on
0.100 g, using a mixture of 20 volumes of formamide R and
25 volumes of methanol R as solvent.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g in a platinum crucible.
ASSAY D. 6-chloro-1-(4-fluorophenyl)-7-(4-methylpiperazin-1-yl)-4-
Dissolve 0.150 g in 5 mL of anhydrous formic acid R and add oxo-1,4-dihydroquinoline-3-carboxylic acid,
50 mL of acetic anhydride R. Titrate with 0.1 M perchloric
acid, determining the end-point potentiometrically (2.2.20).
Read the volume added at the 2nd point of inflexion.
1 mL of 0.1 M perchloric acid is equivalent to 21.79 mg of
C21H20ClF2N3O3.
IMPURITIES
Specified impurities : G.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general E. 7-chloro-1-(4-fluorophenyl)-6-(4-methylpiperazin-1-yl)-4-
acceptance criterion for other/unspecified impurities and/or oxo-1,4-dihydroquinoline-3-carboxylic acid,

General Notices (1) apply to all monographs and other texts 2047
Digitoxin EUROPEAN PHARMACOPOEIA 8.0

B. Examine the chromatograms obtained in the test for related

substances. The principal spot in the chromatogram
obtained with the test solution is similar in position,
colour and size to the principal spot in the chromatogram
obtained with reference solution (a).
C. Suspend about 0.5 mg in 0.2 mL of alcohol (60 per
cent V/V) R. Add 0.1 mL of dinitrobenzoic acid solution R
and 0.1 mL of dilute sodium hydroxide solution R. A violet
colour develops.
F. 6-fluoro-N,1-bis(4-fluorophenyl)-7-(4-methylpiperazin-1- D. Dissolve about 0.5 mg in 1 mL of glacial acetic acid R,
yl)-4-oxo-1,4-dihydroquinoline-3-carboxamide, heating gently, allow to cool and add 0.05 mL of ferric
chloride solution R1. Cautiously add 1 mL of sulfuric acid R,
avoiding mixing the two liquids. A brown ring develops
at the interface and on standing a green, then blue colour
passes to the upper layer.

TESTS
Appearance of solution. Dissolve 50 mg in a mixture of
equal volumes of methanol R and methylene chloride R and
dilute to 10 mL with the same mixture of solvents. The
solution is clear (2.2.1) and colourless (2.2.2, Method I).
G. 7-chloro-6-fluoro-1-(4-fluorophenyl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid. Specific optical rotation (2.2.7). Dissolve 0.25 g in
chloroform R and dilute to 10.0 mL with the same solvent. The
specific optical rotation is + 16.0 to + 18.5.
01/2008:0078 Related substances. Examine by thin-layer chromatography
corrected 6.0 (2.2.27), using a TLC silica gel G plate R.
Test solution. Dissolve 20 mg of the substance to be examined
DIGITOXIN in a mixture of equal volumes of methanol R and methylene
chloride R and dilute to 2 mL with the same mixture of
Digitoxinum solvents.
Reference solution (a). Dissolve 20 mg of digitoxin CRS in
a mixture of equal volumes of methanol R and methylene
chloride R and dilute to 2 mL with the same mixture of
solvents.
Reference solution (b). Dilute 0.5 mL of reference solution (a)
to 50 mL with a mixture of equal volumes of methanol R and
methylene chloride R.
Reference solution (c). Dissolve 10 mg of gitoxin CRS with
stirring in a mixture of equal volumes of methanol R and
methylene chloride R and dilute to 50 mL with the same
mixture of solvents.
Reference solution (d). Dilute 1 mL of reference solution (b)
to 2 mL with a mixture of equal volumes of methanol R and
methylene chloride R.
Reference solution (e). Mix 1 mL of reference solution (a) and
1 mL of reference solution (c).
C41H64O13 Mr 765 Apply to the plate 5 μL of each solution. Develop immediately
[71-63-6] over a path of 15 cm using a mixture of 15 volumes of
DEFINITION methanol R, 40 volumes of cyclohexane R and 90 volumes of
methylene chloride R. Dry the plate in a stream of cold air for
Digitoxin contains not less than 95.0 per cent and 5 min. Repeat the development and dry the plate in a stream
not more than the equivalent of 103.0 per cent of of cold air for 5 min. Spray with a mixture of 1 volume of
3β-[(O-2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-O-2,6- sulfuric acid R and 9 volumes of alcohol R and heat at 130 °C
dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D-ribo- for 15 min. Examine in daylight.
hexopyranosyl)oxy]-14-hydroxy-5β,14β-card-20(22)-enolide,
calculated with reference to the dried substance. Gitoxin. Any spot corresponding to gitoxin in the
chromatogram obtained with the test solution is not more
CHARACTERS intense than the spot in the chromatogram obtained with
A white or almost white powder, practically insoluble in water, reference solution (c) (2.0 per cent).
freely soluble in a mixture of equal volumes of methanol Other glycosides. Any spot in the chromatogram obtained with
and methylene chloride, slightly soluble in alcohol and in the test solution, apart from the principal spot and the spot
methanol. corresponding to gitoxin, is not more intense than the spot
in the chromatogram obtained with reference solution (b)
IDENTIFICATION (1.0 per cent).
First identification : A. The test is not valid unless the chromatogram obtained
Second identification : B, C, D. with reference solution (e) shows clearly separated spots
A. Examine by infrared absorption spectrophotometry corresponding to digitoxin, gitoxin and other glycosides
(2.2.24), comparing with the spectrum obtained with and the spot in the chromatogram obtained with reference
digitoxin CRS. solution (d) is clearly visible.

2048 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Digoxin

Loss on drying (2.2.32). Not more than 1.5 per cent, TESTS
determined on 0.500 g by drying in an oven at 105 °C for 2 h. Appearance of solution. The solution is clear (2.2.1) and
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined colourless (2.2.2, Method I).
on the residue from the test for loss on drying. Dissolve 50 mg in a mixture of equal volumes of methanol R
and methylene chloride R and dilute to 10 mL with the same
ASSAY mixture of solvents.
Dissolve 40.0 mg in alcohol R and dilute to 50.0 mL with the Specific optical rotation (2.2.7) : + 13.9 to + 15.9 (dried
same solvent. Dilute 5.0 mL of the solution to 100.0 mL with substance).
alcohol R. Prepare a reference solution in the same manner, Dissolve 0.50 g in a mixture of equal volumes of methanol R
using 40.0 mg of digitoxin CRS. To 5.0 mL of each solution and methylene chloride R and dilute to 25.0 mL with the same
add 3.0 mL of alkaline sodium picrate solution R, allow to mixture of solvents.
stand protected from bright light for 30 min and measure
the absorbance (2.2.25) of each solution at the maximum at Related substances. Liquid chromatography (2.2.29).
495 nm, using as the compensation liquid a mixture of 5.0 mL Test solution. Dissolve 50.0 mg of the substance to be
of alcohol R and 3.0 mL of alkaline sodium picrate solution R examined in 100.0 mL of methanol R.
prepared at the same time. Reference solution (a). Dissolve 10.0 mg of digoxin CRS in
Calculate the content of C41H64O13 from the absorbances methanol R and dilute to 20.0 mL with the same solvent.
measured and the concentrations of the solutions. Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with methanol R.
STORAGE Reference solution (c). Dissolve 2.5 mg of digoxigenin CRS
Store protected from light. (impurity C) in methanol R and dilute to 5.0 mL with the
same solvent. Dilute 1.0 mL of the solution to 50.0 mL with
methanol R. Dilute 1.0 mL of this solution to 10.0 mL with
methanol R.
01/2008:0079 Reference solution (d). Dissolve 50.0 mg of lanatoside C R
corrected 7.0 (impurity H) in methanol R and dilute to 100.0 mL with the
same solvent. To 1.0 mL of this solution, add 1.0 mL of the
test solution and dilute to 20.0 mL with methanol R.
DIGOXIN Reference solution (e). Dissolve 5.0 mg of digoxin for peak
identification CRS in methanol R and dilute to 10.0 mL with
Digoxinum the same solvent.
Column :
– size : l = 0.15 m, Ø = 3.9 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase :
– mobile phase A : acetonitrile R, water R (10:90 V/V) ;
– mobile phase B : water R, acetonitrile R (10:90 V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-5 78 22

5 - 15 78 → 30 22 → 70

Flow rate : 1.5 mL/min.

Detection : spectrophotometer at 220 nm.
Injection : 10 μL of the test solution and reference solutions (b),
(c), (d) and (e).
C41H64O14 Mr 781
[20830-75-5] Identification of impurities : use the chromatogram supplied
with digoxin for peak identification CRS and the chromatogram
DEFINITION obtained with reference solution (e) to identify the peaks due
to impurities A, B, C, E, F, G and K.
3β-[(2,6-Dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-
dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D- Relative retention with reference to digoxin (retention
ribo-hexopyranosyl)oxy]-12β,14-dihydroxy-5β-card-20(22)- time = about 4.3 min) : impurity C = about 0.3 ;
enolide. impurity E = about 0.5 ; impurity F = about 0.6 ;
impurity G = about 0.8 ; impurity L = about 1.4 ;
Content : 96.0 per cent to 102.0 per cent (dried substance). impurity K = about 1.6 ; impurity B = about 2.2 ;
impurity A = about 2.6.
CHARACTERS
System suitability : reference solution (d) :
Appearance : white or almost white powder, or colourless – resolution : minimum 1.5 between the peaks due to
crystals. impurity H and digoxin.
Solubility : practically insoluble in water, soluble in a mixture Limits :
of equal volumes of methanol and methylene chloride, slightly – impurity F : not more than 2.5 times the area of the principal
soluble in ethanol (96 per cent). peak in the chromatogram obtained with reference
solution (b) (2.5 per cent) ;
IDENTIFICATION
– impurity C : not more than 5 times the area of the
Infrared absorption spectrophotometry (2.2.24). corresponding peak in the chromatogram obtained with
Comparison : digoxin CRS. reference solution (c) (1.0 per cent) ;

General Notices (1) apply to all monographs and other texts 2049
Digoxin EUROPEAN PHARMACOPOEIA 8.0

– impurities E, K : for each impurity, not more than the area

of the principal peak in the chromatogram obtained with
reference solution (b) (1.0 per cent) ;
– impurity G : not more than 0.8 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.8 per cent) ;
– impurities A, B : for each impurity, not more than 0.5 times
the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.5 per cent) ;
– impurity L : not more than 0.3 times the area of the principal
peak in the chromatogram obtained with reference
solution (b) (0.3 per cent) ;
– any other impurity : for each impurity, not more than
0.2 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.2 per cent) ;
– sum of impurities other than A, B, C, E, F, G, K, L : not A. R1 = R2 = R3 = R4 = H : 3β-[(2,6-dideoxy-β-
D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D-
more than 0.7 times the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.7 per ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β- D-ribo-
cent) ; hexopyranosyl)oxy]-14-hydroxy-5β-card-20(22)-enolide
(digitoxin),
– total : not more than 3.5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(3.5 per cent) ;
B. R1 = R3 = R4 = H, R2 = OH : 3β-[(2,6-dideoxy-
– disregard limit : 0.05 times the area of the principal peak β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D-
in the chromatogram obtained with reference solution (b) ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D-ribo-
(0.05 per cent). hexopyranosyl)oxy]-14,16β-dihydroxy-5β-card-20(22)-
The thresholds indicated under Related Substances enolide (gitoxin),
(Table 2034.-1) in the general monograph Substances for
pharmaceutical use (2034) do not apply.
Loss on drying (2.2.32) : maximum 1.0 per cent, determined
E. R1 = R2 = OH, R3 = R4 = H : 3β-[(2,6-dideoxy-
on 0.500 g by drying in vacuo in an oven.
β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D-
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D-ribo-
the residue obtained in the test for loss on drying. hexopyranosyl)oxy]- 12β,14,16β-trihydroxy-5β-card-
20(22)-enolide (diginatin),
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification. H. R1 = OH, R2 = H, R3 = CO-CH3, R4 = Glu :
3β-[(β-D-glucopyranosyl-(1→4)-3-O-acetyl-2,6-
Injection : test solution and reference solution (a). dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-
Calculate the percentage content of C41H64O14 from the β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D-ribo-
declared content of digoxin CRS. hexopyranosyl)oxy]-12β,14-dihydroxy-5β-card-20(22)-
enolide (lanatoside C),
STORAGE
Protected from light.
I. R1 = OH, R2 = R4 = H, R3 = CO-CH3 :
3β-[(3-O-acetyl-2,6-dideoxy-β-D-ribo-hexopyranosyl-
IMPURITIES (1→4)-2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-
dideoxy-β-D-ribo-hexopyranosyl)oxy]-12β,14-dihydroxy-
Specified impurities : A, B, C, E, F, G, K, L. 5β-card-20(22)-enolide (α-acetyldigoxin),
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities. It J. R1 = OH, R2 = R3 = H, R4 = CO-CH3 :
is therefore not necessary to identify these impurities for 3β-[(4-O-acetyl-2,6-dideoxy-β-D-ribo-hexopyranosyl-
demonstration of compliance. See also 5.10. Control of (1→4)-2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-
impurities in substances for pharmaceutical use) : D, H, I, J. dideoxy-β-D-ribo-hexopyranosyl)oxy]-12β,14-dihydroxy-
5β-card-20(22)-enolide (β-acetyldigoxin),

K. R1 = OH, R2 = R3 = H, R4 = Dig : 3β-[(2,6-

dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-
β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D-
ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D-ribo-
hexopyranosyl)oxy]-12β,14-dihydroxy-5β-card-20(22)-
enolide (digoxigenin tetrakisdigitoxoside),

2050 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dihydralazine sulfate, hydrated

Related substances. Liquid chromatography (2.2.29). Prepare

the solutions immediately before use.
Test solution. Dissolve 50.0 mg of the substance to be
examined in a 6 g/L solution of glacial acetic acid R and dilute
to 50.0 mL with the same solution.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase containing 0.5 g/L of sodium
edetate R. Dilute 1.0 mL of this solution to 10.0 mL with the
mobile phase containing 0.5 g/L of sodium edetate R.
C. R = H : 3β,12β,14-trihydroxy-5β-card-20(22)-enolide Reference solution (b). Dilute 1.0 mL of the test solution to
(digoxigenin), 50.0 mL with the mobile phase containing 0.5 g/L of sodium
edetate R.
D. R = Dig : 3β-(2,6-dideoxy-β-D-ribo-hexopyranosyloxy)- Reference solution (c). Dissolve 5 mg of dihydralazine for
12β,14-dihydroxy-5β-card-20(22)-enolide (digoxigenin system suitability CRS in a 6 g/L solution of glacial acetic
monodigitoxoside), acid R and dilute to 5.0 mL with the same solution.
F. R = Dig-(1→4)-Dig : 3β-[(2,6-dideoxy-β-D-ribo- Column :
hexopyranosyl-(1→4)-2,6-dideoxy-β-D-ribo- – size : l = 0.25 m, Ø = 4.6 mm ;
hexopyranosyl)oxy]-12β,14-dihydroxy-5β-card- – stationary phase : nitrile silica gel for chromatography R
20(22)-enolide (digoxigenin bisdigitoxoside), (5 μm).
G. R = Gdd-(1→4)-Dig-(1→4)-Dig : 3β-[(2,6-dideoxy- Mobile phase : mix 22 volumes of acetonitrile R1 and
β-D-arabino-hexopyranosyl-(1→4)-2,6-dideoxy-β- 78 volumes of a solution containing 1.44 g/L of sodium
D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D-ribo- laurilsulfate R and 0.75 g/L of tetrabutylammonium bromide R,
hexopyranosyl)oxy]-12β,14-dihydroxy-5β-card-20(22)- then adjust to pH 3.0 with 0.05 M sulfuric acid.
enolide (neodigoxin), Flow rate : 1.5 mL/min.
L. unknown structure. Detection : spectrophotometer at 230 nm.
Injection : 20 μL.
Run time : twice the retention time of dihydralazine.
01/2008:1310 Relative retention with reference to dihydralazine :
corrected 6.1 impurity A = about 0.8.
System suitability : reference solution (c) :
DIHYDRALAZINE SULFATE, – the peaks due to impurity A and dihydralazine are
HYDRATED baseline separated as in the chromatogram supplied with
dihydralazine for system suitability CRS.
Limits :
Dihydralazini sulfas hydricus – impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (b)
(2 per cent) ;
– impurity C : not more than the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.1 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
C8H12N6O4S,2½H2O Mr 333.3 with reference solution (a) (0.10 per cent) ;
[7327-87-9] – sum of impurities other than A : not more than 5 times the
DEFINITION area of the principal peak in the chromatogram obtained
with reference solution (a) (0.5 per cent) ;
(Phthalazine-1,4(2H,3H)-diylidene)dihydrazine sulfate
2.5-hydrate. – disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
Content : 98.0 per cent to 102.0 per cent (dried substance). (0.01 per cent).
CHARACTERS Impurity B. Liquid chromatography (2.2.29). Prepare the
Appearance : white or slightly yellow, crystalline powder. solutions immediately before use.
Solubility : slightly soluble in water, practically insoluble in Test solution. Dissolve 40.0 mg of hydrazine sulfate R
anhydrous ethanol. It dissolves in dilute mineral acids. (impurity B) in water R and dilute to 100.0 mL with the same
solvent. Dilute 1.0 mL of the solution to 25.0 mL with water R.
IDENTIFICATION To 0.50 mL of this solution, add 0.200 g of the substance to be
A. Infrared absorption spectrophotometry (2.2.24). examined and dissolve in 6 mL of dilute hydrochloric acid R,
then dilute to 10.0 mL with water R. In a centrifuge tube with
Comparison : Ph. Eur. reference spectrum of dihydralazine a ground-glass stopper, place immediately 0.50 mL of this
sulfate hydrated. solution and 2.0 mL of a 60 g/L solution of benzaldehyde R in
B. Dissolve about 50 mg in 5 mL of dilute hydrochloric acid R. a mixture of equal volumes of methanol R and water R. Shake
The solution gives reaction (a) of sulfates (2.3.1). for 90 s. Add 1.0 mL of water R and 5.0 mL of heptane R.
Shake for 1 min and centrifuge. Use the upper layer.
TESTS
Reference solution. Dissolve 40.0 mg of hydrazine sulfate R
Appearance of solution. The solution is clear (2.2.1) and not (impurity B) in water R and dilute to 100.0 mL with the same
more intensely coloured than reference solution BY6 (2.2.2, solvent. Dilute 1.0 mL of the solution to 25.0 mL with water R.
Method II). To 0.50 mL of this solution, add 6 mL of dilute hydrochloric
Dissolve 0.20 g in dilute nitric acid R and dilute to 10 mL with acid R and dilute to 10.0 mL with water R. In a centrifuge tube
the same acid. with a ground-glass stopper, place 0.50 mL of this solution

General Notices (1) apply to all monographs and other texts 2051
Dihydrocodeine hydrogen tartrate EUROPEAN PHARMACOPOEIA 8.0

and 2.0 mL of a 60 g/L solution of benzaldehyde R in a mixture 01/2008:1776

of equal volumes of methanol R and water R. Shake for 90 s.
Add 1.0 mL of water R and 5.0 mL of heptane R. Shake for DIHYDROCODEINE HYDROGEN
1 min and centrifuge. Use the upper layer.
TARTRATE
Blank solution. Prepare in the same manner as for the
reference solution but replacing the 0.50 mL of hydrazine
sulfate solution by 0.50 mL of water R. Dihydrocodeini hydrogenotartras
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : 0.3 g/L solution of sodium edetate R,
acetonitrile R (30:70 V/V).
Flow rate : 1 mL/min. C22H29NO9 Mr 451.5
Detection : spectrophotometer at 305 nm. [5965-13-9]
Injection : 20 μL. DEFINITION
Relative retention with reference to benzaldehyde : 4,5α-Epoxy-3-methoxy-17-methylmorphinan-6α-ol hydrogen
benzaldehyde azine (benzalazine) corresponding to (2R,3R)-2,3-dihydroxybutanedioate.
impurity B = about 1.8. Content : 98.5 per cent to 101.0 per cent (anhydrous substance).
Limit : CHARACTERS
– impurity B : the area of the peak due to benzaldehyde azine Appearance : white or almost white, crystalline powder.
is not greater than twice the area of the corresponding peak Solubility : freely soluble in water, sparingly soluble in alcohol,
in the chromatogram obtained with the reference solution practically insoluble in cyclohexane.
(10 ppm).
Iron (2.4.9) : maximum 20 ppm. IDENTIFICATION
To the residue obtained in the test for sulfated ash add 0.2 mL First identification : A.
of sulfuric acid R and heat carefully until the acid is almost Second identification : B, C, D.
completely eliminated. Allow to cool and dissolve the residue A. Infrared absorption spectrophotometry (2.2.24).
with heating in 5.5 mL of hydrochloric acid R1. Filter the Comparison: Ph. Eur. reference spectrum of dihydrocodeine
hot solution through a filter previously washed 3 times with hydrogen tartrate.
dilute hydrochloric acid R. Wash the crucible and the filter
B. To about 0.1 g add 1 mL of sulfuric acid R and 0.05 mL
with 5 mL of water R. Combine the filtrate and the washings
of ferric chloride solution R1 and heat on a water-bath. A
and neutralise with about 3.5 mL of strong sodium hydroxide
brownish-yellow colour develops. Add 0.05 mL of dilute
solution R. Adjust to pH 3-4 with acetic acid R and dilute to
nitric acid R. The colour does not become red.
20 mL with water R. Prepare the standard with 5 mL of iron
standard solution (2 ppm Fe) R and 5 mL of water R. C. To 1 mL of solution S (see Tests) add 5 mL of picric acid
solution R. Heat on a water-bath until a clear solution is
Loss on drying (2.2.32) : 13.0 per cent to 15.0 per cent, obtained. Allow to cool. A precipitate is formed. Filter,
determined on 1.000 g by drying in an oven at 50 °C at a wash with 5 mL of water R and dry at 100-105 °C. The
pressure not exceeding 0.7 kPa for 5 h. crystals melt (2.2.14) at 220 °C to 223 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on D. It gives reaction (b) of tartrates (2.3.1).
1.0 g.
TESTS
ASSAY Solution S. Dissolve 2.50 g in carbon dioxide-free water R and
Dissolve 60.0 mg in 25 mL of water R. Add 35 mL of dilute to 25.0 mL with the same solvent.
hydrochloric acid R and titrate slowly with 0.05 M potassium Appearance of solution. Solution S is clear (2.2.1) and not
iodate, determining the end-point potentiometrically (2.2.20), more intensely coloured than reference solution BY5 (2.2.2,
using a calomel reference electrode and a platinum indicator Method II).
electrode.
pH (2.2.3) : 3.2 to 4.2 for solution S.
1 mL of 0.05 M potassium iodate is equivalent to 7.208 mg Specific optical rotation (2.2.7) : − 70.5 to − 73.5 (anhydrous
of C8H12N6O4S. substance).
IMPURITIES Dilute 10.0 mL of solution S to 20.0 mL with water R.
Specified impurities : A, B, C. Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 10.0 mg of the substance to be
examined in the mobile phase and dilute to 10.0 mL with the
mobile phase.
Reference solution (a). Dissolve 2.0 mg of codeine phosphate R
in 2.0 mL of the test solution and dilute to 25.0 mL with the
mobile phase.
Reference solution (b). Dilute 1.0 mL of the test solution to
A. R = NH2 : 4-hydrazinophthalazin-1-amine, 200 mL with the mobile phase.
Column :
C. R = H : (phthalazin-1-yl)hydrazine (hydralazine), – size : l = 0.25 m, Ø = 4.6 mm,
– stationary phase : octylsilyl silica gel for chromatography R
B. H2N-NH2 : hydrazine. (5 μm).

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EUROPEAN PHARMACOPOEIA 8.0 Dihydroergocristine mesilate

Mobile phase : to 1.0 g of sodium heptanesulfonate R, add

10.0 mL of glacial acetic acid R and 4.0 mL of a solution of
5.0 mL of triethylamine R diluted to 25.0 mL with a mixture of
equal volumes of water R and acetonitrile R. Add 170 mL of
acetonitrile R and dilute to 1000 mL with water R.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 284 nm. C. 4,5α-epoxy-3-methoxy-17-methylmorphinan-6-one
Injection : 20 μL. (hydrocodone),
Run time : 5 times the retention time of dihydrocodeine.
Retention time : dihydrocodeine = about 14 min.
System suitability: reference solution (a) :
– resolution : minimum of 2 between the peaks due to
dihydrocodeine and to impurity A.
Limits : D. 4,5α-epoxy-3,6α-dimethoxy-17-methylmorphinan
– impurity A : not more than the area of the principal peak (tetrahydrothebaine).
in the chromatogram obtained with reference solution (b)
(0.5 per cent), 07/2013:1416
– any other peak : not more than 0.6 times the area of
the principal peak in the chromatogram obtained with DIHYDROERGOCRISTINE MESILATE
reference solution (b) (0.3 per cent),
– total : not more than twice the area of the principal peak Dihydroergocristini mesilas
in the chromatogram obtained with reference solution (b)
(1 per cent); disregard any peak due to tartaric acid (relative
retention with reference to dihydrocodeine = about 0.25),
– disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Water (2.5.12) : maximum 0.7 per cent, determined on 1.00 g.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. C36H45N5O8S Mr 708
[24730-10-7]
ASSAY
DEFINITION
Dissolve 0.350 g in 60 mL of anhydrous acetic acid R. (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-Benzyl-10b-hydroxy-
Titrate with 0.1 M perchloric acid determining the end-point 2-(1-methylethyl)-3,6-dioxo-octahydro-8H-oxazolo[3,2-
potentiometrically (2.2.20). a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10,10a-
1 mL of 0.1 M perchloric acid is equivalent to 45.15 mg of octahydroindolo[4,3-fg]quinoline-9-carboxamide
C22H29NO9. methanesulfonate.
Content : 98.0 per cent to 102.0 per cent (dried substance).
STORAGE
PRODUCTION
Protected from light. It is considered that alkylsulfonate esters are genotoxic
and are potential impurities in dihydroergocristine
IMPURITIES mesilate. The manufacturing process should be developed
taking into consideration the principles of quality risk
management, together with considerations of the quality
of starting materials, process capability and validation.
The general methods 2.5.37. Methyl, ethyl and isopropyl
methanesulfonate in methanesulfonic acid, 2.5.38. Methyl,
ethyl and isopropyl methanesulfonate in active substances and
2.5.39. Methanesulfonyl chloride in methanesulfonic acid are
available to assist manufacturers.
CHARACTERS
A. 7,8-didehydro-4,5α-epoxy-3-methoxy-17-
methylmorphinan-6α-ol (codeine), Appearance : white or almost white, fine crystalline powder.
Solubility : slightly soluble in water, soluble in methanol.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs.
Comparison: dihydroergocristine mesilate CRS.
B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 0.10 g of the substance to be
examined in a mixture of 1 volume of methanol R and
B. 7,8-didehydro-4,5α-epoxy-17-methylmorphinan-3,6α-diol 9 volumes of methylene chloride R and dilute to 5 mL with
(morphine), the same mixture of solvents.

General Notices (1) apply to all monographs and other texts 2053
Dihydroergocristine mesilate EUROPEAN PHARMACOPOEIA 8.0

Reference solution. Dissolve 0.10 g of dihydroergocristine with water R. Dilute 6.0 mL of the solution to 50.0 mL with a
mesilate CRS in a mixture of 1 volume of methanol R and mixture of 20 volumes of acetonitrile R, 20 volumes of a 1.0 g/L
9 volumes of methylene chloride R and dilute to 5 mL with solution of phosphoric acid R and 60 volumes of water R.
the same mixture of solvents. Column :
Plate : TLC silica gel F254 plate R. – size : l = 0.25 m, Ø = 4.6 mm ;
Mobile phase : concentrated ammonia R, dimethylforma- – stationary phase : octadecylsilyl silica gel for
mide R, ether R (2:15:85 V/V/V). chromatography R (5 μm) with a pore size of
Application : 5 μL. 10 nm and a carbon loading of 19 per cent.
Development : over 2/3 of the plate protected from light. Mobile phase :
Drying : in a current of cold air for 5 min. – mobile phase A : mix 100 volumes of acetonitrile R
Detection : spray with dimethylaminobenzaldehyde with 900 volumes of water R and add 10 volumes of
solution R7 and dry in a current of hot air for 2 min. triethylamine R ;
Results : the principal spot in the chromatogram obtained – mobile phase B : mix 100 volumes of water R with
with the test solution is similar in position, colour and size 900 volumes of acetonitrile R and add 10 volumes of
to the principal spot in the chromatogram obtained with triethylamine R ;
the reference solution. Time Mobile phase A Mobile phase B
C. Thin-layer chromatography (2.2.27). (min) (per cent V/V) (per cent V/V)
Test solution. Dissolve 0.20 g of the substance to be 0-5 75 25
examined in a mixture of 1 volume of methanol R and 5 - 20 75 → 25 25 → 75
9 volumes of methylene chloride R and dilute to 5 mL with
the same mixture of solvents. Flow rate : 1.2 mL/min.
Reference solution. Dissolve 0.20 g of methanesulfonic Detection : spectrophotometer at 280 nm.
acid R in a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R and dilute to 5 mL with Injection : 10 μL.
the same mixture of solvents. Dilute 1 mL of the solution Relative retention with reference to dihydroergocristine
to 10 mL with a mixture of 1 volume of methanol R and (retention time = about 13.7 min) : impurity F = about 0.8 ;
9 volumes of methylene chloride R. impurity H = about 0.9 ; impurity I = about 1.02.
Plate : TLC silica gel F254 plate R. System suitability : reference solution :
Mobile phase : water R, concentrated ammonia R, butanol R, – the chromatogram shows 4 peaks ;
acetone R (5:10:20:65 V/V/V/V). – resolution : minimum 1 between the peaks due to
Application : 10 μL. dihydroergocristine and impurity I.
Development : over a path of 10 cm protected from light. Limits :
Drying : in a current of cold air for not more than 1 min. – any impurity : not more than the area of the peak due to
Detection : spray with a 1 g/L solution of bromocresol dihydroergocristine in the chromatogram obtained with
purple R in methanol R, adjusting the colour to violet-red the reference solution (1 per cent) ;
with one drop of dilute ammonia R1 and dry the plate in a – total : not more than twice the area of the peak due to
current of hot air at 100 °C. dihydroergocristine in the chromatogram obtained with
Results : the principal spot in the chromatogram obtained the reference solution (2 per cent) ;
with the test solution is similar in position, colour and size – disregard limit : 0.1 times the area of the peak due to
to the principal spot in the chromatogram obtained with dihydroergocristine in the chromatogram obtained with
the reference solution. the reference solution (0.1 per cent).
Loss on drying (2.2.32) : maximum 3.0 per cent, determined
TESTS on 0.500 g by drying under high vacuum at 80 °C.
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution B7 (2.2.2, ASSAY
Method II). Dissolve 0.300 g in 60 mL of pyridine R. Pass a stream of
Dissolve 0.50 g in methanol R and dilute to 25.0 mL with the nitrogen R over the surface of the solution and titrate with
same solvent. 0.1 M tetrabutylammonium hydroxide, determining the
end-point potentiometrically (2.2.20). Note the volume used
pH (2.2.3) : 4.0 to 5.0.
at the second point of inflexion.
Dissolve 0.10 g in carbon dioxide-free water R and dilute to
1 mL of 0.1 M tetrabutylammonium hydroxide is equivalent
20 mL with the same solvent.
to 35.39 mg of C36H45N5O8S.
Specific optical rotation (2.2.7) : − 43 to − 37 (dried
substance). STORAGE
Dissolve 0.250 g in anhydrous pyridine R and dilute to 25.0 mL Store protected from light.
with the same solvent.
IMPURITIES
Related substances. Liquid chromatography (2.2.29). Carry
out the test and preparation of the solutions protected from
bright light.
Test solution. Dissolve 75.0 mg of the substance to be
examined in 10 mL of acetonitrile R. Add 10 mL of a 1.0 g/L
solution of phosphoric acid R and dilute to 50.0 mL with
water R.
Reference solution. Dissolve 20.0 mg of codergocrine A. (6aR,9R,10aR)-7-methyl-4,6,6a,7,8,9,10,10a-
mesilate CRS in 10 mL of acetonitrile R. Add 10 mL of a octahydroindolo[4,3-fg]quinoline-9-carboxamide
1.0 g/L solution of phosphoric acid R and dilute to 50.0 mL (6-methylergoline-8β-carboxamide),

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EUROPEAN PHARMACOPOEIA 8.0 Dihydroergocristine mesilate

B. (6aR,9S,10aS)-7-methyl-4,6,6a,7,8,9,10,10a-
octahydroindolo[4,3-fg]quinoline-9-carboxamide G. (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-2-ethyl-
(6-methylisoergoline-8α-carboxamide), 10b-hydroxy-3,6-dioxooctahydro-8H-oxazolo[3,2-
a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10,10a-
octahydroindolo[4,3-fg]quinoline-9-carboxamide
(dihydroergostine),

C. (6aR,9R,10aR)-N-[(2S,5S,10aS,10bS)-5-benzyl-10b-
hydroxy-2-(1-methylethyl)-3,6-dioxooctahydro-8H-
oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl- H. (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-2-(1-
4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9- methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro-
carboxamide (2′-epidihydroergocristine), 8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9-
carboxamide (α-dihydroergocryptine),

D. (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-
2-methyl-5-(1-methylethyl)-3,6-dioxooctahydro-8H-
oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl- I. (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-10b-hydroxy-
4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9- 2-(1-methylethyl)-5-[(1RS-1-methylpropyl]-3,6-
carboxamide (dihydroergosine), dioxooctahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-
2-yl]-7-methyl-4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-
fg]quinoline-9-carboxamide (β-dihydroergocryptine or
epicriptine),

E. (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-
hydroxy-2-methyl-3,6-dioxooctahydro-8H-oxazolo[3,2- J. (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-
a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10,10a- hydroxy-2-[(1RS)-1-methylpropyl]-3,6-dioxooctahydro-
octahydroindolo[4,3-fg]quinoline- 9-carboxamide 8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
(dihydroergotamine), 4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9-
carboxamide (dihydroergosedmine),

F. (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)10b-hydroxy-2,5- K. (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-
bis(1-methylethyl)-3,6-dioxooctahydro-8H-oxazolo[3,2- hydroxy-2-(1-methylethyl)-3,6-dioxooctahydro-8H-
a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10,10a- oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-
octahydroindolo[4,3-fg]quinoline-9-carboxamide 4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-
(dihydroergocornine), carboxamide (ergocristine),

General Notices (1) apply to all monographs and other texts 2055
Dihydroergotamine mesilate EUROPEAN PHARMACOPOEIA 8.0

Specific absorbance at the absorption maximum at 281 nm :

95 to 105 (dried substance).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: dihydroergotamine mesilate CRS.
C. Thin-layer chromatography (2.2.27). Prepare the reference
solution and the test solution immediately before use.
Solvent mixture : methanol R, methylene chloride R
(10:90 V/V).
L. (6aR,7RS,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-
hydroxy-2-(1-methylethyl)-3,6-dioxooctahydro-8H- Test solution. Dissolve 5 mg of the substance to be
oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl- examined in the solvent mixture and dilute to 2.5 mL with
4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9- the solvent mixture.
carboxamide 7-oxide (dihydroergocristine 6-oxide). Reference solution. Dissolve 5 mg of dihydroergotamine
mesilate CRS in the solvent mixture and dilute to 2.5 mL
with the solvent mixture.
07/2013:0551 Plate : TLC silica gel G plate R.
Mobile phase : concentrated ammonia R, methanol R, ethyl
DIHYDROERGOTAMINE MESILATE acetate R, methylene chloride R (1:6:50:50 V/V/V/V).
Application : 5 μL.
Dihydroergotamini mesilas Development : protected from light, over a path of 15 cm ;
dry in a current of cold air for not longer than 1 min and
repeat the development protected from light over a path of
15 cm using a freshly prepared amount of the mobile phase.
Drying : in a current of cold air.
Detection : spray abundantly with dimethylaminobenzalde-
hyde solution R7 and dry in a current of hot air for about
2 min.
Results : the principal spot in the chromatogram obtained
C34H41N5O8S Mr 680 with the test solution is similar in position, colour and size
[6190-39-2] to the principal spot in the chromatogram obtained with
the reference solution.
DEFINITION
D. To 0.1 g of the substance to be examined, add 5 mL of
(6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-Benzyl-10b-hydroxy- dilute hydrochloric acid R and shake for about 5 min. Filter,
2-methyl-3,6-dioxooctahydro-8H-oxazolo[3,2-a]pyrrolo[2,1- then add 1 mL of barium chloride solution R1. The filtrate
c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10,10a- remains clear. Mix 0.1 g of the substance to be examined
octahydroindolo[4,3-fg]quinoline-9-carboxamide with 0.4 g of powdered sodium hydroxide R, heat to fusion
methanesulfonate. and continue to heat for 1 min. Cool, add 5 mL of water R,
Content : 98.0 per cent to 101.0 per cent (dried substance). boil and filter. Acidify the filtrate with hydrochloric acid R1
and filter again. The filtrate gives reaction (a) of sulfates
PRODUCTION (2.3.1).
It is considered that alkylsulfonate esters are genotoxic
and are potential impurities in dihydroergotamine TESTS
mesilate. The manufacturing process should be developed Appearance of solution. The solution is clear (2.2.1) and not
taking into consideration the principles of quality risk more intensely coloured than reference solution Y7 or BY7
management, together with considerations of the quality (2.2.2, Method II).
of starting materials, process capability and validation. Dissolve 0.10 g in a mixture of 0.1 mL of a 70 g/L solution of
The general methods 2.5.37. Methyl, ethyl and isopropyl methanesulfonic acid R and 50 mL of water R.
methanesulfonate in methanesulfonic acid, 2.5.38. Methyl,
ethyl and isopropyl methanesulfonate in active substances and pH (2.2.3) : 4.4 to 5.4.
2.5.39. Methanesulfonyl chloride in methanesulfonic acid are Dissolve 0.10 g in carbon dioxide-free water R and dilute to
available to assist manufacturers. 100 mL with the same solvent.
CHARACTERS Specific optical rotation (2.2.7) : − 47 to − 42 (dried
substance).
Appearance : white or almost white, crystalline powder or
colourless crystals. Dissolve 0.250 g in anhydrous pyridine R and dilute to 25.0 mL
with the same solvent.
Solubility : slightly soluble in water, sparingly soluble in
methanol, slightly soluble in ethanol (96 per cent). Related substances. Liquid chromatography (2.2.29). Carry
out the test protected from light.
IDENTIFICATION Solvent mixture : acetonitrile R, water R (50:50 V/V).
First identification : B, C. Test solution. Dissolve 70 mg of the substance to be examined
Second identification : A, C, D. in the solvent mixture and dilute to 100.0 mL with the solvent
A. Ultraviolet and visible absorption spectrophotometry mixture.
(2.2.25). Reference solution (a). Dilute 1.0 mL of the test solution
Test solution. Dissolve 5.0 mg in methanol R and dilute to to 10.0 mL with the solvent mixture. Dilute 1.0 mL of this
100.0 mL with the same solvent. solution to 100.0 mL with the solvent mixture.
Spectral range : 250-350 nm. Reference solution (b). Dissolve 7 mg of the substance to be
examined and 6.8 mg of ergotamine tartrate CRS (impurity A)
Absorption maxima : at 281 nm and 291 nm.
(equivalent to 7 mg of ergotamine mesilate) in the solvent
Shoulder : at 275 nm. mixture and dilute to 100 mL with the solvent mixture. Dilute
Absorbance : negligible above 320 nm. 5 mL of this solution to 10 mL with the solvent mixture.

2056 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dihydroergotamine mesilate

Reference solution (c). Dissolve 5 mg of dihydroergotamine 1 mL of 0.1 M perchloric acid is equivalent to 68.00 mg
for peak identification CRS (containing impurities A, B, C, D of C34H41N5O8S.
and E) in the solvent mixture, add 100 μL of dilute sulfuric
acid R and dilute to 5 mL with the solvent mixture. STORAGE
Column : Protected from light.
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : spherical end-capped octadecylsilyl silica IMPURITIES
gel for chromatography R (3 μm) ; Specified impurities : A, B, C, D, E.
– temperature : 25 °C.
Mobile phase :
– mobile phase A : 3 g/L solution of sodium heptanesulfonate
monohydrate R adjusted to pH 2.0 with phosphoric acid R ;
– mobile phase B : mobile phase A, acetonitrile for
chromatography R (20:80 V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 15 58 → 40 42 → 60
A. (6aR,9R)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-hydroxy-
Flow rate : 1.5 mL/min. 2-methyl-3,6-dioxooctahydro-8H-oxazolo[3,2-
Detection : spectrophotometer at 220 nm. a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9-
hexahydroindolo[4,3-fg]quinoline-9-carboxamide
Injection : 5 μL. (ergotamine),
Identification of impurities : use the chromatogram supplied
with dihydroergotamine for peak identification CRS and the
chromatogram obtained with reference solution (c) to identify
the peaks due to impurities A, B, C, D and E.
Relative retention with reference to dihydroergotamine
(retention time = about 6.5 min) : impurity D = about 0.7 ;
impurity C = about 0.86 ; impurity A = about 0.95 ;
impurity B = about 1.2 ; impurity E = about 1.4.
System suitability : reference solution (b) :
– resolution : minimum 1.5 between the peaks due to B. (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-2-ethyl-
impurity A and dihydroergotamine. 10b-hydroxy-3,6-dioxooctahydro-8H-oxazolo[3,2-
Limits : a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10,10a-
– correction factors : for the calculation of content, multiply octahydroindolo[4,3-fg]quinoline-9-carboxamide
the peak areas of the following impurities by the (9,10-dihydroergostine),
corresponding correction factor : impurity A = 1.3 ;
impurity C = 1.3 ;
– impurities B, E : for each impurity, not more than 5 times the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.5 per cent) ;
– impurity C : not more than 3 times the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.3 per cent) ;
– impurities A, D : for each impurity, not more than 1.5 times
the area of the principal peak in the chromatogram C. (6aR,9S,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-
obtained with reference solution (a) (0.15 per cent) ; 10b-hydroxy-2-methyl-3,6-dioxooctahydro-
– unspecified impurities : for each impurity, not more than the 8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-
area of the principal peak in the chromatogram obtained yl]-9-hydroxy-7-methyl-4,6,6a,7,8,9,10,10a-
with reference solution (a) (0.10 per cent) ; octahydroindolo[4,3-fg]quinoline-9-carboxamide
(8-hydroxy-9,10-dihydroergotamine),
– total : not more than 10 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(1.0 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 4.0 per cent, determined
on 0.500 g by drying at 105 °C at a pressure not exceeding
0.1 kPa for 5 h.

ASSAY D. (6aR,9R,10aR)-N-[(2S,5S,10aS,10bS)-5-benzyl-10b-
Dissolve 0.500 g in a mixture of 10 mL of anhydrous acetic hydroxy-2-methyl-3,6-dioxooctahydro-8H-oxazolo[3,2-
acid R and 70 mL of acetic anhydride R. Titrate with 0.1 M a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10,10a-
perchloric acid, determining the end-point potentiometrically octahydroindolo[4,3-fg]quinoline-9-carboxamide
(2.2.20). (2′-epi-9,10-dihydroergotamine),

General Notices (1) apply to all monographs and other texts 2057
Dihydroergotamine tartrate EUROPEAN PHARMACOPOEIA 8.0

D. Suspend about 15 mg in 1 mL of water R. 0.1 mL of the

suspension gives reaction (b) of tartrates (2.3.1).
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution Y7 or BY7
(2.2.2, Method II).
Dissolve 0.1 g in alcohol (85 per cent V/V) R warming carefully
E. (6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b- in a water-bath at 40 °C and dilute to 50 mL with the same
hydroxy-2-(1-methylethyl)-3,6-dioxo-octahydro-8H- solvent.
oxazolo[3,2-a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl- pH (2.2.3) : 4.0 to 5.5 for the clear supernatant.
4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline-9- Suspend 50 mg in 50 mL of carbon dioxide-free water R and
carboxamide (dihydroergocristine). shake for 10 min. Allow to stand.
Specific optical rotation (2.2.7) : − 52 to − 57 (dried
01/2008:0600 substance).
corrected 6.0 Dissolve 0.250 g in anhydrous pyridine R and dilute to 25.0 mL
with the same solvent.
DIHYDROERGOTAMINE TARTRATE Related substances. Thin-layer chromatography (2.2.27).
Prepare the reference solutions and the test solutions
Dihydroergotamini tartras immediately before use and in the order indicated.
Reference solution (a). Dissolve 20 mg of dihydroergotamine
tartrate CRS in a mixture of 1 volume of methanol R and
9 volumes of chloroform R and dilute to 10 mL with the same
mixture of solvents.
Reference solution (b). Dilute 2.5 mL of reference solution (a)
to 50 mL with a mixture of 1 volume of methanol R and
9 volumes of chloroform R.
Reference solution (c). Dilute 2 mL of reference solution (b) to
5 mL with a mixture of 1 volume of methanol R and 9 volumes
of chloroform R.
Test solution (a). Dissolve 0.10 g of the substance to be
examined in a mixture of 1 volume of methanol R and
C70H80N10O16 Mr 1317 9 volumes of chloroform R and dilute to 5 mL with the same
[5989-77-5] mixture of solvents.
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
DEFINITION with a mixture of 1 volume of methanol R and 9 volumes of
Bis[(6aR,9R,10aR)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b- chloroform R.
hydroxy-2-methyl-3,6-dioxooctahydro-8H-oxazolo[3,2- Plate : TLC silica gel G plate R.
a]pyrrolo[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9,10,10a- Mobile phase : concentrated ammonia R, methanol R, ethyl
octahydroindolo[4,3-fg]quinoline-9-carboxamide] acetate R, methylene chloride R (1:6:50:50 V/V/V/V).
(2R,3R)-2,3-dihydroxybutanedioate. Application : 5 μL.
Content : 98.0 per cent to 101.0 per cent (dried substance). Development : protected from light over a path of 15 cm. Dry
CHARACTERS the plate in a current of cold air for not longer than 1 min.
Repeat the development protected from light over a path of
Appearance : white or almost white, crystalline powder or 15 cm using a freshly prepared amount of the mobile phase.
colourless crystals.
Drying : in a current of cold air.
Solubility : very slightly soluble in water, sparingly soluble in
Detection : spray the plate abundantly with dimethylamino-
alcohol.
benzaldehyde solution R7 and dry in a current of hot air for
IDENTIFICATION about 2 min.
First identification : B, C. Limits : in the chromatogram obtained with test solution (a) :
Second identification : A, C, D. – any impurity : any spot, apart from the principal spot, is not
more intense than the principal spot in the chromatogram
A. Dissolve 5.0 mg in methanol R and dilute to 100.0 mL
obtained with reference solution (b) (0.5 per cent) and
with the same solvent. Examined between 250 nm
not more than 2 such spots are more intense than the
and 350 nm (2.2.25), the solution shows 2 absorption
principal spot in the chromatogram obtained with reference
maxima, at 281 nm and 291 nm, and a shoulder at 275 nm.
solution (c) (0.2 per cent).
Above 320 nm the absorbance is negligible. The specific
absorbance at the maximum at 281 nm is 95 to 115 (dried Loss on drying (2.2.32) : maximum 5.0 per cent, determined
substance). on 0.200 g by drying in an oven at 105 °C.
B. Infrared absorption spectrophotometry (2.2.24). ASSAY
Preparation : discs. Dissolve 0.250 g in 50 mL of anhydrous acetic acid R. Titrate
Comparison : dihydroergotamine tartrate CRS. with 0.05 M perchloric acid, determining the end-point
C. Examine the chromatograms obtained in the test for potentiometrically (2.2.20).
related substances. 1 mL of 0.05 M perchloric acid is equivalent to 32.93 mg of
Results : the principal spot in the chromatogram obtained C70H80N10O16.
with test solution (b) is similar in position, colour and size
to the principal spot in the chromatogram obtained with STORAGE
reference solution (a). Protected from light.

2058 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dihydrostreptomycin sulfate for veterinary use

04/2010:0485 Test solution. Dissolve 10 mg of the substance to be

examined in water R and dilute to 10 mL with the same
DIHYDROSTREPTOMYCIN SULFATE solvent.
Reference solution (a). Dissolve the contents of a vial of
FOR VETERINARY USE dihydrostreptomycin sulfate CRS in 5.0 mL of water R.
Reference solution (b). Dilute 1.0 mL of reference
Dihydrostreptomycini sulfas ad usum solution (a) to 5.0 mL with water R.
veterinarium Reference solution (c). Dissolve 10 mg of kanamycin
monosulfate CRS and 10 mg of neomycin sulfate CRS
in water R, add 2.0 mL of reference solution (a), mix
thoroughly and dilute to 10 mL with water R.
Plate : TLC silica gel plate R.
Mobile phase : 70 g/L solution of potassium dihydrogen
phosphate R.
Application : 10 μL.
Development : over 2/3 of the plate.
Drying : in a current of warm air.
Detection : spray with a mixture of equal volumes of a 2 g/L
solution of 1,3-dihydroxynaphthalene R in ethanol (96 per
cent) R and a 460 g/L solution of sulfuric acid R ; heat at
150 °C for 5-10 min.
System suitability : reference solution (c):
– the chromatogram shows 3 clearly separated spots.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
reference solution (b).
C. Dissolve 0.1 g in 2 mL of water R and add 1 mL of
[5490-27-7] α-naphthol solution R and 2 mL of a mixture of equal
DEFINITION volumes of strong sodium hypochlorite solution R and
water R. A red colour develops.
Main compound : bis[N,N′′′-[(1R,2R,3S,4R,5R,6S)-4-[[5-
deoxy-2-O-[2-deoxy-2-(methylamino)-α-L-glucopyranosyl]- D. Dissolve 10 mg in 5 mL of water R and add 1 mL of 1 M
3-C-(hydroxymethyl)-α-L-lyxofuranosyl]oxy]-2,5,6- hydrochloric acid. Heat in a water-bath for 2 min. Add
trihydroxycyclohexane-1,3-diyl]diguanidine] trisulfate. 2 mL of a 5 g/L solution of α-naphthol R in 1 M sodium
hydroxide and heat in a water-bath for 1 min. A violet-pink
Sulfate of a substance obtained by catalytic hydrogenation of colour is produced.
streptomycin or by any other means.
E. It gives reaction (a) of sulfates (2.3.1).
Semi-synthetic product derived from a fermentation product.
Stabilisers may be added. TESTS
Content : Solution S. Dissolve 2.5 g in carbon dioxide-free water R and
dilute to 10 mL with the same solvent.
– sum of the percentage contents of dihydrostreptomycin
sulfate and streptomycin sulfate : 95.0 per cent to 102.0 per Appearance of solution. Solution S is not more intensely
cent (dried substance) ; coloured than intensity 5 of the range of reference solutions of
– streptomycin sulfate : maximum 2.0 per cent (dried the most appropriate colour (2.2.2, Method II). Allow to stand
protected from light at about 20 °C for 24 h ; solution S is not
substance).
more opalescent than reference suspension II (2.2.1).
PRODUCTION pH (2.2.3) : 5.0 to 7.0 for solution S.
The method of manufacture is validated to demonstrate that Specific optical rotation (2.2.7) : − 83.0 to − 91.0 (dried
the product, if tested, would comply with the following test. substance).
Abnormal toxicity (2.6.9). Inject into each mouse 1 mg Dissolve 0.200 g in water R and dilute to 10.0 mL with the
dissolved in 0.5 mL of water for injections R. same solvent.
CHARACTERS Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 50.0 mg of the substance to be
Appearance : white or almost white, hygroscopic powder.
examined in water R and dilute to 10.0 mL with the same
Solubility : freely soluble in water, practically insoluble in solvent.
acetone, in ethanol (96 per cent) and in methanol.
Reference solution (a). Dissolve the contents of a vial of
IDENTIFICATION dihydrostreptomycin sulfate CRS (containing impurities A, B
and C) in 5.0 mL of water R.
First identification : A, E.
Reference solution (b). Dilute 1.0 mL of the test solution to
Second identification : B, C, D, E. 100.0 mL with water R.
A. Examine the chromatograms obtained in the assay. Reference solution (c). Dilute 5.0 mL of reference solution (b)
Results : the principal peak in the chromatogram obtained to 50.0 mL with water R.
with the test solution is similar in retention time and size Reference solution (d). Dissolve 10 mg of streptomycin
to the principal peak in the chromatogram obtained with sulfate CRS in water R and dilute to 20 mL with the same
reference solution (a). solvent. Mix 0.1 mL of this solution with 1.0 mL of reference
B. Thin-layer chromatography (2.2.27). solution (a).

General Notices (1) apply to all monographs and other texts 2059
Dihydrostreptomycin sulfate for veterinary use EUROPEAN PHARMACOPOEIA 8.0

Reference solution (e). Dilute 1.0 mL of reference solution (a) Sulfated ash (2.4.14) : maximum 1.0 per cent, determined on
to 100.0 mL with water R. 1.0 g.
Column : Bacterial endotoxins (2.6.14) : less than 0.50 IU/mg, if
– size : l = 0.25 m, Ø = 4.6 mm ; intended for use in the manufacture of parenteral preparations
without a further appropriate procedure for removal of
– stationary phase : octadecylsilyl silica gel for bacterial endotoxins.
chromatography R (5 μm) ;
– temperature : 45 °C. ASSAY
Mobile phase : solution in water R containing 4.6 g/L Liquid chromatography (2.2.29) as described in the test for
of anhydrous sodium sulfate R, 1.5 g/L of sodium related substances with the following modification.
octanesulfonate R, 120 mL/L of acetonitrile R1 and 50 mL/L Injection : test solution and reference solutions (a) and (e).
of a 27.2 g/L solution of potassium dihydrogen phosphate R
adjusted to pH 3.0 with a 22.5 g/L solution of phosphoric Calculate the percentage content of streptomycin sulfate using
acid R. the chromatogram obtained with reference solution (e) and
the declared content of dihydrostreptomycin sulfate CRS.
Flow rate : 1.0 mL/min.
Calculate the percentage content of dihydrostreptomycin
Detection : spectrophotometer at 205 nm. sulfate using the chromatogram obtained with reference
Injection : 20 μL. solution (a) and the declared content of dihydrostreptomycin
sulfate CRS.
Run time : 1.5 times the retention time of dihydrostreptomycin.
Identification of impurities : use the chromatogram supplied STORAGE
with dihydrostreptomycin sulfate CRS and the chromatogram
In an airtight container, protected from light. If the substance
obtained with reference solution (a) to identify the peaks due
is sterile, store in a sterile, airtight, tamper-proof container.
to streptomycin and impurities A, B and C.
Relative retention with reference to dihydrostreptomycin IMPURITIES
(retention time = about 57 min) : impurity A = about 0.2 ;
impurity B = about 0.8 ; streptomycin = about 0.9 ; Specified impurities : A, B, C.
impurity C = about 0.95. Other detectable impurities (the following substances would,
System suitability : if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
– peak-to-valley ratio (a) : minimum 1.1, where Hp = height acceptance criterion for other/unspecified impurities and/or
above the baseline of the peak due to streptomycin and by the general monograph Substances for pharmaceutical
Hv = height above the baseline of the lowest point of the use (2034). It is therefore not necessary to identify these
curve separating this peak from the peak due to impurity C impurities for demonstration of compliance. See also 5.10.
in the chromatogram obtained with reference solution (d) ; Control of impurities in substances for pharmaceutical use) : D.
– peak-to-valley ratio (b): minimum 5, where Hp = height
above the baseline of the peak due to impurity C and
Hv = height above the baseline of the lowest point of
the curve separating this peak from the peak due to
dihydrostreptomycin in the chromatogram obtained with
reference solution (d) ;
– the chromatogram obtained with reference solution (a)
is similar to the chromatogram supplied with
dihydrostreptomycin sulfate CRS.
Limits :
A. N,N′′′-[(1R,2s,3S,4R,5r,6S)-2,4,5,6-tetrahydroxy-
– correction factor : for the calculation of content, multiply cyclohexane-1,3-diyl]diguanidine (streptidine),
the peak area of impurity A by 0.5 ;
– impurity C : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (b) (2.0 per cent) ;
– impurities A, B : for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
reference solution (b) (1.0 per cent) ;
– any other impurity : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (1.0 per cent) ;
– total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(5.0 per cent) ;
– disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (c) (0.1 per
cent) ; disregard the peak due to streptomycin. B. N,N′′′-[(1S,2R,3R,4S,5R,6R)-2,4,5-trihydroxy-6-[[β-D-
mannopyranosyl-(1→4)-2-deoxy-2-(methylamino)-α-L-
Heavy metals (2.4.8) : 20 ppm. glucopyranosyl-(1→2)-5-deoxy-3-C-(hydroxymethyl)-α-
1.0 g complies with test C. Prepare the reference solution using L-lyxofuranosyl]oxy]cyclohexane-1,3-diyl]diguanidine
2 mL of lead standard solution (10 ppm Pb) R. (dihydrostreptomycin B),
Loss on drying (2.2.32) : maximum 5.0 per cent, determined
on 1.000 g by drying under high vacuum at 60 °C for 4 h. C. unknown structure,

2060 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dihydrotachysterol

Reference solution (b). Dissolve 10.00 mg of

dihydrotachysterol CRS in acetonitrile R and dilute to
50.0 mL with the same solvent.
Reference solution (c). Dilute 5.0 mL of the test solution to
100.0 mL with acetonitrile R. Dilute 5.0 mL of this solution to
50.0 mL with acetonitrile R.
Column :
– size : l = 0.25 m, Ø = 3.0 mm,
– stationary phase : spherical trifunctional end-capped
octadecylsilyl silica gel for chromatography R (4 μm),
– temperature : 40 °C.
Mobile phase : decanol R, water for chromatography R,
acetonitrile for chromatography R (1:25:1000 V/V/V).
D. N,N′′′-[(1R,2R,3S,4R,5R,6S)-4-[[3,5-dideoxy-2-O-
[2-deoxy-2-(methylamino)-α-L-glucopyranosyl]- Flow rate : 0.5 mL/min.
3-(hydroxymethyl)-α-L-arabinofuranosyl]oxy]- Detection : variable-wavelength spectrophotometer capable of
2,5,6-trihydroxycyclohexane-1,3-diyl]diguanidine operating at 251 nm and at 203 nm.
(deoxydihydrostreptomycin). Injection : 5 μL of the test solution and reference solutions (a)
and (c).
01/2008:2014 Run time : twice the retention time of dihydrotachysterol.
Identification of impurities : reference solution (a) :
DIHYDROTACHYSTEROL – use the chromatogram obtained at 203 nm and the
chromatogram obtained at 203 nm supplied with
dihydrotachysterol for system suitability CRS to identify the
Dihydrotachysterolum peak due to impurity A,
– use the chromatogram obtained at 251 nm and the
chromatogram obtained at 251 nm supplied with
dihydrotachysterol for system suitability CRS to identify the
peak due to impurities B and C.
Relative retention with reference to dihydrotachysterol
(retention time = about 15 min) ; impurity B = about 0.9 ;
impurity C = about 1.2 ; impurity A (not visible at 251 nm,
detected at 203 nm) = about 1.2.
System suitability : reference solution (a) :
– peak-to-valley ratio : minimum of 4, where Hp = height
above the baseline of the peak due to impurity B, and
C28H46O Mr 398.7 Hv = height above the baseline of the lowest point of
[67-96-9] the curve separating this peak from the peak due to
DEFINITION dihydrotachysterol in the chromatogram obtained at
251 nm.
(5E,7E,22E)-9,10-Seco-10α-ergosta-5,7,22-trien-3β-ol.
Examine the chromatogram obtained at 203 nm for impurity A
Content : 97.0 per cent to 102.0 per cent.
and the chromatogram obtained at 251 nm for the impurities
CHARACTERS other than A.
Appearance : colourless crystals or white or almost white Limits :
crystalline powder. – impurity A : not more than the area of the principal peak
Solubility : practically insoluble in water, freely soluble in in the chromatogram obtained with reference solution (c)
acetone and hexane, sparingly soluble in ethanol (96 per cent). (0.5 per cent),
It shows polymorphism (5.9). – impurities B, C : for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
IDENTIFICATION reference solution (c) (0.5 per cent),
Infrared absorption spectrophotometry (2.2.24). – any other impurity : for each impurity, not more than
Comparison : dihydrotachysterol CRS. 0.2 times the area of the principal peak in the chromatogram
If the spectra obtained in the solid state show differences, obtained with reference solution (c) (0.1 per cent),
record new spectra using the residues after recrystallisation – total (including A) : not more than twice the area of
from methanol R. the principal peak in the chromatogram obtained with
reference solution (c) at 251 nm (1.0 per cent),
TESTS
– disregard limit : 0.1 times the area of the principal peak in
Specific optical rotation (2.2.7) : + 99 to + 103. the chromatogram obtained with reference solution (c)
Dissolve 0.500 g in ethanol (96 per cent) R and dilute to (0.05 per cent).
25.0 mL with the same solvent. Water (2.5.32) : maximum 0.1 per cent, determined on
Related substances. Liquid chromatography (2.2.29). 40.0 mg.
Test solution. Dissolve 10.00 mg of the substance to be ASSAY
examined in acetonitrile R and dilute to 50.0 mL with the
same solvent. Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications.
Reference solution (a). Dissolve 1.0 mg of dihydrotachysterol
for system suitability CRS (containing impurities A, B and C) Detection : spectrophotometer at 251 nm.
in acetonitrile R and dilute to 5.0 mL with the same solvent. Injection : test solution and reference solution (b).

General Notices (1) apply to all monographs and other texts 2061
Diltiazem hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Calculate the percentage content of C28H46O using DEFINITION

the chromatograms obtained with the test solution Hydrochloride of (2S,3S)-5-[2-(dimethylamino)ethyl]-
and reference solution (b) and the declared content of 2-(4-methoxyphenyl)-4-oxo-2,3,4,5-tetrahydro-1,5-
dihydrotachysterol CRS. benzothiazepin-3-yl acetate.
STORAGE Content : 98.5 per cent to 101.0 per cent (dried substance).
Under an inert gas, in an airtight container, at a temperature CHARACTERS
of 2 °C to 8 °C.
Appearance : white or almost white, crystalline powder.
The contents of an opened container are to be used Solubility : freely soluble in water, in methanol and in
immediately. methylene chloride, slightly soluble in anhydrous ethanol.
IMPURITIES mp : about 213 °C, with decomposition.
Specified impurities : A, B, C. IDENTIFICATION
First identification : A, D.
Second identification : B, C, D.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: diltiazem hydrochloride CRS.
B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 50 mg of the substance to be
examined in methylene chloride R and dilute to 5 mL with
the same solvent.
Reference solution. Dissolve 50 mg of diltiazem
hydrochloride CRS in methylene chloride R and dilute to
A. (7E,22E)-9,10-secoergosta-5(10),7,22-trien-3β-ol 5 mL with the same solvent.
(dihydrovitamin D2-I), Plate : TLC silica gel F254 plate R.
Mobile phase : acetic acid R, water R, methylene chloride R,
anhydrous ethanol R (1:3:10:12 V/V/V/V).
Application : 10 μL.
Development : over 2/3 of the plate.
Drying : in air.
Detection : examine in ultraviolet light at 254 nm.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the
reference solution.
B. (5E,7E,22E)-9,10-secoergosta-5,7,22-trien-3β-ol C. Dissolve 50 mg in 5 mL of water R. Add 1 mL of ammonium
(dihydrovitamin D2-IV), reineckate solution R. A pink precipitate is produced.
D. It gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S. Dissolve 1.00 g in carbon dioxide-free water R and
dilute to 20.0 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
pH (2.2.3) : 4.3 to 5.3.
Dilute 2.0 mL of solution S to 10.0 mL with carbon dioxide-free
water R.
Specific optical rotation (2.2.7) : + 115 to + 120 (dried
C. (5E,7E)-9,10-seco-10α-ergosta-5,7-dien-3β-ol substance).
(dihydrotachysterol4).
Dilute 5.0 mL of solution S to 25.0 mL with water R.
Related substances. Liquid chromatography (2.2.29).
04/2013:1004 Test solution. Dissolve 50 mg of the substance to be examined
in the mobile phase and dilute to 200.0 mL with the mobile
DILTIAZEM HYDROCHLORIDE phase.
Reference solution (a). Dissolve 5 mg of diltiazem for system
suitability CRS (containing impurity A) in the mobile phase
Diltiazemi hydrochloridum and dilute to 20.0 mL with the mobile phase.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10.0 mL with the mobile phase.
Reference solution (c). Dissolve 5 mg of diltiazem
impurity F CRS in the mobile phase and dilute to 100.0 mL
with the mobile phase. Dilute 1.0 mL of the solution to
100.0 mL with the mobile phase.
C22H27ClN2O4S Mr 451.0 Column :
[33286-22-5] – size : l = 0.10 m, Ø = 4.6 mm ;

2062 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dimenhydrinate

– stationary phase : octadecylsilyl silica gel for

chromatography R (3 μm).
Mobile phase : mix 5 volumes of anhydrous ethanol R,
25 volumes of acetonitrile R and 70 volumes of a solution
containing 6.8 g/L of potassium dihydrogen phosphate R and
0.1 mL/L of N,N-dimethyloctylamine R, adjusted to pH 4.5
with dilute phosphoric acid R.
A. (2R,3S)-5-[2-(dimethylamino)ethyl]-2-(4-
Flow rate : 1.5 mL/min. methoxyphenyl)-4-oxo-2,3,4,5-tetrahydro-1,5-
Detection : spectrophotometer at 240 nm. benzothiazepin-3-yl acetate,
Injection : 20 μL.
Run time : 5 times the retention time of diltiazem.
Identification of impurities : use the chromatogram obtained
with reference solution (c) to identify the peak due to
impurity F.
Relative retention with reference to diltiazem
(retention time = about 5 min) : impurity F = about
0.5 ; impurity A = about 0.8. B. (2S,3S)-2-(4-methoxyphenyl)-4-oxo-2,3,4,5-tetrahydro-
1,5-benzothiazepin-3-yl acetate,
System suitability: reference solution (a) :
– resolution : minimum 3.0 between the peaks due to
impurity A and diltiazem ;
– symmetry factor : maximum 2.0 for the peak due to
impurity A ; if necessary, adjust the concentration of
N,N-dimethyloctylamine in the mobile phase.
Limits :
– impurity F : not more than 3 times the area of the principal C. (2S,3S)-5-[2-(dimethylamino)ethyl]-2-(4-hydroxyphenyl)-
peak in the chromatogram obtained with reference 4-oxo-2,3,4,5-tetrahydro-1,5-benzothiazepin-3-yl acetate,
solution (b) (0.3 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.10 per cent) ;
– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.3 per cent) ;
D. (2S,3S)-2-(4-methoxyphenyl)-5-[2-(methylamino)ethyl]-4-
– disregard limit : 0.5 times the area of the principal peak in oxo-2,3,4,5-tetrahydro-1,5-benzothiazepin-3-yl acetate,
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Heavy metals (2.4.8) : maximum 10 ppm.
Dissolve 2.0 g in water R and dilute to 20.0 mL with the same
solvent. 12 mL of the solution complies with test A. Prepare
the reference solution using lead standard solution (1 ppm
Pb) R.
E. (2S,3S)-3-hydroxy-2-(4-methoxyphenyl)-2,3-dihydro-1,5-
Loss on drying (2.2.32) : maximum 0.5 per cent, determined benzothiazepin-4(5H)-one,
on 1.000 g by drying in an oven at 105 °C for 2 h.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY
Dissolve 0.400 g in a mixture of 2 mL of anhydrous formic
acid R and 60 mL of acetic anhydride R. Titrate with 0.1 M
perchloric acid, determining the end-point potentiometrically F. (2S,3S)-5-[2-(dimethylamino)ethyl]-3-hydroxy-2-(4-
(2.2.20). methoxyphenyl)-2,3-dihydro-1,5-benzothiazepin-4(5H)-
one.
1 mL of 0.1 M perchloric acid is equivalent to 45.1 mg
of C22H27ClN2O4S.
07/2009:0601
STORAGE
In an airtight container, protected from light. DIMENHYDRINATE
IMPURITIES Dimenhydrinatum
Specified impurities : F.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of C24H28ClN5O3 Mr 470.0
impurities in substances for pharmaceutical use) : A, B, C, D, E. [523-87-5]

General Notices (1) apply to all monographs and other texts 2063
Dimenhydrinate EUROPEAN PHARMACOPOEIA 8.0

DEFINITION Mobile phase :

Diphenhydramine [2-(diphenylmethoxy)-N,N- – mobile phase A : dissolve 10.0 g of triethylamine R2 in
dimethylethanamine] 8-chlorotheophylline (8-chloro-1,3- 950 mL of water R, adjust to pH 2.5 with phosphoric acid R
dimethyl-3,7-dihydro-1H-purine-2,6-dione). and dilute to 1000 mL with water R ;
Content : – mobile phase B : acetonitrile R1 ;
– diphenhydramine (C17H21NO ; Mr 255.4) : 53.0 per cent to Time Mobile phase A Mobile phase B Flow rate
55.5 per cent (dried substance) ; (min) (per cent V/V) (per cent V/V) (mL/min)
– 8-chlorotheophylline (C7H7ClN4O2 ; Mr 214.6) : 44.0 per cent 0-2 82 18 1.2
to 46.5 per cent (dried substance).
2 - 15 82 → 50 18 → 50 1.2
CHARACTERS 15 - 20 50 → 20 50 → 80 1.2 → 2.0
Appearance : white or almost white, crystalline powder or
20 - 30 20 80 2.0
colourless crystals.
Solubility : slightly soluble in water, freely soluble in ethanolDetection : spectrophotometer at 225 nm.
(96 per cent). Injection : 10 μL.
IDENTIFICATION Identification of impurities : use the chromatogram supplied
First identification : C. with dimenhydrinate for peak identification CRS and the
chromatogram obtained with reference solution (d) to identify
Second identification : A, B, D. the peaks due to impurities A and E ; use the chromatogram
A. Melting point (2.2.14) : 102 °C to 106 °C. obtained with reference solution (c) to identify impurity F.
B. Dissolve 0.1 g in a mixture of 3 mL of water R and 3 mL of Relative retention with reference to diphenhydramine
ethanol (96 per cent) R, add 6 mL of water R and 1 mL of (retention time = about 13 min) : impurity A = about 0.3 ;
dilute hydrochloric acid R and cool in iced water for 30 min, impurity E = about 0.7 ; impurity F = about 0.95.
scratching the wall of the tube with a glass rod if necessary System suitability : reference solution (c) :
to initiate crystallisation. Dissolve about 10 mg of the
precipitate obtained in 1 mL of hydrochloric acid R, add – resolution : minimum 1.5 between the peaks due to
0.1 g of potassium chlorate R and evaporate to dryness in a impurity F and diphenhydramine.
porcelain dish. A reddish residue is obtained that becomes Limits :
violet-red when exposed to ammonia vapour. – impurities A, F : for each impurity, not more than the area
C. Infrared absorption spectrophotometry (2.2.24). of the principal peak in the chromatogram obtained with
Comparison : dimenhydrinate CRS. reference solution (b) (0.2 per cent) ;
D. Dissolve 0.2 g in 10 mL of ethanol (96 per cent) R. Add – impurity E : not more than 0.75 times the area of the
10 mL of picric acid solution R and initiate crystallisation principal peak in the chromatogram obtained with
by scratching the wall of the tube with a glass rod. The reference solution (b) (0.15 per cent) ;
precipitate, washed with water R and dried at 100-105 °C, – unspecified impurities : for each impurity, not more than
melts (2.2.14) at 130 °C to 134 °C. 0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.10 per cent) ;
TESTS – total : not more than 2.5 times the area of the principal peak
Appearance of solution. The solution is clear (2.2.1) and in the chromatogram obtained with reference solution (b)
colourless (2.2.2, Method II). (0.5 per cent) ;
Dissolve 1.0 g in ethanol (96 per cent) R and dilute to 20 mL – disregard limit : 0.25 times the area of the principal peak
with the same solvent. in the chromatogram obtained with reference solution (b)
pH (2.2.3) : 7.1 to 7.6 for the filtrate. (0.05 per cent).
To 0.4 g add 20 mL of carbon dioxide-free water R, shake for Loss on drying (2.2.32) : maximum 0.5 per cent, determined
2 min and filter. on 1.000 g by drying in vacuo.
Related substances. Liquid chromatography (2.2.29). Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
1.0 g.
Solvent mixture : acetonitrile R, water R (18:82 V/V).
Test solution. Dissolve 0.100 g of the substance to be examined ASSAY
in the solvent mixture and dilute to 100.0 mL with the solvent Diphenhydramine. Dissolve 0.200 g in 60 mL of anhydrous
mixture. acetic acid R. Titrate with 0.1 M perchloric acid, determining
Reference solution (a). Dissolve 57 mg of diphenhydramine the end-point potentiometrically (2.2.20).
hydrochloride CRS in the solvent mixture and dilute to 50.0 mL 1 mL of 0.1 M perchloric acid is equivalent to 25.54 mg
with the solvent mixture. of C17H21NO.
Reference solution (b). Dilute 1.0 mL of reference solution (a) 8-Chlorotheophylline. To 0.800 g add 50 mL of water R,
to 100.0 mL with the solvent mixture. Dilute 2.0 mL of this 3 mL of dilute ammonia R1 and 0.6 g of ammonium nitrate R
solution to 10.0 mL with the solvent mixture. and heat on a water-bath for 5 min. Add 25.0 mL of 0.1 M
Reference solution (c). Dissolve 5.0 mg of diphenhydramine silver nitrate and continue heating on a water-bath for 15 min
impurity A CRS (impurity F) in 5.0 mL of reference solution (a) with frequent swirling. Cool, add 25 mL of dilute nitric acid R
and dilute to 50.0 mL with the solvent mixture. and dilute to 250.0 mL with water R. Filter and discard the
Reference solution (d). Dissolve the contents of a vial of first 25 mL of the filtrate. Using 5 mL of ferric ammonium
dimenhydrinate for peak identification CRS (containing sulfate solution R2 as indicator, titrate 100.0 mL of the filtrate
impurities A and E) in 1.0 mL of the solvent mixture. with 0.1 M ammonium thiocyanate until a yellowish-brown
Column : colour is obtained.
– size : l = 0.25 m, Ø = 4.6 mm ; 1 mL of 0.1 M silver nitrate is equivalent to 21.46 mg
of C7H7ClN4O2.
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm) ; IMPURITIES
– temperature : 30 °C. Specified impurities : A, E, F.

2064 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dimercaprol

Other detectable impurities (the following substances would,

if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use) : J. diphenylmethanone (benzophenone).
C, D, G, H, I, J, K.

01/2008:0389

DIMERCAPROL
A. 1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione Dimercaprolum
(theophylline),

C3H8OS2 Mr 124.2
[59-52-9]
DEFINITION
C. 1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (2RS)-2,3-Disulfanylpropan-1-ol.
(caffeine), Content : 98.5 per cent to 101.5 per cent.
CHARACTERS
Appearance : clear, colourless or slightly yellow liquid.
Solubility : soluble in water and in arachis oil, miscible with
ethanol (96 per cent) and with benzyl benzoate.
IDENTIFICATION
D. R1 = CH2-N(CH3)2, R2 = H : N-[2-(diphenylmethoxy)ethyl]- A. Dissolve 0.05 mL in 2 mL of water R. Add 1 mL of 0.05 M
N,N′,N′-trimethylethane-1,2-diamine, iodine. The colour of the iodine is discharged immediately.
G. R1 = H, R2 = CH3 : N,N-dimethyl-2-[(RS)-(4- B. Dissolve 0.1 mL in 5 mL of water R and add 2 mL of copper
methylphenyl)(phenyl)methoxy]ethanamine sulfate solution R. A bluish-black precipitate is formed
(4-methyldiphenhydramine), which quickly becomes dark grey.
C. In a ground-glass-stoppered tube, suspend 0.6 g of sodium
H. R1 = H, R2 = Br : 2-[(RS)-(4-bromophenyl)-
bismuthate R, previously heated to 200 °C for 2 h, in a
(phenyl)methoxy]-N,N-dimethylethanamine
mixture of 2.8 mL of dilute phosphoric acid R and 6 mL
(4-bromodiphenhydramine),
of water R. Add 0.2 mL of the substance to be examined,
mix and allow to stand for 10 min with frequent shaking.
To 1 mL of the supernatant add 5 mL of a 4 g/L solution
of chromotropic acid, sodium salt R in sulfuric acid R and
mix. Heat in a water-bath for 15 min. A violet-red colour
develops.
TESTS
E. 8-chloro-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione
(8-chlorocaffeine), Appearance. It is clear (2.2.1) and not more intensely
coloured than reference solution B6 or BY6 (2.2.2, Method II).
Acidity or alkalinity. Dissolve 0.2 g in carbon dioxide-free
water R and dilute to 10 mL with the same solvent. Add
0.25 mL of bromocresol green solution R and 0.3 mL of 0.01 M
hydrochloric acid. The solution is yellow. Not more than
0.5 mL of 0.01 M sodium hydroxide is required to change the
colour of the indicator to blue.
F. 2-(diphenylmethoxy)-N-methylethanamine Refractive index (2.2.6) : 1.568 to 1.574.
(diphenhydramine impurity A), Halides. To 2.0 g add 25 mL of alcoholic potassium hydroxide
solution R and boil under a reflux condenser for 2 h. Eliminate
the ethanol by evaporation in a stream of hot air. Add 20 mL
of water R and cool. Add 40 mL of water R and 10 mL of
strong hydrogen peroxide solution R, boil gently for 10 min,
cool and filter rapidly. Add 10 mL of dilute nitric acid R
and 5.0 mL of 0.1 M silver nitrate. Using 2 mL of ferric
ammonium sulfate solution R2 as indicator, titrate with 0.1 M
I. R = H : diphenylmethanol (benzhydrol), ammonium thiocyanate until a reddish-yellow colour is
obtained. Carry out a blank titration. The difference between
K. R = CH(C6H5)2 : [oxybis(methanetriyl)]tetrabenzene, the titration volumes is not greater than 1.0 mL.

General Notices (1) apply to all monographs and other texts 2065
Dimethyl sulfoxide EUROPEAN PHARMACOPOEIA 8.0

ASSAY Reference solution. Dissolve 50.0 mg of the substance to be

Dissolve 0.100 g in 40 mL of methanol R. Add 20 mL of 0.1 M examined and 50 mg of dimethyl sulfone R in acetone R,
hydrochloric acid and 50.0 mL of 0.05 M iodine. Allow to add 10.0 mL of the internal standard solution and dilute to
stand for 10 min and titrate with 0.1 M sodium thiosulfate. 100.0 mL with acetone R.
Carry out a blank titration. Column :
1 mL of 0.05 M iodine is equivalent to 6.21 mg of C3H8OS2. – material : glass ;
STORAGE – size : l = 1.5 m, Ø = 4 mm ;
In a well-filled, airtight container, protected from light, at a – stationary phase : diatomaceous earth for gas
temperature of 2 °C to 8 °C. chromatography R (125-180 μm) impregnated with 10 per
cent m/m of polyethyleneglycol adipate R.
Carrier gas : nitrogen for chromatography R.
01/2008:0763 Flow rate : 30 mL/min.
Temperature :
DIMETHYL SULFOXIDE – column : 165 °C ;
– injection port and detector : 190 °C.
Dimethylis sulfoxidum Detection : flame ionisation.
Injection : 1 μL.
Run time : 4 times the retention time of dimethyl sulfoxide.
Elution order : dimethyl sulfoxide, dimethyl sulfone, bibenzyl.
C2H6OS Mr 78.1 Retention time : dimethyl sulfoxide = about 5 min.
[67-68-5]
System suitability :
DEFINITION – resolution : minimum 3 between the peaks due to dimethyl
Sulfinylbismethane. sulfoxide and dimethyl sulfone in the chromatogram
obtained with the reference solution ;
CHARACTERS – in the chromatogram obtained with test solution (a) there
Appearance : colourless liquid or colourless crystals, is no peak with the same retention time as the internal
hygroscopic. standard.
Solubility : miscible with water and with ethanol (96 per cent). Limit :
IDENTIFICATION – total : calculate the ratio R of the area of the peak due
First identification : C. to dimethyl sulfoxide to the area of the peak due to the
internal standard from the chromatogram obtained with
Second identification : A, B, D. the reference solution ; from the chromatogram obtained
A. Relative density (see Tests). with test solution (b), calculate the ratio of the sum of the
B. Refractive index (see Tests). areas of any peaks, apart from the principal peak and the
C. Infrared absorption spectrophotometry (2.2.24). peak due to the internal standard to the area of the peak
due to the internal standard : this ratio is not greater than
Comparison : dimethyl sulfoxide CRS. R (0.1 per cent).
D. Dissolve 50 mg of nickel chloride R in 5 mL of the substance Water (2.5.12) : maximum 0.2 per cent, determined on 10.0 g.
to be examined. The solution is greenish-yellow. Heat
in a water-bath at 50 °C. The colour changes to green or STORAGE
bluish-green. Cool. The colour changes to greenish-yellow. In an airtight, glass container, protected from light.
TESTS
Acidity. Dissolve 50.0 g in 100 mL of carbon dioxide-free
water R. Add 0.1 mL of phenolphthalein solution R1. Not 01/2008:1667
more than 5.0 mL of 0.01 M sodium hydroxide is required to
produce a pink colour.
DIMETHYLACETAMIDE
Relative density (2.2.5) : 1.100 to 1.104.
Refractive index (2.2.6) : 1.478 to 1.479. Dimethylacetamidum
Freezing point (2.2.18) : minimum 18.3 °C.
Absorbance (2.2.25). Purge with nitrogen R for 15 min. The
absorbance, measured using water R as the compensation
liquid, is not more than 0.30 at 275 nm and not more than 0.20
at both 285 nm and 295 nm. Examined between 270 nm and
350 nm, the substance to be examined shows no absorption C4H9NO Mr 87.1
maximum. [127-19-5]
Related substances. Gas chromatography (2.2.28).
DEFINITION
Internal standard solution. Dissolve 0.125 g of bibenzyl R in
acetone R and dilute to 50 mL with the same solvent. N,N-Dimethylacetamide.
Test solution (a). Dissolve 5.0 g of the substance to be CHARACTERS
examined in acetone R and dilute to 10.0 mL with the same
solvent. Appearance : clear, colourless, slightly hygroscopic liquid.
Test solution (b). Dissolve 5.0 g of the substance to be Solubility : miscible with water, with ethanol (96 per cent), and
examined in acetone R, add 1.0 mL of the internal standard with most common organic solvents.
solution and dilute to 10.0 mL with acetone R. bp : about 165 °C.

2066 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dimeticone

IDENTIFICATION – total : maximum 0.3 per cent,

First identification : C. – disregard limit : the area of the peak in the chromatogram
Second identification : A, B, D. obtained with reference solution (b) (0.05 per cent).
A. Relative density (2.2.5) : 0.941 to 0.944. Heavy metals (2.4.8) : maximum 10 ppm.
B. Refractive index (2.2.6) : 1.435 to 1.439. Dilute 4.0 g to 20.0 mL with water R. 12 mL of the solution
C. Infrared absorption spectrophotometry (2.2.24). complies with limit test A. Prepare the reference solution
Preparation : films. using lead standard solution (2 ppm Pb) R.
Comparison : Ph. Eur. reference spectrum of Non-volatile matter : maximum 20 ppm.
dimethylacetamide. Evaporate 50 g to dryness using a rotary evaporator at a
D. Dilute 50 mg with 1 mL of methanol R. Add 1 mL of a 15 g/L pressure not exceeding 1 kPa and on a water-bath. Dry the
solution of hydroxylamine hydrochloride R and mix. Add residue in an oven at 170-175 °C. The residue weighs not
1 mL of dilute sodium hydroxide solution R, mix and allow more than 1 mg.
to stand for 30 min. Add 1 mL of dilute hydrochloric acid R Water (2.5.32) : maximum 0.1 per cent, determined on 0.100 g.
and add 1 mL of a 100 g/L solution of ferric chloride R in
0.1 M hydrochloric acid. A reddish-brown colour develops, STORAGE
reaching a maximum intensity after about 5 min. In an airtight container, protected from light.
TESTS IMPURITIES
Appearance. The substance to be examined is clear (2.2.1)
and not more intensely coloured than reference solution Y7
(2.2.2, Method II).
Acidity. Dilute 50 mL with 50 mL of water R previously A. acetic acid,
adjusted with 0.02 M potassium hydroxide or 0.02 M
hydrochloric acid to a bluish-green colour, using 0.5 mL of
bromothymol blue solution R1 as indicator. Not more than
5.0 mL of 0.02 M potassium hydroxide is required to restore
the initial (bluish-green) colour.
B. R = H : N,N-dimethylformamide,
Alkalinity. To 50 mL add 50 mL of water R previously adjusted
with 0.02 M potassium hydroxide or 0.02 M hydrochloric C. R = C2H5 : N,N-dimethylpropanamide,
acid to a yellow colour, using 0.5 mL of bromothymol blue D. R = CH2-CH2-CH3 : N,N-dimethylbutanamide.
solution R1 as indicator. Not more than 0.5 mL of 0.02 M
hydrochloric acid is required to restore the initial (yellow)
colour. 07/2013:0138
Related substances. Gas chromatography (2.2.28) : use the
normalisation procedure. DIMETICONE
Test solution. The substance to be examined.
Reference solution (a). Dilute a mixture of 1 mL of the Dimeticonum
substance to be examined and 1 mL of dimethylformamide R
to 20 mL with methylene chloride R.
Reference solution (b). Dilute 1 mL of the substance to be
examined to 20.0 mL with methylene chloride R. Dilute 0.1 mL
of the solution to 10.0 mL with methylene chloride R.
[9006-65-9]
Column :
– material : fused silica, DEFINITION
– size : l = 30 m, Ø = 0.32 mm, α-Trimethylsilyl-ω-methylpoly[oxy(dimethylsilanediyl)].
– stationary phase : macrogol 20 000 R (film thickness 1 μm). This poly(dimethylsiloxane) is obtained by hydrolysis
Carrier gas : nitrogen for chromatography R. and polycondensation of dichlorodimethylsilane and
Linear velocity : 30 cm/s. chlorotrimethylsilane. Different grades of dimeticone exist
which are distinguished by a number indicating the nominal
Split ratio : 1:20. kinematic viscosity placed after the name.
Temperature : Their degree of polymerisation (n = 20 to 400) is such that
Time Temperature their kinematic viscosities are nominally between 20 mm2·s− 1
(min) (°C) and 1300 mm2·s− 1.
Column 0 - 15 80 → 200 Dimeticones with a nominal viscosity of 50 mm2·s− 1 or lower
Injection port 250 are intended for external use only.
Detector 250 CHARACTERS
Appearance : clear, colourless liquid of various viscosities.
Detection : flame ionisation.
Solubility : practically insoluble in water, very slightly soluble
Injection : 0.5 μL. or practically insoluble in anhydrous ethanol, miscible with
System suitability : ethyl acetate, with methyl ethyl ketone and with toluene.
– resolution : minimum 5.0 between the peaks due to
dimethylacetamide and impurity B in the chromatogram IDENTIFICATION
obtained with reference solution (a), A. It is identified by its kinematic viscosity at 25 °C (see Tests).
– signal-to-noise ratio : minimum 10 for the principal peak in B. Infrared absorption spectrophotometry (2.2.24).
the chromatogram obtained with reference solution (b). Comparison: dimeticone CRS.
Limits : The region of the spectrum from 850 cm− 1 to 750 cm− 1 is
– any impurity : maximum 0.1 per cent, not taken into account.

General Notices (1) apply to all monographs and other texts 2067
Dimetindene maleate EUROPEAN PHARMACOPOEIA 8.0

C. Heat 0.5 g in a test-tube over a small flame until white The following characteristic may be relevant for dimeticone
fumes begin to appear. Invert the tube over a 2nd tube used as emollient.
containing 1 mL of a 1 g/L solution of chromotropic acid, Viscosity (see Tests).
sodium salt R in sulfuric acid R so that the fumes reach the
solution. Shake the 2nd tube for about 10 s and heat on a
water-bath for 5 min. The solution is violet.
D. In a platinum crucible, prepare the sulfated ash (2.4.14)
using 50 mg. The residue is a white powder that gives the 01/2008:1417
reaction of silicates (2.3.1). corrected 6.0

TESTS DIMETINDENE MALEATE

Acidity. To 2.0 g add 25 mL of a mixture of equal volumes
of anhydrous ethanol R and ether R, previously neutralised
to 0.2 mL of bromothymol blue solution R1, and shake. Not Dimetindeni maleas
more than 0.15 mL of 0.01 M sodium hydroxide is required to
change the colour of the solution to blue.
Viscosity (2.2.9) : 90 per cent to 110 per cent of the nominal
kinematic viscosity stated on the label, determined at 25 °C.
Mineral oils. Place 2 g in a test-tube and examine in ultraviolet
light at 365 nm. The fluorescence is not more intense than
that of a solution containing 0.1 ppm of quinine sulfate R in
0.005 M sulfuric acid examined in the same conditions. C24H28N2O4 Mr 408.5
Phenylated compounds. Dissolve 5.0 g with shaking in 10 mL [3614-69-5]
of cyclohexane R. At wavelengths from 250 nm to 270 nm, the
absorbance (2.2.25) of the solution is not greater than 0.2. DEFINITION
Heavy metals : maximum 5 ppm. N,N-Dimethyl-2-[3-[(RS)-1-(pyridin-2-yl)ethyl]-1H-inden-2-
Mix 1.0 g with methylene chloride R and dilute to 20 mL yl]ethanamine (Z)-butenedioate.
with the same solvent. Add 0.75 mL of a freshly prepared Content : 99.0 per cent to 101.0 per cent (dried substance).
0.02 g/L solution of dithizone R in methylene chloride R,
0.5 mL of water R and 0.5 mL of a mixture of 1 volume of CHARACTERS
dilute ammonia R2 and 9 volumes of a 2 g/L solution of Appearance : white or almost white, crystalline powder.
hydroxylamine hydrochloride R. At the same time, prepare a
reference solution as follows : to 20 mL of methylene chloride R Solubility : slightly soluble in water, soluble in methanol.
add 0.75 mL of a freshly prepared 0.02 g/L solution of
dithizone R in methylene chloride R, 0.5 mL of lead standard IDENTIFICATION
solution (10 ppm Pb) R and 0.5 mL of a mixture of 1 volume Infrared absorption spectrophotometry (2.2.24).
of dilute ammonia R2 and 9 volumes of a 2 g/L solution of
hydroxylamine hydrochloride R. Immediately shake each Preparation : discs.
solution vigorously for 1 min. Any pink colour in the test Comparison: dimetindene maleate CRS.
solution is not more intense than that in the reference solution.
Volatile matter : maximum 0.3 per cent, for dimeticones with TESTS
a nominal viscosity greater than 50 mm2·s− 1, determined on Solution S. Dissolve 0.20 g in methanol R and dilute to
1.00 g by heating in an oven at 150 °C for 2 h. Carry out the 20.0 mL with the same solvent.
test using a dish 60 mm in diameter and 10 mm deep.
Appearance of solution. Solution S is clear (2.2.1) and not
LABELLING more intensely coloured than Y6 (2.2.2, Method II).
The label states : Optical rotation (2.2.7): − 0.10° to + 0.10°, determined on
solution S.
– the nominal kinematic viscosity by a number placed after
the name of the product ; Related substances. Gas chromatography (2.2.28).
– where applicable, that the product is intended for external Solvent mixture : acetone R, methylene chloride R (50:50 V/V).
use. Test solution. Dissolve 50.0 mg of the substance to be
examined in the solvent mixture and dilute to 5.0 mL with the
FUNCTIONALITY-RELATED CHARACTERISTICS solvent mixture.
This section provides information on characteristics that are Reference solution (a). Dilute 1 mL of the test solution to
recognised as being relevant control parameters for one or 100.0 mL with the solvent mixture.
more functions of the substance when used as an excipient
(see chapter 5.15). Some of the characteristics described in Reference solution (b). Dissolve 5.0 mg of 2-ethylpyridine R
the Functionality-related characteristics section may also be (impurity A) in the solvent mixture and dilute to 50.0 mL
present in the mandatory part of the monograph since they with the solvent mixture. Dilute 10.0 mL of this solution to
also represent mandatory quality criteria. In such cases, a 100.0 mL with the solvent mixture.
cross-reference to the tests described in the mandatory part is Column :
included in the Functionality-related characteristics section. – material : fused silica ;
Control of the characteristics can contribute to the quality
of a medicinal product by improving the consistency of the – size : l = 30 m, Ø = 0.32 mm ;
manufacturing process and the performance of the medicinal – stationary phase : polymethylphenylsiloxane R (film
product during use. Where control methods are cited, they are thickness 0.25 μm).
recognised as being suitable for the purpose, but other methods
can also be used. Wherever results for a particular characteristic Carrier gas : helium for chromatography R.
are reported, the control method must be indicated. Linear velocity : about 30 cm/s.

2068 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dinoprost trometamol

Temperature :
Time Temperature
(min) (°C)
Column 0-1 60
1 - 34.3 60 → 260 E. (2RS)-2-[2-(dimethylamino)ethyl]indan-1-one,
34.3 - 46.3 260
Injection port 240

Detector 260

Detection : flame ionisation.

Injection : 2 μL ; inject via a split injector with a split flow of F. R = [CH2]3-CH3 : 2-(3-butyl-1H-inden-2-yl)-N,N-
30 mL/min. dimethylethanamine,
Run time : 1.3 times the retention time of dimetindene. G. R = C6H5 : N,N-dimethyl-2-(3-phenyl-1H-inden-2-
Elution order : impurity A and maleic acid appear during the yl)ethanamine,
first 8 min.
System suitability: reference solution (a) :
– symmetry factor : maximum 1.3 for the principal peak.
Limits :
– impurity A : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (b) (0.1 per cent) ;
– impurities B, C, D, E, F, G, H, I : for each impurity, not H. R = CH = CH2 : 2-[(1RS)-1-(2-ethenyl-1H-inden-3-
more than 0.2 times the area of the principal peak in the yl)ethyl]pyridine,
chromatogram obtained with reference solution (a) (0.2 per I. R = CH2-CH2-NH-CH3 : N-methyl-2-[3-[(1RS)-1-(pyridin-
cent) ; 2-yl)ethyl]-1H-inden-2-yl]ethanamine.
– sum of impurities other than A : not more than 0.5 times the
area of the principal peak in the chromatogram obtained 01/2008:1312
with reference solution (a) (0.5 per cent) ;
– disregard limit : 0.05 times the area of the principal peak DINOPROST TROMETAMOL
in the chromatogram obtained with reference solution (a)
(0.05 per cent) ; disregard the peak due to maleic acid.
Dinoprostum trometamolum
Loss on drying (2.2.32) : maximum 0.1 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 2 h.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY
Dissolve 0.150 g in 80 mL of anhydrous acetic acid R. Titrate C24H45NO8 Mr 475.6
with 0.1 M perchloric acid, determining the end-point [38562-01-5]
potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 20.43 mg DEFINITION
of C24H28N2O4. Trometamol (Z)-7-[(1R,2R,3R,5S)-3,5-dihydroxy-2-[(E)-(3S)-
3-hydroxyoct-1-enyl]cyclopentyl]hept-5-enoate (PGF2α).
STORAGE
Content : 96.0 per cent to 102.0 per cent (anhydrous substance).
Protected from light.
CHARACTERS
IMPURITIES
Appearance : white or almost white powder.
Specified impurities : A, B, C, D, E, F, G, H, I. Solubility : very soluble in water, freely soluble in ethanol
(96 per cent), practically insoluble in acetonitrile.
IDENTIFICATION
A. Specific optical rotation (2.2.7) : + 19 to + 26 (anhydrous
A. 2-ethylpyridine, substance).
Dissolve 0.100 g in ethanol (96 per cent) R and dilute to
10.0 mL with the same solvent.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: dinoprost trometamol CRS.
B. 2-(1H-inden-2-yl)-N,N-dimethylethanamine, TESTS
Related substances. Liquid chromatography (2.2.29).
Solvent mixture : acetonitrile R, water R (23:77 V/V).
Test solution. Dissolve 10.0 mg of the substance to be
examined in the solvent mixture and dilute to 10.0 mL with
the solvent mixture.
Reference solution (a). Degradation of dinoprost trometamol
C. R = C2H5 : ethyl (2RS)-2-benzyl-4-(dimethylamino)- to impurity B. Dissolve 1 mg of the substance to be examined
butanoate, in 1 mL of the mobile phase and heat the solution on a
D. R = H : (2RS)-2-benzyl-4-(dimethylamino)butanoic acid, water-bath at 85 °C for 5 min and cool.

General Notices (1) apply to all monographs and other texts 2069
Dinoprostone EUROPEAN PHARMACOPOEIA 8.0

Reference solution (b). Dilute 2.0 mL of the test solution Detection : spectrophotometer at 200 nm.
to 20.0 mL with the solvent mixture. Dilute 2.0 mL of this Injection : 20 μL.
solution to 20.0 mL with the solvent mixture. Retention time : dinoprost = about 23 min.
Column : System suitability : reference solution :
– size : l = 0.15 m, Ø = 3.9 mm ; – repeatability : maximum relative standard deviation of
– stationary phase : octadecylsilyl silica gel for 2.0 per cent for the peak due to dinoprost after 6 injections.
chromatography R1 (5 μm) with a pore size of Calculate the percentage of dinoprost trometamol from the
10 nm and a carbon loading of 19 per cent. declared content of dinoprost trometamol CRS.
Mobile phase : dissolve 2.44 g of sodium dihydrogen phosphate R
in water R and dilute to 1000 mL with water R ; adjust to IMPURITIES
pH 2.5 with phosphoric acid R (about 0.6 mL) ; mix 770 mL of Specified impurities : A, B, C, D.
this solution with 230 mL of acetonitrile R1.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 200 nm.
Injection : 20 μL.
Run time : 2.5 times the retention time of the principal peak
(to elute degradation products formed during heating) for A. (E)-7-[(1R,2R,3R,5S)-3,5-dihydroxy-2-[(E)-(3S)-3-
reference solution (a) and 10 min after the elution of dinoprost hydroxyoct-1-enyl]cyclopentyl]hept-5-enoic acid
for the test solution and reference solution (b). ((5E)-PGF2α ; 5,6-trans-PGF2α),
Retention time : impurity B = about 55 min ;
impurity A = about 60 min ; dinoprost = about 66 min.
System suitability: reference solution (a) :
– resolution : minimum 1.5 between the peaks due to
impurities B and A and minimum 2.0 between the peaks
due to impurity A and dinoprost ; if necessary, adjust B. (Z)-7-[(1R,2R,3R,5S)-3,5-dihydroxy-2-[(E)-(3R)-3-
the composition of the mobile phase by increasing hydroxyoct-1-enyl]cyclopentyl]hept-5-enoic acid
the concentration of acetonitrile to decrease the ((15R)-PGF2α ;15-epiPGF2α),
retention times ;
– symmetry factor : maximum 1.2 for the peaks due to
impurities A and B.
Limits :
– impurity A : not more than twice the area of the principal
peak obtained with reference solution (b) (2 per cent) ; C. (Z)-7-[(1S,2R,3R,5S)-3,5-dihydroxy-2-[(E)-(3S)-3-
– impurities B, C, D : for each impurity, not more than hydroxyoct-1-enyl]cyclopentyl]hept-5-enoic acid
1.5 times the area of the principal peak obtained with ((8S)-PGF2α ; 8-epiPGF2α),
reference solution (b) (1.5 per cent) and not more than
one such peak has an area greater than 0.5 times the area
of the principal peak obtained with reference solution (b)
(0.5 per cent) ;
– sum of impurities other than A : not more than twice
the area of the principal peak obtained with reference
solution (b) (2 per cent) ; D. (Z)-7-[(1R,2R,3S,5S)-3,5-dihydroxy-2-[(E)-(3S)-3-
hydroxyoct-1-enyl]cyclopentyl]hept-5-enoic acid
– disregard limit : 0.05 times the area of the principal (11β-PGF2α ; 11-epiPGF2α).
peak obtained with reference solution (b) (0.05 per
cent) ; disregard any peak due to trometamol (retention 01/2008:1311
time = about 1.5 min).
Water (2.5.12) : maximum 1.0 per cent, determined on 0.500 g. DINOPROSTONE
ASSAY
Liquid chromatography (2.2.29).
Dinoprostonum
Solvent mixture : acetonitrile R, water R (23:77 V/V).
Test solution. Dissolve 10.0 mg of the substance to be
examined in the solvent mixture and dilute to 10.0 mL with
the solvent mixture.
Reference solution. Dissolve 10.0 mg of dinoprost
trometamol CRS in the solvent mixture and dilute to 10.0 mL C20H32O5 Mr 352.5
with the solvent mixture. [363-24-6]
Column :
DEFINITION
– size : l = 0.15 m, Ø = 3.9 mm ; (Z)-7-[(1R,2R,3R)-3-Hydroxy-2-[(E)-(3S)-3-hydroxyoct-1-
– stationary phase : octadecylsilyl silica gel for enyl]-5-oxocyclopentyl]hept-5-enoic acid (PGE2).
chromatography R1 (5 μm) with a pore size of Content : 95.0 per cent to 102.0 per cent (anhydrous substance).
10 nm and a carbon loading of 19 per cent.
Mobile phase : dissolve 2.44 g of sodium dihydrogen phosphate R CHARACTERS
in water R and dilute to 1000 mL with water R ; adjust to Appearance : white or almost white, crystalline powder or
pH 2.5 with phosphoric acid R (about 0.6 mL) ; mix 730 mL of colourless crystals.
this solution with 270 mL of acetonitrile R1. Solubility : practically insoluble in water, very soluble in
Flow rate : 1 mL/min. methanol, freely soluble in alcohol.

2070 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dinoprostone

The substance degrades at room temperature. – impurity D : not more than twice the area of the principal
peak in the chromatogram obtained with reference
IDENTIFICATION solution (b) (1 per cent),
A. Specific optical rotation (2.2.7) : − 90 to − 82 (anhydrous – impurity E : not more than the area of the principal peak
substance). in the chromatogram obtained with reference solution (b)
Immediately before use, dissolve 50.0 mg in alcohol R and (0.5 per cent),
dilute to 10.0 mL with the same solvent. – any other impurity : not more than the area of the principal
B. Infrared absorption spectrophotometry (2.2.24). peak in the chromatogram obtained with reference
Comparison : dinoprostone CRS. solution (b) (0.5 per cent),
TESTS – total of other impurities : not more than twice the area of
the principal peak in the chromatogram obtained with
Prepare the solutions immediately before use. reference solution (b) (1 per cent),
Related substances. Liquid chromatography (2.2.29). – disregard limit : 0.1 times the area of the principal peak in
Test solution (a). Dissolve 10.0 mg of the substance to be the chromatogram obtained with reference solution (b)
examined in a 58 per cent V/V solution of methanol R2 and (0.05 per cent).
dilute to 2.0 mL with the same solvent. If any peak with a relative retention to dinoprostone of about
Test solution (b). Dissolve 20.0 mg of the substance to be 0.8 is greater than 0.5 per cent or if the total of other impurities
examined in a 58 per cent V/V solution of methanol R2 and is greater than 1.0 per cent, record the chromatogram of test
dilute to 20.0 mL with the same solvent. solution (a) with a detector set at 230 nm. If the area of the
Reference solution (a). Dissolve 1 mg of dinoprostone CRS and peak at 230 nm is twice the area of the peak at 210 nm, multiply
1 mg of dinoprostone impurity C CRS in a 58 per cent V/V the area at 210 nm by 0.2 (correction factor for impurity F).
solution of methanol R2 and dilute to 10.0 mL with the same Water (2.5.12) : maximum 0.5 per cent, determined on 0.50 g.
solvent. Dilute 4.0 mL of the solution to 10.0 mL with a 58 per
cent V/V solution of methanol R2. ASSAY
Reference solution (b). Dilute 0.5 mL of test solution (a) to Prepare the solutions immediately before use.
10.0 mL with a 58 per cent V/V solution of methanol R2.
Dilute 1.0 mL of the solution to 10.0 mL with a 58 per cent V/V Liquid chromatography (2.2.29) as described in the test for
solution of methanol R2. related substances.
Reference solution (c). In order to prepare in situ the Injection : test solution (b) and reference solution (d).
degradation compounds (impurity D and impurity E), dissolve Calculate the percentage content of C20H32O5.
1 mg of the substance to be examined in 100 μL of 1 M sodium
hydroxide (the solution becomes brownish-red), wait 4 min, STORAGE
add 150 μL of 1 M acetic acid (yellowish-white opalescent At a temperature not exceeding - 15 °C.
solution) and dilute to 5.0 mL with a 58 per cent V/V solution
of methanol R2. IMPURITIES
Reference solution (d). Dissolve 20 mg of dinoprostone CRS
in a 58 per cent V/V solution of methanol R2 and dilute to
20.0 mL with the same solvent.
Column :
– size : l = 0.25 m, Ø = 4.6 mm,
– stationary phase : end-capped octadecylsilyl silica gel for A. (Z)-7-[(1R,2R,3R)-3-hydroxy-2-[(E)-(3R)-3-hydroxyoct-
chromatography R, 1-enyl]-5-oxocyclopentyl]hept-5-enoic acid (15-epiPGE2 ;
– temperature : 30 °C. (15R)-PGE2),
Mobile phase : mix 42 volumes of a 0.2 per cent V/V solution
of acetic acid R and 58 volumes of methanol R2.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 210 nm.
Injection : 20 μL ; inject test solution (a) and reference
solutions (a), (b) and (c).
B. (Z)-7-[(1S,2R,3R)-3-hydroxy-2-[(E)-(3S)-3-hydroxyoct-
Relative retention with reference to dinoprostone 1-enyl]-5-oxocyclopentyl]hept-5-enoic acid (8-epiPGE2 ;
(retention time = about 18 min) : impurity C = about 1.2 ; (8S)-PGE2),
impurity D = about 1.8 ; impurity E = about 2.0.
System suitability: reference solution (a) :
– resolution : minimum of 3.8 between the peaks due to
dinoprostone and to impurity C. If necessary adjust the
concentration of the acetic acid solution and/or methanol
(increase the concentration of the acetic acid solution
to increase the retention time for dinoprostone and C. (E)-7-[(1R,2R,3R)-3-hydroxy-2-[(E)-(3S)-3-hydroxyoct-1-
impurity C and increase the concentration of methanol to enyl]-5-oxocyclopentyl]hept-5-enoic acid (5-trans-PGE2 ;
decrease the retention time for both compounds). (5E)-PGE2),
Limits :
– correction factors : for the calculation of contents,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity D = 0.2 ;
impurity E = 0.7,
– impurity C : not more than 3 times the area of the principal
peak in the chromatogram obtained with reference D. (Z)-7-[(1R,2S)-2-[(E)-(3S)-3-hydroxyoct-1-enyl]-5-
solution (b) (1.5 per cent), oxocyclopent-3-enyl]hept-5-enoic acid (PGA2),

General Notices (1) apply to all monographs and other texts 2071
Diosmin EUROPEAN PHARMACOPOEIA 8.0

the contents of the flask immediately after the end of the

combustion to dissolve completely the combustion products.
Continue stirring for 1 h.
Reference solution. Dilute 2.0 mL of a 16.6 g/L solution of
potassium iodide R to 100.0 mL with water R. Dilute 10.0 mL
E. (Z)-7-[2-[(E)-(3S)-3-hydroxyoct-1-enyl]-5-oxocyclopent- of the solution to 100.0 mL with water R.
1-enyl]hept-5-enoic acid (PGB2), Introduce into a beaker 30 mL of a 200 g/L solution of
potassium nitrate R in 0.1 M nitric acid. Immerse the
electrodes and stir for 10 min. The potential of the solution
(nT1) must remain stable. Add 1 mL of the test solution and
measure the potential (nT2).
Introduce into a beaker 30 mL of a 200 g/L solution of
potassium nitrate R in 0.1 M nitric acid. Immerse the
F. (Z)-7-[(1R,2R,3R)-3-hydroxy-2-[(E)-3-oxo-oct-1-enyl]- electrodes and stir for 10 min. The potential of the solution
5-oxocyclopentyl]hept-5-enoic acid (15-oxo-PGE2 ; must remain stable (nR1). Add 80 μL of the reference solution
15-keto-PGE2). and measure the potential (nR2).
The absolute value |nT2 - nT1| is not higher than the absolute
01/2008:1611 value |nR2 - nR1|.
Related substances. Liquid chromatography (2.2.29).
DIOSMIN Test solution. Dissolve 25.0 mg of the substance to be
examined in dimethyl sulfoxide R and dilute to 25.0 mL with
the same solvent.
Diosminum
Reference solution (a). Dissolve 25.0 mg of diosmin CRS in
dimethyl sulfoxide R and dilute to 25.0 mL with the same
solvent.
Reference solution (b). Dilute 5.0 mL of reference solution (a)
to 100.0 mL with dimethyl sulfoxide R.
Reference solution (c). Dissolve 5.0 mg of diosmin for system
suitability CRS in dimethyl sulfoxide R and dilute to 5.0 mL
with the same solvent.
C28H32O15 Mr 609 Column :
[520-27-4] – size : l = 0.10 m, Ø = 4.6 mm,
DEFINITION – stationary phase : octadecylsilyl silica gel for
chromatography R (3 μm),
7-[[6-O-(6-Deoxy-α-L-mannopyranosyl)-β-D-
glucopyranosyl]oxy]-5-hydroxy-2-(3-hydroxy-4- – temperature : 40 °C.
methoxyphenyl)-4H-1-benzopyran-4-one. Mobile phase : acetonitrile R, glacial acetic acid R, methanol R,
Substance obtained through iodine-assisted oxidation water R (2:6:28:66 V/V/V/V).
of (2S)-7-[[6-O-(6-deoxy-α-L-mannopyranosyl)-β- Flow rate : 1.5 mL/min.
D-glucopyranosyl]oxy]-5-hydroxy-2-(3-hydroxy-4- Detection : spectrophotometer at 275 nm.
methoxyphenyl)-2,3-dihydro-4H-1-benzopyran-4-one Injection : 10 μL loop injector ; inject the test solution and
(hesperidin) of natural origin. reference solutions (b) and (c).
Content : 90.0 per cent to 102.0 per cent (anhydrous substance). Run time : 6 times the retention time of diosmin.
CHARACTERS Relative retention with reference to diosmin (retention
Appearance : greyish-yellow or light yellow hygroscopic time = about 4.6 min) : impurity A = about 0.5,
powder. impurity B = about 0.6, impurity C = about 0.8,
impurity D = about 2.2, impurity E = about 2.6,
Solubility : practically insoluble in water, soluble in dimethyl impurity F = about 4.5.
sulfoxide, practically insoluble in alcohol. It dissolves in dilute
solutions of alkali hydroxides. System suitability : reference solution (c) :
– resolution : minimum of 2.5 between the peaks due to
IDENTIFICATION impurities B and C.
A. Infrared absorption spectrophotometry (2.2.24). Limits :
Comparison : diosmin CRS. – correction factors : for the calculation of contents,
B. Examine the chromatograms obtained in the assay. multiply the peak areas of the following impurities by
Results : the principal peak in the chromatogram obtained the corresponding correction factor : impurity A = 0.38 ;
with the test solution is similar in retention time and size impurity F = 0.61,
to the principal peak in the chromatogram obtained with – impurity A : not more than 0.2 times the area of the
reference solution (a). principal peak in the chromatogram obtained with
reference solution (b) (1 per cent),
TESTS – impurity B : not more than the area of the principal peak
Iodine : maximum 0.1 per cent. in the chromatogram obtained with reference solution (b)
Determine the total content of iodine by potentiometry, using (5 per cent),
an iodide-selective electrode (2.2.36), after oxygen combustion – impurity C : not more than 0.6 times the area of the
(2.5.10). principal peak in the chromatogram obtained with
Test solution. Wrap 0.100 g of the substance to be examined in reference solution (b) (3 per cent),
a piece of filter paper and place it in a sample carrier. Introduce – impurity E : not more than 0.6 times the area of the
into the flask 50 mL of a 0.2 g/L solution of hydrazine R. Flush principal peak in the chromatogram obtained with
the flask with oxygen for 10 min. Ignite the filter paper. Stir reference solution (b) (3 per cent),

2072 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Diphenhydramine hydrochloride

– impurity F : not more than 0.6 times the area of the principal
peak in the chromatogram obtained with reference
solution (b) (3 per cent),
– any other impurity : not more than 0.2 times the area of
the principal peak in the chromatogram obtained with
reference solution (b) (1 per cent),
– total of other impurities and impurity A : not more than
0.2 times the area of the principal peak in the chromatogram F. 5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-
obtained with reference solution (b) (1 per cent), benzopyran-4-one (diosmetin).
– total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (b) 01/2008:0023
(10 per cent), corrected 6.0
– disregard limit : 0.02 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.1 per cent).
DIPHENHYDRAMINE
Heavy metals (2.4.8) : maximum 20 ppm.
HYDROCHLORIDE
2.0 g complies with test C. Prepare the reference solution using
4.0 mL of lead standard solution (10 ppm Pb) R. Diphenhydramini hydrochloridum
Water (2.5.12) : maximum 6.0 per cent, determined on 0.300 g.
Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
1.0 g.
ASSAY
Liquid chromatography (2.2.29), as described in the test for
related substances.
C17H22ClNO Mr 291.8
Injection : test solution and reference solution (a). [147-24-0]
STORAGE DEFINITION
In an airtight container. 2-(Diphenylmethoxy)-N,N-dimethylethanamine
IMPURITIES hydrochloride.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : very soluble in water, freely soluble in alcohol.
IDENTIFICATION
A. 1-(3-hydroxy-4-methoxyphenyl)ethanone
(acetoisovanillone), First identification : C, D.
Second identification : A, B, D.
A. Melting point (2.2.14) : 168 °C to 172 °C.
B. Dissolve 50 mg in alcohol R and dilute to 100.0 mL with
the same solvent. Examined between 230 nm and 350 nm,
the solution shows 3 absorption maxima (2.2.25), at
253 nm, 258 nm and 264 nm. The ratio of the absorbance
measured at the maximum at 258 nm to that measured
at the maximum at 253 nm is 1.1 to 1.3. The ratio of the
B. (2S)-7-[[6-O-(6-deoxy-α-L-mannopyranosyl)-β-D- absorbance measured at the maximum at 258 nm to that
glucopyranosyl]oxy]-5-hydroxy-2-(3-hydroxy-4- measured at the maximum at 264 nm is 1.2 to 1.4.
methoxyphenyl)-2,3-dihydro-4H-1-benzopyran-4-one C. Infrared absorption spectrophotometry (2.2.24).
(hesperidin),
Preparation : discs.
Comparison: diphenhydramine hydrochloride CRS.
D. It gives the reactions of chlorides (2.3.1).
TESTS
Solution S. Dissolve 1.0 g in carbon dioxide-free water R and
dilute to 20 mL with the same solvent.
Appearance of solution. Solution S and a fivefold dilution of
C. R1 = R3 = H, R2 = OH : 7-[[6-O-(6-deoxy-α-L- solution S are clear (2.2.1). Solution S is not more intensely
mannopyranosyl)-β-D-glucopyranosyl]oxy]-5-hydroxy-2- coloured than reference solution BY6 (2.2.2, Method II).
(4-hydroxyphenyl)-4H-1-benzopyran-4-one (isorhoifolin),
Acidity or alkalinity. To 10 mL of solution S add 0.15 mL of
D. R1 = OH, R2 = OCH3, R3 = I : 7-[[6-O-(6-deoxy-α-L- methyl red solution R and 0.25 mL of 0.01 M hydrochloric acid.
mannopyranosyl)-β-D-glucopyranosyl]oxy]-5-hydroxy-2- The solution is pink. Not more than 0.5 mL of 0.01 M sodium
(3-hydroxy-4-methoxyphenyl)-6-iodo-4H-1-benzopyran- hydroxide is required to change the colour of the indicator
4-one (6-iododiosmin), to yellow.
E. R1 = R3 = H, R2 = OCH3 : 7-[[6-O-(6-deoxy-α-L- Related substances. Liquid chromatography (2.2.29).
mannopyranosyl)-β-D-glucopyranosyl]oxy]-5-hydroxy-2- Test solution. Dissolve 70 mg of the substance to be examined
(4-methoxyphenyl)-4H-1-benzopyran-4-one (linarin), in the mobile phase and dilute to 20.0 mL with the mobile

General Notices (1) apply to all monographs and other texts 2073
Diphenoxylate hydrochloride EUROPEAN PHARMACOPOEIA 8.0

phase. Dilute 2.0 mL of the solution to 10.0 mL with the B. R = R′ = CH3 : 2-[(RS)-(4-methylphenyl)phenylmethoxy]-
mobile phase. N,N-dimethylethanamine,
Reference solution (a). Dilute 1.0 mL of the test solution to C. R = Br, R′ = CH3 : 2-[(RS)-(4-bromophenyl)phenyl-
10.0 mL with the mobile phase. Dilute 1.0 mL of this solution methoxy]-N,N-dimethylethanamine,
to 20.0 mL with the mobile phase.
Reference solution (b). Dissolve 5 mg of diphenhydramine
impurity A CRS and 5 mg of diphenylmethanol R in the mobile
phase and dilute to 10.0 mL with the mobile phase. To 2.0 mL
of this solution add 1.5 mL of the test solution and dilute to
10.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm, D. R = OH, R′ = H : diphenylmethanol (benzhydrol),
– stationary phase : base-deactivated octylsilyl silica gel for E. R + R′ = O : diphenylmethanone (benzophenone).
chromatography R (5 μm).
Mobile phase : mix 35 volumes of acetonitrile R and 65 volumes
of a 5.4 g/L solution of potassium dihydrogen phosphate R 04/2012:0819
adjusted to pH 3.0 using phosphoric acid R.
Flow rate : 1.2 mL/min. DIPHENOXYLATE HYDROCHLORIDE
Detection : spectrophotometer at 220 nm.
Injection : 10 μL.
Diphenoxylati hydrochloridum
Run time : 7 times the retention time of diphenhydramine.
Relative retention with reference to diphenhydramine
(retention time = about 6 min) : impurity A = about 0.9 ;
impurity B = about 1.5 ; impurity C = about 1.8 ;
impurity D = about 2.6 ; impurity E = about 5.1.
System suitability : reference solution (b) :
– resolution : minimum 2.0 between the peaks due to
diphenhydramine and to impurity A. C30H33ClN2O2 Mr 489.1
Limits : [3810-80-8]
– correction factor : for the calculation of content, multiply
the peak area of impurity D by 0.7, DEFINITION
– impurity A : not more than the area of the principal peak Ethyl 1-(3-cyano-3,3-diphenylpropyl)-4-phenylpiperidine-
in the chromatogram obtained with reference solution (a) 4-carboxylate hydrochloride.
(0.5 per cent), Content : 98.0 per cent to 102.0 per cent (dried substance).
– any other impurity : not more than 0.6 times the area of CHARACTERS
the principal peak in the chromatogram obtained with
reference solution (a) (0.3 per cent), Appearance : white or almost white, crystalline powder.
– total : not more than twice the area of the principal peak Solubility : very slightly soluble in water, freely soluble in
in the chromatogram obtained with reference solution (a) methylene chloride, sparingly soluble in ethanol (96 per cent).
(1.0 per cent), IDENTIFICATION
– disregard limit : 0.1 times the area of the principal peak in A. Infrared absorption spectrophotometry (2.2.24).
the chromatogram obtained with reference solution (a)
(0.05 per cent). Comparison: diphenoxylate hydrochloride CRS.
B. Dissolve about 30 mg in 5 mL of methanol R. Add 0.25 mL
Loss on drying (2.2.32) : maximum 0.5 per cent, determined of nitric acid R and 0.4 mL of silver nitrate solution R1.
on 1.000 g by drying in an oven at 105 °C. Shake and allow to stand. A curdled precipitate is formed.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Centrifuge and rinse the precipitate with 3 quantities, each
1.0 g. of 2 mL, of methanol R. Carry out this operation rapidly
and protected from bright light. Suspend the precipitate
ASSAY in 2 mL of water R and add 1.5 mL of ammonia R. The
Dissolve 0.250 g in 50 mL of alcohol R and add 5.0 mL of precipitate dissolves easily.
0.01 M hydrochloric acid. Carry out a potentiometric titration
(2.2.20), using 0.1 M sodium hydroxide. Read the volume TESTS
added between the 2 points of inflexion. Appearance of solution. The solution is clear (2.2.1) and not
1 mL of 0.1 M sodium hydroxide is equivalent to 29.18 mg of more intensely coloured than reference solution Y6 (2.2.2,
C17H22ClNO. Method II).
STORAGE Dissolve 1.0 g in methylene chloride R and dilute to 10 mL
with the same solvent.
Protected from light.
Related substances. Liquid chromatography (2.2.29).
IMPURITIES Solution A. Adjust 900 mL of water R to pH 2.3 with phosphoric
Specified impurities : A, B, C, D, E. acid R and dilute to 1000.0 mL with water R.
Solvent mixture : acetonitrile R1, solution A (50:50 V/V).
Test solution. Dissolve 25 mg of the substance to be examined
in 20 mL of the solvent mixture, sonicate for 2 min, cool and
dilute to 25.0 mL with the solvent mixture.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
A. R = R′ = H : 2-(diphenylmethoxy)-N-methylethanamine, solution to 10.0 mL with the solvent mixture.

2074 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dipivefrine hydrochloride

Reference solution (b). Dissolve 2 mg of diphenoxylate for

system suitability CRS (containing impurity A) in 2.0 mL of
the solvent mixture.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm). A. 1-(3-cyano-3,3-diphenylpropyl)-4-phenylpiperidine-4-
carboxylic acid (diphenoxylic acid),
Mobile phase :
– mobile phase A : solution A ;
– mobile phase B : acetonitrile R1 ;
Time Mobile phase A Mobile phase B B. 1-cyanomethanamide,
(min) (per cent V/V) (per cent V/V)
0-5 75 25

5 - 40 75 → 15 25 → 85

Flow rate : 2.0 mL/min.

Detection : spectrophotometer at 210 nm.
C. 4-bromo-2,2-diphenylbutanenitrile.
Injection : 20 μL.
Relative retention with reference to diphenoxylate (retention
time = about 16 min) : impurity A = about 0.8. 01/2008:1719
System suitability : reference solution (b) : corrected 7.0
– resolution : minimum 5.0 between the peaks due to
impurity A and diphenoxylate. DIPIVEFRINE HYDROCHLORIDE
Limits :
– impurity A : not more than 1.5 times the area of the
Dipivefrini hydrochloridum
principal peak in the chromatogram obtained with
reference solution (a) (0.15 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
– total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ; C19H30ClNO5 Mr 387.9
– disregard limit : 0.5 times the area of the principal peak in [64019-93-8]
the chromatogram obtained with reference solution (a)
(0.05 per cent). DEFINITION
Loss on drying (2.2.32) : maximum 0.5 per cent, determined Hydrochloride of 4-[(1RS)-1-hydroxy-2-(methylamino)ethyl]-
on 1.000 g by drying in an oven at 105 °C. 1,2-phenylene bis(2,2-dimethylpropanoate).
Content : 97.5 per cent to 102.0 per cent (dried substance).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. CHARACTERS
Appearance : white or almost white, crystalline powder.
ASSAY
Solubility : freely soluble in water, very soluble in methanol,
Dissolve 0.400 g in 40 mL of ethanol (96 per cent) R and add freely soluble in ethanol (96 per cent) and in methylene
5.0 mL of 0.01 M hydrochloric acid. Carry out a potentiometric chloride.
titration (2.2.20), using 0.1 M ethanolic sodium hydroxide. mp : about 160 °C.
Read the volume added between the 2 points of inflexion.
1 mL of 0.1 M ethanolic sodium hydroxide is equivalent to IDENTIFICATION
48.91 mg of C30H33ClN2O2. A. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs.
STORAGE Comparison: dipivefrine hydrochloride CRS.
Protected from light. B. It gives reaction (a) of chlorides (2.3.1).
IMPURITIES TESTS
Specified impurities : A. Impurities A and B. Liquid chromatography (2.2.29).
Other detectable impurities (the following substances would, Test solution. Dissolve 0.100 g of the substance to be examined
if present at a sufficient level, be detected by one or other of in 0.01 M hydrochloric acid and dilute to 10.0 mL with the
the tests in the monograph. They are limited by the general same acid.
acceptance criterion for other/unspecified impurities and/or Reference solution. Dissolve 10.0 mg of adrenaline R and
by the general monograph Substances for pharmaceutical use 10.0 mg of adrenalone hydrochloride R in 0.01 M hydrochloric
(2034). It is therefore not necessary to identify these impurities acid and dilute to 100.0 mL with the same acid. Dilute 1.0 mL
for demonstration of compliance. See also 5.10. Control of of this solution to 10.0 mL with 0.01 M hydrochloric acid.
impurities in substances for pharmaceutical use) : B, C. Protect this solution from light.

General Notices (1) apply to all monographs and other texts 2075
Dipivefrine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Column : Limits :
– size : l = 0.15 m, Ø = 4.6 mm ; – correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
– stationary phase : end-capped polar-embedded octadecylsilyl
corresponding correction factor : impurities C and D = 0.5 ;
amorphous organosilica polymer R (5 μm).
impurity E = 0.06 ;
Mobile phase : – sum of impurities C and D : not more than 0.3 times the
– mobile phase A : 0.1 per cent V/V solution of anhydrous area of the principal peak in the chromatogram obtained
formic acid R ; with reference solution (a) (0.3 per cent) ;
– mobile phase B : methanol R2, acetonitrile R (40:60 V/V) ; – impurities E, F : for each impurity, not more than 0.1 times
the area of the principal peak in the chromatogram
Time Mobile phase A Mobile phase B obtained with reference solution (a) (0.1 per cent) ;
(min) (per cent V/V) (per cent V/V)
– unspecified impurities : for each impurity, not more than
0-3 100 0
0.1 times the area of the principal peak in the chromatogram
3-5 100 → 40 0 → 60 obtained with reference solution (a) (0.10 per cent) ;
5 - 10 40 60 – total : not more than 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
Flow rate : 1 mL/min. (0.5 per cent) ;
Detection : spectrophotometer at 260 nm. – disregard limit : 0.05 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
Injection : 10 μL. (0.05 per cent); disregard any peak with a mass distribution
Retention times : impurity A = about 2.2 min ; ratio less than 0.5.
impurity B = about 3.2 min. Loss on drying (2.2.32) : maximum 1.0 per cent, determined
System suitability: reference solution : on 1.000 g by drying in vacuo at 60 °C for 6 h.
– resolution : minimum 2.0 between the peaks due to Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
impurities A and B. 1.0 g.
Limits : ASSAY
– impurities A, B : for each impurity, not more than the area Liquid chromatography (2.2.29) as described in the test for
of the corresponding peak in the chromatogram obtained related substances with the following modification.
with the reference solution (0.1 per cent).
Injection : 20 μL of reference solutions (a) and (c).
Related substances. Liquid chromatography (2.2.29). System suitability : reference solution (c) :
Solvent mixture. Mix 40 volumes of methanol R2 and – symmetry factor : maximum 2.0 for the peak due to
60 volumes of acetonitrile R. Mix 55 volumes of this mixture dipivefrine.
and 45 volumes of 0.01 M hydrochloric acid.
Calculate the percentage content of C19H30ClNO5 using the
Test solution. Dissolve 50.0 mg of the substance to be chromatograms obtained with reference solutions (a) and (c)
examined in the solvent mixture and dilute to 5.0 mL with the and the declared content of dipivefrine hydrochloride CRS.
solvent mixture.
Reference solution (a). Dilute 1.0 mL of the test solution to IMPURITIES
100.0 mL with the solvent mixture. Specified impurities : A, B, C, D, E, F.
Reference solution (b). Dissolve 5 mg of dipivefrine for system
suitability CRS (containing impurities C, D and E) in the
solvent mixture and dilute to 2.0 mL with the solvent mixture.
Reference solution (c). Dissolve 5.0 mg of dipivefrine
hydrochloride CRS in the solvent mixture and dilute to 2.0 mL
with the solvent mixture. Dilute 1.0 mL of this solution to A. R1 = R2 = R3 = H : 4-[(1RS)-1-hydroxy-2-
25.0 mL with the solvent mixture. (methylamino)ethyl]benzene-1,2-diol ((±)-adrenaline),
Column :
C. R1 = R3 = H, R2 = CO-C(CH3)3 : 2-hydroxy-5-
– size : l = 0.15 m, Ø = 4.6 mm ; [(1RS)-1-hydroxy-2-(methylamino)ethyl]phenyl
– stationary phase : end-capped polar-embedded octadecylsilyl 2,2-dimethylpropanoate,
amorphous organosilica polymer R (5 μm).
D. R1 = CO-C(CH3)3, R2 = R3 = H : 2-hydroxy-4-
Mobile phase : mix 45 volumes of a 2.7 g/L solution of [(1RS)-1-hydroxy-2-(methylamino)ethyl]phenyl
concentrated ammonia R adjusted to pH 10.0 with dilute 2,2-dimethylpropanoate,
acetic acid R and 55 volumes of a mixture of 40 volumes of
methanol R2 and 60 volumes of acetonitrile R. F. R1 = R2 = CO-C(CH3)3, R3 = C2H5 : 4-[(1RS)-2-
Flow rate : 1 mL/min. (ethylmethylamino)-1-hydroxyethyl]-1,2-phenylene
bis(2,2-dimethylpropanoate),
Detection : spectrophotometer at 260 nm.
Injection : 10 μL.
Run time : 2.5 times the retention time of dipivefrine.
Relative retention with reference to dipivefrine (retention
time = about 7 min) : impurities C and D = about 0.4 ;
impurity E = about 1.3 ; impurity F = about 2.0. B. R = H : 1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanone
System suitability : reference solution (b) : (adrenalone),
– resolution : minimum 3.0 between the peaks due to E. R = CO-C(CH3)3 : 4-[(methylamino)acetyl]-1,2-phenylene
dipivefrine and impurity E. bis(2,2-dimethylpropanoate) (adrenalone dipivalate ester).

2076 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dipotassium clorazepate

01/2008:0898 immediately with 2 quantities, each of 5.0 mL, of methylene

chloride R. Combine the organic layers and dilute to 10.0 mL
DIPOTASSIUM CLORAZEPATE with methylene chloride R.
Reference solution (a). Dissolve 10 mg of aminochlorobenzo-
Dikalii clorazepas phenone R in methylene chloride R and dilute to 100.0 mL with
the same solvent. Dilute 5.0 mL of the solution to 25.0 mL
with methylene chloride R.
Reference solution (b). Dissolve 5 mg of nordazepam CRS
in methylene chloride R and dilute to 25.0 mL with the
same solvent. Dilute 5.0 mL of the solution to 25.0 mL with
methylene chloride R.
Reference solution (c). Dilute 10.0 mL of reference solution (b)
to 20.0 mL with methylene chloride R.
C16H11ClK2N2O4 Mr 408.9 Reference solution (d). Dissolve 5 mg of nordazepam CRS and
[57109-90-7] 5 mg of nitrazepam CRS in methylene chloride R and dilute to
25 mL with the same solvent.
DEFINITION
Plate : TLC silica gel F254 plate R.
Potassium (3RS)-7-chloro-2-oxo-5-phenyl-2,3-dihydro-1H-
1,4-benzodiazepine-3-carboxylate compound with potassium Mobile phase : acetone R, methylene chloride R (15:85 V/V).
hydroxide (1:1). Application : 5 μL.
Content : 99.0 per cent to 101.0 per cent (dried substance). Development : over 2/3 of the plate.
CHARACTERS Drying : in air.
Appearance : white or light yellow, crystalline powder. Detection A : examine in ultraviolet light at 254 nm.
Solubility : freely soluble to very soluble in water, very slightly System suitability : the chromatogram obtained with reference
soluble in alcohol, practically insoluble in methylene chloride. solution (d) shows 2 clearly separated spots.
Solutions in water and in alcohol are unstable and are to be Limits A :
used immediately.
– impurity B : any spot due to impurity B is not more intense
IDENTIFICATION than the spot in the chromatogram obtained with reference
First identification : B, E. solution (b) (0.2 per cent),
Second identification : A, C, D, E. – any other impurity : any spot, apart from any spot due
A. Dissolve 10.0 mg in a 0.3 g/L solution of potassium to impurity B, is not more intense than the spot in the
carbonate R and dilute to 100.0 mL with the same solution chromatogram obtained with reference solution (c) (0.1 per
(solution A). Dilute 10.0 mL of solution A to 100.0 mL with cent).
a 0.3 g/L solution of potassium carbonate R (solution B). Detection B : spray with a freshly prepared 10 g/L solution
Examined between 280 nm and 350 nm (2.2.25), solution A of sodium nitrite R in dilute hydrochloric acid R. Dry in
shows a broad absorption maximum at about 315 nm. The a current of warm air and spray with a 4 g/L solution of
specific absorbance at the absorption maximum at 315 nm naphthylethylenediamine dihydrochloride R in alcohol R.
is 49 to 56. Examined between 220 nm and 280 nm (2.2.25), Limits B :
solution B shows an absorption maximum at 230 nm. The
specific absorbance at the absorption maximum at 230 nm – impurity A : any spot due to impurity A is not more intense
is 800 to 870. than the spot in the chromatogram obtained with reference
solution (a) (0.1 per cent).
B. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in vacuo at 60 °C for 4 h.
Comparison : Ph. Eur. reference spectrum of dipotassium
clorazepate. ASSAY
C. Dissolve about 20 mg in 2 mL of sulfuric acid R. Observed Dissolve 0.130 g in 10 mL of anhydrous acetic acid R. Add
in ultraviolet light at 365 nm, the solution shows yellow 30 mL of methylene chloride R. Titrate with 0.1 M perchloric
fluorescence. acid, determining the 2 points of inflexion by potentiometry
D. Dissolve 0.5 g in 5 mL of water R. Add 0.1 mL of thymol (2.2.20).
blue solution R. The solution is violet-blue. At the 2nd point of inflexion, 1 mL of 0.1 M perchloric acid is
E. Place 1.0 g in a crucible and add 2 mL of dilute sulfuric equivalent to 13.63 mg of C16H11ClK2N2O4.
acid R. Heat at first on a water-bath, then ignite until all
black particles have disappeared. Allow to cool. Take up STORAGE
the residue with water R and dilute to 20 mL with the same In an airtight container, protected from light.
solvent. The solution gives reaction (b) of potassium (2.3.1).
TESTS IMPURITIES
Appearance of solution. The solution is clear (2.2.1) and not Specified impurities : A, B.
more intensely coloured than reference solution GY5 (2.2.2,
Method II).
Rapidly dissolve 2.0 g with shaking in water R and dilute to
20.0 mL with the same solvent. Observe immediately.
Related substances. Thin-layer chromatography (2.2.27).
Prepare the solutions immediately before use and carry out the
test protected from light.
Test solution. Dissolve 0.20 g of the substance to be examined A. (2-amino-5-chlorophenyl)phenylmethanone
in water R and dilute to 5.0 mL with the same solvent. Shake (aminochlorobenzophenone),

General Notices (1) apply to all monographs and other texts 2077
Dipotassium phosphate EUROPEAN PHARMACOPOEIA 8.0

Sodium : maximum 0.1 per cent, if intended for use in the

manufacture of parenteral preparations.
Atomic emission spectrometry (2.2.22, Method I).
Test solution. Dissolve 1.00 g in water R and dilute to 100.0 mL
with the same solvent.
Reference solutions. Prepare the reference solutions using
sodium standard solution (200 ppm Na) R, diluted as necessary
B. 7-chloro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2- with water R.
one (nordazepam). Wavelength : 589 nm.
Loss on drying (2.2.32) : maximum 2.0 per cent, determined
01/2008:1003 on 1.000 g by drying in an oven at 125-130 °C.
corrected 7.0 Bacterial endotoxins (2.6.14) : less than 1.1 IU/mg, if intended
for use in the manufacture of parenteral preparations without
DIPOTASSIUM PHOSPHATE a further appropriate procedure for the removal of bacterial
endotoxins.
Dikalii phosphas ASSAY
Dissolve 0.800 g (m) in 40 mL of carbon dioxide-free water R
K2HPO4 Mr 174.2 and add 10.0 mL of 1 M hydrochloric acid. Carry out a
[7758-11-4] potentiometric titration (2.2.20) using 1 M sodium hydroxide.
Read the volume added at the 1st inflexion point (n1 mL).
DEFINITION Continue the titration to the 2nd inflexion point (total volume
Content : 98.0 per cent to 101.0 per cent (dried substance). of 1 M sodium hydroxide required, n2 mL).
Calculate the percentage content of K2HPO4 from the
CHARACTERS following expression :
Appearance : white or almost white powder or colourless
crystals, very hygroscopic.
Solubility : very soluble in water, very slightly soluble in
ethanol (96 per cent).
d = percentage loss on drying.
IDENTIFICATION
A. Solution S (see Tests) is slightly alkaline (2.2.4). STORAGE
B. Solution S gives reaction (b) of phosphates (2.3.1). In an airtight container.
C. Solution S gives reaction (a) of potassium (2.3.1). LABELLING
TESTS The label states, where applicable, that the substance is suitable
for use in the manufacture of parenteral preparations.
Solution S. Dissolve 5.0 g in distilled water R and dilute to
50 mL with the same solvent. 01/2012:0486
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II). DIPROPHYLLINE
Reducing substances. To 5 mL of solution S add 5 mL
of dilute sulfuric acid R and 0.25 mL of 0.02 M potassium Diprophyllinum
permanganate and heat on a water-bath for 5 min. The
solution remains faintly pink.
Monopotassium phosphate : maximum 2.5 per cent.
From the volume of 1 M hydrochloric acid (10.0 mL) and of
1 M sodium hydroxide (n1 mL and n2 mL) used in the assay,
calculate the following ratio :

C10H14N4O4 Mr 254.2
[479-18-5]
This ratio is not greater than 0.025.
DEFINITION
Chlorides (2.4.4) : maximum 200 ppm.
7-[(2RS)-2,3-Dihydroxypropyl]-1,3-dimethyl-3,7-dihydro-
To 2.5 mL of solution S add 10 mL of dilute nitric acid R and
1H-purine-2,6-dione.
dilute to 15 mL with water R.
Content : 98.5 per cent to 101.0 per cent (dried substance).
Sulfates (2.4.13) : maximum 0.1 per cent.
To 1.5 mL of solution S add 2 mL of dilute hydrochloric acid R CHARACTERS
and dilute to 15 mL with distilled water R. Appearance : white or almost white, crystalline powder.
Arsenic (2.4.2, Method A) : maximum 2 ppm, determined on Solubility : freely soluble in water, slightly soluble in ethanol
5 mL of solution S. (96 per cent).
Iron (2.4.9): maximum 10 ppm, determined on solution S. IDENTIFICATION
Heavy metals (2.4.8) : maximum 10 ppm. Infrared absorption spectrophotometry (2.2.24).
Dissolve 2.0 g in 8 mL of water R. Acidify with about 6 mL of Comparison: diprophylline CRS.
dilute hydrochloric acid R (pH 3-4) and dilute to 20 mL with
water R. 12 mL of this solution complies with test A. Prepare TESTS
the reference solution using lead standard solution (1 ppm Solution S. Dissolve 2.5 g in carbon dioxide-free water R and
Pb) R. dilute to 50 mL with the same solvent.

2078 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dipyridamole

Appearance of solution. Solution S is clear (2.2.1) and IMPURITIES

colourless (2.2.2, Method II). Other detectable impurities (the following substances would,
Acidity or alkalinity. To 10 mL of solution S add 0.25 mL if present at a sufficient level, be detected by one or other of
of bromothymol blue solution R1. The solution is yellow or the tests in the monograph. They are limited by the general
green. Not more than 0.4 mL of 0.01 M sodium hydroxide is acceptance criterion for other/unspecified impurities and/or
required to change the colour of the indicator to blue. by the general monograph Substances for pharmaceutical use
Related substances. Liquid chromatography (2.2.29). (2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
Test solution. Dissolve 50 mg of the substance to be examined impurities in substances for pharmaceutical use) : A, B, C, D.
in water R and dilute to 50.0 mL with the same solvent.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with water R. Dilute 1.0 mL of this solution to
10.0 mL with water R.
Reference solution (b). Dissolve 5 mg of etofylline CRS
(impurity C) in water R and dilute to 50.0 mL with the same
solvent. Dilute 0.5 mL of the solution to 20.0 mL with the A. N-methyl-5-(methylamino)-1H-imidazole-4-carboxamide
test solution. (theophyllidine),
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography with polar incorporated groups R (3 μm) ;
– temperature : 30 °C.
Mobile phase : methanol R, water R (10:90 V/V).
B. 1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
Flow rate : 0.7 mL/min.
(theophylline),
Detection : spectrophotometer at 272 nm.
Injection : 10 μL.
Run time : 3 times the retention time of diprophylline.
Relative retention with reference to diprophylline (retention
time = about 18 min) : impurity C = about 1.1.
System suitability : reference solution (b) :
C. 7-(2-hydroxyethyl)-1,3-dimethyl-3,7-dihydro-1H-purine-
– peak-to-valley ratio : minimum 5, where Hp = height above
2,6-dione (etofylline),
the baseline of the peak due to impurity C and Hv = height
above the baseline of the lowest point of the curve
separating this peak from the peak due to diprophylline.
Limits :
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
– total : not more than 3 times the area of the principal peak D. 7-[(2RS)-2-hydroxypropyl]-1,3-dimethyl-3,7-dihydro-1H-
in the chromatogram obtained with reference solution (a) purine-2,6-dione (proxyphylline).
(0.3 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a) 01/2014:1199
(0.05 per cent).
Chlorides (2.4.4): maximum 400 ppm. DIPYRIDAMOLE
Dilute 2.5 mL of solution S to 15 mL with water R.
Heavy metals (2.4.8) : maximum 20 ppm.
Dipyridamolum
12 mL of solution S complies with test A. Prepare the reference
solution using lead standard solution (1 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY
In order to avoid overheating in the reaction medium, mix
thoroughly throughout and stop the titration immediately after C24H40N8O4 Mr 504.6
the end-point has been reached. [58-32-2]
Dissolve 0.200 g in 3.0 mL of anhydrous formic acid R and add
50.0 mL of acetic anhydride R. Titrate with 0.1 M perchloric DEFINITION
acid, determining the end-point potentiometrically (2.2.20). 2,2′,2″,2′′′-[[4,8-Di(piperidin-1-yl)pyrimido[5,4-
1 mL of 0.1 M perchloric acid is equivalent to 25.42 mg of d]pyrimidine-2,6-diyl]dinitrilo]tetraethanol.
C10H14N4O4. Content : 98.5 per cent to 101.5 per cent (dried substance).
STORAGE CHARACTERS
Protected from light. Appearance : bright yellow, crystalline powder.

General Notices (1) apply to all monographs and other texts 2079
Dipyridamole EUROPEAN PHARMACOPOEIA 8.0

Solubility : practically insoluble in water, freely soluble in – impurities D, E : for each impurity, not more than twice the
acetone, soluble in anhydrous ethanol. It dissolves in dilute area of the principal peak in the chromatogram obtained
mineral acids. with reference solution (a) (0.2 per cent) ;
– unspecified impurities : for each impurity, not more than the
IDENTIFICATION area of the principal peak in the chromatogram obtained
Infrared absorption spectrophotometry (2.2.24). with reference solution (a) (0.10 per cent) ;
Preparation : discs of potassium bromide R. – total : not more than 10 times the area of the principal peak
Comparison : dipyridamole CRS. in the chromatogram obtained with reference solution (a)
(1.0 per cent) ;
TESTS – disregard limit : 0.5 times the area of the principal peak in
Related substances. Liquid chromatography (2.2.29). Prepare the chromatogram obtained with reference solution (a)
the solutions immediately before use. (0.05 per cent).
Test solution. Dissolve 0.100 g of the substance to be examined Chlorides (2.4.4) : maximum 200 ppm.
in methanol R and dilute to 50 mL with the same solvent. To 0.250 g add 10 mL of water R and shake vigorously. Filter,
Reference solution (a). Dilute 1.0 mL of the test solution to rinse the filter with 5 mL of water R and dilute to 15 mL with
100.0 mL with methanol R. Dilute 1.0 mL of this solution to water R.
10.0 mL with methanol R. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Reference solution (b). Dissolve the contents of a vial on 1.000 g by drying in an oven at 105 °C.
of dipyridamole for peak identification CRS (containing Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
impurities A, B, C, D, E and F) in 1 mL of methanol R. 1.0 g.
Column :
– size : l = 0.10 m, Ø = 4.0 mm ; ASSAY
– stationary phase : spherical end-capped octadecylsilyl silica Dissolve 0.400 g in 70 mL of methanol R. Titrate with 0.1 M
gel for chromatography R (5 μm) ; perchloric acid, determining the end-point potentiometrically
(2.2.20).
– temperature : 45 °C.
1 mL of 0.1 M perchloric acid is equivalent to 50.46 mg
Mobile phase :
of C24H40N8O4.
– mobile phase A : dissolve 1.0 g of potassium dihydrogen
phosphate R in 900 mL of water R, adjust to pH 7.0 with STORAGE
0.5 M sodium hydroxide and dilute to 1000 mL with
Protected from light.
water R ;
– mobile phase B : methanol R ; IMPURITIES
Time Mobile phase A Mobile phase B Specified impurities : A, B, C, D, E.
(min) (per cent V/V) (per cent V/V) Other detectable impurities (the following substances would,
0-5 40 60 if present at a sufficient level, be detected by one or other of
5 - 19 40 → 5 60 → 95 the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
19 - 24 5 → 40 95 → 60 by the general monograph Substances for pharmaceutical use
24 - 29 40 60 (2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
Flow rate : 1.2 mL/min. impurities in substances for pharmaceutical use) : F, G.
Detection : spectrophotometer at 295 nm.
Injection : 5 μL.
Identification of impurities : use the chromatogram supplied
with dipyridamole for peak identification CRS and the
chromatogram obtained with reference solution (b) to identify
the peaks due to impurities A, B, C, D, E and F.
Relative retention with reference to dipyridamole
(retention time = about 8 min): impurity B = about 0.2 ;
impurity F = about 0.3 ; impurity D = about 0.9 ;
impurity E = about 1.3 ; impurity C = about 1.6 ;
impurity A = about 2.2. A. 2,2′-[[4,6,8-tri(piperidin-1-yl)pyrimido[5,4-d]pyrimidin-
System suitability : reference solution (b) : 2-yl]nitrilo]diethanol,
– resolution : minimum 2.0 between the peaks due to
impurity D and dipyridamole ;
– peak-to-valley ratio : minimum 2.0, where Hp = height
above the baseline of the peak due to impurity F and
Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to impurity B.
Limits :
– correction factor : for the calculation of content, multiply
the peak area of impurity B by 1.7 ;
– impurities A, B, C : for each impurity, not more than
5 times the area of the principal peak in the chromatogram B. 2,2′,2″,2′′′,2′′′′,2′′′′′[[8-(piperidin-1-yl)pyrimido[5,4-
obtained with reference solution (a) (0.5 per cent) ; d]pyrimidine-2,4,6-triyl]trinitrilo]hexaethanol,

2080 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dirithromycin

01/2008:1313
corrected 6.1

DIRITHROMYCIN
Dirithromycinum

C. 2,2′-[[6-chloro-4,8-di(piperidin-1-yl)pyrimido[5,4-
d]pyrimidin-2-yl]nitrilo]diethanol,

C42H78N2O14 Mr 835
[62013-04-1]
DEFINITION
(1R,2S,3R,6R,7S,8S,9R,10R,12R,13S,15R,17S)-9-
D. 2,2′-[[6-[(2-hydroxyethyl)amino]-4,8-di(piperidin-1- [[3-(Dimethylamino)-3,4,6-trideoxy-β-D-xylo-
yl)pyrimido[5,4-d]pyrimidin-2-yl]nitrilo]diethanol, hexopyranosyl]oxy]-3-ethyl-2,10-dihydroxy-15-[(2-
methoxyethoxy)methyl]-2,6,8,10,12,17-hexamethyl-7-[(3-C-
methyl-3-O-methyl-2,6dideoxy-α-L-ribo-hexopyranosyl)oxy]-
4,16-dioxa-14azabicyclo[11.3.1]heptadecan-5-one (or
(9S)-9,11-[imino[(1R)-2-(2-methoxyethoxy)ethylidene]oxy]-
9-deoxo-11-deoxyerythromycin).
Semi-synthetic product derived from a fermentation product.
Content : 96.0 per cent to 102.0 per cent for the sum of
the percentage contents of C42H78N2O14 and dirithromycin
15S-epimer (anhydrous substance).
CHARACTERS
Appearance : white or almost white powder.
E. 2,2′,2′′,2′′′-[[6,8-di(piperidin-1-yl)pyrimido[5,4- Solubility : very slightly soluble in water, very soluble in
d]pyrimidine-2,4-diyl]dinitrilo]tetraethanol, methanol and in methylene chloride.
It shows polymorphism (5.9).
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: dirithromycin CRS.
B. Examine the chromatograms obtained in the assay.
Results : the principal peak in the chromatogram obtained
with test solution (a) is similar in retention time and size
to the principal peak in the chromatogram obtained with
reference solution (a).
TESTS
F. 2,2′,2′′,2′′′-[[4-[(2-hydroxyethyl)amino]-8- Related substances. Liquid chromatography (2.2.29).
(piperidin-1-yl)pyrimido[5,4-d]pyrimidine-2,6-
diyl]dinitrilo]tetraethanol, Solvent mixture : methanol R, acetonitrile R1 (30:70 V/V).
Test solution (a). Dissolve 20.0 mg of the substance to be
examined in the solvent mixture and dilute to 10.0 mL with
the solvent mixture.
Test solution (b). Dissolve 0.10 g of the substance to be
examined in the solvent mixture and dilute to 10.0 mL with
the solvent mixture.
Reference solution (a). Dissolve 20.0 mg of dirithromycin CRS
in the solvent mixture and dilute to 10.0 mL with the solvent
mixture.
Reference solution (b). Dilute 5.0 mL of reference solution (a)
to 50.0 mL with the solvent mixture.
Reference solution (c). Dissolve 20 mg of dirithromycin CRS in
G. 2,6-dichloro-4,8-di(piperidin-1-yl)pyrimido[5,4- the mobile phase and dilute to 10 mL with the mobile phase.
d]pyrimidine. Allow to stand for 24 h before use.

General Notices (1) apply to all monographs and other texts 2081
Disodium edetate EUROPEAN PHARMACOPOEIA 8.0

Column : System suitability : reference solution (a) :

– size : l = 0.25 m, Ø = 4.6 mm ; – repeatability : maximum relative standard deviation of
– stationary phase : octadecylsilyl silica gel for 1.0 per cent after 6 injections.
chromatography R (5 μm) ; IMPURITIES
– temperature : 40 °C. Specified impurities : A.
Mobile phase : mix 9 volumes of water R, 19 volumes of Other detectable impurities (the following substances would,
methanol R, 28 volumes of a solution containing 1.9 g/L of if present at a sufficient level, be detected by one or other of
potassium dihydrogen phosphate R and 9.1 g/L of dipotassium the tests in the monograph. They are limited by the general
hydrogen phosphate R adjusted to pH 7.5 if necessary with a acceptance criterion for other/unspecified impurities and/or
100 g/L solution of potassium hydroxide R, and 44 volumes by the general monograph Substances for pharmaceutical use
of acetonitrile R1. (2034). It is therefore not necessary to identify these impurities
Flow rate : 2.0 mL/min. for demonstration of compliance. See also 5.10. Control of
Detection : spectrophotometer at 205 nm. impurities in substances for pharmaceutical use) : B, C, D, E.
Injection : 10 μL of test solution (b) and reference solutions (b)
and (c).
Run time : 3 times the retention time of dirithromycin.
Relative retention with reference to dirithromycin :
impurity A = about 0.7 ; 15S-epimer = about 1.1.
System suitability: reference solution (c) :
– resolution : minimum 2.0 between the peaks due to
dirithromycin and its 15S-epimer ; if necessary, adjust the
concentration of the organic modifiers in the mobile phase.
Limits :
– impurity A : not more than 0.75 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (1.5 per cent) ;
– any other impurity : for each impurity, not more than A. (9S)-9-amino-9-deoxoerythromycin,
0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (1 per cent) ; B. R = H : (9S)-9-amino-3-de(2,6-dideoxy-3-C-methyl-3-O-
– disregard limit : disregard the peak due to the 15S-epimer. methyl-α-L-ribo-hexopyranosyl)-9-deoxoerythromycin,
Dirithromycin 15S-epimer. Liquid chromatography (2.2.29)
as described in the test for related substances with the
following modifications.
Injection : test solution (b) and reference solution (b).
System suitability : reference solution (b) :
– repeatability : maximum relative standard deviation of
5.0 per cent after 6 injections.
Limit :
– 15S-epimer : maximum 1.5 per cent.
Acetonitrile (2.4.24, System A) : maximum 0.1 per cent.
Prepare the solutions using dimethylformamide R instead of
water R. C. R = CH2-O-CH2-CH2-O-CH3, R′ = H, R2 = H, R3 = CH3 :
(9S)-9,11-[imino[(1RS)-2-(2-methoxyethoxy)ethylidene]-
Sample solution. Dissolve 0.200 g of the substance to be oxy]-9-deoxo-11,12-dideoxyerythromycin
examined in dimethylformamide R and dilute to 20.0 mL with (dirithromycin B),
the same solvent.
Static head-space injection conditions that may be used : D. R = CH2-O-CH2-CH2-O-CH3, R′ = H, R2 = OH, R3 = H :
(9S)-9,11-[imino[(1RS)-2-(2-methoxyethoxy)ethylidene]-
– equilibration temperature : 120 °C ; oxy]-3′-O-demethyl-9-deoxo-11-deoxyerythromycin
– equilibration time : 60 min ; (dirithromycin C),
– transfer-line temperature : 125 °C. E. R = CH3, R′ = CH3, R2 = OH, R3 = CH3 : 9,11-[imino(1-
Heavy metals (2.4.8) : maximum 20 ppm. methylethylidene)oxy]-9-deoxo-11-deoxyerythromycin.
Dissolve 1.0 g in 20 mL of a mixture of equal volumes of
methanol R and water R. 12 mL of the solution complies with
test B. Prepare the reference solution using lead standard 01/2008:0232
solution (1 ppm Pb) obtained by diluting lead standard
solution (100 ppm Pb) R with a mixture of equal volumes of DISODIUM EDETATE
methanol R and water R.
Water (2.5.12) : maximum 1.0 per cent, determined on 1.00 g. Dinatrii edetas
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications.
Injection : test solution (a) and reference solution (a). C10H14N2Na2O8,2H2O Mr 372.2

2082 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Disodium phosphate, anhydrous

DEFINITION System suitability : reference solution :

Disodium dihydrogen (ethylenedinitrilo)tetraacetate – resolution : minimum 7 between the peaks due to the iron
dihydrate. complex of impurity A and the iron complex of edetic acid,
Content : 98.5 per cent to 101.0 per cent. – signal-to-noise ratio : minimum 50 for the peak due to
impurity A.
CHARACTERS Limit :
Appearance : white or almost white, crystalline powder. – impurity A : not more than the area of the corresponding
Solubility : soluble in water, practically insoluble in ethanol peak in the chromatogram obtained with the reference
(96 per cent). solution (0.1 per cent).
Iron (2.4.9) : maximum 80 ppm.
IDENTIFICATION Dilute 2.5 mL of solution S to 10 mL with water R. Add 0.25 g
First identification : A, B, D. of calcium chloride R to the test solution and the standard
Second identification : B, C, D. before the addition of the thioglycollic acid R.
A. Infrared absorption spectrophotometry (2.2.24). Heavy metals (2.4.8) : maximum 20 ppm.
Preparation : discs. 1.0 g complies with test F. Prepare the reference solution using
2 mL of lead standard solution (10 ppm Pb) R.
Comparison : disodium edetate CRS.
B. Dissolve 2 g in 25 mL of water R, add 6 mL of lead nitrate ASSAY
solution R, shake and add 3 mL of potassium iodide Dissolve 0.300 g in water R and dilute to 300 mL with the
solution R. No yellow precipitate is formed. Make alkaline same solvent. Add 2 g of hexamethylenetetramine R and 2 mL
to red litmus paper R by the addition of dilute ammonia R2. of dilute hydrochloric acid R. Titrate with 0.1 M lead nitrate,
Add 3 mL of ammonium oxalate solution R. No precipitate using about 50 mg of xylenol orange triturate R as indicator.
is formed. 1 mL of 0.1 M lead nitrate is equivalent to 37.22 mg
C. Dissolve 0.5 g in 10 mL of water R and add 0.5 mL of of C10H14N2Na2O8,2H2O.
calcium chloride solution R. Make alkaline to red litmus
paper R by the addition of dilute ammonia R2 and add 3 mL STORAGE
of ammonium oxalate solution R. No precipitate is formed. Protected from light.
D. It gives the reactions of sodium (2.3.1). IMPURITIES
TESTS Specified impurities : A.
Solution S. Dissolve 5.0 g in carbon dioxide-free water R and
dilute to 100 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
pH (2.2.3): 4.0 to 5.5 for solution S. A. nitrilotriacetic acid.
Impurity A. Liquid chromatography (2.2.29). Carry out the
test protected from light. 01/2008:1509
Solvent mixture. Dissolve 10.0 g of ferric sulfate pentahydrate R corrected 7.2
in 20 mL of 0.5 M sulfuric acid and add 780 mL of water R.
Adjust to pH 2.0 with 1 M sodium hydroxide and dilute to DISODIUM PHOSPHATE,
1000 mL with water R.
Test solution. Dissolve 0.100 g of the substance to be examined
ANHYDROUS
in the solvent mixture and dilute to 25.0 mL with the solvent
mixture. Dinatrii phosphas anhydricus
Reference solution. Dissolve 40.0 mg of nitrilotriacetic acid R
in the solvent mixture and dilute to 100.0 mL with the solvent Na2HPO4 Mr 142.0
mixture. To 1.0 mL of the solution add 0.1 mL of the test [7558-79-4]
solution and dilute to 100.0 mL with the solvent mixture. DEFINITION
Column : Content : 98.0 per cent to 101.0 per cent (dried substance).
– size : l = 0.10 m, Ø = 4.6 mm,
CHARACTERS
– stationary phase : spherical graphitised carbon for
chromatography R1 (5 μm) with a specific surface area of Appearance : white or almost white powder, hygroscopic.
120 m2/g and a pore size of 25 nm. Solubility : soluble in water, practically insoluble in ethanol
(96 per cent).
Mobile phase : dissolve 50.0 mg of ferric sulfate pentahydrate R
in 50 mL of 0.5 M sulfuric acid and add 750 mL of water R. IDENTIFICATION
Adjust to pH 1.5 with 0.5 M sulfuric acid or 1 M sodium
A. Solution S (see Tests) is slightly alkaline (2.2.4).
hydroxide, add 20 mL of ethylene glycol R and dilute to
1000 mL with water R. B. Loss on drying (see Tests).
Flow rate : 1 mL/min. C. Solution S gives reaction (b) of phosphates (2.3.1).
Detection : spectrophotometer at 273 nm. D. Solution S gives reaction (a) of sodium (2.3.1).
Injection : 20 μL ; filter the solutions and inject immediately. TESTS
Run time : 4 times the retention time of the iron complex of Solution S. Dissolve 5.0 g in distilled water R and dilute to
impurity A. 100.0 mL with the same solvent.
Retention times : iron complex of impurity A = about 5 min ; Appearance of solution. Solution S is clear (2.2.1) and
iron complex of edetic acid = about 10 min. colourless (2.2.2, Method II).

General Notices (1) apply to all monographs and other texts 2083
Disodium phosphate dihydrate EUROPEAN PHARMACOPOEIA 8.0

Reducing substances. To 10 mL of solution S add 5 mL D. Solution S gives reaction (a) of sodium (2.3.1).
of dilute sulfuric acid R and 0.25 mL of 0.02 M potassium
permanganate and heat on a water-bath for 5 min. The colour TESTS
of the permanganate is not completely discharged. Solution S. Dissolve 5.0 g in distilled water R and dilute to
Monosodium phosphate : maximum 2.5 per cent. 100 mL with the same solvent.
From the volume of 1 M hydrochloric acid (25 mL) and of Appearance of solution. Solution S is clear (2.2.1) and
1 M sodium hydroxide (n1 mL and n2 mL) used in the assay, colourless (2.2.2, Method II).
calculate the following ratio : Reducing substances. To 5 mL of solution S add 5 mL
of dilute sulfuric acid R and 0.25 mL of 0.02 M potassium
permanganate and heat on a water-bath for 5 min. The colour
of the permanganate is not completely discharged.
This ratio is not greater than 0.025.
Monosodium phosphate : maximum 2.5 per cent.
Chlorides (2.4.4) : maximum 200 ppm. From the volume of 1 M hydrochloric acid (25 mL) and of
Dilute 5 mL of solution S to 15 mL with dilute nitric acid R. 1 M sodium hydroxide (n1 mL and n2 mL) used in the assay,
Sulfates (2.4.13) : maximum 500 ppm. calculate the following ratio :
To 6 mL of solution S add 2 mL of dilute hydrochloric acid R
and dilute to 15 mL with distilled water R.
Arsenic (2.4.2, Method A) : maximum 2 ppm, determined on This ratio is not greater than 0.025.
10 mL of solution S.
Chlorides (2.4.4) : maximum 400 ppm.
Iron (2.4.9): maximum 20 ppm, determined on solution S.
To 2.5 mL of solution S add 10 mL of dilute nitric acid R and
Heavy metals (2.4.8) : maximum 10 ppm. dilute to 15 mL with water R.
12 mL of solution S complies with test A. Prepare the referenceSulfates (2.4.13) : maximum 0.1 per cent.
solution using 5 mL of lead standard solution (1 ppm Pb) R
and 5 mL of water R. To 3 mL of solution S add 2 mL of dilute hydrochloric acid R
and dilute to 15 mL with distilled water R.
Loss on drying (2.2.32) : maximum 1.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 4 h. Arsenic (2.4.2, Method A) : maximum 4 ppm, determined on
5 mL of solution S.
ASSAY Iron (2.4.9) : maximum 40 ppm.
Dissolve 1.600 g (m) in 25.0 mL of carbon dioxide-free water R Dilute 5 mL of solution S to 10 mL with water R.
and add 25.0 mL of 1 M hydrochloric acid. Carry out a
potentiometric titration (2.2.20) using 1 M sodium hydroxide. Heavy metals (2.4.8) : maximum 20 ppm.
Read the volume added at the 1st inflexion point (n1 mL). 12 mL of solution S complies with test A. Prepare the reference
Continue the titration to the 2nd inflexion point (total volume solution using lead standard solution (1 ppm Pb) R.
of 1 M sodium hydroxide required, n2 mL). Loss on drying (2.2.32) : 19.5 per cent to 21.0 per cent,
Calculate the percentage content of Na2HPO4 from the determined on 1.000 g by drying in an oven at 130 °C.
following expression :
ASSAY
Dissolve 2.000 g (m) in 50 mL of water R and add 25.0 mL of
1 M hydrochloric acid. Carry out a potentiometric titration
(2.2.20) using 1 M sodium hydroxide. Read the volume added
d = percentage loss on drying. at the 1st inflexion point (n1 mL). Continue the titration to
the 2nd inflexion point (total volume of 1 M sodium hydroxide
STORAGE required, n2 mL).
In an airtight container. Calculate the percentage content of Na2HPO4 from the
following expression :
01/2008:0602
corrected 7.2

DISODIUM PHOSPHATE DIHYDRATE d = percentage loss on drying.

Dinatrii phosphas dihydricus

04/2008:0118
Na2HPO4,2H2O Mr 178.0 corrected 7.2
[10028-24-7]
DEFINITION DISODIUM PHOSPHATE
Content : 98.0 per cent to 101.0 per cent (dried substance). DODECAHYDRATE
CHARACTERS
Appearance : white or almost white powder or colourless
Dinatrii phosphas dodecahydricus
crystals.
Na2HPO4,12H2O Mr 358.1
Solubility : soluble in water, practically insoluble in ethanol [10039-32-4]
(96 per cent).
DEFINITION
IDENTIFICATION
Content : 98.5 per cent to 102.5 per cent.
A. Solution S (see Tests) is slightly alkaline (2.2.4).
B. Loss on drying (see Tests). CHARACTERS
C. Solution S gives reaction (b) of phosphates (2.3.1). Appearance : colourless, transparent crystals, very efflorescent.

2084 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Disopyramide

Solubility : very soluble in water, practically insoluble in 01/2008:1006

ethanol (96 per cent).
DISOPYRAMIDE
IDENTIFICATION
Disopyramidum
A. Solution S (see Tests) is slightly alkaline (2.2.4).
B. Water (see Tests).
C. Solution S gives reaction (b) of phosphates (2.3.1).
D. Solution S gives reaction (a) of sodium (2.3.1).

TESTS
Solution S. Dissolve 5.0 g in distilled water R and dilute to C21H29N3O Mr 339.5
50 mL with the same solvent. [3737-09-5]
Appearance of solution. Solution S is clear (2.2.1) and DEFINITION
colourless (2.2.2, Method II).
Disopyramide contains not less than 98.5 per cent and not
Reducing substances. To 5 mL of solution S add 5 mL more than the equivalent of 101.5 per cent of (2RS)-4-[bis(1-
of dilute sulfuric acid R and 0.25 mL of 0.02 M potassium methylethyl)amino]-2-phenyl-2-(pyridin-2-yl)butanamide,
permanganate and heat on a water-bath for 5 min. The colour calculated with reference to the dried substance.
of the permanganate is not completely discharged.
CHARACTERS
Monosodium phosphate : maximum 2.5 per cent.
A white or almost white powder, slightly soluble in water,
From the volume of 1 M hydrochloric acid (25 mL) and of freely soluble in methylene chloride, soluble in alcohol.
1 M sodium hydroxide (n1 mL and n2 mL) used in the assay,
IDENTIFICATION
calculate the following ratio :
First identification : B.
Second identification : A, C.
A. Dissolve 40.0 mg in a 5 g/L solution of sulfuric acid R
in methanol R and dilute to 100.0 mL with the same
This ratio is not greater than 0.025. solution. Dilute 5.0 mL of this solution to 50.0 mL with a
Chlorides (2.4.4) : maximum 200 ppm. 5 g/L solution of sulfuric acid R in methanol R. Examined
between 240 nm and 350 nm (2.2.25), the solution shows an
To 2.5 mL of solution S add 10 mL of dilute nitric acid R and absorption maximum at 269 nm and a shoulder at 263 nm.
dilute to 15 mL with water R. The specific absorbance at the maximum is 190 to 210.
Sulfates (2.4.13) : maximum 500 ppm. B. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
To 3 mL of solution S add 2 mL of dilute hydrochloric acid R disopyramide CRS. Examine the substances as discs
and dilute to 15 mL with distilled water R. prepared by placing 50 μL of a 50 g/L solution in methylene
chloride R on a disc of potassium bromide R. Dry the discs
Arsenic (2.4.2, Method A) : maximum 2 ppm, determined on
at 60 °C for 1 h before use.
5 mL of solution S.
C. Examine the chromatograms obtained in the test for related
Iron (2.4.9) : maximum 20 ppm. substances in ultraviolet light at 254 nm. The principal
Dilute 5 mL of solution S to 10 mL with water R. spot in the chromatogram obtained with test solution (b)
is similar in position and size to the principal spot in the
Heavy metals (2.4.8) : maximum 10 ppm. chromatogram obtained with reference solution (a). Spray
with dilute potassium iodobismuthate solution R. Examine
12 mL of solution S complies with test A. Prepare the reference in daylight. The principal spot in the chromatogram
solution using lead standard solution (1 ppm Pb) R. obtained with test solution (b) is similar in position,
Water (2.5.12) : 57.0 per cent to 61.0 per cent, determined colour and size to the principal spot in the chromatogram
on 50.0 mg. Use a mixture of 10 volumes of anhydrous obtained with reference solution (a).
methanol R and 40 volumes of formamide R1 as solvent.
TESTS
Related substances. Examine by thin-layer chromatography
ASSAY (2.2.27), using silica gel GF254 R as the coating substance.
Test solution (a). Dissolve 0.20 g of the substance to be
Dissolve 4.00 g (m) in 25 mL of water R and add 25.0 mL of examined in methanol R and dilute to 10 mL with the same
1 M hydrochloric acid. Carry out a potentiometric titration solvent.
(2.2.20) using 1 M sodium hydroxide. Read the volume added
st
at the 1 inflexion point (n1 mL). Continue the titration to Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
the 2 inflexion point (total volume of 1 M sodium hydroxide with methanol R.
nd

required, n2 mL). Reference solution (a). Dissolve 20 mg of disopyramide CRS in

methanol R and dilute to 10 mL with the same solvent.
Calculate the percentage content of Na2HPO4,12H2O from the Reference solution (b). Dilute 0.5 mL of test solution (b) to
following expression : 20 mL with methanol R.
Apply to the plate 10 μL of each solution. Develop over a
path of 15 cm using a mixture of 1 volume of concentrated
ammonia R, 30 volumes of acetone R and 30 volumes of
cyclohexane R. Dry the plate in a current of warm air and

General Notices (1) apply to all monographs and other texts 2085
Disopyramide phosphate EUROPEAN PHARMACOPOEIA 8.0

examine in ultraviolet light at 254 nm. Any spot in the DEFINITION

chromatogram obtained with test solution (a), apart from Disopyramide phosphate contains not less than 98.0 per
the principal spot, is not more intense than the spot in the cent and not more than the equivalent of 102.0 per cent of
chromatogram obtained with reference solution (b) (0.25 per (2RS)-4-[bis(1-methylethyl)amino]-2-phenyl-2-(pyridin-
cent). 2-yl)butanamide dihydrogen phosphate, calculated with
Heavy metals (2.4.8). 2.0 g complies with test C for heavy reference to the dried substance.
metals (10 ppm). Prepare the reference solution using 2 mL
of lead standard solution (10 ppm Pb) R. CHARACTERS
Loss on drying (2.2.32). Not more than 0.5 per cent, A white or almost white powder, soluble in water, sparingly
determined on 1.000 g by drying at 80 °C over diphosphorus soluble in alcohol, practically insoluble in methylene chloride.
pentoxide R at a pressure not exceeding 0.7 kPa for 2 h. IDENTIFICATION
Sulfated ash (2.4.14). Not more than 0.2 per cent, determined First identification : B.
on 1.0 g.
Second identification : A, C, D.
ASSAY A. Dissolve 50.0 mg in a 5 g/L solution of sulfuric acid R
Dissolve 0.130 g in 30 mL of anhydrous acetic acid R. Add in methanol R and dilute to 100.0 mL with the same
0.2 mL of naphtholbenzein solution R. Titrate with 0.1 M solution. Dilute 5.0 mL of this solution to 50.0 mL with a
perchloric acid until the colour changes from yellow to green. 5 g/L solution of sulfuric acid R in methanol R. Examined
1 mL of 0.1 M perchloric acid is equivalent to 16.97 mg of between 240 nm and 350 nm (2.2.25), the solution shows an
C21H29N3O. absorption maximum at 269 nm and a shoulder at 263 nm.
The specific absorbance at the maximum is 147 to 163.
STORAGE B. Examine by infrared absorption spectrophotometry
Store protected from light. (2.2.24), comparing with the spectrum obtained with
disopyramide phosphate CRS. Examine the substances
IMPURITIES prepared as discs.
C. Examine the chromatograms obtained in the test for related
substances in ultraviolet light at 254 nm. The principal
spot in the chromatogram obtained with test solution (b)
is similar in position and size to the principal spot in the
chromatogram obtained with reference solution (a). Spray
with dilute potassium iodobismuthate solution R. Examine
in daylight. The principal spot in the chromatogram
obtained with test solution (b) is similar in position,
colour and size to the principal spot in the chromatogram
A. R = CN, R′ = CH(CH3)2 : (2RS)-4-[bis(1- obtained with reference solution (a).
methylethyl)amino]-2-phenyl-2-(pyridin-2- D. Solution S (see Tests) gives reaction (a) of phosphates
yl)butanenitrile (di-isopyronitrile), (2.3.1).
B. R = H, R′ = CH(CH3)2 : (3RS)-N,N-bis(1-methylethyl)-3-
phenyl-3-(pyridin-2-yl)propan-1-amine, TESTS
Solution S. Dissolve 1.0 g in carbon dioxide-free water R and
C. R = CO-NH2, R′ = H : (2RS)-4-[(1-methylethyl)amino]-2- dilute to 20 mL with the same solvent.
phenyl-2-(pyridin-2-yl)butanamide,
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
pH (2.2.3). The pH of solution S is 4.0 to 5.0.
Related substances. Examine by thin-layer chromatography
(2.2.27), using silica gel GF254 R as the coating substance.
Test solution (a). Dissolve 0.25 g of the substance to be
examined in methanol R and dilute to 10 mL with the same
D. (RS)-phenyl(pyridin-2-yl)acetonitrile (pyronitrile). solvent.
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
01/2008:1005 with methanol R.
corrected 6.0 Reference solution (a). Dissolve 25 mg of disopyramide
phosphate CRS in methanol R and dilute to 10 mL with the
DISOPYRAMIDE PHOSPHATE same solvent.
Reference solution (b). Dilute 1 mL of test solution (b) to
20 mL with methanol R.
Disopyramidi phosphas
Apply to the plate 10 μL of each solution. Develop over a
path of 15 cm using a mixture of 1 volume of concentrated
ammonia R, 30 volumes of acetone R and 30 volumes of
cyclohexane R. Dry the plate in a current of warm air and
examine in ultraviolet light at 254 nm. Any spot in the
chromatogram obtained with test solution (a), apart from
the principal spot, is not more intense than the spot in the
chromatogram obtained with reference solution (b) (0.5 per
cent).
Heavy metals (2.4.8). 2.0 g complies with test C for heavy
C21H32N3O5P Mr 437.5 metals (10 ppm). Prepare the reference solution using 2 mL
[22059-60-5] of lead standard solution (10 ppm Pb) R.

2086 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Disulfiram

Loss on drying (2.2.32). Not more than 0.5 per cent, A. Melting point (2.2.14) : 70 °C to 73 °C.
determined on 1.000 g by drying in an oven at 105 °C. B. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
ASSAY disulfiram CRS. Examine the substances prepared as discs.
Dissolve 0.180 g in 30 mL of anhydrous acetic acid R. Add C. Examine the chromatograms obtained in the test for
0.2 mL of naphtholbenzein solution R. Titrate with 0.1 M related substances. The principal spot in the chromatogram
perchloric acid until the colour changes from yellow to green. obtained with test solution (b) is similar in position and
1 mL of 0.1 M perchloric acid is equivalent to 21.88 mg of size to the principal spot in the chromatogram obtained
C21H32N3O5P. with reference solution (a).
STORAGE D. Dissolve about 10 mg in 10 mL of methanol R. Add 2 mL
of a 0.5 g/L solution of cupric chloride R in methanol R. A
Store protected from light. yellow colour develops which becomes greenish-yellow.
IMPURITIES TESTS
Related substances. Examine by thin-layer chromatography
(2.2.27), using as the coating substance a suitable silica gel with
a fluorescent indicator having an optimal intensity at 254 nm.
Test solution (a). Dissolve 0.20 g of the substance to be
examined in ethyl acetate R and dilute to 10 mL with the same
solvent.
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
with ethyl acetate R.
A. R = CN, R′ = CH(CH3)2 : (2RS)-4-[bis(1- Reference solution (a). Dissolve 10 mg of disulfiram CRS in
methylethyl)amino]-2-phenyl-2-(pyridin-2- ethyl acetate R and dilute to 5 mL with the same solvent.
yl)butanenitrile (di-isopyronitrile), Reference solution (b). Dilute 1 mL of test solution (b) to
20 mL with ethyl acetate R.
B. R = H, R′ = CH(CH3)2 : (3RS)-N,N-bis(1-methylethyl)-3- Apply to the plate 10 μL of each solution. Develop over a path
phenyl-3-(pyridin-2-yl)propan-1-amine, of 15 cm using a mixture of 30 volumes of butyl acetate R
C. R = CO-NH2, R′ = H : (2RS)-4-[(1-methylethyl)amino]-2- and 70 volumes of hexane R. Allow the plate to dry in air
phenyl-2-(pyridin-2-yl)butanamide, and examine in ultraviolet light at 254 nm. Any spot in the
chromatogram obtained with test solution (a), apart from
the principal spot, is not more intense than the spot in the
chromatogram obtained with reference solution (b) (0.5 per
cent).
Diethyldithiocarbamate. Dissolve 0.20 g in 10 mL of
peroxide-free ether R, add 5 mL of buffer solution pH 8.0 R and
shake vigorously. Discard the upper layer and wash the lower
layer with 10 mL of peroxide-free ether R. Add to the lower
D. (RS)-phenyl(pyridin-2-yl)acetonitrile (pyronitrile). layer 0.2 mL of a 4 g/L solution of copper sulfate R and 5 mL of
cyclohexane R. Shake. Any yellow colour in the upper layer is
not more intense than that of a standard prepared at the same
01/2008:0603 time using 0.2 mL of a freshly prepared 0.15 g/L solution of
sodium diethyldithiocarbamate R (150 ppm).
DISULFIRAM Heavy metals (2.4.8). 1.0 g complies with test C for heavy
metals (20 ppm). Prepare the reference solution using 2 mL
of lead standard solution (10 ppm Pb) R.
Disulfiramum Loss on drying (2.2.32). Not more than 0.5 per cent,
determined on 1.000 g by drying in vacuo at 50 °C.
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
on 1.0 g.
ASSAY
Dissolve 0.450 g in 80 mL of acetone R and add 20 mL of a
C10H20N2S4 Mr 296.5 20 g/L solution of potassium nitrate R. Titrate with 0.1 M
[97-77-8] silver nitrate. Determine the end-point potentiometrically
DEFINITION (2.2.20), using a silver electrode and a silver-silver chloride
double-junction electrode saturated with potassium nitrate.
Disulfiram contains not less than 98.5 per cent and
not more than the equivalent of 101.0 per cent of 1 mL of 0.1 M silver nitrate is equivalent to 59.30 mg of
tetraethyldisulfanedicarbothioamide, calculated with C10H20N2S4.
reference to the dried substance. STORAGE
CHARACTERS Store protected from light.
A white or almost white, crystalline powder, practically IMPURITIES
insoluble in water, freely soluble in methylene chloride,
sparingly soluble in alcohol.
IDENTIFICATION
First identification : A, B.
Second identification : A, C, D. A. diethylthiocarbamic thioanhydride (sulfiram),

General Notices (1) apply to all monographs and other texts 2087
Dithranol EUROPEAN PHARMACOPOEIA 8.0

TESTS
Related substances
A. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.200 g of the substance to be
B. diethyldithiocarbamate. examined in 20 mL of methylene chloride R, add 1.0 mL of
glacial acetic acid R and dilute to 100.0 mL with hexane R.
Reference solution. Dissolve 5.0 mg of anthrone R
01/2008:1007 (impurity A), 5.0 mg of dantron R (impurity B), 5.0 mg
corrected 6.0 of dithranol impurity C CRS and 5.0 mg of dithranol CRS
in methylene chloride R and dilute to 5.0 mL with the
DITHRANOL same solvent. To 1.0 mL of this solution, add 19.0 mL of
methylene chloride R and 1.0 mL of glacial acetic acid R,
Dithranolum and dilute to 50.0 mL with hexane R.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : silica gel for chromatography R (5 μm).
Mobile phase : glacial acetic acid R, methylene chloride R,
hexane R (1:5:82 V/V/V).
C14H10O3 Mr 226.2
Flow rate : 2 mL/min.
[1143-38-0]
Detection : spectrophotometer at 260 nm.
DEFINITION Injection : 20 μL.
1,8-Dihydroxyanthracen-9(10H)-one. Run time : 1.5 times the retention time of impurity C.
Content : 98.5 per cent to 101.0 per cent (dried substance). Elution order : dithranol, impurity B, impurity A,
impurity C.
CHARACTERS
System suitability : reference solution:
Appearance : yellow or brownish-yellow, crystalline powder.
– resolution : minimum 2.0 between the peaks due to
Solubility : practically insoluble in water, soluble in methylene dithranol and impurity B.
chloride, sparingly soluble in acetone, slightly soluble in
ethanol (96 per cent). It dissolves in dilute solutions of alkali Limits:
hydroxides. – impurities A, B, C : for each impurity, not more than the
Carry out all tests protected from bright light and use freshly area of the corresponding peak in the chromatogram
prepared solutions. obtained with the reference solution (1 per cent).
B. Liquid chromatography (2.2.29).
IDENTIFICATION Test solution. Dissolve 25.0 mg of the substance to be
First identification : A, B. examined in 5 mL of tetrahydrofuran R and dilute to
Second identification : A, C, D. 25.0 mL with the mobile phase.
A. Melting point (2.2.14) : 178 °C to 182 °C. Reference solution. Dissolve 5.0 mg of dithranol
B. Infrared absorption spectrophotometry (2.2.24). impurity D CRS and 5.0 mg of dithranol CRS in 5 mL of
tetrahydrofuran R and dilute to 10.0 mL with the mobile
Comparison : dithranol CRS. phase. Dilute 1.0 mL of this solution to 20.0 mL with the
C. Thin-layer chromatography (2.2.27). mobile phase.
Test solution. Dissolve 10 mg of the substance to be Column :
examined in methylene chloride R and dilute to 10 mL with – size : l = 0.20 m, Ø = 4.6 mm ;
the same solvent.
– stationary phase : octadecylsilyl silica gel for
Reference solution (a). Dissolve 10 mg of dithranol CRS in chromatography R (5 μm).
methylene chloride R and dilute to 10 mL with the same
solvent. Mobile phase : glacial acetic acid R, tetrahydrofuran R,
water R (2.5:40:60 V/V/V).
Reference solution (b). Dissolve about 5 mg of dantron R in
5 mL of reference solution (a). Flow rate : 0.9 mL/min.
Plate : TLC silica gel plate R. Detection : spectrophotometer at 254 nm.
Mobile phase : hexane R, methylene chloride R (50:50 V/V). Injection : 20 μL.
Application : 10 μL. Run time : 3 times the retention time of dithranol.
Development : over a path of 12 cm. System suitability : reference solution:
Drying : in air. – resolution : minimum 2.5 between the peaks due to
impurity D and dithranol.
Detection : place the plate in a tank saturated with ammonia
vapour until the spots appear. Examine in daylight. Limit :
System suitability: reference solution (b) : – impurity D : not more than the area of the corresponding
peak in the chromatogram obtained with the reference
– the chromatogram shows 2 clearly separated spots. solution (2.5 per cent).
Results : the principal spot in the chromatogram obtained Total (tests A + B) : maximum 3.0 per cent for the sum of the
with the test solution is similar in position, colour and size contents of all impurities.
to the principal spot in the chromatogram obtained with
reference solution (a). Chlorides (2.4.4) : maximum 100 ppm.
D. To 5 mg add 0.1 g of anhydrous sodium acetate R and 1 mL Shake 1.0 g with 20 mL of water R for 1 min and filter. Dilute
of acetic anhydride R. Boil for 30 s. Add 20 mL of ethanol 10 mL of the filtrate to 15 mL with water R.
(96 per cent) R. Examined in ultraviolet light at 365 nm, the Loss on drying (2.2.32) : maximum 0.5 per cent, determined
solution shows a blue fluorescence. on 1.000 g by drying in an oven at 105 °C.

2088 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dobutamine hydrochloride

Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Test solution. Dissolve 20.0 mg in methanol R and dilute
1.0 g. to 100.0 mL with the same solvent. Dilute 10.0 mL of this
solution to 100.0 mL with methanol R.
ASSAY
Spectral range : 220-300 nm.
Dissolve 0.200 g in 50 mL of anhydrous pyridine R. Titrate
with 0.1 M tetrabutylammonium hydroxide under nitrogen R. Absorption maxima : at 223 nm and 281 nm.
Determine the end-point potentiometrically (2.2.20), using a Absorbance ratio : A281 / A223 = 0.34 to 0.36.
glass indicator electrode and a calomel reference electrode C. Infrared absorption spectrophotometry (2.2.24).
containing, as the electrolyte, a saturated solution of potassium Comparison: dobutamine hydrochloride CRS.
chloride R in methanol R.
D. Thin-layer chromatography (2.2.27).
1 mL of 0.1 M tetrabutylammonium hydroxide is equivalent to
22.62 mg of C14H10O3. Solvent mixture: glacial acetic acid R, methanol R
(50:50 V/V).
STORAGE Test solution. Dissolve 10 mg of the substance to be
Protected from light. examined in the solvent mixture and dilute to 10 mL with
the solvent mixture.
IMPURITIES
Reference solution (a). Dissolve 10.0 mg of dobutamine
Specified impurities : A, B, C, D. hydrochloride CRS in the solvent mixture and dilute to
10 mL with the solvent mixture.
Reference solution (b). Dissolve 5.0 mg of dopamine
hydrochloride CRS in 5 mL of the test solution.
Plate : TLC silica gel G plate R.
Mobile phase : water R, glacial acetic acid R, ether R,
A. R1 = R2 = H, X = H2 : anthracen-9(10H)-one (anthrone), butanol R (5:15:30:45 V/V/V/V).
B. R1 = R2 = OH, X = O : 1,8-dihydroxyanthracene-9,10-dione Application : 10 μL.
(dantron), Development : over 2/3 of the plate.
D. R1 = OH, R2 = H, X = H2 : 1-hydroxyanthracen-9(10H)-one, Drying : in air.
Detection : spray with a 1 g/L solution of potassium
permanganate R.
System suitability : reference solution (b) :
– the chromatogram shows 2 clearly separated spots.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
reference solution (a).
E. It gives reaction (a) of chlorides (2.3.1) using a mixture of
C. 4,4′,5,5′-tetrahydroxy-9,9′-bianthracenyl-10,10′(9H,9′H)- equal volumes of methanol R and water R.
dione.
TESTS
07/2010:1200 Acidity or alkalinity. Dissolve 0.1 g in water R with gentle
heating and dilute to 10 mL with the same solvent. Add
DOBUTAMINE HYDROCHLORIDE 0.1 mL of methyl red solution R and 0.2 mL of 0.01 M sodium
hydroxide. The solution is yellow. Add 0.4 mL of 0.01 M
Dobutamini hydrochloridum hydrochloric acid. The solution is red.
Optical rotation (2.2.7) : − 0.05° to + 0.05°.
Dissolve 0.50 g in methanol R and dilute to 10.0 mL with the
same solvent.
Absorbance (2.2.25) : maximum 0.04 at 480 nm.
Dissolve 0.5 g in a mixture of equal volumes of methanol R and
C18H24ClNO3 Mr 337.9 of water R with heating, if necessary, at 30-35 °C and dilute
[49745-95-1] to 25 mL with the same mixture of solvents. Cool quickly.
Examine immediately.
DEFINITION
Related substances. Liquid chromatography (2.2.29).
(RS)-4-[2-[[3-(4-Hydroxyphenyl)-1-
methylpropyl]amino]ethyl]benzene-1,2-diol hydrochloride. Solvent mixture : mobile phase B, mobile phase A (35:65 V/V).
Content : 98.5 per cent to 101.0 per cent (dried substance). Test solution. Dissolve 0.10 g of the substance to be examined
in the solvent mixture and dilute to 20.0 mL with the solvent
CHARACTERS mixture.
Appearance : white or almost white, crystalline powder. Reference solution (a). Dilute 4.0 mL of the test solution to
Solubility : sparingly soluble in water, soluble in methanol, 100.0 mL with a 0.05 g/L solution of anisaldehyde R in the
sparingly soluble in ethanol (96 per cent). solvent mixture. Dilute 1.0 mL of this solution to 10.0 mL
with the solvent mixture.
IDENTIFICATION
Reference solution (b). Dilute 5.0 mL of the test solution to
First identification : C, E. 100.0 mL with the solvent mixture. Dilute 1.0 mL of this
Second identification : A, B, D, E. solution to 10.0 mL with the solvent mixture.
A. Melting point (2.2.14) : 189 °C to 192 °C. Reference solution (c). Dissolve the contents of a vial of
B. Ultraviolet and visible absorption spectrophotometry dobutamine impurity mixture CRS (impurities A, B and C) in
(2.2.25). 1.0 mL of the solvent mixture.

General Notices (1) apply to all monographs and other texts 2089
Docetaxel, anhydrous EUROPEAN PHARMACOPOEIA 8.0

Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm). A. 4-(2-aminoethyl)benzene-1,2-diol (dopamine),
Mobile phase :
– mobile phase A : dissolve 2.60 g of sodium octanesulfonate R
in 1000 mL of water R, add 3 mL of triethylamine R and
adjust to pH 2.5 with phosphoric acid R ;
– mobile phase B : acetonitrile R, methanol R (18:82 V/V) ;
B. 4-(4-hydroxyphenyl)butan-2-one,
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-5 65 35

5 - 20 65 → 20 35 → 80

20 - 25 20 80
C. (2RS)-N-[2-(3,4-dimethoxyphenyl)ethyl]-4-(4-
Flow rate : 1 mL/min. methoxyphenyl)butan-2-amine.
Detection : spectrophotometer at 280 nm.
Injection : 20 μL. 07/2012:2593
Identification of impurities : use the chromatogram supplied
with dobutamine impurity mixture CRS and the chromatogram DOCETAXEL, ANHYDROUS
obtained with reference solution (c) to identify the peaks due
to impurities A, B and C.
Relative retention with reference to dobutamine
Docetaxelum anhydricum
(retention time = about 12 min) : impurity A = about 0.3 ;
impurity B = about 0.5 ; impurity C = about 1.4.
System suitability: reference solution (a) :
– resolution : minimum 4.0 between the peaks due to
dobutamine and anisaldehyde.
Limits :
– correction factor : for the calculation of content, multiply
the peak area of impurity B by 1.4 ;
– impurities A, B, C : for each impurity, not more than the
area of the principal peak in the chromatogram obtained C43H53NO14 Mr 808
with reference solution (b) (0.5 per cent) ; [114977-28-5]
– unspecified impurities : for each impurity, not more than
0.2 times the area of the principal peak in the chromatogram DEFINITION
obtained with reference solution (b) (0.10 per cent) ; 5β,20-Epoxy-1,7β,10β-trihydroxy-9-oxotax-11-ene-
– total : not more than twice the area of the principal peak in 2α,4,13α-triyl 4-acetate 2-benzoate 13-[(2R,3S)-3-
the chromatogram obtained with the reference solution (b) [[(1,1-dimethylethoxy)carbonyl]amino]-2-hydroxy-3-
(1 per cent) ; phenylpropanoate].
– disregard limit : 0.1 times the area of the principal peak in Content : 97.5 per cent to 102.0 per cent (anhydrous substance).
the chromatogram obtained with reference solution (b) CHARACTERS
(0.05 per cent).
Appearance : white or almost white, crystalline, hygroscopic
Heavy metals (2.4.8) : maximum 10 ppm. powder.
2.0 g complies with test C. Prepare the reference solution using Solubility : practically insoluble in water, freely soluble in
2 mL of lead standard solution (10 ppm Pb) R. anhydrous ethanol, soluble in methylene chloride.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C. IDENTIFICATION
A. Specific optical rotation (see Tests).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. B. Infrared absorption spectrophotometry (2.2.24).
Comparison: anhydrous docetaxel CRS.
ASSAY
In order to avoid overheating in the reaction medium, mix TESTS
thoroughly throughout and stop the titration immediately after Appearance of solution. The solution is not more opalescent
the end-point has been reached. than reference suspension II (2.2.1) and not more intensely
Dissolve 0.250 g in 10 mL of anhydrous formic acid R. Add coloured than reference solution B5 (2.2.2, Method I).
50 mL of acetic anhydride R. Titrate with 0.1 M perchloric Dissolve 1.0 g in anhydrous ethanol R and dilute to 20 mL
acid, determining the end-point potentiometrically (2.2.20). with the same solvent.
1 mL of 0.1 M perchloric acid is equivalent to 33.79 mg Specific optical rotation (2.2.7) : − 41.5 to − 38.5 (anhydrous
of C18H24ClNO3. substance).
STORAGE Dissolve 0.250 g in methanol R and dilute to 25.0 mL with
the same solvent.
Protected from light.
Related substances. Liquid chromatography (2.2.29).
IMPURITIES Solvent mixture: acetic acid R, acetonitrile R1, water R
Specified impurities : A, B, C. (0.05:50:50 V/V/V).

2090 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Docetaxel, anhydrous

Test solution. Dissolve 50.0 mg of the substance to be – disregard limit : 0.5 times the area of the principal peak in
examined in 2.5 mL of anhydrous ethanol R and dilute to the chromatogram obtained with reference solution (b)
50.0 mL with the solvent mixture. (0.05 per cent).
Reference solution (a). Dissolve 50.0 mg of docetaxel Heavy metals (2.4.8) : maximum 20 ppm.
trihydrate CRS in 2.5 mL of anhydrous ethanol R and dilute to Solvent mixture : water R, dimethylformamide R (15:85 V/V).
50.0 mL with the solvent mixture.
Dissolve, using sonication, 1.0 g in the solvent mixture and
Reference solution (b). Dilute 1.0 mL of the test solution to dilute to 20 mL with the solvent mixture. 12 mL of the solution
100.0 mL with the solvent mixture. Dilute 1.0 mL of this complies with test B. Prepare the reference solution using
solution to 10.0 mL with the solvent mixture. lead standard solution (1 ppm Pb), obtained by diluting lead
Reference solution (c). Dissolve 5 mg of docetaxel for system standard solution (100 ppm Pb) R with the solvent mixture.
suitability CRS (containing impurities A, B and C) in 0.25 mL Water (2.5.32) : maximum 1.5 per cent.
of anhydrous ethanol R and dilute to 5.0 mL with the solvent
mixture. Inject 800 μL of a 25 mg/mL solution of the substance to be
examined in methanol R.
Reference solution (d). Dissolve 5 mg of docetaxel
impurity E CRS in 2.5 mL of anhydrous ethanol R and dilute Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
to 50.0 mL with the solvent mixture. Dilute 1.0 mL of the 1.0 g.
solution to 100.0 mL with the solvent mixture. Bacterial endotoxins (2.6.14) : less than 0.3 IU/mg, if intended
Column : for use in the manufacture of parenteral preparations without
a further appropriate procedure for the removal of bacterial
– size : l = 0.15 m, Ø = 4.6 mm ; endotoxins.
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (3.5 μm) ; ASSAY
– temperature : 45 °C. Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Mobile phase :
Injection : 10 μL of the test solution and reference solution (a).
– mobile phase A : water R ;
Calculate the percentage content of C43H53NO14 taking into
– mobile phase B : acetonitrile R1 ; account the assigned content of docetaxel trihydrate CRS.
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
STORAGE
0-9 72 28 Protected from light, in an airtight container.
9 - 39 72 → 28 28 → 72 IMPURITIES
Specified impurities : A, B, C, E.
Flow rate : 1.2 mL/min. Other detectable impurities (the following substances would,
Detection : spectrophotometer at 232 nm. if present at a sufficient level, be detected by one or other of
Injection : 10 μL of the test solution and reference solutions (b), the tests in the monograph. They are limited by the general
(c) and (d). acceptance criterion for other/unspecified impurities and/or
Identification of impurities : use the chromatogram supplied by the general monograph Substances for pharmaceutical use
with docetaxel for system suitability CRS and the chromatogram (2034). It is therefore not necessary to identify these impurities
obtained with reference solution (c) to identify the peaks due for demonstration of compliance. See also 5.10. Control of
to impurities A, B and C ; use the chromatogram obtained with impurities in substances for pharmaceutical use) : D, F, G.
reference solution (d) to identify the peak due to impurity E.
Relative retention with reference to docetaxel (retention
time = about 27 min) : impurity E = about 0.2 ;
impurity A = about 0.97 ; impurity B = about 1.08 ;
impurity C = about 1.13.
System suitability: reference solution (c) :
– resolution : minimum 3.0 between the peaks due to
impurity A and docetaxel.
Limits :
– correction factor : for the calculation of content, multiply A. 5β,20-epoxy-1,7β,10β-trihydroxy-9-oxotax-11-
the peak area of impurity A by 1.6 ; ene-2α,4,13α-triyl 4-acetate 13-[(2R,3S)-3-[[(1,1-
– impurity B : not more than 3 times the area of the principal dimethylethoxy)carbonyl]amino]-2-hydroxy-3-
peak in the chromatogram obtained with reference phenylpropanoate] 2-[(2E)-2-methylbut-2-enoate]
solution (b) (0.3 per cent) ; (2-O-desbenzoyl-2-O-tiglyldocetaxel),
– impurity A : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (b) (0.2 per cent) ;
– impurity C : not more than 1.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.15 per cent) ;
– impurity E : not more than 1.5 times the area of the
corresponding peak in the chromatogram obtained with
reference solution (d) (0.15 per cent) ;
– unspecified impurities : for each impurity, not more than the B. 5β,20-epoxy-1,7β-dihydroxy-9,10-dioxotax-
area of the principal peak in the chromatogram obtained 11-ene-2α,4,13α-triyl 4-acetate 2-benzoate
with reference solution (b) (0.10 per cent) ; 13-[(2R,3S)-3-[[(1,1-dimethylethoxy)carbonyl]amino]-2-
– total : maximum 0.8 per cent ; hydroxy-3-phenylpropanoate] (10-deoxy-10-oxodocetaxel),

General Notices (1) apply to all monographs and other texts 2091
Docetaxel trihydrate EUROPEAN PHARMACOPOEIA 8.0

07/2012:2449

DOCETAXEL TRIHYDRATE
Docetaxelum trihydricum

C. 5β,20-epoxy-1,7α,10β-trihydroxy-9-oxotax-
11-ene-2α,4,13α-triyl 4-acetate 2-benzoate
13-[(2R,3S)-3-[[(1,1-dimethylethoxy)carbonyl]amino]-2-
hydroxy-3-phenylpropanoate] (7-epi-docetaxel),

C43H53NO14,3H2O Mr 862
[148408-66-6]
DEFINITION
5β,20-epoxy-1,7β,10β-Trihydroxy-9-oxotax-11-ene-
2α,4,13α-triyl 4-acetate 2-benzoate 13-[(2R,3S)-3-
[[(1,1-dimethylethoxy)carbonyl]amino]-2-hydroxy-3-
phenylpropanoate] trihydrate.
D. 5β,20-epoxy-1,7α-dihydroxy-9,10-dioxotax-11-ene- Content : 97.5 per cent to 102.0 per cent (anhydrous substance).
2α,4,13α-triyl 4-acetate 2-benzoate 13-[(2R,3S)-3- CHARACTERS
[[(1,1-dimethylethoxy)carbonyl]amino]-2-hydroxy-3-
phenylpropanoate] (10-deoxy-10-oxo-7-epi-docetaxel), Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, freely soluble in
anhydrous ethanol, soluble in methylene chloride.
IDENTIFICATION
A. Specific optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: docetaxel trihydrate CRS.
TESTS
Appearance of solution. The solution is not more opalescent
than reference suspension II (2.2.1) and not more intensely
E. 5β,20-epoxy-4-(acetyloxy)-1,7β,10β,13α-tetrahydroxy-9- coloured than reference solution B5 (2.2.2, Method I).
oxotax-11-en-2α-yl benzoate (10-desacetyl-baccatin III),
Dissolve 1.0 g in anhydrous ethanol R and dilute to 20 mL
with the same solvent.
Specific optical rotation (2.2.7) : − 41.5 to − 38.5 (anhydrous
substance).
Dissolve 0.250 g in methanol R and dilute to 25.0 mL with
the same solvent.
Related substances. Liquid chromatography (2.2.29).
Solvent mixture: acetic acid R, acetonitrile R1, water R
(0.05:50:50 V/V/V).
Test solution. Dissolve 50.0 mg of the substance to be
examined in 2.5 mL of anhydrous ethanol R and dilute to
F. 5β,20-epoxy-1,7β-dihydroxy-9-oxotax-11-ene- 50.0 mL with the solvent mixture.
2α,4,10β,13α-tetrayl 4,10-diacetate 2-benzoate 13-[(2R,3S)- Reference solution (a). Dissolve 50.0 mg of docetaxel
3-(benzoylamino)-2-hydroxy-3-phenylpropanoate] trihydrate CRS in 2.5 mL of anhydrous ethanol R and dilute to
(paclitaxel), 50.0 mL with the solvent mixture.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Reference solution (c). Dissolve 5 mg of docetaxel for system
suitability CRS (containing impurities A, B and C) in 0.25 mL
of anhydrous ethanol R and dilute to 5.0 mL with the solvent
mixture.
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (3.5 μm) ;
G. 5β,20-epoxy-1,7β-dihydroxy-9-oxotax-11-ene-
2α,4,10β,13α-tetrayl 4,10-diacetate 2-benzoate – temperature : 45 °C.
13-[(2R,3S)-3-[[(1,1-dimethylethoxy)carbonyl]amino]-2- Mobile phase :
hydroxy-3-phenylpropanoate] (10-acetyldocetaxel). – mobile phase A : water R ;

2092 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Docetaxel trihydrate

– mobile phase B : acetonitrile R1 ; IMPURITIES

Time Mobile phase A Mobile phase B Specified impurities : A, B, C.
(min) (per cent V/V) (per cent V/V) Other detectable impurities (the following substances would,
0-9 72 28 if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
9 - 39 72 → 28 28 → 72
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
Flow rate : 1.2 mL/min.
use (2034). It is therefore not necessary to identify these
Detection : spectrophotometer at 232 nm. impurities for demonstration of compliance. See also 5.10.
Injection : 10 μL of the test solution and reference solutions (b) Control of impurities in substances for pharmaceutical use) : D.
and (c).
Identification of impurities : use the chromatogram
supplied with docetaxel for system suitability CRS and the
chromatogram obtained with reference solution (c) to identify
the peaks due to impurities A, B and C.
Relative retention with reference to docetaxel (retention
time = about 27 min) : impurity A = about 0.97 ;
impurity B = about 1.08 ; impurity C = about 1.13.
System suitability: reference solution (c) :
– resolution : minimum 3.0 between the peaks due to
impurity A and docetaxel. A. 5β,20-epoxy-1,7β,10β-trihydroxy-9-oxotax-11-
Limits : ene-2α,4,13α-triyl 4-acetate 13-[(2R,3S)-3-[[(1,1-
dimethylethoxy)carbonyl]amino]-2-hydroxy-3-
– correction factor : for the calculation of content, multiply phenylpropanoate] 2-[(2E)-2-methylbut-2-enoate]
the peak area of impurity A by 1.6 ; (2-O-desbenzoyl-2-O-tiglyldocetaxel),
– impurity A : not more than 5 times the area of the principal
peak in the chromatogram obtained with reference
solution (b) (0.5 per cent) ;
– impurities B, C : for each impurity, not more than 3 times
the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.3 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.10 per cent) ;
– total : not more than 10 times the area of the principal peak
in the chromatogram obtained with reference solution (b) B. 5β,20-epoxy-1,7β-dihydroxy-9,10-dioxotax-
(1.0 per cent) ; 11-ene-2α,4,13α-triyl 4-acetate 2-benzoate
– disregard limit : 0.5 times the area of the principal peak in 13-[(2R,3S)-3-[[(1,1-dimethylethoxy)carbonyl]amino]-2-
the chromatogram obtained with reference solution (b) hydroxy-3-phenylpropanoate] (10-deoxy-10-oxodocetaxel),
(0.05 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
Solvent mixture : water R, dimethylformamide R (15:85 V/V).
Dissolve, using sonication, 1.0 g in the solvent mixture and
dilute to 20 mL with the solvent mixture. 12 mL of the solution
complies with test B. Prepare the reference solution using
lead standard solution (1 ppm Pb) obtained by diluting lead
standard solution (100 ppm Pb) R with the solvent mixture.
Water (2.5.32) : 5.0 per cent to 7.0 per cent.
Inject 200 μL of a 100 mg/mL solution of the substance to be
examined in dimethylformamide R. C. 5β,20-epoxy-1,7α,10β-trihydroxy-9-oxotax-
11-ene-2α,4,13α-triyl 4-acetate 2-benzoate
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on 13-[(2R,3S)-3-[[(1,1-dimethylethoxy)carbonyl]amino]-2-
1.0 g. hydroxy-3-phenylpropanoate] (7-epi-docetaxel),
Bacterial endotoxins (2.6.14) : less than 0.3 IU/mg, if intended
for use in the manufacture of parenteral preparations without
a further appropriate procedure for the removal of bacterial
endotoxins.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : 10 μL of the test solution and reference solution (a).
Calculate the percentage content of C43H53NO14 taking into
account the assigned content of docetaxel trihydrate CRS. D. 5β,20-epoxy-1,7α-dihydroxy-9,10-dioxotax-11-ene-
2α,4,13α-triyl 4-acetate 2-benzoate 13-[(2R,3S)-3-
STORAGE [[(1,1-dimethylethoxy)carbonyl]amino]-2-hydroxy-3-
Protected from light. phenylpropanoate] (10-deoxy-10-oxo-7-epi-docetaxel).

General Notices (1) apply to all monographs and other texts 2093
Docusate sodium EUROPEAN PHARMACOPOEIA 8.0

07/2013:1418 Carrier gas : nitrogen for chromatography R.

Flow rate : 30 mL/min.
DOCUSATE SODIUM Temperature :
– column : 230 °C ;
Natrii docusas – injection port and detector : 280 °C.
Detection : flame ionisation.
Injection : 1 μL.
Run time : 2.5 times the retention time of the internal standard.
System suitability : there is no peak with the same retention
time as the internal standard in the chromatogram obtained
C20H37NaO7S Mr 444.6 with test solution (b).
[577-11-7] Limits : test solution (a) :
DEFINITION – any impurity : for each impurity, not more than the area of
Sodium 1,4-bis[(2-ethylhexyl)oxy]-1,4-dioxobutane-2- the peak due to the internal standard (0.4 per cent).
sulfonate. Chlorides : maximum 350 ppm.
Content : 98.0 to 101.0 per cent (anhydrous substance). Dissolve 5.0 g in 50 mL of ethanol (50 per cent V/V) R.
Titrate with 0.01 M silver nitrate, determining the end-point
CHARACTERS potentiometrically (2.2.20).
Appearance : white or almost white, waxy masses or flakes, 1 mL of 0.01 M silver nitrate is equivalent to 0.3545 mg of Cl.
hygroscopic.
Sodium sulfate : maximum 2 per cent.
Solubility : sparingly soluble in water, freely soluble in ethanol
(96 per cent) and in methylene chloride. Dissolve 0.25 g in 40 mL of a mixture of 20 volumes of water R
and 80 volumes of 2-propanol R. Adjust the pH to between
IDENTIFICATION 2.5 and 4.0 using perchloric acid solution R. Add 0.4 mL of
A. Infrared absorption spectrophotometry (2.2.24). naphtharson solution R and 0.1 mL of a 0.125 g/L solution of
methylene blue R. Not more than 1.5 mL of 0.025 M barium
Preparation : place about 3 mg of the substance to be perchlorate is required to change the colour of the indicator
examined on a sodium chloride plate, add 0.05 mL of from yellowish-green to yellowish-pink.
acetone R and immediately cover with another sodium
chloride plate. Rub the plates together to dissolve the Heavy metals (2.4.8) : maximum 10 ppm.
substance to be examined, slide the plates apart and allow Dissolve 4.0 g in ethanol (80 per cent V/V) R and dilute to
the acetone to evaporate. 20 mL with the same solvent. 12 mL of the solution complies
Comparison : docusate sodium CRS. with test B. Prepare the reference solution using lead standard
B. In a crucible, ignite 0.75 g in the presence of dilute sulfuric solution (2 ppm Pb) obtained by diluting lead standard
acid R, until an almost white residue is obtained. Allow to solution (100 ppm Pb) R with ethanol (80 per cent V/V) R.
cool and take up the residue with 5 mL of water R. Filter. Water (2.5.12) : maximum 3.0 per cent, determined on 0.250 g.
2 mL of the filtrate gives reaction (a) of sodium (2.3.1).
ASSAY
TESTS To 1.000 g in a 250 mL conical flask fitted with a reflux
Alkalinity. Dissolve 1.0 g in 100 mL of a mixture of equal condenser add 25.0 mL of 0.5 M alcoholic potassium hydroxide
volumes of methanol R and water R, previously neutralised to and heat on a water-bath under reflux for 45 min. Allow to
methyl red solution R. Add 0.1 mL of methyl red solution R. cool. Add 0.25 mL of phenolphthalein solution R1 and titrate
Not more than 0.2 mL of 0.1 M hydrochloric acid is required with 0.5 M hydrochloric acid until the red colour disappears.
to change the colour of the indicator to red. Carry out a blank titration.
Related non-ionic substances. Gas chromatography (2.2.28). 1 mL of 0.5 M alcoholic potassium hydroxide is equivalent to
0.1112 g of C20H37NaO7S.
Internal standard solution. Dissolve 10 mg of methyl
behenate R in hexane R and dilute to 50 mL with the same STORAGE
solvent. In an airtight container.
Test solution (a). Dissolve 0.10 g of the substance to be
examined in 2.0 mL of the internal standard solution and 01/2008:2078
dilute to 5.0 mL with hexane R. Pass the solution, at a rate
of about 1.5 mL/min, through a column 10 mm in internal DODECYL GALLATE
diameter, packed with 5 g of basic aluminium oxide R and
previously washed with 25 mL of hexane R. Elute with 5 mL of Dodecylis gallas
hexane R and discard the eluate. Elute with 20 mL of a mixture
of equal volumes of ether R and hexane R. Evaporate the eluate
to dryness and dissolve the residue in 2.0 mL of hexane R.
Test solution (b). Prepare as described for test solution (a) but
dissolving 0.10 g of the substance to be examined in hexane R,
diluting to 5.0 mL with the same solvent, and using a new
column.
Reference solution. Dilute 2.0 mL of the internal standard C19H30O5 Mr 338.4
solution to 5.0 mL with hexane R. [1166-52-5]
Column : DEFINITION
– material : glass ; Dodecyl 3,4,5-trihydroxybenzoate.
– size : l = 2 m, Ø = 2 mm ; Content : 97.0 per cent to 103.0 per cent (dried substance).
– stationary phase : silanised diatomaceous earth for gas
chromatography R impregnated with 3 per cent m/m of CHARACTERS
polymethylphenylsiloxane R. Appearance : white or almost white, crystalline powder.

2094 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Domperidone

Solubility : very slightly soluble or practically insoluble in STORAGE

water, freely soluble in ethanol (96 per cent), slightly soluble In a non-metallic container, protected from light.
in methylene chloride.
IMPURITIES
IDENTIFICATION
Specified impurities : A.
A. Determine the melting point (2.2.14) of the substance to be
examined. Mix equal parts of the substance to be examined
and dodecyl gallate CRS and determine the melting point
of the mixture. The difference between the melting points
(which are about 96 °C) is not greater than 2 °C.
B. Examine the chromatograms obtained in the test for A. 3,4,5-trihydroxybenzoic acid (gallic acid).
impurity A.
Results : the principal spot in the chromatogram obtained 07/2011:1009
with test solution (b) is similar in position, colour and size
to the principal spot in the chromatogram obtained with
reference solution (a).
DOMPERIDONE
TESTS Domperidonum
Impurity A. Thin-layer chromatography (2.2.27).
Test solution (a). Dissolve 0.20 g of the substance to be
examined in acetone R and dilute to 10 mL with the same
solvent.
Test solution (b). Dilute 1.0 mL of test solution (a) to 20 mL
with acetone R.
Reference solution (a). Dissolve 10 mg of dodecyl gallate CRS C22H24ClN5O2 Mr 425.9
in acetone R and dilute to 10 mL with the same solvent. [57808-66-9]
Reference solution (b). Dissolve 20 mg of gallic acid R in
DEFINITION
acetone R and dilute to 20 mL with the same solvent.
5-Chloro-1-[1-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1-
Reference solution (c). Dilute 1.0 mL of reference solution (b) yl)propyl]piperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-
to 10 mL with acetone R. one.
Reference solution (d). Dilute 1.0 mL of reference solution (b) Content : 99.0 per cent to 101.0 per cent (dried substance).
to 5 mL with test solution (a).
Plate : TLC silica gel plate R. CHARACTERS
Mobile phase : anhydrous formic acid R, ethyl formate R, Appearance : white or almost white powder.
toluene R (10:40:50 V/V/V). Solubility : practically insoluble in water, soluble in
Application : 5 μL of test solutions (a) and (b) and reference dimethylformamide, slightly soluble in ethanol (96 per cent)
solutions (a), (c) and (d). and in methanol.
Development : over 2/3 of the plate. IDENTIFICATION
Drying : in air for 10 min. First identification : A, B.
Detection : spray with a mixture of 1 volume of ferric chlorideSecond identification : A, C, D.
solution R1 and 9 volumes of ethanol (96 per cent) R. A. Melting point (2.2.14) : 244 °C to 248 °C.
System suitability : reference solution (d) : B. Infrared absorption spectrophotometry (2.2.24).
– the chromatogram shows 2 clearly separated principal Comparison: domperidone CRS.
spots. C. Thin-layer chromatography (2.2.27).
Limit : test solution (a) : Test solution. Dissolve 20 mg of the substance to be
– impurity A : any spot due to impurity A is not more intense examined in methanol R and dilute to 10 mL with the same
than the spot in the chromatogram obtained with reference solvent.
solution (c) (0.5 per cent). Reference solution (a). Dissolve 20 mg of domperidone CRS
Chlorides (2.4.4) : maximum 100 ppm. in methanol R and dilute to 10 mL with the same solvent.
To 1.65 g add 50 mL of water R. Shake for 5 min. Filter. 15 mL Reference solution (b). Dissolve 20 mg of domperidone CRS
of the filtrate complies with the test. and 20 mg of droperidol CRS in methanol R and dilute to
10 mL with the same solvent.
Heavy metals (2.4.8) : maximum 10 ppm.
Plate : TLC octadecylsilyl silica gel plate R.
2.0 g complies with limit test C. Prepare the reference solution
using 2 mL of lead standard solution (10 ppm Pb) R. Mobile phase : ammonium acetate solution R, dioxan R,
methanol R (20:40:40 V/V/V).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined Application : 5 μL.
on 1.000 g by drying in an oven at 70 °C.
Development : over 3/4 of the plate.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Drying : in a current of warm air for 15 min.
1.0 g.
Detection : expose to iodine vapour until the spots appear ;
ASSAY examine in daylight.
Dissolve 0.100 g in methanol R and dilute to 250.0 mL with the System suitability : reference solution (b) :
same solvent. Dilute 5.0 mL of the solution to 200.0 mL with – the chromatogram shows 2 clearly separated spots.
methanol R. Measure the absorbance (2.2.25) at the absorption Results : the principal spot in the chromatogram obtained
maximum at 275 nm. with the test solution is similar in position and size to
Calculate the content of C19H30O5 taking the specific the principal spot in the chromatogram obtained with
absorbance to be 321. reference solution (a).

General Notices (1) apply to all monographs and other texts 2095
Domperidone EUROPEAN PHARMACOPOEIA 8.0

D. It gives the reaction of non-nitrogen substituted ASSAY

barbiturates (2.3.1). Dissolve 0.300 g in 50 mL of a mixture of 1 volume of
anhydrous acetic acid R and 7 volumes of methyl ethyl ketone R.
TESTS Titrate with 0.1 M perchloric acid until the colour changes
Appearance of solution. The solution is clear (2.2.1) and not from orange-yellow to green using 0.2 mL of naphtholbenzein
more intensely coloured than reference solution Y6 (2.2.2, solution R as indicator.
Method II). 1 mL of 0.1 M perchloric acid is equivalent to 42.59 mg
Dissolve 0.20 g in dimethylformamide R and dilute to 20.0 mL of C22H24ClN5O2.
with the same solvent.
Related substances. Liquid chromatography (2.2.29). Prepare STORAGE
the solutions immediately before use. Protected from light.
Test solution. Dissolve 0.10 g of the substance to be examined
in dimethylformamide R and dilute to 10.0 mL with the same IMPURITIES
solvent. Specified impurities : A, B, C, D, E, F.
Reference solution (a). Dissolve 10.0 mg of domperidone CRS
and 15.0 mg of droperidol CRS in dimethylformamide R and
dilute to 100.0 mL with the same solvent.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with dimethylformamide R. Dilute 5.0 mL of this
solution to 20.0 mL with dimethylformamide R.
Column : A. 5-chloro-1-(piperidin-4-yl)-1,3-dihydro-2H-
benzimidazol-2-one,
– size : l = 0.1 m, Ø = 4.6 mm ;
– stationary phase : base-deactivated octadecylsilyl silica gel for
chromatography R (3 μm).
Mobile phase :
– mobile phase A : 5 g/L solution of ammonium acetate R ;
– mobile phase B : methanol R ;
B. 4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)-1-
Time Mobile phase A Mobile phase B formylpiperidine,
(min) (per cent V/V) (per cent V/V)
0 - 10 70 → 0 30 → 100

10 - 12 0 100

Flow rate : 1.5 mL/min.

Detection : spectrophotometer at 280 nm.
Injection : 10 μL.
Relative retention with reference to domperidone C. cis-4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazol-
(retention time = about 6.5 min) : impurity A = about 0.4 ; 1-yl)-1-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1-
impurity B = about 0.65 ; impurity C = about 0.7 ; yl)propyl]piperidine 1-oxide,
droperidol = about 1.1 ; impurity D = about 1.15 ;
impurity E = about 1.2 ; impurity F = about 1.3.
System suitability: reference solution (a) :
– resolution : minimum 2.0 between the peaks due to
domperidone and droperidol.
Limits :
– impurities A, B, C, D, E, F : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.25 per cent) ;
– unspecified impurities : for each impurity, not more than
0.4 times the area of the principal peak in the chromatogram D. 5-chloro-3-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1-
obtained with reference solution (b) (0.10 per cent) ; yl)propyl]-1-[1-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-
– total : not more than twice the area of the principal peak 1-yl)propyl]piperidin-4-yl]-1,3-dihydro-2H-benzimidazol-
in the chromatogram obtained with reference solution (b) 2-one,
(0.5 per cent) ;
– disregard limit : 0.2 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test D. Prepare the reference solution
using 2 mL of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined E. 1-[3-[4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazol-
on 1.000 g by drying in an oven at 105 °C. 1-yl)piperidin-1-yl]propyl]-3-[3-(2-oxo-2,3-dihydro-1H-
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on benzimidazol-1-yl)propyl]-1,3-dihydro-2H-benzimidazol-
1.0 g. 2-one,

2096 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Domperidone maleate

Plate : TLC octadecylsilyl silica gel plate R.

Mobile phase : ammonium acetate solution R, dioxan R,
methanol R (20:40:40 V/V/V).
Application : 5 μL.
Development : over a path of 15 cm.
Drying : in a current of warm air for 15 min.
Detection : expose to iodine vapour until the spots appear.
Examine in daylight.
System suitability : reference solution (b) :
– the chromatogram shows 2 clearly separated spots.
Results : the principal spot in the chromatogram obtained
F. 1,3-bis[3-[4-(5-chloro-2-oxo-2,3-dihydro-1H- with the test solution is similar in position and size to
benzimidazol-1-yl)piperidin-1-yl]propyl]-1,3-dihydro-2H- the principal spot in the chromatogram obtained with
benzimidazol-2-one. reference solution (a).
C. Triturate 0.1 g with a mixture of 1 mL of strong sodium
hydroxide solution R and 3 mL of water R. Shake with
01/2008:1008 3 quantities, each of 5 mL, of ether R. To 0.1 mL of the
corrected 6.0 aqueous layer add a solution of 10 mg of resorcinol R in
3 mL of sulfuric acid R. Heat on a water-bath for 15 min.
DOMPERIDONE MALEATE No colour develops. To the remainder of the aqueous layer
add 2 mL of bromine solution R. Heat on a water-bath for
15 min and then heat to boiling. Cool. To 0.1 mL of this
Domperidoni maleas solution add a solution of 10 mg of resorcinol R in 3 mL of
sulfuric acid R. Heat on a water-bath for 15 min. A violet
colour develops.
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution Y6 (2.2.2,
Method II).
C26H28ClN5O6 Mr 542.0 Dissolve 0.20 g in dimethylformamide R and dilute to 20.0 mL
[83898-65-1] with the same solvent.
DEFINITION Related substances. Liquid chromatography (2.2.29). Prepare
5-Chloro-1-[1-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1- the solutions immediately before use.
yl)propyl]piperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2- Test solution. Dissolve 0.10 g of the substance to be examined
one hydrogen (Z)-butenedioate. in dimethylformamide R and dilute to 10.0 mL with the same
Content : 99.0 per cent to 101.0 per cent (dried substance). solvent.
Reference solution (a). Dissolve 10.0 mg of domperidone
CHARACTERS maleate CRS and 15.0 mg of droperidol CRS in
Appearance : white or almost white powder. dimethylformamide R and dilute to 100.0 mL with the same
Solubility : very slightly soluble in water, sparingly soluble in solvent.
dimethylformamide, slightly soluble in methanol, very slightly Reference solution (b). Dilute 1.0 mL of the test solution to
soluble in ethanol (96 per cent). 100.0 mL with dimethylformamide R. Dilute 5.0 mL of this
It shows polymorphism (5.9). solution to 20.0 mL with dimethylformamide R.
IDENTIFICATION Column :
First identification : A. – size : l = 0.1 m, Ø = 4.6 mm ;
Second identification : B, C. – stationary phase : base-deactivated octadecylsilyl silica gel for
chromatography R (3 μm).
A. Infrared absorption spectrophotometry (2.2.24).
Mobile phase :
Preparation : discs.
Comparison : domperidone maleate CRS. – mobile phase A : 5 g/L solution of ammonium acetate R ;
If the spectra obtained show differences, dissolve the – mobile phase B : methanol R ;
substance to be examined and the reference substance Time Mobile phase A Mobile phase B
separately in the minimum volume of 2-propanol R, (min) (per cent V/V) (per cent V/V)
evaporate to dryness on a water-bath and record new 0 - 10 70 → 0 30 → 100
spectra using the residues.
10 - 12 0 100
B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 20 mg of the substance to be Flow rate : 1.5 mL/min.
examined in methanol R and dilute to 10 mL with the same
solvent. Detection : spectrophotometer at 280 nm.
Reference solution (a). Dissolve 20 mg of domperidone Equilibration : with methanol R for at least 30 min and then
maleate CRS in methanol R and dilute to 10 mL with the with the mobile phase at the initial composition for at least
same solvent. 5 min.
Reference solution (b). Dissolve 20 mg of domperidone Injection : 10 μL ; inject dimethylformamide R as a blank.
maleate CRS and 20 mg of droperidol CRS in methanol R Retention time : domperidone = about 6.5 min ;
and dilute to 10 mL with the same solvent. droperidol = about 7 min.

General Notices (1) apply to all monographs and other texts 2097
Dopamine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

System suitability: reference solution (a) :

– resolution : minimum 2.0 between the peaks due to
domperidone and droperidol ; if necessary, adjust the
concentration of methanol in the mobile phase or adjust
the time programme for the linear gradient.
Limits : D. 5-chloro-3-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1-
– impurities A, B, C, D, E, F : for each impurity, not more yl)propyl]-1-[1-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-
than the area of the principal peak in the chromatogram 1-yl)propyl]piperidin-4-yl]-1,3-dihydro-2H-benzimidazol-
obtained with reference solution (b) (0.25 per cent) ; 2-one,
– total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.5 per cent) ;
– disregard limit : 0.2 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent) ; disregard any peak due to the blank E. 1-[3-[4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazol-
and any peak due to maleic acid at the beginning of the 1-yl)piperidin-1-yl]propyl]-3-[3-(2-oxo-2,3-dihydro-1H-
chromatogram. benzimidazol-1-yl)propyl]-1,3-dihydro-2H-benzimidazol-
Heavy metals (2.4.8) : maximum 20 ppm. 2-one,
1.0 g complies with test D. Prepare the reference solution
using 2 mL of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. F. 1,3-bis[3-[4-(5-chloro-2-oxo-2,3-dihydro-1H-
benzimidazol-1-yl)piperidin-1-yl]propyl]-1,3-dihydro-2H-
ASSAY benzimidazol-2-one.
Dissolve 0.400 g in 50 mL of anhydrous acetic acid R. Using
0.2 mL of naphtholbenzein solution R as indicator, titrate 01/2008:0664
with 0.1 M perchloric acid until the colour changes from
orange-yellow to green.
DOPAMINE HYDROCHLORIDE
1 mL of 0.1 M perchloric acid is equivalent to 54.20 mg
of C26H28ClN5O6. Dopamini hydrochloridum
STORAGE
Protected from light.

IMPURITIES
C8H12ClNO2 Mr 189.6
Specified impurities : A, B, C, D, E, F. [62-31-7]
DEFINITION
4-(2-Aminoethyl)benzene-1,2-diol hydrochloride.
Content : 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : freely soluble in water, soluble in ethanol (96 per
cent), sparingly soluble in acetone and in methylene chloride.
IDENTIFICATION
A. 5-chloro-1-(piperidin-4-yl)-1,3-dihydro-2H- First identification : B, E.
benzimidazol-2-one, Second identification : A, C, D, E.
A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Dissolve 40.0 mg in 0.1 M hydrochloric acid
and dilute to 100.0 mL with the same acid. Dilute 10.0 mL
of this solution to 100.0 mL with 0.1 M hydrochloric acid.
B. 4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)-1-
Spectral range : 230-350 nm.
formylpiperidine,
Absorption maximum : at 280 nm.
Specific absorbance at the absorption maximum : 136 to 150.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: dopamine hydrochloride CRS.
C. Dissolve about 5 mg in a mixture of 5 mL of 1 M
hydrochloric acid and 5 mL of water R. Add 0.1 mL of
C. cis-4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazol- sodium nitrite solution R containing 100 g/L of ammonium
1-yl)-1-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1- molybdate R. A yellow colour develops which becomes red
yl)propyl]piperidine 1-oxide, on the addition of strong sodium hydroxide solution R.

2098 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dopexamine dihydrochloride

D. Dissolve about 2 mg in 2 mL of water R and add 0.2 mL Heavy metals (2.4.8) : maximum 20 ppm.
of ferric chloride solution R2. A green colour develops 1.0 g complies with test C. Prepare the reference solution using
which changes to bluish-violet on the addition of 0.1 g of 2 mL of lead standard solution (10 ppm Pb) R.
hexamethylenetetramine R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
E. It gives reaction (a) of chlorides (2.3.1). on 1.000 g by drying in an oven at 105 °C for 2 h.
TESTS Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Appearance of solution. The solution is clear (2.2.1) and 1.0 g.
not more intensely coloured than reference solution B6 or Y6 ASSAY
(2.2.2, Method II). In order to avoid overheating in the reaction medium, mix
Dissolve 0.4 g in water R and dilute to 10 mL with the same thoroughly throughout the titration and stop the titration
solvent. immediately after the end-point has been reached.
Acidity or alkalinity. Dissolve 0.5 g in carbon dioxide-free Dissolve 0.150 g in 10 mL of anhydrous formic acid R. Add
water R and dilute to 10 mL with the same solvent. Add 50 mL of acetic anhydride R. Titrate with 0.1 M perchloric
0.1 mL of methyl red solution R and 0.75 mL of 0.01 M sodium acid, determining the end-point potentiometrically (2.2.20).
hydroxide. The solution is yellow. Add 1.5 mL of 0.01 M 1 mL of 0.1 M perchloric acid is equivalent to 18.96 mg
hydrochloric acid. The solution is red. of C8H12ClNO2.
Related substances. Liquid chromatography (2.2.29). Protect
the solutions from light. STORAGE
In an airtight container, under nitrogen, protected from light.
Buffer solution. Dissolve 21 g of citric acid R in 200 mL of 1 M
sodium hydroxide and dilute to 1000 mL with water R. To IMPURITIES
600 mL of this solution add 400 mL of 0.1 M hydrochloric acid. Other detectable impurities (the following substances would,
Test solution. Dissolve 50 mg of the substance to be examined if present at a sufficient level, be detected by one or other of
in mobile phase A and dilute to 25 mL with mobile phase A. the tests in the monograph. They are limited by the general
Reference solution (a). Dilute 1.0 mL of the test solution to acceptance criterion for other/unspecified impurities and/or
100.0 mL with mobile phase A. Dilute 1.0 mL of this solution by the general monograph Substances for pharmaceutical use
to 10.0 mL with mobile phase A. (2034). It is therefore not necessary to identify these impurities
Reference solution (b). Dissolve 10 mg of 3-O-methyldopamine for demonstration of compliance. See also 5.10. Control of
hydrochloride R (impurity B) and 10 mg of 4-O- impurities in substances for pharmaceutical use): A, B, C.
methyldopamine hydrochloride R (impurity A) in mobile
phase A and dilute to 100 mL with mobile phase A. Dilute
6 mL of this solution to 25 mL with mobile phase A.
Column : A. R = CH3, R′ = H : 5-(2-aminoethyl)-2-methoxyphenol
– size : l = 0.15 m, Ø = 3.9 mm ; (4-O-methyldopamine),
– stationary phase : spherical end-capped octadecylsilyl silica B. R = H, R′ = CH3 : 4-(2-aminoethyl)-2-methoxyphenol
gel for chromatography R (4 μm). (3-O-methyldopamine),
Mobile phase :
C. R = R′ = CH3 : 2-(3,4-dimethoxyphenyl)ethanamine.
– mobile phase A : dissolve 1.08 g of sodium octanesulfonate R
in 880 mL of the buffer solution and add 50 mL of 01/2008:1748
methanol R and 70 mL of acetonitrile R ; corrected 7.0
– mobile phase B : dissolve 1.08 g of sodium octanesulfonate R
in 700 mL of the buffer solution and add 100 mL of DOPEXAMINE DIHYDROCHLORIDE
methanol R and 200 mL of acetonitrile R ;
Time Mobile phase A Mobile phase B Dopexamini dihydrochloridum
(min) (per cent V/V) (per cent V/V)
0-5 90 10

5 - 20 90 → 40 10 → 60

20 - 25 40 60

Flow rate : 1.0 mL/min.

Detection : spectrophotometer at 280 nm.
C22H34Cl2N2O2 Mr 429.4
Injection : 10 μL. [86484-91-5]
Retention time : dopamine = about 5 min.
DEFINITION
System suitability : reference solution (b) :
4-[2-[[6-[(2-Phenylethyl)amino]hexyl]amino]ethyl]benzene-
– resolution : minimum 5.0 between the peaks due to 1,2-diol dihydrochloride.
impurities B and A.
Content : 98.5 per cent to 101.0 per cent (anhydrous substance).
Limits :
– unspecificied impurities : for each impurity, not more CHARACTERS
than the area of the principal peak in the chromatogram Appearance : white or almost white, crystalline powder.
obtained with reference solution (a) (0.10 per cent) ; Solubility : soluble in water, sparingly soluble in ethanol (96 per
– total : not more than twice the area of the principal peak cent) and in methanol, practically insoluble in acetone.
in the chromatogram obtained with reference solution (a)
(0.2 per cent) ; IDENTIFICATION
– disregard limit : 0.5 times the area of the principal peak in A. Infrared absorption spectrophotometry (2.2.24).
the chromatogram obtained with reference solution (a) Comparison: dopexamine dihydrochloride CRS.
(0.05 per cent). B. It gives reaction (a) of chlorides (2.3.1).

General Notices (1) apply to all monographs and other texts 2099
Dopexamine dihydrochloride EUROPEAN PHARMACOPOEIA 8.0

TESTS – impurities A, B, C, D, E, F, I, K : for each impurity, not more

Appearance of solution. The solution is clear (2.2.1) and not than the area of the principal peak in the chromatogram
more intensely coloured than reference solution BY7 (2.2.2, obtained with reference solution (a) (0.1 per cent) ;
Method II). – unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
Dissolve 0.10 g in 0.1 M hydrochloric acid and dilute to 10 mL with reference solution (a) (0.10 per cent) ;
with the same acid.
– total : not more than 5 times the area of the principal peak
pH (2.2.3) : 3.7 to 5.7. in the chromatogram obtained with reference solution (a)
Dissolve 0.20 g in carbon dioxide-free water R and dilute to (0.5 per cent) ;
20 mL with the same solvent. – disregard limit : 0.5 times the area of the principal peak in
Related substances. Liquid chromatography (2.2.29). the chromatogram obtained with reference solution (a)
(0.05 per cent).
Test solution. Dissolve 0.100 g of the substance to be examined
in mobile phase A and dilute to 10.0 mL with mobile phase A. Impurity J. Liquid chromatography (2.2.29) as described in
the test for related substances with the following modification.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with mobile phase A. Dilute 1.0 mL of this solution Detection : spectrophotometer at 210 nm.
to 10.0 mL with mobile phase A. Limit :
Reference solution (b). Dissolve 5 mg of the substance to be – impurity J : not more than the area of the principal peak
examined and 5 mg of dopexamine impurity B CRS in mobile in the chromatogram obtained with reference solution (a)
phase A and dilute to 10.0 mL with mobile phase A. (0.1 per cent).
Reference solution (c). Dissolve 5 mg of dopexamine Heavy metals (2.4.8) : maximum 10 ppm.
impurity F CRS in mobile phase A and dilute to 100 mL with Dissolve 0.50 g in water R and dilute to 20 mL with the same
mobile phase A. solvent. 12 mL of the solution complies with test A. Prepare
Column : the reference solution using lead standard solution (0.25 ppm
Pb) R. For the evaluation of the results, filter the solutions
– size : l = 0.15 m, Ø = 4.6 mm ; through a membrane filter (nominal pore size 0.45 μm).
– stationary phase : octadecylsilyl silica gel for Water (2.5.12) : maximum 0.5 per cent, determined on 1.00 g.
chromatography R (5 μm) ; Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
– temperature : 45 °C. 1.0 g.
Mobile phase : Bacterial endotoxins (2.6.14) : less than 10 IU/mg.

– mobile phase A : mix 5 volumes of buffer solution pH 2.5 R ASSAY

and 95 volumes of water R ;
Carry out the titration immediately after preparation of the test
– mobile phase B : mix 5 volumes of buffer solution solution. In order to avoid overheating in the reaction medium,
pH 2.5 R and 95 volumes of a 60 per cent V/V solution of mix thoroughly throughout and stop the titration immediately
acetonitrile R ; after the end-point has been reached.
Time Mobile phase A Mobile phase B Dissolve 0.150 g in 10 mL of anhydrous formic acid R. Add
(min) (per cent V/V) (per cent V/V)
50 mL of acetic anhydride R. Titrate with 0.1 M perchloric
acid, determining the end-point potentiometrically (2.2.20).
0 - 10 81 → 77 19 → 23
1 mL of 0.1 M perchloric acid is equivalent to 21.47 mg
10 - 25 77 → 50 23 → 50
of C22H34Cl2N2O2.
25 - 30 50 50
STORAGE
Flow rate : 1 mL/min. Protected from light.
Detection : spectrophotometer at 280 nm.
IMPURITIES
Preconditioning of the column : rinse for 5 min with a mixture
of 19 volumes of mobile phase B and 81 volumes of mobile Specified impurities : A, B, C, D, E, F, I, J, K.
phase A. Other detectable impurities (the following substances would,
Injection : 20 μL. if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
Relative retention with reference to dopexamine acceptance criterion for other/unspecified impurities and/or
(retention time = about 5 min) : impurity A = about 0.5 ; by the general monograph Substances for pharmaceutical use
impurity B = about 2.0 ; impurity C = about 2.3 ; (2034). It is therefore not necessary to identify these impurities
impurity D = about 2.8 ; impurity E = about 2.9 ; for demonstration of compliance. See also 5.10. Control of
impurity F = about 3.0 ; impurity I = about 3.6 ; impurities in substances for pharmaceutical use) : G, H.
impurity J = about 5.0 ; impurity K = about 5.9.
System suitability : reference solution (b) :
– resolution : minimum 2 between the peaks due to
dopexamine and impurity B.
Limits :
– correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
corresponding correction factor : impurity A = 1.4 ; A. 4,4′-[hexane-1,6-diylbis(iminoethylene)]dibenzene-1,2-
impurity F = 0.7 ; diol,

2100 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dorzolamide hydrochloride

01/2008:2359

DORZOLAMIDE HYDROCHLORIDE
Dorzolamidi hydrochloridum
B. R1 = OH, R2 = OCH3, R3 = H : 2-methoxy-4-[2-[[6-[(2-
phenylethyl)amino]hexyl]amino]ethyl]phenol,

C. R1 = OCH3, R2 = OH, R3 = H : 2-methoxy-5-[2-[[6-[(2-

phenylethyl)amino]hexyl]amino]ethyl]phenol,

F. R1 = R2 = OH, R3 = Cl : 4-chloro-5-[2-[[6-[(2- C10H17ClN2O4S3 Mr 360.9

phenylethyl)amino]hexyl]amino]ethyl]benzene-1,2-diol, [130693-82-2]
DEFINITION
H. R1 = R2 = OCH3, R3 = H : N-[2-(3,4-dimethoxyphenyl)- (4S,6S)-4-(Ethylamino)-6-methyl-5,6-dihydro-4H-thieno[2,3-
ethyl]-N′-(2-phenylethyl)hexane-1,6-diamine, b]thiopyran-2-sulfonamide 7,7-dioxide hydrochloride.
Content : 99.0 per cent to 101.0 per cent (dried substance).
J. R1 = R2 = R3 = H : N,N′-bis(2-phenylethyl)hexane-1,6-
diamine, CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : soluble in water, slightly soluble in methanol, very
slightly soluble in anhydrous ethanol.
It shows polymorphism (5.9).
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: dorzolamide hydrochloride CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
substance separately in methanol R, evaporate to dryness
and record new spectra using the residues.
D. R = H, R′ = OH : 4,4′-methylenebis[5-[2-[[6-[(2- B. It complies with the test for impurity A (see Tests).
phenylethyl)amino]hexyl]amino]ethyl]benzene-1,2-diol], C. It gives reaction (a) of chlorides (2.3.1).
TESTS
E. R = OH, R′ = H : 3-[4,5-dihydroxy-2-[2-[[6-[(2-
phenylethyl)amino]hexyl]amino]ethyl]benzyl]-4-[2-[[6- Impurity A. Liquid chromatography (2.2.29).
[(2-phenylethyl)amino]hexyl]amino]ethyl]benzene-1,2- Solvent mixture: acetonitrile R, glacial acetic acid R,
diol, 1,1-dimethylethyl methyl ether R (3:10:87 V/V/V).
Test solution. In a centrifuge tube, dissolve 20.0 mg of the
substance to be examined in 4 mL of dilute ammonia R4,
add 4 mL of ethyl acetate R, and mix. Separate the organic
layer and transfer it to a separate centrifuge tube. Add 4 mL
of ethyl acetate R to the aqueous layer, mix, separate the
organic layer, and combine it with the 1st extract. Evaporate
the combined organic layers to dryness in a water-bath at
50 °C under a stream of nitrogen R. Dissolve the residue in
G. bis(2-phenylethyl) hexanedioate, 3 mL of acetonitrile R, add 0.06 mL of (S)-(−)-α-methylbenzyl
isocyanate R, and heat in a water-bath at 50 °C for 5 min.
Evaporate to dryness in a water-bath at 50 °C under a stream
of nitrogen R. Dissolve the residue in 10 mL of the solvent
mixture.
Reference solution. In a centrifuge tube, dissolve 18.0 mg of
dorzolamide hydrochloride CRS and 2.0 mg of dorzolamide
impurity A CRS in 4 mL of dilute ammonia R4, and proceed
as indicated for the test solution beginning with “add 4 mL
of ethyl acetate R, and mix”.
I. 1-[6-[(2-phenylethyl)amino]hexyl]-2,3-dihydro-1H-
indole5,6-dione (dopexamine aminochrome), Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : silica gel for chromatography R (5 μm).
Mobile phase : water R, acetonitrile R, heptane R,
1,1-dimethylethyl methyl ether R (0.2:2:35:63 V/V/V/V).
Flow rate : 2 mL/min.
Detection : spectrophotometer at 254 nm.
Injection : 10 μL.
K. 1-[6-[(2-phenylethyl)amino]hexyl]-1H-indole-5,6-diol. Run time : 3 times the retention time of dorzolamide.

General Notices (1) apply to all monographs and other texts 2101
Dorzolamide hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Relative retention with reference to dorzolamide (retention – total : not more than 3 times the area of the principal peak
time = about 10 min) : impurity A = about 1.4. in the chromatogram obtained with reference solution (a)
System suitability: reference solution : (0.3 per cent) ;
– resolution : minimum 4.0 between the peaks due to – disregard limit : 0.5 times the area of the principal peak in
dorzolamide and impurity A. the chromatogram obtained with reference solution (a)
(0.05 per cent).
Calculate the percentage content of impurity A using the
following expression : Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
A = area of the peak due to impurity A in the
chromatogram obtained with the test solution; ASSAY
B = area of the peak due to dorzolamide in the Dissolve 0.150 g in a mixture of 5.0 mL of 0.01 M hydrochloric
chromatogram obtained with the test solution. acid and 50 mL of ethanol (96 per cent) R, using sonication if
necessary. Carry out a potentiometric titration (2.2.20), using
Limit :
0.1 M sodium hydroxide. Read the volume added between the
– impurity A : maximum 0.5 per cent. 1st and the 3rd points of inflexion.
Related substances. Liquid chromatography (2.2.29). 1 mL of 0.1 M sodium hydroxide is equivalent to 18.05 mg
Test solution. Dissolve 30.0 mg of the substance to be of C10H17N2O4S3Cl.
examined in mobile phase A and dilute to 50.0 mL with
mobile phase A. IMPURITIES
Reference solution (a). Dissolve 1.0 mL of the test solution to
Specified impurities : A, C.
100.0 mL with mobile phase A. Dilute 1.0 mL of this solution
to 10.0 mL with mobile phase A. Other detectable impurities (the following substances would,
Reference solution (b). Dissolve 2 mg of dorzolamide for system if present at a sufficient level, be detected by one or other of
suitability CRS (containing impurity C) in 2 mL of mobile the tests in the monograph. They are limited by the general
phase A. acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
Column : (2034). It is therefore not necessary to identify these impurities
– size : l = 0.25 m, Ø = 4.6 mm ; for demonstration of compliance. See also 5.10. Control of
– stationary phase : end-capped octadecylsilyl silica gel for impurities in substances for pharmaceutical use) : B, D.
chromatography R (5 μm) ;
– temperature : 35 °C.
Mobile phase :
– mobile phase A : mix 65 mL of acetonitrile R and 935 mL of
a 3.7 g/L solution of potassium dihydrogen phosphate R ;
– mobile phase B : acetonitrile R ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V) A. (4R,6R)-4-(ethylamino)-6-methyl-5,6-dihydro-4H-thieno-
0 - 15 100 0 [2,3-b]thiopyran-2-sulfonamide 7,7-dioxide,
15 - 30 100 → 50 0 → 50

30 - 37 50 → 100 50 → 0

37 - 44 100 0

Flow rate : 1.5 mL/min.

Detection : spectrophotometer at 254 nm.
Injection : 10 μL.
Identification of impurities : use the chromatogram supplied B. (4RS,6SR)-4-(ethylamino)-6-methyl-5,6-dihydro-4H-
with dorzolamide for system suitability CRS and the thieno[2,3-b]thiopyran-2-sulfonamide 7,7-dioxide,
chromatogram obtained with reference solution (b) to identify
the peak due to impurity C.
Relative retention with reference to dorzolamide (retention
time = about 11 min) : impurity C = about 0.9.
System suitability : reference solution (b) :
– resolution : minimum 2.0 between the peaks due to
impurity C and dorzolamide.
Limits : C. R = CH2-CH2-B(OH)2 : [2-[[(4S,6S)-6-methyl-7,7-dioxo-2-
– impurity C : not more than 1.5 times the area of the sulfamoyl-4,5,6,7-tetrahydro-7λ6-thieno[2,3-b]thiopyran-
principal peak in the chromatogram obtained with 4-yl]amino]ethyl]boronic acid,
reference solution (a) (0.15 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained D. R = H : (4S,6S)-4-amino-6-methyl-5,6-dihydro-4H-thieno-
with reference solution (a) (0.10 per cent) ; [2,3-b]thiopyran-2-sulfonamide 7,7-dioxide.

2102 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dosulepin hydrochloride

01/2008:1314 Column :
corrected 6.0 – size : l = 0.25 m, Ø = 4.6 mm,
– stationary phase : nitrile silica gel for chromatography R1
DOSULEPIN HYDROCHLORIDE (5 μm),
– temperature : 35 °C.
Dosulepini hydrochloridum Mobile phase : 0.83 per cent V/V solution of perchloric acid R,
propanol R, methanol R, water R (1:10:30:60 V/V/V/V).
Flow rate : 1 mL/min.
Detection : spectrophotometer at 229 nm.
Injection : 5 μL.
Run time : 2.5 times the retention time of dosulepin
((E)-isomer).
Relative retention with reference to dosulepin ((E)-isomer ;
C19H22ClNS Mr 331.9 retention time = about 25 min) : impurity E = about 0.9.
[897-15-4] System suitability : reference solution (b) :
DEFINITION – peak-to-valley ratio : minimum 4, where Hp = height
(E)-3-(Dibenzo[b,e]thiepin-11(6H)-ylidene)-N,N- above the baseline of the peak due to impurity E and
dimethylpropan-1-amine hydrochloride. Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to dosulepin
Content : 98.0 per cent to 101.0 per cent (dried substance). ((E)-isomer).
CHARACTERS Limits :
Appearance : white or faintly yellow, crystalline powder. – impurity E : not more than 5 per cent of the sum of the
areas of the peak due to impurity E and the principal peak
Solubility : freely soluble in water, in alcohol and in methylene in the chromatogram obtained with the test solution,
chloride.
– impurity A : not more than the area of the principal peak
IDENTIFICATION in the chromatogram obtained with reference solution (a)
First identification : B, D. (0.25 per cent),
Second identification : A, C, D. – any other impurity : not more than 0.4 times the area of
the principal peak in the chromatogram obtained with
A. Dissolve 25.0 mg in a 1 g/L solution of hydrochloric reference solution (a) (0.1 per cent),
acid R in methanol R and dilute to 100.0 mL with the
– total of other impurities and impurity A : not more than
same solution. Dilute 2.0 mL to 50.0 mL with a 1 g/L
twice the area of the principal peak in the chromatogram
solution of hydrochloric acid R in methanol R. Examined
obtained with reference solution (a) (0.5 per cent),
between 220 nm and 350 nm (2.2.25), the solution shows 2
absorption maxima at 231 nm and 306 nm and a shoulder – disregard limit : 0.2 times the area of the principal peak in
at about 260 nm. The specific absorbance at the maximum the chromatogram obtained with reference solution (a)
at 231 nm is 660 to 730. (0.05 per cent).
B. Infrared absorption spectrophotometry (2.2.24). Heavy metals (2.4.8) : maximum 20 ppm.
Preparation : discs. 1.0 g complies with test C. Prepare the reference solution using
2 mL of lead standard solution (10 ppm Pb) R.
Comparison : dosulepin hydrochloride CRS.
C. Dissolve about 1 mg in 5 mL of sulfuric acid R. A dark red Loss on drying (2.2.32) : maximum 0.5 per cent, determined
colour is produced. on 1.000 g by drying in an oven at 105 °C.
D. It gives reaction (b) of chlorides (2.3.1). Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
TESTS
ASSAY
Appearance of solution. The solution is clear (2.2.1) and not Dissolve 0.250 g in a mixture of 5 mL of anhydrous acetic
more intensely coloured than reference solution Y5 (2.2.2, acid R and 35 mL of acetic anhydride R. Titrate with 0.1 M
Method II). perchloric acid, determining the end-point potentiometrically
Dissolve 1 g in water R and dilute to 20 mL with the same (2.2.20).
solvent. 1 mL of 0.1 M perchloric acid is equivalent to 33.19 mg
pH (2.2.3) : 4.2 to 5.2. of C19H22ClNS.
Dissolve 1 g in carbon dioxide-free water R and dilute to 10 mL STORAGE
with the same solvent.
Protected from light.
Impurity E and related substances. Liquid chromatography
(2.2.29). Prepare the solutions immediately before use and IMPURITIES
protect from light.
Test solution. Dissolve 50.0 mg of the substance to be
examined in 5 mL of methanol R and dilute to 100.0 mL with
the mobile phase.
Reference solution (a). Dissolve 12.5 mg of dosulepin
impurity A CRS in 5 mL of methanol R and dilute to 50.0 mL
with the mobile phase. Dilute 0.5 mL to 100.0 mL with the
mobile phase. A. X = SO : (E)-3-(5-oxo-5λ4-dibenzo[b,e]thiepin-11(6H)-
ylidene)-N,N-dimethylpropan-1-amine,
Reference solution (b). Dissolve 10.0 mg of dosulepin
hydrochloride CRS in 5 mL of methanol R and dilute to D. X = SO2 : (E)-3-(5,5-dioxo-5λ6-dibenzo[b,e]thiepin-11(6H)-
20.0 mL with the mobile phase. ylidene)-N,N-dimethylpropan-1-amine,

General Notices (1) apply to all monographs and other texts 2103
Doxapram hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Results : the principal spot in the chromatogram obtained

with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
the reference solution.
C. It gives reaction (a) of chlorides (2.3.1).
TESTS
B. R + R′ = O : dibenzo[b,e]thiepin-11(6H)-one,
Solution S. Dissolve 1.000 g in carbon dioxide-free water R
C. R = OH, R′ = [CH2]3-N(CH3)2 : (11RS)-11-[3- and dilute to 50.0 mL with the same solvent.
(dimethylamino)propyl]-6,11-dihydrodibenzo[b,e]thiepin-
Appearance of solution. The solution is clear (2.2.1) and
11-ol,
colourless (2.2.2, Method II).
Dilute 10 mL of solution S to 25 mL with water R.
pH (2.2.3) : 3.5 to 5.0.
Dilute 5 mL of solution S to 25 mL with carbon dioxide-free
water R.
Optical rotation (2.2.7): − 0.10° to + 0.10°, determined on
solution S.
E. (Z)-3-(dibenzo[b,e]thiepin-11(6H)-ylidene)-N,N-
dimethylpropan-1-amine. Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
Test solution. Dissolve 10.0 mg of the substance to be
01/2014:1201
examined in the mobile phase and dilute to 10.0 mL with the
mobile phase.
DOXAPRAM HYDROCHLORIDE Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase.
Doxaprami hydrochloridum Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 5.0 mL with the mobile phase.
Reference solution (c). Dissolve 5 mg of doxapram
impurity B CRS in the mobile phase and dilute to 5.0 mL with
the mobile phase. To 1.0 mL of the solution, add 1.0 mL of the
test solution and dilute to 100.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : spherical end-capped octadecylsilyl silica
C24H31ClN2O2,H2O Mr 433.0 gel for chromatography R (5 μm) with a carbon loading of
[7081-53-0] 14 per cent, a specific surface area of 350 m2/g and a pore
DEFINITION size of 10 nm.
Mobile phase : mix 50 volumes of acetonitrile R and 50 volumes
(4RS)-1-Ethyl-4-[2-(morpholin-4-yl)ethyl]-3,3- of a 0.82 g/L solution of sodium acetate R adjusted to pH 4.5
diphenylpyrrolidin-2-one hydrochloride. with glacial acetic acid R.
Content : 98.0 per cent to 100.5 per cent (dried substance). Flow rate : 1.5 mL/min.
CHARACTERS Detection : spectrophotometer at 214 nm.
Appearance : white or almost white, crystalline powder. Injection : 20 μL.
Solubility : sparingly soluble in water, soluble in ethanol Run time : 4 times the retention time of doxapram.
(96 per cent) and in methylene chloride. Retention time : doxapram = about 6 min.
IDENTIFICATION System suitability : reference solution (c) :
– resolution : minimum 3.0 between the peaks due to
First identification : A, C. doxapram and impurity B.
Second identification : B, C. Limits :
A. Infrared absorption spectrophotometry (2.2.24). – any impurity : not more than the area of the principal peak
Comparison : doxapram hydrochloride CRS. in the chromatogram obtained with reference solution (b)
(0.2 per cent),
B. Thin-layer chromatography (2.2.27).
– total : not more than the area of the principal peak in the
Test solution. Dissolve 10 mg of the substance to be chromatogram obtained with reference solution (a) (1.0 per
examined in methanol R and dilute to 10 mL with the same cent),
solvent.
– disregard limit : 0.05 times the area of the principal peak
Reference solution. Dissolve 10 mg of doxapram in the chromatogram obtained with reference solution (a)
hydrochloride CRS in methanol R and dilute to 10 mL with (0.05 per cent).
the same solvent.
Heavy metals (2.4.8) : maximum 20 ppm.
Plate : TLC silica gel plate R.
Dissolve 2.0 g in 20 mL of a mixture of 15 volumes of water R
Mobile phase : solution of ammonia R containing 17 g/L of
and 85 volumes of methanol R. 12 mL of the solution complies
NH3, 2-propanol R, 2-methylpropanol R (10:10:80 V/V/V).
with test B. Prepare the reference solution using lead standard
Application : 10 μL. solution (2 ppm Pb) obtained by diluting lead standard
Development : over 2/3 of the plate. solution (100 ppm Pb) R with a mixture of 15 volumes of
Drying : in air. water R and 85 volumes of methanol R.
Detection : spray with dilute potassium iodobismuthate Loss on drying (2.2.32) : 3.0 per cent to 4.5 per cent,
solution R and examine immediately. determined on 1.000 g by drying in an oven at 105 °C.

2104 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Doxazosin mesilate

Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Solubility : slightly soluble in water, soluble in a mixture of
1.0 g. 15 volumes of water and 35 volumes of tetrahydrofuran,
slightly soluble in methanol, practically insoluble in acetone.
ASSAY
It shows polymorphism (5.9), some forms may be hygroscopic.
Dissolve 0.300 g in a mixture of 10 mL of 0.01 M hydrochloric
acid and 50 mL of ethanol (96 per cent) R. Carry out IDENTIFICATION
a potentiometric titration (2.2.20) using 0.1 M sodium Infrared absorption spectrophotometry (2.2.24).
hydroxide. Read the volume added between the 2 points of Comparison: doxazosin mesilate CRS.
inflexion. If the spectra obtained in the solid state show differences,
1 mL of 0.1 M sodium hydroxide is equivalent to 41.50 mg mix 1 part of the substance to be examined and 1 part of the
of C24H31ClN2O2. reference substance separately with 10 parts of anhydrous
ethanol R and heat to boiling. Continue heating the suspension
IMPURITIES under a reflux condenser for about 3 h. Cool and filter. Record
new spectra using the previously dried residues on the filters.
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution BY6 (2.2.2,
Method II).
Dissolve 1.0 g in a mixture of 15 mL of water R and 35 mL of
tetrahydrofuran R.
A. (4RS)-4-(2-chloroethyl)-1-ethyl-3,3-diphenylpyrrolidin-
2-one, Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be
examined in 5 mL of mobile phase B, adding water R, and
dilute to 50.0 mL with water R.
Reference solution (a). Dilute 5.0 mL of the test solution to
100.0 mL with water R. Dilute 2.0 mL of this solution to
100.0 mL with water R.
Reference solution (b). Dissolve 5 mg of doxazosin
impurity D CRS and 5 mg of doxazosin impurity F CRS in
B. (4RS)-1-ethyl-4-[2-[(2-hydroxyethyl)amino]ethyl]-3,3- 5 mL of mobile phase B, adding water R, and dilute to 50.0 mL
diphenylpyrrolidin-2-one. with water R. Dilute 10.0 mL of this solution to 50.0 mL with
water R.
Reference solution (c). Dilute 5.0 mL of reference solution (a)
07/2013:2125 to 10.0 mL with water R.
Reference solution (d). Dissolve 25.0 mg of doxazosin
DOXAZOSIN MESILATE mesilate CRS in 5 mL of mobile phase B, adding water R, and
dilute to 50.0 mL with water R.
Doxazosini mesilas Column :
– size : l = 0.25 m, Ø = 4.0 mm ;
– stationary phase : base-deactivated octylsilyl silica gel for
chromatography R (5 μm) ;
– temperature : 35 °C.
Mobile phase :
– mobile phase A : 10 g/L solution of phosphoric acid R ;
– mobile phase B : 10 g/L solution of phosphoric acid R in
acetonitrile R1 ;
C24H29N5O8S Mr 547.6
[77883-43-3] Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
DEFINITION 0-5 90 10
1-(4-Amino-6,7-dimethoxyquinazolin-2-yl)-4-[(2RS)- 5 - 40 90 → 50 10 → 50
2,3-dihydro-1,4-benzodioxin-2-ylcarbonyl]piperazine
methanesulfonate. 40 - 45 50 50
Content : 98.0 per cent to 102.0 per cent (anhydrous substance).
Flow rate : 0.8 mL/min.
PRODUCTION Detection : spectrophotometer at 210 nm.
It is considered that alkylsulfonate esters are genotoxic and are Injection : 10 μL of the test solution and reference solutions (a),
potential impurities in doxazosin mesilate. The manufacturing (b) and (c).
process should be developed taking into consideration Relative retention with reference to doxazosin (retention
the principles of quality risk management, together with time = about 30 min) : impurity D = about 0.5 ;
considerations of the quality of starting materials, process impurity F = about 0.6.
capability and validation. The general methods 2.5.37. Methyl, System suitability : reference solution (b) :
ethyl and isopropyl methanesulfonate in methanesulfonic
acid, 2.5.38. Methyl, ethyl and isopropyl methanesulfonate – resolution : minimum 4.5 between the peaks due to
in active substances and 2.5.39. Methanesulfonyl chloride in impurities D and F.
methanesulfonic acid are available to assist manufacturers. Limits :
– unspecified impurities : for each impurity, not more than the
CHARACTERS area of the principal peak in the chromatogram obtained
Appearance : white or almost white crystalline powder. with reference solution (a) (0.10 per cent) ;

General Notices (1) apply to all monographs and other texts 2105
Doxepin hydrochloride EUROPEAN PHARMACOPOEIA 8.0

– total : not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.3 per cent) ;
– disregard limit : the area of the principal peak in the
chromatogram obtained with reference solution (c) F. 2-chloro-6,7-dimethoxyquinazolin-4-amine,
(0.05 per cent).
Water (2.5.12) : maximum 1.5 per cent, determined on 0.500 g.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY
Liquid chromatography (2.2.29) as described in the test for G. 6,7-dimethoxy-2-(piperazin-1-yl)quinazolin-4-amine,
related substances with the following modification.
Injection : test solution and reference solution (d).
Calculate the percentage content of C24H29N5O8S using the
chromatogram obtained with reference solution (d) and the
assigned content of doxazosin mesilate CRS.
STORAGE
In an airtight container.
IMPURITIES
Other detectable impurities (the following substances would, H. 2,2′-(piperazine-1,4-diyl)bis(6,7-dimethoxyquinazolin-4-
if present at a sufficient level, be detected by one or other of amine).
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or 04/2009:1096
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these DOXEPIN HYDROCHLORIDE
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use) : Doxepini hydrochloridum
A, B, C, D, E, F, G, H.

A. (2RS)-2,3-dihydro-1,4-benzodioxine-2-carboxylic acid, C19H22ClNO Mr 315.8

[1229-29-4]
DEFINITION
(E)-3-(Dibenzo[b,e]oxepin-11(6H)-ylidene)-N,N-
dimethylpropan-1-amine hydrochloride.
Content : 98.0 per cent to 101.0 per cent of C19H22ClNO (dried
substance).
B. 1-[(2RS)-2,3-dihydro-1,4-benzodioxin-2-
ylcarbonyl]piperazine, CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : freely soluble in water, in ethanol (96 per cent) and
in methylene chloride.
IDENTIFICATION
First identification : C, E.
Second identification : A, B, D, E.
C. 1,4-bis(2,3-dihydro-1,4-benzodioxin-2-ylcarbonyl)- A. Melting point (2.2.14) : 185 °C to 191 °C.
piperazine,
B. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Dissolve 50.0 mg in a 1 g/L solution of
hydrochloric acid R in methanol R and dilute to 100.0 mL
with the same acid solution. Dilute 5.0 mL to 50.0 mL with
a 1 g/L solution of hydrochloric acid R in methanol R.
D. 6,7-dimethoxyquinazoline-2,4(1H,3H)-dione, Spectral range : 230-350 nm.
Absorption maximum : at 297 nm.
Specific absorbance at the absorption maximum : 128 to 142.
C. Infrared absorption spectrophotometry (2.2.24).
Comparison: doxepin hydrochloride CRS.
D. Dissolve about 5 mg in 2 mL of sulfuric acid R. A dark red
E. 2,4-dichloro-6,7-dimethoxyquinazoline, colour is produced.

2106 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Doxepin hydrochloride

– unspecified impurities : for each impurity, not more than the

E. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
area of the principal peak in the chromatogram obtained
TESTS with reference solution (a) (0.10 per cent) ;
Solution S. Dissolve 1.5 g in carbon dioxide-free water R and – total : not more than 3 times the area of the principal peak
dilute to 30 mL with the same solvent. in the chromatogram obtained with reference solution (a)
Appearance of solution. Dilute 10 mL of solution S to 25 mL (0.3 per cent) ;
with water R. The solution is clear (2.2.1) and colourless – disregard limit : 0.5 times the area of the principal peak in
(2.2.2, Method II). the chromatogram obtained with reference solution (a)
Acidity. To 10 mL of solution S add 0.1 mL of methyl red (0.05 per cent).
solution R. Not more than 0.1 mL of 0.1 M sodium hydroxide (Z)-Isomer. Liquid chromatography (2.2.29).
is required to change the colour of the indicator to yellow. Test solution. Dissolve 20.0 mg of the substance to be
Related substances. Liquid chromatography (2.2.29). Prepare examined in the mobile phase and dilute to 20.0 mL with the
the solutions immediately before use and protect them from mobile phase. Dilute 1.0 mL of this solution to 10.0 mL with
light. the mobile phase.
Phosphate buffer solution. Dissolve 1.42 g of anhydrous Column :
disodium hydrogen phosphate R in water R, adjust to pH 7.7 – size : l = 0.12 m, Ø = 4 mm ;
with dilute phosphoric acid R and dilute to 1000 mL with – stationary phase : spherical octylsilyl silica gel for
water R. chromatography R (5 μm) with a specific surface area of
Solvent mixture. Mix 1 volume of 1 M sodium hydroxide and 220 m2/g and a pore size of 80 nm ;
250 volumes of the mobile phase. – temperature : 50 °C.
Test solution. Dissolve 50 mg of the substance to be examined Mobile phase : mix 30 volumes of methanol R and 70 volumes
in the solvent mixture and dilute to 50.0 mL with the solvent of a 30 g/L solution of sodium dihydrogen phosphate R
mixture. previously adjusted to pH 2.5 with phosphoric acid R.
Reference solution (a). Dilute 1.0 mL of the test solution to Flow rate : 1 mL/min.
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture. Detection : spectrophotometer at 254 nm.
Reference solution (b). Dissolve the contents of a vial of Injection : 20 μL.
doxepin for system suitability CRS (containing impurities A, B System suitability :
and C) in 1.0 mL of mobile phase. – resolution : minimum 1.5 between the peaks due to the
Column: (E)-isomer (1st peak) and to the (Z)-isomer (2nd peak).
– size : l = 0.25 m, Ø = 4.6 mm ; Results :
– stationary phase : end-capped octadecylsilyl silica gel for – calculate the ratio of the area of the peak due to the
chromatography R (5 μm) ; (E)-isomer to the area of the peak due to the (Z)-isomer :
this ratio is 4.4 to 6.7 (13.0 per cent to 18.5 per cent of the
– temperature : 30 °C.
(Z)-isomer).
Mobile phase : acetonitrile R1, phosphate buffer solution,
methanol R1 (20:30:50 V/V/V). Heavy metals (2.4.8) : maximum 20 ppm.
Dissolve 2.0 g in water R and dilute to 20 mL with the same
Flow rate : 1.0 mL/min.
solvent. 12 mL of the solution complies with test A. Prepare
Detection : spectrophotometer at 215 nm. the reference solution using lead standard solution (2 ppm
Injection : 20 μL. Pb) R.
Run time : 1.5 times the retention time of doxepin. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Identification of impurities : use the chromatogram supplied on 1.000 g by drying in an oven at 105 °C.
with doxepin for system suitability CRS and the chromatogram Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
obtained with reference solution (b) to identify the peaks due 1.0 g.
to impurities A, B and C.
ASSAY
Relative retention with reference to doxepin (retention
time = about 18 min) : impurity A = about 0.5 ; Dissolve 0.250 g in a mixture of 5 mL of anhydrous acetic
impurity C = about 0.6 ; impurity B = about 0.7 ; the peak due acid R and 35 mL of acetic anhydride R. Using 0.2 mL of crystal
to doxepin might show a shoulder caused by the (Z)-isomer violet solution R as indicator, titrate with 0.1 M perchloric acid
(impurity D). until the colour changes from blue to green.
System suitability : reference solution (b) : 1 mL of 0.1 M perchloric acid is equivalent to 31.58 mg of
C19H22ClNO.
– resolution : minimum 1.5 between the peaks due to
impurities A and C, and minimum 1.5 between the peaks STORAGE
due to impurities C and B ;
Protected from light.
– the chromatogram obtained is similar to the chromatogram
supplied with doxepin for system suitability CRS. IMPURITIES
Limits : Specified impurities : A, B, C, D.
– correction factor : for the calculation of content, multiply
the peak area of impurity B by 1.7 ;
– impurities A, B : for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
reference solution (a) (0.1 per cent) ;
– impurity C : not more than twice the area of the principal
peak in the chromatogram obtained with reference
solution (a) (0.2 per cent) ; A. dibenzo[b,e]oxepin-11(6H)-one (doxepinone),

General Notices (1) apply to all monographs and other texts 2107
Doxorubicin hydrochloride EUROPEAN PHARMACOPOEIA 8.0

B. Dissolve about 10 mg in 0.5 mL of nitric acid R, add 0.5 mL

of water R and heat over a flame for 2 min. Allow to
cool and add 0.5 mL of silver nitrate solution R1. A white
precipitate is formed.
TESTS
pH (2.2.3) : 4.0 to 5.5.
B. (11RS)-11-[3-(dimethylamino)propyl]-6,11- Dissolve 50 mg in carbon dioxide-free water R and dilute to
dihydrodibenzo[b,e]oxepin-11-ol (doxepinol), 10 mL with the same solvent.
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
Test solution (a). Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with the
mobile phase.
Test solution (b). Dilute 10.0 mL of test solution (a) to
100.0 mL with the mobile phase.
C. (E)-3-(dibenzo[b,e]oxepin-11(6H)-ylidene)-N- Reference solution (a). Dissolve 10.0 mg of doxorubicin
methylpropan-1-amine (desmethyldoxepin), hydrochloride CRS and 10 mg of epirubicin hydrochloride CRS
in the mobile phase and dilute to 50.0 mL with the mobile
phase. Dilute 10.0 mL of the solution to 100.0 mL with the
mobile phase.
Reference solution (b). Dilute 5.0 mL of reference solution (a)
to 20.0 mL with the mobile phase.
Reference solution (c). Dissolve 50.0 mg of doxorubicin
hydrochloride CRS in the mobile phase and dilute to 50.0 mL
with the mobile phase. Dilute 10.0 mL of the solution to
D. (Z)-3-(dibenzo[b,e]oxepin-11(6H)-ylidene)-N,N- 100.0 mL with the mobile phase.
dimethylpropan-1-amine. Column :
– size : l = 0.25 m, Ø = 4.0 mm,
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
01/2008:0714 Mobile phase : mix equal volumes of acetonitrile R and a
solution containing 2.88 g/L of sodium laurilsulfate R and
DOXORUBICIN HYDROCHLORIDE 2.25 g/L of phosphoric acid R.
Flow rate : 1 mL/min.
Doxorubicini hydrochloridum Detection : spectrophotometer at 254 nm.
Injection : 5 μL ; inject test solution (a) and reference
solutions (a) and (b).
Run time : 3.5 times the retention time of doxorubicin.
Retention time : doxorubicin = about 8 min.
System suitability : reference solution (a) :
– resolution : minimum of 2.0 between the peaks due to
doxorubicin and to epirubicin.
Limits :
– any impurity : not more than the area of the peak due to
doxorubicin in the chromatogram obtained with reference
C27H30ClNO11 Mr 580.0 solution (b) (0.5 per cent),
[25316-40-9]
– disregard limit : 0.1 times the area of the peak due to
DEFINITION doxorubicin in the chromatogram obtained with reference
solution (b) (0.05 per cent).
(8S,10S)-10-[(3-Amino-2,3,6-trideoxy-α-L-lyxo-
hexopyranosyl)oxy]-6,8,11-trihydroxy-8-(hydroxyacetyl)- Ethanol (2.4.24, System B) : maximum 1.0 per cent.
1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione Water (2.5.12) : maximum 4.0 per cent, determined on 0.100 g.
hydrochloride. Bacterial endotoxins (2.6.14) : less than 2.2 IU/mg, if intended
Substance produced by certain strains of Streptomyces for use in the manufacture of parenteral preparations without
coeruleorubidus or Streptomyces peucetius or obtained by any a further appropriate procedure for the removal of bacterial
other means. endotoxins.
Content : 98.0 per cent to 102.0 per cent (anhydrous substance). ASSAY

CHARACTERS Liquid chromatography (2.2.29) as described in the test for

related substances.
Appearance : orange-red, crystalline powder, hygroscopic.
Injection : test solution (b) and reference solution (c).
Solubility : soluble in water, slightly soluble in methanol.
Calculate the percentage content of C27H30ClNO11.
IDENTIFICATION STORAGE
A. Infrared absorption spectrophotometry (2.2.24). In an airtight container. If the substance is sterile, store in a
Comparison : doxorubicin hydrochloride CRS. sterile, airtight, tamper-proof container.

2108 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Doxycycline hyclate

IMPURITIES CHARACTERS
Appearance : yellow, hygroscopic, crystalline powder.
Solubility : freely soluble in water and in methanol, sparingly
soluble in ethanol (96 per cent). It dissolves in solutions of
alkali hydroxides and carbonates.
IDENTIFICATION
A. Examine the chromatograms obtained in the assay.
Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time and size
A. (8S,10S)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo- to the principal peak in the chromatogram obtained with
hexopyranosyl)oxy]-6,8,11-trihydroxy-1-methoxy-7,8,9,10- reference solution (a).
tetrahydrotetracene-5,12-dione (daunorubicin), B. To about 2 mg add 5 mL of sulfuric acid R. A yellow colour
develops.
C. It gives reaction (a) of chlorides (2.3.1).
TESTS
pH (2.2.3) : 2.0 to 3.0.
Dissolve 0.1 g in carbon dioxide-free water R and dilute to
10 mL with the same solvent.
Specific optical rotation (2.2.7) : − 120 to − 105 (anhydrous
and ethanol-free substance).
B. R = OCH3 : (8S,10S)-10[(3-amino-2,3,6-trideoxy-α-L-lyxo- Dissolve 0.250 g in a mixture of 1 volume of 1 M hydrochloric
hexopyranosyl)oxy]-8-(2-bromo-1,1-dimethoxyethyl)- acid and 99 volumes of methanol R and dilute to 25.0 mL with
6,8,11-trihydroxy-1-methoxy-7,8,9,10-tetrahydrotetracene- the same mixture of solvents. Carry out the measurement
5,12-dione, within 5 min of preparing the solution.
Specific absorbance (2.2.25): 300 to 335, determined at the
C. R + R = O : (8S,10S)-10[(3-amino-2,3,6-trideoxy-α-L-lyxo- absorption maximum at 349 nm (anhydrous and ethanol-free
hexopyranosyl)oxy]-8-(bromoacetyl)-6,8,11-trihydroxy-1- substance).
methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione, Dissolve 25.0 mg in a mixture of 1 volume of 1 M hydrochloric
acid and 99 volumes of methanol R and dilute to 25.0 mL with
the same mixture of solvents. Dilute 1.0 mL of the solution to
100.0 mL with a mixture of 1 volume of 1 M hydrochloric acid
and 99 volumes of methanol R. Carry out the measurement
within 1 h of preparing the solution.
Light-absorbing impurities. The absorbance (2.2.25)
D. (8S,10S)-6,8,10,11-tetrahydroxy-8-(hydroxyacetyl)- determined at 490 nm is not greater than 0.07 (anhydrous and
1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione ethanol-free substance).
(doxorubicin aglycone, doxorubicinone). Dissolve 0.10 g in a mixture of 1 volume of 1 M hydrochloric
acid and 99 volumes of methanol R and dilute to 10.0 mL with
the same mixture of solvents. Carry out the measurement
01/2008:0272 within 1 h of preparing the solution.
corrected 7.4 Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
DOXYCYCLINE HYCLATE Test solution. Dissolve 20.0 mg of the substance to be
examined in 0.01 M hydrochloric acid and dilute to 25.0 mL
Doxycyclini hyclas with the same acid.
Reference solution (a). Dissolve 20.0 mg of doxycycline
hyclate CRS in 0.01 M hydrochloric acid and dilute to 25.0 mL
with the same acid.
Reference solution (b). Dissolve 20.0 mg of 6-epidoxycycline
hydrochloride CRS (impurity A) in 0.01 M hydrochloric acid
and dilute to 25.0 mL with the same acid.
Reference solution (c). Dissolve 20.0 mg of metacycline
hydrochloride CRS (impurity B) in 0.01 M hydrochloric acid
C22H25ClN2O8,½C2H6O,½H2O Mr 512.9 and dilute to 25.0 mL with the same acid.
[24390-14-5] Reference solution (d). Mix 4.0 mL of reference solution (a),
1.5 mL of reference solution (b) and 1.0 mL of reference
DEFINITION solution (c) and dilute to 25.0 mL with 0.01 M hydrochloric
Hydrochloride hemiethanol hemihydrate acid.
of (4S,4aR,5S,5aR,6R,12aS)-4-(dimethyl- Reference solution (e). Mix 2.0 mL of reference solution (b)
amino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-di- and 2.0 mL of reference solution (c) and dilute to 100.0 mL
oxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide. with 0.01 M hydrochloric acid.
Substance obtained from oxytetracycline or metacycline or by Column :
any other means. – size : l = 0.25 m, Ø = 4.6 mm ;
Semi-synthetic product derived from a fermentation product. – stationary phase : styrene-divinylbenzene copolymer R
Content : 95.0 per cent to 102.0 per cent of C22H25ClN2O8 (8 μm) ;
(anhydrous and ethanol-free substance). – temperature : 60 °C.

General Notices (1) apply to all monographs and other texts 2109
Doxycycline hyclate EUROPEAN PHARMACOPOEIA 8.0

Mobile phase : weigh 60.0 g of 2-methyl-2-propanol R and Heavy metals (2.4.8) : maximum 50 ppm.
transfer to a 1000 mL volumetric flask with the aid of 200 mL 0.5 g complies with test C. Prepare the reference solution using
of water R ; add 400 mL of buffer solution pH 8.0 R, 50 mL of 2.5 mL of lead standard solution (10 ppm Pb) R.
a 10 g/L solution of tetrabutylammonium hydrogen sulfate R
adjusted to pH 8.0 with dilute sodium hydroxide solution R, Water (2.5.12) : 1.4 per cent to 2.8 per cent, determined on
and 10 mL of a 40 g/L solution of sodium edetate R adjusted 1.20 g.
to pH 8.0 with dilute sodium hydroxide solution R ; dilute to Sulfated ash (2.4.14) : maximum 0.4 per cent, determined on
1000.0 mL with water R. 1.0 g.
Flow rate : 1.0 mL/min. Bacterial endotoxins (2.6.14) : less than 1.14 IU/mg, if
Detection : spectrophotometer at 254 nm. intended for use in the manufacture of parenteral preparations
Injection : 20 μL of the test solution and reference solutions (d) without a further appropriate procedure for the removal of
and (e). bacterial endotoxins.
Relative retention with reference to doxycycline ASSAY
(retention time = about 17 min) : impurity E = about 0.2 ;
Liquid chromatography (2.2.29) as described in the test for
impurity D = about 0.3 ; impurity C = about 0.5 ;
related substances with the following modification.
impurity B = about 0.8 ; impurity A = about 0.85 ;
impurity F = about 1.2. Injection : test solution and reference solution (a).
System suitability : reference solution (d) : Calculate the percentage content of C22H25ClN2O8 (Mr = 480.9).
– resolution : minimum 1.25 between the peaks due to STORAGE
impurities B (1st peak) and A (2nd peak) and minimum 2.0
between the peaks due to impurity A and doxycycline In an airtight container, protected from light. If the substance
(3rd peak) ; if necessary, adjust the 2-methyl-2-propanol is sterile, store in a sterile, airtight, tamper-proof container.
content in the mobile phase ;
IMPURITIES
– symmetry factor : maximum 1.25 for the peak due to
doxycycline. Specified impurities : A, B, C, D, E, F.
Limits :
– impurities A, B : for each impurity, not more than the area
of the corresponding peak in the chromatogram obtained
with reference solution (e) (2.0 per cent) ;
– impurities C, D, E, F : for each impurity, not more than
0.25 times the area of the peak due to impurity A in the
chromatogram obtained with reference solution (e) (0.5 per
cent) ; A. (4S,4aR,5S,5aR,6S,12aS)-4-(dimethylamino)-3,5,10,12,12a-
pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-
– any other impurity : for each impurity, not more than octahydrotetracene-2-carboxamide (6-epidoxycycline),
0.25 times the area of the peak due to impurity A in the
chromatogram obtained with reference solution (e) (0.5 per
cent) ;
– disregard limit : 0.05 times the area of the peak due to
impurity A in the chromatogram obtained with reference
solution (e) (0.1 per cent).
Ethanol. Gas chromatography (2.2.28).
Internal standard solution. Dilute 0.50 mL of propanol R to B. (4S,4aR,5S,5aR,12aS)-4-(dimethylamino)-
1000.0 mL with water R. 3,5,10,12,12a-pentahydroxy-6-methylene-1,11-dioxo-
Test solution (a). Dissolve 0.10 g of the substance to be 1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide
examined in water R and dilute to 10.0 mL with the same (metacycline),
solvent.
Test solution (b). Dissolve 0.10 g of the substance to be
examined in the internal standard solution and dilute to
10.0 mL with the same solution.
Reference solution. Dilute 0.50 mL of anhydrous ethanol R to
100.0 mL with the internal standard solution. Dilute 1.0 mL
of the solution to 10.0 mL with the internal standard solution.
Column : C. (4R,4aR,5S,5aR,6R,12aS)-4-(dimethylamino)-
– size : l = 1.5 m, Ø = 4.0 mm; 3,5,10,12,12apentahydroxy-6-methyl-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide
– stationary phase : ethylvinylbenzene-divinylbenzene (4-epidoxycycline),
copolymer R (150-180 μm).
Carrier gas : nitrogen for chromatography R.
Temperature :
– column : 135 °C;
– injection port and detector : 150 °C.
Detection : flame ionisation.
Calculate the content of ethanol taking the density (2.2.5) at
20 °C to be 0.790 g/mL. D. (4R,4aR,5S,5aR,6S,12aS)-4-(dimethylamino)-
3,5,10,12,12apentahydroxy-6-methyl-1,11-dioxo-
Limit : 1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide
– ethanol : 4.3 per cent to 6.0 per cent. (4-epi-6-epidoxycycline),

2110 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Doxycycline monohydrate

Specific optical rotation (2.2.7) : − 113 to − 130 (anhydrous

substance).
Dissolve 0.250 g in a mixture of 0.5 volumes of hydrochloric
acid R and 99.5 volumes of methanol R and dilute to 25.0 mL
with the same mixture of solvents. Carry out the measurement
within 5 min of preparing the solution.
E. (4S,4aR,5S,5aR,6S,12aS)-4-(dimethylamino)- Specific absorbance (2.2.25) : 325 to 363 determined at the
3,5,6,10,12,12ahexahydroxy-6-methyl-1,11-dioxo- maximum at 349 nm (anhydrous substance).
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dissolve 25.0 mg in a mixture of 0.5 volumes of hydrochloric
(oxytetracycline), acid R and 99.5 volumes of methanol R and dilute to 50.0 mL
with the same mixture of solvents. Dilute 2.0 mL of the
solution to 100.0 mL with a mixture of 0.5 volumes of 1 M
hydrochloric acid and 99.5 volumes of methanol R. Carry out
the measurement within 1 h of preparing the solution.
Light-absorbing impurities. The absorbance (2.2.25)
determined at 490 nm has a maximum of 0.07 (anhydrous
substance).
F. (4S,4aR,5S,5aR,6R,12aS)-2-acetyl-4-(dimethylamino)-
3,5,10,12,12a-pentahydroxy-6-methyl-4a,5a,6,12a- Dissolve 0.10 g in a mixture of 0.5 volumes of hydrochloric
tetrahydrotetracene-1,11(4H,5H)-dione (2-acetyl-2- acid R and 99.5 volumes of methanol R and dilute to 10.0 mL
decarbamoyldoxycycline). with the same mixture of solvents. Carry out the measurement
within 1 h of preparing the solution.
Related substances. Liquid chromatography (2.2.29). Prepare
01/2008:0820 the solutions immediately before use.
corrected 6.0
Test solution. Dissolve 20.0 mg of the substance to be
examined in 0.01 M hydrochloric acid and dilute to 25.0 mL
DOXYCYCLINE MONOHYDRATE with the same acid.
Reference solution (a). Dissolve 20.0 mg of doxycycline
Doxycyclinum monohydricum hyclate CRS in 0.01 M hydrochloric acid and dilute to 25.0 mL
with the same acid.
Reference solution (b). Dissolve 20.0 mg of 6-epidoxycycline
hydrochloride CRS in 0.01 M hydrochloric acid and dilute to
25.0 mL with the same acid.
Reference solution (c). Dissolve 20.0 mg of metacycline
hydrochloride CRS in 0.01 M hydrochloric acid and dilute to
25.0 mL with the same acid.
C22H24N2O8,H2O Mr 462.5 Reference solution (d). Mix 4.0 mL of reference solution (a),
[17086-28-1] 1.5 mL of reference solution (b) and 1.0 mL of reference
solution (c) and dilute to 25.0 mL with 0.01 M hydrochloric
DEFINITION
acid.
(4S,4aR,5S,5aR,6R,12aS)-4-(Dimethylamino)-3,5,10,12,12a- Reference solution (e). Mix 2.0 mL of reference solution (b)
pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a- and 2.0 mL of reference solution (c) and dilute to 100.0 mL
octahydrotetracene-2-carboxamide monohydrate. with 0.01 M hydrochloric acid.
Substance obtained from oxytetracycline or metacycline or by Column :
any other means.
– size : l = 0.25 m, Ø = 4.6 mm,
Semi-synthetic product derived from a fermentation product.
– stationary phase : styrene-divinylbenzene copolymer R
Content : 95.0 per cent to 102.0 per cent (anhydrous substance). (8 μm),
CHARACTERS – temperature : 60 °C.
Appearance : yellow, crystalline powder. Mobile phase : weigh 60.0 g of 2-methyl-2-propanol R and
Solubility : very slightly soluble in water and in alcohol. It transfer into a 1000 mL volumetric flask with the aid of
dissolves in dilute solutions of mineral acids and in solutions 200 mL of water R ; add 400 mL of buffer solution pH 8.0 R,
of alkali hydroxides and carbonates. 50 mL of a 10 g/L solution of tetrabutylammonium hydrogen
sulfate R adjusted to pH 8.0 with dilute sodium hydroxide
IDENTIFICATION solution R and 10 mL of a 40 g/L solution of sodium edetate R
A. Examine the chromatograms obtained in the assay. adjusted to pH 8.0 with dilute sodium hydroxide solution R ;
dilute to 1000.0 mL with water R.
Results : the principal peak in the chromatogram obtained
with the test solution is similar in retention time and size Flow rate : 1.0 mL/min.
to the principal peak in the chromatogram obtained with Detection : spectrophotometer at 254 nm.
reference solution (a). Injection : 20 μL ; inject the test solution and reference
B. To about 2 mg add 5 mL of sulfuric acid R. A yellow colour solutions (d) and (e).
develops. Relative retention with reference to doxycycline :
C. Dissolve 25 mg in a mixture of 0.2 mL of dilute nitric impurity E = about 0.2 ; impurity D = about 0.3 ;
acid R and 1.8 mL of water R. The solution does not give impurity C = about 0.5 ; impurity F = about 1.2.
reaction (a) of chlorides (2.3.1). System suitability : reference solution (d) :
TESTS – resolution : minimum 1.25 between the peaks due to
impurity B (1st peak) and impurity A (2nd peak) and
pH (2.2.3) : 5.0 to 6.5. minimum 2.0 between the peaks due to impurity A
Suspend 0.1 g in carbon dioxide-free water R and dilute to and doxycycline (3rd peak) ; if necessary, adjust the
10 mL with the same solvent. 2-methyl-2-propanol content in the mobile phase,

General Notices (1) apply to all monographs and other texts 2111
Doxylamine hydrogen succinate EUROPEAN PHARMACOPOEIA 8.0

– symmetry factor : maximum 1.25 for the peak due to 01/2013:1589

doxycycline.
Limits : DOXYLAMINE HYDROGEN
– impurity A : not more than the area of the corresponding SUCCINATE
peak in the chromatogram obtained with reference
solution (e) (2.0 per cent), Doxylamini hydrogenosuccinas
– impurity B : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (e) (2.0 per cent),
– any other impurity : not more than 0.25 times the area of
the peak due to impurity A in the chromatogram obtained
with reference solution (e) (0.5 per cent),
– disregard limit : 0.05 times the area of the peak due to
impurity A in the chromatogram obtained with reference
solution (e) (0.1 per cent). C21H28N2O5 Mr 388.5
[562-10-7]
Heavy metals (2.4.8) : maximum 50 ppm.
0.5 g complies with test C. Prepare the reference solution using DEFINITION
2.5 mL of lead standard solution (10 ppm Pb) R. N,N-Dimethyl-2-[(1RS)-1-phenyl-1-(pyridin-2-
Water (2.5.12) : 3.6 per cent to 4.6 per cent, determined on yl)ethoxy]ethanamine hydrogen butanedioate.
0.200 g. Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
Sulfated ash (2.4.14) : maximum 0.4 per cent, determined on CHARACTERS
1.0 g. Appearance : white or almost white powder.
ASSAY Solubility : very soluble in water, freely soluble in ethanol
Liquid chromatography (2.2.29) as described in the test for (96 per cent).
related substances with the following modification. It shows polymorphism (5.9).
Injection : test solution and reference solution (a). IDENTIFICATION
Calculate the percentage content of C22H24N2O8. Infrared absorption spectrophotometry (2.2.24).
STORAGE Comparison: doxylamine hydrogen succinate CRS.
Protected from light. If the spectra obtained show differences, dissolve the substance
to be examined and the reference substance separately in
IMPURITIES methanol R, evaporate to dryness and record new spectra
using the residues.
TESTS
Appearance of solution. The solution is clear (2.2.1) and
colourless (2.2.2, Method II).
Dissolve 0.4 g in water R and dilute to 20 mL with the same
solvent.
A. R1 = NH2, R2 = R5 = H, R3 = N(CH3)2, R4 = CH3 :
(4S,4aR,5S,5aR,6S,12aS)-4-(dimethylamino)-3,5,10,12,12a- Related substances. Gas chromatography (2.2.28).
pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a- Test solution. Dissolve 0.650 g of the substance to be examined
octahydrotetracene-2-carboxamide (6-epidoxycycline), in 20 mL of a 10.3 g/L solution of hydrochloric acid R. Add
3 mL of a 100 g/L solution of sodium hydroxide R and extract
B. R1 = NH2, R2 = H, R3 = N(CH3)2, R4 + R5 = CH2 : with 3 quantities, each of 25 mL, of methylene chloride R.
(4S,4aR,5S,5aR,12aS)-4-(dimethylamino)- Combine the methylene chloride extracts and filter using
3,5,10,12,12a-pentahydroxy-6-methylene-1,11-dioxo- hydrophobic phase-separation filter paper. Rinse the filter
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide with 10 mL of methylene chloride R and combine the rinsings
(metacycline), with the methylene chloride extracts. Evaporate the solvent
under reduced pressure at a temperature not exceeding 40 °C.
C. R1 = NH2, R2 = N(CH3)2, R3 = R4 = H, R5 = CH3 : Dissolve the residue in 20.0 mL of anhydrous ethanol R.
(4R,4aR,5S,5aR,6R,12aS)-4-(dimethylamino)-
Reference solution (a). Dilute 1.0 mL of the test solution to
3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-
100.0 mL with anhydrous ethanol R. Dilute 1.0 mL of this
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide
solution to 10.0 mL with anhydrous ethanol R.
(4-epidoxycycline),
Reference solution (b). Dissolve 50 mg of doxylamine for
D. R1 = NH2, R2 = N(CH3)2, R3 = R5 = H, R4 = CH3 : system suitability CRS (containing impurity C) in 10 mL of
(4R,4aR,5S,5aR,6S,12aS)-4-(dimethylamino)- a 10.3 g/L solution of hydrochloric acid R. Add 1.5 mL of a
3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo- 100 g/L solution of sodium hydroxide R and extract with 3
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide quantities, each of 25 mL, of methylene chloride R. Combine
(4-epi-6-epidoxycycline), the methylene chloride extracts and filter using hydrophobic
phase-separation filter paper. Rinse the filter with 10 mL
E. R1 = NH2, R2 = H, R3 = N(CH3)2, R4 = OH, R5 = CH3 : of methylene chloride R and combine the rinsings with the
oxytetracycline, methylene chloride extracts. Evaporate the solvent under
reduced pressure at a temperature not exceeding 40 °C.
F. R1 = CH3, R2 = R4 = H, R3 = N(CH3)2, R5 = CH3 : Dissolve the residue in 5.0 mL of anhydrous ethanol R.
(4S,4aR,5S,5aR,6R,12aS)-2-acetyl-4-(dimethylamino)-
3,5,10,12,12a-pentahydroxy-6-methyl-4a,5a,6,12a- Column :
tetrahydrotetracene-1,11(4H,5H)-dione (2-acetyl-2- – material : fused silica ;
decarbamoyldoxycycline). – size : l = 30 m, Ø = 0.53 mm ;

2112 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Droperidol

– stationary phase : poly(dimethyl)(diphenyl)siloxane R (film

thickness 1.5 μm).
Carrier gas : helium for chromatography R.
Flow rate : 7 mL/min.
Temperature :
Time Temperature B. (1RS)-1-phenyl-1-(pyridin-2-yl)ethanol,
(min) (°C)
Column 0 - 12 160 → 220

12 - 27 220

Injection port 250

Detector 250
C. N,N-dimethyl-2-[(1RS)-1-phenyl-1-(pyridin-2-
Detection : flame ionisation. yl)methoxy]ethanamine,
Injection : 1 μL.
Identification of impurities : use the chromatogram supplied
with doxylamine for system suitability CRS and the
chromatogram obtained with reference solution (b) to identify
the peak due to impurity C.
Relative retention with reference to doxylamine (retention
time = about 12 min) : impurity C = about 0.96.
D. phenyl(pyridin-2-yl)methanone (2-benzoylpyridine).
System suitability : reference solution (b) :
– resolution : minimum 1.5 between the peaks due to
impurity C and doxylamine. 07/2011:1010
Limits :
– impurity C : not more than 5 times the area of the principal DROPERIDOL
peak in the chromatogram obtained with reference
solution (a) (0.5 per cent) ; Droperidolum
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.10 per cent) ;
– total : not more than 7 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.7 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a) C22H22FN3O2 Mr 379.4
(0.05 per cent). [548-73-2]
Water (2.5.12) : maximum 0.5 per cent, determined on 2.00 g.
DEFINITION
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. 1-[1-[4-(4-Fluorophenyl)-4-oxobutyl]-1,2,3,6-tetrahydropyridin-
4-yl]-1,3-dihydro-2H-benzimidazol-2-one.
ASSAY Content : 99.0 per cent to 101.0 per cent (dried substance).
Dissolve 0.150 g in 50 mL of anhydrous acetic acid R. Titrate CHARACTERS
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20). Appearance : white or almost white powder.
1 mL of 0.1 M perchloric acid is equivalent to 19.43 mg of Solubility : practically insoluble in water, freely soluble in
C21H28N2O5. dimethylformamide and in methylene chloride, sparingly
soluble in ethanol (96 per cent).
IMPURITIES It shows polymorphism (5.9).
Specified impurities : C.
IDENTIFICATION
Other detectable impurities (the following substances would, First identification : A.
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general Second identification : B, C, D.
acceptance criterion for other/unspecified impurities and/or A. Infrared absorption spectrophotometry (2.2.24).
by the general monograph Substances for pharmaceutical use Comparison: droperidol CRS.
(2034). It is therefore not necessary to identify these impurities If the spectra obtained show differences, dissolve the
for demonstration of compliance. See also 5.10. Control of substance to be examined and the reference substance
impurities in substances for pharmaceutical use) : A, B, D. separately in the minimum volume of acetone R, evaporate
to dryness on a water-bath and record new spectra using
the residues.
B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 30 mg of the substance to be
examined in the mobile phase and dilute to 10 mL with
the mobile phase.
A. N,N-dimethyl-2-[(1RS)-1-phenyl-1-(pyridin-4- Reference solution (a). Dissolve 30 mg of droperidol CRS in
yl)ethoxy]ethanamine, the mobile phase and dilute to 10 mL with the mobile phase.

General Notices (1) apply to all monographs and other texts 2113
Droperidol EUROPEAN PHARMACOPOEIA 8.0

Reference solution (b). Dissolve 30 mg of droperidol CRS Relative retention with reference to droperidol
and 30 mg of benperidol CRS in the mobile phase and dilute (retention time = about 7 min) : impurity A = about 0.2 ;
to 10 mL with the mobile phase. impurity B = about 0.85 ; benperidol = about 0.9 ;
Plate : TLC silica gel GF254 plate R. impurity C = about 0.95 ; impurity D = about 1.2 ;
impurity E = about 1.5.
Mobile phase : acetone R, methanol R (10:90 V/V).
System suitability : reference solution (a) :
Application : 10 μL.
– resolution : minimum 2.0 between the peaks due to
Development : over 3/4 of the plate. benperidol and droperidol.
Drying : in air. Limits :
Detection : examine in ultraviolet light at 254 nm. – impurities A, B, C, D, E : for each impurity, not more
System suitability: reference solution (b) : than the area of the principal peak in the chromatogram
– the chromatogram shows 2 clearly separated spots. obtained with reference solution (b) (0.25 per cent) ;
Results : the principal spot in the chromatogram obtained – unspecified impurities : for each impurity, not more than
with the test solution is similar in position and size to 0.4 times the area of the principal peak in the chromatogram
the principal spot in the chromatogram obtained with obtained with reference solution (b) (0.10 per cent) ;
reference solution (a). – total : not more than twice the area of the principal peak
C. Dissolve about 10 mg in 5 mL of anhydrous ethanol R. in the chromatogram obtained with reference solution (b)
Add 0.5 mL of dinitrobenzene solution R and 0.5 mL of (0.5 per cent) ;
2 M alcoholic potassium hydroxide R. A violet colour is – disregard limit : 0.2 times the area of the principal peak in
produced and becomes brownish-red after 20 min. the chromatogram obtained with reference solution (b)
D. Mix about 5 mg with 45 mg of heavy magnesium oxide R (0.05 per cent).
and ignite in a crucible until an almost white residue is Heavy metals (2.4.8) : maximum 20 ppm.
obtained (usually less than 5 min). Allow to cool, add 1.0 g complies with test D. Prepare the reference solution
1 mL of water R, 0.05 mL of phenolphthalein solution R1 using 2 mL of lead standard solution (10 ppm Pb) R.
and about 1 mL of dilute hydrochloric acid R to render the
solution colourless. Filter. To a freshly prepared mixture Loss on drying (2.2.32) : maximum 0.5 per cent, determined
of 0.1 mL of alizarin S solution R and 0.1 mL of zirconyl on 1.000 g by drying in an oven at 105 °C.
nitrate solution R, add 1.0 mL of the filtrate. Mix, allow Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
to stand for 5 min and compare the colour of the solution 1.0 g.
with that of a blank prepared in the same manner. The test
solution is yellow and the blank is red. ASSAY
Dissolve 0.300 g in 50 mL of a mixture of 1 volume of
TESTS anhydrous acetic acid R and 7 volumes of methyl ethyl
Appearance of solution. The solution is clear (2.2.1) and not ketone R. Using 0.2 mL of naphtholbenzein solution R as
more intensely coloured than reference solution BY5 (2.2.2, indicator, titrate with 0.1 M perchloric acid until the colour
Method II). changes from orange-yellow to green.
Dissolve 0.20 g in methylene chloride R and dilute to 20.0 mL 1 mL of 0.1 M perchloric acid is equivalent to 37.94 mg
with the same solvent. of C22H22FN3O2.
Related substances. Liquid chromatography (2.2.29). Prepare STORAGE
the solutions immediately before use. Protected from light.
Test solution. Dissolve 0.10 g of the substance to be examined
in dimethylformamide R and dilute to 10.0 mL with the same IMPURITIES
solvent. Specified impurities : A, B, C, D, E.
Reference solution (a). Dissolve 2.5 mg of droperidol CRS and
2.5 mg of benperidol CRS in dimethylformamide R and dilute
to 100.0 mL with the same solvent.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with dimethylformamide R. Dilute 5.0 mL of this
solution to 20.0 mL with dimethylformamide R. A. 1-(1,2,3,6-tetrahydropyridin-4-yl)-1,3-dihydro-2H-
Column : benzimidazol-2-one,
– size : l = 0.10 m, Ø = 4.6 mm ;
– stationary phase : base-deactivated octadecylsilyl silica gel for
chromatography R (3 μm).
Mobile phase :
– mobile phase A : acetonitrile R ;
– mobile phase B : 10 g/L solution of tetrabutylammonium
hydrogen sulfate R1 ; B. 1-[1-[4-(2-fluorophenyl)-4-oxobutyl]-1,2,3,6-tetrahydro-
Time Mobile phase A Mobile phase B pyridin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one,
(min) (per cent V/V) (per cent V/V)
0 - 15 0 → 40 100 → 60

15 - 20 40 60

20 - 25 40 → 0 60 → 100

Flow rate : 1.5 mL/min.

Detection : spectrophotometer at 275 nm. C. 1-[4-(4-fluorophenyl)-4-oxobutyl]-4-(2-oxo-2,3-dihydro-
Injection : 10 μL. 1H-benzimidazol-1-yl)pyridinium chloride,

2114 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Drospirenone

Reference solution (a). Dilute 1.0 mL of the test solution

to 10.0 mL with the solvent mixture. Use 1.0 mL of this
solution to dissolve the contents of a vial of drospirenone
impurity E CRS.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
D. (1RS)-1-[4-(4-fluorophenyl)-4-oxobutyl]-4-(2-oxo-2,3- Reference solution (c). Dissolve 30.0 mg of drospirenone CRS
dihydro-1H-benzimidazol-1-yl)-1,2,3,6-tetrahydropyridine in the solvent mixture and dilute to 50.0 mL with the solvent
1-oxide, mixture.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : spherical end-capped octadecylsilyl silica
gel for chromatography R (3 μm) ;
– temperature : 35 °C.
Mobile phase :
– mobile phase A : water R ;
– mobile phase B : acetonitrile R ;
Time Mobile phase A Mobile phase B
E. 1-[1-[4-[4-[4-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)- (min) (per cent V/V) (per cent V/V)
3,6-dihydropyridin-1(2H)-yl]-1-oxobutyl]phenyl]-1,2,3,6- 0-2 63 37
tetrahydropyridin-4-yl]-1,3-dihydro-2H-benzimidazol-2-
one. 2 - 16 63 → 52 37 → 48

16 - 23 52 48

07/2009:2404 23 - 31 52 → 20 48 → 80

31 - 39 20 80
DROSPIRENONE
Flow rate : 1.0 mL/min.
Drospirenonum Detection : spectrophotometer at 245 nm.
Injection : 10 μL of the test solution and reference solutions (a)
and (b).
Relative retention with reference to drospirenone (retention
time = about 22 min): impurity E = about 1.1.
System suitability : reference solution (a) :
– resolution : minimum 5.0 between the peaks due to
drospirenone and impurity E.
Limits :
C24H30O3 Mr 366.5
[67392-87-4] – unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
DEFINITION with reference solution (b) (0.10 per cent) ;
3-Oxo-6α,7α,15α,16α-tetrahydro-3′H,3″H-dicyclopropa- – total : not more than 3 times the area of the principal peak
[6,7:15,16]-17α-pregn-4-en-21,17-carbolactone. in the chromatogram obtained with reference solution (b)
Content : 98.0 per cent to 102.0 per cent (dried substance). (0.3 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
CHARACTERS the chromatogram obtained with reference solution (b)
Appearance : white or almost white powder. (0.05 per cent).
Solubility : practically insoluble in water, freely soluble in Loss on drying (2.2.32) : maximum 0.5 per cent, determined
methylene chloride, soluble in methanol, sparingly soluble in on 1.000 g by drying in an oven at 105 °C for 3 h.
ethanol (96 per cent).
ASSAY
IDENTIFICATION Liquid chromatography (2.2.29) as described in the test for
A. Specific optical rotation (see Tests). related substances with the following modification.
B. Infrared absorption spectrophotometry (2.2.24). Injection : 10 μL of the test solution and reference solution (c).
Comparison : drospirenone CRS. Calculate the percentage content of C24H30O3 from the
declared content of drospirenone CRS.
TESTS
Specific optical rotation (2.2.7) : − 187 to − 193 (dried IMPURITIES
substance). Other detectable impurities (the following substances would,
Dissolve 0.100 g in methanol R and dilute to 10.0 mL with if present at a sufficient level, be detected by one or other of
the same solvent. the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
Related substances. Liquid chromatography (2.2.29).
by the general monograph Substances for pharmaceutical
Solvent mixture : acetonitrile R, water R (50:50 V/V). use (2034). It is therefore not necessary to identify these
Test solution. Dissolve 30.0 mg of the substance to be impurities for demonstration of compliance. See also 5.10.
examined in the solvent mixture and dilute to 50.0 mL with Control of impurities in substances for pharmaceutical use) : A,
the solvent mixture. B, C, D, E, F, G, H, I, K.

General Notices (1) apply to all monographs and other texts 2115
Duloxetine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

A. 3-oxo-15α,16α-dihydro-3′H-cyclopropa[15,16]-17α-pregn-
4-ene-21,17-carbolactone (6,7-desmethylenedrospirenone), H. 7β-(chloromethyl)-3-oxo-15α,16α-dihydro-3′H-
cyclopropa[15,16]pregn-4-ene-21,17-carbolactone
(3′-chloro-3′,6-seco-17-epidrospirenone),

B. 7β-(hydroxymethyl)-3-oxo-15α,16α-dihydro-3′H-
cyclopropa[15,16]-17α-pregn-4-ene-21,17-carbolactone
(7β-hydroxymethyl derivative), I. 7β-(hydroxymethyl)-15α,16α-dihydro-3′H-
cyclopropa[15,16]-17α-pregna-3,5-diene-21,17-
carbolactone (7β-hydroxymethyldiene derivative),

C. 6α,7α,15α,16α-tetrahydro-3′H,3″H -dicyclopropa-
[6,7:15,16]androst-4-ene-3,17-dione (17-keto derivative),
K. 3-oxo-6β,7β,15α,16α-tetrahydro-3′H,3″H-dicyclopropa-
[6,7:15,16]-17α-pregn-4-ene-21,17-carbolactone
(6α,7α-drospirenone).

07/2012:2594

DULOXETINE HYDROCHLORIDE
D. 3-oxo-15α,16α-dihydro-3′H-cyclopropa[15,16]-17α-
pregna-4,6-diene-21,17-carbolactone (Δ6-drospirenone),
Duloxetini hydrochloridum

C18H20ClNOS Mr 333.9
E. 3-oxo-6α,7α,15α,16α-tetrahydro-3′H,3″H - [136434-34-9]
dicyclopropa[6,7:15,16]pregn-4-ene-21,17-carbolactone
(17-epidrospirenone), DEFINITION
(3S)-N-Methyl-3-(naphthalen-1-yloxy)-3-(thiophen-2-
yl)propan-1-amine hydrochloride.
Content : 97.5 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white powder.
Solubility : sparingly soluble in water, freely soluble in
methanol, practically insoluble in hexane.
F. 15β-methyl-3-oxo-6α,7α-dihydro-3′H-cyclopropa[6,7]-
17α-pregn-4-ene-21,17-carbolactone (3″-16- IDENTIFICATION
secodrospirenone), Carry out either tests A, B, D or tests B, C, D.
A. Specific optical rotation (2.2.7) : + 119 to + 127 (dried
substance).
Dissolve 0.250 g in methanol R and dilute to 25.0 mL with
the same solvent. Examine within 30 min of preparing the
solution.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: duloxetine hydrochloride CRS.
G. 7β-(chloromethyl)-3-oxo-15α,16α-dihydro-3′H- C. Enantiomeric purity (see Tests).
cyclopropa[15,16]-17α-pregn-4-ene-21,17-carbolactone D. Dissolve 25 mg in 5 mL of methanol R. The solution gives
(3′-chloro-3′,6-secodrospirenone), reaction (a) of chlorides (2.3.1).

2116 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Duloxetine hydrochloride

TESTS the same solution : dissolve 2.9 g (1.7 mL) of phosphoric acid R
Enantiomeric purity. Liquid chromatography (2.2.29). in 900 mL of water R, adjust to pH 2.5 with dilute sodium
hydroxide solution R and dilute to 1000 mL with water R.
Test solution. Dissolve 5.0 mg of the substance to be examined
Mobile phase : acetonitrile R1, propanol R, hexanesulfonate
in the mobile phase and dilute to 100.0 mL with the mobile
solution (13:17:70 V/V/V).
phase.
Flow rate : 1.0 mL/min.
Reference solution (a). Dilute 1.0 mL of the test solution to
200.0 mL with the mobile phase. Detection : spectrophotometer at 230 nm.
Injection : 20 μL of test solution (a) and reference solutions (a)
Reference solution (b). Dissolve 5 mg of duloxetine
and (b).
impurity A CRS and 5 mg of the substance to be examined in
100.0 mL of the mobile phase. Run time : 2.5 times the retention time of duloxetine.
Column : Identification of impurities : use the chromatogram
supplied with duloxetine for system suitability CRS and the
– size : l = 0.25 m, Ø = 4.6 mm ; chromatogram obtained with reference solution (b) to identify
– stationary phase : silica gel OD for chiral separations R the peaks due to impurities C, D and F.
(5 μm) ; Relative retention with reference to duloxetine (retention
– temperature : 40 °C. time = about 16 min) : impurity C = about 0.4 ;
impurity D = about 0.5 ; impurity F = about 1.1.
Mobile phase : add 2.0 mL of diethylamine R to 1000 mL of
a mixture of 17 volumes of 2-propanol R and 83 volumes of System suitability : reference solution (b) :
hexane R. – resolution : minimum 1.5 between the peaks due to
impurities C and D ;
Flow rate : 1.0 mL/min.
– peak-to-valley ratio : minimum 4.0, where Hp = height
Detection : spectrophotometer at 230 nm. above the baseline of the peak due to impurity F and
Injection : 20 μL. Hv = height above the baseline of the lowest point of the
Relative retention with reference to duloxetine (retention curve separating this peak from the peak due to duloxetine.
time = about 7 min) : impurity A = about 1.3. Limits :
System suitability : – impurity F : not more than 4 times the area of the principal
peak in the chromatogram obtained with reference
– resolution : minimum 3.5 between the peaks due to solution (a) (0.4 per cent) ;
duloxetine and impurity A in the chromatogram obtained
with reference solution (b) ; – unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
– signal-to-noise ratio : minimum 10 for the principal peak in with reference solution (a) (0.10 per cent) ;
the chromatogram obtained with reference solution (a). – total : not more than 5 times the area of the principal peak
Limit : in the chromatogram obtained with reference solution (a)
– impurity A : not more than the area of the principal peak (0.5 per cent) ;
in the chromatogram obtained with reference solution (a) – disregard limit : 0.5 times the area of the principal peak in
(0.5 per cent). the chromatogram obtained with reference solution (a)
(0.05 per cent).
Related substances. Liquid chromatography (2.2.29).
Carry out the test protected from light. Prepare the solutions Heavy metals (2.4.8) : maximum 10 ppm.
immediately before use. Solvent : methanol R.
Solvent mixture : acetonitrile R1, water R (25:75 V/V). 0.250 g complies with test H. Prepare the reference solution
Test solution (a). Dissolve 20 mg of the substance to be using 250 μL of lead standard solution (10 ppm Pb) R.
examined in 200.0 mL of the solvent mixture. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Test solution (b). Dissolve 50.0 mg of the substance to be on 1.000 g by drying in an oven at 105 °C for 3 h.
examined in 100.0 mL of the solvent mixture. Dilute 1.0 mL Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
of the solution to 10.0 mL with the solvent mixture. 1.0 g.
Reference solution (a). Dilute 1.0 mL of test solution (a) to ASSAY
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10.0 mL with the mobile phase. Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Reference solution (b). Dissolve 20 mg of duloxetine for system
suitability CRS (containing impurity F) in the mobile phase Injection : test solution (b) and reference solution (c).
and dilute to 200.0 mL with the mobile phase. In order to Calculate the percentage content of C18H20ClNOS taking into
prepare impurities C and D in situ, heat the solution at 60 °C account the assigned content of duloxetine hydrochloride CRS.
for 1 h (solution containing impurities C, D and F).
STORAGE
Reference solution (c). Dissolve 50.0 mg of duloxetine
hydrochloride CRS in 100.0 mL of the solvent mixture. Dilute Protected from light.
1.0 mL of the solution to 10.0 mL with the solvent mixture. IMPURITIES
Column : Specified impurities : A, F.
– size : l = 0.15 m, Ø = 4.6 mm ; Other detectable impurities (the following substances would,
– stationary phase : spherical octylsilyl silica gel for if present at a sufficient level, be detected by one or other of
chromatography R (3.5 μm) ; the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
– temperature : 40 °C. by the general monograph Substances for pharmaceutical use
Hexanesulfonate solution : dissolve 10.3 g of sodium (2034). It is therefore not necessary to identify these impurities
hexanesulfonate monohydrate for ion-pair chromatography R for demonstration of compliance. See also 5.10. Control of
in a solution prepared as follows and dilute to 1000.0 mL with impurities in substances for pharmaceutical use) : B, C, D, E, G.

General Notices (1) apply to all monographs and other texts 2117
Dutasteride EUROPEAN PHARMACOPOEIA 8.0

Content : 97.0 per cent to 102.0 per cent (anhydrous substance).

CHARACTERS
Appearance : white or pale yellow powder.
Solubility : practically insoluble in water, freely soluble in
A. (3R)-N-methyl-3-(naphthalen-1-yloxy)-3-(thiophen-2- methylene chloride, soluble or sparingly soluble in anhydrous
yl)propan-1-amine, ethanol.
IDENTIFICATION
A. Specific optical rotation (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: dutasteride CRS.
B. (1S)-3-(methylamino)-1-(thiophen-2-yl)propan-1-ol,
TESTS
Specific optical rotation (2.2.7) : + 33.0 to + 39.0 (anhydrous
substance).
Dissolve 0.100 g in anhydrous ethanol R and dilute to 20.0 mL
with the same solvent.
Related substances
C. 4-[(1RS)-3-(methylamino)-1-(thiophen-2-
yl)propyl]naphthalen-1-ol, A. Liquid chromatography (2.2.29).
Solvent mixture : water for chromatography R, acetonitrile R1
(40:60 V/V).
Test solution. Dissolve 50.0 mg of the substance to be
examined in the solvent mixture and dilute to 100.0 mL
with the solvent mixture.
D. naphthalen-1-ol, Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Reference solution (b). Dissolve 5 mg of dutasteride for
system suitability CRS (containing impurities A, B, C, E,
F, G, H and I) in the solvent mixture and dilute to 10 mL
with the solvent mixture.
E. 2-[(1RS)-3-(methylamino)-1-(thiophen-2- Reference solution (c). Dissolve 50.0 mg of dutasteride CRS
yl)propyl]naphthalen-1-ol, in the solvent mixture and dilute to 100.0 mL with the
solvent mixture.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm) ;
F. (3S)-N-methyl-3-(naphthalen-1-yloxy)-3-(thiophen-3- – temperature : 35 °C.
yl)propan-1-amine,
Mobile phase : mix 0.25 volumes of trifluoroacetic
acid R, 480 volumes of water for chromatography R and
520 volumes of acetonitrile R1.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 220 nm.
G. 1-fluoronaphthalene. Injection : 20 μL of the test solution and reference
solutions (a) and (b).
01/2014:2641 Run time : 1.6 times the retention time of dutasteride.
Identification of impurities : use the chromatogram
DUTASTERIDE supplied with dutasteride for system suitability CRS and
the chromatogram obtained with reference solution (b) to
Dutasteridum identify the peaks due to impurities A, B, C, E, F and G.
Relative retention with reference to dutasteride (retention
time = about 36 min) : impurity A = about 0.10 ;
impurity B = about 0.11 ; impurity C = about 0.4 ;
impurity E = about 0.9 ; impurity F = about 1.1 ;
impurity G = about 1.2.
System suitability :
– resolution : minimum 1.5 between the peaks due
C27H30F6N2O2 Mr 528.5 to impurity E and dutasteride and minimum 1.5
[164656-23-9] between the peaks due to impurities A and B in the
chromatogram obtained with reference solution (b) ;
DEFINITION – signal-to-noise ratio : minimum 30 for the peak due
N-[2,5-Bis(trifluoromethyl)phenyl]-3-oxo-4-aza-5α-androst- to dutasteride in the chromatogram obtained with
1-ene-17β-carboxamide. reference solution (a).

2118 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dutasteride

Calculation of percentage contents : IMPURITIES

– correction factors : multiply the peak areas of the Specified impurities : A, B, C, E, F, G, H, I.
following impurities by the corresponding correction Other detectable impurities (the following substances would,
factor : impurity B = 0.7 ; impurity F = 3.0 ; if present at a sufficient level, be detected by one or other of
– for each impurity, use the concentration of dutasteride the tests in the monograph. They are limited by the general
in reference solution (a). acceptance criterion for other/unspecified impurities and/or
Limits : by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
– impurity F : maximum 0.4 per cent ; impurities for demonstration of compliance. See also 5.10.
– impurities E, G : for each impurity, maximum 0.3 per Control of impurities in substances for pharmaceutical use) : D.
cent ;
– impurities A, C : for each impurity, maximum 0.2 per
cent ;
– impurity B : maximum 0.15 per cent ;
– unspecified impurities : for each impurity, maximum
0.10 per cent ;
– reporting threshold : 0.05 per cent.
A. 3-oxo-4-aza-5α-androst-1-ene-17β-carboxylic acid,
B. Liquid chromatography (2.2.29) as described in test A for
related substances with the following modifications.
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : phenylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : water for chromatography R, acetonitrile R1
(20:80 V/V).
Injection : 10 μL of the test solution and reference B. N,N-dimethyl-3-oxo-4-aza-5α-androst-1-ene-17β-
solutions (a) and (b). carboxamide,
Run time : 5 times the retention time of dutasteride.
Identification of impurities : use the chromatogram
supplied with dutasteride for system suitability CRS and
the chromatogram obtained with reference solution (b) to
identify the peaks due to impurities H and I.
Relative retention with reference to dutasteride
(retention time = about 4 min) : impurity H = about 3.4 ;
impurity I = about 3.9.
C. ethyl 3-oxo-4-aza-5α-androst-1-ene-17β-carboxylate,
System suitability: reference solution (b) :
– resolution : minimum 2.0 between the peaks due to
impurities H and I.
Calculation of percentage contents :
– for each impurity, use the concentration of dutasteride
in reference solution (a).
Limits :
– impurity I : maximum 0.5 per cent ;
D. N-[2,5-bis(trifluoromethyl)phenyl]-3-oxo-4-azaandrost-
– impurity H : maximum 0.3 per cent ; 1,5-diene-17α-carboxamide,
– unspecified impurities eluting after dutasteride : for each
impurity, maximum 0.10 per cent ;
– reporting threshold : 0.05 per cent.
Limit :
– total for tests A and B : maximum 1.5 per cent.
Water (2.5.32) : maximum 0.2 per cent, determined on 0.100 g
using the evaporation technique :
– temperature : 180 °C ; E. N-[2,5-bis(trifluoromethyl)phenyl]-3-oxo-4-aza-5α-
– heating time : 4 min. androst-1-ene-17α-carboxamide,
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g in a platinum crucible.
ASSAY
Liquid chromatography (2.2.29) as described in test A for
related substances with the following modification.
Injection : 10 μL of the test solution and reference solution (c).
Calculate the percentage content of C27H30F6N2O2 taking into F. N-[2,5-bis(trifluoromethyl)phenyl]-1α-chloro-3-oxo-4-aza-
account the assigned content of dutasteride CRS. 5α-androstane-17β-carboxamide,

General Notices (1) apply to all monographs and other texts 2119
Dydrogesterone EUROPEAN PHARMACOPOEIA 8.0

TESTS
Specific optical rotation (2.2.7) : − 469 to − 485 (dried
substance), measured at 25 °C.
Dissolve 0.100 g in methylene chloride R and dilute to 20.0 mL
with the same solvent.
Related substances. Liquid chromatography (2.2.29).
G. N-[2,5-bis(trifluoromethyl)phenyl]-3-oxo-4-azaandrost- Test solution (a). Dissolve 50.0 mg of the substance to be
1,5-diene-17β-carboxamide, examined in the mobile phase and dilute to 100.0 mL with
the mobile phase.
Test solution (b). Dissolve 20.0 mg of the substance to be
examined in the mobile phase and dilute to 100.0 mL with
the mobile phase.
Reference solution (a). Dissolve 3.0 mg of dydrogesterone
impurity A CRS in the mobile phase and dilute to 20.0 mL with
the mobile phase. Dilute 1.0 mL of this solution to 100.0 mL
with the mobile phase.
Reference solution (b). Dilute 1.0 mL of test solution (a) to
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10.0 mL with the mobile phase.
Reference solution (c). Dissolve 10 mg of the substance to be
examined in 10 mL of reference solution (a).
H. N-[2,5-bis(trifluoromethyl)phenyl]-3-oxo-4-[3-oxo-4-aza- Reference solution (d). Dissolve 10 mg of the substance to be
5α-androst-1-ene-17α-carbonyl]-4-aza-5α-androst-1-ene- examined in 30 mL of ethanol (96 per cent) R. Add 1 mL of a
17β-carboxamide (dutasteride dimer 1), 8.4 g/L solution of sodium hydroxide R and heat at 85 °C for
10 min. Cool to room temperature, add 1 mL of a 20.6 g/L
solution of hydrochloric acid R, add 20 mL of acetonitrile R,
2 mg of dydrogesterone impurity B CRS, dilute to 100 mL with
water R and mix. This solution contains dydrogesterone and
impurities B and C.
Reference solution (e). Dissolve 20.0 mg of dydrogesterone CRS
in the mobile phase and dilute to 100.0 mL with the mobile
phase.
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : spherical end-capped octadecylsilyl silica
gel for chromatography R (3 μm) ;
I. N-[2,5-bis(trifluoromethyl)phenyl]-3-oxo-4-[3-oxo-4-aza- – temperature : 40 °C.
5α-androst-1-ene-17β-carbonyl]-4-aza-5α-androst-1-ene- Mobile phase : acetonitrile R, ethanol (96 per cent) R, water R
17β-carboxamide (dutasteride dimer 2). (21:25:54 V/V/V).
Flow rate : 1.0 mL/min.
01/2009:2357 Detection : spectrophotometer at 280 nm and at 385 nm.
Injection : 10 μL of test solution (a) and reference solutions (a),
DYDROGESTERONE (b), (c) and (d).
Run time : twice the retention time of dydrogesterone.
Dydrogesteronum Relative retention at 385 nm with reference to dydrogesterone
(retention time = about 13 min) : impurity A = about 0.9.
Relative retention at 280 nm with reference to dydrogesterone
(retention time = about 13 min) : impurity B = about 1.1 ;
impurity C = about 1.2.
System suitability :
– resolution at 385 nm : minimum 1.1 between the peaks due
to impurity A and dydrogesterone in the chromatogram
obtained with reference solution (c) ;
C21H28O2 Mr 312.5
[152-62-5] – resolution at 280 nm : minimum 4.5 between the peaks
due to dydrogesterone and impurity B and minimum 1.5
DEFINITION between the peaks due to impurity B and impurity C in the
9β,10α-Pregna-4,6-diene-3,20-dione. chromatogram obtained with reference solution (d).
Content : 98.0 per cent to 102.0 per cent (dried substance). Limits :
– impurity A at 385 nm : not more than the area of the
CHARACTERS corresponding peak in the chromatogram obtained with
Appearance : white or almost white, crystalline powder. reference solution (a) (0.3 per cent) ;
Solubility : practically insoluble in water, soluble in acetone, – impurity B at 280 nm : not more than 1.5 times the area
sparingly soluble in ethanol (96 per cent). of the principal peak in the chromatogram obtained with
reference solution (b) (0.15 per cent) ;
IDENTIFICATION – impurity C at 280 nm : not more than 3 times the area of
Infrared absorption spectrophotometry (2.2.24). the principal peak in the chromatogram obtained with
Comparison : dydrogesterone CRS. reference solution (b) (0.3 per cent) ;

2120 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Dydrogesterone

– unspecified impurities at 280 nm : for each impurity,

not more than the area of the principal peak in the
chromatogram obtained with reference solution (b)
(0.10 per cent) ;
– total at 280 nm : not more than 5 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent) ;
– disregard limit at 280 nm : 0.5 times the area of the principal A. 9β,10α-pregna-4,6,8(14)-triene-3,20-dione,
peak in the chromatogram obtained with reference
solution (b) (0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 3 h.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY
Liquid chromatography (2.2.29) as described in the test for B. pregna-4,6-diene-3,20-dione,
related substances with the following modifications.
Detection : spectrophotometer at 280 nm.
Injection : test solution (b) and reference solution (e).
Calculate the percentage content of C21H28O2 from the
declared content of dydrogesterone CRS.
IMPURITIES
Specified impurities : A, B, C. C. 9β,10α,17α-pregna-4,6-diene-3,20-dione.

General Notices (1) apply to all monographs and other texts 2121
EUROPEAN PHARMACOPOEIA 8.0 Ebastine

01/2008:2015 Limits :
– impurities A, B, C, D, E, F, G : for each impurity, not more
EBASTINE than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.1 per cent),
Ebastinum – any other impurity : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.1 per cent),
– total : not more than 4 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.4 per cent),
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
C32H39NO2 Mr 469.7 Sulfates (2.4.13) : maximum 100 ppm.
[90729-43-4]
Suspend 2.5 g in 25 mL of dilute nitric acid R. Boil under a
DEFINITION reflux condenser for 10 min. Cool and filter. 15 mL of the
1-[4-(1,1-Dimethylethyl)phenyl]-4-[4-(diphenylmethoxy)- filtrate complies with the limit test for sulfates.
piperidin-1-yl]butan-1-one. Water (2.5.12) : maximum 0.5 per cent, determined on 0.500 g.
Content : 99.0 per cent to 101.0 per cent (anhydrous substance). Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
CHARACTERS 1.0 g.
Appearance : white or almost white, crystalline powder. ASSAY
Solubility : practically insoluble in water, very soluble in Dissolve 0.350 g in 50 mL of anhydrous acetic acid R. Titrate
methylene chloride, sparingly soluble in methanol. with 0.1 M perchloric acid, determining the end-point
mp : about 86 °C. potentiometrically (2.2.20).
IDENTIFICATION 1 mL of 0.1 M perchloric acid is equivalent to 46.97 mg
of C32H39NO2.
Infrared absorption spectrophotometry (2.2.24).
Comparison : Ph. Eur. reference spectrum of ebastine. STORAGE
TESTS Protected from light.
Related substances. Liquid chromatography (2.2.29). Keep IMPURITIES
the solutions protected from light.
Solution A. Mix 65 volumes of acetonitrile R and 35 volumes
of a 1.1 g/L solution of phosphoric acid R adjusted to pH 5.0
with a 40 g/L solution of sodium hydroxide R.
Test solution. Dissolve 0.125 g of the substance to be examined
in solution A and dilute to 50.0 mL with the same solution.
Reference solution (a). Dissolve 5.0 mg of ebastine
impurity C CRS and 5.0 mg of ebastine impurity D CRS in A. R1–H : diphenylmethanol (benzhydrol),
solution A and dilute to 20.0 mL with the same solution. B. R2–CH3 : 1-[4-(1,1-dimethylethyl)phenyl]ethanone,
Dilute 1.0 mL of the solution to 100.0 mL with solution A.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with solution A. Dilute 1.0 mL of this solution to
10.0 mL with solution A.
Column : C. 4-(diphenylmethoxy)piperidine,
– size : l = 0.25 m, Ø = 4.6 mm,
– stationary phase : nitrile silica gel for chromatography R
(5 μm).
Mobile phase : mix 35 volumes of acetonitrile R and 65 volumes
of a 1.1 g/L solution of phosphoric acid R adjusted to pH 5.0 D. 1-[4-(1,1-dimethylethyl)phenyl]-4-(4-hydroxypiperidin-1-
with a 40 g/L solution of sodium hydroxide R. Adjust the yl)butan-1-one,
percentage of acetonitrile to between 30 per cent V/V and
40 per cent V/V so that the retention time of ebastine is about
110 min.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 210 nm.
Injection : 10 μL.
Run time : 1.4 times the retention time of ebastine. E. 1-[4-(1,1-dimethylpropyl)phenyl]-4-[4-(diphenyl-
Relative retention with reference to ebastine : methoxy)piperidin-1-yl]butan-1-one,
impurity A = about 0.04 ; impurity B = about 0.05 ;
impurity D = about 0.20 ; impurity C = about 0.22 ;
impurity F = about 0.42 ; impurity G = about 0.57 ;
impurity E = about 1.14.
System suitability: reference solution (a) :
– resolution : minimum 2.0 between the peaks due to F. 1-[4-(1,1-dimethylethyl)phenyl]-4-[cis-4-
impurity D and impurity C. (diphenylmethoxy)-1-oxidopiperidin-1-yl]butan-1-one,

General Notices (1) apply to all monographs and other texts 2125
Econazole EUROPEAN PHARMACOPOEIA 8.0

Flow rate : 1.5 mL/min.

Detection : spectrophotometer at 225 nm.
Injection : 10 μL.
Identification of impurities : use the chromatogram
G. 1-[4-(1,1-dimethylethyl)phenyl]-4-[trans-4- supplied with econazole for system suitability CRS and the
(diphenylmethoxy)-1-oxidopiperidin-1-yl]butan-1-one. chromatogram obtained with reference solution (a) to identify
the peaks due to impurities A, B and C.
Relative retention with reference to econazole (retention
07/2010:2049 time = about 15 min) : impurity A = about 0.2 ;
corrected 7.0 impurity B = about 0.6 ; impurity C = about 1.1.
System suitability : reference solution (a) :
ECONAZOLE – peak-to-valley ratio : minimum 1.5, where Hp = height
above the baseline of the peak due to impurity C and
Hv = height above the baseline of the lowest point of the
Econazolum curve separating this peak from the peak due to econazole.
Limits :
– correction factor : for the calculation of content, multiply
the peak area of impurity A by 1.4 ;
– impurities A, B, C : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.2 per cent) ;
– unspecified impurities : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram
C18H15Cl3N2O Mr 381.7 obtained with reference solution (b) (0.10 per cent) ;
[27220-47-9] – total : not more than 1.5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
DEFINITION (0.3 per cent) ;
1-[(2RS)-2-[(4-Chlorobenzyl)oxy]-2-(2,4-dichlorophenyl)- – disregard limit : 0.25 times the area of the principal peak
ethyl]-1H-imidazole. in the chromatogram obtained with reference solution (b)
Content : 99.0 per cent to 101.0 per cent (dried substance). (0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
CHARACTERS on 1.000 g by drying in vacuo at 60 °C for 4 h.
Appearance : white or almost white powder.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Solubility : practically insoluble in water, very soluble in 1.0 g.
ethanol (96 per cent) and in methylene chloride.
ASSAY
IDENTIFICATION Dissolve 0.300 g in 75 mL of anhydrous acetic acid R. Titrate
A. Melting point (2.2.14) : 88 °C to 92 °C. with 0.1 M perchloric acid, determining the end-point
B. Infrared absorption spectrophotometry (2.2.24). potentiometrically (2.2.20). Carry out a blank titration.
Comparison : econazole CRS. 1 mL of 0.1 M perchloric acid is equivalent to 38.17 mg of
C18H15Cl3N2O.
TESTS
STORAGE
Related substances. Liquid chromatography (2.2.29).
Protected from light.
Test solution. Dissolve 0.100 g of the substance to be examined
in methanol R and dilute to 10.0 mL with the same solvent. IMPURITIES
Reference solution (a). Dissolve 10 mg of econazole for Specified impurities : A, B, C.
system suitability CRS (containing impurities A, B and C) in
methanol R and dilute to 1.0 mL with the same solvent.
Reference solution (b). Dilute 1.0 mL of the test solution to
20.0 mL with methanol R. Dilute 1.0 mL of this solution to
25.0 mL with methanol R.
Column :
– size : l = 0.10 m, Ø = 4.6 mm ;
– stationary phase : base-deactivated octadecylsilyl silica gel for
chromatography R (3 μm) ;
A. (1RS)-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-
– temperature : 35 °C. yl)ethanol,
Mobile phase :
– mobile phase A : methanol R, 0.77 g/L solution of ammonium
acetate R (20:80 V/V) ;
– mobile phase B : methanol R, acetonitrile R (40:60 V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 25 60 → 10 40 → 90

25 - 27 10 90 B. (2RS)-2-[(4-chlorobenzyl)oxy]-2-(2,4-dichlorophenyl)-
ethanamine,

2126 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Econazole nitrate

Mobile phase :
– mobile phase A : methanol R, 0.77 g/L solution of ammonium
acetate R (20:80 V/V) ;
– mobile phase B : methanol R, acetonitrile R (40:60 V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 25 60 → 10 40 → 90

25 - 27 10 90

C. 1-(4-chlorobenzyl)-3-[(2RS)-2-[(4-chlorobenzyl)oxy]-2- Flow rate : 1.5 mL/min.

(2,4-dichlorophenyl)ethyl]imidazolium. Detection : spectrophotometer at 225 nm.
Injection : 10 μL.
Identification of impurities : use the chromatogram
supplied with econazole for system suitability CRS and the
07/2010:0665 chromatogram obtained with reference solution (a) to identify
corrected 7.0 the peaks due to impurities A, B and C.
Relative retention with reference to econazole (retention
ECONAZOLE NITRATE time = about 15 min) : impurity A = about 0.2 ;
impurity B = about 0.6 ; impurity C = about 1.1.
System suitability : reference solution (a) :
Econazoli nitras – peak-to-valley ratio : minimum 1.5, where Hp = height
above the baseline of the peak due to impurity C and
Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to econazole.
Limits :
– correction factor : for the calculation of content, multiply
the peak area of impurity A by 1.4 ;
– impurities A, B, C : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.2 per cent) ;
C18H16Cl3N3O4 Mr 444.7 – unspecified impurities : for each impurity, not more than
[24169-02-6] 0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.10 per cent) ;
DEFINITION
– total : not more than 1.5 times the area of the principal peak
1-[(2RS)-2-[(4-Chlorobenzyl)oxy]-2-(2,4-dichlorophenyl)- in the chromatogram obtained with reference solution (b)
ethyl]-1H-imidazole nitrate. (0.3 per cent) ;
Content : 99.0 per cent to 101.0 per cent (dried substance). – disregard limit : 0.25 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
CHARACTERS (0.05 per cent) ; disregard the peak due to the nitrate ion at
Appearance : white or almost white, crystalline powder. the beginning of the chromatogram.
Solubility : very slightly soluble in water, soluble in methanol, Loss on drying (2.2.32) : maximum 0.5 per cent, determined
sparingly soluble in methylene chloride, slightly soluble in on 1.000 g by drying in an oven at 105 °C for 4 h.
ethanol (96 per cent). Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
mp : about 165 °C, with decomposition. 1.0 g.
ASSAY
IDENTIFICATION Dissolve 0.400 g in 50 mL of anhydrous acetic acid R. Titrate
Infrared absorption spectrophotometry (2.2.24). with 0.1 M perchloric acid, determining the end-point
Comparison : econazole nitrate CRS. potentiometrically (2.2.20). Carry out a blank titration.
1 mL of 0.1 M perchloric acid is equivalent to 44.47 mg of
TESTS C18H16Cl3N3O4.
Related substances. Liquid chromatography (2.2.29). STORAGE
Test solution. Dissolve 0.100 g of the substance to be examined Protected from light.
in methanol R and dilute to 10.0 mL with the same solvent.
IMPURITIES
Reference solution (a). Dissolve 10 mg of econazole for
system suitability CRS (containing impurities A, B and C) in Specified impurities : A, B, C.
methanol R and dilute to 1.0 mL with the same solvent.
Reference solution (b). Dilute 1.0 mL of the test solution to
20.0 mL with methanol R. Dilute 1.0 mL of this solution to
25.0 mL with methanol R.
Column :
– size : l = 0.10 m, Ø = 4.6 mm ;
– stationary phase : base-deactivated octadecylsilyl silica gel for
chromatography R (3 μm) ; A. (1RS)-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-
– temperature : 35 °C. yl)ethanol,

General Notices (1) apply to all monographs and other texts 2127
Edetic acid EUROPEAN PHARMACOPOEIA 8.0

Appearance of solution. Solution S is clear (2.2.1) and

colourless (2.2.2, Method II).
Impurity A. Liquid chromatography (2.2.29). Carry out the
test protected from light.
Solvent mixture. Dissolve 10.0 g of ferric sulfate pentahydrate R
in 20 mL of 0.5 M sulfuric acid and add 780 mL of water R.
Adjust to pH 2.0 with 1 M sodium hydroxide and dilute to
B. (2RS)-2-[(4-chlorobenzyl)oxy]-2-(2,4-dichlorophenyl)- 1000 mL with water R.
ethanamine, Test solution. Dissolve 0.100 g of the substance to be examined
in 1.0 mL of 1 M sodium hydroxide and dilute to 25.0 mL with
the solvent mixture.
Reference solution. Dissolve 40.0 mg of nitrilotriacetic acid R
in the solvent mixture and dilute to 100.0 mL with the solvent
mixture. To 1.0 mL of the solution add 0.1 mL of the test
solution and dilute to 100.0 mL with the solvent mixture.
Column :
– size : l = 0.10 m, Ø = 4.6 mm,
– stationary phase : spherical graphitised carbon for
C. 1-(4-chlorobenzyl)-3-[(2RS)-2-[(4-chlorobenzyl)oxy]-2- chromatography R1 (5 μm) with a specific surface area of
(2,4-dichlorophenyl)ethyl]imidazolium. 120 m2/g and a pore size of 25 nm.
Mobile phase : dissolve 50.0 mg of ferric sulfate pentahydrate R
in 50 mL of 0.5 M sulfuric acid and add 750 mL of water R.
01/2008:1612 Adjust to pH 1.5 with 0.5 M sulfuric acid or 1 M sodium
hydroxide, add 20 mL of ethylene glycol R and dilute to
EDETIC ACID 1000 mL with water R.
Flow rate : 1 mL/min.
Acidum edeticum Detection : spectrophotometer at 273 nm.
Injection : 20 μL ; filter the solutions and inject immediately.
Run time : 4 times the retention time of the iron complex of
impurity A.
Retention time : iron complex of impurity A = about 5 min ;
iron complex of edetic acid = about 10 min.
C10H16N2O8 Mr 292.2 System suitability : reference solution :
[60-00-4] – resolution : minimum 7 between the peaks due to the iron
DEFINITION complex of impurity A and the iron complex of edetic acid,
(Ethylenedinitrilo)tetraacetic acid. – signal-to-noise ratio : minimum 50 for the peak due to
Content : 98.0 per cent to 101.0 per cent. impurity A.
Limit :
CHARACTERS – impurity A : not more than the area of the corresponding
Appearance : white or almost white, crystalline powder or peak in the chromatogram obtained with the reference
colourless crystals. solution (0.1 per cent).
Solubility : practically insoluble in water and in ethanol (96 per Chlorides (2.4.4) : maximum 200 ppm.
cent). It dissolves in dilute solutions of alkali hydroxides.
To 10 mL of solution S add 8 mL of nitric acid R and stir for
IDENTIFICATION 10 min. A precipitate is formed. Filter and wash the filter with
First identification : A. water R. Collect the filtrate and the washings and dilute to
20 mL with water R. Dilute 10 mL of this solution to 15 mL
Second identification : B, C. with water R.
A. Infrared absorption spectrophotometry (2.2.24).
Iron (2.4.9) : maximum 80 ppm.
Preparation : discs, after drying the substance to be
Dilute 2.5 mL of solution S to 10 mL with water R and add
examined in an oven at 100-105 °C for 2 h.
0.25 g of calcium chloride R before adding the thioglycollic
Comparison : sodium edetate R, treated as follows : dissolve acid R. Allow to stand for 5 min. Also add 0.25 g of calcium
0.25 g of sodium edetate R in 5 mL of water R, add 1.0 mL chloride R to the standard.
of dilute hydrochloric acid R. Filter, wash the residue with
2 quantities, each of 5 mL, of water R and dry the residue Heavy metals (2.4.8) : maximum 20 ppm.
in an oven at 100-105 °C for 2 h. 1.0 g complies with test F. Prepare the reference solution using
B. To 5 mL of water R add 0.1 mL of ammonium thiocyanate 2 mL of lead standard solution (10 ppm Pb) R.
solution R and 0.1 mL of ferric chloride solution R1 and Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
mix. The solution is red. Add 0.5 mL of solution S (see 1.0 g.
Tests). The solution becomes yellowish.
C. To 10 mL of solution S add 0.5 mL of calcium chloride ASSAY
solution R. Make alkaline to red litmus paper R by the Dissolve 0.250 g in 2.0 mL of dilute sodium hydroxide
addition of dilute ammonia R2 and add 3 mL of ammonium solution R and dilute to 300 mL with water R. Add 2 g of
oxalate solution R. No precipitate is formed. hexamethylenetetramine R and 2 mL of dilute hydrochloric
acid R. Titrate with 0.1 M zinc sulfate using about 50 mg of
TESTS xylenol orange triturate R as indicator.
Solution S. Dissolve 5.0 g in 20 mL of dilute sodium hydroxide 1 mL of 0.1 M zinc sulfate corresponds to 29.22 mg
solution R and dilute to 100 mL with water R. of C10H16N2O8.

2128 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Emedastine difumarate

STORAGE Relative retention with reference to edrophonium (retention

Protected from light. time = about 3.8 min) : impurity A = about 1.3.
System suitability : reference solution (b) :
IMPURITIES
– resolution : minimum 2.0 between the peaks due to
Specified impurities : A. edrophonium and impurity A.
Limits :
– impurity A : not more than the area of the corresponding
peak in the chromatogram obtained with reference
solution (b) (0.1 per cent),
A. nitrilotriacetic acid. – any other impurity : for each impurity, not more than the
area of the peak due to edrophonium in the chromatogram
01/2008:2106 obtained with reference solution (b) (0.1 per cent),
– total : not more than 5 times the area of the peak due to
EDROPHONIUM CHLORIDE edrophonium in the chromatogram obtained with reference
solution (b) (0.5 per cent),
Edrophonii chloridum – disregard limit : 0.5 times the area of the peak due to
edrophonium in the chromatogram obtained with reference
solution (b) (0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in a desiccator over diphosphorus
pentoxide R at a pressure not exceeding 0.7 kPa for 24 h.
C10H16ClNO Mr 201.7 Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
[116-38-1] 1.0 g.
Bacterial endotoxins (2.6.14) : less than 8.3 IU/mg.
DEFINITION
N-Ethyl-3-hydroxy-N,N-dimethylanilinium chloride. ASSAY
Content : 99.0 per cent to 101.0 per cent (dried substance). Dissolve 0.150 g in 60 mL of a mixture of equal volumes
of acetic anhydride R and anhydrous acetic acid R. Titrate
CHARACTERS with 0.1 M perchloric acid, determining the end-point
Appearance : white or almost white, crystalline powder. potentiometrically (2.2.20).
Solubility : very soluble in water, freely soluble in ethanol 1 mL of 0.1 M perchloric acid is equivalent to 20.17 mg
(96 per cent), practically insoluble in methylene chloride. of C10H16ClNO.
IDENTIFICATION STORAGE
A. Infrared absorption spectrophotometry (2.2.24). Protected from light.
Comparison : edrophonium chloride CRS. IMPURITIES
B. It gives reaction (a) of chlorides (2.3.1). Specified impurities : A.
TESTS
Appearance of solution. The solution is clear (2.2.1) and
colourless (2.2.2, Method II).
Dissolve 0.5 g in water R and dilute to 25 mL with the same
solvent. A. 3-(dimethylamino)phenol.
pH (2.2.3) : 4.0 to 5.0.
Dissolve 1.0 g in carbon dioxide-free water R and dilute to 01/2008:2242
10.0 mL with the same solvent.
Related substances. Liquid chromatography (2.2.29). EMEDASTINE DIFUMARATE
Test solution. Dissolve 50.0 mg in water R and dilute to
50.0 mL with the same solvent. Emedastini difumaras
Reference solution (a). Dissolve 10.0 mg of 3-dimethylami-
nophenol R in acetonitrile R and dilute to 10.0 mL with the
same solvent.
Reference solution (b). Mix 1.0 mL of the test solution and
1.0 mL of reference solution (a) and dilute to 100.0 mL with
water R. Dilute 10.0 mL of this solution to 100.0 mL with
water R. C25H34N4O9 Mr 534.6
Column : [87233-62-3]
– size : l = 0.25 m, Ø = 4.6 mm,
DEFINITION
– stationary phase : styrene-divinylbenzene copolymer R
(8-10 μm). 1-(2-Ethoxyethyl)-2-(4-methylhexahydro-1H-1,4-diazepin-1-
yl)-1H-benzimidazole bis[hydrogen (2E)-butenedioate].
Mobile phase : mix 10 volumes of acetonitrile R and 90 volumes
of a 7.7 g/L solution of tetramethylammonium bromide R Content : 99.0 per cent to 101.0 per cent (dried substance).
previously adjusted to pH 3.0 with phosphoric acid R. CHARACTERS
Flow rate : 1 mL/min. Appearance : white or yellowish powder.
Detection : spectrophotometer at 281 nm. Solubility : soluble in water, sparingly soluble in anhydrous
Injection : 20 μL. ethanol, very slightly soluble in acetone.
Run time : twice the retention time of edrophonium. It shows polymorphism (5.9).

General Notices (1) apply to all monographs and other texts 2129
Emetine hydrochloride pentahydrate EUROPEAN PHARMACOPOEIA 8.0

IDENTIFICATION Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on

Infrared absorption spectrophotometry (2.2.24). 1.0 g.
Comparison : emedastine difumarate CRS. ASSAY
If the spectra obtained in the solid state show differences, Dissolve 0.200 g in 50 mL of glacial acetic acid R. Titrate
dissolve the substance to be examined and the reference with 0.1 M perchloric acid, determining the end-point
substance separately in anhydrous ethanol R, evaporate to potentiometrically (2.2.20).
dryness and record new spectra using the residues.
1 mL of 0.1 M perchloric acid is equivalent to 26.73 mg
TESTS of C25H34N4O9.
Appearance of solution. The solution is clear (2.2.1) and not STORAGE
more intensely coloured than reference solution Y5 (2.2.2, Protected from light.
Method II).
Dissolve 2.50 g in water R and dilute to 50 mL with the same IMPURITIES
solvent. Specified impurities : A, B, C, D, E, F.
pH (2.2.3) : 3.0 to 4.5.
Dissolve 0.20 g in 100 mL of carbon dioxide-free water R.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 10 mg of the substance to be examined
in the mobile phase and dilute to 10 mL with the mobile phase.
Reference solution (a). Dissolve 5 mg of emedastine
impurity E CRS in the mobile phase and dilute to 25 mL with A. 1-(2-ethoxyethyl)-1,3-dihydro-2H-benzimidazol-2-one,
the mobile phase.
Reference solution (b). Dissolve 10 mg of the substance to
be examined in the mobile phase. Add 0.5 mL of reference
solution (a) and dilute to 10 mL with the mobile phase.
Reference solution (c). Dilute 5.0 mL of the test solution to
50.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 100.0 mL with the mobile phase.
B. R = Cl : 2-chloro-1-(2-ethoxyethyl)-1H-benzimidazole,
Column :
– size : l = 0.15 m, Ø = 4.6 mm ; F. R = NH-[CH2]3-NH-CH3 : N-[1-(2-ethoxyethyl)-1H-
benzimidazol-2-yl]-N′-methylpropane-1,3-diamine,
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : dissolve 3.9 g of disodium hydrogen phosphate R
and 2.5 g of sodium dodecyl sulfate R in water R and dilute
to 1000.0 mL with the same solvent. Adjust to pH 2.4 with
phosphoric acid R. Mix 550 volumes of this solution with
450 volumes of acetonitrile R.
Flow rate : 1.0 mL/min. C. R1 = CH2-CH2OH, R2 = CH3 : 2-[2-(4-methylhexahydro-
Detection : spectrophotometer at 280 nm. 1H-1,4-diazepin-1-yl)-1H-benzimidazol-1-yl]ethanol,
Injection : 10 μL of the test solution and reference solutions (b) D. R1 = CH=CH2, R2 = CH3 : 1-ethenyl-2-(4-
and (c). methylhexahydro-1H-1,4-diazepin-1-yl)-1H-
Run time : twice the retention time of emedastine. benzimidazole,
Relative retention with reference to emedastine (retention E. R1 = CH2-CH2-O-C2H5, R2 = H : 1-(2-ethoxyethyl)-2-
time = about 18 min): fumaric acid = about 0.1 ; (hexahydro-1H-1,4-diazepin-1-yl)-1H-benzimidazole.
impurity A = about 0.2 ; impurity B = about 0.3 ;
impurity C = about 0.5 ; impurity D = about 0.7 ;
impurity E = about 0.9 ; impurity F = about 1.4. 01/2008:0081
corrected 6.0
System suitability : reference solution (b) :
– peak-to-valley ratio : minimum 4, where Hp = height above
the baseline of the peak due to impurity E and Hv = height
EMETINE HYDROCHLORIDE
above the baseline of the lowest point of the curve PENTAHYDRATE
separating this peak from the peak due to emedastine.
Limits : Emetini hydrochloridum pentahydricum
– impurities A, B, C, D, E, F : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.1 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (c) (0.1 per cent) ;
– total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (c)
(0.2 per cent) ; C29H42Cl2N2O4,5H2O Mr 644
– disregard limit : 0.5 times the area of the principal peak in DEFINITION
the chromatogram obtained with reference solution (c) Emetine hydrochloride pentahydrate contains not less
(0.05 per cent); disregard the peak due to fumaric acid. than 98.0 per cent and not more than the equivalent of
Loss on drying (2.2.32) : maximum 0.5 per cent, determined 102.0 per cent of (2S,3R,11bS)-2-[[(1R)-6,7-dimethoxy-
on 1.000 g by drying in an oven at 105 °C for 3 h. 1,2,3,4-tetrahydroisoquinolin-1-yl]methyl]-3-ethyl-9,10-

2130 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Enalapril maleate

dimethoxy-1,3,4,6,7,11b-hexahydro-2H-benzo[a]quinolizine ether R and 100 volumes of chloroform R. Allow the plate to

dihydrochloride, calculated with reference to the dried dry in air until the solvent has evaporated. In a well-ventilated
substance. fume cupboard, spray with chloroformic iodine solution R
and heat at 60 °C for 15 min. Examine in ultraviolet light at
CHARACTERS 365 nm. In the chromatogram obtained with the test solution,
A white or slightly yellowish, crystalline powder, freely soluble any spots corresponding to isoemetine and cephaeline are not
in water and in alcohol. more intense than the spots in the chromatograms obtained
with reference solutions (b) and (c) respectively (2.0 per
IDENTIFICATION cent) ; any spot, apart from the principal spot and the spots
First identification : A, E. corresponding to isoemetine and cephaeline, is not more
Second identification : B, C, D, E. intense than the spot in the chromatogram obtained with
A. Examine by infrared absorption spectrophotometry reference solution (d) (1.0 per cent). The test is not valid
(2.2.24), comparing with the spectrum obtained with unless the chromatogram obtained with reference solution (e)
emetine hydrochloride CRS. shows three clearly separated spots.
B. Examine the chromatograms obtained in the test for related Loss on drying (2.2.32). 11.0 per cent to 15.0 per cent,
substances in ultraviolet light at 365 nm. The principal determined on 1.00 g by drying in an oven at 105 °C for 3 h.
spot in the chromatogram obtained with the test solution is Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
similar in position, fluorescence and size to the spot in the on 1.0 g.
chromatogram obtained with reference solution (a).
C. Dissolve about 10 mg in 2 mL of dilute hydrogen peroxide ASSAY
solution R, add 1 mL of hydrochloric acid R and heat. An Dissolve 0.200 g in a mixture of 5.0 mL of 0.01 M hydrochloric
orange colour develops. acid and 50 mL of alcohol R. Carry out a potentiometric
D. Sprinkle about 5 mg on the surface of 1 mL of sulfomolybdic titration (2.2.20), using 0.1 M sodium hydroxide. Read
reagent R2. A bright-green colour develops. the volume added between the two points of inflexion.
E. It gives reaction (a) of chlorides (2.3.1). 1 mL of 0.1 M sodium hydroxide is equivalent to 27.68 mg
of C29H42Cl2N2O4.
TESTS
STORAGE
Solution S. Dissolve 1.25 g in carbon dioxide-free water R and
dilute to 25 mL with the same solvent. Store protected from light.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y5 or BY5 07/2010:1420
(2.2.2, Method II).
pH (2.2.3). Dilute 4 mL of solution S to 10 mL with carbon ENALAPRIL MALEATE
dioxide-free water R. The pH of the solution is 4.0 to 6.0.
Specific optical rotation (2.2.7). Dissolve in water R a Enalaprili maleas
quantity of the substance to be examined corresponding to
1.250 g of dried substance and dilute to 25.0 mL with the same
solvent. The specific optical rotation is + 16 to + 19, calculated
with reference to the dried substance.
Related substances. Examine by thin-layer chromatography
(2.2.27), using a TLC silica gel G plate R. Prepare the solutions
immediately before use. C24H32N2O9 Mr 492.5
[76095-16-4]
Test solution. Dissolve 50 mg of the substance to be examined
in methanol R containing 1 per cent V/V of dilute ammonia R2 DEFINITION
and dilute to 100 mL with the same solvent.
(2S)-1-[(2S)-2-[[(1S)-1-(Ethoxycarbonyl)-3-phenylpropyl]-
Reference solution (a). Dissolve 50 mg of emetine amino]propanoyl]pyrrolidine-2-carboxylic acid
hydrochloride CRS in methanol R containing 1 per cent V/V of (Z)-butenedioate.
dilute ammonia R2 and dilute to 100 mL with the same solvent.
Content : 98.5 per cent to 101.5 per cent (dried substance).
Reference solution (b). Dissolve 10 mg of isoemetine
hydrobromide CRS in methanol R containing 1 per cent V/V CHARACTERS
of dilute ammonia R2 and dilute to 100 mL with the same Appearance : white or almost white, crystalline powder.
solvent. Dilute 5 mL of this solution to 50 mL with methanol R Solubility : sparingly soluble in water, freely soluble in
containing 1 per cent V/V of dilute ammonia R2. methanol, practically insoluble in methylene chloride. It
Reference solution (c). Dissolve 10 mg of cephaeline dissolves in dilute solutions of alkali hydroxides.
hydrochloride CRS in methanol R containing 1 per cent V/V mp : about 144 °C.
of dilute ammonia R2 and dilute to 100 mL with the same
solvent. Dilute 5 mL of this solution to 50 mL with methanol R IDENTIFICATION
containing 1 per cent V/V of dilute ammonia R2. Infrared absorption spectrophotometry (2.2.24).
Reference solution (d). Dilute 1 mL of reference solution (a) to Comparison: enalapril maleate CRS.
100 mL with methanol R containing 1 per cent V/V of dilute
ammonia R2. TESTS
Reference solution (e). To 1 mL of reference solution (a) Solution S. Dissolve 0.25 g in carbon dioxide-free water R and
add 1 mL of reference solution (b) and 1 mL of reference dilute to 25.0 mL with the same solvent.
solution (c).
Appearance of solution. Solution S is clear (2.2.1) and
Apply to the plate 10 μL of the test solution and each of colourless (2.2.2, Method II).
reference solutions (a), (b), (c) and (d) and 30 μL of reference
solution (e). Develop over a path of 15 cm using a mixture of pH (2.2.3) : 2.4 to 2.9 for solution S.
0.5 volumes of diethylamine R, 2 volumes of water R, 5 volumes Specific optical rotation (2.2.7) : − 48 to − 51 (dried
of methanol R, 20 volumes of ethylene glycol monomethyl substance), determined on solution S.

General Notices (1) apply to all monographs and other texts 2131
Enalapril maleate EUROPEAN PHARMACOPOEIA 8.0

Related substances. Liquid chromatography (2.2.29). – unspecified impurities : for each impurity, not more than
Buffer solution A. Dissolve 2.8 g of sodium dihydrogen 0.1 times the area of the principal peak in the chromatogram
phosphate monohydrate R in 950 mL of water R. Adjust to obtained with reference solution (a) (0.10 per cent) ;
pH 2.5 with phosphoric acid R and dilute to 1000 mL with – sum of impurities other than A : not more than the area
water R. of the principal peak in the chromatogram obtained with
Buffer solution B. Dissolve 2.8 g of sodium dihydrogen reference solution (a) (1.0 per cent) ;
phosphate monohydrate R in 950 mL of water R. Adjust to – disregard limit : 0.05 times the area of the principal peak
pH 6.8 with strong sodium hydroxide solution R and dilute to in the chromatogram obtained with reference solution (a)
1000 mL with water R. (0.05 per cent) ; disregard the peak due to maleic acid.
Dissolution mixture. Mix 50 mL of acetonitrile R1 and 950 mL Heavy metals (2.4.8) : maximum 10 ppm.
of buffer solution A. 2.0 g complies with test C. Prepare the reference solution using
Test solution. Dissolve 30 mg of the substance to be examined 2 mL of lead standard solution (10 ppm Pb) R.
in the dissolution mixture and dilute to 100.0 mL with the Loss on drying (2.2.32) : maximum 1.0 per cent, determined
dissolution mixture. on 1.000 g by drying in an oven at 105 °C for 3 h.
Reference solution (a). Dilute 1.0 mL of the test solution to Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
100.0 mL with the dissolution mixture. 1.0 g.
Reference solution (b). Dissolve 3 mg of enalapril for system
suitability CRS (containing impurity A) in the dissolution ASSAY
mixture and dilute to 10.0 mL with the dissolution mixture. Dissolve 0.100 g in carbon dioxide-free water R and dilute
Reference solution (c). Dissolve the contents of a vial of to 30 mL with the same solvent. Titrate with 0.1 M sodium
enalapril impurity mixture CRS (impurities B, C, D, E and H) hydroxide determining the end-point potentiometrically
in 1.0 mL of the dissolution mixture. (2.2.20). Titrate to the 2nd point of inflexion.
Column : 1 mL of 0.1 M sodium hydroxide is equivalent to 16.42 mg
– size : l = 0.15 m, Ø = 4.1 mm ; of C24H32N2O9.
– stationary phase : styrene-divinylbenzene copolymer R STORAGE
(5 μm) ; Protected from light.
– temperature : 70 °C.
IMPURITIES
Mobile phase :
Specified impurities : A, B, C, D, E, H.
– mobile phase A : mix 50 mL of acetonitrile R1 and 950 mL
of buffer solution B ; Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
– mobile phase B : mix 340 mL of buffer solution B and the tests in the monograph. They are limited by the general
660 mL of acetonitrile R1 ; acceptance criterion for other/unspecified impurities and/or
Time Mobile phase A Mobile phase B by the general monograph Substances for pharmaceutical use
(min) (per cent V/V) (per cent V/V) (2034). It is therefore not necessary to identify these impurities
0 - 20 95 → 40 5 → 60 for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : F, G, I.
20 - 25 40 60

Flow rate : 1.0 mL/min.

Detection : spectrophotometer at 215 nm.
Injection : 50 μL.
Identification of impurities :
A. (2S)-1-[(2S)-2-[[(1R)-1-(ethoxycarbonyl)-3-
– use the chromatogram supplied with enalapril impurity phenylpropyl]amino]propanoyl]pyrrolidine-2-carboxylic
mixture CRS and the chromatogram obtained with acid,
reference solution (c) to identify the peaks due to
impurities B, C, D, E and H ;
– use the chromatogram obtained with reference solution (b)
to identify the peak due to impurity A.
Relative retention with reference to enalapril (retention
time = about 11 min) : impurity C = about 0.2 ;
impurity B = about 0.8 ; impurity A = about 1.1 ; B. (2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl]-
impurity H = about 1.3 ; impurity E = about 1.5 ; amino]propanoic acid,
impurity D = about 2.1.
System suitability: reference solution (b):
– peak-to-valley ratio : minimum 10, where Hp = height
above the baseline of the peak due to impurity A and
Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to enalapril.
C. R = H : (2S)-1-[(2S)-2-[[(1S)-1-carboxy-3-phenylpropyl]-
Limits: amino]propanoyl]pyrrolidine-2-carboxylic acid,
– impurity A : not more than the area of the principal
peak in the chromatogram obtained with reference E. R = CH2-CH2-C6H5 : (2S)-1-[(2S)-2-[[(1S)-3-phenyl-1-[(2-
solution (a) (1.0 per cent) ; phenylethoxy)carbonyl]propyl]amino]propanoyl]pyrrolidine-
2-carboxylic acid,
– impurities B, C, D, E, H : for each impurity, not more
than 0.3 times the area of the principal peak in the F. R = C4H9 : (2S)-1-[(2S)-2-[[(1S)-1-(butoxycarbonyl)-3-
chromatogram obtained with reference solution (a) (0.3 per phenylpropyl]amino]propanoyl]pyrrolidine-2-carboxylic
cent) ; acid,

2132 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Enalaprilat dihydrate

TESTS
Appearance of solution. The solution is clear (2.2.1) and
colourless (2.2.2, Method II).
Dissolve 0.10 g in water R and dilute to 100.0 mL with the
same solvent.
Specific optical rotation (2.2.7) : − 53.0 to − 56.0 (anhydrous
D. ethyl (2S)-2-[(3S,8aS)-3-methyl-1,4-dioxo- substance).
octahydropyrrolo[1,2-a]pyrazin-2-yl]-4-phenylbutanoate,
Dissolve 0.200 g in methanol R and dilute to 20.0 mL with
the same solvent.
Related substances. Liquid chromatography (2.2.29). Use
freshly prepared solutions.
Buffer solution. Dissolve 1.36 g of potassium dihydrogen
phosphate R in 950 mL of water R. Adjust to pH 3.0 with
G. (2S)-2-[[(1S)-3-cyclohexyl-1-(ethoxycarbonyl)propyl]- phosphoric acid R and dilute to 1000 mL with water R.
amino]propanoic acid, Solvent mixture. Buffer solution, acetonitrile R1, methanol R1
(1:2:2 V/V/V).
Dissolution mixture. Solvent mixture, buffer solution
(8:92 V/V).
Test solution. Dissolve 25.0 mg of the substance to be
examined in 2.5 mL of methanol R1 and dilute to 25.0 mL
with the dissolution mixture.
H. (2S)-1-[(2S)-2-[[(1S)-3-cyclohexyl-1-(ethoxycarbonyl)-
Reference solution (a). Dilute 1.0 mL of the test solution to
propyl]amino]propanoyl]pyrrolidine-2-carboxylic acid,
100.0 mL with the dissolution mixture. Dilute 5.0 mL of this
solution to 10.0 mL with the dissolution mixture.
Reference solution (b). Dissolve 5 mg of enalaprilat for
system suitability CRS (containing impurity C) in 0.5 mL of
methanol R1 and dilute to 5 mL with the dissolution mixture.
I. 1H-imidazole.
Reference solution (c). Dissolve the contents of a vial of
enalaprilat impurity G CRS in 1 mL of the test solution.
01/2008:1749 Column :
corrected 7.0 – size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
ENALAPRILAT DIHYDRATE chromatography R (5 μm) ;
– temperature : 70 °C.
Enalaprilatum dihydricum Mobile phase :
– mobile phase A : solvent mixture, buffer solution
(10:90 V/V) ;
– mobile phase B : acetonitrile R1 ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
C18H24N2O5,2H2O Mr 384.4 0 - 25 100 0
[84680-54-6]
25 - 50 100 → 90 0 → 10
DEFINITION 50 - 80 90 10
(2S)-1-[(2S)-2-[[(1S)-1-Carboxy-3-phenylpropyl]amino]-
propanoyl)pyrrolidine-2-carboxylic acid dihydrate. Flow rate : 2.0 mL/min.
Content : 98.5 per cent to 101.5 per cent (anhydrous substance). Detection : spectrophotometer at 210 nm.
Injection : 20 μL.
CHARACTERS
Identification of impurities : use the chromatogram
Appearance : white or almost white, hygroscopic, crystalline supplied with enalaprilat for system suitability CRS and
powder. the chromatogram obtained with reference solution (b) to
Solubility : very slightly soluble or slightly soluble in water, identify the peak due to impurity C ; use the chromatogram
sparingly soluble in methanol, practically insoluble in obtained with reference solution (c) to identify the peak due
acetonitrile. to impurity G.
It shows pseudopolymorphism (5.9). Relative retention with reference to enalaprilat (retention
time = about 21 min) : impurity C = about 1.2 ;
IDENTIFICATION impurity G = about 2.9.
A. Specific optical rotation (see Tests). System suitability : reference solution (b) :
B. Infrared absorption spectrophotometry (2.2.24). – peak-to-valley ratio : minimum 2.0, where Hp = height
Preparation : mulls in liquid paraffin R. above the baseline of the peak due to impurity C and
Comparison : enalaprilat dihydrate CRS. Hv = height above the baseline of the lowest point of the
If the spectra obtained show differences, expose the curve separating this peak from the peak due to enalaprilat.
substance to be examined and the reference substance to Limits :
a 98 per cent relative humidity for 3 days using a chamber – impurities C, G : for each impurity, not more than the area
conditioned with a saturated solution of calcium sulfate R. of the principal peak in the chromatogram obtained with
Record new spectra. reference solution (a) (0.5 per cent) ;

General Notices (1) apply to all monographs and other texts 2133
Enilconazole for veterinary use EUROPEAN PHARMACOPOEIA 8.0

– unspecified impurities : for each impurity, not more than

0.2 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.10 per cent) ;
– total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (a)
(1.0 per cent) ; E. (2SR)-1-[[(2SR)-1-[(2SR)-2-[[(1SR)-1-carboxy-
3-phenylpropyl]amino]propanoyl]pyrrolidin-2-
– disregard limit : 0.1 times the area of the principal peak in
yl]carbonyl]pyrrolidine-2-carboxylic acid,
the chromatogram obtained with reference solution (a)
(0.05 per cent).
Heavy metals (2.4.8) : maximum 10 ppm.
2.0 g complies with test G. Prepare the reference solution
using 2 mL of lead standard solution (10 ppm Pb) R.
Water (2.5.12) : 7.0 per cent to 11.0 per cent, determined on
G. (2SR)-2-[(3SR,8aRS)-3-methyl-1,4-dioxohexahydro-
0.100 g.
pyrrolo[1,2-a]pyrazin-2(1H)-yl]-4-phenylbutanoic acid.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. 07/2010:1720
Bacterial endotoxins (2.6.14) : less than 0.1 IU/mg.
ENILCONAZOLE FOR VETERINARY
ASSAY
USE
Dissolve 0.300 g in glacial acetic acid R and dilute to 50 mL
with the same solvent. Titrate with 0.1 M perchloric acid, Enilconazolum ad usum veterinarium
determining the end point potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 34.84 mg
of C18H24N2O5.

STORAGE
In an airtight container.

IMPURITIES
Specified impurities : C, G.
C14H14Cl2N2O Mr 297.2
Other detectable impurities (the following substances would, [35554-44-0]
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general DEFINITION
acceptance criterion for other/unspecified impurities and/or 1-[(2RS)-2-(2,4-Dichlorophenyl)-2-(prop-2-enyloxy)ethyl]-
by the general monograph Substances for pharmaceutical use 1H-imidazole.
(2034). It is therefore not necessary to identify these impurities Content : 98.5 per cent to 101.5 per cent (dried substance).
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : A, B, D, E, F. CHARACTERS
Appearance : clear, yellowish, oily liquid or solid mass.
Solubility : very slightly soluble in water, freely soluble in
ethanol (96 per cent), in methanol and in toluene.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: enilconazole CRS.
A. R = H : (2SR)-2-[[(1SR)-1-carboxyethyl]amino]-4-
phenylbutanoic acid, TESTS
Optical rotation (2.2.7) : − 0.10° to + 0.10°.
F. R = C2H5 : (2SR)-2-[[(1SR)-1-(ethoxycarbonyl)-3- Dissolve 0.1 g in methanol R and dilute to 10 mL with the
phenylpropyl]amino]propanoic acid, same solvent.
Related substances. Gas chromatography (2.2.28). Prepare
the solutions immediately before use and protect from light.
Test solution. Dissolve 0.100 g of the substance to be examined
in toluene R and dilute to 100.0 mL with the same solvent.
Reference solution (a). Dissolve 10.0 mg of enilconazole CRS
B. R1 = R4 = H, R2 = CO2H, R3 = CH3 : (2SR)- and 10.0 mg of enilconazole impurity E CRS in toluene R and
1-[(2RS)-2-[[(1RS)-1-carboxy-3-phenylpropyl]- dilute to 100.0 mL with the same solvent.
amino]propanoyl]pyrrolidine-2-carboxylic acid, Reference solution (b). Dilute 5.0 mL of the test solution to
100.0 mL with toluene R. Dilute 1.0 mL of this solution to
10.0 mL with toluene R.
C. R1 = R3 = H, R2 = CO2H, R4 = CH3 : (2SR)-
1-[(2SR)-2-[[(1RS)-1-carboxy-3-phenylpropyl]- Column :
amino]propanoyl]pyrrolidine-2-carboxylic acid, – material : fused silica ;
– size : l = 25 m, Ø = 0.32 mm ;
D. R1 = CO2H, R2 = R4 = H, R3 = CH3 : (2SR)- – stationary phase : chemically bonded poly(dimethyl)(di-
1-[(2RS)-2-[[(1SR)-1-carboxy-3-phenylpropyl]- phenyl)siloxane R (film thickness 0.52 μm).
amino]propanoyl]pyrrolidine-2-carboxylic acid, Carrier gas : helium for chromatography R.

2134 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Enoxaparin sodium

Flow rate : 1.3 mL/min. B. R1 = H, R2 = CH2-CH=CH2 : N-[(2RS)-2-(2,4-

Split ratio : 1:38. dichlorophenyl)-2-(prop-2-enyloxy)ethyl]prop-2-en-1-
amine,
Temperature :
C. R1 = CHO, R2 = H : N-[(2RS)-2-(2,4-dichlorophenyl)-2-
Time Temperature
(prop-2-enyloxy)ethyl]formamide,
(min) (°C)
Column 0 - 6.4 100 → 260 D. R1 = CHO, R2 = CH2-CH=CH2 : N-[(2RS)-2-(2,4-
6.4 - 14 260 dichlorophenyl)-2-(prop-2-enyloxy)ethyl]-N-(prop-2-
Injection port 250 enyl)formamide,
Detector 300

Detection : flame ionisation.

Injection : 2 μL.
Identification of impurities : use the chromatogram obtained
with reference solution (a) to identify the peak due to
impurity E.
Relative retention with reference to enilconazole
(retention time = about 10 min) : impurity A = about 0.6 ; E. (1RS)-1-(2,4-dichlorophenyl)-2-(-1H-imidazol-1-
impurity B = about 0.7 ; impurity C = about 0.8 ; yl)ethanol,
impurity D = about 0.9 ; impurity E = about 1.03 ;
impurity F = about 1.1.
System suitability: reference solution (a) :
– resolution : minimum 2.5 between the peaks due to
enilconazole and impurity E.
Limits :
– impurities A, B, C, D, E, F : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (b) (1.0 per cent), and not F. 1-[(2RS)-2-(3,4-dichlorophenyl)-2-(prop-2-enyloxy)ethyl]-
more than 1 such peak has an area greater than the area 1H-imidazole.
of the principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent) ;
– unspecified impurities : for each impurity, not more than 01/2008:1097
0.4 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.20 per cent) ; ENOXAPARIN SODIUM
– total : not more than 4 times the area of the principal peak
in the chromatogram obtained with reference solution (b) Enoxaparinum natricum
(2.0 per cent) ;
– disregard limit : 0.1 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in vacuo at 40 °C for 4 h.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY
Dissolve 0.230 g in 50 mL of a mixture of 1 volume of
anhydrous acetic acid R and 7 volumes of methyl ethyl
ketone R. Titrate with 0.1 M perchloric acid using 0.2 mL of
naphtholbenzein solution R as indicator.
1 mL of 0.1 M perchloric acid is equivalent to 29.72 mg of
C14H14Cl2N2O.
STORAGE
In an airtight container, protected from light.
IMPURITIES
Specified impurities : A, B, C, D, E, F. DEFINITION
Enoxaparin sodium is the sodium salt of a low-molecular-mass
heparin that is obtained by alkaline depolymerisation of
the benzyl ester derivative of heparin from porcine
intestinal mucosa. Enoxaparin consists of a complex set
of oligosaccharides that have not yet been completely
characterised. Based on current knowledge, the majority of
the components have a 4-enopyranose uronate structure at
the non-reducing end of their chain. 15 per cent to 25 per
A. R1 = R2 = H : (2RS)-2-(2,4-dichlorophenyl)-2-(prop-2- cent of the components have a 1,6-anhydro structure at the
enyloxy)ethanamine, reducing end of their chain.

General Notices (1) apply to all monographs and other texts 2135
Enoxolone EUROPEAN PHARMACOPOEIA 8.0

Enoxaparin sodium complies with the monograph From the chromatogram obtained with the reference solution,
Low-molecular-mass heparins (0828) with the modifications calculate the ratio (R1) of the height of the peak due to benzyl
and additional requirements below. alcohol to the height of the peak due to the internal standard.
The mass-average relative molecular mass ranges between From the chromatogram obtained with the test solution,
3800 and 5000, with a characteristic value of about 4500. calculate the ratio (R2) of the height of the peak due to benzyl
The degree of sulfatation is about 2 per disaccharide unit. alcohol to the height of the peak due to the internal standard.
The potency is not less than 90 IU and not more than 125 IU Calculate the percentage content m/m of benzyl alcohol using
of anti-factor Xa activity per milligram, calculated with the following expression :
reference to the dried substance. The anti-factor IIa activity is
not less than 20.0 IU and not more than 35.0 IU per milligram,
calculated with reference to the dried substance. The ratio of
anti-factor Xa activity to anti-factor IIa activity is between m = mass of the substance to be examined, in grams.
3.3 and 5.3.
Limit :
PRODUCTION – benzyl alcohol : maximum 0.1 per cent m/m.
Enoxaparin is produced by alkaline depolymerisation of Sodium (2.2.23, Method I) : 11.3 per cent to 13.5 per cent
benzyl ester derivatives of heparin from porcine intestinal (dried substance).
mucosa under conditions that yield a product complying with
the structural requirements stated under Definition. 01/2008:1511
IDENTIFICATION corrected 6.0
Carry out identification test A as described in the monograph
Low-molecular-mass heparins (0828) using enoxaparin ENOXOLONE
sodium CRS.
Carry out identification test C as described in the monograph Enoxolonum
Low-molecular-mass heparins (0828). The following
requirements apply.
The mass-average relative molecular mass ranges between
3800 and 5000. The mass percentage of chains lower than
2000 ranges between 12.0 per cent and 20.0 per cent. The
mass percentage of chains between 2000 and 8000 ranges
between 68.0 per cent and 82.0 per cent.
TESTS
Appearance of solution. The solution is clear (2.2.1) and C30H46O4 Mr 470.7
not more intensely coloured than intensity 6 of the range of [471-53-4]
reference solutions of the most appropriate colour (2.2.2,
Method II). DEFINITION
Dissolve 1.0 g in 10 mL of water R. (20β)-3β-Hydroxy-11-oxo-olean-12-en-29-oic acid.
pH (2.2.3) : 6.2 to 7.7. Content : 98.0 per cent to 101.0 per cent (dried substance).
Dissolve 1.0 g in carbon dioxide-free water R and dilute to CHARACTERS
10.0 mL with the same solvent. Appearance : white or almost white crystalline powder.
Specific absorbance (2.2.25) : 14.0 to 20.0 (dried substance), Solubility : practically insoluble in water, soluble in ethanol,
determined at 231 nm. sparingly soluble in methylene chloride.
Dissolve 50.0 mg in 100 mL of 0.01 M hydrochloric acid. It shows polymorphism (5.9).
Benzyl alcohol. Liquid chromatography (2.2.29).
IDENTIFICATION
Internal standard solution: 1 g/L solution of
First identification : A.
3,4-dimethylphenol R in methanol R.
Second identification : B, C.
Test solution. Dissolve about 0.500 g of the substance to be
examined in 5.0 mL of 1 M sodium hydroxide. Allow to stand A. Examine by infrared absorption spectrophotometry
for 1 h. Add 1.0 mL of glacial acetic acid R and 1.0 mL of the (2.2.24).
internal standard solution and dilute to 10.0 mL with water R. Comparison: enoxolone CRS.
Reference solution. Prepare a 0.25 g/L solution of benzyl If the spectra obtained in the solid state show differences,
alcohol R in water R. Mix 0.50 mL of this solution with 1.0 mL dissolve 0.2 g of the substance to be examined and 0.2 g of
of the internal standard solution and dilute to 10.0 mL with the reference substance separately in 6 mL of ethanol R. Boil
water R. under a reflux condenser for 1 h and add 6 mL of water R.
Precolumn : A precipitate is formed. Cool to about 10 °C and filter with
the aid of vacuum. Wash the precipitate with 10 mL of
– size : l = 0.02 m, Ø = 4.6 mm ;
alcohol R, dry in an oven at 80 °C and record new spectra.
– stationary phase : octylsilyl silica gel for chromatography R B. Thin-layer chromatography (2.2.27).
(5 μm).
Test solution. Dissolve 10 mg of the substance to be
Column : examined in methylene chloride R and dilute to 10 mL with
– size : l = 0.15 m, Ø = 4.6 mm ; the same solvent.
– stationary phase : octylsilyl silica gel for chromatography R Reference solution. Dissolve 10 mg of enoxolone CRS in
(5 μm). methylene chloride R and dilute to 10 mL with the same
Mobile phase : methanol R, acetonitrile R, water R solvent.
(5:15:80 V/V/V). Plate : TLC silica gel plate R.
Flow rate : 1 mL/min. Mobile phase : glacial acetic acid R, acetone R, methylene
Detection : spectrophotometer at 256 nm. chloride R (5:10:90 V/V/V).

2136 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Enrofloxacin for veterinary use

Application : 5 μL. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Development : over 2/3 of the plate. on 1.000 g by drying in an oven at 105 °C for 4 h.
Drying : in air for 5 min. Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
Detection : spray with anisaldehyde solution R and heat at 1.0 g.
100-105 °C for 10 min. ASSAY
Results : the principal spot in the chromatogram obtained Dissolve 0.330 g in 40 mL of dimethylformamide R. Titrate
with the test solution is similar in position, colour and size with 0.1 M tetrabutylammonium hydroxide, determining the
to the principal spot in the chromatogram obtained with end-point potentiometrically (2.2.20). Carry out a blank
the reference solution. titration.
C. Dissolve 50 mg in 10 mL of methylene chloride R. To 2 mL 1 mL of 0.1 M tetrabutylammonium hydroxide is equivalent to
of this solution, add 1 mL of acetic anhydride R and 0.3 mL 47.07 mg of C30H46O4.
of sulfuric acid R. A pink colour is produced.
STORAGE
TESTS Protected from light.
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution Y6 (2.2.2, IMPURITIES
Method II).
Dissolve 0.1 g in ethanol R and dilute to 10 mL with the same
solvent.
Specific optical rotation (2.2.7) : + 145 to + 154 (dried
substance).
Dissolve 0.50 g in dioxan R and dilute to 50.0 mL with the
same solvent.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.10 g of the substance to be examined A. (20β)-3β-hydroxy-11-oxo-18α-olean-12-en-29-oic acid,
in the mobile phaseand dilute to 100.0 mL with the mobile
phase.
Reference solution (a). Dilute 2.0 mL of the test solution to
100.0 mL with the mobile phase.
Reference solution (b). Dilute 5.0 mL of reference solution (a)
to 100.0 mL with the mobile phase.
Reference solution (c). Dissolve 0.1 g of 18α-glycyrrhetinic
acid R in tetrahydrofuran R and dilute to 100.0 mL with the
same solvent. To 2.0 mL of the solution, add 2.0 mL of the test
solution and dilute to 100.0 mL with the mobile phase.
B. (4β,20β)-3β,23-dihydroxy-11-oxo-olean-12-en-29-oic acid.
Column :
– size : l = 0.25 m, Ø = 4.6 mm,
04/2010:2229
– stationary phase : octadecylsilyl silica gel for
corrected 7.0
chromatography R (5 μm),
– temperature : 30 °C.
ENROFLOXACIN FOR
Mobile phase : mix 430 volumes of tetrahydrofuran R and
570 volumes of a 1.36 g/L solution of sodium acetate R adjusted VETERINARY USE
to pH 4.8 with glacial acetic acid R.
Flow rate : 0.8 mL/min. Enrofloxacinum ad usum veterinarium
Detection : spectrophotometer at 250 nm.
Injection : 20 μL loop injector ; inject the test solution and the
reference solutions.
Run time : 4 times the retention time of enoxolone.
System suitability :
– resolution : minimum of 2.0 between the peaks due
to enoxolone and to 18α-glycyrrhetinic acid in the C19H22FN3O3 Mr 359.4
chromatogram obtained with reference solution (c). [93106-60-6]
Limits : DEFINITION
– any impurity : not more than 7 times the area of the 1-Cyclopropyl-7-(4-ethylpiperazin-1-yl)-6-fluoro-4-oxo-1,4-
principal peak in the chromatogram obtained with dihydroquinoline-3-carboxylic acid.
reference solution (b) (0.7 per cent),
Content : 98.5 per cent to 101.5 per cent (dried substance).
– total : not more than the area of the principal peak in the
chromatogram obtained with reference solution (a) (2.0 per CHARACTERS
cent), Appearance : pale yellowish or light yellow, crystalline powder.
– disregard limit : 0.5 times the area of the principal peak in Solubility : practically insoluble in water, freely soluble in
the chromatogram obtained with reference solution (b) methylene chloride, slightly soluble in methanol.
(0.05 per cent).
Heavy metals (2.4.8) : maximum 20 ppm. IDENTIFICATION
1.0 g complies with test F. Prepare the reference solution using Infrared absorption spectrophotometry (2.2.24).
2 mL of lead standard solution (10 ppm Pb) R. Comparison: enrofloxacin CRS.

General Notices (1) apply to all monographs and other texts 2137
Enrofloxacin for veterinary use EUROPEAN PHARMACOPOEIA 8.0

TEST – impurity C : not more than the area of the principal peak
Appearance of solution. The solution is not more opalescent in the chromatogram obtained with reference solution (b)
than reference suspension II (2.2.1) and not more intensely (0.2 per cent) ;
coloured than reference solution GY4 (2.2.2, Method II). – unspecified impurities : for each impurity, not more than the
To 1.0 g of the substance to be examined add about 0.25 g area of the principal peak in the chromatogram obtained
of potassium hydroxide R and 7 mL of water R. Sonicate to with reference solution (b) (0.20 per cent) ;
dissolve and dilute to 10.0 mL with water R. – total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
Impurity A. Thin-layer chromatography (2.2.27). Prepare the
(1.0 per cent) ;
solutions immediately before use.
– disregard limit : 0.5 times the area of the principal peak in
Solvent mixture : methanol R, methylene chloride R (50:50 V/V).
the chromatogram obtained with reference solution (b)
Test solution. Dissolve 0.100 g of the substance to be examined (0.1 per cent).
in the solvent mixture and dilute to 5.0 mL with the solvent
mixture. Heavy metals (2.4.8) : maximum 20 ppm.
Reference solution. Dissolve 5.0 mg of ciprofloxacin Dissolve 1.5 g in a mixture of 5 mL of 2 M acetic acid and
impurity A CRS (enrofloxacin impurity A) in the solvent 10 mL of water R. Filter. 12 mL of the filtrate after adding
mixture and dilute to 50.0 mL with the solvent mixture. Dilute 2 mL of water R (instead of buffer solution) complies with
4.0 mL of this solution to 10.0 mL with the solvent mixture. test E. Prepare the reference solution using 12 mL of lead
standard solution (2 ppm Pb) R.
Plate : TLC silica gel F254 plate R (2-10 μm).
Loss on drying (2.2.32) : maximum 1.0 per cent, determined
Mobile phase : butanol R, water R, anhydrous acetic acid R, on 2.000 g by drying under high vacuum at 120 °C for 6 h.
ethyl acetate R (15:15:20:50 V/V/V/V).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Application : 10 μL.
1.0 g.
Development : over 3/4 of the plate.
Drying : in air. ASSAY
Detection : examine in ultraviolet light at 254 nm. Dissolve 0.250 g in 100 mL of anhydrous acetic acid R and
titrate with 0.1 M perchloric acid determining the end-point
Results :
potentiometrically (2.2.20).
– impurity A : any spot due to impurity A is not more intense
1 mL of 0.1 M perchloric acid is equivalent to 35.94 mg of
than the spot in the chromatogram obtained with the
C19H22FN3O3.
reference solution (0.2 per cent).
Related substances. Liquid chromatography (2.2.29). STORAGE
Test solution. Dissolve 50 mg of the substance to be examined Protected from light.
in the mobile phase and dilute to 50.0 mL with the mobile
phase. IMPURITIES
Reference solution (a). Dissolve 10 mg of enrofloxacin for Specified impurities : A, B, C.
system suitability CRS (containing impurities B and C) and Other detectable impurities (the following substances would,
dilute to 10 mL with the mobile phase. if present at a sufficient level, be detected by one or other of
Reference solution (b). Dilute 1.0 mL of the test solution to the tests in the monograph. They are limited by the general
50.0 mL with the mobile phase. Dilute 1.0 mL of this solution acceptance criterion for other/unspecified impurities and/or
to 10.0 mL with the mobile phase. by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
Column :
for demonstration of compliance. See also 5.10. Control of
– size : l = 0.15 m, Ø = 4.6 mm ; impurities in substances for pharmaceutical use) : E, F, G.
– stationary phase : base-deactivated end-capped octadecylsilyl
silica gel for chromatography R (5 μm) ;
– temperature : 40 °C.
Mobile phase : mix 15 volumes of methanol R and 85 volumes
of a 2.9 g/L solution of phosphoric acid R, previously adjusted
to pH 2.3 with triethylamine R.
Flow rate : 1.5 mL/min.
A. 7-chloro-1-cyclopropyl-6-fluoro-4-oxo-1,4-
Detection : spectrophotometer at 270 nm. dihydroquinoline-3-carboxylic acid,
Injection : 10 μL.
Run time : 3 times the retention time of enrofloxacin.
Identification of impurities : use the chromatogram supplied
with enrofloxacin for system suitability CRS and the
chromatogram obtained with reference solution (a) to identify
the peaks due to impurities B and C.
Relative retention with reference to enrofloxacin
(retention time = about 16 min) : impurity C = about 0.6 ; B. ciprofloxacin,
impurity B = about 0.8.
System suitability: reference solution (a) :
– resolution : minimum 2.0 between the peaks due to
impurity B and enrofloxacin.
Limits :
– impurity B : not more than 2.5 times the area of the
principal peak in the chromatogram obtained with C. 1-cyclopropyl-7-(4-ethylpiperazin-1-yl)-4-oxo-1,4-
reference solution (b) (0.5 per cent) ; dihydroquinoline-3-carboxylic acid,

2138 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Entacapone

Reference solution (a). Dissolve 5 mg of entacapone

impurity A CRS in the solvent mixture, add 5.0 mL of test
solution (a) and dilute to 25.0 mL with the solvent mixture.
Dilute 1.0 mL of the solution to 20.0 mL with the solvent
mixture. Dilute 1.0 mL of this solution to 10.0 mL with the
solvent mixture.
E. 6-chloro-1-cyclopropyl-7-(4-ethylpiperazin-1-yl)-4-oxo- Reference solution (b). Dilute 1.0 mL of test solution (b) to
1,4-dihydroquinoline-3-carboxylic acid, 100.0 mL with the solvent mixture.
Reference solution (c). Dissolve 50.0 mg of entacapone CRS in
the solvent mixture and dilute to 50.0 mL with the solvent
mixture. Dilute 5.0 mL of the solution to 50.0 mL with the
solvent mixture.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
F. 1-cyclopropyl-7-(4-ethylpiperazin-1-yl)-6-fluoroquinolin- – stationary phase : end-capped propyl-2-phenylsilyl
4(1H)-one, amorphous organosilica polymer R (5 μm).
Mobile phase : mix 2 volumes of tetrahydrofuran R, 44 volumes
of methanol R and 54 volumes of a 2.34 g/L solution of sodium
dihydrogen phosphate R previously adjusted to pH 2.1 with
phosphoric acid R.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 300 nm.
Injection : 10 μL of test solution (a) and reference solutions (a)
G. 7-[(2-aminoethyl)amino]-1-cyclopropyl-6-fluoro-4-oxo- and (b).
1,4-dihydroquinoline-3-carboxylic acid.
Run time : 2.5 times the retention time of entacapone.
01/2011:2574 Relative retention with reference to entacapone (retention
corrected 7.3 time = about 17 min) : impurity A = about 0.8.
System suitability : reference solution (a) :
ENTACAPONE – resolution : minimum 3.0 between the peaks due to
impurity A and entacapone.
Entacaponum Limits :
– impurity A : not more than 1.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.15 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.10 per cent) ;
C14H15N3O5 Mr 305.3 – sum of impurities other than A : not more than twice the
[130929-57-6] area of the principal peak in the chromatogram obtained
with reference solution (b) (0.2 per cent) ;
DEFINITION
– disregard limit : 0.5 times the area of the principal peak in
(2E)-2-Cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N- the chromatogram obtained with reference solution (b)
diethylprop-2-enamide. (0.05 per cent).
Content : 98.0 per cent to 102.0 per cent (dried substance). Heavy metals (2.4.8) : maximum 10 ppm.
CHARACTERS Solvent mixture : dimethylformamide R, methanol R
Appearance : greenish-yellow or yellow powder. (25:75 V/V).
Solubility : practically insoluble in water, soluble or sparingly 1.00 g complies with test H. Prepare the reference solution
soluble in acetone, slightly soluble in anhydrous ethanol. using 1.0 mL of lead standard solution (10 ppm Pb) R.
It shows polymorphism (5.9). After filtration, rinse the membrane filter with at least 20 mL
of methanol R.
IDENTIFICATION Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Infrared absorption spectrophotometry (2.2.24). on 1.000 g by drying in vacuo at 60 °C.
Comparison : entacapone CRS. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
If the spectra obtained in the solid state show differences, 1.0 g.
dissolve the substance to be examined and the reference
substance separately in acetone R, evaporate to dryness and ASSAY
record new spectra using the residues. Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
TESTS
Injection : test solution (b) and reference solution (c).
Related substances. Liquid chromatography (2.2.29). Use
Calculate the percentage content of C14H15N3O5 from the
freshly prepared solutions.
declared content of entacapone CRS.
Solvent mixture : tetrahydrofuran R, methanol R (30:70 V/V).
Test solution (a). Dissolve 50.0 mg of the substance to be STORAGE
examined in the solvent mixture and dilute to 50.0 mL with Protected from light.
the solvent mixture.
Test solution (b). Dilute 5.0 mL of test solution (a) to 50.0 mL IMPURITIES
with the solvent mixture. Specified impurities : A.

General Notices (1) apply to all monographs and other texts 2139
Ephedrine, anhydrous EUROPEAN PHARMACOPOEIA 8.0

Other detectable impurities (the following substances would,

if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10. I. propyl (2E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)prop-
Control of impurities in substances for pharmaceutical use) : B, 2-enoate.
C, D, E, F, G, H, I.
01/2008:0488
corrected 6.0

EPHEDRINE, ANHYDROUS

A. (2Z)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N-
Ephedrinum anhydricum
diethylprop-2-enamide,

C10H15NO Mr 165.2
[299-42-3]

B. ethyl (2E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)prop- DEFINITION

2-enoate, Anhydrous ephedrine contains not less than 99.0 per cent
and not more than the equivalent of 101.0 per cent of
(1R,2S)-2-methylamino-1-phenylpropan-1-ol, calculated with
reference to the anhydrous substance.
CHARACTERS
A white or almost white, crystalline powder or colourless
C. 3,4-dihydroxy-5-nitrobenzaldehyde,
crystals, soluble in water, very soluble in alcohol.
It melts at about 36 °C.
IDENTIFICATION
First identification : B, D.
Second identification : A, C, D, E.
A. Specific optical rotation (see Tests).
B. Examine by infrared absorption spectrophotometry
D. (2E)-2-cyano-3-(3-ethoxy-4-hydroxy-5-nitrophenyl)-N,N- (2.2.24), comparing with the spectrum obtained with the
diethylprop-2-enamide, base isolated from ephedrine hydrochloride CRS. Examine
the substances in discs prepared as follows : dissolve 40 mg
of the substance to be examined in 1 mL of water R, add
1 mL of dilute sodium hydroxide solution R and 4 mL of
chloroform R and shake ; dry the organic layer over 0.2 g
of anhydrous sodium sulfate R ; prepare a blank disc using
about 0.3 g of potassium bromide R ; apply dropwise to the
E. 3,5-dinitrobenzene-1,2-diol, disc 0.1 mL of the organic layer, allowing the solvent to
evaporate between applications ; dry the disc at 50 °C for
2 min. Repeat the operations using 50 mg of ephedrine
hydrochloride CRS.
C. Examine the chromatograms obtained in the test for
related substances. The principal spot in the chromatogram
F. (2E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)prop-2- obtained with test solution (b) is similar in position,
enoic acid, colour and size to the principal spot in the chromatogram
obtained with reference solution (a).
D. Dissolve about 10 mg in 1 mL of water R. Add 0.2 mL of
strong sodium hydroxide solution R and 0.2 mL of copper
sulfate solution R. A violet colour is produced. Add 2 mL
of ether R and shake. The ether layer is purple and the
aqueous layer blue.
G. (2E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N- E. Water (see Tests).
methylprop-2-enamide,
TESTS
Appearance of solution. Dissolve 0.25 g in water R and dilute
to 10 mL with the same solvent. The solution is clear (2.2.1)
and colourless (2.2.2, Method II).
Specific optical rotation (2.2.7). Dissolve 2.25 g in 15 mL of
dilute hydrochloric acid R and dilute to 50.0 mL with water R.
H. (2E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(piperidin-1- The specific optical rotation is − 41 to − 43, calculated with
ylcarbonyl)prop-2-ennitrile, reference to the anhydrous substance.

2140 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ephedrine hemihydrate

Related substances. Examine by thin-layer chromatography IDENTIFICATION

(2.2.27), using silica gel G R as the coating substance. First identification : B, D.
Test solution (a). Dissolve 0.2 g of the substance to be Second identification : A, C, D, E.
examined in methanol R and dilute to 10 mL with the same A. Specific optical rotation (see Tests).
solvent.
B. Examine by infrared absorption spectrophotometry
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL (2.2.24), comparing with the spectrum obtained with the
with methanol R. base isolated from ephedrine hydrochloride CRS. Examine
Reference solution (a). Dissolve 25 mg of ephedrine the substances in discs prepared as follows : dissolve 40 mg
hydrochloride CRS in methanol R and dilute to 10 mL with of the substance to be examined in 1 mL of water R, add
the same solvent. 1 mL of dilute sodium hydroxide solution R and 4 mL of
Reference solution (b). Dilute 1.0 mL of test solution (a) to chloroform R and shake ; dry the organic layer over 0.2 g
200 mL with methanol R. of anhydrous sodium sulfate R ; prepare a blank disc using
Apply separately to the plate 10 μL of each solution. Develop about 0.3 g of potassium bromide R ; apply dropwise to the
over a path of 15 cm using a mixture of 5 volumes of disc 0.1 mL of the organic layer, allowing the solvent to
chloroform R, 15 volumes of concentrated ammonia R and evaporate between applications ; dry the disc at 50 °C for
80 volumes of 2-propanol R. Allow the plate to dry in air and 2 min. Repeat the operations using 50 mg of ephedrine
spray with ninhydrin solution R. Heat at 110 °C for 5 min. hydrochloride CRS.
Any spot in the chromatogram obtained with test solution (a), C. Examine the chromatograms obtained in the test for
apart from the principal spot, is not more intense than the related substances. The principal spot in the chromatogram
spot in the chromatogram obtained with reference solution (b) obtained with test solution (b) is similar in position,
(0.5 per cent). Disregard any spot of lighter colour than the colour and size to the principal spot in the chromatogram
background. obtained with reference solution (a).
Chlorides. Dissolve 0.17 g in 10 mL of water R. Add 5 mL of D. Dissolve about 10 mg in 1 mL of water R. Add 0.2 mL of
dilute nitric acid R and 0.5 mL of silver nitrate solution R1. strong sodium hydroxide solution R and 0.2 mL of copper
Allow to stand for 2 min, protected from bright light. Any sulfate solution R. A violet colour is produced. Add 2 mL
opalescence in the solution is not more intense than that in a of ether R and shake. The ether layer is purple and the
standard prepared at the same time and in the same manner aqueous layer blue.
using 10 mL of chloride standard solution (5 ppm Cl) R, 5 mL E. Water (see Tests).
of dilute nitric acid R and 0.5 mL of silver nitrate solution R1
(290 ppm). TESTS
Water (2.5.12). Not more than 0.5 per cent, determined on Appearance of solution. Dissolve 0.25 g in water R and dilute
2.000 g by the semi-micro determination of water. to 10 mL with the same solvent. The solution is clear (2.2.1)
and colourless (2.2.2, Method II).
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
on 1.0 g. Specific optical rotation (2.2.7). Dissolve 2.25 g in 15 mL of
dilute hydrochloric acid R and dilute to 50.0 mL with water R.
ASSAY The specific optical rotation is − 41 to − 43, calculated with
Dissolve 0.200 g in 5 mL of alcohol R and add 20.0 mL of 0.1 M reference to the anhydrous substance.
hydrochloric acid. Using 0.05 mL of methyl red solution R as Related substances. Examine by thin-layer chromatography
indicator, titrate with 0.1 M sodium hydroxide until a yellow (2.2.27), using silica gel G R as the coating substance.
colour is obtained.
Test solution (a). Dissolve 0.2 g of the substance to be
1 mL of 0.1 M hydrochloric acid is equivalent to 16.52 mg of examined in methanol R and dilute to 10 mL with the same
C10H15NO. solvent.
STORAGE Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
Store protected from light. with methanol R.
Reference solution (a). Dissolve 25 mg of ephedrine
01/2008:0489 hydrochloride CRS in methanol R and dilute to 10 mL with
corrected 6.0 the same solvent.
Reference solution (b). Dilute 1.0 mL of test solution (a) to
EPHEDRINE HEMIHYDRATE 200 mL with methanol R.
Apply separately to the plate 10 μL of each solution. Develop
Ephedrinum hemihydricum over a path of 15 cm using a mixture of 5 volumes of
chloroform R, 15 volumes of concentrated ammonia R and
80 volumes of 2-propanol R. Allow the plate to dry in air and
spray with ninhydrin solution R. Heat at 110 °C for 5 min.
Any spot in the chromatogram obtained with test solution (a),
apart from the principal spot, is not more intense than the
spot in the chromatogram obtained with reference solution (b)
C10H15NO,½H2O Mr 174.2 (0.5 per cent). Disregard any spot of lighter colour than the
[50906-05-3] background.
DEFINITION Chlorides. Dissolve 0.18 g in 10 mL of water R. Add 5 mL of
Ephedrine hemihydrate contains not less than 99.0 per dilute nitric acid R and 0.5 mL of silver nitrate solution R1.
cent and not more than the equivalent of 101.0 per cent of Allow to stand for 2 min, protected from bright light. Any
(1R,2S)-2-(methylamino)-1-phenylpropan-1-ol, calculated opalescence in the solution is not more intense than that in a
with reference to the anhydrous substance. standard prepared at the same time and in the same manner
using 10 mL of chloride standard solution (5 ppm Cl) R, 5 mL
CHARACTERS of dilute nitric acid R and 0.5 mL of silver nitrate solution R1
A white or almost white, crystalline powder or colourless (280 ppm).
crystals, soluble in water, very soluble in alcohol. Water (2.5.12) : 4.5 per cent to 5.5 per cent, determined on
It melts at about 42 °C, determined without previous drying. 0.300 g by the semi-micro determination of water.

General Notices (1) apply to all monographs and other texts 2141
Ephedrine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Sulfated ash (2.4.14). Not more than 0.1 per cent, determined Add 2 mL of methylene chloride R and shake. The lower
on 1.0 g. (organic) layer is dark grey and the upper (aqueous) layer
is blue.
ASSAY
E. To 5 mL of solution S (see Tests) add 5 mL of water R. The
Dissolve 0.200 g in 5 mL of alcohol R and add 20.0 mL of 0.1 M solution gives reaction (a) of chlorides (2.3.1).
hydrochloric acid. Using 0.05 mL of methyl red solution R as
indicator, titrate with 0.1 M sodium hydroxide until a yellow TESTS
colour is obtained. Solution S. Dissolve 5.00 g in distilled water R and dilute to
1 mL of 0.1 M hydrochloric acid is equivalent to 16.52 mg of 50.0 mL with the same solvent.
C10H15NO.
Appearance of solution. Solution S is clear (2.2.1) and
STORAGE colourless (2.2.2, Method II).
Store protected from light. Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of
methyl red solution R and 0.2 mL of 0.01 M sodium hydroxide.
01/2008:0487 The solution is yellow. Add 0.4 mL of 0.01 M hydrochloric
corrected 6.0 acid. The solution is red.
Specific optical rotation (2.2.7) : − 33.5 to − 35.5 (dried
EPHEDRINE HYDROCHLORIDE substance).
Dilute 12.5 mL of solution S to 25.0 mL with water R.
Ephedrini hydrochloridum Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 75 mg of the substance to be examined
in the mobile phase and dilute to 10 mL with the mobile phase.
Reference solution (a). Dilute 2.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 10.0 mL with the mobile phase.
C10H16ClNO Mr 201.7 Reference solution (b). Dissolve 5 mg of the substance to be
[50-98-6] examined and 5 mg of pseudoephedrine hydrochloride CRS in
DEFINITION the mobile phase and dilute to 50 mL with the mobile phase.
(1R,2S)-2-(Methylamino)-1-phenylpropan-1-ol Column :
hydrochloride. – size : l = 0.15 m, Ø = 4.6 mm ;
Content : 99.0 per cent to 101.0 per cent (dried substance). – stationary phase : spherical phenylsilyl silica gel for
chromatography R (3 μm).
CHARACTERS Mobile phase : mix 6 volumes of methanol R and 94 volumes of
Appearance : white or almost white, crystalline powder or a 11.6 g/L solution of ammonium acetate R adjusted to pH 4.0
colourless crystals. with glacial acetic acid R.
Solubility : freely soluble in water, soluble in ethanol (96 per Flow rate : 1.0 mL/min.
cent).
Detection : spectrophotometer at 257 nm.
mp : about 219 °C.
Injection : 20 μL.
IDENTIFICATION Run time : 2.5 times the retention time of ephedrine.
First identification : B, E. Relative retention with reference to ephedrine (retention
Second identification : A, C, D, E. time = about 8 min) : impurity B = about 1.1 ;
A. Specific optical rotation (see Tests). impurity A = about 1.4.
B. Infrared absorption spectrophotometry (2.2.24). System suitability : reference solution (b) :
Comparison : ephedrine hydrochloride CRS. – resolution : minimum 2.0 between the peaks due to
C. Thin-layer chromatography (2.2.27). ephedrine and impurity B.
Test solution. Dissolve 20 mg of the substance to be Limits :
examined in methanol R and dilute to 10 mL with the same – correction factor : for the calculation of content, multiply
solvent. the peak area of impurity A by 0.4 ;
Reference solution. Dissolve 10 mg of ephedrine – impurity A : not more than the area of the principal peak
hydrochloride CRS in methanol R and dilute to 5 mL with in the chromatogram obtained with reference solution (a)
the same solvent. (0.2 per cent) ;
Plate : TLC silica gel plate R. – unspecified impurities : for each impurity, not more than
Mobile phase : methylene chloride R, concentrated 0.5 times the area of the principal peak in the chromatogram
ammonia R, 2-propanol R (5:15:80 V/V/V). obtained with reference solution (a) (0.1 per cent) ;
Application : 10 μL. – sum of impurities other than A : not more than 2.5 times the
area of the principal peak in the chromatogram obtained
Development : over 2/3 of the plate.
with reference solution (a) (0.5 per cent) ;
Drying : in air.
– disregard limit : 0.25 times the area of the principal peak
Detection : spray with ninhydrin solution R ; heat at 110 °C in the chromatogram obtained with reference solution (a)
for 5 min. (0.05 per cent).
Results : the principal spot in the chromatogram obtained
Sulfates (2.4.13) : maximum 100 ppm, determined on
with the test solution is similar in position, colour and size
solution S.
to the principal spot in the chromatogram obtained with
the reference solution. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
D. To 0.1 mL of solution S (see Tests) add 1 mL of water R, on 1.000 g by drying in an oven at 105 °C.
0.2 mL of copper sulfate solution R and 1 mL of strong Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
sodium hydroxide solution R. A violet colour is produced. 1.0 g.

2142 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ephedrine hydrochloride, racemic

ASSAY B. Examine by infrared absorption spectrophotometry

Dissolve 0.150 g in 50 mL of ethanol (96 per cent) R and add (2.2.24), comparing with the spectrum obtained with
5.0 mL of 0.01 M hydrochloric acid. Carry out a potentiometric racemic ephedrine hydrochloride CRS. Examine the
titration (2.2.20), using 0.1 M sodium hydroxide. Read substances prepared as discs.
the volume added between the 2 points of inflexion. C. Examine the chromatograms obtained in the test for
1 mL of 0.1 M sodium hydroxide is equivalent to 20.17 mg related substances. The principal spot in the chromatogram
of C10H16ClNO. obtained with test solution (b) is similar in position,
colour and size to the principal spot in the chromatogram
STORAGE obtained with reference solution (a).
Protected from light. D. To 0.1 mL of solution S (see Tests) add 1 mL of water R,
0.2 mL of copper sulfate solution R and 1 mL of strong
IMPURITIES sodium hydroxide solution R. A violet colour is produced.
Specified impurities : A. Add 2 mL of ether R and shake. The ether layer is purple
Other detectable impurities (the following substances would, and the aqueous layer is blue.
if present at a sufficient level, be detected by one or other of E. To 5 mL of solution S add 5 mL of water R. The solution
the tests in the monograph. They are limited by the general gives reaction (a) of chlorides (2.3.1).
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical TESTS
use (2034). It is therefore not necessary to identify these Solution S. Dissolve 5.00 g in distilled water R and dilute to
impurities for demonstration of compliance. See also 5.10. 50.0 mL with the same solvent.
Control of impurities in substances for pharmaceutical use) : B.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of
methyl red solution R and 0.1 mL of 0.01 M sodium hydroxide ;
the solution is yellow. Add 0.2 mL of 0.01 M hydrochloric
A. (–)-(1R)-1-hydroxy-1-phenylpropan-2-one, acid ; the solution is red.
Optical rotation (2.2.7) : + 0.2° to − 0.2°, determined on
solution S.
Related substances. Examine by thin-layer chromatography
(2.2.27), using silica gel G R as the coating substance.
B. (1S,2S)-2-(methylamino)-1-phenylpropan-1-ol Test solution (a). Dissolve 0.20 g of the substance to be
(pseudoephedrine). examined in methanol R and dilute to 10 mL with the same
solvent.
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
with methanol R.
01/2008:0715
Reference solution (a). Dissolve 20 mg of racemic ephedrine
corrected 6.0
hydrochloride CRS in methanol R and dilute to 10 mL with
the same solvent.
EPHEDRINE HYDROCHLORIDE, Reference solution (b). Dilute 1 mL of test solution (a) to
RACEMIC 200 mL with methanol R.
Apply separately to the plate 10 μL of each solution. Develop
Ephedrini racemici hydrochloridum over a path of 15 cm using a mixture of 5 volumes of
chloroform R, 15 volumes of concentrated ammonia R and
80 volumes of 2-propanol R. Allow the plate to dry in air.
Spray with ninhydrin solution R and heat at 110 °C for 5 min.
Any spot in the chromatogram obtained with test solution (a),
apart from the principal spot, is not more intense than the
spot in the chromatogram obtained with reference solution (b)
C10H16ClNO Mr 201.7 (0.5 per cent). Disregard any spot of lighter colour than the
[134-71-4] background.
DEFINITION Sulfates (2.4.13). 15 mL of solution S complies with the limit
test for sulfates (100 ppm).
Racemic ephedrine hydrochloride contains not less than
99.0 per cent and not more than the equivalent of 101.0 per Loss on drying (2.2.32). Not more than 0.5 per cent,
cent of (1RS,2SR)-2-(methylamino)-1-phenylpropan-1-ol determined on 1.000 g by drying in an oven at 105 °C.
hydrochloride, calculated with reference to the dried Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
substance. on 1.0 g.
CHARACTERS ASSAY
A white or almost white, crystalline powder or colourless Dissolve 0.170 g in 30 mL of ethanol (96 per cent) R. Add
crystals, freely soluble in water, soluble in ethanol (96 per 5.0 mL of 0.01 M hydrochloric acid. Carry out a potentiometric
cent). titration (2.2.20), using 0.1 M sodium hydroxide. Read
It melts at about 188 °C. the volume added between the two points of inflexion.
IDENTIFICATION 1 mL of 0.1 M sodium hydroxide corresponds to 20.17 mg of
C10H16ClNO.
First identification : B, E.
Second identification : A, C, D, E. STORAGE
A. Optical rotation (see Tests). Store protected from light.

General Notices (1) apply to all monographs and other texts 2143
Epinastine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

01/2010:2411 Detection : spectrophotometer at 220 nm.

corrected 7.0 Injection : 10 μL.
Identification of impurities : use the chromatogram
EPINASTINE HYDROCHLORIDE supplied with epinastine for system suitability CRS and the
chromatogram obtained with reference solution (b) to identify
Epinastini hydrochloridum the peaks due to impurities A and B.
Relative retention with reference to epinastine (retention
time = about 4 min) : impurity A = about 1.2 ;
impurity B = about 2.0.
System suitability : reference solution (b) :
– peak-to-valley ratio : minimum 2.0, where Hp = height
above the baseline of the peak due to impurity A and
C16H16ClN3 Mr 285.8 Hv = height above the baseline of the lowest point of the
[108929-04-0] curve separating this peak from the peak due to epinastine.
Limits :
DEFINITION – impurity B : not more than 3 times the area of the principal
(13bRS)-9,13b-Dihydro-1H-dibenzo[c,f]imidazo[1,5- peak in the chromatogram obtained with reference
a]azepin-3-amine hydrochloride. solution (a) (0.3 per cent) ;
Content : 99.0 per cent to 101.0 per cent (dried substance). – impurity A : not more than twice the area of the principal
peak in the chromatogram obtained with reference
CHARACTERS solution (a) (0.2 per cent) ;
Appearance : white or almost white, hygroscopic, crystalline – unspecified impurities : for each impurity, not more than the
powder. area of the principal peak in the chromatogram obtained
Solubility : freely soluble in water and in methanol, sparingly with reference solution (a) (0.10 per cent) ;
soluble in methylene chloride, slightly soluble in acetonitrile. – total : not more than 7 times the area of the principal peak
IDENTIFICATION in the chromatogram obtained with reference solution (a)
(0.7 per cent) ;
A. Infrared absorption spectrophotometry (2.2.24).
– disregard limit : 0.5 times the area of the principal peak in
Comparison : epinastine hydrochloride CRS. the chromatogram obtained with reference solution (a)
B. It gives reaction (a) of chlorides (2.3.1). (0.05 per cent).
TESTS Heavy metals (2.4.8) : maximum 20 ppm.
Acidity or alkalinity. Dissolve 1.0 g in carbon dioxide-free Solvent : water R.
water R and dilute to 10 mL with the same solvent. Add 0.250 g complies with test H. Prepare the reference solution
0.1 mL of methyl red mixed solution R and 0.25 mL of 0.01 M using 0.5 mL of lead standard solution (10 ppm Pb) R.
sodium hydroxide. The solution is green. Add 0.5 mL of Loss on drying (2.2.32) : maximum 1.0 per cent, determined
0.01 M hydrochloric acid. The solution is reddish-violet. on 1.000 g by drying in an oven at 105 °C.
Related substances. Liquid chromatography (2.2.29). Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Buffer solution pH 4.4. Dissolve 3.8 g of sodium 1.0 g.
pentanesulfonate monohydrate R and 4.0 g of potassium
dihydrogen phosphate R in water R, adjust to pH 4.4 with ASSAY
phosphoric acid R and dilute to 1000.0 mL with water R. Dissolve 0.200 g in 100 mL of a mixture of 1 volume of
Solvent mixture : mobile phase B, mobile phase A (25:75 V/V). anhydrous acetic acid R and 2 volumes of acetic anhydride R.
Test solution. Dissolve 50 mg of the substance to be examined Titrate with 0.1 M perchloric acid, determining the end-point
in the solvent mixture and dilute to 100.0 mL with the solvent potentiometrically (2.2.20).
mixture. 1 mL of 0.1 M perchloric acid is equivalent to 28.58 mg of
Reference solution (a). Dilute 10.0 mL of the test solution C16H16ClN3.
to 100.0 mL with the solvent mixture. Dilute 1.0 mL of this STORAGE
solution to 100.0 mL with the solvent mixture. In an airtight container.
Reference solution (b). Dissolve 5 mg of epinastine for system
suitability CRS (containing impurities A and B) in 10.0 mL IMPURITIES
of the solvent mixture. Specified impurities : A, B.
Column :
– size : l = 0.10 m, Ø = 3.0 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (3 μm) ;
– temperature : 50 °C.
Mobile phase :
A. 9H-dibenzo[c,f]imidazo[1,5-a]azepin-3-amine,
– mobile phase A : methanol R2, buffer solution pH 4.4
(15:85 V/V) ;
– mobile phase B : methanol R2, acetonitrile R1 (15:85 V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-4 80 20

4 - 13 80 → 30 20 → 70
B. (13bRS)-7-bromo-9,13b-dihydro-1H-dibenzo[c,f]imidazo-
Flow rate : 1.4 mL/min. [1,5-a]azepin-3-amine.

2144 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Epirubicin hydrochloride

01/2008:1590 Reference solution (d). Dilute 1.0 mL of the test solution to

100.0 mL with the mobile phase.
EPIRUBICIN HYDROCHLORIDE Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
Epirubicini hydrochloridum – stationary phase : trimethylsilyl silica gel for
chromatography R (6 μm) ;
– temperature : 35 °C.
Mobile phase : mix 17 volumes of methanol R, 29 volumes
of acetonitrile R and 54 volumes of a solution containing
3.7 g/L of sodium laurilsulfate R and 2.8 per cent V/V of dilute
phosphoric acid R.
Flow rate : 2.5 mL/min.
Detection : spectrophotometer at 254 nm.
Injection : 10 μL of the test solution and reference solutions (b),
C27H30ClNO11 Mr 580.0 (c) and (d).
[56390-09-1] Run time : 3.5 times the retention time of epirubicin.
DEFINITION Identification of impurities : use the 2nd most abundant
(8S,10S)-10-[(3-Amino-2,3,6-trideoxy-α-L-arabino- peak present in the chromatogram obtained with reference
hexopyranosyl)oxy]-6,8,11-trihydroxy-8-(hydroxyacetyl)- solution (c) to identify impurity A.
1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione Relative retention with reference to epirubicin (retention
hydrochloride. time = about 9.5 min) : impurity A = about 0.3 ;
Substance obtained by chemical transformation of a substance impurity B = about 0.4 ; impurity C = about 0.8 ;
produced by certain strains of Streptomyces peucetius. impurity E = about 1.1 ; impurity D = about 1.5 ;
impurity F = about 1.7 ; impurity G = about 2.1.
Content : 97.0 per cent to 102.0 per cent (anhydrous substance).
System suitability : reference solution (b) :
CHARACTERS – resolution : minimum 2.0 between the peaks due to
Appearance : orange-red powder. impurity C and epirubicin.
Solubility : soluble in water and in methanol, slightly soluble in Limits :
anhydrous ethanol, practically insoluble in acetone.
– correction factor : for the calculation of content, multiply
IDENTIFICATION the peak area of impurity A by 0.7 ;
A. Infrared absorption spectrophotometry (2.2.24). – impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (d)
Comparison : epirubicin hydrochloride CRS.
(1.0 per cent) ;
B. Examine the chromatograms obtained in the assay.
– impurity C : not more than the area of the principal peak
Results : the principal peak in the chromatogram obtained in the chromatogram obtained with reference solution (d)
with the test solution is similar in retention time to (1.0 per cent) ;
the principal peak in the chromatogram obtained with
reference solution (a). – any other impurity : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram
C. Dissolve about 10 mg in 0.5 mL of nitric acid R, add 0.5 mL obtained with reference solution (d) (0.5 per cent) ;
of water R and heat over a flame for 2 min. Allow to
cool and add 0.5 mL of silver nitrate solution R1. A white – total : not more than twice the area of the principal peak
precipitate is formed. in the chromatogram obtained with reference solution (d)
(2.0 per cent) ;
TESTS – disregard limit : 0.05 times the area of the principal peak
pH (2.2.3) : 4.0 to 5.5. in the chromatogram obtained with reference solution (d)
Dissolve 50 mg in carbon dioxide-free water R and dilute to (0.05 per cent).
10 mL with the same solvent. Acetone (2.4.24) : maximum 1.5 per cent.
Related substances. Liquid chromatography (2.2.29). Allow Water (2.5.12) : maximum 4.0 per cent, determined on 0.100 g.
the solutions to stand for 3 h before use.
Bacterial endotoxins (2.6.14) : less than 1.1 IU/mg, if intended
Test solution. Dissolve 25.0 mg of the substance to be for use in the manufacture of parenteral preparations without
examined in the mobile phase and dilute to 25.0 mL with the a further appropriate procedure for removal of bacterial
mobile phase. endotoxins.
Reference solution (a). Dissolve 25.0 mg of epirubicin
hydrochloride CRS in the mobile phase and dilute to 25.0 mL ASSAY
with the mobile phase. Liquid chromatography (2.2.29) as described in the test for
Reference solution (b). Dissolve 10 mg of epirubicin related substances with the following modification.
hydrochloride CRS and 10 mg of doxorubicin hydrochloride CRS Injection : test solution and reference solution (a).
in the mobile phase and dilute to 100 mL with the mobile
phase. Calculate the percentage content of C27H30ClNO11.
Reference solution (c). Dissolve 10 mg of doxorubicin
hydrochloride CRS in a mixture of 5 mL of water R and 5 mL STORAGE
of phosphoric acid R. Allow to stand for 30 min. Adjust to In an airtight container, protected from light, at a temperature
pH 2.6 with an 80 g/L solution of sodium hydroxide R. Add of 2 °C to 8 °C. If the substance is sterile, store in a sterile,
15 mL of acetonitrile R and 10 mL of methanol R. Mix. airtight, tamper-proof container.

General Notices (1) apply to all monographs and other texts 2145
Ergocalciferol EUROPEAN PHARMACOPOEIA 8.0

IMPURITIES

A. R = OH : (8S,10S)-6,8,10,11-tetrahydroxy-8-
(hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-
5,12-dione (doxorubicinone),

B. R = H : (8S,10S)-8-acetyl-6,8,10,11-tetrahydroxy-1-
methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione
(daunorubicinone),

G. 8,8′-[(2R,4R)-4-hydroxy-2-(hydroxymethyl)-1,3-dioxolan-
2,4-diyl]bis[(8S,10S)-10-[(3-amino-2,3,6-trideoxy-α-L-
arabino-hexopyranosyl)oxy]-6,8,11-trihydroxy-1-methoxy-
7,8,9,10-tetrahydrotetracene-5,12-dione] (epirubicin
dimer).

01/2008:0082
corrected 6.3
C. (8S,10S)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-
hexopyranosyl)oxy]-6,8,11-trihydroxy-8-(hydroxyacetyl)- ERGOCALCIFEROL
1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione
(doxorubicin), Ergocalciferolum

D. (8S,10S)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-
hexopyranosyl)oxy]-6,8,11-trihydroxy-1-methoxy-7,8,9,10-
tetrahydrotetracene-5,12-dione (daunorubicin), C28H44O Mr 396.7
[50-14-6]
DEFINITION
Ergocalciferol contains not less than 97.0 per cent
and not more than the equivalent of 103.0 per cent of
(5Z,7E,22E)-9,10-secoergosta-5,7,10(19),22-tetraen-3β-ol.
1 mg of ergocalciferol is equivalent to 40 000 IU of antirachitic
activity (vitamin D) in rats.
CHARACTERS
A white or slightly yellowish, crystalline powder or white or
E. (8S,10S)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo- almost white crystals, practically insoluble in water, freely
hexopyranosyl)oxy]-6,8,11-trihydroxy-8-[(1RS)-1- soluble in alcohol, soluble in fatty oils. It is sensitive to air,
hydroxyethyl]-1-methoxy-7,8,9,10-tetrahydrotetracene- heat and light. Solutions in volatile solvents are unstable and
5,12-dione (dihydrodaunorubicin), are to be used immediately.
A reversible isomerisation to pre-ergocalciferol takes place in
solution, depending on temperature and time. The activity is
due to both compounds.
IDENTIFICATION
Examine by infrared absorption spectrophotometry (2.2.24),
comparing with the spectrum obtained with ergocalciferol CRS.
Examine the substances prepared as discs.
TESTS
Specific optical rotation (2.2.7). Dissolve 0.200 g rapidly
F. (8S,10S)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-α-L- and without heating in aldehyde-free alcohol R and dilute to
arabino-hexopyranosyl)oxy]-6,8,11-trihydroxy-1- 25.0 mL with the same solvent. The specific optical rotation,
methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione determined within 30 min of preparing the solution, is + 103
(epi-daunorubicin), to + 107.

2146 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ergocalciferol

Reducing substances. Dissolve 0.1 g in aldehyde-free alcohol R – as mobile phase at a flow rate of 2 mL/min a mixture of
and dilute to 10.0 mL with the same solvent. Add 0.5 mL of a 3 volumes of pentanol R and 997 volumes of hexane R,
5 g/L solution of tetrazolium blue R in aldehyde-free alcohol R – as detector a spectrophotometer set at 254 nm.
and 0.5 mL of dilute tetramethylammonium hydroxide
solution R. Allow to stand for exactly 5 min and add 1.0 mL of An automatic injection device or a sample loop is
glacial acetic acid R. Prepare a reference solution at the same recommended. Inject a suitable volume of reference
time and in the same manner using 10.0 mL of a solution solution (b). Adjust the sensitivity of the system so that the
containing 0.2 μg/mL of hydroquinone R in aldehyde-free height of the principal peak is at least 50 per cent of the full
alcohol R. Measure the absorbance (2.2.25) of the two scale of the recorder. Inject reference solution (b) 6 times.
solutions at 525 nm using as the compensation liquid 10.0 mL When the chromatograms are recorded in the prescribed
of aldehyde-free alcohol R treated in the same manner. The conditions, the approximate relative retention times with
absorbance of the test solution is not greater than that of the reference to cholecalciferol are 0.4 for pre-cholecalciferol
reference solution (20 ppm). and 0.5 for trans-cholecalciferol. The relative standard
deviation of the response for cholecalciferol is not greater
Ergosterol. Examine by thin-layer chromatography (2.2.27), than 1 per cent and the resolution between the peaks due to
using a TLC silica gel G plate R. pre-cholecalciferol and trans-cholecalciferol is not less than
Test solution. Dissolve 0.25 g of the substance to be examined 1.0. If necessary adjust the proportions of the constituents and
in ethylene chloride R containing 10 g/L of squalane R and the flow rate of the mobile phase to obtain this resolution.
0.1 g/L of butylhydroxytoluene R and dilute to 5 mL with the Inject a suitable volume of reference solution (a). Adjust the
same solvent. Prepare immediately before use. sensitivity of the system so that the height of the principal
Reference solution (a). Dissolve 0.10 g of ergocalciferol CRS peak is at least 50 per cent of the full scale of the recorder.
in ethylene chloride R containing 10 g/L of squalane R and Inject the same volume of the test solution and record the
0.1 g/L of butylhydroxytoluene R and dilute to 2 mL with the chromatogram in the same manner.
same solvent. Prepare immediately before use. Calculate the percentage content of ergocalciferol from the
Reference solution (b). Dissolve 5 mg of ergosterol CRS in expression :
ethylene chloride R containing 10 g/L of squalane R and 0.1 g/L
of butylhydroxytoluene R and dilute to 50 mL with the same
solvent. Prepare immediately before use.
Reference solution (c). Mix equal volumes of reference m
solution (a) and reference solution (b). Prepare immediately = mass of the substance to be examined in the test
before use. solution, in milligrams ;
m′ = mass of ergocalciferol CRS in reference solution (a),
Apply to the plate 10 μL of the test solution, 10 μL of reference
solution (a), 10 μL of reference solution (b) and 20 μL of in milligrams ;
reference solution (c). Develop immediately, protected from SD = area (or height) of the peak due to ergocalciferol in
light, over a path of 15 cm using a mixture of equal volumes the chromatogram obtained with the test solution ;
of cyclohexane R and peroxide-free ether R, the mixture S′ D = area (or height) of the peak due to ergocalciferol
containing 0.1 g/L of butylhydroxytoluene R. Allow the plate in the chromatogram obtained with reference
to dry in air and spray three times with antimony trichloride solution (a).
solution R1. Examine the chromatograms for 3 min to 4 min
after spraying. The principal spot in the chromatogram STORAGE
obtained with the test solution is initially orange-yellow and
then becomes brown. In the chromatogram obtained with the Store in an airtight container, under nitrogen, protected from
test solution, any slowly appearing violet spot (corresponding light, at a temperature between 2 °C and 8 °C.
to ergosterol) immediately below the principal spot is not The contents of an opened container are to be used
more intense than the spot in the chromatogram obtained immediately.
with reference solution (b) (0.2 per cent). There is no spot in
the chromatogram obtained with the test solution that does IMPURITIES
not correspond to one of the spots in the chromatograms
obtained with reference solutions (a) and (b). The test is
not valid unless the chromatogram obtained with reference
solution (c) shows two clearly separated spots.

ASSAY
Carry out the operations as rapidly as possible, avoiding
exposure to actinic light and air.
Examine by liquid chromatography (2.2.29).
Test solution. Dissolve 10.0 mg of the substance to be
examined without heating in 10.0 mL of toluene R and dilute
to 100.0 mL with the mobile phase. A. (5E,7E,22E)-9,10-secoergosta-5,7,10(19),22-tetraen-3β-ol
Reference solution (a). Dissolve 10.0 mg of ergocalciferol CRS (trans-vitamin D2),
without heating in 10.0 mL of toluene R and dilute to 100.0 mL
with the mobile phase.
Reference solution (b). Dilute 1.0 mL of cholecalciferol for
system suitability CRS to 5.0 mL with the mobile phase. Heat
in a water-bath at 90 °C under a reflux condenser for 45 min
and cool.
The chromatographic procedure may be carried out using :
– a stainless steel column 0.25 m long and 4.6 mm in internal
diameter packed with a suitable silica gel (5 μm), B. (22E)-ergosta-5,7,22-trien-3β-ol (ergosterol),

General Notices (1) apply to all monographs and other texts 2147
Ergometrine maleate EUROPEAN PHARMACOPOEIA 8.0

IDENTIFICATION
First identification : B, C.
Second identification : A, C, D, E.
A. Dissolve 30 mg in 0.01 M hydrochloric acid and dilute to
100.0 mL with the same acid. Dilute 10.0 mL of the solution
to 100.0 mL with 0.01 M hydrochloric acid. Examined
between 250 nm and 360 nm (2.2.25), the solution shows an
C. (9β,10α,22E)-ergosta-5,7,22-trien-3β-ol (lumisterol2), absorption maximum at 311 nm and a minimum at 265 nm
to 272 nm. The specific absorbance at the maximum is 175
to 195.
B. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
ergometrine maleate CRS. Examine the substances prepared
as discs.
C. Examine the chromatograms obtained in the test for
related substances. The principal spot in the chromatogram
obtained with test solution (b) is similar in position,
colour and size to the principal spot in the chromatogram
obtained with reference solution (a).
D. To 0.1 mL of solution S (see Tests) add 1 mL of glacial
D. (6E,22E)-9,10-secoergosta-5(10),6,8(14),22-tetraen-3β-ol acetic acid R, 0.05 mL of ferric chloride solution R1 and
(iso-tachysterol2), 1 mL of phosphoric acid R and heat in a water-bath at 80 °C.
After about 10 min, a blue or violet colour develops which
becomes more intense on standing.
E. Dissolve 0.1 g in a mixture of 0.5 mL of dilute sulfuric acid R
and 2.5 mL of water R. Add 5 mL of ether R and 1 mL of
strong sodium hydroxide solution R and shake. Separate the
aqueous layer and shake with two quantities, each of 5 mL,
of ether R. To 0.1 mL of the aqueous layer add a solution
of 10 mg of resorcinol R in 3 mL of sulfuric acid R. Heat
on a water-bath for 15 min. No colour develops. To the
rest of the aqueous layer add 1 mL of bromine water R.
Heat on a water-bath for 10 min, then heat to boiling and
cool. To 0.2 mL of this solution add a solution of 10 mg of
resorcinol R in 3 mL of sulfuric acid R. Heat on a water-bath
E. (6E,22E)-9,10-secoergosta-5(10),6,8,22-tetraen-3β-ol for 15 min. A pinkish-violet colour develops.
(tachysterol2).
TESTS
Solution S. Dissolve 0.100 g, without heating and protected
from light, in 9 mL of carbon dioxide-free water R and dilute
to 10.0 mL with the same solvent.
01/2008:0223
corrected 6.0 Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y5 or BY5
(2.2.2, Method II).
ERGOMETRINE MALEATE pH (2.2.3). The pH of solution S is 3.6 to 4.4.
Specific optical rotation (2.2.7) : + 50 to + 56, determined on
Ergometrini maleas solution S and calculated with reference to the dried substance.
Related substances. Examine by thin-layer chromatography
(2.2.27), using silica gel G R as the coating substance. Carry
out all operations as rapidly as possible, protected from light.
Prepare the test and reference solutions immediately before use.
Test solution (a). Dissolve 50 mg of the substance to be
examined in a mixture of 1 volume of concentrated ammonia R
and 9 volumes of alcohol (80 per cent V/V) R and dilute to
C23H27N3O6 Mr 441.5 5.0 mL with the same mixture of solvents.
[129-51-1] Test solution (b). Dilute 1.0 mL of test solution (a) to 10.0 mL
with a mixture of 1 volume of concentrated ammonia R and
DEFINITION 9 volumes of alcohol (80 per cent V/V) R.
Ergometrine maleate contains not less than 98.0 per cent and Reference solution (a). Dissolve 10 mg of ergometrine
not more than the equivalent of 101.0 per cent of (6aR,9R)- maleate CRS in a mixture of 1 volume of concentrated
N-[(S)-2-hydroxy-1-methylethyl]-7-methyl-4,6,6a,7,8,9- ammonia R and 9 volumes of alcohol (80 per cent V/V) R and
hexahydro-indolo[4,3-fg]quinoline-9-carboxamide dilute to 10.0 mL with the same mixture of solvents.
(Z)-butenedioate, calculated with reference to the dried Reference solution (b). Dilute 5.0 mL of reference solution (a)
substance. to 50.0 mL with a mixture of 1 volume of concentrated
ammonia R and 9 volumes of alcohol (80 per cent V/V) R.
CHARACTERS Reference solution (c). To 2.0 mL of reference solution (b) add
A white or almost white or slightly coloured, crystalline 2.0 mL of a mixture of 1 volume of concentrated ammonia R
powder, sparingly soluble in water, slightly soluble in alcohol. and 9 volumes of alcohol (80 per cent V/V) R.

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EUROPEAN PHARMACOPOEIA 8.0 Ergotamine tartrate

Apply separately to the plate 5 μL of each solution. Develop minimum at 265 nm to 275 nm. The specific absorbance
immediately over a path of 14 cm using a mixture of 3 volumes at the maximum is 118 to 128, calculated with reference
of water R, 25 volumes of methanol R and 75 volumes of to the dried substance.
chloroform R. Dry the plate in a current of cold air and spray B. Examine by infrared absorption spectrophotometry
with dimethylaminobenzaldehyde solution R7. Dry the plate (2.2.24), comparing with the spectrum obtained with
in a current of warm air for about 2 min. Any spot in the ergotamine tartrate CRS. Examine the substances as
chromatogram obtained with test solution (a), apart from the discs prepared as follows : triturate the substance to be
principal spot, is not more intense than the principal spot examined and the reference substance separately with
in the chromatogram obtained with reference solution (b) 0.2 mL of methanol R and then with potassium bromide R
(1.0 per cent) and at most one such spot is more intense as prescribed in the general method.
than the principal spot in the chromatogram obtained with C. Examine for not more than 1 min in ultraviolet light at
reference solution (c) (0.5 per cent). 365 nm the chromatograms obtained in the test for related
Loss on drying (2.2.32). Not more than 2.0 per cent, substances. The principal spot in the chromatogram
determined on 0.20 g by drying over diphosphorus pentoxide R obtained with test solution (b) is similar in position and
at 80 °C at a pressure not exceeding 2.7 kPa for 2 h. fluorescence to the principal spot in the chromatogram
obtained with reference solution (a). After spraying with
ASSAY dimethylaminobenzaldehyde solution R7, examine in
Dissolve 0.150 g in 40 mL of anhydrous acetic acid R. Titrate daylight. The principal spot in the chromatogram obtained
with 0.05 M perchloric acid, determining the end-point with test solution (b) is similar in position, colour and size
potentiometrically (2.2.20). to the principal spot in the chromatogram obtained with
1 mL of 0.05 M perchloric acid is equivalent to 22.07 mg of reference solution (a).
C23H27N3O6. D. To 0.1 mL of solution S (see Tests) add 1 mL of glacial
acetic acid R, 0.05 mL of ferric chloride solution R1 and
STORAGE 1 mL of phosphoric acid R and heat in a water-bath at 80 °C.
Store in an airtight, glass container, protected from light, at a After about 10 min, a blue or violet colour develops which
temperature of 2 °C to 8 °C. becomes more intense on standing.
E. Dissolve about 10 mg in 1.0 mL of 0.1 M sodium hydroxide.
01/2008:0224 Transfer to a separating funnel and shake with 5 mL of
methylene chloride R. Discard the organic layer. Neutralise
ERGOTAMINE TARTRATE the aqueous layer with a few drops of dilute hydrochloric
acid R. 0.1 mL of this solution gives reaction (b) of tartrates
Ergotamini tartras (2.3.1). Pour the reaction mixture into 1 mL of water R to
observe the colour change to red or brownish-red.
TESTS
Carry out all operations as rapidly as possible, protected from
light.
Solution S. Triturate 30 mg finely with about 15 mg of tartaric
acid R and dissolve with shaking in 6 mL of water R.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y6 (2.2.2,
Method II).
pH (2.2.3). Shake 10 mg, finely powdered, with 4 mL of carbon
dioxide-free water R. The pH of the suspension is 4.0 to 5.5.
C70H76N10O16 Mr 1313 Specific optical rotation (2.2.7). Dissolve 0.40 g in 40 mL
[379-79-3] of a 10 g/L solution of tartaric acid R. Add 0.5 g of sodium
hydrogen carbonate R cautiously in several portions and
DEFINITION mix thoroughly. Shake with four quantities, each of 10 mL,
Ergotamine tartrate contains not less than 98.0 per cent of chloroform R previously washed with five quantities of
and not more than the equivalent of 101.0 per cent of water R, each of 50 mL per 100 mL of chloroform R. Combine
bis[(6aR,9R)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-hydroxy-2- the organic layers. Filter through a small filter moistened with
methyl-3,6-dioxo-octahydro-8H-oxazolo[3,2-a]pyrrolo[2,1- chloroform R previously washed as described above. Dilute
c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9-hexahydroindolo[4,3- the filtrate to 50.0 mL with chloroform R previously washed as
fg]quinoline-9-carboxamide] tartrate, calculated with described above. Measure the angle of rotation.
reference to the dried substance. It may contain two molecules Determine the amount of ergotamine base in the chloroformic
of methanol of crystallisation. solution as follows : to 25.0 mL of the solution add 50 mL of
CHARACTERS anhydrous acetic acid R and titrate with 0.05 M perchloric acid,
determining the end-point potentiometrically (2.2.20).
A white or almost white, crystalline powder or colourless
crystals, slightly hygroscopic, slightly soluble in alcohol. 1 mL of 0.05 M perchloric acid is equivalent to 29.08 mg of
Aqueous solutions slowly become cloudy owing to hydrolysis ; C33H35N5O5.
this may be prevented by the addition of tartaric acid. The specific optical rotation is − 154 to − 165, calculated from
the angle of rotation and the concentration of ergotamine base.
IDENTIFICATION Related substances. Examine by thin-layer chromatography
First identification : B, C. (2.2.27), using a TLC silica gel G plate R. Prepare the reference
Second identification : A, C, D, E. solutions and the test solutions immediately before use and in
A. Dissolve 50 mg in 0.01 M hydrochloric acid and dilute to the order indicated below.
100.0 mL with the same acid. Dilute 10.0 mL of the solution Reference solution (a). Dissolve 10 mg of ergotamine
to 100.0 mL with 0.01 M hydrochloric acid. Examined tartrate CRS in a mixture of 1 volume of methanol R and
between 250 nm and 360 nm (2.2.25), the solution shows 9 volumes of methylene chloride R and dilute to 10.0 mL with
an absorption maximum at 311 nm to 321 nm and a the same mixture of solvents.

General Notices (1) apply to all monographs and other texts 2149
Erythritol EUROPEAN PHARMACOPOEIA 8.0

Reference solution (b). Dilute 7.5 mL of reference solution (a) IDENTIFICATION

to 50.0 mL with a mixture of 1 volume of methanol R and A. Melting point (2.2.14) : 119 °C to 122 °C.
9 volumes of methylene chloride R. B. Infrared absorption spectrophotometry (2.2.24).
Reference solution (c). To 2.0 mL of reference solution (b) add Comparison: erythritol CRS.
4.0 mL of a mixture of 1 volume of methanol R and 9 volumes
of methylene chloride R. TESTS
Test solution (a). Dissolve 50 mg of the substance to be Appearance of solution. The solution is clear (2.2.1) and
examined in a mixture of 1 volume of methanol R and colourless (2.2.2, Method II).
9 volumes of methylene chloride R and dilute to 5.0 mL with Dissolve 5.0 g in water R and dilute to 50 mL with the same
the same mixture of solvents. solvent.
Test solution (b). Dilute 1.0 mL of test solution (a) to 10.0 mL Conductivity (2.2.38) : maximum 20 μS·cm− 1.
with a mixture of 1 volume of methanol R and 9 volumes of
methylene chloride R. Dissolve 20.0 g in carbon dioxide-free water R prepared from
distilled water R and dilute to 100.0 mL with the same solvent.
Apply immediately to the plate 5 μL of each reference solution Measure the conductivity of the solution, while gently stirring
and then 5 μL of each test solution. Expose the points of with a magnetic stirrer.
application immediately to ammonia vapour and for exactly
20 s by moving the line of application from side to side above a Related substances. Liquid chromatography (2.2.29).
beaker 55 mm high and 45 mm in diameter containing about Test solution. Dissolve 0.50 g of the substance to be examined
20 mL of concentrated ammonia R. Dry the line of application in water R and dilute to 10.0 mL with the same solvent.
in a current of cold air for exactly 20 s. Develop immediately Reference solution (a). Dissolve 0.50 g of erythritol CRS in
over a path of 17 cm using a mixture of 5 volumes of water R and dilute to 10.0 mL with the same solvent.
ethanol R, 10 volumes of methylene chloride R, 15 volumes of Reference solution (b). Dilute 2.0 mL of the test solution to
dimethylformamide R and 70 volumes of ether R. Dry the plate 100.0 mL with water R.
in a current of cold air for about 2 min. Examine for not more
than 1 min in ultraviolet light at 365 nm for the identification. Reference solution (c). Dilute 5.0 mL of reference solution (b)
to 100.0 mL with water R.
Spray the plate abundantly with dimethylaminobenzaldehyde
solution R7 and dry in a current of warm air for about 2 min. Reference solution (d). Dissolve 1.0 g of erythritol R and 1.0 g
Any spot in the chromatogram obtained with test solution (a), of glycerol R in water R and dilute to 20.0 mL with the same
apart from the principal spot, is not more intense than the solvent.
principal spot in the chromatogram obtained with reference Column :
solution (b) (1.5 per cent) and at most one such spot is more – size : l = 0.3 m, Ø = 7.8 mm ;
intense than the principal spot in the chromatogram obtained – stationary phase : cation-exchange resin R (9 μm) ;
with reference solution (c) (0.5 per cent).
– temperature : 70 °C.
Loss on drying (2.2.32). Not more than 6.0 per cent, Mobile phase : 0.01 per cent V/V solution of sulfuric acid R.
determined on 0.100 g by drying in vacuo at 95 °C for 6 h.
Flow rate : 0.8 mL/min.
ASSAY Detection : refractometer maintained at a constant temperature.
Dissolve 0.200 g in 40 mL of anhydrous acetic acid R. Titrate Injection : 20 μL ; inject the test solution and reference
with 0.05 M perchloric acid, determining the end-point solutions (b), (c) and (d).
potentiometrically (2.2.20). Run time : 3 times the retention time of erythritol.
1 mL of 0.05 M perchloric acid is equivalent to 32.84 mg of Relative retention with reference to erythritol (retention
C70H76N10O16. time = about 11 min) : impurity A = about 0.77 ;
impurity B = about 0.90 ; impurity C = about 0.94 ;
STORAGE impurity D = about 1.10.
Store in an airtight, glass container, protected from light, at a System suitability : reference solution (d) :
temperature of 2 °C to 8 °C.
– resolution : minimum 2 between the peaks due to erythritol
and impurity D.
01/2009:1803 Limits :
– any impurity : not more than the area of the principal peak
ERYTHRITOL in the chromatogram obtained with reference solution (b)
(2.0 per cent) ;
Erythritolum – total : not more than the area of the principal peak in the
chromatogram obtained with reference solution (b) (2.0 per
cent) ;
– disregard limit : area of the principal peak in the
chromatogram obtained with reference solution (c) (0.1 per
cent).
Lead (2.4.10) : maximum 0.5 ppm.
C4H10O4 Mr 122.1
[149-32-6] Water (2.5.12) : maximum 0.5 per cent, determined on 1.00 g.
Microbial contamination
DEFINITION
If intended for use in the manufacture of parenteral
(2R,3S)-Butane-1,2,3,4-tetrol (meso-erythritol). preparations :
Content : 96.0 per cent to 102.0 per cent (anhydrous substance). – TAMC : acceptance criterion 102 CFU/g (2.6.12).
CHARACTERS If not intended for use in the manufacture of parenteral
Appearance : white or almost white, crystalline powder or preparations :
free-flowing granules. – TAMC : acceptance criterion 103 CFU/g (2.6.12) ;
Solubility : freely soluble in water, very slightly soluble in – TYMC : acceptance criterion 102 CFU/g (2.6.12) ;
ethanol (96 per cent). – absence of Escherichia coli (2.6.13) ;

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EUROPEAN PHARMACOPOEIA 8.0 Erythromycin

– absence of Salmonella (2.6.13). DEFINITION

Bacterial endotoxins (2.6.14). If intended for use in the Mixture of macrolide antibiotics produced by a strain
manufacture of parenteral preparations without a further of Streptomyces erythreus, the main component being
appropriate procedure for the removal of bacterial endotoxins : (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-4-[(2,6-dideoxy-
– less than 4 IU/g for parenteral preparations having a 3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-
concentration of 100 g/L or less of erythritol ; 14-ethyl-7,12,13-trihydroxy-3,5,7,9,11,13-hexamethyl-6-
– less than 2.5 IU/g for parenteral preparations having a [(3,4,6-trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyl)-
concentration of more than 100 g/L of erythritol. oxy]oxacyclotetradecane-2,10-dione (erythromycin A).
Content :
ASSAY
– sum of the contents of erythromycin A, erythromycin B and
Liquid chromatography (2.2.29) as described in the test for erythromycin C : 93.0 per cent to 102.0 per cent (anhydrous
related substances with the following modification. substance) ;
Injection : test solution and reference solution (a). – erythromycin B : maximum 5.0 per cent ;
Calculate the percentage content of erythritol using the
chromatogram obtained with reference solution (a) and the – erythromycin C : maximum 5.0 per cent.
declared content of erythritol CRS. CHARACTERS
LABELLING Appearance : white or slightly yellow powder or colourless or
The label states where applicable, that the substance is suitable slightly yellow crystals, slightly hygroscopic.
for use in the manufacture of parenteral preparations. Solubility : slightly soluble in water (the solubility decreases as
the temperature rises), freely soluble in ethanol (96 per cent),
IMPURITIES soluble in methanol.
IDENTIFICATION
First identification : A.
Second identification : B, C, D.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: erythromycin A CRS.
Disregard any band in the region from 1980 cm− 1 to
A. 4-O-α-D-glucopyranosyl-D-glucitol (D-maltitol),
2050 cm− 1.
If the spectra obtained show differences, dissolve 50 mg
of the substance to be examined and of the reference
substance separately in 1.0 mL of methylene chloride R, dry
at 60 °C at a pressure not exceeding 670 Pa for 3 h and
record new spectra using the residues.
B. D-glucitol (D-sorbitol), B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 10 mg of the substance to be
examined in methanol R and dilute to 10 mL with the same
solvent.
Reference solution (a). Dissolve 10 mg of
C. (2R,3s,4S)-pentane-1,2,3,4,5-pentol (meso-ribitol), erythromycin A CRS in methanol R and dilute to
10 mL with the same solvent.
Reference solution (b). Dissolve 20 mg of spiramycin CRS in
methanol R and dilute to 10 mL with the same solvent.
D. propane-1,2,3-triol (glycerol). Plate : TLC silica gel G plate R.
Mobile phase : mix 4 volumes of 2-propanol R, 8 volumes
01/2012:0179 of a 150 g/L solution of ammonium acetate R previously
adjusted to pH 9.6 with ammonia R and 9 volumes of ethyl
ERYTHROMYCIN acetate R. Allow to settle and use the upper layer.
Application : 10 μL.
Erythromycinum Development : over 2/3 of the plate.
Drying : in air.
Detection : spray with anisaldehyde solution R1 and heat at
110 °C for 5 min.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with reference solution (a) and its position and colour are
different from those of the spots in the chromatogram
obtained with reference solution (b).
C. To about 5 mg add 5 mL of a 0.2 g/L solution of
xanthydrol R in a mixture of 1 volume of hydrochloric
acid R and 99 volumes of acetic acid R and heat on a
water-bath. A red colour develops.
D. Dissolve about 10 mg in 5 mL of hydrochloric acid R1 and
allow to stand for 10-20 min. A yellow colour develops.

General Notices (1) apply to all monographs and other texts 2151
Erythromycin EUROPEAN PHARMACOPOEIA 8.0

TESTS – any impurity : not more than the area of the principal peak
Specific optical rotation (2.2.7) : − 71 to − 78 (anhydrous in the chromatogram obtained with reference solution (d)
substance). (3.0 per cent) ;
Dissolve 1.00 g in ethanol R and dilute to 50.0 mL with the – total : not more than 2.3 times the area of the principal peak
same solvent. The specific optical rotation is determined at in the chromatogram obtained with reference solution (d)
least 30 min after preparing the solution. (7.0 per cent) ;
Related substances. Liquid chromatography (2.2.29). – disregard limit : 0.02 times the area of the principal peak
in the chromatogram obtained with reference solution (d)
Test solution. Dissolve 40.0 mg of the substance to be (0.06 per cent) ; disregard the peaks due to erythromycin B
examined in a mixture of 1 volume of methanol R and and erythromycin C.
3 volumes of phosphate buffer solution pH 7.0 R1 and dilute to
10.0 mL with the same mixture of solvents. Thiocyanate : maximum 0.3 per cent.
Reference solution (a). Dissolve 40.0 mg of erythromycin A CRS Prepare the solutions immediately before use and protect from
in a mixture of 1 volume of methanol R and 3 volumes of actinic light.
phosphate buffer solution pH 7.0 R1 and dilute to 10.0 mL with Compensation liquid. Dilute 1.0 mL of a 90 g/L solution of
the same mixture of solvents. ferric chloride R to 50.0 mL with methanol R.
Reference solution (b). Dissolve 10.0 mg of erythromycin B CRS Test solution. Dissolve 0.100 g (m g) of the substance to be
and 10.0 mg of erythromycin C CRS in a mixture of 1 volume examined in 20 mL of methanol R, add 1.0 mL of a 90 g/L
of methanol R and 3 volumes of phosphate buffer solution solution of ferric chloride R and dilute to 50.0 mL with
pH 7.0 R1 and dilute to 50.0 mL with the same mixture of methanol R.
solvents. Prepare 2 independent reference solutions.
Reference solution (c). Dissolve 5 mg of N-demethylerythro- Reference solution. Dissolve 0.100 g of potassium thiocyanate R,
mycin A CRS in reference solution (b). Add 1.0 mL of reference previously dried at 105 °C for 1 h, in methanol R and dilute
solution (a) and dilute to 25 mL with reference solution (b). to 50.0 mL with the same solvent. Dilute 5.0 mL to 50.0 mL
Reference solution (d). Dilute 3.0 mL of reference solution (a) with methanol R. To 5.0 mL of this solution, add 1.0 mL of
to 100.0 mL with a mixture of 1 volume of methanol R and a 90 g/L solution of ferric chloride R and dilute to 50.0 mL
3 volumes of phosphate buffer solution pH 7.0 R1. with methanol R.
Reference solution (e). Transfer 40 mg of erythromycin A CRS Measure the absorbances (2.2.25) of each reference solution
to a glass vial and spread evenly such that it forms a layer not (A1, A2) and of the test solution (A) at the maximum (about
more than about 1 mm thick. Heat at 130 °C for 4 h. Allow to 492 nm).
cool and dissolve in a mixture of 1 volume of methanol R and Suitability value :
3 volumes of phosphate buffer solution pH 7.0 R1 and dilute to
10 mL with the same mixture of solvents.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ; m1, m2 = mass of potassium thiocyanate used to prepare
– stationary phase : styrene-divinylbenzene copolymer R the respective reference solutions, in grams.
(8 μm) with a pore size of 100 nm ;
The test is not valid unless S is not less than 0.985 and not
– temperature : 70 °C using a water-bath for the column and more than 1.015.
at least one-third of the tubing preceding the column.
Calculate the percentage content of thiocyanate from the
Mobile phase : to 50 mL of a 35 g/L solution of dipotassium following expression :
hydrogen phosphate R adjusted to pH 9.0 ± 0.05 with dilute
phosphoric acid R, add 400 mL of water R, 165 mL of
2-methyl-2-propanol R and 30 mL of acetonitrile R, and dilute
to 1000 mL with water R.
Flow rate : 2.0 mL/min. 58.08 = relative molecular mass of the thiocyanate
Detection : spectrophotometer at 215 nm. moiety ;
Injection : 100 μL of the test solution and reference 97.18 = relative molecular mass of potassium
solutions (c), (d) and (e). thiocyanate.
Run time : 5 times the retention time of erythromycin A. Water (2.5.12) : maximum 6.5 per cent, determined on 0.200 g.
Relative retention with reference to erythromycin A Use a 100 g/L solution of imidazole R in anhydrous methanol R
(retention time = about 15 min) : impurity A = about 0.3 ; as the solvent.
impurity B = about 0.45 ; erythromycin C = about 0.5 ;
impurity C = about 0.9 ; impurity D = about 1.4 ; Sulfated ash (2.4.14) : maximum 0.2 per cent, determined on
impurity F = about 1.5 ; erythromycin B = about 1.8 ; 1.0 g.
impurity E = about 4.3. ASSAY
System suitability: reference solution (c) : Liquid chromatography (2.2.29) as described in the test for
– resolution : minimum 0.8 between the peaks due to related substances with the following modifications.
impurity B and erythromycin C and minimum 5.5 between Injection : test solution and reference solutions (a) and (b).
the peaks due to impurity B and erythromycin A. If
necessary, adjust the concentration of 2-methyl-2-propanol System suitability : reference solution (a) :
in the mobile phase or reduce the flow rate to 1.5 mL or – symmetry factor : maximum 5 ;
1.0 mL/min. – repeatability : maximum relative standard deviation of
Limits : 1.2 per cent after 6 injections.
– correction factors : for the calculation of contents, multiply Calculate the percentage content of erythromycin A using
the peak areas of the following impurities (use the the chromatogram obtained with reference solution (a).
chromatogram obtained with reference solution (e) to Calculate the percentage contents of erythromycin B and
identify them) by the corresponding correction factor : erythromycin C using the chromatogram obtained with
impurity E = 0.09 ; impurity F = 0.15 ; reference solution (b).

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EUROPEAN PHARMACOPOEIA 8.0 Erythromycin

STORAGE
Protected from light.

IMPURITIES

D. (1S,2R,3R,4S,5R,8R,9S,10S,11R,12R,14R)-9-
[(2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-
ribo-hexopyranosyl)oxy]-5-ethyl-3-hydroxy-
2,4,8,10,12,14-hexamethyl-11-[[3,4,6-trideoxy-3-
(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-
6,15,16-trioxatricyclo[10.2.1.11,4]hexadecan-7-one
(anhydroerythromycin A),

A. (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-4-[(2,6-dideoxy-3-
C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-14-
ethyl-7,12,13-trihydroxy-3-(hydroxymethyl)-5,7,9,11,13-
pentamethyl-6-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-
xylo-hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione
(erythromycin F),

E. (2R,3R,4S,5R,8R,9S,10S,11R,12R)-9-[(2,6-dideoxy-3-
C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-
5-ethyl-3,4-dihydroxy-2,4,8,10,12,14-hexamethyl-
11-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-
B. (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-4-[(2,6-dideoxy- hexopyranosyl]oxy]-6,15-dioxabicyclo[10.2.1]pentadec-
3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]- 1(14)-en-7-one (erythromycin A enol ether),
14-ethyl-7,12,13-trihydroxy-3,5,7,9,11,13-hexamethyl-
6-[[3,4,6-trideoxy-3-(methylamino)-β-D-xylo-
hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione
(3″-N-desmethylerythromycin A),

C. (2S,4aR,4′R,5′S,6′S,7R,8S,9R,10R,12R,14R,15R,16S)-
7-ethyl-5′,8,9,14-tetrahydroxy-4′-methoxy- F. (2R,3R,6R,7S,8S,9R,10R)-7-[(2,6-dideoxy-3-C-methyl-
4′,6′,8,10,12,14,16-heptamethyl-15-[[3,4,6-trideoxy- 3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-3-[(1R,2R)-
3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]- 1,2-dihydroxy-1-methylbutyl]-2,6,8,10,12-pentamethyl-
hexadecahydrospiro[5H,11H-1,3-dioxino[5,4- 9-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-
c]oxacyclotetradecin-2,2′-pyrane]-5,11-dione hexopyranosyl]oxy]-4,13-dioxabicyclo[8.2.1]tridec-1(12)-
(erythromycin E), en-5-one (pseudoerythromycin A enol ether).

General Notices (1) apply to all monographs and other texts 2153
Erythromycin estolate EUROPEAN PHARMACOPOEIA 8.0

01/2008:0552 Reference solution (d). Dilute 3.0 mL of reference solution (a)

to 100.0 mL with a mixture of equal volumes of methanol R
ERYTHROMYCIN ESTOLATE and the hydrolysis solution.
Reference solution (e). Dissolve 40 mg of erythromycin A CRS,
previously heated at 130 °C for 3 h, in 10 mL of methanol R
Erythromycini estolas and dilute to 20 mL with the hydrolysis solution (in situ
preparation of impurities E and F).
Reference solution (f). Dissolve 2 mg of erythromycin A CRS
in 10 mL of 0.01 M hydrochloric acid. Allow to stand at room
temperature for 30 min. Dilute to 20 mL with the hydrolysis
solution (in situ preparation of impurity D).
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : styrene-divinylbenzene copolymer R
(8 μm) with a pore size of 100 nm ;
– temperature : 70 °C using a water-bath for the column and
at least one third of the tubing preceding the column.
Mobile phase : to 50 mL of a 35 g/L solution of dipotassium
hydrogen phosphate R adjusted to pH 8.0 with dilute
phosphoric acid R, add 400 mL of water R, 165 mL of
2-methyl-2-propanol R and 30 mL of acetonitrile R, and dilute
to 1000 mL with water R.
Flow rate : 2.0 mL/min.
Detection : spectrophotometer at 215 nm.
DEFINITION
Injection : 200 μL of the test solution and reference
Main component : (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)- solutions (c), (d), (e) and (f).
4-[(2,6-Dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexo- Run time : 5 times the retention time of erythromycin A ; begin
pyranosyl)oxy]-14-ethyl-7,12,13-trihydroxy-3,5,7,9,11,13- integration after the hydrolysis peak.
hexamethyl-6-[[3,4,6-trideoxy-3-(dimethylamino)-2-O-
propionyl-β-D-xylo-hexopyranosyl]oxy]oxacyclotetradecane- Identification of impurities: use the chromatogram obtained
2,10-dione dodecyl sulfate (erythromycin A 2″-propionate with reference solution (e) to identify the peaks due to
dodecyl sulfate). impurities E and F.
Semi-synthetic product derived from a fermentation product. Relative retention with reference to erythromycin A
(retention time = about 15 min) : hydrolysis peak = less
Content : than 0.3 ; impurity A = about 0.3 ; impurity B = about 0.45 ;
– erythromycin estolate : 86.0 per cent to 102.0 per cent erythromycin C = about 0.5 ; impurity C = about 0.9 ;
(anhydrous substance) ; impurity G = about 1.3 ; impurity D = about 1.4 ;
– erythromycin B : maximum 5.0 per cent (anhydrous impurity F = about 1.5 ; erythromycin B = about 1.8 ;
substance) ; impurity E = about 4.3.
– erythromycin C : maximum 5.0 per cent (anhydrous System suitability : reference solution (c) :
substance). – resolution : minimum 0.8 between the peaks due to
impurity B and erythromycin C and minimum 5.5 between
CHARACTERS the peaks due to impurity B and erythromycin A.
Appearance : white or almost white, crystalline powder. Limits :
Solubility : practically insoluble in water, freely soluble in – correction factors : for the calculation of content, multiply
ethanol (96 per cent), soluble in acetone. It is practically the peak areas of the following impurities by the
insoluble in dilute hydrochloric acid. corresponding correction factor : impurity E = 0.09 ;
impurity F = 0.15 ; impurity G = 0.14 ;
IDENTIFICATION
– impurities A, B, C, D, E, F, G : for each impurity, not more
Infrared absorption spectrophotometry (2.2.24). than the area of the principal peak in the chromatogram
Comparison : erythromycin estolate CRS. obtained with reference solution (d) (3.0 per cent) ;
– any other impurity : for each impurity, not more than
TESTS
0.067 times the area of the principal peak in the
Related substances. Liquid chromatography (2.2.29). chromatogram obtained with reference solution (d) (0.2 per
Hydrolysis solution. A 20 g/L solution of dipotassium hydrogen cent) ;
phosphate R adjusted to pH 8.0 with phosphoric acid R. – total : not more than 1.67 times the area of the principal
Test solution. Dissolve 0.150 g of the substance to be examined peak in the chromatogram obtained with reference
in 25 mL of methanol R. Add 20 mL of the hydrolysis solution, solution (d) (5.0 per cent) ;
mix and allow to stand at room temperature for at least 12 h. – disregard limit : 0.02 times the area of the principal peak
Dilute to 50.0 mL with the hydrolysis solution. in the chromatogram obtained with reference solution (d)
Reference solution (a). Dissolve 40.0 mg of erythromycin A CRS (0.06 per cent).
in 10 mL of methanol R and dilute to 20.0 mL with the Free erythromycin. Liquid chromatography (2.2.29). Prepare
hydrolysis solution. the solutions immediately before use.
Reference solution (b). Dissolve 10.0 mg of erythromycin B CRS Test solution. Dissolve 0.250 g of the substance to be examined
and 10.0 mg of erythromycin C CRS in 50.0 mL of methanol R. in the mobile phase and dilute to 50.0 mL with the mobile
Add 5.0 mL of reference solution (a) and dilute to 100.0 mL phase.
with the hydrolysis solution. Reference solution. Dissolve 75.0 mg of erythromycin A CRS in
Reference solution (c). Dissolve 2 mg of N-demethylerythro- the mobile phase and dilute to 50.0 mL with the mobile phase.
mycin A CRS in 20 mL of reference solution (b). Dilute 5.0 mL of the solution to 25.0 mL with acetonitrile R.

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EUROPEAN PHARMACOPOEIA 8.0 Erythromycin estolate

Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : octylsilyl silica gel for chromatography R
(5 μm) ;
– temperature : 30 °C.
Mobile phase : mix 35 volumes of acetonitrile R1 and
65 volumes of a solution containing 3.4 g/L of potassium
dihydrogen phosphate R and 2.75 mL/L of triethylamine R,
adjusted to pH 3.0 with dilute phosphoric acid R.
Flow rate : 1 mL/min. A. R1 = OH, R2 = CH3 : erythromycin F,
Detection : spectrophotometer at 195 nm.
B. R1 = R2 = H : N-demethylerythromycin A,
Injection : 20 μL.
Run time : twice the retention time of erythromycin A for the G. R1 = H, R2 = CO-C2H5 : N-demethyl-N-propanoyl-
reference solution and 4.5 times the retention time of the erythromycin A,
1st peak of erythromycin propionate for the test solution.
Retention time : erythromycin A = about 5 min ; 1st peak of
erythromycin propionate = about 10 min.
Limit :
– free erythromycin: not more than the area of the principal
peak in the chromatogram obtained with the reference
solution (6.0 per cent).
Dodecyl sulfate : 23.0 per cent to 25.5 per cent of C12H26O4S
(anhydrous substance).
Dissolve 0.500 g in 25 mL of dimethylformamide R. Titrate
with 0.1 M sodium methoxide using 0.05 mL of a 3 g/L solution
of thymol blue R in methanol R as indicator. C. erythromycin E,
1 mL of 0.1 M sodium methoxide is equivalent to 26.64 mg
of C12H26O4S.
Water (2.5.12) : maximum 4.0 per cent, determined on 0.300 g.
Use a 100 g/L solution of imidazole R in anhydrous methanol R
as the solvent.
Sulfated ash (2.4.14) : maximum 0.5 per cent, determined on
0.5 g.

ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications. D. anhydroerythromycin A,

Injection : test solution and reference solutions (a) and (b).

System suitability :
– repeatability : maximum relative standard deviation of
1.2 per cent after 6 injections of reference solution (a).
Calculate the percentage content of erythromycin A using the
chromatogram obtained with reference solution (a). Express
the result as erythromycin A estolate by multiplying the
percentage content of erythromycin A by 1.4387.
Calculate the percentage contents of erythromycin B and
erythromycin C using the chromatogram obtained with
reference solution (b). Express the result as erythromycin B E. erythromycin A enol ether,
estolate and as erythromycin C estolate by multiplying
by 1.4387.
For the calculation of content of erythromycin estolate use
the sum of erythromycins A, B and C expressed as estolate as
described above.

STORAGE
Protected from light.

IMPURITIES
Specified impurities : A, B, C, D, E, F, G. F. pseudoerythromycin A enol ether.

General Notices (1) apply to all monographs and other texts 2155
Erythromycin ethylsuccinate EUROPEAN PHARMACOPOEIA 8.0

01/2012:0274 Reference solution (b). Dissolve 10.0 mg of erythromycin B CRS

and 10.0 mg of erythromycin C CRS in 50 mL of methanol R.
ERYTHROMYCIN ETHYLSUCCINATE Add 5.0 mL of reference solution (a) and dilute to 100.0 mL
with the hydrolysis solution.
Reference solution (c). Dissolve 2 mg of N-demethylerythro-
Erythromycini ethylsuccinas mycin A CRS in 20 mL of reference solution (b).
Reference solution (d). Dilute 3.0 mL of reference solution (a)
to 100.0 mL with a mixture of equal volumes of methanol R
and the hydrolysis solution.
Reference solution (e). Dissolve 40 mg of erythromycin A CRS,
previously heated at 130 °C for 3 h, in 10 mL of methanol R
and dilute to 20 mL with the hydrolysis solution.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : styrene-divinylbenzene copolymer R
(8 μm) with a pore size of 100 nm ;
– temperature : 70 °C using a water-bath for the column and
at least one-third of the tubing preceding the column.
Mobile phase : to 50 mL of a 35 g/L solution of dipotassium
hydrogen phosphate R adjusted to pH 8.0 with dilute
phosphoric acid R, add 400 mL of water R, 165 mL of
2-methyl-2-propanol R and 30 mL of acetonitrile R, and dilute
to 1000 mL with water R.
DEFINITION
Flow rate : 2.0 mL/min.
Main component : (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-
Detection : spectrophotometer at 215 nm.
4-[(2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-
hexopyranosyl)oxy]-14-ethyl-7,12,13-trihydroxy- Injection : 200 μL of the test solution and reference
3,5,7,9,11,13-hexamethyl-6-[[3,4,6-trideoxy-3- solutions (a), (c), (d) and (e).
(dimethylamino)-2-O-(4-ethoxy-4-oxobutanoyl)-β-D- Run time : 5 times the retention time of erythromycin A ; begin
xylo-hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione integration after the hydrolysis peak.
(erythromycin A 2″-(ethyl succinate)). Relative retention with reference to erythromycin A (retention
Semi-synthetic product derived from a fermentation product. time = about 15 min) : hydrolysis peak = less than 0.3 ;
Content : impurity B = about 0.45 ; erythromycin C = about 0.5 ;
impurity C = about 0.9 ; impurity G = about 1.3 ;
– sum of erythromycin A, erythromycin B and erythromycin C : impurity D = about 1.4 ; impurity F = about 1.5 ;
minimum 78.0 per cent (anhydrous substance) ; erythromycin B = about 1.8 ; impurity E = about 4.3.
– erythromycin B : maximum 5.0 per cent (anhydrous System suitability : reference solution (c) :
substance) ;
– resolution : minimum 0.8 between the peaks due to
– erythromycin C : maximum 5.0 per cent (anhydrous impurity B and erythromycin C and minimum 5.5 between
substance). the peaks due to impurity B and erythromycin A.
CHARACTERS Limits :
Appearance : white or almost white, crystalline powder, – correction factors : for the calculation of contents,
hygroscopic. multiply the peak areas of the following impurities by
Solubility : practically insoluble in water, freely soluble in the corresponding correction factor : impurity E = 0.09 ;
acetone, in anhydrous ethanol and in methanol. impurity F = 0.15 ; impurity G = 0.14 ; use the chromatogram
obtained with reference solution (e) to identify the peaks
IDENTIFICATION due to impurities E and F ;
Infrared absorption spectrophotometry (2.2.24). – any impurity : not more than the area of the principal peak
in the chromatogram obtained with reference solution (d)
Comparison : erythromycin ethylsuccinate CRS. (3.0 per cent) ;
TESTS – total : not more than 1.67 times the area of the principal
peak in the chromatogram obtained with reference
Specific optical rotation (2.2.7) : − 70 to − 82 (anhydrous solution (d) (5.0 per cent) ;
substance).
– disregard limit : 0.02 times the area of the principal peak
Dissolve 0.100 g in acetone R and dilute to 10.0 mL with the in the chromatogram obtained with reference solution (d)
same solvent. Measure the angle of rotation at least 30 min (0.06 per cent).
after preparing the solution.
Free erythromycin. Liquid chromatography (2.2.29).
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.250 g of the substance to be examined
Hydrolysis solution. A 20 g/L solution of dipotassium hydrogen in acetonitrile R and dilute to 50.0 mL with the same solvent.
phosphate R adjusted to pH 8.0 with phosphoric acid R.
Reference solution. Dissolve 75.0 mg of erythromycin A CRS
Test solution. Dissolve 0.115 g of the substance to be examined in acetonitrile R and dilute to 50.0 mL with the same solvent.
in 25 mL of methanol R. Add 20 mL of the hydrolysis solution, Dilute 5.0 mL of the solution to 25.0 mL with acetonitrile R.
mix and allow to stand at room temperature for at least 12 h.
Dilute to 50.0 mL with the hydrolysis solution. Column :
Reference solution (a). Dissolve 40.0 mg of erythromycin A CRS – size : l = 0.25 m, Ø = 4.6 mm ;
in 10 mL of methanol R and dilute to 20.0 mL with the – stationary phase : octylsilyl silica gel for chromatography R
hydrolysis solution. (5 μm).

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EUROPEAN PHARMACOPOEIA 8.0 Erythromycin ethylsuccinate

Mobile phase : mix 35 volumes of acetonitrile R and 65 volumes

of a solution containing 3.4 g/L of potassium dihydrogen
phosphate R and 2.0 g/L of triethylamine R, adjusted to pH 3.0
with dilute phosphoric acid R.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 195 nm.
Injection : 20 μL.
Run time : twice the retention time of erythromycin A
(retention time = about 8 min) for the reference solution
and twice the retention time of erythromycin ethylsuccinate B. (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-4-[(2,6-dideoxy-
(retention time = about 24 min) for the test solution. 3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-
14-ethyl-7,12,13-trihydroxy-3,5,7,9,11,13-hexamethyl-
Limit : 6-[[3,4,6-trideoxy-3-(methylamino)-β-D-xylo-
hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione
– free erythromycin: not more than the area of the principal (3″-N-desmethylerythromycin A),
peak in the chromatogram obtained with the reference
solution (6.0 per cent).
Water (2.5.12) : maximum 3.0 per cent, determined on 0.30 g.
Use a 100 g/L solution of imidazole R in anhydrous methanol R
as the solvent.
Sulfated ash (2.4.14) : maximum 0.3 per cent, determined on
1.0 g.

ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications. C. (2S,4aR,4′R,5′S,6′S,7R,8S,9R,10R,12R,14R,15R,16S)-
7-ethyl-5′,8,9,14-tetrahydroxy-4′-methoxy-
Injection : test solution and reference solutions (a) and (b). 4′,6′,8,10,12,14,16-heptamethyl-15-[[3,4,6-trideoxy-
3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-
System suitability: reference solution (a) : hexadecahydrospiro[5H,11H-1,3-dioxino[5,4-
– symmetry factor : maximum 5 ; c]oxacyclotetradecin-2,2′-pyrane]-5,11-dione
(erythromycin E),
– repeatability : maximum relative standard deviation of
1.2 per cent after 6 injections.
Calculate the percentage content of erythromycin A using
the chromatogram obtained with reference solution (a).
Calculate the percentage contents of erythromycin B and
erythromycin C using the chromatogram obtained with
reference solution (b).

STORAGE
In an airtight container, protected from light.
D. (1S,2R,3R,4S,5R,8R,9S,10S,11R,12R,14R)-9-
[(2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-
IMPURITIES ribo-hexopyranosyl)oxy]-5-ethyl-3-hydroxy-
2,4,8,10,12,14-hexamethyl-11-[[3,4,6-trideoxy-3-
(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-
6,15,16-trioxatricyclo[10.2.1.11,4]hexadecan-7-one
(anhydroerythromycin A),

A. (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-4-[(2,6-dideoxy-3- E. (2R,3R,4S,5R,8R,9S,10S,11R,12R)-9-[(2,6-dideoxy-3-
C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-14- C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-
ethyl-7,12,13-trihydroxy-3-(hydroxymethyl)-5,7,9,11,13- 5-ethyl-3,4-dihydroxy-2,4,8,10,12,14-hexamethyl-
pentamethyl-6-[[3,4,6-trideoxy-3-(dimethylamino)-β-D- 11-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-
xylo-hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione hexopyranosyl]oxy]-6,15-dioxabicyclo[10.2.1]pentadec-
(erythromycin F), 1(14)-en-7-one (erythromycin A enol ether),

General Notices (1) apply to all monographs and other texts 2157
Erythromycin lactobionate EUROPEAN PHARMACOPOEIA 8.0

Salt of a product obtained by fermentation using a strain of

Streptomyces erythreus.
Content :
– sum of erythromycin A lactobionate, erythromycin B
lactobionate and erythromycin C lactobionate : 93.0 per cent
to 102.0 per cent (anhydrous substance);
– erythromycin B lactobionate : maximum 5.0 per cent
(anhydrous substance);
– erythromycin C lactobionate : maximum 5.0 per cent
(anhydrous substance).
F. (2R,3R,6R,7S,8S,9R,10R)-7-[(2,6-dideoxy-3-C-methyl-
3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-3-[(1R,2R)- CHARACTERS
1,2-dihydroxy-1-methylbutyl]-2,6,8,10,12-pentamethyl- Appearance : white or slightly yellow hygroscopic, powder.
9-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo- Solubility : soluble in water, freely soluble in anhydrous
hexopyranosyl]oxy]-4,13-dioxabicyclo[8.2.1]tridec-1(12)- ethanol and in methanol, very slightly soluble in acetone and
en-5-one (pseudoerythromycin A enol ether), in methylene chloride.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 30 mg of the substance to be
examined in methanol R and dilute to 10 mL with the same
solvent.
Reference solution (a). Dissolve 20 mg of
erythromycin A CRS in methanol R and dilute to
10 mL with the same solvent.
Reference solution (b). Dissolve 10 mg of lactobionic acid R
in water R and dilute to 10 mL with the same solvent.
Plate : TLC silica gel plate R.
G. (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-4-[(2,6-
dideoxy-3-C-methyl-3-O-methyl-α-L-ribo- Mobile phase : glacial acetic acid R, water R, methanol R
hexopyranosyl)oxy]-14-ethyl-7,12,13-trihydroxy- (3:10:90 V/V/V).
3,5,7,9,11,13-hexamethyl-6-[[3,4,6-trideoxy-3-[(4- Application : 5 μL.
ethoxy-4-oxobutanoyl)methylamino]-β-D-xylo- Development : over 3/4 of the plate.
hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione Drying : in air.
(3″-N-desmethyl-3″-N-(ethoxysuccinyl)erythromycin A).
Detection : spray with a 5 g/L solution of potassium
permanganate R in 1 M sodium hydroxide and heat at
110 °C for 5 min.
01/2008:1098
Results : the 2 spots in the chromatogram obtained with the
test solution are similar in position, colour and size, one
ERYTHROMYCIN LACTOBIONATE to the principal spot in the chromatogram obtained with
reference solution (a) and the other to the principal spot in
Erythromycini lactobionas the chromatogram obtained with reference solution (b).
B. To about 5 mg add 5 mL of a 0.2 g/L solution of xanthydrol R
in a mixture of 1 volume of hydrochloric acid R and
99 volumes of acetic acid R. A red colour develops.
C. Dissolve about 10 mg in 5 mL of hydrochloric acid R1. A
yellowish-green colour develops.
TESTS
Appearance of solution. The solution is clear (2.2.1) and
colourless (2.2.2, Method II).
Dissolve 1.0 g in 20 mL of water R.
pH (2.2.3) : 6.5 to 7.5.
Dissolve 0.50 g in carbon dioxide-free water R and dilute to
25 mL with the same solvent.
Related substances. Liquid chromatography (2.2.29). The test
solution and the reference solutions can be used within 24 h if
stored at 2-8 °C.
Solvent mixture : methanol R, phosphate buffer solution
pH 7.0 R (25:75 V/V).
Test solution. Dissolve 60.0 mg of the substance to be
DEFINITION examined in the solvent mixture and dilute to 10.0 mL with
Main component : (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)- the solvent mixture.
4-[(2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo- Reference solution (a). Dissolve 40.0 mg of erythromycin A CRS
hexopyranosyl)oxy]-14-ethyl-7,12,13-trihydroxy- in the solvent mixture and dilute to 10.0 mL with the solvent
3,5,7,9,11,13-hexamethyl-6-[[3,4,6-trideoxy-3- mixture.
(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]- Reference solution (b). Dissolve 10.0 mg of erythromycin B CRS
oxacyclotetradecane-2,10-dione 4-O-β-D-galactopyranosyl-D- and 10.0 mg of erythromycin C CRS in the solvent mixture and
gluconate (erythromycin A lactobionate). dilute to 50.0 mL with the solvent mixture.

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EUROPEAN PHARMACOPOEIA 8.0 Erythromycin lactobionate

Reference solution (c). Dissolve 5 mg of N-demethylerythro- – disregard limit : 0.02 times the area of the principal peak
mycin A CRS (impurity B) in reference solution (b). Add in the chromatogram obtained with reference solution (d)
1.0 mL of reference solution (a) and dilute to 25 mL with (0.06 per cent).
reference solution (b). Free lactobionic acid : maximum 1.0 per cent of C12H22O12
Reference solution (d). Dilute 3.0 mL of reference solution (a) (anhydrous substance).
to 100.0 mL with the solvent mixture. Dissolve 0.400 g in 50 mL of water R. Titrate with 0.1 M sodium
Reference solution (e). Dissolve 40 mg of erythromycin A CRS, hydroxide, determining the end-point potentiometrically
previously heated at 130 °C for 4 h, in the solvent mixture and (2.2.20). Calculate the volume of 0.1 M sodium hydroxide
dilute to 10 mL with the solvent mixture (in situ preparation required per gram of the substance to be examined (n1 mL).
of impurities E and F). Dissolve 0.500 g in 40 mL of anhydrous acetic acid R and
titrate with 0.1 M perchloric acid, determining the end-point
Reference solution (f). Dissolve 2 mg of erythromycin A CRS potentiometrically (2.2.20). Calculate the volume of 0.1 M
in 5 mL of 0.01 M hydrochloric acid. Allow to stand at room perchloric acid required per gram of the substance to be
temperature for 30 min. Dilute to 10 mL with the solvent examined (n2 mL).
mixture (in situ preparation of impurity D).
Calculate the percentage content of C12H22O12 using the
Column : following expression :
– size : l = 0.25 m, Ø = 4.6 mm;
– stationary phase : styrene-divinylbenzene copolymer R
(8 μm) with a pore size of 100 nm; Water (2.5.12) : maximum 5.0 per cent, determined on 0.200 g.
– temperature : 70 °C using a water-bath for the column and Use a 100 g/L solution of imidazole R in anhydrous methanol R
at least 1/3 of the tubing preceding the column. as the solvent.
Mobile phase : to 50 mL of a 35 g/L solution of dipotassium Sulfated ash (2.4.14) : maximum 0.5 per cent, determined on
hydrogen phosphate R adjusted to pH 9.0 with dilute 1.0 g.
phosphoric acid R, add 400 mL of water R, 165 mL of Bacterial endotoxins (2.6.14) : less than 0.35 IU/mg of
2-methyl-2-propanol R and 30 mL of acetonitrile R1, and dilute erythromycin, if intended for use in the manufacture of
to 1000 mL with water R. parenteral preparations without a further appropriate
Flow rate : 2.0 mL/min. procedure for the removal of bacterial endotoxins.
Detection : spectrophotometer at 215 nm.
ASSAY
Injection : 100 μL of the test solution and reference Liquid chromatography (2.2.29) as described in the test for
solutions (a), (c), (d), (e) and (f). related substances with the following modifications.
Run time : 5 times the retention time of erythromycin A. Injection : test solution and reference solutions (a) and (b).
Identification of impurities : use the chromatogram obtained System suitability :
with reference solution (c) to identify the peak due to
impurity B, with reference solution (e) to identify the peaks – repeatability : maximum relative standard deviation of
due to impurities E and F, and with reference solution (f) to 2.0 per cent after 6 injections of reference solution (a).
identify the peak due to impurity D. Calculate the percentage content of erythromycin A using the
Relative retention with reference to erythromycin A chromatogram obtained with reference solution (a). Express
(retention time = about 15 min) : impurity A = about 0.3 ; the result as erythromycin A lactobionate by multiplying the
percentage content of erythromycin A by 1.4877. Calculate the
impurity B = about 0.45 ; erythromycin C = about 0.5 ;
impurity C = about 0.9 ; impurity D = about 1.4 ; percentage contents of erythromycin B and erythromycin C
impurity F = about 1.5 ; erythromycin B = about 1.8 ; using the chromatogram obtained with reference solution (b).
Express the result as erythromycin B lactobionate and as
impurity E = about 4.3.
erythromycin C lactobionate by multiplying by 1.4877.
System suitability: reference solution (c) :
– resolution : minimum 0.8 between the peaks due to STORAGE
impurity B and erythromycin C and minimum 5.5 between In an airtight container. If the substance is sterile, store in a
the peaks due to impurity B and erythromycin A. If sterile, airtight, tamper-proof container.
necessary adjust the concentration of 2-methyl-2-propanol
in the mobile phase or reduce the flow rate to 1.5 mL/min IMPURITIES
or 1.0 mL/min. Specified impurities : A, B, C, D, E, F.
Limits :
– correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
corresponding correction factor : impurity E = 0.09 ;
impurity F = 0.15 ;
– impurities A, B, C, D, E, F : for each impurity, not more
than the area of the principal peak in the chromatogram
obtained with reference solution (d) (3.0 per cent) ;
– any other impurity : for each impurity, not more than
0.067 times the area of the principal peak in the
chromatogram obtained with reference solution (d) (0.2 per
cent) ;
A. R1 = OH, R2 = CH3 : erythromycin F,
– total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (d)
(6.0 per cent) ; B. R1 = R2 = H : N-demethylerythromycin A,

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Erythromycin stearate EUROPEAN PHARMACOPOEIA 8.0

01/2012:0490

ERYTHROMYCIN STEARATE
Erythromycini stearas

C. erythromycin E,

DEFINITION
A mixture of the stearates of erythromycin and stearic
acid. The main component is the octadecanoate of
(3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-4-[(2,6-dideoxy-
3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-
14-ethyl-7,12,13-trihydroxy-3,5,7,9,11,13-hexamethyl-
D. anhydroerythromycin A, 6-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-
hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione
(erythromycin A stearate).
Fermentation product.
Content :
– sum of the contents of erythromycin A, erythromycin B
and erythromycin C : minimum 60.5 per cent (anhydrous
substance) ;
– erythromycin B : maximum 5.0 per cent ;
– erythromycin C : maximum 5.0 per cent.
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, soluble in acetone
and in methanol.
Solutions may be opalescent.
IDENTIFICATION
E. erythromycin A enol ether, A. Infrared absorption spectrophotometry (2.2.24).
Comparison: erythromycin stearate CRS.
B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 28 mg of the substance to be
examined in methanol R and dilute to 10 mL with the same
solvent.
Reference solution (a). Dissolve 20 mg of
erythromycin A CRS in methanol R and dilute to
10 mL with the same solvent.
Reference solution (b). Dissolve 10 mg of stearic acid R in
methanol R and dilute to 10 mL with the same solvent.
Plate : TLC silica gel G plate R.
Mobile phase : mix 4 volumes of 2-propanol R, 8 volumes
of a 150 g/L solution of ammonium acetate R previously
adjusted to pH 9.6 with ammonia R and 9 volumes of ethyl
acetate R. Allow to settle and use the upper layer.
Application : 5 μL.
Development : over 2/3 of the plate.
F. pseudoerythromycin A enol ether. Drying : in air.

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EUROPEAN PHARMACOPOEIA 8.0 Erythromycin stearate

Detection A : spray with a solution containing 0.2 g/L of – stationary phase : styrene-divinylbenzene copolymer R
dichlorofluorescein R and 0.1 g/L of rhodamine B R in (8 μm) with a pore size of 100 nm ;
ethanol (96 per cent) R. Maintain the plate for a few seconds
– temperature : 70 °C using a water-bath for the column and
in the vapour above a water-bath. Examine in ultraviolet
at least one-third of the tubing preceding the column.
light at 365 nm.
Mobile phase : to 50 mL of a 35 g/L solution of dipotassium
Results A : the chromatogram obtained with the test solution
hydrogen phosphate R adjusted to pH 9.0 ± 0.05 with dilute
shows 2 spots, one of which corresponds in position to
phosphoric acid R, add 400 mL of water R, 165 mL of
the principal spot in the chromatogram obtained with
2-methyl-2-propanol R and 30 mL of acetonitrile R, and dilute
reference solution (a) and the other to the principal spot in
to 1000 mL with water R.
the chromatogram obtained with reference solution (b).
Detection B : spray the plate with anisaldehyde solution R1. Flow rate : 2.0 mL/min.
Heat at 110 °C for 5 min and examine in daylight. Detection : spectrophotometer at 215 nm.
Results B : the spot in the chromatogram obtained with Injection : 100 μL of the test solution and reference
the test solution corresponds in position, colour and size solutions (c), (d) and (e).
to the principal spot in the chromatogram obtained with
Run time : 5 times the retention time of erythromycin A.
reference solution (a).
Relative retention with reference to erythromycin A
TESTS (retention time = about 15 min) : impurity A = about 0.3 ;
impurity B = about 0.45 ; erythromycin C = about 0.5 ;
Free stearic acid : maximum 14.0 per cent (anhydrous impurity C = about 0.9 ; impurity D = about 1.4 ;
substance) of C18H36O2. impurity F = about 1.5 ; erythromycin B = about 1.8 ;
Dissolve 0.400 g in 50 mL of methanol R. Titrate with impurity E = about 4.3.
0.1 M sodium hydroxide, determining the end-point System suitability : reference solution (c) :
potentiometrically (2.2.20). Calculate the volume of
0.1 M sodium hydroxide required per gram of the substance to – resolution : minimum 0.8 between the peaks due to
be examined (n1 mL). Dissolve 0.500 g in 30 mL of methylene impurity B and erythromycin C and minimum 5.5 between
chloride R. If the solution is opalescent, filter and shake the peaks due to impurity B and erythromycin A. If
the residue with 3 quantities, each of 25 mL, of methylene necessary, adjust the concentration of 2-methyl-2-propanol
chloride R. Filter, if necessary, and rinse the filter with in the mobile phase or reduce the flow rate to 1.5 mL/min
methylene chloride R. Reduce the volume of the combined or 1.0 mL/min.
filtrate and rinsings to 30 mL by evaporation on a water-bath. Limits :
Add 50 mL of glacial acetic acid R and titrate with 0.1 M
perchloric acid, determining the end-point potentiometrically – correction factors : for the calculation of contents, multiply
(2.2.20). Calculate the volume of 0.1 M perchloric acid the peak areas of the following impurities (use the
required per gram of the substance to be examined (n2 mL). chromatogram obtained with reference solution (e) to
identify them) by the corresponding correction factor :
Calculate the percentage content of C18H36O2 from the impurity E = 0.09 ; impurity F = 0.15 ;
expression :
– any impurity : not more than the area of the principal peak
in the chromatogram obtained with reference solution (d)
(3 per cent) ;
– total : not more than twice the area of the principal peak
h = percentage water content.
in the chromatogram obtained with reference solution (d)
Related substances. Liquid chromatography (2.2.29). (6 per cent) ;
Test solution. Dissolve 55.0 mg of the substance to be – disregard limit : 0.02 times the area of the principal peak
examined in 5.0 mL of methanol R and dilute to 10.0 mL in the chromatogram obtained with reference solution (d)
with buffer solution pH 8.0 R1. Centrifuge and use the clear (0.06 per cent) ; disregard the peaks due to erythromycin B
solution. and erythromycin C.
Reference solution (a). Dissolve 40.0 mg of erythromycin A CRS Water (2.5.12) : maximum 4.0 per cent, determined on 0.300 g.
in 5.0 mL of methanol R and dilute to 10.0 mL with buffer Use a 100 g/L solution of imidazole R in anhydrous methanol R
solution pH 8.0 R1. as the solvent.
Reference solution (b). Dissolve 10.0 mg of erythromycin B CRS Sulfated ash (2.4.14) : maximum 0.5 per cent, determined on
and 10.0 mg of erythromycin C CRS in 25.0 mL of methanol R 1.0 g.
and dilute to 50.0 mL with buffer solution pH 8.0 R1.
Reference solution (c). Dissolve 5 mg of N-demethylerythro- ASSAY
mycin A CRS in reference solution (b). Add 1.0 mL of reference Liquid chromatography (2.2.29) as described in the test for
solution (a) and dilute to 25 mL with reference solution (b). related substances with the following modifications.
Reference solution (d). Dilute 3.0 mL of reference solution (a) Injection : test solution and reference solutions (a) and (b).
to 100.0 mL with a mixture of equal volumes of methanol R
and buffer solution pH 8.0 R1. System suitability : reference solution (a) :
Reference solution (e). Transfer 40 mg of erythromycin A CRS – symmetry factor : maximum 5 ;
to a glass vial and spread evenly such that it forms a layer not – repeatability : maximum relative standard deviation of
more than about 1 mm thick. Heat at 130 °C for 4 h. Allow to 1.2 per cent after 6 injections.
cool and dissolve in a mixture of 1 volume of methanol R and
3 volumes of buffer solution pH 8.0 R1 and dilute to 10 mL Calculate the percentage content of erythromycin A using
with the same mixture of solvents. the chromatogram obtained with reference solution (a).
Calculate the percentage contents of erythromycin B and
Column : erythromycin C using the chromatogram obtained with
– size : l = 0.25 m, Ø = 4.6 mm ; reference solution (b).

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Erythropoietin concentrated solution EUROPEAN PHARMACOPOEIA 8.0

IMPURITIES

D. (1S,2R,3R,4S,5R,8R,9S,10S,11R,12R,14R)-9-
[(2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-
ribo-hexopyranosyl)oxy]-5-ethyl-3-hydroxy-
2,4,8,10,12,14-hexamethyl-11-[[3,4,6-trideoxy-3-
(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-
6,15,16-trioxatricyclo[10.2.1.11,4]hexadecan-7-one
(anhydroerythromycin A),
A. (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-4-[(2,6-dideoxy-3-
C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-14-
ethyl-7,12,13-trihydroxy-3-(hydroxymethyl)-5,7,9,11,13-
pentamethyl-6-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-
xylo-hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione
(erythromycin F),

E. (2R,3R,4S,5R,8R,9S,10S,11R,12R)-9-[(2,6-dideoxy-3-
C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-
5-ethyl-3,4-dihydroxy-2,4,8,10,12,14-hexamethyl-
11-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-
hexopyranosyl]oxy]-6,15-dioxabicyclo[10.2.1]pentadec-
1(14)-en-7-one (erythromycin A enol ether),

B. (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-4-[(2,6-dideoxy-
3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-
14-ethyl-7,12,13-trihydroxy-3,5,7,9,11,13-hexamethyl-
6-[[3,4,6-trideoxy-3-(methylamino)-β-D-xylo-
hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione F. (2R,3R,6R,7S,8S,9R,10R)-7-[(2,6-dideoxy-3-C-methyl-
(3″-N-desmethylerythromycin A), 3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-3-[(1R,2R)-
1,2-dihydroxy-1-methylbutyl]-2,6,8,10,12-pentamethyl-
9-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-
hexopyranosyl]oxy]-4,13-dioxabicyclo[8.2.1]tridec-1(12)-
en-5-one (pseudoerythromycin A enol ether).

01/2008:1316

ERYTHROPOIETIN CONCENTRATED
SOLUTION
Erythropoietini solutio concentrata

C. (2S,4aR,4′R,5′S,6′S,7R,8S,9R,10R,12R,14R,15R,16S)-
7-ethyl-5′,8,9,14-tetrahydroxy-4′-methoxy- Mr approx. 30 600
4′,6′,8,10,12,14,16-heptamethyl-15-[[3,4,6-trideoxy-
3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]- DEFINITION
hexadecahydrospiro[5H,11H-1,3-dioxino[5,4- Erythropoietin concentrated solution is a solution
c]oxacyclotetradecin-2,2′-pyrane]-5,11-dione containing a family of closely-related glycoproteins which
(erythromycin E), are indistinguishable from the naturally occurring human

2162 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Erythropoietin concentrated solution

erythropoietin (urinary erythropoietin) in terms of amino acid Migration : apply a field strength of 143 V/cm (15.4 kV for
sequence (165 amino acids) and average glycosylation pattern, capillaries of 107 cm total length) for 80 min, using CZE
at a concentration of 0.5-10 mg/mL. It may also contain buffer buffer as the electrolyte in both buffer reservoirs.
salts and other excipients. It has a potency of not less than System suitability : in the electropherogram obtained with
100 000 IU/mg of active substance determined using the the reference solution, a pattern of well separated peaks
conditions described under Assay and in the test for protein. corresponding to the peaks in the electropherogram of
erythropoietin supplied with erythropoietin BRP is seen,
PRODUCTION
and the largest peak is at least 50 times greater than the
Erythropoietin is produced in rodent cells in vitro by a method baseline noise. If necessary, adjust the sample load to
based on recombinant DNA technology. give peaks of sufficient height. Identify the peaks due to
Prior to batch release, the following tests are carried out on isoforms 1 to 8. Isoform 1 may not be visible. The peak
each batch of the final product, unless exemption has been due to isoform 8 is detected and the resolution between the
granted by the competent authority. peaks due to isoforms 5 and 6 is not less than 1. Repeat the
Host cell-derived proteins : the limit is approved by the separation at least 3 times. The baseline is stable, showing
competent authority. little drift, and the distribution of peaks is qualitatively
and quantitatively similar to the distribution of peaks in
Host cell- and vector-derived DNA : the limit is approved the electropherogram of erythropoietin supplied with
by the competent authority. erythropoietin BRP. The relative standard deviation of the
CHARACTERS migration time of the peak due to isoform 2 is less than
2 per cent.
Appearance : clear or slightly turbid, colourless solution.
Limits : identify the peaks due to isoforms 1 to 8 in the
IDENTIFICATION electropherogram obtained with the test solution by
A. It gives the appropriate response when examined using the comparison with the electropherogram obtained with
conditions described under Assay. the reference solution. Calculate the percentage content
of each isoform from the corresponding peak area. The
B. Capillary zone electrophoresis (2.2.47). percentages are within the following ranges :
Test solution. Dilute the preparation to be examined
Isoform Content (per cent)
with water R to obtain a concentration of 1 mg/mL.
Desalt 0.25 mL of the solution by passage through a 1 0 - 15
micro-concentrator cartridge provided with a membrane
2 0 - 15
with a molecular mass cut-off of not more than 10 000 Da.
Add 0.2 mL of water R to the sample and desalt again. 3 1 - 20
Repeat the desalting procedure once more. Dilute the
4 10 - 35
sample with water R, determine its protein concentration
as described under Tests and adjust to a concentration of 5 15 - 40
approximately 1 mg/mL with water R.
6 10 - 35
Reference solution. Dissolve the contents of a vial of
erythropoietin BRP in 0.25 mL of water R. Proceed with 7 5 - 25
desalting as described for the test solution. 8 0 - 15
Capillary :
– material : uncoated fused silica ; C. Polyacrylamide gel electrophoresis and immunoblotting.
– size : effective length = about 100 cm, Ø = 50 μm. (a) Polyacrylamide gel electrophoresis (2.2.31)
Temperature : 35 °C. Gel dimensions : 0.75 mm thick, about 16 cm square.
CZE buffer concentrate (0.1 M sodium chloride, 0.1 M Resolving gel : 12 per cent acrylamide.
tricine, 0.1 M sodium acetate). Dissolve 0.584 g of sodium Sample buffer : concentrated SDS-PAGE sample buffer R.
chloride R, 1.792 g of tricine R and 0.820 g of anhydrous Test solution (a). Dilute the preparation to be examined
sodium acetate R in water R and dilute to 100.0 mL with in water R to obtain a concentration of 1.0 mg/mL. To
the same solvent. 1 volume of this solution add 1 volume of sample buffer.
1 M putrescine solution. Dissolve 0.882 g of putrescine R in Test solution (b). Dilute the preparation to be examined
10 mL of water R. Distribute in 0.5 mL aliquots. in water R to obtain a concentration of 0.1 mg/mL. To
CZE buffer (0.01 M tricine, 0.01 M sodium chloride, 0.01 M 1 volume of this solution add 1 volume of sample buffer.
sodium acetate, 7 M urea, 2.5 mM putrescine). Dissolve Reference solution (a). Dissolve the contents of a vial of
21.0 g of urea R in 25 mL of water R by warming in a erythropoietin BRP in 0.25 mL of water R. To 1 volume of
water-bath at 30 °C. Add 5.0 mL of CZE buffer concentrate this solution add 1 volume of sample buffer.
and 125 μL of 1 M putrescine solution. Dilute to 50.0 mL Reference solution (b). Dissolve the contents of a vial
with water R. Using dilute acetic acid R, adjust to pH 5.55 of erythropoietin BRP in water R and dilute with the
at 30 °C and filter through a membrane filter (nominal same solvent to obtain a concentration of 0.1 mg/mL. To
pore size 0.45 μm). 1 volume of this solution add 1 volume of sample buffer.
Detection : spectrophotometer at 214 nm. Reference solution (c). A solution of molecular mass
Set the autosampler to store the samples at 4 °C during markers suitable for calibrating SDS-polyacrylamide gels
analysis. in the range of 10-70 kDa.
Preconditioning of the capillary : rinse the capillary for Reference solution (d). A solution of pre-stained molecular
60 min with 0.1 M sodium hydroxide filtered through mass markers suitable for calibrating SDS-polyacrylamide
a membrane filter (nominal pore size 0.45 μm) and for gels in the range of 10-70 kDa and suitable for the
60 min with CZE buffer. Apply voltage for 12 h (20 kV). electrotransfer to an appropriate membrane.
Between-run rinsing : rinse the capillary for 10 min with Sample treatment : boil for 2 min.
water R, for 5 min with 0.1 M sodium hydroxide filtered Application : 20 μL, in the following order : reference
through a membrane filter (nominal pore size 0.45 μm) solution (c), reference solution (a), test solution (a), empty
and for 10 min with CZE buffer. well, reference solution (b), test solution (b), reference
Injection : under pressure or vacuum. solution (d).

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Erythropoietin concentrated solution EUROPEAN PHARMACOPOEIA 8.0

At the end of the separation, remove the gel-cassette from Time Flow rate Mobile phase A Mobile phase B
the apparatus and cut the gel into 2 parts : the first part (min) (mL/min) (per cent V/V) (per cent V/V)
containing reference solution (c), reference solution (a) 0 - 10 0.75 100 0
and test solution (a) ; the second part containing reference
10 - 125 0.75 100 → 39 0 → 61
solution (b), test solution (b) and reference solution (d).
Detection : by Coomassie staining on the first part of the gel. 125 - 135 1.25 39 → 17 61 → 83

System suitability: reference solution (c): 135 - 145 1.25 17 → 0 83 → 100

– the validation criteria are met. 145 - 150 1.25 100 0

Results : the electropherogram obtained with test Detection : spectrophotometer at 214 nm.
solution (a) shows a single diffuse band corresponding
in position and intensity to the single band seen in the Equilibration : at initial conditions for at least 15 min.
electropherogram obtained with reference solution (a). Carry out a blank run using the above-mentioned gradient.
Injection : 50 μL.
(b) Immunoblotting
System suitability : the chromatograms obtained with the
Transfer the second part of the gel onto a membrane suitable test solution and the reference solution are qualitatively
for the immobilisation of proteins, using commercially similar to the chromatogram of erythropoietin digest
available electrotransfer equipment and following the supplied with erythropoietin BRP.
manufacturer’s instructions. After electrotransfer, incubate
the membrane in a neutral isotonic buffer containing a Results : the profile of the chromatogram obtained with
suitable blocking agent (for example, 50 g/L of dried milk the test solution corresponds to that of the chromatogram
or 10 per cent V/V foetal calf serum), for 1-2 h, followed by obtained with the reference solution.
incubation for 1-14 h in the same blocking solution with E. N-terminal sequence analysis.
a suitable dilution of either a polyclonal or monoclonal The first 15 amino acids are : Ala - Pro - Pro - Arg - Leu
anti-erythropoietin antibody. Detect erythropoietin-bound - Ile - (no recovered peak) - Asp - Ser - Arg - Val - Leu -
antibody using a suitable enzyme- or radiolabelled Glu - Arg - Tyr.
antibody (for example, an alkaline phosphatase-conjugated Perform the Edman degradation using an automated
second antibody). The precise details of blocking agents, solid-phase sequencer, operated in accordance with the
concentrations and incubation times should be optimised manufacturer’s instructions.
using the principles set out in Immunochemical methods Desalt the equivalent of 50 μg of erythropoietin. For
(2.7.1). example, dilute a volume of the preparation to be examined
System suitability : in the electropherogram obtained with equivalent to 50 μg of the active substance in 1 mL of a
reference solution (d), the molecular mass markers are 0.1 per cent V/V solution of trifluoroacetic acid R. Pre-wash
resolved on the membrane into discrete bands, with a linear a C18 reverse-phase sample preparation cartridge according
relationship between distance migrated and logarithm10 to the instructions supplied and equilibrate the cartridge
of the molecular mass. in a 0.1 per cent V/V solution of trifluoroacetic acid R.
Results : the electropherogram obtained with test Apply the sample to the cartridge, and wash successively
solution (b) shows a single broad band corresponding with a 0.1 per cent V/V solution of trifluoroacetic acid R
in position and intensity to the single band seen in the containing 0 per cent, 10 per cent and 50 per cent V/V of
electropherogram obtained with reference solution (b). acetonitrile R according to the manufacturer’s instructions.
Lyophilise the 50 per cent V/V acetonitrile R eluate.
D. Peptide mapping (2.2.55). Liquid chromatography (2.2.29).
Redissolve the desalted sample in 50 μL of a 0.1 per
Test solution. Dilute the preparation to be examined in cent V/V solution of trifluoroacetic acid R and couple to a
tris acetate buffer solution pH 8.5 R to a concentration sequencing cartridge using the protocol provided by the
of 1.0 mg/mL. Equilibrate the solution in tris acetate manufacturer. Run 15 sequencing cycles, using the reaction
buffer solution pH 8.5 R using a suitable procedure conditions for proline when running the 2nd and 3rd cycles.
(dialysis against tris acetate buffer solution pH 8.5 R, or Identify the phenylthiohydantoin (PTH)-amino acids
membrane filtration using the procedure described under released at each sequencing cycle by reverse-phase
Identification B, but reconstituting the desalted sample with liquid chromatography. The procedure may be carried
tris acetate buffer solution pH 8.5 R, are suitable). Transfer out using the column and reagents recommended by
the dialysed solution to a polypropylene centrifuge tube. the manufacturer of the sequencing equipment for the
Freshly prepare a solution of trypsin for peptide mapping R separation of PTH-amino-acids.
at a concentration of 1 mg/mL in water R, and add 5 μL to
0.25 mL of the dialysed solution. Cap the tube and place in The separation procedure is calibrated using :
a water-bath at 37 °C for 18 h. Remove the sample from the – the mixture of PTH-amino acids provided by the
water-bath and stop the reaction immediately by freezing. manufacturer of the sequencer, with the gradient
conditions adjusted as indicated to achieve optimum
Reference solution. Dissolve the contents of a vial of
resolution of all amino acids ;
erythropoietin BRP in 0.25 mL of water R. Prepare as for
the test solution, ensuring that all procedures are carried – a sample obtained from a blank sequencing cycle
out simultaneously, and under identical conditions. obtained as recommended by the equipment
manufacturer.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ; TESTS
– stationary phase : butylsilyl silica gel for chromatography R Protein (2.5.33, Method I) : 80 per cent to 120 per cent of the
(5-10 μm). stated concentration.
Test solution. Dilute the preparation to be examined in a 4 g/L
Mobile phase :
solution of ammonium hydrogen carbonate R.
– mobile phase A : 0.06 per cent V/V solution of Record the absorbance spectrum between 250 nm and 400 nm.
trifluoroacetic acid R ; Measure the value at the absorbance maximum (276-280 nm),
– mobile phase B : to 100 mL of water R add 0.6 mL after correction for any light scattering, measured up to
of trifluoroacetic acid R and dilute to 1000 mL with 400 nm. Calculate the concentration of erythropoietin taking
acetonitrile for chromatography R ; the specific absorbance to be 7.43.

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EUROPEAN PHARMACOPOEIA 8.0 Erythropoietin concentrated solution

Dimers and related substances of higher molecular mass. System suitability :

Size-exclusion chromatography (2.2.30). – the individual replicates agree to within ± 10 per cent of
Test solution. Dilute the preparation to be examined in the each other ;
mobile phase to obtain a concentration of 0.2 mg/mL. – the value obtained from reference solution (a) is between
Reference solution. To 0.02 mL of the test solution add 0.98 mL 1.5 and 3.3 times that obtained with test solution (a).
of the mobile phase. Bacterial endotoxins (2.6.14) : less than 20 IU in the volume
Column : that contains 100 000 IU of erythropoietin.
– size : l = 0.6 m, Ø = 7.5 mm ; ASSAY
– stationary phase : hydrophilic silica gel for chromatography R, The activity of the preparation is compared with that of
of a grade suitable for fractionation of globular proteins in erythropoietin BRP and expressed in International Units (IU).
the relative molecular mass range of 20 000 to 200 000. The estimated potency is not less than 80 per cent and not
Mobile phase : dissolve 1.15 g of anhydrous disodium hydrogen more than 125 per cent of the stated potency. The confidence
phosphate R, 0.2 g of potassium dihydrogen phosphate R limits of the estimated potency (P = 0.95) are not less than
and 23.4 g of sodium chloride R in 1 L of water R (1.5 mM 64 per cent and not more than 156 per cent of the stated
potassium dihydrogen phosphate, 8.1 mM disodium hydrogen potency.
phosphate, 0.4 M sodium chloride, pH 7.4) ; adjust to pH 7.4 Carry out the determination of potency by Method A or B.
if necessary.
A. In polycythaemic mice
Flow rate : 0.5 mL/min.
The activity of the preparation is estimated by examining,
Detection : spectrophotometer at 214 nm. under given conditions, its effect in stimulating the
Injection : 100 μL. incorporation of 59Fe into circulating red blood cells of mice
Run time : minimum 1 h. made polycythaemic by exposure to reduced atmospheric
pressure.
System suitability : the area of the principal peak in the The following schedule, using treatment in a hypobaric
chromatogram obtained with the reference solution is 1.5 per chamber, has been found to be suitable.
cent to 2.5 per cent of the area of the principal peak in the
chromatogram obtained with the test solution. Induce polycythaemia in female mice of the same strain,
weighing 16-18 g. Place the mice in a hypoxic chamber
Limits : and reduce the pressure to 0.6 atmospheres. After
– total of any peaks eluted before the principal peak : not more 3 days at 0.6 atmospheres, further reduce the pressure to
than the area of the principal peak in the chromatogram 0.4-0.5 atmospheres and maintain the animals at this pressure
obtained with the reference solution (2 per cent). for a further 11 days (the partial vacuum is interrupted daily
Sialic acids : minimum 10 mol of sialic acids (calculated as for a maximum of 1 h at about 11:00 a.m., in order to clean
N-acetylneuraminic acid) per mole of erythropoietin. the cages and feed the animals). At the end of the specified
period, return the mice to normal atmospheric conditions.
Test solution (a). Dilute the preparation to be examined Randomly distribute the mice into cages, each containing
in the mobile phase used in the test for dimers and related 6 animals, and mark them.
substances of higher molecular mass to obtain a concentration
of 0.3 mg/mL. Test solution (a). Dilute the substance to be examined in
phosphate-albumin buffered saline pH 7.2 R1 to obtain a
Test solution (b). To 0.5 mL of test solution (a) add 0.5 mL concentration of 0.2 IU/mL.
of the mobile phase used in the test for dimers and related
Test solution (b). Mix equal volumes of test solution (a) and
substances of higher molecular mass.
phosphate-albumin buffered saline pH 7.2 R1.
Reference solution (a). Dissolve a suitable amount of Test solution (c). Mix equal volumes of test solution (b) and
N-acetylneuraminic acid R in water R to obtain a concentration phosphate-albumin buffered saline pH 7.2 R1.
of 0.1 mg/mL.
Reference solution (a). Dissolve erythropoietin BRP in
Reference solution (b). To 0.8 mL of reference solution (a) add phosphate-albumin buffered saline pH 7.2 R1 to obtain a
0.2 mL of water R. concentration of 0.2 IU/mL.
Reference solution (c). To 0.6 mL of reference solution (a) add Reference solution (b). Mix equal volumes of reference
0.4 mL of water R. solution (a) and phosphate-albumin buffered saline pH 7.2 R1.
Reference solution (d). To 0.4 mL of reference solution (a) add Reference solution (c). Mix equal volumes of reference
0.6 mL of water R. solution (b) and phosphate-albumin buffered saline pH 7.2 R1.
Reference solution (e). To 0.2 mL of reference solution (a) add Radiolabelled ferric [59Fe] chloride solution, concentrated.
0.8 mL of water R. Use a commercially available solution of [59Fe]ferric chloride
Reference solution (f). Use water R. (approximate specific activity : 100-1000 MBq/mg of Fe).
Carry out the test in triplicate. Transfer 100 μL of each of the Radiolabelled [59Fe]ferric chloride solution. Dilute the
test and reference solutions to 10 mL glass test tubes. To each concentrated radiolabelled [59Fe]ferric chloride solution in
tube add 1.0 mL of resorcinol reagent R. Stopper the tubes and sodium citrate buffer solution pH 7.8 R to obtain a solution
incubate at 100 °C for 30 min. Cool on ice. To each tube, add with an activity of 3.7 × 104 Bq/mL.
2.0 mL of a mixture of 12 volumes of butanol R and 48 volumes The concentrations of the test solutions and reference
of butyl acetate R. Mix vigorously, and allow the 2 phases to solutions may need to be modified, based on the response
separate. Ensuring that the upper phase is completely clear, range of the animals used.
remove the upper phase, taking care to exclude completely 3 days after returning the animals to atmospheric pressure,
any of the lower phase. Measure the absorbance (2.2.25) of inject each animal subcutaneously with 0.2 mL of one of
all samples at 580 nm. Using the calibration curve generated the solutions. The 6 animals in each cage must each receive
by the reference solutions, determine the content of sialic one of the 6 different treatments (3 test solutions and
acids in test solutions (a) and (b) and calculate the mean. 3 reference solutions), and the order of injection must be
Calculate the number of moles of sialic acids per mole of separately randomised for each cage. A minimum of 8 cages is
erythropoietin assuming that the relative molecular mass of recommended. 2 days after injection of the test or reference
erythropoietin is 30 600 and that the relative molecular mass solution, inject each animal intraperitoneally with 0.2 mL
of N-acetylneuraminic acid is 309. of radiolabelled [59Fe]ferric chloride solution. The order of

General Notices (1) apply to all monographs and other texts 2165
Esketamine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

the injections must be the same as that of the erythropoietin reticulocyte count microfluorometrically in a flow cytometer.
injections, and the time interval between administration of the The percentage of reticulocytes is determined using a
erythropoietin and the radiolabelled ferric chloride solution biparametric histogram : number of cells/red fluorescence
must be the same for each animal. After a further 48 h, (620 nm).
anaesthetise each animal by injection of a suitable anaesthetic, Calculate the potency by the usual statistical methods for a
record body weights and withdraw blood samples (0.65 mL) parallel line assay.
into haematocrit capillaries from the bifurcation of the aorta.
After determining the packed cell volume for each sample, STORAGE
measure the radioactivity. In an airtight container at a temperature below − 20 °C. Avoid
Calculate the response (percentage of iron-59 in total repeated freezing and thawing.
circulating blood) for each mouse using the expression :
LABELLING
The label states :
– the erythropoietin content in milligrams per millilitre ;
As = radioactivity in the sample ; – the activity in International Units per millilitre ;
At = total radioactivity injected ; – the name and the concentration of any other excipients.
7.5 = total blood volume as per cent body weight ;
01/2008:1742
M = body weight, in grams ;
corrected 6.0
Vs = sample volume.
Calculate the potency by the usual statistical methods for a ESKETAMINE HYDROCHLORIDE
parallel line assay. Eliminate from the calculation any animal
where the packed cell volume is less than 54 per cent, or where Esketamini hydrochloridum
the body weight is more than 24 g.
B. In normocythaemic mice
The assay is based on the measurement of stimulation of
reticulocyte production in normocythaemic mice.
The assay may be carried out using the following procedure :
Test solution (a). Dilute the preparation to be examined in C13H17Cl2NO Mr 274.2
phosphate-albumin buffered saline pH 7.2 R1 to obtain a [33795-24-3]
concentration of 80 IU/mL.
Test solution (b). Mix equal volumes of test solution (a) and DEFINITION
phosphate-albumin buffered saline pH 7.2 R1. (2S)-2-(2-Chlorophenyl)-2-(methylamino)cyclohexanone
Test solution (c). Mix equal volumes of test solution (b) and hydrochloride.
phosphate-albumin buffered saline pH 7.2 R1. Content : 99.0 per cent to 101.0 per cent.
Reference solution (a). Dissolve erythropoietin BRP in
phosphate-albumin buffered saline pH 7.2 R1 to obtain a CHARACTERS
concentration of 80 IU/mL. Appearance : white or almost white, crystalline powder.
Reference solution (b). Mix equal volumes of reference Solubility : freely soluble in water and in methanol, soluble
solution (a) and phosphate-albumin buffered saline pH 7.2 R1. in alcohol.
Reference solution (c). Mix equal volumes of reference IDENTIFICATION
solution (b) and phosphate-albumin buffered saline pH 7.2 R1.
A. Specific optical rotation (2.2.7) : + 85.0 to + 95.0.
The exact concentrations of the test solutions and reference
solutions may need to be modified, based on the response Dilute 12.5 mL of solution S (see Tests) to 40.0 mL with
range of the animals used. water R.
At the beginning of the assay procedure, randomly distribute B. Infrared absorption spectrophotometry (2.2.24).
mice of a suitable age and strain (8-week old B6D2F1 mice Comparison: Ph. Eur. reference spectrum of esketamine
are suitable) into 6 cages. A minimum of 8 mice per cage is hydrochloride.
recommended. Inject each animal subcutaneously with 0.5 mL C. It gives reaction (a) of chlorides (2.3.1).
of the appropriate treatment (one solution per cage) and put
the animal in a new cage. Combine the mice in such a way that TESTS
each cage housing the treated mice contains one mouse out Solution S. Dissolve 8.0 g in carbon dioxide-free water R and
of the 6 different treatments (3 test solutions and 3 reference dilute to 50.0 mL with the same solvent.
solutions, 6 mice per cage). 4 days after the injections, collect
Appearance of solution. Solution S is clear (2.2.1) and
blood samples from the animals and determine the number of
colourless (2.2.2, Method II).
reticulocytes using a suitable procedure.
The following method may be employed : pH (2.2.3) : 3.5 to 4.5.
The volume of blood, dilution procedure and fluorescent reagent Dilute 12.5 mL of solution S to 20 mL with carbon dioxide-free
may need to be modified to ensure maximum development and water R.
stability of fluorescence. Impurity D. Liquid chromatography (2.2.29).
Colorant solution, concentrated. Use a solution of thiazole Test solution. Dissolve 25.0 mg of the substance to be
orange suitable for the determination of reticulocytes. Prepare examined in water R and dilute to 100.0 mL with the same
at a concentration twice that necessary for the analysis. solvent.
Proceed with the following dilution steps. Dilute whole Reference solution (a). Dissolve 5 mg of esketamine
blood 500-fold in the buffer used to prepare the colorant impurity D CRS in water R, add 20 mL of the test solution and
solution. Dilute this solution 2-fold in the concentrated dilute to 50 mL with water R. Dilute 10 mL of this solution to
colorant solution. After staining for 3-10 min, determine the 100 mL with water R.

2166 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Esketamine hydrochloride

Reference solution (b). Dilute 5.0 mL of the test solution Relative retention with reference to esketamine :
to 25.0 mL with water R. Dilute 5.0 mL of this solution to impurity A = about 1.6 ; impurity B = about 3.3 ;
50.0 mL with water R. impurity C = about 4.6.
Reference solution (c). Dilute 2.5 mL of reference solution (b) System suitability : reference solution (a) :
to 10.0 mL with water R. Dilute 1.0 mL of this solution to – retention time : esketamine = 3.0 min to 4.5 min,
10.0 mL with water R.
– resolution : minimum 1.5 between the peaks due to
Precolumn : impurity A and esketamine.
– size : l = 0.01 m, Ø = 3.0 mm, Limits :
– stationary phase : silica gel AGP for chiral chromatography R – impurities A, B, C : for each impurity, not more than
(5 μm), 0.4 times the area of the principal peak in the chromatogram
– temperature : 30 °C. obtained with reference solution (b) (0.2 per cent),
– any other impurity : for each impurity, not more than
Column :
0.2 times the area of the principal peak in the chromatogram
– size : l = 0.125 m, Ø = 4.6 mm, obtained with reference solution (b) (0.1 per cent),
– stationary phase : silica gel AGP for chiral chromatography R – total : not more than the area of the principal peak in the
(5 μm), chromatogram obtained with reference solution (b) (0.5 per
cent),
– temperature : 30 °C.
– disregard limit : 0.2 times the area of the principal peak in
Mobile phase : mix 16 volumes of methanol R and 84 volumes the chromatogram obtained with reference solution (b)
of a 6.8 g/L solution of potassium dihydrogen phosphate R (0.1 per cent).
previously adjusted to pH 7.0 with potassium hydroxide R.
Heavy metals (2.4.8) : maximum 20 ppm.
Flow rate : 0.8 mL/min.
Dilute 12.5 mL of solution S to 20 mL with water R. 12 mL
Detection : spectrophotometer at 215 nm. of the solution complies with test A. Prepare the reference
Injection : 20 μL. solution using lead standard solution (2 ppm Pb) R.
Run time : 20 min. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
Relative retention with reference to esketamine (retention
time = about 10 min) : impurity D = about 1.3. ASSAY
System suitability : Dissolve 0.200 g in 50 mL of methanol R and add 1.0 mL of
– resolution : minimum 2.0 between the peaks due to 0.1 M hydrochloric acid. Carry out a potentiometric titration
esketamine and impurity D in the chromatogram obtained (2.2.20), using 0.1 M sodium hydroxide. Read the volume
with reference solution (a), added between the 2 points of inflexion.
– signal-to-noise ratio : minimum 3 for the principal peak in 1 mL of 0.1 M sodium hydroxide is equivalent to 27.42 mg
the chromatogram obtained with reference solution (c). of C13H17Cl2NO.
Limit : STORAGE
– impurity D : not more than the area of the principal peak Protected from light.
in the chromatogram obtained with reference solution (b)
(2.0 per cent). IMPURITIES
Related substances. Liquid chromatography (2.2.29). Specified impurities : A, B, C, D.
Test solution. Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with the
mobile phase.
Reference solution (a). Dissolve 5 mg of ketamine
impurity A CRS in the mobile phase (using ultrasound, if
necessary) and dilute to 10 mL with the mobile phase. To A. X = N-CH3 : 1-[(2-chlorophenyl)(methylimino)methyl]-
1 mL of the solution add 0.5 mL of the test solution and dilute cyclopentanol,
to 100 mL with the mobile phase. Prepare immediately before
use.
C. X = O : (2-chlorophenyl)(1-hydroxycyclopentyl)-
Reference solution (b). Dilute 1.0 mL of the test solution to methanone,
10.0 mL with the mobile phase. Dilute 1.0 mL of this solution
to 20.0 mL with the mobile phase.
Column :
– size : l = 0.125 m, Ø = 4.0 mm,
– stationary phase : spherical octadecylsilyl silica gel for
chromatography R (5 μm). B. (2RS)-2-(2-chlorophenyl)-2-hydroxycyclohexanone,
Mobile phase : dissolve 0.95 g of sodium hexanesulfonate R
in 1000 mL of a mixture of 25 volumes of acetonitrile R and
75 volumes of water R and add 4 mL of acetic acid R.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 215 nm.
Injection : 20 μL. D. (2R)-2-(2-chlorophenyl)-2-(methylamino)cyclohexanone
Run time : 10 times the retention time of esketamine. ((R)-ketamine).

General Notices (1) apply to all monographs and other texts 2167
Esomeprazole magnesium trihydrate EUROPEAN PHARMACOPOEIA 8.0

01/2009:2372 Column :
corrected 6.7 – size : l = 0.125 m, Ø = 4.6 mm ;
– stationary phase : octylsilyl silica gel for chromatography R
ESOMEPRAZOLE MAGNESIUM (5 μm).
TRIHYDRATE Mobile phase : mix 27 volumes of acetonitrile R and 73 volumes
of a 1.4 g/L solution of disodium hydrogen phosphate R
previously adjusted to pH 7.6 with phosphoric acid R.
Esomeprazolum magnesicum trihydricum Flow rate : 1 mL/min.
Detection : spectrophotometer at 280 nm.
Injection : 40 μL.
Run time : 5 times the retention time of esomeprazole.
Identification of impurities :
– use the chromatogram supplied with omeprazole for
peak identification CRS and the chromatogram obtained
with reference solution (b) to identify the peak due to
C34H36MgN6O6S2,3H2O Mr 767.2 impurity E ;
[217087-09-7] – use the chromatogram obtained with reference solution (a)
DEFINITION to identify the peak due to impurity D.
Relative retention with reference to esomeprazole
Magnesium bis[5-methoxy-2-[(S)-[(4-methoxy-3,5- (retention time = about 9 min) : impurity E = about 0.6 ;
dimethylpyridin-2-yl)methyl]sulfinyl]-1H-benzimidazol-1- impurity D = about 0.8.
ide] trihydrate.
System suitability : reference solution (a) :
Content : 98.0 per cent to 102.0 per cent (anhydrous substance).
– resolution : minimum 3.0 between the peaks due to
CHARACTERS impurity D and omeprazole. If necessary, adjust the pH of
Appearance : white or slightly coloured powder, slightly the aqueous part of the mobile phase or its proportion of
hygroscopic. acetonitrile ; an increase in the pH will improve the resolution.
Limits :
Solubility : slightly soluble in water, soluble in methanol,
practically insoluble in heptane. – impurity D : maximum 0.2 per cent ;
– impurity E : maximum 0.1 per cent ;
IDENTIFICATION – unspecified impurities : for each impurity, maximum
Carry out either tests A, B, C or A, B, E or B, C, D or B, D, E. 0.10 per cent ;
A. Specific optical rotation (2.2.7) : − 155 to − 137. – total : maximum 0.5 per cent ;
Dissolve 0.250 g in methanol R and dilute to 25.0 mL with – disregard limit : 0.5 times the area of the principal peak in
the same solvent. the chromatogram obtained with reference solution (c)
(0.05 per cent).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison : esomeprazole magnesium trihydrate CRS. Enantiomeric purity. Liquid chromatography (2.2.29).
Buffer solution pH 6.0. Mix 70 mL of a 156.0 g/L solution of
C. Atomic absorption spectrometry (2.2.23) as described in
sodium dihydrogen phosphate R with 20 mL of a 179.1 g/L
the test for magnesium.
solution of disodium hydrogen phosphate R. Dilute to 1000 mL
The test solution shows the absorption maximum at with water R, then dilute 250 mL of this solution to 1000.0 mL
285.2 nm. with water R.
D. Enantiomeric purity (see Tests). Buffer solution pH 11.0. Mix 11 mL of a 95.0 g/L solution
E. Ignite about 0.5 g of the substance to be examined according of trisodium phosphate dodecahydrate R with 22 mL of a
to the procedure for the sulfated ash test (2.4.14). Dissolve 179.1 g/L solution of disodium hydrogen phosphate R, then
the residue in 10 mL of water R. 2 mL of this solution gives dilute to 1000.0 mL with water R.
the reaction of magnesium (2.3.1). Test solution. Dissolve 40 mg of the substance to be examined
in 5 mL of methanol R and dilute to 25 mL with buffer solution
TESTS pH 11.0. Dilute 1.0 mL of this solution to 50.0 mL with buffer
Absorbance (2.2.25) : maximum 0.20 at 440 nm. solution pH 11.0.
Reference solution (a). Dissolve 2 mg of omeprazole CRS in
Dissolve 0.500 g in methanol R and dilute to 25.0 mL with the buffer solution pH 11.0 and dilute to 10.0 mL with the same
same solvent. Filter the solution through a membrane filter buffer solution. Dilute 1.0 mL of this solution to 50.0 mL with
(nominal pore size 0.45 μm). buffer solution pH 11.0.
Related substances. Liquid chromatography (2.2.29). Use the Reference solution (b). Dilute 1.0 mL of reference solution (a)
normalisation procedure. Use freshly prepared solutions. to 50.0 mL with buffer solution pH 11.0.
Test solution. Dissolve 3.5 mg of the substance to be examined Column :
in the mobile phase and dilute to 25.0 mL with the mobile – size : l = 0.1 m, Ø = 4.0 mm ;
phase.
– stationary phase : silica gel AGP for chiral chromatography R
Reference solution (a). Dissolve 1 mg of omeprazole CRS and (5 μm).
1 mg of omeprazole impurity D CRS in the mobile phase and
dilute to 10.0 mL with the mobile phase. Mobile phase : acetonitrile R, buffer solution pH 6.0
(65:435 V/V).
Reference solution (b). Dissolve 3 mg of the omeprazole for Flow rate : 0.6 mL/min.
peak identification CRS (containing impurity E) in the mobile
phase and dilute to 20.0 mL with the mobile phase. Detection : spectrophotometer at 302 nm.
Reference solution (c). Dilute 1.0 mL of the test solution to Injection : 20 μL.
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution Elution order : impurity F, esomeprazole.
to 10.0 mL with the mobile phase. Retention time : esomeprazole = about 4 min.

2168 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Estradiol benzoate

System suitability : STORAGE

– resolution : minimum 3.0 between the peaks due to In an airtight container, protected from light.
impurity F and esomeprazole in the chromatogram
obtained with reference solution (a) ; IMPURITIES
– signal-to-noise ratio : minimum 10 for the peak due to Specified impurities : D, E, F.
impurity F in the chromatogram obtained with reference Other detectable impurities (the following substances would,
solution (b). if present at a sufficient level, be detected by one or other of
Calculate the percentage content of impurity F using the the tests in the monograph. They are limited by the general
following expression : acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : A, B, C.
ri = area of the peak due to impurity F in the
chromatogram obtained with the test solution ;
rs = sum of the areas of the peaks due to esomeprazole
and impurity F in the chromatogram obtained with
the test solution. A. 5-methoxy-1H-benzimidazole-2-thiol,
Limits :
– impurity F : maximum 0.2 per cent.
Magnesium : 3.30 per cent to 3.55 per cent (anhydrous
substance).
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Dissolve 0.250 g in 20 mL of a 103 g/L solution B. R = H, X = SO : 2-[(RS)-[(3,5-dimethylpyridin-2-
of hydrochloric acid R, adding the acid slowly, and dilute to yl)methyl]sulfinyl]-5-methoxy-1H-benzimidazole,
100.0 mL with water R. Dilute 10.0 mL of this solution to C. R = OCH3, X = S : 5-methoxy-2-[[(4-methoxy-3,5-
200.0 mL with water R. To 10.0 mL of the solution obtained dimethylpyridin-2-yl)methyl]sulfanyl]-1H-benzimidazole
add 4 mL of lanthanum chloride solution R and dilute to (ufiprazole),
100.0 mL with water R.
D. R = OCH3, X = SO2 : 5-methoxy-2-[[(4-methoxy-3,5-
Reference solutions. Prepare the reference solutions using dimethylpyridin-2-yl)methyl]sulfonyl]-1H-benzimidazole
magnesium standard solution (1000 ppm Mg) R, diluted as (omeprazole sulfone),
necessary with a mixture of 1 mL of a 103 g/L solution of
hydrochloric acid R in 1000.0 mL of water R.
Wavelength : 285.2 nm.
Water (2.5.12) : 6.0 per cent to 8.0 per cent, determined on
0.200 g.
ASSAY E. 4-methoxy-2-[[(RS)-(5-methoxy-1H-benzimidazol-2-
Liquid chromatography (2.2.29). yl)sulfinyl]methyl]-3,5-dimethylpyridine 1-oxide.
Buffer solution pH 11.0. Mix 11 mL of a 95.0 g/L solution
of trisodium phosphate dodecahydrate R with 22 mL of a
179.1 g/L solution of disodium hydrogen phosphate R, and
dilute to 100.0 mL with water R.
Test solution. Dissolve 10.0 mg of the substance to be
examined in about 10 mL of methanol R, add 10 mL of buffer
solution pH 11.0 and dilute to 200.0 mL with water R. F. 5-methoxy-2-[(R)-[(4-methoxy-3,5-dimethylpyridin-2-
Reference solution. Dissolve 10.0 mg of omeprazole CRS in yl)methyl]sulfinyl]-1H-benzimidazole ((R)-omeprazole).
about 10 mL of methanol R, add 10 mL of buffer solution
pH 11.0 and dilute to 200.0 mL with water R. 04/2008:0139
Column :
– size : l = 0.125 m, Ø = 4 mm ; ESTRADIOL BENZOATE
– stationary phase : octylsilyl silica gel for chromatography R
(5 μm). Estradioli benzoas
Mobile phase : mix 35 volumes of acetonitrile R with 65 volumes
of a 1.4 g/L solution of disodium hydrogen phosphate R
previously adjusted to pH 7.6 with phosphoric acid R.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 280 nm.
Injection : 20 μL.
Run time : 1.5 times the retention time of esomeprazole.
C25H28O3 Mr 376.5
Retention time : esomeprazole = about 4 min. [50-50-0]
Calculate the percentage content of C34H36MgN6O6S2 from the
declared content of omeprazole CRS. DEFINITION
1 g of omeprazole is equivalent to 1.032 g of esomeprazole 17β-Hydroxyestra-1,3,5(10)-trien-3-yl benzoate.
magnesium. Content : 97.0 per cent to 103.0 per cent (dried substance).

General Notices (1) apply to all monographs and other texts 2169
Estradiol benzoate EUROPEAN PHARMACOPOEIA 8.0

CHARACTERS – impurity C : not more than the area of the principal peak
Appearance : almost white, crystalline powder or colourless in the chromatogram obtained with reference solution (b)
crystals. (0.5 per cent) ;
Solubility : practically insoluble in water, freely soluble in – impurities B, E, G : for each impurity, not more than
methylene chloride, sparingly soluble in acetone, slightly 0.6 times the area of the principal peak in the chromatogram
soluble in methanol. obtained with reference solution (b) (0.3 per cent) ;
It shows polymorphism (5.9). – impurity A : not more than 0.4 times the area of the
principal peak in the chromatogram obtained with
IDENTIFICATION reference solution (b) (0.2 per cent) ;
Infrared absorption spectrophotometry (2.2.24). – unspecified impurities : for each impurity, not more than
0.2 times the area of the principal peak in the chromatogram
Comparison : estradiol benzoate CRS. obtained with reference solution (b) (0.10 per cent) ;
If the spectra obtained in the solid state show differences, – total : not more than twice the area of the principal peak
dissolve the substance to be examined and the reference in the chromatogram obtained with reference solution (b)
substance separately in acetone R, evaporate to dryness and (1.0 per cent) ;
record new spectra using the residues.
– disregard limit : 0.1 times the area of the principal peak in
TESTS the chromatogram obtained with reference solution (b)
(0.05 per cent).
Specific optical rotation (2.2.7) : + 55.0 to + 59.0 (dried
substance). Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Dissolve 0.250 g in acetone R and dilute to 25.0 mL with the on 1.000 g by drying in an oven at 105 °C for 3 h.
same solvent. ASSAY
Related substances. Liquid chromatography (2.2.29). Dissolve 25.0 mg in anhydrous ethanol R and dilute to
Test solution. Dissolve 20 mg of the substance to be examined 250.0 mL with the same solvent. Dilute 10.0 mL of this
in acetonitrile R1 and dilute to 10.0 mL with the same solvent. solution to 100.0 mL with anhydrous ethanol R. Measure the
absorbance (2.2.25) at the absorption maximum at 231 nm.
Reference solution (a). Dissolve 5 mg of estradiol benzoate for
system suitability CRS (containing impurities A, B, C, E and G) Calculate the content of C25H28O3 taking the specific
in acetonitrile R1 and dilute to 2.5 mL with the same solvent. absorbance to be 500.
Reference solution (b). Dilute 0.5 mL of the test solution to IMPURITIES
100.0 mL with acetonitrile R1. Specified impurities : A, B, C, E, G.
Column : Other detectable impurities (the following substances would,
– size : l = 0.25 m, Ø = 4.6 mm ; if present at a sufficient level, be detected by one or other of
– stationary phase : end-capped octylsilyl silica gel for the tests in the monograph. They are limited by the general
chromatography R (5 μm). acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
Mobile phase : (2034). It is therefore not necessary to identify these impurities
– mobile phase A : water R, acetonitrile R1 (40:60 V/V) ; for demonstration of compliance. See also 5.10. Control of
– mobile phase B : acetonitrile R1 ; impurities in substances for pharmaceutical use): D, F, H.
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 20 100 0

20 - 21 100 → 10 0 → 90

21 - 31 10 90

Flow rate : 1.0 mL/min. A. R1 = R2 = R3 = H, R4 = OH : estradiol,

Detection : spectrophotometer at 230 nm.
B. R1 = CO-C6H5, R2 = CH3, R3 = H, R4 = OH :
Injection : 10 μL. 17β-hydroxy-4-methylestra-1,3,5(10)-trien-3-yl benzoate,
Identification of impurities : use the chromatogram supplied
with estradiol benzoate for system suitability CRS and the C. R1 = CO-C6H5, R2 = R3 = H, R4 = O-CO-C6H5 :
chromatogram obtained with reference solution (a) to identify estra-1,3,5(10)-triene-3,17β-diyl dibenzoate,
the peaks due to impurities A, B, C, E and G. E. R1 = CO-C6H5, R2 = R4 = H, R3 = OH :
Relative retention with reference to estradiol benzoate 17α-hydroxyestra-1,3,5(10)-trien-3-yl benzoate,
(retention time = about 19 min) : impurity A = about 0.3 ; G. R1 = CO-C6H5, R2 = H, R3 + R4 = O : 17-oxoestra-
impurity E = about 1.1 ; impurity B = about 1.2 ; 1,3,5(10)-trien-3-yl benzoate (estrone benzoate),
impurity G = about 1.3 ; impurity C = about 1.5.
System suitability: reference solution (a) :
– peak-to-valley ratio : minimum 2.0, where Hp = height
above the baseline of the peak due to impurity E and
Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to estradiol
benzoate.
Limits :
D. R1 = H, R2 = C6H5 : 3-hydroxyestra-1,3,5(10)-trien-17β-yl
– correction factors : for the calculation of content, multiply benzoate,
the peak areas of the following impurities by the
corresponding correction factor : impurity A = 3.3 ; H. R1 = CO-C6H5, R2 = CH3 : estra-1,3,5(10)-triene-3,17β-diyl
impurity C = 0.7 ; 17-acetate 3-benzoate,

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EUROPEAN PHARMACOPOEIA 8.0 Estradiol hemihydrate

D. To about 1 mg add 0.5 mL of freshly prepared sulfomolybdic

reagent R2. A blue colour develops which in ultraviolet
light at 365 nm has an intense green fluorescence. Add
1 mL of sulfuric acid R and 9 mL of water R. The colour
becomes pink with a yellowish fluorescence.
E. Water (see Tests).

F. 17β-hydroxyestra-1,3,5(10),9(11)-tetraen-3-yl benzoate. TESTS

Specific optical rotation (2.2.7) : + 76.0 to + 83.0 (anhydrous
substance).
01/2008:0821
Dissolve 0.250 g in ethanol (96 per cent) R and dilute to
25.0 mL with the same solvent.
ESTRADIOL HEMIHYDRATE
Related substances. Liquid chromatography (2.2.29).
Estradiolum hemihydricum Test solution. Dissolve 25.0 mg of the substance to be
examined in 10 mL of acetonitrile R and dilute to 25.0 mL
with methanol R2.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 2.0 mL of the solution
to 10.0 mL with the mobile phase.
Reference solution (b). Dissolve 2 mg of 17α-estradiol R in
5.0 mL of acetonitrile R. Mix 2.0 mL of this solution with
C18H24O2,½H2O Mr 281.4 1.0 mL of the test solution and dilute to 5.0 mL with the
mobile phase.
DEFINITION
Reference solution (c). Mix equal volumes of a 1 mg/mL
Estra-1,3,5(10)-triene-3,17β-diol hemihydrate. solution of the substance to be examined in methanol R2 and of
Content : 97.0 per cent to 103.0 per cent (anhydrous substance). a 1 mg/mL solution of 2,3-dichloro-5,6-dicyanobenzoquinone R
in methanol R2. Allow to stand for 30 min before injection.
CHARACTERS
Appearance : white or almost white, crystalline powder or Reference solution (d). Dissolve 5 mg of estradiol for peak
colourless crystals. identification CRS (estradiol hemihydrate spiked with
impurities A, B and C at about 0.5 per cent) in 2 mL of
Solubility : practically insoluble in water, soluble in acetone, acetonitrile R and dilute to 5 mL with methanol R2.
sparingly soluble in ethanol (96 per cent), slightly soluble in
methylene chloride. Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
IDENTIFICATION
– stationary phase : end-capped octadecylsilyl silica gel for
First identification : B. chromatography R (5 μm).
Second identification : A, C, D, E. Mobile phase : to 400 mL of acetonitrile R add 50 mL of
A. Melting point (2.2.14) : 175 °C to 180 °C. methanol R2 and 400 mL of water R ; allow to stand for 10 min,
B. Infrared absorption spectrophotometry (2.2.24). dilute to 1000 mL with water R and mix again.
Comparison : estradiol hemihydrate CRS. Flow rate : 1 mL/min.
C. Thin-layer chromatography (2.2.27). Detection : spectrophotometer at 280 nm.
Test solution. Dissolve 50 mg of the substance to be Equilibration : about 60 min.
examined in methanol R and dilute to 50 mL with the same
Injection : 20 μL.
solvent.
Reference solution (a). Dissolve 50 mg of estradiol Run time : twice the retention time of the principal peak.
hemihydrate CRS in methanol R and dilute to 50 mL with Identification of impurities : use the chromatogram
the same solvent. supplied with estradiol for peak identification CRS and the
Reference solution (b). Dissolve 25 mg of chromatogram obtained with reference solution (d) to identify
ethinylestradiol CRS in reference solution (a) and the peaks due to impurities A, B and C. Use the chromatogram
dilute to 25 mL with reference solution (a). obtained with reference solution (c) to identify the peak due
to impurity D.
Plate : TLC silica gel plate R.
Relative retention with reference to estradiol (retention
Mobile phase : ethanol (96 per cent) R, toluene R (20:80 V/V).
time = about 13 min) : impurity D = about 0.9 ;
Application : 5 μL. impurity B = about 1.1 ; impurity A = about 1.4 ;
Development : over 3/4 of the plate. impurity C = about 1.9.
Drying : in air until the solvent has evaporated. System suitability : reference solution (b) :
Detection : heat at 110 °C for 10 min. Spray the hot plate – resolution : minimum 2.5 between the peaks due to estradiol
with alcoholic solution of sulfuric acid R. Heat again at and impurity B.
110 °C for 10 min. Allow to cool. Examine in daylight and
in ultraviolet light at 365 nm. Limits :
System suitability : the chromatogram obtained with – correction factor : for the calculation of content, multiply
reference solution (b) shows 2 spots which may however the peak area of impurity D by 0.4 ;
not be completely separated. – impurities A, B, C, D : for each impurity, not more than
Results : the principal spot in the chromatogram obtained 1.5 times the area of the principal peak obtained with
with the test solution is similar in position, colour in reference solution (a) (0.3 per cent) ;
daylight, fluorescence in ultraviolet light at 365 nm and – unspecified impurities : for each impurity, not more than
size to the principal spot in the chromatogram obtained 0.5 times the area of the principal peak obtained with
with reference solution (a). reference solution (a) (0.10 per cent) ;

General Notices (1) apply to all monographs and other texts 2171
Estradiol valerate EUROPEAN PHARMACOPOEIA 8.0

– total : not more than 2.5 times the area of the principal peak IDENTIFICATION
in the chromatogram obtained with reference solution (a) Infrared absorption spectrophotometry (2.2.24).
(0.5 per cent) ;
Comparison: estradiol valerate CRS.
– disregard limit : 0.25 times the area of the principal peak
in the chromatogram obtained with reference solution (a) TESTS
(0.05 per cent). Solution S. Dissolve 0.500 g in methanol R and dilute to
Water (2.5.12) : 2.9 per cent to 3.5 per cent, determined on 20.0 mL with the same solvent.
0.500 g. Appearance of solution. Solution S is clear (2.2.1) and
ASSAY colourless (2.2.2, Method II).
Dissolve 20.0 mg in ethanol (96 per cent) R and dilute to Specific optical rotation (2.2.7) : + 41 to + 47 (dried
100.0 mL with the same solvent. Dilute 5.0 mL of the solution substance), determined on solution S.
to 50.0 mL with 0.1 M sodium hydroxide. Allow to cool to Related substances. Liquid chromatography (2.2.29).
room temperature. Measure the absorbance (2.2.25) of the Solvent mixture. Mix 15 volumes of water R and 135 volumes
solution at the maximum at 238 nm. of acetonitrile R.
Calculate the content of C18H24O2 taking the specific Test solution. Dissolve 0.100 g of the substance to be examined
absorbance to be 335. in the solvent mixture and dilute to 10.0 mL with the solvent
IMPURITIES mixture.
Specified impurities : A, B, C, D. Reference solution (a). Dissolve 2 mg of estradiol valerate CRS
and 2 mg of estradiol butyrate CRS in the solvent mixture and
dilute to 10 mL with the solvent mixture.
Reference solution (b). Dilute 0.5 mL of the test solution to
100.0 mL with the solvent mixture.
Column :
– size : l = 0.25 m, Ø = 4.6 mm,
– stationary phase : octadecylsilyl silica gel for
A. R1 = H, R2 + R3 = O : 3-hydroxyestra-1,3,5(10)-trien-17-one chromatography R (5 μm),
(estrone), – temperature : 40 °C.
Mobile phase :
B. R1 = R3 = H, R2 = OH : estra-1,3,5(10)-triene-3,17α-diol
(17α-estradiol), – mobile phase A : water R,
– mobile phase B : acetonitrile R,
C. R1 = CH3, R2 = H, R3 = OH : 4-methylestra-1,3,5(10)-
triene-3,17β-diol, Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 15 40 → 0 60 → 100

15 - 25 0 100

25 - 30 40 60

30 = 0 40 60
D. estra-1,3,5(10),9(11)-tetraene-3,17β-diol.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 220 nm.
01/2008:1614 Injection : 10 μL.
corrected 6.0 Relative retention with reference to estradiol valerate (retention
time = about 12 min) : impurity F = about 0.9.
ESTRADIOL VALERATE System suitability : reference solution (a) :
– resolution : minimum of 5.0 between the peaks due to
Estradioli valeras impurity F and to estradiol valerate.
Limits :
– any impurity : not more than the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.5 per cent),
– total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (b)
(1.0 per cent),
C23H32O3 Mr 356.5 – disregard limit : 0.1 times the area of the principal peak in
[979-32-8] the chromatogram obtained with reference solution (b)
(0.05 per cent).
DEFINITION
Loss on drying (2.2.32) : maximum 1.0 per cent, determined
3-Hydroxyestra-1,3,5(10)-trien-17β-yl pentanoate. on 0.500 g by drying in an oven at 105 °C for 3 h.
Content : 97.0 per cent to 103.0 per cent (dried substance).
ASSAY
CHARACTERS Dissolve 25.0 mg in alcohol R and dilute to 250.0 mL with
Appearance : white or almost white, crystalline powder or the same solvent. Measure the absorbance (2.2.25) at the
colourless crystals. maximum at 280 nm.
Solubility : practically insoluble in water, soluble in alcohol. Calculate the content of C23H32O3 taking the specific
mp : about 145 °C. absorbance to be 58.0.

2172 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Estriol

STORAGE Plate : TLC silica gel plate R.

Protected from light. Mobile phase : ethanol (96 per cent) R, toluene R (20:80 V/V).
Application : 5 μL.
IMPURITIES
Development : over 3/4 of the plate.
Drying : in air.
Detection : spray with alcoholic solution of sulfuric acid R.
Heat at 100 °C for 10 min or until the spots appear. Allow to
cool. Examine in daylight and in ultraviolet light at 365 nm.
System suitability : reference solution (b) :
– the chromatogram shows 2 clearly separated spots.
A. R1 = R2 = R3 = H : estradiol, Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour in
B. R1 = CO-[CH2]3-CH3, R2 = R3 = H : 17β-hydroxyestra- daylight, fluorescence in ultraviolet light at 365 nm and
1,3,5(10)-trien-3-yl pentanoate, size to the principal spot in the chromatogram obtained
D. R1 = H,R2 = CH3,R3 = CO-[CH2]3-CH3 : with reference solution (a).
3-hydroxy-4-methylestra-1,3,5(10)-trien-17β-yl
pentanoate, TESTS
Specific optical rotation (2.2.7) : + 60 to + 65 (dried
E. R1 = R3 = CO-[CH2]3-CH3,R2 = H : estra-1,3,5(10)-trien- substance).
3,17β-diyl dipentanoate,
Dissolve 80 mg in anhydrous ethanol R and dilute to 10 mL
F. R1 = R2 = H,R3 = CO-[CH2]2-CH3 : 3-hydroxyestra- with the same solvent.
1,3,5(10)-trien-17β-yl butanoate (estradiol butyrate),
Related substances. Liquid chromatography (2.2.29).
Solvent mixture : 2-propanol R1, heptane R (20:80 V/V).
Test solution. Dissolve 20.0 mg of the substance to be
examined in 5 mL of 2-propanol R1 and dilute to 20.0 mL
with the solvent mixture.
Reference solution (a). Dissolve 5 mg of estriol CRS and 2.0 mg
of estriol impurity A CRS in 5 mL of 2-propanol R1, then dilute
to 10.0 mL with the solvent mixture. Dilute 1.0 mL of this
C. 3-hydroxyestra-1,3,5(10),9(11)-tetraen-17β-yl pentanoate. solution to 20.0 mL with the solvent mixture.
Reference solution (b). Dilute 1.0 mL of the test solution
07/2011:1203 to 10.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
ESTRIOL Column :
– size : l = 0.15 m, Ø = 4.0 mm ;
Estriolum – stationary phase : diol silica gel for chromatography R (5 μm) ;
– temperature : 40 °C.
Mobile phase :
– mobile phase A : heptane R ;
– mobile phase B : 2-propanol R1 ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
C18H24O3 Mr 288.4
0 - 10 95 → 88 5 → 12
[50-27-1]
10 - 20 88 12
DEFINITION
Estra-1,3,5(10)-triene-3,16α,17β-triol. 20 - 30 88 → 95 12 → 5

Content : 97.0 per cent to 103.0 per cent (dried substance). 30 - 35 95 5

CHARACTERS Flow rate : 1.2 mL/min.

Appearance : white or almost white, crystalline powder. Detection : spectrophotometer at 280 nm.
Solubility : practically insoluble in water, sparingly soluble in
Injection : 20 μL.
ethanol (96 per cent). Relative retention with reference to estriol (retention
mp : about 282 °C. time = about 19 min) : impurity B = about 0.4 ;
impurity C = about 0.47 ; impurity D = about 0.5 ;
IDENTIFICATION impurity E = about 0.7 ; impurity F = about 0.75 ;
A. Infrared absorption spectrophotometry (2.2.24). impurity A = about 1.1 ; impurity G = about 1.2. If the
Comparison : estriol CRS. retention times increase, wash the column first with acetone R
B. Thin-layer chromatography (2.2.27). and then with heptane R.
Test solution. Dissolve 10 mg of the substance to be System suitability : reference solution (a) :
examined in methanol R and dilute to 10 mL with the same – resolution : minimum 2.2 between the peaks due to estriol
solvent. and impurity A ; if the resolution decreases, wash the
Reference solution (a). Dissolve 10 mg of estriol CRS in column first with acetone R and then with heptane R.
methanol R and dilute to 10 mL with the same solvent. Limits :
Reference solution (b). Dissolve 5 mg of estradiol – impurity A : not more than 0.5 times the area of the
hemihydrate CRS in reference solution (a) and dilute to corresponding peak in the chromatogram obtained with
5 mL with reference solution (a). reference solution (a) (0.5 per cent) ;

General Notices (1) apply to all monographs and other texts 2173
Estrogens, conjugated EUROPEAN PHARMACOPOEIA 8.0

– impurities B, C, D, E, F, G : for each impurity, not more

than 0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.5 per
cent) ;
– unspecified impurities : for each impurity, not more than
0.1 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.10 per cent) ; E. estra-1,3,5(10)-triene-3,16α,17α-triol (17-epi-estriol),
– sum of impurities other than A : not more than the area
of the principal peak in the chromatogram obtained with
reference solution (b) (1 per cent) ;
– disregard limit : 0.05 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent).
F. estra-1,3,5(10)-triene-3,16β,17β-triol (16-epi-estriol),
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 3 h.

ASSAY
Dissolve 25.0 mg in ethanol (96 per cent) R and dilute to
50.0 mL with the same solvent. Dilute 10.0 mL of this
solution to 50.0 mL with ethanol (96 per cent) R. Measure the
absorbance (2.2.25) at the absorption maximum at 281 nm. G. estra-1,3,5(10)-triene-3,16β,17α-triol (16,17-epi-estriol),
Calculate the content of C18H24O3 taking the specific
absorbance to be 72.5.

IMPURITIES
Specified impurities : A, B, C, D, E, F, G.
Other detectable impurities (the following substances would, H. 3,16α-dihydroxyestra-1,3,5(10)-trien-17-one,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : H, I.
I. 3-hydroxy-17-oxa-D-homoestra-1,3,5(10)-trien-17a-one.

01/2008:1512

ESTROGENS, CONJUGATED
Estrogeni coniuncti
A. estra-1,3,5(10),9(11)-tetraene-3,16α,17β-triol
(9,11-didehydroestriol),

B. 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone), C18H21O5NaS + C18H19O5NaS Mr 372.4 + 370.4

DEFINITION
Mixture of various conjugated forms of estrogens obtained
from the urine of pregnant mares or by synthesis, dispersed in
a suitable powdered diluent.
The 2 principal components are 17-oxoestra-1,3,5(10)-
trien-3-yl sodium sulfate (sodium estrone sulfate) and
C. 3-methoxyestra-1,3,5(10)-triene-16α,17β-diol (estriol 17-oxoestra-1,3,5(10),7-tetraen-3-yl sodium sulfate (sodium
3-methyl ether), equilin sulfate). Concomitants are sodium 17α-estradiol
sulfate, sodium 17α-dihydroequilin sulfate and sodium
17β-dihydroequilin sulfate.
Content (percentages related to the labelled content) :
– sodium estrone sulfate : 52.5 per cent to 61.5 per cent ;
– sodium equilin sulfate : 22.5 per cent to 30.5 per cent ;
– sodium 17α-estradiol sulfate : 2.5 per cent to 9.5 per cent ;
– sodium 17α-dihydroequilin sulfate : 13.5 per cent to 19.5 per
D. estradiol, cent ;

2174 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Estrogens, conjugated

– sodium 17β-dihydroequilin sulfate : 0.5 per cent to 4.0 per N,O-bis(trimethylsilyl)trifluoroacetamide R containing 1 per
cent ; cent of chlorotrimethylsilane R. Immediately cap the tube
– sum of sodium estrone sulfate and sodium equilin sulfate : tightly, mix and allow to stand for 15 min. Add 0.5 mL of
79.5 per cent to 88.0 per cent. toluene R.
Reference solution (b). Prepare as described in reference
CHARACTERS solution (a), but dilute tenfold with anhydrous ethanol R
Appearance : almost white or brownish, amorphous powder. before adding the internal standard.
Column :
IDENTIFICATION
– material : fused silica ;
A. Examine the chromatograms obtained in the assay.
– size : l = 15 m, Ø = 0.25 mm ;
Results : the 2 principal peaks due to estrone and equilin
in the chromatogram obtained with test solution (a) are – stationary phase : poly[(cyanoprop-
similar in retention time and size to the 2 principal peaks yl)(methyl)][(phenyl)(methyl)]siloxane R (film thickness
in the chromatogram obtained with reference solution (a). 0.25 μm).
B. Examine the chromatogram obtained in the test for Carrier gas : hydrogen for chromatography R.
chromatographic profile. Flow rate : 2 mL/min.
Results : the chromatogram obtained with test solution (b) Split ratio : 1:20 to 1:30.
exhibits additional peaks due to 17α-estradiol, Temperature :
17α-dihydroequilin and 17β-dihydroequilin, at relative
– column : 220 °C ;
retentions with reference to 3-O-methylestrone (internal
standard) of about 0.24, 0.30 and 0.35 respectively. – injection port and detector : 260 °C.
Detection : flame ionisation.
TESTS
Injection : 1 μL.
Chromatographic profile. Gas chromatography (2.2.28). Relative retention with reference to 3-O-methylestrone :
Internal standard solution. Dissolve 8 mg of 17α-dihydroequilin = about 0.30 ; estrone = about 0.80 ;
3-O-methylestrone R in 10.0 mL of anhydrous ethanol R. Dilute equilin = about 0.87.
2.0 mL of this solution to 10.0 mL with anhydrous ethanol R. System suitability : reference solution (a) :
Acetate buffer solution pH 5.2. Dissolve 10 g of sodium – resolution : minimum 1.2 between the peaks due to estrone
acetate R in 100 mL of water R and add 10 mL of dilute and equilin ; if necessary, adjust the temperature and the
acetic acid R. Dilute to 500 mL with water R and adjust to flow rate of the carrier gas.
pH 5.2 ± 0.1.
In the chromatogram obtained with reference solution (a),
Test solution (a). Considering the labelled content, transfer an measure the areas of the peaks due to 17α-dihydroequilin,
accurately weighed quantity corresponding to about 2 mg of estrone and 3-O-methylestrone.
conjugated estrogens to a 50 mL centrifuge tube containing
15 mL of the acetate buffer solution pH 5.2 and 1 g of barium In the chromatogram obtained with test solution (a),
chloride R. Cap the tube tightly and shake for 30 min. If locate the peaks with relative retentions with reference to
necessary, adjust to pH 5.0 ± 0.5 with acetic acid R or a 120 g/L 3-O-methylestrone of 1 and about 0.24, 0.29, 0.30, 0.35, 0.56,
solution of sodium acetate R. Sonicate for 30 s, then shake for 0.64, 0.90 and 1.3 and measure their areas.
30 min. Add a suitable sulfatase preparation equivalent to Calculate the percentage content of the components occurring
2500 units and shake mechanically for 10 min in a water-bath as sodium sulfate salts using expression (1) below.
at 50 ± 1 °C. Swirl the tube by hand, then shake mechanically In the chromatogram obtained with reference solution (b),
for 10 min in the water-bath. Allow to cool. Add 15.0 mL measure the areas of the peaks due to estrone and
of ethylene chloride R to the mixture, immediately cap the 3-O-methylestrone.
tube tightly and shake for 15 min. Centrifuge for 10 min or
until the lower layer is clear. Draw out the organic layer to a In the chromatogram obtained with test solution (b),
screw-cap tube, add 5 g of anhydrous sodium sulfate R and locate the peaks with relative retentions with reference to
shake. Allow the solution to stand until clear. Protect the 3-O-methylestrone of about 0.30, 0.80 and 0.87 and measure
solution from any loss due to evaporation. Transfer 3.0 mL the sum of the areas.
of the clear solution to a suitable centrifuge tube fitted with Calculate the percentage content of 17α-dihydroequilin,
a screw cap. Add 1.0 mL of the internal standard solution. estrone and equilin occurring as free steroids using
Evaporate the mixture to dryness with the aid of a stream of expression (2) below.
nitrogen R, maintaining the temperature below 50 °C. To the
dry residue add 15 μL of anhydrous pyridine R and 65 μL of
N,O-bis(trimethylsilyl)trifluoroacetamide R containing 1 per
cent of chlorotrimethylsilane R. Immediately cap the tube
tightly, mix thoroughly and allow to stand for 15 min. Add
0.5 mL of toluene R and mix mechanically.
Test solution (b). Prepare as described in test solution (a), but SI = area of the peak due to the internal standard in the
do not add the sulfatase and use 6.0 mL of the upper layer chromatogram obtained with the corresponding
instead of 3.0 mL. Prepare a blank in the same manner. reference solution ;
Reference solution (a). Dissolve separately 8 mg of estrone CRS, S′I = area of the peak due to the internal standard in the
7 mg of equilin CRS and 5 mg of 17α-dihydroequilin CRS chromatogram obtained with the corresponding
in 10.0 mL of anhydrous ethanol R. Dilute together 2.0 mL, test solution ;
1.0 mL and 1.0 mL respectively of these solutions to SR = area of the peak due to the reference substance
10.0 mL with anhydrous ethanol R. Transfer 1.0 mL of this
(Table 1512.-1) in the chromatogram obtained with
solution and 1.0 mL of the internal standard solution to
the corresponding reference solution ;
a centrifuge tube fitted with a screw cap. Evaporate the
mixture to dryness with the aid of a stream of nitrogen R, S′A = area of the peak due to the analyte in the
maintaining the temperature below 50 °C. To the dry chromatogram obtained with the corresponding
residue add 15 μL of anhydrous pyridine R and 65 μL of test solution ;

General Notices (1) apply to all monographs and other texts 2175
Estrogens, conjugated EUROPEAN PHARMACOPOEIA 8.0

Table 1512.-1
Relative retention Analyte Quantified with reference to CRS Present as
(to 3-O-methylestrone)
0.24 17α-estradiol 17α-dihydroequilin CRS sodium sulfate
0.29 17β-estradiol estrone CRS sodium sulfate
0.30 17α-dihydroequilin 17α-dihydroequilin CRS free steroid, sodium sulfate (assay)
0.35 17β-dihydroequilin 17α-dihydroequilin CRS sodium sulfate
0.56 17α-dihydroequilenin estrone CRS sodium sulfate
0.64 17β-dihydroequilenin estrone CRS sodium sulfate
0.80 estrone estrone CRS free steroid, sodium sulfate (assay)
0.87 equilin equilin CRS free steroid, sodium sulfate (assay)
0.90 8,9-didehydroestrone estrone CRS sodium sulfate
1 3-O-methylestrone (internal standard)
1.3 equilenin estrone CRS sodium sulfate

mR = mass of the reference substance (Table 1512.-1) Calculate the percentage content of sodium estrone sulfate
in the corresponding reference solution, in and sodium equilin sulfate using expression (1).
milligrams ;
m = mass of the substance to be examined in the LABELLING
corresponding test solution, in milligrams ; The label states :
S′FS = sum of the areas of the peaks due to – the name of the substance ;
17α-dihydroequilin, estrone and equilin in the – the content of the substance ;
chromatogram obtained with the corresponding
test solution ; – the nature of the diluent.
SE = area of the peak due to estrone CRS in the IMPURITIES AND CONCOMITANTS
chromatogram obtained with the corresponding
reference solution ;
mE = mass of estrone CRS in the corresponding reference
solution, in milligrams ;
LC = labelled content, in milligrams per gram.
The percentages are within the following ranges :
– sodium 17α-estradiol sulfate : 2.5 per cent to 9.5 per cent ;
A. R1 = OH, R2 = H, R3 = SO3Na : 17α-hydroxyestra-
– sodium 17α-dihydroequilin sulfate : 13.5 per cent to 19.5 per
1,3,5(10)-trien-3-yl sodium sulfate (sodium 17α-estradiol
cent ;
sulfate),
– sodium 17β-dihydroequilin sulfate : 0.5 per cent to 4.0 per
cent ;
D. R1 = H, R2 = OH, R3 = SO3Na : 17β-hydroxyestra-1,3,5(10)-
– sodium 17β-estradiol sulfate : maximum 2.25 per cent ; trien-3-yl sodium sulfate (sodium 17β-estradiol sulfate),
– sodium 17α-dihydroequilenin sulfate : maximum 3.25 per
cent ;
I. R1 + R2 = O, R3 = H : 3-hydroxyestra-1,3,5(10)-trien-17-
– sodium 17β-dihydroequilenin sulfate : maximum 2.75 per one (estrone),
cent ;
– sodium 8,9-didehydroestrone sulfate : maximum 6.25 per
cent ;
– sodium equilenin sulfate : maximum 5.5 per cent ;
– sum of estrone, equilin and 17α-dihydroequilin : maximum
1.3 per cent.
ASSAY
Gas chromatography (2.2.28) as described in the test for B. R1 = OH, R2 = H, R3 = SO3Na : 17α-hydroxyestra-
chromatographic profile with the following modifications. 1,3,5(10),7-tetraen-3-yl sodium sulfate (sodium
17α-dihydroequilin sulfate),
Injection : test solution (a) and reference solution (a).
System suitability: reference solution (a) :
C. R1 = H, R2 = OH, R3 = SO3Na : 17β-hydroxyestra-
– repeatability : maximum relative standard deviation of 1,3,5(10),7-tetraen-3-yl sodium sulfate (sodium
2.0 per cent for the ratio of the area of the peak due to 17β-dihydroequilin sulfate),
estrone to that due to the internal standard after at least
6 injections.
In the chromatogram obtained with reference solution (a), J. R1 + R2 = O, R3 = H : 3-hydroxyestra-1,3,5(10),7-tetraen-
measure the areas of the peaks due to estrone or equilin and 17-one (equilin),
3-O-methylestrone. In the chromatogram obtained with test
solution (a), measure the areas of the peaks due to estrone, K. R1 = OH, R2 = R3 = H : estra-1,3,5(10),7-tetraene-3,17α-
equilin and 3-O-methylestrone. diol (17α-dihydroequilin),

2176 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Etacrynic acid

water R and 0.15 mL of hydrochloric acid R. Examined in

ultraviolet light at 254 nm, the mixture shows an intense
blue fluorescence.
E. Dissolve about 25 mg in 2 mL of a 42 g/L solution of sodium
hydroxide R and heat in a water-bath for 5 min. Cool and
add 0.25 mL of a mixture of equal volumes of sulfuric
E. R1 = OH, R2 = H : 17α-hydroxyestra-1,3,5(10),6,8-pentaen- acid R and water R. Add 0.5 mL of a 100 g/L solution of
3-yl sodium sulfate (sodium 17α-dihydroequilenin sulfate), chromotropic acid, sodium salt R and, carefully, 2 mL of
F. R1 = H, R2 = OH : 17β-hydroxyestra-1,3,5(10),6,8-pentaen- sulfuric acid R. An intense violet colour is produced.
3-yl sodium sulfate (sodium 17β-dihydroequilenin sulfate), TESTS
H. R1 + R2 = O : 17-oxoestra-1,3,5(10),6,8-pentaen-3-yl Related substances. Liquid chromatography (2.2.29).
sodium sulfate (sodium equilenin sulfate),
Solvent mixture : acetonitrile R, water R (40:60 V/V).
Test solution. Dissolve 25 mg of the substance to be examined
in the solvent mixture and dilute to 25.0 mL with the solvent
mixture.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
G. 17-oxoestra-1,3,5(10),8-tetraen-3-yl sodium sulfate
(sodium 8,9-didehydroestrone sulfate). Reference solution (b). Dissolve 5 mg of etacrynic acid for
system suitability CRS (containing impurities A, B and C)
in 5.0 mL of the solvent mixture.
07/2009:0457
Column :
ETACRYNIC ACID – size : l = 0.25 m, Ø = 4.0 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
Acidum etacrynicum chromatography R (5 μm) ;
– temperature : 25 °C.
Mobile phase :
– mobile phase A : 1 per cent V/V solution of triethylamine R
adjusted to pH 6.8 with phosphoric acid R ;
– mobile phase B : acetonitrile R ;
C13H12Cl2O4 Mr 303.1 Time Mobile phase A Mobile phase B
[58-54-8] (min) (per cent V/V) (per cent V/V)
DEFINITION 0-2.5 70 30

[2,3-Dichloro-4-(2-methylenebutanoyl)phenoxy]acetic acid 2.5-3 70→65 30→35

Content : 98.0 per cent to 102.0 per cent (dried substance). 3-6 65 35
CHARACTERS 6-7 65→45 35→55
Appearance : white or almost white, crystalline powder. 7-22 45 55
Solubility : very slightly soluble in water, freely soluble in
ethanol (96 per cent). It dissolves in ammonia and in dilute Flow rate : 0.8 mL/min.
solutions of alkali hydroxides and carbonates. Detection : spectrophotometer at 280 nm.
IDENTIFICATION Injection : 10 μL.
First identification : C. Identification of impurities : use the chromatogram supplied
Second identification : A, B, D, E. with etacrynic acid for system suitability CRS and the
chromatogram obtained with reference solution (b) to identify
A. Melting point (2.2.14) : 121 °C to 124 °C. the peaks due to impurities A, B and C.
B. Ultraviolet and visible absorption spectrophotometry Relative retention with reference to etacrynic acid
(2.2.25). (retention time = about 9 min) : impurity A = about 0.8 ;
Solvent mixture : 103 g/L solution of hydrochloric acid R, impurity B = about 1.3 ; impurity C = about 1.7.
methanol R (1:99 V/V). System suitability : reference solution (b) :
Test solution : Dissolve 50.0 mg in the solvent mixture and – resolution : minimum 4.0 between the peaks due to
dilute to 100.0 mL with the solvent mixture. Dilute 10.0 mL impurity A and etacrynic acid.
of this solution to 100.0 mL with the solvent mixture.
Limits :
Spectral range : 230-350 nm.
– correction factors : for the calculation of contents,
Absorption maximum : at 270 nm. multiply the peak areas of the following impurities by
Shoulder : at about 285 nm. the corresponding correction factor : impurity A = 0.6 ;
Specific absorbance at the absorption maximum : 110 to 120. impurity B = 0.6 ; impurity C = 1.3 ;
C. Infrared absorption spectrophotometry (2.2.24). – impurity C : not more than 3 times the area of the principal
Comparison : etacrynic acid CRS. peak in the chromatogram obtained with reference
D. Dissolve about 30 mg in 2 mL of aldehyde-free alcohol R. solution (a) (0.3 per cent) ;
Dissolve 70 mg of hydroxylamine hydrochloride R in 0.1 mL – impurities A, B : for each impurity, not more than 1.5 times
of water R, add 7 mL of alcoholic potassium hydroxide the area of the principal peak in the chromatogram
solution R and dilute to 10 mL with aldehyde-free alcohol R. obtained with reference solution (a) (0.15 per cent) ;
Allow to stand and add 1 mL of the supernatant to the – unspecified impurities : for each impurity, not more than the
solution of the substance to be examined. Heat the mixture area of the principal peak in the chromatogram obtained
on a water-bath for 3 min. After cooling, add 3 mL of with reference solution (a) (0.10 per cent) ;

General Notices (1) apply to all monographs and other texts 2177
Etamsylate EUROPEAN PHARMACOPOEIA 8.0

– total : not more than 8 times the area of the principal peak Second identification : A, C, D.
in the chromatogram obtained with reference solution (a) A. Melting point (2.2.14) : 127 °C to 134 °C.
(0.8 per cent) ; B. Infrared absorption spectrophotometry (2.2.24).
– disregard limit : 0.5 times the area of the principal peak in Comparison: etamsylate CRS.
the chromatogram obtained with reference solution (a)
(0.05 per cent). C. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Heavy metals (2.4.8) : maximum 20 ppm.
Test solution. Dissolve 0.100 g in water R and dilute to
1.0 g complies with test F. Prepare the reference solution using 200.0 mL with the same solvent. Dilute 5.0 mL of the
2 mL of lead standard solution (10 ppm Pb) R. solution to 100.0 mL with water R. Examine immediately.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined Spectral range : 210-350 nm.
on 2.000 g by drying at 60 °C over diphosphorus pentoxide R Absorption maxima : at 221 nm and 301 nm.
at a pressure of 0.1-0.5 kPa.
Specific absorbance at the absorption maximum at 301 nm :
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on 145 to 151.
1.0 g. D. Into a test-tube, introduce 2 mL of freshly prepared
ASSAY solution S (see Tests) and 0.5 g of sodium hydroxide R.
Warm the mixture and place a wet strip of red litmus
Dissolve 0.250 g in 100 mL of methanol R and add 5 mL of
paper R near the open end of the tube. The colour of the
water R. Titrate with 0.1 M sodium hydroxide, determining
paper becomes blue.
the end-point potentiometrically (2.2.20).
1 mL of 0.1 M sodium hydroxide is equivalent to 30.31 mg TESTS
of C13H12Cl2O4. Solution S. Dissolve 10.0 g in carbon dioxide-free water R and
IMPURITIES dilute to 100 mL with the same solvent.
Specified impurities : A, B, C. Appearance of solution. Solution S, when freshly prepared,
is clear (2.2.1) and colourless (2.2.2, Method II).
pH (2.2.3) : 4.5 to 5.6 for solution S.
Related substances. Liquid chromatography (2.2.29). Keep
all solutions at 2-8 °C.
Buffer solution. Dissolve 1.2 g of anhydrous sodium dihydrogen
A. R = H : (4-butanoyl-2,3-dichlorophenoxy)acetic acid, phosphate R in 900 mL of water for chromatography R. Adjust
B. R = CH2Cl : [2,3-dichloro-4-[2-(chloromethyl)butanoyl]- to pH 6.5 with disodium hydrogen phosphate solution R and
phenoxy]acetic acid, dilute to 1000 mL with water for chromatography R.
Test solution. Dissolve 0.100 g of the substance to be examined
in water R and dilute to 10.0 mL with the same solvent.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with water R. Dilute 1.0 mL of this solution to
10.0 mL with water R.
Reference solution (b). Dissolve 10 mg of the substance to
be examined and 10 mg of hydroquinone R (impurity A) in
C. [4-[2-[4-(carboxymethoxy)-2,3-dichlorobenzoyl]-
water R and dilute to 10 mL with the same solvent. Dilute
2,5-diethyl-3,4-dihydro-2H-pyran-6-yl]-2,3-
1 mL of the solution to 100 mL with water R.
dichlorophenoxy]acetic acid.
Column :
07/2008:1204 – size : l = 0.25 m, Ø = 4.6 mm ;
corrected 7.1 – stationary phase: spherical end-capped octadecylsilyl silica
gel for chromatography R (5 μm).
ETAMSYLATE Mobile phase : acetonitrile R1, buffer solution (10:90 V/V).
Flow rate : 0.8 mL/min.
Etamsylatum Detection : spectrophotometer at 220 nm.
Injection : 10 μL.
Run time : 11 times the retention time of etamsylate.
Relative retention with reference to etamsylate (retention
time = about 6 min) : impurity A = about 1.7.
C10H17NO5S Mr 263.3 System suitability : reference solution (b) :
[2624-44-4] – resolution : minimum 8.0 between the peaks due to
DEFINITION etamsylate and impurity A.
N-Ethylethanamine 2,5-dihydroxybenzenesulfonate. Limits:
Content : 99.0 per cent to 101.0 per cent (dried substance). – correction factor : for the calculation of content, multiply
the peak area of impurity A by 0.5 ;
CHARACTERS – impurity A : not more than the area of the principal peak
Appearance : white or almost white, crystalline powder. in the chromatogram obtained with reference solution (a)
Solubility : very soluble in water, freely soluble in methanol, (0.1 per cent) ;
soluble in anhydrous ethanol, practically insoluble in – unspecified impurities : for each impurity, not more than the
methylene chloride. area of the principal peak in the chromatogram obtained
It shows polymorphism (5.9). with reference solution (a) (0.10 per cent) ;
– total : not more than twice the area of the principal peak
IDENTIFICATION in the chromatogram obtained with reference solution (a)
First identification : B. (0.2 per cent) ;

2178 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ethacridine lactate monohydrate

– disregard limit : 0.5 times the area of the principal peak in C. To 0.5 mL of solution S add 1.0 mL of water R, 0.1 mL of a
the chromatogram obtained with reference solution (a) 10 g/L solution of cobalt chloride R and 0.1 mL of a 50 g/L
(0.05 per cent). solution of potassium ferrocyanide R. The solution is green.
Iron (2.4.9): maximum 10 ppm, determined on solution S. D. To 50 mL of solution S add 10 mL of dilute sodium
hydroxide solution R. Filter. To 5 mL of the filtrate, add
Heavy metals (2.4.8) : maximum 15 ppm.
1 mL of dilute sulfuric acid R. 5 mL of the solution obtained
1.0 g complies with test C. Prepare the reference solution using gives the reaction of lactates (2.3.1).
1.5 mL of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined TESTS
on 1.000 g by drying in vacuo in an oven at 60 °C. Solution S. Dissolve 2.0 g in carbon dioxide-free water R and
dilute to 100.0 mL with the same solvent.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. pH (2.2.3) : 5.5 to 7.0 for solution S.
Related substances. Liquid chromatography (2.2.29).
ASSAY
Test solution. Dissolve 10.0 mg of the substance to be
Dissolve 0.200 g in a mixture of 10 mL of water R and 40 mL examined in the mobile phase and dilute to 25.0 mL with the
of dilute sulfuric acid R. Titrate with 0.1 M cerium sulfate, mobile phase.
determining the end-point potentiometrically (2.2.20).
Reference solution (a). Dilute 1.0 mL of the test solution to
1 mL of 0.1 M cerium sulfate is equivalent to 13.16 mg of 100.0 mL with the mobile phase.
C10H17NO5S.
Reference solution (b). Dilute 1.0 mL of reference solution (a)
STORAGE to 10.0 mL with the mobile phase.
In an airtight container, protected from light. Column :
– size : l = 0.25 m, Ø = 4.6 mm,
IMPURITIES
– stationary phase : octadecylsilyl silica gel for
Specified impurities : A. chromatography R (5 μm).
Mobile phase : dissolve 1.0 g of sodium octanesulfonate R in a
mixture of 300 mL of acetonitrile R and 700 mL of phosphate
buffer solution pH 2.8 R.
Flow rate : 1 mL/min.
A. benzene-1,4-diol (hydroquinone). Detection : spectrophotometer at 268 nm.
Injection : 10 μL.
Run time : 3 times the retention time of ethacridine.
01/2008:1591
corrected 6.3 Retention time : ethacridine = about 15 min.
Limits :
ETHACRIDINE LACTATE – any impurity : not more than 3 times the area of the
principal peak in the chromatogram obtained with
MONOHYDRATE reference solution (b) (0.3 per cent),
– total : not more than the area of the principal peak in the
Ethacridini lactas monohydricus chromatogram obtained with reference solution (a) (1 per
cent),
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent).
Heavy metals (2.4.8) : maximum 50 ppm.
C18H21N3O4,H2O Mr 361.4 1.0 g complies with test F. Prepare the reference solution using
[6402-23-9] 5.0 mL of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : 4.5 per cent to 5.5 per cent,
DEFINITION determined on 1.000 g by drying in an oven in vacuo at 105 °C.
7-Ethoxyacridine-3,9-diamine (2RS)-2-hydroxypropanoate Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
monohydrate. 1.0 g.
Content : 99.0 per cent to 101.0 per cent (dried substance).
ASSAY
CHARACTERS Dissolve 0.270 g in 5.0 mL of anhydrous formic acid R. Add
Appearance : yellow crystalline powder. 60.0 mL of acetic anhydride R and titrate with 0.1 M perchloric
Solubility : sparingly soluble in water, very slightly soluble acid, determining the end-point potentiometrically (2.2.20).
in ethanol (96 per cent), practically insoluble in methylene 1 mL of 0.1 M perchloric acid is equivalent to 34.34 mg of
chloride. C18H21N3O4.
IDENTIFICATION STORAGE
First identification : A. Protected from light.
Second identification : B, C, D. IMPURITIES
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : ethacridine lactate monohydrate CRS.
B. Mix 0.1 mL of solution S (see Tests) and 100 mL of water R.
The solution is greenish-yellow and shows a strong green
fluorescence in ultraviolet light at 365 nm. Add 5 mL of
1 M hydrochloric acid. The fluorescence remains. A. 6-amino-2-ethoxyacridin-9(10H)-one,

General Notices (1) apply to all monographs and other texts 2179
Ethambutol hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Application : 2 μL.
Development : over 2/3 of the plate.
Drying : in air ; heat at 110 °C for 10 min.
Detection : cool then spray with ninhydrin solution R1 ; heat at
B. R = Cl : 6-chloro-2-ethoxyacridin-9-amine, 110 °C for 5 min.
C. R = O-CH2-CH2-OH : 2-[(9-amino-7-ethoxyacridin-3- System suitability : reference solution (b) :
yl)oxy]ethanol. – the chromatogram shows 2 clearly separated spots.
Limit :
04/2008:0553
– impurity A : any spot due to impurity A in the chromatogram
obtained with test solution (a) is not more intense than
ETHAMBUTOL HYDROCHLORIDE the spot in the chromatogram obtained with reference
solution (a) (1.0 per cent).
Ethambutoli hydrochloridum Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
Test solution. Suspend 4.0 mg of the substance to be
examined in 4.0 mL of acetonitrile R1 and add 100 μL of
triethylamine R. Sonicate the mixture for 5 min. Add 15 μL
of (R)-(+)-α-methylbenzyl isocyanate R and heat at 70 °C for
20 min.
C10H26Cl2N2O2 Mr 277.2 Reference solution (a). Dilute 0.50 mL of the test solution to
[1070-11-7] 100.0 mL with acetonitrile R1.
DEFINITION Reference solution (b). Treat 4.0 mg of ethambutol for system
(2S,2′S)-2,2′-(Ethylenediimino)dibutan-1-ol dihydrochloride. suitability CRS (containing impurity B) as described for the
test solution.
Content : 99.0 per cent to 101.0 per cent (dried substance).
Column :
CHARACTERS – size : l = 0.10 m, Ø = 4.6 mm ;
Appearance : white or almost white, crystalline powder, – stationary phase : end-capped octadecylsilyl silica gel for
hygroscopic. chromatography R (3 μm) ;
Solubility : freely soluble in water, soluble in ethanol (96 per – temperature : 40 °C.
cent). Mobile phase :
IDENTIFICATION – mobile phase A : methanol R, water R (50:50 V/V) ;
First identification : A, D, E. – mobile phase B : methanol R ;
Second identification : B, C, D. Time Mobile phase A Mobile phase B
A. Infrared absorption spectrophotometry (2.2.24). (min) (per cent V/V) (per cent V/V)
Comparison : ethambutol hydrochloride CRS. 0 - 30 71 29
B. Examine the chromatograms obtained in the test for 30 - 35 71 → 0 29 → 100
impurity A.
35 - 37 0 100
Results : the principal spot in the chromatogram obtained
with test solution (b) is similar in position, colour and size 37 - 38 0 → 71 100 → 29
to the principal spot in the chromatogram obtained with
reference solution (b). Flow rate : 1.0 mL/min.
C. Dissolve 0.1 g in 10 mL of water R. Add 0.2 mL of copper Detection : spectrophotometer at 215 nm.
sulfate solution R and 0.5 mL of dilute sodium hydroxide Injection : 10 μL.
solution R ; a blue colour is produced. Relative retention with reference to ethambutol (retention
D. It gives reaction (a) of chlorides (2.3.1). time = about 14 min) : impurity B = about 1.3.
E. Related substances (see Tests). System suitability : reference solution (b) :
TESTS – resolution : minimum 4.0 between the peaks due to
ethambutol and impurity B.
pH (2.2.3) : 3.7 to 4.0.
Limits :
Dissolve 0.2 g in 10 mL of carbon dioxide-free water R.
– impurity B : not more than twice the area of the principal
Impurity A. Thin-layer chromatography (2.2.27). peak in the chromatogram obtained with reference
Test solution (a). Dissolve 0.50 g of the substance to be solution (a) (1.0 per cent) ;
examined in methanol R and dilute to 10 mL with the same – unspecified impurities with a relative retention of 0.75 to 1.5
solvent. with reference to ethambutol : for each impurity, not more
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL than 0.2 times the area of the peak due to ethambutol in
with methanol R. the chromatogram obtained with reference solution (a)
Reference solution (a). Dissolve 50.0 mg of aminobutanol R (0.10 per cent);
(impurity A) in methanol R and dilute to 10.0 mL with the – total (impurity B and unspecified impurities with a relative
same solvent. Dilute 1.0 mL of this solution to 10.0 mL with retention of 0.75 to 1.5 with reference to ethambutol) : not
methanol R. more than twice the area of the principal peak in the
Reference solution (b). Dissolve 50 mg of ethambutol chromatogram obtained with reference solution (a) (1.0 per
hydrochloride CRS and 5 mg of aminobutanol R in methanol R cent) ;
and dilute to 10 mL with the same solvent. – disregard limit : 0.1 times the area of the peak due to
Plate : TLC silica gel plate R. ethambutol in the chromatogram obtained with reference
Mobile phase : concentrated ammonia R, water R, methanol R solution (a) (0.05 per cent).
(10:15:75 V/V/V). Impurity D (1,2-dichloroethane) (2.4.24) : maximum 5 ppm.

2180 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ethanol (96 per cent)

Heavy metals (2.4.8) : maximum 10 ppm. Solubility : miscible with water and with methylene chloride.
Dissolve 2.0 g in water R and dilute to 20 mL with the same It burns with a blue, smokeless flame.
solvent. 12 mL of the solution complies with test A. Prepare bp : about 78 °C.
the reference solution using 10 mL of lead standard solution
(1 ppm Pb) R. IDENTIFICATION
Loss on drying (2.2.32) : maximum 0.5 per cent, determined First identification : A, B.
on 0.500 g by drying in an oven at 105 °C for 3 h.
Second identification : A, C, D.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. A. Relative density (see Tests).
B. Infrared absorption spectrophotometry (2.2.24).
ASSAY
Comparison: Ph. Eur. reference spectrum ethanol (96 per
Dissolve 0.200 g in 50 mL of water R and add 1.0 mL of cent).
0.1 M hydrochloric acid. Carry out a potentiometric titration
(2.2.20), using 0.1 M sodium hydroxide. Read the volume C. Mix 0.1 mL with 1 mL of a 10 g/L solution of potassium
added between the 2 points of inflexion. permanganate R and 0.2 mL of dilute sulfuric acid R in a
test-tube. Cover immediately with a filter paper moistened
1 mL of 0.1 M sodium hydroxide is equivalent to 27.72 mg with a freshly prepared solution containing 0.1 g of sodium
of C10H26Cl2N2O2. nitroprusside R and 0.5 g of piperazine hydrate R in 5 mL
STORAGE of water R. After a few minutes, an intense blue colour
appears on the paper and becomes paler after 10-15 min.
In an airtight container.
D. To 0.5 mL add 5 mL of water R, 2 mL of dilute sodium
IMPURITIES hydroxide solution R, then slowly add 2 mL of 0.05 M
Specified impurities : A, B, D. iodine. A yellow precipitate is formed within 30 min.
Other detectable impurities (the following substances would, TESTS
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general Appearance. It is clear (2.2.1) and colourless (2.2.2, Method II)
acceptance criterion for other/unspecified impurities and/or when compared with water R. Dilute 1.0 mL to 20 mL with
by the general monograph Substances for pharmaceutical water R. After standing for 5 min, the dilution remains clear
use (2034). It is therefore not necessary to identify these (2.2.1) when compared with water R.
impurities for demonstration of compliance. See also 5.10. Acidity or alkalinity. To 20 mL add 20 mL of carbon
Control of impurities in substances for pharmaceutical use): C.dioxide-free water R and 0.1 mL of phenolphthalein solution R.
The solution is colourless. Add 1.0 mL of 0.01 M sodium
hydroxide. The solution is pink (30 ppm, expressed as acetic
acid).
A. 2-aminobutan-1-ol, Relative density (2.2.5) : 0.805 to 0.812.
Absorbance (2.2.25) : maximum 0.40 at 240 nm, 0.30 between
250 nm and 260 nm and 0.10 between 270 nm and 340 nm.
The absorption curve is smooth.
Examine between 235 nm and 340 nm, in a 5 cm cell using
water R as the compensation liquid.
B. R = CH2-OH, R′ = H : (2R,2′S)-2,2′-(ethylenediimino)- Volatile impurities. Gas chromatography (2.2.28).
dibutan-1-ol (meso-ethambutol),
Test solution (a). The substance to be examined.
C. R = H, R′ = CH2-OH : (2R,2′R)-2,2′-(ethylenediimino)- Test solution (b). Add 150 μL of 4-methylpentan-2-ol R to
dibutan-1-ol ((R,R)-ethambutol), 500.0 mL of the substance to be examined.
Reference solution (a). Dilute 100 μL of anhydrous methanol R
to 50.0 mL with the substance to be examined. Dilute 5.0 mL
D. 1,2-dichloroethane (ethylene chloride). of the solution to 50.0 mL with the substance to be examined.
Reference solution (b). Dilute 50 μL of anhydrous methanol R
and 50 μL of acetaldehyde R to 50.0 mL with the substance to
be examined. Dilute 100 μL of the solution to 10.0 mL with
01/2008:1317 the substance to be examined.
Reference solution (c). Dilute 150 μL of acetal R to 50.0 mL
ETHANOL (96 PER CENT) with the substance to be examined. Dilute 100 μL of the
solution to 10.0 mL with the substance to be examined.
Ethanolum (96 per centum) Reference solution (d). Dilute 100 μL of benzene R to 100.0 mL
with the substance to be examined. Dilute 100 μL of the
DEFINITION solution to 50.0 mL with the substance to be examined.
Content :
Column :
– ethanol (C2H6O ; Mr 46.07) : 95.1 per cent V/V (92.6 per
cent m/m) to 96.9 per cent V/V (95.2 per cent m/m) – material : fused silica ;
at 20 °C, calculated from the relative density using the – size : l = 30 m, Ø = 0.32 mm ;
alcoholimetric tables (5.5) ; – stationary phase : poly[(cyanopropyl)(phenyl)][dimeth-
– water. yl]siloxane R (film thickness 1.8 μm).
CHARACTERS Carrier gas : helium for chromatography R.
Appearance : colourless, clear, volatile, flammable liquid, Linear velocity : 35 cm/s.
hygroscopic. Split ratio : 1:20.

General Notices (1) apply to all monographs and other texts 2181
Ethanol (96 per cent) EUROPEAN PHARMACOPOEIA 8.0

Temperature : IMPURITIES
Time Temperature
(min) (°C)
Column 0 - 12 40
A. 1,1-diethoxyethane (acetal),
12 - 32 40 → 240

32 - 42 240

Injection port 200

Detector 280
B. acetaldehyde,

Detection : flame ionisation.

Injection : 1 μL.
C. propan-2-one (acetone),
System suitability : reference solution (b) :
– resolution : minimum 1.5 between the first peak
(acetaldehyde) and the second peak (methanol).
Limits :
D. benzene,
– methanol in the chromatogram obtained with test
solution (a) : not more than half the area of the
corresponding peak in the chromatogram obtained with
reference solution (a) (200 ppm V/V) ;
E. cyclohexane,
– acetaldehyde + acetal : maximum 10 ppm V/V, expressed
as acetaldehyde.
Calculate the sum of the contents of acetaldehyde and acetal F. methanol,
in parts per million V/V using the following expression :

G. butan-2-one (methyl ethyl ketone),

AE = area of the acetaldehyde peak in the
chromatogram obtained with test solution (a),
AT = area of the acetaldehyde peak in the
chromatogram obtained with reference H. 4-methylpentan-2-one (methyl isobutyl ketone),
solution (b),
CE = area of the acetal peak in the chromatogram
obtained with test solution (a),
I. propan-1-ol (propanol),
CT = area of the acetal peak in the chromatogram
obtained with reference solution (c).
– benzene : maximum 2 ppm V/V.
Calculate the content of benzene in parts per million V/V J. propan-2-ol (isopropyl alcohol),
using the following expression :

K. butan-1-ol (butanol),

BE = area of the benzene peak in the chromatogram

obtained with the test solution (a),
BT = area of the benzene peak in the chromatogram L. butan-2-ol,
obtained with reference solution (d).
If necessary, the identity of benzene can be confirmed using
another suitable chromatographic system (stationary phase
with a different polarity). M. 2-methylpropan-1-ol (isobutanol),
– total of other impurities in the chromatogram obtained with
test solution (b) : not more than the area of the peak due to
4-methylpentan-2-ol in the chromatogram obtained with
test solution (b) (300 ppm),
– disregard limit : 0.03 times the area of the peak due to N. furane-2-carbaldehyde (furfural),
4-methylpentan-2-ol in the chromatogram obtained with
test solution (b) (9 ppm).
Residue on evaporation : maximum 25 ppm m/V.
Evaporate 100 mL to dryness on a water-bath and dry at O. 2-methylpropan-2-ol (1,1-dimethylethyl alcohol),
100-105 °C for 1 h. The residue weighs a maximum of 2.5 mg.

STORAGE
Protected from light. P. 2-methylbutan-2-ol,

2182 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ethanol, anhydrous

TESTS
Appearance. It is clear (2.2.1) and colourless (2.2.2, Method II)
when compared with water R. Dilute 1.0 mL to 20 mL with
Q. pentan-2-ol, water R. After standing for 5 min, the dilution remains clear
(2.2.1) when compared with water R.
Acidity or alkalinity. To 20 mL add 20 mL of carbon
R. pentan-1-ol (pentanol), dioxide-free water R and 0.1 mL of phenolphthalein solution R.
The solution is colourless. Add 1.0 mL of 0.01 M sodium
hydroxide. The solution is pink (30 ppm, expressed as acetic
acid).
S. hexan-1-ol (hexanol), Relative density (2.2.5) : 0.790 to 0.793.
Absorbance (2.2.25) : maximum 0.40 at 240 nm, 0.30 between
250 nm and 260 nm, and 0.10 between 270 nm and 340 nm.
The absorption curve is smooth.
T. heptan-2-ol, Examined between 235 nm and 340 nm in a 5 cm cell using
water R as the compensation liquid.
Volatile impurities. Gas chromatography (2.2.28).
Test solution (a). The substance to be examined.
U. hexan-2-ol, Test solution (b). Add 150 μL of 4-methylpentan-2-ol R to
500.0 mL of the substance to be examined.
Reference solution (a). Dilute 100 μL of anhydrous methanol R
to 50.0 mL with the substance to be examined. Dilute 5.0 mL
of the solution to 50.0 mL with the substance to be examined.
V. hexan-3-ol.
Reference solution (b). Dilute 50 μL of anhydrous methanol R
and 50 μL of acetaldehyde R to 50.0 mL with the substance to
be examined. Dilute 100 μL of the solution to 10.0 mL with
01/2008:1318 the substance to be examined.
Reference solution (c). Dilute 150 μL of acetal R to 50.0 mL
ETHANOL, ANHYDROUS with the substance to be examined. Dilute 100 μL of the
solution to 10.0 mL with the substance to be examined.
Ethanolum anhydricum Reference solution (d). Dilute 100 μL of benzene R to 100.0 mL
with the substance to be examined. Dilute 100 μL of the
solution to 50.0 mL with the substance to be examined.
Column :
C 2H 6O Mr 46.07 – material : fused silica ;
[64-17-5]
– size : l = 30 m, Ø = 0.32 ;
DEFINITION – stationary phase : poly[(cyanopropyl)(phenyl)][dimeth-
Content : not less than 99.5 per cent V/V of C2H6O (99.2 per yl]siloxane R (film thickness 1.8 μm).
cent m/m), at 20 °C, calculated from the relative density using Carrier gas : helium for chromatography R.
the alcoholimetric tables (5.5).
Linear velocity : 35 cm/s.
CHARACTERS Split ratio : 1:20.
Appearance : colourless, clear, volatile, flammable liquid, Temperature :
hygroscopic.
Time Temperature
Solubility : miscible with water and with methylene chloride. (min) (°C)
It burns with a blue, smokeless flame. Column 0 - 12 40
bp : about 78 °C. 12 - 32 40 → 240
IDENTIFICATION 32 - 42 240
First identification : A, B. Injection port 200
Second identification : A, C, D.
Detector 280
A. Relative density (see Tests).
B. Infrared absorption spectrophotometry (2.2.24). Detection : flame ionisation.
Comparison : Ph. Eur. reference spectrum of anhydrous Injection : 1 μL.
ethanol. System suitability : reference solution (b) :
C. Mix 0.1 mL with 1 mL of a 10 g/L solution of potassium – resolution : minimum 1.5 between the first peak
permanganate R and 0.2 mL of dilute sulfuric acid R in a (acetaldehyde) and the second peak (methanol).
test-tube. Cover immediately with a filter paper moistened
with a freshly prepared solution containing 0.1 g of sodium Limits :
nitroprusside R and 0.5 g of piperazine hydrate R in 5 mL – methanol : in the chromatogram obtained with test
of water R. After a few minutes, an intense blue colour solution (a) : not more than half the area of the
appears on the paper and becomes paler after 10-15 min. corresponding peak in the chromatogram obtained with
D. To 0.5 mL add 5 mL of water R, 2 mL of dilute sodium reference solution (a) (200 ppm V/V) ;
hydroxide solution R, then slowly add 2 mL of 0.05 M – acetaldehyde + acetal : maximum of 10 ppm V/V, expressed
iodine. A yellow precipitate is formed within 30 min. as acetaldehyde.

General Notices (1) apply to all monographs and other texts 2183
Ethanol, anhydrous EUROPEAN PHARMACOPOEIA 8.0

Calculate the sum of the contents of acetaldehyde and acetal

in parts per million V/V using the following expression :

G. butan-2-one (methyl ethyl ketone),

AE = area of the acetaldehyde peak in the

chromatogram obtained with test solution (a),
AT = area of the acetaldehyde peak in the H. 4-methylpentan-2-one (methyl isobutyl ketone),
chromatogram obtained with reference
solution (b),
CE = area of the acetal peak in the chromatogram I. propan-1-ol (propanol),
obtained with test solution (a),
CT = area of the acetal peak in the chromatogram
obtained with reference solution (c).
– benzene : maximum 2 ppm V/V.
J. propan-2-ol (isopropyl alcohol),
Calculate the content of benzene in parts per million V/V
using the following expression :

K. butan-1-ol (butanol),

BE = area of the benzene peak in the chromatogram

obtained with the test solution (a),
BT = area of the benzene peak in the chromatogram L. butan-2-ol,
obtained with reference solution (d).
If necessary, the identity of benzene can be confirmed using
another suitable chromatographic system (stationary phase
with a different polarity). M. 2-methylpropan-1-ol (isobutanol),
– total of other impurities in the chromatogram obtained with
test solution (b) : not more than the area of the peak due to
4-methylpentan-2-ol in the chromatogram obtained with
test solution (b) (300 ppm) ;
– disregard limit : 0.03 times the area of the peak due to
4-methylpentan-2-ol in the chromatogram obtained with N. furane-2-carbaldehyde (furfural),
test solution (b) (9 ppm).
Residue on evaporation : maximum 25 ppm m/V.
Evaporate 100 mL to dryness on a water-bath and dry at
100-105 °C for 1 h. The residue weighs a maximum of 2.5 mg. O. 2-methylpropan-2-ol (1,1-dimethylethyl alcohol),
STORAGE
Protected from light.
IMPURITIES P. 2-methylbutan-2-ol,

A. 1,1-diethoxyethane (acetal),
Q. pentan-2-ol,

B. acetaldehyde, R. pentan-1-ol (pentanol),

S. hexan-1-ol (hexanol),
C. propan-2-one (acetone),

T. heptan-2-ol,
D. benzene,

U. hexan-2-ol,
E. cyclohexane,

F. methanol, V. hexan-3-ol.

2184 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ether, anaesthetic

01/2008:0650 01/2008:0367

ETHER ETHER, ANAESTHETIC

Aether Aether anaestheticus

C4H10O Mr 74.1 C4H10O Mr 74.1

[60-29-7] [60-29-7]

DEFINITION
DEFINITION
Diethyl ether.
Diethyl ether.
It may contain a suitable non-volatile antioxidant at an
It may contain a suitable non-volatile antioxidant at a suitable appropriate concentration.
concentration.
CHARACTERS
CHARACTERS Appearance : clear, colourless liquid, volatile, very mobile.
Appearance : clear, colourless liquid, volatile. Solubility : soluble in 15 parts of water, miscible with ethanol
Solubility : soluble in water, miscible with ethanol (96 per (96 per cent) and with fatty oils.
cent), with methylene chloride and with fatty oils. It is highly flammable.
It is highly flammable.
IDENTIFICATION
IDENTIFICATION A. Relative density (see Tests).
A. Relative density (see Tests). B. Distillation range (see Tests).
B. Distillation range (see Tests). TESTS
TESTS Acidity. To 20 mL of ethanol (96 per cent) R add 0.25 mL of
bromothymol blue solution R1 and, dropwise, 0.02 M sodium
Acidity. To 20 mL of ethanol (96 per cent) R add 0.25 mL of hydroxide until a blue colour persists for 30 s. Add 25 mL
bromothymol blue solution R1 and, dropwise, 0.02 M sodium of the substance to be examined, shake and add, dropwise,
hydroxide until a blue colour persists for 30 s. Add 25 mL 0.02 M sodium hydroxide until the blue colour reappears and
of the substance to be examined, shake and add, dropwise, persists for 30 s. Not more than 0.4 mL of 0.02 M sodium
0.02 M sodium hydroxide until the blue colour reappears and hydroxide is required.
persists for 30 s. Not more than 0.4 mL of 0.02 M sodium
hydroxide is required. Relative density (2.2.5) : 0.714 to 0.716.
Relative density (2.2.5) : 0.714 to 0.716. Distillation range (2.2.11). Do not distil if the substance to
be examined does not comply with the test for peroxides. It
Distillation range (2.2.11). Do not distil if the substance to distils completely between 34.0 °C and 35.0 °C. Carry out the
be examined does not comply with the test for peroxides. It test using a suitable heating device and taking care to avoid
distils completely between 34.0 °C and 35.0 °C. Carry out the directly heating the flask above the level of the liquid.
test using a suitable heating device and taking care to avoid
directly heating the flask above the level of the liquid. Acetone and aldehydes. To 10.0 mL in a ground-glass-
stoppered cylinder add 1 mL of alkaline potassium
Aldehydes. To 10.0 mL in a ground-glass-stoppered cylinder tetraiodomercurate solution R and shake for 10 s. Allow to
add 1 mL of alkaline potassium tetraiodomercurate solution R stand for 5 min, protected from light. The lower layer shows
and shake for 10 s. Allow to stand for 5 min, protected from only a slight opalescence.
light. The lower layer may show a yellow or reddish-brown
opalescence but not a grey or black opalescence. If the substance to be examined does not comply with the test,
distil 40 mL, after ensuring that the substance to be examined
Peroxides. Place 8 mL of potassium iodide and starch complies with the test for peroxides, until only 5 mL remains.
solution R in a 12 mL ground-glass-stoppered cylinder about Collect the distillate in a receiver cooled in a bath of iced
15 mm in diameter. Fill completely with the substance to be water and repeat the test described above using 10.0 mL of
examined, mix and allow to stand protected from light for the distillate.
5 min. No colour develops.
Peroxides. Place 8 mL of potassium iodide and starch
Non-volatile matter : maximum 20 mg/L. solution R in a 12 mL ground-glass-stoppered cylinder about
After ensuring that the substance to be examined complies 15 mm in diameter. Fill completely with the substance to be
with the test for peroxides, evaporate 50 mL to dryness on a examined, shake vigorously and allow to stand protected from
water-bath and dry the residue in an oven at 100-105 °C. The light for 30 min. No colour develops.
residue weighs a maximum of 1 mg. Non-volatile matter: maximum 20 mg/L.
Substances with a foreign odour. Moisten a disc of filter After ensuring that the substance to be examined complies
paper 80 mm in diameter with 5 mL of the substance to with the test for peroxides, evaporate 50 mL to dryness on a
be examined and allow to evaporate. No foreign odour is water-bath and dry the residue in an oven at 100-105 °C. The
perceptible immediately after the evaporation. residue weighs a maximum of 1 mg.
Water (2.5.12): maximum 2 g/L, determined on 20 mL. Substances with a foreign odour. Moisten a disc of filter
paper 80 mm in diameter with 5 mL of the substance to
STORAGE be examined and allow to evaporate. No foreign odour is
In an airtight container, protected from light, at a temperature perceptible immediately after the evaporation.
of 8 °C to 15 °C. Water (2.5.12) : maximum 2 g/L, determined on 20 mL.

General Notices (1) apply to all monographs and other texts 2185
Ethinylestradiol EUROPEAN PHARMACOPOEIA 8.0

STORAGE Reference solution (a). Dilute 1.0 mL of the test solution to

In an airtight container, protected from light, at a temperature100.0 mL with the solvent mixture. Dilute 1.0 mL of this
of 8 °C to 15 °C. The contents of a partly filled container may solution to 10.0 mL with the solvent mixture.
deteriorate rapidly. Reference solution (b). Dissolve 2 mg of estrone CRS
(impurity C) in 10.0 mL of the solvent mixture. Dilute 1.0 mL
of the solution to 100.0 mL with the solvent mixture. Use
04/2012:0140 1.0 mL of this solution to dissolve the contents of a vial
of ethinylestradiol for system suitability CRS (containing
ETHINYLESTRADIOL impurities B, F, H, I and K).
Reference solution (c). Dissolve 50.0 mg of ethinylestradiol CRS
Ethinylestradiolum in 30 mL of acetonitrile R1 and dilute to 50.0 mL with water R.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped butylsilyl silica gel for
chromatography R (5 μm) ;
– temperature : 30 °C.
Mobile phase :
C20H24O2 Mr 296.4
– mobile phase A : acetonitrile R1, water R (30:70 V/V) ;
[57-63-6]
– mobile phase B : water R, acetonitrile R1 (25:75 V/V) ;
DEFINITION
Time Mobile phase A Mobile phase B
19-Nor-17α-pregna-1,3,5(10)-trien-20-yne-3,17-diol. (min) (per cent V/V) (per cent V/V)
Content : 97.5 per cent to 102.0 per cent (dried substance). 0 - 35 100 0

CHARACTERS 35 - 65 100 → 0 0 → 100

Appearance : white or slightly yellowish-white, crystalline
powder. Flow rate : 1.5 mL/min.
Solubility : practically insoluble in water, freely soluble in Detection : spectrophotometer at 220 nm.
ethanol (96 per cent). It dissolves in dilute alkaline solutions. Injection : 30 μL of the test solution and reference solutions (a)
It shows polymorphism (5.9). and (b).
Identification of impurities : use the chromatogram supplied
IDENTIFICATION with ethinylestradiol for system suitability CRS and the
A. Infrared absorption spectrophotometry (2.2.24). chromatogram obtained with reference solution (b) to identify
Comparison : ethinylestradiol CRS. the peaks due to impurities B, C, F, H, I and K.
If the spectra obtained in the solid state show differences, Relative retention with reference to ethinylestradiol
dissolve the substance to be examined and the reference (retention time = about 35 min) : impurity F = about 0.2 ;
substance separately in methanol R, evaporate to dryness impurity H = about 0.5 ; impurity I = about 0.8 ;
and record new spectra using the residues. impurity B = about 0.88 ; impurity C = about 0.92 ;
B. Thin-layer chromatography (2.2.27). impurity K = about 1.3.
Solvent mixture : methanol R, methylene chloride R System suitability : reference solution (b) :
(10:90 V/V). – resolution : minimum 1.2 between the peaks due to
Test solution. Dissolve 25 mg of the substance to be impurities I and B.
examined in the solvent mixture and dilute to 25 mL with Limits :
the solvent mixture. – correction factors : for the calculation of content, multiply
Reference solution. Dissolve 25 mg of ethinylestradiol CRS the peak areas of the following impurities by the
in the solvent mixture and dilute to 25 mL with the solvent corresponding correction factor : impurity B = 0.7 ;
mixture. impurity I = 0.4 ;
Plate : TLC silica gel G plate R. – impurity B : not more than 5 times the area of the principal
Mobile phase : ethanol (96 per cent) R, toluene R (10:90 V/V). peak in the chromatogram obtained with reference
Application : 5 μL. solution (a) (0.5 per cent) ;
Development : over 2/3 of the plate. – impurities H, I, K : for each impurity, not more than
twice the area of the principal peak in the chromatogram
Drying : in air until the solvent has evaporated. obtained with reference solution (a) (0.2 per cent) ;
Detection : heat at 110 °C for 10 min, spray the hot plate
– impurities C, F : for each impurity, not more than 1.5 times
with alcoholic solution of sulfuric acid R and heat again at
the area of the principal peak in the chromatogram
110 °C for 10 min. Examine in daylight and in ultraviolet
obtained with reference solution (a) (0.15 per cent) ;
light at 365 nm.
Results : the principal spot in the chromatogram obtained – unspecified impurities : for each impurity, not more than the
with the test solution is similar in position, colour, area of the principal peak in the chromatogram obtained
fluorescence and size to the principal spot in the with reference solution (a) (0.10 per cent) ;
chromatogram obtained with the reference solution. – total : not more than 8 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
TESTS (0.8 per cent) ;
Related substances. Liquid chromatography (2.2.29). – disregard limit : 0.5 times the area of the principal peak in
Solvent mixture : water R, acetonitrile R1 (40:60 V/V). the chromatogram obtained with reference solution (a)
Test solution. Dissolve 50.0 mg of the substance to be (0.05 per cent).
examined in 30 mL of acetonitrile R1 and dilute to 50.0 mL Loss on drying (2.2.32) : maximum 1.0 per cent, determined
with water R. on 0.500 g by drying in an oven at 105 °C for 3 h.

2186 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ethinylestradiol

ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
Injection : test solution and reference solution (c).
Calculate the percentage content of C20H24O2 from the
declared content of ethinylestradiol CRS.
F. 19-nor-17α-pregna-1,3,5(10)-trien-20-yne-3,6β,17-triol
STORAGE (6β-hydroxy-ethinylestradiol),
Protected from light.

IMPURITIES
Specified impurities : B, C, F, H, I, K.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or G. 3,17-dihydroxy-19-nor-17α-pregna-1,3,5(10)-trien-20-yn-
by the general monograph Substances for pharmaceutical 6-one (6-oxo-ethinylestradiol),
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use) :
A, D, E, G, J, L, M.

H. 3,17-dihydroxy-19-nor-17α-pregna-1,3,5(10)-trien-20-yn-
16-one (16-oxo-ethinylestradiol),

A. 19-norpregna-1,3,5(10)-trien-20-yne-3,17-diol
(17β-ethinylestradiol),

I. 19-nor-17α-pregna-1,3,5(10),6-tetraen-20-yne-3,17-diol,

B. 19-nor-17α-pregna-1,3,5(10),9(11)-tetraen-20-yne-3,17-
diol, J. 1-methyl-19-nor-17α-pregna-1,3,5(10)-trien-20-yne-3,17-
diol (1-methyl-ethinylestradiol),

C. 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone),
K. 4-methyl-19-nor-17α-pregna-1,3,5(10)-trien-20-yne-3,17-
diol (4-methyl-ethinylestradiol),

D. estra-1,3,5(10)-triene-3,17β-diol (estradiol),

L. estra-1,3,5(10)-triene-3,17α-diol (17α-estradiol),

E. 19-nor-17α-pregna-1,3,5(10)-trien-20-yne-3,6α,17-triol M. 2-methyl-19-nor-17α-pregna-1,3,5(10)-trien-20-yne-3,17-
(6α-hydroxy-ethinylestradiol), diol (2-methyl-ethinylestradiol).

General Notices (1) apply to all monographs and other texts 2187
Ethionamide EUROPEAN PHARMACOPOEIA 8.0

01/2008:0141 cent) and at most 1 such spot is more intense than the spot
corrected 6.0 in the chromatogram obtained with reference solution (b)
(0.2 per cent).
ETHIONAMIDE Heavy metals (2.4.8). 1.0 g complies with test D for heavy
metals (20 ppm). Prepare the reference solution using 2 mL
Ethionamidum of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32). Not more than 0.5 per cent,
determined on 1.00 g by drying in an oven at 105 °C for 3 h.
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined
on 1.0 g.

C8H10N2S Mr 166.2 ASSAY

[536-33-4] Dissolve 0.150 g in 50 mL of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point
DEFINITION potentiometrically (2.2.20).
Ethionamide contains not less than 98.5 per cent and 1 mL of 0.1 M perchloric acid is equivalent to 16.62 mg of
not more than the equivalent of 101.0 per cent of C8H10N2S.
2-ethylpyridine-4-carbothioamide, calculated with reference
to the dried substance.
01/2008:0764
CHARACTERS
A yellow, crystalline powder or small, yellow crystals,
practically insoluble in water, soluble in methanol, sparingly
ETHOSUXIMIDE
soluble in alcohol.
Ethosuximidum
IDENTIFICATION
First identification : A, C.
Second identification : A, B, D.
A. Melting point (2.2.14) : 158 °C to 164 °C.
B. Dissolve 10.0 mg in methanol R and dilute to 100.0 mL
with the same solvent. Dilute 10.0 mL of the solution to C7H11NO2 Mr 141.2
100.0 mL with methanol R. Examined between 230 nm [77-67-8]
and 350 nm (2.2.25), the solution shows an absorption DEFINITION
maximum at 290 nm. The specific absorbance at the
maximum is 380 to 440. (RS)-3-Ethyl-3-methylpyrrolidine-2,5-dione.
C. Examine by infrared absorption spectrophotometry Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
(2.2.24), comparing with the spectrum obtained with CHARACTERS
ethionamide CRS.
Appearance : white or almost white, powder or waxy solid.
D. Dissolve about 10 mg in 5 mL of methanol R. Add 5 mL
Solubility : freely soluble in water, very soluble in ethanol
of silver nitrate solution R2. A dark-brown precipitate is
(96 per cent) and in methylene chloride.
formed.
It shows polymorphism (5.9).
TESTS
IDENTIFICATION
Appearance of solution. Dissolve 0.5 g in 10 mL of
methanol R, heating to about 50 °C. Allow to cool to room First identification : A, C.
temperature. The solution is not more opalescent than Second identification : A, B, D, E.
reference suspension II (2.2.1). A. Melting point (2.2.14) : 45 °C to 50 °C.
Acidity. Dissolve 2.0 g in 20 mL of methanol R, heating to B. Dissolve 50.0 mg in ethanol (96 per cent) R and dilute to
about 50 °C, and add 20 mL of water R. Cool slightly while 50.0 mL with the same solvent. Examined between 230 nm
shaking until crystallisation begins and then allow to cool and 300 nm (2.2.25), the solution shows an absorption
to room temperature. Add 60 mL of water R and 0.2 mL of maximum at 248 nm. The specific absorbance at the
cresol red solution R. Not more than 0.2 mL of 0.1 M sodium absorption maximum is 8 to 9.
hydroxide is required to change the colour of the indicator C. Infrared absorption spectrophotometry (2.2.24).
to red. Preparation : discs of potassium bromide R.
Related substances. Examine by thin-layer chromatography Comparison: ethosuximide CRS.
(2.2.27), using silica gel GF254 R as the coating substance.
If the spectra obtained in the solid state show differences,
Test solution. Dissolve 0.2 g of the substance to be examined dissolve the substance to be examined and the reference
in acetone R and dilute to 10 mL with the same solvent. substance separately in methylene chloride R, evaporate to
Reference solution (a). Dilute 0.5 mL of the test solution to dryness and record new spectra using the residues.
100 mL with acetone R. D. Dissolve 0.1 g in 3 mL of methanol R. Add 0.05 mL of
Reference solution (b). Dilute 0.2 mL of the test solution to a 100 g/L solution of cobalt chloride R and 0.05 mL of a
100 mL with acetone R. 100 g/L solution of calcium chloride R and add 0.1 mL
Apply separately to the plate 10 μL of each solution. Develop of dilute sodium hydroxide solution R. A purple colour
over a path of 15 cm using a mixture of 10 volumes of develops and no precipitate is formed.
methanol R and 90 volumes of chloroform R. Allow the plate to E. To about 10 mg add 10 mg of resorcinol R and 0.2 mL of
dry in air. Examine in ultraviolet light at 254 nm. Any spot in sulfuric acid R. Heat at 140 °C for 5 min and cool. Add
the chromatogram obtained with the test solution, apart from 5 mL of water R and 2 mL of concentrated ammonia R1.
the principal spot, is not more intense than the spot in the A brown colour is produced. Add about 100 mL of water R.
chromatogram obtained with reference solution (a) (0.5 per A green fluorescence is produced.

2188 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ethosuximide

TESTS Detection : flame ionisation.

Solution S. Dissolve 2.5 g in water R and dilute to 25 mL with Injection : 1 μL.
the same solvent. Run time : 1.5 times the retention time of ethosuximide.
Appearance of solution. Solution S is clear (2.2.1) and Relative retention with reference to the internal standard
colourless (2.2.2, Method II). (retention time = about 8 min) : impurity A = about 0.7 ;
Cyanide. Liquid chromatography (2.2.29). ethosuximide = about 1.1.
Test solution. Dissolve 0.50 g of the substance to be examined System suitability : reference solution (b) :
in water R and dilute to 10.0 mL with the same solvent. – resolution : minimum 5 between the peaks due to the
Reference solution (a). Dissolve 0.125 g of potassium cyanide R internal standard and ethosuximide.
in water R and dilute to 50.0 mL with the same solvent. Dilute Limits :
1.0 mL of the solution to 100.0 mL with water R. Dilute 0.5 mL – impurity A : calculate the ratio (R) of the area of the peak
of this solution to 10.0 mL with water R. due to impurity A to the area of the peak due to the internal
Reference solution (b). Dissolve 0.50 g of the substance to be standard from the chromatogram obtained with reference
examined in water R, add 0.5 mL of reference solution (a) and solution (a) ; from the chromatogram obtained with the
dilute to 10.0 mL with water R. test solution, calculate the ratio of the area of any peak due
to impurity A to the area of the peak due to the internal
Column :
standard : this ratio is not greater than R (0.1 per cent) ;
– size : l = 0.075 m, Ø = 7.5 mm,
– any other impurity : calculate the ratio (R) of half the area
– stationary phase : spherical weak anion-exchange resin R of the peak due to ethosuximide to the area of the peak due
(10 μm). to the internal standard from the chromatogram obtained
Mobile phase : dissolve 2.1 g of lithium hydroxide R and 85 mg with reference solution (b) ; from the chromatogram
of sodium edetate R in water for chromatography R and dilute obtained with the test solution, calculate the ratio of the
to 1000.0 mL with the same solvent. area of any peak, apart from the principal peak and the
Flow rate : 2.0 mL/min. peaks due to impurity A and to the internal standard, to
Detection : electrochemical detector (direct amperometry) the area of the peak due to the internal standard : this ratio
with a silver working electrode, a silver-silver chloride is not greater than R (0.1 per cent) ;
reference electrode, held at + 0.05 V oxidation potential, and a – total : calculate the ratio (R) of the area of the peak due to
detector sensitivity of 20 nA full scale. ethosuximide to the area of the peak due to the internal
Injection : 20 μL of the test solution and reference solution (b). standard from the chromatogram obtained with reference
solution (b) ; from the chromatogram obtained with the
System suitability : reference solution (b) :
test solution, calculate the ratio of the sum of the areas of
– peak-to-valley ratio : minimum 3, where Hp = height above any peaks, apart from the principal peak and the peak due
the baseline of the peak due to cyanide and Hv = height to the internal standard, to the area of the peak due to the
above the baseline of the lowest point of the curve internal standard : this ratio is not greater than R (0.2 per
separating this peak from the peak due to ethosuximide. cent) ;
Limit : – disregard limit : calculate the ratio (R) of 0.25 times the area
– cyanide : not more than 0.5 times the height of the of the peak due to impurity A to the area of the peak due
corresponding peak in the chromatogram obtained with to the internal standard from the chromatogram obtained
reference solution (b) (0.5 ppm). with reference solution (a) ; from the chromatogram
Related substances. Gas chromatography (2.2.28). obtained with the test solution, calculate the ratio of the
Internal standard solution. Dissolve 20 mg of myristyl area of any peak, apart from the principal peak and the
alcohol R in anhydrous ethanol R and dilute to 10.0 mL with peak due to the internal standard, to the area of the peak
the same solvent. due to the internal standard : disregard any peak which has
a ratio less than R (0.025 per cent).
Test solution. Dissolve 1.00 g of the substance to be examined
in anhydrous ethanol R add 1.0 mL of the internal standard Heavy metals (2.4.8) : maximum 10 ppm.
solution and dilute to 20.0 mL with anhydrous ethanol R. 12 mL of solution S complies with test A. Prepare the reference
Reference solution (a). Dissolve 10.0 mg of ethosuximide solution using lead standard solution (1 ppm Pb) R.
impurity A CRS in anhydrous ethanol R and dilute to 5.0 mL Water (2.5.12) : maximum 0.5 per cent, determined on 1.00 g.
with the same solvent. To 0.5 mL of the solution add 1.0 mL Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
of the internal standard solution and dilute to 20.0 mL with 1.0 g.
anhydrous ethanol R.
Reference solution (b). Dissolve 0.500 g of the substance to be ASSAY
examined in anhydrous ethanol R and dilute to 10.0 mL with Dissolve 0.120 g in 20 mL of dimethylformamide R and
the same solvent. Dilute 1.0 mL of the solution to 50.0 mL carry out a potentiometric titration (2.2.20) using 0.1 M
with anhydrous ethanol R. To 2.0 mL of this solution add tetrabutylammonium hydroxide. Protect the solution from
1.0 mL of the internal standard solution and dilute to 20.0 mL atmospheric carbon dioxide throughout the titration. Carry
with anhydrous ethanol R. out a blank titration.
Column : 1 mL of 0.1 M tetrabutylammonium hydroxide is equivalent to
– material : fused silica, 14.12 mg of C7H11NO2.
– size : l = 30 m, Ø = 0.25 mm, STORAGE
– stationary phase : poly(cyanopropyl)(phenylmethyl)silox- Protected from light.
ane R (film thickness 0.25 μm).
Carrier gas : helium for chromatography R. IMPURITIES
Flow rate : 1 mL/min. Specified impurities : A.
Split ratio : 1:67.
Temperature :
– column : 175 °C,
– injection port and detector : 240 °C. A. (2RS)-2-ethyl-2-methylbutanedioic acid.

General Notices (1) apply to all monographs and other texts 2189
Ethyl acetate EUROPEAN PHARMACOPOEIA 8.0

01/2008:0899 Injection : 1 μL.

Limit :
ETHYL ACETATE – total : not more than 0.2 per cent of the area of the principal
peak.
Ethylis acetas Residue on evaporation : maximum 30 ppm.
Evaporate 100.0 g to dryness on a water-bath and dry in an
oven at 100-105 °C. The residue weighs not more than 3 mg.
Water (2.5.12) : maximum 0.1 per cent, determined on
10.0 mL.
C 4H 8O 2 Mr 88.1
[141-78-6] STORAGE
Protected from light, at a temperature not exceeding 30 °C.
DEFINITION
Ethyl ethanoate. IMPURITIES
CHARACTERS
Appearance : clear, colourless, volatile liquid.
Solubility : soluble in water, miscible with acetone, with
ethanol (96 per cent) and with methylene chloride. A. methyl ethanoate (methyl acetate),

IDENTIFICATION
First identification : B. B. ethanol,
Second identification : A, C, D.
A. Boiling point (2.2.12) : 76 °C to 78 °C.
B. Infrared absorption spectrophotometry (2.2.24). C. methanol.
Comparison : Ph. Eur. reference spectrum of ethyl acetate.
C. It gives the reaction of acetyl (2.3.1).
D. It gives the reaction of esters (2.3.1). 01/2008:1319
TESTS
Appearance of solution. The solution is clear (2.2.1) and ETHYL OLEATE
colourless (2.2.2, Method II).
Mix 1 mL of the substance to be examined and 15 mL of Ethylis oleas
water R.
DEFINITION
Acidity. To 10 mL of ethanol (96 per cent) R add 0.1 mL of
Mixture consisting of the ethyl esters of fatty acids, mainly
phenolphthalein solution R and 0.01 M sodium hydroxide until
oleic (cis-9-octadecenoic) acid.
the colour changes to pink. Add 5.5 mL of the substance to
be examined and 0.25 mL of 0.02 M sodium hydroxide. The A suitable antioxidant may be added.
solution remains pink for not less than 15 s.
CHARACTERS
Relative density (2.2.5) : 0.898 to 0.902.
Appearance : clear, pale yellow or colourless liquid.
Refractive index (2.2.6) : 1.370 to 1.373.
Solubility : practically insoluble in water, miscible with
Reaction with sulfuric acid. Carefully add 2 mL to 10 mL ethanol (96 per cent), with methylene chloride and with light
of sulfuric acid R. After 15 min, the interface between the petroleum (bp : 40-60 °C).
2 liquids is not coloured.
Related substances. Gas chromatography (2.2.28). IDENTIFICATION
Test solution. The substance to be examined. A. Relative density (see Tests).
Column : B. Saponification value (see Tests).
– material : glass ; C. Oleic acid (see Tests).
– size : l = 2 m, Ø = 2 mm ; TESTS
– stationary phase : ethylvinylbenzene-divinylbenzene Relative density (2.2.5) : 0.866 to 0.874.
copolymer R (136-173 μm).
Acid value (2.5.1) : maximum 0.5, determined on 10.0 g.
Carrier gas : nitrogen for chromatography R.
Iodine value (2.5.4, Method A) : 75 to 90.
Flow rate : 30 mL/min.
Peroxide value (2.5.5, Method A) : maximum 10.0.
Temperature :
Saponification value (2.5.6) : 177 to 188, determined on 2.0 g.
Time Temperature
(min) (°C)
Oleic acid (2.4.22, Method A) : minimum 60.0 per cent in the
0 - 18.8 90 → 240
fatty acid fraction of the substance to be examined.
Column
Water (2.5.12) : maximum 1.0 per cent, determined on 1.00 g.
18.8 - 26.8 240
Total ash (2.4.16) : maximum 0.1 per cent, determined on
Injection port 240 2.0 g.
Detector 240
STORAGE
Detection : flame ionisation. Protected from light.

2190 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ethyl parahydroxybenzoate

07/2010:0900 Related substances. Liquid chromatography (2.2.29).

Test solution. Dissolve 50.0 mg of the substance to be
ETHYL PARAHYDROXYBENZOATE examined in 2.5 mL of methanol R and dilute to 50.0 mL with
the mobile phase. Dilute 10.0 mL of this solution to 100.0 mL
Ethylis parahydroxybenzoas with the mobile phase.
Reference solution (a). Dissolve 5 mg of 4-hydroxybenzoic
acid R (impurity A), 5 mg of methyl parahydroxybenzoate R
(impurity B) and 5 mg of the substance to be examined in the
mobile phase and dilute to 100.0 mL with the mobile phase.
Dilute 1.0 mL of this solution to 10.0 mL with the mobile
phase.
C9H10O3 Mr 166.2
[120-47-8] Reference solution (b). Dissolve 50.0 mg of ethyl
parahydroxybenzoate CRS in 2.5 mL of methanol R and dilute
DEFINITION to 50.0 mL with the mobile phase. Dilute 10.0 mL of this
Ethyl 4-hydroxybenzoate. solution to 100.0 mL with the mobile phase.
Content : 98.0 per cent to 102.0 per cent. Reference solution (c). Dilute 1.0 mL of the test solution to
20.0 mL with the mobile phase. Dilute 1.0 mL of this solution
CHARACTERS to 10.0 mL with the mobile phase.
Appearance : white or almost white, crystalline powder or Column :
colourless crystals. – size : l = 0.15 m, Ø = 4.6 mm ;
Solubility : very slightly soluble in water, freely soluble in – stationary phase : end-capped octadecylsilyl silica gel for
ethanol (96 per cent) and in methanol. chromatography R (5 μm).
IDENTIFICATION Mobile phase : 6.8 g/L solution of potassium dihydrogen
First identification : A, B. phosphate R, methanol R (35:65 V/V).
Second identification : A, C. Flow rate : 1.3 mL/min.
A. Melting point (2.2.14) : 115 °C to 118 °C. Detection : spectrophotometer at 272 nm.
B. Infrared absorption spectrophotometry (2.2.24). Injection : 10 μL of the test solution and reference solutions (a)
Comparison : ethyl parahydroxybenzoate CRS. and (c).
C. Thin-layer chromatography (2.2.27). Run time : 4 times the retention time of ethyl
parahydroxybenzoate.
Test solution (a). Dissolve 0.10 g of the substance to be
examined in acetone R and dilute to 10 mL with the same Relative retention with reference to ethyl parahydroxybenzoate
solvent. (retention time = about 3.0 min) : impurity A = about 0.5 ;
impurity B = about 0.8.
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL
with acetone R. System suitability : reference solution (a) :
Reference solution (a). Dissolve 10 mg of ethyl – resolution : minimum 2.0 between the peaks due to
parahydroxybenzoate CRS in acetone R and dilute to 10 mL impurity B and ethyl parahydroxybenzoate.
with the same solvent. Limits :
Reference solution (b). Dissolve 10 mg of methyl – correction factor : for the calculation of content, multiply
parahydroxybenzoate R in 1 mL of test solution (a) and the peak area of impurity A by 1.4 ;
dilute to 10 mL with acetone R. – impurity A : not more than the area of the principal peak
Plate : TLC octadecylsilyl silica gel F254 plate R. in the chromatogram obtained with reference solution (c)
Mobile phase : glacial acetic acid R, water R, methanol R (0.5 per cent) ;
(1:30:70 V/V/V). – unspecified impurities : for each impurity, not more than the
Application : 2 μL of test solution (b) and reference area of the principal peak in the chromatogram obtained
solutions (a) and (b). with reference solution (c) (0.5 per cent) ;
Development : over 2/3 of the plate. – total : not more than twice the area of the principal peak
in the chromatogram obtained with reference solution (c)
Drying : in air.
(1.0 per cent) ;
Detection : examine in ultraviolet light at 254 nm.
– disregard limit : 0.2 times the area of the principal peak in
System suitability: reference solution (b) : the chromatogram obtained with reference solution (c)
– the chromatogram shows 2 clearly separated principal (0.1 per cent).
spots. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Results : the principal spot in the chromatogram obtained 1.0 g.
with test solution (b) is similar in position and size to
the principal spot in the chromatogram obtained with ASSAY
reference solution (a). Liquid chromatography (2.2.29) as described in the test for
related substances with the following modification.
TESTS
Injection : test solution and reference solution (b).
Solution S. Dissolve 1.0 g in ethanol (96 per cent) R and dilute
to 10 mL with the same solvent. Calculate the percentage content of C9H10O3 from the declared
content of ethyl parahydroxybenzoate CRS.
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution BY6 (2.2.2, IMPURITIES
Method II). Specified impurities : A.
Acidity. To 2 mL of solution S add 3 mL of ethanol (96 per Other detectable impurities (the following substances would,
cent) R, 5 mL of carbon dioxide-free water R and 0.1 mL of if present at a sufficient level, be detected by one or other of
bromocresol green solution R. Not more than 0.1 mL of 0.1 M the tests in the monograph. They are limited by the general
sodium hydroxide is required to change the colour of the acceptance criterion for other/unspecified impurities and/or
indicator to blue. by the general monograph Substances for pharmaceutical use

General Notices (1) apply to all monographs and other texts 2191
Ethylcellulose EUROPEAN PHARMACOPOEIA 8.0

(2034). It is therefore not necessary to identify these impurities 20 g of ethanol (96 per cent) R and 80 g of toluene R until the
for demonstration of compliance. See also 5.10. Control of substance is dissolved. Determine the viscosity in mPa·s at
impurities in substances for pharmaceutical use) : B, C, D. 25 °C using a capillary viscometer.
Acetaldehyde : maximum 100 ppm.
Introduce 3.0 g into a 250 mL conical flask with a ground-glass
stopper, add 10 mL of water R and stir mechanically for
1 h. Allow to stand for 24 h, filter and dilute the filtrate to
100.0 mL with water R. Transfer 5.0 mL of the filtrate to
A. 4-hydroxybenzoic acid, a 25 mL volumetric flask, add 5 mL of a 0.5 g/L solution
of methylbenzothiazolone hydrazone hydrochloride R and
heat in a water-bath at 60 °C for 5 min. Add 2 mL of ferric
chloride-sulfamic acid reagent R and heat again in a water-bath
at 60 °C for 5 min. Cool and dilute to 25.0 mL with water R.
The solution is not more intensely coloured than a standard
B. methyl 4-hydroxybenzoate (methyl parahydroxybenzoate), prepared at the same time and in the same manner using
instead of the 5.0 mL of filtrate, 5.0 mL of a reference solution
prepared by diluting 3.0 mL of acetaldehyde standard solution
(100 ppm C2H4O) R1 to 100.0 mL with water R.
Chlorides (2.4.4) : maximum 0.1 per cent.
Disperse 0.250 g in 50 mL of water R, heat to boiling and
C. propyl 4-hydroxybenzoate (propyl parahydroxybenzoate), allow to cool, shaking occasionally. Filter and discard the first
10 mL of the filtrate. Dilute 10 mL of the filtrate to 15 mL
with water R.
♦Heavy metals (2.4.8) : maximum 20 ppm.
1.0 g complies with test C. Prepare the reference solution using
D. butyl 4-hydroxybenzoate (butyl parahydroxybenzoate). 2 mL of lead standard solution (10 ppm Pb) R.♦
Loss on drying (2.2.32) : maximum 3.0 per cent, determined
on 1.000 g by drying in an oven at 105 °C for 2 h.
01/2014:0822
Sulfated ash (2.4.14) : maximum 0.5 per cent, determined on
ETHYLCELLULOSE (1) 1.0 g.
ASSAY
Ethylcellulosum Gas chromatography (2.2.28).
DEFINITION CAUTION : hydriodic acid and its reaction by-products are
highly toxic. Perform all steps for preparation of the test and
Partly O-ethylated cellulose. reference solutions in a fume cupboard.
Content : 44.0 per cent to 51.0 per cent of ethoxy (-OC2H5) Internal standard solution. Dilute 120 μL of toluene R to
groups (dried substance). 10 mL with o-xylene R.
♦CHARACTERS Test solution. Transfer 50.0 mg of the substance to be
Appearance : white or yellowish-white powder or granular examined, 50.0 mg of adipic acid R and 2.0 mL of the internal
powder, odourless or almost odourless. standard solution into a suitable 5 mL thick-walled reaction
vial with a pressure-tight septum-type closure. Cautiously add
Solubility : practically insoluble in water, soluble in methylene
2.0 mL of hydriodic acid R, immediately close the vial tightly
chloride and in a mixture of 20 g of ethanol (96 per cent)
and weigh the contents and the vial accurately. Shake the vial
and 80 g of toluene, slightly soluble in ethyl acetate and in
for 30 s, heat to 125 °C for 10 min, allow to cool for 2 min,
methanol, practically insoluble in glycerol (85 per cent)
shake again for 30 s and heat to 125 °C for 10 min. Afterwards
and in propylene glycol. The solutions may show a slight
allow to cool for 2 min and repeat shaking and heating for a
opalescence.♦
3rd time. Allow the vial to cool for 45 min and reweigh. If the
IDENTIFICATION loss is greater than 10 mg, discard the mixture and prepare
A. Infrared absorption spectrophotometry (2.2.24). another. Use the upper layer.
Comparison : ethylcellulose CRS. Reference solution. Transfer 100.0 mg of adipic acid R, 4.0 mL
◊B. It complies with the limits of the assay.◊
of the internal standard solution and 4.0 mL of hydriodic
acid R into a suitable 10 mL thick-walled reaction vial with a
TESTS pressure-tight septum-type closure. Close the vial tightly and
weigh the vial and contents accurately. Afterwards inject 50 μL
Acidity or alkalinity. To 0.5 g add 25 mL of carbon of iodoethane R through the septum with a syringe, weigh the
dioxide-free water R and shake for 15 min. Filter through a vial again and calculate the mass of iodoethane added, by
sintered-glass filter (40) (2.1.2). To 10 mL of the solution add difference. Shake well and allow the layers to separate. Use
0.1 mL of phenolphthalein solution R and 0.5 mL of 0.01 M the upper layer.
sodium hydroxide. The solution is pink. To 10 mL of the
solution add 0.1 mL of methyl red solution R and 0.5 mL of Column :
0.01 M hydrochloric acid. The solution is red. – material : stainless steel ;
Viscosity (2.2.9) : 80.0 per cent to 120.0 per cent of that stated – size : l = 5.0 m, Ø = 2 mm ;
on the label for a nominal viscosity greater than 6 mPa·s ; – stationary phase : diatomaceous earth for gas
75.0 per cent to 140.0 per cent of that stated on the label for a chromatography R (150-180 μm) impregnated with 3 per
nominal viscosity not greater than 6 mPa·s. cent m/m of poly(dimethyl)siloxane R.
Shake a quantity of the substance to be examined equivalent Carrier gas : nitrogen for chromatography R.
to 5.00 g of the dried substance with 95 g of a mixture of Flow rate : 15 mL/min.
(1) This monograph has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.

2192 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ethylenediamine

Temperature : Free ethylene glycol : maximum 5.0 per cent, determined as

– column : 80 °C ; prescribed under Assay.
– injection port and detector : 200 °C. Total ash (2.4.16) : maximum 0.1 per cent, determined on
Detection : flame ionisation. 1.0 g.
Injection : 1 μL. ASSAY
Relative retention with reference to toluene : iodoethane = about Size-exclusion chromatography (2.2.30).
0.6 ; o-xylene = about 2.3. Test solution. Into a 15 mL flask, weigh about 0.2 g (m), to the
System suitability: reference solution : nearest 0.1 mg. Add 5.0 mL of tetrahydrofuran R and shake
– resolution : minimum 2.0 between the peaks due to to dissolve. Heat gently, if necessary. Reweigh the flask and
iodoethane and toluene. calculate the total mass of solvent and substance (M).
Calculate the percentage content of ethoxy groups using the Reference solutions. Into four 15 mL flasks, weigh, to the
following expression : nearest 0.1 mg, about 2.5 mg, 5.0 mg, 10.0 mg and 20.0 mg
of ethylene glycol R. Add 5.0 mL of tetrahydrofuran R and
shake to dissolve. Weigh the flasks again and calculate the
concentration of ethylene glycol in milligrams per gram for
each reference solution.
Q1 = ratio of iodoethane peak area to toluene peak Column :
area in the chromatogram obtained with the test
solution ; – size : l = 0.6 m, Ø = 7 mm,
Q2 = ratio of iodoethane peak area to toluene peak area – stationary phase : styrene-divinylbenzene copolymer R
in the chromatogram obtained with the reference (particle diameter 5 μm and pore size 10 nm).
solution ; Mobile phase : tetrahydrofuran R.
m1 = mass of the substance to be examined used in the Flow rate : 1 mL/min.
test solution, in milligrams ; Detection : differential refractometer.
m2 = mass of iodoethane used in the reference solution, Injection : 40 μL.
in milligrams ; Relative retention with reference to ethylene glycol :
d = percentage loss on drying. diesters = about 0.76, monoesters = about 0.83.
Limits :
LABELLING – free ethylene glycol : from the calibration curve obtained
The label states the nominal viscosity in millipascal seconds with the reference solutions, determine the concentration
for a 5 per cent m/m solution. (C) in milligrams per gram in the test solution and calculate
the percentage content in the substance to be examined
using the following expression :
01/2008:1421

ETHYLENE GLYCOL
MONOPALMITOSTEARATE – monoesters : calculate the percentage content of monoesters
using the following expression :
Ethylenglycoli monopalmitostearas
DEFINITION
Mixture of ethylene glycol mono- and diesters of stearic A = area of the peak due to the monoesters,
(octadecanoic) and palmitic (hexadecanoic) acids, produced
from the condensation of ethylene glycol and stearic acid 50 B = area of the peak due to the diesters,
of vegetable or animal origin (see Stearic acid (1474)). D = percentage content of free ethylene glycol
Content : minimum of 50.0 per cent of monoesters. + percentage content of free fatty acids which may
be determined using the following expression :
CHARACTERS
Appearance : white or almost white, waxy solid.
IA = acid value.
Solubility : practically insoluble in water, soluble in acetone
and in hot alcohol.
STORAGE
IDENTIFICATION Protected from light.
A. Melting point (see Tests).
B. Composition of fatty acids (see Tests). 01/2008:0716
C. It complies with the assay (monoesters content).
TESTS ETHYLENEDIAMINE
Melting point (2.2.15) : 54 °C to 60 °C.
Ethylendiaminum
Acid value (2.5.1) : maximum 3.0, determined on 10.0 g.
Iodine value (2.5.4) : maximum 3.0.
Saponification value (2.5.6) : 170 to 195, determined on 2.0 g.
C 2H 8N2 Mr 60.1
Composition of fatty acids (2.4.22, Method A). The fatty acid [107-15-3]
fraction has the following composition :
– stearic acid : 40.0 per cent to 60.0 per cent, DEFINITION
– sum of contents of palmitic acid and stearic acid : minimum Ethane-1,2-diamine.
90.0 per cent. Content : 98.0 per cent to 101.0 per cent.

General Notices (1) apply to all monographs and other texts 2193
Ethylmorphine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

CHARACTERS 01/2008:0491
Appearance : clear, colourless or slightly yellow liquid,
hygroscopic. ETHYLMORPHINE HYDROCHLORIDE
Solubility : miscible with water and with anhydrous ethanol. Ethylmorphini hydrochloridum
On exposure to air, white fumes are evolved. On heating, it
evaporates completely.

IDENTIFICATION
A. Relative density (2.2.5) : 0.895 to 0.905.
B. Boiling point (2.2.12) : 116 °C to 118 °C.
C19H24ClNO3,2H2O Mr 385.9
C. To 0.2 mL add 0.5 mL of acetic anhydride R. Boil. A
crystalline mass forms after cooling, which dissolves in DEFINITION
5 mL of 2-propanol R with heating. Cool the solution and 7,8-Didehydro-4,5α-epoxy-3-ethoxy-17-methylmorphinan-
add 5 mL of ether R. If necessary, initiate crystallisation by 6α-ol hydrochloride dihydrate.
scratching the walls of the test-tube with a glass rod. Filter Content : 99.0 per cent to 101.0 per cent (anhydrous substance).
through a sintered-glass filter (2.1.2), wash with several
portions of ether R and dry at 100-105 °C. The residue CHARACTERS
melts (2.2.14) at 173 °C to 177 °C. Appearance : white or almost white, crystalline powder.
Solubility : soluble in water and in alcohol, insoluble in
TESTS cyclohexane.
Solution S. Mix 10 g with carbon dioxide-free water R and IDENTIFICATION
dilute to 100 mL with the same solvent. First identification : A, D.
Appearance of solution. Solution S is clear (2.2.1) and not Second identification : B, C, D.
more intensely coloured than the reference solution BY6 A. Infrared absorption spectrophotometry (2.2.24).
(2.2.2, Method II).
Comparison: Ph. Eur. reference spectrum of ethylmorphine
Carbonate. A mixture of 4 mL of solution S and 6 mL of hydrochloride.
calcium hydroxide solution R is not more opalescent than B. In a test-tube, dissolve 0.5 g in 6 mL of water R and add
reference suspension II (2.2.1). 15 mL of 0.1 M sodium hydroxide. Scratch the wall of the
Chlorides (2.4.4): maximum 100 ppm. tube with a glass rod. A white, crystalline precipitate is
formed. Collect the precipitate, wash and dissolve in 20 mL
To 5 mL of solution S add 5 mL of dilute nitric acid R and of water R heated to 80 °C. Filter and cool in iced water.
dilute to 15 mL with water R. The crystals, after drying in vacuo for 12 h, melt (2.2.14)
Ammonia and other bases. Dissolve 1.2 g in 20 mL of ethanol at 85 °C to 89 °C.
(96 per cent) R and add, dropwise with stirring, 4.5 mL of C. To about 10 mg add 1 mL of sulfuric acid R and 0.05 mL
hydrochloric acid R. Evaporate to dryness on a water-bath, of ferric chloride solution R2. Heat on a water-bath. A blue
breaking up any resulting cake with a glass rod, and dry at colour develops. Add 0.05 mL of nitric acid R. The colour
100-105 °C for 1 h. 1 g of the residue is equivalent to 0.4518 g becomes red.
of C2H8N2. Calculate the percentage content of C2H8N2 : it D. Solution S (see Tests) gives reaction (a) of chlorides (2.3.1).
does not vary by more than 0.5 from the percentage content
determined in the assay. TESTS
Iron (2.4.9): maximum 10 ppm, determined on solution S. Solution S. Dissolve 0.500 g in carbon dioxide-free water R
and dilute to 25.0 mL with the same solvent.
Heavy metals (2.4.8) : maximum 10 ppm.
Appearance of solution. Solution S is clear (2.2.1) and not
12 mL of solution S complies with test A. Prepare the reference more intensely coloured than reference solution BY6 (2.2.2,
solution using lead standard solution (1 ppm Pb) R. Method II).
Residue on evaporation : maximum 0.3 per cent. Acidity or alkalinity. To 10 mL of solution S add 0.05 mL of
methyl red solution R and 0.2 mL of 0.02 M hydrochloric acid,
Evaporate 5.00 g to dryness on a water-bath and dry at the solution is red. Add 0.4 mL of 0.02 M sodium hydroxide,
100-105 °C for 1 h. The residue weighs a maximum of 15 mg. the solution becomes yellow.
Specific optical rotation (2.2.7) : − 102 to − 105 (anhydrous
ASSAY substance), determined on solution S.
Related substances. Liquid chromatography (2.2.29).
Place 25.0 mL of 1 M hydrochloric acid and 0.2 mL of methyl
red mixed solution R in a flask. Add 0.600 g of the substance Test solution. Dissolve 50.0 mg of the substance to be
to be examined. Titrate with 1 M sodium hydroxide until the examined in the mobile phase and dilute to 20.0 mL with the
colour changes from violet-red to green. mobile phase.
Reference solution (a). Dilute 1.0 mL of the test solution to
1 mL of 1 M hydrochloric acid is equivalent to 30.05 mg 25.0 mL with the mobile phase. Dilute 1.0 mL of this solution
of C2H8N2. to 20.0 mL with the mobile phase.
Reference solution (b). Dissolve 12.5 mg of codeine R in the
mobile phase and dilute to 5.0 mL with the mobile phase.
STORAGE
Reference solution (c). Dilute 0.5 mL of reference solution (b)
In an airtight container, protected from light. to 100.0 mL with the mobile phase.

2194 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Etidronate disodium

Reference solution (d). To 1.0 mL of the test solution, add

1.0 mL of reference solution (b) and dilute to 50.0 mL with
the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm,
– stationary phase : octylsilyl silica gel for chromatography R
(5 μm), A. R = R′ = C2H5 : 7,8-didehydro-4,5α-epoxy-3,6α-diethoxy-
– temperature : 30 °C. 17-methylmorphinan,

Mobile phase : add 1.25 g of sodium heptanesulfonate R to a B. R = R′ = H : 7,8-didehydro-4,5α-epoxy-17-

mixture of 12.5 mL of glacial acetic acid R and 5 mL of a 20 per methylmorphinan-3,6α-diol (morphine),
cent V/V solution of triethylamine R in a mixture of equal C. R = CH3, R′ = H : 7,8-didehydro-4,5α-epoxy-3-methoxy-
volumes of methanol R and water R. Dilute to 1000 mL with 17-methylmorphinan-6α-ol (codeine),
water R. To 550 mL of this solution add 450 mL of methanol R.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 230 nm.
Injection : 10 μL.
Run time : 4 times the retention time of ethylmorphine.
Relative retention with reference to ethylmorphine D. 7,8-didehydro-4,5α-epoxy-3-ethoxy-17-methylmorphinan-
(retention time = about 6.2 min) : impurity B = about 0.7 ; 6-one (ethylmorphinone).
impurity C = about 0.8 ; impurity D = about 1.3 ;
impurity A = about 2.5. 01/2008:1778
System suitability : reference solution (d) :
– resolution : minimum 5 between the peaks due to ETIDRONATE DISODIUM
ethylmorphine and impurity C.
Limits :
Dinatrii etidronas
– correction factor : for the calculation of content, multiply
the peak area of impurity D by 0.4,
– impurities A, B, D : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.2 per cent), C2H6Na2O7P2 Mr 250.0
– impurity C : not more than the area of the principal peak [7414-83-7]
in the chromatogram obtained with reference solution (c) DEFINITION
(0.5 per cent),
Disodium dihydrogen (1-hydroxyethylidene)bisphosphonate.
– any other impurity : for each impurity, not more than Content : 98.0 per cent to 102.0 per cent (anhydrous substance).
0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.1 per cent), CHARACTERS
– total of impurities other than C : not more than 2.5 times the Appearance : white or yellowish, hygroscopic powder.
area of the principal peak in the chromatogram obtained Solubility : freely soluble in water, practically insoluble in
with reference solution (a) (0.5 per cent), acetone and in ethanol (96 per cent).
– disregard limit : 0.25 times the area of the principal peak IDENTIFICATION
in the chromatogram obtained with reference solution (a) A. Infrared absorption spectrophotometry (2.2.24).
(0.05 per cent). Comparison: etidronate disodium CRS.
Water (2.5.12) : 8.0 per cent to 10.0 per cent, determined on The transmittance at about 2000 cm− 1 (5 μm) is not less
0.250 g. than 40 per cent without compensation.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on B. It gives reaction (a) of sodium (2.3.1).
1.0 g.
TESTS
ASSAY pH (2.2.3) : 4.2 to 5.2.
Dissolve 1.0 g in carbon dioxide-free water R and dilute to
Dissolve 0.300 g in a mixture of 5 mL of 0.01 M hydrochloric 100 mL with the same solvent.
acid and 30 mL of alcohol R. Carry out a potentiometric
titration (2.2.20), using 0.1 M sodium hydroxide. Read the Impurities A and B. Liquid chromatography (2.2.29).
volume added between the 2 points of inflexion. Test solution. Dissolve 20.0 mg of the substance to be
examined in water R and dilute to 10.0 mL with the same
1 mL of 0.1 M sodium hydroxide is equivalent to 34.99 mg
solvent.
of C19H24ClNO3.
Reference solution. To 2.0 mL of a 0.3 g/L solution of
phosphoric acid R add 2.0 mL of a 0.25 g/L solution of
STORAGE phosphorous acid R and dilute to 50.0 mL with water R.
Protected from light. Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
IMPURITIES – stationary phase : anion-exchange resin R (5 μm) ;
Specified impurities : A, B, C, D. – temperature : 35 °C.

General Notices (1) apply to all monographs and other texts 2195
Etilefrine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

Mobile phase : mix 0.2 mL of anhydrous formic acid R and Comparison: etilefrine hydrochloride CRS.
1000 mL of water R ; adjust to pH 3.5 with an 80 g/L solution C. Thin-layer chromatography (2.2.27).
of sodium hydroxide R. Prepare the solutions protected from bright light and develop
Flow rate : 1.0 mL/min. the chromatograms protected from light.
Detection : differential refractometer. Test solution. Dissolve 25 mg of the substance to be
Injection : 100 μL. examined in methanol R and dilute to 5 mL with the same
System suitability: reference solution : solvent.
– resolution : minimum 2.5 between the peaks due to Reference solution (a). Dissolve 25 mg of etilefrine
impurity A and impurity B. hydrochloride CRS in methanol R and dilute to 5 mL with
the same solvent.
Limits :
Reference solution (b). Dissolve 10 mg of phenylephrine
– impurities A, B : for each impurity, not more than the area
hydrochloride CRS in 2 mL of reference solution (a) and
of the corresponding peak in the chromatogram obtained
dilute to 10 mL with methanol R.
with the reference solution (0.5 per cent).
Plate : TLC silica gel plate R.
Heavy metals (2.4.8) : maximum 20 ppm.
Mobile phase : concentrated ammonia R, methanol R,
1.0 g complies with test F. Prepare the reference solution using methylene chloride R (5:25:70 V/V/V).
2 mL of lead standard solution (10 ppm Pb) R.
Application : 5 μL.
Water (2.5.32): maximum 5.0 per cent. Development : over a path of 15 cm.
Dissolve 50.0 mg in a mixture of equal volumes of anhydrous Drying : in a current of warm air.
acetic acid R and formamide R and dilute to 5.0 mL with the
same mixture of solvents. Use 1.0 mL of the solution. Detection : spray with a 10 g/L solution of potassium
permanganate R ; examine in daylight after 15 min.
ASSAY System suitability : reference solution (b) :
Dissolve 0.100 g in 2 mL of formic acid R and dilute to 50 mL – the chromatogram shows 2 clearly separated spots.
with glacial acetic acid R. Titrate with 0.1 M perchloric acid, Results : the principal spot in the chromatogram obtained
determining the end-point potentiometrically (2.2.20). with the test solution is similar in position, colour and size
1 mL of 0.1 M perchloric acid is equivalent to 12.50 mg to the principal spot in the chromatogram obtained with
of C2H6Na2O7P2. reference solution (a).
STORAGE D. To 0.2 mL of solution S (see Tests), add 1 mL of water R,
0.1 mL of copper sulfate solution R and 1 mL of strong
In an airtight container. sodium hydroxide solution R. A blue colour is produced.
IMPURITIES Add 2 mL of ether R and shake. The upper layer is
colourless.
Specified impurities : A, B.
E. Dilute 1 mL of solution S to 10 mL with water R. The
A. H3PO4 : phosphoric acid, solution gives reaction (a) of chlorides (2.3.1).
B. H3PO3 : phosphorous acid. TESTS
Solution S. Dissolve 2.50 g in carbon dioxide-free water R
01/2008:1205 prepared from distilled water R and dilute to 50.0 mL with
corrected 6.0 the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
ETILEFRINE HYDROCHLORIDE colourless (2.2.2, Method II).
Acidity or alkalinity. Dilute 4 mL of solution S to 10 mL
Etilefrini hydrochloridum with carbon dioxide-free water R. Add 0.1 mL of methyl
red solution R and 0.2 mL of 0.01 M sodium hydroxide.
The solution is yellow. Not more than 0.4 mL of 0.01 M
hydrochloric acid is required to change the colour of the
indicator to red.
Optical rotation (2.2.7): − 0.10° to + 0.10°, determined on
C10H16ClNO2 Mr 217.7 solution S.
[943-17-9]
Related substances. Liquid chromatography (2.2.29). Prepare
DEFINITION the solutions immediately before use.
(1RS)-2-(Ethylamino)-1-(3-hydroxyphenyl)ethanol Test solution. Dissolve 50.0 mg of the substance to be
hydrochloride. examined in water R and dilute to 50.0 mL with the same
Content : 98.0 per cent to 101.0 per cent (dried substance). solvent.
Reference solution (a). Dilute 1.0 mL of the test solution
CHARACTERS to 10.0 mL with water R. Dilute 1.0 mL of this solution to
Appearance : white or almost white, crystalline powder or 50.0 mL with water R.
colourless crystals. Reference solution (b). Dissolve 10.0 mg of etilefrine
Solubility : freely soluble in water, soluble in ethanol (96 per impurity A CRS in water R and dilute to 50.0 mL with the same
cent), practically insoluble in methylene chloride. solvent. Dilute 1.0 mL of the solution to 50.0 mL with water R.
Reference solution (c). To 10.0 mL of reference solution (a)
IDENTIFICATION add 5.0 mL of reference solution (b) and dilute to 20.0 mL
First identification : B, E. with water R.
Second identification : A, C, D, E. Column :
A. Melting point (2.2.14) : 118 °C to 122 °C. – size : l = 0.25 m, Ø = 4.6 mm,
B. Infrared absorption spectrophotometry (2.2.24). – stationary phase : octylsilyl silica gel for chromatography R
Preparation : discs of potassium chloride R. (5 μm).

2196 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Etodolac

Mobile phase : mix 35 volumes of acetonitrile R and 65 volumes IMPURITIES

of a 1.1 g/L solution of sodium laurilsulfate R adjusted to Specified impurities : A, B, C, D, E.
pH 2.3 with phosphoric acid R. Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
Flow rate : 1 mL/min.
the tests in the monograph. They are limited by the general
Detection : spectrophotometer at 220 nm. acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
Injection : 20 μL. use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Run time : 5 times the retention time of etilefrine. Control of impurities in substances for pharmaceutical use) : F.

Relative retention with reference to etilefrine (retention

time = about 9 min) : impurity E = about 0.5 ;
impurity C = about 0.8 ; impurity B = about 0.9 ;
impurity A = about 1.2 ; impurity F = about 1.7 ;
impurity D = about 4.5. A. R = H : 2-(ethylamino)-1-(3-hydroxyphenyl)ethanone
(etilefrone),
System suitability: reference solution (c) : D. R = CH2-C6H5 : 2-(benzylethylamino)-1-(3-
hydroxyphenyl)ethanone (benzyletilefrone),
– resolution : minimum 2.5 between the peaks due to
etilefrine and impurity A.

Limits :

– impurity A : not more than the area of the principal peak B. R = CH3 : (1RS)-1-(3-hydroxyphenyl)-2-(methylamino)-
in the chromatogram obtained with reference solution (b) ethanol (phenylephrine),
(0.4 per cent),
C. R = H : (1RS)-2-amino-1-(3-hydroxyphenyl)ethanol
– impurities B, C, D, E : for each impurity, not more than the (norfenefrine),
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.2 per cent),

– any other impurity : for each impurity, not more than

0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.1 per cent), E. 1-(3-hydroxyphenyl)ethanone (3-hydroxyacetophenone),

– sum of impurities other than A : not more than 5 times the

area of the principal peak in the chromatogram obtained
with reference solution (a) (1 per cent),

– disregard limit : 0.1 times the area of the principal peak in F. N-benzylethanamine (benzylethylamine).
the chromatogram obtained with reference solution (a)
(0.02 per cent) ; disregard any peak due to the solvent.
01/2008:1422
Sulfates (2.4.13) : maximum 200 ppm, determined on 15 mL
of solution S. ETODOLAC
Heavy metals (2.4.8) : maximum 20 ppm.
Etodolacum
Dissolve 2.0 g in 20 mL of water R. 12 mL of the solution
complies with limit test A. Prepare the reference solution
using lead standard solution (2 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g. C17H21NO3 Mr 287.4
[41340-25-4]
DEFINITION
ASSAY
2-[(1RS)-1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-
Dissolve 0.150 g in a mixture of 20 mL of anhydrous acetic yl]acetic acid.
acid R and 50 mL of acetic anhydride R. Titrate with 0.1 M Content : 98.0 per cent to 102.0 per cent (anhydrous substance).
perchloric acid, determining the end-point potentiometrically
(2.2.20). CHARACTERS
Appearance : white or almost white, crystalline powder.
1 mL of 0.1 M perchloric acid is equivalent to 21.77 mg Solubility : practically insoluble in water, freely soluble in
of C10H16ClNO2. acetone and in ethanol (96 per cent).
IDENTIFICATION
First identification : B.
STORAGE
Second identification : A, C.
In an airtight container, protected from light. A. Melting point (2.2.14) : 144 °C to 150 °C.

General Notices (1) apply to all monographs and other texts 2197
Etodolac EUROPEAN PHARMACOPOEIA 8.0

B. Infrared absorption spectrophotometry (2.2.24). Identification of impurities : use the chromatogram

supplied with etodolac for peak identification CRS and the
Comparison : etodolac CRS.
chromatogram obtained with reference solution (c) to identify
C. Thin-layer chromatography (2.2.27). the peaks due to impurities A, B, C, D, E, F, G, H, I and K.
Test solution. Dissolve 10 mg of the substance to be Relative retention with reference to etodolac (retention
examined in acetone R and dilute to 10 mL with the same time = about 16.7 min) : impurity A = about 0.68 ;
solvent. impurity B = about 0.83 ; impurity C = about 0.85 ;
Reference solution. Dissolve 10 mg of etodolac CRS in impurity H = about 1.09 ; impurity D = about 1.17 ;
acetone R and dilute to 10 mL with the same solvent. impurity G = about 1.19 ; impurity E = about 1.20 ;
impurity F = about 1.22 ; impurity I = about 1.50 ;
Plate : TLC silica gel GF254 plate R previously activated by impurity K = about 2.37.
heating at 105 °C for 1 h.
System suitability : reference solution (b) :
Place the plate in an unsaturated tank containing a mixture
of 20 volumes of a 25 g/L solution of ascorbic acid R and – resolution : minimum 5.0 between the peaks due to etodolac
80 volumes of methanol R. Allow the solution to ascend and impurity H.
1 cm above the line of application on the plate, remove the Limits :
plate and allow it to dry for at least 30 min.
– impurity C : not more than 5 times the area of the principal
Mobile phase : glacial acetic acid R, anhydrous ethanol R, peak in the chromatogram obtained with reference
toluene R (0.5:30:70 V/V/V). solution (a) (0.5 per cent) ;
Application : 10 μL.
– impurities A, B, D, E, F, G, H, I, K : for each impurity,
Development : 2/3 of the plate. not more than twice the area of the principal peak in the
Drying : in air. chromatogram obtained with reference solution (a) (0.2 per
cent) ;
Detection : examine in ultraviolet light at 254 nm.
– unspecified impurities : for each impurity, not more than the
Results : the principal spot in the chromatogram obtained area of the principal peak in the chromatogram obtained
with the test solution is similar in position and size to the with reference solution (a) (0.10 per cent) ;
principal spot in the chromatogram obtained with the
reference solution. – total : not more than 10 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(1.0 per cent) ;
TESTS
Related substances. Liquid chromatography (2.2.29). – disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
Test solution. Dissolve 20.0 mg of the substance to be (0.05 per cent).
examined in acetonitrile R1 and dilute to 50.0 mL with the
Chlorides : maximum 300 ppm.
same solvent.
Reference solution (a). Dilute 1.0 mL of the test solution to Dissolve 1.0 g of the substance to be examined in 60 mL of
50.0 mL with acetonitrile R1. Dilute 1.0 mL of this solution to methanol R, add 10 mL of water R and 20 mL of dilute nitric
20.0 mL with acetonitrile R1. acid R. Titrate with 0.01 M silver nitrate, determining the
end-point potentiometrically (2.2.20).
Reference solution (b). Dissolve 4 mg of etodolac
impurity H CRS in the test solution and dilute to 10 mL with 1 mL of 0.01 M silver nitrate is equivalent to 0.3545 mg of Cl.
the same solution. Dilute 0.5 mL of this solution to 50 mL Heavy metals (2.4.8) : maximum 10 ppm.
with acetonitrile R1.
2.0 g complies with test C. Prepare the reference solution using
Reference solution (c). Dissolve 4 mg of etodolac for peak 2 mL of lead standard solution (10 ppm Pb) R.
identification CRS (containing impurities A, B, C, D, E, F, G,
H, I and K) in 10 mL of acetonitrile R1. Water (2.5.12) : maximum 0.5 per cent, determined on 1.00 g.

Column : Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on

1.0 g.
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : end-capped butylsilyl silica gel for ASSAY
chromatography R (3.5 μm) ;
Dissolve 0.250 g in 60 mL of methanol R. Titrate with 0.1 M
– temperature : 35 °C. tetrabutylammonium hydroxide determining the end-point
Mobile phase : potentiometrically (2.2.20). Carry out a blank titration.
– mobile phase A : 0.77 g/L solution of ammonium acetate R ; 1 mL of 0.1 M tetrabutylammonium hydroxide is equivalent to
28.74 mg of C17H21NO3.
– mobile phase B : mobile phase A, acetonitrile R1 (10:90 V/V) ;
Time Mobile phase A Mobile phase B IMPURITIES
(min) (per cent V/V) (per cent V/V)
0 - 25 80 → 50 20 → 50
Specified impurities : A, B, C, D, E, F, G, H, I, K.

25 - 42 50 50
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
42 - 48 50 → 80 50 → 20 the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
Flow rate : 1 mL/min. by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
Detection : spectrophotometer at 225 nm.
for demonstration of compliance. See also 5.10. Control of
Injection : 5 μL. impurities in substances for pharmaceutical use): J, L.

2198 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Etofenamate

07/2012:1513

ETOFENAMATE
Etofenamatum
A. R1 = H, R2 = CH2-CH3 : 2-[(1RS)-1-ethyl-1,3,4,9-
tetrahydropyrano[3,4-b]indol-1-yl]acetic acid (8-desethyl
etodolac),

B. R1 = CH3, R2 = CH2-CH3 : 2-[(1RS)-1-ethyl-8-methyl-

1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl]acetic acid
(8-methyl etodolac),

C. R1 = CH2-CH3, R2 = CH3 : 2-[(1RS)-8-ethyl-1-methyl-

1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl]acetic acid C18H18F3NO4 Mr 369.4
(1-methyl etodolac), [30544-47-9]
D. R1 = CH(CH3)2, R2 = CH2-CH3 : 2-[(1RS)-1-ethyl-8-(1- DEFINITION
methylethyl)-1,3,4,9-tetrahydropyrano[3,4-b]indol-1- 2-(2-Hydroxyethoxy)ethyl 2-[[3-(trifluoromethyl)phenyl]-
yl]acetic acid (8-isopropyl etodolac), amino]benzoate.
Content : 98.5 per cent to 101.5 per cent (anhydrous substance).
E. R1 = CH2-CH2-CH3, R2 = CH2-CH3 : 2-[(1RS)-1-ethyl-8-
propyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl]acetic CHARACTERS
acid (8-propyl etodolac), Appearance : yellowish, viscous liquid.
F. R1 = CH2-CH3, R2 = CH(CH3)2 : 2-[(1RS)-8-ethyl-1-(1- Solubility : practically insoluble in water, miscible with ethanol
methylethyl)-1,3,4,9-tetrahydropyrano[3,4-b]indol-1- (96 per cent) and with ethyl acetate.
yl]acetic acid (1-isopropyl etodolac), IDENTIFICATION
G. R1 = CH2-CH3, R2 = CH2-CH2-CH3 : 2-[(1RS)-8-ethyl-1- Infrared absorption spectrophotometry (2.2.24).
propyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl]acetic Comparison: etofenamate CRS.
acid (1-propyl etodolac), Preparation : films.
TESTS
Appearance. The substance to be examined is clear (2.2.1)
and not more intensely coloured than reference solution GY1
(2.2.2, Method II).
H. 2-(7-ethyl-1H-indol-3-yl)ethanol, Impurity F. Gas chromatography (2.2.28).
Internal standard : tetradecane R.
Solution A. Dissolve 6.0 mg of tetradecane R in hexane R and
dilute to 10.0 mL with the same solvent.
Solution B. To 6.0 mg of diethylene glycol R in a 10 mL
volumetric flask add 3 mL of N-methyltrimethylsilyl-
trifluoroacetamide R and heat for 30 min at 50 °C. After
cooling dilute to 10.0 mL with N-methyltrimethylsilyl-
trifluoroacetamide R.
I. (3RS)-3-[7-ethyl-3-(2-hydroxyethyl)-1H-indol-2-yl]-3-(7- Test solution. To 0.200 g of the substance to be
ethyl-1H-indol-3-yl)pentanoic acid (etodolac dimer), examined add 10 μL of solution A. Add 2 mL of
N-methyltrimethylsilyl-trifluoroacetamide R and heat for
30 min at 50 °C.
Reference solution. To 2.0 mL of N-methyltrimethylsilyl-
trifluoroacetamide R add 10 μL of solution A and 10 μL of
solution B.
Column :
– size : l = 25 m, Ø = 0.20 mm ;
J. R = CH3 : (1RS)-1,8-diethyl-1-methyl-1,3,4,9- – stationary phase : poly(dimethyl)(diphenyl)siloxane R (film
tetrahydropyrano[3,4-b]indole (decarboxy etodolac), thickness 0.33 μm).
Carrier gas : hydrogen for chromatography R.
K. R = CH2-CO-O-CH3 : methyl 2-[(1RS)-1,8-diethyl-1,3,4,9-
tetrahydropyrano[3,4-b]indol-1-yl]acetate (etodolac Flow rate : 0.9 mL/min.
methyl ester), Temperature :
Time Temperature Rate
(min) (°C) (°C/min)
Column 0 - 13 60 → 150 7

13 - 19 150 → 300 25

19 - 34 300

Injection port 150

L. (EZ)-3-[7-ethyl-3-(2-hydroxyethyl)-1H-indol-2-yl]pent- Detector 300
3-enoic acid.

General Notices (1) apply to all monographs and other texts 2199
Etofenamate EUROPEAN PHARMACOPOEIA 8.0

Detection : flame ionisation. Limits :

Injection : 0.2 μL. – correction factors : for the calculation of content, multiply
the peak areas of the following impurities by the
Limit: corresponding correction factor : impurity A = 0.62 ;
impurity C = 0.45 ; impurity D = 0.77 ;
– impurity F : maximum 0.1 per cent.
– impurity D : not more than 2.5 times the area of the
Related substances. Liquid chromatography (2.2.29). principal peak in the chromatogram obtained with
Solvent mixture : water R, methanol R (40:60 V/V). reference solution (b) (0.5 per cent) ;
– impurity A : not more than 1.25 times the area of the
Test solution. Dissolve 50.0 mg of the substance to be principal peak in the chromatogram obtained with
examined in 30 mL of methanol R and dilute to 50.0 mL with reference solution (b) (0.25 per cent) ;
water R.
– impurities B, C, E : for each impurity, not more than the
Reference solution (a). Dissolve 10.0 mg of etofenamate area of the principal peak in the chromatogram obtained
impurity G CRS in methanol R and dilute to 20.0 mL with the with reference solution (b) (0.2 per cent) ;
same solvent. Dilute 0.2 mL of the solution to 50.0 mL with – impurity G : not more than the area of the principal peak
the solvent mixture. in the chromatogram obtained with reference solution (a)
Reference solution (b). Dilute 0.2 mL of the test solution to (0.2 per cent) ;
100.0 mL with the solvent mixture. – unspecified impurities : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram
Reference solution (c). To 5.0 mL of reference solution (a), add obtained with reference solution (b) (0.10 per cent) ;
5.0 mL of reference solution (b).
– total : not more than 6 times the area of the principal peak
Reference solution (d). Dissolve 10.0 mg of etofenamate for in the chromatogram obtained with reference solution (b)
peak identification CRS (containing impurities A, B, C, D (1.2 per cent) ;
and E) in 6.0 mL of methanol R and dilute to 10.0 mL with – disregard limit : 0.25 times the area of the principal peak
water R. in the chromatogram obtained with reference solution (b)
Column : (0.05 per cent).
Heavy metals (2.4.8) : maximum 10 ppm.
– size : l = 0.10 m, Ø = 4.0 mm ;
2.0 g complies with test C. Prepare the reference solution using
– stationary phase : end-capped octadecylsilyl silica gel for 2 mL of lead standard solution (10 ppm Pb) R.
chromatography R (3 μm) ;
Water (2.5.12) : maximum 0.5 per cent, determined on 1.00 g.
– temperature : 40 °C. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Mobile phase : 1.0 g.

– mobile phase A : dissolve 1.3 g of ammonium phosphate R ASSAY

and 4.0 g of tetrabutylammonium hydroxide R in 900 mL To 3.000 g add 20 mL of 2-propanol R and 20.0 mL of 1 M
of water R, adjust to pH 8.0 with dilute phosphoric acid R sodium hydroxide and heat under reflux for 2 h. Add 0.1 mL
and dilute to 1000 mL with water R ; of bromothymol blue solution R1. Titrate after cooling with
– mobile phase B : methanol R ; 1 M hydrochloric acid until the colour disappears. Carry out
a blank titration.
Time Mobile phase A Mobile phase B 1 mL of 1 M sodium hydroxide is equivalent to 0.3694 g of
(per cent V/V) (per cent V/V) C18H18F3NO4.
0 - 13 40 60
IMPURITIES
13 - 20 40 → 10 60 → 90
Specified impurities : A, B, C, D, E, F, G.
20 - 25 10 90

Flow rate : 1.2 mL/min.

Detection : spectrophotometer at 286 nm.
Injection : 20 μL.
Identification of impurities : use the chromatogram supplied
with etofenamate for peak identification CRS and the
chromatogram obtained with reference solution (d) to A. 2-[[3-(trifluoromethyl)phenyl]amino]benzoic acid
identify the peaks due to impurities A, B, C, D and E ; use the (flufenamic acid),
chromatogram obtained with reference solution (a) to identify
the peak due to impurity G.
Relative retention with reference to etofenamate
(retention time = about 13 min) : impurity A = about 0.2 ;
impurity C = about 0.7 ; impurity G = about 0.85 ;
impurity E = about 1.5 ; impurity B = about 1.6 ;
impurity D = about 1.7.
System suitability: reference solution (c) :
– resolution : minimum 2.3 between the peaks due to B. butyl 2-[[3-(trifluoromethyl)phenyl]amino]benzoate (butyl
impurity G and etofenamate. flufenamate),

2200 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Etomidate

B. Specific optical rotation (see Tests).

TESTS
Solution S. Dissolve 0.25 g in anhydrous ethanol R and dilute
to 25.0 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
C. N-phenyl-3-(trifluoromethyl)aniline, colourless (2.2.2, Method II).
Specific optical rotation (2.2.7) : + 67 to + 70 (dried
substance), determined on solution S.
Related substances. Liquid chromatography (2.2.29).
Solvent mixture : anhydrous ethanol R, water R (50:50 V/V).
Test solution. Dissolve 0.100 g of the substance to be examined
in the solvent mixture and dilute to 10.0 mL with the solvent
mixture.
D. 2,2′-oxybis(ethylene) bis[2-[[3-(trifluoromethyl)phenyl]- Reference solution (a). Dissolve 5.0 mg of etomidate CRS and
amino]benzoate], 5.0 mg of etomidate impurity B CRS in the solvent mixture,
then dilute to 250.0 mL with the solvent mixture.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 5.0 mL of this
solution to 25.0 mL with the solvent mixture.
Column :
– size : l = 0.1 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (3 μm).
E. 2-(2-butoxyethoxy)ethyl 2-[[3-(trifluoromethyl)phenyl]- Mobile phase :
amino]benzoate,
– mobile phase A : 5 g/L solution of ammonium carbonate R ;
– mobile phase B : acetonitrile R ;
F. 2,2′-oxydiethanol, Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-5 90 → 30 10 → 70

5-6 30 → 10 70 → 90

6 - 10 10 90

Flow rate : 2.0 mL/min.

G. 2-hydroxyethyl 2-[[3-(trifluoromethyl)phenyl]amino]- Detection : spectrophotometer at 235 nm.
benzoate.
Injection : 10 μL.
01/2008:1514 Retention time : impurity B = about 4.5 min ;
corrected 7.0 etomidate = about 5.0 min.
System suitability : reference solution (a) :
ETOMIDATE – resolution : minimum 5.0 between the peaks due to
impurity B and etomidate ; if necessary, adjust the
Etomidatum concentration of ammonium carbonate in the mobile phase
or the time programme of the linear gradient.
Limits :
– impurities A, B, C : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.2 per cent) ;
– total : not more than 1.5 times the area of the principal peak
C14H16N2O2 Mr 244.3 in the chromatogram obtained with reference solution (b)
[33125-97-2] (0.3 per cent) ;
DEFINITION – disregard limit : 0.25 times the area of the principal peak
Ethyl 1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate. in the chromatogram obtained with reference solution (b)
Content : 99.0 per cent to 101.0 per cent (dried substance). (0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
CHARACTERS on 1.000 g by drying in vacuo at 40 °C for 4 h.
Appearance : white or almost white powder. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Solubility : very slightly soluble in water, freely soluble in 1.0 g.
ethanol (96 per cent) and in methylene chloride.
mp : about 68 °C. ASSAY
Dissolve 0.200 g in 50 mL of a mixture of 1 volume of
IDENTIFICATION anhydrous acetic acid R and 7 volumes of methyl ethyl
A. Infrared absorption spectrophotometry (2.2.24). ketone R. Titrate with 0.1 M perchloric acid using 0.2 mL of
Comparison : etomidate CRS. naphtholbenzein solution R as indicator.

General Notices (1) apply to all monographs and other texts 2201
Etoposide EUROPEAN PHARMACOPOEIA 8.0

1 mL of 0.1 M perchloric acid is equivalent to 24.43 mg Plate : silica gel H R as the coating substance.
of C14H16N2O2. Mobile phase : water R, glacial acetic acid R, acetone R,
methylene chloride R (1.5:8:20:100 V/V/V/V).
STORAGE
Application : 5 μL as bands of 10 mm.
Protected from light.
Development : immediately, over 6/7 of the plate.
IMPURITIES Drying : in a current of warm air for 5 min.
Specified impurities : A, B, C. Detection : spray with a mixture of 1 volume of sulfuric
acid R and 9 volumes of ethanol (96 per cent) R and heat at
140 °C for 15 min. Cover the plate immediately with a glass
plate of the same size. Examine in daylight.
Results : the principal zone in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal zone in the chromatogram obtained with
A. R = H : 1-[(1RS)-1-phenylethyl]-1H-imidazole-5-carboxylic the reference solution.
acid, D. In a test-tube dissolve about 5 mg in 5 mL of glacial acetic
acid R and add 0.05 mL of ferric chloride solution R1. Mix
B. R = CH3 : methyl 1-[(1RS)-1-phenylethyl]-1H-imidazole-5- and cautiously add 2 mL of sulfuric acid R. Avoid mixing
carboxylate (metomidate), the 2 layers. Allow to stand for about 30 min ; a pink to
C. R = CH(CH3)2 : 1-methylethyl 1-[(1RS)-1-phenylethyl]- reddish-brown ring develops at the interface and the upper
1H-imidazole-5-carboxylate. layer is yellow.
TESTS
04/2011:0823 Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution Y6 or BY6
ETOPOSIDE (2.2.2, Method II).
Dissolve 0.6 g in a mixture of 1 volume of methanol R and
Etoposidum 9 volumes of methylene chloride R and dilute to 20 mL with
the same mixture of solvents.
Specific optical rotation (2.2.7) : − 106 to − 114 (anhydrous
substance).
Dissolve 50.0 mg in a mixture of 1 volume of methanol R and
9 volumes of methylene chloride R and dilute to 10.0 mL with
the same mixture of solvents.
Related substances. Liquid chromatography (2.2.29).
Solvent mixture : mobile phase A, mobile phase B (50:50 V/V).
Test solution (a). Dissolve 40 mg of the substance to be
C29H32O13 Mr 588.6 examined in the solvent mixture and dilute to 10.0 mL with
[33419-42-0] the solvent mixture.
Test solution (b). Dissolve 50.0 mg of the substance to be
DEFINITION examined in the solvent mixture and dilute to 50.0 mL with
(5R,5aR,8aR,9S)-9-[[4,6-O-[(R)-Ethylidene]-β-D- the solvent mixture.
glucopyranosyl]oxy]-5-(4-hydroxy-3,5-dimethoxyphenyl)- Reference solution (a). Dilute 1.0 mL of test solution (a) to
5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol- 100.0 mL with the solvent mixture. Dilute 1.0 mL of this
6(5aH)-one. solution to 10.0 mL with the solvent mixture.
Content : 98.0 per cent to 101.0 per cent (anhydrous substance). Reference solution (b). Dissolve 4 mg of etoposide for system
suitability CRS (containing impurities B, C, D, E, N and O) in
CHARACTERS 1.0 mL of the solvent mixture.
Appearance : white or almost white, crystalline powder, slightly Reference solution (c). Dissolve 50.0 mg of etoposide CRS in
hygroscopic. the solvent mixture and dilute to 50.0 mL with the solvent
Solubility : practically insoluble in water, sparingly soluble mixture.
in methanol, slightly soluble in ethanol 96 per cent and in Column :
methylene chloride.
– size : l = 0.125 m, Ø = 4.6 mm ;
IDENTIFICATION – stationary phase : octadecylsilyl silica gel for
First identification : A, B. chromatography R (5 μm) ;
Second identification : C, D. – temperature : 40 °C.
A. Specific optical rotation (see Tests). Mobile phase :
B. Infrared absorption spectrophotometry (2.2.24). – mobile phase A : anhydrous formic acid R, triethylamine R,
water R (1:1:998 V/V/V) ;
Comparison : etoposide CRS.
– mobile phase B : anhydrous formic acid R, triethylamine R,
C. Thin-layer chromatography (2.2.27). acetonitrile R (1:1:998 V/V/V) ;
Test solution. Dissolve 10 mg of the substance to be
Time Mobile phase A Mobile phase B
examined in a mixture of 1 volume of methanol R and
(min) (per cent V/V) (per cent V/V)
9 volumes of methylene chloride R and dilute to 2 mL with
the same mixture of solvents. 0 - 7 75 25

Reference solution. Dissolve 10 mg of etoposide CRS in 7 - 23 75 → 27 25 → 73

a mixture of 1 volume of methanol R and 9 volumes of
methylene chloride R and dilute to 2 mL with the same Flow rate : 1 mL/min.
mixture of solvents. Detection : spectrophotometer at 285 nm.

2202 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Etoposide

Injection : 10 μL of test solution (a) and reference solutions (a)

and (b).
Identification of impurities : use the chromatogram supplied
with etoposide for system suitability CRS and the chromatogram
obtained with reference solution (b) to identify the peaks due
to impurities B, C, D, E, N and O.
Relative retention with reference to etoposide (retention
time = about 5 min) : impurity D = about 0.4 ;
impurity E = about 0.8 ; impurity C = about 1.1 ;
impurity B = about 1.2 ; impurity N = about 3.1 ;
impurity O = about 4.2.
System suitability : reference solution (b) :
– peak-to-valley ratio : minimum 2.0, where Hp = height
above the baseline of the peak due to impurity C and
Hv = height above the baseline of the lowest point of the
curve separating this peak from the peak due to etoposide ; A. (5R,5aR,8aR,9S)-5-[4-[[(benzyloxy)carbonyl]oxy]-3,5-
and minimum 3.0, where Hp = height above the baseline dimethoxyphenyl]-9-[[4,6-O-[(R)-ethylidene]-β-D-
of the peak due to impurity B and Hv = height above the glucopyranosyl]oxy]-5,8,8a,9-tetrahydroisobenzofuro-
baseline of the lowest point of the curve separating this [5,6-f][1,3]benzodioxol-6(5aH)-one (4′-carbobenzoyl-
peak from the peak due to impurity C. oxyethylidene-lignan P),
Limits :
– correction factor : for the calculation of content, multiply
the peak area of impurity O by 1.7 ;
– impurities B, C, D, E, N : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent) ;
– impurity O : not more than 1.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (a) (0.15 per cent) ;
B. (5R,5aS,8aR,9S)-9-[[4,6-O-[(R)-ethylidene]-β-D-
– unspecified impurities : for each impurity, not more than the glucopyranosyl]oxy]-5-(4-hydroxy-3,5-dimethoxyphenyl)-
area of the principal peak in the chromatogram obtained 5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-
with reference solution (a) (0.10 per cent) ; 6(5aH)-one (picroethylidene-lignan P ; cis-etoposide),
– total : not more than 10 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(1.0 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.05 per cent) ; disregard any peak due to the solvent.
Heavy metals (2.4.8) : maximum 20 ppm.
0.5 g complies with test G. Prepare the reference solution
using 1 mL of lead standard solution (10 ppm Pb) R.
Water (2.5.12) : maximum 6.0 per cent, determined on 0.250 g. C. (5R,5aR,8aR,9S)-9-[[4,6-O-[(R)-ethylidene]-α-D-
glucopyranosyl]oxy]-5-(4-hydroxy-3,5-dimethoxyphenyl)-
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-
1.0 g. 6(5aH)-one (α-etoposide),
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications.
Injection : test solution (b) and reference solution (c).
System suitability :
– repeatability : maximum relative standard deviation of
1.0 per cent after 6 injections of reference solution (c).
Calculate the percentage content of C29H32O13 from the
declared content of etoposide CRS. D. (5R,5aR,8aR,9S)-9-(β-D-glucopyranosyloxy)-5-(4-hydroxy-
STORAGE 3,5-dimethoxyphenyl)-5,8,8a,9-tetrahydroisobenzofuro-
In an airtight container. [5,6-f][1,3]benzodioxol-6(5aH)-one (lignan P),

IMPURITIES
Specified impurities : B, C, D, E, N, O.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10. E. (5R,5aR,8aR,9S)-9-hydroxy-5-(4-hydroxy-3,5-dimethoxy-
Control of impurities in substances for pharmaceutical use): A, phenyl)-5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzo-
F, G, H, I, J, K, L, M, P, Q, R. dioxol-6(5aH)-one (4′-desmethylepipodophyllotoxin),

General Notices (1) apply to all monographs and other texts 2203
Etoposide EUROPEAN PHARMACOPOEIA 8.0

K. 9,9′-oxybis[(5R,5aR,8aR,9S)-5-(4-hydroxy-3,5-
F. (5R,5aR,8aR,9S)-9-[[4,6-O-[(R)-ethylidene]-β-D- dimethoxyphenyl)-5,8,8a,9-tetrahydroisobenzofuro-
glucopyranosyl]oxy]-5-[4-[(phenoxyacetyl)oxy]- [5,6-f][1,3]benzodioxol-6(5aH)-one] (di-4′-O-
3,5-dimethoxyphenyl]-5,8,8a,9-tetrahydroiso- desmethylepipodophyllotoxin),
benzofuro[5,6-f][1,3]benzodioxol-6(5aH)-one
(4′-phenoxyacetyletoposide),

L. (5R,5aR,8aR,9R)-9-hydroxy-5-(4-hydroxy-3,5-dimethoxy-
phenyl)-5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzo-
dioxol-6(5aH)-one (4′-O-desmethylpodophyllotoxin),
G. (5R,5aR,8aR,9S)-5-[4-[[(benzyloxy)carbonyl]oxy]-
3,5-dimethoxyphenyl]-9-[[4,6-O-[(R)-ethylidene]-
2,3-di-O-formyl-β-D-glucopyranosyl]oxy]-5,8,8a,9-
tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)-
one (4′-carbobenzoyloxydiformylethylidene-lignan P),

M. (5R,5aR,8aR,9R)-9-hydroxy-5-(3,4,5-trimethoxyphenyl)-
5,8,8a,9-tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-
6(5aH)-one (podophyllotoxin),

H. (5R,5aR,8aR,9S)-9-ethoxy-5-(4-hydroxy-3,5-
dimethoxyphenyl)-5,8,8a,9-tetrahydroisobenzofuro-[5,6-
f][1,3]benzodioxol-6(5aH)-one (4′-O-desmethyl-1-O-
ethylepipodophyllotoxin),

N. (5R,5aR,8aR,9S)-9-[[4,6-O-[(R)-ethylidene]-β-
D-glucopyranosyl]oxy]-5-[4-[[(5R,5aR,8aR,9S)-
5-(4-hydroxy-3,5-dimethoxyphenyl)-6-oxo-
I. (5R,5aR,8aR,9S)-9-[[4,6-O-[(R)-ethylidene]-β-D- 5,5a,6,8,8a,9-hexahydroisobenzofuro[5,6-
glucopyranosyl]oxy]-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9- f][1,3]benzodioxol-9-yl]oxy]3,5-dimethoxyphenyl]-
tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol-6(5aH)- 5,8,8a,9-tetrahydroisobenzofuro-[5,6-f][1,3]benzodioxol-
one (4-O-methylethylidene-lignan P), 6(5aH)-one.

J. (5R,5aR,8aR,9S)-5-(4-hydroxy-3,5-dimethoxyphenyl)- O. (5R,5aR,8aR,9S)-9-[[2,3-bis-O-(dichloroacetyl)-4,6-O-[(S)-
9-methoxy-5,8,8a,9-tetrahydroisobenzofuro-[5,6- ethylidene]-β-L-glucopyranosyl]oxy]-5-(4-hydroxy-3,5-
f][1,3]benzodioxol-6(5aH)-one (4′-O-desmethyl-1-O- dimethoxyphenyl)-5,8,8a,9-tetrahydroisobenzofuro[5,6-
methylepipodophyllotoxin), f][1,3]benzodioxol-6(5aH)-one,

2204 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Eugenol

B. Infrared absorption spectrophotometry (2.2.24).

Comparison: eugenol CRS.
C. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 50 μL of the substance to be
examined in ethanol (96 per cent) R and dilute to 25 mL
with the same solvent.
Reference solution. Dissolve 50 μL of eugenol CRS in ethanol
P. 9-hydroxy-5-(4-hydroxy-3,5-dimethoxyphenyl)isobenzo- (96 per cent) R and dilute to 25 mL with the same solvent.
furo[5,6-f][1,3]benzodioxol-6(8H)-one, Plate : TLC silica gel F254 plate R.
Mobile phase : ethyl acetate R, toluene R (10:90 V/V).
Application : 5 μL.
Development : over a path of 15 cm.
Drying : in a current of cold air.
Detection A : examine in ultraviolet light at 254 nm.
Results A : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the
Q. 5-(4-hydroxy-3,5-dimethoxyphenyl)isobenzofuro[5,6- reference solution.
f][1,3]benzodioxol-6(8H)-one, Detection B : spray with anisaldehyde solution R and heat at
100-105 °C for 10 min.
Results B : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
the reference solution.
D. Dissolve 0.05 mL in 2 mL of ethanol (96 per cent) R and add
0.1 mL of ferric chloride solution R1. A dark green colour is
produced which changes to yellowish-green within 10 min.
TESTS
Relative density (2.2.5) : 1.066 to 1.070.
Refractive index (2.2.6) : 1.540 to 1.542.
R. (5R,5aR,8aR,9S)-9-[[4,6-O-[(R)-ethylidene]-β-D-
glucopyranosyl]oxy]-5-[4-[[(5R,5aR,8aR,9R)-5-(4- Dimeric and oligomeric compounds. Dissolve 0.150 g in
hydroxy-3,5-dimethoxyphenyl)-6-oxo-5,5a,6,8,8a,9- anhydrous ethanol R and dilute to 100.0 mL with the same
hexahydroisobenzofuro[5,6-f][1,3]benzodioxol- solvent. The absorbance (2.2.25) of the solution at 330 nm is
9-yl]oxy]-3,5-dimethoxyphenyl]-5,8,8a,9- not greater than 0.25.
tetrahydroisobenzofuro[5,6-f][1,3]benzodioxol- Related substances. Gas chromatography (2.2.28) : use the
6(5aH)-one. normalisation procedure.
Test solution. Dissolve 1.00 g of the substance to be examined
01/2008:1100 in anhydrous ethanol R and dilute to 5.0 mL with the same
solvent.
Reference solution (a). Dilute 1.0 mL of the test solution to
EUGENOL 100.0 mL with anhydrous ethanol R.
Reference solution (b). Dissolve 50 mg of vanillin R
Eugenolum (impurity H) in 1 mL of the test solution and dilute to 5 mL
with anhydrous ethanol R.
Column :
– material : fused silica ;
– size : l = 30 m, Ø = 0.25 mm ;
C10H12O2 Mr 164.2
[97-53-0] – stationary phase : polymethylphenylsiloxane R (film
thickness 0.25 μm).
DEFINITION Carrier gas : helium for chromatography R.
2-Methoxy-4-(prop-2-enyl)phenol. Flow rate : 1 mL/min.
Split ratio : 1:40.
CHARACTERS
Temperature :
Appearance : colourless or pale yellow, clear liquid, darkening
on exposure to air. Time Temperature
(min) (°C)
It has a strong odour of clove.
Column 0-2 80
Solubility : practically insoluble in water, freely soluble in
ethanol (70 per cent V/V), practically insoluble in glycerol, 2 - 27 80 → 280
miscible with ethanol (96 per cent), with glacial acetic acid, 27 - 47 280
with methylene chloride and with fatty oils.
Injection port 250
IDENTIFICATION
Detector 280
First identification : B.
Second identification : A, C, D. Detection : flame ionisation.
A. Refractive index (see Tests). Injection : 1 μL.

General Notices (1) apply to all monographs and other texts 2205
Evening primrose oil, refined EUROPEAN PHARMACOPOEIA 8.0

System suitability : reference solution (b) : J. R1 = H, R2 = OCH3, R3 = CO-CH=CH2 :

– relative retention with reference to eugenol : 1-(4-hydroxy-3-methoxyphenyl)prop-2-enone,
impurity H = minimum 1.1. K. R1 = H, R2 = OCH3, R3 = CH=CH-CHO :
Limits : (E)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enal
– any impurity : for each impurity, maximum 0.5 per cent ; (trans-coniferyl aldehyde),
– sum of impurities with a relative retention greater than 2.0
with reference to eugenol : maximum 1.0 per cent ;
– total : maximum 3.0 per cent ;
– disregard limit : 0.05 times the area of the principal peak
in the chromatogram obtained with reference solution (a) L. 2-methoxy-4-[3-methyl-5-(prop-2-enyl)-2,3-
(0.05 per cent). dihydrobenzofuran-2-yl]phenol (dehydrodi-isoeugenol),
Hydrocarbons. Dissolve 1 mL in 5 mL of dilute sodium
hydroxide solution R and add 30 mL of water R in a stoppered
test-tube. Examined immediately, the solution is yellow and
clear (2.2.1).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
STORAGE M. 3,3′-dimethoxy-5,5′-bis(prop-2-enyl)biphenyl-2,2′-diol
In a well-filled container, protected from light. (dehydrodieugenol),
N. O. 2 further unknown dimeric compounds,
IMPURITIES

P. toluene.

01/2010:2104

A. (1R,4E,9S)-4,11,11-trimethyl-8-methylenebicyclo[7.2.0]- EVENING PRIMROSE OIL, REFINED

undec-4-ene (β-caryophyllene),
Oenotherae oleum raffinatum
DEFINITION
Fatty oil obtained from seeds of Oenothera biennis L. or
Oenothera lamarckiana L. by extraction and/or expression. It
is then refined. A suitable antioxidant may be added.
CHARACTERS
B. (1E,4E,8E)-2,6,6,9-tetramethylcycloundeca-1,4,8-triene Appearance : clear, light yellow or yellow liquid.
(α-humulene, α-caryophyllene), Solubility : practically insoluble in water and in ethanol (96 per
cent), miscible with light petroleum (bp : 40-60 °C).
Relative density : about 0.923.
Refractive index : about 1.478.
IDENTIFICATION
First identification : B.
Second identification : A.
C. (1R,4R,6R,10S)-4,12,12-trimethyl-9-methylene-5- A. Identification of fatty oils by thin-layer chromatography
oxatricyclo[8.2.0.04,6]dodecane (β-caryophyllene oxide), (2.3.2).
Results : the chromatogram obtained is similar to the
corresponding chromatogram shown in Figure 2.3.2.-1.
B. Composition of fatty acids (see Tests).

D. R1 = H, R2 = H, R3 = CH2-CH=CH2 : 4-(prop-2- TESTS

enyl)phenol, Acid value (2.5.1) : maximum 0.5, or maximum 0.3 if intended
for use in the manufacture of parenteral preparations.
E. R1 = CH3, R2 = OCH3, R3 = CH2-CH=CH2 :
1,2-dimethoxy-4-(prop-2-enyl)benzene (eugenol methyl Peroxide value (2.5.5, Method A) : maximum 10.0, or
ether), maximum 5.0 if intended for use in the manufacture of
parenteral preparations.
F. R1 = H, R2 = OCH3, R3 = CH=CH-CH3 (cis) :
2-methoxy-4-[(Z)-prop-1-enyl]phenol (cis-isoeugenol), Unsaponifiable matter (2.5.7) : maximum 2.5 per cent,
determined on 5.0 g.
G. R1 = H, R2 = OCH3, R3 = CH=CH-CH3 (trans) : Alkaline impurities (2.4.19). It complies with the test.
2-methoxy-4-[(E)-prop-1-enyl]phenol (trans-isoeugenol),
Composition of fatty acids (2.4.22, Method A). Use the
H. R1 = H, R2 = OCH3, R3 = CHO : 4-hydroxy-3- mixture of calibrating substances in Table 2.4.22.-3.
methoxybenzaldehyde (vanillin), Composition of the fatty-acid fraction of the oil :
I. R1 = CO-CH3, R2 = OCH3, R3 = CH2-CH=CH2 : – saturated fatty acids of chain length less than C16 : maximum
2-methoxy-4-(prop-2-enyl)phenyl acetate (acetyleugenol), 0.3 per cent ;

2206 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Evening primrose oil, refined

– palmitic acid : 4.0 per cent to 10.0 per cent ; Water (2.5.32) : maximum 0.1 per cent, determined on 1.00 g.
– stearic acid : 1.0 per cent to 4.0 per cent ;
STORAGE
– oleic acid : 5.0 per cent to 12.0 per cent ;
– linoleic acid : 65.0 per cent to 85.0 per cent ; Under an inert gas, in a well-filled, airtight container,
protected from light.
– gamma-linolenic acid : 7.0 per cent to 14.0 per cent ;
– alpha-linolenic acid : maximum 0.5 per cent. LABELLING
Brassicasterol (2.4.23) : maximum 0.3 per cent in the sterol The label states, where applicable, that the oil is suitable for
fraction of the oil. use in the manufacture of parenteral preparations.

General Notices (1) apply to all monographs and other texts 2207
EUROPEAN PHARMACOPOEIA 8.0 Famotidine

04/2013:1012 Mobile phase :

– mobile phase A : mix 6 volumes of methanol R, 94 volumes
FAMOTIDINE of acetonitrile R and 900 volumes of a 1.882 g/L solution of
sodium hexanesulfonate R previously adjusted to pH 3.5
with acetic acid R ;
Famotidinum – mobile phase B : acetonitrile R ;
Time Mobile phase A Mobile phase B Flow rate
(min) (per cent V/V) (per cent V/V) (mL/min)
0 - 23 100 → 96 0→4 1

23 - 27 96 4 1→2

27 - 47 96 → 78 4 → 22 2

C8H15N7O2S3 Mr 337.4 Detection : spectrophotometer at 265 nm.

[76824-35-6]
Injection : 20 μL.
DEFINITION Identification of impurities : use the chromatogram
supplied with famotidine for system suitability CRS and
3-[[[2-[(Diaminomethylidene)amino]thiazol-4-
the chromatogram obtained with reference solution (c) to
yl]methyl]sulfanyl]-N′-sulfamoylpropanimidamide.
identify the peaks due to impurities A, B, C, F and G ; use the
Content : 98.5 per cent to 101.5 per cent (dried substance). chromatogram obtained with reference solution (b) to identify
the peak due to impurity D.
CHARACTERS Relative retention with reference to famotidine (retention
Appearance : white or yellowish-white, crystalline powder or time = about 21 min) : impurity D = about 1.1 ;
crystals. impurity C = about 1.2 ; impurity G = about 1.4 ;
Solubility : very slightly soluble in water, freely soluble in impurity F = about 1.5 ; impurity A = about 1.6 ;
glacial acetic acid, very slightly soluble in anhydrous ethanol, impurity B = about 2.0.
practically insoluble in ethyl acetate. It dissolves in dilute System suitability :
mineral acids. – retention time : famotidine = 19-23 min in all the
It shows polymorphism (5.9). chromatograms ;
– resolution : minimum 3.5 between the peaks due to
IDENTIFICATION famotidine and impurity D in the chromatogram obtained
Infrared absorption spectrophotometry (2.2.24). with reference solution (b).
Comparison : famotidine CRS. Limits :
If the spectra obtained show differences, suspend 0.10 g of the – correction factors : for the calculation of content, multiply
substance to be examined and 0.10 g of the reference substance the peak areas of the following impurities by the
separately in 5 mL of water R. Heat to boiling and allow to corresponding correction factor : impurity A = 1.9 ;
cool, scratching the wall of the tube with a glass rod to initiate impurity B = 2.5 ; impurity C = 1.9 ; impurity F = 1.7 ;
crystallisation. Filter, wash the crystals with 2 mL of iced impurity G = 1.4 ;
water R and dry in an oven at 80 °C at a pressure not exceeding – impurities C, D : for each impurity, not more than 3 times
670 Pa for 1 h. Record new spectra using the residues. the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.3 per cent) ;
TESTS – impurities A, B, F, G : for each impurity, not more than
Appearance of solution. Dissolve 0.20 g in a 50 g/L solution 1.5 times the area of the principal peak in the chromatogram
of hydrochloric acid R, heating to 40 °C if necessary, and dilute obtained with reference solution (a) (0.15 per cent) ;
to 20 mL with the same acid. The solution is clear (2.2.1) – unspecified impurities : for each impurity, not more than the
and not more intensely coloured than reference solution BY7 area of the principal peak in the chromatogram obtained
(2.2.2, Method II). with reference solution (a) (0.10 per cent) ;
Related substances. Liquid chromatography (2.2.29). – total : not more than 8 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
Test solution. Dissolve 12.5 mg of the substance to be
(0.8 per cent) ;
examined in mobile phase A and dilute to 25.0 mL with
mobile phase A. – disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
Reference solution (a). Dilute 1.0 mL of the test solution to (0.05 per cent).
100.0 mL with mobile phase A. Dilute 1.0 mL of this solution
to 10.0 mL with mobile phase A. Heavy metals (2.4.8) : maximum 10 ppm.
Reference solution (b). Dissolve 2.5 mg of famotidine Solvent mixture : dimethylformamide R, water R (30:70 V/V).
impurity D CRS in methanol R and dilute to 10.0 mL with the 0.5 g complies with test H. Prepare the reference solution
same solvent. To 1.0 mL of the solution add 0.50 mL of the using 0.5 mL of lead standard solution (10 ppm Pb) R.
test solution and dilute to 100.0 mL with mobile phase A. Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Reference solution (c). Dissolve 5.0 mg of famotidine for system on 1.000 g by drying in an oven at 80 °C at a pressure not
suitability CRS (containing impurities A, B, C, D, F and G) in exceeding 670 Pa for 5 h.
mobile phase A and dilute to 10.0 mL with mobile phase A. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Column : 1.0 g.
– size : l = 0.25 m, Ø = 4.6 mm ; ASSAY
– stationary phase : end-capped octadecylsilyl silica gel for Dissolve 0.120 g in 60 mL of anhydrous acetic acid R. Titrate
chromatography R (5 μm) ; with 0.1 M perchloric acid, determining the end-point
– temperature : 50 °C. potentiometrically (2.2.20).

General Notices (1) apply to all monographs and other texts 2211
Febantel for veterinary use EUROPEAN PHARMACOPOEIA 8.0

1 mL of 0.1 M perchloric acid is equivalent to 16.87 mg

of C8H15N7O2S3.

STORAGE
Protected from light.

IMPURITIES G. N-cyano-3-[[[2-[(diaminomethylidene)amino]thiazol-4-
Specified impurities : A, B, C, D, F, G. yl]methyl]sulfanyl]propanimidamide,
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of H. [2-[(diaminomethylidene)amino]thiazol-4-yl]methyl
impurities in substances for pharmaceutical use) : E, H, I, J. carbamimidothioate,

I. 3-[[[2-[(diaminomethylidene)amino]thiazol-4-
A. 3-[[[2-[(diaminomethylidene)amino]thiazol-4- yl]methyl]sulfinyl]-N-sulfamoylpropanamide,
yl]methyl]sulfanyl]propanimidamide,

J. methyl 3-[[[2-[(diaminomethylidene)amino]thiazol-4-
yl]methyl]sulfanyl]propanoate.
B. 3,5-bis[2-[[[2-[(diaminomethylidene)amino]thiazol-
4-yl]methyl]sulfanyl]ethyl]-4H-1,2,4,6-thiatriazine
1,1-dioxide, 01/2008:2176
corrected 6.0

FEBANTEL FOR VETERINARY USE

Febantelum ad usum veterinarium

C. 3-[[[2-[(diaminomethylidene)amino]thiazol-4-
yl]methyl]sulfanyl]-N-sulfamoylpropanamide,

C20H22N4O6S Mr 446.5
[58306-30-2]
DEFINITION
D. 3-[[[2-[(diaminomethylidene)amino]thiazol-4-
yl]methyl]sulfanyl]propanamide, Dimethyl N,N′-[[[2-[(methoxyacetyl)amino]-4-
(phenylsulfanyl)phenyl]imino]methylene]dicarbamate.
Content : 97.5 per cent to 102.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, soluble in acetone,
E. 2,2′-[disulfanediylbis(methylenethiazole-4,2- slightly soluble in anhydrous ethanol.
diyl)]diguanidine, It shows polymorphism (5.9).
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison: febantel CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
F. 3-[[[2-[(diaminomethylidene)amino]thiazol-4- substance separately in acetone R, evaporate to dryness and
yl]methyl]sulfanyl]propanoic acid, record new spectra using the residues.

2212 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Felbinac

TESTS
Related substances. Liquid chromatography (2.2.29).
Solvent mixture : acetonitrile R, tetrahydrofuran R (50:50 V/V).
Test solution (a). Dissolve 0.100 g of the substance to be
examined in the solvent mixture and dilute to 10.0 mL with
the solvent mixture. A. methyl [[2-[(methoxyacetyl)amino]-4-(phenylsulfanyl)-
Test solution (b). Dilute 5.0 mL of test solution (a) to 100.0 mL phenyl]carbamimidoyl]carbamate,
with the solvent mixture.
Reference solution (a). Dilute 1.0 mL of test solution (a) to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Reference solution (b). Dissolve 50.0 mg of febantel CRS in B. R = CH2-OCH3 : 2-(methoxymethyl)-5-(phenylsulfanyl)-
the solvent mixture and dilute to 10.0 mL with the solvent 1H-benzimidazole,
mixture. Dilute 5.0 mL of this solution to 50.0 mL with the
solvent mixture. C. R = NH-CO-OCH3 : methyl [5-(phenylsulfanyl)-1H-
benzimidazol-2-yl]carbamate (fenbendazole).
Reference solution (c). Dissolve 5 mg of febantel for system
suitability CRS (containing impurities A, B and C) in 1.0 mL
of the solvent mixture. 01/2008:2304
Column :
– size : l = 0.15 m, Ø = 4.0 mm ; FELBINAC
– stationary phase : spherical end-capped octadecylsilyl silica
gel for chromatography R1 (5 μm). Felbinacum
Mobile phase : dissolve 6.8 g of potassium dihydrogen
phosphate R in 1000 mL of water for chromatography R. Mix
350 mL of acetonitrile R with 650 mL of this solution.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 280 nm.
Injection : 10 μL of test solution (a) and reference solutions (a) C14H12O2 Mr 212.2
and (c). [5728-52-9]
Run time : 1.5 times the retention time of febantel. DEFINITION
Elution order : impurity A, impurity B, impurity C, febantel. (Biphenyl-4-yl)acetic acid.
Retention time : febantel = about 32 min. Content : 99.0 per cent to 101.0 per cent (dried substance).
System suitability: reference solution (c) :
CHARACTERS
– resolution : minimum 3.0 between the peaks due to
impurities A and B and minimum 4.0 between the peaks Appearance : white or almost white, crystalline powder.
due to impurities B and C. Solubility : practically insoluble in water, soluble in methanol,
Limits : sparingly soluble in ethanol (96 per cent).
– impurities A, B, C : for each impurity, not more than the mp : about 164 °C.
area of the principal peak in the chromatogram obtained IDENTIFICATION
with reference solution (a) (0.1 per cent) ;
Infrared absorption spectrophotometry (2.2.24).
– unspecified impurities : for each impurity, not more than
twice the area of the principal peak in the chromatogram Comparison: felbinac CRS.
obtained with reference solution (a) (0.20 per cent) ; TESTS
– total : not more than 5 times the area of the principal peak Related substances. Liquid chromatography (2.2.29). Protect
in the chromatogram obtained with reference solution (a) the solutions from light and inject within 20 min of preparation.
(0.5 per cent) ;
Test solution. Dissolve 0.100 g of the substance to be examined
– disregard limit : 0.5 times the area of the principal peak in in methanol R and dilute to 10.0 mL with the same solvent.
the chromatogram obtained with reference solution (a)
(0.05 per cent). Reference solution. Dissolve 5.0 mg of felbinac impurity A CRS
and 5.0 mg of biphenyl R (impurity B) in methanol R,
Heavy metals (2.4.8) : maximum 20 ppm. add 0.5 mL of the test solution and dilute to 50.0 mL with
1.0 g complies with test F. Prepare the reference solution using methanol R. Dilute 1.0 mL of this solution to 10.0 mL with
2 mL of lead standard solution (10 ppm Pb) R. methanol R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined Column :
on 1.000 g by drying in an oven at 105 °C for 2 h. – size : l = 0.15 m, Ø = 4.6 mm ;
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on – stationary phase : octadecylsilyl silica gel for
1.0 g. chromatography R (5 μm).
ASSAY Mobile phase : mix 45 volumes of a 0.1 per cent V/V solution
of glacial acetic acid R and 55 volumes of methanol R.
Liquid chromatography (2.2.29) as described in the test for Flow rate : 2 mL/min.
related substances with the following modification.
Detection : spectrophotometer at 254 nm.
Injection : test solution (b) and reference solution (b).
Injection : 20 μL.
Calculate the percentage content of C20H22N4O6S from the
declared content of febantel CRS. Run time : 3.5 times the retention time of felbinac.
Relative retention with reference to felbinac (retention
IMPURITIES time = about 15 min) : impurity A = about 1.3 ;
Specified impurities : A, B, C. impurity B = about 2.8.

General Notices (1) apply to all monographs and other texts 2213
Felodipine EUROPEAN PHARMACOPOEIA 8.0

System suitability: reference solution : 01/2008:1013

corrected 6.0
– resolution : minimum 3.0 between the peaks due to felbinac
and impurity A.
Limits :
FELODIPINE
– impurity A : not more than the area of the corresponding Felodipinum
peak in the chromatogram obtained with the reference
solution (0.1 per cent) ;
– impurity B : not more than the area of the peak due to
felbinac in the chromatogram obtained with the reference
solution (0.1 per cent) ;
– unspecified impurities : for each impurity, not more than
the area of the peak due to felbinac in the chromatogram
obtained with the reference solution (0.10 per cent) ;
– total : not more than twice the area of the peak due to C18H19Cl2NO4 Mr 384.3
felbinac in the chromatogram obtained with the reference [72509-76-3]
solution (0.2 per cent) ;
DEFINITION
– disregard limit : 0.5 times the area of the peak due to
felbinac in the chromatogram obtained with the reference Ethyl methyl (4RS)-4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4-
solution (0.05 per cent). dihydropyridine-3,5-dicarboxylate.
Content : 99.0 per cent to 101.0 per cent (dried substance).
Chlorides : maximum 110 ppm.
Dissolve 1.0 g in 40 mL of acetone R, add 6 mL of a 10 per CHARACTERS
cent V/V solution of nitric acid R, dilute to 50.0 mL with Appearance : white or light yellow, crystalline powder.
water R and mix. Pour 15.0 mL of this solution as a single Solubility : practically insoluble in water, freely soluble in
addition into 1 mL of 0.1 M silver nitrate and allow to stand for acetone, in anhydrous ethanol, in methanol and in methylene
5 min protected from light. When viewed horizontally against chloride.
a black background, any opalescence produced is not more
intense than that obtained by treating in the same manner IDENTIFICATION
15.0 mL of a mixture of 1.5 mL of 0.002 M hydrochloric acid, First identification : B.
40 mL of acetone R, 6 mL of 10 per cent V/V solution of nitricSecond identification : A, C, D.
acid R, diluted to 50.0 mL with water R.
A. Ultraviolet and visible absorption spectrophotometry
Sulfates : maximum 130 ppm. (2.2.25).
Dissolve 1.5 g in 40 mL of dimethylformamide R, add 1 mL Test solution. Dissolve 50 mg in methanol R and dilute to
of a 10 per cent V/V solution of hydrochloric acid R, dilute 100 mL with the same solvent. Dilute 3 mL of this solution
to 50.0 mL with dimethylformamide R and mix. To 15.0 mL to 100 mL with methanol R.
of this solution add 2.0 mL of a 120 g/L solution of barium Spectral range : 220-400 nm.
chloride R and allow to stand for 5 min. Any opalescence
Absorption maxima : at 238 nm and 361 nm.
produced is not more intense than that of a standard prepared
in the same manner but using 2.0 mL of 0.001 M sulfuric acid Absorbance ratio : A361 / A238 = 0.34 to 0.36.
instead of the substance to be examined. B. Infrared absorption spectrophotometry (2.2.24).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined Preparation : discs.
on 1.000 g by drying in an oven at 105 °C for 3 h. Comparison: felodipine CRS.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on C. Thin-layer chromatography (2.2.27).
1.0 g. Test solution. Dissolve 10 mg of the substance to be
examined in methanol R and dilute to 10 mL with the same
ASSAY solvent.
Dissolve 0.160 g in 50 mL of methanol R. Titrate with 0.1 M Reference solution (a). Dissolve 10 mg of felodipine CRS in
alcoholic potassium hydroxide determining the end-point methanol R and dilute to 10 mL with the same solvent.
potentiometrically (2.2.20). Reference solution (b). Dissolve 5 mg of nifedipine CRS in
reference solution (a) and dilute to 5 mL with reference
1 mL of 0.1 M alcoholic potassium hydroxide is equivalent to solution (a).
21.23 mg of C14H12O2.
Plate : TLC silica gel F254 plate R.
IMPURITIES Mobile phase : ethyl acetate R, cyclohexane R (40:60 V/V).
Application : 5 μL.
Specified impurities : A, B.
Development : over a path of 15 cm.
Drying : in air.
Detection : examine in ultraviolet light at 254 nm.
System suitability : reference solution (b) :
– the chromatogram shows 2 clearly separated spots.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position, fluorescence
A. R = CO-CH3 : 4-acetyl biphenyl, and size to the principal spot in the chromatogram obtained
with reference solution (a).
D. Ultraviolet and visible absorption spectrophotometry
B. R = H : biphenyl. (2.2.25).

2214 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Felypressin

Test solution. Dissolve 0.150 g in a mixture of 25 mL Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
of 2-methyl-2-propanol R and 25 mL of perchloric acid 1.0 g.
solution R. Add 10 mL of 0.1 M cerium sulfate, allow to
stand for 15 min, add 3.5 mL of strong sodium hydroxide ASSAY
solution R and neutralise with dilute sodium hydroxide Dissolve 0.160 g in a mixture of 25 mL of 2-methyl-2-propanol R
solution R. Shake with 25 mL of methylene chloride R. and 25 mL of perchloric acid solution R. Add 0.05 mL of
Evaporate the lower layer to dryness on a water-bath ferroin R. Titrate with 0.1 M cerium sulfate until the pink
under nitrogen (the residue is also used in the test for colour disappears. Titrate slowly towards the end of the
related substances). Dissolve about 20 mg of the residue titration.
in methanol R and dilute to 50 mL with the same solvent. 1 mL of 0.1 M cerium sulfate is equivalent to 19.21 mg
Dilute 2 mL of this solution to 50 mL with methanol R. of C18H19Cl2NO4.
Spectral range : 220-400 nm.
STORAGE
Absorption maximum : at 273 nm. Protected from light.
TESTS IMPURITIES
Solution S. Dissolve 1.00 g in methanol R and dilute to Specified impurities : B, C.
20.0 mL with the same solvent. Other detectable impurities (the following substances would,
Appearance of solution. Solution S is clear (2.2.1). if present at a sufficient level, be detected by one or other of
Absorbance (2.2.25) : maximum 0.10, determined at 440 nm the tests in the monograph. They are limited by the general
on solution S. acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical
Related substances. Liquid chromatography (2.2.29). use (2034). It is therefore not necessary to identify these
Test solution. Dissolve 25.0 mg of the substance to be impurities for demonstration of compliance. See also 5.10.
examined in the mobile phase and dilute to 50.0 mL with the Control of impurities in substances for pharmaceutical use) : A.
mobile phase.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase.
Reference solution (b). Dilute 1.0 mL of reference solution (a)
to 10.0 mL with the mobile phase.
Reference solution (c). Dissolve 50.0 mg of the residue
obtained in identification test D (impurity A) and 25.0 mg of
felodipine CRS in the mobile phase, then dilute to 50.0 mL
with the mobile phase. Dilute 1.0 mL of this solution to A. ethyl methyl 4-(2,3-dichlorophenyl)-2,6-dimethylpyridine-
100.0 mL with the mobile phase. Dilute 1.0 mL of the solution 3,5-dicarboxylate,
to 10.0 mL with the mobile phase.
Column :
– size : l = 0.125-0.15 m, Ø = 4 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : mix 20 volumes of methanol R, 40 volumes of
acetonitrile R and 40 volumes of a phosphate buffer solution
pH 3.0 containing 0.8 g/L of phosphoric acid R and 8 g/L of
B. R = CH3 : dimethyl 4-(2,3-dichlorophenyl)-2,6-dimethyl-
sodium dihydrogen phosphate R.
1,4-dihydropyridine-3,5-dicarboxylate,
Flow rate : 1 mL/min.
C. R = C2H5 : diethyl 4-(2,3-dichlorophenyl)-2,6-dimethyl-
Detection : spectrophotometer at 254 nm. 1,4-dihydropyridine-3,5-dicarboxylate.
Injection : 20 μL.
Run time : twice the retention time of felodipine. 01/2008:1634
Elution order : impurity B, impurity A, felodipine, impurity C. corrected 7.0
Retention time : felodipine = about 12 min.
System suitability: reference solution (c) : FELYPRESSIN
– resolution : minimum 2.5 between the peaks due to
impurity A and felodipine. Felypressinum
Limits :
– sum of impurities B and C : not more than the area of
the principal peak in the chromatogram obtained with C46H65N13O11S2 Mr 1039
reference solution (a) (1.0 per cent) ; [56-59-7]
– unspecified impurities : for each impurity, not more than the DEFINITION
area of the principal peak in the chromatogram obtained
L-Cysteinyl-L-phenylalanyl-L-phenylalanyl-L-glutaminyl-L-
with reference solution (b) (0.10 per cent) ;
asparaginyl-L-cysteinyl-L-prolyl-L-lysylglycinamide cyclic
– sum of impurities other than B and C : not more than (1,6)-disulfide.
3 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.3 per cent) ; Synthetic nonapeptide having a vasoconstricting activity. It is
available as an acetate.
– disregard limit : 0.2 times the area of the principal peak in
Content : 95.0 per cent to 102.0 per cent (anhydrous and acetic
the chromatogram obtained with reference solution (b)
acid-free substance).
(0.02 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined CHARACTERS
on 1.000 g by drying in an oven at 105 °C for 3 h. Appearance : white or almost white, powder or flakes.

General Notices (1) apply to all monographs and other texts 2215
Felypressin EUROPEAN PHARMACOPOEIA 8.0

Solubility : freely soluble in water, practically insoluble in – resolution : minimum 1.5 between the peaks due to
acetone and ethanol (96 per cent). It dissolves in dilute impurity C and impurity D.
solutions of alkali hydroxides. Limits :
IDENTIFICATION – impurities A, B, C, D, E, F : for each impurity, maximum
A. Examine the chromatograms obtained in the assay. 0.5 per cent,
Results : the principal peak in the chromatogram obtained – any other impurity : for each impurity, maximum 0.1 per
with test solution (b) is similar in retention time and size cent,
to the principal peak in the chromatogram obtained with – total : maximum 3.0 per cent,
the reference solution. – disregard limit : 0.05 per cent.
B. Amino acid analysis (2.2.56). For hydrolysis use Method 1
Acetic acid (2.5.34) : 9.0 per cent to 13.0 per cent.
and for analysis use Method 1.
Express the content of each amino acid in moles. Calculate Test solution. Dissolve 10.0 mg of the substance to be
the relative proportions of amino acids, taking one-seventh examined in a mixture of 5 volumes of mobile phase B and
of the sum of the number of moles of glutamic acid, 95 volumes of mobile phase A and dilute to 10.0 mL with the
aspartic acid, proline, lysine, glycine and phenylalanine as same mixture of mobile phases.
equal to one. The values fall within the following limits : Water (2.5.32) : maximum 7.0 per cent.
aspartic acid : 0.9 to 1.1 ; glutamic acid : 0.9 to 1.1 ; proline : Bacterial endotoxins (2.6.14) : less than 100 IU/mg, if
0.9 to 1.1 ; glycine : 0.9 to 1.1 ; phenylalanine : 1.8 to 2.2 ; intended for use in the manufacture of parenteral preparations
half-cystine : 1.8 to 2.2 ; lysine : 0.9 to 1.1. without a further appropriate procedure for the removal of
TESTS bacterial endotoxins.
Specific optical rotation (2.2.7) : − 35 to − 29, determined at ASSAY
25 °C (anhydrous and acetic acid-free substance). Liquid chromatography (2.2.29) as described in the test for
Dissolve 20.0 mg in a 1 per cent V/V solution of glacial acetic related substances with the following modification.
acid R and dilute to 10.0 mL with the same solution. Injection : 10 μL of test solution (b) and of the reference
Related substances. Liquid chromatography (2.2.29) ; use the solution.
normalisation procedure. The solutions are stable for 24 h at Calculate the content of felypressin (C46H65N13O11S2) from the
room temperature or for 1 week at 2-8 °C. areas of the peaks and the declared content of C46H65N13O11S2
Test solution (a). Dissolve 5.0 mg of the substance to be in felypressin CRS.
examined in 5.0 mL of mobile phase A.
Test solution (b). Dilute 1.0 mL of test solution (a) to 5.0 mL STORAGE
with mobile phase A. In an airtight container, protected from light, at a temperature
Reference solution. Dissolve the contents of a vial of of 2 °C to 8 °C. If the substance is sterile, store in a sterile,
felypressin CRS in mobile phase A to obtain a concentration airtight, tamper-proof container.
of 0.2 mg/mL.
LABELLING
Column :
The label states the mass of peptide in the container.
– size : l = 0.15 m, Ø = 3.9 mm,
– stationary phase : octadecylsilyl silica gel for IMPURITIES
chromatography R (5 μm), Specified impurities : A, B, C, D, E, F.
– temperature : 50 °C.
Mobile phase :
– mobile phase A : dissolve 3.62 g of tetramethylammonium
hydroxide R in 900 mL water R; adjust to pH 2.5 with
phosphoric acid R and dilute to 1000 mL with water R ;
– mobile phase B : dissolve 1.81 g of tetramethylammonium A. S1,S6-bis[(acetylamino)methyl]-(reduced felypressin),
hydroxide R in 450 mL of a 50 per cent V/V solution of
acetonitrile for chromatography R; adjust to pH 2.5 with
phosphoric acid R and dilute to 500 mL with a 50 per
cent V/V solution of acetonitrile for chromatography R ; B. [5-aspartic acid]felypressin,
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 20 80 → 50 20 → 50

20 - 25 50 50
C. bis(reduced felypressin) (1,6′),(1′,6)-bis(disulfide),
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 210 nm.
Injection : 10 μL of test solution (a) and 50 μL of the reference
solution. D. bis(reduced felypressin) (1,1′),(6,6′)-bis(disulfide),
Identification of impurities : use the chromatogram supplied
with felypressin CRS to identify the peaks due to impurities
A to F.
Relative retention with reference to felypressin :
impurity A = about 0.9 ; impurity B = about 1.1 ;
impurity F = about 1.2 ; impurity C = about 1.3 ; E. N1-acetylfelypressin,
impurity D = about 1.4 ; impurity E = about 2.1.
System suitability: reference solution :
– retention time : felypressin = about 7.5 min; F. [4-glutamic acid]felypressin.

2216 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Fenbendazole for veterinary use

01/2014:1208 Flow rate : 1.0 mL/min.

Detection : spectrophotometer at 280 nm.
FENBENDAZOLE FOR VETERINARY Injection : 10 μL.
USE
Identification of impurities: use the chromatogram obtained
with reference solution (b) to identify the peak due to
Fenbendazolum ad usum veterinarium impurity A ; use the chromatogram obtained with reference
solution (c) to identify the peak due to impurity B ; use the
chromatogram obtained with reference solution (d) to identify
the peak due to mebendazole.
Relative retention with reference to fenbendazole
(retention time = about 18 min) : impurity A = about 0.2 ;
C15H13N3O2S Mr 299.4 impurity B = about 0.6 ; mebendazole = about 0.8.
[43210-67-9] System suitability : reference solution (d) :
DEFINITION – resolution : minimum 1.5 between the peaks due to
mebendazole and fenbendazole.
Methyl [5-(phenylsulfanyl)-1H-benzimidazol-2-yl]carbamate.
Limits :
Content : 98.0 per cent to 101.0 per cent (dried substance).
– impurity A : not more than 2.5 times the area of the
CHARACTERS corresponding peak in the chromatogram obtained with
reference solution (b) (0.5 per cent) ;
Appearance : white or almost white powder.
– impurity B : not more than 2.5 times the area of the
Solubility : practically insoluble in water, sparingly soluble in corresponding peak in the chromatogram obtained with
dimethylformamide, very slightly soluble in methanol. reference solution (c) (0.5 per cent) ;
– unspecified impurities : for each impurity, not more than
IDENTIFICATION twice the area of the principal peak in the chromatogram
Infrared absorption spectrophotometry (2.2.24). obtained with reference solution (a) (0.5 per cent) ;
– total : maximum 1.0 per cent ;
Comparison : fenbendazole CRS.
– disregard limit : 0.4 times the area of the principal peak in
TESTS the chromatogram obtained with reference solution (a)
(0.10 per cent).
Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use. Keep the temperature of Heavy metals (2.4.8) : maximum 20 ppm.
the autosampler at 10 °C. 1.0 g complies with test C. Prepare the reference solution using
Test solution. Dissolve 50.0 mg of the substance to be 2 mL of lead standard solution (10 ppm Pb) R.
examined in 10.0 mL of hydrochloric methanol R. Loss on drying (2.2.32) : maximum 1.0 per cent, determined
Reference solution (a). Dissolve 50.0 mg of fenbendazole CRS on 1.000 g by drying in an oven at 105 °C for 3 h.
in 10.0 mL of hydrochloric methanol R. Dilute 1.0 mL of the Sulfated ash (2.4.14) : maximum 0.3 per cent, determined on
solution to 200.0 mL with methanol R. Dilute 5.0 mL of this 1.0 g.
solution to 10.0 mL with hydrochloric methanol R.
Reference solution (b). Dissolve 10.0 mg of fenbendazole ASSAY
impurity A CRS in 100.0 mL of methanol R. Dilute 1.0 mL of Dissolve 0.200 g in 30 mL of anhydrous acetic acid R, warming
the solution to 10.0 mL with hydrochloric methanol R. gently if necessary. Cool and titrate with 0.1 M perchloric acid,
Reference solution (c). Dissolve 10.0 mg of fenbendazole determining the end-point potentiometrically (2.2.20).
impurity B CRS in 100.0 mL of methanol R. Dilute 1.0 mL of 1 mL of 0.1 M perchloric acid is equivalent to 29.94 mg
the solution to 10.0 mL with hydrochloric methanol R. of C15H13N3O2S.
Reference solution (d). Dissolve 10.0 mg of fenbendazole CRS
and 10.0 mg of mebendazole CRS in 100.0 mL of methanol R. STORAGE
Dilute 1.0 mL of the solution to 10.0 mL with hydrochloric Protected from light.
methanol R.
Column : IMPURITIES
– size : l = 0.25 m, Ø = 4.6 mm ; Specified impurities : A, B.
– stationary phase : base-deactivated end-capped octadecylsilyl
silica gel for chromatography R (5 μm).
Mobile phase :
– mobile phase A : anhydrous acetic acid R, methanol R,
water R (1:30:70 V/V/V) ;
A. methyl (1H-benzimidazol-2-yl)carbamate,
– mobile phase B : anhydrous acetic acid R, water R,
methanol R (1:30:70 V/V/V) ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 10 100 → 0 0 → 100

10 - 40 0 100
B. methyl (5-chloro-1H-benzimidazol-2-yl)carbamate.

General Notices (1) apply to all monographs and other texts 2217
Fenbufen EUROPEAN PHARMACOPOEIA 8.0

01/2008:1209 Reference solution (a). Dilute 0.5 mL of the test solution

corrected 7.0 to 50.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
FENBUFEN Reference solution (b). Dissolve 25 mg of fenbufen CRS and
6 mg of ketoprofen CRS in the solvent mixture and dilute to
10 mL with the solvent mixture. Dilute 1 mL of this solution
Fenbufenum to 100 mL with the solvent mixture.
Column :
– size : l = 0.125 m, Ø = 4.0 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase :
C16H14O3 Mr 254.3 – mobile phase A : mix 32 volumes of acetonitrile R and
[36330-85-5] 68 volumes of a mixture of 1 volume of glacial acetic acid R
and 55 volumes of water R ;
DEFINITION – mobile phase B : mix 45 volumes of acetonitrile R and
4-(Biphenyl-4-yl)-4-oxobutanoic acid. 55 volumes of a mixture of 1 volume of glacial acetic acid R
and 55 volumes of water R ;
Content : 98.5 per cent to 101.0 per cent (dried substance).
Time Mobile phase A Mobile phase B
CHARACTERS (min) (per cent V/V) (per cent V/V)
Appearance : white or almost white, fine, crystalline powder. 0 – 15 100 0

Solubility : very slightly soluble in water, slightly soluble in 15 – 20 100 → 0 0 → 100

acetone, in ethanol (96 per cent) and in methylene chloride. 20 – 35 0 100

IDENTIFICATION Flow rate : 2 mL/min.

First identification : B. Detection : spectrophotometer at 254 nm.
Second identification : A, C. Injection : 20 μL.
A. Melting point (2.2.14) : 186 °C to 189 °C. System suitability : reference solution (b) :
B. Infrared absorption spectrophotometry (2.2.24). – resolution : minimum 5.0 between the peaks due to
Comparison : fenbufen CRS. ketoprofen and fenbufen.
C. Thin-layer chromatography (2.2.27). Limits :
Test solution. Dissolve 10 mg of the substance to be – any impurity : for each impurity, not more than the area
examined in methylene chloride R and dilute to 10 mL with of the principal peak in the chromatogram obtained with
the same solvent. reference solution (a) (0.1 per cent) ;
Reference solution (a). Dissolve 10 mg of fenbufen CRS in – total : not more than 5 times the area of the principal peak
methylene chloride R and dilute to 10 mL with the same in the chromatogram obtained with reference solution (a)
solvent. (0.5 per cent) ;
Reference solution (b). Dissolve 10 mg of ketoprofen CRS – disregard limit : 0.2 times the area of the principal peak in
in methylene chloride R and dilute to 10 mL with the same the chromatogram obtained with reference solution (a)
solvent. To 5 mL of this solution, add 5 mL of reference (0.02 per cent).
solution (a). Heavy metals (2.4.8) : maximum 20 ppm.
Plate : TLC silica gel F254 plate R. 1.0 g complies with test C. Prepare the reference solution using
Mobile phase : anhydrous acetic acid R, ethyl acetate R, 2 mL of lead standard solution (10 ppm Pb) R.
hexane R (5:25:75 V/V/V).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Application : 10 μL. on 1.000 g by drying in an oven at 105 °C for 3 h.
Development : over a path of 15 cm. Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
Drying : in air. 1.0 g.
Detection : examine in ultraviolet light at 254 nm. ASSAY
System suitability: reference solution (b) : Dissolve 0.200 g in 75 mL of acetone R previously neutralised
– the chromatogram shows 2 clearly separated spots. with phenolphthalein solution R1 and add 50 mL of water R.
Results : the principal spot in the chromatogram obtained Add 0.2 mL of phenolphthalein solution R1 and titrate with
with the test solution is similar in position and size to 0.1 M sodium hydroxide. Carry out a blank titration.
the principal spot in the chromatogram obtained with 1 mL of 0.1 M sodium hydroxide is equivalent to 25.43 mg
reference solution (a). of C16H14O3.
TESTS IMPURITIES
Related substances. Liquid chromatography (2.2.29).
Solvent mixture : dimethylformamide R, mobile phase A
(40:60 V/V).
Test solution. Dissolve 50.0 mg of the substance to be
examined in the solvent mixture and dilute to 10.0 mL with
the solvent mixture. A. 3-(4-chlorophenyl)-3-oxopropanoic acid,

2218 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Fenofibrate

Column :
– size : l = 0.25 m, Ø = 4.0 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : mix 30 volumes of water R acidified to pH 2.5
B. R = CO-CH=CH-CO2H, R′ = H : 4-(biphenyl-4-yl)-4- with phosphoric acid R and 70 volumes of acetonitrile R.
oxobut-2-enoic acid,
Flow rate : 1 mL/min.
C. R = R′ = H : biphenyl, Detection : spectrophotometer at 286 nm.
D. R = CO-CH2-CH2-CO2H, R′ = OH : 4-(4′-hydroxybiphenyl- Injection : 20 μL of the test solution and reference solution (b).
4-yl)-4-oxobutanoic acid. Run time : twice the retention time of fenofibrate.
Relative retention with reference to fenofibrate :
impurity A = about 0.34 ; impurity B = about 0.36 ;
01/2008:1322 impurity C = about 0.50 ; impurity D = about 0.65 ;
impurity E = about 0.80, impurity F = about 0.85 ;
FENOFIBRATE impurity G = about 1.35.
System suitability : reference solution (b) :
Fenofibratum – resolution : minimum 1.5 between the peaks due to
impurities A and B.
Limits :
– impurities A, B : for each impurity, not more than the area
of the corresponding peak in the chromatogram obtained
with reference solution (b) (0.1 per cent) ;
– impurity G : not more than the area of the corresponding
C20H21ClO4 Mr 360.8 peak in the chromatogram obtained with reference
[49562-28-9] solution (b) (0.2 per cent) ;
– unspecified impurities : for each impurity, not more than the
DEFINITION area of the peak due to fenofibrate in the chromatogram
1-Methylethyl 2-[4-(4-chlorobenzoyl)phenoxy]-2- obtained with reference solution (b) (0.10 per cent) ;
methylpropanoate. – total : not more than 5 times the area of the peak due to
Content : 98.0 per cent to 102.0 per cent (dried substance). fenofibrate in the chromatogram obtained with reference
solution (b) (0.5 per cent) ;
CHARACTERS
– disregard limit : 0.1 times the area of the peak due to
Appearance : white or almost white, crystalline powder. fenofibrate in the chromatogram obtained with reference
Solubility : practically insoluble in water, very soluble in solution (b) (0.01 per cent).
methylene chloride, slightly soluble in ethanol (96 per cent).
Halides expressed as chlorides (2.4.4) : maximum 100 ppm.
IDENTIFICATION To 5 mL of solution S add 10 mL of distilled water R.
A. Melting point (2.2.14) : 79 °C to 82 °C. Sulfates (2.4.13) : maximum 100 ppm, determined on
B. Infrared absorption spectrophotometry (2.2.24). solution S.
Preparation : discs. Heavy metals (2.4.8) : maximum 20 ppm.
Comparison : fenofibrate CRS. 1.0 g complies with test C. Prepare the reference solution using
2 mL of lead standard solution (10 ppm Pb) R.
TESTS
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
Solution S. To 5.0 g, add 25 mL of distilled water R and heat at on 1.000 g by drying in vacuo at 60 °C.
50 °C for 10 min. Cool and dilute to 50.0 mL with the same
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
solvent. Filter. Use the filtrate as solution S.
1.0 g.
Appearance of solution. The solution is clear (2.2.1) and
not more intensely coloured than reference solution BY6 ASSAY
(2.2.2, Method II). Liquid chromatography (2.2.29) as described in the test for
Dissolve 0.50 g in acetone R and dilute to 10.0 mL with the related substances with the following modifications.
same solvent. Injection : 5 μL of the test solution and reference solution (a).
Acidity. Dissolve 1.0 g in 50 mL of ethanol (96 per cent) R System suitability : reference solution (a) :
previously neutralised using 0.2 mL of phenolphthalein – repeatability : maximum relative standard deviation of
solution R1. Not more than 0.2 mL of 0.1 M sodium hydroxide 1.0 per cent after 6 injections.
is required to change the colour of the indicator to pink.
STORAGE
Related substances. Liquid chromatography (2.2.29).
Protected from light.
Test solution. Dissolve 0.100 g of the substance to be examined
in the mobile phase and dilute to 100.0 mL with the mobile IMPURITIES
phase. Specified impurities : A, B, G.
Reference solution (a). Dissolve 25.0 mg of fenofibrate CRS in Other detectable impurities (the following substances would,
the mobile phase and dilute to 25.0 mL with the mobile phase. if present at a sufficient level, be detected by one or other of
Reference solution (b). Dissolve 5.0 mg of fenofibrate CRS, the tests in the monograph. They are limited by the general
5.0 mg of fenofibrate impurity A CRS, 5.0 mg of fenofibrate acceptance criterion for other/unspecified impurities and/or
impurity B CRS and 10.0 mg of fenofibrate impurity G CRS by the general monograph Substances for pharmaceutical use
in the mobile phase and dilute to 100.0 mL with the mobile (2034). It is therefore not necessary to identify these impurities
phase. Dilute 1.0 mL of this solution to 50.0 mL with the for demonstration of compliance. See also 5.10. Control of
mobile phase. impurities in substances for pharmaceutical use) : C, D, E, F.

General Notices (1) apply to all monographs and other texts 2219
Fenoterol hydrobromide EUROPEAN PHARMACOPOEIA 8.0

Test solution. Dissolve 50.0 mg in dilute hydrochloric

acid R1 and dilute to 50.0 mL with the same acid. Dilute
5.0 mL of this solution to 50.0 mL with dilute hydrochloric
acid R1.
A. R-H : (4-chlorophenyl)(4-hydroxyphenyl)methanone, Spectral range : 230-350 nm.
Absorption maximum : at 275 nm.
Shoulder : at about 280 nm.
Specific absorbance at the absorption maximum : 80 to 86.
B. 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoic acid B. Infrared absorption spectrophotometry (2.2.24).
(fenofibric acid), Comparison: fenoterol hydrobromide CRS.
C. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 10 mg of the substance to be
examined in ethanol (96 per cent) R and dilute to 10 mL
with the same solvent.
C. (3RS)-3-[4-(4-chlorobenzoyl)phenoxy]butan-2-one, Reference solution. Dissolve 10 mg of fenoterol
hydrobromide CRS in ethanol (96 per cent) R and dilute to
10 mL with the same solvent.
Plate : TLC silica gel G plate R.
Mobile phase : concentrated ammonia R, water R,
D. methyl 2-[4-(4-chlorobenzoyl)phenoxy]-2- aldehyde-free methanol R (1.5:10:90 V/V/V).
methylpropanoate, Application : 2 μL.
Development : over a path of 15 cm.
Drying : in air.
Detection : spray with a 10 g/L solution of potassium
permanganate R.
E. ethyl 2-[4-(4-chlorobenzoyl)phenoxy]-2- Results : the principal spot in the chromatogram obtained
methylpropanoate, with the test solution is similar in position, colour and size
to the principal spot in the chromatogram obtained with
the reference solution.
D. Dissolve about 10 mg in a 20 g/L solution of disodium
F. (4-chlorophenyl)[4-(1-methylethoxy)phenyl]methanone, tetraborate R and dilute to 50 mL with the same solution.
Add 1 mL of a 10 g/L solution of aminopyrazolone R, 10 mL
of a 2 g/L solution of potassium ferricyanide R and 10 mL
of methylene chloride R. Shake and allow to separate. A
reddish-brown colour develops in the lower layer.
E. It gives reaction (a) of bromides (2.3.1).
G. 1-methylethyl 2-[[2-[4-(4-chlorobenzoyl)phenoxy]-2-
methylpropanoyl]oxy]-2-methylpropanoate. TESTS
Solution S. Dissolve 2.00 g in carbon dioxide-free water R and
01/2008:0901 dilute to 50.0 mL with the same solvent.
corrected 7.1
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution Y7 (2.2.2,
FENOTEROL HYDROBROMIDE Method II).
pH (2.2.3) : 4.2 to 5.2 for solution S.
Fenoteroli hydrobromidum Related substances. Liquid chromatography (2.2.29). Prepare
the solutions immediately before use.
Test solution. Dissolve 24.0 mg of the substance to be
examined in water R and dilute to 20.0 mL with the same
solvent.
Reference solution (a). Dissolve 24.0 mg of fenoterol
hydrobromide CRS (containing impurity A) in water R and
C17H22BrNO4 Mr 384.3 dilute to 20.0 mL with the same solvent.
[1944-12-3]
Reference solution (b). Dissolve the contents of a vial of
DEFINITION fenoterol for peak identification CRS (containing impurities B
(1RS)-1-(3,5-Dihydroxyphenyl)-2-[[(1RS)-2-(4- and C) in 1.0 mL of water R.
hydroxyphenyl)-1-methylethyl]amino]ethanol hydrobromide. Reference solution (c). Dilute 10.0 mL of the test solution
Content : 99.0 per cent to 101.0 per cent (dried substance). to 50.0 mL with water R. Dilute 1.0 mL of this solution to
100.0 mL with water R.
CHARACTERS Column :
Appearance : white or almost white, crystalline powder. – size : l = 0.15 m, Ø = 4.6 mm ;
Solubility : soluble in water and in ethanol (96 per cent). – stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
IDENTIFICATION
Mobile phase. Dissolve 24 g of anhydrous disodium hydrogen
First identification : B, E. phosphate R in 1000 mL of water R. Mix 69 volumes of
Second identification : A, C, D, E. this solution and 1 volume of a 9 g/L solution of potassium
A. Ultraviolet and visible absorption spectrophotometry dihydrogen phosphate R, adjust to pH 8.5 with phosphoric
(2.2.25). acid R and add 35 volumes of methanol R2.

2220 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Fentanyl

Flow rate : 1 mL/min.

Detection : spectrophotometer at 215 nm.
Injection : 20 μL.
Run time : 3 times the retention time of fenoterol.
Relative retention with reference to fenoterol (retention
time = about 7 min) : impurity A = about 1.3 ; B. 1-(3,5-dihydroxyphenyl)-2-[[(1RS)-2-(4-hydroxyphenyl)-
impurity B = about 2.0 ; impurity C = about 2.2. 1-methylethyl]amino]ethanone,
System suitability :
– resolution : minimum 3 between the peaks due to fenoterol
and impurity A in the chromatogram obtained with
reference solution (a) ; minimum 1.5 between the peaks
due to impurities B and C in the chromatogram obtained
with reference solution (b).
C. (1RS)-1-(3,5-dihydroxyphenyl)-2-[[(1RS)-2-(4-hydroxy-3-
Limits :
methylphenyl)-1-methylethyl]amino]ethanol.
– correction factor : for the calculation of content, multiply
the peak area of impurity B by 0.6 ;
– impurity A : maximum 4.0 per cent, calculated from the 01/2013:1210
area of the corresponding peak in the chromatogram
obtained with reference solution (a) and taking into
account the declared content of impurity A in fenoterol FENTANYL
hydrobromide CRS ;
– impurity C : not more than 1.5 times the area of the Fentanylum
principal peak in the chromatogram obtained with
reference solution (c) (0.3 per cent) ;
– impurity B : not more than the area of the principal peak
in the chromatogram obtained with reference solution (c)
(0.2 per cent) ;
– unspecified impurities : for each impurity, not more than
0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.10 per cent) ;
– sum of impurities other than A : not more than 1.5 times the
area of the principal peak in the chromatogram obtained C22H28N2O Mr 336.5
with reference solution (c) (0.3 per cent) ; [437-38-7]
– disregard limit : 0.25 times the area of the principal peak DEFINITION
in the chromatogram obtained with reference solution (c) N-Phenyl-N-[1-(2-phenylethyl)piperidin-4-yl]propanamide.
(0.05 per cent).
Content : 99.0 per cent to 101.0 per cent (dried substance).
Iron (2.4.9) : maximum 10 ppm.
Dissolve the residue obtained in the test for sulfated ash in CHARACTERS
2.5 mL of dilute hydrochloric acid R and dilute to 10 mL with Appearance : white or almost white powder.
water R. Solubility : practically insoluble in water, freely soluble in
Loss on drying (2.2.32) : maximum 0.5 per cent, determined ethanol (96 per cent) and in methanol.
on 1.000 g by drying in an oven at 105 °C. It shows polymorphism (5.9).
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
IDENTIFICATION
1.0 g.
Infrared absorption spectrophotometry (2.2.24).
ASSAY Comparison: Ph. Eur. reference spectrum of fentanyl.
Dissolve 0.600 g in 50 mL of water R and add 5 mL of dilute If the spectrum obtained in the solid state shows differences,
nitric acid R, 25.0 mL of 0.1 M silver nitrate and 2 mL of ferric dissolve the substance to be examined in the minimum volume
ammonium sulfate solution R2. Shake and titrate with 0.1 M of anhydrous ethanol R, evaporate to dryness at room
ammonium thiocyanate until an orange colour is obtained. temperature under an air-stream and record a new spectrum
Carry out a blank titration. using the residue.
1 mL of 0.1 M silver nitrate is equivalent to 38.43 mg
of C17H22BrNO4. TESTS
Related substances. Liquid chromatography (2.2.29).
STORAGE
Test solution. Dissolve 0.100 g of the substance to be examined
Protected from light. in methanol R and dilute to 10.0 mL with the same solvent.
IMPURITIES Reference solution (a). Dissolve 10 mg of fentanyl for system
Specified impurities : A, B, C. suitability CRS (containing impurities A, B, C, D and H) in
1.0 mL of methanol R.
Reference solution (b). Dilute 1.0 mL of the test solution to
100.0 mL with methanol R. Dilute 1.0 mL of this solution to
10.0 mL with methanol R.
Column :
– size : l = 0.1 m, Ø = 3.0 mm ;
A. (1RS)-1-(3,5-dihydroxyphenyl)-2-[[(1SR)-2-(4- – stationary phase : end-capped octadecylsilyl silica gel for
hydroxyphenyl)-1-methylethyl]amino]ethanol, chromatography R (3 μm).

General Notices (1) apply to all monographs and other texts 2221
Fentanyl EUROPEAN PHARMACOPOEIA 8.0

Mobile phase :
– mobile phase A : 5 g/L solution of ammonium carbonate R
in a mixture of 10 volumes of tetrahydrofuran R and
90 volumes of water R ;
– mobile phase B : acetonitrile R1 ;
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 15 90 → 40 10 → 60 A. N-phenyl-N-[cis,trans-1-oxido-1-(2-phenylethyl)piperi-
din-4-yl]propanamide,
15 - 20 40 60

Flow rate : 0.64 mL/min.

Detection : spectrophotometer at 220 nm.
Injection : 10 μL.
Identification of impurities : use the chromatogram supplied
with fentanyl for system suitability CRS and the chromatogram
obtained with reference solution (a) to identify the peaks due B. N-phenyl-N-(piperidin-4-yl)propanamide,
to impurities A, B, C, D and H.
Relative retention with reference to fentanyl (retention
time = about 15 min) : impurity B = about 0.1 ;
impurity A = about 0.3 ; impurity C = about 0.9 ;
impurity D = about 1.1 ; impurity H = about 1.2.
System suitability: reference solution (a) :
– resolution : minimum 3.0 between the peaks due to fentanyl
and impurity D.
Limits : C. N-phenyl-N-[1-(2-phenylethyl)piperidin-4-yl]acetamide,
– impurities A, B, C, D : for each impurity, not more than
2.5 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.25 per cent) ;
– impurity H : not more than 1.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.15 per cent) ;
– unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.10 per cent) ; D. N-phenyl-1-(2-phenylethyl)piperidin-4-amine,
– total : not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.5 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.05 per cent). E. benzaldehyde,
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in vacuo at 50 °C.

ASSAY
Dissolve 0.200 g in 50 mL of a mixture of 1 volume of
anhydrous acetic acid R and 7 volumes of methyl ethyl F. aniline (phenylamine),
ketone R and titrate with 0.1 M perchloric acid, using 0.2 mL
of naphtholbenzein solution R as indicator.
1 mL of 0.1 M perchloric acid is equivalent to 33.65 mg
of C22H28N2O.

STORAGE
Protected from light. G. N-phenylpropanamide,
IMPURITIES
Specified impurities : A, B, C, D, H.
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these impurities
for demonstration of compliance. See also 5.10. Control of H. (2RS)-2-chloro-N-phenyl-N-[1-(2-phenylethyl)piperidin-
impurities in substances for pharmaceutical use) : E, F, G. 4-yl]propanamide.

2222 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Fentanyl citrate

01/2013:1103 Identification of impurities : use the chromatogram supplied

with fentanyl for system suitability CRS and the chromatogram
FENTANYL CITRATE obtained with reference solution (a) to identify the peaks due
to impurities A, B, C and D.
Relative retention with reference to fentanyl (retention
Fentanyli citras time = about 15 min) : impurity B = about 0.1 ;
impurity A = about 0.3 ; impurity C = about 0.9 ;
impurity D = about 1.1.
System suitability : reference solution (a) :
– resolution : minimum 3.0 between the peaks due to fentanyl
and impurity D.
Limits :
– impurities A, B, C, D : for each impurity, not more 2.5 times
than the area of the principal peak in the chromatogram
C28H36N2O8 Mr 528.6 obtained with reference solution (b) (0.25 per cent) ;
[990-73-8] – unspecified impurities : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
DEFINITION with reference solution (b) (0.10 per cent) ;
N-Phenyl-N-[1-(2-phenylethyl)piperidin-4-yl]propanamide – total : not more than 5 times the area of the principal peak
dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate. in the chromatogram obtained with reference solution (b)
Content : 99.0 per cent to 101.0 per cent (dried substance). (0.5 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in
CHARACTERS the chromatogram obtained with reference solution (b)
Appearance : white or almost white powder. (0.05 per cent) ; disregard any peak with a relative retention
with reference to fentanyl of 0.05 or less.
Solubility : soluble in water, freely soluble in methanol,
sparingly soluble in ethanol (96 per cent). Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in vacuo at 60 °C.
mp : about 152 °C, with decomposition.
ASSAY
IDENTIFICATION
Dissolve 0.300 g in 50 mL of a mixture of 1 volume of
Infrared absorption spectrophotometry (2.2.24). anhydrous acetic acid R and 7 volumes of methyl ethyl
Comparison : Ph. Eur. reference spectrum of fentanyl citrate. ketone R. Titrate with 0.1 M perchloric acid, using 0.2 mL of
naphtholbenzein solution R as indicator.
TESTS 1 mL of 0.1 M perchloric acid is equivalent to 52.86 mg
Appearance of solution. The solution is clear (2.2.1) and of C28H36N2O8.
colourless (2.2.2, Method II).
STORAGE
Dissolve 0.2 g of the substance to be examined in water R and
dilute to 20 mL with the same solvent. Protected from light.
Related substances. Liquid chromatography (2.2.29). IMPURITIES
Test solution. Dissolve 0.100 g of the substance to be examined Specified impurities : A, B, C, D.
in methanol R and dilute to 10.0 mL with the same solvent. Other detectable impurities (the following substances would,
Reference solution (a). Dissolve 10 mg of fentanyl for system if present at a sufficient level, be detected by one or other of
suitability CRS (containing impurities A, B, C and D) in the tests in the monograph. They are limited by the general
1.0 mL of methanol R. acceptance criterion for other/unspecified impurities and/or
Reference solution (b). Dilute 1.0 mL of the test solution to by the general monograph Substances for pharmaceutical
100.0 mL with methanol R. Dilute 1.0 mL of this solution to use (2034). It is therefore not necessary to identify these
10.0 mL with methanol R. impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use) : E.
Column :
– size : l = 0.1 m, Ø = 3.0 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (3 μm).
Mobile phase :
– mobile phase A : 5 g/L solution of ammonium carbonate R
in a mixture of 10 volumes of tetrahydrofuran R and
90 volumes of water R ;
– mobile phase B : acetonitrile R1 ; A. N-phenyl-N-[cis,trans-1-oxido-1-(2-phenylethyl)piperi-
din-4-yl]propanamide,
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0 - 15 90 → 40 10 → 60

15 - 20 40 60

Flow rate : 0.64 mL/min.

Detection : spectrophotometer at 220 nm.
Injection : 10 μL. B. N-phenyl-N-(piperidin-4-yl)propanamide,

General Notices (1) apply to all monographs and other texts 2223
Fenticonazole nitrate EUROPEAN PHARMACOPOEIA 8.0

C. Infrared absorption spectrophotometry (2.2.24).

Comparison: fenticonazole nitrate CRS.
D. It gives the reaction of nitrates (2.3.1).
TESTS
Optical rotation (2.2.7) : − 0.10° to + 0.10°.
Dissolve 0.10 g in methanol R and dilute to 10.0 mL with the
same solvent.
C. N-phenyl-N-[1-(2-phenylethyl)piperidin-4-yl]acetamide,
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 25.0 mg of the substance to be
examined in the mobile phase and dilute to 25.0 mL with the
mobile phase.
Reference solution (a). Dilute 1.0 mL of the test solution to
200.0 mL with the mobile phase.
Reference solution (b). Dilute 10.0 mL of reference solution (a)
to 25.0 mL with the mobile phase.
D. N-phenyl-1-(2-phenylethyl)piperidin-4-amine, Reference solution (c). Dilute 1.0 mL of reference solution (a)
to 10.0 mL with the mobile phase.
Reference solution (d). To 5 mL of the test solution add 5.0 mg
of fenticonazole impurity D CRS, dissolve in the mobile phase
and dilute to 100.0 mL with the mobile phase. Dilute 2.0 mL
of this solution to 10.0 mL with the mobile phase.
E. benzaldehyde. Column :
– size : l = 0.25 m, Ø = 4 mm ;
01/2008:1211 – stationary phase : octadecylsilyl silica gel for
corrected 6.0 chromatography R (5-10 μm).
Mobile phase : mix 70 volumes of acetonitrile R1 and
FENTICONAZOLE NITRATE 30 volumes of a phosphate buffer solution prepared by
dissolving 3.4 g of potassium dihydrogen phosphate R in
900 mL of water R, adjusting to pH 3.0 with phosphoric acid R
Fenticonazoli nitras and diluting to 1000 mL with water R.
Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 229 nm.
Injection : 10 μL.
Run time : 5.5 times the retention time of fenticonazole.
System suitability :
– resolution : minimum 2.0 between the peaks due to
impurity D and fenticonazole in the chromatogram
obtained with reference solution (d) ;
C24H21Cl2N3O4S Mr 518.4 – signal-to-noise ratio : minimum 5 for the principal peak in
[73151-29-8] the chromatogram obtained with reference solution (c).
DEFINITION Limits :
1-[(2RS)-2-(2,4-Dichlorophenyl)-2-[[4-(phenylsulfanyl)- – impurities A, B, C, D, E : for each impurity, not more
benzyl]oxy]ethyl]-1H-imidazole nitrate. than the area of the principal peak in the chromatogram
Content : 99.0 per cent to 101.0 per cent (dried substance). obtained with reference solution (b) (0.2 per cent) ;
– total : not more than the area of the principal peak in the
CHARACTERS chromatogram obtained with reference solution (a) (0.5 per
Appearance : white or almost white, crystalline powder. cent) ;
Solubility : practically insoluble in water, freely soluble in – disregard limit : the area of the principal peak in the
dimethylformamide and in methanol, sparingly soluble in chromatogram obtained with reference solution (c)
anhydrous ethanol. (0.05 per cent) ; disregard the peak due to the nitric ion
(which corresponds to the dead volume of the column).
IDENTIFICATION
Toluene. Head-space gas chromatography (2.2.28) : use the
First identification : C, D. standard additions method.
Second identification : A, B, D. Test solution. Disperse 0.2 g of the substance to be examined
A. Melting point (2.2.14) : 134 °C to 137 °C. in a 10 mL vial with 5 mL of water R.
B. Ultraviolet and visible absorption spectrophotometry Reference solution. Mix 4 mg of toluene R with water R and
(2.2.25). dilute to 1000 mL with the same solvent. Place 5 mL of this
Test solution. Dissolve 20.0 mg in anhydrous ethanol R and solution in a 10 mL vial.
dilute to 100.0 mL with the same solvent. Dilute 1.0 mL of Column :
this solution to 10.0 mL with anhydrous ethanol R. – size : l = 25 m, Ø = 0.32 mm ;
Spectral range : 230-350 nm. – stationary phase : poly(cyanopropyl)(7)(phenyl)(7)-
Absorption maximum : at 252 nm. (methyl)(86)siloxane R (film thickness 1.2 μm).
Shoulder : at about 270 nm. Carrier gas : helium for chromatography R.
Absorption minimum : at 236 nm. Split ratio : 1:25.
Specific absorbance at the absorption maximum : 260 to 280. Column head pressure : 40 kPa.

2224 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ferric chloride hexahydrate

Static head-space conditions which may be used :

– equilibration temperature : 90 °C ;
– equilibration time : 1 h.
Temperature :
– column : 80 °C ;
– injection port : 180 °C ;
– detector : 220 °C.
Detection : flame ionisation. E. (RS)-1-[2-(2,4-dichlorophenyl)-2-[4-(phenylsulfanyl)-
benzyloxy]ethyl]-3-[4-(phenylsulfanyl)benzyl]imidazolium
Injection : 1 mL of the gaseous phase. nitrate.
Limit :
– toluene : maximum 100 ppm. 01/2008:1515
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in vacuo at 60 °C. FERRIC CHLORIDE HEXAHYDRATE
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on Ferri chloridum hexahydricum
1.0 g.
FeCl3,6H2O Mr 270.3
ASSAY
[10025-77-1]
Dissolve 0.450 g in 50 mL of a mixture of equal volumes of
anhydrous acetic acid R and methyl ethyl ketone R. Titrate DEFINITION
with 0.1 M perchloric acid, determining the end-point Content : 98.0 per cent to 102.0 per cent.
potentiometrically (2.2.20).
CHARACTERS
1 mL of 0.1 M perchloric acid is equivalent to 51.84 mg
of C24H21Cl2N3O4S. Appearance : crystalline mass or orange-yellow or
brownish-yellow crystals, very hygroscopic.
STORAGE Solubility : very soluble in water and in ethanol (96 per cent),
Protected from light. freely soluble in glycerol.
IDENTIFICATION
IMPURITIES
A. It gives reaction (a) of chlorides (2.3.1).
Specified impurities : A, B, C, D, E.
B. It gives reaction (c) of iron (2.3.1).
TESTS
Solution S. Dissolve 10 g in distilled water R and dilute to
100 mL with the same solvent.
Acidity. In a suitable polyethylene container, dissolve 3.0 g of
potassium fluoride R in 15 mL of water R. Titrate with 0.1 M
sodium hydroxide using 0.1 mL of phenolphthalein solution R
as indicator until a pink colour is obtained. Add 10 mL of
solution S and allow to stand for 3 h. Filter and use 12.5 mL of
A. (RS)-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanol, the filtrate. Not more than 0.30 mL of 0.1 M sodium hydroxide
is required to change the colour of the indicator to pink.
Free chlorine. Heat 5 mL of solution S. The vapour does not
turn starch iodide paper R blue.
Sulfates (2.4.13) : maximum 100 ppm.
Heat 15 mL of solution S on a water-bath and add 5 mL of
strong sodium hydroxide solution R. Allow to cool and filter.
Neutralise the filtrate to blue litmus paper R using hydrochloric
acid R1 and evaporate to 15 mL.
Ferrous ions : maximum 50 ppm.
B. X = SO : 1-[(2RS)-2-(2,4-dichlorophenyl)-2-[[4- To 10 mL of solution S, add 1 mL of water R, and 0.05 mL
(phenylsulfinyl)benzyl]oxy]ethyl]-1H-imidazole, of potassium ferricyanide solution R followed by 4 mL of
phosphoric acid R. After 10 min, any blue colour in the
C. X = SO2 : 1-[(2RS)-2-(2,4-dichlorophenyl)-2-[[4- solution is not more intense than that in a standard prepared
(phenylsulfonyl)benzyl]oxy]ethyl]-1H-imidazole, at the same time and in the same manner using 10 mL of
water R and 1 mL of a freshly prepared 0.250 g/L solution of
ferrous sulfate R.
Heavy metals (2.4.8) : maximum 50 ppm.
Dissolve 1.0 g in 10 mL of hydrochloric acid R1. Add 2 mL
of strong hydrogen peroxide solution R, then evaporate to
5 mL. Allow to cool and dilute to 20 mL with hydrochloric
acid R1 and transfer the solution to a separating funnel. Shake
3 times, for 3 min each time, with 20 mL of methyl isobutyl
ketone R1. Separate the lower phase, reduce to half its volume
by evaporation and dilute to 25 mL with water R. Neutralise
D. (RS)-1-[2-(2,4-dichlorophenyl)-2-hydroxyethyl]-3-[4- 10 mL of the solution with dilute ammonia R1 to red litmus
(phenylsulfanyl)benzyl]imidazolium nitrate, paper R and dilute to 20 mL with water R. 12 mL of the

General Notices (1) apply to all monographs and other texts 2225
Ferrous fumarate EUROPEAN PHARMACOPOEIA 8.0

solution complies with test A. Prepare the reference solution TESTS

using lead standard solution (1 ppm Pb) R. Solution S. Dissolve 2.0 g in a mixture of 10 mL of lead-free
ASSAY hydrochloric acid R and 80 mL of water R, heating slightly
if necessary. Allow to cool, filter if necessary and dilute to
In a conical flask with a ground-glass stopper, dissolve 0.200 g 100 mL with water R.
in 20 mL of water R. Add 10 mL of dilute hydrochloric acid R
and 2 g of potassium iodide R. Allow the stoppered flask to Sulfates (2.4.13) : maximum 0.2 per cent.
stand for 1 h protected from light. Titrate with 0.1 M sodium Heat 0.15 g with 8 mL of dilute hydrochloric acid R and 20 mL
thiosulfate, adding 5 mL of starch solution R towards the end of distilled water R. Cool in iced water, filter and dilute to
of the titration. 30 mL with distilled water R.
1 mL of 0.1 M sodium thiosulfate is equivalent to 27.03 mg Arsenic (2.4.2, Method A) : maximum 5 ppm.
of FeCl3,6H2O.
Mix 1.0 g with 15 mL of water R and 15 mL of sulfuric acid R.
STORAGE Warm to precipitate the fumaric acid completely. Cool and
add 30 mL of water R. Filter. Wash the precipitate with
In an airtight container, protected from light.
water R. Dilute the combined filtrate and washings to 125 mL
with water R. 25 mL of the solution complies with the test.
Ferric ion : maximum 2.0 per cent.
01/2008:0902
corrected 7.0 In a flask with a ground-glass stopper, dissolve 3.0 g in a
mixture of 10 mL of hydrochloric acid R and 100 mL of water R
by heating rapidly to boiling. Boil for 15 s. Cool rapidly, add
FERROUS FUMARATE 3 g of potassium iodide R, stopper the flask and allow to stand
protected from light for 15 min. Add 2 mL of starch solution R
Ferrosi fumaras as indicator. Titrate the liberated iodine with 0.1 M sodium
thiosulfate. Carry out a blank test. The difference between
the volumes used in the 2 titrations corresponds to the amount
of iodine liberated by ferric ion.
C4H2FeO4 Mr 169.9 1 mL of 0.1 M sodium thiosulfate is equivalent to 5.585 mg
[141-01-5] of ferric ion.
DEFINITION Cadmium : maximum 10 ppm.
Iron(II) (E)-butenedioate. Atomic absorption spectrometry (2.2.23, Method I).
Content : 93.0 per cent to 101.0 per cent (dried substance). Test solution. Solution S.
Reference solutions. Prepare the reference solutions using
CHARACTERS cadmium standard solution (0.1 per cent Cd) R and diluting
Appearance : fine, reddish-orange or reddish-brown powder. with a 10 per cent V/V solution of lead-free hydrochloric acid R.
Solubility : slightly soluble in water, very slightly soluble in Source : cadmium hollow-cathode lamp.
ethanol (96 per cent). Wavelength : 228.8 nm.
IDENTIFICATION Atomisation device : air-acetylene flame.
A. Thin-layer chromatography (2.2.27). Chromium : maximum 200 ppm.
Test solution. To 1.0 g add 25 mL of a mixture of Atomic absorption spectrometry (2.2.23, Method I).
equal volumes of hydrochloric acid R and water R and heat Test solution. Solution S.
on a water-bath for 15 min. Cool and filter. Use the filtrate
for identification test C. Wash the residue with 50 mL of Reference solutions. Prepare the reference solutions using
a mixture of 1 volume of dilute hydrochloric acid R and chromium standard solution (0.1 per cent Cr) R and diluting
9 volumes of water R and discard the washings. Dry the with a 10 per cent V/V solution of lead-free hydrochloric acid R.
residue at 100-105 °C. Dissolve 20 mg of the residue in Source : chromium hollow-cathode lamp.
acetone R and dilute to 10 mL with the same solvent. Wavelength : 357.9 nm.
Reference solution. Dissolve 20 mg of fumaric acid CRS in Atomisation device : air-acetylene flame.
acetone R and dilute to 10 mL with the same solvent.
Lead : maximum 20 ppm.
Plate : TLC silica gel F254 plate R.
Atomic absorption spectrometry (2.2.23, Method I).
Mobile phase : anhydrous formic acid R, methylene
chloride R, butanol R, heptane R (12:16:32:44 V/V/V/V). Test solution. Solution S.
Application : 5 μL. Reference solutions. Prepare the reference solutions using lead
Development : in an unsaturated tank, over a path of 10 cm. standard solution (10 ppm Pb) R and diluting with a 10 per
cent V/V solution of lead-free hydrochloric acid R.
Drying : at 105 °C for 15 min.
Source : lead hollow-cathode lamp.
Detection : examine in ultraviolet light at 254 nm.
Wavelength : 283.3 nm.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to the Atomisation device : air-acetylene flame.
principal spot in the chromatogram obtained with the Mercury : maximum 1 ppm.
reference solution. Atomic absorption spectrometry (2.2.23, Method I).
B. Mix 0.5 g with 1 g of resorcinol R. To 0.5 g of the mixture in Test solution. Solution S.
a crucible add 0.15 mL of sulfuric acid R and heat gently.
A dark red semi-solid mass is formed. Add the mass, Reference solutions. Prepare the reference solutions using
with care, to 100 mL of water R. An orange-yellow colour mercury standard solution (10 ppm Hg) R and diluting with a
develops and the solution shows no fluorescence. 25 per cent V/V solution of lead-free hydrochloric acid R.
C. The filtrate obtained during preparation of the test solution Source : mercury hollow-cathode lamp.
in identification test A gives reaction (a) of iron (2.3.1). Wavelength : 253.7 nm.

2226 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ferrous gluconate

Following the recommendations of the manufacturer, Test solution. Dissolve 20 mg of the substance to be
introduce 5 mL of solution S or 5 mL of the reference solutions examined in 2 mL of water R, heating if necessary in a
into the reaction vessel of the cold-vapour mercury assay water-bath at 60 °C.
accessory, add 10 mL of water R and 1 mL of stannous chloride Reference solution. Dissolve 20 mg of ferrous gluconate CRS
solution R1. in 2 mL of water R, heating if necessary in a water-bath
Nickel : maximum 200 ppm. at 60 °C.
Atomic absorption spectrometry (2.2.23, Method I). Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel
plate R (2-10 μm)].
Test solution. Solution S.
Mobile phase : concentrated ammonia R, ethyl acetate R,
Reference solutions. Prepare the reference solutions using water R, ethanol (96 per cent) R (10:10:30:50 V/V/V/V).
nickel standard solution (10 ppm Ni) R and diluting with a
10 per cent V/V solution of lead-free hydrochloric acid R. Application : 1 μL.
Source : nickel hollow-cathode lamp. Development : over 2/3 of the plate.
Drying : at 105 °C for 20 min ; allow to cool.
Wavelength : 232 nm.
Detection : spray with a solution containing 10 g/L of cerium
Atomisation device : air-acetylene flame. sulfate R and 25 g/L of ammonium molybdate R in dilute
Zinc : maximum 500 ppm. sulfuric acid R and heat at 105 °C for about 10 min.
Atomic absorption spectrometry (2.2.23, Method I). Results : after 5 min, the principal spot in the chromatogram
Test solution. Solution S diluted to 10 volumes. obtained with the test solution is similar in position,
colour and size to the principal spot in the chromatogram
Reference solutions. Prepare the reference solutions using zinc
obtained with the reference solution.
standard solution (10 ppm Zn) R and diluting with a 1 per
cent V/V solution of lead-free hydrochloric acid R. B. 1 mL of solution S (see Tests) gives reaction (a) of iron
(2.3.1).
Source : zinc hollow-cathode lamp.
Wavelength : 213.9 nm. TESTS
Atomisation device : air-acetylene flame. Solution S. Dissolve 5.0 g in carbon dioxide-free water R
Loss on drying (2.2.32) : maximum 1.0 per cent, determined prepared from distilled water R and heated to about 60 °C,
on 1.000 g by drying in an oven at 105 °C. allow to cool and dilute to 50 mL with carbon dioxide-free
water R prepared from distilled water R.
ASSAY Appearance of solution. The solution is clear (2.2.1).
Dissolve with slight heating 0.150 g in 7.5 mL of dilute sulfuric Dilute 2 mL of solution S to 10 mL with water R. Examine
acid R. Cool and add 25 mL of water R. Add 0.1 mL of the solution against the light.
ferroin R. Titrate immediately with 0.1 M cerium sulfate until pH (2.2.3) : 4.0 to 5.5 for solution S, measured 3-4 h after
the colour changes from orange to light bluish-green. preparation.
1 mL of 0.1 M cerium sulfate is equivalent to 16.99 mg
Sucrose and reducing sugars. Dissolve 0.5 g in 10 mL of
of C4H2FeO4.
warm water R and add 1 mL of dilute ammonia R1. Pass
STORAGE hydrogen sulfide R through the solution and allow to stand
for 30 min. Filter and wash the precipitate with 2 quantities,
In an airtight container, protected from light.
each of 5 mL, of water R. Acidify the combined filtrate and
washings to blue litmus paper R with dilute hydrochloric
acid R and add 2 mL in excess. Boil until the vapour no longer
01/2013:0493 darkens lead acetate paper R and continue boiling, if necessary,
until the volume is reduced to about 10 mL. Cool, add 15 mL
FERROUS GLUCONATE of sodium carbonate solution R, allow to stand for 5 min and
filter. Dilute the filtrate to 100 mL with water R. To 5 mL of
this solution add 2 mL of cupri-tartaric solution R and boil for
Ferrosi gluconas 1 min. Allow to stand for 1 min. No red precipitate is formed.
Chlorides (2.4.4) : maximum 0.06 per cent.
Dilute 0.8 mL of solution S to 15 mL with water R.
Oxalates. Dissolve 5.0 g in a mixture of 10 mL of dilute
sulfuric acid R and 40 mL of water R. Shake the solution with
50 mL of ether R for 5 min. Separate the aqueous layer and
C12H22FeO14,xH2O Mr 446.1 (anhydrous substance) shake it with 20 mL of ether R for 5 min. Combine the ether
layers, evaporate to dryness and dissolve the residue in 15 mL
DEFINITION of water R. Filter, boil the filtrate until the volume is reduced
Iron(II) bis[(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoate] to 5 mL and add 1 mL of dilute acetic acid R and 1.5 mL of
(iron(II) di(D-gluconate)). calcium chloride solution R. Allow to stand for 30 min. No
precipitate is formed.
Content : 11.8 per cent to 12.5 per cent of iron(II) (dried
substance). Sulfates (2.4.13) : maximum 500 ppm.
It contains a variable quantity of water. To 3.0 mL of solution S add 3 mL of acetic acid R and dilute
to 15 mL with distilled water R. Examine the solutions against
CHARACTERS the light.
Appearance : greenish-yellow or grey powder or granules. Arsenic (2.4.2, Method A) : maximum 2 ppm, determined on
Solubility : freely but slowly soluble in water giving a 0.5 g.
greenish-brown solution, more readily soluble in hot water, Barium. Dilute 10 mL of solution S to 50 mL with distilled
practically insoluble in ethanol (96 per cent). water R and add 5 mL of dilute sulfuric acid R. Allow to stand
for 5 min. Any opalescence in the solution is not more intense
IDENTIFICATION than that in a mixture of 10 mL of solution S and 45 mL of
A. Thin-layer chromatography (2.2.27). distilled water R.

General Notices (1) apply to all monographs and other texts 2227
Ferrous sulfate, dried EUROPEAN PHARMACOPOEIA 8.0

Ferric ions : maximum 1.0 per cent. TESTS

In a ground-glass-stoppered flask, dissolve 5.00 g in a mixture Solution S. Dissolve 2.00 g in a 5 per cent V/V solution of
of 10 mL of hydrochloric acid R and 100 mL of carbon lead-free nitric acid R and dilute to 100.0 mL with the same
dioxide-free water R. Add 3 g of potassium iodide R, close acid.
the flask and allow to stand protected from light for 5 min. pH (2.2.3) : 3.0 to 4.0.
Titrate with 0.1 M sodium thiosulfate, using 0.5 mL of starch
solution R, added towards the end of the titration, as indicator. Dissolve 1.0 g in carbon dioxide-free water R and dilute to
Carry out a blank titration. Not more than 9.0 mL of 0.1 M 20 mL with the same solvent.
sodium thiosulfate is used. Chlorides (2.4.4) : maximum 300 ppm.
Heavy metals (2.4.8) : maximum 20 ppm. Dissolve 2.5 g in water R, add 0.5 mL of dilute sulfuric acid R
Thoroughly mix 2.5 g with 0.5 g of magnesium oxide R1 in and dilute to 50 mL with water R. Dilute 3.3 mL of this solution
a silica crucible. Ignite to dull redness until a homogeneous to 10 mL with water R and add 5 mL of dilute nitric acid R.
mass is obtained. Heat at 800 ± 50 °C for about 1 h, allow to Prepare the standard using a mixture of 10 mL of chloride
cool and take up the residue in 20 mL of hot hydrochloric standard solution (5 ppm Cl) R and 5 mL of dilute nitric acid R.
acid R. Allow to cool. Transfer the liquid to a separating Use 0.15 mL of silver nitrate solution R2 in this test.
funnel and shake for 3 min with 3 quantities, each of 20 mL, Chromium : maximum 100 ppm.
of methyl isobutyl ketone saturated with hydrochloric acid Atomic absorption spectrometry (2.2.23, Method II).
(prepared by shaking 100 mL of freshly distilled methyl Test solution. Solution S.
isobutyl ketone R with 1 mL of hydrochloric acid R). Allow to
stand, separate the aqueous layer, reduce to half its volume Reference solutions. Prepare the reference solutions using
by boiling, allow to cool and dilute to 25 mL with water R. chromium standard solution (100 ppm Cr) R, diluted as
Neutralise 10 mL of this solution to red litmus paper R with necessary with a 5 per cent V/V solution of lead-free nitric
dilute ammonia R1 and dilute to 20 mL with water R. 12 mL acid R.
of the solution complies with test A. Prepare the reference Source : chromium hollow-cathode lamp using a transmission
solution using lead standard solution (1 ppm Pb) R. band preferably of 1 nm.
Loss on drying (2.2.32) : 5.0 per cent to 10.5 per cent, Wavelength : 357.9 nm.
determined on 0.500 g by drying in an oven at 105 °C for 5 h. Atomisation device : air-acetylene flame.
Microbial contamination Copper : maximum 50 ppm.
TAMC : acceptance criterion 103 CFU/g (2.6.12). Atomic absorption spectrometry (2.2.23, Method II).
TYMC : acceptance criterion 102 CFU/g (2.6.12). Test solution. Solution S.
Reference solutions. Prepare the reference solutions using
ASSAY copper standard solution (0.1 per cent Cu) R, diluted as
Dissolve 0.5 g of sodium hydrogen carbonate R in a mixture of necessary with a 5 per cent V/V solution of lead-free nitric
30 mL of dilute sulfuric acid R and 70 mL of water R. When acid R.
the effervescence stops, dissolve 1.00 g of the substance to be Source : copper hollow-cathode lamp using a transmission
examined with gentle shaking. Using 0.1 mL of ferroin R as band preferably of 1 nm.
indicator, titrate with 0.1 M ammonium and cerium nitrate
Wavelength : 324.7 nm.
until the red colour disappears.
Atomisation device : air-acetylene flame.
1 mL of 0.1 M ammonium and cerium nitrate is equivalent
to 5.585 mg of iron(II). Ferric ions : maximum 0.5 per cent.
In a ground-glass-stoppered flask, dissolve 5.00 g in a mixture
STORAGE of 10 mL of hydrochloric acid R and 100 mL of carbon
Protected from light. dioxide-free water R. Add 3 g of potassium iodide R, close the
flask and allow to stand in the dark for 5 min. Titrate the
liberated iodine with 0.1 M sodium thiosulfate, using 0.5 mL
01/2008:2340 of starch solution R, added towards the end of titration, as
corrected 7.2 indicator. Carry out a blank test in the same conditions. Not
more than 4.5 mL of 0.1 M sodium thiosulfate is used.
FERROUS SULFATE, DRIED Manganese : maximum 0.1 per cent.
Atomic absorption spectrometry (2.2.23, Method II).
Ferrosi sulfas desiccatus Test solution. Dilute 1.0 mL of solution S to 20.0 mL with a
5 per cent V/V solution of lead-free nitric acid R.
FeSO4 Mr 151.9 Reference solutions. Prepare the reference solutions using
manganese standard solution (1000 ppm Mn) R, diluted as
DEFINITION
necessary with a 5 per cent V/V solution of lead-free nitric
Hydrated ferrous sulfate from which part of the water of acid R.
hydration has been removed by drying.
Source : manganese hollow-cathode lamp using a transmission
Content : 86.0 per cent to 90.0 per cent. band preferably of 1 nm.
CHARACTERS Wavelength : 279.5 nm.
Appearance : greyish-white powder. Atomisation device : air-acetylene flame.
Solubility : slowly but freely soluble in water, very soluble in Nickel : maximum 100 ppm.
boiling water, practically insoluble in ethanol (96 per cent). Atomic absorption spectrometry (2.2.23, Method II).
It is oxidised in moist air, becoming brown. Test solution. Solution S.
Reference solutions. Prepare the reference solutions using
IDENTIFICATION
nickel standard solution (10 ppm Ni) R, diluted as necessary
A. It gives the reactions of sulfates (2.3.1). with a 5 per cent V/V solution of lead-free nitric acid R.
B. It gives reaction (a) of iron (2.3.1). Source : nickel hollow-cathode lamp using a transmission band
C. It complies with the limits of the assay. preferably of 1 nm.

2228 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Ferrous sulfate heptahydrate

Wavelength : 232.0 nm. Reference solutions. Prepare the reference solutions using
Atomisation device : air-acetylene flame. chromium standard solution (100 ppm Cr) R, diluting with a
5 per cent V/V solution of lead-free nitric acid R.
Zinc : maximum 100 ppm.
Source : chromium hollow-cathode lamp using a transmission
Atomic absorption spectrometry (2.2.23, Method II). band preferably of 1 nm.
Test solution. Solution S. Wavelength : 357.9 nm.
Reference solutions. Prepare the reference solutions using zinc Atomisation device : air-acetylene flame.
standard solution (100 ppm Zn) R, diluted as necessary with a
5 per cent V/V solution of lead-free nitric acid R. Copper : maximum 50 ppm.
Source : zinc hollow-cathode lamp using a transmission band Atomic absorption spectrometry (2.2.23, Method II).
preferably of 1 nm. Test solution. Solution S.
Wavelength : 213.9 nm. Reference solutions. Prepare the reference solutions using
Atomisation device : air-acetylene flame. copper standard solution (0.1 per cent Cu) R, diluting with a
5 per cent V/V solution of lead-free nitric acid R.
ASSAY Source : copper hollow-cathode lamp using a transmission
Dissolve 2.5 g of sodium hydrogen carbonate R in a mixture band preferably of 1 nm.
of 150 mL of water R and 10 mL of sulfuric acid R. When Wavelength : 324.7 nm.
the effervescence ceases, add to the solution 0.140 g of the Atomisation device : air-acetylene flame.
substance to be examined and dissolve with gentle shaking.
Add 0.1 mL of ferroin R and titrate with 0.1 M ammonium Ferric ions : maximum 0.3 per cent.
and cerium nitrate until the red colour disappears. In a ground-glass-stoppered flask, dissolve 5.00 g in a mixture
1 mL of 0.1 M ammonium and cerium nitrate is equivalent of 10 mL of hydrochloric acid R and 100 mL of carbon
to 15.19 mg of FeSO4. dioxide-free water R. Add 3 g of potassium iodide R, close the
flask and allow to stand in the dark for 5 min. Titrate the
STORAGE liberated iodine with 0.1 M sodium thiosulfate, using 0.5 mL
In an airtight container. of starch solution R, added towards the end of the titration, as
indicator. Carry out a blank test in the same conditions. Not
01/2010:0083 more than 2.7 mL of 0.1 M sodium thiosulfate is used, taking
corrected 7.2 into account the blank titration.
Manganese : maximum 0.1 per cent.
FERROUS SULFATE HEPTAHYDRATE Atomic absorption spectrometry (2.2.23, Method II).
Test solution. Dilute 1.0 mL of solution S to 20.0 mL with a
Ferrosi sulfas heptahydricus 5 per cent V/V solution of lead-free nitric acid R.
Reference solutions. Prepare the reference solutions using
FeSO4,7H2O Mr 278.0 manganese standard solution (1000 ppm Mn) R, diluting with
[7782-63-0] a 5 per cent V/V solution of lead-free nitric acid R.
Source : manganese hollow-cathode lamp using a transmission
DEFINITION
band preferably of 1 nm.
Content : 98.0 per cent to 105.0 per cent. Wavelength : 279.5 nm.
CHARACTERS Atomisation device : air-acetylene flame.
Appearance : light green, crystalline powder or bluish-green Nickel : maximum 50 ppm.
crystals, efflorescent in air. Atomic absorption spectrometry (2.2.23, Method II).
Solubility : freely soluble in water, very soluble in boiling water, Test solution. Solution S.
practically insoluble in ethanol (96 per cent). Reference solutions. Prepare the reference solutions using
Ferrous sulfate heptahydrate is oxidised in moist air, becoming nickel standard solution (10 ppm Ni) R, diluting with a 5 per
brown. cent V/V solution of lead-free nitric acid R.
IDENTIFICATION Source : nickel hollow-cathode lamp using a transmission band
preferably of 1 nm.
A. It gives the reactions of sulfates (2.3.1).
Wavelength : 232.0 nm.
B. It gives reaction (a) of iron (2.3.1).
Atomisation device : air-acetylene flame.
C. It complies with the limits of the assay.
Zinc : maximum 50 ppm.
TESTS Atomic absorption spectrometry (2.2.23, Method II).
Solution S. Dissolve 4.0 g in a 5 per cent V/V solution of Test solution. Solution S.
lead-free nitric acid R and dilute to 100.0 mL with the same Reference solutions. Prepare the reference solutions using
solution. zinc standard solution (100 ppm Zn) R, diluting with a 5 per
pH (2.2.3) : 3.0 to 4.0. cent V/V solution of lead-free nitric acid R.
Dissolve 1.0 g in carbon dioxide-free water R and dilute to Source : zinc hollow-cathode lamp using a transmission band
20 mL with the same solvent. preferably of 1 nm.
Chlorides (2.4.4): maximum 200 ppm. Wavelength : 213.9 nm.
Dilute 5 mL of solution S to 10 mL with water R and add 5 mL Atomisation device : air-acetylene flame.
of dilute nitric acid R. Prepare the standard with a mixture
of 2 mL of water R, 5 mL of dilute nitric acid R and 8 mL of ASSAY
chloride standard solution (5 ppm Cl) R. Use 0.15 mL of silver Dissolve 2.5 g of sodium hydrogen carbonate R in a mixture
nitrate solution R2 in this test. of 150 mL of water R and 10 mL of sulfuric acid R. When
the effervescence ceases add to the solution 0.500 g of the
Chromium : maximum 50 ppm. substance to be examined and dissolve with gentle swirling.
Atomic absorption spectrometry (2.2.23, Method II). Add 0.1 mL of ferroin R and titrate with 0.1 M ammonium
Test solution. Solution S. and cerium nitrate until the red colour disappears.

General Notices (1) apply to all monographs and other texts 2229
Fexofenadine hydrochloride EUROPEAN PHARMACOPOEIA 8.0

1 mL of 0.1 M ammonium and cerium nitrate is equivalent Detection : spectrophotometer at 220 nm.
to 27.80 mg of FeSO4,7H2O. Injection : 20 μL.
STORAGE Run time : 1.2 times the retention time of fexofenadine.
In an airtight container. Relative retention with reference to fexofenadine (retention
time = about 20 min) : impurity B = about 0.7.
01/2008:2280 System suitability : reference solution (a) :
– resolution : minimum 3.0 between the peaks due to
FEXOFENADINE HYDROCHLORIDE fexofenadine and impurity B.
Limits :
Fexofenadini hydrochloridum – correction factor : for the calculation of content, multiply
the peak area of impurity B by 1.3 ;
– impurity B : not more than the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.1 per cent).
Related substances. Liquid chromatography (2.2.29).
Buffer solution. Dissolve 6.64 g of sodium dihydrogen
phosphate monohydrate R and 0.84 g of sodium perchlorate R
in water for chromatography R, adjust to pH 2.0 ± 0.1 with
C32H40ClNO4 Mr 538.1 phosphoric acid R and dilute to 1000 mL with water for
[153439-40-8] chromatography R.
Solvent mixture. Mix equal volumes of acetonitrile for
DEFINITION chromatography R and the buffer solution.
2-[4-[(1RS)-1-hydroxy-4-[4-(hydroxydiphenylmethyl)- Test solution (a). Dissolve 25.0 mg of the substance to be
piperidin-1-yl]butyl]phenyl]-2-methylpropanoic acid examined in 25.0 mL of the solvent mixture.
hydrochloride.
Test solution (b). Dilute 3.0 mL of test solution (a) to 50.0 mL
Content : 98.0 per cent to 102.0 per cent (anhydrous substance). with the mobile phase.
CHARACTERS Reference solution (a). Dissolve 25.0 mg of fexofenadine
Appearance : white or almost white powder. hydrochloride CRS in the solvent mixture and dilute to 25.0 mL
with the solvent mixture. Dilute 3.0 mL of this solution to
Solubility : slightly soluble in water, freely soluble in methanol, 50.0 mL with the mobile phase.
very slightly soluble in acetone.
Reference solution (b). Dilute 1.0 mL of test solution (a) to
It shows polymorphism (5.9).
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution
IDENTIFICATION to 10.0 mL with the mobile phase.
A. Infrared absorption spectrophotometry (2.2.24). Reference solution (c). Dissolve 1 mg each of fexofenadine
Comparison : fexofenadine hydrochloride CRS. impurity A CRS and fexofenadine impurity C CRS in 20 mL of
reference solution (a) and dilute to 200.0 mL with the mobile
It the spectra obtained in the solid state show differences, phase.
dissolve the substance to be examined and the reference
substance separately in methanol R, evaporate to dryness Column :
and record new spectra using the residues. – size : l = 0.25 m, Ø = 4.6 mm ;
B. Dissolve 30 mg of the substance to be examined in a – stationary phase : phenylsilyl silica gel for chromatography R
mixture of equal volumes of methanol R and water R ; (5 μm).
sonicate if necessary and dilute to 2 mL with the same Mobile phase : mix 350 volumes of acetonitrile for
mixture of solvents. The solution gives reaction (a) of chromatography R and 650 volumes of the buffer solution ; add
chlorides (2.3.1). 3 volumes of triethylamine R and mix.
TESTS Flow rate : 1.5 mL/min.
Impurity B. Liquid chromatography (2.2.29). Detection : spectrophotometer at 220 nm.
Test solution. Dissolve 50.0 mg of the substance to be Injection : 20 μL of test solution (a) and reference solutions (b)
examined in the mobile phase and dilute to 100.0 mL with and (c).
the mobile phase. Relative retention with reference to fexofenadine
Reference solution (a). Dissolve the contents of a vial of (retention time = about 9 min) : impurity A = about 1.7 ;
fexofenadine impurity B CRS in the test solution and dilute to impurity D = about 2.3 ; impurity C = about 3.2.
2.0 mL with the test solution. Run time : 6 times the retention time of fexofenadine for test
Reference solution (b). Dilute 1.0 mL of the test solution to solution (a) and reference solution (c), twice the retention
100.0 mL with the mobile phase. Dilute 1.0 mL of this solution time of fexofenadine for reference solution (b).
to 10.0 mL with the mobile phase. System suitability : reference solution (c) :
Column : – resolution : minimum 10 between the peaks due to
– size : l = 0.25 m, Ø = 4.6 mm ; fexofenadine and impurity A.
– stationary phase : silica gel BC for chiral chromatography R1 Limits :
(5 μm). – correction factor : for the calculation of content, multiply
Mobile phase : mix 20 volumes of acetonitrile for the peak area of impurity A by 1.4 ;
chromatography R and 80 volumes of a buffer solution – impurities A, C, D : not more than the area of the principal
prepared as follows : to 1.15 mL of glacial acetic acid R add peak in the chromatogram obtained with reference
water for chromatography R, adjust to pH 4.0 ± 0.1 with solution (b) (0.1 per cent) ;
dilute ammonia R1 and dilute to 1000 mL with water for – unspecified impurities : for each impurity, not more than the
chromatography R. area of the principal peak in the chromatogram obtained
Flow rate : 0.5 mL/min. with reference solution (b) (0.10 per cent) ;

2230 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Fibrin sealant kit

– total : not more than 3 times the area of the principal peak D. R = CO-OCH3, R′ = CH3 : methyl 2-[4-[(1RS)-1-hydroxy-4-
in the chromatogram obtained with reference solution (b) [4-(hydroxydiphenylmethyl)piperidin-1-yl]butyl]phenyl]-
(0.3 per cent) ; 2-methylpropanoate,
– disregard limit : 0.5 times the area of the principal peak in F. R = CO2H, R′ = H : 2-[4-[1-hydroxy-4-[4-(hydroxydiphenyl-
the chromatogram obtained with reference solution (b) methyl)piperidin-1-yl]butyl]phenyl]propanoic acid,
(0.05 per cent).
Heavy metals (2.4.8) : maximum 10 ppm.
Dissolve 1.0 g in a mixture of 15 volumes of water R and
85 volumes of methanol R and dilute to 20 mL with the same
mixture of solvents. 12 mL of the solution complies with
test B. Prepare the reference solution using 5 mL of lead
standard solution (1 ppm Pb) R. E. diphenyl(piperidin-4-yl)methanol,
Water (2.5.32): maximum 0.5 per cent.
Dissolve 1.000 g in anhydrous methanol R and dilute to 5.0 mL
with the same solvent. Use 1.0 mL of this solution.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on
1.0 g.
ASSAY
Liquid chromatography (2.2.29) as described in the test for
related substances with the following modifications.
Injection : test solution (b) and reference solution (a). G. 2-[4-[(1RS)-4-[4-(diphenylmethylidene)piperidin-1-yl]-1-
Run time : twice the retention time of fexofenadine. hydroxybutyl]phenyl]-2-methylpropanoic acid.
Calculate the percentage content of fexofenadine
hydrochloride from the declared content of fexofenadine 01/2008:0903
hydrochloride CRS. corrected 7.6
IMPURITIES
FIBRIN SEALANT KIT
Specified impurities : A, B, C, D.
Other detectable impurities (the following substances would, Fibrini glutinum
if present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general DEFINITION
acceptance criterion for other/unspecified impurities and/or Sterile, freeze-dried, frozen or liquid preparation of plasma
by the general monograph Substances for pharmaceutical use protein fractions containing essentially 2 components, namely
(2034). It is therefore not necessary to identify these impurities fibrinogen concentrate (component 1), a protein fraction
for demonstration of compliance. See also 5.10. Control of containing human fibrinogen, and a preparation containing
impurities in substances for pharmaceutical use) : E, F, G. human thrombin (component 2). A fibrin clot is rapidly
formed when the 2 thawed or reconstituted components are
mixed. Other ingredients (for example, human coagulation
factor XIII, a fibrinolysis inhibitor or calcium ions) and
stabilisers (for example, Human albumin solution (0255)) may
be added.
Human constituents are obtained from plasma that
complies with the monograph on Human plasma for
fractionation (0853).
When thawed or reconstituted as stated on the label,
A. 2-[4-[4-[4-(hydroxydiphenylmethyl)piperidin-1- component 1 contains not less than 40 g/L of clottable protein ;
yl]butanoyl]phenyl]-2-methylpropanoic acid, the thrombin activity of component 2 varies over a wide range
(approximately 4-1000 IU/mL).
PRODUCTION
The method of preparation is designed to maintain functional
integrity of the components. It includes a step or steps that
have been shown to remove or to inactivate known agents of
infection ; if substances are used for inactivation of viruses
during production, the subsequent purification procedure
must be validated to demonstrate that the concentration of
B. 2-[3-[(1RS)-1-hydroxy-4-[4-(hydroxydiphenylmethyl)- these substances is reduced to a suitable level and any residues
piperidin-1-yl]butyl]phenyl]-2-methylpropanoic acid, are such as not to compromise the safety of the preparation
for patients.
The constituents or mixtures of constituents are dissolved in
a suitable liquid. No antimicrobial preservative or antibiotic
is added. Constituents or mixtures of constituents are passed
through a bacteria-retentive filter and distributed aseptically
into sterile containers. Containers of freeze-dried constituents
are closed under vacuum or filled with a suitable inert gas,
such as oxygen-free nitrogen, before being closed.
If the human coagulation factor XIII content in component 1
C. R = H, R′ = CH3 : (1RS)-4-[4-(hydroxydiphenylmethyl)- is greater than 10 units/mL, the assay of human coagulation
piperidin-1-yl]-1-[4-(1-methylethyl)phenyl]butan-1-ol, factor XIII is carried out.

General Notices (1) apply to all monographs and other texts 2231
Fibrin sealant kit EUROPEAN PHARMACOPOEIA 8.0

CHARACTERS – detection reagent, containing a suitable factor XIIIa-specific

peptide substrate, such as Leu-Gly-Pro-Gly-Glu-Ser-Lys-
Appearance : Val-Ile-Gly-NH2 and glycine ethyl ester as 2nd substrate in a
– freeze-dried constituents : white or pale yellow, hygroscopic suitable buffer solution ;
powder or friable solid, – NADH reagent, containing glutamate dehydrogenase,
α-ketoglutarate and NADH in a suitable buffer solution.
– frozen constituents : colourless or pale yellow, opaque solid,
After mixing, the absorbance changes (ΔA/min) are measured
– liquid constituents : colourless or pale yellow liquid. at a wavelength of 340 nm, after the linear phase of the
For the freeze-dried or frozen constituents, reconstitute or reaction is reached.
thaw as stated on the label immediately before carrying out the 1 unit of human coagulation factor XIII is equal to the potency
identification and the tests, except those for solubility and water. of 1 mL of human normal plasma.
Calculate the potency of the test preparation by the usual
statistical methods (5.3, for example). The confidence limits
Component 1 (fibrinogen concentrate) (P = 0.95) are not less than 80 per cent and not more than
125 per cent of the estimated potency.
IDENTIFICATION
A. It complies with the limits of the assay of fibrinogen. Component 2 (thrombin preparation)
B. It complies with the limits of the assay of human
IDENTIFICATION
coagulation factor XIII (where applicable).
It complies with the limits of the assay of thrombin.
TESTS
TESTS
Solubility. Freeze-dried concentrates dissolve within 20 min
Solubility. Freeze-dried preparations dissolve within 5 min in
in the volume of liquid and at the temperature stated on the
the volume of liquid stated on the label, forming a colourless,
label, forming an almost colourless, clear or slightly turbid
clear or slightly turbid solution.
solution.
pH (2.2.3) : 5.0 to 8.0.
pH (2.2.3) : 6.5 to 8.0.
Water. Determined by a suitable method, such as semi-micro
Stability of solution. No gel formation appears at determination of water (2.5.12), loss on drying (2.2.32) or
room temperature during 120 min following thawing or near-infrared spectroscopy (2.2.40), the water content is
reconstitution. within the limits approved by the competent authority.
Water. Determined by a suitable method, such as semi-micro Sterility (2.6.1). It complies with the test.
determination of water (2.5.12), loss on drying (2.2.32) or
near-infrared spectroscopy (2.2.40), the water content is
within the limits approved by the competent authority. ASSAY
Sterility (2.6.1). It complies with the test. Thrombin. If necessary, dilute the reconstituted preparation
to be examined to approximately 2-20 IU of thrombin
per millilitre using as diluent a suitable buffer solution
ASSAY (pH 7.3-7.5), such as imidazole buffer solution pH 7.3 R
containing 10 g/L of human albumin R or bovine albumin R.
Fibrinogen (clottable protein). Mix 0.2 mL of the
To a suitable volume of the dilution, add a suitable volume
reconstituted concentrate with 2 mL of a suitable buffer
of fibrinogen solution (1 g/L of clottable protein) warmed to
solution (pH 6.6-7.4) containing sufficient human thrombin R
37 °C and start measurement of the clotting time immediately.
(approximately 3 IU/mL) and calcium (0.05 mol/L). Maintain
Repeat the procedure with each of at least 3 dilutions, in the
at 37 °C for 20 min, separate the precipitate by centrifugation
range stated above, of a reference preparation of thrombin,
at 5000 g for 20 min, wash thoroughly with a 9 g/L solution of
calibrated in International Units.
sodium chloride R and determine the protein as nitrogen by
sulfuric acid digestion (2.5.9). Calculate the clottable protein Calculate the activity of the test preparation by the usual
content by multiplying the result by 6.0. The estimated content statistical methods (5.3, for example). The estimated activity
in milligrams of clottable protein is not less than 70 per cent is not less than 80 per cent and not more than 125 per cent
and not more than 130 per cent of the stated content. If for of the stated activity. The confidence limits (P = 0.95) are
a particular preparation this method cannot be applied, use not less than 80 per cent and not more than 125 per cent of
another validated method for determination of fibrinogen. the estimated activity.
Human coagulation factor XIII. Where the label indicates
that the human coagulation factor XIII potency is greater than STORAGE
10 units/mL, the estimated potency is not less than 80 per cent Protected from light and, for freeze-dried components, in an
and not more than 120 per cent of the stated potency. airtight container.
Make at least 3 suitable dilutions of thawed or reconstituted
concentrate and of human normal plasma (reference LABELLING
preparation) using human coagulation factor XIII-deficient
plasma or another suitable diluent. Add to each dilution The label states :
suitable amounts of the following reagents : – the amount of fibrinogen (milligrams of clottable protein),
thrombin (International Units) per container, and of
– activator reagent, containing bovine or human thrombin,
human coagulation factor XIII, if the latter is greater than
a suitable buffer, calcium chloride and a suitable inhibitor
10 units/mL,
such as Gly-Pro-Arg-Pro-Ala-NH2 which inhibits clotting
of the sample but does not prevent human coagulation – where applicable, the name and volume of liquid to be used
factor XIII activation by thrombin ; to reconstitute the components.

2232 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Filgrastim concentrated solution

07/2010:2206 Test solution. Introduce a volume of the preparation to

corrected 7.6 be examined corresponding to 25 μg of protein into a
polypropylene tube. Add 25 μL of a 0.1 mg/mL solution
FILGRASTIM CONCENTRATED of glutamyl endopeptidase for peptide mapping R. Dilute
to 100 μL with 0.02 M sodium phosphate buffer solution
SOLUTION pH 8.0 R, stopper the tube and incubate at about 37 °C for
17 h. Cool to 2-8 °C until analysis.
Filgrastimi solutio concentrata Reference solution. Prepare at the same time and in the same
manner as for the test solution but using filgrastim CRS
instead of the preparation to be examined.
CHROMATOGRAPHIC SEPARATION. Liquid chromatography
(2.2.29).
Column :
– size : l = 0.10 m, Ø = 2.1 mm ;
C845H1339N223O243S9 Mr 18 799 – stationary phase : octadecylsilyl silica gel for
[121181-53-1] chromatography R (5 μm) with a pore size of 20 nm ;
– temperature : 60 °C.
DEFINITION Mobile phase :
Solution of a protein having the primary structure of the – mobile phase A : dilute 0.5 mL of trifluoroacetic acid R
granulocyte colony-stimulating factor plus 1 additional in 950 mL of water R, add 50 mL of acetonitrile for
amino acid, an N-terminal methionine (r-met HU G-CSF). chromatography R and mix ;
In contrast to its natural counterpart, the protein is not
glycosylated. Human G-CSF is produced and secreted by – mobile phase B : dilute 0.5 mL of trifluoroacetic acid R
endothelium, monocytes and other immune cells. The protein in 50 mL of water R, add 950 mL of acetonitrile for
stimulates the differentiation and proliferation of leucocyte chromatography R and mix ;
stem cells into mature granulocytes. Time Mobile phase A Mobile phase B
Content : minimum 0.9 mg of protein per millilitre. (min) (per cent V/V) (per cent V/V)
0-8 97 → 94 3→6
Potency : minimum 1.0 × 108 IU per milligram of protein.
8 - 25 94 → 66 6 → 34
PRODUCTION
25 - 40 66 → 10 34 → 90
Filgrastim concentrated solution is produced by a method
based on recombinant DNA (rDNA) technology, using 40 - 45 10 90
bacteria as host cells.
Prior to release, the following tests are carried out on each batch Flow rate : 0.2 mL/min.
of the final bulk product, unless exemption has been granted by Detection : spectrophotometer at 215 nm.
the competent authority. Injection : 10 μL.
Host-cell-derived proteins. The limit is approved by the System suitability : the chromatogram obtained with the
competent authority. reference solution is similar to the chromatogram of
filgrastim digest supplied with filgrastim CRS.
Host-cell- or vector-derived DNA. The limit is approved
by the competent authority. Results : the profile of the chromatogram obtained with
the test solution corresponds to that of the chromatogram
CHARACTERS obtained with the reference solution.
Appearance : clear, colourless or slightly yellowish liquid. TESTS
IDENTIFICATION Impurities with molecular masses higher than that of
A. It complies with the requirements described under Assay. filgrastim. Size-exclusion chromatography (2.2.30) : use the
normalisation procedure.
B. Examine the electropherograms obtained in the test for
impurities with charges differing from that of filgrastim. Solution A. Dissolve 4.1 g of sodium acetate R in 400 mL of
water R, adjust to pH 4.0 with acetic acid R and dilute to
Results : the principal band in the electropherogram 500 mL with water R.
obtained with the test solution is similar in position to
the principal band in the electropherogram obtained with Test solution. Dilute the preparation to be examined with
reference solution (a). solution A to obtain a concentration of 0.4 mg/mL.
C. Examine the chromatograms obtained in the test for Reference solution. Dilute filgrastim CRS with solution A to
impurities with molecular masses higher than that of obtain a concentration of 0.4 mg/mL.
filgrastim. Resolution solution. Mix a sample of the reference solution for
Results : the principal peak in the chromatogram obtained about 30 s using a vortex mixer.
with the test solution is similar in retention time to the Column :
principal peak in the chromatogram obtained with the – size : l = 0.3 m, Ø = 7.8 mm ;
reference solution. – stationary phase : hydrophilic silica gel for chromatography R
D. Examine the electropherograms obtained under both (5 μm) of a grade suitable for fractionation of globular
reducing and non-reducing conditions in the test for proteins in the relative molecular mass range of 10 000 to
impurities with molecular masses differing from that of 500 000 ;
filgrastim. – temperature : 30 °C.
Results : the principal band in the electropherogram Mobile phase. Dissolve 7.9 g of ammonium hydrogen
obtained with test solution (a) is similar in position to the carbonate R in 1000 mL of water R and adjust to pH 7.0 with
principal band obtained with reference solution (b). phosphoric acid R ; dilute to 2000 mL with water R.
E. Peptide mapping (2.2.55). Flow rate : 0.5 mL/min.
SELECTIVE CLEAVAGE OF THE PEPTIDE BONDS Detection : spectrophotometer at 215 nm.

General Notices (1) apply to all monographs and other texts 2233
Filgrastim concentrated solution EUROPEAN PHARMACOPOEIA 8.0

Injection : 20 μL. Focusing :

Relative retention with reference to the filgrastim monomer – pH gradient : 4.5-8.0 ;
(retention time = about 19 min) : aggregates = about 0.60 ; – catholyte : 1 M solution of sodium hydroxide R ;
filgrastim oligomer 1 = about 0.75 ; filgrastim – anolyte : 0.04 M solution of glutamic acid R in a 0.0025 per
oligomer 2 = about 0.80 ; filgrastim dimer = about 0.85. cent V/V solution of phosphoric acid R ;
System suitability: resolution solution : – application : 20 μL.
– retention time : filgrastim monomer = 17 min to 20 min ; Detection : as described in 2.2.54.
– resolution : minimum 3 between the peaks due to the System suitability :
filgrastim dimer and the filgrastim monomer. – in the electropherogram obtained with reference
Calculate the percentage content of the dimer, oligomers and solution (c), the relevant isoelectric point markers are
aggregates. distributed along the entire length of the gel ;
Limit : – in the electropherogram obtained with reference
– total of the peaks with retention times less than that of the solution (a), the pI of the principal band is 5.7 to 6.3.
principal peak : maximum 2 per cent. Limit :
Impurities with molecular masses differing from that of – any impurity : no band is more intense than the principal
filgrastim. Polyacrylamide gel electrophoresis (2.2.31) under band in the electropherogram obtained with reference
both reducing and non-reducing conditions. solution (b) (10 per cent).
Gel dimensions : 1 mm thick. Related proteins. Liquid chromatography (2.2.29) : use the
Resolving gel : 13 per cent acrylamide. normalisation procedure.
Sample buffer (non-reducing conditions). Mix equal volumes Solution A. 0.1 M sodium acetate buffer solution pH 4.0 R,
of water R and concentrated SDS-PAGE sample buffer R. containing 0.1 mg/mL of polysorbate 80 R and 50 mg/mL of
Sample buffer (reducing conditions). Mix equal volumes sorbitol R.
of water R and concentrated SDS-PAGE sample buffer for Test solution. Dilute the preparation to be examined with
reducing conditions R containing 2-mercaptoethanol as the solution A to obtain a concentration of 0.2 mg/mL.
reducing agent. Reference solution (a). Dilute filgrastim CRS with solution A
Test solution (a). Dilute the preparation to be examined with to obtain a concentration of 0.2 mg/mL.
sample buffer to obtain a concentration of 100 μg/mL. Reference solution (b). To 500 μL of reference solution (a)
Test solution (b). To 0.20 mL of test solution (a) add 0.20 mL add 2.0 μL of a 4.5 g/L solution of hydrogen peroxide.
of sample buffer. Mix and incubate at 25 °C for 30 min, then add 1.5 mg of
L-methionine R.
Test solution (c). Dilute 0.20 mL of test solution (b) to 1 mL
with sample buffer. Column :
Test solution (d). Dilute 0.20 mL of test solution (c) to 1 mL – size : l = 0.25 m, Ø = 4.6 mm ;
with sample buffer. – stationary phase : butylsilyl silica gel for chromatography R
Test solution (e). To 0.20 mL of test solution (d) add 0.20 mL (5 μm) with a pore size of 30 nm ;
of sample buffer. – temperature : 60 °C.
Reference solution (a). Solution of molecular mass markers Mobile phase :
suitable for calibrating SDS-polyacrylamide gels in the range – mobile phase A : dilute 1.0 mL of trifluoroacetic acid R to
of 14.4-94 kDa. 900 mL with water R, then add 100 mL of acetonitrile R ;
Reference solution (b). Dilute filgrastim CRS with sample – mobile phase B : dilute 1.0 mL of trifluoroacetic acid R to
buffer to obtain a concentration of 100 μg/mL. 200 mL with water R, then add 800 mL of acetonitrile R ;
Sample treatment : boil for 5 min. Time Mobile phase A Mobile phase B
Application : 20 μL. (min) (per cent V/V) (per cent V/V)
Detection : by silver staining. 0 - 35 34 → 27 66 → 73

System suitability : 35 - 50 27 → 10 73 → 90
– reference solution (a) : the validation criteria are met ; 50 - 60 10 → 34 90 → 66
– a band is seen in the electropherogram obtained with test
solution (e) ; Flow rate : 0.6 mL/min.
– a gradation of intensity of staining is seen in the Detection : spectrophotometer at 215 nm.
electropherograms obtained with test solutions (a) to (e). Injection : 50 μL.
Limit : test solution (a) : Relative retention with reference to filgrastim (retention
– impurities with molecular masses lower or higher than that of time = about 28 min) : oxidised form 1 = about 0.85 ; oxidised
filgrastim : no band is more intense than the principal band form 2 = about 0.95 ; deamidated forms = about 1.1.
in the electropherogram obtained with test solution (d) System suitability : reference solution (b):
(2.0 per cent). – resolution : minimum 1.5 between the peaks due to oxidised
Impurities with charges differing from that of filgrastim. form 1 and oxidised form 2.
Isoelectric focusing (2.2.54). Limits :
Test solution. Dilute the preparation to be examined with – any impurity : for each impurity, maximum 2.0 per cent ;
water R to obtain a concentration of 0.3 mg/mL. – total : maximum 3.5 per cent.
Reference solution (a). Dilute filgrastim CRS with water R to Bacterial endotoxins (2.6.14) : less than 2 IU in the volume
obtain a concentration of 0.3 mg/mL. that contains 1.0 mg of protein.
Reference solution (b). Dilute filgrastim CRS with water R to
obtain a concentration of 0.03 mg/mL. ASSAY
Reference solution (c). Use an isoelectric point (pI) calibration Protein. Liquid chromatography (2.2.29) as described in the
solution, in the pI range of 2.5-6.5, prepared according to the test for related proteins with the following modification.
manufacturer’s instructions. Injection : test solution and reference solution (a).

2234 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 8.0 Finasteride

Calculate the content of filgrastim (C845H1339N223O243S9) taking 01/2014:1615

into account the assigned content of C845H1339N223O243S9 in
filgrastim CRS. FINASTERIDE
Potency. The potency of the preparation to be examined
is determined by comparison of the dilutions of the test Finasteridum
preparation with the dilutions of the International Standard
of filgrastim or with a reference preparation calibrated in
International Units.

The International Unit is the activity contained in a stated

amount of the appropriate International Standard. The
equivalence in International Units of the International
Standard is stated by the World Health Organization.
C23H36N2O2 Mr 372.6
Carry out the assay using a suitable method such as the [98319-26-7]
following, which uses the conversion of a tetrazolium
salt (MTS) as a staining method. Alternative methods DEFINITION
of quantifying cell proliferation, such as measurement of N-(1,1-Dimethylethyl)-3-oxo-4-aza-5α-androst-1-ene-17β-
intracellular ATP by luciferase bioluminescence, have also carboxamide.
been found suitable, and may be used as the assay readout, Content : 98.0 per cent to 102.0 per cent (dried substance).
subject to appropriate validation. The assay conditions (for
example, cell concentration, incubation time and dilution CHARACTERS
steps) are then adapted accordingly. Appearance : white or almost white, crystalline powder.
Solubility : practically insoluble in water, freely soluble in
Use an established cell line responsive to filgrastim. M-NFS-60 ethanol and in methylene chloride.
cells (ATCC No. CRL-1838) have been found suitable. It shows polymorphism (5.9).
Incubate with varying dilutions of test and reference
preparations of filgrastim. Then incubate with a solution of IDENTIFICATION
tetrazolium salt R. This cytochemical stain is converted by Infrared absorption spectrophotometry (2.2.24).
cellular dehydrogenases to a coloured formazan product. The
formazan is then measured spectrophotometrically. Comparison: finasteride CRS.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
Add 50 μL of dilution medium to all wells of a 96-well
substance separately in methanol R, evaporate to dryness and
microtitre plate. Add an additional 50 μL of this solution to the
record new spectra using the residues.
wells designed for the blanks. Add 50 μL of each solution to be
tested in triplicate (test preparation and reference preparation TESTS
at a concentration of about 800 IU/mL, plus a series of 10
twofold dilutions to obtain a standard curve). Prepare a Specific optical rotation (2.2.7) : + 12.0 to + 14.0 (dried
5
suspension of M-NFS-60 cells containing 7 × 10 cells per substance).
millilitre. Immediately before use, add 2-mercaptoethanol to a Dissolve 0.250 g in methanol R and dilute to 25.0 mL with
final concentration of 0.1 mM, and add 50 μL of the prepared the same solvent.
cell suspension to each well, maintaining the cells in a uniform Related substances. Liquid chromatography (2.2.29).
suspension during addition. Solvent mixture : acetonitrile R1, water for chromatography R
(50:50 V/V).
Incubate the plate at 36.0-38.0 °C for 44-48 h in a humidified Test solution (a). Dissolve 25.0 mg of the substance to be
incubator using 6 ± 1 per cent CO2. Add 20 μL of a 5.0 g/L examined in the solvent mixture and dilute to 50.0 mL with
sterile solution of tetrazolium salt R to each well and reincubate the solvent mixture.
for 4 h. Estimate the quantity of formazan produced using a Test solution (b). Dissolve 100.0 mg of the substance to be
microtitre well plate reader at 490 nm. examined in the solvent mixture and dilute to 10.0 mL with
the solvent mixture.
Calculate the potency of the preparation to be examined using Reference solution (a). Dissolve 25.0 mg of finasteride CRS in
a suitable statistical method, for example the parallel line the solvent mixture and dilute to 50.0 mL with the solvent
assay (5.3). mixture.
Reference solution (b). Dissolve 10 mg of finasteride for peak
The estimated potency is not less than 80 per cent and not identification CRS (containing impurities A and C) in 1.0 mL
more than 125 per cent of the stated potency. The confidence of the solvent mixture.
limits (P = 0.95) are not less than 74 per cent and not more Reference solution (c). Dilute 1.0 mL of test solution (b) to
than 136 per cent of the estimated potency. 100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Column :
– size : l = 0.25 m, Ø = 4.0 mm ;
LABELLING – stationary phase : base-deactivated end-capped octadecylsilyl
silica gel for chromatography R (5 μm) ;
The label states : – temperature : 60 °C.
Mobile phase : acetonitrile R1, tetrahydrofuran R, water for
– the content, in milligrams of protein per millilitre ; chromatography R (10:10:80 V/V/V).
Flow rate : 1.5 mL/min.
– the potency, in International Units per milligram of protein. Detection : spectrophotometer at 210 nm.

General Notices (1) apply to all monographs and other texts 2235
Fish oil, rich in omega-3 acids EUROPEAN PHARMACOPOEIA 8.0

Injection : 15 μL of test solution (b) and reference solutions (b)

and (c).
Run time : twice the retention time of finasteride.
Identification of impurities : use the chromatogram supplied
with finasteride for peak identification CRS and the
chromatogram obtained with reference solution (b) to identify
the peaks due to impurities A and C.
B. methyl 3-oxo-4-aza-5α-androst-1-ene-17β-carboxylate,
Relative retention with reference to finasteride (retention
time = about 28 min) : impurity A = about 0.9 ;
impurity C = about 1.3.
System suitability :
– signal-to-noise ratio : minimum 40 for the principal peak in
the chromatogram obtained with reference solution (c) ;
– peak-to-valley ratio : minimum 5, where Hp = height
above the baseline of the peak due to impurity A and C. N-(1,1-dimethylethyl)-3-oxo-4-azaandrosta-1,5-diene-
Hv = height above the baseline of the lowest point of the 17β-carboxamide (Δ5-finasteride).
curve separating this peak from the peak due to finasteride
in the chromatogram obtained with reference solution (b). 07/2012:1912
Calculation of percentage contents :
– correction factor : multiply the peak area of impurity A
FISH OIL, RICH IN OMEGA-3 ACIDS
by 2.4 ;
Piscis oleum omega-3 acidis abundans
– for each impurity, use the concentration of finasteride in
reference solution (c). DEFINITION
Limits : Purified, winterised and deodorised fatty oil obtained from
fish of families such as Engraulidae, Carangidae, Clupeidae,
– impurities A, C : for each impurity, maximum 0.3 per cent ; Osmeridae, Scombridae (except the genera Thunnus and
– unspecified impurities : for each impurity, maximum Sarda) and Ammodytidae (type I), or from the genera Thunnus
0.10 per cent ; and Sarda within the family Scombridae (type II). The omega-3
acids are defined as the following acids : alpha-linolenic acid
– total : maximum 0.5 per cent ; (C18:3 n-3), moroctic acid (C18:4 n-3), eicosatetraenoic acid
– reporting threshold : 0.05 per cent. (C20:4 n-3), timnodonic (eicosapentaenoic) acid (C20:5 n-3 ;
EPA), heneicosapentaenoic acid (C21:5 n-3), clupanodonic
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
acid (C22:5 n-3) and cervonic (docosahexaenoic) acid (C22:6
on 1.000 g by drying in an oven at 105 °C.
n-3 ; DHA).
ASSAY Content :
Liquid chromatography (2.2.29) as described in the test for Type I Type II
related substances with the following modification.
EPA, expressed as minimum 13 per cent 4 per cent to 12 per
Injection : test solution (a) and reference solution (a). triglycerides cent
Calculate the percentage content of C23H36N2O2 taking into DHA, expressed as minimum 9 per cent minimum 20 per cent
account the assigned content of finasteride CRS. triglycerides

STORAGE Total omega-3 minimum 28 per cent minimum 28 pe