WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Basavaiah et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 6.647

Volume 6, Issue 9, 1301-1314 Research Article ISSN 2278 – 4357

METHOD DEVELOPMENT AND VALIDATION OF CLEANING

PROCEDURE FOR THE RESIDUAL DETERMINATION OF
FLUNIXIN MEGLUMINE IN BULK DRUG MANUFACTURING OF
ACTIVE PHARMACEUTICALS INGREDIENT BY REVERSE PHASE
HIGH PRESSURE LIQUID CHROMATOGRAPHY

N. H. Eswara Prasad1, D. Rama Devi2, Dr. B. M. Rao3, N. V. N. B. Srinivasa Rao4, Y. V.

Sunil Kumar5 and Dr. K. Basavaiah*1

1
Dept. of Inorganic & Analytical Chemistry, Andhra University, Visakhapatnam-530 003.
2
A.U College of Pharmaceutical Sciences, Andhra University, Visakhapatnam-530003
3
Corporate Quality Control, Dr.Reddy‟s Laboratories Limited.
4
Department of Chemistry, DRG, Govt. Degree College, Tadepalligudem, A.P. India.
5
Quality Control, Lupin Limited, Visakhapatnam, A.P. India.

Article Received on ABSTRACT

08 July 2017, The objective of Cleaning Validation is to establish cleaning
Revised on 29 July 2017,
Accepted on 20 August 2017, procedures and residue limits that are practical, achievable, and
DOI: 10.20959/wjpps20179-10027 verifiable and assure safety. Cleaning of the equipment train, utensil
and / or components are carried out separately or clubbed followed by

*Corresponding Author
visual verification and testing. The purpose is to establish documented
Dr. K. Basavaiah evidence to assure that, cleaning procedure and methods can repeatedly
Dept. of Inorganic & and reproducibly remove residue of the subjected product within the
Analytical Chemistry,
established acceptance limit. The acceptance limit is maximum
Andhra University,
allowable quantity of product residue, which does not affect quality
Visakhapatnam-530 003.
and safety of the subsequent product to be manufactured, by using
same equipment and facility. During development of cleaning validation importance should
be paid to the residue and contaminants. The residue and contaminants shall include the
absence of previously manufactured product, equipment related materials such as equipment
linings, gaskets, filter agents and / or lubricants. Recovery shall be established using the swab
technique and rinse technique and shall be within the acceptable limit. The main study
depicts the development and validation of a RP-HPLC Cleaning method for the residual

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determination of Flunixin Meglumine in veterinary active pharmaceutical ingredient

manufacturing. The method was developed by using the isocratic solvent system, HPLC
grade acetonitrile and Mill-Q water in the ratio of 70:30 (v/v) and water is used as diluent.
Successful elution of the Flunixin Meglumine was achieved on Xbridge shield RP18 column
with 150x4.6 mm internal diameter and 3.5 µm particle size (or) equivalent. The method
validation was successfully applied for routine analysis for cleaning/residual samples. The
developed RP-HPLC method was validated with respect to system suitability, specificity,
linearity, limit of quantitation, limit of detection and recovery study (i.e. rinse recovery &
swab recovery).

KEYWORDS: Flunixin meglumine, Reverse phase high performance liquid

chromatographic (RPHPLC), Validation.

INTRODUCTION
Flunixin meglumine is a non-steroidal anti-inflammatory drug (NSAID). It is used to treat
pain and reduce fever or inflammation. Flunixin meglumine can be used for the treatment of
arthritis, and is FDA approved for use in horses. Flunixin may be given IV, IM or orally. The
precise site and mode of action is unknown. Flunixin meglumine shown in Fig-1 acts via
analgesic and anti-inflammatory mechanisms. Analgesic actions may involve blocking pain
impulse generation via a peripheral action by inhibition of the synthesis of prostaglandins and
possibly inhibition of the synthesis or actions of other substances, which sensitize pain
receptors to mechanical or chemical stimulation. Flunixin may act peripherally in inflamed
tissue, probably by inhibiting the enzyme cyclooxygenase to decrease the formation of
precursors of prostaglandins, and possibly by inhibiting other local mediators of the
inflammatory response.

In horse the Flunixin is four times as potent on an mg per mg basis as phenyl butazone as
measured by the reduction in lameness and swelling in the horse. Plasma half-life in horse
serum is 1.6 hours following a single dose of 1.1 mg/kg. Measurable amounts are detectable
in horse plasma at 8 hours post injection and in the Cattle the Flunixin meglumine is a weak
acid (pKa=5.82) which exhibits a high degree of plasma protein binding (approximately
99%).In healthy cattle, total body clearance has been reported to range from 90 to 151
mL/kg/hr2-5. These studies also report a large discrepancy between the volume of
distribution at steady state (Vss) and the volume of distribution associated with the terminal
elimination phase (Vβ). The discrepancy appears to be attributable to extended drug

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elimination from a deep compartment. The terminal half-life has been shown to vary from
3.14 to 8.12 hours.

In Horse, the recommended dose for musculoskeletal disorders is 0.5 mg per pound (1
mL/100 lbs.) of bodyweight once daily. Treatment may be given by intravenous or
intramuscular injection and repeated for up to five days. Studies show onset of activity is
within 2 hours. Peak response occurs between 12 and 16 hours and duration of activity is 24-
36 hours. The recommended dose for the alleviation of pain associated with equine colic is
0.5 mg per pound of bodyweight. Intravenous administration is recommended for prompt
relief. Clinical studies show pain is alleviated in less than 15 minutes in many cases.
Treatment may be repeated when signs of colic recur. During clinical studies approximately
10% of the horses required one or two additional treatments. The cause of the colic should be
determined and treated with concomitant therapy. In Cattle, the recommended dose for cattle
is 1.1 to 2.2 mg/kg (0.5 to 1 mg/lb.; 1 to 2 mL per 100 lbs.) given by slow intravenous
administration either once a day as a single dose or divided into two doses administered at 12
hour intervals for up to 3 days. The total daily dose should not exceed 2.2 mg/kg (1.0 mg/lb.)
of bodyweight. Avoid rapid intravenous administration of the drug.[1-3]

MATERIALS AND METHODS

Chemicals: Reference standard of Flunixin meglumine and cleaning samples was obtained
from well reputed research laboratories and characterized by use of LCMS, NMR and IR. All
reagents used were of analytical reagent grade unless stated otherwise. Milli.Q-water, HPLC
grade acetonitrile, ortho phosphoric acid was purchased from Merck India. The solutions and
the mobile phase prepared were stored at room temperature. The liquid chromatography
system was equipped with quaternary gradient pumps with auto sampler and column oven,
auto injector connected to a variable wave length programmable ultra violet visible detector
were controlled by open lab software, Agilent technologies with instrument model. no: 1200
series.

Selection of suitable mobile phase, diluent & wave length: The mobile phase for the
analysis of cleaning method validation for residual determination of Flunixin meglumine was
set by injecting different ratios of acetonitrile and orthophosphoric acid in HPLC grade water.
The selected mobile phase ratio was Acetonitrile and orthophosphoric acid in HPLC grade
water is 70:30 (ml/ml). Similarly for the selection of diluent, tried the standard into different
solvents like water, methanol, mobile phase and acetone. Finally, the diluent used was water.

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Similarly for the wave length selection, tried at different nanometres (nm) and observed
maximum response at 254 nm. The selected mobile phase, diluent and wave length resulted
an RSD of six replicates 0.17 (< 5 %).

Instrumentation and analytical chromatographic conditions: The chromatographic

analysis of the cleaning method validation for residual determination of Flunixin meglumine
was carried out on Agilent high pressure liquid chromatography with instrument model
no.1200 series containing quaternary pump, variable wave length programmable ultra violet
visible detector and auto injector with up to 1μl-1000μl loop, column oven modules.
Chromatographic analysis was performed using Xbridge shield RP18 column with 150 x
4.6mm internal diameter and 3.5μm particle size (or) equivalent. Sartorius electronic balance
was used for weighing. Isocratic elution with, acetonitrile, orthophosphoric acid in HPLC
grade water 70:30 (ml /ml) was selected with a flow rate of 1.0 ml/min and injection volume
40 μl. The detection wavelength was set at 254 nm with a runtime of 10 minutes. The mobile
phase was prepared freshly and it was degassed before use. The column was equilibrated for
at least 10 minutes with the mobile phase flowing through the system. The column oven
module and the high pressure liquid chromatography system were kept at 25ºC temperature.

METHOD VALIDATION PROCEDURE

The objective of the method validation is to demonstrate that the method is suitable for its
intended purpose as it is stated in international conference on harmonisation (ICH)
guidelines. The method was validated for system suitability, precision, specificity, linearity,
limit of detection and limit of quantification, recovery.[4-11]

Preparation of System suitability solution: Flunixin meglumine was used as external

standard in the analysis. Different concentrations of the standard were used based on the
range required to plot a suitable calibration curve. About 100mg of the standard Flunixin
meglumine was accurately weighed and transferred in to 10ml volumetric flask. Dissolved
and diluted the volume with water and mixed well.

System suitability
System suitability test was carried out on freshly prepared 10 ppm standard solutions of
Flunixin meglumine and it was calculated by determining the standard deviation of Flunixin
meglumine system suitability solution by injecting in six replicates at 10 minutes interval.
The values of % relative standard deviation (R.S.D) proved that the method is accurate,

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precise. The results were below the acceptance criteria i.e. not more than 5 %. The values
were recorded in Table-1 and the sample recorded chromatogram is in Fig. 2.

Specificity Parameter: Specificity tests were carried out on above prepared 10 ppm system
suitability solution of Flunixin meglumine and it was determining by injecting blank, blank
with swab stick and 10 ppm system suitability solution. As per the details tabulated in Table-
2, proved that the method is specific that there is no interference of blank peaks in Flunixin
meglumine standard solution.

Linearity
The developed method has been validated using the standard solutions of Flunixin
meglumine in the mass concentration range of 0.2 ppm to 15 ppm was injected into the
chromatographic system. The chromatograms were developed and the peak area was
determined for each concentration of the drug solution. Calibration curve of Flunixin
Meglumine was obtained by plotting the peak area ratio versus the applied concentrations of
Flunixin meglumine. The preparation of linearity solutions and results were recorded in
Table-3 and Table-4 respectively. The linearity of Flunixin meglumine was depicted in Fig.3.
Injected each solution once into the HPLC system and calculated the correlation coefficient
by plotting the calibration curve of concentration (mg/ml) on X-axis and peak area on Y-
Axis. Based on the data, the area response against concentration in percentage of Flunixin
meglumine is linear in the range of interest. The correlation coefficient and regression
coefficient was calculated from regular plot and found greater than 0.999. Hence the method
is linear for the determination of Flunixin meglumine.

Limit of detection and limit of quantification

Limit of detection is the lowest amount of analyte in a sample that can be detected, but not
necessarily quantitated, under the stated experimental conditions. Limit of quantitation is the
lowest amount of analyte in a sample that can be quantitated with acceptable precision, under
the stated experimental conditions. The residual and predicted Y are shown in Table-5. The
residual plot graph is depicted in Fig.4. Limit of detection and Limit of quantitation were
calculated using the following formulae and results are shown in Table-6.

Limit of detection = 3.3 X Residual standard deviation

Slope
Limit of quantitation = 10 X Residual standard deviation
Slope

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Preparation of limit of quantification (LOQ) Solution: 0.005 mL of Flunixin meglumine

stock solution taken into 100 ml volumetric flask and diluted up to the mark with water &
mixed well and injected in six replicates. The Limit of quantification (LOQ) experimental
results are recorded in Table-7. Based on the data, the % relative standard deviation was
found 1.67 % against the acceptance criteria of not more than 10 %. Therefore, it can be
concluded that the cleaning method validation is precise at limit of quantification at
concentration 0.0005 ppm and limit of detection (LOD) at concentration 0.0003 ppm level.

Recovery study (or) Accuracy: To study of the reliability, suitability and accuracy of the
method recovery experiments were carried out for cleaning method validation for residual
determination of Flunixin meglumine are broadly classified into two stages.
1) Rinse method 2) Swab method.

Rinse recovery: The rinse recovery of the sampling method is established by spiking a
solution of known concentration on both stainless surface and glass plate. Recovered the
spiked sample from the surface by rinsing the surface with the sampling agent.

Preparation of rinsed spiking solution: Weighed about 100.34 mg of test sample and
transferred into 100 mL volumetric flask. Dissolved and diluted up to the mark with water.
Mixed well. Taken 10 mL of the above solution into 100 mL volumetric flask. Dissolved and
diluted up to the mark with diluent. Mixed well.

Rinse recovery study on stainless plate: Selected three cleaned and dried 10 x 10 cm
surface area stainless steel plate. Spread 10 mL of spiking solution on dried 10 x 10 cm
surface area steel plate, taking utmost care to avoid any spillage.The plates were dried at
room temperature. Study was done using 100 mL of accurately measured diluent to recover
the test sample from 10 x 10 cm surface area stainless steel plate, by gentle swirling. Filtered
and injected the sample into high pressure liquid chromatography in triplicate.

Rinse recovery study on glass plate

Selected three cleaned and dried 10 x 10 cm surface area glass plate. Spread 10 mL of spiking
solution on dried 10 x 10 cm surface area glass plate, taking utmost care to avoid any
spillage. Then the plates were dried at room temperature. Study was done using 100 mL of
accurately measured diluent to recover the test sample from 10 x 10 cm surface area glass
plate, by gentle swirling. Filtered and injected the sample into high pressure liquid

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chromatography in triplicate. Finally, the area of test sample was recorded in the rinse
recovery on stainless plate and glass plate as shown in Table-8.

Swab recovery: The swab recovery of the sampling method is established by spiking a
solution of known concentration on stainless steel surface. The spiked sample was recovered
from the surface by swabbing the surface using swab stick with the sampling agent.

Preparation of swab spiking solution: Weighed about 100.34 mg of test sample and
transferred into 100 mL volumetric flask. Dissolved and diluted up to the mark with water
and then mixed well and 10 mL of the above solution was transferred into 100 mL volumetric
flask. Dissolved and diluted up to the mark with diluent.

Swab recovery study on stainless plate: Selected three cleaned and dried 10 x 10 cm
surface area glass plates. Spread 10 mL of spiking solution on dried 10 x 10 cm surface glass
plates, taking utmost care to avoid any spillage and the plate dried at room temperature.
Study was done using 100 mL of accurately measured diluent to recover the test sample from
10 x 10 cm surface area of glass plate, by gentle swirling. Filtered and injected into high
pressure liquid chromatography in triplicate. The area of test sample was recorded in swab
recovery on stainless plate and glass plate and presented in Table-9. Based on the results, it
can be concluded that percentage (%) of rinse and percentage (%) of swab recovery on
stainless steel plate and glass plate is consistently above 80.0%. The values obtained above
are in good agreement in terms reliability, suitability and accuracy of the proposed method.

Solution stability and mobile phase stability: To determine the stability of sample solution,
the mobile phase and 10 ppm Standard solutions of Flunixin meglumine were prepared and
injected into the high performance liquid chromatographic system with a frequency of
immediately after preparation and at 24 hours. The results from these studies indicated, that
the standard and sample solutions were stable at room temperature for at least 24 hours. The
sampled chromatograms are recorded as below in Fig. 5, Fig. 6, Fig.7 and Fig. 8.

RESULTS AND DISCUSSION

The expectation of regulatory agencies is to have a sensitive analytical method to detect the
residues or contaminations during the cleaning procedure. Hence the detection limit for each
analytical method shall be sufficiently sensitive to detect the established acceptable level of
the residue or contaminant and it is also essential to develop fast, cost-effective, stable,

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precise and sensitive analytical method .The primary target in developing and validate this
RPHPLC method is to determine the residual content of Flunixin meglumine. Based on the
above observed results, the method developed and validated using the RPHPLC for Flunixin
meglumine is valid. The summary and evaluation of results are presented in Table-10.

ILLUSTRATION: (Figures)

Fig.1. Structure of Flunixin Meglumine.

Molecular Formula: C14H11F3N2O2·C7H17NO5

Chemical Name: 2-[[2-Methyl-3-(trifluoromethyl) phenyl] amino]-3-pyridinecarboxylic acid
Meglumine salt, Banamine.

Fig. 2: Chromatogram of 10 ppm standard solution.

Fig. 3. Linearity for Flunixin meglumine.

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Fig.4. Residual plot graph.

Fig. 5. Blank Solution (initial).

Fig.6. Standard Solution (initial).

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Fig.7. Blank after 24 hours injected chromatogram.

Fig. 8. Standard solution after 24hours.

Table.1: System suitability.

Injection No. Area
1 280.46097
2 280.72653
3 280.02744
4 279.70523
5 280.08517
6 279.42972
Average 280.073
Standard deviation 0.4752
Percentage (%) R.S.D 0.17
Acceptance criteria NMT 5.0 %

Table. 2: Specificity parameters.

Peak name Retention time (minutes)
Blank No peak
Blank with swab stick No peak
Standard solution 3.332

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Table. 3: Linearity different levels of concentrations.

Concentration in ppm Stock solution to be added Volume make up to
0.0002 0.02 100
0.0005 0.05 100
0.001 0.10 100
0.003 0.30 100
0.005 0.50 100
0.008 0.80 100
0.010 1.00 100
0.013 1.30 100
0.015 1.50 100

Table. 4: Linearity parameters.

Trial number Actual concentration (ppm) Area response
1 0.0002008 6.46591
2 0.0005020 13.95392
3 0.0010040 28.13911
4 0.0030120 84.10034
5 0.0050200 140.08226
6 0.0080320 224.51443
7 0.0100400 282.21277
8 0.0130520 375.25388
9 0.0150600 426.87277
Slope 28451.4687
Correlation coefficient 0.9999
Regression coefficient 0.9998

Table. 5: Predicated Y and residuals.

Residual Output
Observation Predicted Y Residuals
1 4.658565500 1.807344500
2 13.22814788 0.725772123
3 27.51078517 0.628324827
4 84.64133436 -0.540994357
5 141.7718835 -1.689623541
6 227.4677073 -2.953277316
7 284.5982565 -2.385486500
8 370.2940803 4.959799724
9 427.4246295 -0.551859460

Table. 6: LOD & LOQ results.

Theoretical LOD in mg/ml 0.0003 mg/ml
Theoretical LOQ in mg/ml 0.0005 mg/ml

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Table. 7: LOQ Precision results.

Trial Area
1 14.33640
2 14.21061
3 14.28251
4 14.02512
5 13.74669
6 13.88674
Mean 14.081
Standard deviation 0.2346
Percentage (%) R.S.D 1.67 %
Acceptance criteria NMT 10.0 %

Table. 8: Percentage (%) rinse recovery results.

Percentage (%) Mean (%) Standard Percentage
S. No. Type
recovery recovery deviation (%) R.S.D
1 90.85
2 SS Plate 90.01 89.76 1.2396 1.38
3 88.41
4 90.17
5 Glass plate 88.72 90.01 1.2133 1.35
6 91.13

Table. 9: Percentage (%) swab recovery results.

Percentage Mean (%) Standard Percentage
S. No. Type
(%) recovery recovery deviation (%) R.S.D
1 90.17
2 SS Plate 89.02 89.53 0.5871 0.66
3 89.39
4 89.32
5 Glass plate 88.46 89.46 1.082 1.21
6 90.61

Table. 10: Summary and evaluation of results.

Validation Acceptance criteria Results
parameter
System The % RSD of Flunixin meglumine System suitability parameter meets the criteria.
suitability from six replicate injections of system R.S.D=0.17 %
suitability should be NMT 5.0 %
Specificity The peaks of blank should not The peaks of blank do not interfere with Flunixin
interfere with Flunixin meglumine meglumine peak.
peak Peak Name Retention time (minutes)
Blank No peak
Blank with swab stick No peak
System suitability solution 3.332

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Linearity The correlation coefficient and the The method is linear

regression coefficient between Correlation coefficient=0.9999
concentration and area response Regression coefficient=0.9998
should be NLT 0.995
LOD/LOQ The percentage (%) RSD for area The RSD for area response of Flunixin meglumine from
response of six replicates at LOQ level six replicates at LOQ level.
should be NMT 10.0 % LOQ in mg/mL 0.0005 mg/mL
LOD in mg/mL 0.0003 mg/mL
Recovery Report the % rinse recovery if the % Percentage (%) rinse recovery
study rinse recovery is less than 80.0 % then Type (%) Mean (%) Standard (%)
incorporate the recovery factor to the Recovery Recovery deviation RSD
analytical method. SS 90.85
plates 90.01 89.76 1.2396 1.38
88.41
Glass 90.17
plates 88.72 90.01 1.2133 1.35
91.13
Recovery Report the % swab recovery if the % Percentage (%) swab recovery
study swab recovery is less than 80.0 % then Type (%) Mean (%) Standard (%)
incorporate the recovery factor to the Recovery Recovery deviation RSD
analytical method. SS plates 90.17
89.02 89.53 0.5871 0.66
89.39
Glass 89.32
plates 88.46 89.46 1.082 1.21
90.61

CONCLUSION
Analytical method was validated unless the method employed is included in the relevant
pharmacopoeia or other recognized standard reference and also included the consideration of
characteristics within the ICH guidance‟s on validation of analytical methods. The degree of
analytical method validation performed should reflect the purpose of the analysis and the
stage of the API production process. Finally, the proposed method is found to be specific for
the residual determination of Flunixin meglumine. The method is found to be linear in the
range of interest. The sampling method is found to be precise for rinse and swab recovery. A
system suitability test is established and recorded. Hence, this method stands validated can be
used for routine line clearance samples.

ACKNOWLEDGEMENTS
The author is thankful to the faculty members of Department of Chemistry, Andhra
University Andhra Pradesh, India, for their valuable guidance, advice, technical and moral

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support for the work done and towards the completion of the analytical method validation of
Flunixin meglumine.

REFERENCES
1. Odensvik K, Johansson M, “High-performance liquid chromatography method for
determination of Flunixin in bovine plasma and pharmacokinetics after single and
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meglumine in lactating cattle after single and multiple intramuscular and intravenous
administrations” , Am J Vet Res., 1990; 51: 1464-1467.
3. Odensvik K. “Pharmacokinetics of Flunixin and its effect on prostaglandin F2α
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23 1999).
10. International Conference on Harmonization (ICH) of technical requirements Guide lines
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