(5) R. K. Y. Zee-Cheng, E. G. Podrebarac, C. S. Menon, and C. C. (12) F. Traganos, D. P. Evenson, L. Staiano-Coico, Z.

Darzynkiewicz,
Cheng, J. Med. Chem., 22,501 (1979). and M. R. Melamed, ibid., 40,671 (1980).
(6) K. C. Murdock, R. G. Child, P. F. Fabio, R. B. Angier, R. E. (13) D. P. Evenson, Z. Darzynkiewicz, L. Staiano-Coico,F. Traganos,
Wallace, F. E. Durr, and R. V. Citarella, ibid.,22,1024 (1979). and M. R. Melamed, ibid., 39,2574 (1979).
(7) R. K. Johnson, R. K. Y. Zee-Cheng, W. W. Lee, E. M. Acton, D. (14) D. P. Evenson, F. Traganos, Z. Darzynkiewicz, L. Staiano-Coico,
W. Henry, and C. C. Cheng, Cancer Treat. Rep., 63,425 (1979). and M. R. Melamed, J. Natl. Cancer Znst., 64,857 (1980).
(8) R. E. Wallace, K. C. Murdock, R. B. Angier, and F. E. Durr, (15) C. W. Greenhalgh, Endeauour, 35,134 (1976).
Cancer Res., 39,1570 (1979).
(9) B. F. Kimler, ibid., 40,42 (1980).
(10) T. W. Plumbridge, V. Knight, K. L. Patel, and J. R. Brown, J. ACKNOWLEDGMENTS
Pharm. Pharmacol., 32,78 (1980).
(11) D. D. Von Hoff, E. Pollard, J. Kuhn, E. Murray, and C. A. Colt- Supported by Public Health Service Grant ES-02046 and a grant from
man, Jr., Cancer Res., 40,1516 (1980). the University of Kansas Endowment Association.

Simultaneous Determination of Vitamins B1, Bz, Be, and

Niacinamide in Multivitamin Pharmaceutical
Preparations by Paired-Ion Reversed-Phase
High-pressure Liquid Chromatography

RODERIC P. KWOK *x, W. P. ROSE *, RICK TABOR *, and THOMAS S. PATTISON *

Received March 3,1980, from the *Foremost-McKesson Research and Development Center, Dublin, CA 94566, and the *Forrest C. Shaklee
Research Laboratories, Hayward, CA 94545. Accepted for publication February 20,1981.

Abstract 0 A high-pressure liquid chromatographic procedure for the multivitamin-multimineral tablets by paired-ion re-
simultaneous determination of vitamins B1, B2, Bg, and niacinamide in versed-phase HPLC.
multivitamin pharmaceutical preparations was developed and evaluated.
The method uses paired-ion reversed-phase partition chromatography EXPERIMENTAL
for baseline separation of the four water-soluble vitamins. This method
was applied to the analysis of a multivitamin and multivitamin-mul- Apparatus-The HPLC system included two solvent pumps1, a sol-
timineral tablets, and a technique was developed to reduce vitamin ad- vent programmer2, a UV absorbance detector3 a t 280 nm, and an au-
sorption by the minerals. The results obtained by this method were toinjector4. A pBondapak phenyl(l0 pm) 30-cm X 3.9-mm i.d. column
compared with those obtained by the official methods. It was concluded and a 10-mv full-scale recorder5 were used. Peak areas were determined
that this method is fast, accurate, specific,and suitable for routine quality using a laboratory data system6.The column temperature was regulated
control use. a t 30" by a constant temperature bath7. A high-speed centrifuge was
useds, and the samples were disintegrated in a shaker bathg set a t 60".
Keyphrases Vitamins, water soluble-simultaneous high-pressure
Materials and Reagents-Thiamine hydrochloride, riboflavin, py-
liquid chromatographic analyses in multivitamin preparations 0 High-
ridoxine hydrochloride, and niacinamide were obtained as USP reference
pressure liquid chromatography-simuItaneous analyses of various
standards. 1-Hexanesulfonicacid was used as received'*. Anhydrous citric
water-soluble vitamins in multivitamin preparations Multivitamin
acid" was obtained commercially and used without further purification.
preparations-simultaneous high-pressure liquid chromatographic assay Solvents, all distilled-in-glass12 grade, were obtained commercially.
of various water-soluble vitamins
Commercial multivitamin preparations were obtained from local phar-
macies.
Progress in vitamin preparations is being impeded by Preparation of Mobile Phases-The two pumps used 0.0025 M 1-
methodological problems. Current official methods (1, 2) hexanesulfonic acid in water and in methanol, respectively. Prior to use,
the mobile phases were degassed by vacuum filtration through a 0.5-pm
for the assay of water-soluble vitamins involve a compli- pore, 47-mm diameter filterI3.With a linear solvent program14, a gradient
cated sample workup, tend to reproduce poorly because was run a t a constant flow rate of 2.0 ml/min for 18 min. Solvent condi-
of the instability and pH sensitivity of the color develop- tions varied from 0 to 80%methanol in water. Five minutes was allowed
ment, and require that each vitamin be analyzed individ- between sample injections.
ually. These methods also are subject to interferences from
Model 6000A, Waters Associates, Milford Mass.
various sources when analyzing samples with complex Model 660, Waters Associates, Milford, Mass.
matrixes (3,4). Model 440, Waters Associates, Milford Mass.
WISP model 710A. Waters Associates, Milford Mass.
High-pressure liquid chromatographic (HPLC) proce- Omniscribe, Houston Instrument, Houston. Tex.
6 Model 3353, Hewlett-Packard, Palo Alto, Calif.
dures for the simultaneous determination of water-soluble FK2, Haak Inc., Saddle Brook, N.J.
vitamins in pharmaceutical preparations have been de- Sorvall RC-5B centrifuge fitted with 99-34 motor, DuPont Instruments,
Wilmington, Del.
scribed (5-7), but no mechanism has been developed to Magni-Whirl water bath 15-453-400, Fisher Scientific Co., San Francisco,
control vitamin adsorption by the minerals in multivi- Calif.
lo B-6 PIC reagent, Waters Associates, Milford Mass.
tamin-multimineral tablets. This paper describes an l1 Matheson, Coleman and Bell, Norwood, Ohio.
HPLC procedure for the simultaneous determination of l2 Burdick &Jackson, Muskegon, Mich.
l3 Type LS, 47 mm, Millipore Corp. Bedford, Mass.
vitamins B1, B 1 , B 6 , and niacinamide in multivitamin and Curve 6 on model 660 solvent programmer.

1014 I Journal of Pharmaceutical Sciences 0022-3549/81/0900-1014$01.00/0

Vol. 70, No. 9, September 1981 @ 198 1, American Pharmaceutical Association
Table I-Effect of Ferrous Gluconate and Magnesium Oxide on the Recovery of Pyridoxine Hydrochloride in 100 ml of 0.1 N HCl
Solutions
Solution Composition
Amount of Ferrous Magnesium B6, % RSD,
Sample Be,mg Gluconate, mg Oxide, mg recoverya SD %
Pyridoxine 0.5 - - 101 1.02 1.01
+
Ferrous gluconate magnesium oxide - 40 90 None - -
+
Pyridoxine hydrochloride ferrous gluconate 0.5 40 - 80.43 1.01 1.26
+
Pyridoxine hydrochloride magnesium oxide 0.5 - 90 59.00 5.31 9.00
+
Pyridoxine hydrochloride ferrous gluconate and magnesium oxide 0.5 40 90 91.67 1.03 1.12
art=&

Table 11-Water-Soluble Vitamins Found in Multivitamin-Multimineral Tablets a by Various Modes of Extraction

Percent of Label Claim
1% Citric Acid in
Vitamin, Label Claim 0.1 N HCI Dimethyl Sulfoxide Dimethyl Sulfoxide
Thiamine hydrochloride, 2.1 mg/tablet 96.6 f 1.94 108.5 f 7.54 100 f 1.25
Riboflavin, 2.4 mg/tablet 81.2 f 0.90 104.2 f 1.80 100 f 1.75
Pyridoxine hydrochloride, 2.0 mg/tablet 88.3 f 1.70 84.2 f 0.78 100 f 1.58
Niacinamide. 2.0 mdtablet 101.9 f 3.26 100.6 f 0.85 100 f 3.11
~~

Vita Lea, Shaklee Corp.; each value is the mean f SD of five determinations.
Preparation of Standard Solutions-Stock standard solutions of were placed in a low actinic glass-stoppered erlenmeyer flask. Exactly
thiamine hydrochloride, pyridoxine hydrochloride, and niacinamide (0.5 100 ml of 0.1 N HCl was pipetted into the flask, and the flask was stop-
mg/ml each) were prepared separately by dissolving accurately weighed pered and shaken for 45 min in a constant-temperature bath at 60". The
USP reference standards in 0.1 N HCl. Due to poor solubility, only the sample was allowed to cool to room temperature and then was Centrifuged
riboflavinworking standard solution was prepared in a more diluted form; a t 44,OOOXg for 18 min. Then 10 pl of the clear supernate was injected
<0.1 g accurately weighed USP reference standard was dissolved in lo00 into the liquid chromatograph.
ml of 0.1 N HC1, heated, and constantly stirred in a low actinic volumetric Multivitamin-Multimineral Tablets-A suitable number of tablets
flask. containing no more than 10 mg of riboflavin were placed in a low actinic
Different aliquots from each stock standard vitamin solution were glass-stoppered erlenmeyer flask. Exactly 100 ml of dimethyl sulfoxide
combined and diluted with the riboflavin working standard solution. This containing 1% citric acid (anhydrous) was delivered into the flask. The
procedure resulted in a working standard solution containing all four solution then was shaken for 45 min in a constant-temperaturebath a t
water-soluble vitamins a t the desired concentration. 60". It was allowed to cool to room temperature and then was centrifuged
Preparation of Sample Solutions-Multivitamin Tablets-A at 44,000Xg for 18 min. Then 10 pl of the clear supernate was injected
suitable number of tablets containing no more than 10 mg of riboflavin into the liquid chromatograph.

ill
ul Figure 2-Liquid chromato-
Figure l-Liquid chromato- graphic trace of the extract of
graphic trace of a standard a vitamin R complex tablet.
mixture of niacinamide (A), u1
Key: A, niacinamide; B, pyri-
pyridoxine (B), thiamine (C), a doxine; C, thiamine; and D,
and riboflavin (0). L riboflavin.

I
0
I
5
MINUTES
I
10 15
I
4
0 6
li
MINUTES
10 15

Journal of Pharmaceutical Sciences 1 1015

Vol. 70, No. 9, September 1981
Table 111-Recovery Results on Vitamins BI,Bz, B6, and Niacinamide
~ ~ ~ ~

Percent Recoverya, mean f SD (% RSD)

Sample Bi B2 B6 Niacinamide
Control multivitamin tablets None None None None t
+
Multivitamin tablets added vitamins' 100.8 f 2.23 102.1 f 2.48 104.3 f 6.7 102.1 f 2.78
(2.21) (2.43) (6.43) (2.72)
Control multivitamin-multimineral tablets None None None None
+
Multivitamin-multimineral tablets added vitaminsC 101.6 f 2.21 99.60 f 2.01 99.62 f 2.92 99.97 f 1.45
(2.18) (2.02) (2.93) (1.45)
a Each value is the mean f SD (96 RSD) of six determinations. Control tablets contain the com lete formulation of B-Complex (multivitamin tablets) and Vita Lea
(multivitamin-multimineraltablets) without vitamins B1, Bz,Be, and niacinamide. Same as contrortableta but with vitamins B1, Bz, &, and niacinamide added directly
to the flask.
Table IV-Comparison of USP Method versus HPLC Method for the Analysis of Vitamins B1 and Bz in Multivitamin Products
Vitamin B1 Vitamin Bz
Formulation Found Formulation Found
Product" Amount HPLC SD USP SD Amount HPLC SD USP SD
t
A 66.42 65.91 0.56 66.5 1.83 70.72 70.66 0.27 72.9 1.36
mgk mgk
B 1.178 1.151 0.02 1.248 0.04 1.143 1.172 0.018 1.245 0.026 CI
mghablet mg/tablet
C 9.45 9.63 0.35 8.73 0.90 8.415 8.597 0.110 7.910 0.129
mg/tablet mghablet
D 0.678 ' 0.687 0.003 0.695 0.009 0.718 0.716 0.002 0.674 0.010
mghblet mg/tablet
E 10 10.5c -d -d -d 10 18.0C -d -d -d
mg/tablet mghablet
F 11.6 11.9c -d -d -d 15 14.6= -d -d -d
mg/tablet mghablet
G 3 3.lC -d -d -d 3 3.4c -d -d -d
mghapsule mghapsule
0 Key: A, Vita Lea Premixes (Shaklee);B, Vita Cal (Shaklee);C, B-Complex (Shaklee);D, Vita Lea (Shaklee); E, Super Thera-ViteM (Nature's Blend); F! Stress Formula
(Value Rite); and G, B-Complex Capsules (Nature's Blend). b Each value is a mean f S D of five determinations. Single determmation. Not determined.

RESULTS AND DISCUSSION agent like citric acid in dimethyl sulfoxide can minimize this effect (Table
11). Thus, anhydrous citric acid was dissolved first in dimethyl sulfoxide
HPLC can provide a quick and accurate evaluation of multivitamin to allow for the exposure to the minerals as soon as possible.Table I shows
and multivitamin-multimineral tablets, and paired-ion reversed-phase the effect of ferrous gluconate and magnesium oxide on pyridoxine hy-
HPLC is particularly attractive for determining water-soluble vitamins drochloride in 0.1 N HCl solution. Table I1 demonstrates the percentage
because polar constituents that dissolve in the aqueous mobile phase elute of recovery of all four water-soluble vitamins in multivitamin-multi-
at or near the solvent front. Lipophilic constituents with very limited mineral tablets by different modes of extraction. It is obvious from Table
solubility in the aqueous mobile phase partition with the stationary phase, I1 that 1%citric acid in dimethyl sulfoxide reduced the effect of ferrous
thereby eluting much later. Under these conditions, separation can be gluconate and magnesium oxide to an insignificant level. The extraction
achieved. Thus, sample preparation time is greatly reduced and the ac- of the other vitamins (thiamine, riboflavin, and niacinamide) was not
curacy of the method also is enhanced. impaired by the addition of the anhydrous citric acid in dimethyl sulf-
In the present study, 0.1 N HCl was used to extract the water-soluble oxide.
vitamins from the multivitamin preparations. Dimethyl sulfoxide con- Linearity of response over the range studied can be achieved using
taining anhydrous citric acid was used to disperse the multivitamin- standard solutions. In these laboratories, the ratios of the concentration
multimineral preparations since neither 0.1 N HCl nor dimethyl sulfoxide of the standard solution (expressed in nanograms per 10 microliters) to
alone extracts pyridoxine completely. This may be due to adsorption of the area under the peak were consistent for all four water-solublevitamins
the vitamin by one or more of the minerals. Kwok et al. (8) established studied. These ratios were determined over 6 days, and their relative
that vitamin A acetate adsorbs to ferrous gluconate and that a complexing standard deviations were <1%. This finding indicates that the procedure
agent can minimize this effect. can be used as a single-point standard for each vitamin.
With a similar approach, it was demonstrated in these laboratories that, Chromatographic traces of vitamin B standards, vitamin B complex,
in 0.1 N HCl solution, pyridoxine hydrochloride adsorbs strongly to and the multivitamin-multimineral tablet extracts are presented in Figs.
ferrous gluconate and magnesium oxide (Table I) and that a complexing 1,2, and 3, respectively.Using paired-ion reversed-phase HPLC, baseline
Table V-Comparison of USP Method versus HPLC Method for Analysis of Vitamin B6 and Niacinamide in Multivitamin Products
Vitamin Be Niacinamide
Formulation Found Formulation Found
Product" Amount HPLC SD USP SD Amount HPLC SD USP SD
A 54.21 54.33 1.40 54.9 1.24 None None None
mgk
B None None None None None None
C 11.97 11.37 0.15 9.27 0.23 99.00 98.60 3.31 97.20 4.40
mghablet mg/tablet
D 0.670 0.668 0.013 0.680 0.019 5.49 5.54 0.06 5.18 0.30
mghablet mghablet
E 5 4.7c -d -d -d 100 101.5c -d -d -d
mghablet mg/tablet
F 5 5.lC -d -d -d 100 90.8c -d -d -d
mg/tablet mg/tablet
G 0.5 0.9c -d -d -d 20 26.OC -d -d -d
mghapsule mg/capsule
0 Key: A, Vita Lea Premixes (Shaklee);B, Vita Cal (Shaklee);C, B-Complex (Shaklee);D, Vita Lea (Shaklee!; E, Super Thera-Vite M (Nature's Blend); F , Stress Formula
(Value Rite); and G, B-Complex Capsules (Nature's Blend). Each value is a mean +SD of five determinations. Single determination. Not determined.

1016 I Journal of Pharmaceutical Sciences

Vol. 70, No. 9, September 1981
 oxide extract (Fig. 3).
To validate the selectivity of the procedure, placebo formulations
spiked with known amount of the vitamins were employed. The results
are tabulated in Table 111. All four vitamins were quantitatively recovered
in both multivitamin and multivitamin-multimineral tablets.
To verify the applicability of this procedure further, various com-
mercial multivitamin and multivitamin-multimineral tablets were as-
sayed simultaneously by the HPLC and the USP methods (Tables IV
and V). In each case, the precision of the HPLC procedure was greater
than that of the current official procedure. This result may be attributed
to the less complicated sample workup required by the former procedure.
However, the results of the two methods compared favorably in terms
of accuracy.
The described HPLC method appears to be attractive in terms of
analysis time and should be adapted to various commercial products. The
u1 column lifetimes under these conditions of minimum sample preparation
v)
z Figure %-Liquid chromato- initially was a subject of concern. However, over 500 preparations were
g
v)
graphic trace of the extract of processed without change in the chromatographic characteristics of the
system. This stability may be due in part to the small quantities of sample
w a multivitamin-multimineral
a tablet. Key: A, niacinamide; B, injected and in part to the proper care as recommended by the column
pyridoxine; C, thiamine; and supplier. The methodologyfor the HPLC determination of other vitamins
D, riboflauin. is currently under investigation and will be published later.

REFERENCES
(1) ”The United States Pharmacopeia,” 19th rev., Mack Publishing
Co., Easton, Pa., 1975, pp. 341,429,442,502.
(2) “The National Formulary,” 14th ed., Mack Publishing Co., Easton,

i“
Pa., 1975, pp. 486,984.
(3) V. D. Gupta and D. E. Cadwallader, J . Pharm. Sci., 57, 112
(1968).
(4) S. A. Ismaiel and D. A. Yassa, Analyst, 98,816 (1973).
(5) D. P. Wittmer and W. G. Haney, Jr., J. Pharm. Sci., 63, 588
(1974).

- i
(6) S. P. Sood, D. P. Wittmer, S. A. Ismaiel, and W. G. Haney, ibid,
66,40 (1977).
(7) R. L. Kirchmeier and R. P. Upton, ibid, 67,1444 (1978).
0 5 10 15
MINUTES (8) R. P. Kwok, T. Hudson, and S. Subramanian, in “Application of
High Pressure Liquid Chromatographic Methods for Determination of
separation of all four water-soluble vitamins is possible (Figs. 1and 2). Fat-Soluble Vitamins, A, D, E, and K in Foods & Pharmaceuticals,”
There also is a baseline separation of the vitamins in the dimethyl sulf- Association of Vitamin Chemists, Chicago, Ill., 1978.

In Vivo-In Vitro Correlations with a Commercial

Dissolution Simulator I: Methenamine,
Nitrofurantoin, and Chlorothiazide

MARTIN K. T. YAU and MARVIN C. MEYER”

Received October 20,1980, from the Diuision of Biopharmaceutics and Pharmacokinetics, Department of Pharmaceutics, College of Pharmacy,
Uniuersity of Tennessee Center for the Health Sciences, Memphis, TN 38163. Accepted for publication February 20,1981.

Abstract 0 Dissolution profiles were determined for nine methenamine, rothiazide tablets between the percent of drug dissolved in 1 min or the
14 nitrofurantoin. and six chlorothiazidedosaee forms usine a dissolution
I I
time for 15%dissolution and the maximum excretion rate.
simulator. Various in uiuo-in uitro correlations were examined. The best
correlation for methenamine was between the maximum urinary excre- Keyphrases 0 Dissolution-profiles for methenamine, nitrofurantoin,
tion rate and the time for 15%dissolution. A good correlation for the and chlorothiazide, dissolution simulator Dissolution simulator-
50-mg nitrofurantoin tablets was also found between cumulative percent profiles for methenamine, nitrofurantoin, and chlorothiazide Meth-
of drug excreted in 12 hr and the percent dissolved in 1hr. There were enamine-dissolution profile using dissolution simulator 0 Nitrofu-
no significant correlations for the 100-mg nitrofurantoin dosage forms. rantoin-dissolution profile using dissolution simulator 0 Chlorothia-
Good correlations were also observed for the 250- and 500-mg chlo- zide-dissolution profile using dissolution simulator

Many methods developed to study the i n vitro dissolu- velop in uitro dissolution procedures is to provide data that
tion properties of dosage forms have been reviewed pre- can be related to the performance of oral dosage forms
viously (1). The primary objective of most efforts to de- when administered to human subjects. One sophisticated

0022-354918110900-7077$01.0010 Journal of Pharmaceutical Sciences I 1017

@ 198 1, American Pharmaceutical Association Vol. 70, No. 9, September 1981