JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES® Vol. 27, No. 3, pp. 511-519, 2004
Biotin Dissolution from Pharmaceutical Dosage Forms Using an Automated HPLC System 1
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Hassan Y. Aboul-Enein, '* Rajaa F. Hussein, Mahasen A. Radwan, and Sameer Al-Rawithi 3
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Pharmaceutical Analysis Laboratory, Biological and Medical Research Department, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia Pharmacokinetics and Therapeutic Drug Monitoring Laboratory, Biological and Medical Research Department, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia Department of Clinical Pharmacy, College of Pharmacy, Science & Medical Studies, Department for Women Students, King Saud University, Saudi Arabia 3
ABSTRACT A novel, rapid, accurate, and sensitive automated high-performance liquid chromatographic assay was developed to detcamme btdtin (BI) in
""Correspondence: Hassan Y. Aboul-Enein, Pharmaceutical Analysis Laboratory, Biological and Medical Research Department (MBC 03-65), King Faisal Specialist Hospital and Research Center, P. O. Box 3354, Riyadh 11211, Saudi Arabia; E-mail:
[email protected]. 511 DOI: 10.I081/JLC-120027622 Copyright © 2004 by Marcel Dekkcr, Inc.
1082-6076 (Print); 1520-572X ( O n l i n e ) www.dekker.com
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A b o u l - E n e i n e t al. pharmaceutical d o s a g e f o r m s and to f o l l o w i t s dissolution pattern. An efficient separation of BI w a s performed using a stainless steel Supelcosil L C - 1 8 c o l u m n (25 cm x 4.6 m m ; 5 u,m p a n i c l e size) preceded by a Sentry guard column. The m o b i l e p h a s e consisted of an 8 0 % aqueous solution (pH 2.5 adjusted with phosphoric acid) containing 2 0 % acetonitrile delivered at a flow rate of 1 . 5 m L / m i n . T h e compound of interest w a s detected using a photodiode array detector at 1 9 0 n m . Under t h e s e conditions, the assay run time w a s 6 min since the retention time of BI w a s 3.8 ± 0 . 2 min. T h e detector response w a s linear for BI in alkaline solution (r > 0.999) in the range of 0.01 - 2 . 0 0 p . g / m L . T h e detection and the quantification limits for BI were 0 . 0 0 5 and 0.01 u . g / m L , respectively. T h e dissolution data s h o w e d R S D % of 3 . 6 - 1 2 . 7 % for all BI determined concentrations. No interferences w e r e observed from the tablet's excipients. T h e drug content in e a c h tablet ranged from 100 to 102.5%. T h e dissolution study o f B I O T T N * tablets revealed that B I i n U S P m e d i a (pH 1.2) s h o w e d no dissolution up to 3 nr. H o w e v e r , a first order release kinetic, with dissolution T s o of 14 ± 1.3 m i n , w a s observed in U S P media (pH7.4). %
Key Words:
Biotin; Photodiode array detector; Dissolution; HPLC.
INTRODUCTION B i o t i n ( B I ) , Cis h e x a h y d r o - 2 - o x o - l H - t h i e n o - [ . 3 , 4 - d ] - i m i d a z o l e - 4 - p e n t a n o i c a c i d , k n o w n a s "Vitamin H , i s a v e r y s l i g h t l y w a t e r - s o l u b l e v i t a m i n b e l o n g i n g t o the B - c o m p l e x , w h i c h i s f o u n d i n s m a l l quantities i n all l i v i n g cells. It exists in eight isomer forms, but only D - ( + ) - b i o t i n is biologically active. In the intestine, biotin is absorbed through a saturable transportation s y s t e m , w h i l e at great biotin concentrations; p a s s i v e diffusion p r e d o m i n a t e s . L o w biotin intake has b e e n reported to result in serious biochemical disorders in animal organisms, such as reduced carboxylase activity, inhibition of protein and R N A synthesis, r e d u c e d antibody production, etc. T h e r e are indications of biotin i n v o l v e m e n t in severe animal syndromes, such as the a v i a n fatty liver and k i d n e y s y n d r o m e ( F L K S ) a n d the trout 'blue s l i m e ' disease. - - Diagnosis of biotin deficiency, as well as monitoring of biotin l e v e l s i n b i o l o g i c a l fluids o f p a t i e n t s r e c e i v i n g b i o t i n t r e a t m e n t i s v e r y c r u c i a l . Equally important is the determination of biotin levels in pharmaceutical preparations, as w e l l as in f o o d and f o o d supplement products, w h i c h constitute the main source of biotin in h u m a n s . For this reason, analytical m e t h o d s h a v e b e e n developed, in order to determine biotin in biological fluids, as w e l l as various kinds of food products and pharmaceutical preparation c o n t a i n i n g biotin. M a n y m e t h o d s h a v e b e e n described for the determination of 1 1 1
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biotin, using m i c r o b i o l o g i c a l ~ and, recently, high performance liquid chromatographic ( H P L C ) p r o c e d u r e s . Although the microbiological assays provide excellent sensitivity, these methods are laborious a n d t i m e consuming, with an average incubation time of 18—24 nr. On the other hand, at present, the best H P L C procedures involve electrochemical detection - ' and fluorescent reaction with derivatization. Although these methods are highly sensitive and specific, they are susceptible to interference, have long retention times, or use m o r e sophisticated equipment, w h i c h is n o t normally found in a c o m m o n clinical laboratory. Drug dissolution testing is an integral part ' of pharmaceutical development and in routine quality control monitoring of drug release characteristics. To the best of our knowledge, no method has been reported in the literature for the determination of BI release from pharmaceutical dosage forms. Therefore, the objectives of this investigation w e r e to develop a new, simple, fully automated, rapid, and sensitive procedure, which involves no derivatization H P L C , with photodiode array detectors, for the quantitation of BI in pharmaceutical formulation, tablets, and to evaluate its in vitro dissolution rate. 1
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EXPERIMENTAL Chemicals and Reagents T h e H P L C grade acetonitrile, phosphoric acid 8 5 % , and hydrochloric acid were purchased from Fisher Scientific Co. (Fairlawn, NJ, U S A ) . D i sodium hydrogen phosphate w a s supplied from Fluka (Buchs, Switzerland). H P L C grade water w a s prepared by reverse osmosis and further purified by passing through a Milli-Q System (Millipore Company, Milford, M A , U S A ) . Pure BI w a s purchased from Sigma Chemical Co. (St. Louis, M O , U S A ) , B I O T I N tablets, containing 0.8 mg biotin per tablet, were obtained from the local market. A stock solution of BI (2 | x g / m L ) was prepared in 0.02N disodium hydrogen phosphate ( N a H P 0 4 ) , p H 7 . 4 adjusted with phosphoric acid. This stock solution w a s prepared weekly and further diluted to produce concentrations of BI that ranged from 0.01 to 2.00 M-g/mL ( 0 . 0 1 , 0 . 0 5 , 0 . 1 , 0.2, 0.4, 0 . 6 , 0 . 8 , 1 . 6 , and 2.00 p , g / m L ) . T h e stock solutions were stored in the dark at — 70°C until needed during the week. No instabilities were observed from solutions stored under these conditions compared to fresh daily-diluted ones, since there w a s no observed change in BI peak height or appearance of any impurities. (8,
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Instrument and Chromatographic Conditions A High-Performance Liquid Chromatographic System (HPLC), Waters Alliance, dissolution system (Waters Associates, Inc. Milford, M A . U S A ) consisting of W a t e r s 2 6 9 0 D Separation Module with eight-needle dissolution dispenser, Waters Transfer Module, with eight syringes, one dissolution test bath (Hanson Research SR8-Plus), eight Uni-Probes, one for each dissolution vessel to be sampled, and Waters 996 Photodiode array detector. T h e compound of interest w a s detected at 190 n m . T h e data were collected with a M i l l e n n i u m Chromatography M a n a g e r data collection system, utilizing a Pentium 4 computer connected to Inkjet HP PSC 750 printer. BI determination was performed using a stainless steel Supelcosil LC-18 column (25 cm x 4.6 m m ; 5 p.m particle size) preceded by a Sentry guard column. T h e mobile phase consisted of an 8 0 % aqueous solution (pH 2.S adjusted with phosphoric acid), containing 2 0 % acetonirrile delivered at a flow rate of 1.6 m L / m i n . 32
In Vitro Dissolution Studies T h e dissolution rates of BI from tablets were performed on a Hanson SR8Plus dissolution apparatus (Hanson Research Corp., Chatsworth, C A , U S A ) . Drug release tests were carried out according to conventional U S P 26 dissolution procedures for the single-entity p r o d u c t s , with the use of a paddle-stirrer type of apparatus, in 500 mL of 0.02N di-sodium hydrogen phosphate ( N a H P 0 ) ( p H 7 . 4 using phosphoric acid, to simulate intestinal medium), at a stirring rate of 75 r p m for 5 hr, then at infinity ( 2 5 0 r p m ) for the rest of the dissolution period 5.5 hr. The temperature of the cell was maintained at 37 + 0.5°C by using a thermostatic bath. At each sample time interval, an exact volume of sample was withdrawn from each flask and immediately replaced with an identical volume of fresh medium to maintain a dissolution sink condition. A correction factor was included in the calculations to account for the drug lost in the samples. At predetermined time intervals (0, 0.083, 0 . 1 6 6 , 0 . 3 3 3 , 0.50, 0.75, 1, 1.5, 2 , 3 , 4 , 5 , and 5.5 hr), the concentrations of BI (p,g/mL) in the dissolution medium were determined by the proposed H P L C . 1151
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Analysis of Pharmaceutical Dosage Form Preparation of Biotin Standard Solution
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A stock solution of pure BI (2 u.g/mL) was prepared in water. This stock solution was further diluted weekly with 0.02N di-sodium hydrogen
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p h o s p h a t e ( N a H P 0 ) p H 7.4 adjusted with p h o s p h o r i c acid t o p r o d u c e concentrations of BI that ranged from (0.01 to 2.00 n-g/mL) 0 . 0 1 , 0.05, 0 . 1 , 0 . 2 , 0 . 4 , 0 . 6 , 0 . 8 , 1.6, a n d 2 . 0 0 M-g/mL. 2
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Preparation of Biotin Solution from Tablets T h r e e different s t o c k s o l u t i o n s o f B I O T I N ® t a b l e t s c o n t a i n i n g ( 0 . 8 m g ) biotin was prepared in 500 mL of 0.02N di-sodium hydrogen phosphate (Na HP04) pH 7.4 adjusted w i t h p h o s p h o r i c acid to p r o d u c e concentration of ( 1 . 6 f A g / m L ) . F o r t h e c o n c e n t r a t i o n s 1.6 u , g / m L , w e f o u n d a v e r a g e a m o u n t s p e r tablet (drug content) ± S.D. were 0.81 + 0.01 m g , while the percentage a v e r a g e recovery + S.D. of BI tablets c o m p a r e d to the p u r e BI w e r e 1 0 1 . 2 5 ± 1.25 (n = 3 ) . 2
Data Analysis All data w e r e reported as the m e a n ± S.D. of at least seven parallel studies. T h e results were calculated by linear regression without weighing, u s i n g t h e f o r m u l a : Y = a 4- bX, W h e r e F i s t h e p e a k h e i g h t of t h e d r u g , a is t h e intercept, b is the slope, and X is the concentration of BI. T h e a m o u n t of B I , obtained from the drug dissolution studies, was calculated from the calculated l i n e a r r e g r e s s i o n e q u a t i o n . T h e i n v i t r o d i s s o l u t i o n d a t a ( n = 7 ) w e r e fitted t o P e p p a s ' equation - to determine the kinetic order of release. 116
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as t h r e e t i m e s the level of noise for-BI in b l a n k s a m p l e s , w a s 0.005 | i g / m L , w h i l e t h e quantification limit o f this m e t h o d w a s 0.01 | x g / m L . T o d e m o n s t r a t e t h e utility of the m e t h o d , Fig. 1 d e p i c t s t h r e e representative c h r o m a t o g r a m s i n c l u d i n g a blank s a m p l e (A); a s a m p l e s u p p l e m e n t e d (B) with 0.8 p , g / m L of B I , and t h e third sample ( C ) , calculated c o n c e n t r a t i o n of 1.6 | x g / m L collected 1.5 hr after starting the tablet dissolution.
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Figure 1. Chromatograms of a blank s a m p l e ( A ) ; a sample supplemented ( B ) w i t h 0.8 | x g / m L of pure BI powder; and the third s a m p l e (C), calculated concentration of 1.6 ( j L g / m L collected 1.5 hr after starting the tablet dissolution.
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t a b l e 1 shows the m e a n BI content in the tablets d o s a g e form. F o r t h e t e s t e d t a b l e t s , n = 7 , (0.8 g m / t a b l e t ) , t h e m e a n d r u g c o n t e n t w a s 0.81 + 0 . 0 6 m g . T h e dissolution study of B I O T I N * tablets revealed that BI in U S P m e d i a ( p H 1.2) s h o w e d n o d i s s o l u t i o n u p t o 3 hr, s i n c e B I i s v e r y s l i g h t l y s o l u b l e i n w a t e r b u t it d i s s o l v e s in d i l u t e a l k a l i n e s o l u t i o n s . H o w e v e r , a first o r d e r r e l e a s e k i n e t i c , r = 0 . 9 9 , w i t h d i s s o l u t i o n T s o » of 14 ± 1.3 m i n , w a s o b s e r v e d in U S P media ( p H 7 . 4 ) as s h o w n in Fig. 2. For each plotted point, the m e a n reading of eight tablets is s h o w n expressed as percent BI released. T h e d i s s o l u t i o n d a t a o f B I , a t this p H , s h o w e d a little h i g h e r R S D % (9.3 t o 1 2 . 7 % ) a t l o w e r d r u g c o n c e n t r a t i o n s ( < 1.0 u - g / m L ) . O n t h e o t h e r h a n d , t h e R S D % w a s 3 . 6 - 7 . 2 % f o r BT c o n c B n t r a t i n n s > 1.0 [i.g/rnT. Thf.re.fnny t h e utilized a u t o m a t e d d i s s o l u t i o n - H P L C s y s t e m p r o d u c e d p r e c i s e a n d
Table 1. T h e m e a n * ( + S D ) biotin content in tablet d o s a g e form. L a b e l c l a i m ( m g p e r tablet*) Measured amount (mg/tablet) T h e % total a m o u n t per tablet dosage form
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T i m e (min) Figure 2. T h e in vitro dissolution profile of B I O T I N * c a p s u l e s in U S P intestinal m e d i a ( p H 7 . 4 ) . A v e r a g e of 7 determinations.
r e p r o d u c i b l e results. A l m o s t all t h e d r u g w a s r e l e a s e d (at p H 7 . 4 $ from t h e tablets w i t h i n 1.5 hr. I n c o n c l u s i o n , t h e fully a u t o m a t e d d i s s o l u t i o n - H P L C s y s t e m used w a s rapid, r e p r o d u c i b l e , a n d c o n v e n i e n t t o follow B I after dissolution from p h a r m a c e u t i c a l d o s a g e f o r m s , a n d required n o internal standard. T h e m e t h o d d e s c r i b e d is suitable for quality control of biotin p h a r m a c e u t i c a l formulations.
REFERENCES 1. L i v a n i o u , E.; C o s t o p o u l o u , D . ; Vassiliavou, L.; L e o n d i a d i s , L . ; N y a l a l a , J . O . ; Ithakissios, U.S.; Evangelatos, J . P . A n a l y t i c a l t e c h n i q u e for d e t e r m i n i n g biotin. J. C h r o m a t o g r . A 2 0 0 0 , 881, 3 3 1 - 3 4 3 . 2. M o c k , D . M . Biotin. In Handbook of Vitamins, 3rd E d . ; R u c h e r , R . B . , Suttie, J . W . , M c c o r m i l l e , D . B . , Maachlin, L J . , E d s . ; M a r c e l D e k k e r Inc.: N e w York, 2001. 3 . B o n j o u r , J.P. B i o t i n i n h u m a n nutrition. A n n . N . Y . A c a d . Sci. 1 9 8 5 , 447, 97-104. 4 . W h i t e h e a d , C . C . A s s e s s m e n t o f biotin deficiency i n a n i m a l s . A n n . N . Y . A c a d . S c i . 1 9 8 5 , 447, 8 6 - 9 6 . 5. W a t k i n s , B . A . Influences of biotin deficiency a n d dietary trans-fatty acids on t i s s u e lipids in children. British J. Nutr. 1 9 8 9 , 61, 9 9 - 1 1 1 .
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