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HPLC analysis of digoxin and digitoxin : development of methods for dosage form assay and separation… Desta, Belachew 1982

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HPLC ANALYSIS OF DIGOXIN AND DIGITOXIN: DEVELOPMENT OF METHODS FOR DOSAGE FORM ASSAY AND SEPARATION OF POTENTIAL IMPURITIES AND METABOLITES by BELACHEW DESTA B. Pharm., A d d i s Abeba U n i v e r s i t y A d d i s Abeba, E t h i o p i a , 1964 M . S c , U n i v e r s i t y o f B r i t i s h Columbia Vancouver, B.C. Canada, 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s D i v i s i o n o f P h a r m a c e u t i c a l C h e m i s t r y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1982 © Belachew Desta In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o lumbia, I agre e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f 'Pifrt7?,rt££/5\>~Tt £>tL "5cj£A)<Ur* The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main M a l l Vancouver, Canada V6T 1Y3 ABSTRACT The o b j e c t i v e o f t h i s i n v e s t i g a t i o n was t o d e v e l o p q u a n t i t a t i v e i s o c r a t i c HPLC methods f o r the a n a l y s i s o f d i g o x i n and d i g i t o x i n . An HPLC system t h a t employs a r e v e r s e - p h a s e column, UV d e t e c t i o n a t 220 nm and s o l v e n t systems c o n s i s t i n g o f v a r i o u s p r o p o r t i o n s o f wat e r , m e t h a n o l , i s o p r o p a n o l and d i c h l o r m e t h a n e was d e v e l o p e d f o r t h e s e p a r a t i o n o f d i g o x i n , d i g i t o x i n and t h e i r p o t e n t i a l d e g r a d a t i o n p r o d u c t s and meta-b o l i t e s . HPLC s e p a r a t i o n s o f t h e above compounds by i s o c r a t i c , s o l v e n t s w i t c h o v e r and g r a d i e n t e l u t i o n modes were c a r r i e d out i n c h r o m a t o g r a p h i c times o f 27, 16 and 13 m i n u t e s , r e s p e c t i v e l y . For purposes o f m o n i t o r i n g t h e s e p a r a t i o n o f d i h y d r o m e t a b o l i t e s o f d i g o x i n , a 100% f l u i d r e c o v e r y system was d e v e l o p e d f o r use i n t h e HPLC a n a l y s i s o f d i g o x i n and i t s m e t a b o l i t e s a f t e r f l u o r o g e n i c post-column d e r i v a t i z a t i o n u s i n g t h e a i r - s e g m e n t a t i o n p r o c e s s . As an e v i d e n c e o f s e l e c t i v i t y , t h e i s o c r a t i c HPLC systems were u t i l i z e d f o r the s e p a r a t i o n o f a m i x t u r e o f t e n c l o s e l y r e l a t e d s t e r o i d s and t h e i s o l a t i o n o f d i g i t o x i n from D i g i t a l i s purpurea l e a f . The i s o c r a t i c HPLC systems were found t o be a p p l i c a b l e f o r t h e q u a n t i t a t i v e a n a l y s i s o f d i g o x i n and d i g i t o x i n i n t h e i r r e s p e c t i v e dosage forms. The HPLC a s s a y o f d i g o x i n and d i g i t o x i n dosage forms was c a r r i e d o u t i n l e s s than f o r t y - f i v e m i n u t e s . These methods were found t o be p r e c i s e , a c c u r a t e , s e n s i t i v e enough f o r s i n g l e t a b l e t a s s a y , and c a p a b l e o f s i m u l t a n e o u s l y m o n i t o r i n g t he p o t e n t i a l d e g r a d a t i o n p r o d u c t s o r m e t a b o l i t e s o f d i g o x i n and d i g i t o x i n . A c omparison o f t h e a s s a y o f d i g o x i n and d i g i t o x i n dosage forms i i i . by HPLC and USP methods i n d i c a t e d t h a t : (a) t h e p r e c i s i o n and a c c u r a c y o f both methods were comparable and w i t h i n a c c e p t a b l e l i m i t s ; (2) a n a l y s i s by HPLC can be completed i n l e s s than f o r t y - f i v e m i n u t e s whereas t h e USP methods r e q u i r e o v e r f o u r h o u r s ; and (3) t h e HPLC methods have the advantages o f h i g h e r s e n s i t i v i t y , s e l e c t i v i t y and s i m p l i c i t y o v e r t h e USP methods. The HPLC methods were used f o r t h e s t a b i l i t y s t u d y o f d i g o x i n and d i g i t o x i n i n t h e i r r e s p e c t i v e dosage forms. L a n o x i n and d i g i t o x i n t a b l e t s were found t o be s t a b l e under a l l t h e c o n d i t i o n s o f s t o r a g e used i n t h i s s t u d y . N a t i g o x i n t a b l e t s , L a n o x i n i n j e c t i o n and e l i x i r were found t o be s u b j e c t t o v a r y i n g d e g r e e s and p a t t e r n s o f d e g r a d a t i o n . On the b a s i s o f t h e s t a b i l i t y r e s u l t s i t was o b s e r v e d t h a t t h e a s s o r t m e n t o f pathways t h a t may be i n v o l v e d a t d i f f e r e n t c o n d i t i o n s and times o f s t o r a g e ' w o u l d make i t d i f f i c u l t t o e s t i m a t e d i g o x i n s h e l f - l i f e from d a t a o b t a i n e d by a c c e l e r a t e d a g i n g . From t h e r e s u l t s o f t h i s i n v e s t i g a t i o n , i t was c o n c l u d e d t h a t t h e i s o c r a t i c HPLC methods were s u i t a b l e f o r t h e a s s a y o f d i g o x i n and d i g i t o x i n dosage forms as w e l l as f o r purposes o f s t a b i l i t y t e s t i n g and s i m u l t a n e o u s m o n i t o r i n g o f d e g r a d a t i o n p r o d u c t s or m e t a b o l i t e s . T h i s a b s t r a c t r e p r e s e n t s t he t r u e c o n t e n t s o f the t h e s i s s u b m i t t e d . IV. TABLE OF CONTENTS Page ABSTRACT 1 1 LIST OF TABLES x i i LIST OF FIGURES x v ABBREVIATIONS x x i i INTRODUCTION 1 I . LITERATURE SURVEY 4 1. H i g h - P e r f o r m a n c e L i q u i d Chromatography (HPLC) 5 (A) T h e o r y 5 (a) Mechanism o f R e t e n t i o n 5 (b) T h e o r e t i c a l P l a t e 8 ( c ) Band Widening 1 0 (d) R e s o l u t i o n 1 2 (B) I n s t r u m e n t a t i o n 2 0 (a) S o l v e n t R e s e r v o i r s 2 0 (b) Pumps 20 ( c ) Sample I n t r o d u c t i o n D e v i c e s 2 2 (d) Columns 2 3 (e) D e t e c t o r s 2 8 ( f ) Data P r o c e s s o r s 33 (C) T e c h n i q u e 3 4 (a) HPLC Mode S e l e c t i o n 34 (b) O p t i m i z a t i o n o f R e s o l u t i o n 36 ( c ) Advantages o f HPLC 4 0 (d) D i s a d v a n t a g e s o f HPLC 41 V. Page 2. The T e s t Drugs: D i g o x i n and D i g i t o x i n 42 (A) C h e m i s t r y 42 (a) D e s c r i p t i o n 42 (b) Names 42 (c ) Appearance 43 (d) S y n t h e s i s 43 (e) S o l u b i l i t y 43 ( f ) S t a b i l i t y 44 (g) S t r u c t u r e and B i o l o g i c a l A c t i v i t y 44 (B) Pharmacology 51 (a) Mechanism o f A c t i o n 52 (b) A b s o r p t i o n , Fate and E x c r e t i o n 52 (C) P h a r m a c o k i n e t i c s and T h e r a p y 53 3. Development o f Methods o f A n a l y s i s o f D i g o x i n and D i g i t o x i n 56 (A) A n a l y s i s o f Samples i n P l a n t E x t r a c t s , S t a n d a r d M i x t u r e s and Dosage Forms -> 56 (B) A n a l y s i s o f Samples i n B i o l o g i c a l F l u i d s 63 (a) Methods 63 (b) R a t i o n a l e f o r Measurement o f Serum D i g o x i n and D i g i t o x i n 65 4. C l a s s i f i c a t i o n o f the A n a l y t i c a l Methods o f D i g o x i n and D i g i t o x i n 67 (A) C o l o r i m e t r i c Methods 67 (a) Methods Based on the Sugar M o i e t y , 67 (b) Methods Based on the B u t e n o l i d e M o i e t y 67 (B) F l u o r o m e t r i c Methods 67 (C) C h r o m a t o g r a p h i c Methods 67 (D) B i o c h e m i c a l Methods 68 v i . Page 5. L i m i t a t i o n s o f the A n a l y t i c a l Methods 72 (A) S e n s i t i v i t y 72 (B) S e l e c t i v i t y 73 (C) Time 75 I I . EXPERIMENTAL 76 1. A p p a r a t u s 76 2. M a t e r i a l s 78 3. P r e p a r a t i o n o f HPLC S o l v e n t Systems 85 4. E q u i l i b r a t i o n o f HPLC Column 85 5. D e t e r m i n a t i o n o f R e t e n t i o n Time 85 6. S e p a r a t i o n o f D i g o x i n , D i g i t o x i n and t h e i r M e t a b o l i t e s o r D e g r a d a t i o n P r o d u c t s and I m p u r i t i e s 85 (A) I s o c r a t i c E l u t i o n 85 (B) S o l v e n t S w i t c h o v e r E l u t i o n 86 (C) G r a d i e n t E l u t i o n 86 7. S e p a r a t i o n o f D i g o x i n and i t s M e t a b o l i t e s a f t e r F l u o r o -g e n i c Post-Column D e r i v a t i z a t i o n u s i n g t he A i r -S egmentation P r i n c i p l e w i t h 100 p e r c e n t F l u i d Recovery 87 (A) P r e p a r a t i o n o f Hydrogen P e r o x i d e S o l u t i o n 87 (B) P r e p a r a t i o n o f D e h y d r o a s c o r b i c A c i d S o l u t i o n 87 D (C) P r e p a r a t i o n o f B r i j 35 S o l u t i o n 87 (D) Dual D e t e c t o r M o n i t o r i n g o f the S e p a r a t i o n o f D i g o x i n and i t s M e t a b o l i t e s b e f o r e and a f t e r F l u o r o g e n i c D e r i v a t i z a t i o n 87 (E) HPLC P r o c e d u r e and C o n d i t i o n s 88 8. S e p a r a t i o n o f Nine E q u i n e E s t r o g e n s as E v i d e n c e o f S e l e c t i v i t y 90 v i i . Page 9. I s o l a t i o n o f D i g i t o x i n from D i g i t a l i s p u r p u r e a L e a f 90 (A) E x t r a c t i o n 90 (B) HPLC P r o c e d u r e and C o n d i t i o n s 90 10. A n a l y s i s o f D i g o x i n i n i t s Dosage Forms 91 (A) I n f r a r e d Spectrum o f D i g o x i n 91 (B) S p e c t r a l C h a r a c t e r i s t i c s o f D i g o x i n 91 (C) HPLC P r o c e d u r e and C o n d i t i o n s f o r the A n a l y s i s o f T a b l e t s and I n j e c t i o n 91 (D) HPLC P r o c e d u r e and C o n d i t i o n s f o r the A n a l y s i s o f E l i x i r ;. 94 (E) P r e p a r a t i o n o f I n t e r n a l S t a n d a r d S o l u t i o n s 94 (F) P r e p a r a t i o n o f S t a n d a r d S o l u t i o n s o f D i g o x i n 94 (G) P r e p a r a t i o n o f C a l i b r a t i o n Curves 94 (H) Sample P r e p a r a t i o n o f D i g o x i n Dosage Forms 98 (a) Composite T a b l e t Assay 98 (b) S i n g l e T a b l e t A s s a y 98 ( c ) I n j e c t a b l e F o r m u l a t i o n A s s a y 98 (d) E l i x i r A s s a y 99 ( I ) Q u a n t i t a t i o n 99 (J ) D e t e r m i n a t i o n o f P r e c i s i o n o f T a b l e t Assay 100 (K) D e t e r m i n a t i o n o f P e r c e n t a g e Recovery o f D i g o x i n from T a b l e t s 100 11. A n a l y s i s o f D i g i t o x i n i n i t s Dosage Forms 100 (A) I n f r a r e d Spectrum o f D i g i t o x i n 100 (B) S p e c t r a l C h a r a c t e r i s t i c s o f D i g i t o x i n 101 (C) HPLC P r o c e d u r e and C o n d i t i o n s f o r the A n a l y s i s o f T a b l e t s and I n j e c t i o n 101 (D) P r e p a r a t i o n o f I n t e r n a l S t a n d a r d S o l u t i o n 101 v i i l . Page (E) P r e p a r a t i o n o f S t a n d a r d S o l u t i o n s o f D i g i t o x i n 105 (F) P r e p a r a t i o n o f a C a l i b r a t i o n Curve 105 (G) Sample P r e p a r a t i o n o f D i g i t o x i n Dosage Forms 105 (a) Composite T a b l e t A s s a y 105 (b) S i n g l e T a b l e t A s say 107 (c) I n j e c t a b l e F o r m u l a t i o n A s s a y 107-(H) Q u a n t i t a t i o n 107 ( I ) D e t e r m i n a t i o n o f P r e c i s i o n o f T a b l e t A s say 108 ( J ) D e t e r m i n a t i o n o f P e r c e n t a g e Recovery o f D i g i t o x i n from T a b l e t s 108 12. Comparison o f the A n a l y s i s o f D i g o x i n and D i g i t o x i n Dosage Forms by HPLC and USP XX Methods 109 (A) Brands o f D i g o x i n and D i g i t o x i n T a b l e t s Used 109 (B) Dosage Forms and S t r e n g t h s o f D i g o x i n and D i g i t o x i n Used 109 (C) Sample P r e p a r a t i o n o f D i g o x i n Dosage Forms f o r HPLC A n a l y s i s 109 (D) HPLC P r o c e d u r e f o r Q u a n t i t a t i o n o f D i g o x i n 113 (E) Sample P r e p a r a t i o n o f D i g o x i n Dosage Forms f o r A n a l y s i s by USP Methods 113 (F) USP P r o c e d u r e f o r Q u a n t i t a t i o n o f D i g o x i n 113 (G) Sample P r e p a r a t i o n o f D i g i t o x i n Dosage Forms f o r HPLC A n a l y s i s 113 (H) HPLC P r o c e d u r e f o r Q u a n t i t a t i o n o f D i g i t o x i n 113 ( I ) Sample P r e p a r a t i o n o f D i g i t o x i n Dosage Forms f o r A n a l y s i s by USP Methods 113 ( J ) USP P r o c e d u r e f o r Q u a n t i t a t i o n o f D i g i t o x i n 120 (K) D e t e r m i n a t i o n o f P r e c i s i o n o f t h e USP Method f o r D i g o x i n T a b l e t A s s a y 120 i x . Page (L) D e t e r m i n a t i o n o f P r e c i s i o n o f t h e USP Method f o r D i g i t o x i n T a b l e t A s s a y 120 (M) D e t e r m i n a t i o n o f P e r c e n t a g e Recovery o f D i g o x i n from T a b l e t s u s i n g the USP Method 120 (N) D e t e r m i n a t i o n o f P e r c e n t a g e Recovery o f D i g i t o x i n from T a b l e t s u s i n g the USP Method 126 S t a b i l i t y M o n i t o r i n g o f D i g o x i n and D i g i t o x i n i n t h e i r r e s p e c t i v e Dosage Forms 126 (A) Brands o f D i g o x i n and D i g i t o x i n T a b l e t s Used 126 (B) Dosage Forms and S t r e n g t h s o f D i g o x i n and D i g i t o x i n Used 126 (C) C o n d i t i o n s o f S t o r a g e 127 (D) HPLC P r o c e d u r e and C o n d i t i o n s 127 (a) For A n a l y s i s o f D i g o x i n T a b l e t s and I n j e c t i o n ... 127 (b) For A n a l y s i s o f D i g i t o x i n T a b l e t s 127 ( c ) For A n a l y s i s o f D i g o x i n E l i x i r 127 (E) P r e p a r a t i o n o f I n t e r n a l S t a n d a r d S o l u t i o n s 127 (a) For A n a l y s i s o f D i g o x i n T a b l e t s and I n j e c t i o n ... 127 (b) For A n a l y s i s o f D i g o x i n E l i x i r 127 ( c ) For A n a l y s i s o f D i g i t o x i n T a b l e t s 129 (F) P r e p a r a t i o n o f S t a n d a r d S o l u t i o n s o f D i g o x i g e n i n , D i g o x i g e n i n m o n o d i g i t o x o s i d e , D i g o x i g e n i n b i s d i g i t o -x o s i d e and D i g o x i n 129 (a) For A n a l y s i s o f D i g o x i n and i t s D e g r a d a t i o n P r o d u c t s i n T a b l e t s and I n j e c t i o n 129 (b) For A n a l y s i s o f D i g o x i n and i t s D e g r a d a t i o n P r o d u c t s i n the E l i x i r Dosage Form 129 (G) P r e p a r a t i o n o f S t a n d a r d S o l u t i o n s o f D i g i t o x i g e n i n , D i g i t o x i g e n i n m o n o d i g i t o x o s i d e , D i g i t o x i g e n i n b i s d i g i t o x o s i d e and D i g i t o x i n 129 (H) P r e p a r a t i o n o f C a l i b r a t i o n Curves 130 ( I ) Sample P r e p a r a t i o n 130 (a) D i g o x i n T a b l e t s 130 X . Pa^e (b) D i g o x i n I n j e c t i o n 130 (c) D i g o x i n E l i x i r 130 (d) D i g i t o x i n T a b l e t s 136 ( J ) Q u a n t i t a t i o n 136 (a) A n a l y s i s o f D i g o x i n T a b l e t s and I n j e c t i o n 136 (b) A n a l y s i s o f D i g o x i n E l i x i r . 136 (c) A n a l y s i s o f D i g i t o x i n T a b l e t s 137 (K) D e t e r m i n a t i o n o f pH 137 I I I . RESULTS AND DISCUSSION 139 1. E v o l u t i o n o f the b a s i c HPLC S o l v e n t System f o r t h e A n a l y s i s o f C a r d i a c G l y c o s i d e s 139 2. Development o f HPLC Systems f o r the S e p a r a t i o n o f D i g o x i n , D i g i t o x i n , t h e i r R e s p e c t i v e D e g r a d a t i o n P r o d u c t s o r M e t a b o l i t e s and R e l a t e d Compounds 144 (A) S e p a r a t i o n by I s o c r a t i c E l u t i o n 144 (B) S e p a r a t i o n by G r a d i e n t E l u t i o n 151 (C) S e p a r a t i o n u s i n g a S o l v e n t S w i t c h o v e r System 155 3. Development o f a 100% F l u i d Recovery System f o r t h e HPLC A n a l y s i s o f D i g o x i n and i t s M e t a b o l i t e s a f t e r F l u o r o g e n i c Post-Column D e r i v a t i z a t i o n U s i n g t he A i r - S e g m e n t a t i o n P r o c e s s 158 4. S e p a r a t i o n o f Nine E q u i n e E s t r o g e n s u s i n g t he HPLC System, as e v i d e n c e o f S e l e c t i v i t y 163 5. I s o l a t i o n o f D i g i t o x i n from D i g i t a l i s p u r p u r e a L e a f 168 6. Development o f HPLC Methods f o r t h e A n a l y s i s o f D i g o x i n i n i t s Dosage Forms 170 7. Development o f an HPLC Method f o r the A n a l y s i s o f D i g i t o x i n i n i t s Dosage Forms 185 . 8. Comparison o f t h e A n a l y t i c Data o f D i g o x i n and D i g i t o x i n Dosage Forms as O b t a i n e d by HPLC and USP XX Methods 192 9. S t a b i l i t y Study o f D i g o x i n and D i g i t o x i n i n t h e i r R e s p e c t i v e Dosage Forms u s i n g HPLC Methods 200 x i . Page IV. SUMMARY AND CONCLUSIONS 232 V. REFERENCES • 239 x i i . LIST OF TABLES T a b l e Page I Name, F u n c t i o n a l i t y and S u r f a c e Area o f Commer-c i a l l y a v a i l a b l e A d s o r b e n t s used i n High P e r formance L i q u i d Chromatography 27 II Summary o f Some C h a r a c t e r i s t i c s o f Normal and Reverse Phase Columns 31 I I I D e s i g n and O p e r a t i o n a l Parameters A s s o c i a t e d w i t h A n a l y t i c a l and P r e p a r a t i v e HPLC 32 IV G e n e r a l Guide f o r HPLC Mode S e l e c t i o n 37 V P a r t i a l L i s t i n g o f S o l v e n t s Used i n H i g h -P e r formance L i q u i d Chromatography i n Order o f D e c r e a s i n g P o l a r i t y 39 VI Genins o f the C a r d e n o l i d e S e r i e s o f D i g i t a l i s G l y c o s i d e s 49 VII Chemical S t r u c t u r e s o f Some C a r d i a c G l y c o s i d e s o f t he C a r d e n o l i d e S e r i e s 79 V I I I R e l a t i v e H u m i d i t y V a l u e s O b t a i n e d w i t h Aqueous S u l f u r i c A c i d S o l u t i o n s '. 128 IX Response F a c t o r s O b t a i n e d f o r t h e A n a l y s i s o f D i g o x i n , D i g i t o x i n and t h e i r P o t e n t i a l D e g r a d a t i o n P r o d u c t s u s i n g 17a-ethynyl e s t r a d i o l ( a ) , h y d r o c o r t i s o n e (b) and 1 7 a - m e t h y l t e s t o s t e r o n e (c) as I n t e r n a l S t a n d a r d s 138 X R e t e n t i o n Times ( t r ) and C a p a c i t y R a t i o V a l u e s ( K 1 ) o f the C a r d i a c G l y c o s i d e s and A g l y c o n e s used i n t h i s Study as O b t a i n e d under the C o n d i t i o n s D e f i n e d by the C o r r e s p o n d i n g F i g u r e s 157 XI R e s u l t s o f the HPLC A n a l y s i s o f Composite Samples o f D i g o x i n T a b l e t s 175 XII P r e c i s i o n Data f o r the HPLC A n a l y s i s o f D i g o x i n T a b l e t s 177 X I I I R e s u l t s o f HPLC S i n g l e T a b l e t Assay o f D i g o x i n T a b l e t s 178 XIV Recovery Data f o r D i g o x i n T a b l e t A s s a y 179 x i i i . T a b l e Page XV R e s u l t s o f the HPLC A n a l y s i s o f D i g o x i n I n j e c t i o n and E l i x i r 181 XVI R e s u l t s o f the HPLC A n a l y s i s o f D i g i t o x i n T a b l e t s and I n j e c t i o n 190 XVII Recovery Data f o r the HPLC A n a l y s i s o f D i g i t o x i n T a b l e t s 191 XVIII R e s u l t s o f t h e A n a l y s i s o f Composite Samples o f D i g o x i n T a b l e t s by HPLC and the USP Method 194 XIX R e s u l t s o f the A n a l y s i s o f D i g o x i n I n j e c t i o n and E l i x i r by HPLC and t h e USP Method 195 XX Re c o v e r y Data f o r D i g o x i n T a b l e t A s s a y by HPLC and t h e USP Method 196 XXI R e s u l t s o f the A n a l y s i s o f Composite Samples o f D i g i t o x i n T a b l e t s by HPLC and the USP Method 198 XXII R e c o v e r y Data f o r D i g i t o x i n T a b l e t A s s a y by HPLC and the USP Method 199 XXIII R e s u l t s o f the S t a b i l i t y Study o f D i g o x i n T a b l e t s S t o r e d a t 60°C and 70.4% R e l a t i v e H u m i d i t y 215 XXIV R e s u l t s o f the S t a b i l i t y Study o f D i g o x i n T a b l e t s S t o r e d a t 80°C and 37.1% R e l a t i v e H u m i d i t y 218 XXV R e s u l t s o f the S t a b i l i t y Study o f L a n o x i n R I n j e c t i o n 0.05 mg/ml s t o r e d a t 60°C and 70.4% R e l a t i v e H u m i d i t y 219 XXVI R e s u l t s o f S t a b i l i t y Study o f L a n o x i n R I n j e c t i o n 0.05 mg/ml S t o r e d a t 80°C and 37.1% R e l a t i v e H u m i d i t y 220 XXVII R e s u l t s o f the S t a b i l i t y Study o f L a n o x i n R E l i x i r 0.05 mg/ml S t o r e d a t 60°C and 70.4% R e l a t i v e H u m i d i t y 222 XXVIII R e s u l t s o f the S t a b i l i t y Study o f L a n o x i n R E l i x i r S t o r e d a t 80 C and 37.1% R e l a t i v e H u m i d i t y 223 XI V . T a b l e Page XXIX R e s u l t s o f the S t a b i l i t y Study o f D i g i t o x i n T a b l e t s S t o r e d a t 60°C and 70.4% R e l a t i v e H u m i d i t y and 80°C and 70.4% R e l a t i v e H u m i d i t y 228 XV . LIST OF FIGURES F i g u r e Page 1 Diagrammatic R e p r e s e n t a t i o n o f Branches o f Chromatography 6 2 An I d e a l i z e d E l u t i o n Chromatogram o f a S i n g l e . Component 9 3 Chromatograms showing: (a) Appearance o f a chromato-g r a p h i c band a f t e r m i g r a t i n g 1, 2 and 4 u n i t s from t h e o r i g i n i n an even c h r o m a t o g r a p h i c bed; and (b) S e p a r a t i o n o f a m i x t u r e o f s u b s t a n c e s (A) and (B) 11 4 Chromatogram showing the p a r a m e t e r s used f o r the c a l c u l a t i o n o f r e s o l u t i o n (Rs) 13 5 Chromatogram showing the parameters used f o r the c a l c u l a t i o n o f E f f i c i e n c y , S e l e c t i v i t y and C a p a c i t y F a c t o r 15 6 Chromatogram d e p i c t i n g the parameters t h a t a r e employed f o r c a l c u l a t i n g R e t e n t i o n , S e l e c t i v i t y and T h e o r e t i c a l P l a t e s 19 7 S c h e m a t i c Diagram o f a G r a d i e n t H i g h - P e r f o r m a n c e L i q u i d Chromatograph 21 8 Diagrammatic R e p r e s e n t a t i o n o f a S i x - P o r t I n j e c t i o n V a l v e 24 9 Diagrammatic R e p r e s e n t a t i o n o f the S t r u c t u r e s o f Column P a c k i n g M a t e r i a l s used i n A n a l y t i c a l and P r e p a r a t i v e S e p a r a t i o n s 26 10 Diagrammatic R e p r e s e n t a t i o n o f the S t r u c t u r e and F u n c t i o n a l i t y o f Bonded-Packing M a t e r i a l and the S e p a r a t i o n P r o c e s s 29 11 Diagrammatic R e p r e s e n t a t i o n o f I o n - P a i r i n g f o r R e v e rse Phase S e p a r a t i o n s 30 12 T r i a n g l e o f R e s o l u t i o n , Speed and C a p a c i t y r e p r e s e n t i n g t h e B a l a n c e n e c e s s a r y f o r C h r o m a t o g r a p h i c E f f i c i e n c y 35 x v i . F i g u r e Page 13 Diagrammatic R e p r e s e n t a t i o n o f t h e E f f e c t s o f V a r y i n g C a p a c i t y , E f f i c i e n c y and S e l e c t i v i t y F a c t o r s on R e s o l u t i o n 38 14 Chemical S t r u c t u r e o f D i g o x i n 45 15 Chemical S t r u c t u r e o f D i g i t o x i n 46 16 Chemical S t r u c t u r e s o f the C a r d e n o l i d e and B u f a d i e n o l i d e Groups o f Genins 47 17 Diagrammatic R e p r e s e n t a t i o n o f the P r o c e d u r e f o r Radioimmunoassay o f D i g i t a l i s G l y c o s i d e s 69 18 Diagrammatic R e p r e s e n t a t i o n o f t h e Assay o f D i g o x i n by I n h i b i t i o n o f 86R d T r a n s p o r t by the Red B l o o d C e l l 70 19 Diagrammatic R e p r e s e n t a t i o n o f the Assay o f D i g o x i n by E n z y m a t i c I s o t o p e D i s p l a c e m e n t 70 20 Chemical S t r u c t u r e o f D i g o x i n and D i h y d r o -d i g o x i g e n i n 80 21 Chemical S t r u c t u r e s o f Equine E s t r o g e n s 84 22 S c h e m a t i c Diagram o f the Post-Column F l u o r o g e n i c D e r i v a t i z a t i o n System u s i n g t h e A i r - S e g m e n t a t i o n P r i n c i p l e and 100% F l u i d Recovery Set-up 89 23 The I n f r a r e d S p e c t r a (KBr) o f D i g o x i n as o b t a i n e d w i t h the Beckman IR-10 92 24 A S p e c t r a l - A b s o r b a n c e Curve f o r D i g o x i n i n 35% M e t h a n o l . . 93 25 A C a l i b r a t i o n Curve f o r D i g o x i n i n 35% Methanol as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 47/40/9/4 95 26 A C a l i b r a t i o n Curve f o r D i g o x i n i n 35% Methanol as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 51/42/5/2. Area and w e i g h t r a t i o s a r e i n terms o f d i g o x i n / i n t e r n a l s t a n d a r d ( 1 7 a - e t h y n y l e s t r a d i o l ) 96 27 A C a l i b r a t i o n Curve f o r D i g o x i n i n 35% Methanol as o b t a i n e d w i t h a S o l v e n t System o f water/ m e t h a n o l / i s o p r o p a n o l / d i ch1oromethane: 51/42/5/2. Ar e a and w e i g h t r a t i o s a r e i n terms o f d i g o x i n / i n t e r n a l s t a n d a r d ( 1 7 a - d i h y d r o e q u i l i n ) 97 x v i i . F i g u r e Page 28 The I n f r a r e d S p e c t r a ( k B r ) o f D i g i t o x i n as o b t a i n e d w i t h t he Beckman IR-10 102 29 A S p e c t r a l - A b s o r b a n c e Curve f o r D i g i t o x i n i n 35% Methanol 104 30 A C a l i b r a t i o n Curve f o r D i g i t o x i n i n 35% Methanol as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 45/38 / lVB 106 31 Flow C h a r t P r e s e n t a t i o n o f the HPLC Assay o f D i g o x i n T a b l e t s 110 32 Flow C h a r t P r e s e n t a t i o n o f the HPLC Assay o f D i g o x i n I n j e c t i o n I l l 33 Flow C h a r t P r e s e n t a t i o n o f the HPLC Assay o f D i g o x i n E l i x i r 112 34 Flow C h a r t P r e s e n t a t i o n o f the USP P r o c e d u r e f o r Sample P r e p a r a t i o n o f D i g o x i n T a b l e t s 114 35 Flow C h a r t P r e s e n t a t i o n o f the USP P r o c e d u r e f o r t h e Q u a n t i t a t i o n o f D i g o x i n i n T a b l e t s 115 36 Flow C h a r t P r e s e n t a t i o n o f the USP Assay o f D i g o x i n I n j e c t i o n 116 37 Flow C h a r t P r e s e n t a t i o n o f the USP Assay o f D i g o x i n E l i x i r 117 38 Flow C h a r t P r e s e n t a t i o n o f the HPLC Assay o f D i g i t o x i n T a b l e t s 118 39 Flow C h a r t P r e s e n t a t i o n o f the HPLC As s a y o f D i g i t o x i n I n j e c t i o n 119 40 Flow C h a r t P r e s e n t a t i o n o f the USP P r o c e d u r e f o r Sample P r e p a r a t i o n o f D i g i t o x i n T a b l e t s (Composite T a b l e t A s s a y ) 121 41 Flow C h a r t P r e s e n t a t i o n o f t h e USP P r o c e d u r e f o r t h e Q u a n t i t a t i o n o f D i g i t o x i n i n T a b l e t s (Composite T a b l e t A s s a y ) 122 42 Flow C h a r t P r e s e n t a t i o n o f the USP P r o c e d u r e f o r Sample P r e p a r a t i o n i n D i g i t o x i n S i n g l e T a b l e t A s s a y 123 43 Flow C h a r t P r e s e n t a t i o n o f the USP P r o c e d u r e f o r t h e Q u a n t i t a t i o n o f D i g i t o x i n i n S i n g l e T a b l e t s 124 xv i i i . F i g u r e Page 44 Flow C h a r t P r e s e n t a t i o n o f t h e USP A s s a y o f D i g i t o x i n I n j e c t i o n 125 45 C a l i b r a t i o n C u r v e s f o r D i g o x i g e n i n , D i g o x i g e n i n m o n o d i g i t o x o s i d e , D i g o x i g e n i n b i s d i g i t o x o s i d e and D i g o x i n i n 35% Methanol as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 47/40/9/4 131 46 C a l i b r a t i o n C u r v e s f o r D i g o x i g e n i n , D i g o x i g e n i n m o n o d i g i t o x o s i d e , D i g o x i g e n i n b i s d i g i t o x o s i d e and D i g o x i n i n 35% Methanol as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 51/43/5/1 133 47 C a l i b r a t i o n Curves f o r D i g i t o x i g e n i n , D i g i t o -x i g e n i n m o n o d i g i t o x o s i d e , D i g i t o x i g e n i n b i s d i g i t o x o s i d e and D i g i t o x i n i n 35% Methanol as o b t a i n e d w i t h a S o l v e n t System o f water/ m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 45/38/11/6 135 48 A Chromatogram f o r t h e HPLC S e p a r a t i o n o f a s t a n d a r d M i x t u r e o f D i g o x i g e n i n m o n o d i g i t o -x o s i d e , D i g o x i g e n i n b i s d i g i t o x o s i d e and D i g o x i n as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l : 60/40 140 49 A Chromatogram f o r t h e HPLC S e p a r a t i o n o f a S t a n d a r d M i x t u r e o f D i g o x i g e n i n m o n o d i g i t o x o s i d e , D i g o x i g e n i n b i s d i g i t o x o s i d e and D i g o x i n as o b t a i n e d w i t h a S o l v e n t System o f m e t h a n o l / w a t e r : 60/40 140 50 A Chromatogram f o r the I s o c r a t i c HPLC S e p a r a t i o n o f a S t a n d a r d M i x t u r e o f D i g o x i n , D i g i t o x i n and t h e i r M e t a b o l i t e s as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o -methane: 46/39/10/5 143 51 A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a S t a n d a r d M i x t u r e o f D i g o x i n , D i g i t o x i n and t h e i r M e t a b o l i t e s as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o -methane: 47/40/9/4 146 52 A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a S t a n d a r d M i x t u r e o f D i g o x i n , D i g i t o x i n , and t h e i r M e t a b o l i t e s o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h i o r o m e t h a n e : 45/37/12/6 147 x i x . F i g u r e Page 53 A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a S t a n d a r d M i x t u r e o f t h e D i g i t o x i n S e r i e s from t h e D i g o x i n S e r i e s 148 54 A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a S t a n d a r d M i x t u r e o f D i g o x i g e n i n ; D i g o x i g e n i n m o n o d i g i t o x o s i d e , D i g o x i g e n i n b i s d i g i t o x o s i d e and D i g o x i n as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h i o r o m e t h a n e : 51/42/5/2 150 55 A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a S t a n d a r d M i x t u r e o f D i g o x i n , D i g i t o x i n , t h e r e s p e c t i v e M e t a b o l i t e s and G i t o x i n 152 56 A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a S t a n d a r d M i x t u r e a and B - a c e t y l d i g o x i n from D i g o x i n and i t s M e t a b o l i t e s 153 57 A Chromatogram f o r the HPLC S e p a r a t i o n o f a M i x t u r e o f D i g o x i n , D i g i t o x i n and t h e i r M e t a b o l i t e s by G r a d i e n t E l u t i o n 154 58 A Chromatogram f o r the HPLC S e p a r a t i o n o f a M i x t u r e o f D i g o x i n , D i g i t o x i n and t h e i r M e t a b o l i t e s by S o l v e n t S w i t c h o v e r e l u t i o n 156 59 A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a M i x t u r e o f D i g o x i n , D i h y d r o d i g o x i g e n i n and th e o t h e r D i g o x i n M e t a b o l i t e s as o b t a i n e d by Dual D e t e c t o r M o n i t o r i n g 162 60 A Chromatogram f o r the I s o c r a t i c S e p a r a t i o n o f a M i x t u r e o f n i n e Equine E s t r o g e n s and 17a-e t h y n y l e s t r a d i o l 164 61 A Chromatogram f o r the I s o c r a t i c HPLC S e p a r a t i o n o f a M i x t u r e o f n i n e E q u i n e E s t r o g e n s as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 49/41/7/3 166 62 A Chromatogram f o r Complete I s o c r a t i c HPLC S e p a r a t i o n o f a M i x t u r e o f n i n e E quine E s t r o g e n s as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 52/43/3/2 157 63 A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f D i g i t o x i n from o t h e r Components o f D i g i t a l i s p u r p u r e a L e a f 169 XX . F i g u r e Page 64 A R e p r e s e n t a t i v e Chromatogram f o r the I s o c r a t i c HPLC A n a l y s i s o f D i g o x i n T a b l e t s 172 65 A Chromatogram f o r the I s o c r a t i c HPLC S e p a r a t i o n o f D i g o x i n , i t s p r o b a b l e D e g r a d a t i o n P r o d u c t s , 1 7 a - e t h y n y l e s t r a d i o l ( i n t e r n a l s t a n d a r d ) and g i t o x i n 173 66 A R e p r e s e n t a t i v e Chromatogram f o r the I s o c r a t i c HPLC A n a l y s i s o f D i g o x i n I n j e c t i o n 180 67 A R e p r e s e n t a t i v e Chromatogram f o r the I s o c r a t i c HPLC A n a l y s i s o f D i g o x i n E l i x i r as o b t a i n e d w i t h a s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e 183 68 A R e p r e s e n t a t i v e Chromatogram f o r the I s o c r a t i c HPLC A n a l y s i s o f D i g o x i n E l i x i r u s i n g 173-d i h y d r o e q u i l i n as the I n t e r n a l S t a n d a r d 184 69 A R e p r e s e n t a t i v e Chromatogram f o r the I s o c r a t i c HPLC A n a l y s i s o f D i g i t o x i n T a b l e t s and I n j e c t i o n 187 70 A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f D i g i t o x i n , D i g o x i n , and t h e i r p o t e n t i a l D e g r a d a t i o n p r o d u c t s and 1 7 a - m e t h y l t e s t o s t e r o n e ( i n t e r n a l s t a n d a r d ) 189 71 A R e p r e s e n t a t i v e Chromatogram f o r the S t a b i l i t y M o n i t o r i n g o f D i g o x i n T a b l e t s by HPLC 202 72 A R e p r e s e n t a t i v e Chromatogram f o r the S t a b i l i t y M o n i t o r i n g o f D i g o x i n I n j e c t i o n by HPLC 203 73 A R e p r e s e n t a t i v e Chromatogram f o r t h e S t a b i l i t y M o n i t o r i n g o f N a t i g o x i n R T a b l e t s by HPLC 204 74 A R e p r e s e n t a t i v e Chromatogram f o r t h e S t a b i l i t y M o n i t o r i n g o f D i g i t o x i n T a b l e t s by HPLC 205 D 75 A Chromatogram o f a Sample o f L a n o x i n E l i x i r s p i k e d w i t h D i g o x i n and i t s p o t e n t i a l D e g r a d a t i o n P r o d u c t s 206 76 A Chromatogram o f an E x t r a c t e d Sample o f L a n o x i n E l i x i r s p i k e d w i t h d i g o x i g e n i n and t h e mono-and b i s d i g i t o x o s i d e s 208 77 A Chromatogram o f an E x t r a c t e d Sample o f L a n o x i n E l i x i r a f t e r p r e l i m i n a r y a d d i t i o n o f Sodium C a r b o n a t e and s p i k i n g w i t h D i g o x i n and i t s p o t e n t i a l D e g r a d a t i o n P r o d u c t s 209 xx i . F i g u r e Page 78 A Chromatogram o f a Sample o f L a n o x i n E l i x i r s t o r e d a t room t e m p e r a t u r e , a f t e r p r e l i m i n a r y a d d i t i o n o f Sodium C a r b o n a t e and e x t r a c t i o n w i t h D i c h l o r o m e t h a n e 210 79 A Chromatogram o f an E x t r a c t e d Sample o f L a n o x i n E l i x i r a f t e r P r e l i m i n a r y a d d i t i o n o f Sodium C a r b o n a t e and s p i k i n g w i t h H y d r o c o r t i s o n e ( i n t e r n a l s t a n d a r d ) , D i g o x i n and i t s p o t e n t i a l D e g r a d a t i o n P r o d u c t s 212 80 A R e p r e s e n t a t i v e Chromatogram f o r the S t a b i l i t y M o n i t o r i n g o f L a n o x i n ^ E l i x i r by HPLC 213 81 A Chromatogram f o r t h e 1% l e v e l s o f t h e p o t e n t i a l D e g r a d a t i o n P r o d u c t s o f D i g o x i n 214 82 A Chromatogram f o r the 1% l e v e l s o f the p o t e n t i a l D e g r a d a t i o n P r o d u c t s o f D i g i t o x i n 214 83 The pH P r o f i l e o f Powdered T a b l e t M a t e r i a l ( e q u i v a l e n t t o 20 t a b l e t s ) suspended i n 20 ml o f D i s t i l l e d Water, a f t e r g r a d u a l a d d i t i o n o f 0.005 N HCL 217 84 A Chromatogram f o r the I s o c r a t i c HPLC S e p a r a t i o n o f t he D e g r a d a t i o n P r o d u c t s o f m e t h y l p a r a b e n i n a sample o f L a n o x i n ^ E l i x i r s t o r e d f o r s i x weeks a t 80°C and 37.1% R e l a t i v e H u m i d i t y 225 p 85 A Chromatogram o f a sample o f L a n o x i n E l i x i r s t o r e d under ambient c o n d i t i o n s 226 86 Mass Spectrum o f T M S - d e r i v a t i v e o f p - h y d r o x y b e n z o i c a c i d a f t e r GLC s e p a r a t i o n o f a sample o f L a n o x i n ^ E l i x i r S t o r e d f o r s i x weeks a t 80°C and 37.1% R e l a t i v e H u m i d i t y 227 87 S c h e m a t i c Diagram o f Pathways o f D i g o x i n D e g r a d a t i o n 230 xx i i . ABBREVIATIONS a.u. - a b s o r b a n c e u n i t s a . u . f . s . - a b s o r b a n c e u n i t s f u l l s c a l e °C - d e g r e e s c e n t i g r a d e cm - c e n t i m e t e r ECD - e l e c t r o n c a p t u r e d e t e c t o r eV - e l e c t r o n v o l t F i g . - f i g u r e g - gram GLC - g a s - l i q u i d chromatography HFB - h e p t a f l u o r o b u t y r a t e HPLC - h i g h - p e r f o r m a n c e l i q u i d chromatography i . d . - i n t e r n a l d i a m e t e r IR - i n f r a r e d LC - l i q u i d chromatography m - meter M - m o l a r meg - microgram min. - minute ml. - m i l l i l i t e r mm - m i l l i m e t e r MS - mass s p e c t r o m e t e r ng - nanogram nm - nanometer ODS - o c t a d e c y l s i l a n e xx i i i . P s i - pounds p e r s q u a r e i n c h (= 0.07 atmosphere) RIA - radioimmunoassay T^ - b i o l o g i c a l h a l f - l i f e TLC - t h i n - l a y e r chromatography u - m i c r o n (= 10"^ cm) um - m i c r o m e t e r USP - U n i t e d S t a t e s Pharmacopeia UV - u l t r a v i o l e t x x i v . ACKNOWLEDGEMENTS I am g r e a t l y i n d e b t e d t o Dr. J.H. M c N e i l l , Dean R i e d e l and Dr. T.H. Brown w i t h o u t whose s u p p o r t and g u i d a n c e t h i s work would not have been c o m p l e t e d . My e s p e c i a l thanks go to Dr. F.S. A b b o t t and Dr. B.D. R o u f o g a l i s f o r t h e i r encouragement, g u i d a n c e and i n t e l l e c t u a l s t i m u l a t i o n . I would l i k e t o thank Dr. W. G o d o l p h i n and Dr. J . O r r who, as members o f my g u i d a n c e committee, gave g e n e r o u s l y o f t h e i r t ime and e x p e r t i s e t h r o u g h o u t t h e c o u r s e o f t h i s work. I am g r a t e f u l t o Dr. A. Goodeve f o r h i s h e l p f u l s u g g e s t i o n s and d i s c u s s i o n s on c a r d i a c g l y c o s i d e s and f o r k i n d l y p r o v i d i n g me w i t h a sample o f D i g i t a l i s l e a v e s . I would a l s o l i k e t o thank Mr. Roland W. Bur t o n f o r h i s k i n d l y h e l p i n many t e c h n i c a l d i f f i c u l t i e s . Due acknowledgement s h o u l d go t o my a d v i s o r , Dr. K.M. M c E r l a n e f o r s u g g e s t i n g t h e t o p i c as one o f the p o s s i b i l i t i e s . F i n a l l y I would l i k e t o e x p r e s s my g r a t i t u d e t o my f r i e n d s who hel p e d me m a i n t a i n t he c o n v i c t i o n t h a t each day's t i n y , l i t t l e o b s e r v a t i o n would add up t o something w o r t h w h i l e . F i n a n c i a l s u p p o r t i n t h e form o f a F e l l o w s h i p from t he World H e a l t h O r g a n i z a t i o n i s g r a t e f u l l y acknowledged. XXV. DEDICATED TO THE PHILOSOPHY OF ETHICS IN THE AFFAIRS OF MAN 1. INTRODUCTION E v e r s i n c e t h e p u b l i c a t i o n i n 1785 o f W i l l i a m W i t h e r i n g ' s t r e a t i s e e n t i t l e d , "An A c c o u n t o f the F o x g l o v e and some o f i t s M e d i c a l Uses" ( W i t h e r i n g ( 1 9 3 7 ) ) , t h e r e has been a g r a d u a l i n c r e a s e i n the use o f d i g i t a l i s g l y c o s i d e s . D i g o x i n and d i g i t o x i n a r e c a r d i a c g l y c o s i d e s o b t a i n e d i n p u r i f i e d form from the l e a v e s o f D i g i t a l i s l a n a t a and D i g i t a l i s p u r p u r e a , r e s p e c t i v e l y and a r e commonly used i n the t r e a t m e n t o f c o n g e s t i v e h e a r t f a i l u r e . These drugs b e l o n g t o the C a r d e n o l i d e C and A s e r i e s , r e s p e c t i v e l y , and a r e members o f a l a r g e c l a s s o f c l o s e l y r e l a t e d compounds c o l l e c t i v e l y known as the d i g i t a l i s g l y c o s i d e s . A n a t i o n a l p r e s c r i p t i o n s u r v e y i n t h e U n i t e d S t a t e s has been q u o t e d ( D o h e r t y and Kane (1975)) t o have found t h a t d i g o x i n , d i g i t o x i n and d i g i t a l i s l e a f were, r e s p e c t i v e l y , f o u r t h , s i x t e e n t h and n i n e t e e n t h among t h e most f r e q u e n t l y p r e s c r i b e d drugs i n the c o u n t r y i n 1971. The s i t u a t i o n might a l s o be the same i n Canada. The e x t r e m e l y low u n i t d o s ages, narrow t o x i c t o t h e r a p e u t i c dosage r a t i o s a l o n g w i t h i n t e r s u b j e c t v a r i a t i o n s o f s e n s i t i v i t y r e q u i r e a h i g h degree o f c o n t e n t u n i f o r m i t y o f d i g o x i n and d i g i t o x i n dosage forms, - e s p e c i a l l y i n t a b l e t f o r m u l a t i o n s . The p r e s e n c e o f p h a r m a c e u t i c a l e x c i p i e n t s and p r o b a b l e f o r m a t i o n o f d e g r a d a t i o n p r o d u c t s i n t r o d u c e a d d i t i o n a l r e q u i r e m e n t s o f a s s a y s p e c i f i c i t y . Hence, a n a l y s i s o f d i g o x i n and d i g i t o x i n i n t h e i r r e s p e c t i v e dosage forms c a l l s f o r methods t h a t a r e s e n s i t i v e enough t o m o n i t o r u n i t dose amounts o f d i g o x i n (0.125 mg per t a b l e t ) and d i g i t o x i n (0.1 mg per t a b l e t ) w i t h s u f f i c i e n t s e l e c t i v i t y t o p r e v e n t any i n t e r f e r e n c e from o t h e r compounds. I t would a l s o be 2. a dvantageous i f t h e methods c o u l d a l l o w t h e s i m u l a t n e o u s d e t e r m i n a t i o n o f p o s s i b l e d e g r a d a t i o n p r o d u c t s . Most o f t h e e a r l i e r methods r e p o r t e d f o r t h e q u a n t i t a t i v e a n a l y s i s o f t h e s e drugs were e i t h e r c o l o r i m e t r i c o f f l u o r o m e t r i c . Many i n v e s t i g a t o r s have l a t e r used t h i n - l a y e r , g a s - l i q u i d and h i g h - p e r f o r m a n c e l i q u i d chroma-t o g r a p h y methods. The U n i t e d S t a t e s P harmacopeial methods a l s o employ c o l o r i m e t r i c , f l u o r o m e t r i c and c h r o m a t o g r a p h i c t e c h n i q u e s . The c o l o r i m e t r i c methods g e n e r a l l y l a c k s e n s i t i v i t y . M oreover, t h e y have no s e l e c t i v i t y because t h e c o l o r f o r m i n g d e r i v a t i z i n g r e a g e n t s r e a c t w i t h the s ugar m o i e t y o r t h e l a c t o n e r i n g , both o f which a r e p r e s e n t i n a l l c a r d e n o l i d e s . S i n c e t h e f l u o r o m e t r i c methods a r e based on t h e r e a c t i o n o f the d e r i -v a t i z i n g a gents w i t h the s t e r o i d m o i e t y o f t h e g l y c o s i d e m o l e c u l e , t h e y a r e n o n - s p e c i f i c w i t h r e s p e c t to o t h e r d i g i t a l i s g l y c o s i d e s . Gas-c h r o m a t o g r a p h i c methods i n v o l v e d e r i v a t i z a t i o n p r o c e d u r e s t h a t a g a i n r e n d e r them n o n - s p e c i f i c . The l a c k o f p r e c i s e and r e a d i l y q u a n t i f i a b l e parameters o f t h e r a -p e u t i c r e s p o n s e o f d i g o x i n and d i g i t o x i n has made i t n e c e s s a r y t h a t i n d i v i d u a l t i t r a t i o n s be done on each p a t i e n t . T h i s r e q u i r e s h i g h l y s e n s i t i v e and s p e c i f i c methods o f a n a l y s i s t h a t would be a b l e t o i n d i c a t e c o n c e n t r a t i o n / t h e r a p e u t i c r e s p o n s e c o r r e l a t i o n s by m o n i t o r i n g serum con-c e n t r a t i o n s o f t h e d rugs i n the p r e s e n c e o f t h e i r m e t a b o l i t e s . A v a r i e t y o f a n a l y t i c a l t e c h n i q u e s , i n c l u d i n g a number o f HPLC methods have been r e p o r t e d i n t h e l i t e r a t u r e c u l m i n a t i n g i n a method t h a t employs a t w o - step p r o c e d u r e i n which the c a r d i a c g l y c o s i d e i s s e p a r a t e d by HPLC and q u a n t i -t a t e d by radioimmunoassay ( R I A ) . The l i t e r a t u r e i n d i c a t e s t h a t t h i s t wo-step p r o c e d u r e has been n e c e s s a r y because o f t h e n o n - s p e c i f i c i t y o f t h e RIA t e c h n i q u e and t h e l a c k o f a s e n s i t i v e HPLC method f o r the 3. a n a l y s i s o f d i g o x i n and d i g i t o x i n i n b i o l o g i c a l f l u i d s . M oreover, i t would be n e c e s s a r y t h a t t he HPLC method s h o u l d e n s u r e n o n - i n t e r f e r e n c e from a l l p o s s i b l e m e t a b o l i t e s . In o r d e r t o a s s u r e i d e n t i t y , s a f e t y , e f f i c a c y and m o n i t o r s t a b i l i t y o f d i g o x i n and d i g i t o x i n , i t i s c l e a r t h a t t h e r e i s a need f o r methods o f a n a l y s i s t h a t a r e s u f f i c i e n t l y s e n s i t i v e t o a l l o w s i n g l e t a b l e t a s s a y and s e l e c t i v e enough t o pe r m i t s i m u l t a n e o u s a n a l y s i s o f d e g r a d a t i o n p r o d u c t s . T h i s i n v e s t i g a t i o n , t h e r e f o r e , w i l l a t t empt t o p r o v i d e some HPLC da t a which may h e l p t o s a t i s f y t h i s need. Attempts w i l l a l s o be made t o e s t a b l i s h t h e r e l a t i v e m e r i t s o f t h e HPLC methods i n comparison w i t h t h o s e o f USP XX. 4. I. LITERATURE SURVEY In a s t u d y o f the e v o l u t i o n o f t h e a n a l y t i c a l methods o f a d r u g , one would, o f n e c e s s i t y , have t o examine the n a t u r e and u n i q u e c h a r a c -t e r i s t i c s o f b o t h the t e c h n i q u e and the drug so as t o a p p r o p r i a t e l y l o c a t e them i n t h e o v e r a l l p i c t u r e o f r e l a t e d f a c t s . A n a l y s i s i s a s t e p - w i s e p r o c e s s t h a t may be d i v i d e d i n t o : t h e p r e p a r a t o r y p r o c e s s , the s e p a r a t o r y p r o c e s s and t h e d e t e r m i n a t i o n p r o c e s s . By f a r t h e h i g h e s t p r o p o r t i o n o f t h e time o f a n a l y s i s i s taken by the s e p a r a t o r y s t e p e.g. e x t r a c t i o n , p r e c i p i t a t i o n , f i l t r a t i o n , c e n t r i f u g a t i o n e t c . The major t e c h n i q u e s i n the s e p a r a t i o n p r o c e d u r e s have been e x t r a c t i o n and chroma-t o g r a p h y . S i n c e i t s d i s c o v e r y by T s w e t t ( 1 9 0 3 ) , the development o f chromato-graphy has p r o g r e s s i v e l y f a c i l i t a t e d t h e s e p a r a t i o n p r o c e s s . Many have c o n t r i b u t e d t o t h i s development. These i n c l u d e : L.S. Palmer (1922) who r e p r e s e n t s the l i n k between T s w e t t and the next g e n e r a t i o n ; Edgar L e d e r e r (1972) who f i f t y y e a r s ago b r o u g h t back the t e c h n i q u e from o b l i v i o n (Kuhn et a l .. 1931); L a s z l o Z e c h m e i s t e r (1 937) who p r o b a b l y d i d the most to make c l a s s i c a l column chromatography a s i m p l e t o o l , e a s i l y a c c e s s i b l e t o e v e r y b o d y ; E r i k a Cremer (1950) who b u i l t t h e f i r s t gas chromatograph s i m i l a r t o our p r e s e n t day s y s t e m s ; and M a r t i n and Synge (1941) who l a i d t h e t h e o r e t i c a l f o u n d a t i o n s on which l i q u i d - l i q u i d p a r t i t i o n chromatography i s b a s e d . H i g h - p e r f o r m a n c e l i q u i d chromatography (HPLC) i s j u s t one more r e c e n t r e f i n e m e n t o f the c h r o m a t o g r a p h i c t e c h n i q u e . 5. 1. High Performance L i q u i d Chromatography Chromatography i s a p h y s i c a l p r o c e s s o f s e p a r a t i o n o f the components o f a m i x t u r e due t o t h e d i f f e r e n c e s i n t h e i r e q u i l i b r i u m d i s t r i b u t i o n between a m o b i l e phase and a s t a t i o n a r y phase. The v a r i o u s b r a n c h e s o f chromatography a r e shown i n t h e s c h e m a t i c d i a g r a m i n F i g . 1 . HPLC encompasses t h e t e c h n i q u e s t h a t a r e shown i n t h e s o l i d b o x es. Hence i n e s s e n c e i t i s a r e f i n e m e n t o f what was f o r m e r l y known as l i q u i d o r column chromatography. HPLC may now be d e f i n e d as a l i q u i d c h r o m a t o g r a p h i c t e c h n i q u e i n which the l i q u i d m o b i l e phase i s pumped t h r o u g h a column o f m i c r o p a r t i c u l a t e s t a t i o n a r y m a t e r i a l , under h i g h p r e s s u r e , and t h e s e p a r a t e d components a r e d e t e c t e d and r e c o r d e d i n the form o f i n d i v i d u a l peaks. (A) T h e o r y (a) Mechanism o f R e t e n t i o n The b a s i s o f HPLC s e p a r a t i o n s can be due t o any o f t h e f o l l o w i n g mechanisms o f r e t e n t i o n : ( i ) P a r t i t i o n i n g . T h i s i s the d i s t r i b u t i o n o f the sample between a l i q u i d m o b i l e phase and a l i q u i d s t a t i o n a r y phase i n which the l a t e r l i q u i d i s c o a t e d on s o l i d s u p p o r t m a t e r i a l . The r e t e n t i o n o f the sample by t h e l i q u i d s t a t i o n a r y phase i s dependent on t h e p a r t i t i o n c o e f f i c i e n t o f the sample. The d i s t r i b u t i o n e q u i l i b r i u m i s : m o b i l e _ phase ^ s t a t i o n a r y — phase (Eq. 1) 6. CHROMATOGRAPHY GAS CHROMATOGRAPHY L IQUID CHROMATOGRAPHY G A S - L I Q U I D GAS-SOLID ION EXCLUSION EXCHANGE PARTIT ION L IQUID SOLID CHROMATOGRAPHY ADSORPTION 1 PAPER CHROMATOGRAPHY 1 T H I N LAYER F i g . 1. Diagrammatic R e p r e s e n t a t i o n o f Branches o f Chromatography ( F a r r i s (1976) )  7. ( i i ) A d s o r p t i o n . T h i s i s the r e t e n t i o n o f a sample by a s o l i d s t a t i o n a r y phase ( u n - c o a t e d ) due t o a d s o r p t i o n o f the sample o n t o the s o l i d s u p p o r t m a t e r i a l . The d i s t r i b u t i o n e q u i l i b r i u m o f t h e sample i s due t o a d s o r p t i o n - d e s o r p t i o n : A d s o r p t i o n ». D e s o r p t i o n ( E q . 2) ( s t a t i o n a r y ^ ( m o b i l e phase) phase) ( i i i ) Ion Exchange. T h i s i s the r e t e n t i o n o f a sample i o n by an i o n - e x c h a n g e r r e s i n ( s t a t i o n a r y phase) due t o s o r p t i o n o f t h e sample i o n onto the r e s i n i n exchange f o r the m o b i l e i o n . The d i s t r i b u t i o n e q u i l i b r i u m i s : S o r p t i o n _ D e s o r p t i o n ( E q . 3) ( o n t o t h e Exchanger ( i n t o the l i q u i d r e s i n ) m o b i l e phase) + + + + e.g. RH + sample i o n R-sample i o n + H ( c a t i o n i c e x c h a n g e r r e s i n ) ( i v ) Gel p e r m e a t i o n ( E x c l u s i o n ) . T h i s i s the r e t e n t i o n o f a sample by a porous s t a t i o n a r y phase due t o the r e l a t i v e m o l e c u l a r s i z e o f the sample. L a r g e r m o l e c u l e s e x c l u d e d from a l l o r a p o r t i o n o f the p o r e s , by v i r t u e o f t h e i r p h y s i c a l s i z e , e l u t e from t h e column b e f o r e t h e s m a l l e r m o l e c u l e s . The d i s t r i b u t i o n e q u i l i b r i u m i s : P e r m e a t i o n * D i f f u s i o n ( E q . 4) ( i n t o t he p o r e s ( i n t o t he m o b i l e o f t he s t a t i o n a r y phase) phase) 8. The e q u i l i b r i u m c o n s t a n t i n E q u a t i o n s 1, 2, 3 and 4 may g e n e r a l l y be e x p r e s s e d ( F a r r i s (1976)) a s : C C m = c o n c e n t r a t i o n o f sample i n m o b i l e phase (b) T h e o r e t i c a l P l a t e The path o f a sample m o l e c u l e t h r o u g h a c h r o m a t o g r a p h i c bed may be d e p i c t e d as a number o f minute jumps between the s t a t i o n a r y and m o b i l e p h a s e s . These jumps a p p r o x i m a t e a " d r u n k a r d ' s walk" as a m a t h e m a t i c a l model. I t f o l l o w s from s t a t i s t i c a l a n a l y s i s t h a t a G a u s s i a n d i s t r i b u t i o n (normal d i s t r i b u t i o n ) s h o u l d be o b s e r v e d f o r a c h r o m a t o g r a p h i c band, and t h i s i s f o u n d e x p e r i m e n t a l l y t o be t r u e w i t h i n l i m i t s . U s i n g t h i s a n a l y s i s , a c c o r d i n g t o M a r t i n and Synge ( 1 9 4 1 ) , g i v e n an e x p e r i m e n t a l l y d e t e r m i n e d c h r o m a t o g r a p h i c band as shown i n F i g . 2, t h e number o f t h e o r e -t i c a l p l a t e s N, can be c a l c u l a t e d from t h e r e t e n t i o n volume R*, and t h e band h a i f - w i d t h , a , a s shown i n E q u a t i o n 6 and 7. The s i g n i f i c a n c e o f ( E q . 5) where, K = e q u i l i b r i u m c o n s t a n t C g = c o n c e n t r a t i o n o f sample i n s t a t i o n a r y phase • N = ( — ) ( E q . 6) R • 2 o r , N = 16 ( w ( E q . 7) • where R = r e t e n t i o n volume i a = band h a l f - w i d t h a t 0.607 the h e i g h t o f t h e band (= s t a n d a r d d e v i a t i o n ) 9. 2 * E L U T I O N F i g . 2. An I d e a l i z e d E l u t i o n Chromatogram o f a S i n g l e Component. R1 = r e t e n t i o n t i m e ; w = b a s e l i n e w i d t h o f t h e band; q = band h a l f - w i d t h a t 0.607 o f peak h e i g h t (Walton (1975)) 10. N = number o f t h e o r e t i c a l p l a t e s w = b a s e l i n e w i d t h o f t h e band (w = 4a) t h e number o f t h e o r e t i c a l p l a t e s i s t h a t i t i s equal t o the a v e r a g e number o f e q u i l i b r a t i o n s o f the sample between t h e m o b i l e and s t a t i o n a r y p h ases. Hence t h e g r e a t e r the number o f p l a t e s ( o r c y c l e s ) t h e s h a r p e r t h e band. F a c t o r s t h a t a f f e c t t h e number o f t h e o r e t i c a l p l a t e s i n c l u d e : t y p e and s i z e o f a d s o r b a n t , n a t u r e and f l o w r a t e o f s o l v e n t and d i m e n s i o n s o f the column. A term t h a t i s u s u a l l y e n c o u n t e r e d i n the l i t e r a t u r e r e l a t i n g column l e n g t h (L) t o number o f t h e o r e t i c a l p l a t e s (N) i s known as h e i g h t e q u i v a -l e n t t o a t h e o r e t i c a l p l a t e (HETP). T h i s r e l a t i o n s h i p i s e x p r e s s e d a s : HETP = {=- ( E q . 8) (c) Band w i d e n i n g The T h e o r e t i c a l P l a t e model o f M a r t i n and Synge can be used f o r t h e e x p l a n a t i o n o f the e f f e c t s o f band w i d e n i n g d u r i n g sample m i g r a t i o n . S t a t i s t i c a l a n a l y s i s p r e d i c t s t h a t a band, i n moving a l o n g an even c h r o m a t o g r a p h i c bed, w i l l d o u b l e i n w i d t h when i t s m i g r a t i o n d i s t a n c e i s q u a d r u p l e d . The upper h a l f o f F i g . 3(a) shows t h e a ppearance o f a band o f s u b s t a n c e A, a f t e r i t has m i g r a t e d 1, 2 and 4 u n i t s t h r o u g h a bed w i t h c o r r e s p o n d i n g band w i d t h s o f 0.25, 0.35 ( i . e . 0.25 x /2) and 0.5 ( i . e . 0.25 x /4) u n i t s , r e s p e c t i v e l y . In F i g . 3 ( b ) , one can see the r e s u l t o f c h r o m a t o g r a p h i n g a m i x t u r e o f t h e same s u b s t a n c e A, w i t h a n o t h e r s u b s t a n c e B, where the l a t t e r moves 0.75 u n i t s when A has t r a v e l e d t h e i n i t i a l u n i t . A t t h i s p o i n t , t h e bands o v e r l a p b a d l y , b u t on m i g r a t i n g 11 2 o < cr 2cr=0 25 2o-=0-35 2cr=05 LU O z o o (b) E L U T I O N F i g . 3. Chromatogram showing (a) Appearance o f a C h r o m a t o g r a p h i c band a f t e r m i g r a t i n g 1, 2 and 4 u n i t s from t h e o r i g i n i n an even c h r o m a t o g r a p h i c bed; and (b) S e p a r a t i o n o f a m i x t u r e o f s u b s t a n c e s (A) and ( B ) . 2a = band w i d t h a t 0.607 o f peak h e i g h t .  f o u r times t h e i n i t i a l d i s t a n c e the bands c o r r e s p o n d i n g t o A and B a r e almo s t c o m p l e t e l y s e p a r a t e d . T h i s i s b e c a u s e the band c e n t e r s have moved a p a r t by a f a c t o r o f f o u r , whereas they have widened by o n l y a f a c t o r o f two ( G i d d i n g s , 1975). T h i s f a c t i s the fundamental p r i n c i p l e b e h i n d c h r o m a t o g r a p h i c s e p a r a t i o n s . (d) R e s o l u t i o n R e s o l u t i o n i s the degree o f s e p a r a t i o n o f two a d j a c e n t peaks and i s e x p r e s s e d by E q u a t i o n 9 w i t h r e f e r e n c e t o F i g . 4. d - d S L[O^ + o^) where, R $ = r e s o l u t i o n ( o r r e s o l u t i o n f a c t o r ) d-| = d i s t a n c e t r a v e l e d by peak 1 &2 = d i s t a n c e t r a v e l e d by peak 2 a-| = band h a l f - w i d t h o f peak 1 02 = band h a l f - w i d t h o f peak 2 The u n i t s f o r the d i s t a n c e between bands can be i n terms e i t h e r o f volume o r o f l i n e a r d i m e n s i o n s p r o v i d e d t h a t t he w i d t h s 2a a r e a l s o g i v e n i n t h e same u n i t s . F i g . 4 i l l u s t r a t e s t h e case where R g i s eq u a l t o 1, i . e . , when the band s e p a r a t i o n - ) i s j u s t equal t o the sum o f t h e band w i d t h s (2a-| + 2 0 2 ) . A f r a c t i o n c u t a t the minima between the peaks c o n t a i n s each component i n a l m o s t 98% p u r i t y . I f the v a l u e o f the r e s o l u t i o n f a c t o r ( R g ) i s i n c r e a s e d t o 1.5, i t would mean t h a t t h e components can be i s o l a t e d i n 99.9% p u r i t y ( S n y d e r and K i r k l a n d ( 1 9 7 9 ) ) . The d i s t a n c e between bands i s r e l a t e d t o t h e r a t i o o f the p a r t i t i o n c o e f f i c i e n t s o r t h e d i s t r i b u t i o n c o e f f i c i e n t s i n g e n e r a l o f the c o r r e s -E L U T I O N F i g . 4. Chromatogram showing t h e parameters used f o r t h e c a l c u l a t i o n o f r e s o l u t i o n (R s).. di = r e t e n t i o n v a l u e o f component 1; d 2 = r e t e n t i o n v a l u e o f component 2; h = peak h e i g h t ; a = h a l f band-width. p o n d i n g compounds. By s u b s t i t u t i n g t h i s and c e r t a i n o t h e r r e l a t i o n s i n t o E q u a t i o n 9, i t i s p o s s i b l e t o show t h a t the r e s o l u t i o n f a c t o r ( R $ ) i s a p r o d u c t o f t h r e e d i f f e r e n t t e r m s : R = h ^ (SLZ_L) ( J< ) ( E q . 10) S K + 1 (a) (b) ( c ) where, a = the column e f f i c i e n c y term (random d i s p e r s i o n ) b = the column s e l e c t i v i t y term c = the c a p a c i t y r a t i o f a c t o r N = number o f t h e o r e t i c a l p l a t e s a = s e l e c t i v i t y K 1 = c a p a c i t y f a c t o r ( r e t e n t i o n ) ( i ) E f f i c i e n c y (number o f t h e o r e t i c a l p l a t e s , N) T h i s i s a measure o f the s h a r p n e s s o f a c h r o m a t o g r a p h i c band (se e F i g . 5) and i t s q u a n t i t a t i v e e x p r e s s i o n i s g i v e n i n the e q u a t i o n : T R 2 T R 2 T 2 N = = 16(-£) = 5.50 ( E q . 11) h where N = e f f i c i e n c y (number o f t h e o r e t i c a l p l a t e s ) T R = r e t e n t i o n time a = s t a n d a r d d e v i a t i o n o f the band (measured a t 0.607 o f peak h e i g h t ) w = peak w i d t h a t b a s e l i n e w^ = band w i d t h a t h a l f peak 15. F i g . 5. Chromatogram showing t h e parameters used f o r t h e c a l c u l a t i o n o f E f f i c i e n c y , S e l e c t i v i t y and C a p a c i t y F a c t o r . TR-| = r e t e n t i o n t i m e o f compound 1; TR? = r e t e n t i o n time o f compound 2; T'p-j - a d j u s t e d r e t e n t i o n time o f compound 1; "Top = a d j u s t e d r e t e n t i o n time o f compound 2; a = band h a l f - w i d t h a t 0.607 o f peak h e i g h t . 16. E q u a t i o n 11 i s s i m i l a r t o E q u a t i o n s 6 and 7 e x c e p t t h a t the d i s t a n c e i n t h i s c a s e i s measured i n terms o f r e t e n t i o n t i m e . ( E q u a t i o n s 5-11 a r e from F a r r i s ( 1 9 7 6 ) ) . ( i i ) S e l e c t i v i t y (a) S e l e c t i v i t y i s a measure o f the e l u t i o n time o f a compound r e l a t i v e t o t h a t o f t h e o t h e r compound ( a d j a c e n t b a n d ) , as shown i n F i g . 5. T h i s i s e x p r e s s e d i n E q u a t i o n 12 ( F a 1 1 i c k ( 1 9 7 5 ) ) : P\2 m K2 a = T r T " = 7 T _ = K ~ (Eq. 12) where, a = s e l e c t i v i t y T R = r e t e n t i o n time o f compound 1 TR = r e t e n t i o n time o f compound 2 T ™ = s o l v e n t f r o n t i n d i m e n s i o n s o f time ' m T'R = a d j u s t e d r e t e n t i o n time o f compound 1 T'R = a d j u s t e d r e t e n t i o n time o f compound 2 K-j = d i s t r i b u t i o n c o e f f i c i e n t o f compound 1 l<2 = d i s t r i b u t i o n c o e f f i c i e n t o f compound 2 S e l e c t i v i t y i s a f u n c t i o n o f the s t a t i o n a r y phase and the s o l v e n t s y s t e m and can be improved by c h a n g i n g the d i s t r i b u t i o n c o e f f i c i e n t K and/or the s t a t i o n a r y phase volume V s , as shown i n the E q u a t i o n 13 (Yan e t a l . (1979)) V R = V m + KV S ( E q . 13) where, = r e t e n t i o n volume o f a compound V m = m o b i l e phase i n t e r s t i t i a l volume ( v o i d volume) and dead volume o f t h e i n s t r u m e n t ( t m x f l o w r a t e ) 17. K = d i s t r i b u t i o n c o e f f i c i e n t V g = - s t a t i o n a r y phase volume ( P a r t i t i o n ) o r , - pore volume ( e x c l u s i o n ) o r , - s u r f a c e a r e a ( a d s o r p t i o n ) o r , - i o n - e x c h a n g e c a p a c i t y ( i o n exchange) For p u r p o s e s o f e f f e c t i n g b e t t e r s e l e c t i v i t y , the d i s t r i b u t i o n c o e f f i c i e n t (K) i n E q u a t i o n 13 can be a l t e r e d by: - c h a n g i n g t h e m o b i l e phase, which may i n c r e a s e o r d e c r e a s e t h e p o l a r i t y , pH o r i o n i c s t r e n g t h - c h a n g i n g s t a t i o n a r y phase which may mean c h a n g i n g pore s i z e o f g e l s , m o d i f y i n g s u r f a c e s o f a d s o r b a n t s , o r c h a n g i n g the l i q u i d u sed as s t a t i o n a r y phase. - c h a n g i n g t e m p e r a t u r e - c h a n g i n g t h e n a t u r e o f t h e s o l u t e s : e.g., by e l i m i n a t i n g the c h a r g e o f an amino a c i d by c h a n g i n g t h e pH o f the m o b i l e phase so as t o reduce a f f i n i t y f o r i o n - e x c h a n g e r e s i n s ; o r by f o r m i n g an i o n p a i r . I t i s t o be n o t e d t h a t the f a c t o r o f s e l e c t i v i t y c o n f e r s on HPLC a d e c i d e d advantage o v e r g a s - l i q u i d chromatography ( G L C ) , b e c a u s e the m o b i l e phase i n the l a t t e r t e c h n i q u e i s i n e r t . ( i i i ) C a p a c i t y r a t i o f a c t o r ( K 1 ) T h i s i s a measure o f t h e degree o f r e t e n t i o n o f a compound and i s e x p r e s s e d as ( F a l l i c k ( 1 9 7 5 ) ) : 18. Where K = c a p a c i t y r a t i o f a c t o r T R = r e t e n t i o n time Tm = r e t e n t i o n time o f u n - r e t a i n e d compound ( s o l v e n t f r o n t ) VR = r e t e n t i o n volume m v o i d volume ( m o b i l e phase) K = d i s t r i b u t i o n c o e f f i c i e n t V s = s t a t i o n a r y phase volume R e t e n t i o n and r e s o l u t i o n can be i n c r e a s e d by i n c r e a s i n g the amount o f s t a t i o n a r y phase ( V g ) . T h i s i s done by: - c h a n g i n g the l o a d i n g o f l i q u i d phase i n p a r t i t i o n p a c k i n g s ; - c h a n g i n g t h e c h a r g e d e n s i t y i n i o n - e x c h a n g e r e s i n s ; - c h a n g i n g the pore volume i n e x c l u s i o n g e l s ; - c h a n g i n g t h e s u r f a c e a r e a o f a d s o r b a n t s A c c o r d i n g t o the l i t e r a t u r e , c a p a c i t y r a t i o v a l u e s o f 2 t o 6 a r e a s s o c i a t e d w i t h t h e g r e a t e s t e f f i c i e n c y o f HPLC columns. A more c o n c i s e g r a p h i c a l and m a t h e m a t i c a l p r e s e n t a t i o n o f t h e o r e t i c a l p l a t e s , s e l e c t i v i t y and r e t e n t i o n i s shown i n F i g . 6. 0 1 2 •v0-H RETENTION, k' v i - v 0 SELECTIVITY, a -k'2 v 2 - v 0 PLATES . N - 16 1 — - ) \ W 1 / k*1 - v 0 V , \ 2 6. Chromatogram d e p i c t i n g t h e parameters t h a t a r e employed f o r c a l c u l a t i n g R e t e n t i o n , S e l e c t i v i t y , and T h e o r e t i c a l P l a t e s . Vg = s o l v e n t f r o n t ; V-j = r e t e n t i o n volume o f compound 1; V 2 = r e t e n t i o n volume o f compound 2; w = band w i d t h a t b a s e l i n e ( F a l l i c k ( 1 9 7 5 ) ) . (B) I n s t r u m e n t a t i o n The h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h , as s c h e m a t i c a l l y shown i n F i g . 7, i s b a s i c a l l y a column chromatograph w i t h a c c e s s o r i e s t h a t have been i n t r o d u c e d f o r purposes o f s o l v e n t d e l i v e r y and m i x i n g , sample i n j e c t i o n and d e t e c t i o n and d a t a p r o c e s s i n g . (a) S o l v e n t r e s e r v o i r C o n t a i n e r s t h a t a r e used as s o l v e n t r e s e r v o i r a r e made o f t y p e 304 o r 316 s t a i n l e s s s t e e l , g l a s s o r an i n e r t polymer l i k e p o l y t e t r a f l u o r o -e t h y l e n e . The s o l v e n t u s u a l l y l e a v e s t h e r e s e r v o i r v i a a s t a i n l e s s s t e e l f r i t f i l t e r . (b) Pumps S o l v e n t d e l i v e r y i n HPLC can be a c h i e v e d by u s i n g a v a r i e t y o f pumping mechanisms t h a t a r e c l a s s i f i e d as f o l l o w s : ( i ) M e c h a n i c a l pumps S c r e w - d r i v e n s y r i n g e t y p e . T h i s i s a mechanism i n which the s o l v e n t c o n t a i n e d i n a s y r i n g e i s s l o w l y pushed by means o f a p l u n g e r r e s u l t i n g i n a p u l s e - f r e e f l o w o f s o l v e n t . However, the l i m i t e d s o l v e n t c a p a c i t y o f the s y r i n g e n e c e s s i t a t e s f r e q u e n t s t o p p i n g s f o r puposes o f r e f i l l i n g and r e s u l t i n g . - R e c i p r o c a t i n g p i s t o n . T h i s s e t - u p c o n s i s t s o f a s a p p h i r e o r ruby p l u n g e r t h a t has slow f o r w a r d and f a s t backward movements t h a t r e s u l t i n a p u l s a t i n g b u t c o n t i n u o u s s u p p l y o f s o l v e n t . P u l s a t i o n s cause p e r i o d i c " n o i s e " t h a t i s S O L V E N T R E S E R V O I R A r G R A D I E N T MIXER INJECTOR C O L U M N r P U M P A P U M P B D E T E C T O R S O L V E N T R E S E R V O I R B ELECTROMETER D A T A P R O C E S S O R -» E L U A T E EX IT F i g . 7. Schematic Diagram o f a G r a d i e n t H i g h - P e r f o r m a n c e L i q u i d Chromatograph. e s p e c i a l l y d i s t u r b i n g t o the b u l k - p r o p e r t y d e t e c t o r s . A dampening d e v i c e , c o n s i s t i n g o f a narrow-bore c o i l o f s t a i n l e s s s t e e l t u b i n g , i s u s e d t o r e d u c e d e t e c t o r " n o i s e " by e l i m i n a t i n g t h e p u l s a t i o n s . A n o t h e r v a r i a t i o n o f t h e r e c i p r o c a t i n g p i s t o n i s known as the r e c i p r o c a t i n g diaphragm pump which a l s o s u p p l i e s a p u l s a t i n g m o b i l e phase. ( i i ) Pneumatic Pumps These pumps use g a s - p r e s s u r e which i s a p p l i e d on a s u i t a b l e c o l l a p s i b l e c o n t a i n e r o r p i s t o n t h a t p r e s s u r i z e s t h e m o b i l e phase r e s u l t i n g i n a p u l s e - f r e e f l o w . ( c ) Sample I n t r o d u c t i o n D e v i c e s Mechanisms by which t he sample i s i n t r o d u c e d i n t o the l i q u i d c hromatograph f a l l i n t o the f o l l o w i n g c a t e g o r i e s : ( i ) I n j e c t i o n p o r t s . These a r e c l a s s i f i e d i n t o on-column i n j e c t i o n p o r t s and swept i n j e c t i o n p o r t s . The f o r m e r i s a s e t - u p i n which t he s y r i n g e n e e d l e e x t e n d s t h r o u g h the septum i n t o the column p a c k i n g where i t d e p o s i t s t h e sample. The l a t t e r t y p e i n v o l v e s t h e d e p o s i t i o n o f the sample j u s t b e f o r e the column i n l e t a f t e r which t he sample i s swept i n t o t h e p a c k i n g by the m o b i l e phase. ( i i ) Sample v a l v e ( s i x - p o r t i n j e c t i o n v a l v e ) . Sample i n t r o d u c t i o n u t i l i z e s a v a l v e arrangement i n which t h e p o r t s t h a t l e a d i n t o t h e l o o p and waste a r e c o n n e c t e d w h i l e b e i n g c u t o f f from t h e p o r t t h a t i n t r o d u c e s t h e s o l v e n t and t h e p o r t t h a t l e a d s i n t o the column. Sample i n j e c t i o n i n t o t h e f l o w s y s t e m i s done by c l o s i n g t h e p o r t t h a t i n t r o d u c e s the sample and the o t h e r p o r t t h a t l e a d s i n t o the w a s t e , i n which case t h e s o l v e n t w i l l f l o w t h r o u g h t h e l o o p i n t o the column. T h i s t y p e o f i n j e c t i o n v a l v e i s used i n s i t u a t i o n s where t h e s o l v e n t i s pumped a t p r e s s u r e s o f more than 2000 pounds per s q u a r e i n c h ( p s i ) . A s c h e m a t i c d i a g r a m o f a s i x - p o r t i n j e c t i o n v a l v e i s shown i n F i g . 8. (d) Columns ( i ) Column M a t e r i a l . Most HPLC columns a r e c o n s t r u c t e d o f D p r e c i s i o n - b o r e s t a i n l e s s s t e e l o r T r u b o r e g l a s s . Even though o r d i n a r y s t a i n l e s s s t e e l t u b i n g can be u s e d , i t has been shown by K i r k l a n d (1969) t h a t t h e smooth i n s i d e s u r f a c e o f the w a l l o f t h e p r e c i s i o n - b o r e s t a i n l e s s s t e e l and T r u b o r e g l a s s a p p a r e n t l y r e d u c e s the band s p r e a d i n g due t o w a l l e f f e c t s . ( i i ) S o l i d S u p p o r t M a t e r i a l s . The s o l i d s u p p o r t m a t e r i a l s w i t h which HPLC columns a r e packed a r e a l u m i n a o r s i l i c i o u s i n n a t u r e and a r e c o m m e r c i a l l y a v a i l a b l e i n a v a r i e t y o f p a r t i c l e s i z e s . These p a c k i n g m a t e r i a l s may be grouped i n t o two c a t e g o r i e s . - Small Porous P a r t i c l e s . These a r e t y p i c a l l y 5-10 m i c r o n s i n d i a m e t e r and f u l l y porous and t h e r e f o r e o f f e r r e l a t i v e l y h i g h s u r f a c e a r e a . P r o v i d e d t h a t t h e y a r e u n i f o r m l y packed t h e s e m a t e r i a l s make good columns w i t h s m a l l i n t e r - p a r t i c l e v o i d s p a c e s and o u t s t a n d i n g e f f i c i e n c y . However, the s m a l l p a r t i c l e s have t h e d i s a d v a n t a g e s o f h i g h r e s i s t a n c e t o l i q u i d f l o w and d i f f i c u l t y i n p a c k i n g . INJECT F i g . 8. Diagrammatic R e p r e s e n t a t i o n o f a S i x - P o r t I n j e c t i o n V a l v e ( F a r r i s ( 1 9 7 6 ) ) . - P e l l i c u l a r S u p p o r t s . These a r e non-porous s u p p o r t p a r t i c l e s u n i f o r m l y c o a t e d w i t h t h i n porous l a y e r s o f l i q u i d phase o r i o n exchange r e s i n . The porous s u r f a c e makes t h e s t a t i o n a r y phase r e a d i l y a v a i l a b l e f o r s o l u t e i n t e r a c t i o n . These m a t e r i a l s u s u a l l y have p a r t i c l e diameters o f 37-44 m i c r o n s and the c o a t i n g a c c o u n t s f o r 1/30th o f the r a d i u s o f the p a r t i c l e . P e l l i c u l a r s u p p o r t s a r e a l s o known i n t h e l i t e r a t u r e as Porous L a y e r Beads (PLB) o r C o n t r o l l e d S u r f a c e P o r o s i t y P a c k i n g s ( C S P ) . A d i a g r a m a t i c p r e s e n t a t i o n o f the s t r u c t u r e s o f the p a c k i n g m a t e r i a l s d i s c u s s e d i s shown i n F i g . 9. HPLC columns a r e commonly c l a s s i f i e d i n t o Normal Phase (Forward Phase) and R e v e r s e Phase t y p e s depending upon the n a t u r e o f t h e p a c k i n g m a t e r i a l . - Normal Phase Column. T h i s t y p e o f column i s packed w i t h s i l i c o n o r a l u m i n a and has p o l a r c h a r a c t e r i s t i c s . Examples o f commer-c i a l l y a v a i l a b l e a d s o r b e n t s a l o n g w i t h t h e i r f u n c t i o n a l i t y and s u r f a c e a r e a a r e g i v e n i n T a b l e I. - Reverse Phase Columns. The s o l i d s u p p o r t m a t e r i a l s i n t h i s c a s e a r e c l a s s i f i e d i n t o two: - P h y s i c a l l y Bound. The p a c k i n g i n t h i s column c o n s i s t s o f s i l i c a o r a l u m i n a m a t e r i a l t h a t has been c o a t e d ( p h y s i c a l l y bound) w i t h l i q u i d s t a t i o n a r y phases l i k e t r i e t h y l e n e g l y c o l , e t h y l e n e g l y c o l , h y d r o c a r b o n polymers e t c . - C h e m i c a l l y Bonded. T h i s t y p e o f s t a t i o n a r y phases i s c u r r e n t l y a v a i l a b l e i n two d i f f e r e n t forms which a r e known as E s t e r i f i e d S i l i c i o u s S u p p o r t s and C h e m i c a l l y Bonded S i l i c o n e P o l y m e r s . The f o r m e r a r e p r e p a r e d by e s t e r i f i c a t i o n o f s i l i c i o u s s u p p o r t s 26. PREPARATIVE SEPARATIONS FULLY POROUS HIGH CAPACITY ANALYTICAL SEPARATIONS PELLICULAR HIGH EFFICIENCY ANALYTICAL & PREPARATIVE SEPARATIONS FULLY POROUS HIGH SPEED HIGH EFFICIENCY PORASIL STYRAGEL DURAPAK/PORASIL BONDAPAK/PORASIL CORASIL DURAPAK/CORASIL BONDAPAK/CORASIL pPORASIL /iSTYRAGEL pBONDAPAK F i g . 9. Diagrammatic R e p r e s e n t a t i o n o f the S t r u c t u r e s o f Column P a c k i n g M a t e r i a l s used i n A n a l y t i c a l and P r e p a r a t i v e S e p a r a t i o n s (Waters A s s o c i a t e s P u b l i c a t i o n s Ref. No. 2584).  27. TABLE I. NAME, FUNCTIONALITY AND SURFACE AREA OF COMMERCIALLY AVAILABLE ADSORBENTS USED IN HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY.3 ADSORBENTS NAME FUNCTIONALITY SURFACE AREA (m /^g) PORASIL A, B, C, D f E. F PORASIL T pPORASIL -SiOH -SiOH -SiOH 400 to 2 300 350 CORASIL 1 CORASIL II -SiOH -SiOH 12-15 25-30 WO ELM ALUMINA ACID AI-CI 1 0 1 AI-CI 200 WO ELM ALUMINA NEUTRAL Al 1 \ 0 0 1 / Al 200 WOELM BASIC BASIC Al-O-Na 1 • 0 1 Al-O-Na 200 a W a t e r s A s s o c i a t e s P u b l i c a t i o n s Ref. No. 2571. w i t h a monomolecular o r g a n i c l a y e r o f a l c o h o l s , as d e s c r i b e d by H a l a s z and S e b a s t i a n ( 1 9 6 9 ) . The l a t t e r a r e o b t a i n e d by r e a c t i n g s i l a n e r e a g e n t s w i t h t h e s u r f a c e o f t h e porous s h e l l D o f Z i p a x s u p p o r t and then p o l y m e r i z i n g t he r e a g e n t s t o g i v e t h e d e s i r e d s i l i c o n e c o a t i n g , as r e p o r t e d by K i r k l a n d and D e s t e f a n o ( 1 9 7 0 ) . S i l i c i o u s s u p p o r t m a t e r i a l s bonded w i t h l o n g c h a i n h y d r o c a r b o n s ( e . g . , o c t a d e c y l s i l a n e ) a r e a t p r e s e n t amongst t h e most commonly used r e v e r s e phase columns. A diagram t h a t shows t h e s t r u c t u r e o f bonded p a c k i n g m a t e r i a l , f u n c t i o n a l i t y and t h e s e p a r a t i o n p r o c e s s i s p r e s e n t e d i n F i g . 10. The v e r s a t i l i t y o f a r e v e r s e phase column i s d e m o n s t r a t e d i n F i g . 11 i n which m a n i p u l a t i o n o f pH and i n t r o d u c t i o n o f i o n i c r e a g e n t s l i k e t e t r a b u t y l ammonium h y d r o x i d e can f a c i l i t a t e s e p a r a t i o n as a r e s u l t o f i o n - p a i r f o r m a t i o n . A summary o f some c h a r a c t e r i s t i c s o f Normal and Reverse Phase Columns i s g i v e n i n T a b l e I I . D e s i g n and o p e r a t i o n a l parameters t h a t a r e a s s o c i a t e d w i t h A n a l y t i c a l and P r e p a r a t i v e HPLC a r e l i s t e d i n T a b l e I I I . (e) D e t e c t o r s The d e t e c t o r i s a d e v i c e t h a t c o n t i n u o u s l y m o n i t o r s t he c o n c e n t r a t i o n o f t he s o l u t e as i t l e a v e s t he column. The d e t e c t o r s commonly used i n HPLC f a l l i n t o two c a t e g o r i e s : B u l k p r o p e r t y d e t e c t o r s and s o l u t e p r o p e r t y d e t e c t o r s . ( i ) B u l k P r o p e r t y D e t e c t o r s . These d e v i c e s m o n i t o r t he change i n t h e o v e r a l l p h y s i c a l p r o p e r t y o f t h e m o b i l e phase and a r e a l s o known as u n i v e r s a l d e t e c t o r s . Examples o f t h i s t y p e o f d e t e c t o r s a r e r e f r a c t i v e 29. AC ETON IT RIL E/W AT E R F i g . 10. Diagrammatic R e p r e s e n t a t i o n o f t h e S t r u c t u r e and F u n c t i o n a l i t y o f Bonded-Packing M a t e r i a l and the S e p a r a t i o n P r o c e s s (Waters A s s o c i a t e s P u b l i c a t i o n s Ref. No. 2248) MOBILE PHASE Acidic Solution O F i g . 11. Diagrammatic R e p r e s e n t a t i o n o f I o n - P a i r i n g f o r Reverse Phase S e p a r a t i o n s (Waters A s s o c i a t e s P u b l i c a t i o n s Ref. No. 3100). 31. TABLE I I . SUMMARY OF SOME CHARACTERISTICS OF NORMAL AND REVERSE PHASE COLUMNS 3 CHARACTERISTICS NORMAL P H A S E REVERSE P H A S E PACKING POLARITY SOLVENT POLARITY SAMPLE ELUTION ORDER EFFECT OF INCREASING SOLVENT POLARITY High Low to Medium Least Polar First Reduces Elution Time Low Medium to High Most Polar First Increased Elution Time a W a t e r s A s s o c i a t e s P u b l i c a t i o n s Ref. No. 2583. TABLE I I I . DESIGN AND OPERATIONAL PARAMETERS ASSOCIATED WITH ANALYTICAL AND PREPARATIVE HPLC a Design Parameters A Analytical H P L C B Preparative (High Sample Capacity) Length, cm 2 5 - 1 0 0 2 5 - 2 0 0 i A., mm Shape Support: 2 to 4 Straight ~8 mm Straight Diameter, microns Surface area Mobile Phase Stationary Phase 5 - 4 0 Moderate to high Nonviscous Spectrograde 1 0 - 2 0 u High Volatile Spectrograde j Partition Thin F i lm Higher loading bonded j Adsorption j Ion Exchange 1 Uniform activity Th in fi lm phase Higher surface area Higher capacity, less i 1 Exclusion Rigid gel cross-linking Porous qel i Operational Parameters i J'Jdient ''ow rate, ml/hr ''•'Mure, psig '•mperature Sample size, pg Complex samples only 3 0 - 1 2 0 5 0 0 - 5 0 0 0 Optimize for repro-ducibil ity and resolution 0.050 to 500 Often higher than opt imum Complex samples only 2 0 0 - 4 0 0 5 0 0 - 5 0 0 0 psi Increase for solubility Up to 500 milligrams per injection a P a s c o t t (1976) i n d e x , c o n d u c t i v i t y and d i e l e c t r i c c o n s t a n t m e a s u r i n g d e v i c e s . A l l o f t h e s e d e t e c t o r s a r e t e m p e r a t u r e s e n s i t i v e and most l a c k the s e n s i t i v i t y needed f o r HPLC. ( i i ) S o l u t e P r o p e r t y D e t e c t o r s . S e l e c t o r s o f t h i s t y p e measure / p h y s i c a l p r o p e r t i e s o f t h e s o l u t e which a r e n o t e x h i b i t e d t o any s i g n i -f i c a n t e x t e n t by t h e m o b i l e phase. UV a b s o r p t i o n , f 1 u o r e s c e n c e , p o l a r o -g r a p h i c and r a d i o a c t i v i t y d e t e c t o r s a r e examples o f t h i s t y p e . The two most w i d e l y used d e t e c t o r s a r e the UV a b s o r p t i o n and the r e f r a c t i v e i n d e x . The f o r m e r i s one o f the most s e n s i t i v e d e t e c t o r s i n l i q u i d chromatography and can d e t e c t samples i n nanogram ran g e . The l a t t e r t y p e i s f a i r l y easy t o use and can d e t e c t s o l u t e s i n the microgram r a n g e . U n l i k e the UV a b s o r p t i o n d e t e c t o r , however, the r e f r a c t i v e i n d e x d e t e c t o r i s s e n s i t i v e t o changes i n t e m p e r a t u r e and s o l v e n t f l o w . ( f ) Data P r o c e s s o r s D e t e c t o r r e s p o n s e i n HPLC i s n o r m a l l y p r e s e n t e d i n the form o f a chromatogram showing a number o f peaks c o r r e s p o n d i n g t o d i f f e r e n t compounds t h a t have been s e p a r a t e d . The chromatogram may be o b t a i n e d by u s i n g a s i m p l e pen r e c o r d e r , d i g i t a l i n t e g r a t o r and p r i n t e r o r e l e c t r o n i c c o m p u t e r - c a l c u l a t o r . Q u a n t i t a t i o n o f peak a r e a o r peak h e i g h t measure-ments i s done by t h e l a t t e r t y p e s . E l e c t r o n i c d a t a p r o c e s s o r s w i t h c o m p u t a t i o n a l f e a t u r e s t h a t make them c a p a b l e o f p r i n t i n g o u t d i r e c t l y i n c o n c e n t r a t i o n terms a r e now a v a i l a b l e . (C) T e c h n i q u e The p r i m a r y purpose o f HPLC, as i n a l l c h r o m a t o g r a p h i c work, i s s e p a r a t i o n ( r e s o l u t i o n ) o f the components o f a m i x t u r e . Two o t h e r f a c t o r s t h a t s h o u l d a l s o be c o n s i d e r e d a r e speed and c a p a c i t y (sample l o a d ) . I t i s the a p p r o p r i a t e b a l a n c e o f the above t h r e e f a c t o r s - the so c a l l e d chroma-t o g r a p h e r ' s t r i a n g l e , t h a t r e s u l t s i n an o p t i m a l chromatogram. The t r i a n g l e , as shown i n F i g . 12, r e p r e s e n t s t h e p o s s i b i l i t y t h a t h i g h speed and h i g h c a p a c i t y , h i g h c a p a c i t y and good r e s o l u t i o n , and good r e s o l u t i o n and h i g h speed may be a t the expense o f r e s o l u t i o n , speed and c a p a c i t y , r e s p e c t i v e l y . The u n i q u e c h a r a c t e r i s t i c s o f HPLC t h a t e n a b l e i t t o o p t i m i z e t h e s e f a c t o r s so as t o o b t a i n s a t i s f a c t o r y chroma-tograms a r e : - porous m i c r o p a r t i c u l a t e p a c k i n g s w i t h d i m e n s i o n s o f 5-37 m i c r o n s . - s m a l l b o r e columns (2.6 - 4.0 mm i . d . ) - i n t r o d u c t i o n o f h i g h - p r e s s u r e pumps - a v a i l a b i l i t y o f r e l a t i v e l y h i g h - s e n s i t i v i t y o n - l i n e d e t e c t o r s (a) HPLC Mode S e l e c t i o n A knowledge o f t h e m o l e c u l a r w e i g h t range, s o l u b i l i t y and f u n c t i o n a l groups o f t h e compounds t o be a n a l y s e d by HPLC i s e s s e n t i a l f o r the a p p r o -p r i a t e c h o i c e o f HPLC mode. A m o l e c u l a r w e i g h t o f o v e r 2000 would r e q u i r e e x c l u s i o n HPLC w i t h an aqueous o r non-aqueous phase depending on whether t h e compounds a r e s o l u b l e o r i n s o l u b l e i n w a t e r . For compounds t h a t have m o l e c u l a r w e i g h t s o f l e s s than 2000 and a r e w a ter i n s o l u b l e , the c h o i c e can be p a r t i t i o n , a d s o r p t i o n o r e x c l u s i o n HPLC depending on whether th e y a r e homologs, i s o m e r s o r o f d i f f e r e n t s i z e s . Water s o l u b l e compounds 35. Fig. 12. Triangle of Resolution, Speed and Capacity representing the Balance necessary for Chromatographic Efficiency. o f low m o l e c u l a r w e i g h t may r e q u i r e r e v e r s e phase, e x c l u s i o n , i o n - e x c h a n g e o r r e v e r s e - p h a s e i o n - p a i r p a r t i t i o n HPLC a c c o r d i n g t o whether t h e y a r e n o n - i o n i c , i o n i c o r b o t h . A s c h e m a t i c g u i d e t o HPLC mode s e l e c t i o n i s g i v e n i n T a b l e IV. (b) O p t i m i z a t i o n o f R e s o l u t i o n ( i ) I s o c r a t i c E l u t i o n . T h i s i s a p r o c e s s i n which one s o l v e n t o r a s o l v e n t s y s t e m o f f i x e d c o m p o s i t i o n i s used as the m o b i l e phase. In o r d e r t o c o n v e n i e n t l y l o c a t e the peaks i n a chromatogram h a v i n g r e a s o n a b l y s h o r t r e t e n t i o n t i m e s , i t would f i r s t be n e c e s s a r y t o m a n i p u l a t e t he c a p a c i t y r a t i o f a c t o r ( K * ) , as shown i n F i g . 13. T h i s i s n o r m a l l y a c h i e v e d by c h a n g i n g t he s o l v e n t s t r e n g t h o f the m o b i l e phase. Then e f f i c i e n c y (N) can be a l t e r e d t o see i f t h e r e can be b e t t e r s e p a r a t i o n by making peaks s h a r p e r . T h i s i s b r o u g h t about by c h o o s i n g t h e r i g h t t y p e ( h a v i n g h i g h sample r e t e n t i o n c a p a c i t y ) and l e n g t h o f column. F i n a l l y t h e s e l e c t i v i t y f a c t o r (a) may be changed t o s e l e c t i v e l y move one o f t h e peaks and e f f e c t s e p a r a t i o n . M a n i p u l a t i o n s o f s e l e c t i v i t y , w h i l e more o r l e s s m a i n t a i n i n g t h e same c a p a c i t y f a c t o r , may be a c c o m p l i s h e d by s l i g h t a l t e r a t i o n s o f p o l a r i t y . S e l e c t i v i t y and c a p a c i t y r a t i o f a c t o r a r e f u n c t i o n s o f mo b i l e phase c h a r a c t e r i s t i c s l i k e p o l a r i t y , v i s c o s i t y , s o l u b i l i t y f a c t o r s e t c . Changes i n the above f a c t o r s a r e b r o u g h t about by changes i n the t y p e o f s o l v e n t o r c o m p o s i t i o n o f s o l v e n t s y s t e m s . The s o l v e n t s t h a t a r e used as t h e m o b i l e phase a r e u s u a l l y chosen from a t a b l e where they a r e l i s t e d on t h e b a s i s o f e m p i r i c a l s c a l e s o f r e l a t i v e s o l v e n t p o l a r i t i e s . An example o f such a s c a l e , t h a t o f Macek and P r o c h a z k a , i s shown i n T a b l e V, as r e p o r t e d by Ha i s and Macek ( 1 9 6 3 ) . The r o l e o f t h e m o b i l e phase i n l i q u i d chromatography has been d i s c u s s e d TABLE IV. GENERAL GUIDE FOR HPLC MODE SELECTION 9 MW > 2000 Sample-Water-soluble—4- Exclusion, Aqueous Phase •-Wate r insoluble-^- Exclusion, Nonaqueous Phase MW < 2000 Homologs—(Par t i t i on Chromatography Water- insoluble*Isomers— I Liquid-Solid Chromatography •-Size Differences- •[ Exclusion [Reverse-Phase Partit ion Chromatography Water-soluble nonionic 'Exclus ion, Aqueous Mobile Phase Water-soluble ionic p Basic -WAcidic-• [Cation Ion Exchange • (Anion ion Exchange • W a , e r - I o l u b l e - | - R e v e r s e - P h a s e lon-Pair Partit ion ionic and nonionic Baumann (1971) F i g . 13. Diagrammatic R e p r e s e n t a t i o n o f E f f e c t s o f V a r y i n g C a p a c i t y , E f f i c i e n c y and S e l e c t i v i t y F a c t o r s on R e s o l u t i o n (Snyder and K i r k l a n d ( 1 9 7 9 ) ) . TABLE V. PARTIAL LISTING OF SOLVENTS USED IN HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY IN ORDER OF DECREASING POLARITY 3 Water (most polar) CH2CI2 Formamide CHCI3 Acetonitrile 1,2-Dichloroethane Methanol Bromobenzene Acetic acid Ethyl bromide Ethanol Benzene Isopropanol Propyl chloride Acetone Toluene Dloxane Xylene Tetrahydrofuran ecu t.-Butanol CS2 Methyl ethyl ketone Cyclohexane Phenol Hexane n-Butanol Heptane n-Pentanol Keroslne (least polar) Ethyl acetate Ethyl ether n-Butyl acetate Nltromethane Isopropyl ether a H a i s and Macek (1963) a t l e n g t h by S nyder ( 1 9 7 1 ) . ( i i ) G r a d i e n t E l u t i o n . T h i s t y p e o f e l u t i o n i n v o l v e s a g r a d u a l change o f s o l v e n t c o m p o s i t i o n t o e f f e c t a c o r r e s p o n d i n g change ( i n c r e a s e o r d e c r e a s e ) i n the p o l a r i t y o f the m o b i l e phase, which i n t u r n r e s u l t s i n a s i g n i f i c a n t r e d u c t i o n o f the r e t e n t i o n times o f s l o w l y e l u t i n g components. G r a d i e n t e l u t i o n , which may be programmed i n a l i n e a r o r n o n - l i n e a r mode, i s e s p e c i a l l y u s e f u l f o r a m i x t u r e o f compounds w i t h a wide range o f p o l a r i t i e s . In the case o f UV d e t e c t o r s , even though t h i s method o f e l u t i o n does i n c r e a s e d e t e c t i o n s e n s i t i v i t y f o r l a t e -e l u t i n g components, i t can o n l y be used i n c a s e s where the s o l v e n t s do n o t have any s i g n i f i c a n t UV a b s o r b a n c e . Moreover, f o l l o w i n g s e p a r a t i o n by g r a d i e n t e l u t i o n , t h e l a s t p o r t i o n o f the s o l v e n t g r a d i e n t has t o be washed o u t and t h e column has t o be r e - e q u i l i b r a t e d w i t h the s o l v e n t o f i n i t i a l c o m p o s i t i o n . The d e t a i l s o f e x p e r i m e n t a l o p t i m i z a t i o n o f g r a d i e n t e l u t i o n have been r e p o r t e d by Snyder and Saunders (19 6 9 ) . O t h e r methods t h a t have been employed f o r p u rposes o f o p t i m i z i n g r e s o l u t i o n i n c l u d e f l o w programming, r e p e a t e d s e p a r a t i o n s and c o u p l e d columns. (c ) Advantages o f HPLC The advantages o f HPLC o v e r l i q u i d and g a s - l i q u i d chromatography may be summarized as f o l l o w s -o v e r l i q u i d chromatography: - speed and r e s o l u t i o n - s e n s i t i v i t y o f o n - l i n e d e t e c t o r s - r e - u s a b l e columns - easy sample r e c o v e r y - s o l v e n t programming o v e r g a s - l i q u i d c h r o m atography: - n o n - d e s t r u c t i v e t o sample - amenable t o b i o l o g i c a l s y s t e m s , s i n c e t h e column p a c k i n g can be n e u t r a l , a n i o n i c o r c a t i o n i c - t h e s e l e c t i v i t y f a c t o r o f the l i q u i d m o b i l e phase - i d e a l f o r l a r g e m o l e c u l e s ( n o n - v o l a t i l e compounds) (d) D i s a d v a n t a g e s o f HPLC Even though HPLC o f f e r s many advantages as i n d i c a t e d above, i t has a l s o drawbacks t h a t i n c l u d e t h e f o l l o w i n g : - h i g h equipment c o s t - l i m i t e d s e n s i t i v i t y o f a v a i l a b l e d e t e c t o r s towards c e r t a i n t y p e s o f compounds - t h e e x p e r i e n c e n e c e s s a r y t o o b t a i n good r e s u l t s The development o f new and more s e n s i t i v e HPLC d e t e c t o r systems may b r i n g f u r t h e r advances i n r e s o l u t i o n , speed and c o n v e n i e n c e . Computers i n t e r f a c e d w i t h HPLC can p l a y a s i g n i f i c a n t r o l e i n the s e l e c t i o n o f LC systems as a p p r o p r i a t e f a c t o r s can be i n c o r p o r a t e d i n t o m a t h e m a t i c a l models s u i t a b l e f o r computer c a l c u l a t i o n s . I t a p p e a r s , t h e r e f o r e , t h a t HPLC w i l l p l a y an i n c r e a s i n g l y i m p o r t a n t r o l e i n t h e s e p a r a t o r y and q u a n t i t a t i o n s t e p s o f a n a l y s i s . 42. 2. The T e s t Drugs: D i g o x i n and D i g i t o x i n (A) C h e m i s t r y (a) D e s c r i p t i o n D i g o x i n and d i g i t o x i n a r e c a r d i o t o n i c s e c o n d a r y g l y c o s i d e s o b t a i n e d i n p u r i f i e d form from the l e a v e s o f D i g i t a l i s l a n a t a E h r h a r t and D i g i t a l i s  p u r p u r e a L i n n e , r e s p e c t i v e l y . These d i f f i c u l t l y p u r i f i e d g l y c o s i d e s o c c u r v e r y w i d e l y i n a v a r i e t y o f p l a n t f a m i l i e s as w e l l as i n the venoms o f c e r t a i n t o a d s , and more than one hundred and f i f t y i n d i v i d u a l compounds b e l o n g i n g t o the d i g i t a l i s - s t r o p h a n t h u s - group have been d e s c r i b e d ( W i l s o n , 1960). Commonly employed c a r d i a c g l y c o s i d e s a r e o b t a i n e d from d i g i t a l i s , s t r o p h a n t h u s and s q u i l l . However, the term d i g i t a l i s i s o f t e n used t o d e s i g n a t e t h e e n t i r e group o f c a r d i a c g l y c o s i d e s r a t h e r than those from d i g i t a l i s a l o n e . (b) Names D i g o x i n i s known by names t h a t i n c l u d e the f o l l o w i n g (Merck Index, 1976): C o r d i o x i l , D a v o x i n , D i g a c i n ; D i l a n a c i n , D i x i n a , L a n o c a r d i n , L a n i c o r , L a n o x i n , Rougoxin, V a n o x i n . D i g i t o x i n has been known by r e g i s t e r e d names t h a t i n c l u d e t h e f o l l o w i n g ( J a k o v l j e v i c , 1974): C a r d i g i n , C r y s t o d i g i n , D i g i c o r y l , D i g i l o n g , D i g i m e r c k , D i g i p a n , D i g i s i d i n , D i g i t a l i n e N a t i v e l l e , D i g i t o r a , D i g i t o x o s i d e , D i g i t r i n , L a n a t o x i n , p u r o d i g i n , p u r p u r e n , p u r p u r i d . The c h e m i c a l name o f d i g o x i n i s : 3 p - [ ( 0 - 2 , 6 - D i d e o x y - g - D - r i b o -h e x o p y r a n o s y l - ( l->-4)-0-2,6-dideoxy-g-D-ribo-hexopyranosyl -(l->4) - 2 , 6 - d i d e o x y -g - D - r i b o - h e x o p y r a n o s y 1 ) o x y 3 - 1 2 B , 1 4 - d i h y d r o x y - 5 g - c a r d - 2 0 ( 2 2 ) - e n o 1 i d e . S i m i l a r l y , t h e c h e m i c a l d e s i g n a t i o n o f d i g i t o x i n i s : 3 g - [ ( 0 - 2 , 6-Di deoxy-8 - D - r i b o - h e x o p y r a n o s y l - ( 1 + 4 ) - 0 - 2 , 6 - d i d e o x y - g - D - r i b o - h e x o p y r a n o s y l - ( 1 + 4 ) - 2 , 6 d i d e o x y - e - D - r i b o - h e x o p y r a n o s y l ) o x y ] - 1 4 g - h y d r o x y - 5 e - c a r d - 2 0 ( 2 2 ) - e n o l i d e . (c ) Appearance D i g o x i n i s an o d o r l e s s , w h i t e c r y s t a l l i n e powder and i t s c r y s t a l s appear as r a d i a l l y a r r a n g e d , f o u r - a n d f i v e - s i d e d t r i c l i n i c p l a t e s from d i l u t e a l c o h o l o r d i l u t e p y r i d i n e . D i g i t o x i n i s a w h i t e o r p a l e b u f f c r y s t a l l i n e powder, and the c r y s t a l s from d i l u t e a l c o h o l a r e v e r y s m a l l e l o n g a t e d , r e c t a n g u l a r p l a t e s . (d) S y n t h e s i s S u c c e s s f u l s y n t h e s i s o f d i g o x i n and d i g i t o x i n has n o t y e t been r e p o r t e d and t h e r e f o r e t h e s e drugs a r e c o m m e r c i a l l y o b t a i n e d by e t h a n o l i c e x t r a c t i o n o f d i g i t a l i s l e a v e s and subsequent c h r o m a t o g r a p h i c p u r i f i c a t i o n (e) S o l u b i l i t y . D i g o x i n i s s o l u b l e i n p y r i d i n e , d i l u t e a l c o h o l o r a m i x t u r e o f c h l o r o f o r m and a l c o h o l ; a l m o s t i n s o l u b l e i n e t h e r , a c e t o n e , e t h y l a c e t a t e , c h l o r o f o r m ; and p r a c t i c a l l y i n s o l u b l e i n w a t e r (Merck Index, 1976) . S o l u b i l i t y o f d i g o x i n and i t s m e t a b o l i t e s has been r e p o r t e d t o be maximum i n s o l v e n t s such as a l c o h o l s , c h l o r o f o r m and methylene c h l o r i d e ( G a u l t e t a l . , 1976). A c c o r d i n g t o Merck Index ( 1 9 7 6 ) , one gram o f d i g i t o x i n d i s s o l v e s i n about 40 ml c h l o r o f o r m , i n a b o u t 60 ml a l c o h o l and i n about 400 ml e t h y l a c e t a t e . D i g i t o x i n i s s o l u b l e i n a c e t o n e , amyl a l c o h o l , p y r i d i n e ; and s p a r i n g l y s o l u b l e i n e t h e r , p e t r o l e u m e t h e r , w a t e r (1 g/100 l i t e r a t 2 0 ° C ) . ( f ) S t a b i l i t y D i g o x i n has been r e p o r t e d t o be s t a b l e f o r an i n d e f i n i t e p e r i o d o f time when kep t i n w e l l c l o s e d c o n t a i n e r s and p r o t e c t e d from l i g h t ( Foss e t a l . , 1980). I t has a l s o been o b s e r v e d t h a t no d e g r a d a t i o n o c c u r s i n t a b l e t s o r n e u t r a l s o l u t i o n s o f d i g o x i n i n e t h a n o l and p r o p y l e n e g l y c o l f o r p e r i o d s o f up t o f i v e y e a r s . S t o r a g e o f d i g o x i n under i n t e n s i v e l i g h t f o r l o n g p e r i o d s o f time may r e s u l t i n d e g r a d a t i o n due t o o p e n i n g o f t h e l a c t o n e r i n g . D i g i t o x i n i s a l s o r e p o r t e d t o be r e l a t i v e l y s t a b l e under o p t i m a l c o n d i t i o n s . I t has been shown t h a t no d e g r a d a t i o n o f d i g i t o x i n i n t a b l e t s , i n j e c t i o n s o r s o l u t i o n s was found when s t o r e d f o r f i v e y e a r s i n the dark a t t e m p e r a t u r e s o f up t o 30°C (Samuelson, 1964). (g) C hemical S t r u c t u r e ( i ) F o r m u l a , M o l e c u l a r Weight and C l a s s i f i c a t i o n The e m p i r i c a l f o r m u l a s , m o l e c u l a r s t r u c t u r e s , c o n f o r m a t i o n a l a r r a n g e -ments and m o l e c u l a r w e i g h t s o f d i g o x i n and d i g i t o x i n a r e shown i n F i g s . 14 and 15. L i k e any o f t h e c a r d i a c g l y c o s i d e s , t h e d i g o x i n o r d i g i t o x i n m o l e c u l e i s made o f a s u g a r p o r t i o n and an a g l y c o n e ( g e n i n ) p o r t i o n , as i n d i c a t e d i n t h e m o l e c u l a r s t r u c t u r e . C a r d i a c g l y c o s i d e s a r e g e n e r a l l y c l a s s i f i e d a c c o r d i n g t o the ty p e o f g e n i n . Two t y p e s o f g e n i n may be d i s t i n g u i s h e d a c c o r d i n g t o whether t h e r e i s a f i v e - o r six-membered l a c t o n e r i n g . These g e n i n s , as shown i n F i g . 16, a r e known r e s p e c t i v e l y as c a r d e n o l i d e s ( e . g . d i g o x i g e n i n o r d i g i t o x i g e n i n ) and b u f a d i e n o l i d e s F i g . 14. Chemical S t r u c t u r e o f D i g o x i n . THE CONFORMATIONAL ARRANGEMENT F i g . 15. Chemical S t r u c t u r e o f D i g i t o x i n . 47. Cardenolide Bufadienolide F i g . 16. Chemical S t r u c t u r e s o f the C a r d e n o l i d e and B u f a d i e n o l i d e Groups o f G e n i n s . o ( b u f a n o l i d e s , e.g. s c i l l a r e n i n ) . D i g i t a l i s l a n a t a and D i g i t a l i s p u r p u r e a l e a v e s c o n t a i n a m i x t u r e o f c a r d i o a c t i v e g l y c o s i d e s which a r e c l a s s i f e d i n t o f i v e c a r d e n o l i d e s e r i e s on the b a s i s o f the i n d i v i d u a l a g l y c o n e . The c a r d e n o l i d e "C" and "A" s e r i e s o f c a r d i a c g l y c o s i d e s , among which d i g o x i n and d i g i t o x i n a r e two s e c o n d a r y g l y c o s i d e s , a r e the most abundant and i m p o r t a n t . As shown i n T a b l e V I , a s t e p w i s e h y d r o l y s i s o f the p r i m a r y g l y c o s i d e l a n a t o s i d e C, b r o u g h t about by r e a g e n t s o r enzymes, y i e l d s a number o f s e c o n d a r y g l y c o s i d e s ( i n c l u d i n g d i g o x i n ) and v a r y i n g number o f d i g i t o x o s e s u g a r s . C l e a v a g e o f the l a s t d i g i t o x o s e m o l e c u l e r e s u l t s i n t h e a g l y c o n e , d i g o x i g e n i n . D i g i t o x i g e n i n can a l s o be o b t a i n e d from l a n a t o s i d e A, i n t h e same way. In g e n e r a l , m i l d a l k a l i n e h y d r o l y s i s r e s u l t s i n t h e l o s s o f the a c e t y l group o f the l a n a t o s i d e m o l e c u l e ; e n z y m a t i c h y d r o l y s i s c l e a v e s g l u c o s e ; and sub s e q u e n t a c i d h y d r o l y s i s s p l i t s o f f d i g i t o x o s e y i e l d i n g t he c o r r e s p o n d i n g a g l y c o n e s (Cobb (1976) ) . ( i i ) S t r u c t u r e - A c t i v i t y R e l a t i o n s h i p The b a s i c s t r u c t u r e o f the a g l y c o n e p o r t i o n o f d i g o x i n o r d i g i t o x i n , l i k e a l l o f the o t h e r c a r d i a c g l y c o s i d e s , i s a c y c l o p e n t a n e - p h e n a n t h r e n e n u c l e u s t o which i s a t t a c h e d a l a c t o n e r i n g . And t h e e s s e n t i a l s t r u c t u r e s f o r b i o l o g i c a l a c t i v i t y a r e c o n t a i n e d i n the a g l y c o n e p o r t i o n . The u n i q u e s t r u c t u r a l c h a r a c t e r i s t i c s o f c a r d i a c g l y c o s i d e s may be summarized as f o l l o w s ( W i l s o n e t a l . ( 1 9 7 1 ) ) : - a l l o f the c a r d i a c g l y c o s i d e s a r e complex s t e r o i d s - i n a l l known c a s e s , one o r more c a r b o h y d r a t e r e s i d u e s a r e a t t a c h e d t h r o u g h t h e oxygen a t p o s i t i o n 3 o f the s t e r o i d n u c l e u s , and a l m o s t w i t h o u t e x c e p t i o n t he oxygen bond t o t h e s t e r o i d n u c l e u s i s i n the e - c o n f i g u r a t i o n 49. TABLE V I . GENINS OF THE CARDENOLIDE SERIES OF DIGITALIS GLYCOSIDES (Cobb (1976))  Aflycunc Compound GlycmidicaJly Imkril refill ne at Ci Di fo i tecum U i j o i i r ' H i n monodiiiitiitixuli DiymiKuriin buditMloKOtuIr D l l f o n n • At<l) Idijroii'' 0-Acx'tyl<ii*o(in LftnalotiUt C } - D - D - D -D-I>-r> -D-l>-D Ac - D - D - D - C I Ac Difitocigcmn Gitotifenm DiftnAtin CtUloKin p o s i t i o n 17 o f t h e s t e r o i d n u c l e u s i s i n v a r i a b l y s u b s t i t u t e d w i t h an u n s a t u r a t e d l a c t o n e r i n g , a l s o i n the 3 - c o n f i g u r a t i o n a l l a c t i v e compounds c a r r y a g - h y d r o x y l group on c a r b o n 14 u n l i k e most o t h e r s t e r o i d s , the C/D r i n g j u n c t i o n i n the c a r d i a c g l y c o s i d e s i s always c i s ; w i t h the e x c e p t i o n o f some g l y c o s i d e s ( e . g . u z a r i g e n i n , u r e z i g e n i n , e t c . ) r i n g j u n c t i o n A/B i s always c i s . i n a l l c a s e s t h e B/C r e l a t i o n s h i p i s t r a n s , most o f the c a r d i a c g l y c o s i d e s may be r e l a t e d t o the c o p r o s t a n e s e r i e s , whereas t h o s e o f the u z a r i g e n i n group a r e r e l a t e d t o the c h o l e s t a n e s e r i e s . t h e a , B - u n s a t u r a t e d , five-membered l a c t o n e r i n g ( b u t e n o l i d e ) i n p o s i t i o n 17 i s a d i s t i n g u i s h i n g f e a t u r e o f the d i g i t a l i s -s t r o p h a n t h u s g r o up. most o f t h e d i g i t a l i s g l y c o s i d e s c o n t a i n a n g u l a r methyl groups a t p o s i t i o n s 10 and 13, b u t an a l d e h y d e o r p r i m a r y a l c o h o l a t p o s i t i o n 10 i s c h a r a c t e r i s t i c o f the s t r o p h a n t h u s g l y c o s i d e s . (B) Pharmacology D i g o x i n and d i g i t o x i n , l i k e any o f the d i g i t a l i s g l y c o s i d e s and c e r t a i n c l o s e l y a l l i e d d r u g s , have a s p e c i f i c and p o w e r f u l a c t i o n on the myocardium t h a t i s u n r i v a l e d i n v a l u e f o r the t r e a t m e n t o f c o n g e s t i v e h e a r t f a i l u r e . The main pharmacodynamic p r o p e r t y o f t h e s e drugs i s t h e i r a b i l i t y t o i n c r e a s e t h e f o r c e o f m y o c a r d i a l c o n t r a c t i o n . The u s e f u l e f f e c t s o f t h e drugs i n c o n g e s t i v e h e a r t f a i l u r e - i n c r e a s e d c a r d i a c o u t p u t ; d e c r e a s e d h e a r t s i z e , venous p r e s s u r e and b l o o d volume; d i u r e s i s and r e l i e f o f edema - a r e a l l e x p l a i n e d on the b a s i s o f i n c r e a s e d c o n t r a c t i l e f o r c e , a p o s i t i v e i n o t r o p i c a c t i o n . As a r e s u l t o f t h e c l i n i c a l o b s e r v a t i o n s o f many workers i n the f i e l d (Wenckebach, 1910; P r a t t , 1918; C h r i s t i a n , 1919; L u t e u , 1924 e t c . ) i t has been e s t a b l i s h e d t h a t t h e d i g i t a l i s g l y c o s i d e s a r e e f f e c t i v e i n c o n g e s t i v e h e a r t f a i l u r e r e g a r d l e s s o f c a r d i a c rhythm, and t h a t they b r i n g r e l i e f not by v i r t u e o f c a r d i a c s l o w i n g b u t by t h e i r d i r e c t a c t i o n t o i n c r e a s e t h e . f o r c e o f m y o c a r d i a l c o n t r a c t i o n . I t has a l s o been p o i n t e d o u t t h a t c a r d i a c g l y c o s i d e s e x e r t t h e i r i n o t r o p i c s t i m u l a t i o n by i n c r e a s i n g the r a t e a t which t e n s i o n o r f o r c e i s d e v e l o p e d , and not by p r o l o n g i n g the d u r a t i o n o f the c o n t r a c t i l e p r o c e s s ( W a l l a c e e t a l . 1963). D i g i t a l i s g l y c o s i d e s reduce t h e v e n t r i -c u l a r r a t e i n a t r i a l f i b r i l l a t i o n t h r o u g h v a g a l and e x t r a v a g a l i n f l u e n c e s t h a t i n c r e a s e t h e e f f e c t i v e r e f r a c t o r y p e r i o d o f the A-V t r a n s m i s s i o n s ystem and t h r o u g h a v a g a l l y m e d i a t e d i n c r e a s e i n the a t r i a l f r e q u e n c y . Hence, the d e c r e a s e i n h e a r t r a t e upon d i g i t a l i z a t i o n , i s s e c o n d a r y t o the improvement o f t h e c i r c u l a t i o n , and i s not the p r i m a r y t h e r a p e u t i c a c t i o n o f t h e d r u g . Some e v i d e n c e s u g g e s t s t h a t " t h e r a p e u t i c " c o n c e n t r a t i o n s o f d i g i t a l i s p o t e n t i a t e t h e a c t i v i t y o f t h e Na-K-dependent ATPase enzyme system (Palmer e t a l . , 1966). A t p r e s e n t , c i r c u m s t a n t i a l e v i d e n c e seems t o i n d i c a t e t h a t d i g i t a l i s t o x i c i t y i s r e l a t e d t o i n h i b i t i o n o f ATP-ase (Moe and F a r a h , 1967). (a) Mechanism o f A c t i o n As s u g g e s t e d by Kahn (1963) and Page ( 1 9 6 4 ) , c a r d i a c g l y c o s i d e s may e x e r t a d i r e c t e f f e c t on the myocardium by c o n c e i v a b l y a c t i n g a t one o r more o f the f o l l o w i n g s i t e s o f t h e c a r d i a c m uscle: the c e l l membrane, the T s y s t e m , the s a r c o p l a s m i c r e t i c u l u m , d i r e c t l y on p r o t e i n s a s s o c i a t e d w i t h c o n t r a c t i l e s t r u c t u r e s , m i t o c h o n d r i a , the n u c l e u s , s o l u b l e enzymes and e v e n , p e r h a p s , unknown but i m p o r t a n t s t r u c t u r e s w i t h i n the c e l l . The u n ique c o m b i n a t i o n o f i n h i b i t i o n o f a t r i o v e n t r i c u l a r i m p u l s e t o the o t h e r w i s e s a l u t o r y i n o t r o p i c e f f e c t o f d i g o x i n (and o t h e r c a r d i a c g l y c o s i d e s ) s t i l l remains to be an u n f o r t u n a t e d i s a d v a n t a g e . (b) A b s o r p t i o n , Fate and E x c r e t i o n O r a l l y a d m i n i s t e r e d d i g o x i n i s a d e q u a t e l y a b s o r b e d from the i n t e s t i n a l t r a c t , even i n the' p r e s e n c e o f v a s c u l a r c o n g e s t i o n o f the e n t e r i c mucosa, h y p o x i a and d i a r r h e a - c o n d i t i o n s t h a t may e x i s t i n p a t i e n t s w i t h c a r d i a c f a i l u r e . By a c o mparison o f the o r a l and i n t r a v e n o u s d ose, d i g o x i n i s found t o be a b s o r b e d t o the e x t e n t o f f i f t y p er c e n t o r more; and g a s t r o -i n t e s t i n a l a b s o r p t i o n i s u s u a l l y c o m p l e t e d w i t h i n a p p r o x i m a t e l y two hours (Moe and F a r a h , 1967). M e t a b o l i c t r a n s f o r m a t i o n o f d i g o x i n o c c u r s c h i e f l y , b u t n o t s o l e l y i n t h e l i v e r (Abel e t a l . , 1965; Katzung and Meyers, 1966). And d i g o x i n i s s a i d t o be e x c r e t e d l a r g e l y i n unchanged form, c h i e f l y t h r o u g h the k i d n e y s ( A s h l e y e t a l . , 1958). I t i s g e n e r a l l y r e c o g n i z e d t h a t l i p i d s o l u b l e d i g i t o x i n i s 100% a b s o r b e d ( T a k a n a s h i e t a l . , 1978). In c o n t r a s t t o d i g o x i n , d i g i t o x i n i s bound t o serum a l b u m i n t o the e x t e n t o f o v e r 90% ( L u c a s and M a r t i n o , 1969). I t i s a l s o w i d e l y acknowledged t h a t o v e r 90% o f a b s o r b e d d i g i t o x i n i s m e t a b o l i z e d i n t h e l i v e r ( D o h e r t y , 1973) and e x c r e t e d m a i n l y by the k i d n e y s . Because o f e n t e r o h e p a t i c r e c i r c u l a t i o n , a p p r o x i m a t e l y 25% o f the m e t a b o l i c p r o d u c t s appear i n the s t o o l . The c o n v e r s i o n o f d i g i t o x i n i n t o w a t e r s o l u b l e compounds i n the l i v e r o c c u r s t h r o u g h two pathways ( D o h e r t y , 1973): (1) c o n v e r s i o n t o d i g o x i n by 1 2 6 - h y d r o x y l a t i o n and (2) s u g a r c l e a v a g e by h y d r o l y s i s f o l l o w e d by c o n j u g a t i o n r e a c t i o n s . (C) P h a r m a c o k i n e t i c s and Therapy P h a r m a c o k i n e t i c s t u d i e s w i t h d i g o x i n have shown t h a t i t s d i s t r i b u t i o n i s r a p i d and c o r r e s p o n d s t o an open two-compartment model (Nyberg e t a l . , 1974). Doherty (1968) r e p o r t e d d i g o x i n h a l f - l i v e s o f a p p r o x i m a t e l y 33 h o u r s . The r e s u l t s o f many e x p e r i m e n t s a g r e e s u f f i c i e n t l y t o i n d i c a t e a mean tj o f t h e t e r m i n a l s l o p e f o l l o w i n g i n t r a v e n o u s a d m i n i s t r a t i o n o f d i g o x i n o f 40-50 hours (Koup e t a l . , 1975; Kramer e t a l . , 1976). Some e v i d e n c e s u g g e s t s t h a t the p h a r m a c o k i n e t i c s o f d i g o x i n f o l l o w l i n e a r p r o c e s s e s ( O t t e n e t a l . , 1976; Bodem e t a l . , 1977). The t, o f u r i n a r y e x c r e t i o n o b t a i n e d from a m u l t i p l e dose s t u d y o f d i g o x i n was r e p o r t e d t o be 51.2 hours ( D e n g l e r e t a l . , 1978). The l a t t e r i n v e s t i -g a t o r s have r e p o r t e d a c l e a r a n c e o f 102.5 m i l l i l i t e r s per m i n u t e ; and a volume o f d i s t r i b u t i o n ( a t s t e a d y s t a t e ) o f 421 l i t e r s . I t has been r e p o r t e d t h a t d i g o x i n i s bound o n l y t o the e x t e n t o f 23 p e r c e n t ; and t h a t t h e p r o t e i n b i n d i n g o f the d i g o x i n m e t a b o l i t e s i s lower than d i g o x i n (Smith e t a l . , 1978). T h i s would, t h e n , mean t h a t the e f f e c t o f albumin c o n c e n t r a t i o n on d i g o x i n plasma l e v e l i s l e s s i m p o r t a n t than i n the c a s e o f d i g i t o x i n . D u r i n g m a intenance t h e r a p y , t h e d a y - t o - d a y v a r i a t i o n o f d i g o x i n b l o o d l e v e l s and o f u r i n a r y d i g o x i n e x c r e t i o n show f l u c t u a t i o n t o an e x t e n t t h a t can n o t be e x p l a i n e d by a n a l y t i c o r o t h e r e x p e r i m e n t a l e r r o r s . These a r e not c o n v i n c i n g l y e x p l a i n e d and need f u r t h e r i n v e s t i g a t i o n ( D e n g l e r e t a l . , 1978). Mean serum o r plasma d i g o x i n c o n c e n t r a t i o n s i n groups o f p a t i e n t s w i t h o u t e v i d e n c e o f t o x i c i t y a v e r a g e about 1.4 ng per m i l l i l i t e r ( f r o m d a t a i n v o l v i n g w e l l i n e x c e s s o f 1000 p a t i e n t s - Smith e t a l . , 1978). The above a u t h o r s r e p o r t t h a t mean serum c o n c e n t r a t i o n s t e n d t o be two t o t h r e e t i m e s h i g h e r i n p a t i e n t s w i t h c l i n i c a l e v i d e n c e o f d i g o x i n t o x i c i t y ; and the d i f f e r e n c e i n mean l e v e l s was s t a t i s t i c a l l y s i g n i f i c a n t i n t h e v a s t m a j o r i t y o f c a s e s . I t has a l s o been r e p o r t e d , however, t h a t o v e r l a p o f l e v e l s between groups w i t h and w i t h o u t e v i d e n c e o f t o x i c i t y was o b s e r v e d i n most s e r i e s and t h a t i t tends t o be more pronounced i n p r o s p e c t i v e , b l i n d s t u d i e s than i n r e t r o s p e c t i v e s t u d i e s ( B e l l e r e t a l . , 1971). S i n c e as much as 97% o f d i g i t o x i n i s bound t o plasma a l b u m i n (Lukas and M a r t i n o , 1969), i t s t o t a l plasma c o n c e n t r a t i o n i s from 15 t o 20 times h i g h e r than t h a t o f d i g o x i n a t comparable t h e r a p e u t i c l e v e l s ( S m i t h and Haber, 1973). D i g i t o x i n has a t h e r a p e u t i c plasma c o n c e n t r a t i o n o f 14-26 ng/ml and i s u s u a l l y r e p o r t e d t o have a plasma h a l f - l i f e o f 5-7 d a y s . T o t a l d i g i t a l i z i n g o r a l dose f o r d i g o x i n and d i g i t o x i n i s u s u a l l y i n the range o f 2-3 mg and 1.2-1.6 mg, r e s p e c t i v e l y . And the d a i l y o r a l maintenance dose range f o r d i g o x i n and d i g i t o x i n i s 0.25-0.75 mg and 0.05-0.2 mg, r e s p e c t i v e l y . A c c o r d i n g t o the e x p e r i e n c e o f many i n v e s t i g a t o r s i n the f i e l d , i t i s a p p a r e n t t h a t no serum c o n c e n t r a t i o n o f d i g o x i n and d i g i t o x i n can be s e l e c t e d t h a t c l e a r l y s e p a r a t e s t o x i c and n o n - t o x i c s t a t e s i n the u s u a l c l i n i c a l s e t t i n g (Smith e t a l . , 1978). Hence, judgement o f o p t i m a l doses and serum c o n c e n t r a t i o n s must be b a s e d , t o a l a r g e e x t e n t , on a s s e s s m e n t o f each i n d i v i d u a l c l i n i c a l r e s p o n s e . A c c o r d i n g t o Smith e t a l . ( 1 9 7 8 ) , the f o l l o w i n g f a c t o r s may i n f l u e n c e i n d i v i d u a l s e n s i t i v i t y t o c a r d i a c g l y c o s i d e s : ( i ) Type and s e v e r i t y o f u n d e r l y i n g c a r d i a c d i s e a s e , ( i i ) Serum e l e c t r o l y t e derangements - h y p o k a l e m i a and h y p e r k a l e m i a - hypomagnesemia - h y p e r c a l e m i a - h y p o n a t r e m i a ( i i i ) A c i d - b a s e imbalance ( i v ) C o n c o m i t a n t drug a d m i n i s t r a t i o n - a n a e s t h e t i c s - c a t e c h o l a m i n e s and sympathomimetics - a n t i a r r h y t h m i c a g e n t s (v) T h y r o i d s t a t u s ( v i ) Renal f u n c t i o n ( v i i ) Autonomic nervous system tone ( v i i i ) R e s p i r a t o r y d i s e a s e 56. 3. Development o f Methods o f A n a l y s i s o f D i g o x i n and D i g i t o x i n (A) A n a l y s i s o f Samples i n P l a n t E x t r a c t s , S t a n d a r d M i x t u r e s and Dosage  Forms As f a r back as t h e n i n e t e e n t h c e n t u r y , a c o l o r i m e t r i c method was p u b l i s h e d by L a f o n (1885) who used equal amounts o f s u l p h u r i c a c i d and e t h a n o l w i t h the a d d i t i o n o f f e r r i c c h l o r i d e . Amongst t h e e a r l i e s t c h e m i c a l methods o f a n a l y s i s o f d i g o x i n t h a t s u b s e q u e n t l y appeared were p r o b a b l y t h o s e o f B a l j e t ( 1 9 1 8 ) , Morel (1935) and Warren e t a l . ( 1 9 4 8 ) , i n which a l k a l i n e p i c r a t e , m - d i n i t r o b e n z e n e and 2 - n a p h t h o q u i n o n e - 4 - s u l p h o n a t e were used as r e a g e n t s , r e s p e c t i v e l y . V a r i o u s o t h e r c o l o r i m e t r i c a s s a y s f o r d i g o x i n have a l s o been r e p o r t e d . 1,3,5-t r i n i t r o b e n z e n e i n a l k a l i n e medium was used as r e a g e n t by Kimura ( 1 9 5 1 ) ; X a n t h y d r o l by Pesez (1952); a c e t o n e - p h o s p h o r i c a c i d by Dequeker and Loobuyck (1955); 3 , 5 - d i n i t r o b e n z o i c a c i d by T a t t j e (1957); 2 , 4 - d i n i t r o -d i p h e n y l s u i f o n e by T a t t j e ( 1 958); m - d i n i t r o b e n z e n e by Houk e t a l . (1959); and t h i o b a r b i t u r i c a c i d by Mesnard and Devaux (1961). A l o n g w i t h the development o f c o l o r i m e t r i c p r o c e d u r e s , f l u o r o m e t r i c methods o f a n a l y s i s were r e p o r t e d by P e t i t e t a l . (1 950) and Jensen (1 952) who used s y r u p y p h o s p h o r i c a c i d and h y d r o c h l o r i c a c i d - g l y c e r o l ( 1 : 1 ) , as r e a g e n t s , r e s p e c t i v e l y . Hydrogen p e r o x i d e , h y d r o c h l o r i c a c i d and methanol were used as r e a g e n t s by Jensen (1953) and W e l l s e t a l . ( 1 9 6 1 ) . T a t t j e (1954) used a m i x t u r e o f s u l p h u r i c and p h o s p h o r i c a c i d s w i t h the a d d i t i o n o f f e r r i c c h l o r i d e . The f l u o r o p h o r o b t a i n e d w i t h a m i x t u r e o f a c e t i c a n h y d r i d e , a c e t y l c h l o r i d e and t r i f l u o r o a c e t i c a c i d has been r e p o r t e d by J a k o v l j e v i c ( 1 9 6 3 ) , t o g i v e a low y i e l d o f a h i g h l y c o n j u g a t e d compound o f s u b s t i t u t e d , 3,4-benzpyrene. 57. An automated method based on a c i d i n d u c e d f l u o r e s c e n c e was proposed by Khoury (1967). A n o t h e r automated f l u o r o g e n i c p r o c e d u r e u s i n g a s t a n d a r d T e c h n i c o n a u t o m a t i c a n a l y z e r system f o r the u n i t dose a n a l y s i s o f d i g o x i n and d i g i t o x i n i n t a b l e t s was r e p o r t e d by C u l l i n e t a l . ( 1 9 7 0 ) . Meanwhile, the development o f c h r o m a t o g r a p h i c methods o f a n a l y s i s has t r e m e n d o u s l y f a c i l i t a t e d the q u a l i t a t i v e and q u a n t i t a t i v e d e t e r m i n a -t i o n s o f c a r d i a c g l y c o s i d e s . And chromatography was l a t e r t o be r e c o g n i z e d as the most v a l u a b l e t o o l i n the r e s o l u t i o n and q u a n t i t a t i v e e v a l u a t i o n o f c a r d i a c g l y c o s i d e m i x t u r e s . The method o f Z a f f a r o n i e t a l . (1949) f o r the s e p a r a t i o n o f s t e r o i d a l m i x t u r e s on f i l t e r paper impregnated w i t h formamide o r e t h y l e n e g l y c o l was used by R e i c h s t e i n and S c h i n d l e r (1951) f o r the chromatography o f D i g i t a l i s g l y c o s i d e s . Many i n v e s t i g a t o r s ( J e n s e n , 1956; T a n t i v a t a n a and W r i g h t , 1958; P o t t e r , 1963; Wolf and K a r a c s o n y , 1 963; and Dzyuba e t a l . , 1971) have s u b s e q u e n t l y used paper chromatography t o d e t e r m i n e q u a n t i t a t i v e l y d i g i t a l i s g l y c o s i d e s by e l u t i o n o f the r e s o l v e d compounds f o l l o w e d by v a r i o u s c o l o r i m e t r i c a s s a y p r o c e d u r e s . R a b i t z s c h e t a l . (1969) have r e p o r t e d a b u t e n o l i d e r i n g s p e c i f i c method o f q u a n t i t a t i v e paper c h r o m a t o g r a p h i c a n a l y s i s o f c a r d i a c g l y c o s i d e s u s i n g 2 , 4 , 2 ' , 4 ' - t e t r a n i t r o d i p h e n y l as r e a g e n t . T h i n - l a y e r chromatography on s i l i c a gel was used by S t a h l and m i x t u r e s . T h i n - l a y e r c h r o m a t o g r a p h i c s e p a r a t i o n s have a l s o been r e p o r t e d by Heusser (1 965); Hauser e t a l . (1 968) and P o t t e r e t a l . (1 972) i n which the s e p a r a t e d components were e l u t e d and q u a n t i t a t e d u s i n g c o l o r i m e t r i c methods. Evans e t a l . (1974) r e s o l v e d t h e components o f e x t r a c t s o f D i g i t a l i s p u r p u r e a by h y d r o l y s i n g the g l y c o s i d e s t o t h e p a r e n t a g l y c o n e s , K a l t e n b a c h (1961) i n o r d e r c a r d i a c g l y c o s i d e 7 < E 4 ^ An automated method based on a c i d i n d u c e d f l u o r e s c e n c e was proposed by Khoury (1967). A n o t h e r automated f l u o r o g e n i c p r o c e d u r e u s i n g a s t a n d a r d T e c h n i c o n a u t o m a t i c a n a l y z e r system f o r the u n i t dose a n a l y s i s o f d i g o x i n and d i g i t o x i n i n t a b l e t s was r e p o r t e d by C u l l i n e t a l . ( 1 9 7 0 ) . Meanwhile, the development o f c h r o m a t o g r a p h i c methods o f a n a l y s i s has t r e m e n d o u s l y f a c i l i t a t e d t he q u a l i t a t i v e and q u a n t i t a t i v e d e t e r m i n a -t i o n s o f c a r d i a c g l y c o s i d e s . And chromatography was l a t e r t o be r e c o g n i z e d as the most v a l u a b l e t o o l i n t h e r e s o l u t i o n and q u a n t i t a t i v e e v a l u a t i o n o f c a r d i a c g l y c o s i d e m i x t u r e s . The method o f Z a f f a r o n i e t a l . (1949) f o r the s e p a r a t i o n o f s t e r o i d a l m i x t u r e s on f i l t e r paper i m p r e g n a t e d w i t h formamide o r e t h y l e n e g l y c o l was used by R e i c h s t e i n and S c h i n d l e r (1951) f o r the chromatography o f D i g i t a l i s g l y c o s i d e s . Many i n v e s t i g a t o r s ( J e n s e n , 1956; T a n t i v a t a n a and W r i g h t , 1958; P o t t e r , 1963; Wolf and K a r a c s o n y , 1 963 ; and Dzyuba e t a l . , 1971) have s u b s e q u e n t l y used paper chromatography to d e t e r m i n e q u a n t i t a t i v e l y d i g i t a l i s g l y c o s i d e s by e l u t i o n o f t h e , r e s o l v e d compounds f o l l o w e d by v a r i o u s c o l o r i m e t r i c a s s a y p r o c e d u r e s . R a b i t z s c h e t a l . (1969) have r e p o r t e d a b u t e n o l i d e r i n g s p e c i f i c method o f q u a n t i t a t i v e paper c h r o m a t o g r a p h i c a n a l y s i s o f c a r d i a c g l y c o s i d e s u s i n g 2 , 4 , 2 ' , 4 ' - t e t r a n i t r o d i p h e n y l as r e a g e n t . T h i n - l a y e r chromatography on s i l i c a gel was used by S t a h l and K a l t e n b a c h (1961) i n o r d e r t o s e p a r a t e low l o a d i n g s o f c a r d i a c g l y c o s i d e m i x t u r e s . T h i n - l a y e r c h r o m a t o g r a p h i c s e p a r a t i o n s have a l s o been r e p o r t e d by Heusser ( 1 9 6 5 ) ; Hauser e t a l . (1968) and P o t t e r e t a l . (1 972) i n which the s e p a r a t e d components were e l u t e d and q u a n t i t a t e d u s i n g c o l o r i m e t r i c methods. Evans e t a l . (1974) r e s o l v e d t h e components o f e x t r a c t s o f D i g i t a l i s p u r p u r e a by h y d r o l y s i n g the g l y c o s i d e s t o t h e p a r e n t a g l y c o n e s , which t h e y then measured by d e n s i t o m e t r y o f t h i n - l a y e r chromatograms. Hoeke e t a l . (1969) r e p o r t e d a r a p i d s e p a r a t i o n o f d i g o x i n from d i g i -t o x i n and a c e t y l d i g i t o x i n u s i n g k i e s e l g e l G p l a t e s , a s o l v e n t system o f c h l o r o f o r m / m e t h a n o l (9:1) and h y d r o c h l o r i c a c i d as a d e t e c t i n g a g e n t . S e p a r a t i o n o f d i g o x i n from d i g i t o x i n by the use o f s i l i c a Gel G p l a t e s , f o l l o w e d by d e t e c t i o n w i t h one per c e n t i o d i n e i n c h l o r o f o r m , e x t r a c t i o n and s p e c t r o p h o t o m e t r y q u a n t i t a t i o n a f t e r t r e a t m e n t w i t h d i x a n t h y l u r e a r e a g e n t was d e s c r i b e d b y . B i c a n - F i s t e r e t a l . (1969) and J o h n s t o n e t a l . ( 1 9 6 6 ) . O t h e r TLC methods i n v o l v i n g t h e f o r m a t i o n o f c o l o r e d d e r i v a t i v e s have been r e p o r t e d by B e l l and K r a n t z (1948); Jensen (1973); Zurkowska and Ozarowski (1964); M y r i c k (1969); and W i c h t l and D e x l e r (1966). C a r v a l h a s and F i g u e i r a (1973) d e s c r i b e d a c o m p a r a t i v e s t u d y o f t h i n -l a y e r c h r o m a t o g r a p h i c t e c h n i q u e s ( v a r i o u s s o l v e n t systems) f o r s e p a r a t i o n o f d i g o x i n , d i g i t o x i n and t h e i r main m e t a b o l i t e s . A s p e c t r o f l u o r o m e t r i c method f o r the d i r e c t q u a n t i t a t i v e e v a l u a t i o n o f d i g o x i n , d i g i t o x i n and a c e t y l d i g i t o x i n on TLC u s i n g h y d r o c h l o r i c a c i d as t h e f l u o r o g e n i c r e a g e n t was r e p o r t e d by F r i j n s (1970). The s e n s i t i v i t y m entioned was about 0.25 meg.. S e p a r a t i o n o f d i g o x i n and d i g i t o x i n from t h e i r 2 0 , 2 2 - d i h y d r o d e r i v a -t i v e s , by m u l t i p l e TLC on c e l l u l o s e f i l m s was a c c o m p l i s h e d by R a b i t z s c h ( 1 9 6 8 ) . E a r l i e r , J e l l i f f e e t a l . (1 967) had d e s c r i b e d an u l t r a m i c r o -f l u o r e s c e n t s p r a y r e a g e n t f o r d e t e c t i o n and q u a n t i t a t i o n o f d i g i t o x i n and o t h e r c a r d i a c g l y c o s i d e s on TLC. T h e i r r e a g e n t c o n s i s t e d o f a s c o r b i c a c i d , m e t h a n o l , h y d r o c h l o r i c a c i d and hydrogen p e r o x i d e . They c l a i m e d a l i m i t o f d e t e c t i o n o f 0.01 meg.. Jensen ( 1 9 5 4 ) ; W e l l s e t a l . (1961); Lugt (1973); and B r i t t e n and Njau (1975) have r e p o r t e d o t h e r TLC methods i n v o l v i n g t h e f o r m a t i o n o f f l u o r e s c i n g p r o d u c t s o f c a r d i a c g l y c o s i d e s . Sabatka e t a l . (1 976) d e s c r i b e d t he s e p a r a t i o n o f d i g o x i n and d i h y d r o -d i g o x i n by t h i n - l a y e r and paper chromatography and t h e i r subsequent q u a n t i -t a t i o n a f t e r f l u o r o g e n i c d e r i v a t i z a t i o n . They r e p o r t e d l i m i t s o f d e t e c t i o n o f one nanogram and t e n nanograms f o r d i g o x i n and d i h y d r o d i g o x i n , r e s p e c -t i v e l y . A t w o - d i m e n s i o n a l t h i n - l a y e r chromatography o f d i g i t a l i s c a r d e n -o l i d e s u s i n g a c o n t i n u o u s development t e c h n i q u e has r e c e n t l y been r e p o r t e d by C l a r k e and Cobb (1 979). G a s - L i q u i d c h r o m a t o g r a p h i c methods o f s e p a r a t i n g s i m p l e m i x t u r e s o f c a r d i a c g l y c o s i d e s a f t e r f o r m i n g t r i m e t h y l s i l y l d e r i v a t i v e s were f i r s t r e p o r t e d by J e l l i f e and B l a n k e n h o r n (1963) and s u b s e q u e n t l y , by W i l s o n e t a l . (1967) and W i l s o n e t a l . ( 1 9 6 9 ) . Tan (1969) d e v e l o p e d a gas c h r o m a t o g r a p h i c method o f i d e n t i f i c a t i o n o f d i g i t a l i s c a r d e n o l i d e s as t h e i r anhydro d e r i v a t i v e s . He p r e s e n t e d s p e c t r a l e v i d e n c e showing t h a t t he t e r t i a r y 14 3-OH group i s n e i t h e r a f f e c t e d by e s t e r i f i c a t i o n nor e t h e r i f i c a t i o n . B h a n d a r i and Walker (1969) have used g a s - l i q u i d chromatography q u a l i t a t i v e l y as a means o f i d e n t i f y i n g c a r d i a c g l y c o s i d e s . Watson e t a l . (1972) have d e s c r i b e d i d e n t i f i c a t i o n o f submicrogram amounts o f d i g o x i n , d i g i t o x i n and t h e i r m e t a b o l i c p r o d u c t s by GLC-ECD a n a l y s i s o f g e n i n - d i h e p t a f l u o r o b u t y r a t e (-HFB) d e r i v a t e s a f t e r s e p a r a t i o n by paper c hromatography. They r e p o r t e d a s e n s i t i v i t y o f 25 pg. f o r both d i g o x i g e n i n - H F B and d i g i t o x i g e n i n - H F B . A r a p i d q u a n t i t a t i v e a n a l y s i s o f d i g o x i n was r e p o r t e d by Kibbe and A r a u j o ( 1 9 7 3 ) , by u s i n g g a s - l i q u i d chromatography w i t h a flame i o n i z a t i o n d e t e c t o r . They used no d e r i v a t i -z a t i o n and c l a i m e d a s e n s i t i v i t y o f about 1 mg. per m i l l i l i t e r . I d e n t i f i c a t i o n by g as-chromatography - mass s p e c t r o s c o p y o f d i h y d r o -d i g o x i n - a m e t a b o l i t e o f d i g o x i n i n man, has been r e p o r t e d by Watson et a l . ( 1 9 7 3 ) . S t r o l l e t a l . (1951) used t h e d i f f e r e n c e s i n p o l a r i t y o f the c a r d i a c g l y c o s i d e s t o s e p a r a t e them on s i l i c a - g e l columns, and column chromato-graphy has been used f o r t h e e n r i c h m e n t o f c a r d i a c g l y c o s i d e e x t r a c t s p r i o r t o t h e i r e v a l u a t i o n by paper chromatography ( K a i s e r , 1966) o r t h i n - l a y e r chromatography (Hauser e t a l . , 1 969). The a d v e n t o f h i g h - p e r f o r m a n c e l i q u i d chromatography (HPLC) has i n t r o d u c e d a p o w e r f u l t e c h n i q u e f o r the r e s o l u t i o n o f complex m i x t u r e s o f compounds. Evans (1974) has used i o n - e x c h a n g e HPLC i n o r d e r t o s e p a r a t e t h e components o f the "A" s e r i e s c a r d e n o l i d e s , and L o t s h e r e t a l . (1 975) have d e s c r i b e d t h e use o f r e v e r s e d phases f o r the s e p a r a t i o n o f a number o f c a r d i a c g l y c o s i d e s and a g l y c o n e s . C a s t l e (1975) has r e p o r t e d t h e use o f HPLC f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f low l e v e l s o f d i g o x i n , d i g i t o x i n and t h e i r m e t a b o l i t e s . He d e s c r i b e d : ( l ) i s o c r a t i c s e p a r a t i o n o f (a) d i g o x i g e n i n , d i g o x i g e n i n m o n o - d i g i t o x o s i d e , d i g o x i g e n i n b i s - d i g i t o x o s i d e and d i g o x i n ; (b) d i g i -t o x i g e n i n , d i g i t o x i g e n i n m o n o - d i g i t o x o s i d e , d i g i t o x i g e n i n b i s - d i g i t o x o s i d e and d i g i t o x i n ; (2) g r a d i e n t e l u t i o n s e p a r a t i o n o f (a) d i g o x i n , d i g i t o x i n and t h e i r m e t a b o l i t e s and (b) g i t o x i n from d i g o x i n and i t s m e t a b o l i t e s . He u t i l i z e d a v a r i a b l e w a v e l e n g t h d e t e c t o r s e t a t 220 nanometers and a r e v e r s e phase column w i t h v a r i o u s m i x t u r e s o f a c e t o n i t r i l e and water as t h e m o b i l e phase. T h i s paper i n d i c a t e s a minimum d e t e c t a b l e amount o f d i g o x i n o f 39 ng ( i s o c r a t i c c o n d i t i o n s ) and 14 ng ( g r a d i e n t c o n d i t i o n s ) a t a s i g n a l t o n o i s e r a t i o o f two and a t t e n u a t i o n o f 0.01 a . u . f . s . . T o t a l s e p a r a t i o n i s c l a i m e d t o be complete i n l e s s than t h i r t y m i n u t e s . L i n d n e r and F r e i (1976) have d e s c r i b e d p a r t i t i o n h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c systems f o r t h e s e p a r a t i o n o f d i g i t a l i s g l y c o s i d e s o f the c a r d e n o l i d e group on s i l i c a g e l . They used n o n - p o l a r s o l v e n t systems o f v a r i a b l e c o m p o s i t i o n and an u l t r a - v i o l e t d e t e c t o r s e t a t 220 nm. A s e n s i t i t i v y o f about 15 ng and a s e p a r a t i o n time o f f o u r t o twenty minutes have been r e p o r t e d t h e r e i n . A h i g h - r e s o l u t i o n p r o c e d u r e f o r d i g i t a l i s g l y c o s i d e a n a l y s i s by d e r i v a t i z a t i o n ( w i t h 4 - n i t r o b e n z o y l c h l o r i d e ) l i q u i d chromatography was p u b l i s h e d by Nachtman e t a l . ( 1 9 7 6 ) , u s i n g normal phase column, and non-p o l a r s o l v e n t systems o f v a r y i n g c o m p o s i t i o n s . The s e p a r a t e d compounds were g l y c o s i d e s and a g l y c o n e s o f the A, B and C c a r d e n o l i d e s e r i e s . The r e p o r t e d minimum q u a n t i f i a b l e amounts o f d i g o x i n and d i g i t o x i n were 11.0 ng p e r m i l l i l i t e r and 11.1 ng per m i l l i l i t e r , r e s p e c t i v e l y . S e p a r a t i o n o f d i g o x i n and i t s m e t a b o l i t e s u s i n g Sephadex LH-20 column chromatography was r e p o r t e d by G a u l t e t a l . ( 1 9 7 6 ) . Cobb (1976) r e p o r t e d normal phase HPLC s e p a r a t i o n s o f (1) d i g i t o x i g e n i n g i t o x i g e n i n and d i g o x i g e n i n ; (2) d i g i t o x i n , g i t o x i n d i g o x i n , d i g i n a t i n and g i t a l o x i n ( C a r d e n o l i d e s s e r i e s A, B, C, D and E, r e s p e c t i v e l y ) ; (3) d i g o x i g e n i n , d i g o x i g e n i n m o n o - d i g i t o x o s i d e , d i g o x i g e n i n b i s - d i g i t o x o s i d e and d i g o x i n ; and (4) B - a c e t y l d i g o x i n , a - a c e t y l d i g o x i n and d i g o x i n . Q u a n t i t a t i o n was c a r r i e d o u t by u s i n g s u i p h a m e t h o x a z o l e as an i n t e r n a l s t a n d a r d and a U V - d e t e c t o r s e t a t 265 nm. and 234 nm. A comparison o f r e v e r s e d - p h a s e and p a r t i t i o n h i g h - p e r f o r m a n c e l i q u i d c hromatography o f some d i g i t a l i s g l y c o s i d e s was p u b l i s h e d by E r n i e and F r e i ( 1 9 7 7 ) . They used 3.7% and 8.0% methanol i n methylene c h l o r i d e s a t u r -a t e d w i t h w a t e r ( f o r normal phase HPLC) and 37% a c e t o n i t r i l e i n w a ter ( f o r r e v e r s e phase HPLC) as s o l v e n t s y s t e m s . The minimum q u a n t i f i a b l e amount a t the d e t e c t i o n w a v e l e n g t h o f 220 nm. was r e p o r t e d to be w i t h i n the range o f 10 t o 100 ng. G f e l l e r e t a l . (1977) r e p o r t e d a p r o c e d u r e f o r p o s t column d e r i v a t i -z a t i o n i n h i g h p e r formance l i q u i d chromatography u s i n g the a i r s e g m e n t a t i o n p r i n c i p l e . They d e s c r i b e d s e p a r a t i o n s o f d i g o x i n , d i g i t o x i g e n i n , l a n a t o -s i d e C and d e s a c e t y l 1 a n a t o s i d e C on a r e v e r s e phase column. Minimum d e t e c t i o n l i m i t s o f 0.5 ng ( f o r d e s a c e t y l l a n a t o s i d e C, a t a s i g n a l t o n o i s e r a t i o o f 4:1) were r e p o r t e d . The above workers have a l s o i n d i c a t e d t h a t t h e i r f l u o r o m e t r i c p r o c e d u r e i s a t l e a s t a hundred t i m e s more s e n s i t i v e than UV d e t e c t i o n w i t h o u t d e r i v a t i z a t i o n . In a s t u d y o f the k i n e t i c s o f d i g o x i n s t a b i l i t y i n aqueous s o l u t i o n , S t e r n s o n and S h a f f e r (1978) have used r e v e r s e phase HPLC w i t h UV d e t e c t i o n a t 225 nm and a s o l v e n t system o f m e t h a n o l / w a t e r (55/45) f o r t h e s e p a r a t i o n o f d i g o x i n and i t s d e g r a d a t i o n p r o d u c t s . The g e n i n and t h e m o n o d i g i t o x o s i d e , however, were n o t c o m p l e t e l y s e p a r a t e d . The p r a c t i c a l d e t e c t i o n l i m i t f o r d i g o x i n was r e p o r t e d t o be 25 ng. F u g i i e t a l . (1980) have d e s c r i b e d the s e p a r a t i o n o f d i g i t a l i s g l y c o s i d e s by m i c r o h i g h - p e r f o r m a n c e l i q u i d chromatography (MHPLC). The above a u t h o r s have r e p o r t e d the r e s o l u t i o n o f : (1) a m i x t u r e o f d i g i t o x i n , g i t o x i n , d i g o x i n , l a n a t o s i d e A and l a n a t o s i d e B; (2) d i g i t o x i n and i t s m e t a b o l i t e s ; (3) g i t o x i n and i t s m e t a b o l i t e s and (4) d i g o x i n and i t s m e t a b o l i t e s u s i n g a r e v e r s e - p h a s e column (16.5 cm x 0.5 mm i . d . ) , UV d e t e c t i o n a t 220 nm and s o l v e n t s y s t e m c o n s i s t i n g o f a c e t o n i t r i l e / m e t h a n o l / w a t e r and v a r i o u s m i x t u r e s o f methanol and w a t e r . S e p a r a t i o n times were w i t h i n 30 t o 45 m i n u t e s . In a s t u d y o f d i g o x i n d e g r a d a t i o n i n a c i d i c d i s s o l u t i o n medium, Sonobe e t a l . (1980) have used a r e v e r s e - p h a s e column (5 um, 15 cm x 4 mm i . d . 63. a s o l v e n t s y s t e m o f 25% a c e t o n i t r i l e and UV d e t e c t i o n a t 220 nm f o r the HPLC s e p a r a t i o n o f d i g o x i n and i t s d e g r a d a t i o n p r o d u c t s . The d e t e c t i o n l i m i t f o r d i g o x i n was r e p o r t e d t o be 50 ng. (B) A n a l y s i s o f Samples i n B i o l o g i c a l F l u i d s (a) Methods The i n t r o d u c t i o n o f the b i o a s s a y o f d i g i t a l i s answered the need f o r a p r e p a r a t i o n o f known po t e n c y f o r c l i n i c a l u s e . T h i s t e c h n i q u e was r e f i n e d i n i t s u l t i m a t e f o r m by Friedman e t a l . (1947) i n an embryonic duck h e a r t p r e p a r a t i o n . The e a r l i e s t a p p r o a c h e s to the a n a l y s i s o f c a r d i a c g l y c o s i d e s from b i o l o g i c a l f l u i d s i n v o l v e d c o l o r i m e t r i c d e t e r m i n a t i o n s a f t e r s e p a r a t i o n by paper chromatography (Brown e t a l . , 1957; A s h l e y e t a l . , 1958). In 14 3 t h e p a s t decade, t h e e x t e n s i v e use o f r a d i o l a b e l e d C and H - c a r d i a c g l y c o s i d e s has y i e l d e d b a s i c i n f o r m a t i o n as w e l l as d a t a o f c l i n i c a l i m p o r t a n c e ( D o h e r t y , 1961). M e t a b o l i c p r o d u c t s have been g e n e r a l l y i d e n t i f i e d by c o mparison w i t h Rf v a l u e s o f a u t h e n t i c compounds a f t e r s e p a r a t i o n by such t e c h n i q u e s as p a p e r , column and TLC ( D o h e r t y and P e r k i n s , 1962; Marcus e t a l ., 1966). Most o f t h e e x p e r i m e n t a l s t u d i e s on t h e m e t a b o l i s m o f c a r d i a c g l y c o -s i d e s have been c a r r i e d o u t w i t h l a b e l l e d compounds u s i n g TLC ( G r i f f i n e t a l . , 1971; Kolenda e t a l . , 1971; Stohs e t a l . , 1971; V o i g t l a n d e r , 1972; and Beerman, 1972). Faber (1977) r e p o r t e d q u a n t i t a t i o n o f c a r d i a c g l y c o -s i d e s w i t h h i g h - p e r f o r m a n c e t h i n - l a y e r chromatography u t i l i z i n g programmed m u l t i p l e development w i t h h i g h - p e r f o r m a n c e m i c r o - t h i n - l a y e r m a t e r i a l . The a u t h o r c l a i m s t h a t t h i s method i s b e t t e r than p r e v i o u s TLC t e c h n i q u e s . The u n d e s i r a b i l i t y o f u s i n g r a d i o a c t i v e t r a c e r s r o u t i n e l y i n human s u b j e c t s c o u p l e d w i t h t h e low r e s o l u t i o n o f t h e above mentioned chroma-t o g r a p h i c t e c h n i q u e s n e c e s s i t a t e d t h e development o f a l t e r n a t i v e methods o f a n a l y s i s o f m e t a b o l i t e s o f c a r d i a c g l y c o s i d e s . The need f o r a c c u r a t e c h a r a c t e r i z a t i o n s o f t h e p h a r m a c o k i n e t i c p arameters o f d i g o x i n has a l s o g i v e n a d d i t i o n a l impetus f o r the s e a r c h f o r b e t t e r a n a l y t i c a l t e c h n i q u e s . The c u r r e n t a n a l y t i c a l methods f o r the a n a l y s i s o f d i g o x i n and d i g i -t o x i n ( t h e two w i d e l y used c a r d e n o l i d e s ) i n plasma a r e : (1) i n h i b i t i o n o f 86 Rb t r a n s p o r t by the r e d b l o o d c e l l ( L o w e n s t e i n and C o r i l l , 1966); (2) e n z y m a t i c i s o t o p e d i s p l a c e m e n t ( B r o o k e r and J e l l i f e , 1969); and (3) radioimmunoassay ( 0 1 i v e r e t a l . , 1968; Smith e t a l . , 1969). The n o n - s e l e c t i v i t y o f t h e above methods, however, has r e n d e r e d them u n f i t f o r t h e q u a n t i t a t i o n o f m e t a b o l i c p r o d u c t s . Watson e t a l . (1973) r e p o r t e d a gas c hromatographic-mass s p e c t r o -s c o p i c a n a l y s i s o f d i h y d r o d i g o x i n - a m e t a b o l i t e o f d i g o x i n i n man. A f t e r e x t r a c t i o n from u r i n e and plasma, d i h y d r o d i g o x i n and d i g o x i n were d e r i v a t i z e d w i t h h e p t a f l u o r o b u t y r i c a n h y d r i d e t o form the b i s h e p t a f l u o r o b u t y r a t e e s t e r s . The d e r i v a t i v e s c o u l d then be used f o r i d e n t i f i c a t i o n by gas-chromatography - m a s s - s p e c t r o m e t r y o r q u a n t i t a t e d a t the sub-nanogram l e v e l by gas chroma-t o g r a p h y w i t h e l e c t r o n c a p t u r e d e t e c t i o n . B o g u s l a s k i and Schwartz (1975) d e s c r i b e d a column radioimmunoassay method f o r t h e d e t e r m i n a t i o n o f d i g i t o x i n i n which they used a column o f i m m o b i l i z e d a n t i b o d y which a c t e d as both r e a c t i o n chamber and s e p a r a t i o n d e v i c e . They r e p o r t e d a s e n s i t i v i t y o f about 150 pg.. Sun and S p i e h l e r (1976) compared radioimmunoassay (RIA) and enzyme immunoassay f o r d e t e r m i n a t i o n o f d i g o x i n and s u g g e s t e d t h a t the l a t t e r method c o u l d g i v e comparable r e s u l t s . Loo e t a l . (1977) used normal phase HPLC ( L i c h r o s o r b S i 6 0 ) f o r t h e s e p a r a t i o n o f d i g o x i n and i t s main m e t a b o l i t e s ( D i g o x i g e n i n and t h e mono-and b i s - d i g i t o x o s i d e s ) u t i l i z i n g a four-component n o n - p o l a r s o l v e n t system. The r e p o r t e d d i g o x i g e n i n q u a n t i t a t i o n was c a r r i e d o u t by radioimmunoassay i n which t h e d e t e c t i o n l i m i t was i n d i c a t e d t o be 0.15 ng per m i l l i l i t e r . T h i s t w o - step HPLC-RIA c o m b i n a t i o n o f s e p a r a t i o n and d e t e c t i o n o f d i g o x i n and i t s m e t a b o l i t e s from b i o l o g i c a l f l u i d s , a p p e a r s to be the f i n a l a n a l y t i c s t a t e o f the a r t f o r the p r e s e n t t i m e . An emergency ( f a s t ) p r o c e d u r e f o r d i g o x i n r a d i o a s s a y was r e p o r t e d by Chen e t a l . (1978) i n w hich t h e y c l a i m t h a t the c o n d i t i o n s f o r a n t i g e n -a n t i b o d y i n t e r a c t i o n and f o r s e p a r a t i o n o f bound and f r e e f r a c t i o n s have been o p t i m i z e d . Comparisons ( B e r g d a h l e t a l ., 1979; Kubasik e t a l ., 1979; and e v a l u a t i o n s o f v a r i o u s k i t s f o r plasma d i g o x i n radioimmunoassay (RIA) and improvements o f RIA methodology (O'Leary e t a l . , 1979; H a l p e r n and B o r d e n s , 1979; W e i l e r and Z e n i k , 1979) have r e c e n t l y been r e p o r t e d . (b) R a t i o n a l e f o r Measurement o f Serum D i g o x i n and D i g i t o x i n The j u s t i f i c a t i o n f o r the a n a l y s i s o f d i g o x i n and d i g i t o x i n i n b i o l o g i c a l f l u i d s i s d e r i v e d from a c o n s i d e r a t i o n o f the f o l l o w i n g : ( i ) Both i n o t r o p i c and t o x i c e f f e c t s o f c a r d i a c g l y c o s i d e s a r e known to be d o s e - r e l a t e d phenomena. A l a r g e number o f s t u d i e s have shown i n c r e a s i n g serum d i g i t a l i s c o n c e n -t r a t i o n s w i t h i n c r e a s i n g dosage (Smith and Haber, 1973) so t h a t a t l e a s t a s t a t i s t i c a l c o r r e l a t i o n s h o u l d e x i s t between plasma l e v e l s and c l i n i c a l s t a t e . 66. ( i i ) A number o f i n v e s t i g a t o r s have documented a r e l a t i v e l y c o n s t a n t r a t i o o f d i g o x i n c o n c e n t r a t i o n i n serum o r plasma t o t h a t i n t h e myocardium, both i n e x p e r i m e n t a l animal s t u d i e s and human s u b j e c t s ( D o h e r t y e t a l . , 1967; G u l n e r e t a l . , 1974; H a r t e l e t a l . , 1976). ( i i i ) E v i d e n c e c o n t i n u e s t o a c c u m u l a t e i n d i c a t i n g t h a t ( N a + + K + ) -ATP-ase i s i n v o l v e d i n a t l e a s t some o f the a c t i o n s o f c a r d i a c g l y c o s i d e s ( S c h w a r t z , 1976). T h i s plasma membrane enzyme t r a n s p o r t system i s i n f l u e n c e d by c a r d i a c g l y c o s i d e s o n l y when t h e s e a g e n t s a r e p r e s e n t a t the o u t e r c e l l s u r f a c e ( C a l d w e l l and Keynes, 1959; Hoffman, 1966). Thus the p r e s e n c e o f a t l e a s t one c a r d i a c g l y c o s i d e r e c e p t o r i n c l o s e p r o x i m i t y t o the e x t r a c e l l u l a r compartment, p r o v i d e s a b a s i s f o r t h e t r a n s i t i o n o f plasma c o n c e n t r a t i o n t o m y o c a r d i a l e f f e c t . 67. 4. C l a s s i f i c a t i o n o f A n a l y t i c a l Methods o f D i g o x i n and D i g i t o x i n (A) C o l o r i m e t r i c Methods The methods f o r t h e c o l o r i m e t r i c d e t e r m i n a t i o n o f d i g o x i n (and o t h e r g l y c o s i d e s ) can be d i v i d e d i n t o : (a) methods based on t h e s u g a r m o i e t y , i n which a wide v a r i e t y o f r e a g e n t s a r e used t o r e a c t w i t h t h e d i g i t o x o s e s u g a r t o g i v e a c o l o u r e d d e r i v a t i v e ( e . g . t h e f e r r i c c h l o r i d e methods o f the B.P. and U.S.P.). (b) methods based on the b u t e n o l i d e ( c a r d e n o l i d e ) m o i e t y , i n which r e a g e n t s a r e r e a c t e d w i t h the c a r d e n o l i d e p o r t i o n r e s u l t i n g i n a c o l o r e d d e r i v a t i v e ( e . g . t h e p i c r a t e method o f t h e I n t e r n a t i o n a l P h a r m a c o p o e i a ) . (B) F l u o r o m e t r i c Methods These methods a r e based upon the r e a c t i o n i n the s t e r o i d m o i e t y , and a r e m a i n l y d e h y d r a t i o n type o f r e a c t i o n s t h a t g i v e r i s e to h i g h l y c o n j u g a t e d p r o d u c t s . A number o f r e a g e n t s have been d e v e l o p e d f o r t h i s t e c h n i q u e . (C) C h r o m a t o g r a p h i c Methods Pap e r , t h i n - l a y e r and column chromatography have been w i d e l y used f o r t h e s e p a r a t i o n o f v a r i o u s c a r d i a c g l y c o s i d e s and t h e i r a g l y c o n e s p r i o r t o q u a n t i t a t i o n by c o l o r i m e t r i c o r f l u o r o m e t r i c methods. HPLC and g a s - l i q u i d c h r o m a t o g r a p h i c t e c h n i q u e s have both been u t i l i z e d f o r the s e p a r a t i o n and q u a n t i t a t i o n o f c a r d i a c g l y c o s i d e s . 68. (D) B i o c h e m i c a l Methods ( i ) Radioimmunoassay By v i r t u e o f i t s h i g h s e n s i t i v i t y , t h i s t e c h n i q u e ranks h i g h e s t i n the f r e q u e n c y o f usage f o r the a n a l y s i s o f c a r d i a c g l y c o s i d e s i n b i o l o g i c a l f l u i d s . The b a s i s o f the a n a l y s i s may be s c h e m a t i c a l l y d e p i c t e d as shown i n F i g . 17. Unbound d i g o x i n i s a d s o r b e d onto d e x t r a n - c o a t e d c h a r c o a l * and the d i g o x i n - a n t i b o d y complex i n the s u p e r n a t a n t p o r t i o n i s d e t e r m i n e d u s i n g a l i q u i d s c i n t i l l a t i o n c o u n t e r . oc ( i i ) I n h i b i t i o n o f Rb t r a n s p o r t ( u p t a k e ) by the r e d b l o o d c e l l T h i s method i n v o l v e s e x t r a c t i o n o f the g l y c o s i d e from plasma w i t h methylene d i c h l o r i d e ( d i c h l o r o m e t h a n e ) , e v a p o r a t i o n t o d r y n e s s , ^ c o n s t i -t u t i o n w i t h normal s a l i n e ( c o n t a i n i n g 85 mg. per c e n t g l u c o s e ) , a d d i t i o n o f 8 6 R b and i n c u b a t i o n w i t h r e d c e l l s a t 37°C f o r 2 h o u r s . The 8 6 R b uptake i s then measured i n a s c i n t i l l a t i o n c o u n t e r . The e s s e n t i a l s o f t h i s t e c h n i q u e a r e s c h e m a t i c a l l y shown i n F i g . 18. A s e n s i t i v i t y o f l e s s than 1 ng has been r e p o r t e d ( L o w e n s t e i n and C o r r i l , 1966). ( i i i ) E n z y m a t i c i s o t o p e d i s p l a c e m e n t Serum samples are p r e p a r e d by methanol e x t r a c t i o n , Zn-Ba p r e c i p i t a t i o n and s i l i c a g e l column chromatography. A f t e r i n c u b a t i o n w i t h enzyme, the m i x t u r e i s c e n t r i f u g e d and the unbound s u p e r n a t a n t r a d i o a c t i v e d i g o x i n i s c o u n t e d . A s e n s i t i v i t y o f 0.15 ng has been r e p o r t e d ( B r o o k e r and J e l l i f e , 1969). The p r o c e d u r e i s s c h e m a t i c a l l y p r e s e n t e d i n F i g . 19. ( i v ) Double i s o t o p e - d i l u t i o n d e r i v a t i v e a s s a y T h i s method i n v o l v e s the f o l l o w i n g s t e p s : (a) a d d i t i o n o f t r i t i u m - l a b e l e d d i g i t o x i n t o plasma o r u r i n e sample 69. Serum D i g o x i n D i g o x i n * ( R a d i o l a b e l e d ) A n t i b o d y D i g o x i n * + D i g o x i n + A n t i b o d y D i g o x i n * - A n t i b o d y + D i g o x i n - A n t i b o d y F i g . 17. Diagrammatic R e p r e s e n t a t i o n o f the P r o c e d u r e f o r Radioimmunoassay o f D i g i t a l i s G l y c o s i d e s . 70. Serum Red Digoxin C e l l s i n c u b a t i o n w i t h 86 Rb (glucose i n serum) 86 Rb - Red C e l l - r r F i g . 18. Diagrammatic R e p r e s e n t a t i o n o f t h e A s s a y o f D i g o x i n by I n h i b i t i o n o f °^Rb T r a n s p o r t by t h e Red Blo o d C e l l . Na-K ATP-ase Digox i n * + Digoxin ATP (Mg, Na) bound D i g o x i n * + bound Digoxin + |unbound Digo x i n * F i g . 19. Diagrammatic R e p r e s e n t a t i o n o f the A s s a y o f D i g o x i n by Enzymatic I s o t o p e D i s p l a c e m e n t . (b) e x t r a c t i o n and p r e l i m i n a r y p u r i f i c a t i o n o f d i g i t o x i n by l i q u i d - l i q u i d p a r t i t i o n and paper chromatography 14 (c) a c e t y l a t i o n w i t h a c e t i c a n h y d r i d e - 1 - C (d) i s o l a t i o n and p u r i f i c a t i o n o f d i g i t o x i n t r i a c e t a t e by f o u r d i f f e r e n t p a p er chromatography systems 14 (e) measurement o f t r i t i u m and C a c t i v i t y by l i q u i d s c i n t i l l a -t i o n s p e c t r o m e t r y . A s e n s i t i v i t y o f l e s s than 1 ng has been r e p o r t e d (Lucas and P e t e r s o n , 1966). 5. L i m i t a t i o n s o f t h e A n a l y t i c a l Methods (A) S e n s i t i v i t y I f 0.75 mg o f d i g o x i n i s g i v e n o r a l l y ( t h e u s u a l l y recommended l o a d i n g dose) as one b o l u s o r i n d i v i d e d doses o f 0.5 mg and 0.25 mg a d m i n i s t e r e d 4-6 hours a p a r t , t h e r e would p r o b a b l y be d i g o x i n l e v e l s o f a b o u t 1 ng per m i l l i l i t e r , a t the end o f 24 h o u r s , i n most p a t i e n t s (Smith e t a l . ( 1 9 7 8 ) ) . The u s u a l t h e r a p e u t i c range f o r serum d i g o x i n c o n c e n t r a t i o n s i s 0.7 t o 1.5 ng per m i l l i l i t e r (Smith e t a l . , 1969). I t i s t h e r e f o r e e v i d e n t t h a t (1) m o n i t o r i n g serum d i g o x i n l e v e l s f o r o p t i m i z i n g d i g o x i n t h e r a p y and (2) p h a r m a c o k i n e t i c s t u d i e s o f d i g o x i n r e q u i r e a n a l y t i c t e c h n i q u e s t h a t can p r o v i d e a s e n s i t i v i t y o f a t l e a s t 0.5 ng per m i l l i l i t e r . Minimum l i m i t s o f d e t e c t i o n o f d i g o x i n o f : (1) 10 ng, by t h i n - l a y e r chromatography ( J e l l i f f e e t _ a l _ . , 1967); (2) 25 pg., by g a s - l i q u i d chroma-t o g r a p h y - e l e c t r o n c a p t u r e d e t e c t i o n o f t h e h e p t a f l u o r o b u t y r a t e d e r i v a -t i v e ( K i b b e and A r a u j o , 1973); (3) 11 ng per ml., by h i g h - p e r f o r m a n c e l i q u i d chromatography - a f t e r d e r i v a t i z a t i o n w i t h 4 - n i t r o b e n z o y l c h l o r i d e (Nachtmann e t a l . , 1976); (4) 0.2 ng per m i l l i l i t e r , by radioimmunoassay (Smith e t a l . , 1969) have so f a r been r e p o r t e d i n the l i t e r a t u r e . Of the two methods t h a t have s e n s i t i v i t i e s which a r e w i t h i n the d e s i r a b l e plasma d i g o x i n c o n c e n t r a t i o n r a n g e , t h e GLC-ECD method i n v o l v e s c o n v e r s i o n o f d i g o x i n t o d i g o x i g e n i n - d i h e p t a f l u o r o b u t y r a t e and hence r e q u i r e s a p r e l i m i n a r y s e p a r a t o r y s t e p , i n the p r e s e n c e o f m e t a b o l i t e s . The post-column d e r i v a t i z a t i o n - H P L C method o f G f e l l e r e t a l . (1977) has been r e p o r t e d t o have a s e n s i t i v i t y ( a l b e i t , f o r d e s a c e t y l l a n a t o s i d e C, which has a m o l e c u l a r w e i g h t h i g h e r than t h a t o f d i g o x i n ) o f 0.5 ng. I t a p p e a r s , however, t h a t o p t i m i z a t i o n o f t h e c o n d i t i o n s o f t h i s f l u o r o -m e t r i c method may p r o v i d e comparable s e n s i t i v i t y f o r d i g o x i n . Radioimmunoassay i s the most s e n s i t i v e t e c h n i q u e and i s , t h e r e f o r e , the o n l y method t h a t i s w i d e l y used f o r t h e a n a l y s i s o f d i g o x i n and d i g i t o x i n i n b i o l o g i c a l f l u i d s . (B) S e l e c t i v i t y D i g o x i n and i t s m e t a b o l i t e s a r e n o r m a l l y e x c r e t e d a l m o s t e n t i r e l y i n t h e u r i n e , w i t h o n l y a s m a l l p e r c e n t a g e r e p o r t e d t o be c o n v e r t e d t o m e t a b o l i t e s . These i n c l u d e d i g o x i g e n i n and i t s mono and b i s - d i g i t o x o s i d e s and d i h y d r o d i g o x i n (Marcus e t a l . , 1964; L u c h i and G r u b e r , 1968; Doherty e t a l . , 1971; C l a r k and Kalman, 1974). However, t h e r e a r e r e p o r t s s u g g e s t i n g t h a t d i g o x i n m e t a b o l i s m can be c l i n i c a l l y i m p o r t a n t , and i n two p a t i e n t s , 57 p e r c e n t ( L u c h i and G r u b e r , 1968) and 60 per c e n t ( C l a r k and Kalman, 1974) o f i n g e s t e d d i g o x i n a r e b e l i e v e d t o have been e x c r e t e d as m e t a b o l i t e s . T h e r e i s a l s o l i m i t e d e v i d e n c e i n a n i m a l s ( K o l e n d a e t a l . , 1971) and i n man (Marcus e t a l . , 1964; C l a r k and Kalman, 1974; Beerman e t a l . , 1972) f o r c o n v e r s i o n o f some d i g o x i n t o water s o l u b l e m e t a b o l i t e s such as g l u c u r o n i d e s . M e t a b o l i s m o f d i g i t o x i n i s known t o be much more e x t e n s i v e ( D o h e r t y , 1973). Hence, i t i s a g a i n e v i d e n t t h a t m o n i t o r i n g serum d i g o x i n and d i g i t o x i n l e v e l s r e q u i r e s an a n a l y t i c a l method t h a t can s e l e c t i v e l y a c c o u n t f o r t h e drugs and each o f t h e i r m e t a b o l i t e s . The i n h e r e n t s e p a r a t o r y f u n c t i o n o f t h e c h r o m a t o g r a p h i c methods makes them u n i q u e l y s e l e c t i v e . The h i g h - r e s o l u t i o n c a p a b i l i t y o f h i g h -p e r f o r m a n c e l i q u i d chromatography does, i n d e e d , make i t s u p e r b l y s u i t a b l e f o r s e l e c t i v e m o n i t o r i n g o f d i g o x i n , d i g i t o x i n and t h e i r m e t a b o l i t e s . N e v e r t h e l e s s , t h e c o u p l i n g o f t h i s s e l e c t i v i t y w i t h t h e d e s i r a b l e s e n s i t i -v i t y i s y e t t o be s e e n . On the o t h e r hand, t h e e x c l u s i v e r o l e o f radioimmunoassay's h i g h s e n s i t i v i t y a p p e a r s t o be t a r n i s h e d by i t s i n h e r e n t l a c k o f s e l e c t i v i t y . D i g o x i n radioimmunoassay (RIA) has been r e p o r t e d t o show c r o s s - r e a c t i o n w i t h d i g i t o x i n ( B o i n k e t a l . , 1977; Zeegers e t a l . , 1973), S p i r o n o l a c t o n e ( B o i n k e t a l . , 1977; M a r i s s 1979) and d i h y d r o d i g o x i n (Kramer e t a l . , 1976). A number o f o t h e r i n v e s t i g a t o r s have a l s o r e p o r t e d v a r i o u s f a c t o r s a f f e c t i n g t he r e s u l t s o f d i g o x i n radioimmunoassay. Some o f t h e s e f a c t o r s a r e : (a) q u e n c h i n g ( C e r c e o and E l l o s o , 1972); (b) c h e m i l u m i n i s c e n c e o f serum ( B u t l e r , 1971); ( c ) low i n t r i n s i c a s s o c i a t i o n c o n s t a n t o f the a n t i b o d y - a n t i g e n complex ( S m i t h and Haber, 1973); and (d) e f f e c t s o f a l b u m i n on t h i s complex ( V o s h a l l e t a l . , 1975). Holtzman e t a l . (1974) have r e p o r t e d t h a t t h e r e i s a group o f p a t i e n t s f o r whom the d e t e r m i n a t i o n o f serum d i g o x i n c o n c e n t r a t i o n s (by RIA) w i l l g i v e an e r r o n e o u s l y low v a l u e . In the c l i n i c a l a p p l i c a t i o n o f the RIA method, i t has been i n d i c a t e d t h a t t h e r e i s a s u b s t a n t i a l o v e r l a p i n the d i g o x i n l e v e l s o b s e r v e d f o r t h e i n t o x i c a t e d and n o n - i n t o x i c a t e d p a t i e n t s ( S m i t h e t a l . , 1970). S e v e r a l workers have s u g g e s t e d t h a t some o f t h e s e o v e r l a p s c o u l d be a r e s u l t o f the a s s a y i t s e l f not g i v i n g the t r u e v a l u e f o r the serum c o n c e n t r a t i o n s (Anggard e t a l . , 1972; Fogelman e t a l . , 1971; B u r n e t t e t a l . , 1973). K l i n k e t a l . (1974) have shown t h a t i f aqueous t e s t samples (su c h as u r i n e o r a l c o h o l i c s o l u t i o n s ) and the samples used f o r c o n s t r u c t i n g t h e s t a n d a r d c u r v e do not c o n t a i n e q u i v a l e n t amounts o f serum and p r o t e i n , t h e q u a n t i t a t i v e r e s u l t s so d e r i v e d a r e s u b j e c t t o c o n s i d e r a b l e e r r o r . B o i n k e t a l . (1977) have d e s c r i b e d some f a c t o r s a f f e c t i n g a commercial k i t f o r radioimmunoassay o f d i g o x i n : (1) e r r o n e o u s l y h i g h a s s a y v a l u e s i n serum o f (a) p r e g n a n t women, (b) p a t i e n t s t a k i n g s p i r o n o -l a c t o n e and ( c ) t h o s e samples t h a t c o n t a i n e t h a n o l ; (2) t oo low a s s a y v a l u e s i f haemacel i s p r e s e n t i n t h e sample. (C) Time By v i r t u e o f t h e i r s e l e c t i v i t y o r s e n s i t i v i t y , t he most u s e f u l methods o f a n a l y s i s o f d i g o x i n a r e chromatography and radioimmunoassay. The q u a n t i t a t i v e g a s - c h r o m a t o g r a p h i c methods o f a n a l y s i s r e p o r t e d r e l y on t h e p r o d u c t i o n o f d e r i v a t i v e s and r e q u i r e much time as w e l l as l a b o r i o u s m a n i p u l a t i v e s t e p s . For i n s t a n c e , Watson and Kalman's (1971) g a s - c h r o m a t o g r a p h i c method o f d i g o x i n a s s a y from plasma t a k e s f i v e h o u r s . D i g o x i n and d i g i t o x i n radioimmunoassay have become r o u t i n e l y a v a i l a b l e methods because o f the r e c e n t development o f many commercial r a d i o a s s a y k i t s . U n f o r t u n a t e l y , however, use o f most o f t h e s e k i t s r e q u i r e s two t o t h r e e h ours from r e c e i p t o f specimen t o c o m p l e t i o n o f the r e p o r t (Chen e t a l . , 1978). 76. I I . EXPERIMENTAL 1. A p p a r a t u s (a) Beckman Model 322 G r a d i e n t L i q u i d Chromatograph e q u i p p e d w i t h : - modular d u a l pump syste m (Model 100A and 11 OA pumps) - m i c r o p r o c e s s o r system c o n t r o l l e r (Model 420) - d y n a m i c a l l y s t i r r e d g r a d i e n t m i x i n g chamber - Model 210 sample i n j e c t i o n v a l v e (20 y l l o o p ) - Model 100-10 v a r i a b l e w a v e l e n g t h UV d e t e c t o r - Shimadzu Chromatopac e l e c t r o n i c d a t a p r o c e s s o r (Model C-R1A) - U l t r a s p h e r e 0DS (C-18) r e v e r s e - p h a s e column (25 cm x 4.6 mm i . d . ) w i t h a p a r t i c l e s i z e o f 5 ym. (b) Waters L i q u i d Chromatograph e q u i p p e d w i t h : - Model 6000 s o l v e n t d e l i v e r y system - Model 450 v a r i a b l e w a v e l e n g t h d e t e c t o r - U6K i n j e c t o r - O m i n i s c r i b e r e c o r d e r (Model B-5000) - S p h e r i s o r b 0DS column (25 cm x 3.2 mm i . d . ) w i t h a p a r t i c l e s i z e o f 10 ym. (c) A l t e x H i g h - p e r f o r m a n c e L i q u i d Chromatograph e q u i p p e d w i t h : - Model 110 pump - 20 y l l o o p i n j e c t i o n v a l v e - Model 153 UV d e t e c t o r - U l t r a s p h e r e 0DS (C-18) r e v e r s e - p h a s e column (25 cm x 4.6 mm i . d . ) w i t h a p a r t i c l e s i z e o f 5 ym - O m n i s c r i b e Model B-5000 dual pen r e c o r d e r P o s t - c o l u m n F l u o r o g e n i c D e r i v a t i z a t i o n Set-up c o n s i s t i n g o f t h e f o l l o w i n g : - T e c h n i c o n p e r i s t a l t i c pump - S o l v a f l e x pump t u b e s ( T e c h n i c o n c o l o r c o d e s , o r a n g e / g r e e n and o r a n g e / b l u e ) - S o l v a f l e x d e l i v e r y tube (1 mm i . d . ) - A c i d f l e x pump tubes ( T e c h n i c o n c o l o r code, w h i t e ) - A c i d f l e x d e l i v e r y tube (1 mm i . d . ) - T e c h n i c o n D2 c o n n e c t o r s - M o d i f i e d T e c h n i c o n C4 d e b u b b l e r w i t h s i d e tube o f 1 mm i . d . - M i x i n g c o i l s (120 x 0.1 cm i . d . and 240 x 0.1 cm i . d . ) - C o o l i n g c o i l (180 x 0.1 cm i . d . ) - R e a c t i o n c o i l (1000 x 0.1 cm i . d . ) - Haake T h e r m o r e g u l a t o r (Type E51) - Waters Model 420 f l u o r e s c e n c e d e t e c t o r Accumet Model 220 pH Meter ( F i s h e r ) T h e l c o oven ( G C A / P r e c i s i o n S c i e n t i f i c ) L a b - L i n e oven ( L a b - L i n e I n s t r u m e n t s Inc.) F l a s h E v a p o r a t o r ( B u c h l e r I n s t r u m e n t s ) Cahn E l e c t r o b a l a n c e ( V e n t r o n I n s t r u m e n t s Corp.) ( j ) S p e c t r o p h o t o m e t e r s : - Beckman Models 24 and 25 - Beckman IR-10 (k) Mass s p e c t r o m e t e r (MAT-111) 2. M a t e r i a l s (a) C a r d i a c g l y c o s i d e s and a g l y c o n e s : - D i g o x i n - D i g i t o x i n - D i g o x i g e n i n - D i g o x i g e n i n m o n o d i g i t o x o s i d e - D i g o x i g e n i n b i s d i g i t o x o s i d e - D i g i t o x i g e n i n x - D i g i t o x i g e n i n m o n o d i g i t o x o s i d e - D i g i t o x i g e n i n b i s d i g i t o x o s i d e - a - a c e t y l d i g o x i n - B - a c e t y l d i g o x i n - G i t o x i n - D i h y d r o d i g o x i g e n i n A l l o f t h e above items were o f A n a l y t i c a l Grade and were o b t a i n e d f r o m B o e h r i n g e r Mannheim C o r p . , Mannheim, G.F.R. D i g o x i n and d i g i t o x i n were i d e n t i f i e d by i n f r a r e d s p e c t r o p h o t o m e t r y and USP XX methods. The c h e m i c a l s t r u c t u r e s o f t h e g l y c o s i d e s and a g l y c o n e s a re shown i n T a b l e VI The c h e m i c a l s t r u c t u r e o f d i h y d r o d i g o x i g e n i n i n comparison w i t h t h a t o f d i g o x i n i s p r e s e n t e d i n F i g . 20. TABLE V I I . CHEMICAL STRUCTURES OF SOME CARDIAC GLYCOSIDES OF THE CARDENOLIDE SERIES O ^ O ( B a s i c C a r d e n o l i d e S t r u c t u r e of C a r d i a c g l y c o s i d e s ) C a r d e n o l i d e S e r i e s Compound B C H H II H H OH OK OH OH OH H H H H OH H H H H H H D i g i t o x i g e n i n D D i g i t o x i g e n i n m o n o d i g i t o x o s i d e D-D D i g i t o x i g e n i n b i s d i g i t o x o s i d e D-D-D D i g i t o x i n D-D-D G i t o x i n D i g o x i g e n i n H D D-D D i g o x i g e n i n m o n o d i g i t o x o s i d e D i g o x i g e n i n b i s d i g i t o x o s i d e D-D-D D i g o x i n •DJ <4 - A c e t j (cy j£- A c e t y l d i g o x i n D-D- ) «< e t y l d i g o x i n / c D= DIGITOXOSE A s A C E T Y L Fig. 20. Chemical Structuresof Digoxin and Dihydrodigoxigenin. (b) 1 7 a - e t h y n y l e s t r a d i o l , S i g m a Chemical Co., A n a l y t i c a l Grade (c) 1 7 a - m e t h y l t e s t o s t e r o n e , Sigma Chemical Co., A n a l y t i c a l Grade (d) H y d r o c o r t i s o n e , S e l - W i n C h e m i c a l s L t d . , A n a l y t i c a l Grade (e) A s c o r b i c a c i d , B r i t i s h Drug Houses (Canada) L t d . , A n a l y t i c a l Grade ( f ) Hydrogen p e r o x i d e , 30%, B r i t i s h Drug Houses (Canada) L t d . , A n a l y t i c a l Grade (g) H y d r o c h l o r i c a c i d , American S c i e n t i f i c and Chemical Co., Reagent Grade (h) B r i j 35, A t l a s Chemical I n d u s t r i e s Canada L t d . ( i ) E t h a n o l , Stanchem (Canada) L t d . , Reagent Grade ( j ) n - p r o p y l a l c o h o l , C a l e d o n L a b o r a t o r i e s L t d . , Reagent Grade (k) C h l o r o f o r m , C a l e d o n L a b o r a t o r i e s L t d . , Reagent Grade (1) S u l f u r i c a c i d , American S c i e n t i f i c and Chemical Co., Reagent Grade (m) Phosphorus p e n t o x i d e , B r i t i s h Drug Houses (Canada) L t d . , Reagent Grade (n) F e r r i c C h l o r i d e , The N i c h o l s Chemical Co. L t d . , Reagent Grade (o) Sodium C a r b o n a t e , Anachemia C h e m i c a l s L t d . , Reagent Grade (p) Formamide, M a l l i n c k r o d t Chemical Works, Reagent Grade (q) C h r o m a t o g r a p h i c S i l i c e o u s E a r t h ( C e l i t e 5 4 5 ), S u p e l c o , I n c . , A n a l y t i c a l Grade ( 82. ( r ) Benzene, C a l e d o n L a b o r a t o r i e s L t d . , Reagent Grade ( s ) P i c r i c a c i d , Matheson Coleman and B e l l Co., Reagent Grade ( t ) Whatman No. 1 f i l t e r p a per, W. and R. B a l s t o n L t d . (u) HPLC s o l v e n t s : - Methanol - D i c h l o r o m e t h a n e - I s o p r o p a n o l - Water The above s o l v e n t s were o f HPLC Grade and were p u r c h a s e d from F i s h e r S c i e n t i f i c Co., P i t t s b u r g h , PA., U.S.A. (v) HPLC s o l v e n t s ystems: - w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 4 7 / 4 0 / 9 / 4 45/37/12/6 43/35/15/7 51/42/5/2 49/41/7/3 41/34/17/8 38/32/20/10 46/39/10/5 45/38/11/6 48/40/8/4 52/43/3/2 51/43/4/2 H II II n II H H H n II II H H II H II II n n it n i i II n II II H n II II H II II II II n n II II II n n H II (w) E q u i n e e s t r o g e n s : - E s t r o n e - E q u i l i n - E q u i l e n i n - 1 7 a - e s t r a d i o l - 17e-estradi.ol - 1 7 a - d i h y d r o e q u i 1 i n - 1 7 3-dihydroequi 1 i n - 1 7 a - d i h y d r o e q u i l e n i n - 1 7 3 - d i h y d r o e q u i l e n i n The above e s t r o g e n s were o f A n a l y t i c a l Grade and were o b t a i n e d from D i v i s i o n o f A y e r s t McKenna and H a r r i s o n L t d . , M o n t r e a l , Quebec. T h e i r c h e m i c a l s t r u c t u r e s a r e g i v e n i n F i g . 21. (x) Dosage Forms: D - L a n o x i n t a b l e t s , 0.125 mg p - L a n o x i n t a b l e t s , 0.25 mg D - L a n o x i n i n j e c t i o n , 0.05 mg/ml p - L a n o x i n i n j e c t i o n , 0.25 mg/ml D - L a n o x i n E l i x i r , 0.05 mg/ml The above dosage forms were p u r c h a s e d from Wellcome M e d i c a l D i v i s i o n , Burroughs Wellcome I n c . - N a t i g o x i n t a b l e t s , 0.25 mg, Compagne Pharmacentique V i t a (LTEE) p - D i g i t o x i n ^ t a b l e t s . 0 . 1 mg, Wyeth L t d . (Canada) - C r y s t o d i g i n i n j e c t i o n , 0.2 mg/ml, E l i L i l l y and Company (y) D i g i t a l i s p u r p u r e a l e a v e s , o b t a i n e d from Dr. A. Goodeve's p r i v a t e garden i n the Vancouver a r e a . F i g . 21. Chemical S t r u c t u r e s o f Equine E s t r o g e n s . I , II = 173-and a - d i h y d r o e q u i l e n i n ; I I I , IV = 176- and a - d i h y d r o -e q u i l i n ; V, VI = 176- and a - e s t r a d i o l ; VII = e q u i l e n i n ; V I I I = e q u i l i n ; IX = e s t r o n e . 3. P r e p a r a t i o n o f HPLC S o l v e n t Systems F r e s h l y p r e p a r e 500 ml o f the HPLC s o l v e n t systems by m i x i n g a c c u r a t e l y measured p r o p o r t i o n s (as shown i n t h e r e s p e c t i v e chromatograms) o f w a t e r , m e t h a n o l , i s o p r o p a n o l and d i c h l o r o m e t h a n e . 4. E q u i l i b r a t i o n o f t h e HPLC Column E q u i l i b r a t e the HPLC column by pumping the f r e s h l y p r e p a r e d s o l v e n t system f o r about one hour. M o n i t o r t h e s t a b i l i t y o f t h e b a s e l i n e u s i n g t h e a p p r o p r i a t e d e t e c t o r and r e c o r d e r . 5. D e t e r m i n a t i o n o f R e t e n t i o n Time I n j e c t a sample o f a s o l u t i o n o f each compound i n t o the L i q u i d Chromatograph and r e c o r d t h e r e t e n t i o n t i m e . Repeat t h e i n j e c t i o n t h r e e t i m e s and c a l c u l a t e t h e a v e r a g e r e t e n t i o n t i m e . 6. S e p a r a t i o n o f D i g o x i n , D i g i t o x i n and t h e i r M e t a b o l i t e s o r D e g r a d a t i o n  P r o d u c t s and I m p u r i t i e s (A) I s o c r a t i c E l u t i o n E q u i l i b r a t e the column w i t h the s o l v e n t system. D i s s o l v e a m i x t u r e o f t h e compounds i n the e l u t i n g s o l v e n t system and i n j e c t a sample i n t o t h e L i q u i d Chromatograph which has been a d j u s t e d p r e v i o u s l y t o c o n d i t i o n s o f f l o w r a t e o f 1.2 ml/min; UV d e t e c t i o n a t 220 nm, r a n g e o f 0.02; and a c h a r t speed o f 0.5 cm/min. Record t h e chromatogram. S o l v e n t S w i t c h o v e r E l u t i o n D i s s o l v e a m i x t u r e o f t h e compounds i n t h e i n i t i a l s o l v e n t system ( w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 49/41/7/3) and i n j e c t a sample i n t o t he L i q u i d Chromatograph which has p r e v i o u s l y been a d j u s t e d t o c o n d i t i o n s o f : f l o w r a t e o f 1.2 ml/min; UV d e t e c t i o n a t 220 nm, range o f 0.1; and a c h a r t speed o f 0.5 cm/min. S w i t c h o v e r t o a second s o l v e n t system ( w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 41/38/17/8) a t a p o i n t i n t i m e c o r r e s p o n d i n g t o t h e peak o f d i g o x i -g e n i n b i s d i g i t o x o s i d e . Record t h e chromatogram. A t the end o f the c h r o m a t o g r a p h i c r u n , r e - e q u i l i b r a t e t h e column u s i n g the i n i t i a l s o l v e n t s y s t e m . G r a d i e n t E l u t i o n E q u i l i b r a t e t h e column w i t h the i n i t i a l s o l v e n t system ( w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 49/41/7/3). D i s s o l v e the m i x t u r e o f the compounds i n the i n i t i a l s o l v e n t system and i n j e c t a sample i n t o th e G r a d i e n t L i q u i d Chromatograph which has been a d j u s t e d t o t h e f o l l o w i n g c o n d i t i o n s : f l o w r a t e o f 1.2 ml/min; UV d e t e c t i o n a t 220 nm, range o f 0.1; a c h a r t speed o f 0.5 cm/min; and a l i n e a r g r a d i e n t o f 0 t o 100% o f t h e second s o l v e n t system ( w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 38/32/20/10) programmed between t h e c h r o m a t o g r a p h i c times o f 2.5 and 3 m i n u t e s . Record t h e chromatogram. R e - e q u i l i b r a t e t h e column u s i n g t h e i n i t i a l s o l v e n t system. The c h e m i c a l s t r u c t u r e s o f t h e g l y c o s i d e s and a g l y c o n e s t h a t were employed i n t h i s s t u d y a r e p r e s e n t e d i n T a b l e V I I . 87. 7. S e p a r a t i o n o f D i g o x i n and i t s M e t a b o l i t e s a f t e r Fluorogem'c P o s t - Column D e r i v a t i z a t i o n u s i n g t h e A i r - S e g m e n t a t i o n P r i n c i p l e w i t h  100% F l u i d R e c o v e r y (A) P r e p a r a t i o n o f Hydrogen P e r o x i d e S o l u t i o n Add 1 ml o f hydrogen p e r o x i d e s o l u t i o n (30% v/v) t o 200 ml o f water and mix. (B) P r e p a r a t i o n o f D e h y d r o a s c o r b i c A c i d S o l u t i o n Weigh 100 mg o f a s c o r b i c a c i d and d i s s o l v e i n 200 ml o f w a t e r . Add 5 ml o f t h e hydrogen p e r o x i d e s o l u t i o n d r o p w i s e w i t h c o n t i n u o u s s t i r r i n g . (C) P r e p a r a t i o n o f B r i 3 5 S o l u t i o n R Weigh 1 g o f B r i j 35 and add t o 99 g o f water and mix. (D) Dual D e t e c t o r M o n i t o r i n g o f t h e S e p a r a t i o n o f D i g o x i n and i t s Meta-b o l i t e s b e f o r e and a f t e r F l u o r o g e n i c D e r i v a t i z a t i o n . P o s i t i o n s o l v a f l e x o r a n g e - g r e e n and o r a n g e - b l u e and a c i d f l e x w h i t e pump tubes on t h e T e c h n i c o n pump to d e l i v e r a i r , d e h y d r o a s c o r b i c a c i d s o l u t i o n and h y d r o c h l o r i c a c i d , r e s p e c t i v e l y . Segment the stream o f h y d r o c h l o r i c a c i d by c o n n e c t i n g t h e a c i d and a i r d e l i v e r y tubes w i t h a m i n i a t u r i z e d D2 c o n n e c t o r ( w i t h s i d e arms o f 1 mm i . d . ) . Connect t h e tubes d e l i v e r i n g t h e a i r - s e g m e n t e d h y d r o c h l o r i c a c i d and the HPLC column e f f l u e n t t h a t has passed t h r o u g h a UV d e t e c t o r , u s i n g a m i n i a -t u r i z e d D2 c o n n e c t o r and pass t h e m i x t u r e t h r o u g h a m i x i n g c o i l (120 x 0.1 cm i . d . ) . Connect t h e d e l i v e r y tube l e a d i n g o u t o f the f i r s t m i x i n g c o i l t o t h e d e h y d r o s c o r b i c a c i d s o l u t i o n d e l i v e r y tube u s i n g a m i n i a t u r i z e d D 2 c o n n e c t o r and shunt t h e m i x t u r e i n t o a second m i x i n g c o i l (240 x 0.1 cm i . d . ) . Pass t h e segmented and mixed s o l u t i o n t h r o u g h a r e a c t i o n c o i l (1000 x 0.1 cm i . d . ; 5 5 ° C ) , c o o l i n g c o i l (180 x 0.1 cm i . d . ) , d e b u b b l i n g , s e t - u p , f l u o r e s c e n t d e t e c t o r b e f o r e f i n a l l y d i r e c t i n g i t i n t o a waste r e c e p t a c l e . Assemble the d e b u b b l i n g s e t - u p f o r 100% f l u i d r e c o v e r y as f o l l o w s : U s i n g a c i d f l e x t u b i n g c o n n e c t t h e v e r t i c a l e x i t o f t h e d e b u b b l e r t o t h e t a p e r i n g end (1 mm i . d . ) o f a g l a s s tube (84 x 1.1 cm i . d . ) . P o s i t i o n t h e g l a s s tube as h i g h as p o s s i b l e so t h a t when i t i s t h r e e - q u a r t e r s f u l l w i t h t h e c i r c u l a t i n g f l u i d t h e r e w i l l be s u f f i c i e n t h y d r o s t a t i c p r e s s u r e f o r t h e f l u i d e n t e r i n g t h e d e b u b b l e r t o pass t h r o u g h t h e h o r i z o n t a l e x i t i n t o t h e f l u o r e s c e n c e d e t e c t o r , w h i l e the a i r segments escape t h r o u g h t h e v e r t i c a l e x i t as b u b b l e s . O p t i m i z e t h e d e b u b b l i n g p r o c e s s t o a c h i e v e 100% f l u i d r e c o v e r y by c a r e f u l up and down a d j u s t m e n t s o f the p o s i t i o n o f t h e waste r e c e p t a c l e . A d j u s t the f l o w r a t e o f the f l u i d coming o u t o f t h e f l u o r e s c e n c e d e t e c t o r to about 1 ml/min. (A s c h e m a t i c diagram o f t h e post-column f l u o r o g e n i c d e r i v a t i z a t i o n system i s p r e s e n t e d i n F i g . 22.) HPLC P r o c e d u r e and C o n d i t i o n s E q u i l i b r a t e t h e column w i t h t h e s o l v e n t system ( w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 50/41/6/3). D i s s o l v e a m i x t u r e o f d i h y d r o d i g o x i g e n i n , d i g o x i n , d i g o x i g e n i n m o n o d i g i t o x o s i d e and d i g o -x i g e n i n b i s d i g i t o x o s i d e i n the e l u t i n g s o l v e n t system. I n j e c t a sample o f t h e s o l u t i o n i n t o t h e L i q u i d Chromatograph which has p r e v i o u s l y been a d j u s t e d t o t h e f o l l o w i n g c o n d i t i o n s : f l o w r a t e o f 0.4 ml/min; f l u o r e s c e n t d e t e c t i o n a t A „ o f 360 nm and A exc. em. ( c u t o f f ) o f 460 nm; UV d e t e c t i o n a t 254 nm; and a c h a r t speed o f 0.5 cm/min. Record t h e chromatogram. UQ8QMETEB «-360nm mt HPLC COLUMN U V TECTOR 254 nm) WASTE (0-4 ml/mm) HYDROCHLORIC ACID AIR -DEHYDROASCORBIC ACID PUMP Fig. 22. Schematic Diagram of the Post-Column Fluorogenic Derivatization System using the Air-Sementation Principle and 100% Fluid Recovery Set-up. 90. 8. S e p a r a t i o n o f Nine Equine E s t r o g e n s as E v i d e n c e o f S e l e c t i v i t y E q u i l i b r a t e t h e column w i t h t h e s o l v e n t system. D i s s o l v e a m i x t u r e o f t h e e q u i n e e s t r o g e n s i n 50% e t h a n o l . I n j e c t a sample o f the s o l u t i o n i n t o t h e L i q u i d Chromatograph which has p r e v i o u s l y been a d j u s t e d t o t h e f o l l o w i n g c o n d i t i o n s : f l o w r a t e o f 1.2 ml/min; UV d e t e c t i o n a t 220 nm; and c h a r t speed o f 0.5 cm/min. Record t h e chromatogram. 9. I s o l a t i o n o f D i g i t o x i n from D i g i t a l i s purpurea L e a f (A) E x t r a c t i o n M a c e r a t e 10 g o f the d r i e d l e a f i n 50 ml o f 20% v/v e t h a n o l f o r 48 hours i n a s t o p p e r e d c o n i c a l f l a s k . Add 50 ml o f 20% e t h a n o l , s t o p p e r t h e f l a s k and a g i t a t e f o r f o u r hours u s i n g a m e c h a n i c a l s h a k e r . F i l t e r t h e s l u r r y t h r o u g h a f u n n e l plugged w i t h c o t t o n wool. Wash the r e s i d u e w i t h a d d i t i o n a l p o r t i o n s o f 20% e t h a n o l u n t i l 200 ml o f f i l t r a t e has been c o l l e c t e d . Add 20 ml o f 0.35 M sodium h y d r o x i d e s o l u t i o n , s t i r and s e t a s i d e f o r 15 min u t e s t o e n s u r e d e - a c e t y l a t i o n o f any a c e t y l -d i g i t o x i n t h a t may be p r e s e n t . A d j u s t t he pH o f t h e s o l u t i o n t o ab o u t 6.5 w i t h 0.35 M h y d r o c h l o r i c a c i d s o l u t i o n . E x t r a c t t he l i q u o r w i t h f o u r , 20 ml p o r t i o n s o f d i c h l o r o m e t h a n e and d r y each e x t r a c t by p a s s i n g i t t h r o u g h t he same 10 g o f anhydrous sodium s u l f a t e , packed i n t o a f i l t e r f u n n e l t h a t i s f i t t e d w i t h a p l u g o f g l a s s w o o l . E v a p o r a t e t he d i c h l o r o m e t h a n e e x t r a c t t o d r y n e s s and d i s s o l v e t h e r e s i d u e i n 20 ml o f m e t h a n o l . (B) HPLC P r o c e d u r e and C o n d i t i o n s E q u i l i b r a t e t h e column w i t h t h e s o l v e n t system ( w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 45/38/11/6) and i n j e c t a sample o f t h e m e t h a n o l i c s o l u t i o n i n t o t h e L i q u i d Chromatograph which has been p r e v i o u s l y a d j u s t e d t o t h e f o l l o w i n g c o n d i t i o n s : f l o w r a t e o f 1.1 m l / min; UV d e t e c t i o n a t 220 nm and a c h a r t speed o f 0.5 cm/min. Record t h e chromatogram. 10. A n a l y s i s o f D i g o x i n i n i t s Dosage Forms (A) I n f r a r e d s p ectrum o f D i g o x i n P r e p a r e a KBr p e l l e t and r e c o r d the spectrum on Beckman IR-10 s p e c t r o -photometer . The IR s p e c t r u m o b t a i n e d was compared w i t h a S a d t l e r r e f e r e n c e s p ectrum and found t o have t h e same c h a r a c t e r i s t i c a b s o r p t i o n bands ( F i g . 2 3 ) . (B) S p e c t r a l C h a r a c t e r i s t i c s o f D i g o x i n U s i n g a Cahn e l e c t r o b a l a n c e weigh a c c u r a t e l y 10.0 mg o f d i g o x i n and t r a n s f e r i n t o a 100 ml v o l u m e t r i c f l a s k w i t h the a i d o f about 50 ml o f b o i l i n g m e t h a n o l . D i s s o l v e , c o o l t o room t e m p e r a t u r e and make t o volume w i t h m e t h a n o l . D i l u t e 10 ml o f the above s o l u t i o n t o 100 ml w i t h 35% m e t h a n o l . Record the spectrum on a Beckman Model 24 s p e c t r o p h o t o m e t e r u s i n g 35% methanol as t h e b l a n k . The s p e c t r a l a b s o r b a n c e c u r v e t h a t was o b t a i n e d i s shown i n F i g . 24. The maximum a b s o r p t i o n o f r a d i a n t e n e r g y was found to o c c u r a t 220 nm. (C) HPLC P r o c e d u r e and C o n d i t i o n s f o r t h e A n a l y s i s of, T a b l e t s and I n j e c t i o n E q u i l i b r a t e the column w i t h the s o l v e n t system ( w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 47/40/9/4). I n j e c t a 20 pl sample i n t o t h e L i q u i d Chromatograph which has been p r e v i o u s l y a d j u s t e d t o t h e f o l l o w i n g c o n d i t i o n s : f l o w r a t e o f 1.2 ml/min; UV d e t e c t i o n a t 220 nm, r a n g e o f 0.02; and a c h a r t speed o f 0.5 cm/min. Record t h e chromato-gram. 28.(a) The I n f r a r e d S p e c t r a (KBr) o f D i g o x i n as o b t a i n e d w i t h t h e Beckman IR-10; and (b) S a d t l e r R e f e r e n c e IR S p e c t r a . 0 2 5 0 1 2 5 t-+-.-4-^4--44 H-0 0 2 5 trt 2 0 0 i i i i 8 2 4 0 W A V E L E N G T H (nm) 2 8 0 F i g . 24. A S p e c t r a l A b s o r b a n c e Curve f o r D i g o x i n i n 35% M e t h a n o l . (D) HPLC P r o c e d u r e and C o n d i t i o n s f o r the A n a l y s i s o f E l i x i r P r o ceed as i n (C) u s i n g i n t h i s c a s e a s o l v e n t system o f water/ m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 51/42/5/2. (E) P r e p a r a t i o n o f I n t e r n a l S t a n d a r d S o l u t i o n s Weigh a c c u r a t e l y 100.0 mg o f 1 7 a - e t h y n y l e s t r a d i o l and t r a n s f e r i n t o a 100 ml .• v o l u m e t r i c f l a s k w i t h t h e a i d o f about 50 ml o f m e t h a n o l . D i s s o l v e , make t o volume w i t h methanol and mix. S i m i l a r l y , p r e p a r e a s o l u t i o n o f 100.0 mg o f 1 7 8 - d i h y d r o e q u i l i n i n 100 ml o f m e t h a n o l . (F) P r e p a r a t i o n o f S t a n d a r d S o l u t i o n s o f D i g o x i n A c c u r a t e l y weigh, u s i n g a Cahn e l e c t r o b a l a n c e , 20.0 mg o f d i g o x i n and t r a n s f e r i n t o a 100 ml v o l u m e t r i c f l a s k w i t h the a i d o f about 80 ml o f b o i l i n g m e t h a n o l . D i s s o l v e , c o o l t o room t e m p e r a t u r e and make t o volume w i t h m e t h a n o l . T r a n s f e r a l i q u o t s o f 0.5, 1.0, 2.0, 5.0, 10.0 and 12.5 ml o f the above s t o c k s o l u t i o n t o 100 ml v o l u m e t r i c f l a s k s . To each f l a s k , add 2.5 ml o f t h e i n t e r n a l s t a n d a r d s o l u t i o n and an amount o f methanol s u f f i c i e n t t o b r i n g the volume t o 35 ml. D i l u t e the s o l u t i o n i n each f l a s k to 100 ml w i t h d i s t i l l e d water and mix. The s i x s o l u t i o n s w i l l , t h e r e f o r e , have d i g o x i n c o n c e n t r a t i o n s o f 1, 2, 4, 10, 20 and 25 ng/ul w h i l e t h e c o n c e n t r a t i o n o f the i n t e r n a l s t a n d a r d i n each s o l u t i o n w i l l be 25 n g / u l . (G) P r e p a r a t i o n o f C a l i b r a t i o n Curves I n j e c t a 20 y l sample o f each s t a n d a r d s o l u t i o n i n t o the L i q u i d Chromatograph and o b t a i n a r e a v a l u e s f o r d i g o x i n and the i n t e r n a l s t a n d a r d . Make s i x d e t e r m i n a t i o n s f o r each s o l u t i o n . The c a l i b r a t i o n c u r v e s t h a t were o b t a i n e d u s i n g 1 7 a - e t h y n y l e s t r a d i o l and 1 7 8 - d i h y d r o e q u i l i n as i n t e r n a l s t a n d a r d s a r e p r e s e n t e d i n F i g s . 25, 26, and 27 and i n d i c a t e l i n e a r r e l a t i o n s h i p s w i t h l i n e s p a s s i n g t h r o u g h t h e o r i g i n . —I— 06 0 8 — i 10 W E I G H T R A T I O F i g . 25. A C a i i b r a t i o n . Curve f o r D i g o x i n i n 35% Methanol as o b t a i n e d w i t h a S o l v e n t System o f water/ m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 47/40/9/4 Ar e a and we i g h t r a t i o s a r e i n terms o f d i g o x i n / i n t e r n a l s t a n d a r d ( 1 7 a - e t h y n y l e s t r a d i o l ) . The l e a s t s q u a r e s l i n e o f b e s t f i t i s : y = 0.7137x-0.0026. The c o e f f i c i e n t o f d e t e r m i n a t i o n ( r ^ ) 0.9976. 96. 08 0 0.2 04 0 6 0 8 10 W E I G H T R A T I O F i g . 26. A C a l i b r a t i o n Curve f o r D i g o x i n i n 35% Methanol as o b t a i n e d w i t h a S o l v e n t System o f water/ m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 51/42/5/2. Ar e a and we i g h t r a t i o s a r e i n terms o f d i g o x i n / i n t e r n a l s t a n d a r d ( 1 7 a - e t h y n y l e s t r a d i o l ) . The l e a s t s q u a r e s l i n e o f b e s t f i t i s d e f i n e d fay the e q u a t i o n , y = 0.7199x + 0.0049. The c o e f f i c i e n t o f d e t e r m i n a t i o n ( r 2 ) = 0.9924. 08 W E I G H T R A T I O 27. A C a l i b r a t i o n Curve f o r D i g o x i n i n 35% Methanol as o b t a i n e d w i t h a S o l v e n t System o f wa t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 51/42/5/2. A r e a and weight r a t i o s a r e i n terms o f d i g o x i n / i n t e r n a l s t a n d a r d ( 1 7 8 - d i h y d r o e q u i l i n ) . The l e a s t s q u a r e s l i n e o f b e s t f i t i s d e f i n e d by t h e e q u a t i o n , y = 0.7368x + 0.0092. The c o e f f i c i e n t o f d e t e r m i n a t i o n ( r 2 ) = 0.9972. 98. Sample P r e p a r a t i o n o f D i g o x i n Dosage Forms Composite T a b l e t A s s a y Weigh not l e s s than 30 d i g o x i n t a b l e t s t h a t a r e s e l e c t e d a t random, and t r i t u r a t e t o a f i n e powder. A c c u r a t e l y weigh an amount o f the powdered t a b l e t m a t e r i a l e q u i v a l e n t to 1.25 mg o f d i g o x i n and q u a n t i -t a t i v e l y t r a n s f e r i n t o a 100 ml v o l u m e t r i c f l a s k . Add 10 ml o f d i s t i l l e d w ater and s w i r l t h e f l a s k f o r 2-3 m i n u t e s . Add 32.5 ml o f methanol and m e c h a n i c a l l y shake t h e m i x t u r e f o r about 15 m i n u t e s . F i l t e r the s u s p e n s i o n u s i n g a No. 1 Whatman f i l t e r paper and wash the r e s i d u e w i t h t h r e e , 5 ml p o r t i o n s o f d i s t i l l e d w a ter. C o l l e c t the f i l t r a t e and washings i n a 100 ml v o l u m e t r i c f l a s k . Add a 2.5 ml a l i q u o t o f 1 7 a - e t h y n y l e s t r a d i o l i n t e r n a l s t a n d a r d s o l u t i o n , d i l u t e t h e m i x t u r e w i t h d i s t i l l e d water t o a volume o f 100 ml and mix. S i n g l e T a b l e t A s s a y P l a c e one t a b l e t i n a 100 ml v o l u m e t r i c f l a s k and pr o c e e d as d i r e c t e d i n t h e Composite T a b l e t A s s a y above, b e g i n n i n g w i t h "add 10 ml o f d i s t i l l e d water and s w i r l the f l a s k f o r 2-3 m i n u t e s " . I n j e c t a b l e F o r m u l a t i o n A s s a y Empty the c o n t e n t s o f 20 randomly s e l e c t e d ampules i n t o a 50 ml c o n i c a l f l a s k and mix. T r a n s f e r a 2 ml a l i q u o t o f the above s o l u t i o n i n t o a 10 ml v o l u m e t r i c f l a s k ( f o r t h e 0.05 mg/ml i n j e c t i o n ) o r t o a 50 ml v o l u m e t r i c f l a s k ( f o r the 0.25 mg/ml i n j e c t i o n ) . Add 0.25 ml o r 1.25 ml a l i q u o t s o f 1 7 a - e t h y n y l e s t r a d i o l i n t e r n a l s t a n d a r d s o l u t i o n t o t h e 10 ml o r 50 ml v o l u m e t r i c f l a s k s , r e s p e c t i v e l y . Add 2.8 ml o f methanol i n t o t h e 10 ml v o l u m e t r i c f l a s k o r 14.0 ml o f methanol i n t o t h e 50 ml v o l u m e t r i c f l a s k . D i l u t e t h e s o l u t i o n t o volume w i t h d i s t i l l e d water and mix. E l i x i r A s s a y T r a n s f e r a 20 ml a l i q u o t o f the e l i x i r i n t o a 100 ml v o l u m e t r i c f l a s k . Add a 2.5 ml a l i q u o t o f the i n t e r n a l s t a n d a r d s o l u t i o n ( 1 7 a - e t h y n y1-e s t r a d i o l o r 1 7 B - d i h y d r o e q u i l i n ) and 20 ml o f m e t h a n o l . D i l u t e the s o l u t i o n t o 100 ml w i t h d i s t i l l e d water and mix. Q u a n t i t a t i o n A f t e r a s c e r t a i n i n g t h e l i n e a r i t y o f the r e l a t i o n s h i p o f a r e a r a t i o s v e r s u s w e i g h t r a t i o s , q u a n t i t a t i o n o f d i g o x i n was c a r r i e d o u t by an i n t e r n a l s t a n d a r d s i n g l e - p o i n t a u t o m a t i c c a l i b r a t i o n method u s i n g a Shimadzu Chromatopac C-R1A da t a p r o c e s s o r . Response f a c t o r s were d e t e r m i n e d as f o l l o w s : T r a n s f e r a 5 ml a l i q u o t o f the d i g o x i n s t o c k s o l u t i o n i n t o a 100 ml v o l u m e t r i c f l a s k . Add 2.5 ml o f i n t e r n a l s t a n d a r d s o l u t i o n and 27.5 ml o f methanol and d i l u t e t h e s o l u t i o n t o 100 ml w i t h d i s t i l l e d water and mix. Make t r i p l i c a t e i n j e c t i o n s o f a 20 y l sample i n t o the L i q u i d Chromatograph and o b t a i n t h e r e s p o n s e f a c t o r . The r e s p o n s e f a c t o r s o f d i g o x i n o b t a i n e d by u s i n g 1 7 a - e t h y n y l e s t r a d i o l as i n t e r n a l s t a n d a r d and s o l v e n t systems o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 47/40/94 and 51/42/5/2 were 1.4261 and 1.3864, r e s p e c t i v e l y . The r e s p o n s e f a c t o r o b t a i n e d u s i n g 1 7 B - d i h y d r o e q u i l i n and t h e l a t t e r s o l v e n t system was 1.3652. C a l c u l a t i o n o f amount o f d i g o x i n i n a sample was based on the e q u a t i o n : ^ ( S ) ' ^ ( 1 S ) ' ^ e s P o n s e F a c t o r "IV* ' ''U.S., ( E q - , 5 ) 100. where, W ^ = weig h t o f d i g o x i n = a r e a o f d i g o x i n u ( I . S . ) = w e i g h t o f i n t e r n a l s t a n d a r d ^ ( 1 S ) = a r e a ° ^ 1 n t e r n a ^ s t a n d a r d Peak i d e n t i t y was a u t o m a t i c a l l y m o n i t o r e d by the d a t a p r o c e s s o r . ( J ) D e t e r m i n a t i o n o f P r e c i s i o n o f T a b l e t A s s a y Weigh a t o t a l o f 40 d i g o x i n t a b l e t s and t r i t u r a t e to a f i n e powder. T r a n s f e r s i x a l i q u o t s o f a c c u r a t e l y weighed t a b l e t m a t e r i a l , each e q u i v a l e n t t o 1.25 mg o f d i g o x i n , i n t o s i x 100 ml v o l u m e t r i c f l a s k s . Proceed as d i r e c t e d i n the Composite T a b l e t A s s a y , b e g i n n i n g w i t h "add 10 ml o f d i s t i l l e d water and s w i r l f o r 2-3 m i n u t e s " . Make t h r e e i n j e c t i o n s f o r each sample. (K) D e t e r m i n a t i o n o f P e r c e n t a g e R e c o v e r y o f D i g o x i n from T a b l e t s Weigh a t o t a l o f 60 d i g o x i n t a b l e t s and t r i t u r a t e to a f i n e powder. T r a n s f e r n i n e a l i q u o t s o f a c c u r a t e l y weighed t a b l e t m a t e r i a l , each e q u i v a l e n t t o 1.25 mg o f d i g o x i n i n t o n i n e 100 ml v o l u m e t r i c f l a s k s . Add an a c c u r a t e l y weighed a l i q u o t o f d i g o x i n r e f e r e n c e s t a n d a r d , e q u i v a l e n t t o 0.625 mg, i n t o each o f s i x f l a s k s . T r e a t each o f the n i n e samples as d i r e c t e d i n t h e Composite T a b l e t A s s a y , b e g i n n i n g w i t h "add 10 ml o f d i s t i l l e d water and s w i r l f o r 2-3 m i n u t e s " . Make t h r e e i n j e c t i o n s f o r each sample. 11. A n a l y s i s o f D i g i t o x i n i n i t s Dosage Forms (A) I n f r a r e d Spectrum o f D i g i t o x i n P r e p a r e a KBr p e l l e t and r e c o r d the spectrum on a Beckman IR-10 s p e c t r o m e t e r . 101 . The IR spectrum o b t a i n e d was compared w i t h a S a d t l e r r e f e r e n c e spectrum and found to have the same c h a r a c t e r i s t i c a b s o r p t i o n bands ( F i g . 2 8 ) . (B) S p e c t r a l C h a r a c t e r i s t i c s o f D i g i t o x i n Weigh a c c u r a t e l y , u s i n g a Cahn e l e c t r o b a l a n c e , 10.0 mg o f d i g i t o x i n and t r a n s f e r i n t o a 100 ml v o l u m e t r i c f l a s k , w i t h the a i d o f about 50 ml o f b o i l i n g m e t h a n o l . D i s s o l v e , c o o l t o room t e m p e r a t u r e and make t o volume w i t h m e t h a n o l . D i l u t e 10 ml o f t h e above s o l u t i o n to 100 ml w i t h 35% m e t h a n o l . Record the spectrum on a Beckman Model 24 s p e c t r o p h o t o m e t e r u s i n g 35% methanol as t h e b l a n k . A s p e c t r a l a b s o r b a n c e c u r v e t h a t was o b t a i n e d i s shown i n F i g . 29. The maximum a b s o r p t i o n o f r a d i a n t e n e r g y was found to o c c u r a t a w a v elength o f 220 nm: (C) HPLC P r o c e d u r e and C o n d i t i o n s f o r the A n a l y s i s o f T a b l e t s and I n j e c t i o n E q u i l i b r a t e the column w i t h s o l v e n t system, w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 45/38/11/6. I n j e c t a 20 y l sample i n t o a L i q u i d Chromatograph which has been p r e v i o u s l y a d j u s t e d t o the f o l l o w i n g c o n d i t i o n s : f l o w r a t e o f 1.1 ml/min; UV d e t e c t i o n a t 220 nm, range o f 0.02; and a c h a r t speed o f 0.5 cm/min. Record the chromatogram. (D) P r e p a r a t i o n o f I n t e r n a l S t a n d a r d S o l u t i o n A c c u r a t e l y weigh 100.0 mg o f 1 7 a - m e t h y l t e s t o s t e r o n e and t r a n s f e r i n t o a 100 ml v o l u m e t r i c f l a s k w i t h the a i d o f about 50 ml o f m e t h a n o l . D i s s o l v e , make to volume w i t h methanol and mix. F i q 28 (a ) The I n f r a r e d S p e c t r a ( K B r ) o f D i g i t o x i n as o b t a i n e d w i t h t h e 9 " Beckman IR-10; and (b) S a d t l e r r e f e r e n c e IR s p e c t r a . o 103. F i g . 29. A S p e c t r a l - A b s o r b a n c e Curve f o r D i g i t o x i n i n 35% M e t h a n o l . 025 240 WAVE LENGTH (nm) 280 105. (E) P r e p a r a t i o n o f S t a n d a r d S o l u t i o n s o f D i g i t o x i n Weigh a c c u r a t e l y , u s i n g a Cahn e l e c t r o b a l a n c e , 10.0 mg o f d i g i t o x i n and t r a n s f e r i n t o a 100 ml v o l u m e t r i c f l a s k w i t h t h e a i d o f about 60 ml o f b o i l i n g m e t h a n o l . D i s s o l v e , c o o l t o room t e m p e r a t u r e , make t o volume w i t h methanol and mix. T r a n s f e r a l i q u o t s o f 1.0, 2.0, 5.0, 10.0, 20.0 and 30.0 ml o f the above s o l u t i o n to 100 ml v o l u m e t r i c ( f l a s k s . To each f l a s k add 1.0 ml o f i n t e r n a l s t a n d a r d s o l u t i o n and an amount o f methanol s u f f i c i e n t t o b r i n g the volume t o 35 ml. D i l u t e t h e s o l u t i o n i n each f l a s k t o 100 ml w i t h d i s t i l l e d water and mix. (The s t a n d a r d s o l u t i o n s w i l l , t h e r e f o r e , have d i g i t o x i n c o n c e n t r a t i o n s o f 1, 2, 5, 10, 20 and 30 ng/ml w h i l e t h e c o n c e n t r a t i o n o f the i n t e r n a l s t a n d a r d i n each s o l u t i o n w i l l be 10 ng/ul.) (F) P r e p a r a t i o n o f a C a l i b r a t i o n Curve I n j e c t a 20 y l sample o f each s t a n d a r d s o l u t i o n i n t o the L i q u i d Chromatograph and o b t a i n a r e a v a l u e s f o r d i g i t o x i n and the i n t e r n a l s t a n d a r d . Make s i x d e t e r m i n a t i o n s f o r each s o l u t i o n . The c a l i b r a t i o n c u r v e o b t a i n e d u s i n g 1 7 a - m e t h y l t e s t o s t e r o n e as i n t e r n a l s t a n d a r d i n d i c a t e s a l i n e a r r e l a t i o n s h i p w i t h the l i n e p a s s i n g t h r o u g h t h e o r i g i n ( F i g . 3 0 ) . (G) Sample P r e p a r a t i o n o f D i g i t o x i n F o r m u l a t i o n s (a) Composite T a b l e t A s s a y Weigh not l e s s than 40 d i g i t o x i n t a b l e t s t h a t were s e l e c t e d a t random, and t r i t u r a t e t o a f i n e powder. Weigh a c c u r a t e l y an amount o f powdered t a b l e t m a t e r i a l e q u i v a l e n t t o 1.0 mg o f d i g i t o x i n and q u a n t i t a t i v e l y t r a n s f e r i n t o a 100 ml v o l u m e t r i c f l a s k . Add 10 ml 30. A C a l i b r a t i o n Curve f o r D i g i t o x i n i n 35% Methanol as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o -methane: 45/38/1Vfe. Area and weight r a t i o s a r e i n terms o f d i g i t o x i n / i n t e r n a l s t a n d a r d ( 1 7 a - m e t h y l t e s t o s t e r o n e ) . The l e a s t s q u a r e s l i n e o f b e s t f i t i s d e f i n e d by t h e e q u a t i o n y = 1.0848x + 0.0042; and t h e c o e f f i c i e n t o f d e t e r m i n a t i o n ( r 2 ) i s 0.9957. 107. o f d i s t i l l e d water and s w i r l t h e f l a s k f o r 2-3 m i n u t e s . Add 34 ml o f methanol and m e c h a n i c a l l y shake t he m i x t u r e f o r about 15 m i n u t e s . F i l t e r t h e s u s p e n s i o n u s i n g a No. 1 Whatman f i l t e r paper and wash the r e s i d u e w i t h t h r e e 5 ml p o r t i o n s o f d i s t i l l e d w a t e r . C o l l e c t the f i l t r a t e and washings i n a 100 ml v o l u m e t r i c f l a s k . Add 1 ml o f 1 7 a - m e t h y l t e s t o s t e r o n e i n t e r n a l s t a n d a r d s o l u t i o n , d i l u t e the m i x t u r e w i t h d i s t i l l e d water t o a volume o f 100 ml and mix. (b) S i n g l e T a b l e t A s s a y P l a c e one t a b l e t i n a 10 ml v o l u m e t r i c f l a s k . Add 1 ml o f d i s t i l l e d w ater and s w i r l t h e f l a s k f o r 2-3 m i n u t e s . Add 3.4 ml o f methanol and m e c h a n i c a l l y shake t h e m i x t u r e f o r about 15 m i n u t e s . F i l t e r t h e s u s p e n s i o n u s i n g a No. 1 Whatman f i l t e r paper and wash the r e s i d u e w i t h t h r e e 1 ml. p o r t i o n s o f d i s t i l l e d w a t e r . C o l l e c t the f i l t r a t e and washings i n a 10 ml o f v o l u m e t r i c f l a s k . Add a 0.1 ml a l i q u o t o f 1 7 a - m e t h y l t e s t o s t e r o n e i n t e r n a l s t a n d a r d s o l u t i o n , d i l u t e t h e m i x t u r e w i t h d i s t i l l e d water t o a volume o f 10 ml and mix. ( c ) I n j e c t a b l e F o r m u l a t i o n A s s a y T r a n s f e r a 1 ml a l i q u o t o f i n j e c t i o n i n t o a 10 ml v o l u m e t r i c f l a s k . Add a 0.1 ml a l i q u o t o f 1 7 a - m e t h y l t e s t o s t e r o n e i n t e r n a l s t a n d a r d s o l u t i o n and 3.3 ml o f m e t h a n o l . D i l u t e t h e s o l u t i o n to volume w i t h d i s t i l l e d water and mix. (H) Q u a n t i t a t i o n S i n c e t he c a l i b r a t i o n c u r v e ( F i g . 30) i n d i c a t e s l i n e a r i t y w i t h the l i n e p a s s i n g t h r o u g h t he o r i g i n , q u a n t i t a t i o n was c a r r i e d o u t by an i n t e r n a l s t a n d a r d s i n g l e - p o i n t a u t o m a t i c c a l i b r a t i o n method u s i n g a Shimadzu Chromatopac C-R1A d a t a p r o c e s s o r . The r e s p o n s e f a c t o r was d e t e r m i n e d as f o l l o w s : 108. T r a n s f e r a 10 ml a l i q u o t o f the d i g i t o x i n s t o c k s o l u t i o n i n t o a 100 ml v o l u m e t r i c f l a s k . Add 1 ml o f i n t e r n a l s t a n d a r d s o l u t i o n and 25 ml o f methanol and d i l u t e t h e s o l u t i o n t o 100 ml w i t h d i s t i l l e d w a t e r and mix. Make t r i p l i c a t e i n j e c t i o n s o f a 20 p l sample i n t o the L i q u i d Chromatograph and o b t a i n t h e r e s p o n s e f a c t o r . The r e s p o n s e f a c t o r was found t o be 0.9348 and d e t e r m i n a t i o n o f the amount o f d i g i t o x i n was on the b a s i s o f E q u a t i o n 15. Peak i d e n t i t y was a u t o m a t i -c a l l y m o n i t o r e d by the d a t a p r o c e s s o r . ( I ) D e t e r m i n a t i o n o f P r e c i s i o n o f T a b l e t A s s a y Weigh a t o t a l o f s e v e n t y t a b l e t s and t r i t u r a t e t o a f i n e powder. T r a n s f e r s i x a l i q u o t s o f a c c u r a t e l y weighed t a b l e t m a t e r i a l , each e q u i v a l e n t t o 1.0 mg o f d i g i t o x i n , i n t o s i x 100 ml v o l u m e t r i c f l a s k s . P r o c e e d as d i r e c t e d i n t h e d i g i t o x i n Composite T a b l e t A s s a y , b e g i n n i n g w i t h "add 10 ml o f d i s t i l l e d w a t er and s w i r l f o r 2-3 m i n u t e s " . Make t h r e e i n j e c t i o n s f o r each sample. ( J ) D e t e r m i n a t i o n o f P e r c e n t a g e Recovery o f D i g i t o x i n from T a b l e t s Weigh a t o t a l o f 80 t a b l e t s and t r i t u r a t e t o a f i n e , powder. T r a n s f e r seven a l i q u o t s o f a c c u r a t e l y weighed t a b l e t m a t e r i a l , each e q u i v a -l e n t t o 1.0 mg o f d i g i t o x i n , i n t o seven 100 ml v o l u m e t r i c f l a s k s . Add an a c c u r a t e l y weighed a l i q u o t o f d i g i t o x i n r e f e r e n c e s t a n d a r d , e q u i v a l e n t t o 0.5 mg, i n t o each o f f o u r f l a s k s . T r e a t each o f the seven samples as d i r e c t e d i n the d i g i t o x i n Composite T a b l e t A s s a y , b e g i n n i n g w i t h "add 10 ml o f d i s t i l l e d w a t e r and s w i r l f o r 2-3 m i n u t e s " . Make t h r e e i n j e c t i o n s f o r each sample. 109. 12. Comparison o f the A n a l y s i s o f D i g o x i n and D i g i t o x i n Dosage Forms  by HPLC and USP XX Methods (A) Brands o f D i g o x i n and D i g i t o x i n T a b l e t s u s e d : R (a) L a n o x i n . T a b l e t s (Brand A) R (b) N a t i g o x i n T a b l e t s (Brand B) ( c ) D i g o x i n T a b l e t s (B.D.H.) (Brand C) R (d) P u r o d i g i n T a b l e t s ( D i g i t o x i n ) (B) Dosage Forms and S t r e n g t h s o f D i g o x i n and D i g i t o x i n u s e d : R (a) L a n o x i n T a b l e t s 0.125 mg R (b) L a n o x i n T a b l e t s 0.25 mg R ( c ) L a n o x i n I n j e c t i o n 0.05 mg/ml R (d) L a n o x i n I n j e c t i o n 0.25 mg/ml R (e) L a n o x i n E l i x i r 0.05 mg/ml R ( f ) N a t i g o x i n T a b l e t s 0.25 mg (g) D i g o x i n (B.D.H.) T a b l e t s 0.25 mg R (h) P u r o d i g i n T a b l e t s 0.1 mg R ( i ) C r y s t o d i g i n I n j e c t i o n 0.2 mg/ml ( D i g i t o x i n ) The HPLC and USP a s s a y p r o c e d u r e s a r e p r e s e n t e d i n the form o f f l o w c h a r t s i n o r d e r t h a t t h e r e s p e c t i v e number o f s t e p s and times o f a n a l y s i s can be b e t t e r v i s u a l i z e d . (C) Sample P r e p a r a t i o n o f D i g o x i n Dosage Forms f o r HPLC A n a l y s i s The p r o c e d u r e f o r sample p r e p a r a t i o n o f d i g o x i n t a b l e t s ( i n c o m p o s i t e t a b l e t s and s i n g l e t a b l e t a s s a y i s p r e s e n t e d i n the f l o w c h a r t shown i n F i g . 31. A more d e t a i l e d p r o c e d u r e i s g i v e n i n S e c t i o n 1 0 ( H ) : (a) and (b) o f t h i s C h a p t e r . The p r o c e d u r e s f o r the i n j e c t i o n and e l i x i r a r e g i v e n i n F i g s . 32 and 33; and the d e t a i l s o f the sample p r e p a r a t i o n s a r e p r e s e n t e d i n S e c t i o n 10 ( H ) : ( c ) and ( d ) . D i g o x i n T a b l e t s Sample P r e p a r a t i o n Sample e q u i v a l e n t t o 1.25 mg o f d i g o x i n i n a 100 ml. v o l u m e t r i c f l a s k - add 10 ml. o f ^ 0 and s w i r l f o r 2-3 minutes - add 32.5 ml o f MeOH and shake f o r 10 minutes f i l t e r t h r o u g h a #1 Whatman paper i n t o a 100 ml v o l u m e t r i c f l a s k wash the r e s i d u e w i t h t h r e e , 5 ml. p o r t i o n s o f d i s t i 1 l e d w a ter add 2.5 ml. o f 17«-ethynylestradiol s o l u t i o n ( i n t e r n a l s t a n d a r d , 1 mg/ml i n MeOH) d i l u t e t h e s o l u t i o n w i t h H2O t o volume and mix Pr o c e d u r e f o r Q u a n t i t a t i o n - i n j e c t a 20 Ml sample S i n g l e T a b l e t Assay - same as above F i g . 31. Flow C h a r t P r e s e n t a t i o n o f the HPLC Assay o f D i g o x i n T a b l e t s  D i g o x i n I n j e c t i o n Sample P r e p a r a t i o n Sample e q u i v a l e n t t o 0.1 mg o f D i g o x i n i n a 10 ml. v o l u m e t r i c f l a s k - add 0.25 ml. o f 1 7 a - e t h y n y l e s t r a d i o l s o l u t i o n - add 2.8 ml. o f MeOH and d i l u t e t he s o l u t i o n w i t h H 90 t o volume P r o c e d u r e f o r Quanti t a t i o n same as i n D i g o x i n T a b l e t s F i g . 32. Flow C h a r t P r e s e n t a t i o n o f the HPLC Assay o f D i g o x i n T a b l e t s .  D i g o x i n E l i x i r Sample P r e p a r a t i o n sample e q u i v a l e n t t o 1 mg. o f d i g o x i n i n a 100 ml v o l u m e t r i c f l a s k - add 2.5 ml o f 1 7 a - e t h y n y l e s t r a d i o l s o l u t i o n and 20 ml. o f methanol - d i l u t e t h e s o l u t i o n w i t h h^O t o volume P r o c e d u r e f o r Q u a n t i t a t i o n - same as i n D i g o x i n T a b l e t s F i g . 33. Flow C h a r t P r e s e n t a t i o n o f the HPLC Assay o f D i g o x i n E l i x i r .  113. (D) HPLC P r o c e d u r e f o r Q u a n t i t a t i o n o f D i g o x i n T h i s i s i n d i c a t e d i n the f l o w c h a r t s shown i n F i g s . 31, 32 and 33; and t h e d e t a i l s have been d e s c r i b e d i n S e c t i o n 10 ( I ) o f t h i s C h a p t e r . (E) Sample P r e p a r a t i o n o f D i g o x i n Dosage Forms f o r A n a l y s i s by USP Methods The p r o c e d u r e f o r sample p r e p a r a t i o n o f d i g o x i n t a b l e t s ( i n c o m p o s i t e t a b l e t s and s i n g l e t a b l e t a s s a y ) i s shown i n the f l o w c h a r t p r e s e n t e d i n F i g . 34. The f l o w c h a r t s d e p i c t i n g p r o c e d u r e s o f sample p r e p a r a t i o n f o r t h e i n j e c t i o n and e l i x i r a r e shown i n F i g s . 36 and 37. (F) USP P r o c e d u r e f o r Q u a n t i t a t i o n o f D i g o x i n The USP q u a n t i t a t i o n p r o c e d u r e i s p r e s e n t e d i n F i g . 35. (G) Sample P r e p a r a t i o n o f D i g i t o x i n Dosage Forms f o r HPLC A n a l y s i s The p r o c e u d r e f o r sample p r e p a r a t i o n o f d i g i t o x i n t a b l e t s ( i n compo-s i t e t a b l e t s and s i n g l e t a b l e t 'assay) i s shown i n F i g . 38. The d e t a i l e d p r o c e d u r e i s g i v e n i n S e c t i o n 11 ( G ) : (a) and (b) o f t h i s C h a p t e r . The p r o c e d u r e f o r t h e i n j e c t i o n i s g i v e n i n F i g . 39 and the d e t a i l s o f the sample p r e p a r a t i o n a r e p r e s e n t e d i n S e c t i o n 11 ( G ) : ( c ) . (H) HPLC P r o c e d u r e f o r Q u a n t i t a t i o n o f D i g i t o x i n T h i s i s i n d i c a t e d i n t h e f l o w c h a r t s shown i n F i g s . 38 and 39 and the d e t a i l s have been d e s c r i b e d i n S e c t i o n 11 (H) o f t h i s C h a p t e r . ( I ) Sample P r e p a r a t i o n o f D i g i t o x i n Dosage Forms f o r A n a l y s i s by USP Methods The p r o c e d u r e f o r sample p r e p a r a t i o n o f d i g i t o x i n t a b l e t s ( i n D i g o x i n T a b l e t s Sample P r e p a r a t i o n S t a n d a r d P r e p a r a t i o n : D i g o x i n S o l u t i o n (Et.OH) (25 mcg/ml.) Sample e q u i v a l e n t t o 2.5 mg o f D i g o x i n i n a b e a k e r - add 5 ml. o f b o i l i n g n - p r o p y l a l c o h o l - c o o l f o r 20 minutes t r a n s f e r t o a s e p a r a t o r w i t h 30 ml. CHCl^ and 20 ml. H 20 I - add 1 ml. d i l u t e d HgSO^ and shake - s e p a r a t e lower l a y e r t r a n s f e r l o w e r l a y e r t o a secon d s e p a r a t o r - wash w i t h 5 ml. H o0 f i l t e r CHC1 3 l a y e r t h r o u g h a p l e d g e t o f c o t t o n i n t o a 100 ml. v o l u m e t r i c f l a s k y - r e p e a t t he e x t r a c t i o n and washing p r o c e d u r e u s i n g two 30 ml. p o r t i o n s o f a m i x t u r e o f 5 volumes o f CHCI3 and 1 volume o f n - p r o p y l a l c o h o l - d i l u t e t h e combined e x t r a c t s w i t h MeOH t o volume and mix. F i g . 34. Flow C h a r t P r e s e n t a t i o n o f the USP P r o c e d u r e f o r Sample P r e p a r a t i o n o f D i g o x i n T a b l e t s . P r o c e d u r e f o r Q u a n t i t a t i o n - P i p e t 10 ml. o f each o f the a s s a y p r e p a r a t i o n and s t a n d a r d p r e p a r a t i o n i n t o s e p a r a t e s m a l l c o n i c a l f l a s k s e v a p o r a t e t o d r y n e s s v - Cool i n a vacuum d e s i c c a t o r o v e r phosphorous p e n t o x i d e f o r 60 minutes - add 5 ml. o f a c i d - f e r r i c c h l o r i d e T.S. and a l l o w t o s t a n d , p r o t e c t e d from l i g h t , f o r 10 minutes a t <30°C f i l t e r i f n e c e s s a r y - d e t e r m i n e the a b s o r b a n c e o f t o a r e a g e n t b l a n k a t X590, a t 2-minute i n t e r v a l s u n t i l each s o l u t i o n , r e l a t i v e r e p e a t i n g the measurement maximum a b s o r b a n c e i s o b t a i n e d S i n g l e T a b l e t Assay - same as above e x c e p t t h a t the a b s o r b a n c e f o r the b l a n k i s sub-t r a c t e d from t h a t o f the sample. F i g . 35. Flow C h a r t P r e s e n t a t i o n o f the USP P r o c e d u r e f o r t he Q u a n t i t a t i o n o f D i g o x i n i n T a b l e t s . D i g o x i n I n j e c t i o n Sample P r e p a r a t i o n sample e q u i v a l e n t t o about 2.5 mg D i g o x i n i n s e p a r a t o r - d i l u t e w i t h H^O t o 50 ml. - add 1 ml. o f d i l u t e d ^ S O ^ - e x t r a c t w i t h a 35 ml. p o r t i o n o f a m i x t u r e o f 5 volumes o f c h l o r o f o r m and 1 volume o f n - p r o p y l a l c o h o l t r a n s f e r lower l a y e r t o second s e p a r a t o r wash w i t h 5 ml. o f w a t e r f i l t e r w i t h CHClg l a y e r t h r o u g h a p l e d g e t o f c o t t o n i n t o a 100 ml. v o l u m e t r i c f l a s k r e p e a t the e x t r a c t i o n and washing p r o c e d u r e u s i n g two 30 ml. p o r t i o n s o f a m i x t u r e o f 5 volumes o f CHC1 3 and 1 volume o f n - p r o p y l a l c o h o l d i l u t e the combined e x t r a c t s w i t h MeOH t o volume and mix P r o c e d u r e f o r Q u a n t i t a t i o n same as i n t a b l e t s F i g . 36. Flow C h a r t P r e s e n t a t i o n o f the USP Assay o f D i g o x i n I n j e c t i o n .  D i g o x i n E l i x i r Sample P r e p a r a t i o n sample e q u i v a l e n t t o 2.5 mg d i g o x i n i n s e p a r a t o r - add 10 ml. o f C C l ^ , shake, and a l l o w to s e p a r a t e - d i s c a r d t h e CC1 4 - add 2 ml. o f N a 2 C 0 3 TS - e x t r a c t w i t h f o u r 20 ml. p o r t i o n s o f CHCl^ combine the CHC13 e x t r a c t s i n a 100 ml. v o l u m e t r i c f l a s k v - d i l u t e combined e x t r a c t s w i t h CHC13 t o volume and mix P r o c e d u r e f o r Quanti t a t i o n - same as i n t a b l e t s F i g . 37. Flow C h a r t P r e s e n t a t i o n o f the USP Assay o f D i g o x i n E l i x i r .  Dig i tox in T a b l e t s Sample P r e p a r a t i o n sample e q u i v a l e n t t o 1.0 mg o f d i g i t o x i n i n a 100 ml v o l u m e t r i c f l a s k - add 10 ml. o f H 2 0 and s w i r l f o r 2-3 minutes - add 34 ml. o f MeOH and shake f o r 10 minutes f i l t e r t h r o u g h Whatman #1 paper i n t o a 100 ml. v o l u m e t r i c f l a s k - wash t h e r e s i d u e w i t h t h r e e , 5 ml p o r t i o n s o f d i s t i l l e d w a t e r - add 1 ml. o f 1 7 a - m e t h y l t e s t o s t e r o n e s o l u t i o n ( i n t e r n a l s t a n d a r d , 1 mg/ml i n MeOH) - d i l u t e t h e s o l u t i o n w i t h H 2 0 t o volume and mix P r o c e d u r e f o r Q u a n t i t a t i o n - i n j e c t a 20 y l sample S i n g l e T a b l e t A s s a y same as above F i g . 38. Flow C h a r t P r e s e n t a t i o n o f the HPLC A s s a y o f D i g i t o x i n T a b l e t s .  D i g i t o x i n I n j e c t i o n Sample P r e p a r a t i o n sample e q u i v a l e n t t o 0.2 mg. o f D i g i t o x i n i n a 100 ml. v o l u m e t r i c f l a s k - add 0.1 ml. o f 1 7 a - m e t h y t e s t o s t e r o n e s o l u t i o n - add 3.3 ml. o f MeOH and d i l u t e t h e s o l u t i o n w i t h H o0 t o volume P r o c e d u r e f o r Quanti t a t i o n same as i n D i g i t o x i n T a b l e t s F i g . 39. Flow C h a r t P r e s e n t a t i o n o f the HPLC A s s a y o f D i g i t o x i n I n j e c t i o n .  120. c o m p o s i t e t a b l e t s and s i n g l e t a b l e t a s s a y ) and i n j e c t i o n a r e shown i n t h e f l o w c h a r t s p r e s e n t e d i n F i g s . 40, 42 and 44. ( J ) USP P r o c e d u r e f o r Q u a n t i t a t i o n o f D i g i t o x i n The USP q u a n t i t a t i o n p r o c e d u r e s f o r t h e a n a l y s i s o f c o m p o s i t e t a b l e t s , s i n g l e t a b l e t s and i n j e c t i o n , a r e p r e s e n t e d i n F i g s . 41, 43 and 44, r e s p e c t i v e l y . (K) D e t e r m i n a t i o n o f P r e c i s i o n o f the USP Method f o r D i g o x i n T a b l e t A s s a y Weigh a t o t a l o f 70 t a b l e t s and t r i t u r a t e t o a f i n e powder. T r a n s f e r s i x a l i q u o t s o f a c c u r a t e l y weighed t a b l e t m a t e r i a l , each e q u i v a l e n t t o 2.5 mg o f d i g o x i n , i n t o s i x 50 ml b e a k e r s . P r o c e e d as d i r e c t e d i n the f l o w c h a r t s shown i n F i g s . 34 and 35. Make t h r e e d e t e r m i n a t i o n s f o r each sample. (L) D e t e r m i n a t i o n o f P r e c i s i o n o f the USP Method f o r D i g i t o x i n T a b l e t . A s s a y Weigh a t o t a l o f 70 t a b l e t s and t r i t u r a t e t o a f i n e powder. T r a n s f e r t h r e e a l i q u o t s o f a c c u r a t e l y weighed t a b l e t m a t e r i a l , e q u i v a l e n t t o 2.0 mg o f d i g i t o x i n , i n t o t h r e e 100 ml b e a k e r s . P r o c e e d as d i r e c t e d i n t h e f l o w c h a r t s shown i n F i g s . 40 and 41. Make t h r e e d e t e r m i n a t i o n s f o r each sample. (M) D e t e r m i n a t i o n o f P e r c e n t a g e R e c o v e r y o f D i g o x i n from T a b l e t s u s i n g t h e USP Method Weigh a t o t a l o f 100 t a b l e t s and t r i t u r a t e t o a f i n e powder. T r a n s -f e r n i n e a l i q u o t s o f a c c u r a t e l y weighed t a b l e t m a t e r i a l , each D i g i t o x i n T a b l e t s Sample P r e p a r a t i o n S t a n d a r d P r e p a r a t i o n : D i g i t o x i n S o l u t i o n (EtOH) (40 mcg/ml) sample e q u i v a l e n t t o 2 mg. o f D i g i t o x i n i n a b e a k e r - mix w i t h 2 ml. o f h^O - add 4 ml. o f formamide, s t i r , c o v e r the be a k e r w i t h a watch g l a s s and h e a t on a steam bath f o r 20 minutes - c o o l , add 2 ml. o f H 20 and ab o u t 8 g o f a d s o r b e n t ( c h r o m a t o g r a p h i c s i l i c e o u s e a r t h ) and s t i r t o u n i f o r m c o n s i s t e n c y t r a n s f e r t o a p r e p a r e d c h r o m a t o g r a p h i c column y - e l u t e t h e d i g i t o x i n w i t h B e n z e n e - C h l o r o f o r m m i x t u r e (3:1) a t a r a t e n o t e x c e e d i n g 4 ml/min. - c o l l e c t n e a r l y 250 ml. o f e l u a t e i n a 250 m v o l u m e t r i c f l a s k , add C H C U t o volume and mix F i g . 40. Flow C h a r t P r e s e n t a t i o n o f the USP P r o c e d u r e f o r Sample P r e p a r a t i o n o f D i g i t o x i n Composite T a b l e t s . P r o c e d u r e f o r Q u a n t i t a t i o n - t r a n s f e r 25 ml. o f the e l u a t e and 5 ml. o f the s t a n d a r d p r e p a r a t i o n t o s e p a r a t e , 50 ml. c o n i c a l f l a s k s e v a p o r a t e t o d r y n e s s - m o i s t e n each r e s i d u e w i t h about 0.5 ml. o f a l c o h o l a g a i n e v a p o r a t e t o d r y n e s s - c o o l , p i p e t 5 ml. o f a l c o h o l i n t o each f l a s k , s t o p p e r and a l l o w t o s t a n d f o r 15 m i n u t e s a t 25 ± 3°C - p i p e t 5 ml. o f a l c o h o l i n t o a s i m i l a r f l a s k t o s e r v e as a b l a n k , and t r e a t a l l t h r e e f l a s k s as f o l l o w s : - add 3 ml. o f a l k a l i n e p i c r a t e T.S., mix, m a i n t a i n a t 25 ± 3°C and p r o t e c t t h e m i x t u r e from i n t e n s e l i g h t - a f t e r 8 m inutes d e t e r m i n e the a b s o r b a n c e s o f the s o l u t i o n s r e l a t i v e t o the b l a n k , a t 495 nm. Repeat the measurements a t 2-minute i n t e r v a l s u n t i l maximum a b s o r b a n c e i s r e a c h e d F i g . 41. Flow C h a r t P r e s e n t a t i o n o f the USP P r o c e d u r e f o r t h e Q u a n t i t a t i o n o f D i g i t o x i n Composite T a b l e t s . 123. Sample P r e p a r a t i o n f o r D i g i t o x i n S i n g l e T a b l e t A s s a y S t a n d a r d P r e p a r a t i o n : D i g i t o x i n S o l u t i o n (EtOH) (5 mcg/ml) - p l a c e 1 t a b l e t i n a b e a k e r - add 5 t o 10 drops o f w a t e r & a l l o w t o d i s i n t e g r a t e - add 5 ml. o f a c e t o m ' t r i l e , h e a t on a steam b a t h f o r 5 mi n u t e s and c o o l t r a n s f e r the s o l u t i o n t o a s e p a r a t o r w i t h 30 ml o f CHC1 3 and 20 ml. o f H 20 v - shake t h e s e p a r a t o r - s e p a r a t e t h e CHCl^ l a y e r , wash i n a second s e p a r a t o r c o n t a i n i n g 5 ml. o f NaHCO^ s o l u t i o n f i l t e r t h r o u g h a p l e d g e t o f c o t t o n i n t o a s u i t a b l e c o n t a i n e r - r e p e a t t h e e x t r a c t i o n and w a s h i n g , u s i n g two 30 ml. p o r t i o n s o f CHC l ^ - e v a p o r a t e t o d r y n e s s - d i s s o l v e t h e r e s i d u e i n 80% a l c o h o l by s h a k i n g f o r 20 minutes t o o b t a i n a s o l u t i o n h a v i n g a c o n c e n t r a t i o n o f ab o u t 5 mcg/ml F i g . 42. Flow C h a r t P r e s e n t a t i o n o f t h e USP P r o c e d u r e f o r Sample P r e p a r a t i o n i n D i g i t o x i n S i n g l e T a b l e t A s s a y .  P r o c e d u r e f o r Q u a n t i t a t i o n - p i p e t i n t o 3, s e p a r a t e g l a s s - s t o p p e r e d 25 ml. f l a s k s 2 ml. o f t h e s t a n d a r d s o l u t i o n , 2 ml. o f s o l u t i o n from the t a b l e t s and 2 ml. o f 80% a l c o h o l t o p r o v i d e t h e r e a g e n t b l a n k , r e s p e c t i v e l y t r e a t each f l a s k as f o l l o w s - add 10 ml. o f a s o l u t i o n f r e s h l y p r e p a r e d by d i s s o l v i n g 35 mg. o f a s c o r b i c a c i d i n 25 m l . o f methanol and c a u t i o u s l y a d d i n g the s o l u t i o n t o 100 ml. o f h y d r o c h l o r i c a c i d - mix, and add 1 ml. o f a s o l u t i o n f r e s h l y p r e p a r e d by d i l u t i n g 1 ml. o f 30% hydrogen p e r o x i d e s o l u t i o n w i t h w a ter t o 500 ml. and d i l u t i n g 1 volume o f the r e s u l t i n g s o l u t i o n w i t h 20 volumes o f w a t e r - a l l o w t o s t a n d f o r 30 minutes t r a n s f e r t o c u v e t t e s - d e t e r m i n e f l u o r e s c e n c e r e a d i n g s o f t h e s o l u t i o n from the T a b l e t and S t a n d a r d S o l u t i o n a t x e x c (395 nm) and xem (580 nm) a g a i n s t the r e a g e n t b l a n k F i g . 43. Flow C h a r t P r e s e n t a t i o n o f the USP P r o c e d u r e f o r t h e Q u a n t i t a t i o n o f D i g i t o x i n i n S i n g l e T a b l e t s . D i g i t o x i n I n j e c t i o n Sample P r e p a r a t i o n sample e q u i v a l e n t t o 2 mg o f D i g i t o x i n i n a s e p a r a t o r - add 40 ml. o f H 20 and 1 ml. o f d i l u t e d H 2 S 0 4 - e x t r a c t w i t h f o u r 25 ml. p o r t i o n s o f CHC13, washing the CHC1, e x t r a c t s i n s u c c e s s i o n w i t h 10 ml. o f H o0 f i l t e r t h e washed CHC1 3 e x t r a c t through CHCl 3-washed c o t t o n i n t o a 250 ml. b e a k e r I - e v a p o r a t e t o d r y n e s s - add 4 ml. o f formamide and warm on a steam bath f o r 20 m i n u t e s , w h i l e s t i r r i n g - c o o l , add 4 ml. o f w a t e r and about 8 g o f a d s o r b e n t and s t i r t o u n i f o r m c o n s i s t e n c y t r a n s f e r t o a p r e p a r e d c h r o m a t o g r a p h i c column v - e l u t e t h e d i g i t o x i n w i t h B e n z e n e - C h l o r o f o r m m i x t u r e (3:1) a t a r a t e n o t e x c e e d i n g 4 ml./min. - c o l l e c t n e a r l y 250 ml. o f e l u a t e i n a 250 ml. v o l u m e t r i c f l a s k , add CHC1, t o volume and mix P r o c e d u r e f o r Q u a n t i t a t i o n same as i n D i g i t o x i n T a b l e t A s s a y F i g . 44. Flow C h a r t P r e s e n t a t i o n o f the USP Assay o f D i g i t o x i n I n j e c t i o n .  126. e q u i v a l e n t t o 2.5 mg o f d i g o x i n , i n t o n i n e 50 ml b e a k e r s . Add an a c c u r a t e l y weighed a l i q u o t o f d i g o x i n r e f e r e n c e s t a n d a r d , e q u i v a l e n t t o 1.25 mg, i n t o each o f s i x b e a k e r s . T r e a t each of. the n i n e samples as d i r e c t e d i n the d i g o x i n t a b l e t a s s a y shown i n F i g s . 34 and 35. Make t h r e e d e t e r m i n a t i o n s f o r each sample. (N) D e t e r m i n a t i o n o f P e r c e n t a g e R e c o v e r y o f D i g i t o x i n from T a b l e t s u s i n g t h e USP Method Weigh a t o t a l o f 150 t a b l e t s and t r i t u r a t e t o a f i n e powder. T r a n s f e r seven a l i q u o t s o f a c c u r a t e l y weighed t a b l e t m a t e r i a l , each e q u i v a l e n t t o 2.0 mg o f d i g i t o x i n , i n t o seven 100 ml b e a k e r s . Add an a c c u r a t e l y weighed amount o f d i g i t o x i n r e f e r e n c e s t a n d a r d , e q u i v a l e n t to 1.0 mg i n t o each o f f o u r b e a k e r s . T r e a t each o f the seven samples as d i r e c t e d i n the d i g i t o x i n t a b l e t a s s a y shown i n F i g s . 40 and 41. Make t h r e e d e t e r m i n a t i o n s f o r each sample. 13. S t a b i l i t y M o n i t o r i n g o f D i g o x i n and D i g i t o x i n i n t h e i r R e s p e c t i v e  Dosage Forms (A) Brands o f D i g o x i n and D i g i t o x i n T a b l e t s Used: (a) L a n o x i n T a b l e t s ( d i g o x i n ) (b) N a t i g o x i n T a b l e t s ( d i g o x i n ) ( c ) P u r o d i g i n T a b l e t s ( d i g i t o x i n ) (B) Dosage Forms and S t r e n g t h s o f D i g o x i n and D i g i t o x i n Used: (a) L a n o x i n T a b l e t s 0.125 mg (b) L a n o x i n T a b l e t s 0.25 mg ( c ) L a n o x i n I n j e c t i o n 0.05 mg/ml (d) L a n o x i n E l i x i r 0.05 mg/ml 127. (e) N a t i g o x i n T a b l e t s 0.25 mg ( f ) P u r o d i g i n T a b l e t s 0.1 mg (C) C o n d i t i o n s o f S t o r a g e T r i p l i c a t e samples o f the same ba t c h o f drug s u b s t a n c e s and dosage forms were s t o r e d i n t h e i r o r i g i n a l c o n t a i n e r s a t ambient c o n d i t i o n s ; 60°C and 70.4% R e l a t i v e H u m i d i t y ; and 80°C and 37.1% R e l a t i v e H u m i d i t y , r e s p e c t i v e l y . C o n c e n t r a t i o n s o f aqueous s u l f u r i c a c i d and the c o r r e s -ponding r e l a t i v e h u m i d i t y v a l u e s a r e g i v e n i n T a b l e V I I I . (D) HPLC P r o c e d u r e and C o n d i t i o n s (a) For a n a l y s i s o f D i g o x i n T a b l e t s and I n j e c t i o n P r o c e e d as d i r e c t e d i n S e c t i o n 10 (C) o f t h i s C h a p t e r . (b) For A n a l y s i s o f D i g o x i n E l i x i r E q u i l i b r a t e the column w i t h s o l v e n t system, w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 51/43/5/1. I n j e c t the sample, u s i n g a Waters U6K i n j e c t o r , i n t o a Beckman L i q u i d Chromatograph which has been p r e v i o u s l y a d j u s t e d t o t h e f o l l o w i n g c o n d i t i o n s : f l o w r a t e o f 1.2 ml per m i n u t e , UV d e t e c t i o n a t 220 nm, range o f 0.02; a t t e n u a t i o n o f 6; and a c h a r t speed o f 0.5 cm per m i n u t e . Record the chromatogram. ( c ) For A n a l y s i s o f D i g i t o x i n T a b l e t s Proceed as d i r e c t e d i n S e c t i o n 11 ( G ) : a o f t h i s C h a p t e r . (E) P r e p a r a t i o n o f I n t e r n a l S t a n d a r d S o l u t i o n s (a) For A n a l y s i s o f D i g o x i n T a b l e t s and I n j e c t i o n Proceed as d i r e c t e d i n S e c t i o n 10 (E) o f t h i s C h a p t e r . (b) For A n a l y s i s o f D i g o x i n E l i x i r A c c u r a t e l y weigh 100.0 mg o f h y d r o c o r t i s o n e and t r a n s f e r i n t o a T a b l e V I I I . R e l a t i v e H u m i d i t y V a l u e s O b t a i n e d w i t h Aqueous S u l f u r i c A c i d S o l u t i o n s 9  D e n s i t y o f % H 2 S 0 4 R e l a t i v e Vapour H 2 S 0 4 S o l u t i o n i n Aq. S o l u t i o n H u m i d i t y (%) a t lo 'C^rrm Hg) 1.25 75.0 70.4 12.2 1.40 81.0 37.1 6.5 Handbook o f C h e m i s t r y and P h y s i c s , 57th e d . , E46. 129. 100 ml v o l u m e t r i c f l a s k w i t h the a i d o f about 50 ml o f m e t h a n o l . D i s s o l v e , make t o volume w i t h methanol and mix. ( c ) For A n a l y s i s o f D i g i t o x i n T a b l e t s Proceed as d i r e c t e d i n S e c t i o n 11 (D) o f t h i s C h a p t e r . (F) P r e p a r a t i o n o f S t a n d a r d S o l u t i o n s o f D i g o x i g e n i n , D i g o x i g e n i n mono-d i g i t o x o s i d e , D i g o x i g e n i n b i s d i g i t o x o s i d e and D i g o x i n (a) For A n a l y s i s o f D i g o x i n and i t s D e g r a d a t i o n P r o d u c t s i n T a b l e t s and I n j e c t i o n Weigh a c c u r a t e l y , u s i n g a Cahn E l e c t r o b a l a n c e , 10.0 mg o f d i g o x i n and t r a n s f e r i n t o a 50 ml v o l u m e t r i c f l a s k w i t h t h e a i d o f about 40 ml o f b o i l i n g m e t h a n o l . D i s s o l v e , c o o l t o room t e m p e r a t u r e , make t o volume w i t h m e t h a n o l , and mix. T r a n s f e r a l i q u o t s o f 2.5, 5.0, 10.0, 15.0, 20.0 and 25.0 ml o f the above s t o c k s o l u -t i o n t o s i x 100 ml v o l u m e t r i c f l a s k s . To each f l a s k and 2.5 ml o f 1 7 a - e t h y n y l e s t r a d i o l i n t e r n a l s t a n d a r d s o l u t i o n add an amount o f methanol s u f f i c i e n t t o b r i n g t he volume t o 35 ml. D i l u t e the s o l u t i o n i n each f l a s k t o 100 ml w i t h d i s t i l l e d water and mix. S i m i l a r l y p r e p a r e s t o c k and s t a n d a r d s o l u t i o n s o f d i g o x i g e n i n , d i g o x i g e n i n m o n o d i g i t o x o s i d e and d i g o x i g e n i n b i s d i g i t o x o s i d e . The s i x s t a n d a r d s o l u t i o n s p r e p a r e d f o r each o f the f o u r compounds w i l l , t h e r e f o r e , have c o n c e n t r a t i o n s o f 5, 10, 20, 30, 40 and 50 n g / u l . (b) F o r A n a l y s i s o f D i g o x i n and i t s D e g r a d a t i o n P r o d u c t s i n the E l i x i r Dosage Form Pro c e e d as i n (a) a d d i n g 2 ml o f h y d r o c o r t i s o n e i n t e r n a l s t a n d a r d s o l u t i o n i n s t e a d o f 2.5 ml o f 1 7 a - e t h y n y l e s t r a d i o l s o l u t i o n . (G) P r e p a r a t i o n o f S t a n d a r d S o l u t i o n s o f D i g i t o x i g e n i n , D i g i t o x i g e n i n m o n o d i g i t o x o s i d e , D i g i t o x i g e n i n b i s d i g i t o x o s i d e and D i g i t o x i n . 1 3 0 . P r o c e e d as d i r e c t e d i n S e c t i o n ( F ) , (a) a d d i n g 1 ml o f 1 7 a - m e t h y l -t e s t o s t e r o n e i n t e r n a l s t a n d a r d s o l u t i o n i n s t e a d o f 2 . 5 ml o f 1 7 a - e t h y n y l e s t r a d i o l s o l u t i o n . (H) P r e p a r a t i o n o f C a l i b r a t i o n Curves I n j e c t a 2 0 y l sample o f each s t a n d a r d s o l u t i o n o f the e i g h t compounds ( D i g o x i n , D i g i t o x i n and t h e i r D e g r a d a t i o n P r o d u c t s ) i n t o a L i q u i d Chromatograph and o b t a i n a r e a v a l u e s o f t h e compound and the c o r r e s -ponding i n t e r n a l s t a n d a r d . Make s i x d e t e r m i n a t i o n s f o r each s o l u t i o n . The c a l i b r a t i o n c u r v e s (Area r a t i o v e r s u s Weight r a t i o ) o b t a i n e d f o r d i g o x i n and i t s d e g r a d a t i o n p r o d u c t s , u s i n g 1 7 a - e t h y n y l e s t r a d i o l as the i n t e r n a l s t a n d a r d a r e shown i n F i g . 4 5 . The c a l i b r a t i o n c u r v e s o f d i g o x i n and i t s d e g r a d a t i o n p r o d u c t s u s i n g h y d r o c o r t i s o n e as the i n t e r n a l s t a n d a r d a r e g i v e n i n F i g . 4 6 . The c a l i b r a t i o n c u r v e s o b t a i n e d f o r d i g i t o x i n and i t s d e g r a d a t i o n p r o d u c t s a r e shown i n F i g . 4 7 . ( I ) Sample P r e p a r a t i o n (a) D i g o x i n T a b l e t s Proceed as d i r e c t e d i n S e c t i o n 1 0 ( H ) : (a) o f t h i s C h a p t e r . (b) D i g o x i n I n j e c t i o n Proceed as d i r e c t e d i n S e c t i o n 10 ( H ) : ( c ) o f t h i s C h a p t e r . ( c ) D i g o x i n E l i x i r T r a n s f e r a 1 0 ml a l i q u o t o f t h e e l i x i r i n t o a s e p a r a t o r y f u n n e l and add 2 ml o f sodium c a r b o n a t e T.S. E x t r a c t w i t h f o u r , 15 ml p o r t i o n s o f d i c h l o r o m e t h a n e and f i l t e r each e x t r a c t t h r o u g h the same No. 1 Whatman f i l t e r paper plugged w i t h c o t t o n wool t h a t has been p r e v i o u s l y washed w i t h d i c h l o r o m e t h a n e . A f t e r f i l t r a t i o n o f t h e f o u r t h e x t r a c t , wash the f i l t e r paper and p l u g o f c o t t o n 131 . i i i ~\ 0 6 1.2 1 8 2 4 W E I G H T R A T I O F i g . 45. C a l i b r a t i o n Curves f o r D i g o x i g e n i n (y), D i g o x i g e n i n m o n o d i g i t o x o s i d e ( • ) , D i g o x i g e n i n b i s d i g i t o x o s i d e ( • ) and D i g o x i n ( A ) i n 35% Methanol as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h i o r o m e t h a n e : 47/40/9/4. Area and we i g h t r a t i o s a r e i n terms o f c o m p o u n d / i n t e r n a l s t a n d a r d (17a-ethyny1 -e s t r a d i o l ) . The r e s p e c t i v e l e a s t s q u a r e s l i n e s o f b e s t f i t a r e d e f i n e d by t h e e q u a t i o n s : ( T j y = 1.3770x + 0.0117, r 2 = 0.9937); y = 1.0614x + 0.0088 ( • ) , ( r 2 = 0.9955); y = 0.8910x + 0.0603,0) r 2 = 0.9974; and y = 0.7049x + 0.01401 ( A ) , r 2 = 0.9933. 132. F i g . 46. C a l i b r a t i o n Curves f o r D i g o x i g e n i n ( A ) , D i g o x i g e n i n m o n o d i g i t o x o s i d e ( • ) , D i g o x i g e n i n b i s d i g i t o x o s i d e (y) and D i g o x i n (4) i n 35% Methanol as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 51/43/5/1. A r e a and we i g h t r a t i o s a r e i n terms o f c o m p o u n d / i n t e r n a l s t a n d a r d ( h y d r o c o r t i s o n e ) . The r e s p e c t i v e l e a s t s q u a r e s l i n e s o f b e s t f i t a r e d e f i n e d by t h e e q u a t i o n s : y = 2.1709x + 0.0010 ( A ) , r 2 = 0.9974; y = 1 .8442x + 0.0105 ( • ) , r 2 = 0.9999; y = 1 .6065x + 0.0084 ( y ) , r 2 = 0.9967; and y = 1 .4356x + 0.0034 (*>), r Z = 0.9919. 133. 4 2 0 12 24 W E I G H T R A T I O 134. F i g . 47. C a l i b r a t i o n Curves f o r D i g i t o x i g e n i n ( A ) , D i g i t o x i g e n i n m o n o d i g i t o x o s i d e ( • ) , D i g i t o x i g e n i n b i s d i g i t o x o s i d e ( • ) and D i g i t o x i n ( • ) i n 35% Methanol as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 45/38/11/6. A r e a and weight r a t i o s a r e i n terms o f c o m p o u n d / i n t e r n a l s t a n d a r d ( 1 7 a - m e t h y l t e s t o s t e r o n e ) . The r e s p e c t i v e l e a s t s q u a r e s l i n e s o f b e s t f i t a r e d e f i n e d by t h e e q u a t i o n s : y = 2.0758 + 0.0042 ( A ) , r 2 = 0.9936; y = 1.5759x + 0.0060 ( • ) , r2 = 0.9968; y = 1 .3093x + 0.0074 ( • ) , r 2 = 0.9972; and y = 1 .0793x - 0.0969 ( • ) , r 2 = 0.9942. W E I G H T R A T I O 136. wool w i t h two, 5 ml p o r t i o n s o f d i c h l o r o m e t h a n e . C o l l e c t the e x t r a c t and washings i n a 100 ml round-bottom f l a s k , e v a p o r a t e t o d r y n e s s u s i n g a r o t a r y e v a p o r a t o r a t low h e a t (about 40°C) and c o o l . Add 12.5 ml o f m e t h a n o l , 12.5 ml o f d i s t i l l e d water and s w i r l f o r about f i v e m i n u t e s . T r a n s f e r the s o l u t i o n to a 50 ml v o l u m e t r i c f l a s k . R i n s e t h e round-bottom f l a s k w i t h two, 5 ml p o r t i o n s o f d i s t i l l e d water and add the washings i n t o the v o l u m e t r i c f l a s k . Add 1 ml o f h y d r o c o r t i s o n e i n t e r n a l s t a n d a r d s o l u t i o n , d i l u t e t o volume w i t h d i s t i l l e d water and mix. (d) D i g i t o x i n T a b l e t s Proceed as d i r e c t e d i n S e c t i o n 11 ( G ) : (a) o f t h i s C h a p t e r . ( J ) Q u a n t i t a t i o n Q u a n t i t a t i o n o f d i g o x i n , d i g i t o x i n and t h e i r d e g r a d a t i o n p r o d u c t s was c a r r i e d o u t by an i n t e r n a l s t a n d a r d s i n g l e - p o i n t a u t o m a t i c c a l i b r a t i o n method u s i n g a Shimadzu Chromatopac C-R1A d a t a p r o c e s s o r . Response f a c t o r s were d e t e r m i n e d as f o l l o w s : (a) For A n a l y s i s o f D i g o x i n T a b l e t s and I n j e c t i o n : T r a n s f e r a 5 ml a l i q u o t o f each o f the s t o c k s o l u t i o n s o f d i g o x i -g e n i n , d i g o x i g e n i n m o n o d i g i t o x o s i d e , d i g o x i g e n i n b i s d i g i t o x o s i d e and d i g o x i n i n t o a 100 ml v o l u m e t r i c f l a s k . Add 2.5 ml o f 1 7 a - e t h y n y l e s t r a d i o l i n t e r n a l s t a n d a r d s o l u t i o n and 27.5 ml o f m e t h a n o l . D i l u t e the s o l u t i o n w i t h d i s t i l l e d water and mix. Make t r i p l i c a t e i n j e c t i o n s o f a 20 y l sample i n t o a L i q u i d Chromatograph and o b t a i n the r e s p o n s e f a c t o r s . (b) For A n a l y s i s o f D i g o x i n E l i x i r P r o ceed as i n (a) u s i n g 2 ml o f h y d r o c o r t i s o n e i n t e r n a l s t a n d a r d s o l u t i o n i n s t e a d o f 2.5 ml o f 1 7 a - e t h y n y l e s t r a d i o l s o l u t i o n . ( c ) For A n a l y s i s o f D i g i t o x i n T a b l e t s Proceed as i n (a) u s i n g s t o c k s o l u t i o n s o f d i g i t o x i g e n i n , d i g t o x i g e n i n m o n o d i g i t o x o s i d e , d i g i t o x i g e n i n b i s d i g i t o x o s i d e and d i g i t o x i n i n s t e a d o f the d i g o x i n s e r i e s ; and a d d i n g 1 ml o f 1 7 a - m e t h y l t e s t o s t e r o n e i n t e r n a l s t a n d a r d s o l u t i o n . The r e s p o n s e f a c t o r s t h a t were o b t a i n e d a r e p r e s e n t e d i n T a b l e 9 and d e t e r m i n a t i o n o f the amount o f each compound was based on E q u a t i o n 15. (K) D e t e r m i n a t i o n o f pH Cool t h e sample t o room t e m p e r a t u r e and measure the pH u s i n g a pH meter t h a t has been c a l i b r a t e d p r e v i o u s l y u s i n g a b u f f e r s o l u t i o n o f pH 7.0. 138. T a b l e IX. Response F a c t o r s o b t a i n e d f o r t h e A n a l y s i s o f D i g o x i n , D i g i t o x i n and t h e i r p o t e n t i a l D e g r a d a t i o n P r o d u c t s u s i n g ; 1 7 a - e t h y n y l e s t r a d i o l ( a ) , H y d r o c o r t i s o n e ( b ) , and 17a-methyltestosterone ( c ) as I n t e r n a l S t a n d a r d s S e r i e s Compound Response F a c t o r (a) (b) (c) 1 D i g o x i g e n i n 0 .7176 0, .4469 2 D i g o x i g e n i n m o n o d i g i t o x o s i d e 0 .9403 0, .5862 3 D i g o x i g e n i n b i s d i g i t o x o s i d e 1. 1365 0. .7084 4 D i g o x i n 1 .3890 0. .8656 5 D i g i t o x i g e n i n 0 .4864 6 D i g i t o x i g e n i n m o n o d i g i t o x o s i d e 0 .6351 7 D i g i t o x i g e n i n b i s d i g i t o x o s i d e 0 .7702 8 D i g i t o x i n 0 .9385 I I I . RESULTS AND DISCUSSION 1. E v o l u t i o n o f t h e B a s i c HPLC S o l v e n t System f o r t h e A n a l y s i s o f C a r d i a c  G l y c o s i d e s D i g o x i n , d i g i t o x i n and t h e i r m e t a b o l i t e s a r e n o n - i o n i c , o f medium to low p o l a r i t y , s p a r i n g l y s o l u b l e i n water and have m o l e c u l a r w e i g h t s o f l e s s than 1000. T h e r e f o r e , as shown i n the g e n e r a l g u i d e f o r HPLC mode s e l e c t i o n ( T a b l e I V ) , a r e v e r s e - p h a s e column ( S p h e r i s o r b ODS, 25 cm x 3.2 mm, p a r t i c l e s i z e 10 ym) was s e l e c t e d f o r the i n i t i a l s t u d y . On t h e b a s i s o f t h e i r r e l a t i v e l y h i g h p o l a r i t y c h a r a c t e r i s t i c s , water and methanol were chosen f o r the p r e p a r a t i o n o f the p r e l i m i n a r y s o l v e n t system. In an e f f o r t t o o b t a i n s e p a r a t i o n o f the d i g o x i n s e r i e s i n a r e a s o n a b l e p e r i o d o f time v a r i o u s m i x t u r e s o f methanol and water were t r i e d . A s o l v e n t system o f m e t h a n o l / w a t e r : 60/40 r e s u l t e d i n a 7 m inute chromatogram ( F i g . 48) i n which t h e compounds e l u t e d i n the o r d e r o f d i g o x i g e n i n m o n o d i g i t o x o s i d e , d i g o x i g e n i n b i s d i g i t o x o s i d e and d i g o x i n . I t was e v i d e n t t h a t d i g o x i g e n i n (which was not a v a i l a b l e a t t h i s s t a g e ) i f s e p a r a t e d , would e l u t e b e f o r e d i g o x i g e n i n m o n o d i g i t o x o s i d e whose r e t e n t i o n time was q u i t e c l o s e t o the s o l v e n t f r o n t . T h e r e f o r e , i t was n e c e s s a r y t o push t h e f i r s t peak a l i t t l e b i t f u r t h e r from t h e s o l v e n t f r o n t i n o r d e r t o make room f o r d i g o x i g e n i n . T h i s was a c c o m p l i s h e d w i t h a s o l v e n t system o f w a t e r / m e t h a n o l : 60/40, as shown i n F i g . 49. The t o t a l c h r o m a t o g r a p h i c time i n t h i s c a s e was about 23 m i n u t e s . T h i s s o l v e n t system, however, was found t o be u n f i t f o r t h e s e p a r a t i o n o f d i g i t o x i n and i t s m e t a b o l i t e s , because o f t h e e x t r e m e l y l o n g chromatograph t i m e . 140. 1 ; j t ! i M ; i i i-i • ' A 1 : ! n j 11 11 1 I 1 11 ! 1 tl 0 4 8 M I N U T E S F i g . 48. A Chromatogram f o r the HPLC S e p a r a t i o n o f a Stan d a r d M i x t u r e o f D i g o x i g e n i n m o n o d i g i t o x o s i d e (peak 1 ) , D i g o x i g e n i n b i s -d i g i t o x o s i d e (peak 2) and D i g o x i n (peak 3 ) . HPLC c o n d i t i o n s : S p h e r i s o r b ODS column; Solvent system, w a t e r / m e t h a n o l : 40/60, f l o w r a t e = 1.5 ml/min; UV d e t e c t i o n a t 254 nm, range = 0.2; C h a r t speed = 0.5 cm/min; t h e compounds were d i s s o l v e d i n m e t h a n o l . i j -j- ; • A : 0 L . 5 ••' 1 ' • 1 : . 1 0 15 20 25 M I N U T E S F i g . 49. A Chromatogram f o r t h e HPLC S e p a r a t i o n o f a St a n d a r d M i x t u r e o f D i g o x i g e n i n m o n o d i g i t o x o s i d e (peak 1 ) . D i g o x i g e n i n b i s -d i g i t o x o s i d e (peak 2) and D i g o x i n (peak 3) as o b t a i n e d w i t h a s o l v e n t system o f meth a n o l / w a t e r : 40/60. Other HPLC c o n d i t i o n s were t h e same as i n F i g . 48. In o r d e r t o o p t i m i z e the c a p a c i t y f a c t o r ( r e t e n t i o n ) o f both the d i g o x i n and d i g i t o x i n s e r i e s o f compounds, w h i l e m a i n t a i n i n g the sequence o f e l u t i o n , a v a r i e t y o f r e l a t i v e l y n o n - p o l a r s o l v e n t s was i n t r o d u c e d f o r the p r e p a r a t i o n o f t e r t i a r y o r q u a t e r n a r y s o l v e n t systems. The chromatogram shown i n F i g . 50(a) was o b t a i n e d w i t h a s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 4 6 / 3 9 / 1 0 / 5 . T h i s s o l v e n t system was b a s i c a l l y found t o be most c o n v e n i e n t f o r t h e i s o c r a t i c r e s o l u t i o n o f d i g o x i n , d i g i t o x i n and t h e i r m e t a b o l i t e s i n one chromatogram. The s h a r p n e s s o f the peaks and s h o r t c h r o m a t o g r a p h i c time i n d i c a t e t h e h i g h d i f f e r e n t i a t i n g c a p a b i l i t y and s t r e n g t h o f t h e s o l v e n t system. The 12 m i n u t e chromatogram ( F i g . 5 0 ( a ) ) shows s e p a r a t i o n o f a s t a n d a r d m i x t u r e o f seven compounds w i t h an e l u t i o n o r d e r o f d i g o x i g e n i n m o n o d i g i t o x o s i d e , d i g o x i g e n i n b i s -d i g i t o x o s i d e , d i g o x i n , d i g i t o x i g e n i n , d i g i t o x i g e n i n m o n o d i g i t o x o s i d e , d i g i t o x o g e n i n b i s d i g i t o x o s i d e and d i g i t o x i n , as m o n i t o r e d by a UV d e t e c t o r s e t a t a w a v e l e n g t h o f 220 nm. The chromatogram i n F i g . 50(b) was o b t a i n e d under t h e same c o n d i t i o n s e x c e p t t h a t UV d e t e c t i o n was c a r r i e d out a t a w a v e l e n g t h o f 254 nm. The p r e s e n c e o f a r e l a t i v e l y s m a l l amount o f d i c h l o r o m e t h a n e i n t h e s o l v e n t system c o n t r i b u t e s t o a b s o r p t i o n o f r a d i a n t energy a t a wavelength o f 220 nm and t h e r e f o r e r a i s e s the b a s e l i n e . However, t h e p r e s e n c e o f a c o n s i d e r a b l y l a r g e amount o f water m i n i m i z e s the a b s o r b a n c e e f f e c t o f d i c h l o r o m e t h a n e . The problem o f m i s c i b i l i t y o f water and d i c h l o r o m e t h a n e i s r e s o l v e d by the p r e s e n c e o f methanol and i s o p r o p a n o l . Moreover, s i n c e t h e r e i s s u f f i c i e n t d i f f e r e n t i a l a b s o r b a n c e c o n t r i b u t e d by d i g o x i n and t h e o t h e r c a r d e n o l i d e s , a t the wavelength o f 220 nm, i t has been c o n s i s t -e n t l y shown t h a t no problem a r i s e s from the p r e s e n c e o f d i c h l o r o m e t h a n e . The advantages o f t h i s w a v e l e n g t h (220 nm) i n terms o f i n c r e a s e d s e n s i -t i v i t y can be o b s e r v e d from a comparison o f F i g . 50 (a) and ( b ) . The unique q u a t e r n a r y s o l v e n t system c o n s i s t i n g o f w a t e r , m e t h a n o l , 142. F i g . 50. A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a S t a n d a r d m i x t u r e o f D i g o x i n , D i g i t o x i n and t h e i r M e t a b o l i t e s . Sequence o f e l u t i o n : 1 = d i g o x i g e n i n m o n o d i g i t o x o s i d e ; 2 = d i g o x i g e n i n b i s d i g i t o x o s i d e ; 3 = d i g o x i n ; 4 = d i g i t o x i g e n i n ; 5 = d i g i t o x i g e n i n m o n o d i g i t o x o s i d e ; 6 = d i g i t o x i g e n i n b i s d i g i t o x o s i d e ; 7 = d i g i t o x i n . HPLC c o n d i t i o n s : S p h e r i s o r b column; s o l v e n t system, w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 46/39/10/5, f l o w r a t e = 1.5 ml/min; C h a r t speed = 0.5 cm/min; UV d e t e c t i o n a t 220 nm (a) and 254 nm ( b ) , range = 0.02. The compounds were d i s s o l v e d i n the e l u t i n g s o l v e n t system. 0 5 10 MINUTES 144. i s o p r o p a n o l and d i c h l o r o m e t h a n e , as d e s c r i b e d above, was employed as t h e b a s i c m o b i l e phase f o r a l l HPLC s e p a r a t i o n s r e p o r t e d i n t h i s i n v e s t i g a t i o n . O p t i m i z a t i o n o f t h e s e l e c t i v i t y f a c t o r f o r p a r t i c u l a r HPLC s e p a r a t i o n s o f any o f t h e compounds used i n t h i s s t u d y was a c c o m p l i s h e d by v a r i o u s a l t e r a t i o n s o f t h e r e l a t i v e p r o p o r t i o n o f the f o u r components o f the s o l v e n t system. 2. Development o f HPLC Systems f o r the S e p a r a t i o n o f D i g o x i n , D i g i t o x i n , t h e i r r e s p e c t i v e M e t a b o l i t e s o r D e g r a d a t i o n P r o d u c t s and r e l a t e d  Compounds (A) S e p a r a t i o n s by I s o c r a t i c E l u t i o n I t i s g e n e r a l l y r e c o g n i z e d t h a t d i g o x i n can be a m e t a b o l i c p r o d u c t o f d i g i t o x i n (Kramer e t a l . , 1976). I t has a l s o been o b s e r v e d t h a t both d i g o x i n and d i g i t o x i n can undergo d e g r a d a t i o n t o produce the r e s p e c t i v e g e n i n s and t h e i r mono-and b i s d i g i t o x o s i d e s (Kuhlman e t a l . , 1973). Hence a method t h a t would s e p a r a t e d i g o x i n , d i g i t o x i n and t h e i r m e t a b o l i t e s , i n one chromatogram, would be d e s i r a b l e . HPLC s e p a r a t i o n s have been r e p o r t e d f o r m i x t u r e s o f a wide v a r i e t y o f d i g i t a l i s g l y c o s i d e s . The o n l y paper ( C a s t l e , 1975) t h a t has a d d r e s s e d i t s e l f t o t h e problem o f s e p a r a t i n g d i g o x i n , d i g i t o x i n and t h e i r m e t a b o l i t e s , r e p o r t s g r a d i e n t e l u t i o n . However, s i n c e a n a l y s i s o f d i g o x i n and d i g i t o x i n r e q u i r e s h i g h s e n s i t i v i t y d e t e c t o r s e t t i n g s , t h e b a s e l i n e f l u c t u a t i o n s t h a t may be a s s o c i a t e d w i t h g r a d i e n t e l u t i o n can i n t r o d u c e d i f f i c u l t i e s i n q u a n t i t a t i o n . T h e r e f o r e the need f o r an i s o c r a t i c HPLC system t h a t would s e p a r a t e t h e s e compounds i n one chromatogram i s q u i t e a p p a r e n t . For purposes o f a t t a i n i n g g r e a t e r s e n s i t i v i t y , t h e i s o c r a t i c system s h o u l d 145. be c o m p a t i b l e w i t h aqueous media so t h a t i t may be used a l o n g w i t h the p o st-column f l u o r o g e n i c d e r i v a t i z a t i o n p r o c e s s . I f such an i s o c r a t i c system i s p o s s i b l e , t h e chances o f i t s b e i n g s e l e c t i v e enough to a l l o w the a n a l y s i s o f d i g o x i n o r d i g i t o x i n i n t h e p r e s e n c e o f i t s m e t a b o l i t e s , d e g r a d a t i o n p r o d u c t s , i m p u r i t i e s , endogenous s t e r o i d s and drugs commonly p r e s c r i b e d f o r c a r d i a c p a t i e n t s w i l l , i n d e e d , be h i g h . I t i s w i t h i n t h i s c o n t e n t t h a t t h e f o l l o w i n g i s o c r a t i c s e p a r a t i o n s were d e v e l o p e d . The i s o c r a t i c s e p a r a t i o n o f d i g o x i n , d i g i t o x i n and t h e i r m e t a b o l i t e s i s p r e s e n t e d i n F i g . 51, as o b t a i n e d w i t h a s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 47/40/9/4. The 27-minute chromatogram i n d i c a t e s e l u t i o n o f t h e compounds i n t h e o r d e r o f d i g o x i g e n i n , d i g o x i -g e n i n m o n o d i g i t o x o s i d e , d i g o x i g e n i n b i s d i g i t o x o s i d e , d i g o x i n , d i g i t o x i g e n i n , d i g i t o x i g e n i n m o n o d i g i t o x o s i d e , d i g i t o x i g e n i n b i s d i g i t o x o s i d e and d i g i t o x i n . P r o v i d e d t h a t t h e n e c e s s a r y s e n s i t i v i t y i s a c h i e v e d , t h i s s o l v e n t system can be u s e f u l f o r m o n i t o r i n g t h e l e v e l s o f d i g i t o x i n , d i g o x i n and t h e i r m e t a b o l i t e s i n b i o l o g i c a l specimens. S e p a r a t i o n by HPLC f o r subsequent q u a n t i t a t i o n by radioimmunoassay i s one p o s s i b i l i t y . A s l i g h t l y a l t e r e d s o l v e n t system ( w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 45/37/12/6) o f f e r s a c h r o m a t o g r a p h i c t i m e o f about 13 m i n u t e s ( F i g . 52) i n which a l l components e x c e p t d i g o x i g e n i n and d i g o x i g e n i n m o n o d i g i t o x o s i d e have b a s e l i n e r e s o l u t i o n . I t can be o b s e r v e d t h a t t h i s s o l v e n t system may be a p p l i c a b l e f o r t h e a n a l y s i s o f d i g i t o x i n and i t s m e t a b o l i t e s i n c l u d i n g d i g o x i n . In o r d e r t o o p t i m i z e t i m e , c a p a c i t y f a c t o r and r e s o l u t i o n o f the s e p a r a t i o n o f t h e d i g i t o x i n s e r i e s , f u r t h e r a l t e r a t i o n s o f t h e p r o p o r t i o n o f t h e m o b i l e phase components were t r i e d . A more n o n - p o l a r s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 43/35/15/7 r e s u l t e d i n t h e chromatogram shown i n F i g . 53. T h i s chromatogram r e s o l v e s a l l o f 146. 14 F i g . 51. A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a S t a n d a r d M i x t u r e o f D i g o x i n , D i g i t o x i n and t h e i r M e t a b o l i t e s . Sequence o f e l u t i o n : 1 = d i g o x i g e n i n ; 2 = d i g o x i g e n i n m o n o d i g i t o x o s i d e ; 3 = d i g o x i g e n i n b i s d i g i t o x o s i d e ; 4 = d i g o x i n ; 5 = d i g i t o x i g e n i n ; 6 = d i g i t o x i g e n i n m o n o d i g i t o x o s i d e ; 7 = d i g i t o x i g e n i n b i s d i g i t o x o s i d e ; 8 = d i g i t o x i n . HPLC c o n d i t i o n s : U l t r a s p h e r e ODS column; s o l v e n t system, water/ m e t h a n o l / i s o p r o p a n o l / d i c h i o r o m e t h a n e : 47/40/9/4; f l o w r a t e = 1.2 ml/min; UV d e t e c t i o n a t 220 nm, range = 0.02, a t t e n u a t i o n = 6; C h a r t speed = 0.5 cm/min. The compounds were d i s s o l v e d i n t h e e l u t i n g s o l v e n t system. 147. IAJULJL -L 5 10 1 5 MINUTES F i g . 52. A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a St a n d a r d M i x t u r e o f D i g o x i n , D i g i t o x i n and t h e i r M e t a b o l i t e s o b t a i n e d w i t h a S o l v e n t System o f water/ m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 45/37/12/5. Other HPLC c o n d i t i o n s : same as i n F i g . 51. 148. 0 5 1 0 MINUTES F i g . 53. A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a S t a n d a r d M i x t u r e o f the D i g i t o x i n S e r i e s from t h e D i g o x i n S e r i e s . Sequence o f e l u t i o n : 1,2 = d i g o x i g e n i n and d i g o x i g e n i n m o n o d i g i t o x o s i d e , 3 = d i g o x i g e n i n b i s d i g i t o x o s i d e , 4 = d i g o x i n , 5 = d i g i t o x i g e n i n , 6 = d i g i t o x i g e n i n m o n o d i g i t o x o s i d e , 7 = d i g o x i g e n i n b i s d i g i t o x o s i d e and 8 = d i g i t o x i n . HPLC c o n d i t i o n s : same as i n F i g . 51 e x c e p t t h a t t he s o l v e n t system i s w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 43/35/15/7. 149. the d i g i t o x i n s e r i e s o f compounds and d i g o x i n i n about e i g h t minutes w h i l e t h e peaks o f t h e d i g o x i n m e t a b o l i t e s a r e c l u s t e r e d t o g e t h e r a t the i n i t i a l p o r t i o n o f t h e chromatogram w i t h t h e d i g o x i g e n i n and d i g o x i g e n i n m o n o d i g i t o x o s i d e peaks c o - e l u t i n g . T h i s chromatogram ( F i g . 53) r e p r e s e n t s t h e f a s t e s t s e p a r a t i o n o f t h e d i g i t o x i n s e r i e s and i s o n l y l i m i t e d by the r e s o l u t i o n x o f d i g i t o x i g e n i n and d i g i t o x i g e n i n m o n o d i g i t o x o s i d e . The HPLC system used f o r t h i s s e p a r a t i o n a p p e a r s t o be u s e f u l f o r a f a s t s i m u l t a n e o u s a n a l y s i s o f d i g i t o x i n and i t s m e t a b o l i t e s i n c l u d i n g d i g o x i n . S i n c e the sequence o f e l u t i o n i s kept c o n s t a n t i n a l l s e p a r a t i o n s a more n o n - p o l a r s o l v e n t system w i l l be a b l e t o i s o l a t e d i g i t o x i n i n a much s h o r t e r p e r i o d o f t i m e . Such a system would be u s e f u l i n s i t u a t i o n s where the p r i m a r y c o n c e r n i s t h e i s o l a t i o n and q u a n t i t a t i o n o f d i g i t o x i n . In s i t u a t i o n s where the compounds o f i n t e r e s t a r e d i g o x i n and i t s m e t a b o l i t e s , t h e i n i t i a l p o r t i o n o f t h e chromtogram shown i n F i g . 51 can be expanded i n such a way t h a t b a s e l i n e s e p a r a t i o n o f t h e s e compounds can be a c h i e v e d . The p o s s i b i l i t y o f such a chromatogram was i n v e s t i g a t e d by v a r y i n g t h e p r o p o r t i o n s o f the m o b i l e phase components i n o r d e r t o o b t a i n a r e l a t i v e l y more p o l a r s o l v e n t system w i t h s u f f i c i e n t d i f f e r e n t i a t i n g c a p a c i t y . A s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h i o r o m e t h a n e : 51/42/5/2 r e s u l t e d i n a b a s e l i n e s e p a r a t i o n o f d i g o x i g e n i n , d i g o x i g e n i n m o n o d i g i t o x o s i d e , d i g o x i g e n i n b i s d i g i t o x o s i d e and d i g o x i n i n a chromato-g r a p h i c t i m e o f about n i n e m i n u t e s ( F i g . 5 4 ) . T h i s i s o c r a t i c system i s f a s t e r than the g r a d i e n t system r e p o r t e d ( C a s t l e , 1975) f o r t h e r e s o l u t i o n o f d i g o x i n and i t s m e t a b o l i t e s . G i t o x i n i s known t o be a common i m p u r i t y o f d i g o x i n , and the USP monograph on d i g o x i n i n c l u d e s a t e s t f o r g i t o x i n . T h e r e f o r e , the p o s s i b i -l i t y o f i t s s e p a r a t i o n from t h e d i g o x i n s e r i e s was i n v e s t i g a t e d . I t was 150. 4 3 I 1 2 MINUTES F i g . 54. A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a S t a n d a r d M i x t u r e o f D i g o x i g e n i n (peak 1 ) ; D i g o x i g e n i n m o n o d i g i t o x o s i d e (peak 2 ) ; D i g o x i g e n i n b i s d i g i t o x o s i d e (peak 3) and D i g o x i n (peak 4) as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 51/42/5/2. Othe r HPLC c o n d i t i o n s : same as i n F i g . 51. found t h a t the s o l v e n t system, w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 47/40/9/4 e l u t e s g i t o x i n a t a c h r o m a t o g r a p h i c time o f about 12 m i n u t e s , s e p a r a t i n g i t from d i g o x i n , d i g i t o x i n and t h e i r r e s p e c t i v e m e t a b o l i t e s ( F i g . 5 5 ) . P r e v i o u s a t t e m p t s (Cobb, 1975) t o s e p a r a t e a and B - a c e t y l d i g o x i n have not been f u l l y s u c c e s s f u l . The p o s s i b i l i t y o f t h e i r r e s o l u t i o n by any o f t h e systems d e s c r i b e d above w a s , - t h e r e f o r e , s t u d i e d . The complete s e p a r a t i o n o f a and 8 - a c e t y l d i g o x i n as w e l l as d i g o x i n and i t s m e t a b o l i t e s was found t o be p o s s i b l e ( F i g . 56) u s i n g t h e s o l v e n t system w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 51/42/5/2. T h i s system, which i s i d e n t i c a l t o t h a t used f o r the r e s o l u t i o n o f t h e d i g o x i n s e r i e s ( F i g . 54) e l u t e s a and B - a c e t y l d i g o x i n i n a c h r o m a t o g r a p h i c time o f about 14 and 20 m i n u t e s , r e s p e c t i v e l y . (B) S e p a r a t i o n by G r a d i e n t E l u t i o n Even though i t was p o s s i b l e t o r e s o l v e a l l o f t h e components o f a s t a n d a r d m i x t u r e o f d i g o x i n , d i g i t o x i n and t h e i r m e t a b o l i t e s by i s o c r a t i c e l u t i o n , the p o s s i b i l i t y o f a r e d u c e d c h r o m a t o g r a p h i c time w i t h g r a d i e n t e l u t i o n was i n v e s t i g a t e d . A f t e r a number o f attempts t o o b t a i n an optimum g r a d i e n t system, a s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o -methane: 49/41/7/3 was found t o be most c o n v e n i e n t f o r the i n i t i a l p o r t i o n o f t h e chromatogram ( F i g . 5 7 ) . A l i n e a r g r a d i e n t o f 0 t o 100% o f a second s o l v e n t system ( w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 38/32/20/10) o b t a i n e d between t h e c h r o m a t o g r a p h i c times o f 2.5 t o 3 m i n u t e s and m a i n t a i n e d t i l l t h e end o f the r u n , r e s u l t e d i n a complete s e p a r a t i o n o f a l l compo-nents i n about 13 minutes ( F i g . 5 7 ) . I t can be o b s e r v e d from the chroma-togram t h a t t h i s method i s much f a s t e r than t h e g r a d i e n t s e p a r a t i o n ( i n 21.5 m i n u t e s ) t h a t was p r e v i o u s l y r e p o r t e d ( C a s t l e , 1975). In o r d e r 152. F i g . 55. A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a S t a n d a r d M i x t u r e o f D i g o x i n , D i g i t o x i n , t h e r e s p e c t i v e M e t a b o l i t e s and G i t o x i n . Sequence o f e l u t i o n : 1 = unknown i m p u r i t y ; 2 = d i g o x i g e n i n ; 3 = d i g o x i g e n i n m o n o d i g i t o x o s i d e ; 4 = d i g o x i g e n i n b i s d i g i t o x o s i d e ; 5 = d i g o x i n ; 6 = d i g i t o x i g e n i n ; 7 = d i g i t o x i g e n i n m o n o d i g i t o x o s i d e ; 8 = g i t o x i n ; 9 = d i g i t o x i g e n i n b i s d i g i t o x o s i d e and 10 = d i g i t o x i n . HPLC c o n d i t i o n s : same as i n F i g . 51. 153. F i g . 56. A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a and B - a c e t y l d i g o x i n from D i g o x i n and i t s M e t a b o l i t e s . Sequence o f e l u t i o n : 1 = d i g o x i g e n i n ; 2 = d i g o x i g e n i n m o n o d i g i t o x o s i d e ; 3 = d i g o x i g e n i n b i s d i g i t o x o s i d e ; 4 = d i g o x i n ; 5 = a - a c e t y l d i g o x i n ; and 6 = 8 - a c e t y l -d i g o x i n . HPLC c o n d i t i o n s : same as i n F i g . 54. 154. MINUTES F i g . 57. A Chromatogram f o r t h e HPLC S e p a r a t i o n o f a M i x t u r e o f D i g o x i n , D i g i t o x i n and t h e i r M e t a b o l i t e s by G r a d i e n t E l u t i o n . Sequence o f e l u t i o n : same as i n F i g . 53. HPLC c o n d i t i o n s : same as i n F i g . 53 w i t h t h e f o l l o w i n g e x c e p t i o n s - A s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 49/41/7/3 i s used as the i n i t i a l e l u t i n g s o l v e n t and a l i n e a r g r a d i e n t from 0 t o 100% o f a second s o l v e n t system ( w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 38/32/20/10) o b t a i n e d a t t h e c h r o m a t o g r a p h i c time o f 2.5 t o 3 mi n u t e s i s m a i n t a i n e d u n t i l t h e end o f t h e r u n . Range = 0.1; a t t e n u a t i o n = 9; column r e - e q u i l i b r a t i o n t a k e s about 15 m i n u t e s . 155. t o m i n i m i z e t h e b a s e l i n e f l u c t u a t i o n s t h a t a r e a s s o c i a t e d w i t h g r a d i e n t e l u t i o n , low d e t e c t o r s e t t i n g s and a r e l a t i v e l y h i g h sample l o a d were employed. As d i s c u s s e d b e f o r e , t h e c o n s t r a i n t s o f low d e t e c t o r s e n s i t i v i t y may p r e c l u d e the use o f g r a d i e n t e l u t i o n f o r t h e a n a l y s i s o f c a r d i a c g l y c o s i d e s . N e v e r t h e l e s s , t h e f a s t g r a d i e n t s e p a r a t i o n can be u s e f u l f o r qual i t a t i v e HPLC work w i t h t h e s e compounds. (C) S e p a r a t i o n u s i n g a S o l v e n t S w i t c h - o v e r System The i n i t i a l use o f a s o l v e n t system t h a t would be a b l e t o r e s o l v e a l l o f t h e d i g o x i n s e r i e s a t t h e e a r l i e r p a r t o f t h e chromatogram, w i t h a s u bsequent change-over t o a r e l a t i v e l y l e s s p o l a r s o l v e n t t h a t would r a p i d l y e l u t e t h e d i g i t o x i n s e r i e s i n the same chromatogram, w h i l e m a i n t a i n i n g b a s e l i n e r e s o l u t i o n was t r i e d . A s w i t c h - o v e r from an i n i t i a l s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 49/41/7/3 t o a second s o l v e n t system w i t h a p r o p o r t i o n o f 41/34/17/8 a t a chroma-t o g r a p h i c time t h a t c o r r e s p o n d s w i t h the peak o f d i g o x i g e n i n b i s d i g i t o -x o s i d e r e s u l t e d i n a 16-minute chromatogram t h a t shows c o m p l e t e s e p a r a t i o n o f a l l t h e compounds ( F i g . 5 8 ) . I t can be seen t h a t t h i s chromatogram c l o s e l y a p p r o x i m a t e s the s e p a r a t i o n o b t a i n e d by g r a d i e n t e l u t i o n . The s o l v e n t s w i t c h - o v e r method, t h e r e f o r e , can be u s e f u l f o r q u a l i t a t i v e HPLC work i n s i t u a t i o n s where t h e l i q u i d chromatograph does not have g r a d i e n t c a p a b i l i t y . As i n d i c a t e d e a r l i e r , changes i n the p r o p o r t i o n o f the s o l v e n t s employed i n t h e four-component s o l v e n t system have been shown to b r i n g a bout an e x p a n s i o n o r c o n t r a c t i o n o f t h e d i s t a n c e s between a d j a c e n t peaks and hence r e s u l t i n g i n t h e d e s i r e d s e p a r a t i o n s . T h i s c o u l d be due t o changes i n any one o r a c o m b i n a t i o n o f the f o l l o w i n g f a c t o r s : p o l a r i t y , s o l u b i l i t y e f f e c t and d i f f u s i o n e f f e c t ( v i s c o s i t y ) . I t can a l s o be 156. 7 I I I I I 0 5 10 15 20 MINUTES Fig. 58. A Chromatogram for the HPLC Separation of a mixture of Digoxin, Digitoxin and thei r Metabolites by Solvent Switchover elution. Sequence of elution and HPLC conditions: same as in Fig. 57 with the following exception: A solvent system of water/methanol/ isopropanol/dichloromethane: 49/41/7/3 i s used as the i n i t i a l eluting solvent and a switchover to a second solvent system (water/methanol/isopropanol/ dichloromethane: 41/34/17/8 i s made at a point in time that corresponds to the peak of digoxigenin bisdigitoxoside. Table X. Retention Times (tr, in minutes) and Capacity Ratio Values (<') of the Compojnds Studied, under the Conditions Defined by the Corresponding Figures Compound Fig tr . 51 K' Fig. tr 52 K' Fig. tr 53 K1 Fig. tr 54 K' . F i 9 . tr 55 K' Fig. tr 56 K' Fig. tr 57 K' Fig. tr 58 V Digoxigenin 2.63 0.31 2.40 0.20 2.32 0.16 3.15 0.57 2.78 0.35 2.60 0.30 3.37 0.68 2.95 0.47 Digoxigenin monodigitoxoside 2.87 0.43 2.51 0.25 2.32 0.16 3.76 0.88 3.04 0.46 3.30 0.65 4.00 1.00 3.36 0.68 Digoxigenin bisdigitoxoside 3.62 0.81 2.91 0.45 2.58 0.25 5.81 1.90 3.88 0.80 5.20 1.60 6.03 1 .01 4.59 1.29 Digoxin 4.72 1.36 3.43 0.71 2.90 0.45 9.14 3.57 5.14 1.46 9.00 3.50 8.27 3.31 6.78 2.39 Digitoxigenin 7.29 2.64 5.21 1.60 4.15 1.07 9.73 3.86 10.11 4.05 Digitoxigenin mono-dlgttoxoside 10.35 4.17 6.45 2.22 4.62 1.31 10.83 4.41 12.28 5.14 Digitoxigenin bisdigitoxoside 15.93 6.96 8.72 3.36 5.75 1.87 11.70 4.85 13.81 5.90 Digitoxin 27.38 12.69 12.99 5.49 7.65 2.82 12.90 5.45 15.68 6.84 Gitoxin 12.48 5.08 a-Acetyldigoxin 14.00 6.00 B-Acetyldigoxin 20.43 9.21 158. o b s e r v e d t h a t t h e sequence o f e l u t i o n o f t h e compounds remains the same i n a l l c a s e s . For purposes o f co m p a r i s o n o f t h e v a r i o u s chromatograms t h a t have been o b t a i n e d , v a l u e s f o r r e t e n t i o n time and c a p a c i t y r a t i o f a c t o r ( K 1 ) a r e p r e s e n t e d i n T a b l e X. I t can perhaps be s u r m i s e d , t h e r e f o r e , t h a t t h e v e r s a t i l i t y o f t h i s s o l v e n t system i s such t h a t i t may be a p p l i -c a b l e f o r t h e s e p a r a t i o n o f o t h e r c a r d i a c g l y c o s i d e s and even s t e r o i d s i n g e n e r a l . Moreover, under t i g h t l y c o n t r o l l e d c o n d i t i o n s , t h e s p e c i f i -c i t y o f t h e r e t e n t i o n t i m e s may w a r r a n t t h e use o f t h e s e methods f o r c o n f i r m a t o r y i d e n t i f i c a t i o n o f any o f t h e c a r d i a c g l y c o s i d e s . 3. Development o f a 100% F l u i d R e c o v e r y System f o r the HPLC A n a l y s i s o f  D i g o x i n and i t s M e t a b o l i t e s a f t e r F l u o r o g e n i c Post-Column D e r i v a t i - z a t i o n u s i n g t h e A i r - s e g m e n t a t i o n P r o c e s s The p r e s e n c e o f d i h y d r o d e r i v a t i v e s o f d i g o x i n , d i g o x i g e n i n and th e mono- and b i s d i g i t o x o s i d e s as m e t a b o l i c p r o d u c t s o f d i g o x i n has been r e p o r t e d ( G a u l t e t a l . , 1980). The d i h y d r o p r o d u c t s a r e formed by the r e d u c t i o n o f the - d o u b l e bond o f the l a c t o n e r i n g and t h e r e f o r e do not a b s o r b UV r a d i a n t e n e r g y . Hence HPLC s e p a r a t i o n o f t h e s e compounds can not be m o n i t o r e d u s i n g a UV d e t e c t o r . For purposes o f a t t a i n i n g i n c r e a s e d s e n s i t i v i t y by f l u o r e s c e n c e d e t e c t i o n G f e l l e r e t a l . (1977) have r e p o r t e d a post-column f l u o r o g e n i c d e r i v a t i z a t i o n p r o c e d u r e u s i n g t h e a i r - s e g m e n t a t i o n p r o c e s s f o r the HPLC a n a l y s i s o f some c a r d i a c g l y c o -s i d e s . The p o s s i b i l i t y o f u s i n g t h i s f l u o r o g e n i c d e r i v a t i z a t i o n p r o c e d u r e f o r m o n i t o r i n g t h e HPLC s e p a r a t i o n o f d i g o x i n and i t s m e t a b o l i t e s i n c l u d i n g some o f t h e d i h y d r o p r o d u c t s was t h e r e f o r e i n v e s t i g a t e d . The post-column d e r i v a t i z a t i o n s e t - u p was m o d i f i e d f o r purposes o f a t t a i n i n g 100% f l u i d r e c o v e r y . A s c h e m a t i c diagram o f the m o d i f i e d p o s t - c o l u m d e r i v a t i z a t i o n s e t - u p i s shown i n F i g . 22. The pump t u b e s ; t he D 2 c o n n e c t o r s ; t h e m i x i n g r e a c t i o n and c o o l i n g c o i l s and d e b u b b l e r were the same as i n t h e s e t - u p d e s c r i b e d by G f e l l e r e t a l . (1977) e x c e p t t h a t : (1) the D 2 c o n n e c t o r s and d e b u b b l e r were r e p l a c e d by m i n i a t u r i z e d ones (1 mm i . d . ) ; (2) the v e r t i c a l e x i t o f the d e b u b b l e r was c o n n e c t e d by means o f an a c i d f l e x t u b i n g , t o an o v e r h a n g i n g g l a s s t u b e ; and (3) t h e r e a c t i o n chamber was m a i n t a i n e d a t 55°C. The HPLC e l u a t e was passed t h r o u g h a UV d e t e c t o r , t he r e a c t i o n system, the d e b u b b l e r s e t - u p and t h e f l u o r o m e t e r b e f o r e i t was d i r e c t e d i n t o the waste r e c e p t a c l e The s e t - u p f o r t h e 100% f l u i d r e c o v e r y system c o n s i s t s o f an 84 cm l o n g g l a s s tube (1.1 cm i . d . ) t a p e r i n g a t i t s lower end to 1 mm i . d . where i t i s c o n n e c t e d t o a m o d i f i e d C^ d e b u b b l e r which has s i d e tubes o f 1 mm i . d . The g l a s s tube i s p o s i t i o n e d as h i g h as p o s s i b l e so t h a t when t h r e e - q u a r t e r s f u l l w i t h t h e c i r c u l a t i n g f l u i d , i t e x e r t s hydro-s t a t i c p r e s s u r e on the s u r f a c e o f t h e l i q u i d i n the d e b u b b l e r . The a i r -segmented f l u i d e n t e r i n g t he d e b u b b l e r , t h e r e f o r e , passes t h r o u g h t h e h o r i z o n t a l e x i t i n t o t h e f l u o r e s c e n c e d e t e c t o r , whereas the segments o f a i r escape t h r o u g h t h e v e r t i c a l e x i t as b u b b l e s . In o r d e r t o a t t a i n 100% f l u i d r e c o v e r y , t he d e b u b b l i n g p r o c e s s i s o p t i m i z e d by c a r e f u l up and down a d j u s t m e n t s o f the p o s i t i o n o f t h e waste r e c e p t a c l e u n t i l the upward p r e s s u r e ( i n t h e d e b u b b l e r ) e x e r t e d by the pump i s e q u a l i z e d by the h y d r o s t a t i c p r e s s u r e o f t h e f l u i d i n the suspended g l a s s t u b e . The f l u i d coming o u t o f t h e f l u o r e s c e n c e d e t e c t o r had a f l o w r a t e o f about 1 ml per minute and pumping t h e waste f l u i d o u t o f the d e t e c t o r was not found t o bre necessary-, HPLC s e p a r a t i o n o f d i g o x i n and i t s m e t a b o l i t e s u s i n g UV d e t e c t i o n and t h e f l u o r o g e n i c d e r i v a t i z a t i o n p r o c e d u r e d e s c r i b e d above, i s p r e s e n t e d 160. i n F i g . 59. The UV and f l u o r e s c e n c e d e t e c t o r r e s p o n s e s were o b t a i n e d w i t h a dual pen r e c o r d e r a f t e r one sample i n j e c t i o n . The i n i t i a l p o r t i o n o f t h e chromatogram ( F i g . 5 9 ( a ) ) shows t h e s e p a r a t i o n o f d i g o x i n (Peak 4 ) , d i g o x i -g e n i n (Peak 1) and the mono- and b i s d i g i t o x o s i d e s (Peaks 2 and 3, r e s p e c t i v e l y ) a f t e r UV d e t e c t i o n a t a w a v e l e n g t h o f 254 nm. Even though the sample c o n t a i n s d i h y d r o d i g o x i g e n i n , t h i s compound does not have a peak i n t h e UV m o n i t o r e d chromatogram s i n c e i t does not have UV a b s o r b a n c e . A f t e r o n - l i n e p o s t -column f l u o r o g e n i c d e r i v a t i z a t i o n , however, a l l o f the f i v e compounds a r e d e t e c t e d by the f l u o r o m e t e r a f t e r a t o t a l c h r o m a t o g r a p h i c and d e r i v a t i z a t i o n p e r i o d o f 36 m i n u t e s . T h i s i s shown i n t h e l a t t e r p o r t i o n o f the chromato-gram ( F i g . 5 9 ( b ) ) where d i h y d r o d i g o x i g e n i n and d i g o x i g e n i n e l u t e t o g e t h e r (Peaks 5, 6) w h i l e d i g o x i g e n i n m o n o d i g i t o x o s i d e (Peak 7 ) , d i g o x i g e n i n b i s -d i g i t o x o s i d e (Peak 8) and d i g o x i n (Peak 9) a r e s e p a r a t e d . The s i m u l t a n e o u s e l u t i o n o f d i h y d r o d i g o x i g e n i n and d i g o x i g e n i n i s e v i d e n t i n t h e g r e a t e r magnitude o f Peak 5, 6 r e l a t i v e t o Peak 7 as compared to t h e r e l a t i v e s i z e s o f Peaks 1 and 2. S e p a r a t i o n o f d i h y d r o d i g o x i g e n i n (Peak 1) and d i g o x i g e n i n m o n o d i g i t o x o s i d e (Peak 2) i s shown i n the i n s e t o f F i g . 59. I t can be o b s e r v e d t h a t f l u o r o m e t r i c m o n i t o r i n g o f d i g o x i n and i t s m e t a b o l i t e s r e s u l t s i n a chromatogram w i t h w e l l d e f i n e d s y m m e t r i c a l peaks. I t has been shown t h a t the s o l v e n t system ( w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 50/41/6/3) used f o r g e n e r a t i n g t h e chromatogram i n F i g . 59 i s c o m p a t i b l e w i t h the aqueous media o f the d e r i v a t i z a t i o n p r o c e s s . The d e b u b b l i n g system t h a t was d e v e l o p e d f o r 100% f l u i d r e c o v e r y p r i o r t o f l u o r o m e t r i c d e t e c t i o n was found t o be s t a b l e o v e r l o n g p e r i o d s o f t i m e . Complete f l u i d r e c o v e r y w i t h o u t t h e p r e s e n c e o f any v i s i b l e a i r b u b b l e s c o u l d c o n t i n u o u s l y be m a i n t a i n e d f o r as l o n g as e i g h t h o u r s . I t i s t o be n o t e d t h a t the 100% f l u i d r e c o v e r y s y s t e m , by v i r t u e o f p r e v e n t i n g sample l o s s , can be u s e f u l f o r p u r p o s e s o f c o l l e c t i n g t h e r e l a t i v e l y s m a l l 161. F i g . 59. A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a M i x t u r e o f D i g o x i n , D i h y d r o d i g o x i g e n i n and th e o t h e r D i g o x i n M e t a b o l i t e s as o b t a i n e d by Dual D e t e c t o r M o n i t o r i n g . P e a k i d e n t i t i e s : (a) UV d e t e c t i o n (A254)- (1) d i g o x i g e n i n , (2) d i g o x i g e n i n m o n o d i g i t o x o s i d e , (3) d i g o x i g e n i n b i s d i g i t o x o s i d e , (4) d i g o x i n ; (b) F l u o r e s c e n c e d e t e c t i o n ( A e x c _ = nm and Aem. c u t o f f = 460) - (5,6) d i g o x i g e n i n and d i h y d r o d i g o x i g e n i n , (7) d i g o x i g e n i n m o n o d i g i t o x o s i d e , (8) d i g o x i g e n i n b i s d i g i t o x o s i d e , (9) d i g o x i n . HPLC c o n d i t i o n s : U l t r a s p h e r e ODS column; s o l v e n t system, w a t e r / m e t h a n o l / i s o p r o p a n o l / d i e h i o r o m e t h a n e : 50/41/6/3; C h a r t speed 0.5 cm/min. The compounds were d i s s o l v e d i n t h e e l u t i n g s o l v e n t . I n s e t : A chromatogram f o r the s e p a r a t i o n o f d i h y d r o d i g o x i g e n i n c O and d i g o x i g e n i n m o n o d i g i t o x o s i d e # u n d e r t he c o n d i t i o n s o f ( b ) . 162. M I N U T E S 163. amounts o f s e p a r a t e d d i h y d r o m e t a b o l i t e s o f d i g o x i n f o r s u b s e q u e n t q u a n t i t a -t i o n u s i n g more s e n s i t i v e methods. U s i n g the s e t - u p , the minimum d e t e c t a b l e amount o f d i g o x i n ( a t a r e s p o n s e t o n o i s e r a t i o o f 2:1) was found t o be 10 ng. 4. S e p a r a t i o n o f Nine E q u i n e E s t r o g e n s u s i n g the HPLC System, as e v i d e n c e  o f S e l e c t i v i t y In t h e s e a r c h f o r i n t e r n a l s t a n d a r d s t o be used f o r t h e HPLC a n a l y s i s o f c a r d i a c g l y c o s i d e s . , v a r i o u s s t e r o i d s i n c l u d i n g the e q u i n e e s t r o g e n s were c o n s i d e r e d . T h r e e e q u i n e e s t r o g e n s were found to s a t i s f y t he r e q u i r e d c h r o m a t o g r a p h i c c o n d i t i o n s . D u r i n g t h e c o u r s e o f t h i s s t u d y i t was a l s o o b s e r v e d t h a t most o f the n i n e e q u i n e e s t r o g e n s t h a t were t e s t e d had somewhat d i f f e r e n t r e t e n t i o n t i m e s . An i n s p e c t i o n o f the c h e m i c a l s t r u c t u r e s o f t h e s e compounds ( F i g . 21) i n d i c a t e s t h a t t h e y a r e c l o s e l y r e l a t e d and d i f f e r o n l y on t h e b a s i s o f whether o r n o t t h e y have (1) a - h y d r o x y l , s-hydroxyl o r a k e t o group on C-17 and (2) two, one o r no d o u b l e bond on r i n g B o f the s t e r o i d m o i e t y . The s t r u c t u r a l d i f f e r e n c e s among t h e c a r d e n o l i d e s shown i n T a b l e VII appear t o be more s i g n i f i c a n t compared t o the v a r i a t i o n s among the e s t r o g e n s . I f t h e HPLC system t h a t has been d e v e l o p e d c o u l d s e p a r a t e a m i x t u r e o f compounds w i t h such m i n o r s t r u c t u r a l d i f f e r e n c e s i t would be an a d d i t i o n a l e v i d e n c e o f s e l e c t i v i t y . The p o s s i b i l i t y o f such a s e p a r a t i o n was, t h e r e f o r e , i n v e s t i g a t e d as a t e s t f o r t h e a b i l i t y o f the s o l v e n t system t o d i f f e r e n t i a t e r e l a t e d s t e r o i d s . The s e p a r a t i o n o f a s t a n d a r d m i x t u r e o f t h e n i n e e q u i n e e s t r o g e n s and 1 7 a - e t h y n y l e s t r a d i o l , o b t a i n e d w i t h a s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 48/40/8/4, i s shown i n F i g . 60. I t can be o b s e r v e d t h a t a l l components a r e s u f f i c i e n t l y d i f f e r e n t i a t e d t o w a r r a n t peak h e i g h t q u a n t i t a t i o n i n a c h r o m a t o g r a p h i c time o f about 16 m i n u t e s . E q u i l i n (peak 10) and e s t r o n e (peak 11) which a r e t h e r a p e u t i c a l l y t h e 164. F i g . 60. A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a m i x t u r e o f n i n e E q u i n e E s t r o g e n s and 17a-e t h y n y l e s t r a d i o l . Sequence o f e l u t i o n : (1) s o l v e n t peak; (2) 1 7 8 - d i h y d r o e q u i l e n i n ; (3) 1 7 a - d i h y d r o e q u i l e n i n ; (4) 1 7 6 - d i h y d r o e q u i l i n ; (5) 1 7 a - d i h y d r o e q u i l i n ; (6) 1 7 a - e t h y n y l e s t r a d i o l ; (7) 1 7 8 - e s t r a d i o l ; (8) 1 7 a - e s t r a d i o l ; (9) e q u i l e n i n ; (10) e q u i l i n ; (11) e s t r o n e . HPLC c o n d i t i o n s : U l t r a s p h e r e ODS column; s o l v e n t system, w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 48/40/8/4; f l o w r a t e , 1.2 ml/min; UV d e t e c t i o n a t 220 nm; C h a r t s p e e d , 0.5 cm/min. The compounds were d i s s o l v e d i n 35% m e t h a n o l . 165. most i m p o r t a n t e s t r o g e n s , a r e c o m p l e t e l y s e p a r a t e d . A 22-minute chromatogram t h a t shows b a s e l i n e s e p a r a t i o n o f 176-d i h y d r o e q u i l e n i n (peak 2 ) , 1 7 a - e s t r a d i o l (peak 7 ) , e q u i l e n i n (peak 8 ) , e q u i l i n e (peak 9) and e s t r o n e (peak 10) as w e l l as a f a i r l y good s e p a r a t i o n o f 1 7 a - d i h y d r o e q u i l e n i n (peak 3) from 1 7 6 - d i h y d r o e q u i l i n (peak 4) and 1 7 a - d i h y d r o e q u i l i n (peak 5) from 1 7 6 - e s t r a d i o l (peak 6) i s p r e s e n t e d i n F i g . 61. T h i s chromatogram was o b t a i n e d w i t h a s o l v e n t system o f w a t er/ m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 49/41/7/3. The complete b a s e l i n e s e p a r a t i o n o f t h e n i n e e q u i n e e s t r o g e n s , i n about 34 m i n u t e s ( F i g . 62) was found to be p o s s i b l e w i t h a more p o l a r s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 52/43/3/2. Peak 1 and t h e n e g a t i v e peaks a r e due t o s o l v e n t e f f e c t . The n i n e e s t r o -gens e l u t e i n the f o l l o w i n g sequence: 1 7 6 - d i h y d r o e q u i l e n i n (peak 2 ) , 1 7 a - d i h y d r o e q u i l e n i n (peak 3 ) , 1 7 6 - d i h y d r o e q u i l i n (peak 4 ) , 1 7 a - d i h y d r o -e q u i l i n (peak 5 ) , 1 7 a - e s t r a d i o l (peak 6 ) , e q u i l e n i n (peak 7 ) , 1 7 6 - e s t r a d i o l (peak 8 ) , e q u i l i n (peak 9) and e s t r o n e (peak 1 0 ) . I t i s i n t e r e s t i n g to o b s e r v e t h a t 1 7 6 - e s t r a d i o l i s r e l a t i v e l y more r e t a i n e d and t h e r e f o r e shows a r e v e r s a l i n i t s sequence o f e l u t i o n w i t h r e s p e c t t o 1 7 a - e s t r a d i o l and e q u i l e n i n . The sequence o f e l u t i o n o f the o t h e r s , however, remains the same. I t i s perhaps because o f t h i s e x t r a - s e n s i t i v i t y t o p o l a r i t y changes t h a t 1 7 6 - e s t r a d i o l was a major s o u r c e o f problems i n r e s o l u t i o n (Johnson e t a l . , 1975). The HPLC s e p a r a t i o n s shown i n F i g s . 60, 61 and 62 were o b t a i n e d w i t h s o l v e n t systems t h a t c o n s i s t o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e i n t h e p r o p o r t i o n s o f : 52/43/3/2, 49/41/7/3 and 48/40/8/4, r e s p e c t i v e l y . I t can t h e r e f o r e be o b s e r v e d t h a t the s e l e c t i v i t y and c a p a c i t y r a t i o f a c t o r s were o p t i m i z e d by s l i g h t changes i n t h e r e l a t i v e p r o p o r t i o n s o f the s o l v e n t s . 166. F i g . 61. A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f a m i x t u r e o f n i n e Equine E s t r o g e n s as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 49/41/7/3. Sequence o f e l u t i o n : (1) s o l v e n t peak; (2) 1 7 8 - d i h y d r o e q u i l e n i n ; (3) 1 7 a - d i h y d r o e q u i l e n i n ; (4) 1 7 0 - d i h y d r o e q u i l i n ; (5) 17a-dihydroequi1 i n ; (6) 1 7 g - e s t r a d i o l ; (7) 1 7 a - e s t r a d i o l ; (8) e q u i l e n i n ; (9) e q u i l i n ; (10) e s t r o n e . Other HPLC c o n d i t i o n s : same as i n F i g . 60 e x c e p t t h a t t h e compounds were d i s s o l v e d i n 50% e t h a n o l . MINUTES F i g . 62. A Chromatogram f o r Complete I s o c r a t i c HPLC S e p a r a t i o n o f a m i x t u r e o f n i n e Equine E s t r o g e n s as o b t a i n e d w i t h a S o l v e n t System o f w a t e r / m e t h a n o l / i s o p r o -p a n o l / d i c h l o r o m e t h a n e : 52/43/3/2. Sequence o f e l u t i o n : (1) s o l v e n t peak; (2) 1 7 6 - d i h y d r o e q u i l e n i n ; (3) 1 7 a - d i h y d r o e q u i l e n i n ; (4) 1 7 8 - d i h y d r o e q u i l i n ; (5) 17a-dihydroequil i n ; (6) 1 7 a - e s t r a d i o l ; (7) e q u i l e n i n ; (8) 1 7 8 - e s t r a d i o l ; (9) e q u i l i n ; (10) e s t r o n e . Other HPLC c o n d i t i o n s : same as i n F i g . 61 e x c e p t t h a t t he f l o w r a t e was 1.3 ml/min. _ cn 168. The c u r r e n t USP a s s a y p r o c e d u r e f o r C o n j u g a t e d E s t r o g e n s , and E s t e r i f i e d E s t r o g e n s and t h e i r r e s p e c t i v e t a b l e t dosage forms i s b a s i c a l l y c o l o r i -m e t r i c a f t e r column c h r o m a t o g r a p h i c s e p a r a t i o n and a s e r i e s o f l o n g and t e d i o u s e x t r a c t i o n s t e p s . Compared t o t h e slow s e p a r a t i o n p r o c e s s and o f f - l i n e d e t e c t i o n o f t e n a s s o c i a t e d w i t h column chromatography and the d e r i v a t i z a t i o n s t e p t h a t i s i n h e r e n t i n GLC, HPLC appears to be the t e c h n i q u e o f c h o i c e f o r t h e a n a l y s i s o f c o n j u g a t e d and e s t e r i f i e d e s t r o g e n s . T h i s i s so because the HPLC method d e s c r i b e d above i s f a s t e r and more s e l e c t i v e than column chromatography, a v o i d s the d e r i v a t i z a t i o n s t e p o f GLC and t h e r e f o r e has a d e f i n i t e advantage w i t h r e s p e c t t o time and s i m p l i c i t y o f a n a l y s i s . 5. I s o l a t i o n o f D i g i t o x i n from D i g i t a l i s p u r p u r e a L e a f D i g i t a l i s p u r p u r e a l e a f c o n t a i n s a m i x t u r e o f d i t i t o x i n , g i t o x i n , g i t a l o x i n , o t h e r r e l a t e d g l y c o s i d e s and p l a n t c o n s t i t u e n t s . The p u r i f i e d form o f d i g i t o x i n and the o t h e r c a r d i a c g l y c o s i d e s a r e o b t a i n e d from p l a n t e x t r a c t s a f t e r c h r o m a t o g r a p h i c s e p a r a t i o n and p u r i f i c a t i o n p r o c e s s e s . The f e a s i b i l i t y o f u s i n g the s o l v e n t system t h a t has been d e v e l o p e d , f o r t h e i s o l a t i o n o f d i g i t o x i n from the l e a f e x t r a c t was, t h e r e f o r e , s t u d i e d . A f t e r a number o f o p t i m i z a t i o n s , t h e chromatogram shown i n F i g . 63 was o b t a i n e d u s i n g a s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 45/38/11/6. I t can be o b s e r v e d t h a t peak 6 (which 169. M I N U T E S F i g . 63. A Chromatogram f o r the I s o c r a t i c HPLC S e p a r a t i o n o f D i g i t o x i n from Other Components o f D i g i t a l i s p u r p u r e a L e a f . Peak i d e n t i t i e s : ( 1 , 2, 4, 5) unknowns; (3) g i t o x i n ; (6) d i g i t o x i n . HPLC c o n d i t i o n s : s o l v e n t system, w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o -methane: 45/38/11/6; f l o w r a t e , 1.1 ml/min; UV d e t e c t i o n a t 220 nm; C h a r t speed, 0.5 cm/min. 170. has the same r e t e n t i o n time as t h a t o f an a u t h e n t i c d i g i t o x i n sample) i s v e r y w e l l s e p a r a t e d from any o f t h e o t h e r components. Peak 3 was found t o r e p r e s e n t g i t o x i n . Peaks 1, 2, 4 and 5, however, were not i d e n t i f i e d . D i g i t a l i s powder, which i s c u r r e n t l y o f f i c i a l i n the USP c o n t a i n s a h i g h p r o p o r t i o n o f d i g i t o x i n and a b i o a s s a y method i s used t o m o n i t o r i t s p o t e n c y . I t a p p e a r s , t h e r e f o r e , t h a t t h i s HPLC method can t e u s e d : (1) a t l e a s t as a s u p p l e m e n t a r y t e c h n i q u e f o r f a s t m o n i t o r i n g o f the d i g i t o x i n c o n t e n t i n D i g i t a l i s powder and (2) f o r p r e p a r a t i v e HPLC work i n v o l v i n g i s o l a t i o n and p u r i f i c a t i o n o f d i g i t o x i n drug s u b s t a n c e . 6. Development o f HPLC Methods f o r t h e A n a l y s i s o f D i g o x i n i n i t s  Dosage Forms The a n a l y t i c methods r e p o r t e d i n t h e l i t e r a t u r e , d e a l i n g w i t h d i g o x i n t a b l e t f o r m u l a t i o n s a r e e s s e n t i a l l y c o l o r i m e t r i c ( M y r i c k , 1969; USP XX, 1980), f l u o r o m e t r i c ( C u l l e n t e t a l . , 1970; Nyberg e t a l . , 1974) and g a s - l i q u i d c h r o m a t o g r a p h i c ( K i b b e and A r a u j o , 1973) t e c h n i q u e s . The c o l o r i m e t r i c methods have t h e d i s a d v a n t a g e o f i n s u f f i c i e n t s e n s i t i v i t y and/or l a c k s e l e c t i v i t y . The f l u o r o m e t r i c method i s based on t h e d e t e r m i n a t i o n o f the d e h y d r a t i o n p r o d u c t s o f t h e s t e r o i d m o i e t y o f t h e d i g o x i n m o l e c u l e , a f t e r r e a c t i o n w i t h c o n c e n t r a t e d h y d r o c h l o r i c a c i d i n t h e p r e s e n c e o f hydrogen p e r o x i d e and a s c o r b i c a c i d . Even though the f l u o r o m e t r i c methods have t h e advantage o f h i g h s e n s i t i v i t y , t h e y a r e non s p e c i f i c . The gas l i q u i d c h r o m a t o g r a p h i c methods, i n g e n e r a l , i n v o l v e t h e p r o d u c t i o n o f d e r i v a t i v e s and r e q u i r e a g r e a t d e a l o f t i m e as w e l l as l a b o r i o u s m a n i p u l a t i v e s t e p s . The s e n s i t i v e GLC method r e p o r t e d by Watson e t a l . (1972) employs e l e c t r o n c a p t u r e d e t e c t i o n a f t e r d e r i v a t i z a t i o n t o g e n i n - d i h e p t a f l u o r o b u t y r a t e . Any d e g r a d a t i o n p r o d u c t o f d i g o x i n t h a t may be p r e s e n t , however, w i l l not be d i f f e r e n t i a t e d from d i g o x i n . The GLC method ( K i b b e and A r a n j o , 1973) f o r t h e a n a l y s i s o f d i g o x i n t a b l e t s i s a g a i n n o n - s p e c i f i c ( s i n c e d i g o x i n i s h y d r o l y z e d t o d i g o x i g e n i n p r i o r t o d e t e c t i o n ) and not s e n s i t i v e enough f o r s i n g l e t a b l e t a s s a y . The l a c k o f a m o b i l e phase t h a t has s u f f i c i e n t UV t r a n s p a r e n c y a t t h e w a v e l e n g t h o f maximum a b s o r p t i o n o f d i g o x i n (220 nm) and a s o l v e n t t h a t i s a p p r o p r i a t e f o r sample p r e p a r a t i o n , a p p e a r s t o have p r e v e n t e d the use o f HPLC i n t h e a n a l y s i s ( a f t e r UV d e t e c t i o n ) o f d i g o x i n dosage forms. I t i s c l e a r t h a t t h e r e i s a need f o r methods o f a n a l y s i s o f d i g o x i n i n i t s dosage f o r m s , t h a t a r e s e l e c t i v e enough t o p r e c l u d e a s s a y i n t e r f e r e n c e from d e g r a d a t i o n p r o d u c t s and s u f f i c i e n t l y s e n s i t i v e t o a l l o w s i n g l e t a b l e t a s s a y . The p o s s i b i l i t y f o r such methods, u s i n g HPLC, was t h e r e f o r e i n v e s t i g a t e d . The r e p r e s e n t a t i v e chromatogram ( F i g . 64) f o r the a n a l y s i s o f d i g o x i n i n t a b l e t dosage forms shows t h e peaks o f d i g o x i n and 1 7 a - e t h y n y l e s t r a d i o l ( i n t e r n a l s t a n d a r d ) w i t h r e t e n t i o n times o f 4.7 and 8.9 m i n u t e s , r e s p e c t -i v e l y . T h i s chromatogram was o b t a i n e d w i t h a s o l v e n t system o f w a t er/ m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 47/40/9/4. I t can be seen t h a t the d i g o x i n and i n t e r n a l s t a n d a r d peaks a r e f r e e from i n t e r f e r e n c e by t a b l e t e x c i p i e n t s . Peak 1 was found t o be an i m p u r i t y from t h e f i l t e r p aper. The two n e g a t i v e peaks i m m e d i a t e l y a f t e r peak 1 and the s m a l l n e g a t i v e peak between the d i g o x i n and i n t e r n a l s t a n d a r d peaks were due t o s o l v e n t e f f e c t . The b a s e l i n e p e r t u r b a t i o n t h a t o c c u r s between the peaks o f d i g o x i n and t h e i n t e r n a l s t a n d a r d was found t o appear i n t h e 172. J L 0 5 10 MINUTES Fig. 64. A Representative Chromatogram for the Isocratic HPLC Analysis of Digoxin Tablets. Peak i d e n t i t y : (1) unknown impurity; (2) digoxin; and (3) 17a-ethynylestradiol (internal standard)-HPLC condi-tions: solvent system, water/methanol/isopropanol/ dichloromethane: 47/40/9/4; flow rate, 1.2 ml/min; UV detection at 220 nm; Chart speed, 0.5 cm/min. 173. I I I I 0 5 10 li MINUTES F i g . 65. A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f D i g o x i n , i t s p r o b a b l e D e g r a d a t i o n P r o d u c t s , 1 7 a - e t h y n y l e s t r a d i o l ( i n t e r n a l s t a n d a r d ) and g i t o x i n : sequence o f e l u t i o n : (1) d i g o x i g e n i n ; (2) d i g o x i g e n i n m o n o d i g i t o x o s i d e ; (3) d i g o x i g e n i n b i s d i g i t o x o s i d e ; ( 4 ) d i g o x i n ; (5) 1 7 a - e t h y n y l e s t r a d i o l ; and (6) g i t o x i n . HPLC c o n d i t i o n s : same as i n F i g . 64. form o f a peak o r a b a s e l i n e d e p r e s s i o n d e p e n d i n g upon t he p r o p o r t i o n o f water and methanol i n the s o l v e n t used f o r sample p r e p a r a t i o n . I t was ob s e r v e d t h a t a methanol c o n t e n t o f about 35% v/v i n the h y d r o a l c o h o l i c s o l v e n t i n which t h e sample was d i s s o l v e d p r i o r t o i n j e c t i o n i n the l i q u i d c h r o m a t o g r a p h , e l i m i n a t e d t h e p e r t u r b a t i o n e f f e c t and m a i n t a i n e d b a s e l i n e s t a b i l i t y . A p r o p o r t i o n o f methanol t h a t was h i g h e r o r lower than about 35% v/v i n t r o d u c e d a n e g a t i v e o r p o s i t i v e peak, r e s p e c t i v e l y . The peak t h a t i s a s s o c i a t e d w i t h a h i g h e r water c o n t e n t a p p e a r s t o be due t o the f o r m a t i o n o f an e m u l s i o n between t h e m o b i l e phase and i n j e c t e d sample a t the p o i n t o f f i r s t c o n t a c t . Whereas the b a s e l i n e d e p r e s s i o n t h a t o c c u r s w i t h a h i g h e r methanol c o n t e n t seems t o be due t o a d i l u t i o n e f f e c t o f the i n j e c t e d sample s o l u t i o n on the m o b i l e phase a t the p o i n t o f c o n t a c t , thus m o m e n t a r i l y r e d u c i n g the b a s e l i n e a b s o r b a n c e l e v e l . The HPLC method f o r d i g o x i n t a b l e t a s s a y was found t o be c a p a b l e o f a c c o u n t i n g f o r each o f the p r o b a b l e d e g r a d a t i o n p r o d u c t s o f d i g o x i n as w e l l as g i t o x i n which i s a common c o n t a m i n a n t o f d i g o x i n , as shown i n F i g . 65. D i g o x i g e n i n , d i g o x i g e n i n m o n o d i g i t o x o s i d e , d i g o x i g e n i n b i s -d i g i t o x o s i d e (peaks 1, 2 and 3, r e s p e c t i v e l y ) e l u t e p r i o r t o d i g o x i n (peak 4) and g i t o x i n (peak 6) appears a f t e r 1 7 a - e t h y n y l e s t r a d i o l (peak 5 ) . For purposes o f a s s a y v a l i d a t i o n , common t a b l e t e x c i p i e n t s ( e . g . s t a r c h , l a c t o s e , c e l l u l o s e , s t e a r a t e l u b r i c a n t s ) were t a k e n t h r o u g h the a s s a y and found t o be n o n - i n t e r f e r i n g . The c a l i b r a t i o n c u r v e o f d i g o x i n ( F i g . 25) o b t a i n e d f o r the a n a l y s i s o f d i g o x i n ( F i g . 25) dosage forms was found t o be l i n e a r w i t h t h e l i n e p a s s i n g t h r o u g h t h e o r i g i n , and a r e s p o n s e f a c t o r o f 1.4261 was d e r i v e d f o r th e q u a n t i t a t i o n s t e p . The r e s u l t s o f t h e c o m p o s i t e sample a n a l y s i s o f d i g o x i n t a b l e t s a r e shown i n T a b l e X I . Average l a b e l c l a i m v a l u e s (n = 4) T a b l e X I. R e s u l t s o f the HPLC A n a l y s i s o f Composite Samples o f D i g o x i n T a b l e t s  N P e r c e n t o f L a b e l C l a i m Brand A Brand B Brand C 0.125 mg 0.25 mg 0.25 mg 0.25 mg 1 98.0 94.0 98.8 101.6 2 101.2 96.8 97.2 100.0 3 99.2 95.2 98.0 104.8 4 101.2 95.2 97.6 98.4 Mean 99.9 95.3 97.9 101.2 176. o f 95.3%, 97.9% and 101.1% were o b t a i n e d f o r Brands A, B and C, a l l o f which were 0.25 mg t a b l e t s . The a v e r a g e l a b e l c l a i m v a l u e f o r the 0.125 mg t a b l e t (Brand A) was found t o be 99.9%. S i n c e t h e range o f l a b e l c l a i m v a l u e s t h a t t h e USP a l l o w s f o r d i g o x i n t a b l e t s i s 92-108%, i t can be o b s e r v e d t h a t a l l brands meet t h e a s s a y r e q u i r e m e n t s . The p r e c i s i o n d a t a i n T a b l e XII i n d i c a t e t h a t t h e a n a l y s i s o f the t a b l e t s was accom-p l i s h e d w i t h a r e l a t i v e s t a n d a r d d e v i a t i o n o f 1.5% (n = 6 ) . The r e s u l t s o f the HPLC s i n g l e t a b l e t a s s a y o f d i g o x i n t a b l e t s (0.125 mg per t a b l e t and 0.25 mg per t a b l e t ) o f Brand A, a r e p r e s e n t e d i n T a b l e X I I I . A v e r age l a b e l c l a i m v a l u e s o f 97.1% and 96.6% were o b t a i n e d f o r 0.125 mg and 0.25 mg t a b l e t s , r e s p e c t i v e l y , w i t h a c o r r e s -ponding p r e c i s i o n o f 5.1% and 4.6%. The r e c o v e r y d a t a f o r d i g o x i n t a b l e t a s s a y are p r e s e n t e d i n T a b l e XIV. The a v e r a g e r e c o v e r y v a l u e o f d i g o x i n from t a b l e t m a t e r i a l was found t o be 99.8% w i t h a r e l a t i v e s t a n d a r d d e v i a t i o n o f 3.2% (n = 6 ) . The r e p r e s e n t a t i v e chromatogram f o r the a n a l y s i s o f d i g o x i n i n j e c t i o n ( F i g . 66) i n d i c a t e s t h a t d i g o x i n (peak 2) and the i n t e r n a l s t a n d a r d (peak 3) a r e f r e e from i n t e r f e r e n c e by p h a r m a c e u t i c a l e x c i p i e n t s . Peak 1 and t h e n e g a t i v e peaks a t the i n i t i a l p o r t i o n o f the chromatogram and th e s m a l l n e g a t i v e peak between the d i g o x i n and i n t e r n a l s t a n d a r d peaks a r e due t o s o l v e n t e f f e c t s . T h i s chromatogram i s e s s e n t i a l l y s i m i l a r t o t h a t o b t a i n e d f o r t h e t a b l e t a s s a y and was g e n e r a t e d u s i n g the s o l v e n t s ystem, w a t e r / m e t h a n o l / i s o p r o a n o l / d i c h l o r o m e t h a n e : 47/40/9/4. The r e s u l t s o f t h e a n a l y s i s o f d i g o x i n i n j e c t i o n ( T a b l e XV) i n d i c a t e a v e r a g e l a b e l c l a i m v a l u e s o f 99.4% and 99.1%, f o r the 0.05 mg/ml and 0.25 mg/ml i n j e c t i o n s , r e s p e c t i v e l y . These v a l u e s were o b t a i n e d w i t h r e l a t i v e T a b l e X I I . P r e c i s i o n Data f o r t h e HPLC A n a l y s i s o f D i g o x i n T a b l e t s 9  N P e r c e n t o f L a b e l C l a i m 1 98.0 2 99.4 3 98.2 4 98.0 5 101.4 6 100.5 Mean (x) 99.2 R.S.D., % 1.45 a 0.25 mg d i g o x i n t a b l e t s , Brand A k R e l a t i v e s t a n d a r d d e v i a t i o n T a b l e X I I I . R e s u l t s o f HPLC S i n g l e T a b l e t A s say o f D i g o x i n T a b l e t s  N P e r c e n t o f Lab e l 0.125 mg C l a i m3 0.25 mg 1 90.0 92.8 2 91.4 96.4 3 105.8 92.6 4 102.4 106.0 5 93.9 102.0 6 96.8 94.8 7 94.6 98.6 8 96.8 96.9 9 101.2 93.2 10 98.4 92.8 Mean (x) 97.1 96.6 R.S.D. , % 5.1 4.6 3 Brand A 13 R e l a t i v e s t a n d a r d d e v i a t i o n 179. T a b l e XIV. Recovery Data f o r D i g o x i n T a b l e t A s s a y 9 N T h e o r e t i c a l Amount o f D i g o x i n (mg) Amount o f D i g o x i n Added (mg) % Recovery 1 1.25 0.625 99.7 2 1.25 0.625 101.3 3 1.25 0.625 96.8 4 1.25 0.625 102.6 5 1.25 0.625 95.2 6 1.25 0.625 103.4 Mean (x) 99.8 R.S.D. %b 3.2 95% C o n f i d e n c e limits(±) 3.4 a 0.25 mg d i g o x i n t a b l e t ; Brand A k R e l a t i v e s t a n d a r d d e v i a t i o n 180. .002 A U 5 10 MINUTES F i g . 66. A R e p r e s e n t a t i v e Chromatogram f o r the I s o c r a t i c HPLC A n a l y s i s o f D i g o x i n I n j e c t i o n . Peak I d e n t i t y and HPLC c o n d i t i o n s : same as i n F i g . 64. T a b l e XV. R e s u l t s o f the HPLC A n a l y s i s o f D i g o x i n I n j e c t i o n and E l i x i r  N P e r c e n t o f Labe l C l a i m I n j e c t i o n E l i x i r 0.05 mg/ml 0.25 mg/ml 0.05 mg/ml 1 102.5 98.4 100.5 2 98.5 102.4 98.6 3 97.5 99.2 97.4 4 99.0 98.9 101.2 5 100.0 97.6 -6 99.0 98.4 -Mean (x) 99.4 99.1 99.4 R.S.D. 3, % 1.7 1.6 1.8 a R e l a t i v e s t a n d a r d d e v i a t i o n 182. s t a n d a r d d e v i a t i o n s o f 1.7% and 1.6%, r e s p e c t i v e l y . A n a l y s i s o f d i g o x i n e l i x i r c o u l d not be c a r r i e d out u s i n g t h e s o l v e n t system u t i l i z e d f o r t h e a s s a y o f d i g o x i n t a b l e t s and i n j e c t i o n because o f e x c i p i e n t i n t e r f e r e n c e w i t h t h e d i g o x i n peak. V a r i o u s a l t e r a t i o n s i n the p r o p o r t i o n o f the components o f the s o l v e n t system were made i n o r d e r t o o b t a i n a r e l a t i v e l y more p o l a r m o b i l e phase t h a t would have g r e a t e r d i f f e r -e n t i a t i n g p r o p e r t i e s . A s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 51/42/5/2 r e s u l t e d i n a chromatogram ( F i g . 67) t h a t r e s o l v e s d i g o x i n (peak 5) and t h e i n t e r n a l s t a n d a r d ( 1 7 a - e t h y n y l e s t r a d i o l , peak 7) from any o f the peaks due t o p h a r m a c e u t i c a l e x c i p i e n t s . Peaks 1-4 and 6 were due t o p h a r m a c e u t i c a l e x c i p i e n t s . Peak 4 .was found to be m e t h y l p a r a b e n ; t h e o t h e r peaks, however, were not i d e n t i f i e d . I t can be o b s e r v e d t h a t t h e i n t e r n a l s t a n d a r d , 1 7 a - e t h y n y l e s t r a d i o l , which has a r e t e n t i o n t i m e o f about 18 m i n u t e s , c a u s e s a r e l a t i v e l y l o n g chromato-g r a p h i c t i m e . In an a t tempt t o r e d u c e the c h r o m a t o g r a p h i c t i m e , a number o f s t e r o i d s were t e s t e d f o r use as i n t e r n a l s t a n d a r d s . 1 7 8 - d i h y d r o e q u i l i n was found t o s a t i s f y the c h r o m a t o g r a p h i c r e q u i r e m e n t s as shown i n F i g . 68. Peaks 6 and 9 r e p r e s e n t d i g o x i n and 1 7 6 - d i h y d r o e q u i l i n , r e s p e c t i v e l y w h i l e the o t h e r peaks a r e due t o e l i x i r e x c i p i e n t s . The r e s u l t s o f the a n a l y s i s o f d i g o x i n e l i x i r , however, were o b t a i n e d w i t h 1 7 a - e t h y n y l e s t r a d i o l as t h e i n t e r n a l s t a n d a r d , s i n c e i t has the advantage o f r e a d y a v a i l a b i l i t y . The c a l i b r a t i o n c u r v e o f d i g o x i n o b t a i n e d f o r the a n a l y s i s o f d i g o x i n e l i x i r ( F i g . 26) was found t o be l i n e a r w i t h the l i n e p a s s i n g t h r o u g h the o r i g i n ; and a r e s p o n s e f a c t o r o f 1.3652 was used f o r q u a n t i t a t i o n . The r e s u l t s o f the a n a l y s i s o f d i g o x i n e l i x i r ( T a b l e XV) show an a v e r a g e l a b e l c l a i m v a l u e o f 99.4% w i t h a r e l a t i v e s t a n d a r d d e v i a t i o n o f 1.8%. I t i s t o be noted t h a t the a n a l y s i s o f d i g o x i n e l i x i r was a c h i e v e d by d i r e c t d i l u t i o n o f t h e e l i x i r sample w i t h o u t h a v i n g to r e s o r t t o any p r e - e x t r a c t i o n 183. L U U z < co CH o CD < .002 A U 5 10 15 MINUTES 20 F i g . 67. A R e p r e s e n t a t i v e Chromatogram f o r the I s o c r a t i c HPLC A n a l y s i s o f D i g o x i n E l i x i r as o b t a i n e d w i t h a s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 51/42/5/2. Peak i d e n t i t y : ( 1-3, 6) unknown; (4) m e t h y l p a r a b e n ; (5) d i g o x i n ; and (7) 1 7 a - e t h y n y l e s t r a d i o l ( i n t e r n a l s t a n d a r d ) . Other HPLC c o n d i t i o n s : same as i n F i g . 64. 184. L U u Z < CQ o CQ < 3 6 ? _L K) MINUTES 15 F i g . 68. A R e p r e s e n t a t i v e Chromatogram f o r the I s o c r a t i c HPLC A n a l y s i s o f D i g o x i n E l i x i r u s i n g 176-d i h y d r o e q u i l i n as the I n t e r n a l S t a n d a r d . Peak i d e n t i t y : (1-3, 5, 7, 8) unknown; (6) d i g o x i n ; and (9) 1 7 6 - d i h y d r o e q u i l i n . HPLC c o n d i t i o n s : same as i n F i g . 67. 185. s t e p . The r e s u l t s o f the HPLC a n a l y s i s , as d e s c r i b e d above, i n d i c a t e t h a t t h e methods a r e f a s t , s e l e c t i v e , a c c u r a t e , s e n s i t i v e , r e l a t i v e l y s i m p l e and t h e r e f o r e , c o n v e n i e n t f o r t h e a s s a y o f d i g o x i n dosage forms. 7. Development o f an HPLC Method f o r the A n a l y s i s o f D i g i t o x i n i n i t s  Dosage Forms Due t o the r e l a t i v e l y l o n g h a l f - l i f e o f d i g i t o x i n i n the body, the u n i t t a b l e t dose i s v e r y low ( u s u a l l y 0.1 mg per t a b l e t ) . Hence a s s u r a n c e o f c o n t e n t u n i f o r m i t y o f t a b l e t s would n e c e s s i t a t e an a n a l y t i c a l method o f h i g h s e n s i t i v i t y t h a t would a l l o w s i n g l e t a b l e t a s s a y . F u r t h e r m o r e , the p r e s e n c e o f p h a r m a c e u t i c a l e x c i p i e n t s and p r o b a b l e f o r m a t i o n o f d e g r a d a t i o n p r o d u c t s and/or c o n t a m i n a n t s would r e q u i r e a h i g h d e g r e e o f s e l e c t i v i t y . Most o f the e a r l i e r methods r e p o r t e d f o r t h e q u a n t i t a t i v e a n a l y s i s o f d i g i t o x i n have been c o l o r i m e t r i c (James e t a l . , 1947; B e l l and K r a n t z , 1948; Soos, 1948; Kennedy, 1950; Rowson, 1952; T a t t j e , 1954; T a t t j e , 1957; USP, 1980) o r f l u o r o m e t r i c ( S c i a r i n i and S a l t e r , 1951; J a k o v l j e v i c , 1 963; W e l l s e t a l . , 1961; Khowrz, 1 967; C u l l e n e t a l . , 1 970). Many i n v e s t i g a t o r s have l a t e r used t h i n - l a y e r ( J e l l i f e , 1967; B i c a n -F i s t e r and Merkas, 1 969; F r i g n s , 1970; Evans e t a l ., 1974), g a s - l i q u i d (Watson e t a l . , 1972) and h i g h - p e r f o r m a n c e l i q u i d ( C a s t l e , 1 975; L i n d n e r and F r e i , 1976; Nachtmann e t a l . , 1976; Cobb, 1976; E r n i e and F r e i , 1977) c h r o m a t o g r a p h i c methods f o r t h e s e p a r a t i o n and q u a n t i t a t i v e d e t e r m i n a t i o n o f d i g i t o x i n . The c o l o r i m e t r i c methods g e n e r a l l y l a c k s e n s i t i v i t y . Moreover, t h e y have no s e l e c t i v i t y because the c o l o r - f o r m i n g d e r i v a t i z i n g r e a g e n t s r e a c t w i t h t h e d i g i t o x o s e s ugar m o i e t y o r the l a c t o n e r i n g o f the d i g i t o x i n m o l e c u l e , both o f which a r e s h a r e d by a l l c a r d e n o l i d e s . S i n c e the fluorometric methods are based on the reaction of the derivatizing agent with the steroid moiety of the di g i t o x i n molecule, they are non-specific with respect to other d i g i t a l i s glycosides. Even though the gas chroma-tographic-ECD method reported by Watson et a l . i s highly sensitive, i t is again non-specific. The electron capture detection requires d e r i v a t i -zation to genin-diheptafluorobutyrate, and hence any degradation product that may be present w i l l not be differentiated from d i g i t o x i n . Even though HPLC separations and in some cases minimum quantifiable amounts of d i g i t o x i n have been reported, application of any of these methods to the analysis of di g i t o x i n dosage forms has not been shown. Quanti-tativ e analysis of di g i t o x i n dosage forms would need an appropriate sample preparation that would ensure a high percentage of sample recovery The p o s s i b i l i t y of an HPLC method that f u l f i l l s the requirements of s u f f i c i e n t UV transparency and sample recovery in the analysis of dig i t o x i n in i t s dosage forms was, therefore, investigated. A solvent system of water/methanol/isopropanol/dichloromethane: 45/38/11/6 resulted in the representative chromatogram (Fig. 69) that was used for the analysis of digi t o x i n in tablet and injection dosage forms. This chromatogram indicates the separation of 17a-methyltesto-sterone (internal standard, peak 2) and digitoxin (peak 3) without any interference from pharmaceutical excipients. The internal standard and dig i t o x i n elute at retention times of 12.7 and 14.9 minutes, respectively Peak 1 i s an impurity from the f i l t e r paper and the two negative peaks are due to solvent e f f e c t . In order to test the s e l e c t i v i t y of the HPLC system, a sample of a solution of a standard mixture of the internal standard, d i g i t o x i n , 187. .002 A U < CO Crl CD < » ^ 5 10 15 MINUTES Fig. 69. A Representative Chromatogram for the Isocratic HPLC Analysis of Digitoxin Tablets and Injection. Peak id e n t i t y : (1) unknown; (2) 17a-methyltesto-sterone (internal standard); and (3) d i g i t o x i n . HPLC conditions: solvent system, water/methanol/ isopropanol/dichloromethane: 45/38/11/6; flow rate, 1.2 ml/min; UV detection at 220 nm; Chart speed, 0.5 cm/min. 188. d i g o x i n , and the d e g r a d a t i o n p r o d u c t s o f the l a t t e r two was i n j e c t e d i n t o t h e l i q u i d c h r o m a t o g r a p h . The chromatogram t h a t was o b t a i n e d ( F i g . 70) shows the r e s o l u t i o n o f a l l t he components, thus i n d i c a t i n g the p o s s i b i l i t y o f s i m u l t a n e o u s l y q u a n t i t a t i n g a l l o f t h e compounds i n a c h r o m a t o g r a p h i c t i m e o f ab o u t 15 m i n u t e s . D i g o x i n and i t s d e g r a d a t i o n p r o d u c t s / m e t a b o l i t e s were i n c l u d e d i n t h i s s e p a r a t i o n i n o r d e r t o i n d i c a t e the p o s s i b i l i t y o f a p p l y i n g t h i s method f o r m o n i t o r i n g t he m e t a b o l i t e s o f d i g i t o x i n i n b i o l o g i c a l samples. The c a l i b r a t i o n c u r v e ( F i g . 30) o f d i g i t o x i n , o b t a i n e d f o r the a n a l y s i s o f t a b l e t and i n j e c t i o n dosage forms was found t o be l i n e a r w i t h t he l i n e p a s s i n g t h r o u g h t h e o r i g i n and a r e s p o n s e f a c t o r o f 0.9348 was used f o r q u a n t i t a t i o n . The r e s u l t s o f the HPLC a n a l y s i s o f d i g i t o x i n t a b l e t and i n j e c t i o n dosage forms a r e p r e s e n t e d i n T a b l e XVI. I t can be o b s e r v e d t h a t t h e a v e r a g e l a b e l c l a i m v a l u e f o r the c o m p o s i t e sample a n a l y s i s o f d i g i t o x i n t a b l e t s i s 97.2% w i t h a r e l a t i v e s t a n d a r d d e v i a t i o n o f 1.4% (n = 6 ) . The d a t a f o r t he s i n g l e t a b l e t a s s a y o f d i g i t o x i n i n d i c a t e an a v e r a g e l a b e l c l a i m v a l u e o f 98.8% w i t h a r e l a t i v e s t a n d a r d d e v i a t i o n o f 4.8% (n = 1 0 ) . The r e c o v e r y d a t a f o r the a n a l y s i s o f d i g i t o x i n t a b l e t s ( T a b l e XVII) i n d i c a t e s an a c c u r a c y o f 99.7% w i t h a r e l a t i v e s t a n d a r d d e v i a t i o n o f 3.1% (n = 4 ) . The r e s u l t s o f the a n a l y s i s o f d i g i t o x i n i n j e c t i o n ( T a b l e XVI) show an ave r a g e p e r c e n t a g e l a b e l c l a i m o f 96.9 w i t h a r e l a t i v e s t a n d a r d d e v i a t i o n o f 3.3% (n = 3 ) . S i n c e t h e p e r c e n t a g e l a b e l c l a i m range a l l o w e d by t h e USP i s 90-110; i t can be seen t h a t t h e d i g i t o x i n dosage forms comply w i t h t he o f f i c i a l a s s a y r e q u i r e m e n t s . The r e s u l t s o f the HPLC a n a l y s i s as d e s c r i b e d above, i n d i c a t e t h a t 189. L U U z < CQ CQ < 8 J J J L 5 10 MINUTES 15 F i g . 70. A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f D i g i t o x i n , D i g o x i n and t h e i r p o t e n t i a l D e g r a d a t i o n P r o d u c t s and 1 7 a - m e t h y l t e s t o s t e r o n e ( i n t e r n a l s t a n d a r d ) . Peak i d e n t i t y : (1) d i g o x i g e n i n ; (2) d i g o x i g e n i n m o n o d i g i t o x o s i d e ; (3) d i g o x i g e n i n b i s d i g i t o x o s i d e ; (4) d i g o x i n ; (5) d i g i t o x i g e n i n ; (6) d i g i t o x i g e n i n m o n o d i g i t o x o s i d e ; (7) d i g i t o x i -g e n i n b i s d i g i t o x o s i d e ; (8) 1 7 a - m e t h y l t e s t o s t e r o n e ; and (9) d i g i t o x i n . HPLC c o n d i t i o n s : same as i n F i g . 69. T a b l e XVI. R e s u l t s o f t h e HPLC A n a l y s i s o f D i g i t o x i n T a b l e t s and I n j e c t i o n  N P e r c e n t o f L a b e l C l a i m T a b l e t Composite A s s a y 3 S i n g l e T a b l e t A s s a y 3 I n j e c t a b l e *3 1 95.1 94.8 95.9 2 97.1 104.6 94.3 3 96.8 92.8 100.5 4 99.0 98.4 -5 98.4 96.5 -6 96.8 94.8 -7 - 97.9 -8 107.0 -9 - 98.2 -10 - 103.8 -Mean 97.2 98.8 96.9 R . S . D . ( % ) C 1.4 4.8 3.3 a 0.1 mg T a b l e t b 0.2 mg/ml c R e l a t i v e s t a n d a r d d e v i a t i o n T a b l e XVII. R e c o v e r y Data f o r the HPLC A n a l y s i s o f D i g i t o x i n T a b l e t s 3 N T h e o r e t i c a l Amount o f D i g i t o x i n (mg) Amount Added (mg) % R e c o v e r y 1 1.0 0.5 98.6 2 1.0 0.5 102.6 3 1.0 0.5 95.8 4 1 .0 0.5 101 .9 Mean 99.7 R.S.D. b 3.1 95% C o n f i d e n c e l i m i t s (±) 4.9 a 0.1 mg t a b l e t b R e l a t i v e s t a n d a r d d e v i a t i o n 192. the method f u l f i l l s the r e q u i r e m e n t s o f s e n s i t i v i t y , s e l e c t i v i t y , r e p r o -d u c i b i l i t y , a c c u r a c y , s i m p l i c i t y and s h o r t o f time o f a n a l y s i s , f o r t h e a s s a y o f d i g i t o x i n dosage forms. 8. Comparison o f t h e A n a l y t i c Data o f D i g o x i n and D i g i t o x i n Dosage Forms  by HPLC and USP XX Methods In o r d e r t o d e t e r m i n e the r e l a t i v e m e r i t s o f the HPLC methods t h a t were d e v e l o p e d f o r t h e a n a l y s i s o f d i g o x i n and d i g i t o x i n i n t h e i r r e s p e c -t i v e dosage f o r m s , c o m p a r a t i v e s t u d i e s were c a r r i e d out w i t h r e f e r e n c e t o the USP methods. The same b a t c h e s o f d i g o x i n and d i g i t o x i n dosage forms were a n a l y s e d by HPLC and the USP methods. The HPLC and USP p r o c e d u r e s were e v a l u a t e d i n terms o f time o f a n a l y s i s , s e n s i t i v i t y , s e l e c t i v i t y and c o n v e n i e n c e . The data f o r the a n a l y t i c a l r e s u l t s were compared w i t h r e g a r d t o t h e i r p r e c i s i o n and a c c u r a c y . As shown i n the f l o w c h a r t s o f F i g u r e s 34, 35, 36 and 37, the c u r r e n t USP a s s a y s f o r d i g o x i n t a b l e t s , i n j e c t i o n and e l i x i r i n v o l v e e x t r a c t i o n o f the d r u g , e v a p o r a t i o n t o d r y n e s s , c o o l i n g i n a vacuum d e s i c c a t o r o v e r phosphorus p e n t o x i d e f o r 60 m i n u t e s , d e r i v a t i z a t i o n w i t h a c i d - f e r r i c c h l o r i d e and d e t e r m i n a t i o n o f maximum a b s o r b a n c e a f t e r r e p e a t e d measurements a t two-minute i n t e r v a l s . I t has been o b s e r v e d t h a t the USP j p r o c e d u r e s i n v o l v e l a b o r i o u s and m a n i p u l a t i v e s t e p s t h a t r e q u i r e p e r i o d s o f about f o u r hours f o r c o m p l e t i o n o f one a s s a y . The t e d i o u s n e s s o f the p r o c e d u r e i s p a r t i c u l a r l y s i g n i f i c a n t i n the USP s i n g l e t a b l e t a s s a y ( c o n t e n t u n i f o r m i t y t e s t ) i n which the a n a l y s i s o f 20 i n d i v i d u a l t a b l e t s i s r e q u i r e d . The HPLC methods, on the o t h e r hand, have c h r o m a t o g r a p h i c t i m e s o f l e s s than twenty m i n u t e s and sample p r e p a r a t i o n s can be done i n about 15 m i n u t e s . Thus t h e a n a l y s e s can be c o m p l e t e d i n a p e r i o d o f 193. l e s s t h a n f o r t y m i n u t e s . The USP methods f o r the a n a l y s i s o f c o m p o s i t e t a b l e t s , i n j e c t i o n and e l i x i r r e q u i r e samples e q u i v a l e n t t o 2.5 mg o f d i g o x i n . Whereas, HPLC a n a l y s i s can be c a r r i e d o u t w i t h c o m p o s i t e t a b l e t , i n j e c t i o n and e l i x i r samples o f 1.25 mg, 0.1 mg and 1.0 mg o f d i g o x i n , r e s p e c t i v e l y . The minimum amount o f d i g o x i n t h a t i s q u a n t i f i e d u s i n g the USP p r o c e d u r e i s about 25 meg, w h i l e the HPLC method has been used f o r the q u a n t i t a t i v e d e t e r m i n a t i o n o f about 50 ng o f the d r u g . D i g o x i n q u a n t i t a t i o n u s i n g the USP method i s based on the r e a c t i o n o f the d e r i v a t i z i n g a g e n t w i t h the s ugar m o i e t y t o form a c o l o r e d d e r i v a -t i v e . T h e r e f o r e , any d i g i t o x o s i d e s t h a t may be p r e s e n t as d e g r a d a t i o n p r o d u c t s w i l l not be d i f f e r e n t i a t e d from d i g o x i n ; whereas the HPLC methods have been shown to a c c o u n t f o r the p r o b a b l e d e g r a d a t i o n p r o d u c t s ( F i g . 6 5 ) . R e s u l t s o f the a n a l y s i s o f c o m p o s i t e samples o f t h r e e d i f f e r e n t brands o f d i g o x i n t a b l e t s by HPLC and the USP method a r e shown i n T a b l e X V I I I . I t can be seen t h a t w h i l e t h e mean p e r c e n t a g e l a b e l c l a i m v a l u e s o b t a i n e d by both methods a r e q u i t e comparable t h e HPLC da t a show b e t t e r p r e c i s i o n . HPLC and USP r e s u l t s f o r the a n a l y s i s o f d i g o x i n i n j e c t i o n and e l i x i r ( T a b l e XIX) i n d i c a t e t h a t the mean p e r c e n t a g e l a b e l c l a i m and r e l a t i v e s t a n d a r d d e v i a t i o n v a l u e s o b t a i n e d w i t h the HPLC method compare f a v o u r a b l y w i t h t h o s e d e r i v e d from the USP d a t a . The a c c u r a c y d a t a f o r the a n a l y s i s o f d i g o x i n t a b l e t s as o b t a i n e d by HPLC and the USP method(Table XX) i n d i c a t e mean r e c o v e r y v a l u e s o f 99.8% and 101.2%, r e s p e c t i v e l y w i t h a c o r r e s p o n d i n g r e l a t i v e s t a n d a r d d e v i a t i o n o f 3.3% and 3.2%. The above d a t a , t h e r e f o r e , i n d i c a t e t h a t the a c c u r a c y o f t h e HPLC method compares f a v o u r a b l y w i t h t h a t o f the USP method. Table XVIII. Results of the Analysis of Composite Samples of Digoxin Tablets by HPLC and the USP XX Method  Percent of Label Claim N Brand A Brand B Brand C 0.125 mg 0.25 mg 0.25 mg 0.25 mg HPLC Method USP XX Method HPLC Method USP XX Method HPLC Method USP XX Method HPLC Method USP XX Method 1 98.0 103.0 94.0 105.6 98.8 101 .4 101 .6 97.7 2 101.2 96.1 96.8 96.2 97.2 98.8 100.0 102.7 3 99.2 94.9 95.2 98.4 98.0 103.0 104.8 103.2 4 101.2 - 95.2 - 97.6 - 98.4 -Mean 99.9 98.0 95.3 100.1 97.9 101 .0 101 .2 101 .2 R.S.D.3 % 1 .6 4.4 1 .1 4.9 0.7 2.7 2.7 3.0 a Relative standard deviation T a b l e XIX. R e s u l t s o f t h e A n a l y s i s o f D i g o x i n I n j e c t i o n and E l i x i r by HPLC and t he U.S.P. XX Method N P e r c e n t o f Label C l a i m I n j e c t i o n E l i x i r 0.05 mg/ml 0.25 mg/ml 0.05 mg/ml HPLC USP XX HPLC USP XX HPLC USP XX Method Method Method Method Method Method 1 102.5 103.2 98.4 101.2 100.5 101 .6 2 98.5 101.2 102.4 101.9 98.6 97.4 3 97.5 99.6 99.2 102.7 97.4 98.2 4 99.0 - 98.9 - 101.2 -5 100.0 97.6 - - -6 99.0 - 98.4 - - -Mean (x) 99.4 101 .3 99.1 101.9 99.4 99.1 R.S.D. ,% 1.7 1.8 1 .6 0.8 1 .8 2.2 R e l a t i v e s t a n d a r d d e v i a t i o n T a b l e XX. Recovery Data f o r D i g o x i n T a b l e t 9 A s s a y by HPLC and t h e USP XX Method T h e o r e t i c a l Amount o f Weight o f D i g o x i n R e c o v e r y N D i g o x i n (mg) Added (mg) % HPLC D USP C HPLC USP HPLC USP 1 1.25 2.5 0.625 1.25 99.7 101.3 2 1.25 2.5 0.625 1.25 101.3 102.7 3 1.25 2.5 0.625 1.25 96.8 96.8 4 1.25 2.5 0.625 1.25 102.6 101.9 5 1.25 2.5 0.625 1.25 95.2 98.6 6 1.25 2.5 0.625 1.25 103.4 105.8 Mean (x) 99.8 101 .2 R.S.D. d, % 3.3 3.2 95% C o n f i d e n c e 3.5 3.4 limits(±) 0.25 mg D i g o x i n T a b l e t s , Brand A D A c c o r d i n g t o p r e l i m i n a r y a s s a y , p e r c e n t l a b e l c l a i m i s 100.1 c A c c o r d i n g t o p r e l i m i n a r y a s s a y , p e r c e n t l a b e l c l a i m i s 99.7 d R e l a t i v e s t a n d a r d d e v i a t i o n 197. The USP p r o c e d u r e f o r the a s s a y o f d i g i t o x i n t a b l e t s and i n j e c t i o n g e n e r a l l y i n v o l v e s e x t r a c t i o n , column c h r o m a t o g r a p h i c s e p a r a t i o n , e v a p o r a t -i o n o f the e l u e n t , r e c o n s t i t u t i o n i n a l c o h o l , d e r i v a t i z a t i o n w i t h a l k a l i n e p i c r a t e r e a g e n t and d e t e r m i n a t i o n o f maximum a b s o r b a n c e a f t e r r e p e a t e d measurements a t i n t e r v a l s o f two m i n u t e s . The USP s i n g l e t a b l e t a s s a y ( c o n t e n t u n i f o r m i t y t e s t ) c o m p r i s e s e x t r a c t i o n , e v a p o r a t i o n o f the s o l v e n t , r e c o n s t i t u t i o n i n 80% a l c o h o l , f l u o r o g e n i c d e r i v a t i z a t i o n and f l u o r o -m e t r i c d e t e r m i n a t i o n . I t can be o b s e r v e d t h a t the methods a r e l a b o u r i n t e n s i v e and c o m p l e t i o n o f one a s s a y g e n e r a l l y r e q u i r e s a p e r i o d o f o v e r f o u r h o u r s . The HPLC p r o c e d u r e f o r the a s s a y o f d i g i t o x i n t a b l e t s and i n j e c t i o n , however, r e q u i r e s l e s s than f o r t y - f i v e m i n u t e s f o r c o m p l e t i o n o f one a s s a y . The USP a s s a y s f o r d i g i t o x i n c o m p o s i t e t a b l e t s and i n j e c t i o n r e q u i r e samples e q u i v a l e n t t o 2 mg o f d i g i t o x i n . The USP c o l o r i m e t r i c method used f o r c o m p o s i t e t a b l e t a s s a y i s not s e n s i t i v e enough t o be employed i n s i n g l e t a b l e t a s s a y . T h e r e f o r e , t he USP method f o r d i g i t o x i n s i n g l e t a b l e t a s s a y employs f l u o r o m e t r i c measurement. The HPLC method f o r the a n a l y s i s o f d i g i t o x i n c o m p o s i t e t a b l e t s , s i n g l e t a b l e t s and i n j e c t i o n r e q u i r e s samples e q u i v a l e n t t o 1 mg, 0.1 mg and 0.2 mg, r e s p e c t i v e l y . M oreover, the a s s a y p r o c e d u r e f o r s i n g l e t a b l e t a s s a y i s the same as i n t he c o m p o s i t e t a b l e t a s s a y . Both the c o l o r i m e t r i c and f l u o r o m e t r i c d e t e r m i n a t i o n s o f d i g i t o x i n w i l l n o t be a b l e t o d i f f e r e n t i a t e between the dr u g and i t s p r o b a b l e d e g r a d a t i o n p r o d u c t s . Whereas, the HPLC method has been shown ( F i g . 70) t o be s p e c i f i c t o d i g i t o x i n and a l s o c a p a b l e o f a c c o u n t i n g f o r each o f th e p r o b a b l e d e g r a d a t i o n p r o d u c t s . R e s u l t s o f the a n a l y s i s o f d i g i t o x i n c o m p o s i t e t a b l e t s by HPLC and T a b l e XXI. R e s u l t s o f the A n a l y s i s o f Composite Samples o f D i g i t o x i n T a b l e t s 3 by HPLC and the USP XX Method N P e r c e n t o f L a b e l C l a i m HPLC USP 1 95.1 103.1 2 97.1 101 .1 3 96.8 97.4 4 99.0 -5 98.4 -6 96.8 -Mean (x) 97.2 100.5 R. S. D. , % 1 .4 2.9 a 0.1 mg per t a b l e t k R e l a t i v e s t a n d a r d d e v i a t i o n T a b l e X X I I . Recovery Data f o r D i g i t o x i n T a b l e t 3 A s s a y by HPLC and t h e USP XX Method T h e o r e t i c a l Amount o f Weight o f D i g o x i n R e c o v e r y D i g o x i n (mg) Added (mg) % HPLC USP HPLC USP HPLC USP 1 1.0 2.0 0.5 1 .0 98.6 105.2 2 1.0 2.0 0.5 1 .0 102.6 101 .7 3 1.0 2.0 0.5 1 .0 95.8 96.0 4 1.0 2.0 0.5 1 .0 101 .9 102.4 Mean (x) 99.7 101 .3 R.S.D. , % 3.1 3.8 95% C o n f i d e n c e 4.9 6.2 l i m i t s (±) 3 0.1 mg per t a b l e t k R e l a t i v e s t a n d a r d d e v i a t i o n 200. USP methods ( T a b l e XXI) i n d i c a t e mean l a b e l c l a i m v a l u e s o f 97.2% and 100.5% w i t h r e l a t i v e s t a n d a r d d e v i a t i o n s o f 1.4% and 2.9% r e s p e c t i v e l y . C o m p a r a t i v e d a t a f o r d i g i t o x i n i n j e c t i o n a r e not p r e s e n t e d because o f i n s u f f i c i e n t sample. The mean r e c o v e r y v a l u e s o f d i g i t o x i n as o b t a i n e d by HPLC and the USP method ( T a b l e XXII) were found to be 99.7% and 101.3%, r e s p e c t i v e l y . In c o m p a r i s o n w i t h the USP p r o c e d u r e s f o r the a n a l y s i s o f d i g o x i n and d i g i t o x i n i n t h e i r r e s p e c t i v e dosage forms, the HPLC methods appear t o be more advantageous from the s t a n d p o i n t s o f s e l e c t i v i t y , s e n s i t i v i t y , s i m p l i c i t y , c o n v e n i e n c e and time o f a n a l y s i s . 9. S t a b i l i t y Study o f D i g o x i n and D i g i t o x i n i n t h e i r r e s p e c t i v e  Dosage Forms u s i n g HPLC Methods I t has been r e p o r t e d (Foss and B e n e z r a , 1980) t h a t d i g o x i n i s s t a b l e i n d e f i n i t e l y i f i t i s kept i n the d a r k i n t i g h t l y c l o s e d c o n t a i n e r s . The above a u t h o r s have a l s o r e p o r t e d t h a t d i g o x i n t a b l e t s and n e u t r a l s o l u t i o n s o f d i g o x i n i n e t h y l a l c o h o l and p r o p y l e n e g l y c o l a r e s t a b l e f o r p e r i o d s o f up t o f i v e y e a r s . A c c o r d i n g t o J a k o v l j e v i c ( 1 9 7 4 ) , no d e g r a d a t i o n o f d i g i t o x i n i n t a b l e t s , i n j e c t a b l e s o r s o l u t i o n s was o b s e r v e d when s t o r e d f o r f i v e y e a r s i n the d a r k a t t e m p e r a t u r e s o f up t o 30°C. I t i s g e n e r a l l y known, however, t h a t both d i g o x i n and d i g i t o x i n undergo h y d r o l y s i s under a c i d i c c o n d i t i o n s . S t e r n s o n and S h a f f e r (1978) have r e p o r t e d the k i n e t i c s o f i n v i t r o d i g o x i n d e g r a d a t i o n i n a c i d i c s o l u t i o n s , as m o n i t o r e d by HPLC. Even though the s t a b i l i t y o f d i g o x i n and d i g i t o x i n has been s t u d i e d u s i n g c o l o r i m e t r i c and c h r o m a t o g r a p h i c t e c h n i q u e s , i t a p p e a r s t h a t no HPLC methods have been r e p o r t e d f o r the s t u d y o f the d e g r a d a t i o n o f t h e s e p r o d u c t s i n dosage forms. The HPLC methods d e v e l o p e d f o r t he a s s a y o f d i g o x i n and d i g i t o x i n dosage forms have been shown to be c a p a b l e o f a c c o u n t i n g f o r each o f t h e p o t e n t i a l d e g r a d a t i o n p r o d u c t s . The p o s s i b i l i t y o f a p p l y i n g t h e s e methods f o r m o n i t o r i n g the s t a b i l i t y o f d i g o x i n and d i g i t o x i n i n t h e i r r e s p e c t i v e dosage forms and d e t e r m i n a t i o n o f each o f t h e d e g r a d a t i o n p r o d u c t s was, t h e r e f o r e , i n v e s t i g a t e d . Samples o f dosage forms o f d i g o x i n and d i g i t o x i n s t o r e d a t ambient c o n d i t i o n s ; 60°C and 70.4% r e l a t i v e h u m i d i t y ; and 80°C and 37.1% r e l a t i v e h u m i d i t y were p e r i o d i c a l l y a n a l y s e d . R e p r e s e n t a t i v e chromatograms f o r the s t a b i l i t y s t u d y o f L a n o x i n t a b l e t s ( F i g . 71) and L a n o x i n i n j e c t i o n ( F i g . 72) show t h a t d i g o x i n (peak 2) and 1 7 a - e t h y n y l e s t r a d i o l ( i n t e r n a l s t a n d a r d , peak 3) a r e w e l l resolved w i t h o u t any i n t e r f e r i n g peaks from e x c i p i e n t s . Both o f the above chromatograms were o b t a i n e d w i t h a s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 47/40/9/4 which i s the same as t h a t used f o r the a n a l y s i s o f d i g o x i n t a b l e t s and i n j e c t i o n . The r e p r e s e n t a t i v e chromatogram o b t a i n e d f o r the s t a b i l i t y s t u d y o f N a t i g o x i n t a b l e t s i s shown i n F i g . 73. The r e p r e s e n t a t i v e chromatogram f o r the s t a b i l i t y m o n i t o r i n g o f d i g i t o x i n t a b l e t s ( F i g . 74) shows the r e s o l u t i o n o f d i g i t o x i n (peak 4) and 1 7 a - m e t h y l t e s t o s t e r o n e ( i n t e r n a l s t a n d a r d , peak 3) w i t h o u t any i n t e r f e r e n c e from t a b l e t e x c i p i e n t s . T h i s chromatogram was o b t a i n e d w i t h a s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 45/38/11/6 and i s s i m i l a r to t h a t p r e v i o u s l y used f o r the a n a l y s i s o f d i g i t o x i n dosage forms. The s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 51/42/5/2 which was p r e v i o u s l y used f o r the a s s a y o f L a n o x i n e l i x i r was t r i e d f o r t h e s t a b i l i t y s t u d y o f d i g o x i n i n the e l i x i r . The chromatogram t h a t was o b t a i n e d ( F i g . 75) f o r a sample o f e l i x i r s p i k e d w i t h d i g o x i n and i t s d e g r a d a t i o n p r o d u c t s shows a m a s s i v e e x c i p i e n t peak (peak 1) and 202. 3 0 5 10 15 MINUTES P i g . 71. A R e p r e s e n t a t i v e Chromatogram f o r the S t a b i l i t y M o n i t o r i n g o f D i g o x i n T a b l e t s by HPLC. Peak i d e n t i t y : (1) unknown; (2) d i g o x i n ; and (3) 17a-e t h y n y l e s t r a d i o l . HPLC c o n d i t i o n s : same as i n F i g . 64. 203. Fig. 72. A Representative Chromatogram for the S t a b i l i t y Monitoring of Digoxin Injection by HPLC. Peak iden t i t y : (1) unknown; (2) digoxin; and (3) 17a-ethynylestradiol. HPLC conditions: same as in Fig. 64. -002 A U M I N U T E S F i g . 73. A R e p r e s e n t a t i v e Chromatogram f o r the S t a b i l i t y M o n i t o r i n g o f N a t i g o x i n ^ T a b l e t s by HPLC. Peak i d e n t i t y : (1) unknown; (2) d i g o x i g e n i n ; (3) d i g o x i -g e n i n b i s d i g i t o x o s i d e ; (4) d i g o x i n ; and (5) 17^-e t h y n y l e s t r a d i o l . HPLC c o n d i t i o n s : same as i n F i g . 64. I n s e t : A chromatogram o f a s t a n d a r d sample o f d i g o x i g e n i n ( 1 ) , d i g o x i g e n i n m o n o d i g i t o x o s i d e ( 2 ) , d i g o x i g e n i n b i s d i g i t o x o s i d e ( 3 ) , d i g o x i n (4) and 1 7 a - e t h y n y l e s t r a d i o l (6) o b t a i n e d under the same HPLC c o n d i t i o n s . Peak 5 i s due t o s o l v e n t p e r t u r b a t i o n , 205. F i g . 74. A R e p r e s e n t a t i v e Chromatogram f o r the S t a b i l i t y M o n i t o r i n g o f D i g i t o x i n T a b l e t s by HPLC. Peak i d e n t i t y : (1) unknown; (2) s o l v e n t e f f e c t ; (3) 1 7 a - m e t h y l t e s t o s t e r o n e ; and (4) D i g i t o x i n . HPLC c o n d i t i o n s : same as i n F i g . 69. 206. 10 15 M I N U T E S 20 25 F i g . 75. A Chromatogram o f a Sample o f L a n o x i n E l i x i r s p i k e d w i t h D i g o x i n and i t s p o t e n t i a l D e g r a d a t i o n P r o d u c t s . Peak i d e n t i t y : (1) unknown; (2) d i g o x i g e n i n ; (3) d i g o -x i g e n i n m o n o d i g i t o x o s i d e ; (4) m e t h y l p a r a b e n ; (5) d i g o x i -g e n i n b i s d i g i t o x o s i d e ; (6) unknown; (7) d i g o x i n ; and (8) 1 7 a - e t h y n y l e s t r a d i o l . HPLC c o n d i t i o n s : same as i n F i g . 67. 0 207. peaks 2, 3, 4, 5, 6, 7 and 8 r e p r e s e n t i n g d i g o x i g e n i n , d i g o x i g e n i n mono-d i g i t o x o s i d e , m e t h y l p a r a b e n , d i g o x i g e n i n b i s d i g i t o x o s i d e , unknown, d i g o x i n and 1 7 a - e t h y n y l e s t r a d i o l ( i n t e r n a l s t a n d a r d ) , r e s p e c t i v e l y . I t can be o b s e r v e d t h a t t h e peaks f o r t h e mono- and b i s d i g i t o x o s i d e s a r e i n t e r f e r e d w i t h . T h e r e f o r e , t h e d i r e c t d i l u t i o n method o f sample p r e p a r a t i o n used f o r t h e a s s a y o f L a n o x i n e l i x i r c o u l d not be a p p l i e d t o the s i m u l t a n e o u s m o n i t o r i n g o f d i g o x i n and i t s d e g r a d a t i o n p r o d u c t s . P r e l i m i n a r y e x t r a c t i o n o f t h e e l i x i r sample (which was s p i k e d w i t h d i g o x i g e n i n and t h e mono- and b i s d i g i t o x o s i d e s ) w i t h d i c h l o r o m e t h a n e and r e c o n s t i t u t i o n w i t h the e l u t i n g s o l v e n t system r e s u l t e d i n a chromatogram ( F i g . 76) which shows t h e e l i m i n a t i o n o f a l l e x c i p i e n t and unknown peaks e x c e p t peak 3 ( m e t h y l p a r a b e n ) and peak 5 (unknown). P r e l i m i n a r y a d d i t i o n o f sodium c a r b o n a t e s o l u t i o n , d i c h l o r o m e t h a n e e x t r a c t i o n o f the e l i x i r sample ( s p i k e d w i t h d i g o x i n and i t s p o t e n t i a l d e g r a d a t i o n p r o d u c t s ) and su b s e q u e n t sample p r e p a r a t i o n as p r e v i o u s l y d e s c r i b e d f o r the a n a l y s i s o f d i g o x i n e l i x i r r e s u l t e d i n the chromatogram shown i n F i g . 77. In t h e chromatogram o b t a i n e d f o r a sample o f e l i x i r s t o r e d a t room t e m p e r a t u r e ( F i g . 7 8 ) , i t can be seen t h a t a l l e x c i p i e n t peaks e x c e p t t h a t o f m e t h y l -paraben a r e e l i m i n a t e d . The peak r e p r e s e n t i n g d i g o x i g e n i n b i s d i g i t o x o s i d e ( F i g . 77, peak 4 ) , however, i s i n t e r f e r e d w i t h by t h e s o l v e n t p e r t u r b a t i o n e f f e c t a t t h e r e t e n t i o n time o f about 6.7 m i n u t e s . T h e r e f o r e , t h e s o l v e n t system p r e v i o u s l y used f o r the a n a l y s i s o f d i g o x i n e l i x i r c o u l d not be a p p l i e d f o r t h e s i m u l t a n e o u s m o n i t o r i n g o f d i g o x i n and i t s d e g r a d a t i o n p r o d u c t s i n the e l i x i r . I t can be o b s e r v e d t h a t t h e problem o f i n t e r f e r e n c e w i t h t h e d i g o x i g e n i n b i s d i g i t o x o s i d e peak can be s o l v e d i f the m e t h y l p a r a b e n and b i s d i g i t o x o s i d e peaks c o u l d be pushed f o r w a r d so t h a t t h e former would c o i n c i d e w i t h t h e b a s e l i n e p e r t u r b a t i o n and the l a t t e r would a p p e a r 208. F i g . 76. A Chromatogram o f an E x t r a c t e d Sample o f L a n o x i n " E l i x i r s p i k e d w i t h d i g o x i g e n i n and the mono- and b i s d i g i t o x o s i d e s . Peak i d e n t i t y : (1) d i g o x i g e n i n ; (2) d i g o x i g e n i n m o n o d i g i t o x o s i d e ; (3) m e t h y l p a r a b e n ; (4) d i g o x i g e n i n b i s d i g i t o x o s i d e ; (5) unknown; (6) d i g o x i n ; and (7) 1 7 a - e t h y n y l e s t r a d i o l . HPLC c o n d i t i o n s : same as i n F i g . 67. 0 5 1 0 M I N U T E S 15 F i g . 77. A Chromatogram o f an E x t r a c t e d Sample o f L a n o x i n E l i x i r a f t e r p r e l i m i n a r y a d d i t i o n o f Sodium Ca r b o n a t e and s p i k i n g w i t h D i g o x i n and i t s p o t e n t i a l D e g r a d a t i o n P r o d u c t s . Peak i d e n t i t y : (1) d i g o x i g e n i n ; (2) d i g o x i g e n i n m o n o d i g i t o x o s i d e ; (3) m e t h y l p a r a b e n ; (4) d i g o x i g e n i n b i s d i g i t o x o s i d e ; and (5) d i g o x i n . HPLC c o n d i t i o n s : same as i n F i g . 67. 1 n F i g . 78. A Chromatogram o f a Sample o f L a n o x i n E l i x i r s t o r e d a t room t e m p e r a t u r e , a f t e r p r e l i m i n a r y a d d i t i o n o f sodium c a r b o n a t e and e x t r a c t i o n w i t h d i c h l o r o m e t h a n e . Peak i d e n t i t y : ( i ) m e t h y l p a r a b e n ; (2) d i g o x i n . HPLC c o n d i t i o n s : same as i n F i g . 67 e x c e p t t h a t t h e f l o w r a t e was 0.6 ml/min. a t a l a t e r r e t e n t i o n t i m e thus d i s e n g a g i n g i t s e l f from the i n t e r f e r e n c e . T h i s was a c c o m p l i s h e d w i t h a s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 51/43/4/2 as shown i n F i g . 79. T h i s chromatogram shows the complete s e p a r a t i o n o f d i g o x i g e n i n (peak 2 ) , d i g o x i g e n i n m o n o d i g i t o x o -s i d e (peak 3 ) , m e t h y l p a r a b e n (peak 4 ) , d i g o x i g e n i n b i s d i g i t o x o s i d e (peak 5 ) , h y d r o c o r t i s o n e ( i n t e r n a l s t a n d a r d , peak 6) and d i g o x i n (peak 7) i n a c h r o m a t o g r a p h i c time o f about 14 m i n u t e s . The r e t e n t i o n times i n F i g . 79 a r e l a r g e r t h a n would be e x p e c t e d from the s l i g h t m o d i f i c a t i o n o f s o l v e n t c o m p o s i t i o n p r o b a b l y due t o a change i n t h e s t a t u s o f the column. A r e p r e s e n t a t i v e chromatogram f o r the s t a b i l i t y s t u d y o f L a n o x i n e l i x i r i s shown i n F i g . 80. The chromatogram obtained f o r 1% l e v e l s o f the p o t e n t i a l d e g r a d a t i o n p r o d u c t s o f d i g o x i n under the HPLC c o n d i t i o n s o f the a s s a y o f t a b l e t s (and i n j e c t i o n ) i s p r e s e n t e d i n F i g . 81. The chromatogram o b t a i n e d f o r 1% l e v e l s o f t h e p o t e n t i a l d e g r a d a t i o n p r o d u c t s o f d i g i t o x i n under the HPLC c o n d i t i o n s o f d i g i t o x i n t a b l e t a s s a y i s shown i n F i g . 82. The r e s u l t s o f the s t a b i l i t y s t u d y o f d i g o x i n t a b l e t s s t o r e d a t 60°C and 70.4% r e l a t i v e h u m i d i t y are p r e s e n t e d i n T a b l e X X I I I . I t can be seen t h a t d i g o x i n i n L a n o x i n t a b l e t s does not show any d e g r a d a t i o n f o r s t o r a g e p e r i o d s o f up t o 16 weeks. D i g o x i n i n N a t i g o x i n t a b l e t s , however, undergoes g r a d u a l d e g r a d a t i o n t o a l e v e l o f 73.4% w i t h 22.3% and 10.8% a p p e a r i n g as d i g o x i g e n i n and d i g o x i g e n i n b i s d i g i t o x o s i d e , r e s p e c t i v e l y , a f t e r 16 weeks o f s t o r a g e . I t i s i n t e r e s t i n g t o note t h a t no d i g o x i g e n i n m o n o d i g i t o x o s i d e was d e t e c t e d as a d e g r a d a t i o n p r o d u c t . The s t a b i l i t y d a t a f o r d i g o x i n t a b l e t s s t o r e d a t 80°C and 37.1% I ! L 0 5 7 10 15 M I N U T E S . 79. A Chromatogram o f an E x t r a c t e d sample o f L a n o x i n E l i x i r a f t e r p r e l i m i n a r y a d d i t i o n o f Sodium C a r b o n a t e and s p i k i n g w i t h H y d r o c o r t i s o n e ( i n t e r n a l s t a n d a r d ) , D i g o x i n and i t s p o t e n t i a l D e g r a d a t i o n P r o d u c t s . Peak i d e n t i t y : (1) unknown; (2) d i g o x i g e n i n ; (3) d i g o x i g e n i n m o n o d i g i t o x o s i d e ; (4) m e t h y l p a r a b e n ; (5) d i g o x i g e n i n b i s d i g i t o x o s i d e ; (6) h y d r o c o r t i s o n e and (7) d i g o x i n . S o l v e n t system: w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 51/43/4/2. Oth e r HPLC c o n d i t i o n s : same as i n F i g . 67. n 5 1 0 MINUTES 1 5 F i g . 80. A R e p r e s e n t a t i v e Chromatogram f o r t h e S t a b i l i t y M o n i t o r i n g o f L a n o x i n ^ E l i x i r by HPLC. Peak i d e n t i t y : (1) d i g o x i g e n i n ; (2) d i g o x i g e n i n m o n o d i g i t o x o s i d e ; (3) m e t h y l p a r a b e n ; (4) h y d r o -c o r t i s o n e ; and (5) d i g o x i n . S o l v e n t system: w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h i o r o m e t h a n e : 51/43/4/2, Other HPLC c o n d i t i o n s : same as i n F i g . 67. 214. o 12 0 5 M I N U T E S F i g . 81. A Chromatogram f o r t h e 1% l e v e l s (10 ng) o f t h e p o t e n t i a l D e g r a d a t i o n P r o d u c t s o f D i g o x i n . Peak i d e n t i t y : (1) d i g o x i g e n i n ; (2) d i g o x i g e n i n mono-d i g i t o x o s i d e ; and (3) d i g o x i g e n i n b i s d i g i t o x o s i d e , HPLC c o n d i t i o n s : same as i n F i g . 64. O f 5 10 15 M I N U T E S F i g . 82. A Chromatogram f o r t h e 1% l e v e l s (20 ng) o f t h e p o t e n t i a l D egradation' P r o d u c t s o f D i g i t o x i n . Peak i d e n t i t y : (1) d i g i t o x i g e n i n ; (2) d i g i t o x i g e n i n m o n o d i g i t o x o s i d e ; and (3) d i g i t o x i g e n i n b i s d i g i t o x o s i d e . HPLC c o n d i t i o n s : same as i n F i g . 69. T a b l e X X I I I . R e s u l t s o f the S t a b i l i t y S tudy o f D i g o x i n T a b l e t s s t o r e d a t 60°C and 70.4% R e l a t i v e H u m i d i t y  P r o d u c t S t o r a g e Time (weeks) Assay (ng) D i g o x i g e n i n D i g o x i g e n i n m o n o - d i g i t o x o s i d e D i g o x i g e n i n b i s - d i g i t o x o s i d e D i g o x i n ^ D i g o x i n D i g o x i n D i g o x i n Amount E q u i v a l e n t Amount E q u i v a l e n t Amount E q u i v a l e n t Amount L a n o x i n 0.25 mg 4 - - - - 101.2 L a n o x i n 0.125 mg 4 - - - - 98.9 N a t i g o x i n 0.25 mg 4 - - 7.8 8.9 90.1 L a n o x i n 0.25 mg 8 - - - - 103.2 L a n o x i n 0.125 mg 8 - - - - 96.9 N a t i g o x i n 0.25 mg 8 8.0 16.0 8.2 9.4 84.0 L a n o x i n 0.25 mg 12 - - - - 105.6 L a n o x i n 0.125 mg 12 - - - - 101.3 N a t i g o x i n 0.25 mg 12 10.6 21.2 8.8 10.0 79.3 L a n o x i n 0.25 mg 16 - - - - 99.7 L a n o x i n 0.125 mg 16 - - - - 95.4 N a t i g o x i n 0.25 mg 16 11.2 22.3 9.5 10.8 73.4 a P r e l i m i n a r y a s s a y v a l u e s f o r L a n o x i n t a b l e t s 0.25 mg, Lan o x i n t a b l e t s 0.125 mg and n a t i g o x i n t a b l e t s 0.25 mg a r e 101.6%, 99.6% and 104.5%, r e s p e c t i v e l y . b A c c o r d i n g t o l a b e l c l a i m , t h e o r e t i c a l amount i s 100 ng. r e l a t i v e h u m i d i t y ( T a b l e XXIV) i n d i c a t e s t h e same p a t t e r n o f d i g o x i n d e g r a d a t i o n ( i n N a t i g o x i n t a b l e t s ) as i n t h e r e s u l t s shown i n T a b l e X X I I I . The i n i t i a l d e g r a d a t i o n i s marked by t h e ap p e a r a n c e o f t h e b i s d i g i t o x o s i d e w i t h t h e sub s e q u e n t f o r m a t i o n o f a r e l a t i v e l y h i g h e r p e r c e n t a g e o f t h e g e n i n . D i g o x i n i s degraded t o a l e v e l o f 64.5% w i t h the f o r m a t i o n o f 11.5% o f d i g o x i g e n i n b i s d i g i t o x o s i d e and 32.4% o f d i g o x i g e n i n . The a p p e a r a n c e o f d e g r a d a t i o n p r o d u c t s o n l y i n N a t i g o x i n t a b l e t s may be due t o the use o f s u l f u r i c a c i d s o l u t i o n s t o c o n t r o l h u m i d i t y . T h i s a s s umption a p p e a r s t o be s u p p o r t e d by d i f f e r e n c e s i n the b u f f e r c a p a c i t y o f L a n o x i n and N a t i g o x i n t a b l e t s ( F i g . 8 3 ) . I t was o b s e r v e d t h a t L a n o x i n t a b l e t s 0.25 mg and N a t i g o x i n t a b l e t s 0.25 mg had b u f f e r c a p a c i t i e s o f 0.0013 and 0.0004 gram - e q u i v a l e n t , r e s p e c t i v e l y . The h i g h e r b u f f e r c a p a c i t y o f L a n o x i n t a b l e t s (0.125 mg and 0.25 mg) may a c c o u n t f o r t h e a b i l i t y o f t h e s e f o r m u l a t i o n s t o w i t h s t a n d any p r o b a b l e e f f e c t s o f s u l f u r i c a c i d . The d a t a f o r t h e s t a b i l i t y s t u d y o f L a n o x i n i n j e c t i o n s t o r e d a t 60°C and 70.4% r e l a t i v e h u m i d i t y i s shown i n T a b l e XXV. D i g o x i n d e g r a d a t i o n b e g i n s a t about 8 weeks o f s t o r a g e showing a d i g o x i n l e v e l o f 75.1% w i t h 22.0% and 14.0% a p p e a r i n g as b i s d i g i t o x o s i d e and d i g o x i g e n i n , r e s p e c t i v e l y , i n a p e r i o d o f 12 weeks. The p a t t e r n o f d e g r a d a t i o n i s s i m i l a r t o t h a t o f N a t i g o x i n t a b l e t s i n the sense t h a t d i g o x i g e n i n m o n o d i g i t o x o s i d e i s not p r e s e n t as a d e g r a d a t i o n p r o d u c t . The r e s u l t s f o r t h e s t a b i l i t y s t u d y o f L a n o x i n i n j e c t i o n s t o r e d a t 80°C and 37.1% r e l a t i v e h u m i d i t y ( T a b l e XXVI) i n d i c a t e t h a t d i g o x i n d e g r a d a t i o n b e g i n s a t about f o u r weeks and r e s u l t s i n a d i g o x i n l e v e l o f 37.0% w i t h 28.1%, 11.2% and 34.5% r e s p e c t i v e l y a p p e a r i n g as t h e b i s d i g i t o x o s i d e , m o n o d i g i t o x o s i d e and g e n i n i n a p e r i o d o f 12 weeks o f s t o r a g e . The p a t t e r n o f d e g r a d a t i o n i n t h i s c a s e i s q u i t e d i f f e r e n t from t h a t shown i n T a b l e XXV i n t h a t d i g o x i g e n i n m o n o d i g i t o x o s i d e i s • \ 1 I I " 1 2 4 6 8 V O L U M E O F o o o 5 N H C L (ml.) 83. The pH P r o f i l e o f powdered T a b l e t M a t e r i a l ( e q u i v a l e n t t o 20 t a b l e t s ) suspended i n 20 ml o f d i s t i l l e d w a t e r , a f t e r g r a d u a l a d d i t i o n o f 0.005 N HCL. L a n o x i n t a b l e t s 0.125 mg (-x-); L a n o x i n t a b l e t s 0.25 mg ( — ) ; N a t i g o x i n t a b l e t s ( - - ) . T a b l e XXIV. R e s u l t s o f the S t a b i l i t y Study o f D i g o x i n T a b l e t s s t o r e d a t 80°C and 37.1% R e l a t i v e H u m i d i t y  P r o d u c t S t o r a g e Assay (ng) D i g o x i g e n i n D i g o x i g e n i n ~ 1 ; D i g 0 x i g e r r j n m o n o - d i g i t o x o s i d e b i s - d i g i t o x o s i d e D i g o x i n D i g o x i n D i g o x i n D i g o x i n Amount E q u i v a l e n t Amount E q u i v a l e n t Amount E q u i v a l e n t Amount L a n o x i n 0.25 mg ' 4 - - - - - - 94.0 L a n o x i n 0.125 mg 4 - - - - 101.3 N a t i g o x i n 0.25 mg 4 - - - 8.1 9.2 88.0 L a n o x i n 0.25 mg 8 - - - - - 96.0 L a n o x i n 0.125 mg 8 - - - - - - 97.7 N a t i g o x i n 0.25 mg 8 8.3 16.5 - - 8.2 9.3 77.2 L a n o x i n 0.25 mg 12 - - - - - 98.4 L a n o x i n 0.125 mg 12 - - - - - - 95.2 N a t i g o x i n 0.25 mg 12 15.4 30.7 - - 9.1 10.4 69.6 L a n o x i n 0.25 mg 16 - - - - - - 96.2 L a n o x i n 0.125 mg 16 - - - - - 102.4 N a t i g o x i n 0.25 mg 16 16.3 32.4 - - 10.1 11.5 64.5 a A c c o r d i n g t o l a b e l c l a i m , t h e o r e t i c a l amount i s 100 ng. fV> oo T a b l e XXV. R e s u l t s o f the S t a b i l i t y Study o f L a n o x i n I n j e c t i o n 0.05 mg/ml S t o r e d a t 60°C and 70.4% R e l a t i v e H u m i d i t y  Time (weeks) A s s a y (ng) D i g o x i g e n i n D i g o x i g e n i n m o n o - d i g l t o x o s i d e D i g o x i g e n i n B i s - d i g i t o x o s i d e D i q o x i n a ' b Amount D i g o x i n Equi v a l e n t D i g o x i n Amount E q u i v a l e n t Amount D i g o x i n E q u i v a l e n t Amount 2 - - - - - 103.2 4 - - - - - 101.8 6 - - - - - 96.0 8 3.7 7.4 - 15.9 18.1 87.4 10 5.1 10.2' - 15.1 17.2 80.6 12 7.0 14.0 - 19.3 22.0 75.1 a A c c o r d i n g t o l a b e l c l a i m t h e o r e t i c a l amount i s 100 ng. b I n i t i a l l a b e l c l a i m i s 95.2%. T a b l e XXVI. R e s u l t s o f the S t a b i l i t y Study o f L a n o x i n I n j e c t i o n 0.05 mg/ml s t o r e d a t 80°C and 37.1% R e l a t i v e H u m i d i t y  Time (weeks) Assay (ng) D i g o x i g e n i n D i g o x i s e n i n m o n o - d i g i t o x o s i d e D i g o x i g e n i n B i s - d i g i t o x o s i d e D i g o x i n 3 Amount D i g o x i n E q u i v a l e n t Amount D i g o x i n E q u i v a l e n t Amount D i g o x i n E q u i v a l e n t Amount 2 - - - - - - 94.8 4 4.6 9.2 - - 10.5 12.0 80.0 6 7.0 14.0 - - 16.8 19.2 70.8 8 10.1 20.3 7.0 10.1 23.2 26.4 56.3 10 11.5 23.0 5.9 8.6 27.7 31.6 44.2 12 17.3 34.5 7.7 11.2 24.6 28.1 37.0 3 A c c o r d i n g to l a b e l c l a i m , t h e o r e t i c a l amount i s 100 ng. p r e s e n t as a d e g r a d a t i o n p r o d u c t . Moreover, t h e r e l a t i v e amounts o f the d e g r a d a t i o n p r o d u c t s show a marked s h i f t towards d i g o x i g e n i n . The s t a b i l i t y d a t a f o r L a n o x i n e l i x i r s t o r e d a t 60°C and 70.4% r e l a t i v e h u m i d i t y ( T a b l e XXVII) show t h a t d i g o x i n d e g r a d a t i o n b e g i n s a t about t h r e e weeks w i t h t h e a p p e a r a n c e o f d i g o x i g e n i n . A t about f i v e weeks t h e d e g r a d a t i o n o f d i g o x i n i s c h a r a c t e r i z e d by the f o r m a t i o n o f both t h e g e n i n and t h e m o n o d i g i t o x o s i d e . By t h e end o f e i g h t weeks o f s t o r a g e d i g o x i n i s r e d u c e d t o a l e v e l o f 88.2% w i t h 14.6% and 5.9% a p p e a r i n g as d i g o x i g e n i n and d i g o x i g e n i n m o n o d i g i t o x o s i d e . I t can be seen t h a t t h e pH o f the e l i x i r shows a g r a d u a l d e c l i n e from 6.9 to 6.7 i n a p e r i o d o f f i v e weeks. The r e s u l t s o f t h e s t a b i l i t y s t u d y o f L a n o x i n e l i x i r s t o r e d a t 80°C and 37.1% r e l a t i v e h u m i d i t y ( T a b l e XXVIII) i n d i c a t e t h a t d i g o x i n d e g r a d a -t i o n f o l l o w s t h e same p a t t e r n as shown i n T a b l e XXVII e x c e p t t h a t t he r a t e o f d e g r a d a t i o n i s much h i g h e r i n t h i s c a s e . D i g o x i n i s r e d u c e d t o 16.4% w i t h i n t h r e e weeks w i t h 28.8% and 48.9% a p p e a r i n g as d i g o x i -g e n i n and d i g o x i g e n i n m o n o d i g i t o x o s i d e , r e s p e c t i v e l y . Even though no d i g o x i n i s d e t e c t a b l e by t h e f o u r t h week, t h e s t u d y was c o n t i n u e d up t o a p e r i o d o f s i x weeks i n o r d e r t o f i n d o u t i f t h e r e would be a s h i f t i n t h e r e l a t i v e amounts o f t h e d e g r a d a t i o n p r o d u c t s . The d a t a f o r t h e s i x t h week i n d i c a t e an e v e n t u a l d e c l i n e i n the r e l a t i v e amount o f d i g o x i g e n i n m o n o d i g i t o x o s i d e p o s s i b l y due t o i t s breakdown i n t o t h e g e n i n s p e c i e s . I t i s to be noted t h a t d i g o x i g e n i n b i s d i g i t o x o s i d e was not d e t e c t e d i n any o f t h e e l i x i r samples and t h e r e f o r e , t h e p a t t e r n o f d e g r a d a t i o n i s q u i t e d i f f e r e n t from t h a t o b s e r v e d i n t h e s t a b i l i t y s t u d y o f d i g o x i n t a b l e t s and i n j e c t i o n . The d a t a i n T a b l e XXVIII show t h a t t h e pH o f t h e e l i x i r d r ops from an i n i t i a l v a l u e o f 6.5 t o 3.9 o v e r a p e r i o d o f s i x weeks i n d i c a t i n g T a b l e XXVII. R e s u l t s o f t h e S t a b i l i t y Study o f L a n o x i n E l i x i r s t o r e d a t 60°C and 70.4% R e l a t i v e H u m i d i t y  Time (weeks) Assay (ng) PH D i g o x i g e n i n D i g o x i g e n i n m o n o - d i g i t o x o s i d e D i g o x i g e n i n b i s - d i g i t o x o s i d e a D i g o x i n Amount D i g o x i n E q u i v a l e n t Amount D i g o x i n E q u i v a l e n t D i g o x i n Amount E q u i v a l e n t Amount 3 3.8 7.5 - - - • 100.3 6.9 4 3.9 7.8 - - - 103.0 6.9 5 4.0 8.0 3.0 4.5 - 93.0 6.8 6 6.8 13.6 4.2 6.3 - 90.9 6.7 8 7.3 14.6 3.9 5.9 - 88.2 6.7 a A c c o r d i n g t o l a b e l c l a i m t h e o r e t i c a l amount i s 100 ng T a b l e XXVIII. R e s u l t s o f the S t a b i l i t y Study o f L a n o x i n E l i x i r s t o r e d a t 80°C and 37.1 % R e l a t i v e H u m i d i t y  Time (weeks) Assay (ng) pH D i g o x i g e n i n D i g o x i g e n i n m o n o - d i q i t o x o s i d e D i g o x i g e n i n b i s - d i g i t o x o s i de D i g o x i n Amount D i g o x i n E q u i v a l e n t Di g o x i n Amount E q u i v a l e n t Amount E q u i v a l e n t Amount 1 7.4 14.8 18.5 27.7 - 60.0 6.5 2 12.2 24.4 34.1 51.1 - 30.2 6.0 3 14.4 28.8 34.9 48.9 - 16.4 5.6 4 21.7 43.4 43.1 64.8 - - 5.1 5 23.9 47.8 48.6 72.9 - - 4.5 6 27.1 54.2 31.1 46.6 - - 3.9 a A c c o r d i n g t o l a b e l c l a i m t h e o r e t i c a l amount i s 100 ng. b I n i t i a l l a b e l c l a i m i s 101.2%. 224. t h e g r a d u a l f o r m a t i o n o f an a c i d i c s p e c i e s . An attempt was t h e r e f o r e made t o i d e n t i f y the compound t h a t was m a i n l y r e s p o n s i b l e f o r the i n c r e a s e i n a c i d i t y . A sample o f t h e e l i x i r which had been s t o r e d f o r s i x weeks a t 80°C and 37.1% r e l a t i v e h u m i d i t y was p r e p a r e d u s i n g the p r o c e d u r e d e s c r i b e d f o r sample p r e p a r a t i o n o f d i g o x i n e l i x i r . The chromatogram o b t a i n e d f o r t h i s sample u s i n g a s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 47/40/9/4 as m o n i t o r e d w i t h a UV d e t e c t o r s e t a t t h e wave l e n g t h o f 280 nm i s shown i n F i g . 84. Peak 1 was found t o be p - h y d r o x y b e n z o i c a c i d , peaks 2, 3 and 5 were unknown and peak 4 was i d e n t i f i e d as m e t h y l p a r a b e n . The chromatogram o b t a i n e d under the same c o n d i t i o n s f o r a sample o f L a n o x i n e l i x i r which was s t o r e d a t room t e m p e r a t u r e ( F i g . 85) shows a v e r y small peak (peak 1) and a l a r g e peak (peak 3) c o r r e s p o n d i n g t o p - h y d r o x y b e n z o i c a c i d and m e t h y l p a r a b e n , r e s p e c t i v e l y . The f o r m a t i o n o f a r e l a t i v e l y h i g h e r p r o p o r t i o n of p - h y d r o x y b e n z o i c a c i d as shown i n F i g . 84 c o r r e s p o n d s w i t h the drop i n pH shown i n T a b l e X X V I I I . The p r e s e n c e o f s u l f u r i c a c i d i n the s t o r a g e chambers may a l s o c o n t r i b u t e t o t h e i n c r e a s e i n a c i d i t y o b s e r v e d i n T a b l e s XXVII and XXVIII. The f o r m a t i o n o f p - h y d r o x y b e n z o i c a c i d was c o n f i r m e d by mass s p e c t r o m e t r i c d a t a ( F i g . 86) t h a t matches the r e f e r e n c e s p e c t r a i n t h e EPA/NIH Mass S p e c t r a l Data Base ( 1 9 7 8 ) . The r e s u l t s o f t h e s t a b i l i t y s t u d y o f d i g i t o x i n t a b l e t s s t o r e d a t 80°C and 37.1% r e l a t i v e h u m i d i t y ; and 60°C and 70.4% r e l a t i v e h u m i d i t y a r e shown i n T a b l e XXIX. The d a t a i n d i c a t e t h a t d i g i t o x i n i s s t a b l e o v e r t h e p e r i o d o f s i x t e e n weeks under both c o n d i t i o n s o f s t o r a g e and none o f t h e p o t e n t i a l d e g r a d a t i o n p r o d u c t s were d e t e c t e d . 225. 4 F i g . 84. A Chromatogram f o r t h e I s o c r a t i c HPLC S e p a r a t i o n o f the D e g r a d a t i o n P r o d u c t s o f m e t h y l p a r a b e n i n a sample o f L a n o x i n ^ E l i x i r s t o r e d f o r s i x weeks a t 80°C and 37.1% R e l a t i v e H u m i d i t y . Peak i d e n t i t y : (1) p-hydroxy-b e n z o i c a c i d ; (2,3 and 5) unknown; and (4) m e t h y l p a r a b e n HPLC c o n d i t i o n s : s o l v e n t system, w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 47/40/9/4; UV d e t e c t i o n a t 280 nm; f l o w r a t e , 1.2 ml/min; C h a r t speed, 0.5 cm/min. 0 5 10 MINUTES F i g . 85. A Chromatogram f o r a sample o f L a n o x i n E l i x i r s t o r e d under ambient c o n d i t i o n s . Peak i d e n t i t y (1) p - h y d r o x y b e n z o i c a c i d ; (2) unknown; and (3) m e t h y l p a r a b e n . HPLC c o n d i t i o n s : same as i n F i g . 83. 100 2 50 0 .].. ll I .i, I l.li, I,", ilif', i!"' ,I,I'" n- I.I.-.".,— „I;;;I„,'-- , r m/e "* ! * , T a b l e XXIX. R e s u l t s o f the S t a b i l i t y Study o f D i g i t o x i n T a b l e t s 3 ' 1 3 s t o r e d a t 80°C and 37.1% R e l a t i v e H u m i d i t y and 60°C and 70.4% R e l a t i v e H u m i d i t y S t o r a g e t i m e (weeks) P e r c e n t o f Label Cl aim 80°C & 37.1% R.H. 60°C & 70.4% R.H. 4 104.2 99.6 8 105.9 101 .2 12 98.4 103.5 16 99.2 98.3 3 i n i t i a l p e r c e n t o f l a b e l c l a i m i s 101.8 k no d e g r a d a t i o n p r o d u c t s were o b s e r v e d 229. I t i s known t h a t d i g o x i n has a number o f p o s s i b l e pathways o f d e g r a -d a t i o n ( S t e r n s o n and S h a f f e r , 1978). A s c h e m a t i c diagram o f the pathways o f d i g o x i n d e g r a d a t i o n i s shown i n F i g . 87. A c c o r d i n g t o the r e s u l t s o f t h e s t a b i l i t y s t u d y , d e g r a d a t i o n o f d i g o x i n i n N a t i g o x i n t a b l e t s may f o l l o w pathways 1, 5 and 3. D i g o x i n d e g r a d a t i o n i n the i n j e c t i o n s t o r e d a t 60°C a p p e a r s t o f o l l o w pathways 1 and 3. S i n c e t h e r e l a t i v e amounts o f t h e g e n i n and b i s d i g i t o x o s i d e d e t e r m i n e d a t d i f f e r e n t s t o r a g e times r emains t h e same, i t a p p e a r s t h a t pathway 5 i s i n o p e r a t i v e . In t h e c a s e o f t h e i n j e c t i o n sample s t o r e d a t 80°C, however, t h e p r e s e n c e o f a l l t h r e e d e g r a d a t i o n p r o d u c t s appears to> i n d i c a t e t h a t d i g o x i n breakdown f o l l o w s pathways 1, 2 and 3. The r e l a t i v e d e c r e a s e i n t h e amounts o f t h e b i s - and m o n o d i g i t o x o s i d e s a t t h e l a t e r p a r t o f t h e s t u d y seems t o i n d i c a t e t h a t pathways 4, 5 and 6 may a l s o be o p e r a t i v e as p a r a l l e l r o u t e s . In t he c a s e o f t h e e l i x i r sample s t o r e d a t 60°C, the r e s u l t s i n d i -c a t e t h a t d i g o x i n d e g r a d a t i o n f o l l o w s pathways 3 and 2 and t h a t t he former i s the major r o u t e . The d a t a f o r the e l i x i r sample s t o r e d a t 80°C show t h a t pathway 2 i s i n i t i a l l y t h e major r o u t e o f d e g r a d a t i o n w i t h pathway 3 a l s o b e i n g o p e r a t i v e as a p a r a l l e l r o u t e . The r e l a t i v e l y g r e a t e r amounts o f d i g o x i g e n i n o b s e r v e d a t the end o f the s t o r a g e p e r i o d seem to s u g g e s t t h a t pathway 6 may a l s o be o p e r a t i v e . From t h e above d i s c u s s i o n i t can be o b s e r v e d t h a t : (1) d i g o x i n d e g r a d a t i o n may f o l l o w a l l o f t h e t h r e e o r any two pathways; (2) d i f f e r e n t s t o r a g e c o n d i t i o n s f o r the same sample may r e s u l t i n d i f f e r e n t d e g r a d a t i o n pathways e s p e c i a l l y i n t h e i n j e c t i o n ; and (3) d i f f e r e n t p e r i o d s o f s t o r a g e o f t h e same sample may be a s s o c i a t e d w i t h d i f f e r e n t pathways o f d e g r a d a t i o n . I t a p p e a r s , t h e r e f o r e , t h a t t h e a s s o r t m e n t o f pathways t h a t may be o p e r a -t i v e a t d i f f e r e n t c o n d i t i o n s and times o f s t o r a g e and t h e p r o b a b l e e f f e c t s 230 . ( 4 ) I DIGOXIN I I DIGOXIGENIN BISDIGITOXOSIDE I I I DIGOXIGENIN MONODIGITOXOSIDE IV DIGOXIGENIN F i g . 87. Schematic Diagram o f Pathways o f D i g o x i n D e g r a d a t i o n . 231 o f s u l f u r i c a c i d t h a t was used t o c o n t r o l h u m i d i t y would make i t i m p r a c t i c a l t o e s t i m a t e d i g o x i n s h e l f - l i f e from d a t a o b t a i n e d by t h i s s t u d y . Never-t h e l e s s , t h e d a t a i n t h i s s t a b i l i t y s t u d y demonstrate t he p o s s i b i l i t y o f s i m u l t a n e o u s l y m o n i t o r i n g a l l o f t h e p r o b a b l e d e g r a d a t i o n p r o d u c t s o f d i g o x i n and d i g i t o x i n by HPLC. The HPLC methods d e s c r i b e d i n t h i s i n v e s t i g a t i o n c a n be u s e f u l f o r t h e s t a b i l i t y m o n i t o r i n g o f d i g o x i n and d i g i t o x i n i n dosage forms t h a t may be s t u d i e d under ambient c o n d i t i o n s o f s t o r a g e . The absence o f d i g o x i n d e g r a d a t i o n p r o d u c t s i n La n o x i n and d i g i t o x i n t a b l e t s , under t he c o n d i t i o n s o f r e l a t i v e l y h i g h t e m p e r a t u r e s used i n t h i s s t u d y , c o n f i r m s l i t e r a t u r e r e p o r t s o f h i g h s t a b i l i t y o f t h e s e p r o d u c t s . 232. IV. SUMMARY AND CONCLUSIONS 1. An HPLC system t h a t employs a r e v e r s e - p h a s e column; UV d e t e c t i o n a t 220 nm and s o l v e n t systems c o n s i s t i n g o f v a r i o u s p r o p o r t i o n s o f w a t e r , m e t h a n o l , i s o p r o p a n o l and d i c h l o r o m e t h a n e was d e v e l o p e d f o r t h e s e p a r a t i o n o f a m i x t u r e o f d i g o x i n , d i g i t o x i n and t h e i r p o t e n t i a l d e g r a d a t i o n p r o d u c t s and m e t a b o l i t e s . S e p a r a t i o n o f t h e above compounds by i s o c r a t i c , s o l v e n t s w i t c h o v e r and g r a d i e n t e l u t i o n modes was c a r r i e d out i n a c h r o m a t o g r a p h i c time o f 27, 16 and 13 m i n u t e s , r e s p e c t i v e l y . I s o c r a t i c s e p a r a t i o n o f : (1) d i g o x i n and i t s m e t a b o l i t e s ; (2) d i g i t o x i n and i t s m e t a b o l i t e s ; (3) g i t o x i n , d i g o x i n and i t s m e t a b o l i t e s and (4) a and 8 - a c e t y l d i g o x i n , d i g o x i n and i t s m e t a b o l i t e s was a c h i e v e d i n l e s s than 15 minutes i n most c a s e s . 2. HPLC s e p a r a t i o n o f d i g o x i n and i t s m e t a b o l i t e s i n c l u d i n g d i h y d r o d i g o -x i g e n i n was m o n i t o r e d a f t e r f l u o r o g e n i c post-column d e r i v a t i z a t i o n u s i n g t h e a i r s e g m e n t a t i o n p r i n c i p l e w i t h 100% f l u i d r e c o v e r y . The HPLC s o l v e n t system c o n s i s t i n g o f wa t e r , m e t h a n o l , i s o p r o p a n o l and d i c h l o r o m e t h a n e was found t o be c o m p a t i b l e w i t h the aqueous media o f t h e r e a g e n t s used f o r f l u o r o g e n i c d e r i v a t i z a t i o n . 3. Nine e s t r o g e n s t e r o i d s were r e s o l v e d w i t h a s o l v e n t system c o n s i s t -i n g o f w a t e r , m e t h a n o l , i s o p r o p a n o l and d i c h l o r o m e t h a n e i n a t o t a l e l u t i o n t i m e o f about 35 m i n u t e s . 233. 4. The HPLC solvent system mentioned in (1) was used to examine an extract of d i g i t a l i s leaf. A peak appeared at a retention time corresponding to that of authentic d i g i t o x i n . 5. Isocratic HPLC systems were developed for the quantitative analysis of digoxin in tab l e t s , injectables and e l i x i r . (a) A solvent system of water/methanol/isopropanol/dichloromethane: 47/40/9/4 and an internal standard of 17a-ethynylestradiol were used for the assay of digoxin tablets and injection in a chromatographic time of less than 10 minutes. (b) The HPLC method for the analysis of digoxin tablets and injection was found to be capable of accounting for gitoxin and each of the probable degradation products of digoxin. (c) The ca l i b r a t i o n curve of digoxin obtained for the assay of tablets and injection dosage forms was found to be linea r with the l i n e passing close to the o r i g i n . (d) The mean label claim values for the composite tablet analysis of 0.25 mg digoxin tablets of Brands A, B and C were found to be 95.3%, 97.9% and 101.1%, respectively. The composite assay of 0.125 mg digoxin tablets (Brand A) resulted in a mean label c l a i m v a l u e o f 99.9%. D i g o x i n c o m p o s i t e t a b l e t a n a l y s i s was a c c o m p l i s h e d w i t h a r e l a t i v e s t a n d a r d d e v i a t i o n o f 1.5%. The r e s u l t s o f s i n g l e t a b l e t a s s a y o f 0.125 mg and 0.25 d i g o x i n t a b l e t s i n d i c a t e d mean l a b e l c l a i m v a l u e s o f 97.1% and 96.6% w i t h r e l a t i v e s t a n d a r d d e v i a t i o n s o f 5.1% and 4.6%, r e s p e c t i v e l y . (e) The ave r a g e r e c o v e r y v a l u e o f d i g o x i n from t a b l e t m a t e r i a l was found t o be 99.8% w i t h a r e l a t i v e s t a n d a r d d e v i a t i o n o f 3.2%. ( f ) The HPLC a n a l y s i s o f 0.05 mg/ml and 0.25 mg/ml d i g o x i n i n j e c t a b l e s r e s u l t e d i n mean l a b e l c l a i m v a l u e s o f 99.4% and 99.1% w i t h r e l a t i v e s t a n d a r d d e v i a t i o n s o f 1.7% and 1.6%, r e s p e c t i v e l y . (g) A s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 51/42/5/2 was used f o r the a s s a y o f d i g o x i n e l i x i r . The c h r o m a t o g r a p h i c time was found t o be 18 m i n u t e s , u s i n g 17a-e t h y n y l e s t r a d i o l as the i n t e r n a l s t a n d a r d . The HPLC a n a l y s i s was performed by d i r e c t i n j e c t i o n o f the d i l u t e d e l i x i r sample (h) The c a l i b r a t i o n c u r v e o f d i g o x i n o b t a i n e d f o r t h e a n a l y s i s o f the e l i x i r was found t o be l i n e a r w i t h t h e l i n e p a s s i n g c l o s e t o the o r i g i n . ( i ) The HPLC a s s a y o f d i g o x i n e l i x i r r e s u l t e d i n a mean l a b e l c l a i m v a l u e o f 99.4% w i t h a r e l a t i v e s t a n d a r d d e v i a t i o n o f 1.8%. The r e s u l t s o f t h e HPLC a n a l y s i s i n d i c a t e t h a t t h e methods a r e f a s t , s e l e c t i v e , a c c u r a t e , s e n s i t i v e and, t h e r e f o r e , c o n v e n i e n t f o r t h e a s s a y o f d i g o x i n dosage forms. An i s o c r a t i c HPLC system was d e v e l o p e d f o r the q u a n t i t a t i v e a n a l y s i s o f d i g i t o x i n i n t a b l e t and i n j e c t i o n dosage f o r m s . (a) a s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e : 45/38/11/6 and an i n t e r n a l s t a n d a r d o f 1 7 a - m e t h y l t e s t o s t e r o n e were used f o r the a s s a y o f d i g i t o x i n t a b l e t s and i n j e c t i o n i n a c h r o m a t o g r a p h i c time o f ab o u t 15 m i n u t e s . (b) The HPLC method was found t o be c a p a b l e o f a c c o u n t i n g f o r each o f t h e p r o b a b l e d e g r a d a t i o n p r o d u c t s o f d i g i t o x i n . ( c ) The c a l i b r a t i o n c u r v e o f d i g i t o x i n was found t o be l i n e a r w i t h the l i n e p a s s i n g c l o s e t o the o r i g i n . (d) The r e s u l t s o f the a s s a y o f d i g i t o x i n t a b l e t s ( c o m p o s i t e t a b l e t a n a l y s i s ) and i n j e c t i o n i n d i c a t e d mean l a b e l c l a i m v a l u e s o f 97.2% and 96.9% w i t h r e l a t i v e s t a n d a r d d e v i a t i o n s o f 1.4% and 3.3%, r e s p e c t i v e l y . (e) The mean l a b e l c l a i m v a l u e f o r the d i g i t o x i n s i n g l e t a b l e t a s s a y was found t o be 98.8% w i t h a r e l a t i v e s t a n d a r d d e v i a t i o n o f 4.8%. ( f ) The mean r e c o v e r y v a l u e o f d i g i t o x i n from t a b l e t m a t e r i a l was found t o be 99.7% w i t h a r e l a t i v e s t a n d a r d d e v i a t i o n o f 3.1%. The a n a l y t i c d a t a , t h e r e f o r e , i n d i c a t e t h a t t h e HPLC method has the d e s i r a b l e c h a r a c t e r i s t i c s o f s e n s i t i v i t y , s e l e c t i v i t y , r e p r o -d u c i b i l i t y , a c c u r a c y , s i m p l i c i t y and s h o r t time o f a n a l y s i s f o r th e a s s a y o f d i g i t o x i n dosage forms. Comparison o f t h e a n a l y s i s o f d i g o x i n and d i g i t o x i n dosage forms by HPLC and t h e USP methods was made w i t h r e f e r e n c e t o the f o l l o w i n g f a c t o r s : (a) Time. The HPLC and USP a s s a y p r o c e d u r e s f o r d i g o x i n dosage forms were found t o r e q u i r e p e r i o d s o f l e s s than 40 minutes and about 4 h o u r s , r e s p e c t i v e l y . The times o f a n a l y s i s r e q u i r e d f o r the a s s a y o f d i g i t o x i n dosage forms by HPLC and t h e USP method were found t o be l e s s than 45 minutes and o v e r 4 h o u r s , r e s p e c t i v e l y . (b) S e n s i t i v i t y . The USP methods f o r t h e a n a l y s i s o f co m p o s i t e t a b l e t s , i n j e c t i o n and e l i x i r r e q u i r e d samples e q u i v a l e n t t o 2.5 mg o f d i g o x i n . HPLC a n a l y s i s c o u l d be c a r r i e d o u t w i t h c o m p o s i t e t a b l e t , i n j e c t i o n and e l i x i r samples e q u i v a l e n t t o 1.25 mg, 0.1 mg and 1.0 mg o f d i g o x i n , r e s p e c t i v e l y . The USP c o l o r i m e t r i c a s s a y s f o r d i g i t o x i n c o m p o s i t e t a b l e t s and i n j e c t i o n were c a r r i e d o u t w i t h samples e q u i v a l e n t t o 2.0 mg o f d i g i t o x i n and, t h e r e f o r e , were n o t a p p l i c a b l e f o r s i n g l e t a b l e t a s s a y . The HPLC methods f o r the a s s a y o f d i g i t o x i n composite t a b l e t s , s i n g l e t a b l e t s and i n j e c t i o n r e q u i r e d samples e q u i v a l e n t t o 1.0 mg, 0.1 mg and 0.2 mg o f d i g i t o x i n , r e s p e c t i v e l y . ( c ) S e l e c t i v i t y . U n l i k e t he c o l o r i m e t r i c and f l u o r o m e t r i c methods o f t h e USP, the HPLC methods f o r t h e a n a l y s i s o f d i g o x i n and d i g i t o x i n dosage forms were found t o be c a p a b l e o f a c c o u n t i n g f o r each o f the p r o b a b l e d e g r a d a t i o n p r o d u c t s o f both d r u g s . (d) P r e c i s i o n . The r e l a t i v e s t a n d a r d d e v i a t i o n s o b t a i n e d f o r the a n a l y s i s o f d i g o x i n and d i g i t o x i n dosage forms u s i n g HPLC and USP methods were found t o be comparable. (e) A c c u r a c y . The mean r e c o v e r y v a l u e s o f d i g o x i n and d i g i t o x i n from t a b l e t m a t e r i a l as o b t a i n e d by HPLC were found t o be comparable w i t h t h o s e d e r i v e d from t h e USP d a t a . In c o m p a r i s o n w i t h t he USP p r o c e d u r e s , t h e HPLC methods f o r the a n a l y s i s o f d i g o x i n and d i g i t o x i n i n t h e i r dosage forms appear t o be more advantageous from t h e s t a n d p o i n t s o f s e l e c t i v i t y , s e n s i t i v i t y , s i m p l i c i t y , and t i m e o f a n a l y s i s . The HPLC systems d e v e l o p e d f o r t h e a s s a y o f t a b l e t s and i n j e c t a b l e o f both d i g o x i n and d i g i t o x i n may be a p p l i c a b l e f o r the s t a b i l i t y s t u d y o f t h e s e dosage forms. (a) A s o l v e n t system o f w a t e r / m e t h a n o l / i s o p r o p a n o l / d i c h l o r o m e t h a n e 51/43/4/2 was used f o r t h e s t a b i l i t y s t u d y o f d i g o x i n e l i x i r . The c h r o m a t o g r a p h i c t i m e was found t o be l e s s than 15 minutes u s i n g h y d r o c o r t i s o n e as t h e i n t e r n a l s t a n d a r d . (b) The HPLC methods were found t o be s u i t a b l e f o r s i m u l t a n e o u s q u a n t i t a t i o n o f d i g o x i n , d i g i t o x i n and t h e i r r e s p e c t i v e d e g r a d a t i o n p r o d u c t s . ( c ) L a n o x i n and d i g i t o x i n t a b l e t s were found t o be s t a b l e under p a l l t h e c o n d i t i o n s o f s t o r a g e used i n t h i s s t u d y . N a t i g o x i n p t a b l e t s , L a n o x i n i n j e c t i o n and e l i x i r were found t o be s u b j e c t t o v a r y i n g degrees and p a t t e r n s o f d e g r a d a t i o n . (d) The s t a b i l i t y d a t a were found t o i n d i c a t e t h a t d i g o x i n d e g r a d a t i o n may f o l l o w a l l o f t h e t h r e e o r any two pathways. The d e g r a d a t i o n o f some samples i s p r o b a b l y a r e s u l t o f the s u l f u r i c a c i d atmosphere. I t appears t h a n t the La n o x i n t a b l e t f o r m u l a t i o n i s a b l e t o r e s i s t t h i s e f f e c t . T h i s s t u d y d e m o n s t r a t e d t h a t t h e HPLC method d e v e l o p e d c o u l d be u s e f u l f o r c o n v e n t i o n a l a c c e l e r a t e d s t a b i l i t y t e s t i n g f o r t h e d i g o x i n and d i g i t o x i n f o r m u l a t i o n s s t u d i e d . IV. REFERENCES A b e l , R.M., L u c h i , R.J., P e s k i n , G.W., Conn, H.L., J r . and M i l l e r , L.D. ( 1 9 6 5 ) . J . P h a r m a c o l . Exp. T h e r . , 150_, 463. A s h l e y , J . J . , Brown, B.T., O k i t a , G.T. and K l r i g h t , S.E. ( 1 9 5 8 ) . J . B i o l . Chem., 232, 315. Anggard, E.E., Chen, L.P. and Kalman, S.M. ( 1 9 7 2 ) . N. E n g l . J . Med., 287, 935. B a l j e t , H. ( 1 9 1 8 ) . Schewiz Apoth. Z t g . , 56, 71 and 84; t h r o u g h Chem. A b s t r . , 12, 1336 ( 1 9 1 8 ) . B a r r , I . , S m i t h , T.W., K l e i n , M.D., H a g e m e i j e r , F. and Lown, B. (1972). J . P h a r m a c o l . Exp. T h e r . , 180, 710. Bauman, F. " B a s i c L i q u i d Chromatography", V a r i a n A e r o g r a p h , Walnut C r e e k , C a l i f o r n i a , 1971, p. 10. Beerman, B. ( 1 9 7 2 ) . E u r . J . C l i n . P h a r m a c o l . , 5_, 28. Beerman, B., H e l l s t r o m , K. and Rosen, A. ( 1 9 7 2 ) . C l i n . S c i . , 43, 507. B e l l , F.K. and K r a n t z , A.C. ( 1 9 4 8 ) . J . Amer. Pharm. A s s . , S c i . Ed., 37, 297. B e l l e r , G.A., S m i t h , T.W., Abelman, W.H., Haber, E. and Hood, W.B., J r . ( 1 9 7 1 ) . N. E n g l . J . Med., 284, 989. B e r g d a h l , B., Mohn, L., L i n d w a l l , L., D a h l s t r o m , G., S c h e r l i n g , I. and B e r t l e r , A. ( 1 9 7 9 ) . C l i n . Chem., 25_, 305. B i c a n - F i s t e r , T. and Merkas, J . ( 1 9 6 9 ) . J . Chromatog., 41_(1), 91. Bodem, G., G i f r i c h , H.J., A n l e p p , H., Ochs, H.R., D e n g l e r , H.J. ( 1 9 7 7 ) . K l i n . Wschr., 55_, 13. B o g u s l a s k i , R.C. and S c h w a r t z , C.L. ( 1 9 7 5 ) . A n a l . Chem., 47, 1583. B o i n k , A.B.T.J., K r u y s w i j k , H.H., W i l l e b r a n d s , A.F. and Maas, A.H.J. ( 1 9 7 7 ) . J . C l i n . Chem. C l i n . Biochem., 15_, 261. B r i t t e n , A.Z. and N j a u , E . ( 1 9 7 5 ) . A n a l . Chem. A c t a , 76_, 409. B r o o k e r , G. and J e l l i f e , R.W. (1969). Fed. P r o c , 28, 608. Brown, B.T., W r i g h t , S.E. and O k i t a , G.T. ( 1 9 5 7 ) . Nature ( L o n d o n ) , 180, 607. B u r n e t t , G.H., C o n k l i n , R.L. and Wasson, G.W. ( 1 9 7 3 ) . C l i n . Chem., 19, 725. 240. B u t l e r , V.P. ( 1 9 7 1 ) . L a n c e t , 1_, 186. B u t l e r , V.P. ( 1 9 7 8 ) . E v a l u a t i o n s o f D i f f e r e n t Methods f o r D e t e r m i n i n g Serum C o n c e n t r a t i o n s o f C a r d i a c G l y c o s i d e s : I n : G. Bodem and H.J. D e n g l e r ( e d s . ) : C a r d i a c G l y c o s i d e s . S p r i n g e r - V e r l a g , B e r l i n , H e i d e l b e r g , New Y o r k , p. 2. C a l d w e l l , P.C. and Keynes, R.D. (1959). J . P h y s i o l . ( L o n d o n ) , 148, 89. C a r v a l h a s , M.L. and F i g u e i r a , M.A. ( 1 9 7 3 ) . J . Chromatogr., 86, 254. C a s t l e , M.C. ( 1 9 7 5 ) . J . Chromatogr., 115_, 437. C e r c e o , E. and E l l o s o , C A . ( 1 9 7 2 ) . C l i n . Chem., 18, 539. Chen, I.W., S p e r l i n g ' , M., V o i l e , C. and Maxon, H.R. ( 1 9 7 8 ) . I b i d . , 24, 1564. — C h r i s t i a n , H.A. ( 1 9 1 9 ) . Am. J . Med. S c i . , ] 5 7 , 593. C l a r k , D.R. and Kalman,. S.M. ( 1 9 7 4 ) . Drug M e t a b o l i s m and D i s p o s i t i o n , 2, 148. C l a r k e , C.J. and Cobb, P.H. (1979). J . Chromatogr., 1 6 8 ( 2 ) , 541. Cobb, P.H. ( 1 9 7 6 ) . A n a l y s t , 101_, 768. Cremer, E. ( 1 9 5 0 ) . O e s t e r r . Chem. Z t g . , 5J_, 98. C u l l e n , L . F., Packman, D.L. and P a p r i e l l o , G.J. ( 1 9 7 0 ) . J . Pharm. S c i . , 5 9 ( 5 ) , 697. D e n g l e r , H.J., Bodem, G. and G i l f r i c h , H.J. ( 1 9 7 8 ) . D i g o x i n Pharmacokine-t i c s and T h e i r R e l a t i o n t o C l i n i c a l Dosage P a r a m e t e r s . I n : G. Bodem and H.J. D e n g l e r ( e d s . ) : C a r d i a c G l y c o s i d e s . S p r i n g e r - V e r l a g , B e r l i n , H e i d e l b e r g , New York, p. 214. Dequeker, R. and Loobuyck, M. (1955). J . Pharm. P h a r m a c o l . , 7_, 522; t h r o u g h Chem. A b s t r . , 50, 531b ( 1 9 5 6 ) . D o h e r t y , J . E . and P e r k i n s , W.H. ( 1 9 6 2 ) . Amer. H e a r t J . , 63_, 528. D o h e r t y , J . E . , P e r k i n s , W.H. and F l a n i g a n , W.J. ( 1 9 6 7 ) . Ann. I n t e r n . Med., 66, 116. D o h e r t y , J . E . ( 1 9 6 8 ) . Amer. J . Med. S c i . , 255_, 382. D o h e r t y , J . E . , H a l l , W.H., Murphy, M.L. and B e a r d , O.W. ( 1 9 7 1 ) . C h e s t , 59, 433. D o h e r t y , J . E . ( 1 9 7 3 ) . Ann. I n t . Med., 79, 229. 241. D o h e r t y , J . E . and Kane, J . J . ( 1 9 7 5 ) . Ann. Rev. Med., 26, 159. Dzyuba, N.P. e t a l , ( 1 9 7 1 ) . Pharm. Zh. ( K i e v . ) , 2 6 ( 3 ) , 42. E r n i e , F. and F r e i , R.W. (1977). , J . Chromatogr., 130, 169. Evans, F . J . , Flemons, P.A., Duignan, C F . and Cowley, P.S. (1974). I b i d . , 88, 341. Faber, D.B. (19 7 7 ) . I b i d . , 142, 421. Fa1 l i c k , G.J. P r a c t i c a l Methods o f H i g h - s p e e d L i q u i d Chromatography. I n : J.C. G i d d i n g s , E. Gru s h k a , R.A. K e l l e r and J . Cazes ( e d s ) : Advances i n Chromatography. Marcel Dekker, New York, 1975, V o l . 12. F a r r i s , N.A. " I n s t r u m e n t a l L i q u i d Chromatography", E l s e v i e r S c i e n t i f i c , Amsterdam, 1976, Chaps. 2 and 4. Fogelman, A.M., Lamont, J.T., F i n k e l s t e i n , S. e t a l . ( 1 9 7 1 ) . L a n c e t , 2, 727. F o s s , P.R.B. and B e n e z r a , S.A. D i g o x i n . I n : K. F l o r e y ( e d . ) : A n a l y t i c a l P r o f i l e s o f Drug S u b s t a n c e s . Academic P r e s s , New York, 1980, p. 207. Friedman, M. and B i n e , R. J r . ( 1 9 4 7 ) . P r o c . Soc. Exp. B i o l . Med., 64, 162. F r i j n s , J.M.G.J. ( 1 9 7 0 ) . Pharm. Weekblad, J 0 5 , 209. F u j i i , Y., Fukuda, H., S a i t o , Y. and Yamazaki, M. (1980). J . Chromatogr., 202, 139. G a u l t , M.H., Ahmed, M., Symes, A.L. and Vance, J . (19 7 6 ) . C l i n . Biochem., 9, 46. G a u l t , M.H., Ahmed, M., T i b b o , N., L o n g e r i c h , L. and Sugden, D. ( 1 9 8 0 ) . J . Chromatogr., 182, 465. G f e l l e r , J . C , F r e y , G. and F r e i , R.W. ( 1 9 7 7 ) . I b i d . , 142, 271. G i d d i n g s , J.C. Dynamics o f Chromatography, P a r t I , P r i n c i p l e s and T h e o r y . I n : J.C. G i d d i n g s and R.A. K e l l e r ( e d s . ) : C h r o m a t o g r a p h i c S c i e n c e S e r i e s , V o l . I , Dekker, New York, 1965. G r i f f i n , C.L., P e n d l e t o n , R. and B u r s t e i n , S. (19 7 1 ) . Biochem. P h a r m a c o l . , 20, 97. G u l l n e r , H.G., S t i n s o n , E.B., H a r r i s o n , D.C. and Kalman, S.M. ( 1 9 7 4 ) . C i r c u l a t i o n , 50_, 653. H a i s , I.M. and Macek, K. "Paper Chromatography", Academic P r e s s , New Y o r k , 1963, p. 115. H a l a s z , I . and S e b a s t i a n , I . (1 969). Angew. Chem., I n t e r n . Ed., 8, 453 . H a l p e r n , E.P. and Bozdens, R.W. (1979). C l i n . Chem., 25, 67. H a r t e l , G., K y e l o n e n , K., M a r i k a l l i o , E ., O j a l a , K., Manninen, V. and R e i s s e l l , P. ( 1 9 7 6 ) . C l i n . P h a r m a c o l . T h e r . , 1_9, 153. Hauser, W., K a r t n i g , T. and V e r d i n o , G. ( 1 9 6 8 ) . S c i e n t i a Pharm. 36_, 237 Hauser, W., K a r t n i g , T. and V e r d i n o , G. ( 1 9 6 9 ) . I b i d . , 37, 149. H e l l e r , S.R. and M i l n e , G.W. ( e d s . ) . EPA/NIH Mass S p e c t r a l Data Base. U.S. Government P r i n t i n g O f f i c e , 1978, p. 2064. Heuser, D. ( 1 9 6 5 ) . D t . A p o t h z t g . , 103, 1101. Hocke, M. et a l . ( 1 9 6 9 ) . Pharm. Weekblad, 104, 877. Hoffman, J . F . ( 1 9 6 6 ) . Am. J . Med., 41_, 666. Holtzman, J . L . , S h a f e r , R.B. and E r i c k s o n , R.R. ( 1 9 7 4 ) . C l i n . Chem., 20, 1194. Houk, A.E.H., A l e x a n d e r , T.G. and Banes, D. (1959). J . Amer. Pharm. A s s o c . , S c i . Ed ., 48, 217 . J a k o v l j e v i c , I.M. ( 1 9 6 3 ) . A n a l . Chem., 3_5(10), 1513. J a k o v l j e v i c , I.M. D i g i t o x i n . I n : K. F l o r e y ( e d . ) . A n a l y t i c a l P r o f i l e s Drug S u b s t a n c e s . Academic P r e s s , New York, 1974, p. 152. James, A.E., L a q u e r , F.0. and M c l n t y r e , J.D. (1947). J . Amer. Pharm. A s s . , S c i . Ed., 36, 1. J e l l i f f e , R.W. and B l a c k e n h o r n , D.H. ( 1 9 6 3 ) . J . Chromatogr., 1_2, 268 . J e l l i f f e , R.W. (196 7 ) . I b i d . , 27, 172. J e n s e n , K.B. ( 1 9 5 2 ) . A c t a P h a r m a c o l . T o x i c o l . , 8, 101. J e n s e n , K.B. ( 1 9 5 3 ) . I b i d . , 9, 66. J e n s e n , K.B. ( 1 9 6 5 ) . I b i d . , 1_2, 27. J e n s e n , K.B. (197 3 ) . A r c h . Pharm. Chem., S c i . Ed., 1_, 55. Johnson, R., Masserano, R., H a r i n g , R., Kho, B. and S c h i l l i n g , G. (1975) J . Pharm. S c i . , 64, 1007. J o h n s t o n , E . J . and J a c o b s , A.L. ( 1 9 6 6 ) . I b i d . , 5 5 ( 5 ) , 531. Kahn, J . C a r d i a c G l y c o s i d e s and Ion T r a n s p o r t . I n : F i r s t I n t e r n a t i o n a l Congress o f Pharmacology, London, V o l . 3, New York, Pergamon, 1963, pp. 111-135. 243. K a i s e r , F. (196 6 ) . A r c h . P h a r m . B e r l . , 299, 263. Ka t z u n g , B. and Meyers, F.H. ( 1 9 6 6 ) . J . Pharmac o l . Exp. T h e r . , 154, 575: ' Kennedy, E.E. (1950). J . Amer. Pharm. A s s . , S c i . Ed., 39_, 25. Khoury, A . J . ( 1 9 6 7 ) . I n : A u t o m a t i o n i n A n a l y t i c a l C h e m i s t r y , T e c h n i c o n Symposium, 1966, V o l . 1, Mediad I n c . , White P l a i n s , N.Y., pp. 192-195. K i b b e , A.H. and A r a u j o , I.E. (197 3 ) . J . Pharm. S c i . , 62, 1703. K i r k l a n d , J . J . ( 1 9 6 9 ) . J . Chromatogr. S c i . , 7_, 361. K i r k l a n d , J . J . and D e s t e f a n o , J . J . (1970). I b i d . , 8, 309. K l i n k , P.R., Po u s t , R.I., C o l a i z z i , J . L . and McDonald, R.H., J r . (1974). J . Pharm. S c i . , 6^, 1231 . Ko l e n d a , K.D., L u l l m a n n , H. and P e t e r s , T. (1971). B r i t . J . Pharmacol., 41_, 661. Koup, J.R., G r e e n b l a t t , D.J., J u s k o , W.J., Smith, Th.W. and Koch-Weser, J.W. (197 5 ) . J . Pharmacokin. Biopharm., 3_> 181. Kramer, W.G., B a t h a l a , M.S. and Reuning, R.H. (1976). Res. Commun. Chem. Pa t h . P h a r m a c o l . , 1_4, 83. Kramer, W.G., L e w i s , R.P., Cobb, T . C , F o r r e s t e r , W.F., J r . , V i s c o n t i , J.A., Wanke, L.A., Boxenbaum, H.G. and Reuning, R.H. (197 4 ) . J . Pharmacokin. Biopharm., 2, 299. Ku b a s i k , N.P., Warren, K. and S i n e , H.E. (1979). C l i n . Chem., 25, 813. Kuhlman, J . , Abshagen, U. and R i e t b r o c k , J . (1973). Naunyn-Schmiedeberg's A r c h . P h a r m a c o l . , 276, 149. Kuhn, R., W i n t e r s t e i n , A., L e d e r e r , E., Hoppe S e y l e r s , Z. ( 1 9 3 1 ) . P h y s i o l . Chem., 197, 141 . L a f o n , P. (1885). Compt. Rend., 1_00, 1463. L i n d n e r , W. and F r e i , R.W. (1976). I b i d . , 117, 81. Loo, J.C.K., M c G i l v e r a y , J . and J o r d a n , N. (1977). Commun. Chem. P a t h . P h a r m a c o l . , ] ] _ , 497. L o w e n s t e i n , J.M. and C o r r i l l , E.M. (1966). J . Lab. C l i n . Med., 6_7, 1048. L u c h i , R.J. and Gr u b e r , J.W. (1968). Amer. J . Med., 45, 322. L u g t , Ch.B. (1973). P l a n t a M e d i c a , 23, 176. 244. Lukas, D.S. and P e t e r s o n , R.E. (1966). J . C l i n . I n v e s t . , 45, 782. Lukas , D.S. and M a r t i n o , A.G. (1969). I b i d . , 48, 1041. L u t e n , B. (192 4 ) . A r c h . I n t e r n . Med., 33, 251. Marcus, F., K a p a d i a , G.J. and K a p a d i a , G.G. (1964). J . Pharmac o l . Exp. T h e r . , 1_45, 203. Marcus, F . I . , P e t e r s e n , A., S a l e l , A., S c u l l y , J . and K a p a d i a , G.G. (196 6 ) . I b i d . , 1 5 2 , 372. M a r t i n , A.J.P. and Synge, R.L.M. (1941). Biochem. J . , 35_, 1358. M a r i s s , P. (197 9 ) . D e u t s c h e M e d i z i n i s c h e W o c h e n s c h r i f t , 1_04, 1000. M a r v i n , H.M. (192 6 ) . J . C l i n . I n v e s t . , 3, 521. Merck Index, N i n t h E d i t i o n , 1976. Mesnard, P. and Devaux, G. (1961). Compt. Rend., 253, 497. Moe, G.K. and F a r a h , A.E. C a r d i o v a s c u l a r Drugs. I n : The P h a r m a c o l o g i c a l B a s i s o f T h e r a p e u t i c s , 4 t h ed. L.S. Goodman and A. Gilman (Eds.)-. C o l l i n - M a c M i H a n L t d . , T o r o n t o , 1970, p. 697. M o r e l , A. (193 5 ) . B u l l . Soc. Chim. F r a n c e , 5_, 949. M y r i c k , J.W. (1969). J . Pharm. S c i . , 58, 1018. Nachtmann, F., S p t i z y , H. and F r e i , R.W. (1 976). J . Chromatogr. 122, 293. Nyberg, L., A n d e r s s o n , K.E. and B e r t l e r , A. (1974). A c t a Pharm. S u e c i c a , 2 1 , 459. O'Lea r y , T.D., Howe, L.A. and Geary, T.D. (1979). C l i n . Chem., 25, 332. O l i v e r , G.C., P a r k e r , B.M., B r a s f i e l d , D.L. and P a r k e r , C.W. (196 8 ) . J . C l i n . I n v e s t . , £ 7 , 1035. O t t e n , H., Ochs, H.R., Konen, W. and Bodem, G. (197 6 ) . V e r h a n d l g . Deutsche G e s s e l s c h . f . Tnnere Med., 82, 1720. Page, E. (1964). C i r c u l a t i o n , 30, 237. Palmer, L.S., " C a r o t e n o i d s and R e l a t e d Pigments", Chemical C a t a l o g Co., New York, 1922. Palmer, R.F., L a s s e t e r , K.C. and M e l v i n , S. (196 6 ) . A r c h s . Biochem. B i o p h y s . , 113, 629. P a s c o t t , R.P. "Contemporary L i q u i d Chromatography", W i l e y - I n t e r s c i e n c e , New York, 1976, p. 248. Pes e z , M. (1952). Ann. Pharm. F r a n c , TO , 104; t h r o u g h Chem. A b s t r . , 46, 7000e (1952). P e t i t , A. e t a l . (1950). B u l l . Soc. Chim. F r a n c e , 17, 288. P o t t e r , H. (1 963). Pharmazie, 18., 554. P o t t e r , H. and B a e r i s c h , H. (1972). I b i d . , 27, 315. P r a t t , J.H. (191 8 ) . J . Am. Med. A s s . , 71_, 618. R a b i t z s c h , G. (1968). J . Chromatogr., 35, 122. R a b i t z s c h , G. and J u n g l i n g , S. (1969). I b i d . , 41_, 96. R e i c h s t e i n , T. and S c h i n d l e r , 0. (1951). H e l v . Chim.Acta., 34, 108. Rowson, J.M. (1952). J . Pharm. Pharmacol., 4, 814. S a b a t k a , J . J . , B r e n t , D.A., Murphy, J . , C h a r l e s , J . , Vance, J . and G a u l t , M.H. (197 6 ) . J . Chromatogr., 125_, 523. Samuelson, G. (1964). A c t a Pharm. S u e c i c a , 1_(6), 227. Sc h w a r t z , A. (1976). C i r c u l a t i o n Res., _39, 2. S c i a r i n i , L . J . and S a l t e r , W.T. (1951). J . Pharmacol. Exp. T h e r . , 101, 167. S i e g e l , J.H. and S o n n e n b l i c k , E.H. (196 3 ) . C i r c u l a t i o n Res., 1_2, 597. Smi t h , T.W.-, B u t l e r , V.P. and Haber, E. (1969). N. E n g l . J . Med. 281, 1212. S m i t h , T.W., B u t l e r , V.P. and Haber, E. (1970). J . C l i n . I n v e s t . , 49, 2377. S m i t h , T.W. and Haber, E. (1973). Pharmacol. Rev., 25, 219. Sm i t h , T.W. and Haber, E. The C u r r e n t S t a t u s o f C a r d i a c G l y c o s i d e A s s a y T e c h n i q u e s . I n : P.N. T a i and J . F . Goodwin ( e d s . ) . P r o g r e s s i n C a r d i o l o g y . P h i l a d e l p h i a , Lea and F e b i g e r , 1973, v o l . I I . S m i t h , T.W., Green, L.H. and Curfman, G.D. C l i n i c a l i n t e r p r e t a t i o n o f Serum C o n c e n t r a t i o n s o f C a r d i a c G l y c o s i d e s . I n : G. Bodem and H.J. D e n g l e r ( e d s . ) . C a r d i a c G l y c o s i d e s . S p r i n g e r - V e r l a g , B e r l i n , H e i d e l b e r g , New York, 1978, p. 227. Sonobe, T., Hasumi, S., Y o s h i n o , T., K o b a y a s h i , Y., Kawata, H. and N a g a i , T. (1980). J . Pharm. S c i . , 69, 410. Soos, E. (194 8 ) . S c i . Pharm., 1_6, 29. Sn y d e r , L.R. and S a u n d e r s , D.L. (1 969). J . Chromatogr. S c i . , 7_, 195. Sn y d e r , L.R. (197 1 ) . I n : "Modern P r a c t i c e s o f L i q u i d Chromatograph", K i r k l a n d , J . J . ( e d . ) , W i l e y - I n t e r s c i e n c e , New York, Chap. 4, pp. 125-157. Snyd e r , L.R. and K i r k l a n d , J . J . " I n t r o d u c t i o n t o Modern L i q u i d Chroma-t o g r a p h y " , 2nd ed., W i l e y - I n t e r s c i e n c e , New York, 1979, Chap. 2. S t a h l , E. and K a l t e n b a c h , U. (1961). J . Chromatogr., 5, 458. S t e r n s o n , L.A. and S h a f f e r , R.D. (1978). J . Pharm. S c i . , 67, 327. S t o h s , S . J . , R e i n k e , L.A. and El-Olemy, M.M. (1971). Biochem. Pharmacol. 20, 437. S t o l l , A., A n g l i k e r , E., B a r f u s s , F., Kussmaul , W. and Renz, J . (195 1 ) . H e l v . Chim. A c t a , 34, 1460. Sun, L. and S p i e h l e r , V. (1976). C l i n . Chem., 22, 2029. T a k a n a s h i , T., K a t s h , T., Takeda, H., Tokuaka, T., Hamamoto, H. and Ki t a m u r a , K. (1 978). Jap. C i r c . J . , 4j?, 849. Tan, L. (196 9 ) . J . Chromatogr., 45_, 68. T a n t i v a t a n a , P. and W r i g h t , S.E. (1958). J . Pharm. Pharmacol., 1_0, 189. T a t t j e , D.H.E. (1954). I b i d . , 6, 476. T a t t j e , D.H.E. (1957). I b i d . , 9, 29. T a t t j e , D.H.E. (1958). I b i d . , 10, 493. T s w e t t , M. (1903). P r o c . Warsaw Soc. Nat. S c i . B i o l . S e c , 14, No. 6. " U n i t e d S t a t e s Pharmacopeia", 20th Rev., Mack P u b l i s h i n g Co., E a s t o n , PA, 1980. V o i g t l a n d e r , W. (1972). Naunyn-Schmiedeberg 1s A r c h . Pharmacol. 272, 46. V o s h a l l , D.L., Hunter, L. and Grady, H.J. (197 5 ) . C l i n . Chem., 21, 402. — W a l l a c e , A.G., M i t c h e l l , J.H., S k i n n e r , N.S. and S a r n o f f , S . J . .(1963). C i r c u l a t i o n Res., J_2, 611. Walt o n , H.F. P r i n c i p l e s o f Ion Exchange. I n : E. Heftmann ( e d . ) : Chroma-t o g r a p h y , 3rd ed., Van N o s t r a n d R e i n h o l d , New York, 1975, p. 335. 247. Warren, A.T. e t a l . (1948). J . Am. Pharm. A s s o c . , S c i . Ed., 37_, 186. Waters A s s o c i a t e s L i q u i d Chromatography, Waters A s s o c i a t e s I n c . , M i l f o r d , M a s s a c h u s e t t s 01757, 1976. Watson, E., T r a m e l l , P. and Kalman, S.M. (1972). J . Chromatogr. 69, 157. Watson, E., C l a r k , D.R. and Kalman, S.M. (1973). J . Pharmacol. Exp. T h e r . , 184, 424. W e i l e r , E.W. and Z e n i k , M.H. (1 979). C l i n . Chem., 25_, 44. W e l l s , D., Katzung, B. and Meyers, F.H. (1961). J . Pharm. Pharmacol., 12, 389. Wenckebach, K.F. (1910). Br. Med. J . , 2, 1600. W i l s o n , C O . G i s v o l d , 0. and Doerge, R.F. "Textbook o f O r g a n i c M e d i c i n a l and P h a r m a c e u t i c a l C h e m i s t r y " , 6th ed., L i p p i n c o t t , P h i l a d e l p h i a , 1971, pp. 825-829. W i l s o n , J.W. C a r d i a c G l y c o s i d e s . I n : A. Bur g e r ( e d . ) : M e d i c i n a l C h e m i s t r y , 2nd ed. I n t e r s c i e n c e P u b l i s h e r s I n c . , New York, 1960, p. 627. W i l s o n , W.E., Johnson, S.A., P e r k i n s , W.H. and R i p l e y , J . E . (196 7 ) . A n a l . Chem., 39, 40. W i l s o n , W.E. and R i p l e y , J . E . (1 969). I b i d . , 41_(6), 810. W i t h e r i n g , W. An a c c o u n t o f the F o x g l o v e and some o f i t s m e d i c a l uses -w i t h p r a c t i c a l remarks on d r o p s y and o t h e r d i s e a s e s . Birmingham, E n g l a n d , M. Swiney, 1785. R e p r i n t e d i n Med. C l a s s i c s , 1937, 2, 295. Wolf, L. and Ka r a c s o n y , E.M. (1 963). P l a n t a Med., 1J_, 432. Yau, W.W., K i r k l a n d , J . J . and B l y , D.D. "Modern S i z e - E x c l u s i o n L i q u i d Chromatography", W i l e y - I n t e r s c i e n c e , New York, 1979, p. 22. Z a f f a r o n i , A., B u r t o n , R.B. and Keutmann, H.E. (1949). J . B i o l . Chem., 177, 109. Z e c h m e i s t e r , L. and Ch o l n o k y , L. " D i e C h r o m a t o g r a p h i s c h e A d s o r p t i o n s m e t h o d e " , S p r i n g e r , V i e n n a , 1937. Z e e g e r s , J . J . W., Maas, A.H.J., W i l l e b r a n d s , A.F., K r u y s w i j k , H.H. and Jambroes, G. (197 3 ) . C l i n . Chim. A c t a , 44, 109. 

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