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UBC Theses and Dissertations

Synthesis and applications of deuterated methadone and metabolites to biotransformation and disposition… Kang, Gun-Il 1981

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SYNTHESIS AND APPLICATIONS OF DEUTERATED METHADONE AND METABOLITES TO BIOTRANSFORMATION AND DISPOSITION STUDIES by GUN I L KANG B.Sc. S e o u l N a t i o n a l U n i v e r s i t y , K o r e a , 1967 M.Sc. U n i v e r s i t y o f B r i t i s h C olumbia, 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES 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 , 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 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 the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA October 1981 © Gun I I Kang, 1981 I n 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 an 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 l u m b i a , I a g r e 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 agree 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 p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r 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 n o t 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 . Gun I I Kang Pharmaceutical Sciences Department o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V ancouver, Canada V6T 1W5 ^ . Dec. 2nd, 1981 Date ' DE-6 (2/79) i i ABSTRACT Deuterium l a b e l e d methadone and d e u t e r i u m l a b e l e d meta-b o l i t e s were s y n t h e s i z e d t o use i n gas chromatography mass s p e c t r o m e t r y (GCMS) s t u d i e s o f the m e t a b o l i c pathways of metha-done i n r a t s . These compounds were a l s o u s e f u l t o de v e l o p sen-s i t i v e and s e l e c t i v e a n a l y t i c a l methods t o study the pharmaco-k i n e t i c s and d i s p o s i t i o n o f methadone. S y n t h e s i s of the d e u t e r i u m l a b e l e d compounds was m a i n l y a c h i e v e d by u s i n g known p r o c e d u r e s w i t h s p e c i a l t r e a t m e n t s r e q u i r e d t o p r o v i d e l a b e l e n r i c h m e n t . U s i n g the l a b e l e d and u n l a b e l e d d e r i v a t i v e s , mass f r a g -m e n t a t i o n p r o c e s s e s t h a t a re common t o methadone and i t s metabo-l i t e s were d e f i n e d . A r y l r i n g m i g r a t i o n was ob s e r v e d i n a f r a g -m e n t a t i o n p r o c e s s f o r 2 - e t h y l i d e n e - l , 5 - d i m e t h y l - 3 , 3 - d i p h e n y l p y r r o l i d i n e (EDDP). T h i s a r y l r i n g m i g r a t i o n was not a f a v o r -a b l e p r o c e s s f o r r i n g s u b s t i t u t e d EDDP a n a l o g s . V a r i o u s a s p e c t s r e g a r d i n g the o p t i m i z a t i o n o f the s e l e c t e d i o n m o n i t o r i n g (SIM) a n a l y s i s o f methadone and i t s m e t a b o l i t e s i n b i o l o g i c a l f l u i d s a r e d e s c r i b e d . The SIM a n a l -y s i s u s i n g d e u t e r i u m l a b e l e d compounds as i n t e r n a l s t a n d a r d s g e n e r a l l y p r o v e d t o be s e l e c t i v e b u t not as s e n s i t i v e as ex p e c t e d u s i n g e l e c t r o n impact i o n i z a t i o n (EI) c o n d i t i o n s o f GCMS. One advantage o f u s i n g SIM over GC a n a l y s i s was de-s c r i b e d i n terms o f r a t i o a n a l y s i s . Q u a n t i t a t i o n o f methadone i n human plasma and s a l i v a u s i n g SIM gave a lower l i m i t of s e n s i t i v i t y o f 20 ng/0.5 ml of sample by m o n i t o r i n g the base peak, m/e 72. The mean methadone r a t i o s of s a l i v a t o plasma f o r two p a t i e n t s were 0.55 ± 0.15 ( s t a n d a r d d e v i a t i o n ) and 0.48 ± 0.10 ( s t a n d a r d d e v i a t i o n ) . Methadone m e t a b o l i s m s t u d i e s emphasized the d e t e c t i o n o f minor m e t a b o l i t e s u s i n g s p e c i a l e x t r a c t i o n methods f o r r a t b i l e and u s i n g l a b e l e d and u n l a b e l e d compounds. Comparison of t h e mass s p e c t r a from t o t a l i o n c u r r e n t (TIC) p r o f i l e s of m e t a b o l i t e s from u n l a b e l e d compounds w i t h those from l a b e l e d compounds run as s e p a r a t e e x p e r i m e n t s gave GCMS e v i d e n c e f o r methadone n i t r o n e ( N - m e t h y l e n e - l - m e t h y l - 3 , 3 - d i p h e n y l - 4 - o x o -hexanamine-oxide) • P o s s i b i l i t i e s f o r t h e m e t a b o l i c f o r m a t i o n of N-hydroxynormethadone and t h e p h a r m a c o l o g i c a l s i g n i f i c a n c e o f t h e d e t e c t i o n o f methadone n i t r o n e were d e s c r i b e d . A p r o p o s a l f o r m e t a b o l i c s t u d i e s t o examine the p o t e n t i a l forma-t i o n o f o t h e r methadone m e t a b o l i t e s r e s u l t i n g from m e t a b o l i c o x i d a t i o n o f n i t r o g e n was p r e s e n t e d . S t r u c t u r a l e v i d e n c e f o r the methadone n i t r o n e m o l e c u l e was o b t a i n e d i n d i r e c t l y by c h e m i c a l o x i d a t i o n s t u d i e s of metha done m e t a b o l i t e s . m - C h l o r o p e r b e n z o i c a c i d t r e a t m e n t of EDDP p e r c h l o r a t e gave t h r e e p r o d u c t s : methadone n i t r o n e , 4,4-diphen y l - 2 , 5 - h e p t a n e d i o n e ( d i k e t o n e ) , and 2 - a c e t y l - 5 - m e t h y l - 3 , 3 - d i -p h e n y l - l - p y r r o l i n e . These compounds were i d e n t i f i e d from t h e i r IR, NMR and mass s p e c t r a l d a t a . Mass f r a g m e n t a t i o n p r o -c e s s e s were d e f i n e d f o r t h e methadone n i t r o n e . P o s s i b l e mecha nisms f o r the f o r m a t i o n o f methadone n i t r o n e and d i k e t o n e from i v c h e m i c a l o x i d a t i o n of EDDP a r e proposed. S i n c e diazepam i s a drug w i d e l y abused by methadone maintenance p a t i e n t s , methadone-diazepam i n t e r a c t i o n s t u d i e s were d e s i g n e d t o a n a l y z e m e t a b o l i t e s u s i n g d e u t e r i u m l a b e l e d a u t h e n t i c compounds as i n t e r n a l s t a n d a r d s . M e t a b o l i t e s i n the c o n j u g a t e d f r a c t i o n o f r a t b i l e were a n a l y z e d u s i n g d e u t e r i u m l a b e l e d b i o s y n t h e t i c i n t e r n a l s t a n d a r d s . Diazepam (5 mg/kg) was g i v e n t o r a t s t h r o u g h a c a n n u l a t e d j u g u l a r v e i n and a sub-cutaneous dose of methadone (10 mg/kg) was g i v e n . B i l e was c o l l e c t e d t h r o u g h the c a n n u l a t e d b i l e d u c t over a p e r i o d o f 24 h o u r s . The d e u t e r i u m l a b e l was found t o be s t a b l e even under s e v e r e c o n d i t i o n s o f i n c u b a t i o n t e m p e r a t u r e and t i m e . SIM a n a l y s i s o f b i l e sample e x t r a c t s showed t h a t c o n c o m i t a n t a d m i n i s t r a t i o n o f diazepam w i t h methadone d i d n o t a f f e c t b i l i -a r y e x c r e t i o n o f EDDP nor t h e c o n j u g a t e d m e t a b o l i t e s . T h i s i n d i c a t e s t h a t diazepam does n ot i n t e r a c t w i t h methadone a t the h e p a t i c m e t a b o l i s m l e v e l and w i t h t r a n s p o r t of t h e metabo-: l i t e s by t h e b i l i a r y e x c r e t i o n r o u t e . A p p l i c a t i o n of t h e use of a b i o s y n t h e t i c i n t e r n a l s t a n d a r d t o drug m e t a b o l i s m and p h a r m a c o k i n e t i c s t u d i e s by means o f r a t i o a n a l y s i s was d e s c r i b e d w i t h examples. S i g n a t u r e o f T h e s i s S u p e r v i s o r V TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES x i LIST OF FIGURES x i i ABBREVIATIONS x i v ACKNOWLEDGEMENTS x v i INTRODUCTION 1 M e t a b o l i c pathways o f methadone 2 S y n t h e s i s o f methadone and i t s a n a l o g s 4 S y n t h e s i s o f methadone m e t a b o l i t e s 7 Attempted s y n t h e s i s o f normethadone 9 A n a l y s i s o f methadone and m e t a b o l i t e s i n b i o l o g i c a l samples 10 P h a r m a c o k i n e t i c a s p e c t s o f methadone 12 Methadone and dr u g i n t e r a c t i o n s 13 Deuterium l a b e l e d compounds f o r s t u d i e s o f p h a r m a c o k i n e t i c s and drug m e t a b o l i s m 15 OBJECTIVES OF THE RESEARCH 18 S y n t h e s i s o f d e u t e r a t e d methadone and m e t a b o l i t e s 19 Mass f r a g m e n t a t i o n s t u d i e s 20 SIM a n a l y s i s o f methadone and m e t a b o l i t e s i n b i o l o g i c a l samples 20 v i S t u d i e s o f methadone-diazepam i n t e r a c t i o n 21 D e t e c t i o n o f new methadone m e t a b o l i t e s 22 EXPERIMENTAL 2 3 1. M a t e r i a l s 23 G e n e r a l c h e m i c a l s and r e a g e n t s 23 S o l v e n t s 23 M a t e r i a l s f o r a n i m a l s u r g e r y 24 2. A n a l y t i c a l methods 24 GCMS r e p e t i t i v e s c a n n i n g 24 GCMS s e l e c t e d i o n m o n i t o r i n g 24 Gas chromatography 25 Che m i c a l i o n i z a t i o n GCMS 25 High r e s o l u t i o n mass s p e c t r o m e t r y 25 High performance l i q u i d chromatography.... 25 NMR s p e c t r o s c o p y 2 6 I n f r a r e d s p e c t r o s c o p y 26 3. C h e m i c a l s t u d i e s 26 S y n t h e s i s o f d e u t e r i u m l a b e l e d methadone and m e t a b o l i t e s 2 6 2 S y n t h e s i s o f methadone- H^Q 26 S y n t h e s i s o f 4-dimethylamino-2,2-d i p h e n y l p e n t a n o i c a c i d - 2 H i 0 ( 5 , 2 A r = 2 H 1 0 ) and DDP- 2H 1 0 (6, 2Ar= 2Hio) ' 2 7 S y n t h e s i s o f EDDP- 2Hi 0 (3, 2 A r = 2 H i 0 ) and EDDP- 2H 3 28 S y n t h e s i s o f EMDP- 2H 1 0 (2, 2 A r = 2 H 1 0 ) and EMDP- 2H 3 28 S y n t h e s i s o f 2-dimethylamino-4,4-diphe-nyl-5-nonanone p e r c h l o r a t e 28 v i i C h e m i c a l o x i d a t i o n s t u d i e s 29 4. S e l e c t e d i o n m o n i t o r i n g (SIM) a n a l y s i s o f methadone and m e t a b o l i t e s i n human plasma, s a l i v a , and u r i n e samples 29 Samples 2 9 E x t r a c t i o n p r o c e d u r e s and s t a n d a r d c u r v e p r e p a r a t i o n 30 Plasma and s a l i v a 30 U r i n e s 31 GC a n a l y s i s . ; 32 S t a b i l i t y o f EDDP. . 32 5. D e t e c t i o n o f methadone m e t a b o l i t e s 33 Ani m a l e x p e r i m e n t s 3 3 Human e x p e r i m e n t s 33 Sample work up p r o c e d u r e s 33 6. Methadone-diazepam i n t e r a c t i o n s t u d i e s 34 Treatment o f a n i m a l s 34 Sample p r e p a r a t i o n p r o c e d u r e s 36 S t a b i l i t y e x p e r i m e n t s 37 Nonconjugated m e t a b o l i t e s 37 Conj u g a t e d m e t a b o l i t e s 38 7. P h a r m a c o k i n e t i c and s t a t i s t i c a l a n a l y s i s ... 38 RESULTS AND DISCUSSION 40 1. S y n t h e s i s and mass s p e c t r o m e t r y o f d e u t e r -a t e d methadone and m e t a b o l i t e s 40 D e u t e r a t e d d i p h e n y l a c e t o n i t r i l e 40 D e u t e r a t e d methadone 4 3 D e u t e r a t e d methadone m e t a b o l i t e s 46 v i i i S p e c t r o s c o p i c o b s e r v a t i o n s 52 2. SIM a n a l y s i s of methadone and m e t a b o l i t e s i n b i o l o g i c a l samples . 55 S e l e c t e d i o n m o n i t o r i n g 55 A n a l y s i s o f methadone i n plasma and s a l i v a . 58 A n a l y s i s o f methadone and m e t a b o l i t e s / : i n , - u r i n e 62 S t a b i l i t y of EDDP 6 6 3. D e t e c t i o n o f methadone m e t a b o l i t e s 6 9 E x t r a c t i o n p r o c e d u r e s f o r r a t b i l e 69 Nonconjugated f r a c t i o n 72 Co n j u g a t e d f r a c t i o n 73 Ri n g h y d r o x y l a t i o n pathways of methadone me t a b o l i s m 78 N - h y d r o x y l a t e d m e t a b o l i c pathway of methadone 81 P o s s i b i l i t i e s f o r the m e t a b q l i c f o r m a t i o n o f N-hydroxy m e t a b o l i t e s o f methadone . 83 P h a r m a c o l o g i c a l s i g n i f i c a n c e of the d e t e c -t i o n of methadone n i t r o n e 87 P o s s i b l e m e t a b o l i c pathways o f methadone proposed i n F i g . 13 89 4. C h e m i c a l o x i d a t i o n s t u d i e s 92 Ch e m i c a l o x i d a t i o n o f methadone m e t a b o l i t e s 92 Che m i c a l o x i d a t i o n o f EDDP p e r c h l o r a t e .... 93 I d e n t i f i c a t i o n o f methadone n i t r o n e by mass s p e c t r o m e t r y 9 5 New p r o p o s a l f o r f r a g m e n t a t i o n pathways f o r methamphetamine n i t r o n e 102 NMR o f methadone n i t r o n e 10 5 i x IR s p e c t r o s c o p y o f methadone n i t r o n e and r e l a t e d compounds 1 0 8 F o r m a t i o n o f d i k e t o n e (61.) from o x i d a t i o n o f EDDP p e r c h l o r a t e 1 0 9 Proposed mechanism f o r the c h e m i c a l forma-t i o n o f methadone n i t r o n e (5_2) o r o x a z i r i d i n e ( 1 2 8 ) , and d i k e t o n e (6^.) 1 1 2 Proposed s t r u c t u r e f o r compound A ( F i g . 1 3 ) 1 1 4 S y n t h e s i s o f EMDP o x a z i r a n (184) 1 1 5 5. I n t e r a c t i o n o f methadone and diazepam 1 1 8 S t a b i l i t y s t u d i e s . 1 1 8 B i l e c o l l e c t i o n 1 2 5 A n a l y s i s o f non c o n j u g a t e d m e t a b o l i t e s from r a t b i l e 1 2 5 A n a l y s i s o f c o n j u g a t e d m e t a b o l i t e s from r a t b i l e 1 2 9 D i s c u s s i o n o f t h e methadone-diazepam i n t e r a c t i o n 1 3 7 P o t e n t i a l a p p l i c a t i o n s o f r a t i o a n a l y s i s t o d r u g m e t a b o l i s m and pharmaco-k i n e t i c s t u d i e s 1 4 0 P h a r m a c o k i n e t i c s t u d i e s 1 4 0 K i n e t i c approach t o t h e s t u d i e s o f h y d r o x y l a t i o n mechanism 1 4 1 SUMMARY AND CONCLUSIONS 1 4 4 REFERENCES 1 4 8 APPENDIX NMR S p e c t r a o f D e u t e r a t e d Compounds 1 6 2 Spectrum 1 P a r t i a l l y d e u t e r a t e d d i p h e n y l a c e t o -n i t r i l e - 2 H 1 0 1 6 3 2 Spectrum 2 E n r i c h e d d i p h e n y l a c e t o n i t r i l e - H^Q 1 6 3 2 Spectrum 3 Methadone n i t r i l e - H-^ Q 1 6 4 Spectrum 4 4 - D i m e t h y l a m i n o - 2 , 2 - d i p h e n y l p e n t a n o i c 2 a c i d - H N n 1 6 4 X 2 Spectrum 5 Methadone- H 1 Q 165 2 Spectrum 6 DDP- H 1 Q. 166 Spectrum 7 EMDP- 2H 1 Q 166 2 Spectrum 8 EDDP- H 3 16 7 2 Spectrum 9 EDDP- n 16 7 x i LIST OF TABLES Number Page I NMR o f C-4 P r o t o n s o f Methadone M e t a b o l i t e s 54 I I E f f e c t o f D i l u t i o n and I n j e c t i o n Volumes upon t h e Observed Ion R a t i o s 6 3 I I I C a l i b r a t i o n E q u a t i o n s f o r Methadone and EDDP f o r U r i n e A n a l y s i s 6 4 IV Mass S p e c t r a l Data o f TIC O b t a i n e d from Diazomethane T r e a t e d Conjugate F r a c t i o n .... 75 V High R e s o l u t i o n Mass F r a g m e n t a t i o n Data f o r Methadone N i t r o n e (5_2) 96 VI NMR o f C-5 and gem i n a l p r o t o n s o f methadone n i t r o n e (5_2) and 3,4-dimethoxyamphetamine n i t r o n e (127) 10 7 V I I S t a b i l i t y / o f D e u t e r a t e d Methadone, EDDP and EMDP 119 2 V I I I S t a b i l i t y o f Deuterium i n HOEMDP- H g 121 2 IX S t a b i l i t y o f Deuterium i n DiHOEMDP- H Q 122 X E f f e c t o f Diazepam Treatment on the B i l e Flow o f Rats 126 XI E x c r e t i o n o f EDDP from Methadone and Methadone-Diazepam T r e a t e d Rats 130 X I I C a l i b r a t i o n Curve Data f o r Conjugate M e t a b o l i t e s 134 X I I I E x c r e t i o n o f Monohydroxy EMDP from Methadone and Methadone-diazepam T r e a t e d Rats 135 XIV E x c r e t i o n o f D i h y d r o x y EMDP from Methadone and Methadone-diazepam T r e a t e d Rats 136 XV P h a r m a c o k i n e t i c e q u a t i o n s f o r the f o r m a t i o n of t he monohydroxy and d i h y d r o x y m e t a b o l i t e s . 143 x i i LIST OF FIGURES Number Page 1 M e t a b o l i c Pathways o f Methadone 3 2 Mass s p e c t r a o f d e u t e r a t e d d i p h e n y l a c e t o n i - .'. t r i l e s (a) p r e p a r e d u s i n g an e x c e s s o f b e n z e n e - 2 E U ; (b) 50% d e u t e r a t e d p r e p a r e d by : -\ t h e method o f Hachey e t a l . (19); (c) a f t e r e n r i c h m e n t 41 3 Mass s p e c t r a o f (a) methadone and (b) metha-done- 2!^ 0 44 4 Mass s p e c t r a o f (a) DDP and (b) DDP- 2H 1 Q 49 2 5 Mass s p e c t r a o f (a) EDDP, (b) EDDP- H 1 Q , and (c| EDDP- 2H 3 50 6 Mass s p e c t r a o f (a) EMDP and (b) EMDP- 2H 1 Q ... 53 7 SIM chromatogram (m/e 72) o f methadone from s a l i v a 60 8 Methadone c o n c e n t r a t i o n i n plasma and s a l i v a o f a maintenance p a t i e n t (90 mg/day dosage) 60 9 D e c o m p o s i t i o n of EDDP base t o DDP ( EDDP, DDP) 6 7 10 E x t r a c t i o n P r o c e d u r e f o r Rat B i l e 70 11 TIC p r o f i l e o f diazomethane t r e a t e d c o n j u g a t e f r a c t i o n . (GCMS: 150-280°C, 6°/min, h o l d a t 280°C) 74 12 Mass spectrum o f m/e 72 c o n t a i n i n g m e t a b o l i t e 77 13 P o s s i b l e m e t a b o l i c pathways f o r methadone .... 84 14 TIC p r o f i l e (a: GCMS, 200-280°C 8°/min.) and HPLC (b) of EDDP p e r c h l o r a t e o x i d i z e d p r o d u c t s 94 x i i i Number Page 15 NMR spectrum o f methadone n i t r o n e 106 16 IR o f methadone n i t r o n e 110 17 NMR (a) and IR (b) o f 4,4-diphenyl-2,5'-heptanedione (6J.) I l l 18 Proposed mechanisms f o r the f o r m a t i o n o f methadone n i t r o n e (5_2) and 4 , 4 - d i p h e n y l -3,6-heptanedione (61.) by MCPBA o x i d a t i o n o f EDDP p e r c h l o r a t e 113 19 SIM chromatograms f o r EMDP ( a ) , EDDP ( b ) , and methadone (c) 127 20 SIM chromatograms f o r (a) monohydroxy EMDP and (b) d i h y d r o x y EMDP a n a l y s i s 131 21 TIC p r o f i l e (a) and Mass chromatogram (b) of the c o n j u g a t e f r a c t i o n o b t a i n e d from methadone dosed r a t b i l e b e f o r e back e x t r a c t i o n and TIC p r o f i l e (c) a f t e r back e x t r a c t i o n 133 x i v ABBREVIATIONS Ar - Phenyl a.mli'u. a t o m i c mass u n i t CI c h e m i c a l i o n i z a t i o n DDP 1 , 5 - d i m e t h y l - 3 , 3 - d i p h e n y l - 2 - p y r r o l i d o n e DMSO d i m e t h y l s u l f o x i d e dR d e o x y r i b o s e EDDP 2 - e t h y l i d e n e - l , 5 - d i m e t h y l - 3 , 3 - d i p h e n y l p y r r o l i d i n e E I e l e c t r o n impact EMDP 2 - e t h y l - 5 - m e t h y l - 3 , 3 - d i p h e n y l - l - p y r r o l i n e EtOH e t h a n o l GC gas chromatography GCMS gas chromatography mass s p e c t r o m e t r y GSH reduced g l u t a t h i o n e HPLC h i g h p r e s s u r e (performance) 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 I.P. i n t r a p e r i t o n e a l IR i n f r a r e d s p e c t r o s c o p y ( s p e c t r o p h o t o m e t e r ) I.S. i n t e r n a l s t a n d a r d i . v . i n t r a v e n o u s MCPBA m - c h l o r o p e r b e n z o i c a c i d MeOH methanol mp m e l t i n g p o i n t XV NMR n u c l e a r magnetic resonance ( s p e c t r o s c o p y ) o.d. e x t e r n a l d i a m e t e r PFK p e r f l u o r o k e r o s e n e s.c. subcutaneous SD s t a n d a r d d e v i a t i o n SIM s e l e c t e d i o n m o n i t o r i n g *"l/2 b i o l o g i c a l h a l f - l i f e TIC t o t a l i o n chromatogram TLC t h i n l a y e r chromatography TMCS t r i m e t h y l c h l o r o s i l a n e TMS t e t r a m e t h y l s i l a n e UV u l t r a v i o l e t Note: Methadone n i t r o n e i s used f o r convenience t o d e s c r i b e the n i t r o n e found i n me t a b o l i s m s t u d i e s o f methadone and c h e m i c a l o x i d a t i o n o f EDDP p e r c h l o r a t e . S p e c i f i c a l l y the c h e m i c a l i s normethadone n i t r o n e o r N-methylene-1-m e t h y l - 3 . 3 - d i p h e n y l - 4 - o x o - h e x a n a m i n e - o x i d e . x v i ACKNOWLEDGEMENTS T h i s t h e s i s has been made p o s s i b l e by t h e h e l p o f Dr. Frank A b b o t t . The a u t h o r t h a n k s Mr. R. B u r t o n and M i s s S. Ferguson f o r t h e i r t e c h n i c a l a s s i s t a n c e . The a u t h o r i s i n d e b t e d t o Dr. S. Chan o f t h e Department of C h e m i s t r y f o r NMR s p e c t r o s c o p y , Dr. G. E i g e n d o r f o f the Department o f C h e m i s t r y f o r h i g h r e s o l u t i o n mass s p e c t r o m e t r y , Dr. K. McErlane f o r HPLC, and Dr. G. Gudauskas o f the B.C. Cancer Research C e n t e r f o r c h e m i c a l i o n i z a t i o n mass s p e c t r o m e t r y . The a u t h o r would l i k e t o thank Dr. B. R i e d e l , Dr. J . M c N e i l l , Dr. G. B e l l w a r d , Dr. T. Brown, Dr. D. Godin, and Dr. J . Orr f o r h e l p f u l d i s c u s s i o n s . The h e l p o f Dr. J . A x e l s o n and Dr. Y.K. Tam i n a n i m a l e x p e r i m e n t s i s g r e a t l y a p p r e c i a t e d . x v i i DEDICATION To my w i f e and d a u g h t e r s 1 INTRODUCTION Methadone (1) was f i r s t s y n t h e s i z e d by a German c h e m i s t d u r i n g World War I I and the drug was made c l i n i c a l l y a v a i l a b l e i n 194 7 as a morphine s u b s t i t u t e . The p h a r m a c o l o g i c a l p r o p e r -t i e s o f methadone are q u a l i t a t i v e l y i d e n t i c a l t o t h o s e o f morphine. The dose o f methadone as an a n a l g e s i c i s i n the same 1 range as t h a t o f morphine. The drug has marked s e d a t i v e e f f e c t s i n some p a t i e n t s upon r e p e a t e d a d m i n i s t r a t i o n and t h e r e f o r e l i m i t e d use was o n l y a l l o w e d . Methadone shows a c o n s i d e r a b l e degree of a n a l g e s i c a c t i o n when g i v e n o r a l l y . W h i l e the w i t h d r a w a l syndrome of methadone i s s i m i l a r t o t h a t o f morphine, i t d e v e l o p s s l o w l y and i s l e s s i n t e n s e and more p r o l o n g e d . The drug has a l o n g d u r a t i o n o f a c t i o n w i t h an average °f 25 hours com-p a r e d w i t h 2.5-3 hours f o r morphine. The p o s s i b i l i t y o f 2 methadone t r e a t m e n t f o r r e h a b i l i t a t i o n o f h e r o i n a d d i c t s was f i r s t r e p o r t e d i n 1965 by Dole and Nyswander. By 1977, i n the U n i t e d S t a t e s a l o n e , over 90,000 p a t i e n t s were e n r o l l e d i n metha-done maintenance program w i t h c o s t s between $4.00 and $5.50 a day p e r p a t i e n t ( 1 ) . M e t a b o l i c pathways o f methadone S t u d i e s on m e t a b o l i c pathways o f methadone have been performed t o dete r m i n e c o n t r i b u t i o n s o f s p e c i f i c pathways t o t h e k i n e t i c s o f methadone and t o c o r r e l a t e t h e f o r m a t i o n o f s p e c i f i c m e t a b o l i t e s t o a c t i v i t y and t o x i c i t y o f t h i s d r u g . B e c k e t t e t a l . ( 2 ) , P o h l a n d e t a l . ( 3 ) , S u l l i v a n e t a l . ( 4 - 6 ) , Lynn e t a l . ( 7 ) , and Angg&rd e t a l . (8) showed t h a t methadone undergoes de-m e t h y l a t i o n t o form EDDP (_3) and i n t u r n EMDP (4J which i s then o x i d i z e d t o r i n g h y d r o x y l a t e d m e t a b o l i t e s . Methadone i s reduced t o methadol (7_) and N-demethylmethadol. F i g . 1 i s a summary o f the m e t a b o l i c pathways o f methadone as found i n r a t s and i n humans. Methadone and i t s major m e t a b o l i t e , EDDP have been a n a l y z e d t o d e s c r i b e the p h a r m a c o k i n e t i c s o f methadone. Conju-gated m e t a b o l i t e s (3_, 1_0, 11) were a l s o c o n s i d e r e d t o be im-p o r t a n t w i t h r e s p e c t t o i n t e r a c t i o n s a r i s i n g from c o n c o m i t a n t d r u g a d m i n i s t r a t i o n w i t h methadone ( 9 ) . No one has f u l l y e x p l a i n e d t h e a c t i v i t y and t o x i c i t y o f methadone i n r e l a t i o n t o i t s m e t a b o l i s m . EDDP and EMDP were found t o be i n a c t i v e as a n a l g e s i c s ( 3 ) . Methadol and demethylmethadol 3 CHOH-CH2CHj CH2-CH-N(CHj)2 CH3 o II C—OH CH2-(JH-N(CH3)2 CH, A C K— CH, \ / CH- -?H CH, U C D 2-CH, V \\ CH2-CH-NHCHj CH, V CHCH, I 5 C N—CH, If CK- CH 1 2 AH. HO / 0 I C-CH2CHj HO C^Kg—CH-N(CHj)2 " CHj CHCH, si X N —CH, / 3 CH I CHj CHnCH-z I 2 3 A C N \ / CH^  -CH 4-CH2CHj <M\ CH —CH CHj hO HC C N / -CH CY 11 F i g . 1. M e t a b o l i c Pathways o f Methadone 4 account f o r o n l y p a r t o f t h e a n a l g e s i c a c t i v i t y ( 5 ) . The p o s t u -l a t e t h a t t h e mechanism o f a c t i o n o f methadone might be e x p l a i n e d i n r e l a t i o n t o the b i n d i n g o f methadone and -me-tnadone . metabo-l i t e s t o c e l l u l a r components (10) has not been pro v e d . Kreek e t a l . (11-12) e v a l u a t e d the c h r o n i c t o x i c i t y o f methadone and r e p o r t e d t h a t methadone t r e a t m e n t caused m i n i m a l s i d e e f f e c t s w i t h o u t t o x i c e f f e c t s . M e d i c a l c o m p l i c a t i o n s ( 1 3 ) and h e p a t i c damage (14) were s t u d i e d i n r e l a t i o n t o n a r c o t i c a d d i c -t i o n . T h i s phenomenon i s not l i m i t e d t o methadone but a s s o -c i a t e d w i t h n a r c o t i c s i n g e n e r a l . A s p e c i f i c m e t a b o l i c r o u t e does n ot appear t o be i n v o l v e d i n t h e t o x i c i t y o f methadone. S y n t h e s i s o f methadone and i t s a n a l o g s Two methods are commonly a v a i l a b l e f o r the s y n t h e s i s o f methadone. The s y n t h e t i c method o u t l i n e d by S c h u l t z e t a l . (15) used a c o n d e n s a t i o n r e a c t i o n o f d i p h e n y l a c e t o n i t r i l e (12) w i t h 2 - d i m e t h y l a m i n o i s o p r o p y l c h l o r i d e (1T3) i n the p r e s -ence o f NaNH 2 t o form two i s o m e r s , methadone n i t r i l e (1_4) and isomethadone n i t r i l e (15). Methadone n i t r i l e was s e p a r a t e d by f r a c t i o n a l r e c r y s t a l l i z a t i o n from hexane. 5 Anot h e r method d e s c r i b e d by E a s t o n e t a l . (16) i n v o l v e d the r e a c t i o n o f p r o p y l e n e o x i d e w i t h d i p h e n y l a c e t o n i t r i l e t o g i v e 3 , 3 - d i p h e n y l - 5 - m e t h y l t e t r a h y d r o - 2 - f u r a n o n e imine (16) which i s f u r t h e r c o n v e r t e d t o 4-bromo-2,2-diphehylpentane n i t r i l e (_17) . The bromo compound, 1_7 r e a c t e d w i t h d i m e t h y l a m i n e t o g i v e methadone n i t r i l e (_14) . . NaNH2 12 ( C 6 H 5 ) 2 C H - C N + CH, C H — CH, > . x PBr. 1 6 ( C ^ ^ C ~ C = NH ^ ( C 6 H 5 ) 2 C — CN - C H 2 - C H ^ - C H 3 i H — C H - C H 3 ±1 Br ™(CK3)2 ( C 6 H 5 ) 2 C — CN C H 2 — C H - N ( C H 3 ) 2 1± CH, *3 The n i t r i l e , _14 was c o n v e r t e d t o methadone and i t s an a l o g s (18) by a G r i g n a r d r e a c t i o n . 6 C H ^ CH-N (CH 3 ) 2 ( ^ ? C H 2 — CH-N (CH 3 ) 2 CH 3 18 2 Two d e u t e r i u m l a b e l e d methadones, methadone- (18, 2 2 I^CE^CD^) and methadone - H,. (1, Ar= H^) have appeared i n t h e l i t e r a t u r e . B oth o f them were s y n t h e s i z e d by the method o f 2 S c h u l t z e t a l . (15). Methadone- was p r e p a r e d f o r use i n measuring plasma l e v e l s and d e t e r m i n i n g s t e a d y s t a t e k i n e t i c s 2 o f methadone (17). A p o s s i b l e use o f methadone- as an i n v i v o marker f o r m o n i t o r i n g t h e methadone i n t a k e o f a m a i n t e -2 nance p a t i e n t was a l s o suggested (18). Methadone- was used as t h e i n t e r n a l s t a n d a r d i n s e l e c t e d i o n m o n i t o r i n g (SIM) a n a l y s i s t o d e t e r m i n e plasma and u r i n a r y l e v e l s o f methadone (19) and t o s t u d y s t e r e o s p e c i f i c m e t a b o l i s m o f methadone (20). Kreek e t a l . dosed d e u t e r i u m l a b e l e d R-(-)-methadone and S-(+)-methadone t o methadone maintenance p a t i e n t s and m o n i t o r e d s p e c i f i c a l l y l a b e l e d methadone, o b s e r v i n g t h a t t h e a c t i v e R - ( - ) - e n a n t i o m e r has a l o n g e r h a l f l i f e t h a n t h e l e s s a c t i v e S-(+)-enantiomer. 2 The s y n t h e t i c scheme adopted t o o b t a i n methadone-was u n u s u a l i n t h a t CD^CD 2Br i n s t e a d o f CT^CH^Br was used, f o l -lowed by a KOH c a t a l y z e d ' exchange•to y i e l d the d e s i r e d compound (18, R=CH 2CD 3). T h i s c o u l d be due t o an e c o n o m i c a l c o n s i d e r a t i o n i n t h e s y n -2 t h e s i s . The s y n t h e s i s o f methadone- H,. was a c h i e v e d w i t h 2 2 d i p h e n y l a c e t o n i t r i l e - H,. (1_2, Ar= H^) which was o b t a i n e d by 2 F r i e d e l - C r a f t s r e a c t i o n o f benzene- H,. and bromophenylaceto-b n i t r i l e i n , C S 2 s o l v e n t . Two methadone a n a l o g s , the octanone (1J3, R=CH 2CH 2CH 3) (17) and t h e nonanone (1J3, R=CH 2CH 2CH 2CH 3) (21) were p r e p a r e d as the i n t e r n a l s t a n d a r d s f o r the a n a l y s i s o f methadone. The s y n t h e s i s o f t h e s e compounds was s i m p l e , u s i n g e i t h e r p r o p y l bromide o r b u t y l bromide f o r the G r i g n a r d r e a c t i o n s . S y n t h e s i s o f methadone m e t a b o l i t e s S y n t h e t i c methods f o r 4-dimethylamino-2, 2 - d i p h e n y l -p e n t a n o i c a c i d (5_) and DDP (6_) were p u b l i s h e d by Gardner e t a l . (22). H y d r o l y s i s of methadone n i t r i l e forms t h e a c i d , 5_ which i s d e m e t h y l a t e d by S0C1 2 t r e a t m e n t t o g i v e the c y c l i z e d p r o d u c t , DDP. rr""\1 H 2 S 0 4 H 20 CH 5-CH-N(CH,) 3 ' 2 COOH / // CH-CH-N(CH CH, S 0 C 1 7 > X c . ^ N-CH, \\ C H 2 — C H I - CH, 11 I i DDP i s used as an i n t e r m e d i a t e t o s y n t h e s i z e EDDP and EMDP. Treatment o f DDP w i t h C 2 H 5 L i g i v e s EDDP. EDDP i s de m e t h y l a t e d by HI and heat t o produce EMDP. P e r c h l o r a t e 8 f o r m a t i o n o f 3_ l e d t o the c o n v e r s i o n o f t h e e x o c y c l i c s t r u c t u r e , 3_ t o the e n d o c y c l i c s t r u c t u r e , 19_ as shown by NMR s t u d i e s (3, 23) . EMDP (_4) and i t s s a l t (20_) b o t h c o n t a i n an e n d o c y c l i c 4 20 double bond (3, 2 3 ) . Methadol J_7) , a m e t a b o l i t e o f methadone and an i n t e r -mediate f o r t h e s y n t h e s i s o f 1 - a - a c e t y l m e t h a d o l (LAAM) was c h e m i c a l l y p r e p a r e d by p l a t i n u m o x i d e h y d r o g e n a t i o n , L i A l H ^ r e d u c t i o n , o r sodium-propanol reduction (2 4) o f methadone. The r a t i o o f isomers v a r i e d depending upon t h e r e a g e n t s w i t h p r e f e r a b l e f o r m a t i o n o f the a-isomer by t h e use o f Adams c a t -a l y s t o r L i A l H ^ . S y n t h e s i s o f normethadol and d i n o r m e t h a d o l was a c h i e v e d by c h e m i c a l N - d e m e t h y l a t i o n p r o c e d u r e s (25). Methadone N-oxide was p r e p a r e d by m i l d o x i d a t i o n o f methadone. Treatment o f methadone w i t h 2.5 mol o f m-ch l o r o -p e r b e n z o i c a c i d gave EDDP (26). Permanganate o x i d a t i o n o f methadone was r e p o r t e d t o produce a m i x t u r e o f DDP and 2_1 (27). Methoxymethadone (2_2) was s y n t h e s i z e d by u s i n g a n i s o l e i n s t e a d 9 Ph H H' CH • i s -H Ph 1-0 •0 CH30 CH, 0 I! ^C-CH 2CH 3 CH-CH-N (CH,), I CH, 21 22 o f benzene i n t h e F r i e d e l - C r a f t s t e p o f the r e a c t i o n t o s y n t h e -s i z e methadone (28). No r e p o r t has appeared on the s y n t h e s i s o f d e u t e r i u m l a b e l e d methadone m e t a b o l i t e s . T h i s i s perhaps because o f t h e f a c t t h a t EDDP which has t h e most i m p o r t a n t i m p l i c a t i o n s f o r the p h a r m a c o k i n e t i c s o f methadone, can be a n a l y z e d by gas chromato-graphy . A t tempted s y n t h e s i s o f normethadone P o h l a n d e t a l . (25) t r e a t e d DDP w i t h E t L i i n an attempt t o o b t a i n normethadone. The i s o l a t e d p r o d u c t was EDDP because /CH 2-MeCH •CPh, \ s BrCN COEt N-Me I Me /CH2-MeCH Me2N*CN - CPh. v • C-CHMe 0 H ' + -Me2NCN H + CH— CPh, / 2 \ MeCH C=CHMe 10 of spontaneous c y c l i z a t i o n o f normethadone. Cyanogen bromide d e m e t h y l a t i o n (29) of methadone d i d n o t g i v e normethadone b u t y i e l d e d 2 - e t h y l i d e n e - 5 - m e t h y l - 3 , 3 - d i p h e n y l t e t r a h y d r o f u r a n (23) ( 30). A n a l y s i s o f methadone and m e t a b o l i t e s i n b i o l o g i c a l samples Gas chromatography and SIM a n a l y s i s a r e two major methods t o a n a l y z e methadone and i t s m e t a b o l i t e s i n human samples. The s e n s i t i v i t y o f gas chromatography f o r methadone i n plasma and u r i n e and EDDP i n u r i n e i s o f t h e o r d e r o f 5-15 ng/ml (21, 31, 32). On t h e o t h e r hand, EMDP, a minor metabo-l i t e , c o u l d n o t be q u a n t i f i e d i n human u r i n e because o f a l a c k o f s e n s i t i v i t y and s e l e c t i v i t y o f gas chromatography (33). A SIM a s s a y by gas chromatography-mass s p e c t r o m e t r y (GCMS) under e l e c t r o n impact c o n d i t i o n s (EI) was d e s c r i b e d f o r t he q u a n t i t a t i o n o f methadone by m o n i t o r i n g m/e 294 (metha-2 done), m/e 297 (methadone- H^), and m/e 308 f o r t h e i n t e r n a l s t a n d a r d , 2-dimethylamino-4, 4 - d i p h e n y l - 5 - o c t a n o n e (1J3, R= C^C^CH^) (17). By t h i s method, methadone i n plasma was 2 ass a y e d w i t h a s e n s i t i v i t y l i m i t o f 5 ng/ml. Methadone- H,. has been u t i l i z e d f o r the SIM as s a y o f methadone i n human plasma u s i n g c h e m i c a l i o n i z a t i o n (CI) (19). The s e n s i t i v i t y l i m i t o f t h i s method was s i m i l a r t o t h a t r e p o r t e d u s i n g the EI-SIM. 11 In a n i m a l e x p e r i m e n t s a n a l y t i c a l d i f f i c u l t i e s a r e f r e q u e n t l y e n c o u n t e r e d because o f the s m a l l s i z e o f samples, i n w hich t h e methods u s u a l l y r e q u i r e t e n t i m e s t h e s e n s i t i v i t y compared t o t h o s e f o r human e x p e r i m e n t s . Use o f a r a d i o i s o t o p i c method i s t h e method o f c h o i c e i n a n i m a l e x p e r i m e n t s . F o r example, t h e k i n e t i c s o f methadone i n r a t plasma was s t u d i e d 14 by u s i n g C - l a b e l e d methadone (34) because o f a l a c k o f sen-s i t i v i t y g e n e r a l l y shown i n t h e use o f gas chromatography (35). S e n s i t i v e a n a l y t i c a l methods such as radioimmunoassay a r e s t i l l b e i n g d e v e l o p e d t o a n a l y z e methadone i n plasma ( 3 6 ) , by which a lo w e r l i m i t o f s e n s i t i v i t y o f 3 ng/ml w i t h a sample s i z e o f 0.05 ml has been a c h i e v e d . C o n j u g a t e d m e t a b o l i t e s ' a r e i m p o r t a n t from b o t h a q u a l i t a t i v e and q u a n t i t a t i v e p o i n t o f view. When drug i n t e r -a c t i o n s a r e s t u d i e d t h e i n t e r a c t i o n may be r e f l e c t e d i n q u a n t i -t a t i v e changes o f t h e c o n j u g a t e d m e t a b o l i t e ( s ) ( 9 ) . T h e r e f o r e , i t i s n e c e s s a r y t o have a means t o a n a l y z e c o n j u g a t e d metabo-l i t e s a c c u r a t e l y . Q u a l i t a t i v e a n a l y s i s o f c o n j u g a t e d metabo-l i t e s as i n t a c t m o l e c u l e s has' been r e c e n t l y r e v i e w e d (37). Q u a n t i t a t i v e a n a l y s i s i s however a c h i e v e d o n l y by h y d r o l y z i n g t h e c o n j u g a t e p o r t i o n s . The h y d r o l y z e d m e t a b o l i t e s a r e ex-t r a c t e d q u a n t i t a t i v e l y i n t o o r g a n i c s o l v e n t s and chromato-graphed u s i n g GC o r TLC. In t h e case o f r a d i o i s o t o p i c methods, TLC i s t h e s e p a r a t i o n method o f c h o i c e . In most c a s e s , au-t h e n t i c compounds o f c o n j u g a t e d m e t a b o l i t e s a re n o t a v a i l a b l e . When c o m p l i c a t e d b i l e samples a r e chromatographed by TLC, com-p l e t e s e p a r a t i o n i s a problem as emphasized by R o e r i g e t a l . 12 (38) and Whitehouse e t a l . (39) i n t h e i r s t u d i e s o f methadone met a b o l i s m . Depending upon t h e s o l v e n t system, marked v a r i a -t i o n i n t h e amount of c o n j u g a t e d and n o n c o n j u g a t e d m e t a b o l i t e s has been o b s e r v e d . P h a r m a c o k i n e t i c a s p e c t s o f methadone S y n t h e s i s o f methadone a n a l o g s c o n t i n u e d from th e 1940's t o t h e e a r l y 1960's. The use o f methadone f o r m a i n t e -nance of a d d i c t s i s based on accumulated p h a r m a c o l o g i c a l d a t a t o the m i d d l e o f t h e 1960's. S t u d i e s o f m e t a b o l i t e d e t e c t i o n were i n i t i a t e d i n t h e mid 1960's and c o n t i n u e d f o r t e n y e a r s t o t h e mid 1970's. From 1970 t o 1979, many r e f e r e n c e s were p u b l i s h e d on t h e b i o p h a r m a c e u t i c and c l i n i c a l p h a r m a c o k i n e t i c a s p e c t s o f methadone. S t u d i e s o f methadone k i n e t i c s i n humans were aimed a t a d j u s t m e n t of dosage l e v e l s o f maintenance p a t i e n t s . Holm-s t r a n d e t a l . (40) r e p o r t e d t h a t the b e s t r e c o r d o f r e h a b i l i t a -t i o n was a c h i e v e d w i t h s t e a d y - s t a t e plasma c o n c e n t r a t i o n s above 200 ng/ml of methadone i n d i c a t i n g t h a t a p h a r m a c o k i n e t i c -a l l y o p t i m i z e d dosage regimen would be u s e f u l i n i n c r e a s i n g the e f f e c t i v e n e s s o f methadone maintenance t r e a t m e n t . Horns e t a l . (41) r e p o r t e d t h a t t h e r e was no r e l a t i o n s h i p between plasma methadone l e v e l and p a t i e n t ' s s u b j e c t i v e symptom com-p l a i n t s . 13 The mechanism of t o l e r a n c e t o methadone was p a r t l y sought i n m e t a b o l i c t o l e r a n c e by means o f k i n e t i c s t u d i e s of methadone. C h r o n i c a d m i n i s t r a t i o n o f methadone t o man s h o r t e n s t h e h a l f l i f e o f t h e d r u g . The a c u t e p r i m a r y o f 14.3 h o u r s • i n c o m b i n a t i o n w i t h t h e a c u t e secondary °f 54.8 hours was ' l o n g e r than the s i n g l e e x p o n e n t i a l c h r o n i c o f 22.2 hours (42). A r e c e n t paper by L i u e t a l . (43) r e p o r t e d t h a t t o l e r a n c e t o methadone a n a l g e s i a i s due t o b o t h i n c r e a s e d methadone m e t a b o l i s m and c e l l u l a r a d a p t a t i o n t o t h e drug i n the b r a i n . The e f f e c t o f d i s e a s e s t a t e s on methadone k i n e t i c s i n maintenance p a t i e n t s was s t u d i e d . No e v i d e n c e was found f o r t h e a c c u m u l a t i o n o f e i t h e r methadone o r i t s m e t a b o l i t e s w i t h r e n a l d i s e a s e , s u g g e s t i n g t h a t methadone i s an a p p r o p r i a t e n a r c o t i c t o use i n p a t i e n t s w i t h r e n a l i n s u f f i c i e n c y ( 4 4 ) . On t h e o t h e r hand, a d e c r e a s e d u r i n a r y e x c r e t i o n o f methadone and i t s m e t a b o l i t e s was ob s e r v e d .in p a t i e n t s w i t h l i v e r d i s -ease (45) . Methadone and drug i n t e r a c t i o n s A c c o r d i n g t o r e p o r t e d s u r v e y s (46-48)/ t h e drugs which are abused w i t h methadone a r e e t h a n o l , diazepam, o t h e r o p i a t e s , amphetamines, and b a r b i t u r a t e s . The i n t e r a c t i o n s o f methadone w i t h t h e s e d r ugs were s t u d i e d e i t h e r w i t h human o r w i t h a n i m a l models. 14 An a g o n i s t - a n t a g o n i s t i n t e r a c t i o n o c c u r r e d i n m a i n t e -nance p a t i e n t s between methadone and n a l o x o n e , w i t h no changes i n methadone d i s p o s i t i o n (49). S i g n i f i c a n t changes i n methadone d i s p o s i t i o n o c c u r r e d d u r i n g combined t r e a t m e n t w i t h r i f a m p i n (49) , which was due t o t h e enzyme i n d u c t i o n p r o p e r t y o f r i f a m p i n (50) . Enhanced m e t a b o l i s m of methadone by d i p h e n y l h y d a n t o i n was o b s e r v e d i n s t u d i e s of methadone maintenance p a t i e n t s (51). T h i s i s s i m i l a r t o t h e e f f e c t o f d i p h e n y l h y d a n t o i n on dexametha-sone ( 5 2 ) . . - B i l i a r y e x c r e t i o n o f c o n j u g a t e d methadone m e t a b o l i t e s was i n c r e a s e d by p h e n o b a r b i t a l p r e t r e a t m e n t o f r a t s (3.8) . A t doses o f d i s u l f i r a m s u i t a b l e f o r t h e management of a l c o h o l i s m t h e r e was no s i g n i f i c a n t i n t e r a c t i o n between d i s u l f i r a m and methadone (53 ) . Desipramine showed i n h i b i t i o n o f methadone m e t a b o l i s m i n t h e r a t l i v e r ( 5 4 ) . Three papers appeared on methadone-ethanol i n t e r a c t i o n s . The a d m i n i s t r a t i o n o f e t h a n o l t o r a t s r e s u l t e d i n i n c r e a s e d b r a i n and l i v e r c o n c e n t r a t i o n s o f methadone and d e c r e a s e d b i l i a r y o u t p u t of methadone (39, 5 5 ) . No s i g n i f i c a n t a c u t e i n t e r a c t i o n was found between methadone and e t h a n o l i n s t u d i e s o f maintenance p a t i e n t s (56). Diazepam was shown t h r o u g h i n v i t r o s t u d i e s t o be an e f f e c t i v e i n h i b i t o r o f t h e N - d e m e t h y l a t i o n of methadone whi c h may e x p l a i n i n p a r t the enhanced e f f e c t o f methadone o b s e r v e d i n n a r c o t i c a d d i c t s when diazepam i s t a k e n i n combina-t i o n w i t h methadone (57). Four i n v i v o s t u d i e s o f methadone-diazepam i n t e r a c t i o n s were published.: one w i t h r a t s ( 3 4 ) , an-o t h e r w i t h r h e s u s monkeys (58) , and t h e o t h e r s w i t h mice 15 (59, 6 0 ) . A c u t e a d m i n i s t r a t i o n o f diazepam t o t h e r a t p r o -l o n g e d t h e d u r a t i o n o f methadone a n a l g e s i a , i n c r e a s e d the b r a i n 14 c o n c e n t r a t i o n s o f t o t a l C and d e c r e a s e d t h e p e r c e n t o f t o t a l 14 C i n t h e l i v e r o r u r i n e (34). B e h a v i o r a l d e p r e s s i o n a f t e r diazepam was p r o l o n g e d s u b s t a n t i a l l y i n methadone m a i n t a i n e d monkeys. B l o o d l e v e l s o f diazepam and m e t a b o l i t e s were n o t i n c r e a s e d o r p r o l o n g e d i n t h o s e a n i m a l s (58). Enhanced h e p a t i c l e v e l s o f methadone f o r up t o t h r e e hours by diazepam i n d i c a t e d an i n t e r f e r e n c e w i t h methadone m e t a b o l i s m by t h i s agent i n mice (59). On t h e o t h e r hand, Shannon (60) r e p o r t e d t h a t diazepam f a i l e d t o enhance b r a i n and plasma methadone l e v e l s i n mice. Deuterium l a b e l e d compounds f o r s t u d i e s o f  p h a r m a c o k i n e t i c s and drug m e t a b o l i s m The s t e a d y s t a t e k i n e t i c s o f a drug can be s t u d i e d by a method i n which s t a b l e • i s o t o p e l a b e l e d and u n l a b e l e d drugs a r e a d m i n i s t e r e d and c o l l e c t e d samples a r e a n a l y z e d c o n c o m i t a n t l y by u s i n g a t h i r d i n t e r n a l s t a n d a r d . Such an approach was demonstrated f o r methadone (61) and propoxyphene. (62) . The same approach was a p p l i e d t o b i o a v a i l a b i l i t y s t u d -i e s o f N - a c e t y l p r o c a i n a m i d e i n humans (63). The method can p r o v i d e ease i n t h e a n a l y s i s and compensate f o r d i f f e r e n c e s i n h e p a t i c e x t r a c t i o n r e s u l t i n g from d i f f e r e n t h e p a t i c b l o o d 16 f l o w s a t d i f f e r e n t t i m e s and i n d i f f e r e n t i n d i v i d u a l s . The h e p a t i c b l o o d f l o w i s v a r i a b l e even w i t h i n a h e a l t h y p o p u l a -t i o n and v a r i e s between 0.5 and 3 1/min. (64). When s t a b l e i s o t o p e l a b e l e d a n a l o g s a re used f o r pharma-c o k i n e t i c and m e t a b o l i c s t u d i e s o f a d r u g , b i o e q u i v a l e n c e of t h e l a b e l e d a n a l o g s s h o u l d be e s t a b l i s h e d . T h i s was done 2 by H s i a e t a l . (18) f o r methadone and methadone- H^- An o t h e r 13 "I 5 r e p o r t o f [\\ C"f IS^] - d i p h e n y l h y d a n t o i n p u b l i s h e d by Browne. e t a l . (65) a l s o showed c a r e f u l c o n s i d e r a t i o n f o r k i n e t i c e q u i v a l e n c e . B i o e q u i v a l e n c e i s f i r s t s t u d i e d u s i n g s m a l l a n i m a l s , t h e r e s u l t s o f which become a b a s i s o f the use o f l a b e l e d compounds f o r human s t u d i e s . R e c e n t l y p u b l i s h e d papers have i g n o r e d such a c a r e f u l t r e a t m e n t o f t h e t o x i c o l o g i c a l and k i n e t i c e q u i v a l e n c e of s t a b l e i s o t o p e l a b e l e d compounds. F o r example, N - a c e t y l p r o -13 c a i n a m i d e - C was used w i t h o u t such a t e s t (63) . Hachey e t a l . dosed p e n t a d e u t e r a t e d methadone t o st u d y s t e r e o s e l e c t i v e 2 d i s p o s i t i o n of: methadone i n man (20). B e n o x a p r o f e n - was used o n l y w i t h a c u t e t o x i c i t y d a t a e q u i v a l e n t t o u n l a b e l e d compound (66). In o r d e r t o see m e t a b o l i c e q u i v a l e n c e , m a i n l y h e p a t i c u p t a k e , t h e l i v e r p e r f u s i o n method which was used t o st u d y h e p a t i c uptake o f p r o p a n o l o l (67) c o u l d be used. D i f f e r e n c e s o f p h a r m a c o l o g i c a l a c t i o n s between d e u t e -r i u m l a b e l e d and p r o t i o drugs a r e m a i n l y e x p l a i n e d by d i f f e r -ences i n t h e b i o t r a n s f o r m a t i o n o f the d r u g s . I f t h e l a b e l i n g i s a t t h e s i t e where r a t e l i m i t i n g m e t a b o l i s m o c c u r s , an 17 i s o t o p e e f f e c t i s o b s e r v e d . M a r c u c c i e t a l . (68) s t u d i e d the m e t a b o l i s m and a n t i c o n v u l s a n t a c t i v i t y o f d e u t e r a t e d N-demethyldiazepam and c o n c l u d e d t h a t a s i g n i f i c a n t s h o r t e n i n g o f a n t i c o n v u l s a n t a c t i v i t y was due t o a reduced C ^ - h y d r o x y l a -t i o n o f the compounds. S i m i l a r r e s u l t s were o b s e r v e d i n the m e t a b o l i s m and t o x i c i t y o f p h e n a c e t i n where hydrogen a t the p o s i t i o n b e i n g o x i d i z e d was s u b s t i t u t e d w i t h d e u t e r i u m . I t was found t h a t the l a b e l i n g d e c r e a s e d h e p a t i c n e c r o s i s due t o an o x i d i z e d m e t a b o l i t e and i n c r e a s e d methemoglobinemia r e s u l t i n g from the m e t a b o l i t e s o f the n o n d e e t h y l a t e d pathway (69) . I n some cases i t i s d i f f i c u l t t o c o n c l u d e whether the d i f f e r e n t p h a r m a c o l o g i c a l a c t i o n s between d e u t e r i u m l a b e l e d and u n l a b e l e d d r ug a r e due t o m e t a b o l i c d i f f e r e n c e s o r d i f f e r -ences i n t h e p h y s i c a l p r o p e r t i e s which c o u l d i n f l u e n c e t r a n s p o r t t o the r e c e p t o r s i t e and r e c e p t o r b i n d i n g o f t h e d r u g . A s i g n i f i c a n t r e d u c t i o n i n t o x i c i t y and a d e c r e a s e i n s p o n t a -neous l o c o m o t o r a c t i v i t y was o b s e r v e d f o r h i g h l y e n r i c h e d d e u t e r a t e d amphetamine (70). F u r t h e r r e p o r t s showing p o s s i b l e r e a s o n s f o r t h i s have not been p u b l i s h e d . An example o f the p a r t i c i p a t i o n o f the l a b e l e d s i t e i n r e c e p t o r b i n d i n g was p r e s e n t e d f o r N-CD^ morphine. An e x p l a n a t i o n f o r the d e c r e a s e d a c t i v i t y o f N-CD^ morphine compared t o N-CH^ morphine was g i v e n by a c l a s t i c b i n d i n g c o n c e p t (71). 18 OBJECTIVES OF THE RESEARCH The o b j e c t i v e s o f the r e s e a r c h were t o u t i l i z e s t a b l e i s o t o p e a n a l o g s o f methadone and i t s m e t a b o l i t e s t o g e t h e r w i t h GCMS t e c h n i q u e s t o i n v e s t i g a t e methadone d i s p o s i t i o n and m e t a b o l i s m . Methadone i s a dr u g g e n e r a l l y used on a l o n g term b a s i s . T h e r e f o r e , a complete p i c t u r e o f m e t a b o l i c pathways i s e s s e n t i a l t o a s c e r t a i n t he p o t e n t i a l l o n g term t o x i c i t y , p o s s i b l y t h a t a r i s i n g from minor m e t a b o l i t e s . E x t e n s i v e m e t a b o l i c s t u d i e s i n r a t s u s i n g d e u t e r i u m c o n t a i n i n g a n a l o g s were t o c o n c e n t r a t e on d e t e c t i n g and i d e n t i f y i n g new m e t a b o l i t e s t h a t might be i m p l i c a t e d i n p o t e n t i a l t o x i c i t i e s . S e v e r a l p h a r m a c o k i n e t i c s t u d i e s o f methadone have been completed i n man t o determine the n a t u r e o f the v a r i e d dose r e q u i r e m e n t s and whether b l o o d l e v e l s o f the drug c o r r e l a t e w i t h good performance o f the p a t i e n t . Most o f the d i s p o s i -t i o n a l s t u d i e s a r e however i n c o m p l e t e because o f d e f i c i e n t a n a l y t i c a l methods. T h e r e f o r e , the SIM methods d e v e l o p e d w i t h l a b e l e d compounds and GCMS were t o be a p p l i e d t o pharma-c o k i n e t i c s t u d i e s . S a l i v a s a m p l i n g and a n a l y s i s were t o be i n v e s t i g a t e d as a n o n i n v a s i v e t e c h n i q u e f o r p h a r m a c o k i n e t i c s t u d i e s and f o r m o n i t o r i n g methadone l e v e l s . A SIM method w i t h d e u t e r a t e d a n a l o g s as i n t e r n a l s t a n d a r d s was t o be used i n a d r u g i n t e r a c t i o n study i n r a t s o f diazepam and methadone 19 t o s e l e c t i v e l y i d e n t i f y any m e t a b o l i c changes o f methadone t h a t might o c c u r . S y n t h e s i s o f d e u t e r a t e d methadone and m e t a b o l i t e s The s y n t h e s e s o f m e t h a d o n e - 2 ! ^ , EDDP- 2H 1 Q, EMDP- 2H 1 Q, 2 2 DDP- H^Q, 4-dimethylamino-2, 2 - d i p h e n y l p e n t a n o i c a c i d - H^Q, 2 2 EDDP- H^/ and EMDP- were d e s i g n e d . The p r i m a r y c h o i c e f o r the s y n t h e s i s o f methadone and m e t a b o l i t e s where b o t h a r o m a t i c r i n g s a r e d e u t e r i u m l a b e l e d was based on t h e f o l l o w i n g r e a s o n s . F i r s t , t h e l a b e l was n o t l i k e l y t o be l o s t s i n c e most metabo-l i t e s o f methadone can be ex p e c t e d t o r e t a i n t h e p h e n y l r i n g s . S e c o n d l y , under E I c o n d i t i o n s major fragment i o n s o f m e t a b o l i t e s w i l l f r e q u e n t l y c o n t a i n a l l o r a p o r t i o n of the l a b e l , and t h i r d l y , t h e c o s t s o f t h e s y n t h e s i s appeared r e a s o n a b l e . The f i r s t p o i n t i s i m p o r t a n t t o d e t e c t m e t a b o l i t e s and t h e second p o i n t f o r SIM a n a l y s i s o f methadone and metabo-l i t e s under E I c o n d i t i o n s . The compound l a b e l e d on one r i n g 2 (1, Ar= H R ) whi c h was used f o r s t e r e o s e l e c t i v e m e t a b o l i s m — o of methadone (19) has .two diastere'omers w h i c h can : c o m p l i c a t e m e t a b o l i c s t u d i e s . The l a b e l e d compounds were a l s o c o n s i d e r e d t o be use-f u l f o r two f u t u r e r e s e a r c h p r o j e c t s . A c c o r d i n g t o t h e n a r c o t i c r e c e p t o r model, w h i c h shows p a r t i c i p a t i o n of t h e r i n g i n t h e r e c e p t o r b i n d i n g ( 7 2 ) , r i n g l a b e l e d methadone c o u l d be a good model t o study such b i n d i n g . The l a b e l e d 20 compounds c o u l d be used t o i n v e s t i g a t e r i n g o x i d a t i o n mechanisms. A study o f i s o t o p e e f f e c t s would r e v e a l whether o x i d a t i o n o c c u r s v i a arene o x i d e f o r m a t i o n such as i n the case o f d i p h e n y l -h y d a n t o i n (73) o r by d i r e c t oxygen i n s e r t i o n as shown i n mono-o-d e m e t h y l a t i o n o f p _ - t r i d e u t e r e o m e t h o x y a n i s o l e (74) . Mass f r a g m e n t a t i o n s t u d i e s S t a b l e i s o t o p e l a b e l e d compounds were c o n s i d e r e d t o be most u s e f u l i n d e f i n i n g fragment i o n s i n mass s p e c t r o m e t r y . By comparing the mass s p e c t r a o f the l a b e l e d and u n l a b e l e d d e r i v a t i v e s , f r a g m e n t a t i o n p r o c e s s e s can be d e s c r i b e d i n more d e t a i l t h a n p r e v i o u s l y a v a i l a b l e ( 6 ) . F r a g m e n t a t i o n s t u d i e s can a l s o be used t o det e r m i n e the s t r u c t u r e of the new metabo-l i t e s w h i c h may have v e r y s i m i l a r s t r u c t u r e s t o known s y n t h e -s i z e d m e t a b o l i t e s . I n a d d i t i o n t o the f r a g m e n t a t i o n i n f o r m a -t i o n o f mass s p e c t r o m e t r y , s p e c t r o s c o p i c s t u d i e s such as IR and NMR can be d e s c r i b e d i n more d e t a i l u s i n g d e u t e r i u m l a b e l e d compounds. SIM a n a l y s i s o f methadone and m e t a b o l i t e s  i n b i o l o g i c a l samples The GCMS, V a r i a n MAT 111 was remodeled t o do s e l e c t e d i o n m o n i t o r i n g . The a n a l y t i c a l methods f o r methadone and 21 m e t a b o l i t e s were d e v e l o p e d w i t h t h e use o f l a b e l e d methadone and m e t a b o l i t e s as the i n t e r n a l s t a n d a r d s and w i t h a computer program w h i c h was improved t o ensure h i g h s e n s i t i v i t y and h i g h p r e c i s i o n . •: . ... Human samples were used t o i n v e s -t i g a t e t h e a p p l i c a b i l i t y o f t h e d e v e l o p e d methodology t o pharma-c o k i n e t i c s t u d i e s o f methadone. Emphasis was a l s o p l a c e d on t h e use o f a h i g h abundance i o n of methadone t o improve th e s e n s i t i v i t y o f methadone a n a l y s i s i n human or a n i m a l s t u d i e s . The methodology was a l s o a p p l i e d t o t h e i n v e s t i g a t i o n o f the s a l i v a - p l a s m a r e l a t i o n s h i p o f methadone. S t u d i e s of methadone-diazepam i n t e r a c t i o n Methadone-diazepam i n t e r a c t i o n s t u d i e s were d e s i g n e d which i n c l u d e d a methodology f o r t h e use o f b i o l o g i c a l l y formed i n t e r n a l s t a n d a r d s ( b i o s y n t h e t i c i n t e r n a l s t a n d a r d s ) . The use o f b i o s y n t h e t i c i n t e r n a l s t a n d a r d s f o r p h a r m a c o k i n e t i c and drug m e t a b o l i s m e x p e r i m e n t s i s p o s s i b l e o n l y w i t h d e u t e r i u m l a b e l e d compounds and SIM methodology. I n view of the methadone-diazepam i n t e r a c t i o n s t u d y , t h e method was e x p e c t e d t o a c c u r a t e l y q u a n t i t a t e methadone and i t s m e t a b o l i t e s , e s p e c i a l l y c o n j u g a t e d m e t a b o l i t e s . A r e s o l u t i o n p r o blem i n t h e TLC o f t h e m e t a b o l i t e s was encoun-t e r e d i n p r e v i o u s s t u d i e s (38, 3 9 ) . 22 D e t e c t i o n o f new methadone m e t a b o l i t e s S t u d i e s d i r e c t e d toward the d e t e c t i o n o f methadone m e t a b o l i t e s have been performed o v e r the p a s t 15 y e a r s . Empha-s i s was t h e r e f o r e p l a c e d on t h e d e t e c t i o n o f minor m e t a b o l i t e s making use o f d e u t e r a t e d methadone and m e t a b o l i t e s w h i c h have no t p r e v i o u s l y been used f o r such a s t u d y . S p e c i a l e x t r a c t i o n p r o c e d u r e s f o r r a t b i l e samples were d e s i g n e d t o ensure good r e c o v e r i e s o f the m e t a b o l i t e s and t o have a r e l a t i v e l y complete s e p a r a t i o n o f endogenous m a t e r i a l s from m e t a b o l i t e s . GCMS w i t h the use o f l a b e l e d and unlabeled' ' compounds p r o v i d e s s e p a r a t i o n o f the m e t a b o l i t e s from endogenous m a t e r i a l s . Comparison o f the mass s p e c t r a i n peaks a r i s i n g from u n l a b e l e d compounds w i t h t h o s e from l a b e l e d compounds c o u l d p r o v i d e i m p o r t a n t e v i d e n c e o f t h e f o r m a t i o n o f new m e t a b o l i t e s . 23 EXPERIMENTAL 1. M a t e r i a l s G e n e r a l c h e m i c a l s and r e a g e n t s 2 Benzene- (99.5% D, Merck Sharp & Dohme, Canada), 2 - D i m e t h y l a m i n o i s o p r o p y l c h l o r i d e HC1 ( A l d r i c h C h e m i c a l ) , D 20 (99.7% D, SIGMA), D 2 S ° 4 ( m i n i m u m i s o t o p i c p u r i t y 99 atom 2 % D, Merck Sharp & Dohme, Canada), C H^-CI^Br (minimum i s o t o p i c p u r i t y 99% D, Merck Sharp & Dohme, Canada), G l u c u r a s e ( 3 - g l u c u -p r o n i d a s e , 5,000 sigma u n i t / m l , SIGMA), G l u s u l a s e ( 6 - g l u c u r o n i -dase and a r y l s u l f a t a s e , Erido p r o d u c t s I n c . , New Y o r k ) , m-Chlor-o p e r b e n z o i c a c i d ( t e c h n i c a l grade 85%, A l d r i c h C h e m i c a l ) , Diazepam ( a c t i v e s u b s t a n c e o f V a l i u m , Hoffman-La Roche, M o n t r e a l , Canada), 0.1 M sodium a c e t a t e b u f f e r (pH 4.5), 0.1 M d i s o d i u m c i t r a t e b u f f e r (pH 2.0), 0.25 M b o r a t e b u f f e r (pH 9.0). Diazomethane was p r e p a r e d by the method o f L e v i t t ( 75). S o l v e n t s C h l o r o f o r m , hexane, methylene c h l o r i d e , methanol; D i s t i l l e d i n g l a s s , Caledon L a b o r a t o r i e s L t d . , O n t a r i o . M e t h a n o l , H 20; HPLC grade, F i s h e r S c i e n t i f i c Co. E t h e r ; e t h e r a b s o l u t e , Caledon L a b o r a t o r i e s . 24 M a t e r i a l s f o r a n i m a l s u r g e r y H e p a r i n (168 u n i t s / m g , SIGMA), S i l a s t i c (Dow C o r n i n g , 0.020 i n . i.'d., 0.037 in.o.d.), P o l y e t h y l e n e t u b i n g - 1 0 ( C l a y Adams, 0.011 i n . i;d., 0.024 i n . o ; d ) , P o l y e t h y l e n e t u b i n g - 5 0 ( C l a y Adams, 0.023 i n . i.d., 0.038 in.o.d.). 2. A n a l y t i c a l methods GCMS r e p e t i t i v e s c a n n i n g GCMS s p e c t r a l d a t a were o b t a i n e d u s i n g a V a r i a n MAT 111 gas chromatograph-mass s p e c t r o m e t e r . Mass s p e c t r a l d a t a were r e c o r d e d and p r o c e s s e d u s i n g a V a r i a n 620L computer. The e l e c t r o n i o n i z a t i o n v o l t a g e was 70 eV w i t h a sour c e tem-p e r a t u r e o f 285°C. A g l a s s column (1.6 m x 2 mm i . d . ) packed w i t h 3% OV-17 on 80-10 0 mesh Chromosorb W (HP) was used w i t h c a r r i e r gas (He) a t 20 ml/min. GCMS s e l e c t e d i o n m o n i t o r i n g SIM was performed u s i n g a V a r i a n MAT 111 gas chroma-tograph-mass s p e c t r o m e t e r w i t h a c c e l e r a t i n g v o l t a g e s u p p l y of t h e mass s p e c t r o m e t e r m o d i f i e d t o p e r m i t s c a n n i n g t h e a c c e l -e r a t i n g v o l t a g e u s i n g a V a r i a n 620L computer. E l e c t r o n i o n i z a t i o n v o l t a g e , s o u r c e t e m p e r a t u r e , and column c o n d i t i o n s were t h e same as thos e used f o r the r e p e t i t i v e s c a n n i n g . 25 Gas chromatography The GC a n a l y s i s was c a r r i e d out u s i n g a H e w l e t t - P a c k a r d 5830A model equipped w i t h a hydrogen flame d e t e c t o r . The g l a s s column, 1.8 m x 2 mm i . d . was packed w i t h 3% OV-17 on 80-100 mesh Chromosorb W (HP). I n j e c t i o n t emperature was 205°C, oven tem p e r a t u r e 210°C, and d e t e c t o r t e m p e r a t u r e 300°C. The c a r r i e r gas (He) f l o w r a t e was 50 ml/min. C h e m i c a l i o n i z a t i o n GCMS A F i n n i g a n c h e m i c a l i o n i z a t i o n GCMS (Model 4000) was employed. The GC c o n d i t i o n s were the same as thos e d e s c r i b e d f o r r o u t i n e a n a l y s i s e x c e p t f o r the s i z e o f t h e g l a s s column (1.6 m x 2 mm i . d . ) and t h a t methane was used as a c a r r i e r and re a g e n t gas ( f l o w r a t e 40 ml/min). H i g h r e s o l u t i o n mass s p e c t r o m e t r y The KRATOS MS 50 h i g h performance mass s p e c t r o m e t e r ( r e s o l u t i o n 10,000) was used. I o n i z a t i o n v o l t a g e was 70 eV and s o u r c e t e m p e r a t u r e , ^150°C. High performance l i q u i d chromatography An ALTEX MODEL 15 3 h i g h performance l i q u i d chromato-graph equipped w i t h 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 254 nm was employed. The column was a 4.6 mm i . d . x 25 cm packed w i t h U l trasphere-ODS (octadecylsilane)' • \. r'< (dp, 5u) . Pumping p r e s s u r e and f l o w r a t e were 2000-3000 p s i and 1 ml/min, r e s p e c -t i v e l y . The e l u t i o n s o l v e n t was a m i x t u r e o f methanol and H 20 ( 3 : 1 ) . 2 6 NMR s p e c t r o s c o p y The NMR s p e c t r o s c o p y was performed w i t h t h e f o l l o w i n g i n s t r u m e n t s : V a r i a n X L - 1 0 0 , B r u k e r WP-80, and N i c o l e t - O x f o r d H-2 7 0 . TMS was used as t h e i n t e r n a l s t a n d a r d . D e u t e r a t e d methanol and d e u t e r a t e d c h l o r o f o r m were used as s o l v e n t s . I n f r a r e d s p e c t r o s c o p y A Beckman I R - 1 0 i n f r a r e d s p e c t r o m e t e r and a Unicam SP 1 0 0 0 i n f r a r e d s p e c t r o m e t e r (Pye Unicam) were used f o r IR s p e c t r o s c o p y . The s p e c t r a were r e c o r d e d as the l i q u i d f i l m s o r as. KBr d i s c s . 3. C h e m i c a l s t u d i e s S y n t h e s i s o f d e u t e r i u m l a b e l e d methadone and m e t a b o l i t e s The degree o f d e u t e r a t i o n o f t h e s y n t h e s i z e d compounds was d e t e r m i n e d by NMR. 2 S y n t h e s i s o f methadone- H^Q: A g e n e r a l p r o c e d u r e f o r t h e s y n t h e s i s o f d e u t e r a t e d d i p h e n y l a c e t o n i t r i l e (1_2) was t a k e n from 2 the l i t e r a t u r e ( 7 6 ) . Benzene- was used i n p l a c e o f benzene t o o b t a i n p a r t i a l l y d e u t e r a t e d 12^ (spectrum 1 , p. 1 6 3 ) . To ob-2 2 t a i n e n r i c h e d d i p h e n y l a c e t o n i t r i l e - (1J2, 2Ar= H-^Q) (spectrum 2 2 , p. 1 6 3 ) , benzene- Hg ( 1 0 0 gm) and D 2 0 (4 drops) were added t o a m i x t u r e o f p a r t i a l l y d e u t e r a t e d _12 ( 1 2 . 2 gm, 0 . 0 6 mol) and anhydrous A l C l ^ ( 1 2 g, 0 . 0 9 m o l ) . The m i x t u r e was r e f l u x e d on a steam b a t h f o r 2 0 h o u r s . A l C l ^ was d e s t r o y e d by a d d i n g H 2 0 . The s e p a r a t e d benzene l a y e r was d r i e d o v e r anhydrous Na„SO.. 27 F l a s h e v a p o r a t i o n o f t h e s o l v e n t gave a b r o w n i s h compound (11.68 gm, 95.7%) w h i c h was used f o r subsequent r e a c t i o n s . 2 Methadone n i t r i l e - H^Q was p r e p a r e d from e n r i c h e d d i p h e n -2 y l a c e t o n i t r i l e - H-^ Q by the method o f A t t e n b u r r o w e t a l . (77) w i t h minor m o d i f i c a t i o n s . A s o l u t i o n o f e n r i c h e d d i p h e n y l a c e t o n i t r i l e (9.0 gm, 0.047 mol) i n d r y benzene (75 ml) was t r e a t e d w i t h NaH (1.92 gm,_0.08 m o l ) . The m i x t u r e was heated on a steam b a t h f o r 10 minutes and a drop o f DMSO added as c a t a l y s t . 2-Dimethylamino-i s o p r o p y l c h l o r i d e (7.59 gm, 0.048 mol) i n benzene (90 ml) was p r e p a r e d as d e s c r i b e d (76) and added t o the m i x t u r e . The m i x t u r e was s t i r r e d f o r 45 hours a t room t e m p e r a t u r e . Work up d i d not r e q u i r e a d i s t i l l a t i o n but ' the e x t r a c t i o n r e s i d u e upon s t a n d i n g 2 gave a m i x t u r e o f methadone n i t r i l e - H^Q and isomethadone n i t r i l e -2 H-^ Q as a s l i g h t l y y e l l o w c r y s t a l l i n e mass. The s e p a r a t i o n o f 2 methadone n i t r i l e - H-^ Q (4.7 gm, 36%) (spectrum 3, p. 164) from 2 isomethadone n i t r i l e - was o b t a i n e d by f r a c t i o n a l r e c r y s t a l -2 l i z a t i o n from hexane. The s y n t h e s i s o f methadone- H^Q {1, 2Ar= 2 2 H-^ Q ) from methadone n i t r i l e - was a l s o d e s c r i b e d by A t t e n - v burrow e t a l . (77). The p r o d u c t c o n t a i n e d 97% l a b e l l i n g o f the a r o m a t i c p r o t o n s as d e t e r m i n e d by NMR (spectrum 5, p. 165). 2 S y n t h e s i s o f 4-dimethylamino-2 , 2 - d i p h e n y l p e n t a n o i c a c i d - H-^ Q 2 2 2 (_5, 2Ar= H 1 Q and DDP- H 1 Q (£, 2Ar= H 1 Q) .The method was s i m i l a r t o t h a t o f Gardner e t a l . (22). P a r t i a l l y d e u t e r a t e d methadone 2 n i t r i l e - H^Q o b t a i n e d from the r e a c t i o n o f p a r t i a l l y d e u t e r a t e d d i p h e n y l a c e t o n i t r i l e w i t h l - d i m e t h y l a m i n o - 2 - c h l o r o p r o p a n e and NaH was h y d r o l y z e d u s i n g D^O and D^SO^ i n a screw capped r e a c t i o n b o t t l e t o g i v e t h e b i s u l f a t e o f 4 - d i m e t h y l a m i n o - 2 , 2 - d i p h e n y l -28 2 p e n t a n o i c a c i d - H-^ Q . H y d r o l y s i s o f t h e b i s u l f a t e u s i n g 5% NaOH gave t h e f r e e ' a c i d , w h i c h showed 97% l a b e l l i n g on the a r o m a t i c r i n g s (spectrum 4, p. 1 6 4 ) . The a c i d was d r i e d a t 100°C f o r 30 2 minutes p r i o r t o c o n v e r s i o n t o the p y r r o l i d o n e , DDP- H-^ Q . The degree o f d e u t e r a t i o n o f p y r r o l i d o n e was a l s o 97% (spectrum 6, p. 1 6 6 ) . 2 2 2 S y n t h e s i s o f EDDP- H 1 Q (3, 2Ar= H 1 Q ) and EDDP- H 3 — A known 2 2 pr o c e d u r e (3) was used. S t a r t i n g w i t h DDP- H^ Q, EDDP- was o b t a i n e d w i t h t h e a r o m a t i c r i n g s '96% d e u t e r a t e d (spectrum 9, p. 1 6 7 ) . DDP r e a c t e d w i t h C 2 H 3 - G H 2 B r t o g i v e EDDP- 2H 3 ( 9 9 % D) (spectrum 8, p. 1 6 7 ) . 2 2 2 S y n t h e s i s o f EMDP- H 1 Q (2, 2Ar= H 1 Q ) and EMDP- H 3 — The p r o c e -2 2 dure o f Pohland e t a l . (3) was used. EMDP- H 1 Q and EMDP- H 3 were 2 2 o b t a i n e d from EDDP- H^Q and EDDP- H 3 , r e s p e c t i v e l y . The degree 2 o f r i n g d e u t e r a t i o n o f EMDP- H^Q was 9 6 % (spectrum 7, p. 1 6 6 ) . 2 EMDP- H 3 showed more th a n 9 9 % d e u t e r a t i o n . S y n t h e s i s o f 2-dimethylamino-4,4-diphenyl-5-nonanone  p e r c h l o r a t e 2-Dimethylamino-4,4-diphenyl-5-nonanone was p r e p a r e d by t h e method o f Lynn e t a l . (21). The p r o d u c t was not d i s -t i l l e d b ut c r y s t a l l i z e d from e t h e r s o l u t i o n as the p e r c h l o r a t e s a l t . R e c r y s t a l l i z a t i o n from ether-EtOH gave c r y s t a l s , mp 137-139°C. Chemical o x i d a t i o n s t u d i e s The compounds (EMDP H C l , EMDP base, EDDP p e r c h l o r a t e , EDDP base) were t r e a t e d a t 0-5°C w i t h m - c h l o r o p e r b e n z o i c a c i d i n c h l o r o f o r m . F o r EMDP H C l , EMDP base, and EDDP base 20% exc e s s o f m - c h l o r o p e r b e n z o i c a c i d was used. The r e a c t i o n m i x t u r e was k e p t o v e r n i g h t a t 0°C. The optimum molar r a t i o o f m - c h l o r o p e r b e n z o i c a c i d and r e a c t i o n time were s t u d i e d w i t h EDDP p e r c h l o r a t e . Three methods were used t o work up the samples and a l l gave the same products-,* 1) The CHC1 3 s o l u t i o n was washed w i t h 10% Na 2SQ 3 u n t i l the p r e s e n c e o f p e r o x i d e was not d e t e c t e d . The s o l u t i o n was f u r t h e r washed w i t h s a t u r a t e d NaHCO^. F i n a l washing was made w i t h H 20. 2) The CHCl^ s o l u t i o n was washed w i t h s a t u r a t e d NaHCO^. a) The CHCl^ s o l u t i o n was d i r e c t l y a n a l y z e d by GCMS. 4. S e l e c t e d i o n m o n i t o r i n g (SIM) a n a l y s i s o f methadone and m e t a b o l i t e s i n human plasma,  s a l i v a , and u r i n e samples Samples Plasma, s a l i v a , and u r i n e samples were o b t a i n e d from a p h a r m a c o k i n e t i c s t u d y o f f o u r female methadone maintenance pa-t i e n t s w hich was conducted by t h e A l c o h o l and Drug Commission, Vancouver. Maintenance dosage l e v e l s were f o r p a t i e n t A, 30 mg 30 B, 40 mg; C, 30 mg; and D, 90 mg/day. Plasma and s a l i v a samples were t a k e n a t 0, 2, 4,-6, 8, 11, 12 and 24 hours a f t e r the u s u a l dose. U r i n e samples were o b t a i n e d a t 1, 3, 5, 7, 9, 13, and 24 hour s . A f t e r the o r a l dose o f methadone was t a k e n , the p a t i e n t s were i n s t r u c t e d t o r i n s e the mouth w i t h 2 50 ml o f water t o remove t r a c e s o f drug from the o r a l c a v i t y . P a t i e n t s were n ot a l l o w e d t o e a t o r d r i n k j u s t p r i o r t o p r o v i d i n g a s a l i v a sample. The mouth was a g a i n r i n s e d w i t h water b e f o r e the sample was ta k e n t o reduce c o n t a m i n a t i o n from fo o d s u b s t a n c e s . A l l samples were s t o r e d f r o z e n u n t i l a n a l y z e d . E x t r a c t i o n p r o c e d u r e s and s t a n d a r d c u r v e p r e p a r a t i o n Plasma and s a l i v a . S a l i v a was c e n t r i f u g e d i n o r d e r t o remove s o l i d s . To plasma o r s a l i v a samples (0.5 ml) was added 0.2 ml o f i n t e r n a l s t a n d a r d ( I . S . ) , 2-dimethylamino-4, 4,-diphenyl-5-nonanone p e r c h l o r a t e (a s t o c k s o l u t i o n o f I.S. was p r e p a r e d t o c o n t a i n 10 mg/ml i n methanol w h i c h was d i l u t e d w i t h w a t e r t o make a s o l u t i o n e q u i v a l e n t t o 200 ng i n 0.2 ml H„0) .• The s o l u t i o n was d i l u t e d t o 3 ml w i t h H-0 and 0.1 ml of 1 N NaOH was added. A f t e r a d d i n g methylene c h l o r i d e (15 m l ) , the s o l u t i o n was v o r t e x mixed f o r 3 m i n u t e s . The aqueous: l a y e r was a s p i r a t e d o f f and the methylene c h l o r i d e l a y e r was d r i e d o v er anhydrous sodium s u l f a t e . D r i e d methylene c h l o r i d e (10 ml) was t a k e n and e v a p o r a t e d under • The r e s i d u e was d i s s o l v e d i n 50-100 u l o f CH^OH. A 2-5 u l a l i q u o t was i n j e c t e d onto t h e GCMS. S t a n d a r d c u r v e s were p r e p a r e d by s p i k i n g con-t r o l samples o f plasma and s a l i v a (0.5 ml) w i t h methadone i n 31 t h e amounts o f 0, 20, 40, 100, 200, and 500 ng. The peak a r e a r a t i o s o f m e t h a d o n e / i n t e r n a l s t a n d a r d o b t a i n e d by m o n i t o r i n g m/e 72 were p l o t t e d v s . t h e c o n c e n t r a t i o n o f methadone. U r i n e s . A f t e r t h a w i n g the sample, 1 ml o f u r i n e was t a k e n , t o w h i c h was added 0.2 ml o f s o l u t i o n c o n t a i n i n g t h e i n t e r -2 n a l s t a n d a r d s a t c o n c e n t r a t i o n s o f 20 ug methadone- H^Q, 10 ug 2 2 2 EDDP- H 3, 10 ug EMDP- H 1 Q and 10 ug o f DDP- H 1 Q / m l . The m i x t u r e was d i l u t e d t o 5 ml w i t h d i s t i l l e d w a ter and t h e pH was a d j u s t e d t o 7-8 w i t h 0.1 N NaOH. The m i x t u r e was e x t r a c t e d by v o r t e x mix-i n g f o r 2 minutes w i t h methylene c h l o r i d e (15 m l ) . The methylene c h l o r i d e e x t r a c t (13 ml) was d r i e d o v e r anhydrous Na 2S0^ and t a k e n t o d r y n e s s u s i n g N 2 . The r e s i d u e was t a k e n up i n MeOH (0.1-0.4 ml) and a 2-5 u l a l i q u o t was i n j e c t e d onto t h e GCMS. F o r s t a n d a r d c a l i b r a t i o n s , v a r y i n g amounts o f methadone (0.5 ug t o 50 u g ) , EDDP (0.5 ug t o 50 u g ) , EMDP (0.05 ug t o 1 u g ) , and DDP (0.05 ug t o 1 ug) were added t o c o n t r o l u r i n e s (1 m l ) . B l a n k samples c o n t a i n i n g o n l y t h e d e u t e r a t e d i n t e r n a l s t a n d a r d s i n c o n t r o l u r i n e (1 ml) were a l s o p r e p a r e d i n o r d e r t o s u b t r a c t background i n t e r f e r e n c e s r e s u l t i n g from i s o t o p i c i m p u r i t y and column b l e e d i n g . C a l i b r a t i o n c u r v e s were p r e p a r e d by p l o t t i n g the peak a r e a r a t i o s o f u n l a b e l e d / l a b e l e d compound a t each o f t h e i o n p a i r s m o n i t o r e d v s . the known c o n c e n t r a t i o n r a t i o o f u n l a b e l e d compound t o i t s c o r r e s p o n d i n g l a b e l e d i n t e r n a l s t a n d a r d . M o n i t o r i n g i o n s were m/e 223/m/e 233 (methadone), m/e 277/m/e 280 (EDDP), m/e 208/m/e 218 (EMDP), and m/e 265/m/e 275 (DDP). 32 GC a n a l y s i s F o r t h e GC a n a l y s i s o f methadone and EDDP i n u r i n e s 2-dimethylamino-4,4-diphenyl-5-nonanone p e r c h l o r a t e (10 ug i n 0.2 ml H 20) was used as t h e i n t e r n a l s t a n d a r d . The e x t r a c -t i o n p r o c e d u r e s were e s s e n t i a l l y t h e same as thos e d e s c r i b e d f o r t h e SIM a n a l y s i s o f u r i n e samples. S t a b i l i t y o f EDDP D i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y was used t o dete r m i n e mp o f EDDP p e r c h l o r a t e ; mp 175°C, when r e c r y s t a l l i z e d from d i e t h y l ether-EtOH ( l i t . (3) mp 167-168°C). EDDP p e r c h l o r a t e s t o c k s o l u t i o n (1.5 ml, 1 mg/ml i n MeOH) was e v a p o r a t e d under N"2 . A f t e r a d j u s t i n g t o pH 12 w i t h lN-NaOH, e t h e r (25 ml) was added t o e x t r a c t t h e f r e e base. An a l i q u o t o f t h e e t h e r l a y e r (20 ml) was t r a n s f e r r e d t o a 25 ml v o l u m e t r i c f l a s k and made up.to 2 5 ml w i t h t h e same s o l v e n t . Samples (0.2 ml) were t a k e n f o r t h e a n a l y s i s a t 0, 1, 2, and 3 days a f t e r p r e p a r a t i o n o f the sample w h i c h was ke p t on the bench a t room t e m p e r a t u r e . An i n t e r n a l s t a n d a r d s t o c k s o l u t i o n was p r e p a r e d w i t h MeOH t o c o n t a i n 10 ug o f 2 2 DDP- H 1 Q and 20 ug o f EDDP- H 3 p e r c h l o r a t e p e r ml. Sample s o l u t i o n (0.2 ml) was mixed w i t h i n t e r n a l s t a n d a r d s o l u t i o n (0.2 ml) and t h e m i x t u r e was a n a l y z e d f o r DDP and EDDP by m o n i t o r i n g m/e 265 and m/e 275 f o r DDP and m/e 277 and m/e 280 f o r EDDP. The s t a n d a r d c u r v e s were p r e p a r e d u s i n g v a r i o u s c o n c e n t r a t i o n s o f EDDP p e r c h l o r a t e and DDP d i s s o l v e d i n methanol. 33 The c o n c e n t r a t i o n s o f EDDP were e x p r e s s e d as t h e f r e e base. 5. D e t e c t i o n o f methadone m e t a b o l i t e s A n i m a l e x p e r i m e n t s Male W i s t a r r a t s (250-350 gm) were a n e s t h e s i z e d w i t h e t h e r d u r i n g t h e s u r g i c a l p r o c e d u r e s . The common b i l e d u c t was i s o l a t e d t h r o u g h a m i d l i n e abdominal i n c i s i o n and cannu-l a t e d w i t h p o l y e t h y l e n e t u b i n g - 1 0 f o r b i l e c o l l e c t i o n . The s u r g i c a l a r e a o f t h e abdomen was s u t u r e d . The r a t was p l a c e d i n a r e s t r a i n i n g cage. A f t e r r e c o v e r y from e t h e r a n e s t h e s i a , t h e r a t was g i v e n t h e a p p r o p r i a t e d r ug s.c. a t a dose o f 20 mg/kg (methadone, m e t a b o l i t e s , d e u t e r a t e d methadone, d e u t e r a t e d m e t a b o l i t e s ) . The b i l e was c o l l e c t e d o v e r a p e r i o d o f 24 ho u r s . Human ex p e r i m e n t s . The twenty f o u r hour u r i n e samples were o b t a i n e d from methadone maintenance p a t i e n t s who were on - dosages, of 90 mg/day. Samples were s u p p l i e d by the A l c o h o l and Drug Com-m i s s i o n L a b o r a t o r i e s , Vancouver. Sample work up p r o c e d u r e s A b i l e sample (10 ml) was . - d i l u t e d w i t h H 20 (10 ml) and t h e m i x t u r e c e n t r i f u g e d t o remove s o l i d s u b s t a n c e s . The b i l e sample was the n e x t r a c t e d w i t h methylene c h l o r i d e (50 34 ml x 2) . The methylene c h l o r i d e l a y e r s were combined and kept f o r the a n a l y s i s o f nonconjugated m e t a b o l i t e s . The aqueous l a y e r was f r e e z e d r i e d . B u f f e r (pH 4.5, 10 ml) was added t o the f r e e z e d r i e d sample ( f i n a l pH 5.0) and the m i x t u r e w a s i i n c u b a t e d (37°C) i n R R the p r e sence o f G l u c u r a s e o r G l u s u l a s e (1 ml) f o r 24 h o u r s . A f t e r i n c u b a t i o n , the pH o f t h e s o l u t i o n was a d j u s t e d t o 8.0-8.5 by a d d i n g pH 9.0 b o r a t e b u f f e r (10 m l ) . The s o l u t i o n was e x t r a c t e d w i t h methylene c h l o r i d e (75 ml x 3 ) . The combined methylene c h l o r i d e e x t r a c t s were f l a s h e v a p o r a t e d and the r e s i d u e r e d i s s o l v e d w i t h methanol (1 m l ) . T h i s s o l u t i o n was t r e a t e d w i t h diazomethane. 6. Methadone-diazepam i n t e r a c t i o n s t u d i e s Treatment o f a n i m a l s Male W i s t a r r a t s (200-300 gm) o b t a i n e d from Canadian B i o - B r e e d i n g Farm L a b o r a t o r i e s ( M o n t r e a l , Quebec) were used t h r o u g h o u t . They were m a i n t a i n e d on a s t a n d a r d d i e t o f P u r i n a Lab Chow ( R a l s t o n P u r i n a Co. o f Canada) and water ad l i b i t u m . The r a t was j u g u l a r v e i n c a n n u l a t e d by the method o f Upton (78). The e x t e r n a l j u g u l a r v e i n was exposed u s i n g t i s s u e f o r c e p s . A 3 cm p i e c e o f SILASTIC t u b i n g was connected t o 3 cm o f p o l y -e t h y l e n e t u b i n g - 5 0 by means o f a p i e c e o f 22 gauge hypodermic n e e d l e . The v e i n was c u t u s i n g a sharp b l a d e o r m o r i a s p r i n g s c i s s o r s . The h e p a r i n i z e d s a l i n e was normal s a l i n e c o n t a i n i n g 35 h e p a r i n a t a c o n c e n t r a t i o n o f 20 u n i t s / m l . A f t e r c o m p l e t i o n o f j u g u l a r v e i n c a n n u l a t i o n , b i l e d u c t c a n n u l a t i o n was performed as d e s c r i b e d by Lambert (79). I r i s f o r c e p s were used t o h o l d t h e b i l e d u c t and t o i n s e r t the c a n n u l a below t h e j u n c t i o n o f the r i g h t and l e f t h e p a t i c d u c t s . A c u t was made i n t h e b i l e d u c t w i t h a r a z o r b l a d e o r m o r i a s p r i n g s c i s s o r s . P o l y e t h y l e n e t u b i n g - 1 0 was i n s e r t e d i n t o t h e common b i l e d u c t . The t o t a l t i me spent f o r j u g u l a r v e i n and b i l e d u c t c a n n u l a t i o n was 20-30 m i n u t e s . A f t e r b i l e d u c t c a n n u l a t i o n , diazepam o r v e h i c l e o n l y was g i v e n t h r o u g h t h e j u g u l a r v e i n a t an i n f u s i o n r a t e o f 2 ml/hour. The time of i n f u s i o n s t a r t was r e c o r d e d . W h i l e t h e i n f u s i o n was b e i n g p e r f o r m e d , t h e s u r g i c a l a r e a o f t h e b i l e d u c t c a n n u l a t i o n was s u t u r e d . A f t e r i n f u s i o n , t h e PE-50 t u b i n g was c u t s h o r t and the a n i m a l was r e s u t u r e d w i t h t h e t u b i n g i n s i d e t h e l a y e r o f t h e s k i n . S h o r t l y b e f o r e methadone d o s i n g , one hour from t h e s t a r t of thai i n f u s i o n , t h e r a t was p l a c e d i n a r e s t r a i n i n g cage. Metha-done HCl (10 mg/Kg i n 2 ml s a l i n e ) was g i v e n s . c . B i l e was c o l l e c t e d from t h e c a n n u l a t e d b i l e d u c t i n preweighed s c i n t i l l a t i o n v i a l s a t d i f f e r e n t t i m e s (1, 2, 5, 11, and 23 h o u r s ) . The b i l e w h i c h was c o l l e c t e d b e f o r e t h e methadone dose s e r v e d as t h e b l a n k . The diazepam was d i s s o l v e d i n a s o l u t i o n o f p r o p y l e n e g l y c o l ( 4 0 % ) , e t h a n o l ( 1 0 % ) , b e n z y l a l c o h o l ( 1 . 5 % ) , and sodium benzoate (5%) i n wa t e r . C o n t r o l a n i m a l s r e c e i v e d an e q u a l volume o f t h e v e h i c l e . 36 Sample p r e p a r a t i o n p r o c e d u r e s The c o n j u g a t e d i n t e r n a l s t a n d a r d s were p r e p a r e d by 2 d o s i n g 3 r a t s w i t h 20 mg/Kg EMDP- H 1 Q s.c. B i l e was c o l l e c t e d f o r 24 h o u r s . The b i l e was e x t r a c t e d once w i t h methylene 2 c h l o r i d e t o remove EMDP- H 1 Q . The e x t r a c t e d b i l e was d i l u t e d t o 100 ml w i t h water and f r o z e n i n an Erlenmeyer f l a s k a t -20°C u n t i l used. T h i s s o l u t i o n was used d i r e c t l y as the i n t e r n a l s t a n d a r d t o measure c o n j u g a t e d m e t a b o l i t e s . The b i l e sample (0.2-0.4 ml) was mixed w i t h 0.2 ml o f 2 the n o n c o n j u g a t e d i n t e r n a l s t a n d a r d s o l u t i o n (20 ug EDDP- H^, 2 2 2 ug EMDP- H 1 Q , 5 ug methadone- i n 0.2 ml H 20) and 1 ml o f the c o n j u g a t e d 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 m i x t u r e (pH 9.0-9.5) was e x t r a c t e d w i t h methylene c h l o r i d e (15 m l ) . The aqueous l a y e r was k e p t f o r the a n a l y s i s o f c o n j u g a t e d metabo-l i t e s . The methylene c h l o r i d e f r a c t i o n was e v a p o r a t e d under N 2 . A f t e r a d j u s t i n g the pH o f the r e s i d u e w i t h pH 2.0 b u f f e r (2.0 m l ) , the m i x t u r e was b r i e f l y e x t r a c t e d w i t h CHCl^ (0.5 ml) t o remove c o l o r e d m a t e r i a l s . The aqueous l a y e r was made a l k a l i n e by a d d i n g 1 N NaOH (0.5 ml) and was e x t r a c t e d w i t h methylene c h l o r i d e (10 m l ) . The methylene c h l o r i d e l a y e r was d r i e d o v e r anhydrous Na 2SO^ and e v a p o r a t e d under N 2. The f i n a l samples f o r SIM a n a l y s i s were p r e p a r e d w i t h methanol. 2 M o n i t o r i n g i o n s were m/e 277 (EDDP), m/e 280 (EDDP- H 3 ) , m/e 208 (EMDP), m/e 218 (EMDP- 2H 1 Q), m/e 294 (methadone), and 2 m/e 29 7 (methadone- H_). 37 The aqueous l a y e r was f r e e z e d r i e d . B u f f e r , pH 4.5 (1.5 ml) was added t o the r e s i d u e ( f i n a l pH 5.0) and t h e mixture was i n c u b a t e d w i t h G l u c u r a s e (0.2 ml) f o r 24 hours a t 3 7°C. A f t e r i n c u b a t i o n , t h e pH was a d j u s t e d w i t h pH 9.0 b o r a t e b u f f e r (2 ml) t o pH 8.5-9.0. The m i x t u r e was e x t r a c t e d w i t h methylene c h l o r i d e (15 m l ) . The methylene c h l o r i d e was d r i e d o ver anhydrous Na^O^ and t a k e n t o d r y n e s s . The r e s i d u e was d i s -s o l v e d i n 1 ml o f methanol and t r e a t e d w i t h diazomethane. A f t e r m e t h y l a t i o n ( s u s t a i n e d y e l l o w c o l o r ) , methanol was evapo-r a t e d and t h e m i x t u r e was v o r t e x mixed w i t h hexane (5 ml) a f t e r a d j u s t i n g the pH w i t h pH 2.0 b u f f e r (1.5 ml) t o remove endogenous m a t e r i a l s . The pH o f the aqueous p a r t was a d j u s t e d t o a l k a l i n e w i t h 1 N NaOH (0.5 ml) and e x t r a c t e d w i t h methylene c h l o r i d e (10 m l ) . The methylene c h l o r i d e was d r i e d o ver Na 2SO^ and t a k e n t o d r y n e s s under N 2 . The f i n a l r e s i d u e was d i s s o l v e d i n s u i t a b l e volume o f methanol f o r SIM a n a l y s i s . I o n s , m/e 247, 246, 238, and 237 were m o n i t o r e d f o r CH 30EMDP and m/e 245, 244, 237, and 236 f o r DiCH^OEMDP. The r a t i o s , m/e 238/ m/e 247. anid m/e 237i/m/e 245 were s e l e c t e d f o r t h e a n a l y s i s o f HOEMDP and DIHOEMDP, r e s p e c t i v e l y . S t a b i l i t y e x p e r i m e n t s Noncohjugated m e t a b o l i t e s A s t o c k s o l u t i o n was 2 2 p r e p a r e d t o c o n t a i n 1 mg each o f EDDP, EDDP- H 1 Q , EDDP- H 3, 2 2 2 EMDP, EMDP- H l Q , methadone methadone- H 1 Q , methadone- H 3, DDP, 2 and DDP- H-^ Q i n 1 ml o f methanol. Stock s o l u t i o n (1 ml) was d i l u t e d t o 50 ml w i t h methanol i n a v o l u m e t r i c . f l a s k and 38 2.5 ml o f t h e s o l u t i o n (50 ug each) was t a k e n t o d r y n e s s . U s i n g t h e s e d r i e d samples s o l u t i o n s were made up t o 5 ml w i t h water a t d i f f e r e n t pHs (1 N HC1, pH 2,0, pH 4.5, pH 9.0, and 0.1 N NaOH). A f t e r i n c u b a t i o n a t d i f f e r e n t t i m e s , t h e pH o f each s o l u t i o n was a d j u s t e d t o 9.0 and t h e s o l u t i o n e x t r a c t e d w i t h 10 ml o f methylene c h l o r i d e . The methylene c h l o r i d e f r a c t i o n was d r i e d o v er anhydrous N a 2 S 0 4 and e v a p o r a t e d under N 2• The r e s i d u e was d i s s o l v e d i n methanol and i n j e c t e d i n t o the GCMS. I o n s , m/e 277 (EDDP), m/e 287 ( E D D P - 2 H 1 Q ) , m/e 280 (EDDP- 2H 3), m/e 208 (EMDP), m/e 218 (EMDP- 2H 1 0), m/e 223 (metha-• 2 2 done), m/e 226 (methadone- H^), and m/e 233 (methadone- H^Q) were m o n i t o r e d . Conjugated m e t a b o l i t e s The c o n j u g a t e d i n t e r n a l s t a n -d a r d (2 ml) was i n c u b a t e d w i t h G l u c u r a s e (0.5 ml) a t d i f f e r e n t t i m e s (12, 24, 48, and 96 hours) and t h e samples worked up f o l -l o w i n g t h e same p r o c e d u r e s f o r t h e c o n j u g a t e d f r a c t i o n . o f t h e b i l e samples. S p e c i f i c i o n s , m/e 247, 246, 245, 244 and m/e 247, 246, 238, 237 f o r CH 30 EMDP- 2H g; m/e 247, 246, 245, 244 and m/e 2 245, 244, 237, 236 f o r ( C H 3 0 ) 2 EMDP- Hg were m o n i t o r e d which • would i n d i c a t e s t a b i l i t y o f t h e d e u t e r i u m l a b e l i n g . See Ta b l e V I I I and T a b l e IX f o r d e t a i l s . 7. P h a r m a c o k i n e t i c and s t a t i s t i c a l a n a l y s i s The apparent e l i m i n a t i o n r a t e c o n s t a n t s and h a l f l i v e s were c a l c u l a t e d by u s i n g t h e NONLIN program (80). The. use o f 39 b i o s y n t h e t i c i n t e r n a l s t a n d a r d s f o r p h a r m a c o k i n e t i c s t u d i e s was. i n v e s t i g a t e d by u s i n g t h e same program. Slope and i n t e r -c e p t v a l u e s o f c a l i b r a t i o n e q u a t i o n s were c a l c u l a t e d by means of t h e computer program, T r i a n g u l a r R e g r e s s i o n Package, Cora-.. p u t i n g C e n t e r , t h e U n i v e r s i t y of B r i t i s h C o lumbia. The s t a t i s t i c a l a n a l y s i s was performed by S t u d e n t ' s t t e s t . 40 RESULTS AND DISCUSSION 1. S y n t h e s i s and mass s p e c t r o m e t r y of d e u t e r -a t e d methadone and m e t a b o l i t e s : , D e u t e r a t e d d i p h e n y l a c e t o n i t r i l e 2 Treatment o f p h e n y l a c e t o n i t r i l e w i t h benzene- H g by the method of Robb and Schult'z (76) r e s u l t e d i n t h e i s o l a t i o n o f d i p h e n y l a c e t o n i t r i l e l a b e l e d i n b o t h p h e n y l r i n g s . As shown i n the mass spectrum ( F i g . 2 a ) , d e u t e r a t i o n of t h e p h e n y l r i n g s was n o t complete. NMR a n a l y s i s i n d i c a t e d 86% d e u t e r a t i o n . L a b e l l i n g of b o t h p h e n y l r i n g s i s p r o b a b l y t h e r e s u l t of two p r o c e s s e s . A c i d c a t a l y z e d and or aluminum c h l o r i d e 2 c a t a l y z e d exchange (81) between benzene- and the r i n g p r o -t o n s o f p h e n y l a c e t o n i t r i l e o r d i p h e n y l a c e t o n i t r i l e c o u l d a c c o u n t f o r the o b s e r v e d l a b e l l i n g . W h i l e t h i s exchange p r o c e s s i s i n d e e d a c o n t r i b u t i n g f a c t o r i t does n o t t o t a l l y d e termine the end r e s u l t . F o r example, when u n l a b e l e d d i p h e n y l a c e t o n i -2 t r i l e was t r e a t e d w i t h benzene- H, under the r e a c t i o n c o n d i -6 t i o n s ( A l C l ^ r r e f l u x , 2 h o u r s ) , 55-60% o f the e x p e c t e d exchange as measured by NMR had o c c u r r e d . More th a n 20 hours r e f l u x was r e q u i r e d b e f o r e t h e exchange approached t h e p r o p o r t i o n s a c h i e v e d d u r i n g t h e a l k y l a t i o n r e a c t i o n . On t h e o t h e r hand, 2 exchange between benzene and benzene- H, ( A l C l - . , r e f l u x ) 41 wo-60H > < (a) CM—CN 173 93 120 JU,#,i...,J 202 100 200 300 100-1 SOH < -J U i EC (b) |92 118 100 1S8 200 300 100 z U J I- 60H < (c) JL 173 93 1 2 1 203 M< 100 200 300 F i g , Mass s p e c t r a o f d e u t e r a t e d d i p h e n y l a c e t o n i t r i l e s (a) p r e p a r e d u s i n g an e x c e s s o f b e n z e n e - 2 H 6 (b) 50% d e u t e r a t e d p r e p a r e d by the method o f Hachey e t a l . (19) (c) a f t e r e n r i c h m e n t . 42 re a c h e d e q u i l i b r i u m w i t h i n 20 minutes (as measured by mass s p e c t r o m e t r y ) . To e x p l a i n t h e e x t e n t o f l a b e l l i n g a c h i e v e d by t h e a l k y l a t i o n r e a c t i o n , i t i s suggested t h a t t h e r e v e r s i b l e n a t u r e of t h e F r i e d e l C r a f t s r e a c t i o n (82) i s an a d d i t i o n a l f a c t o r t o the exchange p r o c e s s e s t h a t o c c u r . A suggested i n t e r m e d i a t e f o r t h e p r o c e s s i l l u s t r a t e s t h a t r a p i d exchange between benzene and l a b e l e d benzene p l u s t h e r e v e r s i b i l i t y o f t h e r e a c t i o n l e a d s t o a p r o d u c t w i t h t h e e x p e c t e d p e r c e n t o f l a b e l a t e q u i l i b r i u m . CeD6 ' C6H5D The r e s u l t s a r e c o n t r a r y t o a r e p o r t t h a t d i p h e n y l a c e -t o n i t r i l e s y n t h e s i z e d u s i n g e q u i m o l a r c o n c e n t r a t i o n s o f p h e n y l -2 a c e t o n i t r i l e and benzene- i n CS^ as s o l v e n t gave a p r o d u c t w i t h t h e f i v e d e u t e r i u m atoms c o n t a i n e d i n one r i n g (19). T h i s e x p e r i m e n t was r e p e a t e d as d e s c r i b e d and t h e p r o d u c t o b t a i n e d was found t o be 50% d e u t e r a t e d by NMR. The mass spectrum ( F i g . 2b) shows a c l u s t e r o f the m o l e c u l a r i o n s f o r 2 2 the isomers from HQ t o H-^ i n a p p r o x i m a t e l y t h e e x p e c t e d d i s t r i b u t i o n f o r 50% u n i f o r m l a b e l l i n g i n b o t h r i n g s . D i p h e n y l a c e t o n i t r i l e - ' ' H ^ Q e n r i c h e d w i t h d e u t e r i u m 2 was p r e p a r e d u s i n g aluminum c h l o r i d e c a t a l y s t and benzene-as a d e u t e r i u m source (81). A 1:20 molar r a t i o o f d i p h e n y l a c e -2 2 t o n i t r i l e - H-,~ (86% r i n g d e u t e r a t i o n ) t o benzene- H, gave ± U b the p r o d u c t w i t h 98% r i n g d e u t e r a t i o n as d e t e r m i n e d by NMR and mass s p e c t r o m e t r y . Assuming e q u i l i b r i u m was a c h i e v e d 2 between the benzene- and p a r t i a l l y l a b e l e d d i p h e n y l a c e t o n i -t r i l e , 98.5% r i n g d e u t e r a t i o n would be e x p e c t e d . The mass spectrum i s shown i n F i g . 2c. D e u t e r a t e d d i p h e n y l a c e t o n i t r i l e p r e p a r e d u s i n g exchange r e a c t i o n s was found t o be p r e f e r a b l e t o u s i n g t h e a l k y l a t i o n r e a c t i o n . The exchange r e a c t i o n i s s i m p l e w i t h few i f any s i d e p r o d u c t s produced. Methadone n i t r i l e can n o t be l a b e l e d u s i n g t h e s e c o n d i t i o n s . D e u t e r a t e d methadone 2 Methadone- H^Q p r e p a r e d s t a r t i n g from e n r i c h e d d i p h e -n y l a c e t o n i t r i l e r e t a i n e d t h e same degree of l a b e l l i n g as i n 2 t h e s t a r t i n g m a t e r i a l . The mass spectrum of methadone- H^Q ( F i g . 3b) c o r r e s p o n d s t o t h a t f o r methadone (83), t h e m o l e c u l a r i o n a p p e a r i n g a t m/e 319. Two i o n s t h a t might be used i n SIM appear a t m/e 233 and m/e 304 c o r r e s p o n d i n g t o m/e 223 (25) and m/e 294 (27) f o r methadone ( F i g . 3 a ) . S u l l i v a n e t a l . 2 (17) r e p o r t e d t h a t the a p p l i c a t i o n o f methadone- t o plasma d e t e r m i n a t i o n s o f methadone by m o n i t o r i n g m/e 2 94 f o r methadone 2 and m/e 297 f o r methadone- H^- While n e i t h e r o f t h e s e fragment i o n s i s i d e a l f o r SIM s t u d i e s , under our c o n d i t i o n s o f mass s p e c t r o m e t r y , m/e 223 showed s l i g h t l y h i g h e r abundance (1.6%) 44 4 5 2 t h a n t h a t of m/e 294 (1.0%) ( F i g . 3 a ) . U s i n g methadone- H 1 Q as i n t e r n a l s t a n d a r d , i o n s a t m/e 223 and m/e 233 have formed th e b a s i s f o r a s u i t a b l e i o n m o n i t o r i n g assay o f methadone i n u r i n e samples. + CH= N(CH3), I CH3 24 m/e 72 m/e 294 2 H 3 m/e 297 The i o n a t m/e 72 (2_4) was s e l e c t e d t o q u a n t i t a t e methadone i n human s a l i v a and plasma samples, and t h e i o n a t m/e 294 (27) f o r r a t b i l e samples as d e s c r i b e d i n the e x p e r i m e n t a l . By comparing t h e mass spectrum i n F i g . 3a and 3b i t becomes e v i d e n t t h a t b e s i d e s t h e fragments a t m/e 294 and 223 minor peaks a t m/e 208, 193, 179, 178, and 165 appear as t y p i c a l i o n s c o n t a i n i n g t h e two p h e n y l r i n g s . The proposed 46 pathways t o t h e s e fragment i o n s (31-35, 26) a r e d e s c r i b e d as p a r t o f t h e f r a g m e n t a t i o n p r o c e s s e s o f EMDP and DDP However, the p o s s i b i l i t y t h a t m/e 165 (26J might be d e r i v e d from m/e 223 (25^) can n o t be e x c l u d e d . D i p h e n y l a c e t o n i t r i l e which can n o t form m/e 208 a l s o gave t h e i o n m/e 165 (28). m/e 165 H 1 Q m/e 173 M 'We 193 2 H 1 0 m/e 203 m/e 116, H 1 Q m/e 121 29 28 <7 m + CH /e 89 "H m/e 93 30 D e u t e r a t e d methadone m e t a b o l i t e s When p a r t i a l l y d e u t e r a t e d methadone n i t r i l e was h y d r o l y z e d w i t h E^O and H2SC>4, a marked d e c r e a s e i n t h e degree of d e u t e r a t i o n from 86% t o 40% was o b s e r v e d i n t h e p r o d u c t , 4-dimethylamino-2, 2 - d i p h e n y l p e n t a n o i c a c i d . On t h e o t h e r hand, t r e a t m e n t o f p a r t i a l l y d e u t e r a t e d methadone n i t r i l e w i t h D 20 and D 2 S 0 4 produced an i n c r e a s e i n t h e degree o f d e u t e r a t i o n i n t h e a c i d p r o d u c t t o 97%. S t a r t i n g w i t h un-l a b e l e d methadone n i t r i l e , h y d r o l y s i s w i t h D„0 and D„S0. 4 7 34 ... m/e 165 V „ m/e 174 4"8 gave p r o d u c t c o n t a i n i n g o n l y 6 0 - 7 0 % d e u t e r a t i o n as d e t e r m i n e d by NMR and i t was t h e r e f o r e c o n c l u d e d t h a t maximum d e u t e r a t i o n of s y n t h e s i z e d m e t a b o l i t e s c o u l d not be a c h i e v e d by t h i s l a t t e r method. The l a b e l e d a c i d was decomposed i n t h e GC i n l e t t o the d e c a r b o x y l a t e d p r o d u c t , 1 , l - d i p h e n y l - 3 - d i m e t h y l a m i n o b u t a n e -2 H-^ Q as d e t e r m i n e d from t h e mass f r a g m e n t a t i o n p a t t e r n s w i t h peaks a t m/e 7 2 (base p e a k ) , m/e 2 6 3 (M +, 0 . 9 % ) , m/e 1 7 7 ( 3 . 0 % ) , 2 and m/e 1 7 3 ( 1 . 3 % ) and DDP- as d e t e r m i n e d from i t s r e t e n -t i o n time v a l u e and mass spectrum. 2 DDP- H 1 Q s y n t h e s i z e d from the a c i d , 5_ by t h i o n y l c h l o -r i d e d e m e t h y l a t i o n f u l l y r e t a i n e d t h e d e u t e r i u m l a b e l . The s p e c t r a o f DDP i s shown i n F i g . 4. The f r a g m e n t a t i o n p a t t e r n s f o r DDP were s i m i l a r t o t h o s e f o r EMDP b e i n g d e r i v e d m a i n l y from t h e m/e 2 0 8 fragment. 2 Mass s p e c t r a a re shown i n F i g . 5 f o r EDDP, EDDP- H^Q 2 ( 9 7 % l a b e l ) , and EDDP- H 3 ( 9 9 % l a b e l ) . The r e l a t i v e abundance of m/e 2 0 8 ( 3 1 ) f o r EDDP was 0. 9 % . T h i s i m p l i e d t h a t t h e f r a g m e n t a t i o n t o produce m/e 20 8 which would i n v o l v e the l o s s o f C H 3 - N = C = C H C H 3 ~~I from t h e m o l e c u l a r i o n i s no l o n g e r a f a v o r e d p r o c e s s . The f o r m a t i o n o f m/e 1 0 5 (3J7) on the o t h e r hand i s unique t o EDDP and r e q u i r e s t h e m i g r a t i o n o f one p h e n y l r i n g t o the u n s a t u r a t e d s i d e c h a i n . T h i s was r e c o g n i z e d from t h e appearance o f t h e c o r r e s p o n d i n g i o n s of m/e 1 1 0 and m/e 2 2 1 0 8 f o r EDDP- H 1 Q and EDDP- H 3 , r e s p e c t i v e l y . The mechanism of p h e n y l m i g r a t i o n i s s i m i l a r t o t h a t r e p o r t e d f o r the compound, 3 8 ( 8 4 ) . 4 9 51 A n o t h e r p r o c e s s a p p a r e n t l y unique t o EDDP i s the l o s s o f CH3-CH=CH2~1^ t o form m/e 235 (_40) whi c h t h e n can e i t h e r form m/e 220 (£1, 21%) o r by f u r t h e r l o s s , o f a CH 3 form 2 the fragment m/e 69 (4_2, 21%) . In b o t h c a s e s EDDP- H 3 p r o v e d v a l u a b l e i n d e t e r m i n i n g the n a t u r e o f t h e s e i o n s . .52 40 CHCH3 I' C N-CH, > CH3CH= C= N—CH 3 42 m/e 69 ^ m/e 235 2H1Qm/e 245, \rnfe 238 H 3 m / e 7 2 m/e 220 TJ Q m/e 230 41 Mass s p e c t r a f o r EMDP- H^Q and EMDP a r e shown i n F i g . 6 . EMDP gave f r a g m e n t a t i o n s r e s u l t i n g m a i n l y from m/e 2 20 8. S i n c e EMDP- l o s e s the l a b e l e d m e t h y l group when the m/e 208 fragment i s formed, no d i f f e r e n c e i n t h e mass s p e c t r a was o b s e r v e d between EMDP and EMDP- H. S p e c t r o s c o p i c o b s e r v a t i o n s NMR d a t a f o r EDDP and EMDP w e r e ' d e s c r i b e d e a r l i e r by Pohland e t a l . ( 3 ) . NMR s p e c t r a were shown f o r EDDP p e r c h l o -r a t e and EMDP HCl by B e c k e t t e t a l . (2) w i t h o u t assignment of c h e m i c a l s h i f t v a l u e s . The NMR d a t a o b t a i n e d f o r t h e methadone m e t a b o l i t e s i s summarized i n Table I w i t h a compar-i s o n o f t h e C-4 p r o t o n s which d e s c r i b e an AB system. The 5 3 100-i CO z UJ U J > U J GC 50H (a) 115 130 91 193 179 165 208 100 m/e 200 C H , N N / CH, CH, 263 M* 300 100 n CO z U J t-5 50 U J > UJ (b) 135 120 96 203 188 174 100 218 A , A CH, N X CH, CH, 273 M-200 300 m/e F i g . 6. Mass s p e c t r a o f (a) EMDP and (b) EMDP- H 1 0 TABLE I . NMR of C-4 P r o t o n s o f Methadone M e t a b o l i t e s EDDP .(3) (t r a n s ) EDDP (3) ( c i s ) EDDP p e r c h l o -r a t e (19) EMDP (_4) EMDP HCl DDP (6) (20) 6 Ha J Ha-He 6 Hb J Hb-Hc J Ha-Hb 2.24 8.5 2.56 5.3 11.0 2.24 10.0 2.52 7.0 12.0 2.60 (dd) 7.0 3.43 (dd) 8.0 14.0 2.23 (dd) 8.7 2.67 (dd) 6.5 13.4 2.51 (dd) 2.20 (dd) 7.0 9.0 3.17 (dd) 2.95 (dd) 8.0 6.0 14.0 13.0 3, £ as r e p o r t e d by Pohland e t a l . (3) 19, 20 S p e c t r a o b t a i n e d w i t h the V a r i a n XL-100 6 S p e c t r a o b t a i n e d w i t h the Bruker WP-80 The s o l v e n t i n a l l cases was CDC1 3. \ Key"-CH, 55 c h e m i c a l s h i f t v a l u e s f o r the C-4 p r o t o n s i n DDP a r e d i f f e r e n t from t h a t i n the r e f e r e n c e by S i n g h e t a l (27), who r e p o r t e d t h a t C-4 p r o t o n s appeared a t 2.05-2.40 as m u l t i p l e t s i n CDCl^ as s o l v e n t . I n the IR, l a b e l e d methadone and m e t a b o l i t e s e x h i b i t a C-D s t r e t c h i n g v i b r a t i o n a t 2270 cm 1 . The appearance o f the C-D peak and d i s a p p e a r a n c e o f the a r o m a t i c C-H v i b r a t i o n c o u l d be used t o i n d i c a t e the e x t e n t o f d e u t e r i u m e n r i c h m e n t (85). 2. SIM a n a l y s i s o f methadone and m e t a b o l i t e s  i n b i o l o g i c a l samples S e l e c t e d i o n m o n i t o r i n g The computer program f o r SIM u s i n g the MAT 111 GCMS was d e v e l o p e d by Roland B u r t o n i n the 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 . The e f f e c t i v e mass range f o r the i n s t r u m e n t was de s i g n e d t o be ± 7% o f the H a l l probe mass s e t t i n g . The l i m i -t a t i o n o f the mass range r e s u l t s from the f a c t t h a t i o n o p t i c s and i o n abundance are i n f l u e n c e d by a t t e n u a t i o n o f t h e a c c e l e r -a t i n g v o l t a g e when a magnetic s e c t o r i n s t r u m e n t i s used i n SIM mode (86). I n p r a c t i c e , i t was found t h a t the m o n i t o r i n g o f i o n s was l i m i t e d depending upon the t o t a l number and the 2 masses o f t h e i o n s of i n t e r e s t . When EMDP, EMDP- H^ Q, metha-2 done, and methadone- H-^ Q were b e i n g m o n i t o r e d w i t h a magnetic f i e l d s e t t i n g o f m/e 220, f o c u s s i n g o f t h e i o n s was i m p o s s i b l e a l t h o u g h the i o n s b e i n g m o n i t o r e d , m/e 208, 218, 223, and 233 56 ar e a l l i n t h e e f f e c t i v e mass range. I t was a l s o found t h a t 2 methadone- i s a p r e f e r a b l e i n t e r n a l s t a n d a r d t o methadone-2 H-^ Q . S w i t c h i n g o f t h e a c c e l e r a t i n g v o l t a g e o ver a l a r g e mass range r e s u l t s i n l a r g e s t a n d a r d d e v i a t i o n s i n t h e a n a l y s i s . 2 Hence, EDDP- H^ a l s o p r o v e d t o be a b e t t e r i n t e r n a l s t a n d a r d 2 tha n EDDP- H 1 Q f o r t h e a n a l y s i s o f EDDP. The i n i t i a l window f o r an i o n b e i n g m o n i t o r e d was s e t w i t h PFK. F i n a l a d j u s tment was made by i n j e c t i n g a u t h e n t i c samples t o c o r r e c t f o r mass d e f e c t . In p r a c t i c e , i t was found t h a t i o n peak p o s i t i o n s k e p t c h a n g i n g . The most i m p o r t a n t a s p e c t i n t h e computer program i s t o d e t e c t s m a l l changes i n peak p o s i t i o n and t o c o r r e c t f o r them. A method t o a d j u s t window p o s i t i o n s t o t h e peak p o s i t i o n h a v i n g t h e g r e a t e s t i o n c u r r e n t as measured by computer program appears i d e a l (87). The method Roland B u r t o n used f o r t h i s program i s as f o l l o w s : Each i o n i s scanned from 0.5 amu below t o 0.5 amu above i t s n o m i n a l mass. T h i s scan i s f u r t h e r s u b d i v i d e d i n t o q u a r t e r s . I n t e n s i t y i s c a l c u l a t e d as t h e sum of t h e i n n e r two q u a r t e r s , minus t h e sum o f t h e o u t e r two q u a r t e r s t o c o r -r e c t f o r the b a s e l i n e change o r a d j a c e n t peak i o n s . The window p o s i t i o n i n f o r m a t i o n i s o b t a i n e d by s u b t r a c t i n g t h e second q u a r t e r from the t h i r d ; i f the number i s p o s i t i v e , the window i s t o o f a r t o t h e l e f t . The window i s moved t o c o r r e c t a c c o r d i n g l y . Ion peak p o s i t i o n was a l s o found t o change t o t h e peak a r i s i n g from column b l e e d i n g . T h e r e f o r e , a u t o f o c u s s i n g was made t o f u n c t i o n o v e r c e r t a i n peak s t r e n g t h s . When we 57 found a sudden l a r g e change o f the window p o s i t i o n , r e a d j u s t -ment o f the f u n c t i o n o f a u t o f o c u s s i n g was made. In most c a s e s the a u t o f o c u s s i n g was checked c a r e f u l l y a t a l l t i m e s . The s e n s i t i v i t y o f t h e a n a l y s i s i s dependent on t h e i o n abundance o f i o n s b e i n g m o n i t o r e d and the c o n t r i b u t i o n of background peaks. S e v e r a l c o n s i d e r a t i o n s were made t o reduce background. C l e a n i n g t h e i o n sour c e r a i s e d t h e s e n s i t i v -i t y o f t h e a n a l y s i s , b u t i n our e x p e r i e n c e , s t a n d a r d c o n d i t i o n s f o r c l e a n up o f the i o n source and c h a n g i n g f i l a m e n t s were d i f f i c u l t t o o b t a i n . Scan mode o p e r a t i o n d u r i n g a s e r i e s o f SIM a n a l y s e s 'should be a v o i d e d i n o r d e r t o i n c r e a s e s e n s i -t i v i t y . S e l e c t i o n o f column p a c k i n g m a t e r i a l s was a l s o c o n s i d e r -ed. I t was found t h a t m/e 209 from t h e b l e e d o f an OV-17 column s e r i o u s l y i n t e r f e r e d i n t h e EMDP a n a l y s i s where m/e 208 was m o n i t o r e d . T h e r e f o r e , a w e l l c o n d i t i o n e d column was o n l y used f o r t h e a n a l y s i s o f EMDP. The s t r u c t u r e of OV-17 i s shown w i t h p o s s i b l e f r a g m e n t a t i o n i o n s . When m/e 72 was 58 m o n i t o r e d f o r methadone a n a l y s i s , i n t e r f e r e n c e from s i l a n i z i n g agent w h i c h c o n t a i n s the m/e 73 i o n was m i n i m i z e d by e x t e n s i v e c o n d i t i o n i n g o f t h e column. Any s o l v e n t used i n t h e e x t r a c -t i o n and which remained i n the sample a l s o c o n t r i b u t e d t o the background. Methylene c h l o r i d e was c o m p l e t e l y e v a p o r a t e d when m/e 72 was b e i n g m o n i t o r e d f o r methadone a n a l y s i s . One o f t h e advantages o f t h e use o f l a b e l e d i n t e r n a l s t a n d a r d s w i t h t h e SIM system was r e p o r t e d t o be a c a r r i e r e f f e c t o f t h e l a b e l e d compound which c o u l d reduce column a d s o r p -t i o n and d e c o m p o s i t i o n o f the u n l a b e l e d compound t o be d e t e r -mined. D i p h e n o x y l a t e (88) i s such a c a s e . C o n t r a r y t o t h i s f i n d i n g , a l a c k o f a c a r r i e r e f f e c t was o b s e r v e d i n t h e case 2 of octopamine- (89). G e n e r a l l y i m p u r i t i e s due t o i n c o m p l e t e l a b e l l i n g l i m i t s t h e use o f a l a r g e amount of i n t e r n a l s t a n d a r d t o a c t as c a r r i e r . T h i s was c o n s i d e r e d t o be t r u e i n case of t h e [\o] l a b e l e d compounds. Moreover, t h e h i g h c o n c e n t r a -t i o n s r e q u i r e d f o r t h e a n a l y s i s o f methadone u s i n g methadone-2 H^Q as i n t e r n a l s t a n d a r d p r e v e n t e d any attempt t o s t u d y a c a r r i e r e f f e c t . A n a l y s i s o f methadone i n plasma and s a l i v a Methadone l e v e l s i n plasma and s a l i v a samples o f methadone maintenance p a t i e n t s were f r e q u e n t l y found t o be t o o low t o use t h e s e l e c t i v e i o n a t m/e 22 3 f o r m o n i t o r i n g s i n c e w i t h E I t h e r e l a t i v e abundance of t h i s i o n i s o n l y 1.6%. In o r d e r t o enhance t h e s e n s i t i v i t y o f t h e method th e l e s s s e l e c t i v e b u t s t r o n g peak a t m/e 72 was chosen f o r m o n i t o r i n g . 59 As shown i n F i g . 7, SIM a t m/e 72 showed h i g h s e l e c t i v i t y f o r methadone w i t h 2-dimethylamino-4,4-diphenyl-5-nonanone p e r c h l o r a t e as the i n t e r n a l s t a n d a r d , t h e base peak o f wh i c h i s a l s o m/e 72. The lower l i m i t o f r e p r o d u c i b l e q u a n t i t a t i o n o f methadone i n 0.5 ml o f plasma o r s a l i v a t a k e n f o r e x t r a c t i o n was 20 ng. The s e n s i t i v i t y o b t a i n e d by t h i s method i s comparable t o the methods w h i c h are r o u t i n e l y used t o a n a l y z e methadone i n human plasma. The d i s a p p o i n t i n g s e n s i t i v i t y o b s e r v e d i s c o n t r a r y t o e x p e c t a t i o n s o f u s i n g t h e base peak a t m/e 72 f o r m o n i t o r i n g and i s due t o i n s t r u m e n t a l c o n d i t i o n s , e s p e c i a l l y i o n s ource c o n d i t i o n s , v a r i a t i o n s o f w h i c h were found t o s e r i -o u s l y l i m i t s e n s i t i v i t y . S p e c i a l c a r e was t h e r e f o r e t a k e n t o reduce background. The t e m p e r a t u r e s o f s e p a r a t o r , i n l e t l i n e , and i o n sou r c e were e l e v a t e d o v e r n i g h t t o remove r e t a i n e d i m p u r i t i e s b e f o r e f r e s h samples were a n a l y z e d . I n t h i s way the method was found t o be more t h a n adequate t o a n a l y z e p a t i e n t s a l i v a o r plasma samples, t h e c o n c e n t r a t i o n s o f wh i c h were found t o v a r y o v e r a wide range between 0.0 5-1.0 ug/ml d u r i n g t h e 24 hour p e r i o d o f a s t u d y . M o n i t o r i n g d rug c o n c e n t r a t i o n s i n s a l i v a might b e t t e r r e f l e c t t h e time c o u r s e o f a drug a t the r e c e p t o r s i t e . T h i s was found t o be t h e case f o r t h e drug p r o c a i n a m i d e f o r wh i c h a p a r a l l e l r e l a t i o n s h i p between time c o u r s e o f t h e drug i n s a l i v a and c a r d i a c a c t i o n o f t h e d r u g was o b s e r v e d (90). T h i s s h o u l d e s p e c i a l l y be t r u e f o r drugs w h i c h a c t on t h e cen-t r a l nervous system. F o r drugs such as methadone wh i c h a r e 60 INTERNAL STANDARD 200 220 TEMPERATURE C 240 260 F i g . 7. SIM chromatogram (m/e 72) o f methadone from s a l i v a F i g . 8. Methadone c o n c e n t r a t i o n i n plasma and s a l i v a o f a maintenance p a t i e n t (90 mg/day dosage) 61 e x t e n s i v e l y bound t o plasma p r o t e i n , m o n i t o r i n g drug c o n c e n t r a -t i o n s i n s a l i v a c o u l d b e t t e r d e f i n e t h e a c t i v i t y o f a drug because t h e c o n c e n t r a t i o n o f a drug i n s a l i v a r e f l e c t s t h e unbound f r a c t i o n o f t h e drug which c r o s s e s the b l o o d b r a i n b a r r i e r . I t appeared t h e r e f o r e u s e f u l t o i n i t i a t e m o n i t o r i n g o f s a l i v a l e v e l s o f t h e drug i n s t e a d y s t a t e maintenance p a t i e n t s e s p e c i a l l y i n v i e w o f t h e f a c t t h a t a l a c k i n c o r r e l a t i o n between methadone c o n c e n t r a t i o n s i n plasma and symptom complaints o f p a t i e n t s have been r e p o r t e d (41). The r e s u l t s f o r p a t i e n t s A and B were not o b t a i n e d because problems w i t h p a t i e n t s ' c o m p l i a n c e t o t h e p r o t o c o l were e n c o u n t e r e d . The mean r a t i o o f s a l i v a t o plasma of p a t i e n t C was 0.55 ± 0.15 (SD) w i t h a range o f 0.40-0.79. P a t i e n t D showed a mean r a t i o o f s a l i v a t o plasma o f 0.48 ± 0.10 (SD) w i t h a range o f 0.30-0.58. S a l i v a r y pH c o u l d p o s s i b l y a c c o u n t f o r the i n t r a i n d i v i d u a l v a r i a t i o n i n t h e r e s u l t s b u t pH v a l u e s were not a v a i l a b l e . P a t i e n t C had d i f f i c u l t y i n p r o v i d i n g s a l i v a samples and chewing gum (Dentyne, Adams Brands Inc.) was used t o s t i m u l a t e s a l i v a p r o d u c t i o n . I n t e r f e r e n c e from t h e chewing gum i n t h e a n a l y s i s was not o b s e r v e d . A d s o r p -t i o n o f methadone t o the gum was n o t p r o v e n b u t was c o n s i d e r e d t o be m i n i m a l . I f s a l i v a r y c o n c e n t r a t i o n s o f methadone i n th e s e two p a t i e n t s a r e a r e f l e c t i o n o f methadone i n plasma t h e n our r e s u l t s agree w e l l w i t h t h o s e r e p o r t e d by Horns e t a l . ( 4 1 ) , i n which 50% b i n d i n g o f methadone t o plasma was r e p o r t e d . T h i s c o n t r a s t s w i t h t h e r e s u l t s r e p o r t e d by Lynn e t a l . (91) where s a l i v a r y c o n c e n t r a t i o n s were found t o be 62 much h i g h e r t h a n t h o s e o b t a i n e d i n whole b l o o d . The r e s u l t however was not adequate t o draw p h a r m a c o k i n e t i c c o n c l u s i o n s because of t h e l i m i t e d sample s i z e and t h e number of samples. A n a l y s i s o f methadone and m e t a b o l i t e s i n u r i n e The a n a l y t i c a l c o n d i t i o n s were t h e same as t h o s e used f o r t h e a n a l y s i s o f methadone i n plasma o r s a l i v a samples. R e t e n t i o n t i m e s by GCMS (SIM) o f methadone and m e t a b o l i t e s a r e 3.24 (EMDP), 4.0 (EDDP), 4.78 (methadone), and 6.23 min. (DDP). I f we c o n s i d e r the f a c t t h a t many d i f f e r e n t samples are d e a l t w i t h when we a n a l y z e methadone and m e t a b o l i t e s i n u r i n e s , SIM might be a time consuming method compared w i t h gas c h r o m a t o g r a p h i c methods. F o r example, a s e p a r a t e i n j e c t i o n o f t h e sample was r e q u i r e d t o a n a l y z e each m e t a b o l i t e i n SIM a n a l y s i s . On the o t h e r hand, methadone and m e t a b o l i t e s were a n a l y z e d by one i n j e c t i o n o f the sample u s i n g one i n t e r n a l s t a n d a r d w i t h GC. SIM w i t h d e u t e r i u m l a b e l e d i n t e r n a l s t a n d a r d s however p r o v i d e s ease i n work up p r o c e d u r e s . Sample d i l u t i o n s and the s i z e o f t h e i n j e c t i o n . volume d i d not a f f e c t t h e ob s e r v e d i o n r a t i o s w hich were used t o c a l c u l a t e the amount of metha-done (Table I I ) . E x t r a c t i b i l i t y o f l a b e l e d and u n l a b e l e d compounds was found t o be the same i n C I ^ C ^ s o l v e n t . A t pH 7.5 (n=4, 2.5 ug each i n 1.0 m l ) , p e r c e n t r e c o v e r y o f methadone, m e t h a d o n e - 2 H 1 Q , EDDP and EDDP- 2H 3 was 84.9 ± 2.6, 83.6 ± 0.2, 2 94.2 + 3.8 and 93.9 ± 1.1, r e s p e c t i v e l y . EDDP and EDDP-63 were a n a l y z e d i n the r e c o v e r y s t u d i e s u s i n g EDDP- E^ as the 2 i n t e r n a l s t a n d a r d ; methadone and methadone- H^Q u s i n g methadone-2 as i i n t e r n a l s t a n d a r d . TABLE I I . E f f e c t o f D i l u t i o n and I n j e c t i o n Volumes Upon the Observed Ion R a t i o s Methadone/ml MeOH I n j e c t i o n Volume (n=6) 1 y l 5 y l 75 yg 2 H Q , 50 yg 2 H 1 Q 1.91 ± 0.14 2.03 ± 0. 06 15 2 2 yg HQ, 10 yg H 1 Q 1.99 ± 0. 08 I n j e c t i o n was c a r r i e d o u t by m o n i t o r i n g m/e 223 and m/e 233. The numbers denote m/e 223/m/e 233 (SD). Crude windows were s e t w i t h PFK by m o n i t o r i n g m/e 231 wh i c h was l a t e r e r a s e d . M a g n e t i c H a l l s e t t i n g was m/e 223. The c a l i b r a t i o n e q u a t i o n s p r e p a r e d f o r methadone and EDDP are shown i n Ta b l e I I I . The r e s u l t s i n d i c a t e t h a t c a l i b r a -t i o n e q u a t i o n s can be e x p r e s s e d by u s i n g o n l y s l o p e v a l u e s because i n t e r c e p t v a l u e s were found t o be not s i g n i f i c a n t . I f we a l s o know t h e r a t i o o f u n l a b e l e d t o l a b e l e d compound q u a n t i t a t i o n o f t h e drug m o n i t o r e d i s a c h i e v e d by m u l t i p l y i n g t h e s t a n d a r d r a t i o x r e c i p r o c a l v a l u e o f t h e amount o f added i n t e r n a l s t a n d a r d x t h e ob s e r v e d r a t i o . Slope v a l u e s o f 64 TABLE I I I . C a l i b r a t i o n E q u a t i o n s f o r Methadone and EDDP f o r U r i n e A n a l y s i s . Methadone EDDP Slope 0 . 326 0.483 Slope S t a n d a r d E r r o r 0.0574 0.0033 I n t e r c e p t s 0.0513 0.0054 I n t e r c e p t S t a n d a r d E r r o r 0.0503 0.0117 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) 0.9957 0.9990 St a n d a r d R a t i o ( S . D . ) b (n=6) 1.305 ± 0.1272 0. 968 ± 0.0484 Slope c a l c u l a t e d from S t a n d a r d R a t i o 0 0. 326 0.484 a. Sl o p e and i n t e r c e p t v a l u e s a r e c a l c u l a t e d by means o f com-p u t e r program ( T r i a n g u l a r R e g r e s s i o n Package , Computing C e n t r e , The U n i v e r s i t y o f B r i t i s h ' C o l u m b i a ) . . Methadone (n=7), EDDP (n=10) . R a t i o ( D r u g / I n t e r n a l Standard) = Slope x Drug c o n c e n t r a t i o n + i n t e r c e p t 2 2 I n t e r n a l S t a n d a r d s a re Methadone- H^Q (4 jag) and EDDP- H-^ Q (2 u g ). b. R a t i o s o f e q u a l amount o f Drug t o I n t e r n a l S t a n d a r d (Standard R a t i o ) . c S t a n d a r d R a t i o x 1 / I n t e r n a l S t a n d a r d . r65 c a l i b r a t i o n c u r v e s and the s l o p e s c a l c u l a t e d from s t a n d a r d r a t i o were found t o be the same. The c o n c e n t r a t i o n of EDDP was s i m p l y c a l c u l a t e d by m u l t i p l y i n g the r a t i o o f m/e 277 2 t o m/e 280 w i t h t h e c o n c e n t r a t i o n o f EDDP- added as t h e i n t e r n a l s t a n d a r d . The c a l i b r a t i o n c u r v e method was however used f o r t h e a n a l y s i s f o r methadone because of i n s t a b i l i t y o f t h e measured r a t i o which o c c u r r e d when i o n s o f 10 mass u n i t s d i f f e r e n c e o r more were m o n i t o r e d . The r a t i o was found t o be changeable depending upon SIM c o n d i t i o n s and i n p a r t i c -u l a r t o t h e s t a b i l i t y of t h e magnetic f i e l d . The c o e f f i c i e n t o f v a r i a t i o n o f the measured methadone s t a n d a r d r a t i o was t w i c e (9.7%) t h a t o f EDDP ( 5 . 2 % ) . SIM a n a l y s i s o f methadone and EDDP i n u r i n e samples was compared w i t h GC a n a l y s i s . The two methods were found t o be w e l l c o r r e l a t e d . Methadone (0.4 ug-6.8 ug/ml) was a n a l -y z e d f o r p a t i e n t B and C, EDDP (8 ug-32 ug/ml) f o r A and D. 2 The c o r r e l a t i o n 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 ) o f t h e a n a l y s i s of methadone (n=14) and EDDP (n=14) by SIM compared t o GC were 0.963 and 0.962, r e s p e c t i v e l y . P r e v i o u s e x p e r i m e n t s w i t h t h e GC a n a l y s i s o f EMDP i n t h e u r i n e o f maintenance p a t i e n t s i n d i c a t e d t h a t the EMDP peak i s f r e q u e n t l y o v e r l a p p e d e i t h e r w i t h c a f f e i n e o r w i t h h y d r o x y c o t i n i n e , a m e t a b o l i t e o f n i c o t i n e (33). When an att e m p t was made t o a n a l y z e maintenance p a t i e n t u r i n e s f o r EMDP by m o n i t o r i n g m/e 208 f o r EMDP and m/e 218 f o r the i n t e r -2 n a l s t a n d a r d , EMDP- H 1 n , i t was found t h a t the amount of EMDP 66 appears to.be l e s s than 100 ng/ml. Because o f the s m a l l amount o f t h i s m e t a b o l i t e , d e t e r m i n a t i o n o f EMDP was not neces-s a r y t o s t u d y the d e m e t h y l a t i o n mechanisms o f methadone. S t a b i l i t y o f EDDP In an attempt t o o b t a i n i n f o r m a t i o n on the minor meta-b o l i c pathways of methadone, t h e l e v e l of DDP i n u r i n e was de t e r m i n e d by m o n i t o r i n g m/e 265 f o r DDP and m/e 275 f o r DDP-2 H-^ as i n t e r n a l s t a n d a r d . When t h e pH o f t h e u r i n e was a d j u s t e d t o 10.4 f o r e x t r a c t i o n , i n c o n s i s t e n c y i n the r e p r o d u c -i b i l i t y o f t h e c o n c e n t r a t i o n o f DDP i n a u r i n e sample was found. To i n v e s t i g a t e t h i s , EDDP p e r c h l o r a t e s o l u t i o n was made a l k a l i n e and l e f t t o s t a n d a t room t e m p e r a t u r e . A TIC p r o f i l e o f t h e methylene c h l o r i d e e x t r a c t o f t h e s o l u t i o n showed DDP as a s i g n i f i c a n t p r o d u c t of EDDP d e c o m p o s i t i o n . DDP was i d e n t i f i e d by TLC w i t h an R f 0.8 on S i l i c a G e l F 254 (Brinkman) s h e e t s w i t h EtOH, HAC, E^O (6:3:1) as s o l v e n t . F u r t h e r s t u d i e s r e v e a l e d t h a t EDDP f r e e base i n e t h e r s o l u t i o n i s e a s i l y decomposed t o DDP. S t o i c h i o m e t r i c c o n v e r s i o n o f EDDP t o DDP was o b s e r v e d when EDDP and DDP i n e t h e r s o l u t i o n were measured u s i n g l a b e l e d compounds as i n t e r n a l s t a n d a r d s ( F i g . 9 ) . A s i m i l a r r e s u l t f o r t h e c o n v e r s i o n of EDDP t o DDP by ap p a r e n t o x i d a t i o n r e a c t i o n s was s e p a r a t e l y r e p o r t e d by Bowen e t a l . ( 9 2 ) . A 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 methadone t o 2, 2 - d i p h e n y l - 4 - d i m e t h y l a m i n o p e n t a n o i c a c i d (5_) and f u r t h e r m e t a b o l i s m t o DDP was suggested ( 6 ) . 2, 2 - D i p h e n y l - 4 - d i m e t h y l -a m i n o p e n t a n o i c a c i d was n o t d e t e c t e d i n any o f the u r i n e s o f 6 7 F i g . 9. D e c o m p o s i t i o n o f EDDP base t o DDP ( EDDP, — DDP) -68 maintenance p a t i e n t s u s i n g mass chromatograms i n which m/e 167 (C,Hr-)„C+H f o r the a c i d and m/e 225 ( C C H C )„ C +COOCH Q f o r b D Z b D Z J t h e d i a z o m e t h a n e - t r e a t e d a c i d were m o n i t o r e d . The r e s u l t s s t r o n g l y s u ggest t h a t DDP i s a metabonate and t h a t extreme pH a d j u s t m e n t s s h o u l d be a v o i d e d when b i o l o g i c a l samples a r e e x t r a c t e d t o d e t e c t new m e t a b o l i t e s . 69 3. D e t e c t i o n o f methadone m e t a b o l i t e s E x t r a c t i o n p r o c e d u r e s f o r r a t b i l e The p o l a r s o l v e n t (methylene c h l o r i d e ) was used f o r e x t r a c t i o n o f r a t b i l e samples. Clean-up p r o c e d u r e s u s i n g column chromatography such as A m b e r l i t e XAD-2 and back e x t r a c -t i o n .were n ot used. The sample was e x t r a c t e d and any p r e c i p i -t a t e was removed by c e n t r i f u g a t i o n , f o l l o w e d by a d i r e c t i n -j e c t i o n o f the sample e x t r a c t i n t o t he GCMS. A f t e r i n i t i a l e v i d e n c e o f the pr e s e n c e o f m e t a b o l i t e s was found, the sample was back e x t r a c t e d t o see any d i f f e r e n c e between b e f o r e and a f t e r t h e c l e a n up p r o c e d u r e and t o see any compounds which were t r a n s f e r r e d t o the hexane l a y e r used i n the back e x t r a c -t i o n ( F i g . 10) . A r a d i o i s o t o p e l a b e l e d compound and XAD-2 column chromatography f o r sample e x t r a c t i o n was d e s c r i b e d i n the d e t e c t i o n o f methadone m e t a b o l i t e s from r a t u r i n e and b r a i n (93). An XAD-2 column was a l s o used t o d e t e c t methadone meta-b o l i t e s from man and r a t u r i n e by S u l l i v a n e t a l . ( 4 ) . In t h i s e x p e r i m e n t , use of column 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 was n o t c o n s i d e r e d a p p r o p r i a t e t o d e t e c t minor m e t a b o l i t e s because r e c o v e r y of the me t a b o l i t e s " ; c o u l d n o t be m o n i t o r e d . 70 B i l e H 20 C e n t r i f u g e pH 9.0 E x t r a c t i o n w i t h C H 2 C 1 2 ^ N o n c o n j u g a t e f r a c t i o n I Back e x t r a c t i o n , D e r i v a -t i z a t i o n . F r e e z e d r y Residue /Add MeOH, C e n t r i f u g e , \ \Evaporate ) B u f f e r , pH 5.0, 3 - g l u c u r o n i d a s e , 37°C pH 8.0-8.5 E x t r a c t w i t h C H 2 C 1 2 Conjugate f r a c t i o n D e r i v a t i z e : " w i t h CH'2N2 Back e x t r a c t i o n F i g . 10. E x t r a c t i o n P r o c e d u r e f o r Rat B i l e . 71 The r a t b i l e e x t r a c t i o n method i s s i m i l a r t o t h a t used f o r d e t e c t i o n o f methadone m e t a b o l i t e s from human u r i n e s by S u l l i v a n e t a l . ( 6 ) . D i r e c t e x t r a c t i o n , d e r i v a t i z a t i o n , and GCMS a n a l y s i s were the methods used. When r a t b i l e sam-p l e s were a n a l y z e d , the b i l e s a l t s were removed by a c i d p r e -c i p i t a t i o n (94). In our e x p e r i m e n t s , the amount o f b i l e s a l t and p r o t e i n was m i n i m i z e d by c e n t r i f u g a t i o n and s o l v e n t (MeOH) p r e c i p i t a t i o n . Two s e p a r a t e e x p e r i m e n t s w i t h and w i t h o u t a c i d p r e c i p i t a t i o n gave t h e same r e s u l t s . B e c k e t t e t a l . (2) c a r r i e d o u t the d e t e c t i o n o f metha-done m e t a b o l i t e s from human u r i n e by u s i n g normal s o l v e n t e x t r a c t i o n and TLC s e p a r a t i o n . When a u t h e n t i c m e t a b o l i t e samples a r e not a v a i l a b l e , d e t e c t i o n o f minor m e t a b o l i t e s among l a r g e amount o f endogenous m a t e r i a l by TLC i s alm o s t i m p o s s i b l e . The use o f c o l o r d e v e l o p i n g methods t o d e t e c t s p e c i f i c m e t a b o l i t e s has- been atte m p t e d f o r methadone meta-b o l i t e s (95) and f o r N - o x i d a t i o n m e t a b o l i t e s (96). The ap-pro a c h however d i d n ot guarantee d i r e c t i d e n t i f i c a t i o n o f the s t r u c t u r e o f m e t a b o l i t e s . T h e r e f o r e , TLC and c o l o r spray methods were n ot used i n our e x p e r i m e n t s . The GCMS computer t e c h n i q u e s a r e a p o w e r f u l t o o l t o i d e n t i f y t he m o l e c u l a r s t r u c t u r e o f compounds i n m i x t u r e s . T h i s t e c h n i q u e i s f u r t h e r a i d e d by t h e use o f s t a b l e i s o t o p e l a b e l e d compounds i n the m e t a b o l i c s t u d i e s . The t w i n i o n t e c h n i q u e ( i o n d o u b l e t t e c h n i q u e ) has been 'used i n the d e t e c t i o n o f m e t a b o l i t e s f o r v a r i o u s d r u g s . The H 1 0 - l a b e l e d compounds s y n t h e s i z e d f o r t h e metha-done work d i s p l a y a d i f f e r e n c e i n p h y s i c a l p r o p e r t i e s from the u n l a b e l e d compounds. The d i f f e r e n c e i n t h e r e t e n t i o n time o b s e r v e d by GC i s presumably due t o a d e c r e a s e i n p o l a r i t y o f t h e d e u t e r a t e d m a t e r i a l . A s i m i l a r d i f f e r e n c e o f r e t e n t i o n "2 t i m e s f o r u n l a b e l e d and H 1 6 b u t y l a t e d h y d r o x y t o l u e n e was ob s e r v e d when a 3% 0 ^ 2 2 5 GC column was used (97) . The d e u t e -r i u m l a b e l e d methadone and m e t a b o l i t e s were found t o have s l i g h t l y s h o r t e r r e t e n t i o n t i m e s t h a n the u n l a b e l e d compounds. T h e r e f o r e , t w i n i o n s were not r e a d i l y o b s e r v e d i n the GCMS scan d a t a o f e x t r a c t s c o n t a i n i n g l a b e l e d and u n l a b e l e d methadone and/or m e t a b o l i t e s . Comparison o f t h e mass s p e c t r a of GC peaks r e s u l t i n g from u n l a b e l e d compounds w i t h t h o s e from l a -b e l e d compounds r u n as s e p a r a t e e x p e r i m e n t s gave i m p o r t a n t e v i d e n c e f o r the f o r m a t i o n o f new m e t a b o l i t e s . Nonconjugated f r a c t i o n The non c o n j u g a t e d f r a c t i o n i s o l a t e d from r a t b i l e c o n t a i n e d m a i n l y EDDP and a s m a l l amount of DDP. Trace amounts of methadone and EMDP were a l s o found. Kreek e t a l . (98) adso r e p o r t e d the same r e s u l t w i t h t r a c e amounts o f methadone and EMDP found i n human b i l e . The presence o f DDP was a c c o u n t -ed f o r by d e c o m p o s i t i o n o f EDDP. The nonc o n j u g a t e d f r a c t i o n s o f r a t b i l e and human u r i n e were d r i e d i n a vacuum d e s i c c a t o r . S i l a n i z a t i o n o f t h e 73 samples (150 u l TMCS, 50 u l p y r i d i n e , 70°C f o r one hour) f o l l o w e d by GCMS d i d not i n d i c a t e t h e presence o f any d i h y -d r o d i o l . C o n j ugated f r a c t i o n F i g . 11 shows the TIC p r o f i l e o f the e x t r a c t o f the c o n j u g a t e d f r a c t i o n i s o l a t e d from r a t b i l e . T a b l e IV i s a summary o f t h e major fragments and i o n abundance v a l u e s f o r endogenous b i l e components and r e c o v e r e d m e t a b o l i t e s . The pres e n c e o f monohydroxy EMDP, d i h y d r o x y EMDP, and hydroxy EDDP was e v i d e n t from mass f r a g m e n t a t i o n s t u d i e s o f the GC CH30. CH,0 c \ CH, CH2CH3 N / - CH CH, 44 CH30 I J CH,CH, ,C 0 V - f ^ / CH, 47 1 m/e 238 H CH-'CH. 0 m/e 247 V 45 m/e 207 H CH-CH3 U 46 m/e 216 HQ m/e 237 H CH-CH3 U 48 m/e 245 H„ ~~]t m/e 268 H^ CH-CH3' U 49 m/e 276 HQ o Scan numbers F i g . 11, ( C H - J O J E M D P M e t a b o l i t e (base peak, m/e 72) 50 100 150 200 250 300 350 400 450 TIC p r o f i l e o f diazomethane t r e a t e d c o n j u g a t e f r a c t i o n ; f r o m b i l e o f methadone : dosed r a t s . (GCMS: 150-280 °C, 6°/min,hold at.280°C) TABLE IV. Mass S p e c t r a l Data o f TIC O b t a i n e d from Diazomethane T r e a t e d c o n j u g a t e f r a c t i o n from b i l e o f methadone dosed r a t s Peak No. Rt (Min) M/e ( R e l a t i v e I n t e n s i t y )  1 11 .2 87 (50) , 75 (19) , 74(100), 5 5 ( 2 9 ) , 43(36) / 41(33) 2 12 .8 73 (87) , 60 (83) , 57(8 0 ) , 5 5 ( 6 7 ) , 43 (100) , 41(73) 3 16 .0 109 (25) , 95 (56) , 80(8 5 ) , 6 7 ( 1 0 0 ) , 55 (87) , 41(90) 4 19 .7 93 (49) , 91(63) , 79(100), 6 7 ( 7 7 ) , 55(43) , 41(70) 5 24 .5 93 (44) , 91(7 5 ) , 79(100), 6 7 ( 7 0 ) , 55(39) , 41(87) 6 27 .6 270 (16) , 149 (10) , 134(100), 1 2 1 ( 1 1 ) , 119 (14), 91(4) 7 34 .7 326 (5) , 205 (4) , 15 9 ( 5 ) , 147 ( 9 ) , 122 (100) , 91(15) 8 37 .5 107 (43) , 95(4 7 ) , 81 (57), 57 (63), 55 (80), 43 (100) CH3OEMDP 21.5 CH_OEMDP-2H. 2 3 CH OEDDP- H g 24.1 238(100), 223(37), 207(48), 130(39) 247(100), 232 (42), 217(61), 165(23) 316(63), 247(100), 232(59), 216 (67) 11 5 ( 3 5 ) , 91 (25) 135 ( 4 6 ) , 96(18) 20 3 ( 3 1 ) , 135(55) (CH-,0) EMDP 25.3 2 (CH 30) 2EMDP- H g M e t a b o l i t e M e t a b o l i t e - ^ H 28.2 10 268(58), 253(28), 237(100), 129(38) 276(58), 261(36), 245(100), 230(10) 11 5 ( 2 8 ) , 91(21) 1 3 3 ( 2 2 ) , 83(18) 253(42), 207(68), 129(33), 72(100), 5 7 ( 2 0 ) , 44(36) 263 (23), 217(46), 134 (13), 72(100), 5 7 ( 2 6 ) , 44 (40) 76 peaks r e s u l t i n g from the m e t a b o l i s m o f u n l a b e l e d and l a b e l e d methadone (44-49). Ions m/e 238 and m/e 24 7 were s e l e c t e d t o 2 m o n i t o r d e r i v a t i z e d HOEMDP and HOEMDP- H g. Ions m/e 2 37 and m/e 245 were used t o m o n i t o r d e r i v a t i z e d DiHOEMDP and . : : /'M 1-2 DiHOEMDP- Hg. SIM a n a l y s i s o f c o n j u g a t e d m e t a b o l i t e s u s i n g t h e s e i o n s was d e s c r i b e d i n the study o f methadone-diazepam i n t e r a c t i o n . I n a d d i t i o n t o r i n g h y d r o x y l a t e d m e t a b o l i t e s o f metha-done, the c o n j u g a t e d f r a c t i o n from r a t b i l e c o n t a i n e d a peak h a v i n g an abundant m/e 72 i o n ( F i g . 11, Ta b l e I V ) . Diazomethane t r e a t m e n t d i d not s h i f t t he GC peak p o s i t i o n , i m p l y i n g t h a t the compound does n o t c o n t a i n a p h e n y l - h y d r o x y 1 group. The c o n j u -g a t e d f r a c t i o n was made a c i d i c (pH 2.0) and e x t r a c t e d w i t h hexane. The compound was t r a n s f e r r e d t o the hexane l a y e r , i m p l y i n g low water s o l u b i l i t y and the absence o f a b a s i c n i t r o -gen i n the m o l e c u l e . The mass spectrum o f t h i s GC peak i s shown i n F i g u r e 12. When the ex p e r i m e n t f o r the d e t e c t i o n o f m e t a b o l i t e s 2 was r e p e a t e d w i t h methadone- H^Q, d e u t e r a t e d hydroxy EMDP, d i h y d r o x y EMDP, and hydroxy EDDP were o b s e r v e d and the m/e 72 c o n t a i n i n g GC peak (Rt 28.2 min.) was found t o r e t a i n d e u t e r i u m on the benzene r i n g . The r e t e n t i o n t ime o f the com-ound i s shown i n Ta b l e IV w i t h comparison t o the o t h e r m e t a b o l i t e s . The peak w i t h the abundant m/e 72 i o n was not o b s e r v e d when EMDP and EDDP were dosed t o r a t s . U r i n e samples from methadone maintenance p a t i e n t s d i d n o t i n d i c a t e the pr e s e n c e o f the m/e 72 compound. Nonconjugate f r a c t i o n from r a t b i l e was 72 207 44 UL 129 57 J 253 309(M +) J m/e F i g . 12. Mass spectrum of m/e 72 c o n t a i n i n g m e t a b o l i t e 78 s u b j e c t e d t o the same work-up p r o c e d u r e s as thos e f o r the c o n j u -g a t e d m e t a b o l i t e s . T h i s p r o c e d u r e was t o examine the p o s s i b i l -i t y t h a t methadone N-oxide o r o t h e r m e t a b o l i t e s w h i c h were n ot e x t r a c t e d from the b i l e i n the f i r s t e x t r a c t i o n s t e p might be the source o f the m/e 72 c o n t a i n i n g peak. The f i n a l e x t r a c t s d i d n o t g i v e the m/e 72 c o n t a i n i n g peak. R i n g h y d r o x y l a t i o n pathways o f methadone m e t a b o l i s m The p r e s e n c e o f monohydroxy EMDP and d i h y d r o x y EMDP r e s u l t i n g from methadone m e t a b o l i s m was p r e v i o u s l y r e p o r t e d by S u l l i v a n e t a l . ( 4 ) , Lynn e t a l . ( 7 ) , and Angg&rd e t a l . ( 8 ) . R o e r i g e t a l . (38) c a l l e d the compounds "water s o l u b l e m e t a b o l i t e s " w i t h o u t d i f f e r e n t i a t i n g between the mono- and dihydroxy-compounds. The peak h e i g h t o f d i h y d r o x y EMDP i n two e x p e r i m e n t s was found t o be 1/3 t h a t o f monohydroxy EMDP. When EDDP was a d m i n i s t e r e d t o a r a t (20 mg/Kg), c o n j u g a t e d m e t a b o l i t e s were e x c r e t e d i n the b i l e i n l e s s e r amounts than when a d m i n i s t e r i n g methadone. The r e s u l t i s s i m i l a r t o the r e p o r t o f R o e r i g e t a l . (99) and p o s s i b l e r e a s o n s c o u l d be sought i n the d i f f e r e n t m e t a b o l i s m o r e x c r e t i o n mechanism o f methadone compared!>to EDDP. An i s o t o p e e f f e c t i n the m e t a b o l i c h y d r o x y l a t i o n o f l a b e l e d methadone was not s t u d i e d m a i n l y because methods t o c a l c u l a t e t h e s m a l l i s o t o p e e f f e c t were not a v a i l a b l e . The 79 mechanism o f f o r m a t i o n o f t h e r i n g h y d r o x y l m e t a b o l i t e s o f methadone i . e . whether an ep o x i d e i n t e r m e d i a t e (5JD) i s i n v o l v e d (3->-50-»-10 o r 1A) , or d i r e c t i n s e r t i o n (3->-10->-ll) i s t h e o x i d i z i n g mechanism was t h e r e f o r e n o t s t u d i e d . The f a c t t h a t t h e ' d i h y d r o -d i o l (5_1) was not d e t e c t e d does not n e c e s s a r i l y i m p l y t h a t the e p o x i d e i n t e r m e d i a t e (5_0) i s not i n v o l v e d i n the me t a b o l i s m of methadone. I f e p o x i d e i n t e r m e d i a t e f o r m a t i o n i s a mechanism 80 t h e r e i s a p o s s i b i l i t y f o r the f o r m a t i o n o f a r e a c t i v e t o x i c i n t e r m e d i a t e as d e s c r i b e d f o r a n t i c o n v u l s a n t s (100). An attempt was made t o stu d y s t e r e o c h e m i c a l a s p e c t s o f the r i n g h y d r o x y l a t e d m e t a b o l i t e s s i n c e the c o n f i g u r a t i o n o f h y d r o x y - m e t a b o l i t e s has not been examined. I t appeared u s e f u l t o f i n d a s t e r e o s p e c i f i c f r a g m e n t a t i o n p r o c e s s (101) f o r methoxy EDDP. The major -fragments t o observe were t h e p h e n y l r i n g r e a r r a n g e d fragments analogous t o m/e 105 (37) o f EDDP. Mass s p e c t r a d i d not show any m/e 135 (CH 3OC 6H 4CHCH 3"11) o r m/e 105, i n d i c a t i n g t h a t p h e n y l r i n g m i g r a t i o n i s not a f a v o r a b l e f r a g m e n t a t i o n p r o c e s s f o r methoxy EDDP. S t e r e o s e l e c t i v e h y d r o x y l a t i o n and c o n j u g a t i o n have been s t u d i e d f o r drugs such as d i p h e n y l h y d a n t o i n (102), m e t h y l p h e n y t o i n (103), and h e x o b a r b i t a l (104). S t e r e o -s e l e c t i v i t y i n . t h e met a b o l i s m o f EMDP t o i t s r i n g h y d r o x y l a t e d m e t a b o l i t e s i s a l s o v e r y l i k e l y b u t t h i s a s p e c t o f the work remains t o be done. EMDP was found t o be p h a r m a c o l o g i c a l l y i n a c t i v e ( 3 ) . The importance o f hydroxy m e t a b o l i t e s i n the p h a r m a c o l o g i c a l a c t i o n o f methadone appears t h e r e f o r e t o be m i n i m a l b u t may not be e x c l u d e d . The m e t a b o l i t e p_-hydroxyamphetamine f o r example t a k e s p a r t i n the nerve t e r m i n a l a c c u m u l a t i o n o f t h e amine (10 5 ) . I r r e v e r s i b l e b i n d i n g o f e s t r o g e n s and 2-hydroxy-e s t r o g e n s t o mi c r o s o m a l p r o t e i n s r e q u i r e s o x i d a t i o n t o the c a t e c h o l n u c l e u s (106), i n d i c a t i n g t h e importance- o f d i h y d r o x y 81 m o l e c u l e s i n the i n t e r a c t i o n w i t h b i o m o l e c u l e s . A semiquinone type i n t e r a c t i o n o f a d r i a m y c i n t o r a t l i v e r m i c r o s o m a l p r o t e i n s was a l s o r e p o r t e d (107). N - h y d r o x y l a t e d m e t a b o l i c pathway o f methadone The m e t a b o l i t e which was i d e n t i f i e d by GCMS i n the c o n j u g a t e d f r a c t i o n o f r a t b i l e w i t h m/e 72 f o r a base peak was t e m p o r a r i l y a s s i g n e d as N - m e t h y l e n e - l - m e t h y l - 3 , 3 - d i p h e n y l -4-oxo-hexanamine o x i d e (52) . S i n c e the p o s t u l a t e d n i t r o n e was found i n the c o n j u g a t e d f r a c t i o n , t h e r e was a s t r o n g p o s s i b i l -i t y t h a t the N-hydroxy m e t a b o l i t e (5_3) was d e t e c t e d as the methadone n i t r o n e . Methods t o i d e n t i f y N-hydroxy m e t a b o l i t e s have been e x t e n s i v e l y s t u d i e d by B e c k e t t e t a l . (108) and n o r m a l l y i n -v o l v e d e r i v a t i z a t i o n o f t h e N-hydroxy group w i t h TMCS and a n a l y s i s o f the s i l a n i z e d sample w i t h GCMS. An attempt t o 82 d e t e c t the N-hydroxy m e t a b o l i t e (53) u s i n g such methods was not s u c c e s s f u l meaning t h a t t h i s m e t a b o l i t e was absent or p r e s e n t i n c o n c e n t r a t i o n s below the l e v e l o f d e t e c t i o n . S p e c i f -i c c o l o r r e a c t i o n s f o r N-hydroxy m e t a b o l i t e s were d e s c r i b e d by C o u t t s e t a l . (96) b u t i n t h i s work were found t o be d i f f i -c u l t t o use i n t h e p r e s e n c e o f many endogenous m a t e r i a l s and w i t h o u t an a u t h e n t i c sample. t h a t secondary h y d r o x y l a m i n e s a r e o x i d i z e d i n aqueous s o l u t i o n s by a t m o s p h e r i c oxygen t o n i t r o n e s , which s u g g e s t s t h a t methadone n i t r o n e (_52) i s a decomposed or a i r o x i d i z e d p r o d u c t o f N-hydroxynormethadpne . (5_3) . The v u l n e r a b i l i t y of N-hydroxyamphe-tamine and N-hydroxypheritermine t o a i r o x i d a t i o n was s t u d i e d by L i n d e k e e t a l . (110). C o u t t s e t a l . ( I l l ) d e s c r i b e d d e t e c t i o n o f n i t r o n e m e t a b o l i t e s (5_4) as c y c l i z e d forms (5_5) and by means of mass f r a g m e n t a t i o n s t u d i e s and d i f f e r e n t TLC R f v a l u e s . A p p l i c a t i o n o f t h i s c y c l i z a t i o n method t o t e s t f o r the p r e s -ence of methadone n i t r o n e (_52) i n r a t b i l e was a l s o c o n s i d e r e d t o be d i f f i c u l t because o f t h e s m a l l q u a n t i t y o f m e t a b o l i t e p r e s e n t i n a sample. T h e r e f o r e , the t e n t a t i v e assignment o f E a r l i e r s t u d i e s by B e c k e t t e t a l . (109) d e s c r i b e d + 54 55 8 3 the new m e t a b o l i t e t o a methadone n i t r o n e s t r u c t u r e i s based on gas c h r o m a t o g r a p h i c r e t e n t i o n time d a t a and mass fragmenta-t i o n p a t t e r n s o f the compound i n GCMS s t u d i e s o f u n l a b e l e d and l a b e l e d methadone dosed r a t b i l e . A d d i t i o n a l e v i d e n c e f o r the methadone n i t r o n e s t r u c -t u r e was o b t a i n e d from c h e m i c a l o x i d a t i o n s t u d i e s o f EDDP p e r c h l o r a t e by m - c h l o r o p e r b e n z o i c a c i d . A compound was o b t a i n e d w i t h the same f r a g m e n t a t i o n p a t t e r n s and the same GC c h a r a c -t e r i s t i c s as the compound i s o l a t e d from the c o n j u g a t e d f r a c t i o n o f b i l e from methadone dosed r a t s . D e t a i l e d d e t e r m i n a t i o n o f the n i t r o n e s t r u c t u r e i s d i s c u s s e d i n the s e c t i o n d e s c r i b i n g the c h e m i c a l f o r m a t i o n o f methadone n i t r o n e . P o s s i b i l i t i e s f o r the m e t a b o l i c f o r m a t i o n  o f N-hydroxy m e t a b o l i t e s o f methadone The f o r m a t i o n o f n i t r o n e , 5_2 i s shown as one p a r t o f p o s s i b l e m e t a b o l i c r o u t e s f o r methadone ( F i g . 1 3 ) . C o n j u g a t i o n o f t h e N-hydroxy m e t a b o l i t e o f normethadone, 5_3, i s a s t r o n g p o s s i b i l i t y based on o t h e r examples r e p o r t e d f o r N-hydroxy m e t a b o l i t e s (96). Normethadone (2_) which i s formed from methadone (1) may not a l l be s p o n t a n e o u s l y c y c l i z e d t o EDDP. The c y c l i z a t i o n i s f a v o r e d a t pHs h i g h e r than the pKa o f normethadone because the p r o t o n a t e d amine does not f u n c t i o n as a n u c l e o p h i l e . A l -though the pH a t the s i t e o f e n z y m a t i c N - o x i d a t i o n i s not known, t h e r e i s a s t r o n g p o s s i b i l i t y t h a t under p h y s i o l o g i c a l c o n d i t i o n s 0 c XCHrCH-N(CHJ)2 1 I CH., -CHgCHj f^Y ^CH^CH-NCCH-j^  C-CHgCHj CH2-CH-NHCH CH, a C^-CHgCHj "CH2-CH-!jl-CH3 CHjOH 52 CHj o-65 61 C-CH2CH3 CH2-C=0 3 N C \ / CH I CH, % 8 C^-CH2CH3 -^,. , CHg-CH—N-OH " V CH3 \ CH2-CH —M=0 CH3 CH-CH, 20 ,C NH \ CH„ / •CH CH, 64 0 C-CH„CH 2W'3 <y T H^- —NH, 2 I 2 CH, \Jl /C-CH2Ch3 .'66 ^ c x CH2-CH—N05 CH, F i g . 13. P o s s i b l e m e t a b o l i c pathways f o r methadone 00, normethadone i s not a l l c y c l i z e d t o EDDP. A s i m i l a r analogy can be found i n t h e b e n z o d i a z e p i n e s K o n i s h i e t a l . (113) r e p o r t e d t h a t 5_7 formed from 56 i s not s p o n t a n e o u s l y c y c l i z e d t o 5_8 but a l s o forms 5_9 b e f o r e c y c l i z a t i o n o c c u r s . At p h y s i o l o g i c a l pH o f ^7.0 b e n z o d i a z e p i n e s (oxazepam and diazepam) f a v o r c y c l i z a t i o n o v e r h y d r o l y s i s o f t h e azomethine l i n k a g e (114). At pH v a l u e s below i t s pKa, (6,8) i s n°t s u b j e c t t o c y c l i z a t i o n . On the o t h e r hand, i n the h i g h e r pH r e g i o n , f a c i l e c y c l i z a t i o n o c c u r s . Demoxepam (115) and n i t r a z e p a m (116) a l s o form t h e same e q u i l i b r i u m w i t h t h e i r h y d r o l y z e d p r o d u c t s . T h i s means t h e r e f o r e t h a t the pH a t t h e m e t a b o l i c s i t e i s i m p o r t a n t f o r t h e r a t e o f f o r m a t i o n o f c e r t a i n m e t a b o l i t e s such as 59. 86 A n o t h e r s i m i l a r example was r e p o r t e d w i t h N - h y d r o x y l -n a p h thylamine w h i c h forms adducts w i t h DNA, one o f which has the s t r u c t u r e 69 o r 7_0 (117) . Under a c i d i c c o n d i t i o n s , 7_0 i s the major s p e c i e s and the two forms were r e p o r t e d t o be i n t e r c o n v e r t i b l e by c h a n g i n g pH c o n d i t i o n s . I n F i g . 13 a p r o p o s a l was made f o r an e q u i l i b r i u m be-tween normethadone and EDDP (_3) a t p h y s i o l o g i c a l c o n d i t i o n s . The d e m e t h y l a t e d p r o d u c t , 2_ can then be o x i d i z e d t o 5_3 whi c h i s c o n j u g a t e d b e f o r e e x c r e t i o n . F u t u r e work i s r e q u i r e d t o 87 s t u d y a p o s s i b l e e q u i l i b r i u m between N-demethylmethadone and EDDP, perhaps by u s i n g UV o r NMR and under d i f f e r e n t pH c o n d i -t i o n s . S i m i l a r work has been done by H a s s a l e t a l . (118) f o r b e n z o d i a z e p i n e d e r i v a t i v e s . P h a r m a c o l o g i c a l s i g n i f i c a n c e o f t h e d e t e c t i o n o f methadone n i t r o n e The d e t e c t i o n o f an N-hydroxy m e t a b o l i t e o f methadone and p o t e n t i a l f o r m a t i o n o f methadone n i t r o n e by a m e t a b o l i c p r o c e s s from t h e N-hydroxy m e t a b o l i t e s h o u l d be c o n s i d e r e d i n terms o f the p o s s i b l e b i n d i n g o f t h e s e m e t a b o l i t e s and any m e t a b o l i t e s w h i c h a r e f u r t h e r formed frommthe m e t a b o l i t e t o c e l l u l a r components. P h y s i c a l d a t a on t h e compound formed from EDDP o x i d a t i o n and thought t o be a methadone n i t r o n e has a h i g h p a r t i t i o n c o e f f i c i e n t f o r t h i s compounddwith h i g h s o l u b i l i t y i n hexane, a l o n g r e t e n t i o n time on an OV-17 GC column r e l a t i v e t o methadone o r any o f i t s o t h e r m e t a b o l i t e s , and a l o n g r e t e n t i o n time w i t h r e v e r s e d phase HPLC. N - d e m e t h y l a t i o n o c c u r s i n t h e b r a i n i n t h e case o f morphine (119) where N - d e m e t h y l a t i o n o f methadone and forma-t i o n o f N-hydroxynormethadone o r methadone n i t r o n e might a l s o o c c u r . N-hydroxynormethadone c o u l d be o x i d i z e d t o n i t r o n e and o t h e r m e t a b o l i t e s i n the body as are o t h e r h y d r o x y l a m i n e s (120). The formed m e t a b o l i t e s , e.g. methadone n i t r o n e may th e n b i n d t o p r o t e i n s . T h i s b i n d i n g o f the n i t r o n e t o p r o t e i n s 88 i s l i k e l y t o be an e l e c t r o p h i l i c r e a c t i o n as i n t h e r e a c t i o n o f GSH w i t h the n i t r o n e , 7 1 ( 1 2 1 ) . The b i n d i n g o f N-hydroxy compounds and t h e i r m e t a b o l i t e s t o p r o t e i n .. has been e x t e n s i v e l y s t u d i e d i n r e l a t i o n t o the c a r c i n o g e n i c i t y o f N - h y d r o x y - l - n a p h t h y l a m i n e (7_3) ( 1 1 7 ) and N-hydroxy-N-methyl-4-aminoazobenzene ( 7 4 J ( 1 1 2 ) . N i t r o and n i t r o x i d e m e t a b o l i t e s a re known t o p a r t i c i p a t e i n cytochrome P-;4 50 complex f o r m a t i o n ( 1 2 1 ) . \ / 0 H N 73 The p h a r m a c o l o g i a l a c t i o n r e s u l t i n g from N-hydroxy-normethadone o r i t s m e t a b o l i t e s , e.g. methadone n i t r o n e b i n d i n g t o p r o t e i n , one of w h i c h might be c o v a l e n t b o n d i n g , i s o n l y s p e c u l a t i v e a t the moment. The b i n d i n g may cause c a r c i n o -g e n i c i t y . The p r o l o n g e d p h a r m a c o l o g i c a l a c t i v i t y o f N-hydroxy m e t a b o l i t e s compared t o the p a r e n t drugs as r e p o r t e d by Gorrod 89 e t a l . ( 1 2 3 ) c o u l d mean t h a t t h e s e m e t a b o l i t e s a re s t r o n g l y bound t o r e c e p t o r s t r u c t u r e s . The t i g h t b i n d i n g o f l a b e l e d m a t e r i a l - t o r a t b r a i n t i s s u e i n s t u d i e s o f methadone by M i s r a e t a l . ( 1 0 ) might suggest i n v o l v e m e n t o f N-hydroxynormethadone o r methadone n i t r o n e o r t h e i r subsequent m e t a b o l i c p r o d u c t s i n the t o l e r a n c e o r o t h e r p h a r m a c o l o g i c a l a c t i o n s o f methadone. P o s s i b l e m e t a b o l i c pathways o f methadone proposed i n F i g . 1 3 Among the p o s s i b l e m e t a b o l i t e s o f methadone, N-hydroxy-normethadone (5_3) , n i t r o n e s (5_2, 6_0) d i k e t o n e (61) , EMDP N-oxide ( 6 2 ) , N-hydroxyamino compound (6J3) , amino compound (6_4) , n i t r o s o -heptanone (6_5) , and n i t r o h e p t a n o n e (66) were not d e t e c t e d i n t h e work. The l i m i t e d amounts o f t h e sample and d e t e c t i o n l i m i t s p r e -v e n t e d the p o s s i b l e d e t e c t i o n o f m e t a b o l i t e s . T h i s means t h a t p o t e n t i a l l y a c t i v e m e t a b o l i t e s may be formed i n the b i o -l o g i c a l system w i t h o u t b e i n g d e t e c t e d . E v i d e n c e f o r 5_3 based on the p o s t u l a t e d n i t r o n e (5_2) s u g g e s t s t h e p o s s i b i l i t y o f t h e f o r m a t i o n o f i t s f u r t h e r m e t a b o l i z e d p r o d u c t s . I n o r d e r t o examine the p o s s i b l e f o r m a t i o n o f such m e t a b o l i t e s and t h e i r p o t e n t i a l a c t i v i t y , model m e t a b o l i s m s t u d i e s a re suggested i n t h i s t h e s i s . Model m e t a b o l i s m s t u d i e s c o u l d be performed by u s i n g a n i m a l models o r c h e m i c a l r e a c t i o n models f o r w h i c h c h e m i c a l l y s y n t h e s i z e d m e t a b o l i t e s a re e s s e n -t i a l . The m e t a b o l i t e s e.g. N-hydroxynormethadone (5_3) w h i c h c o u l d be o b t a i n e d by r e d u c t i o n o f n i t r o n e (_52) a r e a d m i n i s t e r e d 90 t o a n i m a l s t o s t u d y f u r t h e r m e t a b o l i c pathways. The compounds, 52 and 62 c o u l d a l s o s e r v e as good s t a r t i n g m a t e r i a l s . C h e m i c a l r e a c t i o n p r o c e s s e s are i n v o l v e d i n drug, metab-o l i s m (124) and some o f t h e m e t a b o l i c p r o c e s s e s such as n i t r o n e h y d r o l y s i s and o x i d a t i o n o f N-hydroxy m e t a b o l i t e s f o l l o w s i m i -l a r r e a c t i o n p a t t e r n s t o tho s e o f c h e m i c a l o x i d a t i o n by m - c h l o r o p e r b e n z o i c a c i d . The m e t a b o l i c f o r m a t i o n o f n i t r o and n i t r o s o m e t a b o l i t e s v i a n i t r o n e s and t h e i r h y d r o l y z e d p r o d u c t found i n N-hydroxyphentermine (120) and norbenzphentermine (121) c o u l d be r e p r o d u c e d by MCPBA o x i d a t i o n o f t h e N-hydroxy m e t a b o l i t e s . MCPBA o x i d a t i o n o f methadol may l e a d t o t h e f o r m a t i o n o f methadol n i t r o n e (7_5) and f u r t h e r o x i d a t i o n o f methadol n i t r o n e gave t h e n i t r o h e p t a n o l (7_7) (125). A s i m i l a r example was proposed i n t h e MCPBA o x i d a t i o n o f i s o x a z o l i d i n e , 7j3 t o form o x a z i n e , 19_ w h i c h was f u r t h e r o x i d i z e d t o n i t r o s o , 82^  and n i t r o compound, 8_3 v i a t h e h y d r o l y z e d p r o d u c t , 81. (126). 91 C h e m i c a l h y d r o l y s i s of n i t r o n e s i s common i n n i t r o n e c h e m i s t r y (12 7, 128) . There fore,:.'.hydroxy amino com-pound, 6_3 c o u l d be formed from methadone n i t r o n e , 5_2 and EMDP N- o x i d e , 62. There i s a l s o a p o s s i b i l i t y t h a t ".6 3 c o u l d be formed from amino compound, 6_4 which i s a h y d r o l y z e d p r o d u c t o f p r o t o n a t e d EMDP (2JJ) . 92 4. C h e m i c a l d x i d a t i o n s t u d i e s C h e m i c a l o x i d a t i o n o f methadone m e t a b o l i t e s The f o r m a t i o n o f DDP from a i r o x i d a t i o n o f EDDP base i s c o n s i s t e n t w i t h the f a c t t h a t EDDP base has an e x o c y c l i c d ouble bond ( 3 ) . T h i s does not e x c l u d e the p o s s i b i l i t y t h a t DDP might be d e r i v e d from t h e r m a l c l e a v a g e o f t h e a i r o x i d i z e d EDDP on t h e GC column. D e t e c t i o n o f DDP by TLC i s however more i n d i c a t i v e o f the o x i d a t i v e c l e a v a g e o f the double bond. In o r d e r t o see t h e importance o f the doub l e bond c h a r a c t e r o f EDDP and EMDP a t d i f f e r e n t pH c o n d i t i o n s , the f r e e base and s a l t forms o f EDDP and EMDP were o x i d i z e d w i t h m - c h l o r o p e r b e n z o i c a c i d . O x i d a t i o n o f EDDP base f o l l o w s a mechanism i n v o l v i n g o x i d a t i v e double bond breakage (129) and r e s u l t s i n the forma-t i o n o f DDP. O x i d a t i o n o f EDDP p e r c h l o r a t e r e s u l t e d i n the f o r m a t i o n o f t h r e e major p r o d u c t s i n c l u d i n g t h e methadone n i t r o n e p r o d u c t (5_2) w h i c h i s d i s c u s s e d s e p a r a t e l y i n a l a t e r s e c t i o n . O x i d a t i o n o f EMDP base gave 2 - e t h y l - 5 - m e t h y l - 3 , 3 - ; r d i p h e n y l p y r r o l i d y l - 1 , 2 - o x a z i r a n (84) i d e n t i f i e d by s p e c t r o s c o p i c methods. 93 Four peaks were shown i n the o x i d a t i o n m i x t u r e of EMDP HC1 : GCMS, 150-280°C, 8°/min.; Rt 4 min., d i p h e n y l e t h y l e n e ; 9.4 min., EMDP; 11.6 min., m/e 208 base peak; 12.6 min., m/e 20 8 base peak. At t e m p t s t o i s o l a t e the two unknown compounds u s i n g column chromatography and TLC i n o r d e r t o e l u c i d a t e t h e i r s t r u c t u r e s f a i l e d because of t h e u n s t a b l e n a t u r e o f t h e s e compounds. Ch e m i c a l o x i d a t i o n o f EDDP p e r c h l o r a t e The TIC p r o f i l e o f the o x i d a t i o n p r o d u c t s from EDDP p e r -c h l o r a t e i s shown i n F i g . 14. The same o x i d a t i o n e x p e r i m e n t 2 2 was r e p e a t e d w i t h EDDP- H^Q p e r c h l o r a t e and EDDP- p e r c h l o r -a t e t o c o l l e c t a d d i t i o n a l i n f o r m a t i o n on mass f r a g m e n t a t i o n . Three major components (A, B, and C) from t h e EDDP p e r c h l o r -a t e o x i d a t i o n p r o d u c t s were c o l l e c t e d u s i n g p r e p a r a t i v e GC. H i g h r e s o l u t i o n mass s p e c t r a were o b t a i n e d on t h e s e i s o l a t e d f r a c t i o n s . C h e m i c a l i o n i z a t i o n GCMS of t h e o x i d a t i o n m i x t u r e a l s o gave a d d i t i o n a l d a t a f o r m o l e c u l a r i o n s . F i g . 14. TIC p r o f i l e (a; GCMS, 200-280°C 8°/min.) and HPLC (b) of EDDP p e r c h l o r a t e o x i d i z e d p r o d u c t s 95 Two major components from o x i d a t i o n m i x t u r e were i s o -l a t e d i n pure form u s i n g HPLC ( F i g . 14 B and C ) . Compound A was not i s o l a t e d i n pure form and was t h u s i d e n t i f i e d by GC r e t e n t i o n t ime and mass f r a g m e n t a t i o n p a t t e r n s . A p o s s i b l e s t r u c t u r e f o r A was 2 - a c e t y l - 5 - m e t h y l - 3 , 3 - d i p h e n y l - l - p y r r o l i n e (85). NMR and IR o f compounds, B and C gave p o s i t i v e i d e n t i -f i c a t i o n as 4 , 4 - d i p h e n y l - 2 , 5-heptanedione (6_1) f o r B and metha-done n i t r o n e (52) f o r C. Attempts t o i s o l a t e each component from t h e o x i d a t i o n m i x t u r e u s i n g p r e p a r a t i v e column chroma-t o g r a p h y were not s u c c e s s f u l . I d e n t i f i c a t i o n o f methadone n i t r o n e by mass s p e c t r o m e t r y Mass f r a g m e n t a t i o n d a t a a r e summarized i n T a b l e V f o r methadone n i t r o n e (_52) . The m o l e c u l a r i o n was found t o be m/e 309. The m o l e c u l a r f o r m u l a o b t a i n e d by h i g h r e s o l u t i o n mass s p e c t r o m e t r y was <-20 H23 N <^2* C h e m i c a l i o n i z a t i o n mass s p e c t r o m e t r y gave a q u a s i m o l e c u l a r i o n a t m/e 310. A s t r o n g peak from c h e m i c a l i o n i z a t i o n was m/e 292 a r i s i n g from M+H-H2O. D i r e c t probe mass s p e c t r o m e t r y (EI) a l s o showed a m o l e c u l a r i o n a t m/e 30 9. The methadone n i t r o n e was p o s t u l a t e d t o fragment v i a t h r e e pathways. One proposed pathway d e s c r i b e s the c y c l i z a -t i o n o f _52 t o e i t h e r 8_6 o r 88>, w i t h f u r t h e r f r a g m e n t a t i o n s t o m/e 253 (90 o r 92) , and m/e 73 (91 o r 93) . 96 TABLE V. High R e s o l u t i o n Mass F r a g m e n t a t i o n Data f o r Methadone N i t r o n e (52) Methadone n i t r o n e H_. H m/e 309 C 2 0 H 2 3 N O 2 m/e 312 m/e 319 m/e 253 C 1 7H 1 9NO m/e 253 m/e 263 m/e 208 C 1 6 H 1 6 m/e 207 C 1 6 H 1 5 m/e 207 m/e 217 m/e 193 C 1 5 H 1 5 m/e 181 C 1 4 H 1 3 m/e 180 C 1 4 H 1 2 m/e 179 C 1 4 H 1 1 m/e 178 . ; C 1 4 H 1 0 m/e 165 C 1 3 H 9 m/e 130 C 1 0 H 1 0 m/e 129 C 1 0 H 9 m/e 129 m/e 134 m/e 115 C 9 H 7 m/e 91 C 7 H 7 m/e 73 C 3H 7NO m/e 72 3 6 m/e 57 C,H c0 3 D m/e 60 m/e 44 m/e 44 m/e 44 Comparable i o n fragments as seen by low r e s o l u t i o n GCMS 2 2 are g i v e n f o r t h e H-, and H-, n a n a l o g s . 97 F o r m a t i o n , o f t h e m/e 292 fragment (87_ o r 89) appears t o be the p r e f e r a b l e r o u t e under c h e m i c a l i o n i z a t i o n c o n d i t i o n s whereby p r o t o n a t i o n e a s i l y o c c u r s . The s i x membered i o n , 8j5 appears 98 more s t a b l e than the seven membered i o n , 88> and t h u s the forma-t i o n o f 86^  appears t o be a f a v o r a b l e r o u t e . As an example the r e a c t i o n i n t e r m e d i a t e , - 9 4 o f 3 - c a r b o x y p y r i d i n e r 1 - o x i d e and p r o p i o n i c a n h y d r i d e gave _96, which i s a case o f e l e c t r o p h i l a t t a c k a t the s i t e o f - methylene ca r b o n (130) . S i m i l a r l y , 94 + II H 0 > C-CH3 o-95 96 G02H methadone n i t r o n e (_52) forms c y c l i z e d ' fragment, 86. ; The fragment, -:.-'•'*- • m/e 253 (92) i s i m p o r t a n t i n the \2 1 f a c t t h a t the d e u t e r i u m l a b e l e d - compound was a key t o the i n t e r p r e t a t i o n o f t h i s fragment (Table V ) . The second f r a g m e n t a t i o n pathway f o r t h e methadone n i t r o n e y i e l d s i o n s , m/e 72 (99) , m/e 44 (9_7) , m/e 207 (101) , and m/e 57 (9_8) i n the o r d e r o f r e l a t i v e i n t e n s i t y . Fragmenta-t i o n p r o c e s s e s f o r the f o r m a t i o n o f m/e 72 and m/e 5 7 commonly o c c u r i n methadone a n a l o g s . The pathway l e a d i n g t o the forma-t i o n o f m/e 207 (101) i s a common p r o c e s s f o r n i t r o x i d e d e r i -v a t i v e s . F u r t h e r f r a g m e n t a t i o n l e a d s t o i o n s , m/e 130 (10 2) and m/e 129 (103). 99 1 0 3 The f o r m a t i o n o f fragment i o n m/e 44 i s s i m i l a r t o the o b s e r v e d f r a g m e n t a t i o n o f n i t r o n e s , 104 t o 105 (131) and 106 t o 107 (132). j 100 The t h i r d f r a g m e n t a t i o n pathway p r o v i d e s a d d i t i o n a l i n f o r m a t i o n on t h e f r a g m e n t a t i o n o f d i p h e n y l type compounds. The h i g h r e s o l u t i o n mass s p e c t r o m e t r y d a t a (Table V) p r o v i d e s a b a s i s f o r t h e e x p l a n a t i o n o f t h e f r a g m e n t a t i o n p r o c e s s e s d e s c r i b e d f o r DDP and EMDP (31-35). Such i o n s a r e m/e 193 (108), m/e 180 (32), m/e 181 (111), m/e 115. (109), and m/e 179 (33). The fragments m/e 91 (112) and m/e 89 (113) c o u l d be d e r i v e d from m/e 180 (32); m/e 181 (111) was from m/e 208 (31) . 101 m/e 115 109 113 102 New p r o p o s a l f o r f r a g m e n t a t i o n pathways f o r methamphetamine n i t r o n e Mass f r a g m e n t a t i o n p r o c e s s e s f o r the n i t r o n e metabo-l i t e (54^ o f N-methylamphetamine were shown i n two papers p u b l i s h e d by C o u t t s e t a l . (133, 134). I t was proposed t h a t under E I c o n d i t i o n s the n i t r o n e , 5_4 forms t h e c y c l i z e d i n t e r -mediate o f m/e 163 (115), which f u r t h e r fragments t o m/e 148 (117) and m/e 132 (118). To e x p l a i n the f o r m a t i o n o f m/e 104 (116), a r e t r o D i e l s - A l d e r t y p e f r a g m e n t a t i o n p r o c e s s was proposed. Oxygen e x p u l s i o n was i n v o l v e d i n t h e forma-t i o n o f 118 from 117. These i o n s were d e f i n e d w i t h d e u t e r i u m l a b e l e d amphetamine d e r i v a t i v e s 54 .CH, 1* 114 -CH 3CH=N0H 115 CH„ m/e 104 116 CH, m/e 163 - 0 117 m/e 132 118 m/e 148 103 The f r a g m e n t a t i o n p r o c e s s e s proposed f o r methadone n i t r o n e a r e d i f f e r e n t from th o s e d e s c r i b e d f o r N-methylamphe--' tamine n i t r o n e by G o u t t s e t a l . (134). i t was found t h a t the fragment i o n s r e p o r t e d f o r t h i s amphetamine-nitrone c o u l d a l s o be e x p l a i n e d f o l l o w i n g our p r o p o s a l f o r t h e methadone n i t r o n e f r a g m e n t a t i o n s . Thus, f o r m a t i o n o f the fr a g m e n t s , m/e 148 (120) and m/e 132 (151) f o l l o w s from the c y c l i z e d i n t e r m e d i a t e 119. Fragment 121 has m/e 132 c o r r e s p o n d i n g t o t h e fragment of 118 proposed by C o u t t s e t a l . (134). m/e 132 m/e 148 150 (d , ) 120 134 (d 2 ) — .N-methylamphetamine n i t r o n e fragments t o t h e i o n m/e 118 (122) which i s a s i m i l a r p r o c e s s t o t h e f o r m a t i o n o f m/e 20 7 (101) i n methadone n i t r o n e (5_2) . The i o n m/e 118 (122) was f u r t h e r fragmented t o m/e 91 (124), m/e 117 (125), 104 and m/e 104 (126) . A hydrogen s c r a m b l i n g p r o c e s s i s i n v o l v e d i n t h e f o r m a t i o n of 124, 125, 126 which i s suggested by the r e p o r t e d mass s p e c t r a (134) whi c h were p r e p a r e d by u s i n g un-l a b e l e d and l a b e l e d compounds.. The fragment, m/e 104 (126) was p r e v i o u s l y e x p l a i n e d by a r e t r o D i e l s - A l d e r p r o c e s s . Our p r o p o s a l d e s c r i b e s f o r m a t i o n o f m/e 104 (126) •• from m/e 118 (123) . ' v> CH ™ CH N=CH, CH== N-1 A H 2 C-H *° 54 + CH; CH=CH2 m/e 118 124(d 1 |) 122 CH. 0-+ ^ ^ CH. m/e 117 121 (dO 120(d 3 ) CH 125 CH, CH2 CH« 123 CH. m/e 104 106(d 2 ) CH, 126 124 m/e 91 93(d 2 ) 92(d) 105 NMR o f methadone n i t r o n e The NMR spectrum o f methadone n i t r o n e i s shown i n F i g . 15. The peaks were a s s i g n e d as f o l l o w s : Two d o u b l e t s o f doub-l e t s a t 2.25 6 and 2.73 6 f o r C-5 p r o t o n s are t y p i c a l o f n i -t r o n e t y p e compounds. Methylene p r o t o n s appear a t 3.1 6 and 3.55 6 as two d o u b l e t s . The o t h e r c h e m i c a l s h i f t v a l u e s are shown below t o g e t h e r w i t h t h e s t r u c t u r a l assignment. Peaks from i m p u r i t i e s d e r i v e d from HPLC s o l v e n t s and CHCl^ are shown a t 1.0 - 1.6 6. 1.75 6 (m) \ >. 0.78 6 (d) Ta b l e v i compares the C-5 and C-6 p r o t o n s and m e t h y l -ene p r o t o n s o f methadone n i t r o n e w i t h t h o s e o f N - ( l - ( 3 ' , 4 ' -dimethoxyphenyl) p r o p - 2 - y l ) n i t r o n e (127) p u b l i s h e d by Morgan e t a l . (135). The methylene p r o t o n s o f 5_2 (6 3.1 and 3.55) were shown t o be more s h i e l d e d t h a n t h o s e o f 127 (6 6.0 4 and 6.24) . The c o u p l i n g c o n s t a n t s , JHa-Hc (4.0 Hz) and JHb-Hc (4.0 Hz) suggest a p r e f e r r e d c o n f o r m a t i o n o f 5_2 i n wh i c h the 107 OCH3 127 TABLE V I . NMR o f C-5 and ge m i n a l p r o t o n s of methadone n i t r o n e (5_2) and 3.4-dimethoxyamphetamine n i t r o n e (127) 52 127 6 CH 2 (5) 6 Ha 2.25 (dd) 2.73 (dd) J Ha-Hc 4.0 • 5.6 Ha He 1 1 6 Hb 2.73 (dd) 3.18 (dd) C-— C ,5 ,6 J Hb-Hc 4.0 10.0 Hb CH 3 J Ha-Hb 14.0 14.0 6 He 1.75 (m) 4.05 (m) + / H a N = C I ^Hb 6 6 Ha Hb 3.1 3.55 (d) (d) 6.04 6 . 24 (d) (d) 0 J Ha-Hb 11.0 8.0 oxygen o f t h e ketone group i s o r i e n t e d towards the methylene group. T h i s i n t e r a c t i o n may c o n t r i b u t e t o an i n c r e a s e o f e l e c -t r o n d e n s i t y on t h e methylene group w h i c h c o u l d l e a d t o t h e e l e c t r o n s h i f t c a u s i n g l o s s o f p o l a r i t y o f t h e n i t r o n e n i t r o g e n . T h i s o v e r a l l e f f e c t may e x p l a i n t h e s t a b i l i t y o f t h i s methadone n i t r o n e and the h i g h s h i e l d i n g o b s e r v e d f o r the C-6 p r o t o n 1(6 1.75; 6 4.0 5 f o r 12 7) and t h a t o f the methylene p r o t o n s . 108 He C = T O CH2 52 128 An a l t e r n a t i v e e x p l a n a t i o n o f the h i g h s h i e l d i n g ob-s e r v e d f o r the C-6 p r o t o n and the methylene p r o t o n s i s t h a t i n s t e a d o f s t r u c t u r e 5_2_ t h e compound i s o l a t e d has t h e o x a z i r i -d i n e s t r u c t u r e 128. The methylene p r o t o n s o f o x a z i r i d i n e s a re known t o be more s h i e l d e d t h a n t h o s e o f n i t r o n e s (135). W h i l e the s t r u c t u r e 128 cannot be e x c l u d e d on the b a s i s o f s p e c t r o -s c o p i c e v i d e n c e (MS, MMR, IR) t h e r e i s no precedence f o r t h e f o r m a t i o n o f such a compound by i n v i v o m e tabolism. Upon t h i s b a s i s we w i l l t e n d t o p r e f e r t h e n i t r o n e s t r u c t u r e ^ 52. IR s p e c t r o s c o p y o f methadone n i t r o n e and r e l a t e d compounds The IR spectrum and a summary of t h e d a t a f o r t h e methadone n i t r o n e a r e shown i n F i g . 16. The C-H v i b r a t i o n s d e s c r i b e d a re a s s i g n e d on the b a s i s o f IR 'data c o l l e c t e d from 109 methadone a n a l o g s and t h e i r ' d e u t e r a t e d c o u n t e r p a r t s and s h o u l d be q u i t e u s e f u l f o r d e s c r i b i n g the IR o f o t h e r d i p h e n y l con-t a i n i n g compounds. F o r m a t i o n o f d i k e t o n e (6_1) from o x i d a t i o n o f EDDP p e r c h l o r a t e The compound wh i c h has m/e 4 3 as a base peak ( F i g . 1 4 ( a ) , B and F i g ; 1 4 ( b ) , B) was a s s i g n e d t o t h e d i k e t o n e , 61. NMR and IR s p e c t r a shown i n F i g . 17 and mass f r a g m e n t a t i o n d a t a were c o n s i s t e n t w i t h t h e d i k e t o n e s t r u c t u r e . 61 I s o l a t i o n o f d i k e t o n e , 61 would s u p p o r t t h e proposed mechanism ( F i g . 18) o f m - c h l o r o p e r b e n z o i c a c i d o x i d a t i o n o f EDDP p e r c h l o r a t e . Wavalanglh jx m 3300 3500 3000 2500 2000 1800 1600 1400 1200 1000 600 625 Wuwenumtier _ i v(=CH) 3020, 3040, 1600, 1490 cm <5 (=CH) 1140, 1100, 1030, 930, 760, 700 v (CH) 2960 , 2920 6 (CH,CH 2,CH 3 ) 1450, 1440, 1370, 1340, 820, 800 v(C=0; N=C) 1710 v (N + 0) 1550, 1250 F i g . 16* IR ( l i q u i d f i l m ) o f methadone n i t r o n e i i i J U O u J (b) 10 11 H 13 1< 15 u 2500 2000 800 635 F i g . " 1 7 . (a) NMR and (b) IR (KBr p e l l e t ) o f 4,4-Diphenyl-2 . 5-heptanedione (61.) 112 Proposed mechanism f o r t h e c h e m i c a l f o r m a t i o n o f methadone n i t r o n e (52) o r o x a z i r i d i n e (128) and d i k e t o n e (61) A p o s s i b l e mechanism f o r the f o r m a t i o n o f methadone n i t r o n e (5_2) , o r o x a z i r i d i n e (128) , and d i k e t o n e (61) from th e c h e m i c a l o x i d a t i o n o f EDDP p e r c h l o r a t e i s p r e s e n t e d i n F i g . 18. More t h a n 2 moles o f MCPBA was r e q u i r e d f o r t h e f o r m a t i o n o f 5_2, o r 128 fand 61 whi c h i s i l l u s t r a t e d by t h e proposed i n t e r m e d i a t e , 12 9, formed by N-oxide f o r m a t i o n and an a d d i t i o n o f a second mole o f MCPBA t o t h e doub l e bond. N i t r o n e , 52, o r o x a z i r i d i n e (128) were not formed s e l e c -t i v e l y . The d i k e t o n e , 6_1 can be seen t o be formed t h r o u g h t h e i n t e r m e d i a t e 130. T h i s c h e m i c a l h y d r o l y s i s has a s i m i l a r mechanism t o the m e t a b o l i c f o r m a t i o n o f t h e ketone 132 from the n i t r o n e 131 (136). 0 ~ R - CH„ - C = N - CH_R' •> R - CH 0 - C = 0 Z | + Z Z | CH^ 131 132 113 2 MCPBA 19 CH3 C=0 CH—CH— N CH, 2 j v / 2 \ J CH, CH2 CH3 C= 0 52 CH2—CH—S=;CH2 CH3 128 ' I N — . i -F i g . 18. Proposed mechanisms f o r t h e f o r m a t i o n o f methadone n i t r o n e (5_2) o r o x a z i r i d i n e (128) and 4,4-diphenyl-2,5-heptanedione (6JL) by MCPBA o x i d a t i o n o f EDDP p e r c h l o r a t e . 114 Proposed s t r u c t u r e f o r compound A ( F i g . 13) Hitfh r e s o l u t i o n mass s p e c t r a o f compound A gave f r a g -ments, m/e 208 (24.29%), m/e 207 (12.87%), m/e 193 (22.53%), m/e 179 (13.09%), m/e 178 (14.34%), m/e 165 (11.27%), m/e 130 (19.53%), and m/e 115 (19.82%). These i o n s were common t o the d i p h e n y l butane c o n t a i n i n g compound. The compounds a l s o gave i o n s , m/e 277 ( 1 . 3 2 % ) ( C 1 9 H L G N O ) , m/e 235 (11.63%) ( C^H^NO, the b a s i s o f t h e s e i o n s the compound was a s s i g n e d the s t r u c t u r e , 85 . Compound B was a l s o d e t e c t e d by GCMS when o l d u r i n e samples were a n a l y z e d and proved t o r e s u l t from a i r o x i d a t i o n o f EDDP. When EDDP base was a i r o x i d i z e d i n CHCl^ and a n a l y z e d by GCMS (150 - 280°C a t 10°/min.), t h e major p r o d u c t DDP was e l u t e d a t 245°C and another p r o d u c t e l u t i n g a t 225°C c l o s e t o the EDDP peak was i d e n t i c a l t o t h e a c e t y l compound, 85. A p o s s i b l e mechanism t o t h e f o r m a t i o n o f 85_ e i t h e r by c h e m i c a l o x i d a t i o n o f EDDP p e r c h l o r a t e o r by a i r o x i d a t i o n o f EDDP base i s shown i n 3, 19^ t h r o u g h 8_5 (p. 115). m/e 238)and m/e 43 (base peak)(C„H,0, m/e 46). On 115 85 S y n t h e s i s o f EMDP o x a z i r a n (84.) MCPBA o x i d a t i o n o f EMDP base gave an o x i d i z e d p r o d u c t , w h i c h was e l u t e d a t 250°C (120-270°C a t 10°/min.) by GCMS. Mass f r a g m e n t a t i o n d a t a were compared w i t h d e u t e r i u m l a b e l e d 2, compounds o b t a i n e d by o x i d a t i o n o f EMDP- H^Q. A m o l e c u l a r i o n appeared a t m/e 279 (28.3%) ( m/e 289). M -CHjCHg (m/e 250 (3 6 . 0 % ) , M - p h e n y l (m/e 202 (1 6 . 7 % ) , "H 10 'H 10 10 m/e 260) and m/e 20 7) were major f r a g -ments. The i o n s commonly d e r i v e d from m/e 20 8 were a l s o p r e s e n t w i t h h i g h r e l a t i v e i n t e n s i t y . The s t r u c t u r e was not a b s o l u t e l y p r o v e d but i s most l i k e l y EMDP o x a z i r a n (M) r a t h e r than EMDP N-oxide. (62). H i g h abundant m/e 279,.m/e 250, and m/e 202 i n d i c a t e d t he - . s t a b l e n a t u r e o f the o x y g e n . i n the m o l e c u l e o f _8_4. On the o t h e r hand. N-oxide may e a s i l y l o s e oxygen t o g i v e 'high -abundant m/e M-l6. An attempt t o o b t a i n c y c l i z e d p r o d u c t , 135\ was n o t s u c c e s s f u l . The c h e m i c a l forms o f 6_2 and 8j4 a r e r e p o r t e d t o be i n t e r c o n v e r t i l e e i t h e r by a c i d i c c o n d i t i o n s o r by UV i r r a d i a -t i o n (137). The o x i d i z e d p r o d u c t was h y d r o l y z e d • in - a c i d . • (10% H C l , 70°C ? 2 hours) and a m i x t u r e o f p r o d u c t s which has'peaks ••117 on GCMS c o n t a i n i n g •; m/e 208 fragment as base peak was o b t a i n e d . F u r t h e r a t t e m p t s t o de t e r m i n e the s t r u c t u r e s were n o t performed. NMR d a t a f o r the proposed o x i d i z e d p r o d u c t o f EMDP i s shown below a l o n g w i t h t h e s t r u c t u r e . The c h e m i c a l s h i f t v a l u e s f o r the C-8, C-7, and C-6 p r o t o n s were s i m i l a r t o those of EMDP (C-8 1.07 6 , C-7 2.09 6 , and C-6 1.37 6 ) ( 3 ) . 2.20*6 (q) 0.78 6 ( t ) -3.25 6 (dd,J-, H c'6-:0) 4.85 6 (m) > 0.89 6 (d) 2.30 <5 (dd, J ;T "3.5) Ha-Hc The -IR spectrum o f t h i s compound had s t r o n g peaks a t 1250 and _ i 1550 cm c o r r e s p o n d i n g t o N - 0 s t r e t c h i n g v i b r a t i o n s 118 5. I n t e r a c t i o n o f methadone and diazepam S t a b i l i t y o f the l a b e l i n g o f t h e compounds which were used as i n t e r n a l s t a n d a r d s t o a n a l y z e nonconjugate and c o n j u - " gate m e t a b o l i t e s was examined t o ensure t h e s t a b i l i t y o f the l a b e l i n g d u r i n g i n c u b a t i o n and work up p r o c e d u r e s . P a i r e d e x p e r i m e n t s were performed w i t h f o u r r a t s f o r methadone t r e a t -ment and a n o t h e r f o u r r a t s f o r methadone and diazepam t r e a t m e n t . S t a b i l i t y s t u d i e s The s t a b i l i t y o f the d e u t e r i u m l a b e l e d methadone and the l a b e l e d EDDP and EMDP was e x p l o r e d o v e r a wide range o f pH's and under c o n d i t i o n s o f p r o l o n g e d i n c u b a t i o n (Table V I I ) . As can be seen from the i o n r a t i o s m o n i t o r e d which remain c o n s t a n t , t h e d e u t e r i u m was s t a b l e i n a l l c a s e s . The r e s u l t i m p l i e s t h a t the d e u t e r i u m l a b e l e d compounds wh i c h were used as i n t e r n a l s t a n d a r d s i n t h i s i n t e r a c t i o n s t u d y a re s t a b l e d u r i n g t h e e q u i l i b r a t i o n and work up p r o c e d u r e s f o r the a n a l y s i s . The advantages o f u s i n g d e u t e r i u m l a b e l e d compounds as i n t e r n a l s t a n d a r d s was f u r t h e r demonstrated by EDDP when -11-9 TABLE V I I . S t a b i l i t y o f EDDP and EMDP D e u t e r a t e d Methadone, Methadone 233/223 226/223 EDDP 287/277 280/277 EMDP 218/208 IN HCl 0.633 . 0.954. 0.532 0.972 0.568 pH 2.0 0.643 0.968. 0 .532 1.010 0.571 pH 4.5 0.657 0.946 0.525 0. 984 0 .585 pH 9.0 0.615 0 . 94 5. . 0.513 0.977 0.580 0.1N NaOH 0.656 0. 963' 0.528 0.981 0.561 C o n t r o l 0.673 0.956 0.532 0 . 966 0.580 The v a l u e s demote i o n r a t i o s of i o n s m o n i t o r e d : m/e 22 3 (metha-2 2 done), m/e 2 2 6 (methadone- H^), m/e 2 3 3 (methadone- H-^Q), m/e 2 7 7 (EDDP), m/e 2 8 0 (EDDP- 2H 3), m/e 2 8 7 (E D D P - 2 H 1 Q ) , m/e 2 0 8 2 (EMDP), and m/e 2 1 8 (EMDP- H 1 Q ) . The v a l u e s r e p r e s e n t the mean of t h r e e d e t e r m i n a t i o n s . The aqueous samples were i n c u b a t e d f o r 4 8 hours a t the r e s p e c t i v e pH's. The c o n t r o l was p r e p a r e d i n MeOH. 120 under a l k a l i n e c o n d i t i o n s (Table V I I ) . Because d e u t e r i u m l a b e l e d EDDP c o u l d compensate f o r t h e c h e m i c a l i n s t a b i l i t y of EDDP, t h e r a t i o o f u n l a b e l e d t o l a b e l e d EDDP was the same n o t w i t h s t a n d i n g s i g n i f i c a n t d e c o m p o s i t i o n of EDDP under a l k a l i n e c o n d i t i o n s . T o t a l i n t e g r a t i o n v a l u e s f o r EDDP i n 0.1 N NaOH were l e s s than 1/3 t h a t of c o n t r o l samples p r e p a r e d i n MeOH. T h i s example o f t h e advantage o f u s i n g a d e u t e r i u m l a b e l e d i n t e r n a l s t a n d a r d c o u l d be extended t o t h e a n a l y s i s o f c o n j u g a t e m e t a b o l i t e s . F o r example, the i n s t a b i l i t y o f p h e n o l and c a t e c h o l type m o l e c u l e s c o u l d be compensated f o r by u s i n g d e u t e r i u m l a b e l e d s t a n d a r d s . S t a b i l i t y o f t h e d e u t e r i u m l a b e l was a l s o shown by the c o n j u g a t e m e t a b o l i t e s f o l l o w i n g 96 hours o f i n c u b a t i o n t i m e - ( T a b l e s V I I I , I X ) . Even i n the pr e s e n c e o f an a c t i v a t i n g group such as t h e h y d r o x y l group on the r i n g , the d e u t e r i u m was s t a b l e under a l l c o n d i t i o n s o f i n c u b a t i o n pH and tempera-t u r e . The e f f e c t o f a c t i v a t i n g groups on the- s t a b i l i t y of l a b e l i n g was d e s c r i b e d f o r a h y d r o x y l group (138) and an amino group (139). T h i s r e s u l t i s c o n s i s t e n t w i t h a r e p o r t t h a t t h e d e u t e r i u m l a b e l on t h e r i n g o f £-hydroxyephedrine o b t a i n e d by m e t a b o l i c p r o c e s s e s was found t o be s t a b l e d u r i n g the g l u c u r o n i d e f o r m a t i o n and the subsequent enzyme h y d r o l y s i s o f the c o n j u g a t e (140). The s t a b i l i t y s t u d i e s of c o n j u g a t e m e t a b o l i t e s (Tables X I I I and IX) a l s o gave some i n s i g h t i n t o t h e c o n t r i b u t i o n o f 121 TABLE V I I I . S t a b i l i t y o f Deuterium i n HOEMDP- 2H 9 Time (h) 246/247 238/247 237/247 12 0.435 -0.0062 0.0005 24 0.439 0.0060 0 .0062 48 0 .432 0 .0054 0 .0041 96 0.441 -0.0070 -0.0001 Time (h) 246/247 245/247 244/247 12 0 .446 0.359 0.118-24 0.437 0 . 403 0 .113 48 0.461 0.407 0.113 96 0.437 0.40 7 0.125 Samples were m o n i t o r e d t w i c e w i t h a d i f f e r e n t s e t o f i o n s b e i n g m o n i t o r e d each t i m e . The f i r s t s e t o f i o n s was m/e 247, m/e 246, m/e 238, and m/e 237 and t h e second, m/e 247, m/e 246, m/e 245, and m/e 244. The s t a b i l i t y o f the l a b e l , the p r e s e n c e o f i m p u r i t i e s and t h e s t a b i l i t y of t h e i o n f o c u s s i n g were f o l l o w e d u s i n g the r a t i o o f i o n s , m/e 246 t o m/e 247. The i o n , m/e 238 was chosen f o r CH^OEMDP and m/e 2 24 7 f o r CH 30EMDP- H g. The v a l u e s were the average from two i n c u b a t i o n s a t each i n c u b a t i o n time (12, 24, 48, and 96 h o u r s ) . N e g a t i v e v a l u e s r e s u l t from background s u b t r a c t i o n . .122 TABLE IX. S t a b i l i t y o f Deuterium i n DiHOEMDP- H g Time (h) 12 24 48 96 244/245 0 . 363 0.359 0.364 0.369 237/245 0 .0088 0^0069 0.0059 0.0066 236/245 0 .0091 0.0052 0 .0060 0.0077 Time (h) 12 24 48 96 247/245 0 .0296 0.0341 0.0266 0 .0250 246/245 0.215 0.211 0 . 214 0.207. 244/245 0.359 0.356 0 . 359 0.359. Samples were m o n i t o r e d t w i c e w i t h a d i f f e r e n t s e t o f i o n s b e i n g m o n i t o r e d each t i m e . The f i r s t s e t o f i o n s was m/e 245, m/e 244, m/e 237, and m/e 236 and the second, m/e 247, m/e 246, m/e 245, and m/e 244. The s t a b i l i t y o f the l a b e l , the p r e s e n c e o f i m p u r i t i e s , and t h e s t a b i l i t y o f t h e i o n f o c u s s i n g were f o l l o w e d u s i n g the r a t i o o f i o n s , m/e 244 t o m/e 245. The i o n m/e 237 was chosen f o r DiCHgOEMDP and 2 m/e 245 f o r DiCHgOEMDP- Hg. The v a l u e s were the average from two i n c u b a t i o n s a t each i n c u b a t i o n time (12, 24, 48, and 96 hours) . 123 background t o t h e a n a l y t i c a l p r o c e d u r e . The s t a b i l i t y s t u d i e s o f c o n j u g a t e m e t a b o l i t e s which were performed w i t h d e u t e r i u m l a b e l e d compounds showed t h a t the i o n r a t i o s , m/e 238/247, m/e 237/247, m/e 237/245, and m/e 236/245 were l e s s than 1 p e r c e n t . T h i s meant t h a t t h e r e was an absence of i m p u r i t i e s i n endogenous » b i l e and no c o n t r i b u t i o n from l a b e l e d metabo-l i t e s w hich might i n t e r f e r e i n the a n a l y s i s o f u n l a b e l e d m e t a b o l i t e s . 2 The mass spectrum f o r CH^OEMDP- Hg d i d n ot show any 2 i o n s a t m/e 238, 237 and the mass spectrum o f DiCH^OEMDP- Hg d i d n o t show i o n s a t m/e 237 and m/e 236. A method t o examine t h e p o t e n t i a l o v e r e s t i m a t i o n of methadone c o n j u g a t e m e t a b o l i t e s a r i s i n g from a n a l y t i c a l i n t e r f e r e n c e s such as from endogenous m a t e r i a l s and from c o n j u g a t e m e t a b o l i t e s o f diazepam was a l s o s t u d i e d . I f t h e r e i s no i n t e r f e r e n c e , t h e n m o n i t o r i n g o f c o n s e c u t i v e i o n s s h o u l d g i v e c o n s t a n t r a t i o s . The i o n s m o n i t o r e d were m/e 246/247 2 ? f o r CH-.OEMDP- H n and m/e 244/245 f o r DiCH.OEMDP- H„ . A sudden J y 3 o change of t h e r a t i o s i n d i c a t e d i n most c a s e s d i s r u p t i o n o f the i o n f o c u s s i n g a r i s i n g from e i t h e r power f a i l u r e o r an u n s t a b l e magnetic f i e l d . The f a c t t h a t b l a n k samples w h i c h were c o l l e c t e d f o r t h e f i r s t one hour a f t e r diazepam dose d i d n o t i n t e r f e r e w i t h the i o n r a t i o s i n d i c a t e d an absence o f i n t e r f e r e n c e from diazepam m e t a b o l i t e s . I t has been shown t h a t d u r i n g t h e f i r s t one hour, 45% o f the r a d i o a c t i v i t y i n j e c t e d i n t h e form o f f 5 - ^ C ~ ] - diazepam i n t o t he r a t appeared i n t h e b i l e (141). 124 S t a b i l i t y s t u d i e s were a l s o aimed a t measuring the a c c u r a c y and p r e c i s i o n o f the de v e l o p e d SIM system. Indepen-dent of t h e s e t o f i o n s m o n i t o r e d , '(m/e 247, 246, 245, 244 and m/e 247, 246, 238, 237), the r a t i o o f m/e 246 t o 2 m/e 247 was t h e same (0.43) f o r CH^OEMDP- H g. C o n s e c u t i v e i o n m o n i t o r i n g i . e . m/e 247, 246, 245, 244 and m o n i t o r i n g f o u r s e p a r a t e i o n s i . e . m/e 245, 244, 237, 236 a l s o gave the same m/e 244/245 v a l u e o f 0.36 f o r DiCH 3OEMDP- 2Hg, i m p l y -i n g p r e c i s i o n o f t h e methodology. A c c u r a c y o f t h e i o n m o n i t o r i n g method was d i f f i c u l t t o e v a l u a t e . Ion r a t i o s c a l c u l a t e d from t h e r e l a t i v e i n t e n s i t y o f mass s p e c t r a l scan d a t a were compared w i t h those from i n t e g r a t i o n v a l u e s o f s p e c i f i c i o n b e i n g m o n i t o r e d i n SIM mode. The i o n r a t i o s of m/e 245 t o m/e 247 and m/e 246 t o m/e 247 2 o b t a i n e d by scan mode f o r CH^OEMDP- Hg were 0.4 6 and 0.36, r e s p e c t i v e l y . On t h e o t h e r hand, the r a t i o s o b t a i n e d by 2 SIM were 0.40 and 0.44. In t h e case o f DiCH^OEMDP- Hg, the r a t i o s from scan mode were 0.366 arid 0.220 f o r m/e 244 t o m/e 245 and m/e 246 t o m/e 245, r e s p e c t i v e l y . SIM r a t i o s were 0.360 and 0.215. The d i f f e r e n c e i n the r a t i o s between SIM and r e p e t i t i v e s c a n n i n g i s due t o t h e d i f f e r e n t d a t a a c q u i s i t i o n methods. 125 B i l e c o l l e c t i o n T able X d e s c r i b e s the b i l e volumes c o l l e c t e d from each r a t i n t h e methadone-diazepam i n t e r a c t i o n s t u d y . The t o t a l amount of c o l l e c t e d b i l e averaged 50 ml/kg f o r 2 3 h o u r s , which was a l i t t l e below t h e b i l e f l o w (60-100 ml/kg/24 hour) d e s c r i b e d by Johnson e t a l . (141). T a k i n g i n t o a ccount t h a t f l u i d r e p l a c e m e n t by i n f u s i o n was n o t c a r r i e d o u t , the b i l e f l o w appeared adequate t o o b t a i n r e l i a b l e d a t a f o r the drug i n t e r a c t i o n s t u d i e s . Diazepam d i d n o t i n f l u e n c e b i l e f l o w d u r i n g t h e 2 3 hour t i m e p e r i o d o f the study (p>0.20). A s i m i l a r r e s u l t was r e p o r t e d by E l - H a w a r i e t a l . (143) i n t h e i r s t u d i e s o f the e f f e c t s o f diazepam on b i l i a r y e x c r e t i o n o f d i p h e n y l h y d a n -t o i n . A n a l y s i s o f nonc o n j u g a t e d m e t a b o l i t e s from r a t b i l e As shown i n i n d i v i d u a l SIM chromatograms o f EMDP, methadone, and EDDP ( F i g . 19) p r e p a r e d from s p i k e d samples, a n a l y s i s o f methadone and EDDP had h i g h s p e c i f i c i t y because h i g h masses (m/e 294 and m/e 297 f o r methadone, m/e 277 and m/e 280 f o r EDDP) were m o n i t o r e d . M o n i t o r i n g i o n s , m/e 208 and m/e 218 was found t o l a c k s p e c i f i c i t y f o r EMDP b u t the 12'6 TABLE X. E f f e c t o f Diazepam Treatment on t h e B i l e Flow o f Rats Time (h) 1-M 1-: DM .2 -M 2-DM 3-•M 3-DM 4' -M 4- DM 0-2 3. 85 4 .04 4 .28 4.07 3. 52 4 .41 3 .72 5 . 22 2-11 26. 00 28 .08 13 .81 20.89 23. 18 21 . 92 19 .37 26 . 79 11-23 29. 90 27 .48 23 . 72 17.69 25. 17 25 .66 17 . 97 18 .85 T o t a l 57. 75 59 .60 41 .81 42.65 51. 87 51 .99 41 .06 50 .86 Time (h) M DM P 0-2 3 .842 + 0 . 321 4.435 + 0. 549 >0. 10 2-11 20 . 361 + 5. 627 24.420 + 3. 546 >0. 20 11-23 24 .190 + 4. 915 22.420 + 4. 872 >0 . 20 T o t a l 48 .393 + 8. 900 51.275 + 6. 930 >0 . 20 P a i r e d e x p e r i m e n t s Were performed u s i n g f o u r r a t s f o r metha-done t r e a t m e n t (M) and a n o t h e r f o u r r a t s f o r methadone and diazepam t r e a t m e n t (DM) The v a l u e s denote the amount o f b i l e (g/kg r a t ) c o l l e c t e d o v e r t h e time p e r i o d s 0-2, 2-11, and 11-23 hours f o l l o w i n g the methadone dose. The v a l u e s i n the lower p a r t o f the Ta b l e r e p r e s e n t mean ± SD o b t a i n e d f o r each o f t h e two groups. The v a l u e p was c a l c u l a t e d by u s i n g S t u d e n t ' s t t e s t . ' 12 7 200 210 220 TEMPERATURE 230 240 250 F i g . 19. SIM chromatograms f o r EMDP ( a ) , EDDP (h) and methadone ( c ) . ' ( ' ' 128 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 o f peaks was s u f f i c i e n t l y s e l e c t i v e t o a l l o w , a n a l y s i s o f EMDP. For t h e a n a l y s i s o f nonconjugated m e t a b o l i t e s , authen-t i c samples and d e u t e r i u m l a b e l e d i n t e r n a l s t a n d a r d s were a v a i l a b l e . C a l i b r a t i o n c u r v e s were p r e p a r e d f o r EDDP, metha-done, and EMDP by s p i k i n g EDDP (5-80 u g ) , methadone (0.0 5-0.8 u g ) , and EMDP (0.1-1.6 ug) i n 0.4 ml o f c o n t r o l b i l e and by w o r k i n g up t h e samples as d e s c r i b e d i n the e x p e r i m e n t a l u s i n g 2 2 2 EMDP- R"lQ (2 ug) , methadone- H 3 (5 ug) and EDDP- H 3 (20 ug) as the i n t e r n a l s t a n d a r d s . Under the above c a l i b r a t i o n con-d i t i o n s , 200 ng o f methadone i n 0.4 ml o f b i l e , 400 ng o f EMDP i n 0.4 ml o f b i l e , and 5 ug o f EDDP i n 0.4 ml o f b i l e were r e p r o d u c i b l y a n a l y z a b l e . Methadone e x c r e t e d i n r a t b i l e was not d e t e c t a b l e under the c o n d i t i o n s o f t h e a n a l y s i s . Assuming t h a t t h e l o w e s t c o n c e n t r a t i o n o f methadone t h a t can be p o s i t i v e l y measured i s 200 ng i n 0.4 ml o f b i l e , t h e n l e s s t h a n 0.2% o f t h e a d m i n i s t e r e d methadone dose i s e x c r e t e d i n t o t he b i l e , a t r a c e amount. T h i s r e s u l t was c o n s i s t e n t w i t h a r e p o r t t h a t a v e r y s m a l l amount o f unchanged methadone and EMDP was p r e s e n t i n human b i l e (98) and r a t b i l e (144). T h i s meant t h a t t h e a n a l y s i s o f metha-done -can n ot be used f o r s t u d i e s o f t h e methadone-diazepam i n t e r a c t i o n . EMDP l e v e l s i n r a t b i l e samples were a l s o a n a l y z e d and c o n c e n t r a t i o n s below 400 ng/0.4 ml were o b s e r v e d . T h i s r e s u l t i s the same as t h a t r e p o r t e d by Kreek e t a l . (98) 129 and B a s e l t e t a l . (145). T h e r e f o r e , a n a l y s i s o f EMDP would not be a good i n d i c a t o r o f a m e t a b o l i c i n t e r a c t i o n between methadone and diazepam. The low l e v e l o f methadone and EMDP i n b i l e can be e x p l a i n e d i n t h a t l i v e r has a h i g h i n t r i n s i c a c t i v i t y f o r the m e t a b o l i s m o f methadone and EMDP. The e x t e n t o f b i l i a r y e x c r e t i o n o f a drug i s p r o p o r t i o n a l t o the p o l a r i t y o f the drug which i s r e f l e c t e d by i t s p a r t i t i o n c o e f f i c i e n t . The p e r c e n t a g e s r e p o r t e d e x c r e t e d i n r a t b i l e 4 hours a f t e r t he i.v. i n j e c t i o n were 36% f o r EDDP ( p a r t i t i o n c o e f f i c i e n t 0.04) and 0.2% f o r EMDP ( p a r t i t i o n c o e f f i c i e n t 1 3 . 4 ) ( 9 4 ) . EDDP l e v e l s found i n b i l e samples i n the i n t e r a c t i o n s t u d y a r e shown i n Table X I where 20-30% o f a d m i n i s t e r e d methadone was found t o be e x c r e t e d i n t o t h e b i l e as EDDP. Table X I c l e a r l y i n d i c a t e s t h a t b i l i a r y e x c r e t e d EDDP does n o t show any d i f f e r e n c e between methadone o n l y and methadone-diazepam t r e a t e d r a t s (p >0.10). A n a l y s i s o f c o n j u g a t e d m e t a b o l i t e s from r a t b i l e SIM chromatograms of mono and d i h y d r o x y EMDP i s o l a t e d from r a t b i l e a r e shown i n F i g . 20. The i o n s (4_5, 48) chosen t o m o n i t o r were the base peaks f o r the compounds and had h i g h s e l e c t i v i t y . Diazomethane t r e a t m e n t and s e l e c t i v e e x t r a c t i o n o f the diazomethane t r e a t e d samples p r o v i d e d s e v e r a l advantages 130 TABLE X I . E x c r e t i o n o f EDDP from Methadone and Methadone-Diazepam T r e a t e d Rats Time (h) 1 -M 1-DM 2 -M 2-DM 3-M 3-DM 4 -M 4-DM 0-2 0. 365 0.386 0 . 282 0. 486 0.352 0.395 0. 379 0.496 2-11 1. 967 1. 932 1. 784 1. 758 1. 723 1.785 2. 002 1.727 11-23 0 . 435 0.146 0 . 227 0. 942 0.234 0.330 0. 099 0.204 T o t a l 2. 767 2.464 2 . 293 3. 186 2.309 2.510 2. 480 2.427 Dose % 25 .31 22 . 54 20 .98 29 .15 21.1-2 22.96 22 .69 22.20 Time (h) M DM E 0-2 0.344 ± 0 . 0431 0 .440 ± 0.0582 <0 .05 2-11 1.869 ± 0. 1364 1 . 800 ± 0 .0908 >0 .20 11-23 0.248 ± 0 . 1388 0 . 405 ± 0.3658 >0 .20 T o t a l 2.462 ± 0 . 220 2 .646 ± 0 . 361 •>o .10 P a i r e d e x p e r i m e n t s were performed u s i n g f o u r r a t s f o r metha-. done t r e a t m e n t (M) and an o t h e r f o u r r a t s f o r methadone and diazepam t r e a t m e n t (DM). The v a l u e s denote the amount o f EDDP (shown as EDDP p e r c h l o r a t e mg/kg r a t ) e x c r e t e d o v e r the time p e r i o d s , 0-2, 2-11, 11-23 hours a f t e r the methadone dose. The v a l u e s i n the lower p a r t o f the Table r e p r e s e n t mean ± SD o b t a i n e d f o r each o f the two groups. The v a l u e p was c a l c u l a t e d by u s i n g S t u d e n t ' s t t e s t . , . Dose % r e f e r s t o the p e r c e n t a g e of the dose e x c r e t e d as EDDP. (a) , (b) J 1 1 L 200 220 240 260 200 220 240 260 TEMPERATURE °C TEMPERATURE °C F i g . 20.;.' SIM chromatograms f o r (a) monohydroxy EMDP and (b) d i h y d r o x y EMDP a n a l y s 132 i n t he a n a l y s i s . U n d e r i v a t i z e d d i h y d r o x y EMDP was not d e t e c t a b l e i n the TIC p r o f i l e of the c o n j u g a t e f r a c t i o n . Diazomethane t r e a t m e n t and hexane e x t r a c t i o n of the sample t o t a l l y removed any i n t e r f e r e n c e s ( F i g . 2 1 ) . D e r i v a t i z a t i o n by diazomethane m e t h y l a t i o n a l s o had advantages i n terms o f p r e v e n t i n g o x i d a t i o n o f the p h e n o l i c and c a t e c h o l t ype meta-b o l i t e s . C a l i b r a t i o n c u r v e s were p r e p a r e d by u s i n g one of the b i l e samples i n d i f f e r e n t volumes (Table X I I ) . The r a t i o s o b t a i n e d from n a t u r a l sample a n a l y s i s were w i t h i n the c a l i b r a -t i o n r ange. The r a t i o o f m/e 246 t o m/e 24 7 and t h a t o f m/e 244 t o m/e 245 were 0.43-0.46 and 0.36-0.37, i n d i c a t i n g absence o f i n t e r f e r e n c e i n the a n a l y s i s i . e . the v a l u e s were the same as thos e shown i n T a b l e s V I I I and IX. As shown i n T a b l e s X I I I and XIV, an i n c r e a s e d e x c r e -t i o n o f c o n j u g a t e m e t a b o l i t e s was found d u r i n g t h e f i r s t 2 hours o f b i l e e x c r e t i o n i n the methadone-diazepam t r e a t e d r a t s . F o r HOEMDP 5.7% and 16.8% was e x c r e t e d d u r i n g the f i r s t 2 hours i n c o n t r o l and diazepam t r e a t e d r a t s , r e s p e c t i v e -l y . I n the case o f DiHOEMDP, the p e r c e n t a g e s e x c r e t e d were 5.3% and 10.7% f o r c o n t r o l and diazepam t r e a t e d r a t s , r e s p e c -t i v e l y . However, when the e x c r e t i o n of c o n j u g a t e m e t a b o l i t e s was f o l l o w e d o v er the e n t i r e 2 3 hour p e r i o d , t h e r e was no d i f f e r e n c e o b s e r v e d between the methadone o n l y t r e a t e d and methadone-diazepam t r e a t e d r a t s (HOEMDP, p >0.20, DiHOEMDP p >0.20). 133 (a) (b) m/e (c) F i g . 21. TIC p r o f i l e (a) and Mass chromatogram (b) o f v the c o n j u g a t e f r a c t i o n o b t a i n e d from methadone dosed r a t b i l e b e f o r e back e x t r a c t i o n and TIC p r o f i l e (c) a f t e r back e x t r a c t i o n . 134 TABLE X I I . C a l i b r a t i o n Curve Data f o r Conjugate M e t a b o l i t e s HOEMDP  ml o f sample 1.6 0.8 0.4 0 . 2 0.1 m/e 246/m/e 247 0 .443 0.431 0.461 0.448 0.466 m/e 238/m/e 247 1.440 0 . 712 0 . 359 0 .162 0.092 S l o p e , 0.903; i n t e r c e p t , -0.00683; r , 0.9998 DiHOEMDP  ml o f sample 1.6 0.8 0.4 0 . 2 0.1 m/e 244/m/e 245 0.373 0 . 36.5 0 . 366 0.367 0 . 377 m/e 237/m/e 245 1.204 0.619 0 . 341 0.153 0 .074 S l o p e , 0.748; i n t e r c e p t , 0.014; r , 0.9992 1 ml o f i n t e r n a l s t a n d a r d (as p r e p a r e d i n E x p e r i m e n t a l , p. 36) was added. 135 TABLE X I I I . E x c r e t i o n o f Monohydroxy EMDP from Methadone and Methadone-diazepam T r e a t e d Rats Time(h) 1-M 1-DM 2-M 2-DM 3-M 3-DM 4-M 4-DM ' 0-2 0.447 3.654 0.961 3.275 2.893 5.092 0.874 4.316 2-11 16.545 18.888 10.377 11.650 20.609 15.400 12.647 16.200 11-23 6.270 2.966 9.827 5.469 2.848 4.554 5.995 5.454 T o t a l ' 23.304 25.508 21.165 20.394 26.350 25.046 19.516 25.970 Time(h) M DM £ 0-2 1.293 ± 1.089 4.084 ± 0.797 <0.01 2-11 15.044 ± 4.499 15.534 ± 2.988 >0.20 11-23 6.235 ± 2.853 4.611 ± 1.177 >0.20 T o t a l 22.583 ± 2.951 24.229 ± 2.584 >0.20 P a i r e d e x p e r i m e n t s were performed u s i n g f o u r r a t s ' f o r methadone t r e a t m e n t (M) and ano t h e r f o u r r a t s f o r methadone and diazepam t r e a t m e n t (DM). The v a l u e s denote the r a t i o s ( r a t i o / k g r a t ) o f m/e 238 t o m/e 247 w i t h average o f two d e t e r m i n a t i o n s . The sam-p l e s were c o l l e c t e d o v e r t h e time p e r i o d s 0-2, 2-11, and 11-23 hour a f t e r the" methadone dose and were m o n i t o r e d by GCMS a t m/e 247, m/e 246, m/e 238 and m/e 237. The v a l u e s i n the lower p a r t o f the Table r e p r e s e n t mean .± SD o b t a i n e d f o r each o f t h e two groups. The v a l u e p was c a l c u l a t e d by u s i n g S t u d e n t ' s t t e s t . 136 TABLE XIV. E x c r e t i o n o f Di h y d r o x y EMDP from Methadone and Methadone-diazepam t r e a t e d r a t s Time (h) 1-M 1-DM 2-M 2-DM 3-M 3-DM 4-M 4-DM 0-2 1.794 3.302 0.509 2.009 1.840 2.218 0.726 2.028 2-11 16.510 14.968 10.537 14.160 18.270 14.375 13.511 16.671 11-23 7.079 2.376 11.171 7.526 2.122 5.150 7.243 3.711 T o t a l 25.383 20.646 22.217 23.695 22.23 21.74 21.48 22.41 Time(h) M 0-2 1.217 ± 0.698 2-11 14.707 ± 3.404 11-23 6.903 ± 3.706 T o t a l 22.825 ± 1.783 DM p_ 2.389 ± 0.615 <0.05 14.875 ± 2.356 >0.20 4.690 ± 2.203 >0.20 22.122 ± 1.275 >0.20 P a i r e d e x p e r i m e n t s were performed u s i n g f o u r r a t s f o r methadone t r e a t m e n t (M) and ano t h e r f o u r r a t s f o r methadone and diazepam t r e a t m e n t (DM). The v a l u e s denote t h e r a t i o s ( r a t i o / k g r a t ) o f m/e 237 t o m/e 245 w i t h an average of two d e t e r m i n a t i o n s . The samples were c o l l e c t e d o v er the time p e r i o d s , 0-2, 2-11, and 11-23 hour a f t e r the methadone dose, and m o n i t o r e d by GCMS a t m/e 245, m/e 244, . - - ' m/e 237, and m/e 236. The v a l u e s i n t h e lower p a r t o f the Table r e p r e s e n t mean ± SD o b t a i n e d f o r each o f t h e two groups. The v a l u e p was c a l c u l a t e d by u s i n g S t u d e n t ' s t t e s t . 137 D i s c u s s i o n o f t h e methadone-diazepam i n t e r a c t i o n Drug" i n t e r a c t i o n s a re g e n e r a l l y shown by a n a l y z i n g drug and m e t a b o l i t e l e v e l s i n v a r i o u s b i o l o g i c a l samples, i . e . plasma, u r i n e , b i l e and t i s s u e s such as b r a i n and l i v e r . The a n a l y s i s o f methadone and m e t a b o l i t e s e x c r e t e d t h r o u g h the b i l i a r y r o u t e can be used t o a s s e s s drug i n t e r a c t i o n s a t t h e h e p a t i c l e v e l . The drug i n t e r a c t i o n c o u l d depend on the m e t a b o l i z i n g a c t i v i t y o f t h e l i v e r , t r a n s p o r t o f the drug t o the b i l i a r y r o u t e , h e p a t i c b l o o d f l o w , and drug b i n d i n g t o p r o t e i n (145). E x c r e t i o n o f m e t a b o l i t e s by t h e u r i n a r y r o u t e was c o n s i d e r e d n o t t o be s i g n i f i c a n t t o a n a l y z e because l e s s than 10% o f methadone and m e t a b o l i t e s i s ex-c r e t e d t h r o u g h t h e u r i n a r y r o u t e (144). Methadone l e v e l s i n r a t plasma c o u l d n o t be q u a n t i t a t e d even by m o n i t o r i n g the h i g h abundance m/e 72 i o n because o f a l i m i t e d a n a l y t i c a l s e n s i t i v i t y o f our SIM p r o c e d u r e . The r e s e a r c h was d e s i g n e d t o examine t h e a p p l i c a b i l i t y o f u s i n g r a t i o a n a l y s i s t o t h e drug m e t a b o l i s m s t u d y . A n a l y s i s o f c o n j u g a t e m e t a b o l i t e s by the method of r a t i o measurement was e x p e c t e d t o p r o v i d e a c c u r a t e d a t a on the change i n m e t a b o l i t e l e v e l s r e s u l t i n g from a c o n c o m i t a n t a d m i n i s t r a -t i o n o f a l a r g e dose o f diazepam (5 mg/kg) w i t h methadone (10 mg/kg s . c . ) . A n a l y s i s o f EDDP showed t h e absence o f a methadone-diazepam i n t e r a c t i o n (Table X I ) . T h i s r e s u l t was c o n s i s t e n t 138 w i t h a r e p o r t by R o e r i g e t a l . (38) t h a t methadone l e v e l s i n plasma and methadone and EDDP c o n c e n t r a t i o n s i n r a t u r i n e d i d n o t show any methadone-diazepam i n t e r a c t i o n . Shah e t a l . (59) showed i n t h e i r e x p e r i m e n t u s i n g mice (20 mg/kg i . p . diazepam and 5 mg/kg i . p . methadone) t h a t plasma -and b r a i n l e v e l s o f methadone were i n c r e a s e d by c o n c o m i t a n t a d m i n i s t r a t i o n o f diazepam. T h e i r r e s u l t was c o n t r a d i c t o r y t o t h e r e p o r t o f Shannon e t a l . ( 6 0 ) , whose expe r i m e n t w i t h 10 mg/kg i . p . diazepam and 0.6 mg/kg i . p . methadone f a i l e d t o show any i n t e r a c t i o n i n mice when plasma and b r a i n l e v e l s o f methadone were measured. S i n c e methadone i n plasma was n o t a n a l y z e d i n t h i s s t u d y an a t t e m p t was made t o c a l c u l a t e an apparent e l i m i n a -t i o n r a t e c o n s t a n t u s i n g EDDP d a t a from r a t b i l e (5 d a t a p o i n t s ) by means o f NONLIN (80). A s i m i l a r c a l c u l a t i o n o f a r a t e c o n s t a n t u s i n g b i l e samples was r e p o r t e d f o r t r i p a m i d e m e t a b o l i t e s (146) b u t i n t h i s work r e l i a b i l i t y of t h e c a l c u l a -t i o n was found t o be h e a v i l y dependent on s m a l l d i f f e r e n c e s i n b i l e f l o w w i t h time so t h a t b i l e d a t a c o u l d not be used f o r p h a r m a c o k i n e t i c c a l c u l a t i o n s . The r e s u l t o b t a i n e d by a n a l y z i n g c o n j u g a t e m e t a b o l i t e s by GCMS was q u i t e d i f f e r e n t from t h a t r e p o r t e d by R o e r i g e t a l . ( 3 8 ) , i n t h a t water s o l u b l e m e t a b o l i t e s from methadone i n u r i n e and l i v e r ?.were s i g n i f i c a n t l y d e c r e a s e d by diazepam a d m i n i s t r a t i o n . T h e i r r e s u l t i n d i c a t e d t h a t the drug i n t e r -a c t i o n between methadone and diazepam was r e f l e c t e d i n a d e c r e a s e d c o n c e n t r a t i o n of c o n j u g a t e m e t a b o l i t e s and t h a t 139 t h i s d e c r e a s e o f c o n j u g a t e m e t a b o l i t e s was i n d i r e c t e v i d e n c e f o r an i n c r e a s e o f methadone l e v e l s i n b r a i n . ' 1 The r e a s o n f o r t h e t r a n s i e n t i n c r e a s e o f m e t a b o l i t e s i n the i n i t i a l 2 hour p e r i o d was not sought but c o u l d be due t o c o m p e t i t i o n by diazepam w i t h methadone plasma p r o t e i n b i n d i n g . An e f f e c t o f diazepam on i n c r e a s i n g f r e e d r ug l e v e l s was i n d i c a t e d i n s t u d i e s o f d i a z e p a m - d i p h e n y l h y d a n t o i n (143). But t h e e f f e c t o f an i n c r e a s e o f f r e e d r ug might be m i n i m a l because i n t h e case of the d r ug w h i c h has h i g h h e p a t i c e x t r a c -t i o n , d r u g b i n d i n g t o the plasma i s not a c r i t i c a l f a c t o r i n the h e p a t i c m e t a b o l i s m (147). I n c o n c l u s i o n , t h e c o n c o m i t a n t a d m i n i s t r a t i o n o f diazepam w i t h methadone i n r a t s d i d not a f f e c t b i l i a r y e x c r e -t i o n o f EDDP and c o n j u g a t e m e t a b o l i t e s . Diazepam d i d not i n t e r a c t w i t h methadone a t t h e h e p a t i c m e t a b o l i s m l e v e l nor on the t r a n s p o r t o f m e t a b o l i t e s by the b i l i a r y e x c r e t i o n r o u t e . T h i s r e s u l t i s not c o n s i s t e n t w i t h t h a t r e p o r t e d by R o e r i g e t a l . (38) i n w h i c h a d e c r e a s e o f water s o l u b l e metabo-l i t e l e v e l s i n u r i n e and l i v e r was c o n s i d e r e d t o i n d i c a t e a m e t a b o l i c methadone-diazepam i n t e r a c t i o n . 140 P o t e n t i a l a p p l i c a t i o n s o f r a t i o a n a l y s i s t o drug  m e t a b o l i s m and p h a r m a c o k i n e t i c s t u d i e s A b i o s y n t h e t i c i n t e r n a l s t a n d a r d was used i n t h i s work t o measure r e l a t i v e changes o f t h e c o n j u g a t e d m e t a b o l i t e s o f methadone due t o a co n c o m i t a n t a d m i n i s t r a t i o n o f diazepam. T h i s use o f a s i m p l e measured r a t i o o f a m e t a b o l i t e t o i t s b i o s y n t h e t i c i n t e r n a l s t a n d a r d w i t h o u t knowing t h e a b s o l u t e q u a n t i t y o f a substance can be a v a l u a b l e t e c h n i q u e f o r pharma-c o k i n e t i c and d r u g m e t a b o l i s m s t u d i e s . T h i s method i s e s p e c i a l l y u s e f u l t o a n a l y z e d r ug and m e t a b o l i t e s f o r w h i c h a u t h e n t i c s t a n d a r d s a re not a v a i l a b l e . A s t a b l e i s o t o p e l a b e l e d p r e c u r s o r o f the dr u g produces an i d e a l i n t e r n a l s t a n d a r d t o measure t h e r e l a t i v e c o n c e n t r a t i o n o f t h e i r m e t a b o l i t e s . When the r a d i o i s o t o p e - TLC method f a i l s t o s e p a r a t e i s o l a t e d m e t a b o l i t e s , SIM u s i n g a b i o s y n - ' t h e t i c i n t e r n a l s t a n d a r d i s a v e r y c o n v e n i e n t method t o s e p a r a t e and q u a n t i t a t e the drug and i t s m e t a b o l i t e s . I n a d d i t i o n t o t h i s a p p l i c a t i o n , many m e t a b o l i t e s i n the same m i x t u r e can be a n a l y z e d u s i n g an i n t e r n a l s t a n d a r d m i x t u r e o b t a i n e d by u s i n g a l a b e l e d p r e c u r s o r . T h i s approach i s a p p l i c a b l e t o the b i p h e n y l m e t a b o l i s m s t u d i e s p u b l i s h e d by B e n f o r d e t a l . (148) and Halppap-Wood e t a l . (149). P h a r m a c o k i n e t i c s t u d i e s : When we measure t h e c o n c e n t r a -t i o n o f any dr u g by GCMS, dr u g c o n c e n t r a t i o n = a x peak h e i g h t r a t i o ( u n l a b e l e d / l a b e l e d ) + b. I n the case o f SIM a n a l y s i s 141 when u s i n g l a b e l e d a n a l o g s as i n t e r n a l s t a n d a r d s , b = 0 can be a c h i e v e d by s u b t r a c t i n g the background c o n t r i b u t i o n . T h i s was proved i n t h e SIM a n a l y s i s o f methadone and m e t a b o l i t e s (Table I I I ) . T h e r e f o r e , drug c o n c e n t r a t i o n = a x peak h e i g h t r a t i o . The o p t i m a l dynamic range o f the r a t i o i s u s u a l l y 0.1-10. T h i s range i s f u r t h e r a d j u s t a b l e by c h a n g i n g t h e amount o f l a b e l e d i n t e r n a l s t a n d a r d s . Even i f we do not know the v a l u e o f a, we can use t h e r a t i o o f u n l a b e l e d t o l a b e l e d f o r k i n e t i c s t u d i e s o f a d r u g . In o r d e r t o see the a p p l i c a b i l i t y o f the methodology t o a two-compartment model (150), the e q u a t i o n o f t h e model i s r e w r i t t e n a s : R = — == X ° a a Vc (a- k 2 i ) - a t ( k 2 i - B) ~ 3 t L a - B a - 3 -f o r methadone plasma d a t a r e p o r t e d by Swanson e t a l . (151). Xo A l l k i n e t i c c o n s t a n t s , a, 3, k 2 i , — 7 7 — are c a l c u l a b l e i n d e -ci. V C p e n d e n t l y o f a w i t h r a t i o s (R) a t t i m e s (t) by the NONLIN program (80). K i n e t i c approach t o t h e s t u d i e s o f h y d r o x y l a t i o n mechanism: As an example o f the a p p l i c a t i o n o f r a t i o a n a l y s i s t o p h a r m a c o k i n e t i c s t u d i e s , t h i s method can be used t o s t u d y the mechanism of f o r m a t i o n o f p h e n o l and c a t e c h o l m e t a b o l i t e s . A g e n e r a l scheme f o r the f o r m a t i o n o f hydroxy m e t a b o l i t e s i s shown i n the s e c t i o n o f r i n g h y d r o x y l a t i o n pathways o f methadone me t a b o l i s m (p. 79) I f t h e m e t a b o l i t e s can be a n a l y z e d i n plasma, t h e f o l l o w i n g p r o p o s a l can be made. 142 D euterium l a b e l e d drug A produces d e u t e r i u m l a b e l e d monohydroxy (Ml) and d i h y d r o x y m e t a b o l i t e s (M2). The m i x t u r e s e r v e s as an i n t e r n a l s t a n d a r d . A f t e r c o l l e c t i n g a s e r i e s o f samples from drug A w h i c h i s a m i x t u r e o f monohydroxy and d i h y d r o x y m e t a b o l i t e s a t d i f f e r e n t t i m e s , t h e samples are mixed w i t h e q u a l amount o f l a b e l e d i n t e r n a l s t a n d a r d ( b i o s y n -t h e t i c i n t e r n a l s t a n d a r d ) . A f t e r w o r k i n g up the samples, the r a t i o s R 1 f o r Ml (1£) and R 2 f o r M2 (11) o f t h e m e t a b o l i t e s t o the l a b e l e d i n t e r n a l s t a n d a r d a t d i f f e r e n t t i m e s (6) are o b t a i n e d . The b e s t f i t f o r t h e e q u a t i o n w h i c h shows one o f the pathways i s d e r i v e d by means o f NONLIN program. The equa-t i o n s f o r the f o r m a t i o n o f monohydroxy and d i h y d r o x y metabo-l i t e s by t h r e e p o s s i b l e mechanisms, d i r e c t i n s e r t i o n , e p o x i d e f o r m a t i o n [1]- and a n o t h e r e p o x i d e f o r m a t i o n mechanism [2] are shown i n T a b l e XV. 143 TABLE XV. P h a r m a c o k i n e t i c e q u a t i o n s f o r t h e forma-t i o n ~of monohydroxy and d i h y d r o x y m e t a b o l i t e s Direct insertion mechanism A(3) -> M1(10) M2(11) l l - i " - ( k 2 - k l ) a [e 1 - e 2 J A 0 k l k 2 k l ( k l _ k 2 ) ^ Mx(10) A (3) -*M2 (11) R l ^ i = T i a (k^k^a L R2 = M = A0 k2 r -a (k1+k2)a L Epoxide formation mechanism (2) e - ( k 1 + k 2 ) t -(kl+K_)t e 1 2 A(3) M2(11) I = ^  = A° kl [ e~k3t - e _ ( k l + k 2 ) t l *1 a (k 1+k 2-k 3)a L J £ D k 2 ( k r k 2 > e"k2 t 1 + k 3 _ k l " k 2 eT k3 t + <K2 k3*  k3~ kl~ k2 -(k 1+k 2)tj 144 SUMMARY AND CONCLUSIONS 1. F r i e d e l C r a f t s r e a c t i o n o f b r o m o p h e n y l a c e t o n i t r i l e w i t h 2 benzene- Hg gave d i p h e n y l a c e t o n i t r i l e l a b e l e d i n b o t h p h e n y l r i n g s . The l a b e l i n g p r o c e s s was found t o be a c o m b i n a t i o n o f 2 aluminum c h l o r i d e c a t a l y z e d exchange between benzene- and the r i n g p r o t o n s o f e i t h e r p h e n y l a c e t o n i t r i l e o r d i p h e n y l a c e -t o n i t r i l e and o f the r e v e r s i b l e n a t u r e o f the F r i e d e l C r a f t s r e a c t i o n . 2 2. Methadone- was s y n t h e s i z e d from e n r i c h e d d i p h e n y l -2 2 2 a c e t o n i t r i l e - H 1 Q . The m e t a b o l i t e s , EDDP- H 1 Q , EMDP- H 1 Q , and 2 DDP- H 1 Q were s y n t h e s i z e d from 4 - d i m e t h y l a m i n o - 2 , 2 - d i p h e n y l -2 p e n t a n o i c a c i d - H-^ Q p r e p a r e d u s i n g D2S0^ t o m a i n t a i n l a b e l 2 2 2 e n r i c h m e n t . Methadone- H^, EDDP- H^, and EMDP- were s y n t h e -2 s i z e d u s i n g C H^CH-^Br as s t a r t i n g m a t e r i a l . A l l compounds 2 e x c e p t f o r methadone- a r e new compounds. 3. Methadone and i t s m e t a b o l i t e s fragmented u s i n g E I mass s p e c t r o m e t r y t o g i v e i o n s common t o d i p h e n y l b u t a n e - c o n t a i n i n g compounds. These were u s e f u l t o i d e n t i f y new m e t a b o l i t e s o f methadone. Mass s p e c t r a o f t h e d e u t e r i u m l a b e l e d a n a l o g s p r o v i d e d c o n f i r m a t i o n o f t h e i o n s t r u c t u r e s . A r y l r i n g m i g r a -t i o n was ob s e r v e d i n t h e f r a g m e n t a t i o n p r o c e s s e s f o r EDDP. 145 4. Methadone l e v e l s i n plasma and s a l i v a were a n a l y z e d by m o n i t o r i n g m/e 72 w i t h 2 - d i m e t h y l a m i n o - 4 , 4 - d i p h e n y l - 5 -nonanone p e r c h l o r a t e as the i n t e r n a l s t a n d a r d . The lower l i m i t o f r e p r o d u c i b l e q u a n t i t a t i o n o f methadone i n 0.5 ml o f plasma o r s a l i v a t a k e n f o r e x t r a c t i o n was 20 ng. The mean r a t i o s o f s a l i v a t o plasma f o r two p a t i e n t s were 0.55 ± 0.15 (SD) and 0.48 ± 0.10 (SD). 5. A n a l y s i s o f methadone and EDDP i n p a t i e n t s ' u r i n e showed t h a t the c a l i b r a t i o n e q u a t i o n s p r e p a r e d f o r the a n a l y s i s were c o n s i s t e n t w i t h t h o s e c a l c u l a t e d by u s i n g t he r a t i o s of e q u a l amount o f drug t o i n t e r n a l s t a n d a r d ( s t a n d a r d r a t i o ) , i n d i c a t i n g t h a t t he c o n c e n t r a t i o n o f t h e compounds can be a n a l y z e d w i t h o u t c o n s t r u c t i n g c a l i b r a t i o n c u r v e s . 6. The d e u t e r i u m l a b e l e d a n a l o g s p r o v e d t o be v e r y u s e f u l f o r t he GCMS d e t e c t i o n and i d e n t i f i c a t i o n o f methadone meta-b o l i t e s . The c o n j u g a t e d f r a c t i o n from r a t b i l e was found t o c o n t a i n a new m/e 72 c o n t a i n i n g compound. On the b a s i s o f GCMS d a t a the new m e t a b o l i t e was a s s i g n e d t he s t r u c t u r e , N -methylene-l-methyl-3,3-diphenyl-4-oxo-hexaneamine o x i d e (methadone n i t r o n e ) . The n i t r o n e appeared t o r e s u l t from de-c o m p o s i t i o n o f N-hydroxynormethadone d u r i n g work up p r o c e d u r e s . The d e t e c t i o n o f the n i t r o n e i m p l i e s t h a t methadone i s not a l l s p o n t a n e o u s l y c y c l i z e d t o EDDP a t t h e s i t e o f en z y m a t i c N - o x i d a t i o n . 7. I n d i r e c t e v i d e n c e f o r t h e s t r u c t u r e o f the new metabo-l i t e was o b t a i n e d from c h e m i c a l o x i d a t i o n o f EDDP p e r c h l o r a t e 146 w i t h m - c h l o r o p e r b e n z o i c a c i d i n which 4 , 4 - d i p h e n y l - 2 , 5 -heptanedione and t h e methadone n i t r o n e were o b t a i n e d as p r o d u c t s . Mass s p e c t r a l d a t a , NMR, and IR were used t o i d e n t i f y t h e s e compounds. A mechanism f o r the f o r m a t i o n o f n i t r o n e and the d i k e t o n e from o x i d a t i o n o f EDDP p e r c h l o r a t e was proposed. 8. Three p r o c e s s e s f o r t h e E I f r a g m e n t a t i o n o f methadone n i t r o n e were proposed. The proposed f r a g m e n t a t i o n p r o c e s s e s were a l s o found t o a p p l y t o amphetamine n i t r o n e . 9. A i r o r c h e m i c a l o x i d a t i o n o f EDDP base gave DDP as the main p r o d u c t and the e v i d e n c e s u g g e s t s t h a t DDP i s not a t r u e m e t a b o l i t e o f methadone. MCPBA o x i d a t i o n of EMDP base gave 2 - e t h y l - 5 - m e t h y l - 3 , 3 - d i p h e n y l p y r r o l i d y 1 - 1 , 2 - o x a z i r a n . 10. The f e a s i b i l i t y o f u s i n g b i o s y n t h e t i c i n t e r n a l s t a n d a r d s and SIM was d e r i v e d f o r drug m e t a b o l i s m and k i n e t i c s t u d i e s . Drug c o n c e n t r a t i o n = s l o p e x r a t i o o f drug t o i n t e r n a l s t a n d a r d + i n t e r c e p t , where t h e i n t e r c e p t i s zer o and the s l o p e i s c a l c u l a b l e u s i n g the s t a n d a r d r a t i o which i s a drug dependent v a l u e . T h e r e f o r e , r a t i o = drug c o n c e n t r a t i o n / a (drug dependent c o n s t a n t v a l u e ) . The change o f drug c o n c e n t r a t i o n i s r e f l e c t e d on the r a t i o . P h a r m a c o k i n e t i c c o n s t a n t s can be o b t a i n e d by u s i n g t h e s e r a t i o v a l u e s . 11. A methadone-diazepam i n t e r a c t i o n s t u d y i n r a t s was d e s i g n e d i n which methadone, EDDP, and EMDP were a n a l y z e d by SIM u s i n g d e u t e r i u m l a b e l e d a n a l o g s as i n t e r n a l s t a n d a r d s and c o n j u g a t e d m e t a b o l i t e s u s i n g d e u t e r i u m l a b e l e d b i o s y n t h e t i c i n t e r n a l s t a n d a r d s . The d e u t e r i u m l a b e l i n g of the i n t e r n a l s t a n d a r d s was found t o be s t a b l e o v e r a wide range of pH and 147 under c o n d i t i o n s o f p r o l o n g e d i n c u b a t i o n . 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Ther., 206, 507 (1978). 162 APPENDIX NMR SPECTRA OF DEUTERATED COMPOUNDS 163 T"  1 I | I I I I i I I I " 4 0 0 I "i i i i i i i i i | i i—i—i I—i—i—i—r "i—i—i—i—r~i—i—r I M I I I I i M 0 Hi 5 0 0 3 0 0 I I I 1 i I 2 0 0 ~r~r 1 0 0 1 P a r t i a l l y d e u t e r a t e d d i p h e n y l a c e t o -2 n i t r i l e - H-^ Q S o l v e n t , CDC1 3; 86% l a b e l l i n g . i i i ' i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i 8 . 0 7J) 6 . 0 5.0 » - ' " 4 . 0 3 . 0 "I I I I I I I I I I I I I 'T 1 1 1 1 1 1 1 ~1—I 1 J I I II I I I I I I i L 2 . 0 1.0 0 n—I—I—i—r~r i I -r-|—i—i—r "i—i—i—i—r 'TTT 5 0 0 I • I I 4 0 0 I 3 0 0 I I I ; I I ! 1 I . > | I I 2 0 0 1 0 0 1 I I I 0 Hi E n r i c h e d d i p h e n y l a c e t o n i t r i l e - H S o l v e n t , CDC1 • 98% l a b e l l i n g 10 164 5 I • I • I • 1 I k 5 . Methadone- H 10 S o l v e n t , CD3OD; 97% l a b e l l i n g CTl U l 166 2 6. DDP- H 1 0 S o l v e n t , C D C l 3 ; 97% l a b e l l i n g 7. EMDP- 2H 1 Q S o l v e n t , CD^OD; 96% l a b e l l i n g . 167 

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