"Pharmaceutical Sciences, Faculty of"@en . "DSpace"@en . "UBCV"@en . "Kang, Gun-Il"@en . "2010-04-15T19:34:38Z"@en . "1981"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "Deuterium labeled methadone and deuterium labeled metabolites\r\nwere synthesized to use in gas chromatography mass spectrometry (GCMS) studies of the metabolic pathways of metha-done in rats. These compounds were also useful to develop sensitive\r\nand selective analytical methods to study the pharmacokinetics\r\nand disposition of methadone.\r\nSynthesis of the deuterium labeled compounds was mainly achieved by using known procedures with special treatments required to provide label enrichment.\r\nUsing the labeled and unlabeled derivatives, mass fragmentation\r\nprocesses that are common to methadone and its metabolites\r\nwere defined. Aryl ring migration was observed in a fragmentation\r\nprocess for 2-ethylidene-l,5-dimethyl-3,3-diphenyl pyrrolidine (EDDP). This aryl ring migration was not a favorable\r\nprocess for ring substituted EDDP analogs.\r\nVarious aspects regarding the optimization of the selected ion monitoring (SIM) analysis of methadone and its metabolites in biological fluids are described. The SIM analysis\r\nusing deuterium labeled compounds as internal standards generally proved to be selective but not as sensitive as expected using electron impact ionization (EI) conditions of GCMS. One advantage of using SIM over GC analysis was described\r\nin terms of ratio analysis. Quantitation of methadone\r\n\r\nin human plasma and saliva using SIM gave a lower limit of sensitivity of 20 ng/0.5 ml of sample by monitoring the base peak, m/e 72. The mean methadone ratios of saliva to plasma for two patients were 0.55 \u00B1 0.15 (standard deviation) and 0.48 \u00B1 0.10 (standard deviation).\r\nMethadone metabolism studies emphasized the detection of minor metabolites using special extraction methods for rat bile and using labeled and unlabeled compounds. Comparison of the mass spectra from total ion current (TIC) profiles of metabolites from unlabeled compounds with those from labeled compounds run as separate experiments gave GCMS evidence for methadone nitrone (N-methylene-l-methyl-3,3-diphenyl-4-oxo-hexanamine-oxide). Possibilities for the metabolic formation of N-hydroxynormethadone and the pharmacological significance of the detection of methadone nitrone were described. A proposal for metabolic studies to examine the potential formation\r\nof other methadone metabolites resulting from metabolic oxidation of nitrogen was presented.\r\nStructural evidence for the methadone nitrone molecule was obtained indirectly by chemical oxidation studies of metha done metabolites. m-Chloroperbenzoic acid treatment of EDDP perchlorate gave three products: methadone nitrone, 4,4-diphen yl-2,5-heptanedione (diketone), and 2-acetyl-5-methyl-3,3-di-phenyl-l-pyrroline. These compounds were identified from their IR, NMR and mass spectral data. Mass fragmentation processes\r\nwere defined for the methadone nitrone. Possible mechanisms for the formation of methadone nitrone and diketone from\r\n\r\nchemical oxidation of EDDP are proposed.\r\nSince diazepam is a drug widely abused by methadone maintenance patients, methadone-diazepam interaction studies were designed to analyze metabolites using deuterium labeled authentic compounds as internal standards. Metabolites in the conjugated fraction of rat bile were analyzed using deuterium labeled biosynthetic internal standards. Diazepam (5 mg/kg) was given to rats through a cannulated jugular vein and a subcutaneous\r\ndose of methadone (10 mg/kg) was given. Bile was collected through the cannulated bile duct over a period of 24 hours. The deuterium label was found to be stable even under severe conditions of incubation temperature and time. SIM analysis of bile sample extracts showed that concomitant administration of diazepam with methadone did not affect biliary\r\nexcretion of EDDP nor the conjugated metabolites. This indicates that diazepam does not interact with methadone at the hepatic metabolism level and with transport of the metabo-: lites by the biliary excretion route. Application of the use of a biosynthetic internal standard to drug metabolism and pharmacokinetic studies by means of ratio analysis was described with examples."@en . "https://circle.library.ubc.ca/rest/handle/2429/23616?expand=metadata"@en . "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 \u00C2\u00A9 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 \u00C2\u00B1 0.15 ( s t a n d a r d d e v i a t i o n ) and 0.48 \u00C2\u00B1 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) \u00E2\u0080\u00A2 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\u00C2\u00B0C, 6\u00C2\u00B0/min, h o l d a t 280\u00C2\u00B0C) 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\u00C2\u00B0C 8\u00C2\u00B0/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 \u00C2\u00B0f 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\u00E2\u0080\u0094OH CH2-(JH-N(CH3)2 CH, A C K\u00E2\u0080\u0094 CH, \ / CH- -?H CH, U C D 2-CH, V \\ CH2-CH-NHCHj CH, V CHCH, I 5 C N\u00E2\u0080\u0094CH, If CK- CH 1 2 AH. HO / 0 I C-CH2CHj HO C^Kg\u00E2\u0080\u0094CH-N(CHj)2 \" CHj CHCH, si X N \u00E2\u0080\u0094CH, / 3 CH I CHj CHnCH-z I 2 3 A C N \ / CH^ -CH 4-CH2CHj . x PBr. 1 6 ( C ^ ^ C ~ C = NH ^ ( C 6 H 5 ) 2 C \u00E2\u0080\u0094 CN - C H 2 - C H ^ - C H 3 i H \u00E2\u0080\u0094 C H - C H 3 \u00C2\u00B11 Br \u00E2\u0084\u00A2(CK3)2 ( C 6 H 5 ) 2 C \u00E2\u0080\u0094 CN C H 2 \u00E2\u0080\u0094 C H - N ( C H 3 ) 2 1\u00C2\u00B1 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 \u00E2\u0080\u0094 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\u00E2\u0080\u00A2to 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 \u00E2\u0080\u0094 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 \u00E2\u0080\u00A2 i s -H Ph 1-0 \u00E2\u0080\u00A20 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 \u00E2\u0080\u00A2CPh, \ s BrCN COEt N-Me I Me /CH2-MeCH Me2N*CN - CPh. v \u00E2\u0080\u00A2 C-CHMe 0 H ' + -Me2NCN H + CH\u00E2\u0080\u0094 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 \u00E2\u0080\u00A2 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 \u00C2\u00B0f 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 \u00E2\u0080\u00A2 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 \u00E2\u0080\u0094 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 . \u00E2\u0080\u00A2: . ... 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 \u00C2\u00B0 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\u00C2\u00B0C. 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\u00C2\u00B0C, oven tem p e r a t u r e 210\u00C2\u00B0C, and d e t e c t o r t e m p e r a t u r e 300\u00C2\u00B0C. 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\u00C2\u00B0C. 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)' \u00E2\u0080\u00A2 \. 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\u00E2\u0080\u009ESO.. 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 (\u00C2\u00A3, 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\u00C2\u00B0C 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 \u00E2\u0080\u0094 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 \u00E2\u0080\u0094 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\u00C2\u00B0C. 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\u00C2\u00B0C 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\u00C2\u00B0C. 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\u00E2\u0080\u009E0) .\u00E2\u0080\u00A2 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 \u00E2\u0080\u00A2 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\u00C2\u00B0C, 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\u00C2\u00B0C). 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\u00C2\u00B0C) 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\u00C2\u00B0C 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\u00C2\u00B0C. 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\u00E2\u0080\u00A2 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-\u00E2\u0080\u00A2 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 \u00E2\u0080\u00A2 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\u00E2\u0080\u0094CN 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 \u00C2\u00B1 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\u00E2\u0080\u009E0 and D\u00E2\u0080\u009ES0. 4 7 34 ... m/e 165 V \u00E2\u0080\u009E 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 (\u00C2\u00A31, 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\u00E2\u0080\u0094CH 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, \u00C2\u00A3 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 \u00C2\u00B1 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 \u00C2\u00B1 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 \u00C2\u00B1 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 \u00C2\u00B1 2.6, 83.6 \u00C2\u00B1 0.2, 2 94.2 + 3.8 and 93.9 \u00C2\u00B1 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 \u00C2\u00B1 0.14 2.03 \u00C2\u00B1 0. 06 15 2 2 yg HQ, 10 yg H 1 Q 1.99 \u00C2\u00B1 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 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-\u00C2\u00BB-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\u00E2\u0080\u0094N-OH \" V CH3 \ CH2-CH \u00E2\u0080\u0094M=0 CH3 CH-CH, 20 ,C NH \ CH\u00E2\u0080\u009E / \u00E2\u0080\u00A2CH CH, 64 0 C-CH\u00E2\u0080\u009ECH 2W'3 , 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, -:.-'\u00E2\u0080\u00A2'*- \u00E2\u0080\u00A2 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\u00E2\u0080\u009E 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 ) \u00E2\u0080\u0094 .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) \u00E2\u0080\u00A2\u00E2\u0080\u00A2 from m/e 118 (123) . ' v> CH \u00E2\u0084\u00A2 CH N=CH, CH== N-1 A H 2 C-H *\u00C2\u00B0 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\u00C2\u00AB 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 \u00E2\u0080\u00A2 5.6 Ha He 1 1 6 Hb 2.73 (dd) 3.18 (dd) C-\u00E2\u0080\u0094 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\u00E2\u0080\u009E - C = N - CH_R' \u00E2\u0080\u00A2> R - CH 0 - C = 0 Z | + Z Z | CH^ 131 132 113 2 MCPBA 19 CH3 C=0 CH\u00E2\u0080\u0094CH\u00E2\u0080\u0094 N CH, 2 j v / 2 \ J CH, CH2 CH3 C= 0 52 CH2\u00E2\u0080\u0094CH\u00E2\u0080\u0094S=;CH2 CH3 128 ' I N \u00E2\u0080\u0094 . 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\u00C2\u00B0C a t 10\u00C2\u00B0/min.), t h e major p r o d u c t DDP was e l u t e d a t 245\u00C2\u00B0C and another p r o d u c t e l u t i n g a t 225\u00C2\u00B0C 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\u00E2\u0080\u009EH,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\u00C2\u00B0C (120-270\u00C2\u00B0C a t 10\u00C2\u00B0/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 \u00E2\u0080\u00A2 in - a c i d . \u00E2\u0080\u00A2 (10% H C l , 70\u00C2\u00B0C ? 2 hours) and a m i x t u r e o f p r o d u c t s which has'peaks \u00E2\u0080\u00A2\u00E2\u0080\u00A2117 on GCMS c o n t a i n i n g \u00E2\u0080\u00A2; 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 \u00C2\u00A3-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 \u00C2\u00BB 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\u00E2\u0080\u009E . 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-\u00E2\u0080\u00A2M 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 \u00C2\u00B1 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 \u00C2\u00B1 0 . 0431 0 .440 \u00C2\u00B1 0.0582 <0 .05 2-11 1.869 \u00C2\u00B1 0. 1364 1 . 800 \u00C2\u00B1 0 .0908 >0 .20 11-23 0.248 \u00C2\u00B1 0 . 1388 0 . 405 \u00C2\u00B1 0.3658 >0 .20 T o t a l 2.462 \u00C2\u00B1 0 . 220 2 .646 \u00C2\u00B1 0 . 361 \u00E2\u0080\u00A2>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 \u00C2\u00B1 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 \u00C2\u00B0C TEMPERATURE \u00C2\u00B0C 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 \u00C2\u00A3 0-2 1.293 \u00C2\u00B1 1.089 4.084 \u00C2\u00B1 0.797 <0.01 2-11 15.044 \u00C2\u00B1 4.499 15.534 \u00C2\u00B1 2.988 >0.20 11-23 6.235 \u00C2\u00B1 2.853 4.611 \u00C2\u00B1 1.177 >0.20 T o t a l 22.583 \u00C2\u00B1 2.951 24.229 \u00C2\u00B1 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 .\u00C2\u00B1 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 \u00C2\u00B1 0.698 2-11 14.707 \u00C2\u00B1 3.404 11-23 6.903 \u00C2\u00B1 3.706 T o t a l 22.825 \u00C2\u00B1 1.783 DM p_ 2.389 \u00C2\u00B1 0.615 <0.05 14.875 \u00C2\u00B1 2.356 >0.20 4.690 \u00C2\u00B1 2.203 >0.20 22.122 \u00C2\u00B1 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 \u00C2\u00B1 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 = \u00E2\u0080\u0094 == X \u00C2\u00B0 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 , \u00E2\u0080\u0094 7 7 \u00E2\u0080\u0094 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\u00C2\u00A3) 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\u00C2\u00B0 kl [ e~k3t - e _ ( k l + k 2 ) t l *1 a (k 1+k 2-k 3)a L J \u00C2\u00A3 D k 2 ( k r k 2 > e\"k2 t 1 + k 3 _ k l \" k 2 eT k3 t + | 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 \u00E2\u0080\u00A2 98% l a b e l l i n g 10 164 5 I \u00E2\u0080\u00A2 I \u00E2\u0080\u00A2 I \u00E2\u0080\u00A2 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 "@en . "Thesis/Dissertation"@en . "10.14288/1.0095598"@en . "eng"@en . "Pharmaceutical Sciences"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Synthesis and applications of deuterated methadone and metabolites to biotransformation and disposition studies"@en . "Text"@en . "http://hdl.handle.net/2429/23616"@en .