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

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SYNTHESIS AND APPLICATIONS OF DEUTERATED METHADONE AND METABOLITES TO BIOTRANSFORMATION AND DISPOSITION STUDIES by GUN I L KANG B.Sc.  Seoul National  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 o l u m b i a , 1977  A THESIS SUBMITTED I N PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES D i v i s i o n of Pharmaceutical Chemistry, Faculty  of Pharmaceutical Sciences  We a c c e p t t h i s t h e s i s a s c o n f o r m i n g to the required  standard  THE UNIVERSITY OF B R I T I S H COLUMBIA O c t o b e r 1981 © Gun I I K a n g , 1981  In p r e s e n t i n g  this  thesis i n partial  f u l f i l m e n t of the  r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e of B r i t i s h Columbia, I agree that it  freely  the L i b r a r y s h a l l  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 .  agree that p e r m i s s i o n for  University  f o r extensive  s c h o l a r l y p u r p o s e s may  for  financial  shall  Gun I I Kang  of  Pharmaceutical Sciences  The U n i v e r s i t y o f B r i t i s h 2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5 ^ . Date  DE-6  (2/79)  Dec. 2nd, 1981 '  Columbia  my  It is thesis  n o t be a l l o w e d w i t h o u t my  permission.  Department  thesis  be g r a n t e d by t h e h e a d o f  copying or p u b l i c a t i o n of this  gain  further  copying of t h i s  d e p a r t m e n t 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 . understood that  I  make  written  ii  ABSTRACT  D e u t e r i u m l a b e l e d methadone a n d 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 spectrometry  (GCMS) s t u d i e s o f t h e m e t a b o l i c  done i n r a t s . sitive  mass  pathways o f metha-  T h e s e compounds w e r e a l s o u s e f u l t o d e v e l o p  a n d s e l e c t i v e a n a l y t i c a l methods t o s t u d y  sen-  t h e pharmaco-  k i n e t i c s and d i s p o s i t i o n o f methadone. Synthesis of the deuterium achieved  l a b e l e d compounds was m a i n l y  b y 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  required t o provide Using mentation  label  processes  process  pyrrolidine able process  frag-  t h a t a r e common t o methadone a n d i t s m e t a b o A r y l r i n g m i g r a t i o n was o b s e r v e d  i n a frag-  f o r 2-ethylidene-l,5-dimethyl-3,3-diphenyl  (EDDP). for ring  Various  T h i s a r y l r i n g m i g r a t i o n was n o t a f a v o r s u b s t i t u t e d EDDP  aspects  i nbiological  y s i s using deuterium  analogs.  regarding the optimization ofthe  selected i o n monitoring metabolites  enrichment.  t h e l a b e l e d a n d u n l a b e l e d d e r i v a t i v e s , mass  l i t e s were d e f i n e d . mentation  treatments  (SIM) a n a l y s i s o f methadone a n d i t s fluids are described.  The S I M a n a l -  l a b e l e d compounds a s i n t e r n a l  standards  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 n o t a s s e n s i t i v e a s expected GCMS. scribed  u s i n g e l e c t r o n impact i o n i z a t i o n  One a d v a n t a g e o f u s i n g S I M o v e r i n terms o f r a t i o a n a l y s i s .  (EI) c o n d i t i o n s o f  GC a n a l y s i s was d e -  Q u a n t i t a t i o n o f methadone  i n human p l a s m a and  s a l i v a u s i n g SIM  gave a l o w e r  limit  of  s e n s i t i v i t y o f 20 ng/0.5 ml o f s a m p l e by m o n i t o r i n g t h e p e a k , m/e f o r two 0.48  72.  The  mean methadone r a t i o s o f s a l i v a t o p l a s m a  p a t i e n t s w e r e 0.55  ± 0.10  base  (standard  ± 0.15  (standard d e v i a t i o n )  and  deviation).  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 bile  and  u s i n g l a b e l e d and  u n l a b e l e d compounds.  o f t h e mass s p e c t r a f r o m t o t a l  ion current  (TIC) p r o f i l e s  m e t a b o l i t e s f r o m u n l a b e l e d compounds w i t h t h o s e compounds r u n as  separate  methadone n i t r o n e  experiments  Comparison  from l a b e l e d  g a v e GCMS e v i d e n c e  Possibilities  o f N - h y d r o x y n o r m e t h a d o n e and  f o r the metabolic  the pharmacological  formation  significance  o f t h e d e t e c t i o n o f methadone n i t r o n e w e r e d e s c r i b e d . proposal for metabolic  o x i d a t i o n o f n i t r o g e n was S t r u c t u r a l evidence  from  forma-  metabolic  presented. f o r t h e methadone n i t r o n e  i n d i r e c t l y by c h e m i c a l  done m e t a b o l i t e s .  A  s t u d i e s t o examine the p o t e n t i a l  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  obtained  for  (N-methylene-l-methyl-3,3-diphenyl-4-oxo-  hexanamine-oxide) •  was  of  molecule  o x i d a t i o n s t u d i e s of metha  m-Chloroperbenzoic  a c i d treatment  o f EDDP  p e r c h l o r a t e g a v e t h r e e p r o d u c t s : methadone n i t r o n e , 4 , 4 - d i p h e n yl-2,5-heptanedione  ( d i k e t o n e ) , and  phenyl-l-pyrroline. their  IR, NMR  and  2-acetyl-5-methyl-3,3-di-  T h e s e compounds were i d e n t i f i e d  mass s p e c t r a l d a t a .  from  Mass f r a g m e n t a t i o n  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 . n i s m s 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 and  pro-  P o s s i b l e mecha diketone  from  iv chemical  o x i d a t i o n o f EDDP a r e p r o p o s e d . Since  diazepam i s a drug w i d e l y  a b u s e d by methadone  maintenance p a t i e n t s , methadone-diazepam i n t e r a c t i o n were d e s i g n e d  to analyze  metabolites  a u t h e n t i c compounds a s i n t e r n a l conjugated  was  standards.  standards.  given t o r a t s through a cannulated  c u t a n e o u s d o s e o f methadone  24 h o u r s .  Metabolites using  Diazepam  i n the  deuterium (5 mg/kg)  j u g u l a r v e i n and a sub-  (10 mg/kg) was g i v e n .  c o l l e c t e d through the cannulated  B i l e was  b i l e duct over a p e r i o d of  The d e u t e r i u m l a b e l was f o u n d t o be s t a b l e e v e n  under severe SIM  using deuterium l a b e l 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  labeled biosynthetic internal  studies  c o n d i t i o n s o f i n c u b a t i o n temperature and time.  analysis of bile  s a m p l e e x t r a c t s showed t h a t  concomitant  a d m i n i s t r a t i o n o f d i a z e p a m w i t h methadone d i d n o t a f f e c t a r y e x c r e t i o n o f EDDP n o r t h e c o n j u g a t e d  metabolites.  bili-  This  i n d i c a t e s t h a t d i a z e p a m d o e s n o t 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 a n d w i t h t r a n s p o r t o f t h e metabo-: l i t e s by t h e b i l i a r y  excretion route.  of a b i o s y n t h e t i c i n t e r n a l pharmacokinetic described with  Signature  standard  A p p l i c a t i o n o f t h e use  t o drug metabolism and  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 examples.  of Thesis  Supervisor  V  TABLE OF CONTENTS  ABSTRACT  i  L I S T OF TABLES  i  x i  L I S T OF FIGURES  x i i  ABBREVIATIONS  xiv  ACKNOWLEDGEMENTS  xvi  INTRODUCTION  1  M e t a b o l i c p a t h w a y s o f methadone  2  S y n t h e s i s o f methadone a n d 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 a n d m e t a b o l i t e s i n biological Pharmacokinetic  samples aspects  10 o f methadone  Methadone and d r u g i n t e r a c t i o n s  12 13  D e u t e r i u m l a b e l e d compounds f o r s t u d i e s o f pharmacokinetics  and d r u g m e t a b o l i s m  OBJECTIVES OF THE RESEARCH  18  Synthesis of deuterated  methadone a n d  metabolites Mass f r a g m e n t a t i o n SIM  15  19 studies  20  a n a l y s i s o f methadone a n d 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  vi 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  23  1. M a t e r i a l s General  23 chemicals  and r e a g e n t s  23  Solvents  23  M a t e r i a l s f o r animal  surgery  24  2. A n a l y t i c a l m e t h o d s  24  GCMS r e p e t i t i v e  scanning  24  GCMS s e l e c t e d i o n m o n i t o r i n g Gas  24  chromatography  25  C h e m i c a l i o n i z a t i o n GCMS  25  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  25  High performance l i q u i d chromatography....  25  NMR s p e c t r o s c o p y  26  Infrared spectroscopy  26  3. C h e m i c a l s t u d i e s  26  Synthesis o f deuterium and m e t a b o l i t e s  l a b e l e d methadone 26  2 S y n t h e s i s o f m e t h a d o n e - H^Q  26  Synthesis o f 4-dimethylamino-2,2diphenylpentanoic a c i d - H i ( 5 , 2 A r = H ) and DDP- H (6, 2Ar= Hio) ' 2  0  2  2  1 0  1 0  27  2  Synthesis of EDDP- Hi and E D D P - H 2  (3, 2 A r = H i ) 2  0  0  28  2  3  Synthesis o f EMDP- H and EMDP- H 2  2  3  (2, 2 A r = H ) 2  10  1 0  28  Synthesis o f 2-dimethylamino-4,4-diphenyl-5-nonanone p e r c h l o r a t e 28  vii Chemical 4.  oxidation studies  29  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 a n d 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  29  E x t r a c t i o n procedures  5.  7.  and s t a n d a r d  curve  preparation  30  Plasma and s a l i v a  30  Urines  31  GC a n a l y s i s . ;  32  Stability  o f EDDP. .  32  D e t e c t i o n o f methadone m e t a b o l i t e s  33  Animal  6.  experiments  33  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  Methadone-diazepam i n t e r a c t i o n s t u d i e s  34  Treatment o f animals  34  Sample p r e p a r a t i o n p r o c e d u r e s  36  Stability  37  experiments  Nonconjugated metabolites  37  Conjugated  38  Pharmacokinetic  metabolites and s t a t i s t i c a l  a n a l y s i s ...  RESULTS AND DISCUSSION 1.  29  38 40  S y n t h e s i s a n d mass s p e c t r o m e t r y a t e d methadone  of deuter-  and m e t a b o l i t e s  40  Deuterated  diphenylacetonitrile  40  Deuterated  methadone  43  Deuterated  methadone m e t a b o l i t e s  46  viii Spectroscopic observations 2.  52  S I M a n a l y s i s o f methadone a n d m e t a b o l i t e s in biological Selected  samples  .  ion monitoring  A n a l y s i s o f methadone i n p l a s m a a n d s a l i v a .  55 55 58  A n a l y s i s o f methadone a n d m e t a b o l i t e s / in,- u r i n e  62  :  Stability 3.  o f EDDP  66  D e t e c t i o n o f methadone m e t a b o l i t e s E x t r a c t i o n procedures  for rat bile  69 69  Nonconjugated f r a c t i o n  72  Conjugated  73  fraction  R i n g h y d r o x y l a t i o n p a t h w a y s o f methadone metabolism 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 P o s s i b i l i t i e s f o rthe metabqlic formation o f N - h y d r o x y m e t a b o l i t e s o f methadone .  83  Pharmacological significance of the detect i o n o f methadone n i t r o n e  87  78 81  P o s s i b l e m e t a b o l i c p a t h w a y s o f methadone p r o p o s e d i n F i g . 13 4.  Chemical  oxidation studies  89 92  Chemical  o x i d a t i o n o f methadone m e t a b o l i t e s  92  Chemical  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 b y mass s p e c t r o m e t r y 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 p a t h w a y s 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  95  ix IR s p e c t r o s c o p y o f methadone n i t r o n e r e l a t e d compounds F o r m a t i o n o f d i k e t o n e (61.) f r o m o f EDDP p e r c h l o r a t e  and 108  oxidation 10 9  Proposed mechanism f o r t h e c h e m i c a l format 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  structure  f o r compound  A (Fig.  13)  114  S y n t h e s i s o f EMDP o x a z i r a n 5.  I n t e r a c t i o n o f methadone Stability Bile  (184)  115  and d i a z e p a m  118  studies  .  collection  118 125  A n a l y s i s of nonconjugated m e t a b o l i t e s from r a t b i l e 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 rat bile  129  D i s c u s s i o n o f t h e methadone-diazepam interaction  137  Potential applications of r a t i o analysis t o d r u g m e t a b o l i s m and p h a r m a c o kinetic studies  140  Pharmacokinetic studies  125  140  K i n e t i c approach t o the s t u d i e s of h y d r o x y l a t i o n mechanism SUMMARY AND  141  CONCLUSIONS  144  REFERENCES APPENDIX  148 NMR  S p e c t r a o f D e u t e r a t e d Compounds  Spectrum 1  162  P a r t i a l l y deuterated diphenylacetonitrile- H  163  2  1 0  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  163  2  Spectrum 3 Spectrum 4  M e t h a d o n e n i t r i l e - H-^Q 164 4-Dimethylamino-2,2-diphenylpentanoic 2  acid- H  Nn  164  X  Spectrum 5 Spectrum 6 Spectrum 7 Spectrum 8 Spectrum 9  2 Methadone- H 2 DDP- H . 1Q  EMDP- H 2 EDDP- H 2 EDDP2  1 Q  3  n  1 Q  165 166 166 16 7 16 7  xi  L I S T OF TABLES  Number I II III IV V VI  VII  VIII IX X XI XII XIII XIV XV  Page NMR o f C-4 P r o t o n s o f M e t h a d o n e M e t a b o l i t e s  54  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 V o l u m e s upon t h e O b s e r v e d I o n R a t i o s  63  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  64  Mass S p e c t r a l D a t a o f T I C O b t a i n e d f r o m Diazomethane T r e a t e d Conjugate F r a c t i o n  ....  H i g h R e s o l u t i o n Mass F r a g m e n t a t i o n D a t a f o r M e t h a d o n e N i t r o n e (5_2)  75 96  NMR o f C-5 a n d g e m i n a l p r o t o n s o f methadone n i t r o n e (5_2) a n d 3 , 4 - d i m e t h o x y a m p h e t a m i n e n i t r o n e (127)  10 7  Stability/of EMDP  119  D e u t e r a t e d M e t h a d o n e , EDDP a n d  2 S t a b i l i t y o f D e u t e r i u m i n HOEMDP- H  121  g  2 S t a b i l i t y o f D e u t e r i u m i n DiHOEMDP- H E f f e c t o f Diazepam Treatment on t h e B i l e Flow o f Rats  126  E x c r e t i o n o f EDDP f r o m M e t h a d o n e a n d Methadone-Diazepam T r e a t e d Rats  130  C a l i b r a t i o n Curve Data f o r Conjugate Metabolites  134  E x c r e t i o n o f M o n o h y d r o x y EMDP f r o m M e t h a d o n e and M e t h a d o n e - d i a z e p a m T r e a t e d R a t s  135  E x c r e t i o n o f D i h y d r o x y EMDP f r o m M e t h a d o n e and M e t h a d o n e - d i a z e p a m T r e a t e d R a t s  136  Q  122  Pharmacokinetic equations f o r theformation o f t h e m o n o h y d r o x y a n d d i h y d r o x y m e t a b o l i t e s . 143  xii  L I S T OF  FIGURES  Number  Page  1  Metabolic  Pathways o f Methadone  3  2  M a s s 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 - 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 . ( 1 9 ) ; (c) a f t e r enrichment 2  3  Mass s p e c t r a o f done- !^  (a) methadone and  4  Mass s p e c t r a o f  (a) DDP  5  Mass s p e c t r a o f  (a) EDDP,  (b)  and  (b) D D P - H  49  2  1 Q  2 (b) EDDP- H ,  and  1 Q  50  2  Mass s p e c t r a o f  7  SIM  8  9  41 44  0  6  -\  metha-  2  (c| E D D P - H  :  3  (a) EMDP and  (b) E M D P - H 2  1 Q  ...  53  c h r o m a t o g r a m (m/e 72) o f methadone f r o m saliva M e t h a d o n e c o n c e n t r a t i o n i n p l a s m a and s a l i v a o f a m a i n t e n a n c e p a t i e n t (90 mg/day dosage)  60  D e c o m p o s i t i o n o f EDDP b a s e t o DDP DDP)  67  10  E x t r a c t i o n P r o c e d u r e f o r Rat  11  TIC  (  EDDP,  Bile  70  p r o f i l e of diazomethane t r e a t e d conjugate fraction. (GCMS: 150-280°C, 6°/min, h o l d a t 280°C)  12  Mass s p e c t r u m o f m/e  13  Possible metabolic  14  TIC  60  72 c o n t a i n i n g m e t a b o l i t e p a t h w a y s f o r methadone  ....  p r o f i l e ( a : GCMS, 200-280°C 8°/min.) and HPLC (b) o f EDDP p e r c h l o r a t e o x i d i z e d products  74 77 84  94  xiii Number  Page  15  NMR s p e c t r u m  16  I R o f methadone n i t r o n e  17  NMR  18  19 20 21  o f methadone n i t r o n e  106 110  (a) a n d I R (b) o f 4 , 4 - d i p h e n y l - 2 , 5 ' h e p t a n e d i o n e (6J.)  I l l  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) a n d 4 , 4 - d i p h e n y l 3,6-heptanedione (61.) b y 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  SIM c h r o m a t o g r a m s f o r EMDP and methadone (c)  127  ( a ) , EDDP  (b),  SIM c h r o m a t o g r a m s f o r (a) m o n o h y d r o x y EMDP and (b) d i h y d r o x y EMDP a n a l y s i s  131  T I C p r o f i l e (a) a n d Mass c h r o m a t o g r a m (b) of t h e conjugate f r a c t i o n o b t a i n e d from methadone d o s e d r a t b i l e b e f o r e b a c k e x t r a c t i o n a n d T I C 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  xiv  ABBREVIATIONS  Ar -  Phenyl  a.mli'u.  atomic  CI  chemical  DDP  mass  ionization  1,5-dimethyl-3,3-diphenyl-2-pyrrolidone  DMSO  dimethyl  dR  deoxyribose  EDDP EI EMDP  unit  sulfoxide  2-ethylidene-l,5-dimethyl-3,3-diphenylpyrrolidine electron  impact  2-ethyl-5-methyl-3,3-diphenyl-l-pyrroline  EtOH  ethanol  GC  gas chromatography  GCMS  g a s c h r o m a t o g r a p h y mass  GSH  reduced g l u t a t h i o n e  HPLC  high pressure  i.d.  internal  I.P.  intraperitoneal  IR  infrared  spectroscopy  I.S.  internal  standard  i.v.  intravenous  MCPBA  m-chloroperbenzoic  MeOH  methanol  mp  melting point  spectrometry  (performance)  liquid  chromatography  diameter  acid  (spectrophotometer)  XV NMR  n u c l e a r magnetic resonance  o.d.  external  PFK s.c.  diameter  perfluorokerosene subcutaneous  SD  standard deviation  SIM  selected  *"l/2  biological  TIC  t o t a l i o n chromatogram  TLC  thin  TMCS  trimethylchlorosilane  ion monitoring  layer  half-life  chromatography  TMS  tetramethylsilane  UV  ultraviolet  Note:  (spectroscopy)  Methadone n i t r o n e the  nitrone  i s used f o r convenience t o d e s c r i b e  f o u n d i n m e t a b o l i s m s t u d i e s o f methadone a n d  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 . the  c h e m i c a l i s normethadone n i t r o n e o r  Specifically N-methylene-1-  methyl-3.3-diphenyl-4-oxo-hexanamine-oxide.  xvi  ACKNOWLEDGEMENTS  T h i s t h e s i s h a s b e e n made p o s s i b l e Dr. F r a n k A b b o t t . S. F e r g u s o n  by t h e h e l p o f  The a u t h o r t h a n k s Mr. R. B u r t o n a n d M i s s  for their technical assistance.  The a u t h o r i s  i n d e b t e d t o D r . S. Chan o f t h e D e p a r t m e n t o f 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 , D r . G. E i g e n d o r f o f t h e D e p a r t m e n t o f Chemistry  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 , D r . K.  McErlane  f o r HPLC, a n d D r . G. G u d a u s k a s o f t h e B.C. C a n c e r  Research  Center  The  f o r c h e m i c a l i o n i z a t i o n mass  author would l i k e  t o thank  D r . B. R i e d e l , D r . J . M c N e i l l ,  D r . G. B e l l w a r d , D r . T. B r o w n , D r . D. G o d i n , for helpful discussions.  spectrometry.  and Dr. J . O r r  The h e l p o f D r . J . A x e l s o n a n d  D r . Y.K. Tam i n a n i m a l e x p e r i m e n t s  i sgreatly appreciated.  xvii  DEDICATION  To my w i f e a n d d a u g h t e r s  1  INTRODUCTION  Methadone d u r i n g W o r l d War  (1) was  I I and  first  s y n t h e s i z e d by  t h e d r u g was  i n 194 7 as a m o r p h i n e s u b s t i t u t e .  made c l i n i c a l l y a v a i l a b l e  The  t i e s o f methadone a r e q u a l i t a t i v e l y  a German c h e m i s t  pharmacological  proper-  i d e n t i c a l to those  of  morphine. The  d o s e o f methadone as an a n a l g e s i c i s i n t h e  same  1  r a n g e as t h a t o f m o r p h i n e . effects  The  d r u g has  i n some p a t i e n t s upon r e p e a t e d  t h e r e f o r e l i m i t e d use  was  only  sedative  administration  and  allowed.  M e t h a d o n e shows a c o n s i d e r a b l e a c t i o n when g i v e n o r a l l y .  marked  degree of  While the withdrawal  analgesic  syndrome o f  methadone i s s i m i l a r t o t h a t o f m o r p h i n e , i t d e v e l o p s  slowly  and  a  i s l e s s i n t e n s e and  more p r o l o n g e d .  d u r a t i o n o f a c t i o n w i t h an a v e r a g e p a r e d w i t h 2.5-3  hours f o r morphine.  The °f  The  d r u g has  25 h o u r s compossibility  of  long  2 methadone t r e a t m e n t first  reported  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  i n 1965 b y D o l e a n d N y s w a n d e r .  By 1977,  i n the  United States alone, over  90,000 p a t i e n t s w e r e e n r o l l e d i n m e t h a -  done m a i n t e n a n c e p r o g r a m  w i t h c o s t s b e t w e e n $4.00 a n d $5.50 a  day  per patient(1).  M e t a b o l i c p a t h w a y s o f methadone S t u d i e s on m e t a b o l i c  p a t h w a y s o f methadone h a v e b e e n  performed t o determine 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 a n d 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 metabolites al.  to activity  (2), Pohland  and t o x i c i t y o f t h i s d r u g .  Beckett e t  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 A n g g & r d e t a l . (8) showed t h a t methadone u n d e r g o e s d e methylation  t o f o r m EDDP (_3) a n d i n t u r n EMDP  oxidized to ring hydroxylated metabolites. to methadol the m e t a b o l i c  (7_) and N - d e m e t h y l m e t h a d o l .  (4J w h i c h i s t h e n  Methadone i s r e d u c e d  F i g . 1 i s a summary o f  p a t h w a y s o f methadone a s f o u n d i n r a t s and i n  humans. M e t h a d o n e a n d i t s m a j o r m e t a b o l i t e , EDDP h a v e b e e n analyzed gated  t o describe the pharmacokinetics  metabolites  o f methadone.  (3_, 1_0, 11) w e r e a l s o c o n s i d e r e d  portant 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  Conju-  t o be i m concomitant  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 h a s 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 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 . t o be i n a c t i v e a s a n a l g e s i c s  (3).  and t o x i c i t y o f  EDDP a n d EMDP w e r e  found  M e t h a d o l and demethylmethadol  3  CHOH-CHCHj CH-CH-N(CHj) 2  2  CH  U  C D2  2  3  o II  C—OH CH-(JH-N(CH) CH, 2  32  CH, CH-CH-NHCHj \\ V CH, C IIHCH,5  C  C  K— CH, -H CH,  \CH-  /  If  ?  II-CHCHj C ^CKg—CH-N(CHj) " CHj  1  N—CH,  CK2  CH  0  /  2  2  2  V A  HO  HO  CHCH, X  si  N —CH, / CH CHj  AH.  3  I  CHCHj  CHnCH-z  I3 AH^ -CH C C N 2  \  Fig.  2  4-  <M\  CH —CH CHj  /  1.  hO HC  C N/ CY -CH 11  M e t a b o l i c Pathways o f Methadone  4 account f o r only p a r t of the a n a l g e s i c a c t i v i t y  ( 5 ) . The p o s t u -  l a t e t h a t t h e m e c h a n i s m o f a c t i o n o f methadone m i g h t be e x p l a i n e d i n r e l a t i o n t o t h e b i n d i n g o f methadone a n d -me-tnadone . m e t a b o lites  to cellular  c o m p o n e n t s (10) h a s n o t b e e n p r o v e d .  e t a l . (11-12) e v a l u a t e d and  reported  t h e c h r o n i c t o x i c i t y o f methadone  t h a t methadone t r e a t m e n t  e f f e c t s without  Kreek  toxic effects.  caused minimal  Medical  side  complications(13)  and  h e p a t i c damage (14) w e r e s t u d i e d i n r e l a t i o n t o n a r c o t i c a d d i c tion.  T h i s phenomenon i s n o t l i m i t e d t o methadone b u t a s s o -  ciated with narcotics i n general.  A s p e c i f i c metabolic  route  does n o t appear t o be i n v o l v e d i n t h e t o x i c i t y o f methadone.  Synthesis  o f methadone a n d i t s a n a l o g s  Two m e t h o d s a r e commonly a v a i l a b l e f o r t h e s y n t h e s i s o f methadone. al.  The s y n t h e t i c method o u t l i n e d by S c h u l t z e t  (15) u s e d a c o n d e n s a t i o n  reaction of diphenylacetonitrile  (12) w i t h 2 - d i m e t h y l a m i n o i s o p r o p y l ence o f NaNH  2  t o f o r m two i s o m e r s ,  isomethadone n i t r i l e by  c h l o r i d e (1T3) i n t h e p r e s methadone n i t r i l e  ( 1 5 ) . Methadone n i t r i l e  fractional recrystallization  from hexane.  was  (1_4) a n d  separated  5  A n o t h e r method d e s c r i b e d b y E a s t o n e t a l . (16) i n v o l v e d the r e a c t i o n o f propylene oxide w i t h d i p h e n y l a c e t o n i t r i l e t o give 3,3-diphenyl-5-methyltetrahydro-2-furanone  i m i n e (16)  which i s f u r t h e r converted t o 4-bromo-2,2-diphehylpentane (_17) .  The bromo compound, 1_7 r e a c t e d w i t h d i m e t h y l a m i n e  methadone n i t r i l e (C H ) CH-CN 6  . ( 1  6  C  5  2  +  CH,  NaNH  2  C H — CH,  >  PBr.  x  ^ ^ C ~  -  to give  (_14) . .  12  nitrile  C =  NH  ^  CH -CH^-CH 2  ( C  6  H  5  3  )  2  C —  i  H  —  CN CH-CH  3  ±1  Br ™(CK ) 3  2  (  C  6  H  5  )  2  C —  CN  CH —CH-N(CH ) 2  3  2  1±  CH, *3  The n i t r i l e , _14 was c o n v e r t e d t o methadone a n d i t s analogs  (18) b y a G r i g n a r d  reaction.  6  C H ^ CH-N ( C H ) 3  (  2  ^  ? C H — CH-N ( C H ) 2  3  CH  2  3  18  2 Two d e u t e r i u m l a b e l e d m e t h a d o n e s , m e t h a d o n e 2 2 I^CE^CD^) and methadone - H,. ( 1 , A r = H^) have a p p e a r e d literature.  B o t h o f them w e r e s y n t h e s i z e d  (18, i n the  by t h e m e t h o d o f  2 Schultz  e t a l . ( 1 5 ) . 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 ( 1 7 ) . A p o s s i b l e use o f methadoneas an i n  o f methadone  v i v o m a r k e r f o r m o n i t o r i n g t h e methadone  intake of a mainte2  n a n c e p a t i e n t was a l s o s u g g e s t e d  ( 1 8 ) . Methadone-  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 a n a l y s i s t o determine plasma  was  i o n m o n i t o r i n g (SIM)  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 ( 2 0 ) . K r e e k e t a l . d o s e d d e u t e r i u m l a b e l e d R - ( - ) - m e t h a d o n e and S-(+)-methadone t o methadone m a i n t e n a n c e specifically  p a t i e n t s and m o n i t o r e d  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 a d o p t e d t o o b t a i n  methadone-  was u n u s u a l i n t h a t C D ^ C D B r i n s t e a d o f CT^CH^Br 2  l o w e d by a KOH c a t a l y z e d ' e x c h a n g e • t o (18,  R=CH CD ). 2  3  yield  was u s e d ,  the desired  fol-  compound  T h i s c o u l d be due t o a n 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 The s y n t h e s i s o f m e t h a d o n e - 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, A r = H^) w h i c h was o b t a i n e d b y 2 F r i e d e l - C r a f t s r e a c t i o n o f b e n z e n e - H,. a n d b r o m o p h e n y l a c e t o b thesis.  nitrile  in,CS  solvent.  2  Two methadone a n a l o g s , (17)  a n d t h e nonanone  as t h e i n t e r n a l  (1J3, R = C H C H C H C H ) 2  standards  s y n t h e s i s of these  t h e octanone 2  2  2  (21) w e r e  3  o f methadone  al.  acid  DDP.  rr  4  C H - C H - N ( C H ,3 )' 2 5  ""\  2  //  2  treatment  f o r m s t h e a c i d , 5_  to give the cyclized  1  /  H 0  2-diphenyl-  (6_) w e r e p u b l i s h e d b y G a r d n e r e t  w h i c h i s d e m e t h y l a t e d by S 0 C 1  2  COOH S 0 C 1  7  >  X  c.^  \\  CH-CH-N(CH  11  I  2  I  t o s y n t h e s i z e EDDP  T r e a t m e n t o f DDP w i t h C H L i g i v e s EDDP. 2  CH —CH  i  DDP i s u s e d a s a n i n t e r m e d i a t e EMDP.  N-CH,  - CH,  CH,  and  The  metabolites  (5_) a n d DDP  H S0  prepared  reactions.  ( 2 2 ) . H y d r o l y s i s o f methadone n i t r i l e  product,  3  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  S y n t h e t i c methods f o r 4 - d i m e t h y l a m i n o - 2 , pentanoic  2  f o r t h e a n a l y s i s o f methadone.  bromide o r b u t y l bromide f o r t h e G r i g n a r d  Synthesis  (1J3, R = C H C H C H )  5  d e m e t h y l a t e d b y HI a n d h e a t t o p r o d u c e EMDP.  EDDP i s  Perchlorate  8 formation  o f 3_ l e d t o t h e c o n v e r s i o n  of the exocyclic structure,  3_ t o t h e e n d o c y c l i c s t r u c t u r e , 19_ a s shown by NMR s t u d i e s ( 3 , 23) .  EMDP (_4) a n d 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 d o u b l e bond  20  (3, 23).  M e t h a d o l J_7) , a m e t a b o l i t e  o f methadone  mediate f o r t h e s y n t h e s i s of 1-a-acetylmethadol chemically prepared  by p l a t i n u m  oxide  r e d u c t i o n , o r s o d i u m - p r o p a n o l reduction The r a t i o  alyst or LiAlH^. was a c h i e v e d  hydrogenation,  LiAlH^  (2 4) o f m e t h a d o n e . with  o f t h e a - i s o m e r by t h e u s e o f Adams c a t -  Synthesis  by c h e m i c a l  o f n o r m e t h a d o l and d i n o r m e t h a d o l  N-demethylation procedures (25).  M e t h a d o n e N - o x i d e was p r e p a r e d  perbenzoic  (LAAM) was  o f i s o m e r s v a r i e d d e p e n d i n g upon t h e r e a g e n t s  preferable formation  methadone.  a n d an i n t e r -  by m i l d o x i d a t i o n o f  T r e a t m e n t o f methadone w i t h 2.5 m o l o f m - c h l o r o a c i d gave EDDP  methadone was r e p o r t e d Methoxymethadone  (26).  Permanganate o x i d a t i o n o f  t o produce a mixture  o f DDP a n d 2_1  (27).  (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  CH 0 3  Ph  H  0 I! ^C-CH CH  1-0  2  3  H' CH  • i s H CH,  •0  CH-CH-N (CH,), I CH,  21  22  of benzene i n t h e F r i e d e l - C r a f t s t e p o f t h e r e a c t i o n t o synthes i z e methadone ( 2 8 ) . No r e p o r t h a s a p p e a r e d on t h e 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 .  This  i sperhaps because o f t h e  f a c t t h a t EDDP w h i c h h a s t h e most i m p o r t a n t pharmacokinetics  implications for the  o f m e t h a d o n e , c a n be a n a l y z e d  by gas chromato-  graphy .  A t t e m p t e d 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 t o o b t a i n normethadone. /CH 2  MeCH  •CPh, \ COEt N-Me I Me  BrCN  s  The i s o l a t e d p r o d u c t  CH - - CPh. v MeCH /  2  Me N*CN + 2  • -Me NCN 2  C-CHMe 0  H'  + H  i n an a t t e m p t  was EDDP b e c a u s e C H — CPh, / \ MeCH C=CHMe 2  10 of spontaneous c y c l i z a t i o n demethylation yielded  o f normethadone.  Cyanogen  bromide  (29) o f methadone d i d n o t g i v e n o r m e t h a d o n e b u t  2-ethylidene-5-methyl-3,  3-diphenyltetrahydrofuran  (23) ( 3 0 ) .  A n a l y s i s o f methadone a n d 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 c h r o m a t o g r a p h y a n d S I M a n a l y s i s a r e two m a j o r m e t h o d s t o a n a l y z e methadone a n d 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 g a s c h r o m a t o g r a p h y f o r methadone  i n p l a s m a a n d u r i n e a n d 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  ( 2 1 , 3 1 , 3 2 ) . On t h e o t h e r h a n d , EMDP, a m i n o r  lite,  c o u l d n o t be q u a n t i f i e d  lack of s e n s i t i v i t y (33).  metabo-  i n human u r i n e b e c a u s e o f a  and s e l e c t i v i t y  o f gas chromatography  A SIM a s s a y by gas chromatography-mass  spectrometry  (GCMS) u n d e r e l e c t r o n i m p a c t c o n d i t i o n s ( E I ) was d e s c r i b e d for  t h e q u a n t i t a t i o n o f methadone b y m o n i t o r i n g m/e 2  d o n e ) , m/e standard, C^C^CH^)  297 (methadone- H ^ ) , a n d m/e  294  (metha-  308 f o r t h e i n t e r n a l  2 - d i m e t h y l a m i n o - 4 , 4 - d i p h e n y l - 5 - o c t a n o n e (1J3, R= ( 1 7 ) . By t h i s  assayed with a s e n s i t i v i t y  method,  methadone i n p l a s m a was 2  l i m i t o f 5 ng/ml.  M e t h a d o n e - H,.  h a s b e e n u t i l i z e d f o r t h e SIM a s s a y o f methadone i n human plasma u s i n g chemical l i m i t of this EI-SIM.  ionization  method was s i m i l a r  ( C I ) ( 1 9 ) . The  sensitivity  t o that reported using the  11 In  animal experiments  analytical d i f f i c u l t i e s are  f r e q u e n t l y encountered because o f t h e s m a l l s i z e o f samples, i n w h i c h 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 c o m p a r e d 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 . e x a m p l e , t h e k i n e t i c s o f methadone i n r a t p l a s m a  For  was s t u d i e d  14 by u s i n g sitivity  C - l a b e l e d methadone generally  (34) b e c a u s e o f a l a c k o f s e n -  shown i n t h e u s e o f g a s c h r o m a t o g r a p h y  S e n s i t i v e a n a l y t i c a l methods such a s radioimmunoassay  are  still  (36),  being developed  t o a n a l y z e methadone i n p l a s m a  (35).  by w h i c h a l o 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 s a m p l e s i z e o f 0.05 m l h a s b e e n a c h i e v e d . Conjugated  m e t a b o l i t e s ' a r e important from both 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 d r u g  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  inter-  i n quanti-  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 , it  i s n e c e s s a r y t o h a v e a means t o a n a l y z e c o n j u g a t e d m e t a b o -  lites  accurately.  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-  lites  a s i n t a c t m o l e c u l e s has'  Quantitative analysis  graphed TLC  (37).  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  the conjugate p o r t i o n s . tracted quantitatively  been r e c e n t l y r e v i e w e d  The h y d r o l y z e d m e t a b o l i t e s  are ex-  i n t o o r g a n i c s o l v e n t s and chromato-  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  i s t h e s e p a r a t i o n method o f c h o i c e .  methods,  I n most c a s e s , a u -  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 r e 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 plete  samples a r e chromatographed  s e p a r a t i o n i s a problem  as emphasized  b y TLC, com-  by R o e r i g e t a l .  12 (38)  and  W h i t e h o u s e e t a l . (39)  metabolism. tion has  Depending upon t h e  i n t h e amount o f c o n j u g a t e d been  in their  s t u d i e s o f methadone  s o l v e n t system, marked and  nonconjugated  varia-  metabolites  observed.  Pharmacokinetic  aspects  o f methadone  S y n t h e s i s o f methadone a n a l o g s 1940's t o t h e e a r l y 1960's.  The  use  continued  from  the  o f methadone f o r m a i n t e -  n a n c e o f a d d i c t s i s b a s e d on a c c u m u l a t e d p h a r m a c o l o g i c a l to  the middle  were i n i t i a t e d to  t h e mid  of the 1960's. i n t h e mid  1970's.  Studies of m e t a b o l i t e  1 9 6 0 ' s and  F r o m 1970  t o 1979,  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 aspects  continued  and  f o r ten  were  pharmacokinetic  i n humans w e r e a i m e d  adjustment of dosage l e v e l s of maintenance p a t i e n t s .  s t r a n d e t a l . (40) r e p o r t e d t h a t t h e b e s t r e c o r d o f t i o n was a b o v e 200 ally  achieved  w i t h s t e a d y - s t a t e plasma  concentrations  n g / m l o f methadone i n d i c a t i n g t h a t a  o p t i m i z e d d o s a g e r e g i m e n w o u l d be  a l . (41)  r e p o r t e d t h a t t h e r e was  p l a s m a methadone l e v e l and plaints.  no  Holm-  rehabilita-  pharmacokinetic-  useful in increasing  t h e e f f e c t i v e n e s s o f methadone m a i n t e n a n c e t r e a t m e n t . et  years  o f methadone. S t u d i e s o f methadone k i n e t i c s  at  detection  many r e f e r e n c e s  clinical  data  relationship  Horns  between  p a t i e n t ' s s u b j e c t i v e symptom com-  13 The m e c h a n i s m o f t o l e r a n c e t o methadone was p a r t l y sought i n metabolic of methadone. shortens  t o l e r a n c e by means o f k i n e t i c  Chronic  the half l i f e  a d m i n i s t r a t i o n o f methadone t o man of the drug.  o f 14.3 h o u r s • i n c o m b i n a t i o n 54.8 h o u r s was ' l o n g e r o f 22.2 h o u r s  studies  The a c u t e  w i t h the acute  primary  secondary  than the s i n g l e e x p o n e n t i a l  °f  chronic  ( 4 2 ) . A r e c e n t p a p e r b y 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 methadone m e t a b o l i s m a n d c e l l u l a r  adaptation  increased  t o the 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  i n m a i n t e n a n c e p a t i e n t s was s t u d i e d . for the accumulation with renal disease, narcotic  kinetics  No e v i d e n c e was f o u n d  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 suggesting  t h a t methadone i s a n a p p r o p r i a t e  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 h a n d , 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 ease  i t s metabolites  was o b s e r v e d .in p a t i e n t s w i t h l i v e r  dis-  (45) .  Methadone and d r u g i n t e r a c t i o n s  According  to reported  surveys  (46-48)/  the drugs which  a r e a b u s e d w i t h methadone a r e e t h a n o l , d i a z e p a m , o t h e r amphetamines, and b a r b i t u r a t e s . with these models.  opiates,  The i n t e r a c t i o n s o f methadone  d r u g s w e r e s t u d i e d e i t h e r w i t h human o r w i t h  animal  14 An  agonist-antagonist  i n t e r a c t i o n occurred  n a n c e p a t i e n t s b e t w e e n methadone and i n methadone d i s p o s i t i o n d i s p o s i t i o n occurred (49) , w h i c h was (50) . was This  due  (49).  n a l o x o n e , w i t h no  changes  S i g n i f i c a n t c h a n g e s i n methadone  d u r i n g combined treatment  with  rifampin  t o t h e enzyme i n d u c t i o n p r o p e r t y  E n h a n c e d m e t a b o l i s m o f methadone by  of  rifampin  diphenylhydantoin  o b s e r v e d i n s t u d i e s o f methadone m a i n t e n a n c e p a t i e n t s i s s i m i l a r t o the e f f e c t of diphenylhydantoin  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 was  i n mainte-  i n c r e a s e d by  phenobarbital  on  methadone  pretreatment  of r a t s  d o s e s 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 o f t h e r e was  no  significant  methadone  (53).  (51).  dexamethametabolites  (3.8) .  At  alcoholism  i n t e r a c t i o n between d i s u l f i r a m  and  D e s i p r a m i n e showed i n h i b i t i o n o f methadone  metabolism i n the r a t l i v e r  (54).  T h r e e p a p e r s a p p e a r e d on m e t h a d o n e - e t h a n o l 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 of ethanol  b r a i n and biliary  liver  concentrations  o u t p u t o f methadone  i n t e r a c t i o n was  D i a z e p a m was effective  w h i c h may  o f methadone and  (39, 5 5 ) .  No  increased  decreased  significant  f o u n d b e t w e e n methadone and  of maintenance p a t i e n t s  an  to rats resulted i n  ethanol  acute  in studies  (56).  shown t h r o u g h i n v i t r o  studies to  be  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 o f methadone  e x p l a i n i n p a r t the enhanced e f f e c t of  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 d i a z e p a m i s t a k e n t i o n w i t h methadone  (57).  Four i n v i v o s t u d i e s of  d i a z e p a m i n t e r a c t i o n s w e r e p u b l i s h e d . : one o t h e r w i t h r h e s u s monkeys  ( 5 8 ) , and  with rats  the others w i t h  i n combinamethadone(34), mice  an-  15 (59, 6 0 ) . longed  Acute a d m i n i s t r a t i o n of diazepam t o the r a t pro-  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 t h e b r a i n 14  c o n c e n t r a t i o n s of t o t a l  C and  decreased  the percent of  total  14 C i n the l i v e r d i a z e p a m was monkeys.  or urine  prolonged  Blood  (34).  Behavioral depression  substantially  i n methadone  l e v e l s o f d i a z e p a m and  increased or prolonged  i n those  l e v e l s o f methadone f o r up  (59).  On  diazepam f a i l e d in  maintained  m e t a b o l i t e s were  animals  (58).  t h e o t h e r h a n d , Shannon t o e n h a n c e b r a i n and  not  Enhanced h e p a t i c  t o t h r e e h o u r s by d i a z e p a m  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 mice  after  indicated  by t h i s a g e n t i n  (60) r e p o r t e d t h a t  p l a s m a methadone  levels  mice.  D e u t e r i u m 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 d r u g metabolism  The  steady  s t a t e k i n e t i c s o f a d r u g c a n be  by a method i n w h i c h  s t a b l e • i s o t o p e l a b e l e d and  d r u g s a r e a d m i n i s t e r e d and  collected  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 a p p r o a c h was  samples are  internal  d e m o n s t r a t e d f o r methadone  standard. (61) and  studied unlabeled  analyzed Such  an  propoxyphene.  (62) . The  same a p p r o a c h was  ies of N-acetylprocainamide  applied to b i o a v a i l a b i l i t y  i n humans  p r o v i d e e a s e i n t h e a n a l y s i s and in hepatic extraction resulting  (63).  The  method  studcan  compensate f o r d i f f e r e n c e s from d i f f e r e n t h e p a t i c  blood  16 flows a t d i f f e r e n t times  and i n d i f f e r e n t i n d i v i d u a l s .  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  The  popula-  t i o n a n d v a r i e s b e t w e e n 0.5 and 3 1/min. ( 6 4 ) . 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 c o k i n e t i c and m e t a b o l i c of the l a b e l e d analogs  s t u d i e s of a drug,  a r e used f o r pharmabioequivalence  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 a n d m e t h a d o n e - H^-  Another  13 "I 5 r e p o r t o f [\\ C" 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 b y Browne. f  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 equivalence. animals,  Bioequivalence  is first  studied using  t h e r e s u l t s o f w h i c h become a b a s i s o f t h e u s e 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 p a p e r s have i g n o r e d such treatment stable  small  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  i s o t o p e l a b e l e d compounds.  a careful  equivalence of  F o r example, N - a c e t y l p r o -  13 cainamideC was u s e d w i t h o u t s u c h a t e s t (63) . H a c h e y e t a l . d o s e d p e n t a d e u t e r a t e d methadone t o s t 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 used o n l y w i t h acute  (20). Benoxaprofen-  was  t o x i c i t y data equivalent t o unlabeled  compound ( 6 6 ) . 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 uptake,  hepatic  t h e l i v e r p e r f u s i o n method w h i c h was u s e d t o s t u d y  h e p a t i c uptake of p r o p a n o l o l  (67) c o u l d be u s e d .  Differences of pharmacological  a c t i o n s between  r i u m l a b e l e d and p r o t i o d r u g s a r e m a i n l y  e x p l a i n e d by  deutediffer-  ences i n the b i o t r a n s f o r m a t i o n of the drugs.  I f the 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 isotope effect  i s observed.  t h e m e t a b o l i s m and  Marcucci  anticonvulsant activity  d e m e t h y l d i a z e p a m and  concluded  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 t i o n of the  compounds.  m e t a b o l i s m and  of deuterated  that a significant due  to a reduced  t o x i c i t y of phenacetin  found t h a t the  studied N-  shortening C^-hydroxyla-  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  p o s i t i o n b e i n g o x i d i z e d was was  e t a l . (68)  where hydrogen a t  the  s u b s t i t u t e d with deuterium.  It  l a b e l i n g decreased hepatic necrosis  t o an o x i d i z e d m e t a b o l i t e  the  and  increased  r e s u l t i n g from the m e t a b o l i t e s  due  methemoglobinemia  of the nondeethylated  pathway  (69) . I n some c a s e s i t i s d i f f i c u l t d i f f e r e n t pharmacological and  unlabeled  to conclude whether  a c t i o n s between d e u t e r i u m l a b e l e d  d r u g a r e due  to metabolic  d i f f e r e n c e s or  ences i n the p h y s i c a l p r o p e r t i e s which c o u l d t o the  receptor  s i t e and  n e o u s l o c o m o t o r a c t i v i t y was amphetamine  (70).  and  the p a r t i c i p a t i o n of the presented  f o r N-CD^  observed f o r h i g h l y Further  labeled site morphine.  d e c r e a s e d a c t i v i t y o f N-CD^ was  g i v e n by  influence transport  a decrease i n  An  sponta-  enriched  An  example of  i n receptor  binding  explanation for  m o r p h i n e c o m p a r e d t o N-CH^  a c l a s t i c binding concept  A  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 n o t been p u b l i s h e d .  was  differ-  r e c e p t o r b i n d i n g of the drug.  s i g n i f i c a n t reduction i n t o x i c i t y  deuterated  the  (71).  the morphine  18  OBJECTIVES OF THE RESEARCH  The o b j e c t i v e s o f t h e r e s e a r c h isotope  were t o u t i l i z e  a n a l o g s o f methadone a n d i t s m e t a b o l i t e s  stable  together  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  metabolism.  Methadone i s a d r u g g e n e r a l l y used on a  long term b a s i s .  Therefore,  a complete p i c t u r e o f metabolic  pathways i s e s s e n t i a l t o a s c e r t a i n t h e p o t e n t i a l l o n g 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 Extensive  metabolic  studies  metabolites  metabolites.  i nr a t s using deuterium  a n a l o g s were t o c o n c e n t r a t e  containing  o n d e t e c t i n g a n d i d e n t i f y i n g new  t h a t m i g h t be i m p l i c a t e d i n p o t e n t i a l  Several  term  toxicities.  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 h a v e b e e n  c o m p l e t e d i n man t o d e t e r m i n e t h e n a t u r e o f t h e v a r i e d d o s e r e q u i r e m e n t s and whether b l o o d with  good p e r f o r m a n c e o f t h e p a t i e n t .  tional  Most o f t h e d i s p o s i -  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. with  l e v e l s o f the drug c o r r e l a t e  Therefore,  t h e SIM methods d e v e l o p e d  l a b e l e d compounds a n d GCMS were t o be a p p l i e d t o p h a r m a -  cokinetic  studies.  S a l i v a s a m p l i n g a n d a n a l y s i s w e r e t o be  i n v e s t i g a t e d as a noninvasive s t u d i e s and f o r m o n i t o r i n g with deuterated  technique f o r pharmacokinetic  methadone l e v e l s .  A S I M method  a n a l o g s a s i n t e r n a l s t a n d a r d s was t o be u s e d  i n a d r u g i n t e r a c t i o n s t u d y i n r a t s o f d i a z e p a m a n d methadone  19 to  selectively  that might  i d e n t i f y any m e t a b o l i c  c h a n g e s o f methadone  occur.  S y n t h e s i s o f d e u t e r a t e d methadone a n d m e t a b o l i t e s  The  syntheses  of methadone- !^, 2  EDDP- H 2  2 DDP- H^Q, 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  1 Q  ,  EMDP- H , 2  1 Q  2 a c i d - H^Q,  2  EDDP- H^/ a n d EMDP-  were d e s i g n e d .  The p r i m a r y  choice f o r  t h e s y n t h e s i s o f methadone a n d m e t a b o l i t e s w h e r e b o t h r i n g s are deuterium First,  l a b e l e d was b a s e d o n t h e f o l l o w i n g  t h e l a b e l was n o t l i k e l y  will  t o r e t a i n the phenyl  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  t h e c o s t s o f t h e s y n t h e s i s appeared The f i r s t p o i n t i s i m p o r t a n t  reasonable.  t o detect  metabolites  t h e s e c o n d p o i n t f o r S I M a n a l y s i s o f methadone a n d m e t a b o -  lites (1, —  rings.  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 o f t h e l a b e l , and  thirdly,  and  reasons.  t o b e l o s t s i n c e most m e t a b o -  l i t e s o f methadone c a n be e x p e c t e d Secondly,  aromatic  under E I c o n d i t i o n s .  The compound  l a b e l e d o n one r i n g  2 A r = H ) w h i c h was u s e d f o r s t e r e o s e l e c t i v e m e t a b o l i s m o R  of methadone  (19) h a s .two d i a s t e r e ' o m e r s  which can c o m p l i c a t e :  metabolic studies. The l a b e l e d compounds w e r e a l s o c o n s i d e r e d t o be u s e ful  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 .  According  to the  n a r c o t i c r e c e p t o r m o d e l , w h i c h shows p a r t i c i p a t i o n o f t h e ring  i n the receptor binding  c o u l d be a good model t o s t u d y  ( 7 2 ) , r i n g l a b e l e d methadone such b i n d i n g .  The l a b e l e d  20 compounds c o u l d be u s e d 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 m e c h a n i s m s . 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 occurs v i a arene oxide hydantoin  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 -  (73) o r by d i r e c t o x y g e n i n s e r t i o n a s shown i n mono-o-  demethylation  of p_-trideutereomethoxyanisole  Mass f r a g m e n t a t i o n  (74) .  studies  S t a b l e i s o t o p e l a b e l e d compounds w e r e c o n s i d e r e d t o be m o s t u s e f u l i n d e f i n i n g f r a g m e n t i o n s i n mass  spectrometry.  By c o m p a r i n g t h e mass s p e c t r a o f t h e l a b e l e d and u n l a b e l e d derivatives,  fragmentation processes  d e t a i l than p r e v i o u s l y a v a i l a b l e can  a l s o be u s e d t o d e t e r m i n e  (6). Fragmentation  s t r u c t u r e s t o known  In a d d i t i o n to the fragmentation  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  and NMR  c a n be d e s c r i b e d i n more d e t a i l u s i n g  labeled  compounds.  SIM  studies  t h e s t r u c t u r e o f t h e new m e t a b o -  l i t e s w h i c h may h a v e v e r y s i m i l a r sized metabolites.  c a n be d e s c r i b e d i n more  syntheinformaas I R  deuterium  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 ion monitoring.  111 was r e m o d e l e d t o do s e l e c t e d  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 and  m e t a b o l i t e s as t h e  p r o g r a m w h i c h was  w i t h t h e use  internal  o f l a b e l e d methadone  standards  and w i t h a c o m p u t e r  improved to ensure h i g h  h i g h p r e c i s i o n . •: .  ...  sensitivity  Human s a m p l e s w e r e u s e d t o i n v e s -  t i g a t e the a p p l i c a b i l i t y of the developed cokinetic t h e use  and  s t u d i e s o f methadone.  methodology t o pharma-  E m p h a s i s was  also placed  o f a h i g h a b u n d a n c e i o n o f methadone t o i m p r o v 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 o r a n i m a l The  m e t h o d o l o g y was  a l s o a p p l i e d to the  on  the  studies.  i n v e s t i g a t i o n of  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  interaction  Methadone-diazepam i n t e r a c t i o n  s t u d i e s were  w h i c h i n c l u d e d a m e t h o d o l o g y f o r t h e use internal  standards  of b i o l o g i c a l l y  (biosynthetic internal  of b i o s y n t h e t i c i n t e r n a l standards  designed  standards).  The  use  f o r pharmacokinetic  and  drug  metabolism experiments i s p o s s i b l e only with deuterium compounds and  SIM  and  expected  r e s o l u t i o n p r o b l e m i n t h e TLC i n previous  study,  t o a c c u r a t e l y q u a n t i t a t e methadone  i t s metabolites, e s p e c i a l l y conjugated  tered  labeled  methodology.  In view of the methadone-diazepam i n t e r a c t i o n t h e m e t h o d was  formed  studies  (38,  metabolites.  o f t h e m e t a b o l i t e s was 39).  A  encoun-  22  D e t e c t i o n o f new  methadone  metabolites  S t u d i e s d i r e c t e d t o w a r d t h e d e t e c t i o n o f methadone metabolites s i s was  have been p e r f o r m e d o v e r  the past  15 y e a r s .  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 m i n o r  m a k i n g use  of deuterated  methadone and  Special extraction  samples were d e s i g n e d  r e c o v e r i e s o f t h e m e t a b o l i t e s and  metabolites  m e t a b o l i t e s w h i c h have  not p r e v i o u s l y been used f o r such a s t u d y . procedures f o r r a t b i l e  t o e n s u r e good  t o have a r e l a t i v e l y  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 . the use  o f l a b e l e d and  of the m e t a b o l i t e s  unlabeled' ' compounds p r o v i d e s  from endogenous m a t e r i a l s .  t h e mass s p e c t r a i n p e a k s a r i s i n g w i t h those evidence  metabolites.  complete GCMS w i t h  separation  Comparison of  from u n l a b e l e d  f r o m l a b e l e d compounds c o u l d p r o v i d e  o f t h e f o r m a t i o n o f new  Empha-  compounds important  23  EXPERIMENTAL  1.  Materials  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-  ( 9 9 . 5 % D, M e r c k S h a r p & Dohme, C a n a d a ) ,  2-Dimethylaminoisopropyl D 0 2  ( 9 9 . 7 % D, SIGMA), D  c h l o r i d e HC1 ( A l d r i c h C h e m i c a l ) , S  2  °4  (  m  i  n  i  m  u  isotopic purity  m  99 a t o m  2 % D, M e r c k S h a r p & Dohme, C a n a d a ) , C H ^ - C I ^ B r (minimum i s o t o p i c p u r i t y 9 9 % D, M e r c k S h a r p & Dohme, C a n a d a ) , G l u c u r a s e (3-glucup r o n i d a s e , 5,000 s i g m a u n i t / m l , SIGMA), G l u s u l a s e dase and a r y l  s u l f a t a s e , Erido p r o d u c t s  operbenzoic acid Diazepam  ( t e c h n i c a l grade  (active substance  I n c . , New Y o r k ) , m - C h l o r -  85%, A l d r i c h  Chemical),  o f V a l i u m , Hoffman-La Roche,  M o n t r e a l , C a n a d a ) , 0.1 M s o d i u m a c e t a t e b u f f e r disodium c i t r a t e buffer  (6-glucuroni-  (pH 4 . 5 ) , 0.1 M  (pH 2 . 0 ) , 0.25 M b o r a t e b u f f e r  (pH 9 . 0 ) .  D i a z o m e t h a n e was p r e p a r e d b y t h e method o f L e v i t t ( 7 5 ) . Solvents C h l o r o f o r m , hexane, methylene c h l o r i d e , Distilled  i n g l a s s , Caledon  Methanol,  H 0 ; HPLC g r a d e , F i s h e r S c i e n t i f i c Co.  methanol;  Laboratories Ltd., Ontario.  2  a b s o l u t e , Caledon L a b o r a t o r i e s .  Ether; ether  24  M a t e r i a l s f o r animal Heparin  surgery  (168 u n i t s / m g ,  SIGMA), S i l a s t i c  0.020 i n . i.'d., 0.037 i n . o . d . ) , P o l y e t h y l e n e  (Dow C o r n i n g ,  tubing-10  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  (Clay  tubing-50  ( C l a y Adams, 0.023 i n . i.d., 0.038 i n . o . d . ) .  2.  Analytical  methods  GCMS r e p e t i t i v e  scanning  GCMS s p e c t r a l d a t a w e r e o b t a i n e d u s i n g a V a r i a n MAT 111 g a s c h r o m a t o g r a p h - m a s s s p e c t r o m e t e r . were r e c o r d e d  and p r o c e s s e d  Mass s p e c t r a l  u s i n g a V a r i a n 620L c o m p u t e r .  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 s o u r c e perature  o f 285°C.  data  tem-  A g l a s s c o l u m n (1.6 m x 2 mm i . d . ) p a c k e d  w i t h 3% OV-17 o n 80-10 0 mesh C h r o m o s o r b W (HP) was u s e d  with  c a r r i e r g a s (He) a t 20 m l / m i n .  GCMS s e l e c t e d i o n m o n i t o r i n g SIM was p e r f o r m e d u s i n g a V a r i a n MAT 111 g a s c h r o m a tograph-mass spectrometer o f t h e mass s p e c t r o m e t e r  with accelerating voltage modified  t o permit  scanning  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 c o m p u t e r . i o n i z a t i o n v o l t a g e , source w e r e t h e same a s t h o s e  temperature,  supply the accel-  Electron  and column c o n d i t i o n s  used f o r t h e r e p e t i t i v e  scanning.  25  Gas  chromatography The  GC a n a l y s i s was c a r r i e d o u t u s i n g a H e w l e t t - P a c k a r d  5830A m o d e l e q u i p p e d w i t h a h y d r o g e n column,  The g l a s s  1.8 m x 2 mm i . d . was p a c k e d w i t h 3% OV-17 o n 80-100  mesh C h r o m o s o r b W ( H P ) . temperature gas  flame d e t e c t o r .  I n j e c t i o n t e m p e r a t u r e was 205°C, o v e n  210°C, a n d d e t e c t o r t e m p e r a t u r e  300°C.  The c a r r i e r  (He) f l o w r a t e was 50 m l / m i n .  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. for  The GC c o n d i t i o n s w e r e t h e same a s t h o s e  described  routine a n a l y s i s except f o r the s i z e o f the glass  column  (1.6 m x 2 mm i . d . ) a n d t h a t methane was u s e d a s a c a r r i e r a n d r e a g e n t g a s ( f l o w r a t e 40 m l / m i n ) . 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 p e r f o r m a n c e  ( r e s o l u t i o n 10,000) was u s e d . and  mass s p e c t r o m e t e r  I o n i z a t i o n v o l t a g e was 70 eV  s o u r c e t e m p e r a t u r e , ^150°C.  High performance  liquid  chromatography  An ALTEX MODEL 15 3 h i g h p e r f o r m a n c e  liquid  chromato-  g r a p h e q u i p p e d w i t h a n 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 c o l u m n was a 4.6 mm i . d . x 25 cm p a c k e d  Ultrasphere-ODS  (dp, 5u) .  Pumping  pressure  a n d f l o w r a t e w e r e 2000-3000 p s i a n d 1 m l / m i n ,  respec-  tively.  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 m e t h a n o l a n d  H 0 2  (3:1).  ( o c t a d e c y l s i l a n e ) ' • \. '<  with  r  26  NMR  spectroscopy The  NMR  s p e c t r o s c o p y was  performed  w i t h the  following  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  standard.  m e t h a n o l and d e u t e r a t e d c h l o r o f o r m w e r e u s e d Infrared  as  1000 infrared  spectroscopy. o r as. KBr  3.  spectrometer  The  and a U n i c a m  (Pye Unicam) w e r e u s e d  f o r IR  s p e c t r a w e r e r e c o r d e d as t h e l i q u i d  films  discs.  Chemical s t u d i e s  Synthesis of deuterium The was  solvents.  spectroscopy 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  SP  Deuterated  l a b e l e d methadone and m e t a b o l i t e s  d e g r e e 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  determined  by  NMR.  2 S y n t h e s i s o f m e t h a d o n e - H^Q:  A general procedure  f o r the  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 f r o m 2 the l i t e r a t u r e ( 7 6 ) . Benzenewas u s e d i n p l a c e o f b e n z e n e 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^ ( s p e c t r u m 1 , p. tain enriched diphenylacetonitrile2 2 , p. 1 6 3 ) , b e n z e n e - Hg  ( 1 0 0 gm)  2  2  a steam b a t h f o r 20 hours. The  The  A l C l ^ was  s e p a r a t e d b e n z e n e l a y e r was  ob-  2  and D 0  ( 1 2 g, 0 . 0 9 m o l ) .  To  (1J2, 2Ar= H-^Q) ( s p e c t r u m ( 4 d r o p s ) w e r e 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, anhydrous A l C l ^  163).  0 . 0 6 mol)  m i x t u r e was  and  refluxed  d e s t r o y e d by a d d i n g  on H 0. 2  d r i e d o v e r a n h y d r o u s Na„SO..  27 F l a s h e v a p o r a t i o n o f t h e s o l v e n t gave a b r o w n i s h gm,  95.7%) w h i c h was  used  f o r subsequent 2  Methadone n i t r i l e -  H^Q  was  compound  (11.68  reactions.  prepared  from e n r i c h e d diphen-  2 ylacetonitrile-  H-^Q  by t h e method o f A t t e n b u r r o w  minor m o d i f i c a t i o n s . (9.0 gm, (1.92  0.047 mol)  10 m i n u t e s  and  A s o l u t i o n of enriched d i p h e n y l a c e t o n i t r i l e i n d r y benzene  gm,_0.08 m o l ) .  The  m i x t u r e was  (7.59 gm,  was  heated  0.048 mol)  p r e p a r e d as d e s c r i b e d (76) and stirred  (75 ml)  treated with  f o r 45 h o u r s  i n benzene  2-Dimethylamino(90 ml)  added t o t h e m i x t u r e .  a t room t e m p e r a t u r e .  NaH  on a s t e a m b a t h f o r  a d r o p o f DMSO added as c a t a l y s t .  isopropyl chloride  was  e t a l . (77) w i t h  The  was mixture  Work up d i d n o t  r e q u i r e a d i s t i l l a t i o n but ' t h e 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 i s o m e t h a d o n e 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%) ( s p e c t r u m 3, p. 164) f r o m 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 m e t h a d o n e - H^Q {1, 2Ar= 2 2 H-^Q ) f r o m 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 b u r r o w e t a l . ( 7 7 ) . 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 t h e 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 ( s p e c t r u m 5, p. 1 6 5 ) . 2 S y n t h e s i s 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 p e n t a n o i c a c i d - H-^Q 2 2 2 (_5, 2Ar= H and DDP- H (£, 2Ar= H ) .The method was s i m i l a r 1 Q  to  1 Q  1 Q  t h a t of Gardner e t a l . (22). 2  nitrile-  H^Q  P a r t i a l l y d e u t e r a t e d methadone  o b t a i n e d from the r e a c t i o n of p a r t i a l l y  deuterated  d i p h e n y l a c e t o n i t r i l e with l-dimethylamino-2-chloropropane NaH  was  h y d r o l y z e d u s i n g D^O  and  b o t t l e to give the b i s u l f a t e of  D^SO^  i n a screw  capped  and reaction  4-dimethylamino-2,2-diphenyl-  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 o n t h e a r o m a t i c rings  ( s p e c t r u m 4 , p . 1 6 4 ) . The a c i d was d r i e d a t 1 0 0 ° C f o r 3 0 2  minutes p r i o r t o conversion  t o t h e p y r r o l i d o n e , D D P - H-^Q .  The  d e g r e e 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% ( s p e c t r u m 6, p. 1 6 6 ) . 2 o f EDDP- H  Synthesis  1 Q  2 (3, 2Ar= H  1 Q  2 ) a n d EDDP- H  3  —  A known  2 procedure obtained 167).  (3) was u s e d .  2  S t a r t i n g w i t h DDP- H ^ , EDDP-  was  Q  w i t h t h e a r o m a t i c r i n g s ' 9 6 % d e u t e r a t e d ( s p e c t r u m 9, p.  DDP r e a c t e d w i t h C H - G H B r t o g i v e E D D P - H 2  2  3  2  ( s p e c t r u m 8, p . 1 6 7 ) . 2 2 S y n t h e s i s o f EMDP- H ( 2 , 2Ar= H 1 Q  1 Q  2 ) a n d EMDP- H  3  3  —  ( 9 9 % D)  The p r o c e -  2 2 d u r e o f P o h l a n d e t a l . (3) was u s e d . EMDP- H a n d EMDP- H were 2 2 o b t a i n e d f r o m EDDP- H^Q a n d EDDP- H , r e s p e c t i v e l y . The d e g r e e 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 % ( s p e c t r u m 7 , p . 1 6 6 ) . 2 EMDP- H showed more t h a n 9 9 % d e u t e r a t i o n . 1 Q  3  3  3  Synthesis  of  2-dimethylamino-4,4-diphenyl-5-nonanone  perchlorate 2-Dimethylamino-4,4-diphenyl-5-nonanone by t h e method o f L y n n e t a l . ( 2 1 ) .  The p r o d u c t was n o t d i s -  tilled  but c r y s t a l l i z e d  salt.  R e c r y s t a l l i z a t i o n f r o m e t h e r - E t O H gave c r y s t a l s ,  137-139°C.  from ether  was p r e p a r e d  s o l u t i o n as t h e p e r c h l o r a t e mp  Chemical o x i d a t i o n The  studies  compounds  (EMDP H C l , EMDP b a s e , EDDP p e r c h l o r a t e ,  EDDP b a s e ) w e r e t r e a t e d a t 0-5°C w i t h m - c h l o r o p e r b e n z o i c a c i d in chloroform.  F o r EMDP H C l , EMDP b a s e , and EDDP b a s e 2 0 %  e x c 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 u s e d . m i x t u r e was k e p t o v e r n i g h t a t 0°C.  The r e a c t i o n  The o p t i m u m m o l a r  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 t i m e were w i t h EDDP p e r c h l o r a t e . t h e s a m p l e s a n d a l l gave 1) The C H C 1  3  t h e same products-,*  s o l u t i o n was washed w i t h 1 0 % N a S Q 2  f u r t h e r washed w i t h s a t u r a t e d NaHCO^. 2  NaHCO^.  studied  T h r e e m e t h o d s w e r e u s e d t o work up  t h e p r e s e n c e o f p e r o x i d e was n o t d e t e c t e d .  made w i t h H 0 .  ratio  3  until  The s o l u t i o n was  F i n a l w a s h i n g was  2) The C H C l ^ s o l u t i o n was washed w i t h  a) The C H C l ^ s o l u t i o n was d i r e c t l y  saturated  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 a n d m e t a b o l i t e s i n human p l a s m a , 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 s a m p l e s w e r e o b t a i n e d f r o m 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 f e m a l e methadone m a i n t e n a n c e p a t i e n t s w h i c h was c o n d u c t e d b y t h e A l c o h o l a n d D r u g Vancouver.  Maintenance dosage  Commission,  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; a n d D, 90 mg/day.  Plasma and s a l i v a  samples  w e r e t a k e n a t 0, 2, 4,-6, 8, 1 1 , 12 a n d 24 h o u r s a f t e r t h e u s u a l dose.  U r i n e s a m p l e s w e r e o b t a i n e d a t 1, 3, 5, 7, 9, 1 3 , a n d 24  hours.  A f t e r t h e o r a l d o s e o f methadone was t a k e n , t h e p a t i e n t s  were i n s t r u c t e d  to rinse  t h e mouth w i t h 2 50 m l o f w a t e r t o remove  t r a c e s o f drug from the o r a l c a v i t y . to e a t o r drink  just prior  P a t i e n t s were n o t a l l o w e d  to providing a saliva  sample.  The  mouth was a g a i n r i n s e d w i t h w a t e r b e f o r e t h e s a m p l e was t a k e n to reduce contamination from food substances. stored frozen u n t i l  A l l samples were  analyzed.  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 curve p r e p a r a t i o n Plasma and s a l i v a . o r d e r t o remove s o l i d s .  S a l i v a was c e n t r i f u g e d i n  To p l a s m a o r s a l i v a  was a d d e d 0.2 m l o f i n t e r n a l  standard  4,-diphenyl-5-nonanone p e r c h l o r a t e  samples  (0.5 ml)  (I.S.), 2-dimethylamino-4,  (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 m e t h a n o l 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 n g i n 0.2 m l H„0) .•  The s o l u t i o n was d i l u t e d  o f 1 N NaOH was a d d e d .  t o 3 m l w i t h H-0 a n d 0.1 m l  A f t e r adding methylene c h l o r i d e  t h e s o l u t i o n was v o r t e x m i x e d f o r 3 m i n u t e s .  The a q u e o u s :  l a y e r was a s p i r a t e d o f f a n d t h e m e t h y l e n e c h l o r i d e d r i e d o v e r anhydrous sodium s u l f a t e . (10 m l ) was t a k e n a n d e v a p o r a t e d u n d e r dissolved  i n 50-100 u l o f CH^OH.  o n t o t h e GCMS. trol  l a y e r was  D r i e d methylene •  chloride  The r e s i d u e was  A 2-5 u l a l i q u o t was i n j e c t e d  S t a n d a r d c u r v e s w e r e p r e p a r e d by s p i k i n g  samples o f plasma and s a l i v a  (15 m l ) ,  con-  (0.5 ml) w i t h methadone i n  31 the  amounts o f 0, 2 0 , 40, 1 0 0 , 2 0 0 , a n d 500 n g .  r a t i o s of methadone/internal m/e  standard  obtained  The p e a k  by  area  monitoring  72 w e r e 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 m e t h a d o n e . Urines.  A f t e r t h a w i n g t h e s a m p l e , 1 m l o f u r i n e was  taken,  t o w h i c h was added 0.2 m l 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 m e t h a d o n e - H^Q, 10 ug 2 2 2 EDDP- H , 10 ug EMDP- H a n d 10 u g o f DDP- H / m l . The m i x t u r e 3  was  diluted  1 Q  1 Q  t o 5 m l w i t h d i s t i l l e d w a t e r a n d t h e pH was  t o 7-8 w i t h 0.1 N NaOH.  The m i x t u r e  adjusted  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 m i n u t e s w i t h m e t h y l e n e c h l o r i d e (15 m l ) .  The m e t h y l e n e  chloride extract  2  (13 m l ) was d r i e d o v e r a n h y d r o u s N a S 0 ^ a n d t a k e n  to dryness using N . 2  ml)  The r e s i d u e was t a k e n  a n d a 2-5 u l a l i q u o t was i n j e c t e d o n t o t h e GCMS.  c a l i b r a t i o n s , v a r y i n g amounts o f methadone EDDP  containing only the deuterated urine  i n order  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 bleeding.  C a l i b r a t i o n curves  (1 m l ) .  Monitoring  i o n s w e r e m/e  (EDDP), m/e  samples  i n control background  i m p u r i t y and column by p l o t t i n g t h e p e a k  compound a t e a c h o f t h e i o n  p a i r s m o n i t o r e d v s . t h e known c o n c e n t r a t i o n compound t o i t s c o r r e s p o n d i n g  Blank  to subtract  were p r e p a r e d  area r a t i o s of unlabeled/labeled  For standard  and DDP (0.05  i n t e r n a l standards  (1 m l ) w e r e a l s o p r e p a r e d  (0.1-0.4  (0.5 ug t o 50 u g ) ,  (0.5 ug t o 50 u g ) , EMDP (0.05 ug t o 1 u g ) ,  ug t o 1 ug) w e r e added t o c o n t r o l u r i n e s  280  up i n MeOH  r a t i o of unlabeled  labeled internal  standard.  223/m/e 233 ( m e t h a d o n e ) , m/e  208/m/e 218 (EMDP), a n d m/e  277/m/e  265/m/e 275 (DDP).  32  GC a n a l y s i s For  t h e GC a n a l y s i s o f methadone a n d 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 i n 0.2 m l H 0 ) was u s e d a s t h e i n t e r n a l 2  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 f o r t h e SIM a n a l y s i s o f u r i n e  standard.  (10 ug The e x t r a c -  t h e same a s t h o s e  described  samples.  S t a b i l i t y o f EDDP Differential  scanning  c a l o r i m e t r y was u s e d t o d e t e r m i n e  mp o f EDDP p e r c h l o r a t e ; mp 175°C, when r e c r y s t a l l i z e d d i e t h y l ether-EtOH  (lit.  from  (3) mp 167-168°C).  EDDP p e r c h l o r a t e s t o c k s o l u t i o n (1.5 m l , 1 mg/ml i n MeOH) was e v a p o r a t e d u n d e r N" .  A f t e r a d j u s t i n g t o pH 12 w i t h  2  lN-NaOH, e t h e r An  (25 m l ) was added t o e x t r a c t t h e f r e e b a s e .  a l i q u o t of the ether  25 m l v o l u m e t r i c solvent. 1,  Samples  layer  (20 m l ) was t r a n s f e r r e d t o a  f l a s k a n d made u p . t o 2 5 m l w i t h t h e same (0.2 m l ) w e r e t a k e n  2, a n d 3 d a y s a f t e r p r e p a r a t i o n o f t h e s a m p l e w h i c h was  k e p t o n t h e b e n c h a t room t e m p e r a t u r e . stock 2 DDP- H  s o l u t i o n was p r e p a r e d 1 Q  monitoring  3  perchlorate per ml.  (0.2 m l ) was m i x e d w i t h  (0.2 m l ) a n d t h e m i x t u r e  An i n t e r n a l  standard  w i t h MeOH t o c o n t a i n 10 ug o f  2 a n d 20 u g o f EDDP- H  solution  280  f o r t h e a n a l y s i s a t 0,  i n t e r n a l standard  was a n a l y z e d  Sample solution  f o r DDP a n d EDDP b y  m/e 265 a n d m/e 275 f o r DDP a n d m/e 277 a n d m/e  f o r EDDP.  concentrations  The s t a n d a r d  curves  were p r e p a r e d  using  various  o f EDDP p e r c h l o r a t e a n d DDP d i s s o l v e d i n m e t h a n o l .  33 The  c o n c e n t r a t i o n s o f EDDP w e r e e x p r e s s e d  5.  D e t e c t i o n o f methadone  Animal  metabolites  experiments Male W i s t a r r a t s  (250-350 gm) w e r e a n e s t h e s i z e d  ether during the s u r g i c a l procedures. was  as t h e f r e e base.  i s o l a t e d through  The common b i l e  with duct  a m i d l i n e abdominal i n c i s i o n and cannu-  lated with polyethylene  tubing-10  for bile  collection.  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 . i n a r e s t r a i n i n g cage.  A f t e r recovery  The  The r a t was p l a c e d  from ether  anesthesia,  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 u g s . c . a t a d o s e 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  metabolites).  methadone,  The b i l e was c o l l e c t e d o v e r  deuterated  a p e r i o d o f 24  hours.  Human  experiments. The  t w e n t y f o u r hour u r i n e samples were o b t a i n e d  from  methadone m a i n t e n a n c e p a t i e n t s who w e r e on - dosages, o f 90 mg/day.  S a m p l e s were s u p p l i e d b y t h e A l c o h o l a n d D r u g 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 bile and bile  the mixture  sample  (10 m l ) was . - d i l u t e d w i t h H 0 2  c e n t r i f u g e d t o remove s o l i d  s a m p l e was t h e n  (10 m l )  substances.  The  e x t r a c t e d w i t h m e t h y l e n e c h l o r i d e (50  34 ml x 2) .  The m e t h y l e n e c h l o r i d e l a y e r s w e r e c o m b i n e d and k e p t  for the a n a l y s i s of nonconjugated metabolites.  The a q u e o u s  l a y e r was f r e e z e d r i e d . Buffer sample  (pH 4.5, 10 m l ) was a d d e d t o t h e f r e e z e d r i e d  ( f i n a l pH 5.0) and t h e m i x t u r e  wasiincubated  R the presence o f Glucurase  R or Glusulase  (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 , t h e pH o f t h e s o l u t i o n was a d j u s t e d 8.5 by a d d i n g pH 9.0 b o r a t e  (37°C) i n  buffer  (10 m l ) .  e x t r a c t e d w i t h methylene c h l o r i d e  t o 8.0-  The s o l u t i o n was  (75 m l x 3 ) . The c o m b i n e d  m e t h y l e n e c h l o r i d e e x t r a c t s w e r e f l a s h e v a p o r a t e d and t h e r e s i d u e r e d i s s o l v e d w i t h methanol treated with  6.  (1 m l ) .  diazomethane.  Methadone-diazepam i n t e r a c t i o n  Treatment o f  T h i s s o l u t i o n was  studies  animals  Male W i s t a r  rats  (200-300 gm) o b t a i n e d  Bio-Breeding  Farm L a b o r a t o r i e s  throughout.  They w e r e m a i n t a i n e d  from Canadian  ( M o n t r e a l , Quebec) w e r e u s e d on a standard  diet of Purina  Lab  Chow  ( R a l s t o n P u r i n a Co. o f Canada) and w a t e r 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  The  e x t e r n a l j u g u l a r v e i n was e x p o s e d 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 S I L A S T I C  by t h e method o f U p t o n ( 7 8 ) .  t u b i n g was c o n n e c t e d t o 3 cm o f p o l y -  ethylene  t u b i n g - 5 0 by means o f a p i e c e o f 22 gauge h y p o d e r m i c  needle.  The v e i n was c u t u s i n g a s h a r p b l a d e  scissors.  or moria spring  The h e p a r i n i z e d s a l i n e was n o r m a l 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 completion  of jugular vein cannulation,  bile  d u c t c a n n u l a t i o n was p e r f o r m e d a s d e s c r i b e d b y L a m b e r t ( 7 9 ) . Iris  f o r c e p s were u s e d t o h o l d t h e b i l e  the cannula ducts.  d u c t and t o i n s e r t  below t h e j u n c t i o n o f t h e r i g h t and l e f t  hepatic  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  or moria s p r i n g s c i s s o r s . i n t o t h e common b i l e The  total  Polyethylene  blade  t u b i n g - 1 0 was i n s e r t e d  duct.  t i m e spent f o r j u g u l a r v e i n and b i l e  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 duct  duct  cannulation,  d i a z e p a m o r v e h i c l e o n l y was g i v e n  through the jugular vein  a t an i n f u s i o n r a t e o f 2 ml/hour.  The t i m e o f i n f u s i o n s t a r t  was  recorded.  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 area  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 .  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 a n d t h e a n i m a l  After was  resutured with the tubing inside the layer of the skin. Shortly before thai i n f u s i o n ,  methadone d o s i n g ,  t h e r a t was p l a c e d  one h o u r f r o m t h e s t a r t o f i n a r e s t r a i n i n g cage.  done H C l (10 mg/Kg i n 2 m l s a l i n e ) was g i v e n collected  from t h e cannulated  bile  The glycol  ( 1 , 2, 5, 1 1 , a n d 23  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  dose s e r v e d  t h e methadone  as t h e blank. d i a z e p a m was d i s s o l v e d i n a s o l u t i o n o f  (40%), ethanol  benzoate  B i l e was  duct 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 times hours).  s.c.  Metha-  (10%), benzyl a l c o h o l  (5%) i n w a t e r .  volume o f t h e v e h i c l e .  Control animals  propylene  ( 1 . 5 % ) , and sodium  r e c e i v e d an e q u a l  36  Sample p r e p a r a t i o n The dosing for  procedures  conjugated  3 rats with  24 h o u r s .  internal  s t a n d a r d s were p r e p a r e d by 2 20 mg/Kg EMDP- H s.c. B i l e was c o l l e c t e d 1 Q  The b i l e was e x t r a c t e d o n c e w i t h 2  c h l o r i d e t o remove EMDP- H to  1 Q  .  methylene  The e x t r a c t e d b i l e was d i l u t e d  100 m l w i t h w a t e r and f r o z e n i n an E r l e n m e y e r f l a s k  until  used.  standard  This  s o l u t i o n was u s e d d i r e c t l y  t o measure c o n j u g a t e d The  bile  a t -20°C  as t h e i n t e r n a l  metabolites.  sample  (0.2-0.4 m l ) was m i x e d w i t h 0.2 m l o f 2 t h e 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 u g EDDP- H^, 2 2 2 u g EMDP- H , 5 u g m e t h a d o n e i n 0.2 m l H 0 ) a n d 1 m l o f 1 Q  the  conjugated  2  internal  standard  solution.  The m i x t u r e  9.0-9.5) was e x t r a c t e d w i t h m e t h y l e n e c h l o r i d e  (pH  (15 m l ) .  a q u e o u s l a y e r was k e p t f o r t h e a n a l y s i s o f c o n j u g a t e d  The  metabo-  lites. The N  2  .  m e t h y l e n e 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  A f t e r a d j u s t i n g t h e pH o f t h e r e s i d u e w i t h pH 2.0 b u f f e r  (2.0 m l ) , t h e m i x t u r e ml)  was b r i e f l y  t o remove c o l o r e d m a t e r i a l s .  a l k a l i n e b y a d d i n g 1 N NaOH methylene c h l o r i d e was  e x t r a c t e d w i t h C H C l ^ (0.5 The a q u e o u s l a y e r was made  (0.5 m l ) a n d was e x t r a c t e d  (10 m l ) .  with  The m e t h y l e n e c h l o r i d e l a y e r  d r i e d o v e r anhydrous Na SO^ and e v a p o r a t e d under N .  final  2  2  samples f o r SIM a n a l y s i s were p r e p a r e d  Monitoring 208  under  with  The  methanol. 2 i o n s w e r e m/e 277 (EDDP), m/e 280 (EDDP- H ) , m/e 3  (EMDP), m/e 218 ( E M D P - H ) , m/e 294 ( m e t h a d o n e ) , a n d 2 m/e 29 7 (methadone- H _ ) . 2  1 Q  37 The  a q u e o u s l a y e r was f r e e z e d r i e d .  (1.5 m l ) was a d d e d t o t h e r e s i d u e was  incubated  After  with Glucurase  ( f i n a l pH 5.0) a n d t h e m i x t u r e  (0.2 m l ) f o r 24 h o u r s a t 3 7°C.  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  (2 m l ) t o pH 8.5-9.0. chloride  (15 m l ) .  anhydrous Na^O^ solved  The m i x t u r e  was e x t r a c t e d w i t h  t o dryness.  was v o r t e x m i x e d w i t h h e x a n e  a f t e r a d j u s t i n g t h e pH w i t h pH 2.0 b u f f e r endogenous m a t e r i a l s .  in  taken  (5 m l )  (1.5 m l ) t o  The pH o f t h e a q u e o u s p a r t was  a l k a l i n e w i t h 1 N NaOH  and  diazomethane.  ( s u s t a i n e d y e l l o w c o l o r ) , m e t h a n o l was e v a p o -  r a t e d and t h e m i x t u r e  (10 m l ) .  (0.5 m l ) a n d e x t r a c t e d w i t h  adjusted methylene 2  t o dryness 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  s u i t a b l e volume o f m e t h a n o l f o r SIM a n a l y s i s .  Ions,  2 4 6 , 2 3 8 , a n d 237 w e r e m o n i t o r e d f o r CH 0EMDP  245,  2 4 4 , 2 3 7 , a n d 236 f o r DiCH^OEMDP.  3  247. anid m/e  remove  The m e t h y l e n e c h l o r i d e was d r i e d o v e r N a S O ^  247,  m/e  methylene  The r e s i d u e was d i s -  i n 1 ml o f methanol and t r e a t e d w i t h  chloride  buffer  The m e t h y l e n e c h l o r i d e was d r i e d o v e r and t a k e n  After methylation  to  B u f f e r , pH 4.5  m/e  a n d m/e  The r a t i o s , m/e 2 3 8 /  237i/m/e 245 w e r e 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 a n d DIHOEMDP, r e s p e c t i v e l y .  Stability  experiments  Noncohjugated metabolites  A stock  s o l u t i o n was  2 2 t o c o n t a i n 1 mg e a c h o f EDDP, EDDP- H , EDDP- H , 2 2 2 EMDP, EMDP- H , methadone m e t h a d o n e - H , m e t h a d o n e - H , DDP,  prepared  1 Q  l Q  1 Q  3  3  2 and  DDP- H-^Q i n 1 m l o f m e t h a n o l .  Stock  s o l u t i o n (1 m l ) was  d i l u t e d t o 50 m l w i t h m e t h a n o l i n a v o l u m e t r i c . f l a s k a n d  38 2.5  ml o f t h e s o l u t i o n  Using these d r i e d  (50 u g e a c h ) was t a k e n t o d r y n e s s .  samples  solutions  w e r e made up t o 5 m l w i t h  w a t e r a t d i f f e r e n t pHs (1 N HC1, pH 2,0, pH 4.5, pH 9.0, a n d 0.1  N NaOH).  After  incubation  a t d i f f e r e n t t i m e s , t h e pH o f  e a c h s o l u t i o n was a d j u s t e d t o 9.0 a n d t h e s o l u t i o n w i t h 10 m l o f m e t h y l e n e f r a c t i o n was d r i e d N •  over anhydrous  The r e s i d u e was d i s s o l v e d  2  the  chloride.  GCMS.  The m e t h y l e n e Na S0 2  4  extracted  chloride  and e v a p o r a t e d  i n methanol  and i n j e c t e d  under into  I o n s , m/e 277 (EDDP), m/e 287 ( E D D P - H ) , m/e 280 2  1 Q  ( E D D P - H ) , m/e 208 (EMDP), m/e 218 ( E M D P - H ) , m/e 223 2  2  3  1 0  (metha-  • 2 2 d o n e ) , m/e 226 (methadone- H ^ ) , a n d 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 dard  The c o n j u g a t e d i n t e r n a l  (2 m l ) was i n c u b a t e d w i t h G l u c u r a s e  times  stan-  (0.5 m l ) a t d i f f e r e n t  ( 1 2 , 2 4 , 4 8 , a n d 96 h o u r s ) a n d t h e s a m p l e s  w o r k e d 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 bile  samples.  246,  2 3 8 , 237 f o r C H 0 E M D P - H ; m/e 2 4 7 , 2 4 6 , 2 4 5 , 244 a n d m/e 2  245,  2 4 4 , 2 3 7 , 236 f o r ( C H 0 )  7.  m/e 2 4 7 , 2 4 6 , 2 4 5 , 244 a n d m/e 2 4 7 , 2  3  g  3  would VIII  Specific ions,  indicate  2  EMDP- Hg w e r e m o n i t o r e d w h i c h  s t a b i l i t y of the deuterium l a b e l i n g .  •  See T a b l e  and T a b l e IX f o r d e t a i l s . 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  The  apparent e l i m i n a t i o n  were c a l c u l a t e d  rate  analysis  c o n s t a n t s and h a l f  b y u s i n g t h e NONLIN p r o g r a m  (80).  lives  The. u s e o f  39 biosynthetic  internal  standards f o r pharmacokinetic  was. i n v e s t i g a t e d b y u s i n g t h e same p r o g r a m . cept values of c a l i b r a t i o n  Slope and i n t e r -  e q u a t i o n s were c a l c u l a t e d b y means  of t h e computer program, T r i a n g u l a r puting  R e g r e s s i o n P a c k a g e , Cora-..  Center, the U n i v e r s i t y of B r i t i s h Columbia.  statistical  studies  a n a l y s i s was p e r f o r m e d b y S t u d e n t ' s  The  t test.  40  RESULTS AND  1.  DISCUSSION  S y n t h e s i s a n d 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 a n d m e t a b o l i t e s : ,  Deuterated  diphenylacetonitrile 2  Treatment of p h e n y l a c e t o n i t r i l e the  method o f Robb and Schult'z  of d i p h e n y l a c e t o n i t r i l e  with  (76) r e s u l t e d  labeled  benzene- H  NMR  i n both phenyl rings.  analysis  indicated  L a b e l l i n g of both phenyl r i n g s of  two p r o c e s s e s .  catalyzed  exchange  Acid  catalyzed  is the  indeed a c o n t r i b u t i n g  of the phenyl 86%  and o r aluminum 2  factor  deuteration.  chloride  and t h e r i n g  or d i p h e n y l a c e t o n i t r i l e  account f o r the observed l a b e l l i n g .  As  i s probably the r e s u l t  (81) b e t w e e n b e n z e n e -  tons of p h e n y l a c e t o n i t r i l e  by  i n the i s o l a t i o n  shown i n t h e mass s p e c t r u m ( F i g . 2 a ) , d e u t e r a t i o n r i n g s was n o t c o m p l e t e .  g  pro-  could  While t h i s exchange p r o c e s s  i t does n o t t o t a l l y  determine  end r e s u l t .  F o r e x a m p l e , when u n l a b e l e d diphenylacetoni2 t r i l e was t r e a t e d w i t h b e n z e n e - H, u n d e r t h e r e a c t i o n condi6  tions  ( A l C l ^ r r e f l u x , 2 h o u r s ) , 55-60% o f t h e e x p e c t e d  as m e a s u r e d b y NMR was r e q u i r e d  had o c c u r r e d .  exchange  More t h a n 20 h o u r s r e f l u x  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  achieved during the a l k y l a t i o n reaction. 2 e x c h a n g e b e t w e e n b e n z e n e and b e n z e n e - H,  On t h e o t h e r (AlCl-.,  hand,  reflux)  41  wo-  202  (a)  CM—CN  60H  173  > < 120  93  JU,#,i...,J 100  100-1  300  200  1S8  (b)  SOH  < -J  Ui EC  |92  118  300  200  100  100  203  M<  (c)  z UJ  I-  60H  173  < 93  JL Fig,  100  1 2 1  200  300  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 - H (b) 5 0 % d e u t e r a t e d p r e p a r e d b y t h e method o f H a c h e y e t a l . (19) (c) a f t e r e n r i c h m e n t . 2  6  42 reached e q u i l i b r i u m w i t h i n  20 m i n u t e s (as m e a s u r e d by mass  spectrometry). 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 alkylation reaction,  i t i s suggested t h a t the r e v e r s i b l e  of the 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  the exchange p r o c e s s e s t h a t o c c u r . for the process i l l u s t r a t e s  nature  factor to  A suggested i n t e r m e d i a t e  t h a t r a p i d exchange between  and l a b e l e d b e n z e n e p l u s t h e r e v e r s i b i l i t y  benzene  of the r e a c t i o n  leads t o a product w i t h the expected percent of l a b e l a t equilibrium.  CeD  '  6  CHD 6  5  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 tonitrile  synthesized using equimolar concentrations of phenyl2  a c e t o n i t r i l e and benzene-  i n CS^  a s 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 ( 1 9 ) . This experiment o b t a i n e d was spectrum  was r e p e a t e d a s d e s c r i b e d a n d t h e p r o d u c t  f o u n d t o be 50% d e u t e r a t e d by NMR.  The mass  ( F i g . 2b) shows a c l u s t e r o f t h e m o l e c u l a r i o n s f o r 2 2  the isomers from distribution  HQ t o  H-^  i n approximately the expected  f o r 50% u n i f o r m l a b e l l i n g  i n both  rings.  Diphenylacetonitrile-''H^Q was  prepared  2 t o n i t r i l e - H-,~ ±U  and  (81).  with  98%  and  nitrile  Deuterated  i f any  n o t be  expected.  The  mass  preferable  exchange r e a c t i o n i s  produced.  Methadone  conditions.  methadone 2 prepared  starting material. 3b)  The  side products  n y l a c e t o n i t r i l e r e t a i n e d the  (Fig.  achieved  f o u n d t o be  l a b e l e d using these  M e t h a d o n e - H^Q  the  NMR  Deuterated d i p h e n y l a c e t o n i t r i l e  u s i n g e x c h a n g e r e a c t i o n s was  can  diphenylace-  labeled diphenylacetoni-  r i n g d e u t e r a t i o n w o u l d be  w i t h few  2  2 t o b e n z e n e - H, gave b  partially  to u s i n g the a l k y l a t i o n r e a c t i o n . simple  molar r a t i o of  A s s u m i n g e q u i l i b r i u m was  s p e c t r u m i s shown i n F i g . 2c. prepared  benzene-  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  between the benzene98.5%  A 1:20  (86% r i n g d e u t e r a t i o n )  mass s p e c t r o m e t r y . 2  trile,  deuterium  u s i n g a l u m i n u m c h l o r i d e c a t a l y s t and  as a d e u t e r i u m s o u r c e  the product  enriched with  The  s t a r t i n g from enriched  same d e g r e e o f l a b e l l i n g as i n 2 mass s p e c t r u m o f m e t h a d o n e -  c o r r e s p o n d s t o t h a t f o r methadone  ion appearing  a t m/e  a p p e a r a t m/e  233  319.  and  m/e  diphe-  Two 304  and  m/e  294  (27)  and  m/e  297  f o r m e t h a d o n e - H^-  i o n s t h a t m i g h t be corresponding  t o m/e  molecular  used i n 223  SIM  (25)  Sullivan et a l . 2 (17) r e p o r t e d t h a t t h e a p p l i c a t i o n o f m e t h a d o n e 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  ions  f o r methadone  (83), the  H^Q  i s i d e a l f o r SIM  spectrometry,  m/e  223  ( F i g . 3a).  While n e i t h e r of these  s t u d i e s , under our  fragment  c o n d i t i o n s o f mass  showed s l i g h t l y h i g h e r  abundance  (1.6%)  44  45  t h a n t h a t o f m/e as i n t e r n a l  294  (1.0%)  s t a n d a r d , i o n s a t m/e  the b a s i s f o r a s u i t a b l e urine  ( F i g . 3a).  U s i n g methadone-  223 and m/e  233 h a v e  2  H  1 Q  formed  i o n m o n i t o r i n g a s s a y o f methadone i n  samples. + CH= N(CH ), I CH 3  3  m/e  m/e  294  H  m/e  2  The  i o n a t m/e  72  (2_4) was  3  24  72  297  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 a n d p l a s m a s a m p l e s , and t h e i o n a t m/e (27) f o r r a t b i l e  samples as d e s c r i b e d  294  i n the experimental.  By c o m p a r i n g t h e mass s p e c t r u m 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 f r a g m e n t s a t m/e 223 m i n o r p e a k s a t m/e as t y p i c a l  208, 1 9 3 , 1 7 9 , 178, a n d 165  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 .  294  and  appear The  proposed  46 pathways t o these  fragment  ions  as p a r t o f t h e f r a g m e n t a t i o n However, t h e p o s s i b i l i t y  ( 3 1 - 3 5 , 26) a r e d e s c r i b e d  processes  o f EMDP a n d DDP  t h a t m/e 165 (26J m i g h t be d e r i v e d  f r o m m/e 223 (25^) c a n n o t be e x c l u d e d .  Diphenylacetonitrile  w h i c h c a n n o t f o r m m/e 208 a l s o gave t h e i o n m/e 165  m/e 165 H  1Q  28  m/e 173  + CH  <7 M ' W e 193 2  H  1 0  m/e 116,  m/e 203  H  m/e 121  1Q  (28).  m/e 89 "H  m/e 93  29 30  Deuterated  methadone  When p a r t i a l l y hydrolyzed  metabolites deuterated  methadone n i t r i l e  was  w i t h E^O a n d H SC> , a m a r k e d d e c r e a s e i n t h e d e g r e e 2  4  o f d e u t e r a t i o n f r o m 8 6 % t o 4 0 % 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-diphenylpentanoic hand, treatment  of p a r t i a l l y  w i t h D 0 and D S 0 2  2  4  acid.  deuterated  p r o d u c e d an i n c r e a s e  deuteration i n the a c i d product  t o 97%.  On t h e o t h e r  methadone  nitrile  i n t h e degree o f S t a r t i n g with un-  l a b e l e d methadone n i t r i l e , h y d r o l y s i s w i t h D„0 a n d D„S0.  47  34  ... m/e 165 V „ m/e 174  4"8  gave p r o d u c t  c o n t a i n i n g o n l y 6 0 - 7 0 % d e u t e r a t i o n as  by NMR a n d i t was t h e r e f o r e c o n c l u d e d  determined  t h a t maximum d e u t e r a t i o n  o f 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 n o t be a c h i e v e d b y t h i s method.  latter  The l a b e l e d a c i d was d e c o m p o s e d i n t h e G C i n l e t t o  the d e c a r b o x y l a t e d product,  1,  l-diphenyl-3-dimethylaminobutane-  2  H-^Q  as determined  p e a k s a t m/e  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  (base p e a k ) , m/e  72  263  (M , 0 . 9 % ) , +  m/e  177  (3.0%),  2  and  m/e  173  (1.3%)  a n d DDP-  t i o n t i m e v a l u e a n d mass  as determined  from  i t s reten-  spectrum.  2  DDP- H  1 Q  s y n t h e s i z e d from  ride demethylation  fully  the acid,  r e t a i n e d the deuterium  s p e c t r a o f DDP i s shown i n F i g . 4 . f o r DDP w e r e s i m i l a r t o t h o s e from  t h e m/e  5_ b y t h i o n y l c h l o label.  The f r a g m e n t a t i o n  f o r EMDP b e i n g d e r i v e d  The patterns  mainly  2 0 8 fragment. 2  Mass s p e c t r a a r e shown i n F i g . 5 f o r EDDP, EDDP- H^Q 2  ( 9 7 % l a b e l ) , a n d EDDP- H o f m/e  3  (99% label).  2 0 8 ( 3 1 ) f o r EDDP was 0 . 9 % .  fragmentation  t o p r o d u c e m/e  o f C H - N = C = C H C H ~~I 3  favored  3  from  The r e l a t i v e  abundance  This implied that the  20 8 which would i n v o l v e t h e l o s s  the molecular  i o n i s no l o n g e r a  process. The  f o r m a t i o n o f m/e  105  (3J7) o n t h e o t h e r h a n d i s  u n i q u e t o EDDP a n d 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 to the unsaturated  side chain.  T h i s was r e c o g n i z e d  the appearance o f t h e corresponding 2  1 0 8 f o r EDDP- H  from  1 1 0 a n d m/e  2 1 Q  a n d EDDP- H , r e s p e c t i v e l y . 3  of phenyl m i g r a t i o n i s s i m i l a r 38 ( 8 4 ) .  i o n s o f m/e  ring  The m e c h a n i s m  t o t h a t r e p o r t e d f o r t h e compound,  49  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 u n i q u e t o EDDP i s t h e l o s s o f CH -CH=CH ~1^ t o f o r m m/e 235 (_40) w h i c h t h e n c a n 3  2  e i t h e r f o r m m/e 220 (£1, 21%) o r b y f u r t h e r the  f r a g m e n t m/e 69 (4_2, 21%) .  valuable  loss, o f a CH 2  I n b o t h c a s e s EDDP- H  i n determining the nature o f these  ions.  3  3  form  proved  .52  40  CHCH  3  C  I'  N-CH,  ^  > CH CH= C= N—CH 3  m/e 69  m/e 235 2  H m/e 245, 1Q  \rnfe  42  3  238 3 H  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 a n d EMDP a r e shown i n Fig. 20 8.  6.  EMDP g a v e f r a g m e n t a t i o n s 2  S i n c e EMDP-  r e s u l t i n g mainly  l o s e s the l a b e l e d methyl  f r o m m/e  g r o u p when t h e  m/e 208 f r a g m e n t i s f o r m e d , 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 b e t w e e n EMDP a n d EMDP- H.  Spectroscopic  observations  NMR d a t a Pohland  f o r EDDP a n d EMDP w e r e ' d e s c r i b e d  e t a l . (3).  by  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 a n d EMDP H C l b y B e c k e t t e t a l . (2) w i t h o u t of chemical  earlier  s h i f t values.  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 s u m m a r i z e d i n T a b l e i s o n o f t h e C-4 p r o t o n s  assignment  I w i t h a compar-  w h i c h d e s c r i b e a n AB s y s t e m .  The  53  208  100-i  (a)  193 115  CO z UJ  130  C H ,  N  N  CH,  /  CH,  50H UJ  >  91  179  UJ  165  GC  263 M* 100  300  200  m/e  218  100 n  (b) 203  CO  z  UJ  t5  A,  135  CH,  50  A N  CH, CH,  UJ  >  UJ  X  120  96 188 174 273 M100  Fig.  6.  200  m/e Mass s p e c t r a o f (a) EMDP a n d (b) EMDP- H  300  10  TABLE I .  NMR o f C-4 P r o t o n s o f M e t h a d o n e M e t a b o l i t e s  EDDP .(3) (trans)  EDDP (3) (cis)  6 Ha  2.24  2.24  J Ha-He  8.5  6 Hb  2.56  J Hb-Hc J Ha-Hb  EDDP p e r c h l o r a t e (19)  (_4)  EMDP H C l (20)  DDP (6)  2.60 (dd)  2.23 (dd)  2.51  7.0  8.7  7.0  2.52  3.43 (dd)  2.67 (dd)  3.17  5.3  7.0  8.0  6.5  8.0  6.0  11.0  12.0  14.0  13.4  14.0  13.0  10.0  3, £ as r e p o r t e d b y P o h l a n d e t a l . (3) 19,  EMDP  20 S p e c t r a o b t a i n e d  6 Spectra obtained  with the Varian  with the Bruker  The s o l v e n t i n a l l c a s e s was C D C 1 . 3  \  XL-100  WP-80  Key"CH,  (dd) 2.20 (dd) 9.0 (dd) 2.95 (dd)  55 chemical  s h i f t values  f o r t h e C-4 p r o t o n s  from t h a t i n the r e f e r e n c e t h a t C-4 p r o t o n s as  by S i n g h  i n DDP a r e d i f f e r e n t  e t a l ( 2 7 ) , who r e p o r t e d  a p p e a r e d a t 2.05-2.40 a s m u l t i p l e t s i n C D C l ^  solvent. I n t h e I R , l a b e l e d methadone a n d m e t a b o l i t e s  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  exhibit a  The a p p e a r a n c e o f t h e  C-D p e a k and d i s a p p e a r a n c e o f t h e a r o m a t i c  C-H v i b r a t i o n  be u s e d t o i n d i c a t e t h e 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  2.  (85).  S I M a n a l y s i s o f methadone a n d m e t a b o l i t e s i n b i o l o g i c a l samples  Selected  ion The  was  could  monitoring c o m p u t e r p r o g r a m f o r SIM u s i n g t h e MAT 111 GCMS  d e v e l o p e d by R o l a n d B u r t o n  Sciences. designed  i n the Faculty of  Pharmaceutical  The e f f e c t i v e mass r a n g e f o r t h e i n s t r u m e n t t o be ± 7% o f t h e H a l l p r o b e mass s e t t i n g .  was  The l i m i -  t a t i o n o f t h e mass r a n g e r e s u l t s f r o m t h e f a c t t h a t i o n o p t i c s and  i o n abundance a r e 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 m a g n e t i c s e c t o r i n s t r u m e n t SIM of  mode  (86).  i s used i n  I n p r a c t i c e , i t was f o u n d t h a t t h e m o n i t o r i n g  i o n s was l i m i t e d d e p e n d i n g upon t h e t o t a l  masses o f t h e i o n s o f i n t e r e s t . 2  number a n d t h e  2 When EMDP, EMDP- H ^ , m e t h a Q  d o n e , a n d m e t h a d o n e - H-^Q w e r e b e i n g m o n i t o r e d w i t h a m a g n e t i c field  s e t t i n g o f m/e 2 2 0 , 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  although  t h e i o n s b e i n g m o n i t o r e d , m/e 2 0 8 , 2 1 8 , 2 2 3 , a n d 233  56 are  a l l i n t h e e f f e c t i v e mass r a n g e .  I t was a l s o f o u n d  that  2 methadone2 H-^Q . S w i t c h i n g i  s  a  preferable  i n t e r n a l standard  of the accelerating voltage  range r e s u l t s i n l a r g e standard  t o methadone-  o v e r a l a r g e mass  deviations i n the analysis.  2 H e n c e , 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  standard  2 t h a 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 s e t w i t h PFK.  window  f o r an i o n b e i n g  m o n i t o r e d was  F i n a l a d j u s t m e n t was made b y i n j e c t i n g  s a m p l e s t o c o r r e c t f o r mass d e f e c t .  authentic  I n p r a c t i c e , i t was f o u n d  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  aspect i n t h e computer program i s t o d e t e c t p e a k p o s i t i o n a n d t o c o r r e c t f o r them.  important  s m a l l changes i n  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 p e a k p o s i t i o n h a v i n g  the greatest i o n  c u r r e n t as measured by computer program appears i d e a l ( 8 7 ) . The method R o l a n d B u r t o n u s e d f o r t h i s p r o g r a m i s a s follows:  E a c h i o n i s s c a n n e d f r o m 0.5 amu b e l o w t o 0.5 amu  a b o v e i t s n o m i n a l mass. quarters.  This  scan i s f u r t h e r subdivided  I n t e n s i t y i s c a l c u l a t e d a s t h e sum o f t h e i n n e r  two q u a r t e r s , m i n u s t h e sum o f t h e o u t e r  two q u a r t e r s  r e c t f o r t h e b a s e l i n e change o r a d j a c e n t  peak i o n s .  position quarter  into  information  i s obtained  from the t h i r d ;  is too f a rt o the l e f t .  to corThe window  by s u b t r a c t i n g t h e second  i f t h e number i s p o s i t i v e , t h e window The window  i s moved t o c o r r e c t  accordingly. I o n p e a k p o s i t i o n was a l s o f o u n d t o c h a n g e t o t h e peak a r i s i n g from column b l e e d i n g .  Therefore,  autofocussing  was made t o f u n c t i o n o v e r c e r t a i n p e a k s t r e n g t h s .  When we  57 f o u n d a s u d d e n l a r g e c h a n g e o f t h e window p o s i t i o n , ment o f t h e f u n c t i o n o f a u t o f o c u s s i n g the a u t o f o c u s s i n g The  was made.  readjust-  I n most c a s e s  was c h e c k e d c a r e f u l l y a t a l l t i m e s .  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 d e p e n d e n t 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 t h e c o n t r i b u t i o n of background peaks. reduce background. ity  S e v e r a l c o n s i d e r a t i o n s w e r e made t o Cleaning  t h e i o n source  of the a n a l y s i s , but i n our experience,  f o r c l e a n up o f t h e i o n s o u r c e difficult  to obtain.  raised the sensitivstandard  conditions  and c h a n g i n g f i l a m e n t s were  S c a n 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 S I M a n a l y s e s ' s h o u l d be a v o i d e d  i n order  t o increase sensi-  tivity . S e l e c t i o n o f column p a c k i n g ed.  m a t e r i a l s was a l s o  consider-  I t was f o u n d t h a t m/e 209 f r o m t h e b l e e d o f a n OV-17 c o l u m n  seriously monitored.  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 Therefore,  a w e l l c o n d i t i o n e d c o l u m n was o n l y  u s e d 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 o f 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  ions.  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  a g e n t w h i c h c o n t a i n s t h e m/e  73 i o n was  c o n d i t i o n i n g o f the column.  Any  t i o n and w h i c h r e m a i n e d the background. when m/e  72 was One  m i n i m i z e d by  extensive  s o l v e n t used i n the e x t r a c -  i n t h e sample  also contributed  M e t h y l e n e c h l o r i d e was  to  completely evaporated  b e i n g m o n i t o r e d f o r methadone  analysis.  o f t h e advantages o f t h e use o f l a b e l e d  s t a n d a r d s w i t h t h e SIM s y s t e m was  silanizing  r e p o r t e d t o be a  internal carrier  e f f e c t o f t h e l a b e l e d compound w h i c h c o u l d r e d u c e c o l u m n t i o n and d e c o m p o s i t i o n o f t h e u n l a b e l e d compound t o be mined.  D i p h e n o x y l a t e (88) i s s u c h a c a s e .  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 2 of octopaminelabelling  limits  (89).  adsorp-  deter-  Contrary to this  observed i n the case  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  t h e u s e o f a l a r g e amount o f 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 c a s e l a b e l e d compounds. M o r e o v e r , t h e h i g h c o n c e n t r a of the  [\o]  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 methadone2 H^Q  as i n t e r n a l  carrier  s t a n d a r d p r e v e n t e d any a t t e m p t t o s t u d y a  effect.  A n a l y s i s o f m e t h a d o n e i n p l a s m a and Methadone l e v e l s methadone m a i n t e n a n c e  saliva  i n p l a s m a and s a l i v a  of  p a t i e n t s w e r e f r e q u e n t l y f o u n d 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 In o r d e r t o enhance  samples  ion i s only  t h e s e n s i t i v i t y o f t h e method t h e  s e l e c t i v e b u t s t r o n g p e a k a t m/e  72 was  1.6%.  less  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 f o r methadone w i t h  of  72.  selectivity  2-dimethylamino-4,4-diphenyl-5-nonanone  p e r c h l o r a t e as t h e i n t e r n a l i s a l s o m/e  72 showed h i g h  s t a n d a r d , t h e base peak o f w h i c h  The l o w e r l i m i t o f r e p r o d u c i b l e  methadone i n 0.5 m l o f p l a s m a o r s a l i v a  quantitation  taken f o r e x t r a c t i o n  was 20 n g . The s e n s i t i v i t y to  o b t a i n e d by t h i s  method i s c o m p a r a b l e  t h e m e t h o d s w h i c h a r e r o u t i n e l y u s e d t o a n a l y z e methadone  i n human p l a s m a .  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  observed 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 b a s e p e a k a t m/e 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 , ion  reduce background.  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  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  i o n s o u r c e w e r e e l e v a t e d o v e r n i g h t t o remove  i m p u r i t i e s b e f o r e f r e s h samples the  especially  s o u r c e 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 -  ously l i m i t s e n s i t i v i t y .  and  72 f o r  were a n a l y z e d .  m e t h o d was f o u n d t o be more t h a n a d e q u a t e  patient saliva  o r plasma  samples,  were f o u n d t o v a r y o v e r a w i d e  line,  retained In this  way  to analyze  the c o n c e n t r a t i o n s of which  r a n g e b e t w e e n 0.0 5-1.0  ug/ml  d u r i n g t h e 24 h o u r p e r i o d o f a s t u d y . Monitoring drug c o n c e n t r a t i o n s i n s a l i v a reflect  might  the time course of a drug a t the r e c e p t o r s i t e .  was f o u n d t o be t h e c a s e f o r t h e d r u g p r o c a i n a m i d e a parallel saliva  better This  f o r which  r e l a t i o n s h i p between time course o f t h e drug i n  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 ( 9 0 ) .  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 d r u g s w h i c h a c t o n t h e c e n tral  nervous  system.  For drugs  s u c h a s methadone w h i c h a r e  60  INTERNAL STANDARD  200 220 TEMPERATURE C  240  260  Fig.  7.  SIM c h r o m a t o g r a m (m/e 72) o f methadone f r o m  saliva  Fig.  8.  Methadone c o n c e n t r a t i o n i n p l a s m a and s a l i v a o f a m a i n t e n a n c e p a t i e n t (90 mg/day d o s a g e )  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 tions i n saliva  could better define the a c t i v i t y  drug  concentra-  of 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 d r u g w h i c h c r o s s e s barrier. saliva  the blood  brain  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 monitoring o f  l e v e l s of t h e drug i n steady  especially  s t a t e maintenance p a t i e n t s  i n view of the f a c t that a lack i n c o r r e l a t i o n  b e t w e e n methadone c o n c e n t r a t i o n s  i n p l a s m a a n d symptom  complaints  of p a t i e n t s have been r e p o r t e d ( 4 1 ) . The r e s u l t s f o r p a t i e n t s A a n d B w e r e n o t o b t a i n e d because problems w i t h p a t i e n t s ' compliance 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 p l a s m a o f p a t i e n t  C was 0.55 ± 0.15 (SD) w i t h a r a n g e 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 p l a s m a o f 0.48 ± 0.10  (SD) w i t h a r a n g e 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  account 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 pH v a l u e s w e r e n o t a v a i l a b l e . providing saliva Inc.)  i n the results but  P a t i e n t C had d i f f i c u l t y i n  s a m p l e s a n d c h e w i n g gum  ( D e n t y n e , Adams B r a n d s  was u s e d 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 .  Interference  f r o m t h e c h e w i n g gum i n t h e a n a l y s i s was n o t o b s e r v e d . t i o n o f methadone t o t h e gum was n o t p r o v e n b u t was t o be m i n i m a l . these  I f salivary concentrations  considered  o f methadone i n  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 p l a s m a  then our r e s u l t s agree w e l l w i t h those al.  Adsorp-  r e p o r t e d by Horns e t  ( 4 1 ) , i n w h i c h 5 0 % b i n d i n g o f methadone t o p l a s m a was  reported.  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) w h e r e s a l i v a r y c o n c e n t r a t i o n s  w e r e f o u n d t o be  62 much h i g h e r t h a n t h o s e  obtained  i n whole b l o o d .  h o w e v e r was n o t a d e q u a t e t o d r a w p h a r m a c o k i n e t i c because of t h e l i m i t e d  The r e s u l t conclusions  s a m p l e s i z e a n d t h e number o f s a m p l e s .  A n a l y s i s o f methadone a n d 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 w e r e t h e same a s t h o s e  f o r t h e a n a l y s i s o f methadone i n p l a s m a o r s a l i v a  used  samples.  R e t e n t i o n t i m e s b y GCMS (SIM) o f methadone a n d m e t a b o l i t e s are  3.24  (EMDP), 4.0  (EDDP), 4.78  ( m e t h a d o n e ) , a n d 6.23 m i n .  (DDP). I f we c o n s i d e r t h e f a c t t h a t many d i f f e r e n t  samples  a r e d e a l t w i t h when we a n a l y z e methadone a n d m e t a b o l i t e s i n urines, gas  SIM m i g h t be a t i m e c o n s u m i n g method c o m p a r e d w i t h  chromatographic  methods.  F o r example, a separate  injection  o f t h e s a m p l e was r e q u i r e d t o a n a l y z e e a c h m e t a b o l i t e i n SIM analysis.  On t h e o t h e r h a n d , methadone a n d m e t a b o l i t e s w e r e  a n a l y z e d b y one i n j e c t i o n o f t h e s a m p l e 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  with deuterium  labeled internal  p r o v i d e s e a s e i n work up p r o c e d u r e s .  standards  however  Sample d i l u t i o n s a n d  the s i z e o f t h e i n j e c t i o n . volume d i d n o t a f f e c t t h e o b s e r v e d ion r a t i o s done  w h i c h w e r e u s e d t o c a l c u l a t e t h e amount o f m e t h a -  (Table I I ) .  Extractibility  compounds was f o u n d 7.5  o f l a b e l e d and u n l a b e l e d  t o be t h e same i n C I ^ C ^ s o l v e n t .  (n=4, 2.5 ug e a c h i n 1.0 m l ) , p e r c e n t  m e t h a d o n e - H , EDDP a n d E D D P - H 2  2  1 Q  3  A t pH  r e c o v e r y o f methadone,  was 84.9 ± 2.6, 83.6 ± 0.2, 2  94.2  + 3.8 a n d 93.9 ± 1.1, r e s p e c t i v e l y .  EDDP a n d EDDP-  63 were a n a l y z e d  i n the recovery  s t u d i e s u s i n g EDDP- E^  as t h e  2 i n t e r n a l s t a n d a r d ; methadone and m e t h a d o n e - H^Q u s i n g m e t h a d o n e 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 V o l u m e s Upon the Observed Ion R a t i o s  I n j e c t i o n Volume 1 yl  M e t h a d o n e / m l MeOH  75 yg 15 yg  2  H ,  50 yg  Q  2  HQ,  10 y g  2  H 2  1.91  1 Q  H  ± 0.14  (n=6) 5 yl  2.03 ± 0. 06 1.99  1 Q  ± 0. 08  I n j e c t i o n was c a r r i e d o u t b y m o n i t o r i n g m/e m/e  233.  The numbers d e n o t e m/e  223 and  223/m/e 233 ( S D ) .  C r u d e w i n d o w s w e r e s e t w i t h PFK by m o n i t o r i n g 231 w h i c h was l a t e r e r a s e d . was m/e  EDDP a r e shown i n T a b l e  III.  prepared  The r e s u l t s  c a n be e x p r e s s e d  because i n t e r c e p t I f we a l s o  Hall  f o r methadone and indicate  that c a l i b r a -  by u s i n g o n l y s l o p e  v a l u e s were found  of the drug monitored  values  t o be n o t s i g n i f i c a n t .  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  quantitation  setting  223.  The c a l i b r a t i o n e q u a t i o n s  t i o n equations  Magnetic  m/e  i s a c h i e v e d by  compound multiplying  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 a d d e d internal  standard  x the observed  ratio.  Slope values of  64 TABLE I I I .  C a l i b r a t i o n Equations and EDDP  f o r Urine  Standard  EDDP  0 . 326  0.483  0.0574  0.0033  0.0513  0.0054  0.0503  0.0117  0.9957  0.9990  Error  Intercepts I n t e r c e p t Standard  Analysis.  Methadone Slope Slope  f o r Methadone  Error  Coefficient of Determination <r ) 2  Standard (n=6)  Ratio  (S.D.)  Slope c a l c u l a t e d Standard R a t i o  1.305 ± 0.1272  b  from 0. 326  0  a.  Slope  0. 968 ± 0.0484  and i n t e r c e p t  values are  0.484  c a l c u l a t e d by means o f com-  p u t e r program ( T r i a n g u l a r Regression  Package , Computing  C e n t r e , The U n i v e r s i t y o f B r i t i s h ' C o l u m b i a ) . . M e t h a d o n e (n=7), EDDP Ratio  (n=10) .  ( D r u g / I n t e r n a l Standard) +  I n t e r n a l Standards  = Slope  x Drug c o n c e n t r a t i o n  intercept  2 a r e M e t h a d o n e - H^Q  2 (4 jag) a n d EDDP- H-^Q  (2 u g ) . b.  R a t i o s o f e q u a l amount o f D r u g t o I n t e r n a l (Standard  c  Standard  Ratio). Ratio x 1 / I n t e r n a l Standard.  Standard  r  c a l i b r a t i o n curves and t h e s l o p e s c a l c u l a t e d r a t i o w e r e f o u n d t o be t h e same.  internal  the concentration  standard.  o f EDDP  t h e r a t i o o f m/e 277 2  o f EDDP-  added as t h e  The c a l i b r a t i o n c u r v e method was h o w e v e r  used f o r t h e a n a l y s i s  f o r methadone b e c a u s e o f i n s t a b i l i t y  o f t h e m e a s u r e d r a t i o w h i c h o c c u r r e d when i o n s units difference  standard  The c o n c e n t r a t i o n  was s i m p l y c a l c u l a t e d b y m u l t i p l y i n g t o m/e 280 w i t h  from  65  o r more w e r e m o n i t o r e d .  o f 10 mass  The r a t i o was f o u n d  t o be c h a n g e a b l e d e p e n d i n g upon S I M c o n d i t i o n s ular t o the s t a b i l i t y  and i n p a r t i c -  of t h e magnetic f i e l d .  The c o e f f i c i e n t  o f v a r i a t i o n o f t h e m e a s u r e d methadone s t a n d a r d r a t i o was twice  (9.7%) t h a t o f EDDP SIM  analysis  was c o m p a r e d w i t h  o f methadone a n d EDDP i n u r i n e s a m p l e s  GC a n a l y s i s .  t o be w e l l c o r r e l a t e d . yzed f o r p a t i e n t  (5.2%).  The t w o methods were f o u n d  Methadone  (0.4 ug-6.8 ug/ml) was a n a l -  B a n d C, EDDP (8 ug-32 ug/ml) f o r A a n d D. 2  The c o r r e l a t i o n c o e f f i c i e n t o f d e t e r m i n a t i o n analysis  o f methadone  (n=14) a n d EDDP (n=14) b y S I M c o m p a r e d  t o GC w e r e 0.963 a n d 0.962, Previous experiments in the urine  respectively. with  t h e GC a n a l y s i s  o f maintenance p a t i e n t s  peak i s f r e q u e n t l y hydroxycotinine,  (r ) o ft h e  indicated  overlapped e i t h e r with  a metabolite of nicotine  that  o f EMDP t h e EMDP  caffeine or with ( 3 3 ) . When a n  a t t e m p t was made t o a n a l y z e m a i n t e n a n c e p a t i e n t  urines f o r  EMDP b y m o n i t o r i n g m/e 208 f o r EMDP a n d m/e 218 f o r t h e i n t e r 2 n a l s t a n d a r d , EMDP- H , i t was f o u n d t h a t t h e amount o f EMDP 1 n  66 a p p e a r s t o . b e l e s s t h a n 100 n g / m l .  Because o f t h e s m a l l  amount o f t h i s m e t a b o l i t e , d e t e r m i n a t i o n sary t o study  the demethylation  o f EMDP was n o t n e c e s -  mechanisms o f methadone.  S t a b i l i t y o f EDDP In an a t t e m p t t o o b t a i n i n f o r m a t i o n on t h e minor metab o l i c p a t h w a y s o f m e t h a d o n e , t h e l e v e l o f DDP i n u r i n e was determined by m o n i t o r i n g  m/e 265 f o r DDP a n d m/e 275 f o r DDP-  2 H-^  as i n t e r n a l  adjusted  standard.  When t h e pH o f t h e u r i n e was  t o 10.4 f o r e x t r a c t i o n , i n c o n s i s t e n c y i n t h e r e p r o d u c -  ibility  o f t h e c o n c e n t r a t i o n o f DDP i n a u r i n e s a m p l e was  found.  To i n v e s t i g a t e t h i s ,  made a l k a l i n e a n d l e f t  EDDP p e r c h l o r a t e s o l u t i o n was  t o stand  a t room t e m p e r a t u r e .  A TIC  p r o f i l e o f t h e methylene c h l o r i d e e x t r a c t o f t h e s o l u t i o n showed DDP a s a s i g n i f i c a n t p r o d u c t DDP was i d e n t i f i e d b y TLC w i t h a n R (Brinkman) s h e e t s Further  o f EDDP f  0.8 o n S i l i c a  w i t h E t O H , HAC, E^O  studies revealed  decomposition. G e l F 254  (6:3:1) a s s o l v e n t .  t h a t EDDP f r e e b a s e i n e t h e r s o l u t i o n  i s e a s i l y d e c o m p o s e d t o DDP.  Stoichiometric conversion of  EDDP t o DDP was o b s e r v e d when EDDP a n d DDP i n e t h e r s o l u t i o n were measured u s i n g (Fig.  l a b e l e d compounds a s i n t e r n a l  9). A similar r e s u l t f o r the conversion  o f EDDP t o  DDP b y a p p a r e n t o x i d a t i o n r e a c t i o n s was s e p a r a t e l y by Bowen e t a l . ( 9 2 ) . to  A metabolic  transformation  2, 2 - d i p h e n y l - 4 - d i m e t h y l a m i n o p e n t a n o i c  m e t a b o l i s m t o DDP was s u g g e s t e d  (6).  a m i n o p e n t a n o i c a c i d was n o t d e t e c t e d  2,  acid  standards  reported o f methadone  (5_) a n d f u r t h e r  2-Diphenyl-4-dimethyl-  i n any o f t h e u r i n e s o f  67  Fig.  9.  D e c o m p o s i t i o n o f EDDP b a s e t o DDP  (  EDDP, —  DDP)  -68 maintenance 167  p a t i e n t s u s i n g mass c h r o m a t o g r a m s i n w h i c h  (C,H -)„C H f o r t h e a c i d a n d m/e +  b  r  D  Z  225 ( C H )„ C C O O C H f o r +  b D Z C  s u g g e s t t h a t DDP i s a m e t a b o n a t e  J  C  t h e d i a z o m e t h a n e - t r e a t e d a c i d were m o n i t o r e d . strongly  Q  The r e s u l t s  and t h a t  pH a d j u s t m e n t s s h o u l d be a v o i d e d when b i o l o g i c a l e x t r a c t e d t o d e t e c t new m e t a b o l i t e s .  m/e  extreme  samples a r e  69  3.  D e t e c t i o n o f methadone  metabolites  Extraction procedures f o r r a t b i l e  The p o l a r s o l v e n t extraction of r a t b i l e column  chromatography  t i o n .were n o t u s e d .  ( m e t h y l e n e c h l o r i d e ) was u s e d f o r  samples.  Clean-up procedures using  such as A m b e r l i t e  XAD-2 a n d b a c k  extrac-  The s a m p l e was e x t r a c t e d a n d a n y p r e c i p i -  t a t e was r e m o v e d b y c e n t r i f u g a t i o n ,  f o l l o w e d by a d i r e c t i n -  j e c t i o n o f t h e s a m p l e e x t r a c t i n t o t h e GCMS.  After  initial  e v i d e n c e o f t h e p r e s e n c e o f m e t a b o l i t e s was f o u n d , t h e s a m p l e was  back e x t r a c t e d t o see any d i f f e r e n c e between  b e f o r e and  a f t e r t h e c l e a n up p r o c e d u r e a n d t o s e e a n y compounds w h i c h were t r a n s f e r r e d t o t h e hexane l a y e r used i n t h e back e x t r a c tion  ( F i g . 10) . A r a d i o i s o t o p e l a b e l e d compound a n d XAD-2  chromatography  f o r s a m p l e e x t r a c t i o n was d e s c r i b e d i n t h e  d e t e c t i o n o f methadone m e t a b o l i t e s (93).  column  from r a t u r i n e and b r a i n  A n XAD-2 c o l u m n was a l s o u s e d t o d e t e c t methadone m e t a -  b o l i t e s f r o m man a n d r a t u r i n e b y S u l l i v a n e t a l . ( 4 ) . I n t h i s e x p e r i m e n t , use o f column was  not considered  because  recovery  chromatographic p u r i f i c a t i o n  a p p r o p r i a t e t o d e t e c t minor  metabolites  o f t h e m e t a b o l i t e s " ; c o u l d n o t be m o n i t o r e d .  70  Bile  H 0  Centrifuge  2  pH  9.0  Extraction with CH C1 2  2  ^Nonconjugate f r a c t i o n I Back e x t r a c t i o n , tization.  Deriva-  Freeze d r y Residue /Add  MeOH,  Centrifuge,\  \Evaporate  )  B u f f e r , pH 5.0, 3 - g l u c u r o n i d a s e ,  37°C  pH 8.0-8.5 Extract with  CH C1 2  Conjugate f r a c t i o n  2  D e r i v a t i z e : " w i t h CH' N 2  Back  Fig.  10.  extraction  E x t r a c t i o n Procedure f o r Rat B i l e .  2  71 The r a t b i l e  e x t r a c t i o n method i s s i m i l a r t o t h a t  u s e d f o r d e t e c t i o n o f methadone m e t a b o l i t e s by and  Sullivan e t a l . (6). Direct extraction, d e r i v a t i z a t i o n , GCMS a n a l y s i s w e r e t h e methods u s e d .  p l e s were a n a l y z e d , cipitation and  f r o m human u r i n e s  (94).  theb i l e  sam-  s a l t s were removed b y a c i d  pre-  In our experiments,  p r o t e i n was m i n i m i z e d  precipitation.  When r a t b i l e  by c e n t r i f u g a t i o n and s o l v e n t  Two s e p a r a t e  salt (MeOH)  experiments w i t h and without  a c i d p r e c i p i t a t i o n g a v e t h e same Beckett  t h e amount o f b i l e  results.  e t a l . (2) c a r r i e d o u t t h e d e t e c t i o n o f m e t h a -  done m e t a b o l i t e s  f r o m human u r i n e b y u s i n g n o r m a l  e x t r a c t i o n a n d TLC s e p a r a t i o n .  When a u t h e n t i c  samples a r e n o t a v a i l a b l e , d e t e c t i o n o f minor  solvent  metabolite metabolites  among l a r g e amount o f e n d o g e n o u s m a t e r i a l b y TLC i s a l m o s t impossible.  The u s e o f c o l o r d e v e l o p i n g  s p e c i f i c m e t a b o l i t e s hasbolites  methods t o d e t e c t  b e e n a t t e m p t e d f o r methadone m e t a -  (95) a n d f o r N - o x i d a t i o n m e t a b o l i t e s  p r o a c h however d i d n o t g u a r a n t e e d i r e c t the s t r u c t u r e o f m e t a b o l i t e s .  ( 9 6 ) . The a p -  identification of  Therefore,  TLC a n d c o l o r  spray  methods were n o t used i n o u r e x p e r i m e n t s . The GCMS c o m p u t e r t e c h n i q u e s i d e n t i f y themolecular This technique  s t r u c t u r e o f compounds  tool to  i n mixtures.  i s f u r t h e r a i d e d by t h e use o f s t a b l e i s o t o p e  l a b e l e d compounds technique  are a powerful  i n themetabolic  (iondoublet  technique)  detection of metabolites  studies.  The t w i n i o n  h a s b e e n 'used i n t h e  f o r various  drugs.  The  10  H  - l a b e l e d compounds  done work d i s p l a y a d i f f e r e n c e t h e u n l a b e l e d compounds.  i n physical properties  The d i f f e r e n c e  t i m e o b s e r v e d by GC i s p r e s u m a b l y of the d e u t e r a t e d m a t e r i a l . t i m e s f o r u n l a b e l e d and o b s e r v e d when a 3% 0 ^ 2 2 5  s y n t h e s i z e d f o r t h e metha-  i n the retention  due t o a d e c r e a s e i n p o l a r i t y  A s i m i l a r difference of retention  "2 H  from  16  b u t y l a t e d h y d r o x y t o l u e n e was  GC c o l u m n was u s e d  (97) .  The  deute-  r i u m l a b e l e d methadone and m e t a b o l i t e s w e r e f o u n d t o h a v e slightly  s h o r t e r r e t e n t i o n t i m e s t h a n t h e u n l a b e l e d compounds.  T h e r e f o r e , t w i n i o n s w e r e n o t r e a d i l y o b s e r v e d i n t h e GCMS s c a n d a t a o f e x t r a c t s c o n t a i n i n g l a b e l e d a n d u n l a b e l e d methadone and/or m e t a b o l i t e s . peaks r e s u l t i n g  C o m p a r i s o n o f t h e mass s p e c t r a o f GC  f r o m u n l a b e l e d compounds w i t h t h o s e f r o m l a -  b e l e d compounds r u n a s s e p a r a t e e x p e r i m e n t s g a v e i m p o r t a n t e v i d e n c e f o r 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 .  Nonconjugated  fraction  The n o n c o n j u g a t e d  fraction  i s o l a t e d from r a t b i l e  c o n t a i n e d m a i n l y EDDP and a s m a l l amount o f DDP. o f m e t h a d o n e a n d EMDP were a l s o f o u n d .  Trace  amounts  K r e e k e t a l . (98)  a d s o r e p o r t e d t h e same r e s u l t w i t h t r a c e amounts o f methadone and EMDP f o u n d i n human b i l e .  The p r e s e n c e o f DDP was  account-  ed f o r b y d e c o m p o s i t i o n o f EDDP. The n o n c o n j u g a t e d u r i n e were d r i e d  f r a c t i o n s of r a t b i l e  i n a vacuum d e s i c c a t o r .  and human  S i l a n i z a t i o n of the  73 samples  (150 u l TMCS, 50 u l p y r i d i n e ,  f o l l o w e d b y GCMS d i d n o t i n d i c a t e  70°C f o r o n e h o u r )  t h e presence  o f any d i h y -  drodiol.  Conjugated  fraction  Fig. conjugated  11 shows t h e T I C p r o f i l e o f t h e e x t r a c t o f t h e  fraction  isolated  from r a t b i l e .  summary o f t h e m a j o r f r a g m e n t s  Table  IV i sa  and i o n abundance v a l u e s f o r  endogenous b i l e components and r e c o v e r e d m e t a b o l i t e s . presence  The  o f m o n o h y d r o x y EMDP, d i h y d r o x y EMDP, a n d h y d r o x y  EDDP was e v i d e n t f r o m mass f r a g m e n t a t i o n s t u d i e s o f t h e GC CH 0. 3  1  CH-'CH.  CH,0 CH CH 2  c \ CH,  -  3  N / CH CH,  CH-CH  3  44  CH 0  I  3  CH,CH,  J  CH-CH  ,C  0 ^  V  -  3  m/e 238 H 0 m/e 247 V  45  m/e 207 H  46  U  m/e 216 H  Q  m/e 237 H U  m/e 245 H„  48  f CH,  /  47  ~~]t CH-CH ' 3  m/e 268 ^H U  m/e 276 H o Q  49  (CH-JOJEMDP  Metabolite  Scan numbers F i g . 11,  50  100  150  200  250  300  350  400  (base p e a k , m/e  450  TIC p r o f i l e o f d i a z o m e t h a n e t r e a t e d c o n j u g a t e f r a c t i o n ; f r o m b i l e o f methadone d o s e d r a t s . (GCMS: 150-280 °C, 6°/min,hold at.280°C) :  72)  TABLE I V . Peak No.  Mass S p e c t r a l D a t a o f T I C O b t a i n e d f r o m D i a z o m e t h a n e T r e a t e d c o n j u g a t e f r a c t i o n from b i l e o f methadone d o s e d r a t s  R t (Min)  M/e  (Relative Intensity)  1  11 .2  87 (50) , 75 (19) , 7 4 ( 1 0 0 ) , 5 5 ( 2 9 ) ,  2  12 .8  73 (87) , 60 (83) , 5 7 ( 8 0 ) , 5 5 ( 6 7 ) , 43 (100), 4 1 ( 7 3 )  3  16 .0  109 (25) , 95 (56) , 8 0 ( 8 5 ) , 6 7 ( 1 0 0 ) , 55 (87), 4 1 ( 9 0 )  4  19 .7  93 (49) , 91(63) , 7 9 ( 1 0 0 ) , 6 7 ( 7 7 ) ,  55(43) , 41(70)  5  24 .5  93 (44) , 9 1 ( 7 5 ) , 7 9 ( 1 0 0 ) , 6 7 ( 7 0 ) ,  55(39) , 41(87)  6  27 .6  270 (16) , 149 (10) , 1 3 4 ( 1 0 0 ) ,  7  34 .7  326 (5) , 205 (4) , 1 5 9 ( 5 ) , 147 ( 9 ) , 122 (100) , 9 1 ( 1 5 )  8  37 .5  107 (43) , 9 5 ( 4 7 ) , 81 ( 5 7 ) ,  CH OEMDP  21.5  3  CH_OEMDP- H. 2 CH OEDDP- H 2  43(36) / 41(33)  1 2 1 ( 1 1 ) , 119 ( 1 4 ) , 9 1 ( 4 )  57 ( 6 3 ) ,  55 ( 8 0 ) , 43 (100)  238(100),  223(37), 207(48),  130(39)  1 1 5 ( 3 5 ) , 91 (25)  247(100),  232 ( 4 2 ) , 2 1 7 ( 6 1 ) ,  165(23)  135(46),  96(18)  2 3 2 ( 5 9 ) , 216 (67)  203(31),  135(55)  3  24.1  g  (CH-,0)  EMDP  (CH 0) EMDPMetabolite 3  2  2  Metabolite-^H  25.3 H  g  28.2 10  316(63), 247(100),  268(58), 253(28), 237(100),  129(38)  115(28),  91(21)  276(58), 261(36), 245(100),  230(10)  133(22),  83(18)  253(42), 207(68), 129(33),  72(100),  263 ( 2 3 ) , 2 1 7 ( 4 6 ) , 134 ( 1 3 ) , 7 2 ( 1 0 0 ) ,  57(20),  44(36)  5 7 ( 2 6 ) , 44 (40)  76 peaks r e s u l t i n g f r o m t h e m e t a b o l i s m o f u n l a b e l e d and l a b e l e d methadone  (44-49).  I o n s m/e 238 a n d m/e 24 7 w e r e s e l e c t e d t o  2 m o n i t o r d e r i v a t i z e d HOEMDP a n d HOEMDP- H . g  I o n s m/e 2 37 a n d  m/e 245 w e r e u s e d t o m o n i t o r d e r i v a t i z e d DiHOEMDP a n d . : /'M 1:  2 DiHOEMDP- Hg.  SIM a n a l y s i s o f c o n j u g a t e d m e t a b o l i t e s u s i n g  t h e s e i o n s was d e s c r i b e d  i n t h e study o f methadone-diazepam  interaction. I n a d d i t i o n t o r i n g h y d r o x y l a t e d m e t a b o l i t e s o f methadone, t h e c o n j u g a t e d f r a c t i o n from r a t b i l e c o n t a i n e d a peak h a v i n g an abundant  m/e 72 i o n ( F i g . 1 1 , T a b l e I V ) .  t r e a t m e n t d i d n o t s h i f t t h e GC p e a k p o s i t i o n ,  implying that the  compound d o e s n o t c o n t a i n a p h e n y l - h y d r o x y 1 g r o u p . g a t e d f r a c t i o n was made a c i d i c hexane.  gen  t o t h e hexane  s o l u b i l i t y and t h e absence  i n the molecule.  The c o n j u -  (pH 2.0) a n d e x t r a c t e d  The compound was t r a n s f e r r e d  implying low water  Diazomethane  with  layer,  of a basic  nitro-  The mass s p e c t r u m o f t h i s GC p e a k i s  shown i n F i g u r e 1 2 . When t h e e x p e r i m e n t f o r t h e d e t e c t i o n o f m e t a b o l i t e s 2 was  r e p e a t e d w i t h m e t h a d o n e - H^Q, d e u t e r a t e d h y d r o x y EMDP,  d i h y d r o x y EMDP, a n d h y d r o x y EDDP w e r e o b s e r v e d a n d t h e m/e 72 c o n t a i n i n g GC p e a k  ( R t 28.2 m i n . ) was f o u n d t o r e t a i n  d e u t e r i u m on t h e benzene r i n g .  The r e t e n t i o n t i m e o f t h e com-  o u n d i s shown i n T a b l e I V w i t h c o m p a r i s o n t o t h e o t h e r metabolites. The  peak w i t h t h e abundant  when EMDP a n d EDDP w e r e d o s e d  m/e 72 i o n was n o t o b s e r v e d  to rats.  U r i n e samples  from  methadone m a i n t e n a n c e  p a t i e n t s d i d n o t i n d i c a t e the presence o f  t h e m/e  Nonconjugate  72 compound.  f r a c t i o n f r o m r a t b i l e was  72  207  253  44  129  57  309(M ) +  UL Fig.  J  J  m/e 12.  Mass s p e c t r u m o f m/e 72 c o n t a i n i n g  metabolite  78 s u b j e c t e d t o t h e same w o r k - u p p r o c e d u r e s a s t h o s e gated ity  metabolites.  f o r the conju-  T h i s p r o c e d u r e was t o e x a m i n e t h e p o s s i b i l -  t h a t methadone N - o x i d e o r o t h e r m e t a b o l i t e s w h i c h w e r e n o t  e x t r a c t e d from the b i l e the  source  i n the f i r s t extraction  o f t h e m/e 72 c o n t a i n i n g p e a k .  s t e p m i g h t be  The f i n a l e x t r a c t s  d i d n o t g i v e t h e m/e 72 c o n t a i n i n g p e a k .  R i n g h y d r o x y l a t i o n p a t h w a y s o f methadone  metabolism  The p r e s e n c e o f m o n o h y d r o x y EMDP a n d d i h y d r o x y  EMDP  r e s u l t i n g f r o m methadone m e t a b o l i s m was p r e v i o u s l y r e p o r t e d by  S u l l i v a n e t a l . ( 4 ) , Lynn e t a l . ( 7 ) , and Angg&rd e t a l .  (8).  R o e r i g e t a l . (38) c a l l e d  metabolites" without  t h e compounds  "water s o l u b l e  d i f f e r e n t i a t i n g b e t w e e n t h e mono- a n d  dihydroxy-compounds. The p e a k h e i g h t o f d i h y d r o x y  EMDP i n two e x p e r i m e n t s  was f o u n d t o be 1/3 t h a t o f m o n o h y d r o x y EMDP. administered excreted  i n the b i l e  methadone. al.  t o a r a t (20 mg/Kg),  conjugated  When EDDP was metabolites  were  i n l e s s e r amounts t h a n when a d m i n i s t e r i n g  The r e s u l t  i s similar t o the report of Roerig e t  (99) a n d p o s s i b l e r e a s o n s c o u l d be s o u g h t i n t h e d i f f e r e n t  m e t a b o l i s m o r e x c r e t i o n m e c h a n i s m o f methadone  compared!>to  EDDP. An i s o t o p e e f f e c t  i n the metabolic  l a b e l e d methadone was n o t s t u d i e d m a i n l y  hydroxylation of  b e c a u s e 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 n o t 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 . w h e t h e r an e p o x i d e i n t e r m e d i a t e (3->-50-»-10 o r 1A) , o r d i r e c t  insertion  m e c h a n i s m was t h e r e f o r e n o t s t u d i e d . diol  (5JD) i s i n v o l v e d  (3->-10->-ll) i s t h e o x i d i z i n g The f a c t t h a t  the'dihydro-  (5_1) was n o t d e t e c t e d d o e s n o t n e c e s s a r i l y i m p l y  the epoxide i n t e r m e d i a t e o f methadone.  that  (5_0) i s n o t i n v o l v e d i n t h e m e t a b o l i s m  I f epoxide intermediate formation  i s a mechanism  80 there  i sa possibility  f o rthe formation of a r e a c t i v e toxic  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 An  a t t e m p t was made t o s t u d y  (100).  stereochemical  aspects  of the r i n g hydroxylated metabolites since the c o n f i g u r a t i o n of hydroxy-metabolites useful to find  has n o t been examined.  a stereospecific  f o r m e t h o x y EDDP.  fragmentation  p r o c e s s (101)  The m a j o r - f r a g m e n t s t o o b s e r v e  phenyl  r i n g rearranged  EDDP.  Mass s p e c t r a d i d n o t show a n y m/e  o r m/e  105, i n d i c a t i n g  favorable  I t appeared  were t h e  f r a g m e n t s a n a l o g o u s t o m/e  that phenyl  fragmentation  process  135  105 (37) o f  (CH OC H CHCH "11) 3  6  4  3  ring migration i s not a  f o r m e t h o x y 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 methylphenytoin selectivity  (103), and h e x o b a r b i t a l (104).  (102), Stereo-  i n . t h e m e t a b o l i s m o f EMDP t o i t s r i n g  metabolites i s also very  l i k e l y but this  aspect  hydroxylated  o f t h e work  r e m a i n s t o be d o n e . EMDP was f o u n d The  importance  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 ) .  o f hydroxy m e t a b o l i t e s  i n the pharmacological  a c t i o n o f methadone a p p e a r s t h e r e f o r e t o be m i n i m a l n o t be e x c l u d e d .  b u t may  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 t h e nerve t e r m i n a l a c c u m u l a t i o n amine  (10 5 ) .  estrogens  I r r e v e r s i b l e binding of estrogens  of the  and 2-hydroxy-  t o microsomal 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  catechol nucleus  (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 molecules i n the i n t e r a c t i o n type was  interaction  The conjugated was  (107).  m e t a b o l i c p a t h w a y o f methadone  m e t a b o l i t e w h i c h was i d e n t i f i e d b y GCMS i n t h e fraction  o f r a t b i l e w i t h m/e 72 f o r a b a s e p e a k  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 found i n t h e conjugated ity  A semiquinone  of adriamycin t o r a t l i v e r microsomal p r o t e i n s  also reported  N-hydroxylated  with biomolecules.  (52) .  S i n c e t h e p o s t u l a t e d n i t r o n e was  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 -  t h a t t h e N-hydroxy m e t a b o l i t e  (5_3) was d e t e c t e d a s t h e  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 extensively  s t u d i e d b y B e c k e t t e t a l . (108)  volve d e r i v a t i z a t i o n  and n o r m a l l y i n -  o f t h e N - h y d r o x y g r o u p w i t h TMCS a n d  analysis of the silanized  s a m p l e w i t h GCMS.  An a t t e m p t t o  82 detect  the N-hydroxy m e t a b o l i t e  (53) u s i n g  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 present i n concentrations ic color reactions  below  s u c h methods was  absent or  the l e v e l of d e t e c t i o n .  f o r N-hydroxy m e t a b o l i t e s  were  Specif-  described  by C o u t t s e t a l . (96) b u t i n t h i s work were f o u n d t o be c u l t t o use  diffi-  i n t h e p r e s e n c e o f many e n d o g e n o u s m a t e r i a l s  w i t h o u t an a u t h e n t i c Earlier that  was  and  sample.  s t u d i e s by B e c k e t t  e t a l . (109)  described  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 o x y g e n t o n i t r o n e s , w h i c h s u g g e s t s t h a t methadone nitrone  (_52)  i s a decomposed o r a i r o x i d i z e d p r o d u c t o f  hydroxynormethadpne  . (5_3) .  The  N-  v u l n e r a b i l i t y o f N-hydroxyamphe-  t a m i n e and N - h y d r o x y p h e r i t e r m i n e t o a i r o x i d a t i o n was  studied  by L i n d e k e e t a l . ( 1 1 0 ) . Coutts et a l . ( I l l ) described metabolites  (5_4) a s c y c l i z e d  fragmentation  forms  detection  of  nitrone  (5_5) and by means o f mass  s t u d i e s and d i f f e r e n t TLC  R  f  values.  +  54  55  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 t h e e n c e o f methadone n i t r o n e t o be d i f f i c u l t b e c a u s e p r e s e n t i n a sample.  (_52)  i n r a t b i l e was  also  of the small quantity of  Therefore,  pres-  considered  metabolite  the t e n t a t i v e assignment  of  83  t h e new on gas  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 b a s e d chromatographic  r e t e n t i o n t i m e d a t a and mass  fragmenta-  t i o n p a t t e r n s o f t h e 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 d o s e d r a t b i l e . A d d i t i o n a l evidence t u r e was  f o r t h e methadone n i t r o n e s t r u c -  o b t a i n e d f r o m 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 w i t h the  same f r a g m e n t a t i o n  teristics of b i l e  acid.  A compound was  p a t t e r n s and  t h e same GC  charac-  as t h e compound i s o l a t e d f r o m t h e c o n j u g a t e d  f r o m methadone d o s e d r a t s .  the c h e m i c a l  section describing  formation methadone  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  possible metabolic  r o u t e s f o r methadone  ( F i g . 13).  p a r t of Conjugation  o f t h e N - h y d r o x y m e t a b o l i t e o f n o r m e t h a d o n e , 5_3, i s a possibility  of  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 of N-hydroxy m e t a b o l i t e s of  The  fraction  Detailed determination  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  strong  b a s e d on o t h e r e x a m p l e s r e p o r t e d f o r N - h y d r o x y  metabolites  (96).  Normethadone may  obtained  n o t a l l be  spontaneously  i s f a v o r e d a t pHs the protonated t h o u g h t h e pH  (2_) w h i c h i s f o r m e d f r o m methadone cyclized  h i g h e r t h a n t h e pKa  amine d o e s n o t a t the  The  cyclization  o f normethadone because  f u n c t i o n as a n u c l e o p h i l e .  s i t e of enzymatic  there i s a strong p o s s i b i l i t y  t o EDDP.  (1)  N - o x i d a t i o n i s not  Alknown,  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 CHCH-N(CH) CH., 1 X  I  r  J 2  3  N  -CHgCHj f^Y ^CH^CH-NCCH-j^ C C-CHgCHj CH -CH-NHCH CH,  %  2  8  ^C-CHCH ^- 64 ,. , CHg-CH—N-OH "V CH 2  a  ^C-CHgCHj "CH-CH-!jl-CH CHjOH 2  3  20  /H C CIH,  \  3  3  \  61  13.  0 2 ' 3NH, H „ C <y TT CHC ^ C H — 2CH,I 2 W  C-CHCH CH2-C=0 2  \Jl C-CHCh ^c CH-CH—N0 CH, /  CH-CH —M=0 CH  .'66  2  Fig.  /  52 65  CHj o-  CH-CH, ,C NH \ H CH„ •CCH ,  3  3  P o s s i b l e m e t a b o l i c p a t h w a y s f o r methadone  2  3  x  2  5  00,  normethadone i s n o t a l l c y c l i z e d A similar  analogy  t o EDDP.  c a n be f o u n d  i n the benzodiazepines  K o n i s h i e t a l . (113) r e p o r t e d t h a t 5_7 f o r m e d f r o m 56 i s n o t spontaneously  cyclized  t o 5_8 b u t a l s o f o r m s 5_9 b e f o r e  tion occurs.  A t p h y s i o l o g i c a l pH o f ^7.0  (oxazepam and d i a z e p a m ) f a v o r c y c l i z a t i o n of t h e azomethine l i n k a g e  (6,8)  (114).  t h e h i g h e r pH r e g i o n , f a c i l e (115)  and n i t r a z e p a m  with their  benzodiazepines over h y d r o l y s i s  A t pH v a l u e s b e l o w i t s p K a ,  i s °t subject to c y c l i z a t i o n . n  cycliza  On t h e o t h e r h a n d , i n  cyclization  occurs.  Demoxepam  (116) a l s o f o r m t h e same e q u i l i b r i u m  hydrolyzed products.  t h e pH a t t h e m e t a b o l i c  T h i s means t h e r e f o r e t h a t  s i t e i s important  f o rthe rate of  f o r m a t i o n o f c e r t a i n m e t a b o l i t e s s u c h a s 59.  86  A n o t h e r s i m i l a r e x a m p l e was r e p o r t e d naphthylamine which forms adducts w i t h the  structure  69 o r 7_0 (117) .  i s t h e major species  N-hydroxyl-  DNA, o n e o f w h i c h h a s  U n d e r a c i d i c c o n d i t i o n s , 7_0  a n d t h e two f o r m s were r e p o r t e d  i n t e r c o n v e r t i b l e b y c h a n g i n g pH  I n F i g . 13 a p r o p o s a l t w e e n n o r m e t h a d o n e a n d EDDP The  with  t o be  conditions.  was made f o r a n e q u i l i b r i u m b e (_3) a t p h y s i o l o g i c a l  conditions.  d e m e t h y l a t e d p r o d u c t , 2_ c a n t h e n be o x i d i z e d t o 5_3 w h i c h  i s conjugated before excretion.  F u t u r e work i s r e q u i r e d t o  87 study  a p o s s i b l e e q u i l i b r i u m b e t w e e n N - d e m e t h y l m e t h a d o n e and  EDDP, p e r h a p s by u s i n g UV o r NMR a n d u n d e r d i f f e r e n t pH c o n d i tions.  S i m i l a r w o r k h a s b e e n done by H a s s a l e t a l . (118)  for benzodiazepine  derivatives.  Pharmacological significance of the d e t e c t i o n o f methadone n i t r o n e  The and  d e t e c t i o n o f a n N - h y d r o x y m e t a b o l i t e o f methadone  p o t e n t i a l f o r m a t i o n o f methadone n i t r o n e by a m e t a b o l i c  process  from t h e N-hydroxy m e t a b o l i t e  s h o u l d be  i n terms o f t h e p o s s i b l e b i n d i n g o f these  considered  metabolites  a n d any  m e t a b o l i t e s w h i c h a r e f u r t h e r f o r m e d frommthe m e t a b o l i t e t o cellular  components. P h y s i c a l d a t a o n t h e compound f o r m e d f r o m EDDP o x i d a t i o n  and  t h o u g h t t o be a methadone n i t r o n e h a s a h i g h  coefficient  partition  f o r t h i s compounddwith h i g h s o l u b i l i t y  long r e t e n t i o n time  i n hexane, a  o n a n OV-17 GC c o l u m n r e l a t i v e t o methadone  o r any o f i t s o t h e r m e t a b o l i t e s , and a l o n g r e t e n t i o n time  with  r e v e r s e d p h a s e HPLC. N-demethylation morphine  occurs  i n t h e b r a i n i n t h e case o f  (119) where N - d e m e t h y l a t i o n  o f methadone a n d f o r m a -  t i o n o f N - h y d r o x y n o r m e t h a d o n e o r methadone n i t r o n e m i g h t a l s o occur. and  N - h y d r o x y n o r m e t h a d o n e c o u l d be o x i d i z e d t o n i t r o n e  other metabolites  (120).  i n t h e body as a r e o t h e r  hydroxylamines  The f o r m e d m e t a b o l i t e s , e . g . methadone n i t r o n e may  then bind t o p r o t e i n s .  This binding of the nitrone t o proteins  88 is  likely  t o be an e l e c t r o p h i l i c  o f GSH w i t h  the n i t r o n e ,  The b i n d i n g ..  71  r e a c t i o n as i n t h e r e a c t i o n  (121).  o f N - h y d r o x y compounds and t h e i r  to  protein  has been e x t e n s i v e l y  studied  the  c a r c i n o g e n i c i t y of N-hydroxy-l-naphthylamine  and N - h y d r o x y - N - m e t h y l - 4 - a m i n o a z o b e n z e n e and n i t r o x i d e m e t a b o l i t e s P-;4  50 c o m p l e x f o r m a t i o n  /  \  metabolites  i n r e l a t i o n to (7_3)  (74J (112).  (117) Nitro  a r e known t o p a r t i c i p a t e i n c y t o c h r o m e  (121).  0 H  N  73  The p h a r m a c o l o g i a l a c t i o n r e s u l t i n g f r o m N - h y d r o x y normethadone  or i t smetabolites,  e . g . methadone n i t r o n e  t o p r o t e i n , one o f w h i c h m i g h t be c o v a l e n t speculative genicity. metabolites  a t t h e moment.  The b i n d i n g  may  bonding, i s only cause  The p r o l o n g e d p h a r m a c o l o g i c a l a c t i v i t y compared  binding  carcinoof N-hydroxy  t o t h e p a r e n t d r u g s as r e p o r t e d  by G o r r o d  89 e t a l . ( 1 2 3 ) c o u l d mean t h a t t h e s e m e t a b o l i t e s a r e s t r o n g l y bound t o r e c e p t o r s t r u c t u r e s .  The t i g h t b i n d i n g o f l a b e l e d  m a t e r i a l - t o r a t b r a i n t i s s u e i n s t u d i e s o f methadone by M i s r a et a l . ( 1 0 ) might suggest  involvement  o r methadone n i t r o n e o r t h e i r  o f N-hydroxynormethadone  subsequent metabolic products i n  the t o l e r a n c e o r other pharmacological  a c t i o n s o f methadone.  P o s s i b l e m e t a b o l i c p a t h w a y s o f methadone p r o p o s e d i n F i g . 1 3  Among t h e p o s s i b l e m e t a b o l i t e s o f m e t h a d o n e , N - h y d r o x y normethadone  (5_3) , n i t r o n e s  (5_2, 6_0) d i k e t o n e  ( 6 2 ) , N - h y d r o x y a m i n o compound heptanone  (6J3) , amino compound  (6_5) , a n d n i t r o h e p t a n o n e  The l i m i t e d  vented  the possible detection of metabolites.  logical  (6_4) , n i t r o s o -  (66) were n o t d e t e c t e d  work.  that potentially  (61) , EMDP N - o x i d e  i n the  amounts o f t h e s a m p l e and d e t e c t i o n l i m i t s  pre-  T h i s means  a c t i v e m e t a b o l i t e s may be f o r m e d i n t h e b i o -  system w i t h o u t being d e t e c t e d .  on t h e p o s t u l a t e d n i t r o n e  (5_2) s u g g e s t s  Evidence  f o r 5_3 b a s e d  the p o s s i b i l i t y  of t h e formation o f i t s f u r t h e r metabolized  products.  In o r d e r t o examine t h e p o s s i b l e f o r m a t i o n o f such m e t a b o l i t e s and t h e i r p o t e n t i a l a c t i v i t y , model s t u d i e s are suggested  i n this thesis.  metabolism  Model metabolism  studies  c o u l d be p e r f o r m e d by u s i n g a n i m a l m o d e l s o r c h e m i c a l r e a c t i o n models f o r which c h e m i c a l l y s y n t h e s i z e d m e t a b o l i t e s a r e essential.  The m e t a b o l i t e s e . g . N - h y d r o x y n o r m e t h a d o n e  c o u l d be o b t a i n e d by r e d u c t i o n o f n i t r o n e  (5_3) w h i c h  (_52) a r e a d m i n i s t e r e d  90 to animals t o study f u r t h e r metabolic 52  a n d 62 c o u l d  pathways.  a l s o s e r v e as good s t a r t i n g m a t e r i a l s .  Chemical r e a c t i o n processes are involved olism  (124) a n d some o f t h e m e t a b o l i c  lar reaction patterns  The m e t a b o l i c  via nitrones  found i n N-hydroxyphentermine could  follow simi-  t o t h o s e o f c h e m i c a l o x i d a t i o n by  m-chloroperbenzoic acid. nitroso metabolites  i n drug, metab-  processes such as n i t r o n e  h y d r o l y s i s and o x i d a t i o n o f N-hydroxy m e t a b o l i t e s  (121)  The compounds,  formation  o f n i t r o and  and t h e i r h y d r o l y z e d  product  (120) and n o r b e n z p h e n t e r m i n e  be r e p r o d u c e d b y MCPBA o x i d a t i o n o f t h e N - h y d r o x y  metabolites. MCPBA o x i d a t i o n o f m e t h a d o l may l e a d t o t h e f o r m a t i o n of methadol n i t r o n e nitrone  (7_5) a n d f u r t h e r o x i d a t i o n o f m e t h a d o l  gave t h e n i t r o h e p t a n o l  (7_7) ( 1 2 5 ) .  A s i m i l a r e x a m p l e was p r o p o s e d i n t h e MCPBA o x i d a t i o n of  i s o x a z o l i d i n e , 7j3 t o f o r m o x a z i n e ,  19_ w h i c h was f u r t h e r  o x i d i z e d t o n i t r o s o , 82^ a n d n i t r o compound, 8_3 v i a t h e hydrolyzed  p r o d u c t , 81. ( 1 2 6 ) .  91  Chemical h y d r o l y s i s of nitrones nitrone chemistry  (12 7, 128) .  p o u n d , 6_3 c o u l d be f o r m e d EMDP N - o x i d e , 62. be f o r m e d  There  i s common i n  T h e r e fore,:.'.hydroxy amino com-  f r o m methadone n i t r o n e , 5_2 a n d i salso a p o s s i b i l i t y  f r o m a m i n o compound, 6_4 w h i c h i s a  p r o d u c t o f p r o t o n a t e d EMDP  (2JJ) .  t h a t ".6 3 hydrolyzed  could  92  4.  Chemical d x i d a t i o n  studies  C h e m i c a l o x i d a t i o n o f methadone  The  formation  is consistent with d o u b l e bond  (3).  o f DDP f r o m a i r o x i d a t i o n o f EDDP b a s e  t h e f a c t t h a t EDDP b a s e h a s a n e x o c y c l i c This  DDP m i g h t be d e r i v e d  does n o t e x c l u d e t h e p o s s i b i l i t y  more i n d i c a t i v e o f t h e  character  Detection  o f DDP b y TLC i s h o w e v e r  o x i d a t i v e c l e a v a g e o f the d o u b l e bond.  t o see t h e i m p o r t a n c e o f t h e d o u b l e bond  o f EDDP a n d EMDP a t d i f f e r e n t pH c o n d i t i o n s , t h e  f r e e b a s e a n d s a l t f o r m s o f EDDP a n d EMDP w e r e o x i d i z e d m-chloroperbenzoic Oxidation  t i o n o f DDP.  o f EDDP b a s e f o l l o w s a m e c h a n i s m i n v o l v i n g  Oxidation  of three  nitrone product  with  acid.  o x i d a t i v e d o u b l e bond b r e a k a g e  formation  that  from thermal cleavage o f t h e a i r o x i d i z e d  EDDP on t h e GC c o l u m n .  In order  metabolites  (129) a n d r e s u l t s i n t h e f o r m a -  o f EDDP p e r c h l o r a t e  resulted i n the  m a j o r p r o d u c t s i n c l u d i n g t h e methadone  (5_2) w h i c h i s d i s c u s s e d  separately  i n a later  section. Oxidation  o f EMDP b a s e g a v e 2 - e t h y l - 5 - m e t h y l - 3 , 3 -  d i p h e n y l p y r r o l i d y l - 1 , 2-oxaziran spectroscopic  methods.  (84)  ; r  i d e n t i f i e d by  93  F o u r p e a k s w e r e shown i n t h e o x i d a t i o n m i x t u r e o f EMDP HC1  : GCMS, 150-280°C, 8°/min.; R t 4 m i n . , d i p h e n y l e t h y l e n e ;  9.4  min.,  EMDP; 11.6  20 8 b a s e p e a k .  min.,  Attempts  m/e  208 b a s e p e a k ; 12.6  to isolate  u s i n g c o l u m n c h r o m a t o g r a p h y and  t h e two  TLC  min.,  m/e  unknown compounds  i n order to elucidate  s t r u c t u r e s f a i l e d because of the u n s t a b l e nature of  their  these  compounds.  Chemical  o x i d a t i o n o f EDDP p e r c h l o r a t e  The chlorate  TIC p r o f i l e  o f t h e o x i d a t i o n p r o d u c t s f r o m EDDP p e r -  i s shown i n F i g . 14.  The  same o x i d a t i o n  2 was  2  r e p e a t e d w i t h EDDP- H^Q  ate to c o l l e c t a d d i t i o n a l Three major components  experiment  p e r c h l o r a t e and EDDP-  perchlor-  i n f o r m a t i o n on mass f r a g m e n t a t i o n .  (A, B, and C)  f r o m t h e EDDP p e r c h l o r -  a t e o x i d a t i o n p r o d u c t s w e r e c o l l e c t e d u s i n g p r e p a r a t i v e GC. H i g h r e s o l u t i o n mass s p e c t r a w e r e o b t a i n e d on t h e s e fractions.  Chemical  isolated  i o n i z a t i o n GCMS o f t h e o x i d a t i o n  a l s o gave a d d i t i o n a l d a t a f o r m o l e c u l a r  ions.  mixture  Fig.  14.  T I C p r o f i l e ( a ; GCMS, 200-280°C 8°/min.) a n d HPLC (b) o f EDDP p e r c h l o r a t e o x i d i z e d p r o d u c t s  95 Two lated was  m a j o r components from o x i d a t i o n m i x t u r e were  i n pure  f o r m u s i n g HPLC  not i s o l a t e d  i n pure  ( F i g . 14 B and C ) .  f o r m and was  (85).  NMR  and  Compound A  t h u s i d e n t i f i e d by  r e t e n t i o n t i m e and mass f r a g m e n t a t i o n p a t t e r n s . s t r u c t u r e f o r A was  iso-  A  GC  possible  2-acetyl-5-methyl-3,3-diphenyl-l-pyrroline  IR o f compounds, B and C g a v e p o s i t i v e  f i c a t i o n as 4 , 4 - d i p h e n y l - 2 , 5 - h e p t a n e d i o n e done n i t r o n e (52) f o r C.  Attempts  identi-  (6_1) f o r B and  to i s o l a t e each  metha-  component  from t h e o x i d a t i o n m i x t u r e u s i n g p r e p a r a t i v e column chromat o g r a p h y were n o t  successful.  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  spectrometry  Mass f r a g m e n t a t i o n d a t a a r e s u m m a r i z e d i n T a b l e f o r methadone n i t r o n e (_52) . be m/e  309.  The  The  m o l e c u l a r i o n was  <  -20 23 ^2* H  Chemical  N<  p e a k f r o m c h e m i c a l i o n i z a t i o n was  292  D i r e c t p r o b e mass s p e c t r o m e t r y  a m o l e c u l a r i o n a t m/e The  One  t i o n o f _52 t o e i t h e r 253  arising  mass  A strong from  (EI) a l s o  showed  30 9.  methadone n i t r o n e was  three pathways.  t o m/e  m/e  310.  to  resolution  ionization  s p e c t r o m e t r y gave a q u a s i m o l e c u l a r i o n a t m/e  M+H-H2O.  found  m o l e c u l a r f o r m u l a o b t a i n e d by h i g h  mass s p e c t r o m e t r y was  V  proposed  p o s t u l a t e d to fragment  pathway d e s c r i b e s the  8_6 o r 88>, w i t h f u r t h e r  (90 o r 92) , and m/e  73  via  cycliza-  fragmentations  (91 o r 93) .  96 TABLE V.  H i g h R e s o l u t i o n Mass F r a g m e n t a t i o n D a t a f o r M e t h a d o n e N i t r o n e (52)  Methadone n i t r o n e m/e 309  C  2 0  H  m/e 253  C  1 7  H  m/e 208  C  16 16  m/e 207  C  16 15  m/e 193  C  15 15  m/e 181  C  14 13  m/e 180  C  14 12  m/e 179  C  14 11  m/e 178 . ;  C  14 10  m/e 165  C  13 9  m/e 130  C  10 10  m/e 129  C  10 9  m/e 115  C  9 7  m/e 91  C  7 7  m/e 73  C H NO  m/e 72 m/e 57 m/e 44  Comparable  2 3  1 9  NO  H_.  2  NO  H  m/e 312  m/e 319  m/e 253  m/e 263  m/e 207  m/e 217  m/e 129  m/e 134  H  H  H  H  H  H  H  H  H  H  H  H  3  7  3 6 m/e 60  C,H 0 3 D c  m/e 44  m/e 44  i o n f r a g m e n t s a s s e e n b y l o w r e s o l u t i o n GCMS  2 2 a r e g i v e n f o r t h e H-, a n d H-,  n  analogs.  97  F o r m a t i o n , o f t h e m/e  292  fragment  (87_ o r 89)  a p p e a r s t o be t h e  p r e f e r a b l e r o u t e under c h e m i c a l  ionization  protonation e a s i l y occurs.  s i x membered i o n , 8j5 a p p e a r s  The  c o n d i t i o n s whereby  98 more s t a b l e t h a n t h e s e v e n membered i o n , 88> a n d t h u s t h e f o r m a t i o n o f 86^ a p p e a r s t o be a f a v o r a b l e r o u t e .  A s an e x a m p l e  the r e a c t i o n intermediate,-94 o f 3 - c a r b o x y p y r i d i n e 1 - o x i d e r  and p r o p i o n i c a n h y d r i d e  gave _96, w h i c h  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 t h e s i t e o f - methylene carbon  (130) .  G0 H  >  +  2  C-CH  II H 0  3  o-  96  95  94  methadone n i t r o n e (_52) f o r m s c y c l i z e d ' f r a g m e n t , ;  The f r a g m e n t , -:.-'•'*-  • m/e  The  second  nitrone yields and m/e  t i o n o f m/e  and m/e  fragment  (Table V ) .  f r a g m e n t a t i o n p a t h w a y f o r t h e methadone  i o n s , m/e  72 (99) , m/e  44 (9_7) , m/e  f o r t h e f o r m a t i o n o f m/e  i n methadone a n a l o g s .  vatives.  - compound was a k e y t o  57 (9_8) i n t h e o r d e r o f r e l a t i v e  t i o n processes occur  1  labeled  the i n t e r p r e t a t i o n o f t h i s  intensity. 72 a n d m/e  207 (101) , Fragmenta5 7 commonly  The p a t h w a y l e a d i n g t o t h e f o r m a -  207 (101) i s a common p r o c e s s  Further fragmentation 129 ( 1 0 3 ) .  86.  253 (92) i s i m p o r t a n t i n t h e \2  f a c t that the deuterium  Similarly,  for nitroxide  l e a d s t o i o n s , m/e  deri-  130 (10 2)  99  103  The the observed 106 j  t o 107  f o r m a t i o n o f f r a g m e n t i o n m/e fragmentation (132).  of n i t r o n e s ,  104  44  i s similar  t o 105  (131)  to and  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  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  of d i p h e n y l type  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  data  f o r DDP  a n d EMDP ( 3 1 - 3 5 ) .  ( 1 0 8 ) , m/e  180  179  The f r a g m e n t s m/e  (33).  (32),  be d e r i v e d f r o m m/e (31) .  m/e  180  181  (32);  m/e  processes  115. ( 1 0 9 ) , a n d  (112) a n d m/e 181  provides  S u c h i o n s a r e m/e  ( 1 1 1 ) , m/e 91  compounds.  ( T a b l e V)  a b a s i s f o r the explanation of the fragmentation described  additional  89  (111) was  193 m/e  (113) c o u l d f r o m m/e  208  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 methamphetamine n i t r o n e  pathways f o r  Mass f r a g m e n t a t i o n p r o c e s s e s lite  f o r t h e n i t r o n e metabo-  (54^ o f N - m e t h y l a m p h e t a m i n e w e r e shown i n two p a p e r s  p u b l i s h e d by C o u t t s e t a l . ( 1 3 3 , 1 3 4 ) .  I t was p r o p o s e d t h a t  u n d e r E I c o n d i t i o n s t h e n i t r o n e , 5_4 f o r m s t h e c y c l i z e d m e d i a t e o f m/e (117) 104  and m/e  163 132  ( 1 1 5 ) , w h i c h f u r t h e r f r a g m e n t s t o m/e (118).  To e x p l a i n t h e f o r m a t i o n o f  (116), a r e t r o D i e l s - A l d e r type f r a g m e n t a t i o n  was p r o p o s e d .  inter-  m/e  process  Oxygen e x p u l s i o n was i n v o l v e d i n t h e f o r m a -  t i o n o f 118 f r o m 117. l a b e l e d amphetamine  These i o n s were d e f i n e d w i t h  deuterium  derivatives  114  54  .CH,  1*  -CH CH=N0H  115  3  CH, CH„  m/e 104  m/e 163 116 -  m/e 132  148  118  0  117 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  p r o p o s e d f o r methadone  n i t r o n e a r e d i f f e r e n t f r o m t h 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 f o u n d  that the  fragment ions 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 o u r p r o p o s a l f o r t h e methadone n i t r o n e fragmentations. Thus, f o r m a t i o n o f t h e fragments, m/e  132  (151) f o l l o w s f r o m t h e c y c l i z e d  F r a g m e n t 121 h a s m/e  132 c o r r e s p o n d i n g  m/e  148  (120) a n d  i n t e r m e d i a t e 119. t o the fragment of  118 p r o p o s e d b y C o u t t s e t a l . ( 1 3 4 ) .  m/e 132  m/e 148  134 ( d ) 2  150 (d,)  120  —  .N-methylamphetamine n i t r o n e f r a g m e n t s t o t h e i o n m/e  118  o f m/e (122)  (122) w h i c h i s a s i m i l a r p r o c e s s 20 7  (101) i n methadone n i t r o n e  was f u r t h e r f r a g m e n t e d t o m/e  91  to the formation  (5_2) .  The i o n m/e  ( 1 2 4 ) , m/e  117  118  (125),  104 and  m/e 104 (126) .  in the formation  A hydrogen scrambling  process  i s involved  o f 1 2 4 , 1 2 5 , 126 w h i c h i s s u g g e s t e d b y t h e  r e p o r t e d mass s p e c t r a  (134) w h i c h w e r e p r e p a r e d  by using un-  l a b e l e d a n d l a b e l e d compounds.. The f r a g m e n t , m/e 104  (126)  was p r e v i o u s l y e x p l a i n e d b y a r e t r o D i e l s - A l d e r p r o c e s s . proposal  describes  formation  Our  o f m/e 104 (126) •• f r o m m/e 118  (123) .  '  v>  CH  ™ CH  N=CH, A H C - H *°  CH== N CH;  + CH=CH  122  2  1  m/e 118 124(d )  2  54  0  1|  CH  CH.  -CH. + ^  2  ^  CH  m/e 117 121 (dO 120(d )  CH,  123  CH«  125  3  CH. CH, m/e 104 106(d ) 2  124 126  m/e 91 93(d ) 92(d) 2  105  NMR  o f methadone n i t r o n e  The NMR 15.  spectrum  o f methadone n i t r o n e i s shown i n F i g .  The p e a k s w e r e a s s i g n e d a s f o l l o w s :  l e t s a t 2.25  6 a n d 2.73  t r o n e t y p e compounds. 3.55  6 f o r C-5 p r o t o n s a r e t y p i c a l o f n i Methylene  6 as two d o u b l e t s .  Two d o u b l e t s o f d o u b -  p r o t o n s a p p e a r a t 3.1 6 and  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 a r e  shown b e l o w 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 a s s i g n m e n t . from at  i m p u r i t i e s d e r i v e d f r o m HPLC s o l v e n t s and C H C l ^  1.0  - 1.6  Peaks a r e shown  6.  1.75 6 (m)  \  >.  0.78 6 (d)  T a b l e v i c o m p a r e s t h e 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) et  a l . (135).  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 The m e t h y l e n e p r o t o n s o f 5_2 (6 3.1  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  and  3.55)  (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) s u g g e s t  (4.0 Hz) and JHb-Hc  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 w h i c h t h e  107  OCH  3  127  TABLE V I .  NMR o f C-5 a n d g e m i n a l p r o t o n s o f methadone n i t r o n e (5_2) a n d 3 . 4 - d i m e t h o x y a m p h e t a m i n e n i t r o n e (127) 127  52 6 CH  6 Ha  (5)  2  J Ha-Hc Ha  1  He  1  6 Hb  C-— C  ,5 ,6 CH  Hb  3  /  10.0  J Ha-Hb  14.0  14.0  0  (m)  4.05  (m)  (d)  Ha  6 Ha  3.1 (d)  6.04  ^Hb  6 Hb  3.55  6 . 24 (d)  C  I  3.18 (dd)  (dd)  4.0  1.75  (dd)  • 5.6  4.0 2.73  2.73  (dd)  J Hb-Hc  6 He  + N =  2.25  J Ha-Hb  (d)  8.0  11.0  oxygen o f t h e ketone group i s o r i e n t e d towards t h e methylene group.  This  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  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 electron s h i f t causing This  of elec-  lead to the  loss of polarity of the nitrone  nitrogen.  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  nitrone  and t h e h i g h  s h i e l d i n g o b s e r v e d f o r t h e 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 t h e m e t h y l e n e  protons.  108  He  C =TO  CH  2  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 served  of the high  s h i e l d i n g ob-  f o r t h e C-6 p r o t o n and t h e m e t h y l e n e 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 h a s t h e o x a z i r i dine  s t r u c t u r e 128.  The m e t h y l e n e p r o t o n s o f o x a z i r i d i n e s a r e  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 the  (135).  s t r u c t u r e 128 c a n n o t be e x c l u d e d o n t h e b a s i s o f  scopic evidence formation  (MS, MMR,  IR) t h e r e  tend t o p r e f e r the n i t r o n e  IR s p e c t r o s c o p y o f methadone n i t r o n e  spectro-  i s no p r e c e d e n c e f o r t h e  o f s u c h a compound by i n v i v o m e t a b o l i s m .  b a s i s we w i l l  While  Upon  this  s t r u c t u r e ^ 52.  and r e l a t e d compounds  The IR s p e c t r u m and a summary o f 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. described  are assigned  The C-H v i b r a t i o n s  on t h e b a s i s o f IR 'data c o l l e c t e d f r o m  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 t h e I R o f o t h e r d i p h e n y l taining  con-  compounds.  Formation  of diketone  (6_1) f r o m 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 w h i c h h a s m/e 4 3 a s a b a s e p e a k ( F i g . 1 4 ( a ) , B a n d 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 a n d I R s p e c t r a shown i n F i g . 17 a n d mass  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  fragmentation  structure.  61  I s o l a t i o n o f d i k e t o n e , 61 w o u l d s u p p o r t mechanism  ( F i g . 18) o f m - c h l o r o p e r b e n z o i c  EDDP p e r c h l o r a t e .  the proposed  acid oxidation of  W a v a l a n g l h jx m  3300  3500  3000  2500  2000  1800  1600  1400  1200  1000  600  625  Wuwenumtier  _  v(=CH) 3020, 3040, 1 6 0 0 , 1490 cm v (CH)  2960 , 2920 6 (CH,CH ,CH )  v(C=0; N=C)  2  1710 Fig.  3  i  <5 (=CH) 1 1 4 0 , 1 1 0 0 , 1 0 3 0 , 9 3 0 , 7 6 0 , 1450, 1440, 1370, 1340, 820,  v (N + 0 ) 1 5 5 0 , 1250 16*  IR ( l i q u i d  f i l m ) o f methadone n i t r o n e  800  700  iii  JUOuJ (b) 10  2500  Fig."17.  2000  11  H  13  800  1<  15  u  635  (a) NMR and (b) IR (KBr p e l l e t ) o f 4 , 4 - D i p h e n y l 2 . 5 - h e p t a n e d i o n e (61.)  112  P r o p o s e d m e c h a n i s m 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) a n d d i k e t o n e (61)  A p o s s i b l e m e c h a n i s m f o r t h e f o r m a t i o n o f methadone nitrone  (5_2) , o r o x a z i r i d i n e  (128) , a n d d i k e t o n e  (61) f r o m  t h 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 Fig.  18.  More t h a n  2 moles  f o r m a t i o n o f 5_2, o r 128 a n d f  proposed  o f MCPBA was  required f o r the  61 w h i c h i s i l l u s t r a t e d  i n t e r m e d i a t e , 12 9, f o r m e d b y N - o x i d e  by t h e  f o r m a t i o n and  an a d d i t i o n o f a s e c o n d mole o f MCPBA t o t h e d o u b l e b o n d . Nitrone,  52, o r o x a z i r i d i n e  (128) were n o t f o r m e d  selec-  tively . The  d i k e t o n e , 6_1 c a n be s e e n t o be f o r m e d  the i n t e r m e d i a t e 130.  through  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 t h e m e t a b o l i c  f o r m a t i o n o f t h e k e t o n e 132 f r o m  t h e n i t r o n e 131 ( 1 3 6 ) .  0~  R  -  CH„ - C Z  |  =  N  +  - CH_R' Z  •> R - C H  0  Z  - C = 0 |  CH^  131  132  113  19  2 MCPBA  CH3  C=0 CH—CH— N \ j CH, 2  CH, J 2  v  /  128 CH CH 2  3  C= 0 52 CH —CH—S=;CH ' IN — .  2  CH  3  Fig.  18.  Proposed nitrone  i-  2  m e c h a n i s m s f o r t h e f o r m a t i o n o f methadone (5_2) o r o x a z i r i d i n e  2,5-heptanedione perchlorate.  (128) a n d 4 , 4 - d i p h e n y l -  (6JL) b y MCPBA o x i d a t i o n o f EDDP  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  frag-  m e n t s , m/e 208 ( 2 4 . 2 9 % ) , m/e 207 ( 1 2 . 8 7 % ) , m/e 193 ( 2 2 . 5 3 % ) , m/e 179 ( 1 3 . 0 9 % ) , m/e 178 ( 1 4 . 3 4 % ) , m/e 165 ( 1 1 . 2 7 % ) , m/e 130 ( 1 9 . 5 3 % ) , a n d m/e 115 ( 1 9 . 8 2 % ) .  T h e s e i o n s w e r e common t o  t h e d i p h e n y l b u t a n e c o n t a i n i n g compound. gave i o n s , m/e 277 ( 1 . 3 2 % ) ( C H N O ) , 1 9  L G  The compounds  m/e 235 (11.63%) ( C ^ H ^ N O ,  m/e 2 3 8 ) a n d m/e 43 (base peak)(C„H,0, the b a s i s o f these  also  m/e 4 6 ) .  On  i o n s t h e compound was a s s i g n e d t h e 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 b y GCMS when o l d u r i n e samples were a n a l y z e d and p r o v e d o f EDDP.  to result  When EDDP b a s e was a i r o x i d i z e d  from a i r o x i d a t i o n i n CHCl^  and a n a l y z e d  by GCMS (150 - 280°C a t 10°/min.), t h e m a j o r p r o d u c t DDP was e l u t e d a t 245°C a n d a n o t h e r p r o d u c t e l u t i n g t o t h e EDDP p e a k was i d e n t i c a l  a t 225°C c l o s e  t o t h e a c e t y l compound, 8 5 .  A p o s s i b l e m e c h a n i s m t o t h e f o r m a t i o n o f 85_ e i t h e r b y 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 b y a i r o x i d a t i o n o f EDDP b a s e i s shown i n 3, 19^ t h r o u g h  8_5 ( p . 1 1 5 ) .  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 b a s e gave a n o x i d i z e d w h i c h was e l u t e d  a t 250°C  product,  (120-270°C a t 10°/min.) b y 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  compounds o b t a i n e d b y o x i d a t i o n o f E M D P A molecular m/e 2 8 9 ) .  M -CHjCHg  M -phenyl ments.  (m/e 250 ( 3 6 . 0 % ) ,  (m/e 202 ( 1 6 . 7 % ) ,  H^Q.  "H  10  'H10  10  m/e 260) a n d  m/e 20 7) were m a j o r  frag-  The i o n s commonly d e r i v e d f r o m m/e 20 8 w e r e a l s o  The  High  2,  i o n a p p e a r e d a t m/e 279 (28.3%) (  present with high r e l a t i v e  likely  labeled  intensity.  s t r u c t u r e was n o t a b s o l u t e l y p r o v e d  EMDP o x a z i r a n  (M)  b u t i s most  r a t h e r t h a n EMDP N - o x i d e . (62).  a b u n d a n t m/e 279,.m/e 2 5 0 , a n d m/e 202 i n d i c a t e d t h e - .  stable nature o f the oxygen.in  the molecule  o t h e r h a n d . N - o x i d e may e a s i l y  l o s e o x y g e n t o g i v e 'high -  a b u n d a n t m/e M-l6. was  An a t t e m p t  o f _8_4.  On t h e  t o o b t a i n c y c l i z e d p r o d u c t , 135\  not successful.  The  chemical  f o r m s o f 6_2 a n d 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 b y a c i d i c c o n d i t i o n s o r b y UV i r r a d i a tion  (137).  HCl,  70°C  ?  The o x i d i z e d p r o d u c t 2 hours) and a m i x t u r e  was h y d r o l y z e d • i n - a c i d . • (10% of products which  has'peaks  ••117 on GCMS c o n t a i n i n g Further attempts  •; m/e 208 f r a g m e n t  t o determine  t h e s t r u c t u r e s were n o t p e r f o r m e d .  NMR d a t a f o r t h e p r o p o s e d is  a s b a s e p e a k was o b t a i n e d .  o x i d i z e d p r o d u c t o f EMDP  shown b e l o w 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  shift  v a l u e s f o r t h e C-8, C-7, a n d C-6 p r o t o n s w e r e s i m i l a r t o t h o s e o f EMDP  (C-8 1.07 6 , C-7 2.09 6 , a n d C-6 1.37 6 ) ( 3 ) . 2.20*6 (q) 0.78  6 (t)  4.85 6 (m) > 0.89 -3.25  6 (dd,J-, '6-:0)  The -IR s p e c t r u m 1550  cm  _i  Hc  6 (d)  2.30 <5 (dd, J ; "3.5) Ha-Hc T  o f t h i s compound h a d s t r o n g p e a k s a t 1250 a n d  corresponding t o N - 0 stretching  vibrations  118  5.  I n t e r a c t i o n o f methadone a n d d i a z e p a m  S t a b i l i t y o f t h e l a b e l i n g o f t h e compounds w h i c h used as i n t e r n a l  standards t o analyze nonconjugate  g a t e m e t a b o l i t e s was e x a m i n e d t o e n s u r e  and c o n j u - "  the s t a b i l i t y ofthe  l a b e l i n g d u r i n g i n c u b a t i o n a n d w o r k up p r o c e d u r e s . e x p e r i m e n t s were p e r f o r m e d  Paired  w i t h f o u r r a t s f o r methadone  ment a n d a n o t h e r f o u r r a t s f o r methadone a n d d i a z e p a m  Stability  were  treat-  treatment.  studies  The 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 e d methadone a n d t h e l a b e l e d EDDP a n d EMDP was e x p l o r e d o v e r a w i d e r a n g e o f pH's a n d u n d e r 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 c a n be s e e n f r o m t h e i o n r a t i o s m o n i t o r e d w h i c h 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 .  remain  The r e s u l t  i m p l i e s t h a t t h e d e u t e r i u m l a b e l e d compounds w h i c h w e r e used as i n t e r n a l  standards i n t h i s i n t e r a c t i o n  study are  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 a n d w o r k up p r o c e d u r e s f o r the  analysis. The a d v a n t a g e s  as i n t e r n a l  of using deuterium labeled  s t a n d a r d s was f u r t h e r d e m o n s t r a t e d  compounds b y EDDP when  -11-9  TABLE V I I .  S t a b i l i t y of Deuterated  Methadone,  EDDP a n d EMDP  Methadone 233/223  EDDP  EMDP  226/223  287/277  280/277  218/208  IN H C l  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  Control  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 o f i o n s m o n i t o r e d :  m/e 2 2 3  2  2  d o n e ) , m/e 2 2 6 (methadone- H ^ ) , m/e 2 3 3 (methadone- H-^Q), 277  (metham/e  (EDDP), m/e 2 8 0 ( E D D P - H ) , m/e 2 8 7 ( E D D P - H ) , m/e 2 0 8 2 2  (EMDP), a n d m/e 2 1 8 (EMDP- H of three determinations.  2  3  1 Q  1 Q  ).  The v a l u e s r e p r e s e n t t h e mean  The a q u e o u s s a m p l e s w e r e i n c u b a t e d f o r  4 8 h o u r s a t t h e r e s p e c t i v e p H ' 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 ) .  l a b e l e d EDDP c o u l d c o m p e n s a t e f o r t h e o f EDDP, t h e r a t i o o f u n l a b e l e d notwithstanding  o f EDDP u n d e r f o r EDDP i n  1/3 t h a t o f c o n t r o l s a m p l e s  internal  standard  metabolites.  by u s i n g d e u t e r i u m Stability  the conjugate time-(Tables  IX).  c o u l d be c o m p e n s a t e d  standards.  of the deuterium  VIII,  deuterium  F o r example, t h e i n s t a b i l i t y  labeled  metabolites  prepared  c o u l d be e x t e n d e d t o t h e a n a l y s i s  of 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  l a b e l was a l s o shown b y  f o l l o w i n g 96 h o u r s o f i n c u b a t i o n E v e n i n t h e p r 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 t h e r i n g , was  instability  T h i s example o f t h e advantage o f u s i n g a  of conjugate  for  deuterium  t o l a b e l e d EDDP was t h e same  Total i n t e g r a t i o n values  0.1 N NaOH w e r e l e s s t h a n  labeled  chemical  s i g n i f i c a n t decomposition  alkaline conditions.  i n MeOH.  Because  the deuterium  s t a b l e u n d e r 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 a n d t e m p e r a -  ture.  The e f f e c t o f a c t i v a t i n g g r o u p s o n the- s t a b i l i t y o f  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 g r o u p amino g r o u p  (139).  This r e s u l t deuterium  (138) a n d a n  i s consistent with a report that the  l a b e l o n t h e r i n g o f £-hydroxyephedrine o b t a i n e d  by m e t a b o l i c glucuronide  processes formation  of the conjugate The  was f o u n d t o be s t a b l e d u r i n g t h e a n d t h e s u b s e q u e n t enzyme h y d r o l y s i s  (140).  stability  studies of conjugate  X I I I a n d I X ) a l s o gave some i n s i g h t  metabolites  (Tables  into the contribution of  121  TABLE V I I I .  Stability of  D e u t e r i u m i n HOEMDP- H 2  238/247  9  Time (h)  246/247  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  12  245/247  244/247  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 being monitored each time.  The f i r s t  set of ions  s e t o f i o n s was m/e  247, m/e 2 4 6 , m/e 2 3 8 , a n d m/e 237 a n d t h e s e c o n d , m/e 2 4 7 , m/e 2 4 6 , m/e 2 4 5 , a n d m/e 2 4 4 .  The s t a b i l i t y o f t h e 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 t h e r a t i o o f i o n s , m/e 246 t o m/e 2 4 7 .  The  i o n , m/e 238 was c h o s e n f o r CH^OEMDP a n d m/e 2  24 7 f o r CH 0EMDP- H . 3  g  The v a l u e s  w e r e t h e a v e r a g e f r o m 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 Negative values  ( 1 2 , 24, 4 8 , a n d 96 h o u r s ) .  r e s u l t from background  subtraction.  .122  TABLE I X .  S t a b i l i t y o f D e u t e r i u m i n DiHOEMDP-  g  244/245  237/245  236/245  12  0 . 363  0 .0088  0 .0091  24  0.359  0^0069  0.0052  48  0.364  0.0059  0 .0060  96  0.369  0.0066  0.0077  247/245  246/245  244/245  12  0 .0296  0.215  0.359  24  0.0341  0.211  0.356  48  0.0266  0 . 214  0 . 359  96  0 .0250  0.207.  0.359.  Time  (h)  Time  (h)  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 being monitored each time. 245, m/e m/e  H  244, m/e  2 4 6 , m/e  The f i r s t  2 3 7 , and m/e  2 4 5 , a n d m/e  244.  set of ions  s e t o f i o n s was  236 and t h e s e c o n d , m/e  m/e 247,  The s t a b i l i t y o f t h e 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 t h e r a t i o t o m/e m/e  245.  244  The i o n m/e 237 was c h o s e n f o r DiCHgOEMDP and 2  245 f o r DiCHgOEMDP- Hg.  The v a l u e s w e r e t h e a v e r a g e  two i n c u b a t i o n s a t e a c h i n c u b a t i o n t i m e hours) .  o f i o n s , m/e  from  ( 1 2 , 24, 48, and 96  123 background  t o the a n a l y t i c a l procedure.  of conjugate m e t a b o l i t e s l a b e l e d compounds  The s t a b i l i t y  w h i c h were p e r f o r m e d w i t h  studies  deuterium  showed t h a t t h e i o n r a t i o s , m/e 2 3 8 / 2 4 7 ,  m/e 2 3 7 / 2 4 7 , m/e 2 3 7 / 2 4 5 , a n d m/e 236/245 w e r e l e s s t h a n 1 percent. in  T h i s meant t h a t t h e r e was an a b s e n c e  endogenous » b i l e  l i t e s which might  of impurities  a n d no c o n t r i b u t i o n f r o m l a b e l e d m e t a b o -  interfere i n the analysis of unlabeled  metabolites. 2 The mass s p e c t r u m f o r CH^OEMDP- Hg d i d n o t show a n y 2 i o n s a t m/e 2 3 8 , 237 a n d t h e mass s p e c t r u m o f DiCH^OEMDP- Hg did  n o t show i o n s a t m/e 237 a n d m/e 2 3 6 . A method t o e x a m i n e 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  o f methadone c o n j u g a t e m e t a b o l i t e s  arising  from  analytical  i n t e r f e r e n c e s such a s from endogenous m a t e r i a l s and from conjugate metabolites  o f d i a z e p a m was a l s o s t u d i e d .  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  of consecutive  I f there ions  should  give constant for  ratios. The i o n s m o n i t o r e d were m/e 246/247 2 ? CH-.OEMDP- H a n d m/e 244/245 f o r DiCH.OEMDP- H„ . A s u d d e n J y 3 o n  change o f t h e r a t i o s the  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  i o n focussing arising  unstable  magnetic  field.  f r o m e i t h e r power f a i l u r e o r a n The f a c t t h a t b l a n k  were c o l l e c t e d f o r t h e f i r s t did of  (141).  dose  I t has been  shown  one h o u r , 4 5 % o f t h e r a d i o a c t i v i t y  i n t h e form o f f5-^C~]- diazepam  the b i l e  which  i n d i c a t e d an absence  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 .  injected in  one h o u r a f t e r d i a z e p a m  not interfere with the ion ratios  that during the f i r s t  samples  i n t o t h e r a t appeared  124 Stability  s t u d i e s were a l s o aimed a t measuring t h e  a c c u r a c y and p r e c i s i o n  of the developed  dent of t h e s e t o f i o n s monitored 244 a n d m/e m/e  (0.43) f o r CH^OEMDP- H . g  i o n m o n i t o r i n g i . e . m/e  246 t o  Consecutive  2 4 7 , 246, 2 4 5 , 244 a n d 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 t h e same m/e  Indepen-  , '(m/e 2 4 7 , 246, 2 4 5 ,  2 4 7 , 246, 238, 2 3 7 ) , t h e r a t i o o f m/e 2  247 was t h e same  2 4 5 , 244, 2 3 7 , 236 a l s o g a v e  244/245 v a l u e o f 0.36 f o r D i C H O E M D P - H g , 2  3  ing p r e c i s i o n  to evaluate.  imply-  of the methodology.  Accuracy  o f t h e i o n m o n i t o r i n g method was  Ion r a t i o s  o f mass s p e c t r a l integration  SIM s y s t e m .  calculated  from t h e r e l a t i v e  scan d a t a were compared w i t h t h o s e  values of specific  The i o n r a t i o s  difficult  o f m/e  i o n being monitored  intensity  from i n SIM mode.  245 t o m/e  247 a n d m/e 246 t o m/e 247 2 o b t a i n e d b y s c a n mode f o r CH^OEMDP- Hg w e r e 0.4 6 a n d 0.36, respectively. On t h e o t h e r h a n d , t h e r a t i o s o b t a i n e d by 2 SIM w e r e 0.40 a n d 0.44. ratios m/e  I n t h e c a s e o f DiCH^OEMDP- Hg, t h e  f r o m s c a n mode w e r e 0.366 arid 0.220 f o r m/e  245 a n d m/e  246 t o m/e  w e r e 0.360 a n d 0.215. SIM a n d r e p e t i t i v e acquisition  245, r e s p e c t i v e l y .  The d i f f e r e n c e  SIM r a t i o s  i n t h e r a t i o s between  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  methods.  244 t o  data  125  Bile  collection  Table X d e s c r i b e s t h e b i l e volumes  collected  each r a t i n t h e methadone-diazepam i n t e r a c t i o n total  amount o f c o l l e c t e d b i l e  w h i c h was a l i t t l e  d e s c r i b e d by Johnson e t a l . (141). fluid  study.  a v e r a g e d 50 m l / k g  below t h e b i l e  flow  (60-100  from The  f o r 23 hours,  ml/kg/24  Taking i n t o account  hour) that  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 , t h e b i l e  f l o w appeared adequate interaction  to obtain reliable  data f o r the drug  studies.  Diazepam d i d n o t i n f l u e n c e b i l e hour time p e r i o d o f t h e study  flow during the 2 3  (p>0.20).  A similar  result  was  r e p o r t e d b y E l - H a w a r i e t a l . (143) i n t h e i r  studies of  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 -  toin.  A n a l y s i s o f nonconjugated 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  S I M c h r o m a t o g r a m s o f EMDP,  m e t h a d o n e , a n d EDDP ( F i g . 19) p r e p a r e d f r o m s p i k e d a n a l y s i s o f methadone a n d EDDP h a d h i g h s p e c i f i c i t y h i g h masses  samples, because  (m/e 294 a n d m/e 297 f o r m e t h a d o n e , m/e 277 a n d  m/e 280 f o r EDDP) w e r e m o n i t o r e d . and m/e 218 was f o u n d t o l a c k  M o n i t o r i n g i o n s , m/e 208  specificity  f o r EMDP b u t t h e  12'6  TABLE X.  E f f e c t o f Diazepam Treatment  on t h e  B i l e Flow of Rats  Time  (h)  0-2  1- M  1-:DM  .2 -M  2-DM  3-•M  3- DM  4'-M  4- DM  3. 85  4 .04  4 .28  4.07  3. 52  4 .41  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  57. 75  59 .60  41 .81  42.65  51. 87  51 .99  41 .06 50 .86  Total  Time  (h)  0-2  M  5 . 22  3 .72  DM  P  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  48 .393 + 8. 900  51.275 + 6. 930  >0 . 20  Total  P a i r e d e x p e r i m e n t s Were p e r f o r m e d done t r e a t m e n t diazepam  u s i n g f o u r r a t s f o r metha-  (M) a n d a n o t h e r f o u r r a t s f o r methadone and  treatment  The v a l u e s d e n o t e  (DM) t h e 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 t i m e p e r i o d s 0-2, 2-11, and 11-23 h o u r s t h e methadone d o s e .  following  The v a l u e s i n t h e l o w e r p a r t o f t h e  T a b l e r e p r e s e n t mean ± SD o b t a i n e d f o r e a c h o f t h e two The v a l u e p was c a l c u l a t e d b y u s i n g S t u d e n t ' s t t e s t . '  groups.  12 7  200  210  220  230  240  250  TEMPERATURE Fig.  19.  SIM c h r o m a t o g r a m s f o r EMDP and methadone ( c ) . '  ( ) , EDDP (h) ' ' a  (  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 p e a k s was s u f f i c i e n t l y to  selective  a l l o w , a n a l y s i s o f EMDP. For  tic  t h e a n a l y s i s o f nonconjugated metabolites,  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  available.  C a l i b r a t i o n curves  standards  were p r e p a r e d  a n d EMDP  (0.1-1.6 ug) i n 0.4 m l o f c o n t r o l b i l e  w o r k i n g up t h e s a m p l e s a s d e s c r i b e d 2 EMDP- R"  lQ  2 (2 ug) , m e t h a d o n e - H  as t h e i n t e r n a l s t a n d a r d s . ditions,  3  were  f o r EDDP, m e t h a -  d o n e , a n d EMDP b y s p i k i n g EDDP (5-80 u g ) , methadone ug),  authen-  (0.0 5-0.8 a n d by  i n the experimental  2 (5 ug) and EDDP- H  3  using  (20 ug)  U n d e r t h e above c a l i b r a t i o n  200 n g o f methadone i n 0.4 m l o f b i l e ,  con-  400 n g o f  EMDP i n 0.4 m l o f b i l e , a n d 5 ug o f EDDP i n 0.4 m l o f b i l e were r e p r o d u c i b l y  analyzable.  Methadone e x c r e t e d  i nrat bile  under t h e c o n d i t i o n s o f t h e a n a l y s i s .  was n o t d e t e c t a b l e Assuming t h a t t h e lowest  c o n c e n t r a t i o n o f methadone t h a t c a n be p o s i t i v e l y m e a s u r e d i s 200 n g i n 0.4 m l 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 d o s e i s e x c r e t e d  i n t o t h e b i l e , a t r a c e amount.  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 u n c h a n g e d methadone a n d EMDP was p r e s e n t and  rat bile  (144).  s m a l l amount o f  i n human b i l e (98)  T h i s meant t h a t t h e a n a l y s i s o f m e t h a -  done -can n o t be u s e d f o r s t u d i e s o f t h e m e t h a d o n e - d i a z e p a m interaction. EMDP l e v e l s and  concentrations  result  i nrat bile  samples were a l s o  analyzed  b e l o w 400 ng/0.4 m l w e r e o b s e r v e d .  i s t h e same a s t h a t r e p o r t e d  This  This  by K r e e k e t a l . (98)  129 and  B a s e l t e t a l . (145).  Therefore,  a n a l y s i s o f EMDP w o u l d  n o t be a g o o d i n d i c a t o r o f a m e t a b o l i c  interaction  between  methadone a n d d i a z e p a m . The  l o w l e v e l o f methadone a n d EMDP i n b i l e  e x p l a i n e d i n t h a t l i v e r has a high i n t r i n s i c t h e m e t a b o l i s m o f methadone a n d EMDP.  c a n be  activity  for  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 d r u g w h i c h 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 . percentages  reported excreted  i nrat bile  The  4 hours a f t e r the  i.v. i n j e c t i o n w e r e 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  (partition coefficient  EDDP l e v e l s f o u n d i n b i l e study  a r e shown i n T a b l e  samples i n t h e i n t e r a c t i o n  X I where 2 0 - 3 0 % o f a d m i n i s t e r e d  methadone was f o u n d t o be e x c r e t e d Table  XI clearly  13.4)(94).  into the bile  indicates that b i l i a r y  a s EDDP. excreted  EDDP d o e s n o t show a n y d i f f e r e n c e b e t w e e n methadone o n l y and  methadone-diazepam t r e a t e d r a t s  A n a l y s i s of conjugated  SIM  high  from r a t b i l e  c h r o m a t o g r a m s o f mono a n d d i h y d r o x y  from r a t b i l e to monitor  metabolites  (p >0.10).  a r e shown i n F i g . 2 0 .  The i o n s  EMDP  isolated  (4_5, 48) c h o s e n  were t h e b a s e p e a k s f o r t h e compounds a n d h a d  selectivity. Diazomethane treatment  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 f r o m M e t h a d o n e a n d 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  Total  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  M  Time (h)  DM  E  0-2  0.344 ± 0 .0431  0 .440  ±  0.0582  <0 .05  2-11  1.869 ± 0. 1364  1 . 800  ±  0 .0908  >0 .20  11-23  0.248 ± 0 .1388  0 . 405  ±  0.3658  >0 .20  2.462 ± 0 .220  2 .646  ±  0 . 361  •>o .10  Total  P a i r e d e x p e r i m e n t s were p e r f o r m e d done t r e a t m e n t  (M) a n d a n o t h e r f o u r r a t s f o r methadone a n d  diazepam treatment EDDP  u s i n g f o u r r a t s f o r metha-.  (DM).  The v a l u e s d e n o t e t h e amount o f  (shown a s 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  t h e t i m e p e r i o d s , 0-2, 2 - 1 1 , 11-23 h o u r s a f t e r t h e methadone dose.  The v a l u e s i n t h e l o w e r p a r t o f t h e T a b l e r e p r e s e n t  mean ± SD o b t a i n e d f o r e a c h o f t h e two g r o u p s . was c a l c u l a t e d b y u s i n g S t u d e n t ' s t t e s t . , t o t h e p e r c e n t a g e o f t h e d o s e e x c r e t e d a s EDDP.  The v a l u e p . Dose % r e f e r s  (a)  ,  J  200  220 TEMPERATURE °C Fig.  240  (b)  1  1  260  L  200  220  240  260  TEMPERATURE °C  20.;.' SIM c h r o m a t o g r a m s f o r (a) monohydroxy  EMDP a n d (b) d i h y d r o x y EMDP a n a l y s  132 in  the analysis.  detectable  Underivatized  d i h y d r o x y EMDP was n o t  i n t h e TIC p r o f i l e o f t h e 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 o f t h e sample t o t a l l y removed any i n t e r f e r e n c e s by  diazomethane methylation  preventing  ( F i g . 21).  Derivatization  a l s o had advantages i n terms o f  oxidation of the phenolic  and c a t e c h o l  type meta-  bolites. 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 the b i l e  samples i n d i f f e r e n t volumes  obtained  from n a t u r a l  t i o n range.  one o f  (Table X I I ) .  The r a t i o s  sample a n a l y s i s were w i t h i n t h e c a l i b r a -  The r a t i o o f m/e 246 t o m/e 24 7 a n d t h a t o f  m/e 244 t o m/e 245 w e r e 0.43-0.46 a n d 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 the  i n the analysis  i . e . t h e v a l u e s were  same a s t h o s e shown i n T a b l e s V I I I a n d I X . As  shown i n T a b l e s X I I I a n d X I V ,  t i o n of conjugate metabolites 2 hours o f b i l e rats. first  excretion  an i n c r e a s e d  was f o u n d d u r i n g  the f i r s t  i n t h e methadone-diazepam  F o r HOEMDP 5.7% a n d 16.8%  was e x c r e t e d  excre-  treated  during the  2 hours i n c o n t r o l and diazepam t r e a t e d  rats,  respective-  ly.  I n t h e c a s e o f DiHOEMDP, t h e p e r c e n t a g e s e x c r e t e d  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 -  tively. was  However, when t h e e x c r e t i o n  followed  of conjugate  over t h e e n t i r e 2 3 hour p e r i o d ,  there  d i f f e r e n c e o b s e r v e d b e t w e e n t h e methadone o n l y and  methadone-diazepam t r e a t e d  DiHOEMDP p >0.20).  rats  were  metabolites was no  treated  (HOEMDP, p >0.20,  133  (a)  (b)  m/e  (c)  Fig.  21. v  TIC p r o f i l e  (a) and Mass c h r o m a t o g r a m  (b) o f  t h e 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 f r o m 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 profile  (c) a f t e r b a c k  extraction.  and TIC  134  TABLE X I I .  C a l i b r a t i o n Curve Data f o r Conjugate  Metabolites  HOEMDP ml o f sample  m/e  246/m/e 247  m/e  238/m/e 247  1.6  0 .443  1.440  0.8  0.431  0 . 712  0.4  0.461  0 . 359  0.2  0.448  0 .162  0.1  0.466  0.092  Slope,  0.903; i n t e r c e p t ,  - 0 . 0 0 6 8 3 ; r , 0.9998  DiHOEMDP ml o f sample  m/e  244/m/e 245  m/e  237/m/e 245  1.6  0.373  1.204  0.8  0 . 36.5  0.619  0.4  0 . 366  0 . 341  0.2  0.367  0.153  0.1  0 . 377  0 .074  S l o p e , 0.748; i n t e r c e p t , 1 ml o f i n t e r n a l standard was a d d e d .  0.014; r , 0.9992 (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)  135  TABLE X I I I .  E x c r e t i o n o f M o n o h y d r o x y EMDP f r o m Methadone a n d M e t h a d o n e - d i a z e p a m Treated  Rats  Time(h)  1-M  1-DM  2-M  2-DM  3-M  3-DM  4-M  4-DM  '  0.447  3.654  0.961  3.275  2.893  5.092  0.874  4.316  0-2 2-11  16.545 18.888 10.377 11.650 20.609 15.400 12.647 16.200  11-23 Total'  6.270  5.995  5.454  23.304 25.508 21.165 20.394 26.350 25.046 19.516  25.970  Time(h)  2.966  9.827  5.469  M  0-2  2.848  4.554  DM  1.293 ± 1.089  £  4.084 ± 0.797  <0.01  2-11  15.044 ± 4.499  15.534 ± 2.988  >0.20  11-23  6.235 ± 2.853  4.611 ± 1.177  >0.20  Total  22.583 ± 2.951  P a i r e d e x p e r i m e n t s were p e r f o r m e d  24.229 ± 2.584  using four r a t s ' f o r  treatment  (M) a n d a n o t h e r f o u r r a t s  treatment  (DM). The v a l u e s d e n o t e  >0.20  methadone  f o r methadone a n d d i a z e p a m the ratios  (ratio/kg rat) of  m/e 238 t o m/e 247 w i t h a v e r a g e o f t w o d e t e r m i n a t i o n s .  The sam-  p l e s w e r e c o l l e c t e d o v e r t h e t i m e p e r i o d s 0-2, 2 - 1 1 , a n d 1 1 - 2 3 hour a f t e r  the" methadone d o s e a n d w e r e m o n i t o r e d b y GCMS a t m/e  247, m/e 2 4 6 , m/e 238 a n d m/e 2 3 7 . The v a l u e s i n t h e l o w e r  part  o f t h e T a b l e r e p r e s e n t mean .± SD o b t a i n e d f o r e a c h 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 b y u s i n g S t u d e n t ' s t t e s t .  136  TABLE X I V .  E x c r e t i o n o f D i h y d r o x y EMDP f r o m M e t h a d o n e and M e t h a d o n e - d i a z e p a m t r e a t e d  Time (h) 1-M 0-2  1.794  rats  1-DM  2-M  2-DM  3-M  3-DM  4-M  3.302  0.509  2.009  1.840  2.218  0.726  4-DM 2.028  2-11 16.510 14.968 10.537 14.160 18.270 14.375 13.511 16.671 11-23 Total  7.079  2.376 11.171  7.526  2.122  25.383 20.646 22.217 23.695 22.23  5.150 21.74  7.243  3.711  21.48 22.41  Time(h)  M  DM  p_  0-2  1.217 ± 0.698  2.389 ± 0.615  <0.05  2-11  14.707 ± 3.404  14.875 ± 2.356  >0.20  11-23  6.903 ± 3.706  4.690 ± 2.203  >0.20  Total  22.825 ± 1.783  22.122 ± 1.275  >0.20  P a i r e d e x p e r i m e n t s were p e r f o r m e d  u s i n g f o u r r a t s f o r methadone  treatment  (M) a n d a n o t h e r f o u r r a t s f o r methadone a n d d i a z e p a m  treatment  (DM). The v a l u e s d e n o t e  m/e 237 t o m/e 245 w i t h a n a v e r a g e samples  the ratios  (ratio/kg rat) of  o f two d e t e r m i n a t i o n s . The  w e r e c o l l e c t e d o v e r t h e t i m e p e r i o d s , 0-2, 2 - 1 1 , a n d 1 1 -  23 h o u r a f t e r  t h e methadone d o s e , a n d m o n i t o r e d b y GCMS a t m/e  245, m/e 2 4 4 , . -  - ' m/e 2 3 7 , a n d m/e 2 3 6 . The v a l u e s i n t h e  l o w e r p a r t o f t h e T a b l e r e p r e s e n t mean ± SD o b t a i n e d f o r e a c h o f t h e two g r o u p s . Student's t test.  The v a l u e p was c a l c u l a t e d b y u s i n g  137 D i s c u s s i o n o f t h e methadone-diazepam  interaction  Drug" i n t e r a c t i o n s a r e 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 . p l a s m a , 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 a n d m e t a b o l i t e s e x c r e t e d  the b i l i a r y  r o u t e c a n be u s e d t o a s s e s s d r u g  at the hepatic l e v e l .  The d r u g  t o the b i l i a r y  route, hepatic blood  b i n d i n g t o p r o t e i n (145).  interactions  i n t e r a c t i o n c o u l d depend  on t h e 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 , drug  through  transport of the  f l o w , and drug  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 creted through  1 0 % o f methadone a n d m e t a b o l i t e s i s e x -  the urinary route  (144).  Methadone  levels  i n r a t p l a s m a c o u l d n o t be q u a n t i t a t e d e v e n by m o n i t o r i n g t h e h i g h a b u n d a n c e m/e  72 i o n b e c a u s e o f a l i m i t e d  analytical  s e n s i t i v i t y o f o u r 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  of u s i n g r a t i o a n a l y s i s t o t h e drug metabolism  study.  of conjugate  m e t a b o l i t e s b y t h e method o f r a t i o  was  t o p r o v i d e a c c u r a t e d a t a on t h e change i n  expected  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 concomitant t i o n o f a l a r g e dose o f diazepam  Analysis  measurement  administra-  (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 a b s e n c e o f a m e t h a d o n e 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 i n p l a s m a and d i d not  R o e r i g e t a l . (38)  methadone and  show any  EDDP c o n c e n t r a t i o n s  methadone-diazepam  Shah e t a l . (59) mice  t h a t methadone  in rat urine  interaction.  showed i n t h e i r e x p e r i m e n t  (20 mg/kg i . p . d i a z e p a m and  was  concomitant a d m i n i s t r a t i o n of diazepam.  methadone f a i l e d  t o show any  interaction  Their  result  0.6  mg/kg i . p .  i n m i c e when p l a s m a  b r a i n l e v e l s o f methadone were m e a s u r e d . S i n c e methadone i n p l a s m a was  study  an a t t e m p t was  t i o n rate constant  not  u s i n g EDDP d a t a  from r a t b i l e  (80).  a rate constant  s a m p l e s was  metabolites t i o n was in bile  using b i l e  (146)  but  f o u n d t o be  The GCMS was  reported  i n t h i s work r e l i a b i l i t y h e a v i l y d e p e n d e n t on  (5  data  for  of  tripamide  of the  calcula-  c o u l d n o t be  used  calculations.  r e s u l t obtained  by  a n a l y z i n g conjugate  q u i t e d i f f e r e n t f r o m t h a t r e p o r t e d by  metabolites Roerig  f r o m methadone  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  administration.  data  small differences  et a l . (38), 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 i n u r i n e and  in this  A similar calculation  f l o w w i t h t i m e so t h a t b i l e  for pharmacokinetic  analyzed  made t o c a l c u l a t e an a p p a r e n t e l i m i n a -  p o i n t s ) by means o f NONLIN  by  increased  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  e x p e r i m e n t w i t h 10 mg/kg i . p . d i a z e p a m and  and  using  5 mg/kg i . p . methadone)  t h a t p l a s m a -and b r a i n l e v e l s o f methadone were by  levels  Their r e s u l t  a c t i o n b e t w e e n methadone and  diazepam  i n d i c a t e d t h a t the drug d i a z e p a m was  decreased c o n c e n t r a t i o n of conjugate  reflected  metabolites  and  inter-  in a that  139 t h i s decrease of conjugate for  an  i n c r e a s e o f methadone l e v e l s The  i n the due  metabolites  indirect  i n brain.'  2 h o u r p e r i o d was  to competition An  not  evidence  1  reason f o r the t r a n s i e n t increase of  initial  binding.  was  metabolites  sought but  could  be  by d i a z e p a m w i t h methadone p l a s m a p r o t e i n  e f f e c t o f d i a z e p a m on  i n c r e a s i n g f r e e drug  was  i n d i c a t e d i n s t u d i e s of diazepam-diphenylhydantoin  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 u g m i g h t be  b e c a u s e i n t h e c a s e o f t h e d r u g w h i c h has t i o n , drug b i n d i n g to the plasma i s not the h e p a t i c m e t a b o l i s m  (143).  minimal  high hepatic  a critical  levels  extrac-  factor in  (147).  In c o n c l u s i o n , the concomitant a d m i n i s t r a t i o n of d i a z e p a m w i t h methadone i n r a t s d i d n o t t i o n o f EDDP and  conjugate  metabolites.  affect biliary Diazepam d i d  excrenot  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 on  the t r a n s p o r t of m e t a b o l i t e s  route.  This r e s u l t  R o e r i g e t a l . (38) lite  levels  a metabolic  i s not  by  the b i l i a r y  nor  excretion  consistent with that reported  by  i n w h i c h a d e c r e a s e o f w a t e r s o l u b l e metabo-  i n u r i n e and  l i v e r was  methadone-diazepam  considered  interaction.  to i n d i c a t e  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 standard  was u s e d i n t h i s  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 methadone due t o a c o n c o m i t a n t  metabolites of  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 biosynthetic  internal  standard  q u a n t i t y o f a substance  work  without  to i t s  knowing t h e a b s o l u t e  c a n 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 metabolites  f o r which authentic standards  d r u g and  are not available.  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 t h e d r u g p r o d u c e s an ideal  internal  standard  of t h e i r metabolites. fails  t o separate  thetic  internal  separate  t o measure t h e r e l a t i v e  concentration  When t h e r a d i o i s o t o p e - TLC method  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 - '  standard  i s a very convenient  method t o  and q u a n t i t a t e t h e d r u g 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 t h e same m i x t u r e  c a n be a n a l y z e d  mixture  by u s i n g a l a b e l e d p r e c u r s o r .  obtained  u s i n g an i n t e r n a l  standard  This  approach  i s a p p l i c a b l e t o the biphenyl metabolism studies published by B e n f o r d  e t a l . (148) a n d Halppap-Wood e t a l . ( 1 4 9 ) .  Pharmacokinetic  studies:  When we m e a s u r e t h e c o n c e n t r a -  t i o n o f a n y d r u g b y GCMS, d r u g c o n c e n t r a t i o n = a x p e a k ratio  ( u n l a b e l e d / l a b e l e d ) + b.  height  I n t h e 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 be was  a c h i e v e d by proved  as i n t e r n a l  i n t h e SIM  The  0.1-10.  can  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 . a n a l y s i s o f methadone and  (Table I I I ) . T h e r e f o r e , drug ratio.  standards, b = 0  metabolites  c o n c e n t r a t i o n = a x peak  o p t i m a l dynamic range of the r a t i o  This  height  i s usually  T h i s r a n g e 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  amount o f l a b e l e d i n t e r n a l  standards.  t h e v a l u e o f a, we  the r a t i o of unlabeled to l a b e l e d  for kinetic  c a n use  s t u d i e s of a  In order  t o see  E v e n i f we  the  do n o t know  drug.  the a p p l i c a b i l i t y of the  t o a two-compartment model  methodology  (150), the e q u a t i o n o f the model  i s r e w r i t t e n as: R = — == a a  ° Vc  X  (a- k i ) a - B  -at  2  L  ( k i - B) a - 3 2  ~3t -  f o r methadone p l a s m a d a t a r e p o r t e d by Swanson e t a l . ( 1 5 1 ) . All  k i n e t i c c o n s t a n t s , a,  pendently program  of a w i t h r a t i o s  3, k  2  i ,  Xo  — 7 7 — ci.  V  C  (R) a t t i m e s  are c a l c u l a b l e  ( t ) by t h e NONLIN  (80).  K i n e t i c approach to the  studies  o f h y d r o x y l a t i o n mechanism:  As  an e x a m p l e o f  a p p l i c a t i o n of r a t i o a n a l y s i s to pharmacokinetic t h i s method c a n be of phenol the  inde-  and  used to study  the mechanism o f  catechol metabolites.  the  studies, formation  A g e n e r a l scheme f o r  f o r m 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 i s shown i n t h e 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 p a t h w a y s o f methadone m e t a b o l i s m I f t h e m e t a b o l i t e s c a n be p r o p o s a l c a n be  made.  analyzed  i n plasma, the  (p.  following  79)  142 Deuterium l a b e l e d drug A produces deuterium monohydroxy  (Ml) and  s e r v e s as an  dihydroxy  internal  metabolites  standard.  (M2).  After collecting  of samples from drug A which i s a m i x t u r e  labeled The a  mixture series  o f monohydroxy  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 , the samples m i x e d 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 thetic  internal  the r a t i o s R  1  standard).  f o r Ml  (1£)  to the l a b e l e d i n t e r n a l  A f t e r working and  R  2  standard  f o r M2  standard  up t h e  and  are (biosyn-  samples,  (11) o f t h e  metabolites  at d i f f e r e n t times  (6)  are  obtained. 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  p a t h w a y s i s d e r i v e d by means o f NONLIN p r o g r a m .  of  The  t i o n s f o r t h e f o r m a t i o n o f m o n o h y d r o x y and  dihydroxy  l i t e s by t h r e e p o s s i b l e m e c h a n i s m s , d i r e c t  insertion,  formation  [ 1 ] - and  a r e shown i n T a b l e  another XV.  epoxide  f o r m a t i o n mechanism  the  equametaboepoxide [2]  143  TABLE XV.  Pharmacokinetic  equations  t i o n ~of m o n o h y d r o x y  f o r the forma-  and d i h y d r o x y  metabolites  Direct insertion mechanism  A(3) l  l  -  ->  M (10)  i "-  (k -k  M (11)  1  2  A  2  [e 1  l ) a  2 J  - e  0 l 2 k  k  l  ( k  l  _ k  2  )  k  2 r 2> ( k  k  ^ M (10) x  A (3)  -*M (11) 2  R  ^i a  l  R  2  =  (k^k^a  TLi  0 2 (k +k )a  rL  =  M =  A  a  k  1  2  e  -(k k )t 1 +  2  e-( +K_)t 1 2 kl  Epoxide formation mechanism (2) A(3)  M (11) 2  I =^ *1 a  =  ° l [ e~ 3 (k +k -k )a L A  k  1  £  k  2  -  t  e  _ ( k  l 2 l + k  ) t  J  3  1 + D  e" 2 k  k  T 3 k  e k  3 l" 2 _ k  k  t  +  2 3* 3~ l~ 2 <K  k  k  k  k  -(k +k )tj 1  2  t  144  SUMMARY AND CONCLUSIONS  1.  Friedel Crafts reaction of bromophenylacetonitrile  2 b e n z e n e - Hg gave d i p h e n y l a c e t o n i t r i l e l a b e l e d i n b o t h rings.  The l a b e l i n g p r o c e s s  with  phenyl  was f o u n d t o be a c o m b i n a t i o n  of  2 aluminum c h l o r i d e c a t a l y z e d exchange between benzenethe r i n g p r o t o n s tonitrile  and  of e i t h e r p h e n y l a c e t o n i t r i l e or diphenylace-  and o f t h e r e v e r s i b l e nature  of the F r i e d e l  Crafts  reaction. 2 2. Methadonewas s y n t h e s i z e d f r o m e n r i c h e d d i p h e n y l 2 2 2 acetonitrile- H . The m e t a b o l i t e s , EDDP- H , EMDP- H , a n d 2 DDP- H were s y n t h e s i z e d from 4 - d i m e t h y l a m i n o - 2 , 2 - d i p h e n y l 2 p e n t a n o i c a c i d - H-^Q p r e p a r e d u s i n g D2S0^ t o m a i n t a i n l a b e l 2 2 2 enrichment. M e t h a d o n e - H^, EDDP- H^, a n d EMDPwere s y n t h e 2 s i z e d u s i n g C H^CH-^Br a s s t a r t i n g m a t e r i a l . A l l compounds 2 1 Q  1 Q  1 Q  1 Q  e x c e p t f o r methadone3.  a r e new  compounds.  Methadone and i t s m e t a b o l i t e s  spectrometry  t o g i v e i o n s common t o  f r a g m e n t e d u s i n g E I mass diphenylbutane-containing  compounds.  T h e s e were u s e f u l t o i d e n t i f y new m e t a b o l i t e s o f  methadone.  Mass s p e c t r a o f t h e d e u t e r i u m l a b e l e d a n a l o g s  provided  confirmation of the i o n structures.  t i o n was o b s e r v e d i n t h e f r a g m e n t a t i o n  Aryl ring  processes  migra-  f o r EDDP.  145 4. by  M e t h a d o n e l e v e l s i n p l a s m a a n d s a l i v a were monitoring  m/e 72 w i t h  nonanone p e r c h l o r a t e  2-dimethylamino-4,4-diphenyl-5-  as t h e i n t e r n a l standard.  l i m i t of reproducible  analyzed  The l o w e r  q u a n t i t a t i o n o f methadone i n 0.5 m l o f  p l a s m a 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 n g .  The mean  r a t i o s o f s a l i v a t o p l a s m a f o r t w o p a t i e n t s w e r e 0.55 ± 0.15 (SD) a n d 0.48 ± 0.10 5.  (SD).  A n a l y s i s o f methadone a n d EDDP i n p a t i e n t s ' u r i n e  showed  t h a t t h e 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 t h e a n a l y s i s were c o n s i s t e n t w i t h t h o s e c a l c u l a t e d by u s i n g amount o f d r u g t o i n t e r n a l s t a n d a r d that the concentration  the r a t i o s of equal  (standard  ratio), indicating  o f t h e compounds c a n be a n a l y z e d  without  constructing c a l i b r a t i o n curves. 6.  The d e u t e r i u m l a b e l e d a n a l o g s p r o v e d t o be v e r y  f o r t h e GCMS d e t e c t i o n bolites. contain  a n d i d e n t i f i c a t i o n o f methadone m e t a -  The c o n j u g a t e d f r a c t i o n f r o m r a t b i l e was f o u n d t o a new m/e 72 c o n t a i n i n g  GCMS d a t a t h e new m e t a b o l i t e  compound.  was a s s i g n e d  On t h e b a s i s o f the structure,  N-methylene-l-methyl-3,3-diphenyl-4-oxo-hexaneamine (methadone n i t r o n e ) .  The n i t r o n e  The d e t e c t i o n o f t h e n i t r o n e  oxide  appeared t o r e s u l t from de-  c o m p o s i t i o n o f N-hydroxynormethadone d u r i n g  all  useful  work up p r o c e d u r e s .  i m p l i e s t h a t methadone i s n o t  s p o n t a n e o u s l y c y c l i z e d t o EDDP a t t h e s i t e o f e n z y m a t i c  N-oxidation. 7. lite  I n d i r e c t e v i d e n c e f o r t h e s t r u c t u r e o f t h e new m e t a b o was o b t a i n e d  f r o m c h e m i c a l o x i d a t i o n o f EDDP  perchlorate  146 with m-chloroperbenzoic heptanedione  acid  i n which  4,4-diphenyl-2,5-  a n d t h e methadone n i t r o n e were o b t a i n e d a s p r o d u c t s .  Mass s p e c t r a l d a t a , NMR, a n d I R were u s e d t o i d e n t i f y compounds.  A mechanism f o r t h e f o r m a t i o n o f n i t r o n e  these and  t h e d i k e t o n e f r o m o x i d a t i o n o f EDDP p e r c h l o r a t e was p r o p o s e d . 8.  T h r e e p r o c e s s e s f o r t h e E I f r a g m e n t a t i o n o f methadone  n i t r o n e were p r o p o s e d .  The p r o p o s e d  fragmentation processes  were a l s o f o u n d t o a p p l y t o amphetamine n i t r o n e . 9.  A i r o r c h e m i c a l o x i d a t i o n o f EDDP b a s e g a v e DDP a s t h e  m a i n p r o d u c t a n d t h e e v i d e n c e s u g g e s t s t h a t DDP i s n o t a t r u e m e t a b o l i t e o f methadone.  MCPBA o x i d a t i o n o f EMDP b a s e g a v e  2-ethyl-5-methyl-3,3-diphenylpyrrolidy1-1,2-oxaziran. 10. and  The f e a s i b i l i t y  of using biosynthetic  S I M was d e r i v e d f o r d r u g m e t a b o l i s m  i n t e r n a l standards  and k i n e t i c  studies.  Drug c o n c e n t r a t i o n = s l o p e x r a t i o o f drug t o i n t e r n a l  standard  + i n t e r c e p t , where t h e i n t e r c e p t i s z e r o a n d t h e s l o p e i s c a l c u l a b l e u s i n g the standard r a t i o which value.  i s a drug  dependent  T h e r e f o r e , r a t i o = drug c o n c e n t r a t i o n / a (drug dependent  constant value). on t h e r a t i o .  The c h a n g e o f d r u g c o n c e n t r a t i o n i s r e f l e c t e d  P h a r m a c o k i n e t i c c o n s t a n t s c a n be o b t a i n e d by  using these r a t i o values. 11.  A m e t h a d o n e - d i a z e p a m i n t e r a c t i o n s t u d y i n r a t s was  d e s i g n e d i n w h i c h m e t h a d o n e , EDDP, a n d EMDP w e r e a n a l y z e d b y SIM u s i n g d e u t e r i u m l a b e l e d a n a l o g s a s i n t e r n a l  standards  and c o n j u g a t e d m e t a b o l i t e s u s i n g d e u t e r i u m l a b e l e d internal  standards.  biosynthetic  The d e u t e r i u m l a b e l i n g o f t h e i n t e r n a l  s t a n d a r d s was f o u n d t o be s t a b l e o v e r a w i d e r a n g e o f pH a n d  147 under c o n d i t i o n s of prolonged i n c u b a t i o n . crease i n the during  the  e x c r e t i o n of conjugated m e t a b o l i t e s  first  2 hours of b i l e e x c r e t i o n  diazepam t r e a t e d r a t s . metabolites  was  followed  over the  and  methadone-diazepam t r e a t e d r a t s .  transient  increase  2 hour p e r i o d  of the  c o u l d be  due  i n t e r a c t w i t h methadone a t t h e  route.  there  transport  reason f o r  conjugated metabolites t o the  w i t h methadone p l a s m a p r o t e i n b i n d i n g .  the  conjugated  d i f f e r e n c e o b s e r v e d b e t w e e n t h e methadone o n l y The  of m e t a b o l i t e s  hepatic by  the  found . .  methadone-  23 h o u r s p e r i o d ,  no  on  was  i n the  When t h e e x c r e t i o n o f  was  tial  A significant in-  treated the  i n the  competition  by  Diazepam d i d  diazepam not  metabolism l e v e l biliary  ini-  excretion  nor  148  REFERENCES  1.  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G e r b e r , Seminal e x c r e t i o n , v a g i n a l a b s o r p t i o n , d i s t r i b u t i o n and w h o l e b l o o d k i n e t i c s o f d-methadone i n t h e r a b b i t , J . P h a r m a c o l . Exp. T h e r . , 206, 507 ( 1 9 7 8 ) .  P e r r i e r , Pharmacokinetics, Marcel Y o r k , 1975, p. 48.  162  APPENDIX  NMR  SPECTRA OF DEUTERATED COMPOUNDS  163 i  T" 1 I | I I I I 500  I 400  I  "i i i i i i i i i |1 i i—i—i ~r~r "i—i—i—i—r~i—i—r I iM I I I i I I—i—i—i—r  I"  I  300  200  100  1  I MI I I  P a r t i a l l y deuterated  0 Hi  diphenylaceto-  2 nitrileH-^Q S o l v e n t , C D C 1 ; 86%  labelling.  3  i i i i i i i i i i i i i i ii iii i i i i i ii i'i i i i i i iJi Ii i Ii I I I I I I i n—I—I—i—r~r i I -r-|—i—i—r "I I I II I I I 'T' T1 1 1 1 1 1 1 1 "i—i—i—i—r >| I 8.0  500  7J)  I  I  I  6.0  •  I 400  I I I  I  I  5.0  T  T  300  »-'"  4.0  ~1—I I I  I  3.0  I  2.0  L  0  1.0  I  I ;  I  I  200  I  !  I  I  1 I .  100  1  Enriched  diphenylacetonitrile-  Solvent,  CDC1  • 98%  labelling  H  10  0  Hi  164  5  Ik  I  •  I 5.  •  I  •  1  Methadone- H  10  S o l v e n t , CD OD; 97% 3  labelling  CTl Ul  166  2 6.  DDP-  H  1 0  Solvent, CDCl ; 3  7.  EMDP- H  97%  labelling  2  1 Q  S o l v e n t , CD^OD; 96%  labelling.  167  

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