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Negative ion chemical ionization GCMS analysis of valproic acid and its metabolites Kassahun, Kelem 1987

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NEGATIVE ION CHEMICAL IONIZATION GCMS ANALYSIS OF VALPROIC ACID AND ITS METABOLITES  by  KELEM KASSAHUN B.Sc.  (Pharm.) Addis Ababa U n i v e r s i t y ,  1981  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES F a c u l t y of Pharmaceutical ( D i v i s i o n of Pharmaceutical We accept t h i s to  the  Sciences Chemistry)  t h e s i s as conforming  required  standard  THE UNIVERSITY OF BRITISH COLUMBIA August ©  1987  KELEM KASSAHUN,  1987  In presenting  this thesis in partial fulfilment  of the  requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6(3/81)  -  11  -  ABSTRACT  the  Valproic  a c i d (VPA) i s a major  treatment  o f absence s e i z u r e s .  humans. other  Several  VPA m e t a b o l i t e s  metabolites  hepatotoxicity. was r e q u i r e d  are  possess in  metabolized  anticonvulsant rare  but  used i n  the l a r g e  a method  levels.  and to  method to the d e t e r m i n a t i o n VPA m e t a b o l i t e s .  and  cases  of  fatal  o f VPA and i t s m e t a b o l i t e s for  of t r a c e  a  of a n a l y z i n g  desired  of t h i s  preliminary  VPA l e v e l s  by n e g a t i v e  the  of VPA m e t a b o l i t e s ,  The o b j e c t i v e  make  The s u i t a b i l i t y  GCMS was e v a l u a t e d  number  in  activity  A h i g h l y s e n s i t i v e and more s p e c i f i c a n a l y t i c a l  to analyze  such  VPA i s e x t e n s i v e l y  implicated  which are p r e s e n t at t r a c e develop  a n t i c o n v u l s a n t drug widely  method some of  study was to  application  of  the  and to search f o r new halogenated  ion chemical  sensitivity  derivatives  ionization  (NICI)  and s p e c i f i c i t y .  An  assay was thus developed f o r VPA i n serum and s a l i v a based on NICI-GCMS of  the p e n t a f l u o r o b e n z y l  PFB e s t e r ([M-181]") of  The NIC I  of VPA was dominated by a s i n g l e ion.  detection  [^Hsl-VPA  (PFB) d e r i v a t i v e .  as  fragment  spectrum of the i o n , the m/z 143  When the m/z 143 i o n was monitored  was  2  ng/mL  the  internal  of  VPA  standard,  in  serum the  or  intra-  the lower  limit  saliva.  Using  and  inter-assay  v a r i a t i o n s were l e s s than 10 % at serum VPA c o n c e n t r a t i o n s of 10 t o 800 ng/mL.  L i n e a r i t y was observed over the c o n c e n t r a t i o n range of 10 ng/mL  t o 25 ug/mL. The NICI assay was employed to q u a n t i t a t e VPA i n serum ( t o t a l and free)  and s a l i v a  interaction paired  in  five  study between  healthy  volunteers  who took  VPA and carbamazepine  (CBZ).  part  in  A total  s a l i v a and serum samples were analyzed by NICI-GCMS;  a drug of 63  33 before  the  administration  of  CBZ and 30  average VPA c o n c e n t r a t i o n a f t e r and  48.13  ±  7.70,  respectively.  for  There  average VPA c o n c e n t r a t i o n saliva  to serum f r e e  ± 2.82  f o l l o w i n g CBZ.  2.43% ± 0.86 the  saliva  strong  The  to  related  resulted  that  of  the  derivative  fragment peak  formation  of  ion,  in  the  the  twin i o n s  only  NICI  spectrum  Isolated (deuterated  all  of  PFB  metabolites  chromatographic  Urine  or  ±  the  The average  VPA r a t i o  after  was that  dependent.  0.0450) CBZ  A (r  (after  =  CBZ).  administration  after  CBZ was  not  facile  and  was  studied.  was c a r r i e d of  were  by the  3-keto  derivatized  and undeuterated)  serum  in  serum f r e e  metabolites  being t h a t  comparison of mass s p e c t r a and r e t e n t i o n compounds.  and both  metabolites  ion c u r r e n t  exception  VPA,  of VPA.  VPA of  saliva  b e f o r e CBZ and 16.37%  serum t o t a l  increase  the  13.64,  (p<0.025)  concentration  saliva  fraction  and  fluids.  VPA c o n c e n t r a t i o n  i n uniform d e r i v a t i z a t i o n  [M-181-C02]". of  free  in  36.85 ±  free  VPA (0.9058  not  in  f o l l o w i n g CBZ, i n d i c a t i n g  was  between  decrease  the  3.48,  biological  ± 0.50  % decrease  reduction  s a l i v a to  VPA r a t i o  VPA d i d  NICI mass s p e c t r a most of  ±  was 18.92% ± 6.25  serum t o t a l  to changes in the  PFB  three  The average  and  fraction  suggesting  in a l l  was found  The  serum  significant  VPA r a t i o  serum t o t a l  0.0784)  free  total,  b e f o r e CBZ and 1.67%  correlation  0.9035 ±  a  CBZ.  CBZ was 27.91  serum  was  after  VPA. 3-keto  identified in  the  with  were  run and SIM chromatograms o b t a i n e d .  controls  showed no i n t e r f e r i n g  peaks and the  suitable  f o r a s e n s i t i v e assay of VPA m e t a b o l i t e s .  the  [M-181]" The base VPA the  was help  mass s p e c t r a and by  times with s y n t h e t i c  metabolites  In  reference  analyzed Serum and  analytical  in  one urine  method appears  -  iv  -  The N I C I method employing PFB d e r i v a t i v e s detect The  VPA m e t a b o l i t e s  ratio  of  Z to  in  saliva.  E isomers o f  than i n serum (3.82  vs. 0.458),  Seven  2-ene  was s e n s i t i v e enough  metabolites  were  to  detected.  VPA was much g r e a t e r  in  s u g g e s t i n g d i f f e r e n c e s i n the  saliva  transport  o r plasma p r o t e i n b i n d i n g p r o p e r t i e s of these two i s o m e r s . A new VPA m e t a b o l i t e , detected  in  metabolite  urine.  structure 4'-keto-2-ene  The mass spectrum and  matched  s y n t h e t i c mixture  a s s i g n e d the  that  of  one  retention  compound  containing 4'-keto-2-ene  which  VPA.  which appears to be 2 - ( 2 ' - p r o p e n y l ) - g l u t a r i c  time  was  of  VPA was this  present  in  new a  Another new m e t a b o l i t e  a c i d was a l s o d e t e c t e d  in  urine. The different  synthesis synthetic  dehydrogenation  of  of  methods. the  2-propyl-4-oxopentanoate positional  isomer,  based  the  on  hydroxyheptane  4'-keto-2-ene  0-TMS  p o s s i b l e to i s o l a t e  first  4-keto-2-ene  The  which  ketene in  using  two  involved  the  acetal  the  of  ethyl  formation  second s y n t h e t i c  of  the  route  was  4-carboethoxy-2-ethylenethioketal-5-  produced 4 ' - k e t o - 2 - e n e sufficient  attempted  method  resulted  VPA.  of  was  dialkyl  apparently  dehydration and  The  VPA  VPA.  However,  it  was  product f o r NMR c h a r a c t e r i z a t i o n .  not  -  V -  TABLE OF CONTENTS Page ABSTRACT  ii  LIST OF TABLES  viii  LIST OF FIGURES  x  LIST OF SCHEMES  xiii  LIST OF ABBREVIATIONS  xiv  ACKNOWLEDGEMENT I.  INTRODUCTION A. B. C. D. E. F. G.  H. II.  xvii 1  P h a r m a c o k i n e t i c s o f VPA Metabolism o f VPA A n t i c o n v u l s a n t a c t i v i t y of VPA m e t a b o l i t e s T o x i c i t y o f VPA and i t s m e t a b o l i t e s I n t e r a c t i o n between VPA and carbamazepine A n a l y t i c a l methods f o r VPA and i t s m e t a b o l i t e s Negative i o n chemical i o n i z a t i o n  2 3 5 7 10 11 13  1. 2. 3. 4.  14 14 16  Ion forming r e a c t i o n s i n NICI Negative i o n reagent gas systems S e n s i t i v i t y of NICI F a c t o r s which determine the s e n s i t i v i t y of sample d e t e c t i o n i n NICI  Objectives  16 17  EXPERIMENTAL  18  A.  Chemicals and M a t e r i a l s  18  1.  General  18  2.  VPA m e t a b o l i t e s  B.  and i n t e r n a l  standards  19  Instrumentation  20  1. 2. 3.  20 21 21  Gas chromatography mass spectrometry G C - E l e c t r o n Capture D e t e c t i o n Other instruments  - vi -  Page  C.  Human Study  21  D.  A n a l y s i s of Samples  22  1.  Serum and s a l i v a standards  22  2. 3. 4. 5.  Sample p r e p a r a t i o n Derivatization Serum f r e e l e v e l s Comparison of the s e n s i t i v i t y of EI (t-BDMS) w i t h NICI (PFB, bis-TFMB) I d e n t i f i c a t i o n o f VPA metabol i t e s  24 25 26  6. E.  Chemical S y n t h e s i s 1.  2. 3. 4. III.  26 27 27  Attempted s y n t h e s i s o f 2 - ( 2 ' - o x o p r o p y l ) - 2 pentenoic a c i d ( 4 ' - k e t o - 2 - e n e VPA) v i a ethyl 2-propyl-4-oxopentanoate S y n t h e s i s of 2 - ( 2 - o x o p r o p y l ) - 2 - p e n t e n o i c a c i d s t a r t i n g with a p r o t e c t e d 4-oxopentanoic a c i d S y n t h e s i s of ethyl 2 - p r o p y l - 3 - o x o p e n t a n o a t e S y n t h e s i s of e t h y l 2 - p r o p y l - 3 - h y d r o x y p e n t a n o a t e  27  1  31 35 35  RESULTS AND DISCUSSION  36  A.  NICI-GCMS Assay Development  36  1. 2. 3. 4.  36 37 40  5. B.  Derivatization G C - E l e c t r o n Capture D e t e c t i o n O p t i m i z a t i o n of MS parameters f o r NICI Comparison of the r e l a t i v e s e n s i t i v i t y o f d e r i v a t i z e d VPA by EI and NICI Q u a n t i t a t i v e a n a l y s i s with the PFB d e r i v a t i v e  45 48  VPA l e v e l s i n serum ( f r e e and t o t a l ) and s a l i v a b e f o r e and a f t e r the a d m i n i s t r a t i o n of CBZ  56  1. 2. 3.  60 74 76  E f f e c t of CBZ on serum and s a l i v a l e v e l s of VPA Serum f r e e VPA l e v e l s S a l i v a VPA l e v e l s  -  vii  -  Page C.  I d e n t i f i c a t i o n o f VPA m e t a b o l i t e s u s i n g NICI-GCMS o f t h e i r PFB d e r i v a t i v e s 1. 2. 3. 4. 5. 6.  D.  Negative ion s p e c t r a of PFB d e r i v a t i z e d VPA m e t a b o l i t e s PFB as an e l e c t r o n c a p t u r e NICI-GCMS d e r i v a t i v e f o r VPA m e t a b o l i t e s NICI (PFB) versus EI (t-BDMS) s p e c t r a of VPA m e t a b o l i t e s S e l e c t e d ion chromatograms VPA m e t a b o l i t e s i n s a l i v a D e t e c t i o n of new VPA m e t a b o l i t e s  Synthesis 1.  2.  Attempted s y n t h e s i s o f 2 - ( 2 ' - o x o p r o p y l ) - 2 pentenoic a c i d ( 4 ' - k e t o - 2 - e n e VPA) v i a ethyl 2-propyl-4-oxopentanoate S y n t h e s i s of 4 ' - k e t o - 2 - e n e VPA s t a r t i n g with a p r o t e c t e d 4-oxopentanoic a c i d  93  95 112 113 114 119 123 131  131 135  SUMMARY AND CONCLUSIONS  147  REFERENCES  150  APPENDIX  161  -  viii  -  LIST OF TABLES  Comparison of the r e l a t i v e of three VPA d e r i v a t i v e s  sensitivities  Comparison of [^Hsl-VPA and OA as internal standards Serum VPA l e v e l s U g / m L ) i n two s u b j e c t s on VPA s t e a d y - s t a t e as measured by EI (t-BDMS) and NICI (PFB) Serum t o t a l , serum f r e e and s a l i v a c o n c e n t r a t i o n s (ng/mL) o f VPA and t h e i r r e l a t i o n s h i p to each other in v o l u n t e e r W.T. before the a d m i n i s t r a t i o n of CBZ Serum t o t a l , serum f r e e and s a l i v a c o n c e n t r a t i o n s U g / m L ) of VPA and t h e i r r e l a t i o n s h i p to each other in v o l u n t e e r W.T. a f t e r the a d m i n i s t r a t i o n of CBZ Serum t o t a l , serum f r e e and s a l i v a c o n c e n t r a t i o n s (ug/mL) o f VPA and t h e i r r e l a t i o n s h i p to each other in v o l u n t e e r M.S. b e f o r e the a d m i n i s t r a t i o n of CBZ Serum t o t a l , serum f r e e and s a l i v a c o n c e n t r a t i o n s (ug/mL) of VPA and t h e i r r e l a t i o n s h i p to each other in v o l u n t e e r M.S. a f t e r the a d m i n i s t r a t i o n of CBZ Serum t o t a l , serum f r e e and s a l i v a c o n c e n t r a t i o n s U g / m L ) o f VPA and t h e i r r e l a t i o n s h i p to each other in v o l u n t e e r R.M. before the a d m i n i s t r a t i o n of CBZ Serum t o t a l , serum f r e e and s a l i v a c o n c e n t r a t i o n s U g / m L ) o f VPA and t h e i r r e l a t i o n s h i p to each other in v o l u n t e e r R.M. a f t e r the a d m i n i s t r a t i o n of CBZ Serum t o t a l , serum f r e e and s a l i v a c o n c e n t r a t i o n s U g / m L ) o f VPA and t h e i r r e l a t i o n s h i p to each o t h e r in v o l u n t e e r B . A . b e f o r e the the a d m i n i s t r a t i o n of CBZ Serum t o t a l , serum f r e e and s a l i v a c o n c e n t r a t i o n s U g / m L ) of VPA and t h e i r r e l a t i o n s h i p to each other in v o l u n t e e r B . A . a f t e r the a d m i n i s t r a t i o n of CBZ  -  ix -  LIST OF TABLES (CONT'D) Table  Page  8a  Serum t o t a l , serum f r e e and s a l i v a c o n c e n t r a t i o n s (ug/mL) of VPA and t h e i r r e l a t i o n s h i p to each other in v o l u n t e e r F . A . b e f o r e the a d m i n i s t r a t i o n of CBZ  69  8b  Serum t o t a l , serum f r e e and s a l i v a c o n c e n t r a t i o n s (ug/mL) o f VPA and t h e i r r e l a t i o n s h i p to each o t h e r i n v o l u n t e e r F . A . a f t e r the a d m i n i s t r a t i o n of CBZ  70  9  Time-averaged r a t i o s (6-8 samples) and c o r r e l a t i o n s between serum t o t a l , serum f r e e and s a l i v a VPA c o n c e n t r a t i o n s i n f i v e v o l u n t e e r s before the a d m i n i s t r a t i o n of CBZ  71  10  Time-averaged r a t i o s (6-8 samples) and c o r r e l a t i o n s between serum t o t a l , serum f r e e and s a l i v a VPA c o n c e n t r a t i o n s in f i v e v o l u n t e e r s a f t e r the a d m i n i s t r a t i o n of CBZ  72  11  Decrease (%) i n average VPA c o n c e n t r a t i o n a f t e r CBZ a d m i n i s t r a t i o n  75  12  Ions (m/z) monitored i n NICI mode f o r VPA and [ H6]-VPA metabolites 2  116  X -  LIST OF FIGURES Figure  Page  1  M e t a b o l i c pathways o f v a l p r o i c a c i d i n human  6  2  T o t a l ion c u r r e n t p l o t of the PFB e s t e r s o f VPA (a) and 0A(b) i n the NICI mode  38  3  T y p i c a l chromatograms o f PFB d e r i v a t i v e s o f VPA(a) and 0A(b) o b t a i n e d with GC-ECD  41  4  C a l i b r a t i o n curve f o r PFB d e r i v a t i z e d VPA i n e t h y l a c e t a t e o b t a i n e d u s i n g GC-ECD.  42  5  NICI mass s p e c t r a of the PFB (A) and bis-TFMB (B) d e r i v a t i v e s o f VPA.  46  6  EI mass s p e c t r a of the PFB (A) and bis-TFMB (B) d e r i v a t i v e s o f VPA.  47  7  SIM chromatograms of VPA, OA (m/z 143) and [ He]-VPA (m/z 149) from serum s p i k e d with these s u b s t a n c e s .  50  8  C a l i b r a t i o n curve f o r serum t o t a l VPA.  52  9  C a l i b r a t i o n curve f o r serum f r e e VPA.  53  10  C a l i b r a t i o n curve f o r s a l i v a VPA.  54  11  C a l i b r a t i o n curve f o r EI(t-BDMS) serum t o t a l VPA.  of  55  12  SIM chromatogram of PFB d e r i v a t i z e d VPA o b t a i n e d with 10 pg o f VPA e x t r a c t e d from serum  57  13  R e l a t i o n s h i p between VPA c o n c e n t r a t i o n s (ug/mL) serum determined by EI (t-BDMS) and NICI (PFB)  59  14  Saliva concentration-time p r o f i l e s for five v o l u n t e e r s at steady s t a t e VPA  15  C o n c e n t r a t i o n s time curves f o r seum t o t a l , serum f r e e and s a l i v a VPA in one v o l u n t e e r  2  determination  in  77  78 (M.S.)  16  C o n c e n t r a t i o n - t i m e curve f o r serum f r e e VPA before and a f t e r CBZ a d m i n i s t r a t i o n i n one volunteer (W.T.).  79  17  C o n c e n t r a t i o n - t i m e curve f o r s a l i v a VPA b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n in one v o l u n t e e r ( W . T . ) .  80  18  R e l a t i o n s h i p between serum t o t a l and s a l i v a VPA c o n c e n t r a t i o n s in one v o l u n t e e r ( W . T . ) .  81  -  xi -  LIST OF FIGURES  (CONT'D)  Figure  Page  19  R e l a t i o n s h i p between serum f r e e and s a l i v a VPA c o n c e n t r a t i o n s i n one v o l u n t e e r (W.T.)  82  20  The r e l a t i o n s h i p between serum t o t a l and s a l i v a VPA c o n c e n t r a t i o n s in a l l f i v e v o l u n t e e r s  83  21  The r e l a t i o n s h i p between serum f r e e and s a l i v a VPA c o n c e n t r a t i o n s in a l l f i v e v o l u n t e e r s  84  22  A plot of free f r a c t i o n o f VPA  85  23  T o t a l i o n c u r r e n t p l o t , i n the NICI mode, o f the PFB d e r i v a t i z e d u r i n e e x r a c t from a v o l u n t e e r on VPA steady s t a t e , a l s o given s e l e c t e d doses o f [ ^ l - V P A  94  24  NICI mass spectrum of VPA ( H  96  25  NICI mass spectrum of 3-ene VPA ( H PFB e s t e r s  26  NICI mass spectrum of ( E ) - 2 - e n e VPA ( H Q + %) PFB e s t e r s  98  27  NICI mass spectrum of 2 , 4 - d i e n e VPA ( H PFB e s t e r s  99  28  versus serum t o t a l  2  + %)  PFB e s t e r s  z  0  concentration  + He)  97  2  2  0  2  2  2  NICI mass spectrum of ( E , E ) - 2 , 3 ' - d i e n e H 6 ) PFB e s t e r s  0  + ^ 5 )  VPA ( H  +  100  A) NICI mass spectrum of 4 ' - k e t o - 2 - e n e VPA ( H Q + H3) PFB e s t e r s , B) EI (t-BDMS) mass spectrum of 4 ' - k e t o - 2 - e n e VPA  101  2  0  2  29  2  2  30  NICI mass spectrum of 3 - k e t o VPA ( H Q + H 6 ) PFB e s t e r s 2  102  31  NICI mass spectrum of 3-0H VPA ( H PFB e s t e r s  + %)  103  32  NICI mass spectrum of 4 - k e t o VPA ( H Q + PFB e s t e r s  33  NICI mass spectrum of 4-0H VPA ( H PFB e s t e r s  34  NICI mass spectrum of 5-OH VPA ( H PFB e s t e r s  2  2  2  0  2  ^ 3 )  + H6)  2  105  2  0  2  0  +  2  H  104  5  )  106  -  xii -  LIST OF FIGURES (CONT'D) Figure  Page  35  NICI mass spectrum o f 2-PSA ( H Q + % ) PFB e s t e r s  107  36  NICI mass spectrum o f 2-PGA ( H PFB e s t e r s  108  37  A) Mass chromatograms at m/z 1 4 1 B) NICI mass spectrum o f 4 - e n e VPA PFB e s t e r  109  38  EI(t-BDMS) mass spectrum o f 3-OH VPA  115  39  SIM chromatograms of t h e PFB d e r i v a t i v e s o f VPA and [ H 6 l - V P A m e t a b o l i t e s i n a u r i n e e x t r a c t  117  SIM chromatograms of the PFB d e r i v a t i v e s VPA and [ H e ] - V P A m e t a b o l i t e s  of serum  118  SIM chromatograms of the PFB d e r i v a t i v e s VPA and [ H 6 ] - V P A m e t a b o l i t e s in a s a l i v a e x t r a c t  of  120  2  2  0  + %)  2  40  2  41  2  42  NICI (A) and EI (B) mass s p e c t r a of VPA r e l a t e d m a t e r i a l i n u r i n e t h a t appears to be 2 - ( 2 ' - p r o p e n y l ) g l u t a r i c acid  124  43  EI mass s p e c t r a of A) 4 ' - k e t o - 2 - e n e VPA and B) 4 - k e t o VPA e x t r a c t e d from u r i n e without a l k a l i n e treatment  127  44  NICI mass s p e c t r a of A) 4 ' - k e t o - 2 - e n e VPA and B) 4 - k e t o VPA e x t r a c t e d from u r i n e without a l k a l i n e treatment  128  45  Mass chromatograms (m/z 2 1 3 ) o f the t-BDMS d e r i v a t i v e s of s y n t h e s i z e d 2 - p r o p y l - 4 - o x o - 2 p e n t e n o i c a c i d ( 4 - k e t o - 2 - e n e VPA)  136  46  Mass s p e c t r a of the t-BDMS d e r i v a t i v e s of 2 - p r o p y l - 4 - o x o - 2 - p e n t e n o i c a c i d  137  47  EI mass s p e c t r a of the isomers of the ethyl 4 ' - k e t o - 2 - e n e VPA  48  Mass chromatograms a t m/z 2 1 3 o f t-BDMS d e r i v a t i z e d s y n t h e t i c and n a t i v e 4 ' - k e t o - 2 - e n e VPA  49  Mass s p e c t r a of the t-BDMS d e r i v a t i v e s n a t i v e 4 ' - k e t o - 2 - e n e VPA  50  Mass chromatograms at m/z 1 5 5 o f the PFB d e r i v a t i v e s of s y n t h e t i c and i s o l a t e d 4 ' - k e t o - 2 - e n e VPA  of the isomers  esters  of  of s y n t h e t i c  141  143  and  144  145  xi i i  -  LIST OF SCHEMES Scheme  Page  1  O r i g i n of the m/z 113 anion i n the NICI mass spectrum o f PFB d e r i v a t i z e d 3 - k e t o VPA  111  2  Proposed fragmentation pathway f o r the t-BDMS d e r i v a t i v e of a new VPA m e t a b o l i t e a s s i g n e d the s t r u c t u r e 4 ' - k e t o - 2 - e n e VPA  126  3  S y n t h e t i c route  132  4  Attempted s y n t h e s i s o f 2 - ( 2 ' - o x o p r o p y l ) - 2 - p e n t e n o i c acid  134  5  S y n t h e t i c route f o r 2 - ( 2 ' - o x o p r o p y l ) - 2 - p e n t e n o i c acid  139  for 2-propyl-4-oxopentanoic acid  -  xiv  -  LIST OF ABBREVIATIONS  VPA  Valproic  [ H ]-VPA  [ H6]-Valproic  2 , 3 ' - d i e n e VPA  2-(1 ' - p r o p e n y l ) - 2 - p e n t e n o i c  2 , 4 - d i e n e VPA  2-propyl-2,4-pentadienoic  4 , 4 ' - d i e n e VPA  2-(2'-propenyl)-4-pentenoic  2- ene VPA  2-propyl-2-pentenoic  acid  3- ene VPA  2-propyl-3-pentenoic  acid  4- ene VPA  2-propyl-4-pentenoic  acid  3- keto VPA  2-propyl-3-oxopentanoic  acid  4- keto VPA  2-propyl-4-oxopentanoic  acid  3- OH VPA  2-propyl-3-hydroxypentanoic  acid  4- OH VPA  2-propyl-4-hydroxypentanoic  acid  5- OH VPA  2-propyl-5-hydroxypentanoic  acid  2  2  6  4'-keto-2-ene 4-keto-2-ene  VPA VPA  3'-keto-4-ene 3-keto-4-ene  acid (2-propylpentanoic  VPA VPA  acid)  acid acid acid  2-(2'-oxopropyl)-2-pentenoic 2-propyl-4-oxo-2-pentenoic 2-(1'-oxopropyl)-4-pentenoic 2-propyl-3-oxo-4-pentenoic  acid  acid acid acid acid  4'-0H-2-ene  VPA  2-(2'-hydroxypropyl)-2-pentenoic  acid  4'-0H-4-ene  VPA  2-(2 ' - h y d r o x y p r o p y l ) - 4 - p e n t e n o i c  acid  bis-TFMB Bp  3,5-bi  s(trifluoromethyl)benzyl  boiling  point  CBZ  carbamazepine  CI  chemical  CSF DDQ  ionization  cerebrospinal  fluid  2,3-dichloro-5,6-dicyano-l,4-benzoquinone  -  XV  -  LIST OF ABBREVIATIONS (CONT'D)  DMAP  4-dimethylaminopyridine  E  trans  ECD  e l e c t r o n capture  EI  electron  impact  eV  electron  volts  GC  gas chromatography  GCMS  gas chromatography mass spectrometry  I.D  internal  IR  infrared  LDA  lithium  Lit.  1i t e r a t u r e  m  mul t i pi et  MHz  megahertz  MS  mass  MSTFA  N-methyl-N-trimethyl s i l y l t r i fluoroacetamide  m/z  mass to charge  NICI  negative  NMR  n u c l e a r magnetic  OA  octanoic  PFB  p e n t a f l uorobenzyl  PFBB  p e n t a f l uorobenzyl  q  quadruplet  r  correlation  s  singlet  SIM  s e l e c t e d ion m o n i t o r i n g  6  detection  diameter  diisopropylamide  spectrometer  chemical  ratio  ion chemical  ionization  resonance  acid  bromide  coefficient  shift  -  xv i  -  LIST OF ABBREVIATIONS (CONT'D)  t  t r i pi e t  t-BDMS  tertiarybutyldimethylsilyl  t-BDMSCl  tertiarybutyldimethylsilyl  THF  tetrahydrofuran  TIC  total  TMS  trimethylsilyl  Z  ci s  ion c u r r e n t  chloride  - xvii  -  ACKNOWLEDGEMENT  I  am very  greatful  support  throughout  for  valuable  I  his  like  Dr.  K.  Dr.  F.  course of  the  assistance  am a l s o t h a n k f u l  Burt,  the  to  in  the  S.  Panesar and M.  A.  Vance  for  study.  Special  the Lee.  is. sincerely  various  his  excellent thanks  gas chromatography  to members of my r e s e a r c h  McErlane and Dr. J .  to acknowledge  Abbott  Orr  for  -  committee,  their  service  acknowledged.  The  go to  Dr.  J.  financial  F e l l o w s h i p p r o v i d e d by the World H e a l t h O r g a n i z a t i o n  R.  Axel s o n ,  time and g u i d a n c e .  provided  Mr.  by Mrs. support  I  L e e , G.  S.  Rodgers in  is greatfully  the  work.  Dr.  would  R.  and  Burton  mass spectrometry  a s s i s t a n c e s by my lab mates,  The t y p i n g  supervision  H. also  Slatter, and  form  Mrs. of  acknowledged.  a  -  1 -  I. INTRODUCTION  Valproic is  a major  a c i d ( d i - n - p r o p y l a c e t i c a c i d , 2 - p r o p y l p e n t a n o i c a c i d , VPA)  a n t i c o n v u l s a n t drug now i n use throughout  been known s i n c e 1881 much l a t e r  but i t s  by Meunier e t a l .  f a t t y a c i d and hence i t it  3  - CH  2  - CH  VPA has  a n t i c o n v u l s a n t p r o p e r t i e s were demonstrated (1963). S t r u c t u r a l l y VPA i s a simple branched  differs  from the usual  l a c k s n i t r o g e n and a r i n g s t r u c t u r e .  CH  the w o r l d .  Its  antiepileptic  structural  drugs i n that  formula  is:  2  CH - COOH CH  3  - CH  VPA i s u s e f u l ures  of  partial  the  2  - CH  2  in a variety  and myoclonic  types,  tonic-clonic  s e i z u r e s . The p r e c i s e mode of  action  of VPA remains  although i t  petit  mal  of s e i z u r e s i n c l u d i n g primary g e n e r a l i z e d  has been suggested t h a t VPA e x e r t s  its  seizures  seizand  uncertain,  a c t i o n s through  effects  on gamma-aminobutyric a c i d . VPA i s e x t e n s i v e l y m e t a b o l i z e d i n humans and experimental metabolites  a n i m a l s . The metabolism of VPA i s have been i d e n t i f i e d  very  i n humans. Several  complex; of these  possess a n t i c o n v u l s a n t p r o p e r t i e s and other m e t a b o l i t e s i n v o l v e d with rare but f a t a l In  hepatotoxicity  view of the a n t i c o n v u l s a n t a c t i v i t y  the m e t a b o l i t e s ,  there i s a great deal  olism.  the VPA m e t a b o l i t e s  Some of  trace metabolites  might  17  metabolites  are thought to be  a s s o c i a t e d with VPA t h e r a p y . and/or potential  of i n t e r e s t are  so f a r  found  not have been i d e n t i f i e d  toxicity  of  i n s t u d y i n g VPA metab-  in  minor  quantities  because of  the  lack  and of  -  sensitivity for of  the  of c u r r e n t l y  2 -  used a n a l y t i c a l  development of c o n v e n i e n t ,  a n a l y s i s . The purpose of  this  methods. Hence, there i s a need  highly  s e n s i t i v e and s p e c i f i c  work was to  develop  a highly  methods  sensitive  and s p e c i f i c method of a n a l y s i s and apply the method to measure t r a c e VPA l e v e l s and to search f o r  new VPA m e t a b o l i t e s  t h a t may be present at  trace  levels.  A . P h a r m a c o k i n e t i c s of VPA  The  pharmacokinetics  of  VPA have  been  extensively  studied  both  humans and animals and have been reviewed by G u g l e r and von Unruh Schobben et  al.  (1980),  Morselli  and Richens (1985). A f t e r  oral  and F r a n c o - M o r s e l l i  administration,  (1980),  (1980),  and Rimmer  rapidly  and almost  VPA i s  c o m p l e t e l y a b s o r b e d , peak plasma l e v e l s being a t t a i n e d w i t h i n one to hours  (Schobben  different  al.,  formulations  Therapeutic patients  et  In  terms  levels  plasma  are  generally  concentrations  and W i l d e r ,  1979). There i s a s i g n i f i c a n t  tion  i n the  number of  al.,  1979).  It  plasma  seizures  in  absolute  is  50  curvilinear,  to  excess  and i n c r e a s i n g  i.e.  100 of  bioavailability  ug/mL with 100  u.g/mL  between  the  serum VPA l e v e l s  relationship the  four  bioequivalent.  relationship  has a l s o been found t h a t the  concentration  of  of the drug appear to be  plasma  requiring  1980).  in  plasma  between  some  (Brum' reduc-  (Gram  et  dose and  concentration  to  dose r a t i o decreases with i n c r e a s i n g d o s e s . Studies  in  rodents  have  shown  r e a c h i n g the b r a i n i n a few minutes distribution  is  in  the  range  of 0.1  that  (Vajda, to  0.4  VPA  is  distributed  rapidly,  1983). The apparent  volume of  L/kg  (Gugler  et  al.,  1977;  -  Perucca VPA i s  et  al.,  1978).  distributed  3 -  This  only to  small  the  volume  circulation  c e l l u l a r water.  VPA does not appear  i n b r a i n nor i s  taken up s e l e c t i v e l y  Todoroff, (Gugler plasma  1980). and  protein  of  6%  therapeutic  Antonin, are  dependent  Gugler  which  (Schobben et  al.,  of  hours  18  et  may  be  1980). in  of  plasma  1978).  proteins  the  al.,  free  of  90%)  the  cerebrospinal  high  fluid  of VPA ranges from binding  varies  of  is 0.4  VPA  is  two-fold  within  the  mL/min  (Klotz  and  1986).  VPA ranges  al.,  proteins  (average  because  Protein  fraction  extra-  humans (Goldberg and  Partly  in  exchangable  that  1977).  explained  In by  from 5 children greater  The plasma e l i m i n a t i o n and  antiepileptic  to  is  6  10  higher  clearance  volumes  of  half-life  is  to  12  drugs (Levy e t  distribution i n the  hours al.,  values  when  range  VPA  is  1986).  Metabolism of VPA  Metabolism excretion tered  is  of the  dose  the  (Gugler  pathways  has  sively  studied  Gugler  and  von  major  means  for  the  elimination  unchanged drug accounts f o r et  metabolism of VPA i s lic  to  brain  indicates  intracellular  s a l i v a concentration  monotherapy  a d m i n i s t e r e d with other  B.  bound  and the  of  and r a p i d l y  by the  concentration.  (Levy e t  distribution  be bound to  VPA c o n c e n t r a t i o n  plasma  range  to  Loscher,  plasma and the  1977;  to  1978;  plasma c l e a r a n c e  found  9  highly  binding,  the  concentration  The  is  Mueller,  10% o f t h a t in to  VPA  of  al.,  very  proved  in  1977).  spite  of  complex and complete elusive.  man and v a r i o u s  Unruh  In  less  (1980).  The  More  simple  elucidation of  and was  recently,  in  VPA and  than 5% o f  its  metabolism  animals  of  the  adminis-  structure of  VPA has initially this  renal  its  the  metabo-  been  exten-  reviewed  laboratory,  by the  -  metabolite  4-keto  2-propylmalonic deuterated  VPA  acid  tracers  4 -  was  identified  were  and  metabolic  GCMS  analysis  as  VPA  (Acheampong  dose study i n man 15 t o 20% o f glucuronide  (Bialer  et  and ( o o - l ) - o x i d a t i o n  summarized i n F i g u r e 1.  and g l u c u r o n i d a t i o n are the two primary  et a l . ,  the  administered  acid  metabolites al.,  and using  1983).  VPA  to produce a l a r g e  ( L o s c h e r , 1981a; Granneman e t a l . ,  pathways of VPA are  B-oxidation  2-propylsuccinic  characterized  undergoes g l u c u r o n i d a t i o n , f J - , w number of m e t a b o l i t e s  and  1984a). The human  In both man and  pathways.  In a s i n g l e  dose was e x c r e t e d  as VPA  1985). B - O x i d a t i o n of VPA g i v e s r i s e to  2-ene  VPA, 3-OH VPA and 3 - k e t o VPA with 2-ene VPA and 3 - K e t o VPA being the metabolites urinary  i n plasma (Nau and L o s c h e r ,  metabolite  mitochondrial The  w-  and i s  a-  oxidation  acid.  VPA to  the the  rat,  leads  arise  to 5-0H  (OJ-1)  -  primarily  as  a result  VPA, 2 - p r o p y l g l u t a r i c  oxidation  the  unsaturated  pathway  al.,  1986a).  The OJ and  and B a i l l i e ,  pathway  p r o d u c t i o n of  quantities  a c i d was  results  (^-1)  metabolites  of  VPA  metabolites  Thus 3-ene  produced  VPA and 4-ene  the  the  serum of  pathways  are  be the  terminal  (Granneman 4-ene  of  4-0H  et  al.,  VPA were  VPA and a d m i n i s t r a t i o n  negligible VPA do  in  Products of  administration  and  acid  By a d m i n i s t e r i n g  shown to  metabolites  3-ene  in  (oj-1)  1984).  and s i m i l a r l y  treatment with 4-0H VPA and 5-0H  unsaturated  metabolites.  in minor  (Prickett  w-oxidation  1984a).  observed a f t e r  et  found  2 - p r o p y l gl u t a r i c  in  The  are  (Abbott  VPA r e s u l t e d  these  The  -oxidation  patients  p r o d u c t of  c o n s i d e r e d to  pathway  cytochrome P-450 mediated 5-0H  1984). 3-Keto VPA i s a l s o a major  of 4-OH VPA, 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d .  and ( o ) - l )  epileptic  major  oxidation.  and 2 - p r o p y l m a l o n i c formation  rat,  not  amounts belong  to  of the  not of  hydroxy OJ and  - 5 pathways VPA.  These  and are  thought  unsaturated  to  originate  metabolites  are  further  d i e n e s VPA. One of these diene m e t a b o l i t e s has  been  Abbott  a s s i g n e d the  addition  metabolic example,  to  pathways  the are  Prickett  above  was  suggests  and B a i l l i e  thought that  oxidation.  to  be  a  VPA  to  the  More  recently  displays  a  is  Rettie  and  Acheampong and  late  active  2-ene  in  pathways,  the  of 3 - , 4 -  formed  et  al.  multiple  VPA. For  incubation  VPA with  of  and 5-OH VPA. The 3-OH but  the  above  by cytochrome P-450 (1987)  have  study  dependent  demonstrated  that  of VPA m e t a b o l i t e s  onset  of  antiepileptic  1980)  (Rowan  and a c a r r y - o v e r e f f e c t  (Lockard  metabolites  effects  and L e v y ,  may be  1976).  cleared  from  the  et  al.,  after  drug  These observa-  formed which  accumulate  in  (1982) of the pharmacokinetic and  p r o p e r t i e s of VPA and 2-ene VPA i n the mouse, i t  VPA was  minor  metabolism of  B-oxidation  In a study by Nau and Loscher  pharmacological  plasma  and  brain  p a r e n t drug i n d i c a t i n g t h a t 2-ene VPA may c o n t r i b u t e effect  produce  formation of 4-ene VPA from VPA.  discontinued  suggest t h a t  the b r a i n .  of  3-OH VPA can al so be  administration  that  to  of  serum m e t a b o l i t e  VPA by  showed t h a t  formation  Henriksen and J o h a n n e s s e n ,  tions  operate  product  Anticonvulsant a c t i v i t y  1979;  i s a major  metabolic  (1984)  cytochrome P-450 c a t a l y z e s the  C.  metabolized  (E,E)-2,3'-diene  known  thought  l i v e r microsomes l e d to  VPA  dehydrogenation  (1985).  In  rat  structure  through  slower  to the  was found than  the  anticonvulsant  of c h r o n i c VPA t h e r a p y .  The  anticonvulsant  s t u d i e d using d i f f e r e n t and Nau,  1983;  Keane  activity animal  et  al.,  of  several  VPA  metabolites  has  been  models of e p i l e p s y ( L o s c h e r , 1981b; Loscher 1985;  Loscher  and  Nau,  1985).  In  one  of  CH -CH-CH  CH,-CH,-CH,  2  C l i p CH-CH  2  CHCOOH  CHCOOGlu CH —CH -CH  C K J - C H J - C H J  3  ?  CH =CH-CH  2  ?  VALPROIC ACID CHj—CM —CH ?  2  CHCOOH  y 2  2 M ' - d l e n e VPA  4-ene VPA  2  CHCOOH CHj—CH —CH 2  CH^CH-CH^  CHj-CM-CH^  2  C-COOH  CHCOOH  OH  CHj-CH -CH 2  CH,-CH--CH, CHCOOH  C H j - C H ^ CH^  2  OH CHj-CH-CH  3-ene VPA  2  \  f /  (t) 2.4-dle ne VPA  CHCOOH CHj-CH -CH  6-OH VPA  2  CH _CH -CH^  2  3  CHj—CH=CH^  2  C-COOH  1  A-OH VPA  CH -CH -CH^ 3  HOOC-CH,-CH,  ^C-COOH H CH — C H — CH  2  2  2(t).3'(t)-dlene VPA  2-ene VPA ;CHCOOH  C H j - C M - C H2 2  CHj—C—CH  2  Z-PropylaluUrlc »ctd  2  CHCOOH CHj-CH -CH 2  2  • ,OH CHj-CH —CH^ 2  CH-COOH CHj-C^-CH^  4-Keto VPA  3-OH VPA  CHj-CH^C'^  HOOC-CH, -COOH CHj-CH -CH -CH^ COOH 2  2  2-PronyliMlonlc « c l d  Figure  1.  CHCOOH CHj-CH^CH^  CHCOOH CH -CH -CH  2-Propyltucclnlc «ctd  Metabolic 1986b).  pathways o f v a l p r o i c  J  2  2  /  3-Keto VPA  a c i d i n human  (Abbott et a l . ,  -  these  studies  metabolites for  the  (Loscher,  was  Of  were the most potent  acid,  elevations. several  and  brain  of  the  long both  tions.  the  4-OH  study  1984).  In  a  shown  that  VPA  when  gave  present  in  calculation  was  profile  study  D.  The  gastrointestinal  induced  1.3  it  2-propylglutaric threshold  has shown t h a t of  dogs  and  VPA was in  although  rats  found  times  more  after in  some b r a i n  upon whole  regions  potent  brain  the  than  concentra-  isomer of 2-ene VPA i n d i c a t e  with t h a t of the  an  VPA,  other  the  anticonvulsant  parent  drug without  latter  (Loscher  (unpublished activity  a  the  et  al.,  data)  have  comparable  to  inactive.  of a c t i v e VPA m e t a b o l i t e s  does not  2-ene VPA or 2 , 3 ' - d i e n e  can be shown t h a t e i t h e r  is  not  appear  VPA c o u l d associated  caused by VPA t h e r a p y .  of VPA and i t s  common adverse  be  based  trans  t h a t of VPA. However,  hepatotoxicity  Toxicity  thresholds  significant  2-ene  Acheampong and Abbott  property  be an a l t e r n a t i v e to VPA i f w i t h the  plasma  a s s o c i a t e d with  VPA has  The a n t i c o n v u l s a n t  to  VPA accumulated  t h a t of 2-ene VPA whereas 4 - k e t o VPA was  t o be s u p e r i o r to  the  VPA  2-ene VPA and 4-ene VPA  1983)  VPA only  s t u d i e s with the  2,3'-diene  rise  and was found to  embryotoxicity recent  on  of  pentylenetetrazole  metabolites  also  The 2-ene  comparable a n t i c o n v u l s a n t for  activity  effects  the  ( L o s c h e r and Nau,  treatment  Furthermore,  potential  tested  were  animals.  drug  their  and  term treatment with  chronic  parent  by measuring  3-OH VPA, 3 - k e t o VPA, 5-0H  VPA m e t a b o l i t e s  acute  during  i.e.  VPA and Another  anticonvulsant  d i s p l a y i n g 50 t o 90% o f the potency of VPA. The  tested,  3-ene  the  e l e c t r o c o n v u l s i on  in mice.  metabolites  1981b)  determined  maximal  convulsions  7 -  metabolites  effects  disturbance,  with  VPA  therapy  thrombocytopenia  are  nausea,  and b e h a v i o r a l  vomiting, disturbance  -  8 -  ( S c h m i d t , 1984). P a n c r e a t i t i s et  has a l s o been r e l a t e d  a l . , 1984). The most s e r i o u s t o x i c e f f e c t  which appears to  be an  idiosyncratic  to VPA therapy  of VPA i s the  reaction  in  (Wyllie  hepatotoxicity  a small  population  of  patients. The VPA-induced h e p a t i c t o x i c i t y and B e n t s e n , 1983). The f i r s t enzymes  and  irreversible patients include is  appears liver  to  be  1984).  hepatocellular  forms (Gram  form i s a s s o c i a t e d with an i n c r e a s e i n  dose  damage. Its  (Jaevons,  can assume two d i f f e r e n t  related.  The  other  form  liver  constitutes  an  frequency has been estimated as 1 i n 20,000  The  clinical  symptoms  n e c r o s i s and m i c r o v e s i c u l a r  of  the  liver  steatosis.  toxicity  The  latter  s i m i l a r to t h a t observed i n R e y e ' s syndrome and Jamaican vomiting s i c k -  ness (Gerber et a l . ,  1979).  The h e p a t o t o x i c i t y  of VPA i s b e l i e v e d to  a n d / o r doubly u n s a t u r a t e d m e t a b o l i t e s an  increased  side-effects  formation  of  as opposed to  of VPA.  diunsaturated patients  be a s s o c i a t e d with the mono Kochen e t  metabolites  without  al. in  side-effects.  (1984)  noted  patients  with  The  4,4'-diene  VPA which has never been observed before was d e t e c t e d along with 4-ene VPA in  one p a t i e n t  fatal  hepatotoxicity  and S a n t i l l i , more of that  who d i e d from h e p a t i c  the  cases  1986). toxic  have  been  in  T h i s may be due to  metabolites.  phenobarbital  failure  In  coadministration  (Kochen e t  multiple  drug  al.,  1983).  therapy  an i n c r e a s e d formation  rats,  Granneman e t  caused s i g n i f i c a n t  al.  Most  (Dreifuss of  (1984b)  increases  one or found in  the  plasma l e v e l s of 4-ene VPA and 5-OH VPA. The most l i k e l y 4-ene  VPA. The 4-ene  metabolite VPA i s  to be i n v o l v e d with the  structurally  similar  to  liver the  toxicity  is  metabolite  of  -  hypoglycin  A  4-pentenoic  that  is  9 -  responsible  for  a c i d which produces a f a t t y l i v e r  1984).  The mechanism by which 4-ene  known.  It  their et  has been p o s t u l a t e d  hepatotoxicity  al.,  pathway  by i n h i b i t i o n  prolonged  sequestration and  potent  B-oxidation  result  from  acids  such  interfering  the as  In it  the  mice. A l s o ,  4-ene  Rhesus  VPA i s  the  formation  to  inhibitor  4-keto  VPA  might  of  the  thought of  (Kesterson B-oxidation to  4-ene  biochemical  High  levels  also  affect  cause a VPA-CoA.  several  enzymes  unusual  involved  a r e c e n t study ( T u r n b u l l  1984).  be t o x i c  in  metabolic  d i s t u r b a n c e s because  intermediary  cell  metabolism.  fats  et a l . ,  dose of VPA  and amino a c i d s i n  1986)  infant  i n d i c a t e s t h a t one gram  causes metabolic d i s t u r b a n c e s i n normal humans.  VPA Rettenmeier  monkey  to  by  1983).  in  carbohydrates,  keto  B-oxidation  (1985) have r e p o r t e d t h a t a s i n g l e t h e r a p e u t i c  metabolism of  of  al.,  in  r e a c t i o n s may  of  found  not  cause  of a s p e c i f i c enzyme(s)  VPA and 4-OH VPA have been  terms VPA causes v a r i o u s  a study t h a t  metabolites  due  4-ene  is  metabolites pathway  inhibition  Furthermore, m u l t i p l e  and  4-ene  o f VPA g i v e n o r a l l y In  transient  hepatocytes ( K i n g s l e y e t  Thurston et a l . affects  B-oxidation  and  (Nau and L o s c h e r ,  with the B - k e t o - a c y l t h i o l a s e enzyme (Kesterson et a l . ,  general  inhibits  the  numerous VPA m e t a b o l i t e s . 3-  The m e t a b o l i t e s , c u l t u r e d rat  of  be a potent  system.  in the r a t  sickness,  VPA may cause h e p a t o t o x i c i t y  CoA, w h i l e  inhibition  The 4-ene VPA-CoA might the  of  vomiting  t h a t VPA and u n s a t u r a t e d  1984). VPA causes a m i l d , by  Jamaican  4-ene  sought to et  al.  VPA i n  (1986a).  address the  mechanism of  the  toxicity  of  have d e t e c t e d 3 - 0 H - 4 - e n e VPA as one of  the  the  the  These  perfused  authors  rat  liver  postulate  that  (1985) the  and  in  detection  of  -  3-0H-4-ene VPA i s from 4-ene  VPA which  3-keto-4-ene  highly  reactive  VPA  1985). VPA i s  (Petrere et  al.,  (Dickinson et  bifida  capable  like  other  al.,  (Rimmer but  is  al., the  Brown  et  activated  mitochondrial carbonyl  to  proteins.  compounds  is  major  toxic  the  placenta  effect in  VPA  rabbits,  (Brown  rats  of  fetal  malformations  et  and mice  and can a f f e c t  reports  various  of  the  fetus  malformation including  in  spina  1985). The mechanism of VPA t e r a t o g e n i c i t y  al.  (1985)  have  differs  suggested  that  from t h a t of the  the  is  biochemical  hepatotoxicity.  I n t e r a c t i o n between VPA and carbamazepine  Baciewicz well  as  extent  (1986).  (Levy To  interactions CBZ  (CBZ)  drug i n t e r a c t i o n s  Because  susceptible  expected.  to  and K o c h , date  there  was  for  found  that  and CBZ i s  interactions  have  been  study  steady-state  CBZ l e v e l s  is  reviewed  by  inducible  as  bound  to  a  VPA and CBZ are reports  of  large to  be  possible  (1979) demonstrated a decrease when  (Levy  seven e p i l e p t i c  CBZ  protein  conflicting  concentration  another  of  between  Bowdle e t a l .  steady-state  one week to  have been r e c e n t l y  biotransformation  1982),  between the two.  minimum  was given  the  inhibition,  c o n c o m i t a n t l y with CBZ. In  it  VPA  a d d i t i o n s to g i v e c o v a l e n t adducts  and embroyotoxic  on VPA d e s c r i b e  Carbamazepine  in  alkylating  P u b l i s h e d case  and R i c h e n s ,  of 3 - k e t o - 4 - e n e  VPA i s m e t a b o l i c a l l y  a,B-unsaturated  second  teratogenic  1979).  formation  1982).  mechanism of VPA t e r a t o g e n i c i t y  E.  of  1986). VPA c r o s s e s  mothers  unknown  the  and can undergo Michael  Teratogenicity  epileptic  evidence f o r  is  o f n u c l e o p h i l e s (Eder e t  al.,  -  VPA. They suggest t h a t 4-ene  3-keto-4-ene The  indirect  10  et  patients were  VPA al.,  was  administered  1984a),  where VPA  r e c e i v i n g c h r o n i c CBZ,  reduced  by 3-59%  in  six  -  patients  and unchanged i n  (Pisani  et  al.,  1981;  11  one p a t i e n t .  Brodie et  find significant differences administered  al.  on  VPA  observed  steady-state.  to  13.7  study  will  investigated  the  Analytical  For fluids urine  minimum  Similarly,  is  the  steady-state  Hoffman  in  the  two  1981)  did  not  given  et  al.  CBZ on the  five  healthy  to  normal  (1981)  protein  binding  volunteers  as  a  VPA  volunteers  found  that  hours and c l e a r a n c e given  of  the  increased  together.  In  (in  of VPA  vivo)  part  of  a  the  general  of CBZ on VPA m e t a b o l i s m .  analysis  required,  of  concentrations  drugs were  of  methods f o r VPA and i t s  sample with  extraction  when  effect  study of the e f f e c t  F.  al.,  studies  has a l s o been s t u d i e d . Bowdle  i n c r e a s e d when CBZ was  mt/min  present be  hand s e v e r a l  McKauge e t  of VPA was reduced from 15 t o 6 . 9  from 8.0  other  i n plasma l e v e l s of CBZ when CBZ and VPA were  that  and c l e a r a n c e  half-life  a l . ,1983;  of CBZ on VPA plasma l e v e l s  (1979)  declined  On the  concomitantly.  The e f f e c t et  -  of  VPA,  usually  organic  VPA and  by  separation  of  acidifying  solvents.  its  metabolites  the  and  The most  metabolites  is  ethyl  drug  from  extracting  biological  the  serum  or the  efficient  solvent  for  acetate  (Abbott  et  al.,  1986a). Many methods  have been d e s c r i b e d i n  o f VPA i n b i o l o g i c a l atography 1982; 1985),  fluids.  (Sutheimer  Moody and A l l a n , enzyme  chromatography,  et  al.,  1983;  immunoassay  the  literature  for  These i n c l u d e high-performance 1979;  Alric  Nakamura et (Higgins,  et  al.,  1983;  al., 1984;  Siegmund  1981;  the  l i q u i d chromKline  Kushida and et  al.,  and gas chromatography - mass s p e c t r o m e t r y .  analysis  et  al.,  Ishizaki,  1981),  gas  -  GC d e t e r m i n a t i o n  12  -  of VPA has been by f a r  has been assayed by GC u n d e r i v a t i z e d Freeman and Rawal, quantitated  1980;  Berry  as the methyl  and Roseboom, 1979), al.,1979),  1978).  Kwong e t  Abbott  et  (Loscher,  1981a), and  Kawai,  al.,  derivatives.  Unruh  et  al., and  1980),  methyl  ester  1983),  simultaneous  trimethylsilyl 4-ene  (Gupta  et  1983) The  capture  or  phenacyl detection  1980).  The GCMS methods i n c l u d e the (von  (Hulshoff  phenacyl  (Nishioka 1982)  1980;  The drug has a l s o been  e s t e r of VPA has a l s o been analyzed by GC with e l e c t r o n (Chan,  al.,  ( C a l e n d r i l l o and Reynoso, 1980), butyl  trimethyl s i l y l  (  most common method. VPA  ( L o s c h e r , 1977;  and C l a r k e ,  hexafluoroisopropyl  t-butyl dimethylsilyl  the  VPA as  chemical  ionization  (Granneman e t (Balkon,  al.,  1979)  identification  t-butyl dimethylsilyl analysis  derivatives their  quantitation  of  al.,  trimethylsilyl (CI)  of  eight  1981)  ester  a CI  and a d i r e c t  of  its  et  et  al.,  the al.,  metabolites  (Rettenmeier  GCMS method f o r  ester  using  (Acheampong  and q u a n t i t a t i o n  GCMS assay of the ethyl  1984a),  methyl  VPA m e t a b o l i t e s  derivatives  VPA and  (Nau e t  of VPA as i t s  using  of 2 - 3 -  and  1986b).  A  e s t e r s of VPA m e t a b o l i t e s the  determination  i n s e r t i o n CI method ( S c h i e r e t a l . ,  of VPA  1980)  have  a l s o been r e p o r t e d . The most complete GCMS assay i s enables single  the  simultaneous  chromatographic  monitoring  of  derivatives sensitive  of  and  the the  determination run.  electron drug  specific.  that  and  This  assay  impact its  However,  of  of  Abbott  et  al.  VPA and  12  metabolites  is  based  ionization  metabolites. the  large  of The  number  upon  (1986a)  selected  which in  a  ion  t - b u t y l dimethyl s i l y l assay of  is  relatively  VPA m e t a b o l i t e s ,  -  some  of which  implicated fatty  in  are p r e s e n t  13 -  at  very  hepatotoxicity),  acids,  make  the  low c o n c e n t r a t i o n s  (especially  the p o s s i b l e i n t e r f e r e n c e  search  for  an even  more  those  of endogeneous  sensitive  and s p e c i f i c  method of a n a l y s i s n e c e s s a r y . The above mentioned assay of Abbott e t a l . has a lower l i m i t of d e t e c t i o n metabolites frequently In  near the lower d e t e c t i o n  this  work  a highly  i t s metabolites  on  technique  GCMS.  Because  of of  The  specificity  The be  nature  ionization  in s e n s i t i v i t y would  of n e g a t i v e  overemphasized.  metabolism  of  and more  capture  be  Such  enhanced  i o n chemical  in  In using  a  i o n chemical  number  negative  of  for  the  derivatives.  f o r VPA  laboratories  i o n chemical  determination  The technique  one  order  of  method was e x p e c t e d . because  of  the  soft  ionization.  be v a l u a b l e  small  ionization  animals  method cannot  i n the study  in  order  of i n t e r m e d i a r y  to  of the  elucidate  metabolites  which  of VPA t h e r a p y .  ionization  there  ionization  w i t h GC f o r the a n a l y s i s of c e r t a i n true  least  and s p e c i f i c a n a l y t i c a l  a method w i l l  VPA m e t a b o l i t e s  at  any c u r r e n t  further  pathways and i n the d e t e c t i o n  Negative  analysis  i o n chemical  technique,  may be r e s p o n s i b l e f o r the h e p a t o t o x i c i t y  G.  specific  negative  over  need f o r a more s e n s i t i v e  metabolic  of some VPA  was to be d e v e l o p e d . The method was to be based  this  increase  ionization  The serum l e v e l s  limits.  sensitive  electron  magnitude GCMS  ng/ml.  (4-ene VPA, 3-ene VPA, 2 , 4 - d i e n e VPA, 5-0H VPA, 4-OH VPA) are  and a l l the  of 0.1  of  has been (NICI)  fatty  mass  acids.  prostanoids  a  as  recent  interest  spectrometry  in  coupled  T h i s has been e s p e c i a l l y their  of NICI mass spectrometry  pentafluorobenzyl  is a relatively  new  -  technique  and has been  o n l y d u r i n g the l a s t 1.  Ion  forming  Under  negative mass  in  reactions  the  ions  p r e s s u r e s of  ways (Dougherty, a.  range  (Hunt about  1981;  et  iii  pair  al.,  Ion-pair  Anion/molecule  Negative  CI  and  problems  to  5  10"  and  torr,  formation  7  mechanism and Under  negative  ions  CI can  is  source  dominated  conditions, be  produced  of  by  low  i.e.  at  in  two  reactions capture AB"  »•  (<0.1eV)  capture  A- + B"  (0-15eV)  formation *>  A" + B  +  + e  -  (>10eV)  reactions *  ion reagent  ABC"  or  in  (AB-H)" + HC  gas systems systems  substances play a s i m i l a r result  electrons,  1985):  B r o n s t e d - b a s e reagent  These reagent  and a n a l y t i c a l  70eV  1976 ).  Dissociative electron  AB + C"  positive  ion  •  AB + e"  a.  10"  Watson,  AB + e"  2.  of  1 torr,  AB + e"  b.  conditions,  Resonance e l e c t r o n  ii  structural  in NICI  EI  Electron/molecule i  solve  decade.  i o n s o c c u r s by the  fragment  source  used to  conventional  pressures  14 -  ion/molecule  r o l e as the reactions.  reagent  The  gases  in  Bronsted-base  -  reagent F"  systems  and C T  o r adduct b.  include  (Harrison,  1983).  E l e c t r o n capture  H",  NH ",  They  react  OH ,  reagent  o f near-thermal  source pressure the s i m p l e s t  either  is  where  the  type  reagent  energy  the  negative  generally  electron  capture  of  1983).  of process which l e a d s gas a c t s  only  With  a high  to negative ion  as a moderating  gas to  energy e l e c t r o n s which are captured  collisional  The reagent  stabilization  of  the  gas can a l s o newly  of compounds that  formed  agents  high NICI  (Dougherty,  sensitivity  1981).  agent  base  with  and amines)  derivatization that  endows  aromatic  of  the  the  molecule  amines),  with  with  reagent  anhydride  Pentafluorobenzyl  electron  a  suitable  positive  electron  pentafluorobenzoyl  and t e t r a f l u o r o p h t h a l i c  employed as the d e r i v a t i z i n g  molecule  such as p e n t a f l u o r o b e n z a l d e h y d e  been used f o r NICI mass s p e c t r o m e t r y .  and p r o s t a g l a n d i n s .  Positive  are observed f o r many halogenated compounds, quinones  i s possible. Derivatives  Schiff  intrinsically  o x i d i z i n g and a l k y l a t i n g  capability,  affinity  have  compounds (Howe e t a l . , 1981). For molecules which lack  derivatizing  phenols  capacity  affinities  and n i t r o  a  abstraction  i o n s ( H a s s , 1980).  Classes are  CH3O-,  T  by proton  (Harrison,  by sample molecules with some e l e c t r o n a f f i n i t y . in  02 ,  T  systems  produce a high p o p u l a t i o n o f thermal  act  0 ,  -  2  o f e l e c t r o n s by a molecule i s a resonance process which  electrons  formation  -  formation.  The capture requires  15  (for  (to form  halide  (for  amines)  have  bromide has been  f o r NICI GCMS a n a l y s i s of f a t t y a c i d s  -  3.  Sensitivity  It  16  -  of NICI  can be shown t h a t the  ion c u r r e n t s o b t a i n e d i n CI are g e n e r a l l y  i n t e n s e as those observed i n EI current is  may be c o n c e n t r a t e d  dependent upon k,  reactions  will  Inefficient will  the  show  have lower Compounds  in  a few  or  of  equal  reactions  1983).  ions.  rate constant  better  ionization  (Harrison,  i.e.  The the  Furthermore,  sensitivity CI  those  of  reaction.  sensitivities with  the CI CI  systems  those  smaller  rate  of  affinity  that  are  amenable  to  electron  of  in  the  that,  and l a r g e  electron.  when  cross  This  a molecule section  for  capture  sensitivity 4.  found with other  F a c t o r s which determine  can  as  capture  ionization the  result  possesses electron  spectrum which depends upon e l e c t r o n the  EI.  sensitivities.  mobility  sensitivity  CI  constants  opposed  to  molecule r e a c t i o n s i n NICI can have high r a t e c o n s t a n t s as a r e s u l t high  ion  Efficient  to  as  both  in  the  can e x h i b i t  of  the  extraordinary  a positive  capture,  ion/  electron  negative  up to 100  ion  times  techniques.  sensitivity  of sample  detection  i n NICI. The formation dent  upon  the  of n e g a t i v e  electron  electrons effecting i o n s with n e u t r a l s can be d e t e c t e d , and e x c i t e d 1985). there  anions  addition, detachment  be the  an  ionization  (Dougherty,  can  be  of  the  and the  analyte,  depends upon the  energy  of  the  with which a sample  e x t e n t to which newly  processes can a l s o l e a d to e l e c t r o n e j e c t i o n  and hence  source  reagent  formed  (Chapman,  optimal  by  the  degree of c o l l i s i o n of molecule  1981). The s e n s i t i v i t y  stabilized  i s s t r o n g l y depen-  gas molecules  relative  will  affinity  therefore,  C o l l i sional will  the  i o n s by e l e c t r o n capture  pressure  for  importance of c o l l i s i o n a l  depend  upon  the  internal  maximum  sensitivity.  stabilization  energy  of  the  to  In  electron  reagent  gas  -  molecules  and  temperature as  hence  not  results  be maintained  as  s i n c e the number of response  will  be  Furthermore,  solvents  source and t h i s  -  sensitivity  (Hass, 1980).  halogenated  17  can in  electron  deplete  a drop  the  in  concentration  thermal  electrons  The  sensitive level  main  objective  electrons High  substrate  in  such  the  ion  sensitivities  can  molecules  ion source i s  because n o n - l i n e a r  of  and s p e c i f i c method f o r  VPA and i t s  and  negative  ion  metabolites.  increases  finite.  Linear  response may  start  1984).  chemical  2. (total  The a n a l y t i c a l  the  interaction  ionization  carbamazepine utility  of  on the  free  between  GCMS  to  develop  a  and q u a n t i t a t i o n of  halogenated  were  to  be  to  be used to  highly  of  trace  derivatives  evaluated  healthy  measure VPA i n  v o l u n t e e r s who  VPA and carbamazepine.  fraction  measuring s a l i v a r y  was  for  method.  method was  study  study  detection  and f r e e ) and i n s a l i v a i n f i v e  a drug  study  this  The s u i t a b i l i t y  a c h i e v i n g the d e s i r e d a n a l y t i c a l  of  VPA was  concentration  to  be  participated  The e f f e c t  determined  of VPA i n  serum  a drug  and  of the  interaction  evaluated. 3.  ion  source  Objectives  1.  in  of  by  absorbing i m p u r i t i e s  thermal  i n the  as low as 10 ng i n some cases ( S t o u t ,  H.  influenced  sensitivity.  the  ranges must be determined  strongly  A search f o r  technique  derivatives) derivatives) metabolites standards.  and and  new VPA m e t a b o l i t e s  employing negative  both ion  mass s p e c t r o m e t r y . were  to  be  electron chemical  Potential  synthesized  (as  was to  be done using the  impact  (t-butyldimethylsilyl  ionization  (pentafluorobenzyl  new VPA m e t a b o l i t e s required)  twin  for  use  as  and  known  reference  -  II.  A.  18 -  EXPERIMENTAL  Chemicals and M a t e r i a l s . 1.  General Chemicals were  reagent  grade  and o b t a i n e d  from  the  following  sources. a.  A l d r i c h Chemical C o . (Milwaukee, W i s c o n s i n ) : 3,5-Bis(trifluoromethyl)benzyl etherate, (1.6  M  bromide,  t-Butyldimethyl s i l y l in  hexane),  Boron  chloride,  Calcium  n-Butyllithium  hydride,  18-Crown-6,  2,3-Dichloro-5,6-dicyano-l,4-benzoquinone, amine,  D i i s o p r o p y l e t h y l amine,  1,2-Ethanedithiol, aluminium acid,  b.  chloride,  oil),  (35%  Sodium  Tetrahydrofuran,  dispersion hydride  in  (50%  Pentanoic  mineral  oil),  dispersion  in  T r i e t h y l amine.  bromide  BDH Chemicals ( T o r o n t o , Acetone, Ether Sodium  Acetonitrile,  (anhydrous), hydroxide,  Ontario): Benzene,  Citric  Hydrochloric  acid,  Sodium  sulfate  acid. d.  chloride,  Lithium  A l f a P r o d u c t s (Danvers, M a s s a c h u s e t t s ) : Pentafluorobenzyl  c.  4-Dimethylaminopyridine,  Methanesulfonyl  Potassium hydride  mineral  Di i s o p r o p y l -  Isopropylcyclohexylamine,  hydride,  Propionyl  trifluoride  B r i t i s h Drug House ( P o o l e , Iodoethane,  Pyridine.  U.K.):  acid  (anhydrous),  Potassium  (anhydrous),  iodide, Sulfuric  -  e.  19  Caledon L a b o r a t o r i e s L t d . Dichloromethane,  f.  E t h a n o l , Ethyl  acetoacetate,  Mallinkrodt  i.  New Y o r k ) :  4-0xopentanoic  acid,  Propionaldehyde.  New J e r s e y ) :  Cadmium c a r b o n a t e .  Chemicals ( S t .  Potassium carbonate  Ontario):  acetate.  Fisher S c i e n t i f i c Co. (Fairlawn, Bromine, t - B u t a n o l ,  h.  (Georgetown,  Eastman Kodak Co. ( R o c h e s t e r , Ethyl  g.  -  Louis,  (anhydrous),  Missouri): Sodium b i c a r b o n a t e .  Matheson Coleman and B e l l C o . (Norward, Chlorotrimethylsilane,  Phosphorus  Ohio):  tribromide,  2,4,6-Tri-  methylpyridine. j.  N i c h o l s Chemical Company ( M o n t r e a l , Mercuric  k.  Canada):  chloride,  P i e r c e Chemical Company ( R o c k f o r d ,  Illinois):  N-Methyl-N-trimethylsilyltrifluoroacetamide. 2.  VPA m e t a b o l i t e s  Di-n-propyl acetic Inc. acid  K+K L a b s . (OA)  (Cleveland, [^J-VPA metabolites tory  was  acid  (Plainview, purchased  Ohio). has  and i n t e r n a l  The  been  (VPA)  was  New Y o r k ) . from  reported  obtained The  the  These m e t a b o l i t e s  Biochemicals  standard  internal et  al.,  standards were obtained  s y n t h e s i s has been p u b l i s h e d  ICN  Biochemicals  other  (Acheampong  from  internal  Nutritional  s y n t h e s i s of  used as r e f e r e n c e  and t h e i r  standards  octanoic  Corporation  standard 1984).  The  from t h i s  (Acheampong et  used,  al.,  VPA  labora1983).  i n c l u d e d 4-ene VPA, 3-ene VPA, 2-ene VPA, 4-OH VPA,  5-OH VPA, 2 - p r o p y l g l u t a r i c  a c i d and 2 - p r o p y l s u c c i n i c a c i d .  s i s of 2 , 4 - d i e n e VPA and 2 , 3 ' - d i e n e VPA w i l l  be r e p o r t e d  The synthe-  elsewhere.  -  B.  20  -  Instrumentation  1.  Gas Chromatography Mass Spectrometry a.  Capillary  Capillary 5987A  column GCMS a n a l y s i s was performed on a  gas-chromatograph  Electron ion  Column GCMS  impact  source  mass  spectrometer  s p e c t r a were obtained  pressure  of  1.8  x  IO  with  at  electron  Torr.  - 6  an  EI  Hewlett-Packard  RTE-6 data energy  GCMS  of  system.  70eV,  analysis  and  of  the  t-BDMS d e r i v a t i v e s was done under the f o l l o w i n g c o n d i t i o n s : OV-1701 bonded phase column, 25 m x 0.32 of  0.25  u  temperature,  (Quadrex  240°C;  temperature,  of  the  was  reagent  pressure  50°C  to  at  1  and the mode of  Ionization  oven  100°C to 260°C at 8 ° / m i n ; 250°C;  1 mL/min.  the  source  injection  Negative  instrument  30°/min;  was  conditions 140°C  to  argon-methane;  250°C;  injection  r a t e of  port  ion chemical  1 mL/min.  tuned.  Source  were:  oven  250°C  source  port  value  at  5°/min;  temperature,  temparture,  One uL o f  240°C;  sample was  i n j e c t i o n was s p l i t l e s s .  and  end  products  monitored using a H e w l e t t - P a c k a r d Varian  Connecticut);  Packed Column GCMS  Intermediates  a  thickness  120-170eV depending upon the  ammonia,  a flow  Haven,  Operating  at  interface,  g a s , helium at  b.  rate,  torr.  with a f i l m  interface,  which  140°C  methane,  open s p l i t  injected  split  helium flow  about  gases,  carrier  open  s p e c t r a were recorded at  temperature,  200°C;  240°C;  source  pressure  New  50°C to 100°C at 3 0 ° / m i n ,  temperature,  ionization  Scientific,  mm I.D.  Mat-Ill energy  mass was  and  the  synthetic  reactions  were  5700A gas chromatograph i n t e r f a c e d  spectrometer 70eV  of  via  source  a  variable pressure  slit 5  x  to  separator. 10~6  torr.  -  Scanning  range  seconds.  was  15-750  conditions:  300  100/200  on  GC -  PFB  capture  capillary  20°C/min  to  (column),  of  The  IR  every  system.  with 2% Dexsil  Inc.,  Bellefonte,  50°C,  rate  8°C/min  to  octanoic  acids  were  analyzed  by  Detection and  Temperature min  at  gas chromatograph,  program:  240°C.  150°C  ( h o l d 4 min)  Argon/methane  Column: the  modified  same as i n  flow:  6  then  mL/min  la.  Instruments  film  on  ethyl  2-propyl-4-oxopentanoate  sodium  chloride  disks  using  was a  obtained  Unicam  Proton NMR s p e c t r a were recorded on Bruker WP-80  Nicolet  instruments  Oxford-270 U.B.C.  NMR  at  solvent  the NMR f a c i l i t y was  and  CDCI3  in  and  the Department  the  internal  as  SP-1000  spectrophotometer.  Chemistry,  5  initial  using a HP-5840A  hold 3  spectrum of  liquid  taken  packed  (Supelco,  program:  40 mL/min ( d e t e c t o r ) . Other  scan  V a r i a n 620L computer  m x 2 mm I.D)  valproic  detection  240°C,  one  270°C.  column u s e .  3.  neat  with  Supelcoport  E l e c t r o n Capture  derivatives  electron  a  units  (1.8  Temperature  hold 5 min at  2.  column  mesh  Pennsylvania).  for  mass  Data was processed by an o n - l i n e  Operating  270°C,  21 -  of  standard  tetramethylsilane.  C.  Human Study  The  blood,  identification study between the  M.Sc.  volunteers  urine  and  and q u a n t i t a t i o n carbamazepine  graduate  research  participated  saliva  of VPA were  (CBZ) of  i n the  samples part  used of  for  metabolite  a drug  interaction  and VPA and were c o l l e c t e d  Sukhbinder  Panesar.  study. V o l u n t e e r s  Five  as p a r t  healthy  r e c e i v e d an average  of  male of  -  16.4  mg/kg/day  equal  doses,  of VPA i n  eased to 200 mg f o r received  syrup  one at 8 a.m.  100 mg CBZ twice d a i l y  six  22 -  form.  and the  The drug was administered  other 8 p.m.  was added to  the  doses of  700  two  the  study  dosing regimen which was  incr-  the evening dose on day 16. mg [ H6]-VPA  On day 9 o f  in  One v o l u n t e e r ,  on days 8 ,  2  9,  25,  FA, a l s o  26  (twice)  and 2 7 . Blood was c o l l e c t e d to  the  0.5,  morning  1,  The  1 .5,  2,  samples  dose  of  2.5,  3,  were  in  days 7 5,  7,  Urine  convenient  and a l s o  and a homogenous a l i q u o t Saliva  9, 12, to  samples  after  24,  30,  clot were  an  overnight  and  serum  collected  overnight.  vacutainers fast  36 and 48h a f t e r  Total  in  urine  prior and  the dose.  obtained 2h  at  after  blocks,  volume was  other  recorded  saved.  samples were c o l l e c t e d f o l l o w i n g  acid solution the c i t r i c  non-heparinized  and 23  allowed  centrifugation. blocks  sterile,  and were taken simultaneous  a c i d s o l u t i o n was held i n  stimulation  to  with 5% c i t r i c  blood samples. Four mL o f  the mouth f o r 2 min and spat  out.  The s a l i v a sample (3 t o 5 mL) was then c o l l e c t e d a f t e r 2 m i n . The pH o f the  saliva  However,  during  procedure for  the  a number  -20°C  D.  samples  until  the  was  not  measured  collection  of  blank  pH was measured and found of  such s a m p l i n g s . A l l  immediately  to  saliva  after following  be c o n s t a n t  biological  collection.  at  samples were  the  same  around  7.3  stored  at  analyzed.  A n a l y s i s of Samples  1.  Serum and S a l i v a Standards Stock  solutions  of  VPA (500  ug/mL),  OA (250  ug/mL),  and  [ H ]-VPA 2  6  -  (24  Lig/mL) i n water were made by d i l u t i n g  above  substances  calibration  in  methanol  curves  drug  s o l u t i o n to g i v e e i t h e r assay VPA  23 -  the a p p r o p r i a t e in  serum  to  concentrations  free  provide  in a final  24,  ug/mL VPA standard  saliva.  18,  standards  determine  6,  4,  7.5,  5,  2.5,  a 5  Lig/mL VPA  F o r EI  analysis  volume  from the  and 15 ug/mL VPA  prepared  Lig/mL  or  by  0.5  pipetting  Lig/mL  the  appropriate  VPA standard  in  blank  p o i n t s prepared were 3 , 1 . 5 , 1, 0 . 5 , 0 . 2 5 , 0 . 1 ,  serum  1,  F o r the NICI  and 3  the r e q u i r e d  free  volume o f 1 mL o f s a l i v a .  VPA c o n c e n t r a t i o n s ,  and 2.5 ug/mL i n water were prepared and a l i q u o t s 10,  of VPA  the 30 o r 10 ug/mL  90, 7 5 , 6 0 , 4 5 , 3 0 ,  and 0.05 Lig/mL c o n t a i n e d a f i n a l To  9,  total  volume of 100 uL o f serum.  were  The c a l i b r a t i o n  12,  serum  an a l i q u o t  from e i t h e r  by p i p e t t i n g  to g i v e  concentrations in a final  either  to  For  volume o f 100 \xl o f serum.  150  from  water.  serum was added  volume was taken  standards were prepared  volumes  distilled  150, 30 or 10 ug/mL VPA i n serum.  the  Saliva  with  c o n c e n t r a t e d s o l u t i o n s o f the  and 0.5  ug/mL  VPA s o l u t i o n s taken  concentrations  of 25  to p r o v i d e 15,  i n water.  Final  volume was 100 l i L . For  purposes o f  standards serum  investigating  o f VPA over  were  assayed  the p r e c i s i o n  the c o n c e n t r a t i o n  three  times  on  range  the  day  of  the NICI  of 800 t o of  the  assay,  10 ng/mL  preparation  in of  s t a n d a r d s and repeated on days 4 and 7 . Then the standard d e v i a t i o n s o f the  s l o p e s and  the c o e f f i c i e n t  of v a r i a t i o n  at each c a l i b r a t i o n  point  were c a l c u l a t e d . To saliva  determine samples  standard determined  the r e c o v e r y  spiked  curves using a  were  with  o f VPA i n serum and s a l i v a ,  the  prepared.  same  amounts  These  of  serum and  VPA used  concentrations  standard curve of VPA prepared i n water.  for  were  the then  -  and  For  the  NICI assay  m/z  149  of  the  24 -  the  peaks at  PFB e s t e r  of  m/z 143  [ He]-VPA  of  known  the  area  ratio  concentration  each  serum  analysis.  or  of  of  saliva  VPA to  that  VPA i n  serum or  sample  was  of  the PFB e s t e r  (or  2  e s t e r of OA) were m o n i t o r e d . The c a l i b r a t i o n plot  of  m/z  143  of VPA  of  the  PFB  curves were o b t a i n e d by a  [ H6l-VPA  or OA versus  2  saliva.  obtained  The  using  concentration linear  A new standard curve was prepared p r i o r  the  to  of  regression  the  run of each  b a t c h of serum or s a l i v a samples. 2.  Sample  preparation  To measure serum t o t a l and d i l u t e d  five  times  with  sample was t r a n s f e r r e d  into  standard  a  (40  uL  from  VPA l e v e l s 100 blank  serum.  a 3.5  24  uL o f  mL  serum sample was taken  Then 100  uL o f  the  diluted  screw cap septum v i a l .  ug/ml s o l u t i o n  of  [ H6]-VPA) 2  Internal was  added  f o l l o w e d by 3N NaOH to make the pH 12-13. The samples were subsequently heated  for  1 hour at 6 0 ° C .  was brought  c o o l i n g to  down to 2 u s i n g 4N HC1 and the  room temperature  f o r 10 m i n u t e s .  500  uL o f e t h y l  the  recovery of the  500  uL o f  vial  After  acetate  ethyl  acetate.  containing  volume  derivatized Saliva the  reduced to  extraction  step was repeated with  layer  was  vortexed  2  to g i v e e i t h e r  then  and  uL under N . 2  The  of pH  internal of  the  another  transferred  centrifuged  at  to another v i a l  Finally  the  with  to  a  2000 and  sample was  PFB or t-BDMS d e r i v a t i v e s .  samples were prepared by t a k i n g  addition  solution).  200  at  f o r 20 m i n u t e s . To i n c r e a s e  Na S04,  about  sit  rotation  rpm f o r 20 m i n u t e s . The supernatant was t r a n s f e r r e d the  to  pH  samples were e x t r a c t e d  The o r g a n i c  anhydrous  samples allowed  the  Then the  by g e n t l e  drug the  room temperature  standard samples  (30  was  1 mL o f uL  of  adjusted  12 to  sample f o l l o w e d by ug/mL about  [ He]-VPA 2  2  and  the  -  samples  were  centrifugation  25 -  extracted  with  to  the  break  3  mL  ethyl  emulsion,  acetate  were  and  treated  following  as  for  serum  samples. For serum  the  was  determination  centrifuged  ultrafiltrate extraction  was  was  and  then  done  of  free  100  treated  once  uL  To  the  and  that  ultrafiltrate  as  40  serum  taken.  samples  of  ul  12  1 mL o f The  except  ug/mL  that  [^Hsl-VPA  standard.  Derivatization form  serum or vial  of  exactly  s o l u t i o n was used as an i n t e r n a l 3.  serum VPA c o n c e n t r a t i o n ,  the  pentafluorobenzyl  saliva extract  (PFB)  was t r a n s f e r r e d  derivative into  the  concentrated  a 1 mL c o n i c a l  reaction  and 10 ul o f d i i s o p r o p y l e t h y l amine (neat) was added f o l l o w e d by 10  uL o f  30% p e n t a f l uorobenzyl  bromide  (PFBB)  The sample was then heated i n a h e a t i n g Samples f o r EI adding 50 at 60°C prepared  a n a l y s i s were  uL o f  for  t-BDMSCl  in  block  derivatized pyridine  uL o f  in  ethyl  acetate.  f o r 45 minutes  at  40°C.  to g i v e t-BDMS d e r i v a t i v e s  ( c o n t a i n i n g 5% DMAP) and  4 h o u r s . The TMS d e r i v a t i v e s  by adding 50  solution  of  MSTFA reagent  urinary  heating  metabolites  and h e a t i n g  by  at 6 0 ° C  were  for  30  minutes. For internal Ten ethyl and  GC-ECD  standard the of  ul  each  acetate 60  analysis  ul  18-crown-6)  of  VPA  and 10 0.5%  of  the  PFB e s t e r  following derivatization (from  50  ng/mL  uL o f OA (400 PFBB  to  in  added. The r e a c t i o n  room  for  mixture was p i p e t t e d  into  hour,  10  vial  following  another  using  OA  procedure was iig/mL  benzene  a reaction  potassium a c e t a t e  1  to  VPA  VPA)  vial  solution  (containing  was then  vial  allowed  ul  and the  of  the  the  in  acetate) 1.5  mg/mL  and a few c r y s t a l s  which 65  reaction  as  followed.  ng/mL OA s o l u t i o n i n e t h y l  solution  were t r a n s f e r r e d  temperature  of  to  sit  of at  reaction  volume made to  -  200  uL with  complete  ethyl  dryness  26 -  acetate.  The s o l v e n t  and the  residue  was then  evaporated  reconstituted  with  with  500  ui  N  of  2  to  ethyl  acetate. 4.  Serum f r e e  Serum  free  levels  Ultrafiltrationa  levels  centrifuge  which  VPA were  was c a r r i e d  MPS-1 m i c r o p a r t i t i o n  The  of  out with  system  to  20°C  after  ultrafiltration.  YMT u l t r a f i l t r a t i o n  (Amicon  was a Beckman Model  was e q u i l i b r a t e d  determined  Corp.,  J2-21.  before  Danvers,  membranes  in  Massachusetts).  A 4 5 ° angle  rotor  use. Centrifugation  was used  was  carried  out f o r 20 minutes at 3500 rpm. 5.  Comparison  of  the  sensitivity  of  EI(t-BDMS)  with  NICI (PFB,  bis-TFMB) To  compare  the  sensitivity  3,5-bi s(trifluoromethyl)benzyl t-BDMS NICI way  derivative,  analysis.  a  known  (octanoic  acid  concentration  amount  the  of  reaction  standard) vial  weeks  conditions. time.  EI c a l c u l a t e d .  The samples  The mean area  were  ratios  relative  derivatives to  that  was 5  Each in  derivative  such  ug/mL.  run on were  five  taken  different  and the  ratio  EI  and  was prepared  The t-BDMS under  the  i n the same  a way t h a t  two d e r i v a t i v e s  (PFB,  of  for  of VPA was prepared  d e s c r i b e d above.  was run i n the EI mode and the other NICI  fluorinated  VPA was d e r i v a t i z e d  derivative  used as i n t e r n a l in  the  (bis-TFMB))  The bis-TFMB  as the PFB d e r i v a t i v e  of  the  final  derivative both  days  EI and in  of NICI  two to  -  6.  Identification  For  this on  C H6]-VPA  were  way  the  2  that  VPA  drug  and  included  both  up,  TMS,  each  and  derivatives for to  methyl  ester  were  recorded  above  alkaline  treatment  of  peaks were i d e n t i f i e d  had  urine  after  were  either  mass  mode  urine  other  extracts  samples p r i o r  by i n j e c t i n g  synthetic  in  After the  also  to  both  the  the  usual  t-BDMS, of  all  NICI  mode  In  addition  prepared  extraction.  a  analyzed  times  in  of  such  PFB,  derivatives.  were  doses  Samples  retention obtained  the  labelled,  CBZ. give  from  six  selected  of  The  the  given  mainly  to  spectra  for  sample  unlabelled.  derivatized  derivatized the  been  samples were  serum  administration  was  and  one  also  derivatives.  and EI  samples,  and  predominantly  sample  PFB d e r i v a t i v e s , the  or  and  urine  mL  who  metabolites  and u n l a b e l l e d , before  samples  state  Two  labelled  work  E.  urine  steady  used.  -  of VPA m e t a b o l i t e s  purpose  volunteer  27  without  Metabolite  standards.  Chemical S y n t h e s i s  1.  Attempted s y n t h e s i s of 2 - ( 2 ' - o x o p r o p y l ) - 2 - p e n t e n o i c ( 4 ' - k e t o - 2 - e n e VPA) v i a e t h y l a.  S y n t h e s i s of ethyl  Pentanoic a  reflux  device.  acid  condenser Bromine  (20.4 whose (35  g,  acid  2-propyl-4-oxopentanoate.  2-bromopentanoate  g , 0.2 top 0.22  mol) end  was p l a c e d  was  mol)  in  connected  was  added  a 500 to  a  followed  mL f l a s k gas by  with  absorption 1  mL  of  -  phosphorus  tribromide.  bath  at 7 0 ° C  all  the  The mixture  f o r 30 minutes  bromine  distilled  using  bromide.  The  76-79°C/0.03 mm].  28 -  had a  product  mm.  and then  reacted.  water  The  pump  was  [Lit.  was s t i r r e d  in  then  an o i l  f o r 6 hours by which  reaction  mixture  order  to  distilled  m/z 55  with  at 100°C  (Acheampong,  Mass spectrum:(MW=181)  and heated  remove  under  Ph.D.  (100%),  was  subsequently  residual  reduced  thesis)  bp  138(87%),  time  hydrogen  p r e s s u r e . Bp 102°-105°C/2.5  140(85%),  27(78%),  41(63%) 29(61%), 43(34%), 94(32%), 101(25%). 2-Bromopentanoic refluxing (80  a mixture  mL,1.36  mL)  for  washing ethyl mm.  acid of  hours  the  [Lit.  m/z  acid  Dean-Stark with  was  obtained  (Acheampong,  spectrum:(MW=209)  a  mixture  2-bromopentanoate  to  its  (76g,  ethyl 0.42  (150 mL), and c o n c e n t r a t e d  using  reaction  converted  2-bromopentanoic  m o l ) , benzene  12  was  Ph.D.  29(100%),  water  saturated by  thesis) 55(89%),  mol),  sulfuric  separation NaHC03  60-62°C/3.0  166(22%),  by  ethanol  acid  unit.  and  distillation. bp  ester  (1.7 After  water,  Bp  pure  42-45°C/0.2  mm].  Mass  168(20%),  101(12%)  aluminium  hydride)  129(10%), 140(8%), 138(6%). b. To in  S y n t h e s i s of e t h y l anhydrous  THF (100  a 250 mL f l a s k ,  was  added  0.1  mol)  followed over  15 m i n u t e s , drop  and the  mL)  (dried  sodium h y d r i d e by  a period  ethyl  2-propyl-4-oxopentanoate  dropwise  (5.76  addition  o f 30 m i n u t e s .  2-bromopentanoate  solution  refluxed  with  for 6  g (50% d i s p e r s i o n ) , of  After  (20.9  lithium  ethyl  hours.  acetoacetate  stirring  g , 0.1  0.12  for  an  mol)  (13  g,  additional  mol) was added drop by Distilled  water  (40 mL)  -  was  then  added and the  29 -  resulting  mixture  filtered  under  s u c t i o n . The  o r g a n i c l a y e r was separated and the aqueous phase e x t r a c t e d with  ether.  Na2S04 give  The  and the ethyl  spectrum:  combined  ether  organic  evaporated.  layer  was  The crude  2-propyl-3-acetyl succinate.  dried  product Bp  three  over  times  anhydrous  was d i s t i l l e d  118°C/0.2  mm.  to  Mass  (MW=258) m/z 43(100%), 129(40%), 97(35%), 174(32%), 115(26%),  143(18%), 185(12%), 213(11%). To  obtain  acylsuccinate was  heated  extracted Na2S04-  small  2-propyl-4-oxopentanoic  under  three  times  give of  for  al.,  1983)  8  hours.  with e t h e r  solvent  to  amount  reflux  was  The  a  mixture  resulting  and the e x t r a c t removed  and  2-propyl-4-oxopentanoic  acid  ethyl  then  over  the  crude  refluxing  acid.  bp 1 3 3 - 1 3 6 ° C / 2 . 5  corresponding  ethyl  18-crown-6  (0.3  2-propyl-4-oxopentanoic  acid  THF f o r 6 h o u r s . The mixture was then  the  Mass  fractionated  4-oxopentanoate esterification  which  ester g),  of  ethyl  (0.79  g , 0.005  was f i l t e r e d  by d i s t i l l a t i o n showed  reaction  a single  was a l s o  above  iodide  mol)  a  gave  (MW=158)  m/z  158(1%).  acid  (1.56  was  g,  made  0.01  and K 2 C O 3  (4  by  mol), g)  in  and THF removed. The r e s i d u e  and y i e l d e d peak  carried  with  [ L i t . (Acheampong  spectrum:  the  product  Redistillation  Bp 1 1 5 ° C / 0 . 0 7 mm.  mm].  was  anhydrous  contaminated  43(100%), 58(19%), 101(16%), 83(15%), 73(14%), 140(8%), The  of  mixture  dried  2-propyl-4-oxopentanoate.  pure 2 - p r o p y l - 4 - o x o p e n t a n o i c et  acid  ( 9 . 9 g , 0.04 mol) and c o n c e n t r a t e d HC1 (40 mL, 0 . 4 mol)  The  distilled  the  pure e t h y l  2-propyl-  upon GCMS a n a l y s i s .  out  in  a similar  manner  The to  -  t h a t of ethyl Mass  30  -  2-bromopentanoate.  spectrum:  (MW=186)  m/z  Bp 9 7 ° C / 1 . 7 mm.  43(100%),  129(32%),  101(30%),  141(16%),  29(14%), 73(12%). IR  Spectrum  (neat  film):  2900  cm"l  (0-CH CH ), 2  1735  3  cm"  1  (C=0). NKR Spectrum:  0.9(t,3H,CH -CH2-);1.1-1.6(m,4H,CH2-CH2); 3  1.2(t,3H,-CH );  2.l(s,3H,CH -C0),  3  3  2.3-3(m,3H,CH -CH); 2  4.2(q,2H,0CH ). 2  c.  Introduction  of  0-TMS d i a l k y l  the  double  bond by  acetal  of e t h y l  ketene  dehydrogenation  diisopropylamide  n-butyllithium 0.022 mol) then  (13.5  in  THF (25  cooled in  oxopentanoate 60  min,  added  (1.86  following  dropwise  and  the  0.022  mL)  a dry  ice  at  which  over  under  mixture  bath  to  the  After and  silyl  ether  d i l u t i n g with e t h e r  the  organic extract  period.  -78°C  period.  stirred  (DDQ)  The  for  with  5  g,  aqueous phase  the  the  mL 0.01  (2.95  mL, was  2-propyl-4-  three  of  times  was  dry  mol)  was  allowed  Then  benzene  mixture for  to  distilled  dissolved  in  stirred  for  mol)  benzene.  was  mL)  (1  mixture  0.036  The THF was  10 min t h i s  r e a c t i o n mixture  extracted  g,  temperature  60 m i n .  (2.27  and  N2  dripping  The mixture  and e t h y l  (3.9  2,4,6-trimethylpyridine  under  by  diisopropylamine  to  added dropwise to the DDQ s o l u t i o n . A f t e r to  prepared  added dropwise and allowed to r e a c t  reconstituted  N 2 and  was  chlorotrimethylsilane  dichlorodicyanobenzoquinone benzene  with TMS.  0°C over 20 min  mol)  a 10 min  residue  mol),  acetone  g,0.01  a t t a i n 25°C and the off  mL,  (LDA)  the  2-propyl-4-oxopentano-  a t e with the keto f u n c t i o n p r o t e c t e d Lithium  of  in was  was added 2  hours.  was washed with 1 M NaOH with  ether.  The combined  l a y e r was washed s u c c e s s i v e l y with HC1, NaOH, and water and the dried  over  anhydrous  Na2S04-  GCMS  analysis  of  the  crude  -  product the  indicated  starting  the  presence o f  material.  The other  2-propyl-4-oxopentanoate: 185(55%),  130(44%),  215(8%),  31 -  spectrum:  137(18%),  two were:  97(20%),  258(3%).  2-propyl-4-oxo-2-pentenoate Mass  (MW=256)  109(12%),  components. One o f 1) TMS enol  these was  ether  of  ethyl  Mass spectrum:(MW=258) m/z 73(100%), 75(65%),  115(42%)  243(5%),  three  2) or  m/z  45(18%),  TMS  enol  ethyl  ether  143(15%),  of  the  ethyl  2-(2'-oxopropyl)-2-pentenoate.  73(100%),  183(9%),  213(10%),  167(7%),  75(40%),  95(28%),  43(25%),  213(10%),  227(5%),  256(4%),  241(3%). A small  portion  of the r e a c t i o n mixture  w i t h NaOH and s t i r r e d effect  f o r three  days  at  was taken  room temperature  h y d r o l y s i s of both the TMS enol e t h e r  product.  The mixture  Following  the e v a p o r a t i o n  derivatized capillary  was  to  give  GCMS.  the  then of  acidified  the  t-BDMS  Two peaks  were  ether  and made  derivatives  of  which  with  to  e s t e r of the  extracted  a portion  detected  i n order  and the ethyl and  alkaline  with  the  were  ether.  r e s i d u e was analyzed  mass m/z 213  by  (M-57)  +  and had s i m i l a r mass s p e c t r a . 2.  Synthesis of 2-(2'-oxopropyl)-2-pentenoic a protected 4-oxopentanoic a.  fractional  available  distillation  similar  4-oxopentanoate (dried  to  (11.5  4-oxopentanoic  and was converted  that g,  described 0.08  over c a l c i u m hydride)  mol)  stirred  at  room  for  acid  into  was  its  2-bromopentanoic  was d i s s o l v e d  in  overnight  ester acid.  by in a  Ethyl  dichloromethane 1,2-ethanedithiol  etherate.  and 100  ketal  purified  ethyl  and 10 mL (0.12 mol) of  temperature  with  acid  was added f o l l o w e d by 2 mL o f boron t r i f l u o r i d e was  starting  P r o t e c t i o n of 4 - o x o p e n t a n o i c a c i d through a d i t h i o  Commercially  manner  acid  The s o l u t i o n  mL o f  5% sodium  -  h y d r o x i d e added.  The  and  MgSO^.  dried  over  distillation  of  32  organic  layer  spectrum:  was  Evaporation  the  residue  4-ethylenethioketalpentanoate. Mass  -  (Mw=220)  separated  of  the  and washed with  solvent  afforded  15g  and  water  subsequent  (85%)  of  ethyl  Bp 1 1 2 ° C / 0 . 5 mm.  m/z  119(100%),  59(20%),  175(17%),  29(15%),  61(14%), 87(13%), 115(13%), 220(12%), 87(10%), 205(2%). NMR Spectrum:  6 1.3(t,3H,-CH >;  1.8(s ,3H , C H g - ) ;  3  2.6(t,2H,CH -CH -C0); 2  b.  S y n t h e s i s of  2.25(t,2H,_CH_ -CH -C0); 2  3.3-3.4(m,4H,CH -CH -S);  2  2  2  4.7(q,2H,0CH ).  2  2  4-carboethoxy-2-ethylenethioketal-  5-hydroxyheptane To added  diisopropylamine dropwise  stirred  for  pentanoate  15  min  (11  g,  for  and the  reaction  60  was  (Na2S04). under liquid)  mol) Then  washed  15%  0.06  (38  cooled  mixture  with  After  and  0.05  min.  quenched  extract  mL,  n-butyllithium  stirred  then  (8.2  to  in  mol)  mL, -78°C  THF was  0.06 and  HC1  with  evaporation  to  and  stir  and  the  reduced p r e s s u r e and a f f o r d e d 6  mL THF  mol).  The  dropwise  2.5  saturated  ether g of  the the  the  mol)  was was  ether.  was  The  NaHC03  product  mixture added  The mixture  residue  which was shown to be homogeneous by GCMS.  0°C  mixture  and  hours.  with  at  4-ethylenethioketal-  mL, 0.055  extracted  water of  (4  for  100  ethyl  added  propionaldehyde  allowed  in  was  etheral  and  dried  was  distilled  (thick  yellowish  -  Mass  spectrum:(MW=278)  43(25%),  111(22%),  33 -  m/z  119(100%),  61(21%),  139(20%),  29(35%),  59(30%),  185(27%),  120(15%),  121(14%),  220(10%),  159(8%), 186(7%), 205(4%), 233(2%), 278(1%). c.  Synthesis of  Dehydration was  carried  Hydroxy  of  out  dichloromethane  (40  (1.6  mol)  mixture and  0°C  stirred  the  taken  0.02  and the  following  g , 0.015  reaction  (Na2S04).  GCMS  of  peaks  in  dichloromethane  was  by  the  analysis in  flash  Mass  spectrum:  (Mw=260)  127(100%),  29(58%),  with  added  74(15%),  0.03 at  showed to  these  mol)  room  ether five  the  then at  temperature  neutralized  with  and peaks.  two  with dried Two  geometric  isomers  being  the  identified.  retention  132(22%),  off  g,  was  product  filtered was  hours  of  was  the  The mesylate  extracted  the  dropwise  formed  peaks were not  199(45%),  and  hydride  one  m o l ) , and  added  (1.2 12  mL, 0.02  hydride.  chloride  corresponded  with  three  61(18%),  was  trace  Isomer  187(6%), 260(5%), 215(3%).  for  crude  product  The other  41(19%),  mixture  TIC  desired  stirred  the  (3  evaporation. hydride  potassium  Methanesulfonyl  precipitate  potassium  of  the  component.  99(20%),  0°C.  The  and  triethylamine  cooled  min.  The  chloride  to  mixture  major  m/z  were  unreacted  and w a t e r .  of  mol),  dry THF and potassium  t-butanol  isomers  mL)  removed  which  these  methanesulfonyl  f o r 60  solvent  up i n  4-carboethoxy-2-ethylenethioketal-5-hydroxyheptane  using  compound (4  mL,  4-carboethoxy-2-ethylenethioketal-4-heptene  45(21%),  200(14%),  time  of  19.18 min:  119(21%), 155(10%),  59(20%), 159(9%),  -  Mass  spectrum:  199(100%), 155(25%),  Isomer  127(85%), 61(22%),  34 -  with  the  29(60%),  retention  99(29%),  41(20%),  time  of  200(28%),  45(18%),  59(17%),  20.24  min:  m/z  74(27%),  112(27%),  159(14%),  187(10%),  260(6%), 215(2%). d.  Removal o f the 1 , 3 - d i t h i o l a n e  Cleavage  of  the  dithiolane  group  protecting  group  was accomplished with  mercuric  c h l o r i d e i n the presence o f cadmium c a r b o n a t e . A p o r t i o n of the product mixture water  from above  (c)  stage  additional  were  stirred  mercuric  was f i l t e r e d  ether  potassium  iodide,  removal  the e t h e r  of  at room temperature (0.8  continued  and the acetone  and the e t h e r a l  (100 mL) and  g)  for  g) were  f o r 24 h o u r s . At t h i s  and cadmium carbonate another  72  hours.  (0.8  Then  the  removed. The r e s i d u e was d i s s o l v e d  s o l u t i o n washed s u c c e s s i v e l y with water, 10%  water the  acetone  (2 g ) , and cadmium carbonate(2  chloride  added and s t i r r i n g  mixture in  g) was d i s s o l v e d i n  (10 m L ) , m e r c u r i c c h l o r i d e  added and the mixture  g)  (2.5  and  dried  residue  showed 9 peaks two of which  over  anhydrous  was analyzed  (minor  components)  by GCMS.  Na2S04-  After  The TIC  plot  corresponded to the two  isomers o f the d e s i r e d p r o d u c t . Mass spectrum:  (MW=184) Peak with the r e t e n t i o n  time of 9.06 min: m/z  111(100%), m/z 43(90%), 29(15%), 55(12%), 112(11%), 184(9%). Mass spectrum: Peak with the r e t e n t i o n  time of 9.65 m i n : m/z 111(100%),  43(65%), 55(20%), 29(19%), 184(10%), 112(9%). A portion and s t i r r e d with ether. were taken and EI  of the product  from above was then  f o r 3 days f o l l o w i n g The e t h e r  was evaporated  and d e r i v a t i z e d  analysis,  which  it  t r e a t e d with  was a c i d i f i e d  and two p o r t i o n s  and e x t r a c t e d of the  to g i v e PFB and t-BDMS d e r i v a t i v e s  respectively.  The mass s p e c t r a  were then compared to those from u r i n e  extract.  alkali  residue f o r NICI  and r e t e n t i o n  times  -  3.  Synthesis of ethyl  To at  mixture  0.1 the  n-butyl 1 ithiurn  was s t i r r e d  pentanoate  mol)  for  (13  the  spectrum:  mol)  was  usual  (77  The mixture  quenched  m/z  S y n t h e s i s of e t h y l  diisopropylamine period. cooled was  The  propionaldehyde for  2  hours.  extracted residue  The  with was  9 2 ° C / 4 mm. [ L i t . Mass  Spectrum:  g,  0.12  15%  to  HC1  -78°C  chloride  f o r 25 and  and  (9.3  minutes.  pure  g,  Then  extracted  distillation  mol)  to  stir  yield  (15.6  was  flash ethyl  m/z  for  with ethyl  55(20%),  g,  further  0.12  mol) for  and the  neutralized evaporation  dropwise  0 ° C , over  a  stirred  a  15 in  60  with of  min and  10  mL THF  min.  stirring  101(100%),  Then  continued  15%  the  to  20 min  HC1 and  ether  2-propyl-3-hydroxypentanoate.  (Acheampong e t a l . , 1983) (MW=188)  101(33%),  added  dry THF at  was added  mixture  was  in  mixture  Following to  29(72%),  mol)  mol)  the  0.1  reaction  distilled  continued  with  pentanoate  and  ether.  cooled  and the  157(4%).  allowed  and ethyl  (7.4  dropwise  by p r o p i o n y l  57(100%),  mL,  was  dropwise  mol)  2-propyl-3-hydroxypentanoate  mL, 0.12  mixture  to - 7 8 ° C  added  (77  (12  dry THF (80 mL),  was o b t a i n e d . Bp 8 6 ° C / 0 . 1 mm.  (MW=186)  n-Butyl1ithiurn  in  was  and f r a c t i o n a l  73(19%), 130(10%), 144(9%), 43(8%), 4.  mol)  mL, 0.12  followed  work-up  2-propyl-3-oxopentanoate Mass  min.  g , 0.12  added and s t i r r i n g  mixture  After  15  g , 0.1  i n THF were  reaction  ether.  2-propyl-3-oxopentanoate  i s o p r o p y l c y c l o h e x y l a m i n e (16.9  0 ° C , was added  ethyl  35 -  the BP  bp 7 0 - 7 2 ° C / 0 . 2 mm]. 73(85%),  55(65%),  57(38%), 130(32%), 113(30%), 84(20%), 41(15%), 159(12%), 143(10%).  29(45%),  -  III.  A.  36 -  RESULTS AND DISCUSSION  NICI GCMS assay development  A highly  s e n s i t i v e assay has been developed f o r VPA i n serum and  s a l i v a based on the NICI-GCMS o f the PFB e s t e r . to q u a n t i t a t e VPA i n serum and s a l i v a i n f i v e state.  The assay  quantitation  of  the  chromatographic metabolites obtained.  i s also  applicable  metabolites  characteristics  have  been  The assay development  view of d e r i v a t i v e  formation,  mass  parameters,  spectrometer  the  determined  volunteers  at VPA steady  to VPA m e t a b o l i t e s ,  was made of  The assay was employed  in  present  PFB d e r i v a t i v e s  and  selected  study. of  The  a l l VPA  ion  chromatograms  from  the p o i n t  of  GC-ECD a n a l y s i s , o p t i m i z a t i o n  of  i s discussed  initial  the  although no  and s e l e c t i o n  below  of  a  suitable  internal  standard.  1.  Derivatization  Derivatization method  of  Min e t  prostaglandin Cj  carboxyl  benzylic  E  2  with PFBB was c a r r i e d out by a m o d i f i c a t i o n al.  (1980)  analog.  group  halides.  of To  This  for  the  of  a  trimethyl  i s based upon the o b s e r v a t i o n  prostaglandins form  derivatization  the  PFB  undergoes  facile  derivatives  of  of the  that  reaction VPA  the with  and  its  metabolites  samples were heated at 40°C  f o r 45 minutes  i n the presence  of  of  ethyl  and  10  uL  30%  PFBB  solution  d i i s o p r o p y l e t h y l amine and t h i s Longer  reaction  times  in  acetate  10  uL  of  procedure was used throughout the study.  or higher  temperatures  produced  a yellow  gummy  -  substance.  Initially  t r i e t h y l amine  d i i s o p r o p y l e t h y l amine, ammonium s a l t In  37 -  a  bulky  was  used,  amine,  but  which  of  removed a f t e r  the  NICI  work  the e x t r a c t i o n  acetonitrile  prior  minimizes  to  done  with  step and the  derivatization.  compared  sample.  before  Because  acetate),  With  Removal into  of  derivatization  of  the  acetate; the  presence  GCMS  (see  below).  and  solvent  is  VPA and  and  thought  of  the  to  metabolites,  their  hence  was  its  low m o l e c u l a r  complete result  extracting  solvent  in  loss  solvent  of a small  of  (ethyl  amount of  however t h i s was not a problem.  excess  the  t  intensities diverted  prostaglandins  1973).  r e s i d u e i s r e c o n s t i t u t e d with  there was evidence of the e s t e r i f i c a t i o n  hydrolyzed ethyl  sample  to  with  quaternary  prostaglandins,  such a procedure would not be d e s i r a b l e because of  evaporation  replaced  formation with PFBB (Wickramasinghe et a l . ,  most  weight  was  did  PFBB  not  prior  prove  injection  possible  Because of  scanning commenced 4  to  the  without  excess  minutes  of  after  derivatized  loss  reagent sample  of  peak  solvent injection  was to  reduce the amount of reagent e n t e r i n g the mass spectrometer ion s o u r c e . The  derivatizing  derivatization methyl ate  the  derivatization for  procedure  adopted  s i n c e no a d d i t i o n a l components  of  step with PFBB.  appears  2.  result  in  optimum  peaks were observed upon attempts the  reaction  A typical  the PFB e s t e r s of VPA and OA i n the  Figure  to  total  mixture  to  after  the  ion chromatogram  trace  negative  ion mode i s  shown  in  2.  G C - E l e c t r o n Capture D e t e c t i o n  F i g u r e 3 shows t y p i c a l  (ECD)  GC-ECD chromatograms of the PFB d e r i v a t i v e s  o f VPA and OA. S i n c e GC with ECD i s  in  principle  similar  to  electron  - 38 -  a  b  T 2  i  r~  \  5 TIME  F i g u r e 2.  ~r 6  8  (min)  T o t a l ion c u r r e n t p l o t o f the PFB e s t e r s o f VPA(a) and 0A(b) i n the NICI mode.  -  capture NICI, GC. When the 2 uL o f  it  was of  interest  derivatization  sample i n j e c t e d  the  residue  the  assay with  above was used and 1 or  To remove most of  the  excess PFBB  the  because  b.  step,  with 400  with e t h y l  which  alkali  derivatization  treated  and e x t r a c t e d  alcohol  evaluate  the GC, there was a high background due to  reagent.  Treatment with  After  reagent  initially  procedures were e v a l u a t e d .  a.  the  to  procedure o u t l i n e d  into  the excess d e r i v a t i z i n g following  39 -  the  uL o f  acetate.  product  solvent 3N  was  removed  NaOH a n d / o r  This,  400  however,  did  formed  is  presumably  uL o f  not  of  the  reaction  mixture  at  N  2  ION  and NaOH  remove excess  pentafluorobenzyl  i s s o l u b l e both i n water and most o r g a n i c  Evaporation  under  solvents.  elevated  temperature  (40-60°C). The reagent internal and  standard  conditions  l o s s of the c. To  peak was s i g n i f i c a n t l y peaks.  required  to  Selective  solvent  selectively  remove  hexane,  mixture  was  re-extracted  heptane,  made with  basic, another  t h a t both d e r i v a t i v e d.  is  to  very  evaporate  volatile  excess  relative  reagent  to  and  VPA-PFB  invariably  cause  extraction excess  gel  and  separate  was not p o s s i b l e .  PFBB,  extracted solvent  of  sephadex  were  with  one  differing  and the PFBB reagent  the  different  iso-octane etc.)  Column chromatographic  Silica  not  so were a n a l y t e  sample.  acetate,  attempt  PFBB  reduced but  solvents  employed. of  the  polarity.  were s o l u b l e  The  (ethyl reaction  solvents It  was  in a l l  and found  solvents.  separation  LH-20  derivatives  short from  columns  PFBB;  but,  were again  used  in  an  separation  -  When  a  solution employed, 3.  in  the  it  was  internal  adopted  -  derivatization  presence  An a c c e p t a b l e  as  is  different  40  of  18-crown-6  p o s s i b l e to  obtain  calibration  curve  standard  was  also  from Rubio and G a r l a n d  supposedly advantageous  procedure  in  the for  (1985) that  It the  Optimization  spectrum  VPA i n  ethyl  (Figure  phenols are  not  area  counts obtained  different  NICI,  electron  we experienced for  the  using OA  This  procedure  amount  of PFBB and  of  multiplier.  Efforts  manipulate  etc)  in  be  explained  maintain  reagent  gas  problems  such  filament  pressure  gauge reading  as  flow  by at  the a  did  not  The lack  fact  that  reasonably  sag were  probably  reflect  order  the  it  actual  initial  a factor tended  to of  get  The  VPA on of  to  instrument  100.  destroy  variables  reproducible  reproducibility  was  constant due  our  derivatized  by more than  d i d not prove s u c c e s s f u l .  can  of  influence  sensitivity.  NICI c o n d i t i o n s  to  source p r e s s u r e ,  During  reproducible  times  sag and the  because  acetate.  same c o n c e n t r a t i o n at  Figure  acetate  derivatized  sensitivity.  lack  was  f o r NICI  the  filament  energy,  sensitivities time  hence  runs and days v a r i e d  There was a l s o  (electron  and  PFBB  acetate  shown i n  4.)  uses a l e s s e r  of MS parameters  obtained  with  that  potassium  0.5%  i s known t h a t under CI c o n d i t i o n s a number of f a c t o r s  work  the  used  chromatograms  obtained  o f the use of the l e s s b a s i c potassium  3.  and  that  to  not  possible  value. the  The  fact  at to  other  that  the  the  ion  and with  the  pressure  in  source. With the tune  values  instrument remaining  in apparently reasonably  good working o r d e r ,  constant,  the  effects  of  important  - 41 -  b  T 1  1 3 TIME  F i g u r e 3.  1  r  5  7  (min)  T y p i c a l chromatograms o f the 0A(b) o b t a i n e d with GC-ECD.  PFB d e r i v a t i v e s o f VPA(a) and  - 42 -  Figure  4.  C a l i b r a t i o n curve f o r PFB d e r i v a t i z e d o b t a i n e d u s i n g GC-ECD.  VPA i n e t h y l  acetate  -  mass  spectrometer  determined. Relative  43 -  variables  Source  on  the  temperatures  sensitivities  of  the  sensitivity  tried  were  VPA -PFB peak  to  150,  VPA-PFB 190  were  and  were 7 . 56 ,  2 . 94  240°C. and  1.0  respectively. The  sensitivity  temperature  d e c r e a s e d , but  made between ion  source  Most  of  thus  an optimum becoming  the  temperature  until  analysis  routine  analysis  temperature  was,  increase a  and the  contaminated  therefore,  as  the  source  compromise must  c o n s i d e r a t i o n of  at  lower  carried  low  source pressure s e n s i t i v i t y  pressure  less  at  low  to  be the  temperatures.  out  at  a  source  of 2 0 0 ° C .  highest  pressures  for  excessively  When v a r y i n g the the  appeared  than  tolerated 1  torr,  pressures i.e  by  the  less  the  instrument  sensitivity  than 0.3  was maximal  torr  (1  was  about  torr).  decreased there  at  For  dramatically no s i g n a l  at  all. The h i g h e s t the  highest  electron not  response f o r  electron  energies  energy  less  than  the VPA-PFB  derivative  handled  the  lOOeV  the  obtained  instrument  monitored  ion,  near  (240eV).  m/z  143",  At was  detected. All  example,  the  above  sensitivity  a n i o n s are  instrumental  parameters  are  depends  the  to  upon  s t a b i l i z e d by reagent  internal  energy of  influenced  by  depends  the  source upon  the  reagent  sensitivity will  gas so t h a t  temperature. value  extent  of  The the  interrelated. which  gas m o l e c u l e s , but  a l s o l e a d to e l e c t r o n detachment,  also  by  was  source  formed  s i n c e c o l l i s i o n can  a l s o depend upon the  sensitivity  ionizing  newly  For  will  electron  pressure.  be  highly  energy  Because  used it  is  -  difficult these  to  assess  44 -  a b s o l u t e enhancement of  interdependent  parameters  is  sensitivity,  measured  one parameter w h i l e m a i n t a i n i n g the o t h e r s at The e f f e c t also  of  investigated  Ammonia that  gave  an  reported  resulted  in  reagent using  gases on the  methane,  apparently by  Miyazaki  short  et  filament  al.  life.  approximately some optimal  sensitivity  ammonia,  greater  and  (1984)  by  varying  value.  is  (95:5).  similar  to  prostaglandins,  no  of  of the VPA-PFB was  which  for  was  effect  argon-methane  sensitivity,  There  the  difference  but  between  methane and argon-methane with r e s p e c t to sample response or background ions.  No carbon c o n t a i n i n g ions of higher masses ( i o n / m o l e c u l e  were  observed  with  methane  and  this  gas  was  used  the  reported  adducts)  throughout  all  subsequent e x p e r i m e n t s . In  spite  limited  of  its  high  because i n a d d i t i o n to other  instrument),  poor  reproducibility  mentioned as a r e s t r i c t i n g drawback that  sensitivity  factor  reasons ( e . g . of  NICI  (Oehme e t  of most instruments with n e g a t i v e  important  parameters  spectra  1986).  NICI  is  of  is  the  often  Furthermore,  a  capability  is  ion d e t e c t i o n  such as temperature  of  availablility  mass al.,  use  and p r e s s u r e can only be  measured a p p r o x i m a t e l y .  In  reproducible s e n s i t i v i t y  and some changes i n the mass spectrometer were  required  to  controller  improve  of  the meant  the  our work with VPA we had d i f f i c u l t y  reproducibility.  mass spectrometer  That  values  to  heater  was a l s o made. These changes brought  actual  source source  reagent  was r e p l a c e d with  valve.  the  the  The  pressure pressure.  gauge  A modification a dramatic  r e p r o d u c i b i l i t y and, to a l e s s e r e x t e n t s e n s i t i v i t y .  gas  a simple  indicator to  getting  flow needle  gave  closer  the  source  improvement  in  In our e x p e r i e n c e ,  -  45 -  we found i o n source p r e s s u r e t o be a c r i t i c a l reproducible  4.  parameter  for obtaining  sensitivities.  Comparison of the r e l a t i v e  sensitivity  of  derivatized  VPA by  EI and NICI  Once r e p r o d u c i b l e s e n s i t i v i t i e s were o b t a i n e d , determine  the r e l a t i v e  sensitivity  t h a t o f the t-BDMS d e r i v a t i v e . bis-TFMB  derivatives  fluorinated while  derivatives  the  (M-57)  t-BDMS  ion  +  were  derivative In  spectra,  the  spectra  in  NICI  the base  peaks  represent  o f these  are m/z 181 positive  moieties  derivatives  was an amount  result  of  obtained a f t e r  of a l l  the PFB d e r i v a t i v e t-BDMS d e r i v a t i v e be  about  derivative,  by  EI  The  and NICI modes  and  of  and 6 .  the  both  m/z  201  fluorinated  lack  derivative  5  moieties.  (PFB) and m/z 227  by  derivatization,  specificity.  All  three  concentration  equivalent  to 5  determinations.  derivatives  was s i m i l a r .  Since  the  EI  derivatives  i n the r e a c t i o n  ng/uL  In  the  (bis-TFMB)  of  a n a l y z i n g the samples i s shown i n T a b l e  three  In  i o n s to those o f the NICI.  introduced  area counts are the means of f i v e sensitivity  5  the EI  spectra  were prepared i n a manner t h a t the f i n a l vial  Figures  both  analyzed  to  s p e c t r a of the PFB and  and 3 , 5 - b i s ( t r i f l u o r o m e t h y l ) b e n z y l  which are the complementary ions  was  in  derivatives  the base anion i s m/z 143 c o r r e s p o n d i n g to the l o s s of the  pentafluorobenzyl  these  shown  analyzed  was p o s s i b l e to  two f l u o r i n a t e d  The NICI and EI  VPA are  monitored.  derivatives,  EI  of  of  it  VPA. The 1.  The  the EI mode, the In  the NICI mode,  was found to be 30-50 times more s e n s i t i v e than the by E I .  times  bis-TFMB.  more  The PFB d e r i v a t i v e sensitive  than  (NICI mode) a l s o proved to the  similar  fluorinated  - 46 143  100 _  A)  >l—  C H j - C H 2 C H2 -  v 4  • — i  CO CH3-CH2- C H  -OfCH  2  14:  181  250  350  M/Z B)  100  _  >t—  C  CH^—C  I—I  CO  ^CH-C-O+CH  •z.  C H^-^C^—C H2  LU t—  143_J  UJ  LU  or  242  50  150  250  M/Z  Figure  5.  NICI mass s p e c t r a o f VPA.  o f the  350  PFB(A) AND bis-TFMB(B)  450  derivatives  - 47 -  A)  181  100 _  P|  CH - C H - C H  ^CH-C-0  J  C H  CH -CH -CH " 3  2  2- 6 5 C  F  2  1  — 181  282 240  I il  •il  25  B)  125  325  227  100  >i—  225  M/Z  CH -CH -CH 3  2  0  2  57  cn  II  ,  CH-C-OfCH -C H (CF ) 2  CH -CH -CH 3  2  6  3  3  2  -227  2  >  or  285 328 Ml  50  Figure  JLLl I ••  I  •••  150  6.  I  355  || I M/Z  250  EI mass s p e c t r a o f the VPA.  350  PFB(A) and bis-TFMB(B) d e r i v a t i v e s  of  -  5.  Quantitative  a.  a n a l y s i s with the PFB d e r i v a t i v e  Internal  The  standard  internal  [ H5]-VPA.  standard  The use of  2  48 -  used  seven d i f f e r e n t  the  standard  that  an i n t e r n a l  (OA) was a l s o i n v e s t i g a t e d . with  throughout  The r e s u l t  concentrations  (10 t o 800 ng/mL)  of e i t h e r  i n T a b l e 2.  The r e s u l t s are the means of three  shows t y p i c a l  VPA, [ H 6 ] - V P A As the  seen  from  internal  ion chromatograms  and OA e x t r a c t e d  2  Table  standard  2,  case  of  isotope-labelled used  as  precision stable  standards.  o f assays  due to  accurately  determined  assays  Claeys  curve  with  is  shown Figure  [ H6]-VPA  of  as  2  variability  using  related  et  internal  al.  SIM,  both  compounds  in  stable  have  been  evaluated  the  and concluded  that  (1977)  standard  are  i s lower  standards produced the lowest  manipulation  hand, Lee and M i l l a r d  standard  to t h a t with OA. The i n t e r c e p t s  GCMS  using e i t h e r  sample  of VPA with the  of the PFB d e r i v a t i v e s  calibration  and c h e m i c a l l y  isotope-labelled internal  factors other  analogs  internal  the  In  2  a common ion  determinations.  and the i n t r a - a s s a y  [ H63-VPA.  was  from serum.  is superior  much c l o s e r to the o r i g i n the  or OA as i n t e r n a l  2  selected  gives  study  o f the a n a l y s i s of serum spiked  same amount  7  [ H6]-VPA  present  and instrumental  variance  errors.  On the  (1975) have argued t h a t a substance i s most  using  an i n t e r n a l  standard  giving  a common ion  because o f the advantage of m o n i t o r i n g a s i n g l e ion and hence a gain i n sensitivity extreme can  and s t a b i l i t y .  Under CI c o n d i t i o n s , though, because of the  dependence of the s p e c t r a on source pressure and temperature  be s a i d  that  more  accurate  measurements  can be made with  isotope-label led internal  standard s i n c e i t s physicochemical  closely  o f the a n a l y t e .  this.  approximate  that  Our r e s u l t s  it  stable  properties  seem to  support  - 49 TABLE 1. Comparison of the r e l a t i v e s e n s i t i v i t i e s Derivative  PFB  bis-TFMB  t-BDMS  Mode of ionization  Ion monitored  Area count  EI  181  8,714  NICI  143  412,000  EI  227  8,228  NICI  143  103,500  EI  201  10,184  * Amount i n j e c t e d t o 5 ng o f VPA.  TABLE 2.  in a l l  of three VPA d e r i v a t i v e s * . Relative sensitivity NICI/EI  47  13  cases was an amount of d e r i v a t i v e  Comparison of [ Hg]-VPA and OA as internal 2  standards*  C H6]-VPA  OA  2  Intra-assay Vari ation r  2  y-intercept x-intercept  *  -  < 5%  8-15%  0.9986  0.9960  0.0027  0.1020  2.2483  equivalent  -  12.0155  R e s u l t s obtained with seven d i f f e r e n t c o n c e n t r a t i o n s of VPA ranging from 10 t o 800 ng/mL. The samples were assayed t h r e e times and values shown are means of the three d e t e r m i n a t i o n s .  -  m/z  50  149  TIME Figure  7.  -  (min)  SIM chromatograms o f VPA, OA (m/z 143) and [ H ] - V P A from serum s p i k e d with these s u b s t a n c e s . g  (m/z  149)  -  b. The Abbott  Analytical  volume  al.  -  parameters  extraction  et  51  procedure  employed was  (1986a), modified  (100  uL).  For  procedure was u s e d , acidification.  the  but  to  calibration  curves  standard were o b t a i n e d 149  which  are  the  [ H6]-VPA-PFB, r ,  were  2  greater  saliva. saliva  The VPA  is  shown  curve  for  given i n F i g u r e The  good  an  (coefficient assay  and  of  VPA  in  the  the  for  curves  using  the  The  0.996  in  of  the  intra-and  for  serum  8,  9  EI(t-BDMS)  prior  to  greater  internal  m/z  143  and  VPA-PFB  and  determinations, and  total)  and  free,  and  serum  respectively.  determination  NICI  assay  at  assay  of  The  serum t o t a l  using PFB d e r i v a t i v e s  assay  variation  ug/mL.  standard  deviations  was observed over  The  lower  of  10-800 ng/mL o f VPA i n  limit  of  the  of  less  VPA  was  very  than  10%  serum. The w i t h i n the  slope  respectively  was  2  of  the  (C.V.=0.299%  concentration  detection  range ng/mL  of  10  o f VPA  uL sample of serum. F i g u r e 12 shows the SIM chromatogram  o f VPA o b t a i n e d with 10 pg of VPA e x t r a c t e d is  of  (free  10  at of  total,  and  Linearity  detection  spectra  serum  and 1.92%).  based on a 200  peaks  coefficient  both  Figures  inter-  variation)  between  25  similar  as  2  NICI  curve were 0.000003 and 0.000021,  to  a  alkali  [ H6l-VPA  intense  calibration  ng/mL  serum sample  samples  t r e a t e d with  of  11.  precision  with  for  ions  calibration  are  calibration  than  saliva  that  from w a t e r .  by m o n i t o r i n g  base  small  of  drug from serum and s a l i v a was  respectively.  2  of  samples were not  than 95% compared to drug e x t r a c t e d The  accommodate the  extraction  The recovery of  a modification  similar  to most of  the  reported  from  serum. T h i s  values  for  PFB  limit  of  derivatives  - 52 -  2.5 -i  0  5  10  15  20  25  Concentration (ug/mL serum)  Figure 8.  C a l i b r a t i o n curve f o r serum t o t a l VPA. Peak area r a t i o was o b t a i n e d by m o n i t o r i n g m/z 143 (VPA) and m/z 149 ( [ H , ] - V P A ) . 2  - 53  F i g u r e 9.  C a l i b r a t i o n curve f o r serum f r e e VPA. Peak area r a t i o was o b t a i n e d by m o n i t o r i n g m/z 143 (VPA) and m/z 149 ( [ H ] - V P A ) . 2  f i  - 54 -  F i g u r e 10.  C a l i b r a t i o n curve f o r s a l i v a VPA. Peak area r a t i o was o b t a i n e d by m o n i t o r i n g m/z 143 (VPA) and m/z 149 ( [ H ] - V P A ) . Z  g  - 55 -  F i g u r e 11.  C a l i b r a t i o n curve f o r EI(t-BDMS) d e t e r m i n a t i o n o f serum t o t a l VPA. Peak area r a t i o was o b t a i n e d by m o n i t o r i n g m/z 201 (VPA) and m/z 207 ( [ H ] - V P A ) . 2  g  -  of prostanoids. as  In some c a s e s , with p r o s t a g l a n d i n s l i m i t s  low as 200 f g have  (Miyazaki VPA-PFB fact  et  56 -  been  a l . , 1984).  reported  The lower  with  ammonia  limit  of  i s not as good as the above example  t h a t VPA i s a small  electron  (t-BDMS)  the developed NICI  routinely  serum  samples  the c o r r e l a t i o n one  of  agreement  used of  the  between  for  B.  VPA l e v e l s  in  quantitation  As  values  seen  were  (free  saliva  serum f r e e  and s a l i v a  in Tables 4 to 8.  and s a l i v a ,  as well  are a l s o given i n these  tables.  Tables  saliva  in  our  by both  EI  and F i g u r e 13  by the two methods  Table  3  and  Figure  13, Mean  ( F . A ) and c o r r e l a t i o n s  and s a l i v a  before  levels  before  samples  and  after  between  as the c o r r e l a t i o n s  total,  analyzed  CBZ and 30 a f t e r  The r e l a t i o n s h i p s  and serum  were  of VPA i n the f i v e  9 and 10 show the time-averaged  serum t o t a l ,  an EI  f o r serum VPA i s e x c e l l e n t .  and serum  serum t o t a l ,  free  from  VPA  measured  obtained  and t o t a l )  i n SIM mode; 33 samples  and  of the  of CBZ  o f 63 p a i r e d  presented  of  in Table 3 ,  ( B . A ) and 40.3 vs 40.2  VPA by NICI  serum  with  respectively.  serum  administration  total  serum  EI and NICI methods  were 0.98 and 0 . 9 9 ,  are  because  assay compared with  two v o l u n t e e r s  volunteers.  (r)  A  of the  were 28.4 vs 27.1  the  obtained  probably  and NICI GCMS methods. These values are given  values  detection  molecule and hence with l e s s s u r f a c e area f o r  how well  assay  laboratory,  for  as the reagent gas  capture.  To assess  shows  of detection  ratios  between  between  and s a l i v a  for  CBZ. The volunteers  serum each  serum  and serum  total, other  free free  - 57 -  TIME Figure  12.  (min)  SIM chromatogram o f PFB d e r i v a t i z e d VPA o b t a i n e d with 10 pg o f VPA e x t r a c t e d from serum (the second peak i s background from serum).  -  TABLE 3.  58 -  Serum VPA l e v e l s (ug/mL) in two subjects on VPA steady-state as measured by EI (t-BDMS) and NICI (PFB). BA  EI 40.31 40.56 42.50 25.93 23.20 27.97 25.03 33.80 31.78 27.61 26.05 21.85 16.94 14.35  Mean: 28.42 Correlation(r):  FA  NICI  EI  NICI  42.36 42.44 43.78 20.56 22.27 25.90 21.07 31.05 30.36 26.65 23.60 19.01 16.40 13.26  44.35 58.34 41.20 48.20 48.05 33.41 22.70 51.25 45.27 42.15 34.17 29.02 26.04  44.45 58.77 40.59 46.84 46.06 33.61 24.15 53.24 43.80 42.02 33.34 26.99 28.02  27.053  40.319  40.145  0.9830  0.9913  - 59 -  0.7710  0  10  20  30  40  50  60  El  F i g u r e 13.  R e l a t i o n s h i p between VPA c o n c e n t r a t i o n s (ug/mL) determined by EI(t-BDMS) and NICI ( P F B ) .  i n serum  -  in  three  biological  samples a f t e r  14  the  concentration-time  saliva  F i g u r e 15 the for  11  the  -  while  is  Table  60  concentration  one v o l u n t e e r  time  curves  volunteer. both  for  are  serum t o t a l volunteers  and  and s a l i v a are  Effect  36.85  serum f r e e , (PO.025) decrease for  all  ±  is  the  degree  18  for  and 17  of  9 and  are  and  and  after  19.  serum  free  concentration CBZ in  one  between  saliva  and  for  one  of  relationship  and  In  after  CBZ  The  Figure  and t o t a l VPA  the  and  all  volunteers.  free,  correlation  i n average VPA c o n c e n t r a t i o n  13.64,  and 48.13  higher  for  ±  7.70  respectively.  VPA c o n c e n t r a t i o n serum  except  with decreasing total five  presented. five  saliva,  in  saliva  the  between  for  all  the  of CBZ on serum and s a l i v a l e v e l s of VPA  volunteers  the  is the  in  free  one  (Table  after 11),  CBZ was 27.91  for  serum  There was a s i g n i f i c a n t  all  three  and  saliva  (R.M.).  The  biological compared  greater  f r e e VPA and s a l i v a VPA a f t e r CBZ i s because the  for  concentration  VPA b e f o r e  VPA b e f o r e  Figures  for 16  saliva  and between  and s a l i v a , of  VPA  shown i n F i g u r e s 20 and 2 1 .  The % decrease 3.48,  in  profile  Figures  and  free  given  average  time curves  free  showing  in  CBZ a d m i n i s t r a t i o n  shown.  serum  total  volunteers  1.  are  Curves  serum  % change  serum VPA c o n c e n t r a t i o n s .  total,  reduction  fluids. to  serum  % decrease  ±  of  The % total serum  free f r a c t i o n  declined  The mean f r e e  fraction  s u b j e c t s decreased from 0.1334 t o 0.1072 a f t e r CBZ ( T a b l e s  10). Because  VPA  is  a  highly  undergo drug i n t e r a c t i o n s displace  phenytoin  from  at  protein-bound  the p r o t e i n  its  binding  drug,  VPA  is  likely  to  b i n d i n g l e v e l . VPA i s known to site  on  serum  albumin,  and  TABLE 4a. Serum t o t a l , serum free and s a l i v a concentrations (ug/mL) of VPA and t h e i r relationship to each other i n volunteer W.T. before the administration of CBZ. Time Serum Serum Saliva Saliva: total Saliva: free r a t i o ratio free total (h)  Free: total ratio  1  52.95  6.75  0.891  0.017  0.132  0.127  2  54.57  5.29  1.020  0.019  0.193  0.097  3  47.50  4.60  1.114  0.023  0.242  0.097  5  41.30  4.36  0.756  0.018  0.173  0.106  7  38.50  3.15  0.472  0.012  0.150  0.082  9  32.47  2.45  0.323  0.010  0.132  0.076  12 Mean S.D. C.V.  25.80  1.88  0.345  0.013 0.0160 0.0042 26.25%  0.184 0.1723 0.0361 20.95%  0.073 0.0940 0.0175 18.62%  Correlation  ( r ) : Between serum t o t a l and s a l i v a : Between serum f r e e and s a l i v a : Between serum t o t a l and f r e e  = 0.8945 = 0.8254 = 0.9400  Time (h)  TABLE 4b. Serum t o t a l , serum free and s a l i v a concentrations (ug/mL) of VPA and t h e i r relationship to each other in volunteer W.T. a f t e r the administration of CBZ. S a l i v a : free Serum Serum Saliva: total Saliva ratio free rati o total  Free: total ratio  1  41.26  3.83  0.697  0.017  0.181  0.093  2  35.97  3.19  0.683  0.019  0.214  0.089  3  31.71  3.08  0.499  0.016  0.162  0.097  5  29.75  2.15  0.427  0.014  0.198  0.072  7  23.02  1.62  0.273  0.012  0.168  0.070  9  20.71  1.20  0.219  0.011  0.182  0.058  12 Mean S.D. C.V.  16.44  0.81  -  0.0148 0.0027 18.24%  Correlation(r): : :  Between serum t o t a l and s a l i v a Between serum f r e e and t o t a l Between serum t o t a l and f r e e  = 0.9793 = 0.9615 = 0.9831  0.1841 0.0175 9.50%  0.049 0.0754 0.0169 22.41%  TABLE 5a.  Time (h)  Serum t o t a l , serum free and s a l i v a concentrations (ug/mL) of VPA and t h e i r relationship to each other in volunteer M.S. before the administration of CBZ. Serum Serum Saliva: total S a l i v a : free Saliva free rati o ratio total  1  92.63  8.33  1.533  0.017  0.184  0.090  2  63.60  9.48  1.020  0.016  0.108  0.149  3  65.31  9.25  1.274  0.020  0.138  0.142  5  50.74  6.48  1.093  0.022  0.169  0.128  7  51.06  8.74  1.029  0.020  0.118  0.171  9  51.57  7.94  -  0.154  24 Mean S.D. C.V.  21.48  1.75  Correlation  -  0.343  ( r ) : Between serum t o t a l and s a l i v a : Between serum f r e e and s a l i v a : Between serum t o t a l and f r e e  0.016 0.0185 0.0023 12.43% = 0.9488 = 0.8129 = 0.7463  0.196 0.1522 0.0330 21.68%  Free: total ratio  0.081 0.1307 0.0311 23.78%  Time (h)  TABLE 5b. Serum t o t a l , serum f r e e and s a l i v a c o n c e n t r a t i o n s ( n g / n l ) o f VPA and t h e i r r e l a t i o n s h i p t o each o t h e r 1n v o l u n t e e r M . S . a f t e r the a d m i n i s t r a t i o n o f CBZ. Serum Serum Saliva Saliva: total Saliva: free Free: total total free ratio ratio ratio  roo  O~7\M  007  07121  o.oss  1  57.60  6.81  -  -  -  0.118  2  50.38  3.64  0.670  0.013  0.184  0.072  3  49.69  3.35  0.472  0.010  0.140  0.067  5  41.26  2.31  -  -  -  0.056  7  38.04  2.01  0.612  0.016  0.304  0.053  9  29.64  -  0.411  0.014  12  26.03  1^94  0.355  0.014  0.182  0.075  0.0123 0.0030 24.24%  0.1870 0.0628 33.63%  0.0713 0.0205 28.83%  Mean S.D. C.V. Correlation  (r)  : Between serum t o t a l and s a l i v a : Between serum f r e e and s a l i v a : Between serum t o t a l and f r e e  = 0.8493 = 0.7500 = 0.8332  Time (h)  TABLE 6a. Serum t o t a l , serum free and s a l i v a concentrations (ug/mL) of VPA and t h e i r relationship to each other 1n volunteer R.M. before the administration of CBZ. Serum Serum Saliva Saliva: total S a l i v a : free total free ratio ratio  0  30.33  2.77  0.503  0.017  0.182  0.091  1  47.26  6.72  1.294  0.027  0.193  0.142  2  44.81  5.92  0.870  0.019  0.147  0.132  3  42.01  6.11  0.488  0.012  0.080  0.146  5  38.60  4.32  0.592  0.015  0.137  0.112  7  37.67  3.62  0.662  0.018  0.183  0.096  9  34.40  -  -  -  -  12 Mean S.D. C.V.  29.47  _  _  _  2.89  0.0180 0.0046 25 .66% Correlation  (r)  : Between serum t o t a l and s a l i v a : Between serum f r e e and s a l i v a : Between serum t o t a l and f r e e  = 0.8412 = 0.6500 = 0.9556  0.1536 0.0386 25.15%  Free: total ratio  0.098 0.1167 0.0213 18.30%  Time (h)  TABLE 6b. Serum t o t a l , serum free and s a l i v a concentrations (iig/mL) of VPA and t h e i r relationship to each other 1n volunteer R.M. after the administration of CBZ. Saliva: total S a l i v a : free Serum Serum Saliva ratio ratio free total  0  22.79  1  42.43  2  39.35  6.69  3  0.175  0.008  0.590  0.014  0.388  0.010  6.82  0.757  0.089  Free: total ratio  0.157  0.111  5  30.56  3.10  0.430  0.014  0.139  0.101  7  27.61  2.56  0.268  0.010  0.105  0.092  9  24.10  1.66  12 Mean S.D. C.V.  18.11  1.20  Correlation  (r)  0.069 0.185  : Between serum t o t a l and s a l i v a : Between serum f r e e and s a l i v a : Between serum t o t a l and f r e e  0.010 0.0110 0.0022 20.32% = 0.9011 = 0.9549 = 0.9765  0.154 0.1196 0.0235 19.72%  0.066 0.0970 0.0328 33.835%  Time (h) 1  TABLE 7a. Serum t o t a l , serum free and s a l i v a concentrations (ug/mL) of VPA and t h e i r relationship to each other In volunteer B.A. before the administration of CBZ. Serum Serum Saliva Saliva: total Saliva: free total free ratio ratio 42.36 5.40 0.128  -  Free: total ratio  -  -  2  42.44  5.38  1.064  0.025  0.198  0.127  3  43.78  5.49  1.522  0.035  0.277  0.125  5  20.56  0.611  0.029  -  -  7  22.27  3.39  1.270  0.057  0.375  0.152  9  25.90  2.82  1.233  0.047  0.437  0.109  12 Mean S.D. C.V.  21.07  2.76  0.773  0.035 0.0380 0.0108 28.63%  0.280 0.3134 0.0834 26.61%  0.131 0.1287 0.0126 9.79%  Correlation  -  (r)  : Between serum t o t a l and s a l i v a : Between serum f r e e and s a l i v a : Between serum t o t a l and f r e e  = 0.6017 = 0.4884 = 0.9733  Time (h)  TABLE 7b. Serum t o t a l , serum free and s a l i v a concentrations (iig/mL) of VPA and t h e i r relationship to each other 1n volunteer B.A. a f t e r the administration of CBZ. S a l i v a : free Serum Saliva: total Serum Saliva ratio ratio free total  Free: t o t a l ratio  1  31.05  4.60  0.940  0.030  0.204  0.148  2  30.36  3.80  0.585  0.019  0.154  0.125  3  26.65  0.549  0.021  -  -  5  23.60  3.37  0.529  0.022  0.157  0.142  7  19.01  1.90  0.391  0.021  0.206  0.099  9  16.40  1.62  0.360  0.022  0.222  0.099  12 Mean S.D.  13.26  1.61  0.292  0.022 0.0224 0.0032 14.49%  0.181 0.1873 0.0254 13.56%  0.121 0.1223 0.0189 15.45%  -  c.v. Correlation  (r)  : Between serum t o t a l and s a l i v a : Between serum f r e e and s a l i v a : Between serum t o t a l and f r e e  = 0.8727 = 0.9354 = 0.9614  TABLE 8a. Serum t o t a l , serum free and s a l i v a concentrations (pig/mL) of VPA and t h e i r relationship to each other in volunteer F . A . before the administration of CBZ. Serum Serum Saliva Saliva: total S a l i v a : free Time free ratio ratio (h) total  Free: total ratio  0  44.45  8.21  0.889  0.020  0.108  0.185  1  64.66  13.71  2.963  0.046  0.216  0.212  2  56.58  13.11  2.432  0.043  0.186  0.231  3  58.77  11.04  3.230  0.055  0.293  0.188  5  40.59  9.18  1.218  0.030  0.133  0.226  7  46.84  7.99  1.209  0.026  0.151  0.171  9  46.06  7.88  0.483  0.011  0.061  0.171  12 Mean S.D. C.V.  33.61  6.45  0.559  0.017 0.0310 0.0145 46 .86%  0.087 0.1544 0.0706 45.74%  0.192 0.1979 0.0219 11.10%  Correlation  (r)  : Between serum t o t a l and s a l i v a : Between serum f r e e and s a l i v a : Between serum t o t a l and f r e e  = 0.8933 = 0.8818 = 0.9045  TABLE 8b.  Time (h) 0  Serum t o t a l , serum f r e e and s a l i v a c o n c e n t r a t i o n s (iig/mL) o f VPA and t h e i r r e l a t i o n s h i p t o each o t h e r 1n v o l u n t e e r F . A . a f t e r the a d m i n i s t r a t i o n of CBZ. Serum Serum . Saliva Saliva: total S a l i v a : free total free ratio ratio 4.44 24.15 0.353 0.015 0.080  Free: total ratio 0.183  1  53.24  8.73  -  -  -  0.163  2  43.80  7.17  0.998  0.023  0.139  0.163  3  42.02  7.33  1.294  0.031  0.177  0.174  5  33.34  4.86  0.773  0.023  0.159  0.145  7  26.09  4.61  0.572  0.022  0.124  0.176  9 Mean S.D. C.V.  28.02  5.44  -  _  _  0.0228 0.0051 22.26% Correlation  (r)  : Between serum t o t a l and s a l i v a : Between serum f r e e and s a l i v a : Between serum t o t a l and f r e e  = 0.9265 = 0.9159 = 0.9654  0.1405 0.0331 23.60%  0.194 0.1698 0.0149 8.76%  TABLE 9. Time-averaged r a t i o s (6-8 samples) and c o r r e l a t i o n s between serum t o t a l , serum free and s a l i v a VPA concentrations in f i v e volunteers before the administration of CBZ. Saliva: total ratio  Saliva: free ratio  Free: total ratio  W.T.  0.0160  0.1723  0.0940  0.8945  0.8254  0.9400  M.S.  0.0185  0.1522  0.1307  0.9488  0.8129  0.7463  R.M.  0.0180  0.1536  0.1167  0.8412  0.6500  0 . 9556  F.A.  0.0310  0.1544  0.1970  0.8933  0.8818  0.9045  B.A.  0.0380  0.3134  0.1287  0.6017  0.4884  0.9733  Mean  0.0243  0.1892  0.1334  0.8945*  0.7925*  0.9039  S.D.  0.0086  0.0625  0.0343  C.V.  35.63%  33.06%  25.76%  Volunteer  Correlation Total:  Saliva  Free:  (r)  Saliva  * excluding B . A . ' s  values  Total:  Free  TABLE 10. Time-averaged ratios (6-8 samples) and c o r r e l a t i o n s between serum t o t a l , serum free and s a l i v a VPA concentrations in f i v e volunteers after the administration of CBZ.  Saliva: total ratio  Saliva: free ratio  Free: total ratio  W.T.  0.0148  0.1841  0.0754  0 . 9793  0.9615  0.9831  M.S.  0.0123  0.1870  0.0713  0.8493  0.7500  0.8332  R.M.  0.0110  0.1195  0.0970  0.9011  0.9549  0.9765  F.A.  0.0228  0.1405  0.1698  0 . 9265  0.9159  0.9654  B.A.  0.0224  0.1873  0.1223  0.8727  0 . 9354  0.9614  Mean  0.0167  0.1637  0.1072  0.9058  0.9035  0.9439  S.D.  0.0050  0.0282  0.0362  C.V.  29.96%  17.27%  33.77%  Volunteer  Correlation Total:  Saliva  Free:  (r)  Saliva  Total:  Free  -  salicylate  73 -  i n turn d i s p l a c e s VPA from i t s  and K o c h , 1962). VPA i s a l s o l i k e l y protein  binding  protein  b i n d i n g of CBZ, whereas  the  sites.  serum b i n d i n g  of  CBZ. Our  finding.  In  slightly total  the  reduced  VPA  has  by the in  been  (from  study  0.1334  concentrations  after  normal  a  study  of  subjects,  effects  Bowdle  et  steady-state  levels  of  results  showed  a  also  concentrations free  fraction  reduced  of  two  i n VPA t o t a l  a drug  total  s i n c e the  metabolism the first  free  and was done fraction  of  a general  The  elimination  of  distribution rate  constant  did  1979).  in  the  to  vitro  VPA was  vivo,  on  actually  lower CBZ  in  in  VPA  serum  does  not  kinetics  that  significantly  the  after  VPA  serum  An  and  CBZ. and  1976).  in  an  the  increase  the  rates,  unchanged  However,  Our  saliva  increase  hence  free  decrease  in  to  not  were  measure of  the  any  of  free  effect  CBZ on VPA of  CBZ on  CBZ on VPA metabolism  (M.Sc, change  effect  1987). but  increased  that  It  was  found  clearance  significantly  in  minimum  increased clearance  study on the  effect  s t u d i e d by Sukhbinder Panesar  VPA volume  and L e v y ,  d i d not i n c r e a s e a f t e r CBZ.  specifically  VPA.  change  of  found  observed cannot be due to  T h i s work was p a r t of  reduces  not  of  in  on carbamazepine.  and S e l l e r s ,  for  sites.  decrease  results  VPA  above  because  (1979)  levels,  free f r a c t i o n  the  fraction  Hence,  decreased  usually  (Koch-Weser  levels  al.  weeks  (Levy  of VPA compared  carbamazepine  significant  steady-state  concentrations  fraction  after  VPA  of  that  Patel  confirm  0.1072)  binding  the  i n plasma  CBZ to VPA d i d  affinity  free  CBZ.  vitro  1982;  also  to  d i s p l a c e VPA from plasma p r o t e i n In  al.,  the  in  of  binding  results  present  shown  addition  greater  vivo  sites  to compete with CBZ (75% bound)  of VPA (Mattson et  T h i s was e x p l a i n e d that  It  binding  and  was that the  (p<0.05)  -  in  the  five  volunteers  74  after  -  CBZ a d m i n i s t r a t i o n .  This  suggests that CBZ  induces the metabolism of VPA.  2.  Serum f r e e VPA l e v e l s  The of  serum f r e e  levels  ultrafiltration.  because of i t s obtained  simplicity  and e q u i l i b r i u m  (Levy et  al.,  ug/mL  Lig/mL.  VPA c o n c e n t r a t i o n s  compared  The  free  concentration (e.g.,  to  total  in  VPA  concentrations  over  ranged  concentrations were  highly  of  mean ± S . D . ,  after  CBZ, Table  For  all  except  one  decreased with  a decrease i n t o t a l  For B . A . there  was no change i n  the t o t a l  VPA c o n c e n t r a t i o n  The o v e r a l l ±  dependence  mean f r e e  0.0362 of  t i o n s of l e s s  after  free  than 50  agreement  with  (B.A.)  free  fraction  fraction  was  from  13.71  13.26  with  total  the  possibly  4  10).  less  9  evident  and  to  to  to  The  8) free  concentration  free i.e.  fractions a f t e r CBZ.  because most of  than 30 Lig/mL.  was 0.1334 ± 0.0343 b e f o r e  (Tables  be  1984).  (Tables  VPA c o n c e n t r a t i o n ,  v a l u e s were l e s s  CBZ  fraction  and were more or  volunteer  to  al.,  correlated  r=0.9439 ± 0 . 0 5 ,  to 0.23  found  92.63  studied  from 0.05  dialysis  no a d s o r p t i o n of VPA  range  dependent.  in  study  technique  l e v e l s of VPA  were  concentration  varied  are  dialysis  the  fractions  0.1072  that free  device (Nau et  this  the  equilibrium  1984b). There i s  f i l t r a t i o n membranes or f i l t r a t i o n free  employing  over  and a p r e v i o u s r e p o r t  by u l t r a f i l t r a t i o n  The 0.81  VPA were measured  We chose u l t r a f i l t r a t i o n  strongly correlated t o the  of  10).  even  at  ug/ml. The f r e e  fractions  p u b l i s h e d data  for  This  concentration  VPA t o t a l obtained  epileptic  CBZ and  in  patients  concentrathis  study  (Otten  et  - 75 -  TABLE  11. Decrease [%) 1n average VPA c o n c e n t r a t i o n a f t e r CBZ administration.  Volunteer W.T.  Serum T o t a l 32.15  Serum Free 44.24  Saliva 38.85  M.S.  28.89  54.49  47.21  R.M.  21.94  13.91  40.85  F.A.  29.97  40.13  55.56  B.A.  26.58  31.49  58.20  Mean  27.91  36.85  48.13  S.D.  3.481  13.649  7.701  -  al.,  1984;  Garnett  et  al.,  c o n c e n t r a t i o n and f r e e to free  concentration  before  and  after  concentration  fraction  for  versus f r e e  al.,  all  fraction  In  saturable  addition,  and  free  level  In  spite  free  (Figure  nature  concentrations variability.  is  the  concentrations  that  the  of  plot  of  VPA total  values  total  VPA  appears to curve upwards  assumed  because  study  can  to  be  be  be  situations levels  the  rather  (Levy  fatty  a  do not  total  predicted  and  from  and  of  reflect active  free  VPA,  total  VPA  intra-subject  drug m o n i t o r i n g than the t o t a l  acid  number  pharmacologically  inter-  where  by  VPA l e v e l s  between  high  studies  by f r e e  modified  the  accurately the  and other  affected  correlation  of  the m o n i t o r i n g of the f r e e  this  and  binding,  range of VPA c o n c e n t r a t i o n s used  reasons, total  strong  in  The  22)  VPA b i n d i n g i s  cannot  Hence,  total  of VPA plasma p r o t e i n  in  salicylates,  which of  than  between  dependent b i n d i n g of VPA.  d i s e a s e c o n d i t i o n s . For these  form.  volunteers).  as demonstrated  1986).  concentration  the  The c o r r e l a t i o n  i s not c o n s t a n t over the  therapeutically et  1983).  (0.336 v s . 0 . 7 8 7 ; c a l c u l a t i o n based on a l l  showing the c o n c e n t r a t i o n  free  -  f r a c t i o n s was much lower  CBZ  Because of the  76  is  is  required,  likely  to be  more u s e f u l .  3.  S a l i v a VPA l e v e l s  The  saliva  from 0.175 13.26 ratios  to  concentrations  t o 3.23 92.63  are  VPA i n  ug/mL compared to  ug/mL.  given  of  in  The  4  to  8.  five  serum t o t a l  time-averaged  Tables  the  saliva The  mean  volunteers  ranged  VPA c o n c e n t r a t i o n s to  total  ratios  and for  of  f r e e VPA the  five  - 77 -  O  v  °  n  •„  •  R.M.  •  B.A.  v  F.A.  9 O •  •  O •  "  "  1  1  1  1  1  0  2  4  6  8  10  12  Time, h F i g u r e 14.  Saliva concentration-time steady s t a t e VPA.  profiles  for  five  volunteers  at  - 78 -  Time, h F i g u r e 15.  C o n c e n t r a t i o n - t i m e curves f o r serum t o t a l , s a l i v a VPA i n one v o l u n t e e r (M.S.).  serum f r e e  and  -  79  -  1CH  Time, h F i g u r e 16.  C o n c e n t r a t i o n - t i m e curve f o r serum f r e e VPA b e f o r e and CBZ a d m i n i s t r a t i o n i n one v o l u n t e e r ( W . T . ) .  after  - 80 -  10-i  0.1+  0  6  8  10  Time, h Figure  17.  C o n c e n t r a t i o n - t i m e curve f o r s a l i v a VPA b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n i n one v o l u n t e e r ( W . T . ) .  Figure 18.  R e l a t i o n s h i p between serum t o t a l i n one v o l u n t e e r ( W . T . ) .  and s a l i v a VPA c o n c e n t r a t i o n s  1.2  •  Before CBZ  O After CBZ  S e r u m free V P A , u g / m L  Figure 19.  R e l a t i o n s h i p between serum f r e e and s a l i v a VPA c o n c e n t r a t i o n s i n one v o l u n t e e r ( W . T . ) .  Figure 20.  The r e l a t i o n s h i p between serum t o t a l t r a t i o n s in a l l f i v e volunteers.  and s a l i v a VPA c o n c e n -  3.5-1  2.5-  S e r u m free V P A , u g / m L  Figure 2 1 .  The r e l a t i o n s h i p between serum f r e e and s a l i v a VPA c o n c e n t r a tions in a l l f i v e volunteers.  - 85 -  0.25-1  0.20-  O  0.15-  D  .£  • • ••  0.10-^  • •  •  0.05-  0.00-  i  10  I 20  i  30  i  40  I—  50  i  60  70  Serum total VPA, ug/mL  F i g u r e 22.  A p l o t o f f r e e f r a c t i o n versus serum t o t a l c o n c e n t r a t i o n o f VPA ( a l l values before and a f t e r CBZ f o r a l l f i v e v o l u n t e e r s ) .  -  volunteers  were 0.0243 ±  total  saliva  and  0.0167 ± averaged  to  to  serum  similar  to  ratios  in  The  saliva  total 9).  CBZ ( T a b l e obtained  10)  (0.1723, three  of  four  0.1536,  of  volunteers  the  free  ratios  and d i d  CBZ. However,  this  9).  in  of  These  of  of  time-  volunteers saliva  0.0185,  the  0.0180)  volunteers,  the  to  were  The  as were the  (0.0160,  true  ratios  five  change markedly  was not  saliva  10).  the  0.1544)  four  not  for  CBZ ( T a b l e  0.1522,  were s i m i l a r  before  ratios  ± 0.0625,  (Table  ± 0.0282 a f t e r  free  to  and 0.1892  respectively  0.0050 and 0.1637 saliva  -  0.0086,  free,  were very  (Table  86  after  from the saliva  values  to  total  ratios. The with  saliva  to  serum  p u b l i s h e d data  al.,  1977)  but  (single-dose  for  were  study)  total  healthy higher  saliva  to  al.  (1982)  and Acheampong et  free  ratios  v a l u e s found f o r e p i l e p t i c The c o r r e l a t i o n s and f r e e  a.  Saliva analytical  than  were  patients  between  al.  of  general  those  (multiple-dose  study).  reported also  by Abbott  comparable  et to  1980).  s a l i v a VPA c o n c e n t r a t i o n s the  and both  majority  of  correlation  serum cases  between  and s a l i v a VPA c o n c e n t r a t i o n was a l s o s t r o n g .  Analytical  VPA  aspects of s a l i v a VPA  concentrations  have  been  quantitation  measured  t e c h n i q u e s ; GC (Blom and G u e l e n , 1977;  Acheampong e t  al.,  Gugler et 1982);  no c o r r e l a t i o n  was  al.,  1980);  EMIT  (Monaco  found  between  et  by  saliva  a  Gugler e t  GCMS (Abbott al.,  et  (1982)  al.,  good in  et  (Gugler al.  and were  (Gugler e t  agreement  doses  Abbott  (1984)  to  (1984)  in  on m u l t i p l e  those  similar  al.  were  As seen i n F i g u r e 21 the o v e r a l l  Fung and Ueda, 1982;  assay,  volunteers  c o n c e n t r a t i o n s were very  ( T a b l e s 9 and 1 0 ) . serum f r e e  ratios  and Acheampong et  The  total  VPA  1982). and  et With  number  of  al.,  1977;  al.,  1980;  the  EMIT  plasma VPA and  in  -  more  than  60%  correlations  the  in  high its  serum,  saliva  level  of  it  be h i g h l y  analytical  can  saliva  constant  in  concentration  could  intention  do  factors.  the  near  Gugler  zero.  The  al.  (1977)  of the  saliva  et  variability  The  other  it  and t h a t  al.,  was  of  estimated  VPA i n  b.  Since saliva  the  factor  is  of pH on the  secretion dependent  at  plasma  saliva  In  of  the  is  because  pH and  the  low  measure  a of  hence  concentration  addition,  not  interest  two  equal to  existed,  from  salivary  by  to  find  of  saliva  by  so  was  that  most  use  of  unbound  whether  the  levels.  accurately  the  the  out  incorporating  saliva  It  two  a  unbound was  our  important  ion GCMS assay  down  to  about  reproducible  2  and  of s a l i v a samples.  saliva concentration  relatively  upon  ratio  and there  suggested t h a t  correctly  are  study  serum and  concentration  consistent  s e n s i t i v e and p r e c i s e negative  important  The e f f e c t  a  the  1977).  VPA  the  unbound  obtained,  the  pH. to  of  present  not  ionized  difficult  between be  is  on s a l i v a  et  the  had shown that  erratic,  has been  saliva  levels  We used a h i g h l y  can measure  It  to  be  free  s t a n d a r d i z e d methods of c o l l e c t i o n  into  were  previously  equal  often or  (Gugler  plasma,  relationship  to  into  prove  techniques  concentration  not  be mostly  dependent  may  Although  ng/mL.  VPA i s  VPA w i l l  excreted  concentration  reported  variability.  pKa ( 4 . 9 ) ,  VPA w i l l  that  of  s a l i v a and serum t o t a l  low  levels  and i n t e r - s u b j e c t  work  levels  intersubject  little  saliva  ratios.  concentration  between  cases  a high i n t r a -  The VPA s a l i v a saliva  -  found by Blom and Guelen were poor.  a l s o reported t o plasma  of  87  strong  pH of  acidic  saliva  and  of VPA  and  basic  serum  drugs  (Mucklow,  -  1982),  it  is  generally  concentration highly  of  inflated  effects  assumed t h a t  VPA.  However,  Abbott  those  calculated  using  saliva  pH by c o n v e n t i o n a l saliva  epithelial  cells  diazepam,  VPA.  it  It  et  al.  is  of  the  diazepam c o n c e n t r a t i o n  salicylate,  salivary in  (a weak base) likely  a  to  1980).  does not appear t h a t the drugs  was f e l t t h a t i t stimulation interval be  reflect In  with  rather accurate  salivary  a study of  pH,  reasonably  observed  it  is  citric  get  was  fully  measure  of  possible difference  in  (Koup  not  the by  et  in et  contact al.,  are  al.,  salivary 3),  to  1980).  and c o l l e c t i n g  For  than  the  Since  the  the  case  (Levy  et  salivary  (Abbott, saliva was  constant  by  citric  background,  found  with  that  following  it  over  a given  or  otherwise  the  relationship  time either  would  not  glands.  unpublished data) flow  al.,  s a l i v a c o l l e c t i o n by  sample  acid)  of  excretion  than measuring whole mouth s a l i v a pH which would (affected  in  intra-subject  salivary  this  may  diazepam  In  pH  r o l e of pH i n the  to s t a n d a r d i z e  of  high  saliva  Against  the  1975).  higher  pH. a  with  plasma r a t i o s  concentration  (pKa  related  al.,  accurate  levels  understood.  acid  for  saliva  (Gier  (1984)  1974)  et  an  the  al.  and  diazepam  was more important  the pH i n the  stimulation  saliva  values  s a l i v a to  acid  was  individual  have  obtained  although  affected  variation It  glands  plasma  strong  that  to  pH o f  is 3.3,  be  relatively found  the  found t h a t  free  not  difficult  salivary  good c o r r e l a t i o n ,  is  the  Acheampong e t  (Matin  means because of  show  saliva  affect  (1982)  experimentally equation  compared to  has been  pKa of diazepam  the  Matin's  levels  mixed  of  between  saliva  not  pH w i l l  or f r e e l e v e l s of the d r u g . S i m i l a r l y , discrepancies  of  saliva  s a l i v a l e v e l s of VPA which c o u l d not be e x p l a i n e d by pH  found  pH o f  88 -  the  citric saliva  stimulation  of  acid  and  pH i n by 4  six  mL o f  ensuing  between parotid  volunteers 5% c i t r i c  was acid  -  solution  retained  in  the  p l a t e a u e d between 2-3 protocol the  study,  minutes  in  -  mouth  was thus used f o r  present  89  for  and was i n the  the  the  2 minutes.  range  c o l l e c t i o n of  hope  to  The  parotid of  saliva  minimize  pH 7 . 3 - 7 . 6 .  samples  intra-  saliva  and  pH  This  throughout  inter-subject  variability.  c.  The r e l a t i o n s h i p VPA  In T a b l e s 4 t o 8 i t VPA  values  which  are  decreased c o r r e l a t i o n saliva that  to  total  or  in f o u r of  remarkably  of  the  of  0.1747  and  these between  at  in  standardized For  one  decreased the free  ratios.  five  The of  similar  values the  three  saliva of  the  the  r a t i o s were observed at  saliva  to  free  concentration  ratios  of  thus  these  5.2%.  of  similar  to  to  ratios  free  volunteers  contribute  of to  Table  free  four  11.3%,  those  obtained  could  be  in  9 shows are  for  CBZ with for  four  a mean  the  CBZ.  three  This  two weeks  attributed  is  good apart,  to  the  and p r e c i s i o n of the a s s a y .  al.  (B.A)  the  saliva  CBZ. T h i s  to  free  appears  to  VPA  ratio  contradict  (1984) who r e p o r t e d t h a t low s a l i v a  high serum c o n c e n t r a t i o n s of VPA. In  should  the  volunteers  and  before  to  ratios  Interestingly,  of  saliva  and high v a r i a b i l i t y  were observed a f t e r  0.1873-after  f i n d i n g of Acheampong et  and  occasional  examination  for  variation  volunteers  from 0.3134 t o  value  ratios  are  high  are  the mean s a l i v a  variation  sampling p r o t o c o l of  However,  mean  of  there  and t o t a l )  volunteers  coefficient  agreement least  (both f r e e  coefficient  volunteers,  volunteers  disproportionately  constant.  0.1581 with  s a l i v a and serum c o n c e n t r a t i o n s  can be seen t h a t  free  the  between  not  change  depending  upon  VPA  to  fact, serum  s i n c e any changes in f r e e VPA should b r i n g a p r o p o r t i o n a l  -  change  in  saliva  there  is  ratio  in a l l  levels.  essentially  This  no  subjects  90 -  is  consistent  difference  before  between  and a f t e r  with  the  our  results  saliva  CBZ except  for  to  since  serum  free  B . A . whose  ratio  d e c r e a s e s a f t e r CBZ. The  saliva  exhibited  to  by the  serum t o t a l  s a l i v a to f r e e  CBZ there was  a significant  least  of  in  four  the  total  decrease  concentrations.  decreases  decrease  in  (36.85)  saliva  saliva  and  (Table  9 and 1 0 ) .  Since  or  Similarly,  the  and s a l i v a to f r e e In  serum  summary, free  in  total  between  the  saliva  to  in  a  the  decreased  fraction  serum  higher  concentrations 11)  the %  corresponding %  correlation samples  between after  CBZ  was  less  variability  variability  of s a l i v a  intraindividual  three  of  the  variability  five  the  to  volunteers  s a l i v a VPA to was  reasonably  c o n s t a n t over two d i f f e r e n t sampling p e r i o d s , two weeks a p a r t . s a l i v a VPA to t o t a l  at  total  found at  VPA  the  intraindividual  ratio  CBZ ( T a b l e  The for  total  being  after  agreement  ratios.  despite  ratio  better  to  the  ratios  results  good  and a f t e r CBZ. A f t e r  that  (48.13).  was  a f t e r CBZ. There was no d i f f e r e n c e total  show the  saliva  serum  therefore,  free  the  S.D.)  concentrations  serum t o t a l  in  higher  (Mean ±  and  not  indicating  dependent,  decreased by 27.91S ± 3.48 free  did  serum r a t i o before  volunteers  r a t i o was c o n c e n t r a t i o n serum  ratio  ratios  such a r e l a t i o n s h i p  concentration  and  For  the  was not found because of  accompanying  decrease  in  free  f o l l o w i n g CBZ a d m i n i s t r a t i o n .  From t h i s  work  it  appears  the  time-averaged  saliva  to  free  ratio  once determined  c o u l d be used f o r a s s e s s i n g serum f r e e VPA by measuring  VPA  The  in  saliva.  serum t o t a l  good  correlations  and f r e e VPA c o n c e n t r a t i o n s  found  between  suggest  that  saliva  and  measuring  both  saliva  - 91 -  VPA  by  this  method  would  interaction  studies.  therapeutic  drug  routine NICI.  be  Salivary  monitoring  d.  and van Ginneken salivary  irregularities  observed with  lipophilic  clearance  i s then  independent  dependent  on  blood  decrease with  not a b l e its  to  drugs with clearance  unlikely  with  saliva  levels minutes.  dependent  salivary  of  i n the  and K i d o  levels  drug  value  for  using  and accuracy of to  detect  some a n t i c o n v u l s a n t studied  phenobarbital  after  salivary  (1982)  of  salivary  saliva Such  explain  drugs.  any  have to  introduced some of  permeation  of  a high blood  as  high  flow  and  change p r o p o r t i o n a l l y .  and  al.,  stimulation  pilocarpine,  maximum  values  because  between  Since  salivary  In a study  kinetics  showed  is  consequence  d r u g s , where the with  drug  1979).  a  drug  primidone,  ratio, as  remains the  the  urea  et  of  since  extraction flow  The s a l i v a  s a l i v a flow  and blood at  the is  ratio,  flow  (Bartels  excretion  When a drug  concentration  substances  excretion  have  is rate-determining.  blood and s a l i v a  proportional  and c l e a r a n c e w i l l  were  of  able  i n an attempt to  The  good l i p o p h i l i c i t y  flow  be  of blood flow whereas  between  solubility.  be  to  sensitivity  clearance  membrane  flow.  lipid  will  and  VPA c o u l d be useful  (1982) were not  salivary  increasing  equilibrate  poor  show flow  in  the  of  and hence with a low e x t r a c t i o n  clearance  1982)  is  (1983a)  drug across the e p i t h e l i a l  saliva  it  salivary  the concept of  of  but  pharmacokinetic  s a l i v a samples using GC.  The concept of o f drugs  Zuidema  will  measurement  Fung and Ueda  VPA i n some of t h e i r  the  for  assay methods t h a t do not have the For example,  poorly  suitable  of  (Kido,  the  10  T h i s o b s e r v a t i o n c o u l d not be e x p l a i n e d with M a t i n ' s  drugs  salivary and  30  equation.  -  With  the  addition  phenobarbital Saliva either  of  92 -  the  clearance  l e v e l s were b e t t e r to  active  parameter,  the  explained.  plasma  ratios  greater  than  transport  or  ionization  in  b a s e s ) . An a c t i v e  however,  one  can  saliva  be  explained  (relatively  t r a n s p o r t mechanism appears to be t r u e f o r  by  strong  penicillin  (Zuidema and van G i n n e k e n , 1983b). In  the  with c i t r i c  present acid.  study  saliva  Whereas t h i s  was  obtained  following  helps to e l i m i n a t e  variations  s a l i v a pH between  sampling p e r i o d s , a new v a r i a b l e  saliva  T h i s may p a r t l y  flow  observed.  rate.  VPA  is  poorly  explain  lipophilic  to  drugs such as CBZ, phenytoin and phenobarbital 1980).  However,  in  citric  the  (Abbott,  s i n c e the  acid stimulated  unpublished  transport  concentration  of VPA may be r e s p o n s i b l e f o r  antiepileptic  be  higher  non-stimulated  saliva  factors  the  s a l i v a VPA l e v e l s  other  of VPA was  other  resting  (Goldberg and T o d o r o f f ,  s a l i v a compared to  observation),  in  can be i n t r o d u c e d -  the e r r a t i c  compared  stimulation  found to  such  as  facilitated  inconsistent saliva  levels  o f VPA. It  has  been  because of i t s to  stressed  little  low pKa. On the o t h e r  serum VPA c o n c e n t r a t i o n  Guelen,  that  1977), 0 . 1 1 ,  ratio  (Monaco e t  VPA  1982)  found  in  serum and i t  reflects (West, that  the  1985). following  volunteers the  free  initial  the  In  is  drug the  generally in  plasma.  parotid  stimulation  of  saliva  The  into  normal  saliva  flow very  a c i d (7.46  and  CSF c o n c e n t r a t i o n  pH o f  ± 0.14,  (Blom  unbound c o n c e n t r a -  the  little  (CSF)  ( K i d o , 1982). These  the  with  saliva  fluid  be 0.10  the  ( u n p u b l i s h e d data)  pH v a r i e d  by c i t r i c  to  accepted t h a t  study by Abbott  stimulation  to  and 0.08  c o n c e n t r a t i o n s i n CSF are more or l e s s equal tion  secreted  hand the c e r e b r o s p i n a l  has been  al.,  is  citric  CSF i s it  7.32  was found  acid  in  six  a f t e r 2 minutes n=20).  Thus  of  there  -  is  little  is  almost a 5-10  93  d i f f e r e n c e between s t i m u l a t e d  s a l i v a and CSF pH whereas  there  f o l d d i f f e r e n c e i n the c o n c e n t r a t i o n of VPA i n the  two  tissues. Cornford et entering  out  of  (1985)  the c a p i l l a r i e s  equilibrate albumin  al.  with  in  the  the  brain.  transported  out  i n the  brain  brain  concentration  In  of  concentrations  CSF by  enhanced  in  the  same  of  this  CSF was  range  of  it  the  although f r e e  above  transport  levels  considerations, of  VPA  out  7.6  35.5  were  it of  is  to  Identification  of  in  which  VPA m e t a b o l i t e s  VPA i s  the  that  CSF ( L o s c h e r  humans  ug/mL  determined  conceivable  the  of  from  mechanism  in  that  total  (Vajda  the  plasma et  al.,  the f r e e  fraction,  in  study.  that  this  there  would  to  transported  dogs t h a t  of  found  reflects  to f r e e  dissociation  efflux  150.4  the VPA  capacity  actively  25.0%  to  not  saliva  concentration in s a l i v a r e l a t i v e  C.  anion  was  1981). T h i s CSF c o n c e n t r a t i o n a p p a r e n t l y plasma,  drug  and t h a t VPA i s  the  of  bound form has the  a c i d and probenecid out  spite  VPA  in  of  a fraction  has a l s o been demonstrated  the  1982).  protein  because  transports y-aminobutyric and F r e y ,  suggested t h a t  circulation  It  of  have  is  an  explain  in  From active  the  low  serum and CSF c o n c e n t r a t i o n s .  using  NICI-GCMS  of  their  PFB  derivatives  The urine  total  extract  ion  current  from  one  chromatogram  volunteer  selected  doses  of  [ H5]-VPA  contains  peaks  for  VPA and 14  VPA,  (E)-2-ene  (E,E)-2,3'-diene  2  VPA,  is  at shown  plot  the  VPA  steady  in  Figure  VPA m e t a b o l i t e s :  (E,Z)-2,3'-diene  VPA, a new VPA m e t a b o l i t e  VPA d i a s t e r e o m e r s , 4 - k e t o  of  VPA, (peak  PFB  state  derivatized administered  23.  This  TIC  3-ene  VPA,  (Z)-2-ene  2,410),  diene  plot  VPA,  3 - k e t o VPA, 3-OH  VPA, 4-OH VPA, 5-OH VPA, 2-PSA and 2-PGA. A l l  7  8  9  10 TIME  F i g u r e 23.  Total ion current p l o t ,  12  13 20  21  22  (min)  i n the NICI mode, o f the PFB d e r i v a t i z e d u r i n e e x t r a c t  v o l u n t e e r on VPA steady s t a t e , correspond t o :  11  from a  a l s o g i v e n s e l e c t e d doses o f [ Hg]-VPA. Peak numbers  1= VPA, 2= 3-ene VPA, 3= ( Z ) - 2 - e n e VPA, 4= ( E ) - 2 - e n e  2 , 3 ' - d i e n e VPA, 6= ( E , Z ) - 2 , 3 ' - d i e n e  VPA, 7= 2 , 4 - d i e n e VPA, 8=  VPA, 5=  (E,Z)-[ H ]2  6  (E,E)-[ H J-2,3'-diene 2  6  VPA, 9= ( E , E ) - 2 , 3 ' - d i e n e VPA, 10= 4 ' - k e t o - 2 - e n e VPA, 11= 3 - k e t o VPA, 12= 3-OH VPA, 13= 3-OH VPA, 14= 4 - k e t o VPA, 15= 4-OH VPA, 16= 5-0H VPA, 17= 2 - P S A ,  18= 2-PGA.  -  peaks o f  interest  95  were s u i t a b l y r e s o l v e d except those of 4-ene VPA and  VPA. The 4-ene VPA peak i s  swamped by the  huge VPA peak.  o f the 4-ene VPA peak under the VPA peak was v e r i f i e d mass  chromatograms  retention Figure  time  to  at  m/z  that  of  doublet  negative  injected  fragment  ion  of the ions  spectra  The n e g a t i v e  with  their  synthetic  36.  S i n c e the  standards  urine  in the  Negative  the n e g a t i v e the  ion  stabilized  sample  help  VPA (see  later  in  facilitated  by  metabolites [M-181]  is  of  labelled  (181)]~  of  process  ion  synthetic  their  reference  in  F i g u r e s 24  along  through  deuterated VPA  and u n l a b e l l e d  ions were  f o r the drug and m e t a b o l i t e s .  carried anion.  anion the is  is  by  a  This  formed  24  to  37  as t h e i r  single  highly under  PFB-oxygen bond. dissipated  the  are p r e s e n t e d . A l l -  of  in  ion mass s p e c t r a of VPA and i t s m e t a b o l i t e s  current  by c l e a v a g e  Figures  was  contained mainly  base  negative  the  abundant,  anion,  electron  energy  this  soft i o n i z a t i o n  ion  almost  resonance  dissociative  cleavage,  from  electron process.  spectra  of  and the  parent drug have  the m e t a b o l i t e s peak,  fragment  S i n c e excess  by bond  c a p t u r e NICI t e c h n i q u e i s a h i g h l y e f f i c i e n t  the  identical  undeuterated)  illustrated  analyzed  intensities  carboxylate  ionization  In  had  the  ion s p e c t r a of PFB d e r i v a t i z e d VPA m e t a b o l i t e s  [M-pentafluorobenzyl  the  4-ene  and  the  are  s p e c t r a obtained  capture  peaks  ion s p e c t r a of these u r i n a r y m e t a b o l i t e s  not equal  of  synthetic  with  the  all  these  isolated metabolites  and m e t a b o l i t e s ,  In  of  (deuterated  and  compounds.  1.  One  by o b t a i n i n g  37).  The i d e n t i f i c a t i o n the  141.  The e x i s t e n c e  only  exception  VPA  and  being  its  3-keto  - 96 -  149  100 t . A)  CHj-CHj-CH^  0  >  'CH—C—O-r-CHs-CeF "2 6 5  CH^CH^  -  L  143 ,2, ( \ .  r  149)  d  _1  LU  CC  143  [  0 I. 50  B)  150  r  250 M/Z  143  100  X.  CH^—CH^-Cr^ CH—!!—04CH«-C.F 2 6 5 n  k  r  CH^—CH^—C^ 143-  LU  a:  0  1 .  50  250  150 M/Z  F i g u r e 24.  NICI mass spectrum o f A) VPA-PFB ( H and B) s y n t h e t i c VPA-PFB.  0  - 97 -  A)  147  100 _  CH CH5-CH,  0  r  /  H  -  C  - ° t  C  H  2 -  C  6  F  5  CHj-CH=CH 141 — ( H , 147) Z  6  141  50  150  250  M/Z  B)  100 _  350  141  CH CH CH, "3 ""2 " 2. r  r  n  CHj-CH = CH  /  0 C H - C — O + CH^-CgFj  141-  50  F i g u r e 25.  150  M/Z  ~i 250  350  NICI mass spectrum o f A) 3-ene VPA-PFB ( H 23) and B) s y n t h e t i c 3-ene VPA-PFB. 2  u  + H ) 2  n  C  6  (peak 2 , F i g  -  A)  98 -  147  100  CH5-CH5-CH —C—O-j-CHj-  C  6 5 F  CHj-CH^-CH' 141 ,2, I H , 147) 6  141  LU  cr  D J_ 50  150  250  M/Z  B) 'DO » _  141  CH^—CH^—CHj "2  u  6'5  CHg-CHj-CH  in z  141  LU  UJ  > LU  0  X_  50  F i g u r e 26.  150  M/Z  250  350  NICI mass spectrum o f A) ( E ) - 2 - e n e VPA-PFB ( H F i g . 23) and B) s y n t h e t i c ( E ) - 2 - e n e VPA-PFB.  0  +  H ) 6  (peak  4,  - 99 -  A) 100 _,  144  >i—  CHr-CHr-CH, 3 2 2  CO  N  0 ii C-C-0+CH5-C F " 2 f 5 t  l  CHj=CH-CH  c  139 — ( H , 2  5  144)  en 139  J 50  L  150  250  M/Z  B)  350  139  100  >i—  CH-CH -CH 2  CD  0  2 x  C-C-0  UJ  CH -C F 2  6  5  CH^CH-CH 139  or  50  F i g u r e 27.  150  M/Z  250  350  NICI mass spectrum o f A) 2 , 4 - d i e n e VPA-PFB ( H F i g . 23) and B) s y n t h e t i c 2 , 4 - d i e n e VPA-PFB. 2  + H ) 2  0  5  (peak  7,  100  -  A) 100 _,  -  145  >C-Hr-CH = CH 3  UJ  \  2 C Hj-CH^-CH  0 II C - C - 0 + CHs-C,F 2 "6 5 # r  145-  cc  50  100 _  B)  150  1  250  M/Z  1  350  139  >CHr-CH=CH  CO  3  \  0 II C - C - 0 + CH -C F 2  LU  6  5  CHj-CH^-CH 139-  CC  50  F i g u r e 28.  I  150  M/Z  I  250  1  I  350  NICI mass spectrum o f A) ( E , E ) - 2 , 3 ' - d i e n e VPA-PFB ( H ) 8, F i g . 2 3 ) and B) s y n t h e t i c ( E , E ) - 2 , 3 ' - d i e n e VPA-PFB. fi  (peak  - 101 -  A)  100  X_,  158  0 II CHj-C—CH  2  0 C— C—O-KH^CgFj  CHJ-CHJ-CH  155 /2 ( H , 158) 3  > i— cr _j LU CC  0  X.  ~T 300  ~~1 400  M/Z  100  %_  111 ( M - 1 5 9 )  +  114 ( M - 1 5 9 +  2  H )  +  3  213  to  >  (M-57)  +  ,216 ( M - 5 7 + H ) 2  +  3  171  cx _i LlJ  CC  iee  141  270 ( M ) 0 X.  i  r  200  J  +  . 300  m/z  Figure  29.  2 2 A) NICI mass spectrum o f 4 ' - k e t o - 2 - e n e VPA-PFB ( Hn + H ) (peak 10, F i g . 2 3 ) , B) EI(t-BDMS) mass spectrum o f 4 ' - k e t o - 2 - e n e VPA. 3  -  A)  100  102  -  119  X_,  0 CH^-CH^-C  . p C H - f - C - 0 - C H «r- C 6F , s r  CH5-CHj-CH  2  z  LU  113-1  158  ( Hg, 2  119)  »—  cr _j  LU  CC  163 113 0 t. 50  B)  100  i—  r-  150  M/Z  250  i  1— 350  113  CH^CH^-C \ ,  2  0 I II CH4 c ' — O - C H p C g F j  CHj-CH^^ 113  >  337  cc i—  _J LU CC  0 I. 50  150  250  350  M/Z  Figure  30.  NICI mass spectrum o f A) 3-keto VPA-PFB ( H + F i g . 23) and B) s y n t h e t i c 3-keto VPA-PFB. ° 2  n  2  H ) C  6  fneak  11  -  A)  103 -  165  too t .  OH I CHj-CH^-CH^ CH—C-O+CHy-C^F,  TVs  159 159 ,2, ('H ,  in  6  165)  UJ  0 t_l_ 50  350  250  150  M/Z  159  B)l00 x .  OH CHT-CH5-CH  0 CH—C—0-  CH C F r  6  5  159 -  in z UJ  z  cr _i  UJ  on  0 x_ 50  F i g u r e 31  250  150  350  M/Z  N I C I mass spectrum o f A) 3 - 0 H VPA-PFB ( H Q + H ) and 1 3 , F i g . 2 3 ) and B) s y n t h e t i c 3 - 0 H VPA-PFB. 6  (peaks  12  -  A)  100  104 -  160  X .  I  CHs-C-CH 2  157  >— t  - -CH- c ' - o i •CH^gFg  CHy-CH2-CH  2  157 ,2, ( H . 160)  to  3  CC  0  X.  50  i ,a  ,  L  350  250  150 M/Z  B )  157  I00 X  0 U CHT-C—CH, 3  2  0  \  II  C H — C — 0 + C H 5 5 -LC , rF  "T 6 5  CHJ-CHJ-CHJ  157-  LU  0  X.  50  350  150 M/Z  F i g u r e 32.  2 2 NICI mass spectrum o f A) 4 - k e t o VPA-PFB ( Hg + H ) F i g . 23) and B) s y n t h e t i c 4 - k e t o VPA-PFB. 3  (peak 14,  - 105 -  165  A) 100 _  >OH CD  CHr-CH-CH, •» * \  0 II CH-C-04CH5-C,F "2 6 5 L  CHj-CH^-C^  r  159 ( H , 2  6  165)  or 159  50  1  r  150  250  M/Z  B)  350  159  100 _  >OH I CH CH-CH,  CD  3  r  LU  2  v  0 II C H - C - 0 + CH5-C F "2 "6 5 c  c  CHj- CH^-CH^ 159-  LU  50  i  r  150  M/Z  F i g u r e 33.  250  350  NICI mass spectrum o f A) 4-OH VPA-PFB ( H 23) and B) s y n t h e t i c 4-OH VPA-PFB. 2  n  +  2  H ) fi  (peak  15, F i g .  -  106 -  164  A) 100 _ ,  >I—  OH I CH5-CH5-CH,  CO  z  2  2  2 v  LU  0  v  11  ,  ^6 S  C H - C - O f CH=-C F "2 £  I—  CHj-CH^-CH^  z  r  C  159 LU  ( H , 2  5  164)  cn 159  50  150  M/Z  B)  '0L1  250  350  159  >— t  OH I CHj-CH^-CH,  CO  z  LU  0  „ H —C—0-l-CH=-C F 2 6' 5 CHj-CH^CH^ v  c  159-  50  150  M/Z  Figure  34.  250  350  NICI mass spectrum o f A) 5-0H VPA-PFB ( H 23) and B) s y n t h e t i c 5-0H VPA-PFB. 2  + Hc) 2  n  (peak 16, F i g .  -  A)  107 -  342  100 _ F C ^H -0-C-CH 5  6  0  2  ^CH-C-0 CH -CH -CH 3  2  C H  2- 6 5 C  F  2  339  CO  ( H ,342) 2  3  UJ  LLJ >  UJ  cc 334  144  u  J  100  B)  200  400  300  M/Z  339  100 _, jj  F C -CH -0-C-CH^ 5  6  2  ,CH-C-0  >(—  C H 2  CH -CH -CH 3  CO  2  -V  5  2  339  UJ  UJ  cr UJ  cc  100  F i g u r e 35.  ^  200  I M/Z  I  400  300  NICI mass spectrum o f A) 2 - P S A - d i P F B 23) and B) s y n t h e t i c 2 - P S A - d i P F B .  ( H + 2  n  2  Ho) (peak 1 7 , F i g .  -  A)  108  -  356  100 »_,  F  5 6C  C  35  H  2-°-^  H  2-  C  H  2  N  /  C  CHJ-CHJ-CH  8 , H-d-0-[cH C F R  353 ,2 ( H .  B)  100  250  -i—  450  350 M/Z  5  356)  3  150  6  2  1  I  5 6  2  2  2\  S CHj—CH^—CH^  » CH-C—0+CH=-C,F  "2  V 5 C  353-  150  Figure  36.  250  350 M/Z  i —  450  NICI mass spectrum o f A) 2-PGA-diPFB ( H_ F i g . 23) and B) s y n t h e t i c 2 - P G A - d i P F B . 2  1  + H,) 2  (peak  18,  -  109  -  A)  m/z  141  6.5  7.0  I—  TIME  B)  too _  —I—  7.5  8.0  8.5  (min)  141  |CH -CH -CH 3  2  2  CO  CH-C-O+CH - C , F 2 6 5  z.  C  LU  |CH = 2  CH-CH  2  141-  —  50  150  M/Z  F i g u r e 37.  250  1 350  A) mass chromatograms at m/z 141 from F i g . 23 (peak a = 4-ene V P A ) , B) NICI mass spectrum o f s y n t h e t i c 4-ene VPA-PFB.  -  YPA.  The  complex peak  negative than  is  ion  that of  m/z  113  110  spectrum of similar  (m/z  119,  2  %),  (M-181) and 337  major  113  presence  is  m/z  of  a  (decarboxylation) mechanism of the Under  rather  3-keto of  the  metabolites  reactivity  of  For  via  hydroxyl amine)  and  groups case,  and  into  m/z  that  fragment  ion  of  report  the  the  of S t r i f e  level  the  urinary  157  explained  the  can  be  give  the  a m/z  by  rearrangement 113  anion.  the  of  it  The  be the  group  possible  (using  i.e.  was  less  In  prominent  of VPA the  This  is the  of  two  than  to  obtain  and  PFB  electron  capturing  sensitivity.  the  mono-PFB  ester  In  this  derivative  to l e s s abundant high  The d i c a r b o x y l i c a c i d VPA m e t a b o l i t e s ,  the  with the TIC  metabolite  of 2-PSA a l s o looks h i g h .  metabolite  no  pentafluorobenzyl-  PFB oxime  pathway t h a t gave r i s e  and 3 6 ) .  most  was  h y d r o x y e i c o s a n o i d s towards was  Introduction  sensitivity  35  there  and Murphy (1984) who noted  function  carboxyl  1983).  employed,  only m o n o - d e r i v a t i v e s .  function  6-keto  di-derivative.  (Figures  and  major  o f keto and hydroxy! g r o u p s . Hence, keto  2-PSA and 2-PGA, gave d i - P F B d e r i v a t i v e s  to  other  the  derivatization  hydroxy  the  Blair,  mass i o n s o f the  appears  two  The f a c t t h a t  facilitates  of VPA y i e l d e d  because of a fragmentation  [M-181]"  more  with PFB. The base  are  a molecule should i n c r e a s e d e t e c t i o n  however,  is  (Figure 3 0 ) .  to  6 - o x o - p r o s t a g l andins  di-derivatives  (Waddell  there  (M-l)  group  conditions  i n agreement with the  PFBB.  and  than  evidence of the d e r i v a t i z a t i o n  non-  metabolite  formation o f t h i s anion i s given in Scheme 1.  the  and hydroxyl  particular  compounds d e r i v a t i z e d  f r a g m e n t s ; m/z 157 peak  this  usual  trace  i.e.  (Figure  23),  2-PGA  based on i t s  peak  height  S i n c e 2-PGA i s  enhanced s e n s i t i v i t y  o f the presence of two PFB m o i e t i e s .  base peak  Despite the  not  the  major  observed i s because  increased s e n s i t i v i t y  - Ill -  0 n  Chvchvc 3  2  o \  || CH-C—0-CH C F / c o b o  C H J - C H J - C ^  c  c  0 H  CH-C-0  ^  0 I  chychyc CH  +  C0  /  (m/z  Scheme 1.  o  2  113)  O r i g i n o f the m/z 113 anion i n the d e r i v a t i z e d 3-keto VPA.  NICI mass spectrum o f PFB  -  observed  for  the  two  112  dicarboxylic  dicarboxylic  acid metabolite,  acid  has been c h a r a c t e r i z e d  (2-PMA)  et a l .  acids,  2-PGA  and  2-PSA,  2-PMA, was not d e t e c t e d .  a  third  2-Propylmalonic  as a VPA m e t a b o l i t e  by Acheampong  (1983).  2.  PFB  as  an  electron  capture  NICI  GCMS  derivative  for  VPA  metabol i t e s  The  PFB  derivatives  chromatographic time.  properties  As with other  prostanoids) typified  (Waddell  giving  The  and B l a i r , is  anion.  -  exact  a very  PFB d e r i v a t i v e s molecule  specific  metabolites  peaks  in  a  ion r e s u l t s of  this  specificity.  technique  parameters;  is  (base  have  reasonable  good  GC run  with PFB (mainly  from cleavage of process  peak  is  Because of  s e n s i t i v e and hence ideal The PFB d e r i v a t i v e because with the  latter,  small  fragments  from  Reich  (1980)  In  always the  because  is  not  analysis  gas chromatographic  ion(s).  almost  c a p t u r e NICI and s i n c e there  have  This  mechanism  and the mass o f the monitored  enhanced  sharp  its  is the  known  1983).  out on the b a s i s of two  analyte  and  c a r b o x y l i c compounds d e r i v a t i z e d  [M-181]  bond.  GCMS SIM  with  VPA  the NICI s p e c t r a o f the PFB d e r i v a t i z e d VPA m e t a b o l i t e s  by the  PFB-oxygen  of  addition, directed  intact  the  is l i t t l e  molecule  inherent  from  less  the  time  fragmentation the  1),  sensitivity  fragmentation  carried  retention  since  away  is  of  intact  there  is  electron  system i s very  f o r SIM. is also  the  s u p e r i o r to  the m a j o r i t y  of the  derivatizing  reported  perfluoroalkyl  a detection  ion c u r r e n t  moiety. limit  For of  lfg  derivatives  i s c a r r i e d by  example, by  NICI  Stan  and  for  the  -  heptafluorobutyrate But  the  ions  molecule  fragmentation In  of  were  hydroxy  those  specific.  to  PFB  fatty  arising  acid  from  With b i s - T F M B ,  and  compounds  amines  fragmentation  bis-TFMB,  containing  produce  stability  of  the  methyl  the  derivatizing  however,  an  identical  (Figure 5B).  hydroxy  anions  acyl  groups  with  and/or  virtually  1986). T h i s i s thought  PFB acyl  esters.  pentafluorobenzoyl  free  molecular  (Ramesah and P i c k e t t ,  increased  ester  of  p a t t e r n to t h a t of the PFB was o b t a i n e d  derivatives  the  less  contrast  secondary  derivatives  monitored  and are  113  derivative  no  to be due to  compared  to PFB  derivatives. t h a t 2% ( F i g u r e 5A)  Less  base peak i n the EI  of  the  PFB ion  (m/z  181),  which  is  the  spectrum of PFB d e r i v a t i z e d VPA, i s observed in  the  NICI spectrum. The PFB anion i s p o s s i b l y not as s t a b l e as the resonance stabilized of  the  carboxylate  benzyl  anion  cation  t r o p y l i u m ion i s well the  m/z  181  electrons the  in  in  the  in  1973)  EI,  which  EI  spectrum of  a cycloheptatrienyl  and  and Murphy, rearranges  known and t h i s e x p l a i n s the  (4n+2) n e l e c t r o n  Boyd,  (Strife  rule  hence  a  would  small  amount  NICI (PFB)  versus EI  VPA m e t a b o l i t e s al.,  1986a)  as  have their  characteristic  [M-57]  for  many  VPA  and  +  of  (t-BDMS)  been  fragment the  ion  more  striking  the  m/z  stable  abundance of  the  anion)  stability  number  would  o f TT  not  fit  (Morrison  and  181  anion  was  metabolites.  s p e c t r a of VPA m e t a b o l i t e s  assayed  t-BDMS  of  The  the  be a n t i a r o m a t i c  observed in the NICI s p e c t r a o f VPA and i t s  3.  to  However,  anion ( t r o p y l i u m  and i t  only  VPA-PFB.  1984).  in  our  derivatives which  unsaturated  laboratory by  monitoring  constitutes metabolites.  (Abbott  the  base  et the peak  Nevertheless,  -  the  [M-57]  ion  +  metabolites,  is  not  114  -  the  most  3-OH VPA, 3 - k e t o  derivatizing  38.  In  addition  derivatized analysis  sensitive  the  metabolites  except  the  pyridine,  is  appears  illustrated  all  can g i v e e i t h e r  to  be  3-OH VPA does  not  the t-BDMS method a d e r i v a t i v e  VPA  is  the  is facile  the  the  base  for  the  monitored serum  EI  anion  of PFB (t-BDMS) for a l l  more  s p e c i f i c and  The keto  and hydroxy  of t-BDMS depending  the  t-BDMS  readily  reagent  in  and chromatographs  of 4-OH VPA i s not seen and 4-OH y -lactone.  Finally,  PFB  and the time i s s h o r t compared to the 4 formation.  S e l e c t e d i o n chromatograms  illustrated since  the ion  analysis  S e l e c t e d i o n chromatograms o b t a i n e d by m o n i t o r i n g ions  of  polar  and g i v e s NICI spectrum whereas  underivatized  hours r e q u i r e d f o r t-BDMS d e r i v a t i v e  4.  to  enables  With  derivatize  with  formation  NICI  mono or d i - d e r i v a t i v e s  With PFB, 4-OH VPA d e r i v a t i z e s  derivative  ion i s  VPA m e t a b o l i t e s .  conditions.  as  the  f o r 3-OH VPA i n F i g u r e  superior  VPA and t h i s  derivatization  analyzed  VPA, most  sensitivity,  The [M-181]"  for  for  ions i n c l u d i n g m/z 73 and m/z 75 from  increased  for 3-keto  detection  metabolites  poorly.  to  This  i n many ways.  metabolites  upon  moiety.  ion  VPA and 4 - k e t o  c u r r e n t being c a r r i e d by fragment the  intense  all  VPA m e t a b o l i t e s  in  Figure  sample are  3 9.  VPA and  The deuterated  contained  summarized [ H5]-VPA 2  extracted  [^Hsl-VPA  in Table  12.  metabolites  from  analogs  and i t s Similar are  a  the  urine are  appropriate sample  are  also  presented  metabolites.  The ions  SIM chromatograms  presented  in  Figure  for 40.  -  115  -  75  OH  I  0  CH I 3 -CH-C-0-Si-CH_  CH -CH -CH 3  n  II  2  C H - C H - C H' 3  2  C(CH )  2  147 1 8 7  115 129  JJLL 25  217  ( -57)  +  M  199 159  245  i 1111m 1 125  261  225 M/Z  F i g u r e 38.  EI  (t-BDMS)  mass spectrum o f 3-0H VPA.  325  -  116 -  TABLE 12. Ions (m/z) monitored in NICI mode f o r VPA and [ He]-VPA metabolites derivatized with PFB. 2  COMPOUND  UNDEUTERATED  DEUTERATED  3-keto VPA  113  119  2 , 4 - d i e n e VPA  139  144  ( E , E ) - 2 , 3 ' - d i e n e VPA  139  145  ( Z ) - 2 - e n e VPA  141  147  ( E ) - 2 - e n e VPA  141  147  4-ene VPA  141  146  3-ene VPA  141  147  VPA  143  149  4 ' - k e t o - 2 - e n e VPA  155  158*  4 - k e t o VPA  157  160*  3-OH VPA  159  165  4-OH VPA  159  165  5-OH VPA  159  164  2-PSA  339  342**  2-PGA  353  356**  * When a l k a l i  i s used to h y d r o l y z e c o n j u g a t e s , otherwise 161 and 163  * di-derivatives  -  117 -  M/Z 149  H/Z 139  10  11  12  TIME F i g u r e 39.  13  14  20  21  22  (min)  SIM chromatograms o f the PFB d e r i v a t i v e s o f VPA and [ VPA m e t a b o l i t e s i n a u r i n e e x t r a c t .  Peaks:  H ]g  1= 2 , 4 - d i e n e VPA,  2= ( E , E ) - 2 , 3 ' - d i e n e VPA, 3= 4-ene VPA, 4= 3-ene VPA, 5= 2-ene VPA, 6= ( E ) - 2 - e n e VPA, 7= VPA, 8= [ H ] - 2 , 4 - d i e n e 2  5  9= ( E , E ) - [ H ] - 2 , 3 ' - d i e n e  2  6  [ H ] - 2 - e n e VPA, 12= g  VPA,  VPA, 10= [ H ] - 3 - e n e VPA, 11=(Z)-  2  2  (Z)-  g  (E)-[ H ]-2-ene 2  g  VPA, 13=  [ H ]-VPA, 2  g  14= 4 ' - k e t o - 2 - e n e VPA, 15 and 18= u n i d e n t i f i e d peaks i n t e r f e r i n g w i t h 3 - k e t o VPA p e a k s ,  16= 4 - k e t o VPA, 17=  [ H ]2  3  4 ' - k e t o - 2 - e n e VPA, 19= 3-OH VPA, 20= 3-OH VPA, 21= 4-0H VPA, 22= 5-OH VPA, 23= [ H ] - 4 - k e t o VPA, 24= [ H ] - 5 - 0 H VPA, 2  2  3  5  25= [ H ] - 3 - 0 H VPA, 26= [ H ] - 3 - 0 H VPA, 27= [ H ] - 4 - 0 H VPA, 2  2  g  28= 2-PSA,  2  6  29= [ H ] - 2 - P S A , 30= 2-PGA, 31= 2  3  g  [ H ]-2-PGA 2  3  -  F i g u r e 40.  118 -  SIM chromatograms of t h e PFB d e r i v a t i v e s o f serum VPA and 1= 3 - k e t o VPA, 2--r H g ] - 3 - k e t o  [ H ] - V P A m e t a b o l i t e s . Peaks g  VPA, 3= 2 , 4 - d i e n e VPA, 4 6  (E,E)-  3-ene VPA, 7= ( Z ) - 2 - e n e  10= [ H ] - 2 , 4 - d i e n e 2  5  12= [ H g ] - 3 - e n e  2 , 3 ' - d i e n e VPA, 5= 4-ene VPA,  VPA, 8= ( E ) - 2 - e n e VPA, 9= VPA,  VPA, 11= ( E , E ) - [ H g ] - 2 , 3 ' - d i e n e VPA, 2  VPA, 13= ( Z ) - [ H g ] - 2 - e n e  2  2  VPA, 14=  (E)-[ H ]2  g  2-ene VPA, 15= [ H ] - V P A , 16= 4 ' - k e t o - 2 - e n e VPA, 17= 4 - k e t o 2  C  VPA, 18= r H ] - 4 ' - k e t o - 2 - e n e  VPA, 19= 3-OH VPA, 20= 3-OH VPA  3  21= 4-0H VPA, 22= 5-OH VPA, 23= [ H ] - 4 - k e t o VPA, 24= [ H ] 2  2  3  5  5-OH VPA, 25= [ H g ] - 3 - 0 H VPA, 26= [ H ] 2  2  g  4-OH VPA, 28= 2-PSA, 2-PGA.  3-OH VPA, 27= [ H ] 2  g  29= [ H ] - 2 - P S A , 30= 2-PGA, 31= [ H ] 2  2  3  3  -  Urine  and serum  controls  119  -  showed  no  interfering  peaks,  however,  the  o r i g i n o f the m/z 158 i o n i n the NICI mass spectrum of PFB d e r i v a t i z e d 3-keto  VPA ( F i g u r e 30A) i s not c l e a r  since  this  i o n i s absent  i n the  spectrum o f s y n t h e t i c 3 - k e t o VPA ( F i g u r e 3 0 B ) . In summary GCMS-NICI u s i n g PFB d e r i v a t i v e s appears to be s u p e r i o r , in  terms  of derivative  formation  and s e n s i t i v i t y  to  other  a v a i l a b l e methods f o r the a n a l y s i s o f VPA m e t a b o l i t e s . gave  mono-derivatives  di-derivatives) run.  been  used  formation  to  quantitate  derivatives saliva. VPA,  metabolites,  internal  VPA M e t a b o l i t e s  Because  of it  the  saliva  metabolites and  VPA, ( E , E ) - 2 , 3 ' - d i e n e  that  by i n j e c t i n g of  shown together  it  is  of  the  only  NICI  and i d e n t i f y  a  matter  of  VPA m e t a b o l i t e s  in  VPA, ( Z ) - 2 - e n e  detected by SIM, but i t was a l s o p o s s i b l e to  contained  standards fourteen  with PFB  VPA and 4 - k e t o VPA.  scan c o n d i t i o n s . The l a t t e r  also  method  VPA, 3-ene  VPA, 3 - k e t o  metabolites  were p o s i t i v e l y i d e n t i f i e d  chromatograms are  sensitivity  d e t e c t e d were 4-ene  them under l i n e a r sample  but  The a n a l y s i s has not y e t  standards and a p p l y i n g the method.  was p o s s i b l e to d e t e c t  The m e t a b o l i t e s were f i r s t detect  formed  in S a l i v a  high  The m e t a b o l i t e s  (E)-2-ene  metabolites  and chromatographic c h a r a c t e r i s t i c s have  and SIM chromatograms o b t a i n e d .  s e l e c t i n g appropriate  5.  acid  metabolites  and were analyzed s i m u l t a n e o u s l y i n one chromatographic  The d e r i v a t i v e  been d e f i n e d  (dicarboxylic  All  currently  for  in F i g u r e 4 1 .  [ H6]-VPA. 2  with the help o f t h e i r  retention  salivary  of  was performed on a  time  metabolites  Interestingly,  twin ions  comparison. of  The  The SIM  VPA and [ H63-VPA 2  none of the more p o l a r  -  120  -  Sjn/z 160  m/z 157  —I—  11  10  TIME F i g u r e 41.  (min)  SIM chromatograms o f t h e PFB d e r i v a t i v e s o f VPA and [ H ] - V P A g  metabolites in a s a l i v a e x t r a c t . 3-keto VPA,  VPA, 3=  6=  1= 3 - k e t o  VPA, 2=  [ H ]2  g  ( E , E ) - 2 , 3 ' - d i e n e VPA, 4= 4-ene VPA, 5= 3-ene  (Z)-2-ene  2,3'-diene  Peaks:  VPA, 7= ( E ) - 2 - e n e  VPA, 10=  [ H ] - 4 - e n e VPA, 11= 2  5  ( Z ) - [ H ] - 2 - e n e VPA, 13= 2  g  15= 4 - k e t o VPA, 16=  VPA, 8= VPA, 9=  g  [ H ] - 4 - k e t o VPA. 2  3  2  g  [ H ] - 3 - e n e VPA, 12= 2  g  ( E ) - [ H ] - 2 - e n e VPA, 14= 2  (E,E)-[ H ]-  [ H ]-VPA, 2  g  -  hydroxy  metabolites  metabolites major  appear  VPA  is  al.,1984).  level  is  unlikely these  the  was  properties  the In  much  suggesting  from  serum  (Z)-2-ene.  VPA  of  t h a t there w i l l  was found t h a t of  in  present  greater  in  In  binding  specificity  protein with  the  fatty acids,  binding  fatty  acid  i n c r e a s e s as the  the  strength  of  of  and  the  the  (Nau  Under  a the  metabolites  does not appear VPA,  and  that it  is  the e x t r a c t i o n  of  of  by Abbott  et  is  VPA  et  metabolite  al.(1986a)  was about 30  times  it that  r a t i o of Z to E isomers of 2-ene  than  in  serum or  (3.82  plasma  vs.  0.458),  protein  binding  1984). widely  in plasma i s the most Albumin  has  different  a  important  broad  structures  binding including  and many drugs b i n d i n g with high a f f i n i t y . be dependent  Fletcher,  chain length  given chain length  it  the this  there  of VPA  study  (E)-2-ene  protein  appears to  (Spector  all  isomers.  compounds  bilirubin  of  98%  that  (Z)-2-ene  recent  patients  of  since  (E)-2-ene.  serum,  transport  (Sjoholm,  fatty acids,  a  study the  A l b u m i n , the most abundant drug  extraction  VPA to  of  fact  for  saliva  in  two  the  than  pediatric  the  o f these  excess  be an i n t e r c o n v e r s i o n during  concentration  differences  is  the  levels  surprising  saliva  (E)-2-ene  saliva. of  not in  however,  The  ( E ) - 2 - e n e VPA which i s  is  proteins  intriguing,  a conversion  in  This  plasma  (Z)-2-ene  saliva.  t h a t of  isomers of 2-ene VPA from  compounds  levels  serum.  to  in  than  c o n d i t i o n s employed  i n c l u d i n g the there  is  of  experimental  in  bound  What  detected  to be higher  metabolite  metabolite  higher  were  -121  of  the  presence of  binding  1978). fatty  a single  (oleate  >  upon the The acid cis  structure  strength increases.  of  stearate).  This  of  the  binding  Also  double bond  For  for  a  increases  report  is  not  -  consistent  with  our  122  observation  S p e c t o r and F l e t c h e r  (1978)  to  isomer.  that of  its  trans  and l e s s p r o t e i n not,  however,  has a s i m i l a r et  al.,  likely  elimination Because of  its  drug m e t a b o l i t e  interaction.  The b i n d i n g  (Drayer,  1984;  no  report  to  in  binding  of  acidic  the  high  et  geometric  rat  of  were  ineffective  (Shechter  point  of  contrast  isomers of to  a linear  trans  This  may  explain  why  to  There  to  and  cis  drug (Loscher  and may  other  human  fatty  isomers  and  enantiomers there  protein of  (Vermeulen  and  trans-unsaturated  fatty  From a  a bend  saturated and  at  fatty  Boyd,  structural the  be  less  double  acids  1973).  protein  which  For  or other molecules very  VPA can  is  acids  1984).  (Morrison  plasma  fatty  a c i d s have  with each other cis-2-ene  or  in  examples  cis-unsaturated  and Hem's,  VPA i s  result  knowledge,  isomers  is  bound and  trans-2-ene  some  polar  This  stereoselective  are  saturated  conformation  r e a s o n , c i s isomers f i t  or  To our  respect  adipocytes,  acids  cis  albumin  but  oleate  more  1984).  parent  binding  1983).  trans  whereas  view,  to  with  lipogenesis  in  generally  on VPA b i n d i n g  isomers.  metabolism In  protein  al.,  stimulated  extend  are  VPA,  of c i s  s t e r e o s e l e c t i v e with r e s p e c t to  literature  1983).  in  2-ene  binding  to t h a t of the  effect  van Ginneken  stereoselective  bond  of  parent drugs ( D r a y e r ,  drugs  some extent  of  Breimer,  Drug m e t a b o l i t e s  half-life  a displacing  is  isomers  not compare the  bound than t h e i r  have  proteins  the  t r u e with t r a n s - 2 - e n e VPA which i s more p r o t e i n  1984). to  did  for  this  poorly.  bound  than  t r a n s - 2 - e n e VPA. The well  higher  be due to  facilitated  saliva level  of  a stereoselective  transport  of the  the  cis  isomer  transport  c i s isomer  into  to  of  2-ene  saliva.  VPA might  as  There may be a  s a l i v a or c o n v e r s e l y ,  the  -  trans  123  isomer may be s t e r e o s e l e c t i v e l y t r a n s p o r t e d out o f s a l i v a . In order  level  to d e l i n e a t e  the mechanism of the apparent  o f the c i s isomer o f 2-ene VPA compared to the trans  experiments binding  isomer  are  required,  including  s t u d i e s o f the pure  properties will  6.  also  have  vitro  i s o m e r s . Should the serum p r o t e i n  binding  different,  different  it  and  i s o m e r , more protein  in  is likely  pharmacokinetic  from t h a t o f the trans  that  the c i s  and pharmacodynamic  isomer.  D e t e c t i o n o f new VPA m e t a b o l i t e s  Four d e r i v a t i v e s ,  namely, TMS, methyl  were employed f o r the d e t e c t i o n derivatives  proved  sensitivity including  keto  t-BDMS and PFB (NICI)  o f new VPA m e t a b o l i t e s .  more  useful  diagnostic  metabolite  spectra  urine)  difficult  be  ester,  because  fragment  of  ions.  The l a t t e r two their New  one which appears to be 2 - ( 2 ' - p r o p e n y l ) - g l u t a r i c  and NICI  structural  to  and t y p i c a l  unsaturated  control  in  saliva  vivo  prove to be markedly  properties  EI  higher  that  of  a VPA r e l a t e d  compound  in  urine  for  this  apparent  t h a t the i n t e n s i t y  new  o f the deuterated  The  been  2-(2'-propenyl )-glutaric 4-ene  (absent  VPA i n  acid the  has  Rhesus  monkey  in  acid.  The  is  made  metabolite  those o f other  of  a c i d and an  In F i g u r e 42 a r e shown the  the EI and NICI s p e c t r a are much l e s s than  metabolite  metabolites  appears to be 2 - ( 2 ' - p r o p e n y l ) - g l u t a r i c  assignment by the f a c t  were apparent.  superior  ions i n both metabolites.  characterized (Rettenmeier  as et  a  al.,  1986a). The  mass  characteristic  spectra twin  of  the  fragment  unsaturated  ions that  help  keto  metabolite  reveal  its  contains  identity  with  -  124 -  351  100  >  A  >-  F  5  C 6  -  C H  2-°- C  C H  2- V  0  C  ^CH-C-O-  r— CH =CH-CH  CO  2  C H  2- 6 5 C  F  2  351  ,2 ( H ,353) 2  r—  cx _l LU  331  100  200  353  300  M/Z  400  343 (M-57)  B) 100 _  +  tBDMS-0-C-CH -CH , 2  2  CH-C-O-tBDMS CH =CH-CH 2  / 2  ^  73  147 2,36  LU  0£  B45 115  50  Figure 42.  133 385  150  T 250  M/Z  T 350  I— 450  NICI (A) and EI (B) mass s p e c t r a o f VPA r e l a t e d m a t e r i a l u r i n e t h a t appears to be 2 - ( 2 ' - p r o p e n y l ) - g l u t a r i c a c i d .  i  -  some c e r t a i n t y .  Diagnostic  fragment  s p e c t r a o f the d e r i v a t i v e s of  known m e t a b o l i t e s  in  s p e c t r a o f these d e r i v a t i v e s  are  m/z  ( H3,114)  is  2  213  ( H3, and  +  pathway  metabolite  shown in Scheme 2.  ( H3,  158)  2  apparent [M-57] and  [M-181]  the  six,  twin  three  of  metabolite. having  a  exchange  for  procedure,  the  for  [M-181]"  ion  -  of  the  i o n s were the  compounds  hydrogen urine  2  sample  F i g u r e s 43 and 44 the  derivatives  without a l k a l i n e spectra  of  the  of  are  221), is  at  (157, position  the  in order EI  new  new m e t a b o l i t e  4  and  hence  the  new  spectrum, m/z  155  spectra keto  have  VPA  lost  (Hsia  et  atoms In  is  215 157).  from al.,  of the  1976 )  can  readily  the  work-up  F i g u r e 29 were  to hydrolyze c o n j u g a t e s .  and  sample.  and 4 - k e t o 117),  4-keto  respectively, VPA  obtained  In both the EI and NICI  VPA the  (213,  indicates new  (the  instead  been  in  was  VPA i s  daltons  spectra  it  compound  and NICI mass s p e c t r a  urine  the  of  4-keto  three  metabolite  ((111,  This  of  111 The  solution. the  m/z  and 4 - k e t o  deuterium  alkaline  treatment of the  163)).  the  from which  the  s e p a r a t e d by s i x mass u n i t s  must  the EI mass  ion.  these  of 3 - k e t o  those  NICI mass  +  NICI mass  only  the mass  [M-57] ,  derivative  From  by  In  t-BDMS  methadone^  C H2,  in  and  molecular  the  atoms like  to  EI  the  PFB d e r i v a t i v e  separated  a  to  was an unsaturated  o b t a i n e d was t r e a t e d with a l k a l i  of  In  anion.  deuterium  group  is  the  t-BDMS d e r i v a t i v e s  With keto  270  new m e t a b o l i t e  i o n of the  +  proposed  the  t h a t the  the  Since  is  The  corresponds  m/z  in  were compared to  presented in F i g u r e 29.  fragmentation is  ion d o u b l e t s  identification.  216)  2  [M-159]  i o n s and  o f the new m e t a b o l i t e  aid  spectrum  to  125  219),  that  metabolite  twin (155,  ions  are  161),  the  keto  must  be  a  now (215,  group 4-keto  -  126  -  0  II  CHj-C—CH  0  2  II  ,C-C-0-SiC(CH-)-(CH_) 3'3^"3 2 y  CH -CH -CH 3  2  m/z  270 -C H 4  0  C —CHII  0  II  +  CHj- C — C H  II  CH C-CH  2  r  3  0  f >  C-C-0-SiC(CH3) (CH ) 3  3  0  9  C-C-0-S1(CH )  2  3  CH^CH^CH m/z  g  CH^CH^CH  43  m/z  213  •0-SiC(CH ) (CH ), 3  3  3  0 II  CH=-C-CH 3  2\ 0  CH^CH^CH m/z  //  139  0 in C-C  (not  observed)  -CO 0 CH -C-CH 3  CH^CH^CH m/z  Scheme 2.  111  (base  peak)  Proposed f r a g m e n t a t i o n pathway f o r the t-BDMS d e r i v a t i v e o f a new VPA m e t a b o l i t e a s s i g n e d the s t r u c t u r e 4 ' - k e t o - 2 - e n e VPA.  2  -  75  100 _  A)  127 -  117 0  fl  CHs-C—CH,  >-  CH, , C + - C — 0 — S i — CH,  hCHj-CH^CH  CO  C(CH ) 3  111-  111  ( H  117)  2  >  6 >  219(M-57 H ) 2  +  t— cr _i  3  +  6  149 43  UJ  cn  98  191 129  177  270 213  25  B)  125  225  M/Z  (M+)  276  325  75  100 _ ,  0 I) CHj-C-CH  >— — I  2  Q  \  CO  x  C  H  r  C  H  r  C  H  c  II I CH-C-0-Si-CH 3  2  C  (  C H 3  3  )  3  LU  221  (M-57+ H ) 2  6  LU  cn 193 43 101 ,1  25  Figure 43.  129  148  215  281  I I  125  M/Z  225  325  EI mass s p e c t r a o f A) 4 ' - k e t o - 2 - e n e VPA and B) 4 - k e t o VPA e x t r a c t e d from u r i n e w i t h o u t a l k a l i n e t r e a t m e n t .  -  161  100 _  A)  128 -  0  II  >—  CH -C-CH , 3  2  l o t  CO  C H  2- 6 5 C  F  CH -CH -CH 3  2  155 ,2  155  ( H ,161)  >  6  r—  cr _i  LU  CC  *<  50  150  250  M/Z  B  )  350  163  100 _  0  II  >t—  0  II  CH -C-CH . 3  2  CH-C-OfCH_-C,F  i.  CO  CH -CH -CH 3  2  o _> c  2  157(  cc  6  157  50  150  M/Z  F i g u r e 44.  H , 163)  250  350  NICI mass s p e c t r a o f A) 4 ' - k e t o - 2 - e n e VPA and B) 4 - k e t o VPA e x t r a c t e d from u r i n e without a l k a l i n e t r e a t m e n t .  -  129  -  compound. The double bond cannot be at p o s i t i o n 4 ' evidence  for  metabolite -2-ene  the  loss  of  may be one o f  unsaturated metabolites  level  peak  i n serum ( i . e .  the  Rhesus  monkey  complimentary identification  corresponds structural gave aided the  to  more  the  intact  information fragment  two  GCMS  identifying The  systems  cannot  molecular  and the  were  VPA.  that  and NICI  methods  It  it  was  is  is in an  in  GCMS. The  terms  o f the  be overemphasized. NICI  and abundant anion  less  the  t-BDMS  [M-181]"  one.  characteristic  highly  known VPA m e t a b o l i t e s  new VPA m e t a b o l i t e  is  samples a n a l y z e d .  in  by EI  ionization  was o b t a i n e d ,  ions  o f the major  as a VPA m e t a b o l i t e  1986b),  both  in the p o s t u l a t i o n o f a s t r u c t u r e two  urine  than 2 , 4 - d i e n e  al.,  gave the d i a g n o s t i c  the  unlikely  o f VPA.  o f VPA m e t a b o l i t e s  PFB i n most cases  et  was d e t e c t e d  of  is  i n d u c t i o n by CBZ. The new m e t a b o l i t e  (Rettenmeier  nature  in a l l  VPA which was detected  new m e t a b o l i t e  new  2-ene VPA o r 2 , 3 ' - d i e n e V P A ) .  to be higher  u n s a t u r a t e d keto m e t a b o l i t e The  the  VPA, 4 - k e t o  possibility  be a d e r i v a t i v e  i s no  i n F i g u r e 23 (peak 10) the new m e t a b o l i t e  appears  to 3 ' - k e t o - 4 - e n e  Therefore,  4'-keto-2-ene  VPA. The l a t t e r  d e t e c t e d both b e f o r e and a f t e r related  following:  and has been d e t e c t e d  in u r i n e  atom.  would most l i k e l y  From the peak h e i g h t a prominent  deuterium  the  VPA o r 4 ' - k e t o - 3 - e n e  s i n c e the new m e t a b o l i t e  Its  a  because there  Once  derivative  with  i o n which the  NICI  under  ion, [M-57] , +  EI  which  f o r the unknown compound. Thus,  complimentary  to  each  other  in  and the new VPA m e t a b o l i t e .  appears  to be 4 ' - k e t o - 2 - e n e  this  structural  assignment should be c o n s i d e r e d t e n t a t i v e  D).  F o r the sake o f convenience the new m e t a b o l i t e  VPA although (see s e c t i o n  has been  referred  -  to  as 4 ' - k e t o - 2 - e n e  detected  in  urine  130  VPA i n under  the  text.  linear  scan  The 4 ' - k e t o - 2 - e n e conditions  only.  One p o s s i b l e o r i g i n o f 4 ' - k e t o - 2 - e n e  o f the  saturated  its  in  VPA i s  serum  by  SIM  )-hydroxylation  s i d e c h a i n of 2-ene VPA f o l l o w e d by dehydrogenation of  precursor 4'-0H-2-ene  manner analogous to to  but  VPA c o u l d be  VPA, by the enzyme alcohol dehydrogenase in a  t h a t of the c o n v e r s i o n of (co-l)-hydroxy f a t t y a c i d s  (co-D-keto f a t t y a c i d s (Bjorkhem, 1972). The p o s s i b l e p r e c u r s o r  4'-0H-2-ene  VPA,  however  was  not  detected  p o s s i b l e m e t a b o l i c o r i g i n of 4 ' - k e t o - 2 - e n e by  Rettenmeier  et  al.  (1986a)  found to be a minor m e t a b o l i t e The 4 ' - k e t o - 2 - e n e 3'-double water  bond  of  VPA,  VPA c o u l d a l s o  2,3'-diene  4'-keto-2-ene  however,  no  VPA (Ron L e e ,  arise  VPA f o l l o w e d  confirms that  to  potentially  4'-keto-2-ene  VPA  also  metabolism  which  involves  pathways  such  as  hydroxy!ation.  in  from the by  evidence  one study of  M.Sc.  was  seen  thesis).  apparently  of  Addition  the  In r a t s  for  formation  Regardless,  of  metabolism  1984a). the  the  given of  4'-keto-2-ene  a mono or d i - u n s a t u r a t e d VPA m e t a b o l i t e  oxidation  variety  are  products.  demonstrates a  This report  hydration  oxidation.  unsaturated VPA m e t a b o l i t e s toxic  urine.  VPA was  (Granneman et a l . ,  VPA must be d e r i v e d from e i t h e r and t h i s  or  VPA i s s i m i l a r to the  4'-0H-4-ene  across a double bond was e v i d e n t  2,3'-diene  serum  o f 4-ene VPA in the Rhesus monkey.  o f unsaturated VPA m e t a b o l i t e s  rise  where  in  i.e.  the of  dehydrogenation,  The  complex  enzymes  and  hydration,  likely  to  give  detection nature  of  multiple reduction  of VPA  minor and  -  D.  131  Synthesis  From  the  spectral  considerations, 4'-keto-2-ene  it  VPA  was and  data felt  the  discussed that  the  synthesis  of  above new  this  and  VPA  metabolic  metabolite  compound  was  was  attempted  employing l i t e r a t u r e methods. 1.  Attempted  synthesis  of  2-(2'-oxopropyl)-2-pentenoic  ( 4 ' - k e t o - 2 - e n e VPA) v i a e t h y l 2-Propyl-4-oxopentanoic 2-bromopentanoate prepared  by  adopted  Ethyl  in  the  from  of  acid  al.  iodide  (Fedorynoki  converted the  et  by a c i d ,  used f o r  the  1978). ethyl  according  ethyl  acetoacetate This  (1983)  the  acid  to  is  preparation  was  standard were  synthetic  which  ethyl  then  route  based  on  of Y - k e t o  of the  contaminated yielded  ethyl  of  potassium  in  ester  the  yielded  small  amounts  of  carbonate  in a higher y i e l d .  was e s t a b l i s h e d by GCMS,  by  using  and  keto  presence of  of most of the  its acid. ethyl  18-crown-6  f u n c t i o n was not sulfuric  synthesized acid  The i d e n t i t y  the  product  initially  because the  the  crude  pure 2 - p r o p y l - 4 - o x o p e n t a n o i c  its  However,  with  was  acids.  by d e c a r b o x y l a t i o n and h y d r o l y s i s of  alcohol  esterification  procedure r e s u l t e d oxopentanoate  presence  al.,  affected  to  for  from  acid  Distillation  acid  Redistillation  in  and  al.  synthesized  2-Bromopentanoic  NaH (Scheme 3 ) .  (1962)  intermediate.  was  was  pentanoic  product was obtained  ester.  I,  Acheampong et  2-propyl-4-oxopentanoic  The  of  presence o f  that  acylsuccinate  ethyl  acid,  2-bromopentanoate  method of Lawessen et The f i n a l  2-propyl-4-oxopentanoate.  ethylacetoacetate.  bromination  procedures. condensed  and  acid  of e t h y l  IR and NMR (see  a c i d was since  this  2-propyl-4-  appendix).  -  132  -  CHJ-CH^-CH/J-CH^-C—OH  Br  0  PBr,  0  II  CH CHr-CHr-CH-C-OH c  o r  c i  Br EtOH H  +  0  II  CH-t-CH^CHa-CH-C-OC-H,-  3  2  do  2 I Br  0  0  II  0  M  CHr-C-CH^ C-OC H 0  II  +  C  CH CH^-CH^-CH-C-OC H r  0  c  Br NaH THF  A 0 C00C„H II I 2 5 CH=-C-CH^ 0 \ II CH-C-0C H / CH^— CH^ ""CH2 c  3  o  2  c 5  -  Cone. HC1  A  0  II  CHr-C-CH, CH  Scheme  3.  \  0  II  CH-C-OH  (I)  CH2~ CH2  Synthetic  route  for  2-propyl-4-oxopentanoic  acid.  -  Introduction attempted hydride  by the  of  the  TMS  bond  the  to  the  (Scheme  r e a c t i o n with DDQ. by  Following  the  reaction  indicated  the  presence o f  These  were  a  3:1  the  ratio  the  4).  oxopropyl)-2-pentenoic  moderate  yields  of  The  presence  GCMS a n a l y s i s  the  TMS  oxidation  ether  ether  is  almost  material  all  of  of  enol  the  remaining  time at  Sundberg,  1983).  containing  formed as  di-TMS crude  ether  product  of  ethyl of  enol.  quantities. ethyl  2-propyl-  ethyl  2-(2'-  the  are  generally  incomplete  resulting  end of the  product of 4 - k e t o VPA i s  reaction trapped  s t r u c t u r e s A and B are p o s s i b l e .  0 - TMS CH -C=CHR 3  2  governed  will  ethers  A  is  carbonyls  equal  of  ether  CH =C-CH R  ketones  and  the  enol  0 - TMS  The c o m p o s i t i o n o f  LDA  the  about  enol  products  (Jung and Pan, 1977). When the e n o l a t e  2  The  acid).  in considerable s t a r t i n g  enol  (1977).  TMS  to of  TMS  TMS enol  the  the  was p r e f e r e n t i a l l y  DDQ,  the  using  of  of  was  was observed by GCMS before  mono-TMS enol  material,  (or  and o x i d a t i o n  as a s i l y l  position  1  ether  and carboxyl  the  and  acid  enol  keto  t h r e e components i n  starting  4-oxo-2-pentenoic  The  of  with  2-propyl-4-oxopentanoate,  2  equivalents  The mono -TMS enol  indicated  or  two  compounds, a mono- and di-TMS d e r i v a t i v e s , the  2  trimethylsilyl  use  s i n c e both  enols  at  DDQ a c c o r d i n g to Jung and Pan  was  chlorotrimethyl si lane form  of  reagent  strategy  -  double  oxidation  abstracting  synthetic  the  133  B  the by  regioisomeric kinetic  By a p p r o p r i a t e  or  enolate  mixture  thermodynamic  selection  of  of  factors  experimental  unsymmetrical (Carey  and  conditions  134 0 It  CH=-C-CH,  0  II  CH-C-OH CH^—CH — C H EtI K C0 2  2  18-crown-6 THF  3  0 II  CH=-C—CH,  8  ' C H — C - O C „2"5 H / CH^- C H ^ C H 2  LDA THF TMS CI •78° O-TMS l CH - C - C H „  0—TMS ^ C = C - 0 C „2H5 n  CH^-- C H ^ C H  2  DDQ benzene 0—TMS I  CHs=C—CH  CH^ " C H 2 C H -  C —C —0C H 2"5 o  v  -  2  I OH H  +  0  II  CH C-CH^  3  r  CH — C H ^ C H  Scheme 4.  Attempted s y n t h e s i s  ^  0  II  C-C-OH  (ID  2  of 2-(2 -oxopropyl)-2-pentenoic 1  acid.  135  -  under  which  establish  an  enol ate  either  is  kinetic  s t r o n g and s t e r i c a l l y p r o d u c t formed w i l l  formed  or  a  ketone  thermodynamic  bulky and i f  be the  from  aprotic  product of  is  the  therefore,  the  product  final  p o s i t i o n s 2 or 2 ' . as the  keto  product  function will  of  kinetic will  The formation  A p o r t i o n o f the hydrolysis  of  both  of  the  TMS  was  The mass chromatograms  The mass s p e c t r a  and the  as well  at  were  different  compounds  are  possibly  2-propyl-4-oxo-2-pentenoic  2.  Synthesis  of  4-oxopentanoic  keto to  213  new m e t a b o l i t e  the  acid  from the  major  present  ether  will  conditions  bond  at  either  same c h a i n  [M-57]  times  for  two  of  effect  Following to are  +  give  the  two  in 46.  products  from  urine.  4'-keto-2-ene  geometric  the  shown  in F i g u r e  extracted  that  to  ester.  derivatized  of  spectra  these  the  retention  Since (III),  m/z  is  conjugation.  as the  from  mass  double  c o r r e s p o n d i n g mass s p e c t r a  were d i f f e r e n t the  that  the  these  to  base  was t r e a t e d with a l k a l i  extract  t-BDMS e s t e r .  peaks)  the  In  double bond on the  and  and  (two  Under  the  ether  acidification  F i g u r e 45  extraction  enol  the  dominant enol  be favored because o f  product mixture  If  control.  control.  the  possible  s o l v e n t s are used, the  kinetic  have  is  control.  r e a c t i o n LDA and THF were used and hence, the be A which  it  VPA  isomers  of  (II).  4'-keto-2-ene  VPA  starting  with  a  protected  acid  The 4 - o x o p e n t a n o i c  a c i d was converted  function protected  by means of 1 , 3 - d i t h i o l ane (Scheme 5 ) a c c o r d i n g  Hatch  et  al.  (1978).  The  ethyl  to  its  ethyl  ester  and  4-ethylenethioketalpentanoate  the  was  -  136  -  (b) 10.39  (a) 10.30 m/z  213  8  I  9  10 TIME  Figure  45.  11  12  (min)  Mass chromatograms (m/z 213) o f the t-BDMS d e r i v a t i v e s o f synthesized 2-propyl-4-oxo-2-pentenoic acid (4-keto-2-ene VPA).  -  A)  137  -  213  100 _  (M-57) 0  CH  >t— t—i CO z LU  II  f  0  -C-CHJ  3  3  C-C-O-Si-CH, I C(CH ) 3  CH-CH-CH 3  2  3  2  3  75  > CC  185  _J LU CC  43  95  31  155 171  255(M-15) liLlL 25  125  225  M/Z  325  B) 213  100 _  ( " M  5 7  )"  0  » CH3-C-CH. 0 CH ^C-C-O-Si-CH C H  3  CH -CH -CH  CO z LU  3  2  j  2  j  75 LU  cr _J  LU cc  115 43  .ul 25  171 155  270(Mt)  1"  125  M/Z  F i g u r e 46  185  225  325  Mass s p e c t r a o f A) peak a and B) peak b i n F i g u r e 45.  -  obtained (for  in good y i e l d  NMR spectrum  aldol  propionaldehyde with  methane  synthetic B , 1  see  The to  form  was  1985).  obtained  GCMS.  The  potassium crude  hydride.  spectra  of  After  Since mixture  it  mixture  followed  from  the  the  LDA  was  then  potassium  method  for  Toder,  with  dehydrated  hydride.  preparing 1982;  This  8-hydroxy,  Acheampong  and  and was then  shown  mesylated  dehydration  peaks.  Two of plot  product  to  step, these  the  homogeneous  and  treated  GCMS a n a l y s i s peaks  corresponded to  with  be  (one the  protecting  of two  by  with  of  the  them  was  geometric  group  (for  The three compounds c o u l d not be i d e n t i f i e d  EI from  data. was  not  possible  in  isolate  with mercuric  order  and Nace,  to  to  1959).  cleave The TIC  the  product,  chloride  the  in  the  the  presence  1,3-dithiolane  plot  of  the  product  isomers of the ethyl  ester of 4'-keto-2-ene  shown  Even  Figure 47.  though  c l e a v a g e o f the 1 , 3 - d i t h i o l a n e the  present case the  the  appearance  of  resulting  product  VPA. T h e i r mass s p e c t r a  salts  derivatives  deprotection  additional  Hg(II)  have  been  of  protecting  showed nine peaks, two of which (minor components) were the  in  an  with  condensed  which was  by  and  was  the TIC  desired  carbonate  (Pappas  ester  yield  as such was t r e a t e d  cadmium group  in  see a p p e n d i x ) .  mass s p e c t r a l  B-hydroxy  compound  showed f i v e  the  with  (Kende  a moderate  the major component) isomers  employed was  4-carboethoxy-2-ethylenethioketal-5-hydroxyheptane  hydroxy  product  route  4 - e t h y l e n e t h i o k e t a l pentanoate  chloride  esters  confirmed by GCMS and NMR  synthetic  formed  adopted  The  in  the  identity  The  ethyl  enolate  sulfonyl  route  and i t s  appendix). of  y '-unsaturated  Abbott, was  (85%)  condensation  propionaldehyde.  138  used  for  two are the  of a number of compounds, in  step was i n e f f i c i e n t  compounds. The  and r e s u l t e d  protecting  group  in  itself  -  139  -  0 0 II II C H ^ C - C H j - C H ^ C — OH EtOH + H 0  0 II CH -C-CH -CH -C-OC H II  3  2  2  2  5  HSCH CH SH 2  2  (C H ) 0.BF 2  5  2  3  CH5-CH„ I  2 | 2  S S 0 \ / II CH — C — C H ^ C H ^ C — O C H 3  2  5  LDA C H ^ C H ^ C H THF -78  C  CH^-CH, l 2 | 2 S S \ / CH —C—CH 3  x  CH5-CH5-CH / 3  0  2  21  11  C H - C — OC H 25 0  C  OH MSC1 Et N 3  CH C1 2  Scheme 5.  Synthetic  route for  2  2-(2'-oxopropyl)-2-pentenoic  acid.  -  140 -  Scheme 5. (Continued)  CH5-CH,  I  S  \  2  I  2  S  /  CH-—C—CH  0 CH-C-OCH 2"5  0  CHr-CH^-CH 3 2 1  0S0 CH 2  3  KH THF CH-CH„ I 2 I 2 S \  S /  CH—C-CH 3 2 9  0 n C - C — 0C„H 25  S  n  CH^CH^CH^  HgCl H 0 2  2  Q  II  CdC0„ CHj-C — C H 0 II CH C-CH 3 r  2  0  CH^—CH^—CH  3  0 || C-C-0C„H 2"5  N  I OH !H  +  0 II  CHr-C-CH  0 0  CH^-CH^-CH  II  C—C-OH  (III)  A)  141  111  100 _ 43  CH„-C-CH„  >I—  CH -CH -CH  cn  3  II , C f C - 0 - G2\ H 5  2  111-  1  LU  LU 29  184 (Mt)  55  LL 25  125  225  M/Z B)  111  100 _  0 II  CH -C-CH 3  2  >-  C-0-C" H  i—  2  5  CH -CH -CH  t—)  3  CO  2  43  z  LU I—  111-  1  z  LU > I— cr  LU 29  55  184 (Mt) I ,  25  \  11 I 125  225  M/Z Figure 47.  EI mass s p e c t r a o f the isomers o f the e t h y l 2-ene VPA A) Isomer w i t h the s h o r t e r r e t e n t i o n t i m e . B) Isomer with the l o n g e r r e t e n t i o n t i m e .  ester of  4'-keto-  -  might  have caused a c o m p l i c a t i o n  compounds hydride. After  were  observed  As a r e s u l t ,  hydrolysis of  derivatized  to at  t h a t from the  urine  and  b  from  identical  of  the  new  to  led  formation  In appears both 47)  the  the to  EI be  48.  of  and  of  ion.  VPA. the acid  In  the  4'-keto-2-ene  m/z  155  acid,  the  a  The  mass  along  with  and  same (8.12  111  are  product,  but  are  minutes  which  is that  VPA  the  m/z  111  is  the  base  peak  and  ethyl  qualitative  as  m/z  the  ion in  (Figure well  as  VPA and  VPA).  In  the  mass  111  the  base  peak  ion i s a b s e n t . in  b  intermediates  49)  (4-keto-2-ene and 49)  peaks  and mass s p e c t r a l  mass s p e c t r a of 4 ' - k e t o - 2 - e n e  47  was  used.  (Figure  There  is In  mass  has an i n t e n s i t y  of  addition,  the  spectrum  of  l e s s than 50%  VPA.  mass chromatogram  VPA t h e r e  by GCMS.  mixture  48  peaks  basis of  The m/z  peak  base  of  4'-keto-2-ene  the  that of 4'-keto-2-ene  Figure  synthetic  route  m/z  in  in  almost  VPA on the  (Figures  2-propyl-4-oxo-2-pentenoic  product  unknown  potassium  derivatives.  spectra  one  this  is  with  was analyzed  the chromatographic  ( F i g u r e 46)  ion  step  the  PFB  are  whereas i n the l a t t e r 213  of  shown  synthetic)  in  former  are  synthetic  4'-keto-2-ene  the  and  times  spectra  differences  portion  match  and the  (native  dehydration  mass  Therefore,  2-propyl-4-oxo-2-pentenoic spectrum  +  The  retention  mass  s y n t h e s i s s i n c e three  F i g u r e 4 9 shows the mass s p e c t r a of  a characteristic  of  quantitative  [M-57]  be 4 ' - k e t o - 2 - e n e  t-BDMS  esters  a  t-BDMS  metabolite  considered to i t s  213  minutes).  the  ester, the  Figure  the  crude product mixture  extract.  and t h e i r  v e r s u s 8.15 data  the  m/z  in  after  the  give  chromatograms  a  142  are  four  obtained  by NICI o f  synthesized  peaks whose mass s p e c t r a are  the  same  -  143  -  10.46 A)  m/z  213  (a) 8.12  I  76  8  8.88  9.92  m/z  213  TIME F i g u r e 48.  10.22  (min)  A) Mass chromatograms at m/z 213 of t-BDMS d e r i v a t i z e d t h e t i c 4 ' - k e t o - 2 - e n e VPA. B) Mass chromatograms at m/z 213 o f t-BDMS d e r i v a t i z e d 4 ' - k e t o - 2 - e n e VPA.  synnative  - 144 -  111  100 _  A)  >-  r—  co  0 II CHr-C-CH.  LU-  ? 3 H  V  ;C-(-C-0-Si-CH  CHJ-CH^CH"  3  C(CH ) 3  LU >  3  111-  cc  213(M-57)  75  +  103 43 1  2  7  1  4  ,171185  9  270  2 5 5  I 25  125  225  M/Z  325  1 1 1  100 _  B)  (Mt)  I.  0 II CH C-CH r  0  CO  Cf C-0-Si-CH CHj-CH^CH  LU  I—  C(CH ) 3  3  3  z  »—»  111—I  LU  73  213(M-57)  +  CC 171  43 127 149  25  _L 125  M/Z  Figure 49.  270(M-  185  255  225  —1 325  Mass s p e c t r a o f A) peak a and B) peak b i n F i g u r e 48.  -  145  TIME F i g u r e 50.  -  (min)  A) Mass chromatograms at m/z 155 o f PFB d e r i v a t i z e d s y n t h e t i c 4 ' - k e t o - 2 - e n e VPA. B) Mass chromatogram at m/z 155 o f PFB d e r i v a t i z e d 4 ' - k e t o - 2 ene VPA from a u r i n e e x t r a c t .  -  w i t h m/z 155  as the base peak.  matches t h a t  from  could  be  small  amounts of  the  the  o f the it  of  metabolite larger  urine  a y  extract  positional  is  from  isomer may be the  difficult,  in  from  Figure  urine.  50A  product m i x t u r e .  mass s p e c t r a o f the  50).  to  It  is  be  also  due  side  for 4'-keto-2-ene  this  it  the  VPA.  This structural  until  a successful  data.  availability that  group because of i t s  summary,  the  two  in  the  of  the  reagents  a 1,3-dioxolane  ease of  about  VPA worked well  the  In  the  these  at  at  1,3-dithiolane  that  would  except  time. be  a  From better  removal.  new VPA m e t a b o l i t e  is  most  likely  4'-keto-2-ene  assignment must be c o n s i d e r e d t e n t a t i v e  synthesis provides  the  NICI s p e c t r a .  group because o f  protecting  which  VPA s i n c e  that  an i n f e r e n c e  of the  Because the only ion of s i g n i f i c a n c e in  s t e p . The keto group was p r o t e c t e d with  appears  certain  products  the d e p r o t e c t i o n  experience,  during  time to that of  possible  to  that  as a r e s u l t  say f o r  retention  peaks  possible  VPA or 4 ' - k e t o - 3 - e n e  peaks i n F i g u r e 50 i s 155,  strategy  is  other  derivative  compounds can not be made on the b a s i s o f t h e i r The s y n t h e t i c  it  two  formed  mesylate  however,  could  The  VPA s i n c e  those of 4 ' - k e t o - 2 - e n e  extracted  time o f one of these peaks  (Figure  4'-keto-3-ene  proton It  The r e t e n t i o n  s m a l l e r peak t h a t has i d e n t i c a l  peaks  synthetic  this  step.  peaks are  i s the  the  isomers o f  removal  dehydration  146  sufficient  however,  product to o b t a i n NMR  - 147  SUMMARY AND CONCLUSIONS  The PFB d e r i v a t i v e s [M-181]"  ions  suitable  for  o f VPA and i t s  ([M-181-C0 ]  for  -  2  SIM  metabolites  analysis  as  3-keto well  produced  VPA),  as  intense  which  identification  are of  metabolites.  Three  VPA d e r i v a t i v e s  (PFB, bis-TFMB  with r e s p e c t to d e t e c t i o n derivatives derivative t-BDMS  had  derivative  proved  to  be  by E I .  about  Comparison  of  ([ H5]-VPA)  and an  showed  a  times  times  as  compared  more  NICI,  sensitive (NICI  sensitive  as  the  PFB  than  the  mode) the  three  also  similar  bis-TFMB.  stable  that  In  The PFB d e r i v a t i v e  five  derivative,  (OA)  be 30-50  were  In the EI mode, a l l  sensitivities.  to  fluorinated  2  sensitivity.  similar  was found  and t-BDMS)  isotope-label led  internal the  standard  [ H6]-VPA 2  that was  internal gives  a  a  standard common ion  superior  internal  s t a n d a r d f o r the NICI assay of VPA.  A highly quantitate Serum  s e n s i t i v e and p r e c i s e NICI VPA i n  serum and s a l i v a  VPA c o n c e n t r a t i o n s  agreement  with  those  obtained  obtained  r o u t i n e VPA q u a n t i t a t i o n  in this  assay was developed t h a t can accurately by  by an EI  NICI  down were  (t-BDMS)  laboratory.  to 2 ng/mL. in  excellent  assay  used f o r  -  Paired NICI  saliva  and serum  assay d e v e l o p e d ,  after  CBZ  148  samples  in five  variability,  r a t i o s were remarkably  ratios total  decreased ratio  An a c t i v e  In  after  transport  spite  of  i n three  both  the  saliva  CBZ i n d i c a t i n g  that  the  saliva  found  good c o r r e l a t i o n s  VPA c o n c e n t r a t i o n s method  would  pharmacokinetic  studies.  The  for  behavior  of  determined appears and  the  between  saliva  be s u i t a b l e  derivative  to  and both  for  formation  PFB d e r i v a t i v e s  to be s u p e r i o r  o f serum f r e e )  serum  total  suggest t h a t measuring VPA i n  of  i n terms  currently  drug  of ease available  and the  and  chromatographic have  This analytical of d e r i v a t i v e GCMS  saliva  interaction  VPA m e t a b o l i t e s  and SIM chromatograms o b t a i n e d .  sensitivity  serum  of VPA out of s a l i v a i s invoked to e x p l a i n the  The  conditions  to  dependent.  and CSF c o n c e n t r a t i o n s .  the NICI  free  s a l i v a to serum t o t a l  compared to serum free  by  considerable to serum  lower c o n c e n t r a t i o n of VPA i n s a l i v a (18.92% ± 6 . 2 5  and f r e e  before and  v o l u n t e e r s both before and  The time-averaged  was c o n c e n t r a t i o n  f o r VPA using the  volunteers  the time-averaged  similar  a f t e r CBZ a d m i n i s t r a t i o n .  assayed  healthy  administration.  intra-subject  were  been method  formation  methods  for  the  a n a l y s i s o f VPA m e t a b o l i t e s .  Seven VPA m e t a b o l i t e s  were i d e n t i f i e d  be  plasma  a stereoselective  geometric relative  protein  in s a l i v a . binding  or  There appears to transport  isomers of 2-ene VPA as i n d i c a t e d by t h e i r to those i n serum.  saliva  of the levels  -  10.  A new VPA m e t a b o l i t e  149  -  which from mass s p e c t r a l  data appears to be 4 ' - k e t o - 2 - e n e The  detection  oxidation  of  of  this  VPA  u n s a t u r a t e d VPA m e t a b o l i t e s toxic  oxidation products.  The  synthesis  different  of  4-oxopentonoate starting  deprotection was not data.  yielded  step,  p o s s i b l e to  2-ene  i n human u r i n e .  arising  VPA,  The  through the  however, isolate  was  that  dithio  protected  was  ketal  sufficient  appears  using  route was  in  used  4'-keto-2-ene  inefficient  that  two which  as  VPA.  and c o n s e q u e n t l y ,  product f o r  to  urine.  attempted  synthetic  the  potentially  a c i d was a l s o d e t e c t e d i n  VPA  from  confirms  to give r i s e to  Another new m e t a b o l i t e  routes.  protected  material  or  4'-keto-2-ene  synthetic  apparently  are l i k e l y  be 2 - ( 2 ' - p r o p e n y l ) - g l u t a r i c  11.  VPA was d e t e c t e d  metabolite,  2,3'-diene  and chromatographic  the The it  o b t a i n i n g NMR  -  150  REFERENCES  F . S . A b b o t t , R. B u r t o n , J . O r r , D. Wladichuk, S . Ferguson and T . H . S u n . 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Acta H e l v . , 5 8 , 136 (1983b).  drug  -  161  -  APPENDIX  NMR, IR and mass s p e c t r a of some of the  s y n t h e s i z e d compounds.  IJIICROMETf. U S  i»m)  13 14 i L  16 18 70  75  to  <T>  11  JO  2  4000  3600  JJCO  7U00  ViiJj  ViJwJ  li.OJ  1000  l-i(/0  i  vj  j  i i . L O U l i N C V ( C M ')  IR spectrum of e t h y l  2-propyl-4-oxopentanoate.  1000  y.jo  40U  H-NMR (80 MHz) spectrum of e t h y l  2-propyl-4-oxopentanoate.  -  164  -  73  100  0-TMS CH  >— I  2  =  C  - ™ 2 * :CH-C-0-C H 2  I—H  CH -CH -CH  CO  3  2  5  2  185 LU  115  130  LU  45  97  143  29  Li 25 EI  125  ! 1 5  L_l  L_J  M/Z  spectrum o f the TMS enol e t h e r  243258 ( M t )  of e t h y l  225  325  2-propyl-4-oxopentanoate.  -  100  165  -  73  _  0-TMS  I  0 ||  CH = C - C H  >-  CH - C H - C H  h-  ^  CO LU  cr _i  LU  43  95  137 109 183  29  25 EI  213 241  125  M/Z  spectrum o f the TMS enol e t h e r  of e t h y l  225  256  (Mt)  325  2-propyl-4-oxo-2-pentenoate.  r  CTl  3  | mi | il 111 in i| I I 111 II 11111 \f\ SO i. « 0 J 50 pew 20  A 1  1  1  1  I 40  1  1  1  1  I 35  1  1  1  H-NMR (300 MHz)  1  1  i 30  —'I  1 \  ..J  JL_  i—i—i—I—i—i—t—i—i—i—t—i—i—i—i—t—i—i—i—[—i—i—i—j—i—i—i—i—i—r 2b 20 l b 10 0b 00  spectrum o f e t h y l  4-ethylenethioketalpentanoate.  PPM  -  100  167  -  127  _  I S  >-  I—  i—i  2  I S  2  CH —C—CH 3  cn  0  2  \  LU  H  C—C-0-C„H 2 5 c  i—  CH^CH^CH  29  LU >  199  f— CE _ l LU  260(M ) t  215  25  125  M/Z  225  EI mass spectrum o f 4 - c a r b o e t h o x y - 2 - e t h y 1 e n e t h i o k e t a 1 - 4 - h e p t e n e w i t h the s h o r t e r r e t e n t i o n t i m e ) .  325 (isomer  - 168 -  100  199  _  127  >i—  I  i—i  cn LU  2  I  s s \ /  2  C H , - C — CH  3  2v 9  #  29  0  y  C - C - 0 - C  O  H ,  2 5  CH^CHA-CH  LU  25  4  260(M-) 215  125  1  M/Z  225  EI mass spectrum o f 4 - c a r b o e t h o x y - 2 - e t h y l e n e t h i o k e t a l - 4 - h e p t e n e w i t h the l o n g e r r e t e n t i o n t i m e ) .  325 (isomer  

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