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Determination of an irreversible inhibitor of monoamine oxidase B (MDL 72974A) in human plasma and urine by gas chromatography—positive-ion chemical ionization mass spectrometry
Journal o f Chromatogrtq~hy, 571 ( 199 i ) 241-249 Biomedical ,,Ipplications Elsevier Science Publishers B.V., A m s t e r d a m C H R O M B I O . 6037
Short Communication Determination of an irreversible inhibitor of monoamine oxidase B ( M D L 7 2 9 7 4 A ) in human plasma and urine by gas chromatography-positive-ion chemical ionization mass spectrometry BERTRAND
D. D U L E R Y ,
J O S I A N E S C H O U N and K L A U S D. H A E G E L E *
Departments of Clinical Bioche,,ni.~'try am.i Drug ~4etaholism. Marion Merrell Dow. 16 Rue d'Ankara. B.P. 447 R/9. 67009 Strashourg C~t,:ex (France) (First received M a r c h 26th, 1991; re'¢ised m a n u s c r i p t received June 12th, 1991) ABSTRACT A sensitive a n d specific assay has bcen developed for the quantitative m e a s u r e m e n t in h u m a n plasma a n d urine o f the irreversible inhibitor o f monoanaine oxidase B [(E')-4-fluoro-fl-fluoromethy.'cnebenzcneb u t a n a m i n e HC! salt] (M D L 72974A) (I). This assay is based on gas c h r o m a t o g r a p h y - m a s s spectrometry with a m m o n i a as the chemical ionization reagent gas. A f t e r addition o f l-fluoro-2-(4-chlorobenzene)e t h a n a m i n e HCI salt ( M D L 71946A) as the internal s t a n d a r d , p l a s m a (I ml) and urine (100 Ill) samples were extracted using an a u t o m a t e d solld-liquid extraction procedure on C N columns. T h e eluent was dried with a s t r e a m o f nitrogen, a n d the residue was derivatized with p e n t a f l u o r o p r o p i o n i c anhydride. Selectedion m o n i t o r i n g o f the [ M N H 4 ] + ions m/z 361 (I) and 351 (internal s t a n d a r d ) was use ; lbr quantification. T h e m e t h o d yielded a linear response over the concentration range 0.25-100 p m o i / m l in p l a s m a with a limit o f q u a n t i t a t i o n o f 9.25 p m o l / m l . T h e within-day reproducibility at a c o n c e n t r a t i o n o f 5 p m o l / m l was 4.6% and at a concentration o f 50 p m o l / m l was 1.3%. T h e d a y - t o - d a y reproducibility was 5.2 and 7.0% at c o n c e n t r a t i o n s o f I0 a n d 30 p m o l / m l , respectively. T h e m e t h o d was applied to the quantitication o f I in p l a s m a and urine after the a d m i n i s t r a t i o n o f 12-mg doses o f 1 to a he,'dthy male volunteer.
INTRODUCTION
Recent interest has focused on the possibility that inhibition o f the B form o f m o n o a m i n e oxidase ( M A O ) may delay or prevent the progression o f Parkinson's disease [1-5]. This suggestion is based on two hypotheses o f the pathogenesis o f the disease. The first holds that the destruction o f dopaminergic neurons is initiated by a reactive oxygen species formed during the M A O - c a t a l y s e d metabolism o f d o p a m i n e [6]. Tbe second is based on the possibility that the cause o f Parkinson's disease is related to t h e f o r m a t i o n o f a toxic m e t a b o l i t e [7-9]. In vivo a n d hz vitro studies in a n i m a l s h a v e s h o w n t h a t I [(E)-4-fluoro-fl-fluoro0378-4347/91/$03.50
©
t991 Elsevier Science Publishers B.V. All rights reserved
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methylenebenzenebutanamine] is a potent and selective enzyme-activated, irreversible inhibitor of the B form of MAO. The in vitro ICso (concentration of inhibitor to produce a 50% inhibition of enzyme activity) values for the inhibition of rat brain mitochondrial M A O - A versus M A O - B activity show a selectivity ratio of 190 in favour of M A O - B inhibition for I, compared with only 17 for L-deprenyl. At MAO-B-selective doses in vivo, this drug wa'.¢ orally active, potent, and did not potentiate the cardiovascular effects of intraduodenally administered tyramine [ I 0]. To obtain sufficient sensitivity and specificity, a capillary gas chromatographic-mass spectrometric ( G C - M S ) method was developed for the quantification of I in samples of human plasma and urine. The developed method is based on positive-ion chemical ionization (PICI) G C - M S with ammonia as reagent gas, to quantify with precision and accuracy the low plasma concentrations of I. The assay is performed with I ml of plasma or 100 ltl of urine. The biological extracts were treated with pentafluoropropionic anhydride (PFPA), and the N - P F P derivatives gave intense and characteristic ions at m / z 361 (I) and m / z 351 (internal standard, I.S.) which were recorded by selected-ion monitoring (SIM). The applicability of the method was demonstrated by analysing I in the plasma and urine of a healthy volunteer given I orally. EXPERIMENTAL
Reagents (E)-4-Fluoro-fl-fluoromethylenebenzenebutanamine HCI salt ( M D L 72974A) (I) and 1-fluoro-2-(4-chlorobenzene)ethanamine HCI salt ( M D L 71946A) (I.S.) were :'upplied by Marion Merrell Dow (Strasbourg, France). Methanol and dichloromethane were reagent grade {Merck, Darmstadt, Germany). Acetonitrile was purchased from S.D.S. (Peypin, France). P F P A was purchased from Pierce (Oudbeijerland, Netherlands). Disposable CN extraction columns (3 ml) and n-hexane came from Baker (Deventer, Netherla,'~ds). S t a n d a r d curves Stock solution of I and the !.S. were prepared by dissolving each pure reference compound in methanol at a concentration of 1 #mol/ml and stored at - 2 0 ° C until used. Working solutions obtained from appropriate dilutions in methanol were prepared daily. Aliquots of 1 ml of drug-free human plasma were spiked with 50 pmol of the I.S. and various amounts of I over the concentration range 0.25-100 pmol/ml. F o r urine samples (100 pl) the amount o f I.S. added was the same and the standard curve was prepared in the same range as for plasma samples (0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 25. 50, 75 and 100 pmol). The blank plasma or urine samples were prepared in a similar way by spiking control plasma or urine with the I.S. only.
S H O R T COM M U N I c A T I O N S
243
Sample work-up S a m p l e p r e p a r a t i o n w a s d e v e l o p e d using a n A S P E C ( a u t o m a t i c s a m p l e prepa r a t i o n with e x t r a c t i o n c o l u m n ) system (Gilson M e d i c a l Electronics, Villier-leBel, F r a n c e ) . P l a s m a s a m p l e s (1 ml), spiked with 50 p m o l o f the I.S., were placed in a 4-ml vial. D i s p o s a b l e e x t r a c t i o n c o l u m n s ( C N , 3 rni) were c o n d i t i o n e d with 3 ml o f m e t h a n o l a n d w a s h e d with 2 ml o f distilled water. T h e p l a s m a was a d d e d to the t o p o f the solid phase, a n d p u s h e d t h r o u g h the c o l u m n with 1 ml o f air, then w a s h e d with 2 ml o f distilled w a t e r a n d 2 ml o f m e t h a n o l - w a t e r (60:40, v/v). The c o l u m n was dried with air (10 ml), a n d the c o m p o u n d s were eluted with 1.5 ml o f acetonitrile c o n t a i n i n g 10% 1 AI HCI. T h e c o l u m n was dried with 10 ml o f air, a n d the s a m p l e was e v a p o r a t e d u n d e r a s t r e a m o f n i t r o g e n at r o o m t e m p e r a t u r e .
Derivatization T o the e x t r a c t e d s a m p l e were a d d e d 100 pl o f d i c h l o r o m e t h a n e a n d 50 III o f P F P A , a n d the m i x t u r e w a s allowed to react for 30 rain at r o o m t e m p e r a t u r e . 'i'his w a s folIowed by e v a p o r a t i o n o f the solvent; then the s a m p l e w a s reconstituted in 50 Ill o f n-hexane. A I-td a l i q u o t was injected into the G C c o l u m n for G C - M S analysis.
G C - M S analysis NIS a n a l y s e s were carried o u t with a C a r l o E r b a Q M D 1000 G C - M S system o r a F i n n i g a n T S Q 4 6 G C - M S system coupled on-line to an Incos d a t a system N o v a 4X. T h e c h r o m a t o g r a p h i c c o l u m n w a s a 15 m x 0.32 m m I.D. 5 % phenylmethylsilicone fused-silica capillary c o l u m n ( H e w l e t t P a c k a r d , Les Ulis, F r a n c e ) . T h e film thickness o f the c o l u m n was 0.52 ltm. T h e end o f the c o l u m n was directly i n t r o d u c e d into the ion source via a stainless-steel t r a n s f e r line k e p t at 270°C. S a m p l e s were injected into a splitless injector with heJium used as c a r r i e r gas (inlet p r e s s u r e o f 60 p.s.i.). T h e t e m p e r a t u r e o f the injector was m a i n t a i n e d at 270°C. T h e oven t e m p e r a t u r e was raised f r o m 60 to 120°C at 30°C/min a n d f r o m 120 to 200"C a t 6*C/min, a n d the final t e m p e r a t u r e was held for 1 min. T h e G C - M S system w a s o p e r a t e d in the P I C I m o d e with a n electron energy o f 70 eV, a n emission c u r r e n t o f 25':3 ~ A a n d a source t e m p e r a t u r e o f 160°C for the Q M D 1000 a n d 100*C for the T S Q 4 6 . T h e P I C I m a s s spectra o f the N - P F P derivatives o f M D L 77.974 a n d the !.S. were r e c o r d e d d u r i n g a G C r u n by scanning f r o m 90 to 500 in 0.5 s. Q u a n t i f i c a t i o n o f I w a s p e r f o r m e d by focusing the m a s s s p e c t r o m e t e r in the ion m o n i t o r i n g selected in o r d e r to m e a s u r e m/z 361 a n d m/z 351, the [ M N H 4 ] + ions o f the c o m p o u n d s .
Precision and accuracy T o assess the precision a n d the a c c u r a c y o f the m e t h o d , reproducibility assays were carried o u t at different c o n c e n t r a t i o n s (5, l0 a n d 50 p m o l / m I ) . T h e spiked p l a s m a s a m p l e s were a n a l y s e d the s a m e d a y o r the d a y after. F o r each c o n c e n t r a tion level, the coefficient o f v a r i a t i o n (C.V.) w a s calculated.
244
SHORT COM M ON ICATIONS
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SHORT COMMUNICATIONS
245
Drug administration A healthy v o l u n t e e r was a d m i n i s t e r e d single daily oral doses o f 12 m g o f I o v e r a t e n - d a y period. Blood samples (10 ml) were collected into S a r s t e d t - M o n o v e t t e tubes c o n t a i n i n g p o t a s s s i u m E D T A at 0, 15, 30 a n d 45 min a n d 1,2, 3, 4, 6, 8 a n d 24 h following d r u g a d m i n i s t r a t i o n on the final d a y o f t r e a t m e n t . A 24-h urine s a m p l e w a s collected to d e t e r m i n e the extent o f u r i n a r y excretion. T h e p l a s m a c o n c e n t r a t i o n s were m e a s u r e d to establish the p l a s m a c o n c e n t r a t i o n - t i m e course. RESULTS AND DISCUSSION
Derivatization procedure T h e P1C1 m a s s spectra o f the N - P F P derivatives o f I a n d the I.S. o b t a i n e d with a m m o n i a as reagent gas s h o w e d characteristic ions (Fig. 1). These c o m p o u n d s gave only a p e a k at m/z 361 a n d m/z 351 c o r r e s p o n d i n g to the [ M N H 4 ] + ions o f I a n d the I.S., respectively. Standard
curve
T h e c a l i b r a t i o n curve o b t a i n e d by p l o t t i n g the p e a k - a r e a ratio m/z 361/351 verstts the k n o w n p l a s m a c o n c e n t r a t i o n o f i p r o d u c e d a straight line over the c o n c e n t r a t i o n r a n g e 0 . 2 5 - I 0 0 p m o l / m l . L i n e a r regression analysis gave the e q u a tion ), = 0.0092x - 0.002 with a c o r r e l a t i o n coefficient (r) o f 0.9996.
Precision and accuracy T h e w i t h i n - d a y precision was c h e c k e d by a n a l y s i n g five p l a s m a samples at three different c o n c e n t r a t i o n s o f I (5, 10 a n d 50 p m o l / m l ) . T h e C.V. were less t k a n 4 . 6 % at each o f the c o n c e n t r a t i o n s (Table I). T h e d a y - t o - d a y precision was checked by m e a s u r i n g two c o n c e n t r a t i o n s ( 10.15 a n d 30.45 p m o l / m l ) between d a y s I a n d 15 using the c a l i b r a t i o n curve o b t a i n e d on d a y 1. T h e results o b t a i n e d with this p r o c e d u r e are given in T a b l e 11. A c h r o m a t o g r a m o b t a i n e d a f t e r G C - M S analysis o f a b l a n k p l a s m a s a m p l e is s h o w n in Fig. 2, a n d those o b t a i n e d f r o m a c o n t r o l spiked with 5 a n d 50 p m o l / m l are s h o w n in Fig. 3. T h e r e was no interference f r o m e n d o g e n o u s c o m p o u n d s in
TABLE
!
WITHIN-DAY
PRECISION
AND ACCURACY
FOR I IN SPIKED
PLASMA
Concentration added (pmol/ml)
C o n c e n t r a t i o n fou,~d ( m e a n 4- S . D . . n =~ 3 ) ( p m o l / m l )
C.V. (%)
5 10 50
4.99 4- 0.23 10.09 4- 0.09 49.06 :t: 0.66
4.6 0.9 1.3
SAMPLES
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T A B L E l[ D A Y - T O - D A Y P R E C I S I O N A N D A C C U R A C Y F O R 1 IN S P I K E D P L A S M A S A M P L E S W I T H A C A L I B R A T I O N C U R V E O B T A I N E D ON DAY I D a y o f analysis
A m o u n t found (pmol/mi) 10.15 pmol/ml added
30.45 pmol/ml added
1
I t .22
2 5 9 II 12 15
10.55 10.77 10.05 9.79 10.30 9.75
34.1 I 32.28 29.24 29.69 34.50 29.84 30. i0
Mean S.D. C.V. (%)
10.35 0.54 5.20
31.39 2.22 7.04
the S I M trace oFthe ions, e n a b l i n g the q u a n t i t a t i v e m e a s u r e m e n t o f I for p h a r m a cokinetics. T h e limit o f q u a n t i t a t i o n For I was 250 f m o l / m l in p l a s m a at a signalto-noise ratio o1" 4. A c h r o m a t o g r a m o f a 24-h urine s a m p l e is s h o w n in Fig. 4.
App/icat ion T h e a p p l i c a t i o n o f the G C - M S m e t h o d was s h o w n L,, q u a n t i f i c a t i o n o f I in plasma and urine samples of a healthy male volunteer who had recived daily single oral doses of I over a ten-day period. IOO"
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Fig. 3. Selected-ion monitoring traces obtained from a control plasma sample spiked with (a) 5 pmol/ml 1 and 50 pmol/ml I.S. and (b) 50 pmol/ml I and 50 pmol/ml I.S.
T h e p l a s m a c o n c e n t r a t i o n - t i m e course o b t a i n e d on d a y 10 for the h u m a n v o l u n t e e r is s h o w n in Fig. 5. T h e o b s e r v e d Cm.~x value was 43.1 p m o l / m l , w h i c h w a s a t t a i n e d at 1 h (tm.~x). T h e c o n c e n t r a t i o n o f I in the urine w a s 176.5 p m o l / m l , c o r r e s p o n d i n g to a 24-h u r i n a r y excretion o f 0 . 3 7 % o f the dose. CONCLUSION
T h e present G C - M S a s s a y represents a specific a n d sensitive a n a l y t i c a l m e t h ' od for the m e a s u r e m e n t o f I at the p i c o m o l e level in p l a s m a a n d urine s a m p l e s o f h u m a n origin. T h e N - P F P deriw.tive o f the c o m p o u n d is stable a n d yields high
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SHORT COMMUNICATIONS ~Q
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Fig. 5. Plasma c o n c e n t r a t i o n - t i m e course o f I a f t e r the tenth dose o f 12 m g per day o f I administered orally to a healthy volunteer.
sensitivity with no endogenous interferences. The reproducibility demonstrates that precision and accuracy o f this technique are suitable for routine G C - M S analysis o f I. The method has been successfully applied to the quantification of I in human plasma and urine. ACKNOWLEDGEMENTS
T h e a u t h o r s a c k n o w l e d g e N a d i a le H e n a n f for h e r technical a s s i s t a a c e a n d Muricl R o h f r i t s c h for her secretarial assistance. W e also t h a n k D r . N o r m a n D. H u e b e r t for c o n s t r u c t i v e discussion,
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249
REFERENCES I W. Birkmayer, J. Knoll, P. Ricderer, M. B. Y o u d i m , V. M a r s a n d J. M a r t o n , J. Neural Transm.. 64
(1985) 113. 2 U. K. Rinne, Acta Neurol. Scami.. 68 (1983) 144. 3 L. I. Golbe, A. N. L i c b e r m a n , M, D. Mucntcr, J. E. Ahlskog, G. G o p i n a t h a n , A. N. N e o p h y t i d c s , S. H. F o o and R. C. Duvoisin, Neuropharmacol., t I (1988) 45: 4 T. N. Chase, M. M. M o u r a d i a n , (3. Fabbrini a n d J. L. Juncos, J. Neural Transm., SuppL, 27 (t988) 3. 5 P. K. Sonsalla and L. I. G o l b e , Clin. NeuropharmacoL, I1 (1988) 500. 6 G . Cohen, Adv. NetlroL, 45 ( ! 986) I 19. 7 K. Chiba, A. T r e v o r a n d H. Castagnoli, Biochem. Bioplow. Res. Commun., 120 (t984) 574. 8 R. E. Heikkila, L. M a n z i n o , F. S. C a b b a t a n d R. C. Duvoisin, Naltow, 3I I (1985) 467. 9 J. A. Javitch, R. J. D ' A m a t o a n d S. M. Strittmater, Prof. Natl. Acad. Sci. U.S.A.. 82 (1985) 2173. 10 M. Zreika, J. R. F o z a r d , M. W. Dudley, P. Bey, 1. M c D o n a l d and M. G . Palfreyman, J. Neural Transm. ( P - D Sect.), I (1989) 243.
Short Communication Determination of an irreversible inhibitor of monoamine oxidase B ( M D L 7 2 9 7 4 A ) in human plasma and urine by gas chromatography-positive-ion chemical ionization mass spectrometry BERTRAND
D. D U L E R Y ,
J O S I A N E S C H O U N and K L A U S D. H A E G E L E *
Departments of Clinical Bioche,,ni.~'try am.i Drug ~4etaholism. Marion Merrell Dow. 16 Rue d'Ankara. B.P. 447 R/9. 67009 Strashourg C~t,:ex (France) (First received M a r c h 26th, 1991; re'¢ised m a n u s c r i p t received June 12th, 1991) ABSTRACT A sensitive a n d specific assay has bcen developed for the quantitative m e a s u r e m e n t in h u m a n plasma a n d urine o f the irreversible inhibitor o f monoanaine oxidase B [(E')-4-fluoro-fl-fluoromethy.'cnebenzcneb u t a n a m i n e HC! salt] (M D L 72974A) (I). This assay is based on gas c h r o m a t o g r a p h y - m a s s spectrometry with a m m o n i a as the chemical ionization reagent gas. A f t e r addition o f l-fluoro-2-(4-chlorobenzene)e t h a n a m i n e HCI salt ( M D L 71946A) as the internal s t a n d a r d , p l a s m a (I ml) and urine (100 Ill) samples were extracted using an a u t o m a t e d solld-liquid extraction procedure on C N columns. T h e eluent was dried with a s t r e a m o f nitrogen, a n d the residue was derivatized with p e n t a f l u o r o p r o p i o n i c anhydride. Selectedion m o n i t o r i n g o f the [ M N H 4 ] + ions m/z 361 (I) and 351 (internal s t a n d a r d ) was use ; lbr quantification. T h e m e t h o d yielded a linear response over the concentration range 0.25-100 p m o i / m l in p l a s m a with a limit o f q u a n t i t a t i o n o f 9.25 p m o l / m l . T h e within-day reproducibility at a c o n c e n t r a t i o n o f 5 p m o l / m l was 4.6% and at a concentration o f 50 p m o l / m l was 1.3%. T h e d a y - t o - d a y reproducibility was 5.2 and 7.0% at c o n c e n t r a t i o n s o f I0 a n d 30 p m o l / m l , respectively. T h e m e t h o d was applied to the quantitication o f I in p l a s m a and urine after the a d m i n i s t r a t i o n o f 12-mg doses o f 1 to a he,'dthy male volunteer.
INTRODUCTION
Recent interest has focused on the possibility that inhibition o f the B form o f m o n o a m i n e oxidase ( M A O ) may delay or prevent the progression o f Parkinson's disease [1-5]. This suggestion is based on two hypotheses o f the pathogenesis o f the disease. The first holds that the destruction o f dopaminergic neurons is initiated by a reactive oxygen species formed during the M A O - c a t a l y s e d metabolism o f d o p a m i n e [6]. Tbe second is based on the possibility that the cause o f Parkinson's disease is related to t h e f o r m a t i o n o f a toxic m e t a b o l i t e [7-9]. In vivo a n d hz vitro studies in a n i m a l s h a v e s h o w n t h a t I [(E)-4-fluoro-fl-fluoro0378-4347/91/$03.50
©
t991 Elsevier Science Publishers B.V. All rights reserved
242
SHORT COMMUNICATIONS
methylenebenzenebutanamine] is a potent and selective enzyme-activated, irreversible inhibitor of the B form of MAO. The in vitro ICso (concentration of inhibitor to produce a 50% inhibition of enzyme activity) values for the inhibition of rat brain mitochondrial M A O - A versus M A O - B activity show a selectivity ratio of 190 in favour of M A O - B inhibition for I, compared with only 17 for L-deprenyl. At MAO-B-selective doses in vivo, this drug wa'.¢ orally active, potent, and did not potentiate the cardiovascular effects of intraduodenally administered tyramine [ I 0]. To obtain sufficient sensitivity and specificity, a capillary gas chromatographic-mass spectrometric ( G C - M S ) method was developed for the quantification of I in samples of human plasma and urine. The developed method is based on positive-ion chemical ionization (PICI) G C - M S with ammonia as reagent gas, to quantify with precision and accuracy the low plasma concentrations of I. The assay is performed with I ml of plasma or 100 ltl of urine. The biological extracts were treated with pentafluoropropionic anhydride (PFPA), and the N - P F P derivatives gave intense and characteristic ions at m / z 361 (I) and m / z 351 (internal standard, I.S.) which were recorded by selected-ion monitoring (SIM). The applicability of the method was demonstrated by analysing I in the plasma and urine of a healthy volunteer given I orally. EXPERIMENTAL
Reagents (E)-4-Fluoro-fl-fluoromethylenebenzenebutanamine HCI salt ( M D L 72974A) (I) and 1-fluoro-2-(4-chlorobenzene)ethanamine HCI salt ( M D L 71946A) (I.S.) were :'upplied by Marion Merrell Dow (Strasbourg, France). Methanol and dichloromethane were reagent grade {Merck, Darmstadt, Germany). Acetonitrile was purchased from S.D.S. (Peypin, France). P F P A was purchased from Pierce (Oudbeijerland, Netherlands). Disposable CN extraction columns (3 ml) and n-hexane came from Baker (Deventer, Netherla,'~ds). S t a n d a r d curves Stock solution of I and the !.S. were prepared by dissolving each pure reference compound in methanol at a concentration of 1 #mol/ml and stored at - 2 0 ° C until used. Working solutions obtained from appropriate dilutions in methanol were prepared daily. Aliquots of 1 ml of drug-free human plasma were spiked with 50 pmol of the I.S. and various amounts of I over the concentration range 0.25-100 pmol/ml. F o r urine samples (100 pl) the amount o f I.S. added was the same and the standard curve was prepared in the same range as for plasma samples (0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 25. 50, 75 and 100 pmol). The blank plasma or urine samples were prepared in a similar way by spiking control plasma or urine with the I.S. only.
S H O R T COM M U N I c A T I O N S
243
Sample work-up S a m p l e p r e p a r a t i o n w a s d e v e l o p e d using a n A S P E C ( a u t o m a t i c s a m p l e prepa r a t i o n with e x t r a c t i o n c o l u m n ) system (Gilson M e d i c a l Electronics, Villier-leBel, F r a n c e ) . P l a s m a s a m p l e s (1 ml), spiked with 50 p m o l o f the I.S., were placed in a 4-ml vial. D i s p o s a b l e e x t r a c t i o n c o l u m n s ( C N , 3 rni) were c o n d i t i o n e d with 3 ml o f m e t h a n o l a n d w a s h e d with 2 ml o f distilled water. T h e p l a s m a was a d d e d to the t o p o f the solid phase, a n d p u s h e d t h r o u g h the c o l u m n with 1 ml o f air, then w a s h e d with 2 ml o f distilled w a t e r a n d 2 ml o f m e t h a n o l - w a t e r (60:40, v/v). The c o l u m n was dried with air (10 ml), a n d the c o m p o u n d s were eluted with 1.5 ml o f acetonitrile c o n t a i n i n g 10% 1 AI HCI. T h e c o l u m n was dried with 10 ml o f air, a n d the s a m p l e was e v a p o r a t e d u n d e r a s t r e a m o f n i t r o g e n at r o o m t e m p e r a t u r e .
Derivatization T o the e x t r a c t e d s a m p l e were a d d e d 100 pl o f d i c h l o r o m e t h a n e a n d 50 III o f P F P A , a n d the m i x t u r e w a s allowed to react for 30 rain at r o o m t e m p e r a t u r e . 'i'his w a s folIowed by e v a p o r a t i o n o f the solvent; then the s a m p l e w a s reconstituted in 50 Ill o f n-hexane. A I-td a l i q u o t was injected into the G C c o l u m n for G C - M S analysis.
G C - M S analysis NIS a n a l y s e s were carried o u t with a C a r l o E r b a Q M D 1000 G C - M S system o r a F i n n i g a n T S Q 4 6 G C - M S system coupled on-line to an Incos d a t a system N o v a 4X. T h e c h r o m a t o g r a p h i c c o l u m n w a s a 15 m x 0.32 m m I.D. 5 % phenylmethylsilicone fused-silica capillary c o l u m n ( H e w l e t t P a c k a r d , Les Ulis, F r a n c e ) . T h e film thickness o f the c o l u m n was 0.52 ltm. T h e end o f the c o l u m n was directly i n t r o d u c e d into the ion source via a stainless-steel t r a n s f e r line k e p t at 270°C. S a m p l e s were injected into a splitless injector with heJium used as c a r r i e r gas (inlet p r e s s u r e o f 60 p.s.i.). T h e t e m p e r a t u r e o f the injector was m a i n t a i n e d at 270°C. T h e oven t e m p e r a t u r e was raised f r o m 60 to 120°C at 30°C/min a n d f r o m 120 to 200"C a t 6*C/min, a n d the final t e m p e r a t u r e was held for 1 min. T h e G C - M S system w a s o p e r a t e d in the P I C I m o d e with a n electron energy o f 70 eV, a n emission c u r r e n t o f 25':3 ~ A a n d a source t e m p e r a t u r e o f 160°C for the Q M D 1000 a n d 100*C for the T S Q 4 6 . T h e P I C I m a s s spectra o f the N - P F P derivatives o f M D L 77.974 a n d the !.S. were r e c o r d e d d u r i n g a G C r u n by scanning f r o m 90 to 500 in 0.5 s. Q u a n t i f i c a t i o n o f I w a s p e r f o r m e d by focusing the m a s s s p e c t r o m e t e r in the ion m o n i t o r i n g selected in o r d e r to m e a s u r e m/z 361 a n d m/z 351, the [ M N H 4 ] + ions o f the c o m p o u n d s .
Precision and accuracy T o assess the precision a n d the a c c u r a c y o f the m e t h o d , reproducibility assays were carried o u t at different c o n c e n t r a t i o n s (5, l0 a n d 50 p m o l / m I ) . T h e spiked p l a s m a s a m p l e s were a n a l y s e d the s a m e d a y o r the d a y after. F o r each c o n c e n t r a tion level, the coefficient o f v a r i a t i o n (C.V.) w a s calculated.
244
SHORT COM M ON ICATIONS
tOO
50 >I--¢J3
;=FP .
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Z
t~J I-Z
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Fig. I. A m m o n i a l?lCl mass spectra o f the N - p e n m f l u o r o a c e t y l derivatives oF ! and the I.S.
SHORT COMMUNICATIONS
245
Drug administration A healthy v o l u n t e e r was a d m i n i s t e r e d single daily oral doses o f 12 m g o f I o v e r a t e n - d a y period. Blood samples (10 ml) were collected into S a r s t e d t - M o n o v e t t e tubes c o n t a i n i n g p o t a s s s i u m E D T A at 0, 15, 30 a n d 45 min a n d 1,2, 3, 4, 6, 8 a n d 24 h following d r u g a d m i n i s t r a t i o n on the final d a y o f t r e a t m e n t . A 24-h urine s a m p l e w a s collected to d e t e r m i n e the extent o f u r i n a r y excretion. T h e p l a s m a c o n c e n t r a t i o n s were m e a s u r e d to establish the p l a s m a c o n c e n t r a t i o n - t i m e course. RESULTS AND DISCUSSION
Derivatization procedure T h e P1C1 m a s s spectra o f the N - P F P derivatives o f I a n d the I.S. o b t a i n e d with a m m o n i a as reagent gas s h o w e d characteristic ions (Fig. 1). These c o m p o u n d s gave only a p e a k at m/z 361 a n d m/z 351 c o r r e s p o n d i n g to the [ M N H 4 ] + ions o f I a n d the I.S., respectively. Standard
curve
T h e c a l i b r a t i o n curve o b t a i n e d by p l o t t i n g the p e a k - a r e a ratio m/z 361/351 verstts the k n o w n p l a s m a c o n c e n t r a t i o n o f i p r o d u c e d a straight line over the c o n c e n t r a t i o n r a n g e 0 . 2 5 - I 0 0 p m o l / m l . L i n e a r regression analysis gave the e q u a tion ), = 0.0092x - 0.002 with a c o r r e l a t i o n coefficient (r) o f 0.9996.
Precision and accuracy T h e w i t h i n - d a y precision was c h e c k e d by a n a l y s i n g five p l a s m a samples at three different c o n c e n t r a t i o n s o f I (5, 10 a n d 50 p m o l / m l ) . T h e C.V. were less t k a n 4 . 6 % at each o f the c o n c e n t r a t i o n s (Table I). T h e d a y - t o - d a y precision was checked by m e a s u r i n g two c o n c e n t r a t i o n s ( 10.15 a n d 30.45 p m o l / m l ) between d a y s I a n d 15 using the c a l i b r a t i o n curve o b t a i n e d on d a y 1. T h e results o b t a i n e d with this p r o c e d u r e are given in T a b l e 11. A c h r o m a t o g r a m o b t a i n e d a f t e r G C - M S analysis o f a b l a n k p l a s m a s a m p l e is s h o w n in Fig. 2, a n d those o b t a i n e d f r o m a c o n t r o l spiked with 5 a n d 50 p m o l / m l are s h o w n in Fig. 3. T h e r e was no interference f r o m e n d o g e n o u s c o m p o u n d s in
TABLE
!
WITHIN-DAY
PRECISION
AND ACCURACY
FOR I IN SPIKED
PLASMA
Concentration added (pmol/ml)
C o n c e n t r a t i o n fou,~d ( m e a n 4- S . D . . n =~ 3 ) ( p m o l / m l )
C.V. (%)
5 10 50
4.99 4- 0.23 10.09 4- 0.09 49.06 :t: 0.66
4.6 0.9 1.3
SAMPLES
246
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T A B L E l[ D A Y - T O - D A Y P R E C I S I O N A N D A C C U R A C Y F O R 1 IN S P I K E D P L A S M A S A M P L E S W I T H A C A L I B R A T I O N C U R V E O B T A I N E D ON DAY I D a y o f analysis
A m o u n t found (pmol/mi) 10.15 pmol/ml added
30.45 pmol/ml added
1
I t .22
2 5 9 II 12 15
10.55 10.77 10.05 9.79 10.30 9.75
34.1 I 32.28 29.24 29.69 34.50 29.84 30. i0
Mean S.D. C.V. (%)
10.35 0.54 5.20
31.39 2.22 7.04
the S I M trace oFthe ions, e n a b l i n g the q u a n t i t a t i v e m e a s u r e m e n t o f I for p h a r m a cokinetics. T h e limit o f q u a n t i t a t i o n For I was 250 f m o l / m l in p l a s m a at a signalto-noise ratio o1" 4. A c h r o m a t o g r a m o f a 24-h urine s a m p l e is s h o w n in Fig. 4.
App/icat ion T h e a p p l i c a t i o n o f the G C - M S m e t h o d was s h o w n L,, q u a n t i f i c a t i o n o f I in plasma and urine samples of a healthy male volunteer who had recived daily single oral doses of I over a ten-day period. IOO"
,-.
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Fig. 2. Typical mass c h r o m a t o g r a m obtained from a control h u m a n pl,'tsma sample spiked with 50 pmol/ml I.S.
SHORT COMMUNICATIONS
247
(a)
s.66
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Fig. 3. Selected-ion monitoring traces obtained from a control plasma sample spiked with (a) 5 pmol/ml 1 and 50 pmol/ml I.S. and (b) 50 pmol/ml I and 50 pmol/ml I.S.
T h e p l a s m a c o n c e n t r a t i o n - t i m e course o b t a i n e d on d a y 10 for the h u m a n v o l u n t e e r is s h o w n in Fig. 5. T h e o b s e r v e d Cm.~x value was 43.1 p m o l / m l , w h i c h w a s a t t a i n e d at 1 h (tm.~x). T h e c o n c e n t r a t i o n o f I in the urine w a s 176.5 p m o l / m l , c o r r e s p o n d i n g to a 24-h u r i n a r y excretion o f 0 . 3 7 % o f the dose. CONCLUSION
T h e present G C - M S a s s a y represents a specific a n d sensitive a n a l y t i c a l m e t h ' od for the m e a s u r e m e n t o f I at the p i c o m o l e level in p l a s m a a n d urine s a m p l e s o f h u m a n origin. T h e N - P F P deriw.tive o f the c o m p o u n d is stable a n d yields high
248
SHORT COMMUNICATIONS ~Q
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~ D
m/z
361
L 72974
50t.t.i _1
to
0
-J
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.d :3 h
A
,
la5
m/z
35
50-
4.8Q
4.69
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5.2g
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• .~e
" 5.sl~
6.'mD
6.211
6.4o
rain
Fig. 4. Selected-ion m o n i t o r i n g traces obt:lincd f r o m a urinary extract (50 id) o b t a i n e d on d a y 10 with 50 p m o l / m l I.S..'ldded.
too
._J
5:
I0
.J 0 (3..
I
L~ 0 0 .I
I
0
2 TIME
I
....
4
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i
6
8
( HOURS )
Fig. 5. Plasma c o n c e n t r a t i o n - t i m e course o f I a f t e r the tenth dose o f 12 m g per day o f I administered orally to a healthy volunteer.
sensitivity with no endogenous interferences. The reproducibility demonstrates that precision and accuracy o f this technique are suitable for routine G C - M S analysis o f I. The method has been successfully applied to the quantification of I in human plasma and urine. ACKNOWLEDGEMENTS
T h e a u t h o r s a c k n o w l e d g e N a d i a le H e n a n f for h e r technical a s s i s t a a c e a n d Muricl R o h f r i t s c h for her secretarial assistance. W e also t h a n k D r . N o r m a n D. H u e b e r t for c o n s t r u c t i v e discussion,
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249
REFERENCES I W. Birkmayer, J. Knoll, P. Ricderer, M. B. Y o u d i m , V. M a r s a n d J. M a r t o n , J. Neural Transm.. 64
(1985) 113. 2 U. K. Rinne, Acta Neurol. Scami.. 68 (1983) 144. 3 L. I. Golbe, A. N. L i c b e r m a n , M, D. Mucntcr, J. E. Ahlskog, G. G o p i n a t h a n , A. N. N e o p h y t i d c s , S. H. F o o and R. C. Duvoisin, Neuropharmacol., t I (1988) 45: 4 T. N. Chase, M. M. M o u r a d i a n , (3. Fabbrini a n d J. L. Juncos, J. Neural Transm., SuppL, 27 (t988) 3. 5 P. K. Sonsalla and L. I. G o l b e , Clin. NeuropharmacoL, I1 (1988) 500. 6 G . Cohen, Adv. NetlroL, 45 ( ! 986) I 19. 7 K. Chiba, A. T r e v o r a n d H. Castagnoli, Biochem. Bioplow. Res. Commun., 120 (t984) 574. 8 R. E. Heikkila, L. M a n z i n o , F. S. C a b b a t a n d R. C. Duvoisin, Naltow, 3I I (1985) 467. 9 J. A. Javitch, R. J. D ' A m a t o a n d S. M. Strittmater, Prof. Natl. Acad. Sci. U.S.A.. 82 (1985) 2173. 10 M. Zreika, J. R. F o z a r d , M. W. Dudley, P. Bey, 1. M c D o n a l d and M. G . Palfreyman, J. Neural Transm. ( P - D Sect.), I (1989) 243.