BIOLOGICAL MASS SPECTROMETRY, VOL. 20, 559-564 (1991)
Enantiospecific Quantification of Hexobarbital and its Metabolites in Biological Fluids by Gas Chromatography/ElectronCapture Negative Ion Chemical Ionization Mass Spectrometry Cbandra Prakash, Adedayo Adedoyin, Grant R. Wilkinson and Ian A. Blair7 Department of Pharmacology, Vanderbilt University, Nashville,Tennessee 37232, USA
A highly sensitive and specak: assay based on gas cbromatography/electn capture negative ion chemicpI ionization mass spectrometry has been developed for the analysis of the aantiomers of hexobarbital and its mahr metabolites in human urine and plasmu. s-( )-(5-ZH,)hexobarbitaI aad R-( )-(SZH,)hexolbarbitd were syntbesized for clinical studies along with (fH1$-zH,)bxobarbital and the deuterated major metabolites for me as internal and reference standards. Hexobarbital enantismers and their metabolites were analyzed after pentaeoorob y 1 and trimethylsilyl derivatization, foilowing solid-plmse extraction from plasma and urine, Intense negative ion spectra were observed for all of the derivatives. Tbe base peak ia the spectra corresponded to the M - pentafluorobenzyl mion [ M PFBI- except for 1,5dimethylbnrbiturie acid, where MI' was the most abundant ion. Tbe applicability of the metbod was demonstrated by following the plasma concentration-time profiles and urinary excretion in a male extedve metabolizer of mephenytdn who was given II pseodorncemic oral dose of bexobuMtal containing equal 50 mg amounts of s-( +)-'(H,)hexohrbital a d R-( -)-(zH3)bexobPrbit.l. Muked stereowlective disposition was observed, with the R-(-)enautiomer king more effwiently met.bolizod, p r i d y by a k y c k O X h b t i Q O and M g ChVage.
-
+
-
-~
INTRODUCTION The metabolism of hexobarbital (HB; S-(cyclohexen-1'yl)-l,5-dimethylbarbitu~cacid, 1) has been well studied and the various findings have been recently reviewed.' In humans, alicyclic oxidation to 3'-hydroxy-HB (2) with its subsequent conversion to the 3'-keto (3) metabolite is a major pathway along with Wdimethylbarbituric acid (1,5-DMB, 4) resulting from formation of an intermediate epoxide-diol (Fig. 1). A number of additional minor metabolites (such as N-desmethyl-HB, 5) have also been reported. Because such metabolism is thought to be mediated by the cytochrome P-450-containing mixed-function rnonooxygenase system of the liver, hexobarbital has been widely used as an in uiuo probe to assess oxidative drug-matabolizing ability. Such use has, generally, been limited to the fate of the administered racemic drug despite evidence, particularly in the rat, of stereoselective metabolism. A recent study in humans has also demonstrated pronounced enantioselective disposition, with the oral clearance of R-( -)HB being fivefold higher than that of the S-( +)-enantiomer.2 However, the metabolic pathway(s) responsible for such differential disposition were not defined in this study because the high-performance liquid chromatography (HPLC)-based assay was unable to resolve the enantiomers of the various metabolites. In addition to stereoselective considerations, the metabolism of HB is also of interest because of a
nutative role for cvtochrome P45Qup. This specific knzyme is responsibie for the 4-hydroxylation of i 4 -)mephenytoin and its functional activity is bimodally distributed in the population because of genetic polyanorpl~iism.~ In uitto studies with human liver microsomes and a preliminary clinical study suggests that alicyclic formation of 3'-hydroxy-HJ3 is mediated by cytochrome P-450,, .**' Accordingly, HB disposition might be expected to be different in the extensive and poor metabolizer phenotypes, but the magnitude of such interphenotypic differences and the relative involvement of the two enantiomers is unknown.
5 f Author to whom correspondence should be addressed.
1052-9306/91fmsSu-06 W5.W by John Wiley 8r Sans, Ltd.
Q 1991
Figure 1. Metabolism of hexobarbital.
Received 15 Mmch 1y91 Revised IS May 1991
560
C. PRAKASH, A. ADEDOYIN, G. R. WILKINSON A N D I. A. BLAIR
A useful strategy for investigating stereoselective disposition in uioo is the administration of a pseudoracemate in which the enantiomers are differentially labeled using, for example, a stable isotope.6 Indeed, this approach has been used with HB in the rat;7 however, the sensitivity of the gas chromatography/electron impact mass spectrometry (GC/EIMS) methodology was insufficient for analogous studies to be performed in humans. We have shown previously, however, that electron capture negative ion chemical ionization (NICI) GC/MS of pentafluorobenzyl (PFB) derivatives of lipids can lead to an increase in detection sensitivity of two orders of magnitude.* Such derivatization was therefore applied to the analysis of HB and its metabolites in plasma and urine and the assay's suitability for clinical studies was investigated after administration of an oral dose of HB to an extensive metabolizer of mephenytoin. MATERIALS AND METHODS
Cdneral Bis(trimethylsily1)trifluoroacetamide (BSTFA) was obtained from Supelco (Bellefonte, Pennsylvania), ethyl-lcyclohexenylcyano acetate was from K &L K Laboratories (Cleveland, Ohio) and racemic HB was obtained from Sigma (St Louis, Missouri). C'H,I (isotopic purity > 99 atom%), other reagent-grade chemicals and solvents for synthetic procedures were obtained from Aldrich (Milwaukee, Wisconsin). HPLC-grade solvents were from Fisher (Atlanta, Georgia). Metabolites 3'-OH-HB (2) and 1,5-DMB (4) were synthesized as described previ~usly.~*~' Hexadeuterated analogs were prepared by similar methods using (1,5-'H6)HB (prepared as described below) in place of unlabeled HB. They both contained < O S % protium impurity. Ethyl-(3-2W,)-%cyian~2~l-cyclohexenyl)prop~onate (6)
was heated under reflux at 80°C for 5 h. After cooling the solution to O"C, 6.66 ml acetic acid in 167 ml icecold water was added and the mixture was kept at 0°C for 12h. The solid that separated was filtered, washed with water and dried in a vacuum desiccator overnight. It was crystallized from ethanol to give colorless prismatic needles (8.7 g, 79%), m.p. 105-108 "C.
(5-'H,)Hexobarbital (la) Imine 7 (8.0 g, 33.6 mmol) was mixed with dilute H,SO, (80 g, 20%, v/v) and stirred for 16 h at room temperature and at 60 "C for 2h. It was then cooled to 0 "C and left overnight. The separated solid was filtered and crystallized with ethanol to give colorless needles of racemic (5-'H,)HB (7.1 g, 88%). NMR (6) 8.16 (s, lH, NH), 5.74 (m, lH, olefinis), 3.28 (s, 3H, NCH,), 2.08 and 1.92 (m, 4H, H-3' and H-6) and 1.54 (m, 4H, H-4' and H-5'). Racemic (5-'H,)HB (7 g, 30 mmol) was stirred with a solution of N-methylquininium hydroxide in methanol (170 ml, 0.22 N) for 10 min." The methanol was evaporated under vacuum and the remaining oily residue was dissolved in absolute ethanol (70 ml). Ether was added to the solution until the turbidity persisted. The solution was kept at room temperature and crystallization allowed to proceed. The crystals were isolated and recrystalliied three times from ethanol to give R-( -)-('H,)HB as the N-methylquininium salt. The free HB acid was obtained by treatment of the salt with 2 N H,SO, in ethanol; m.p. 153-154"C, [a];' - 12.2 (c = 0.98; ethanol). The mother liquors were evaporated, the residue recrystallized three times from ethanol and treated with 2 N H'SO, to give S-(+)-('H,)HB; m.p. 153-154"C, [a];' 11.6 (c = 1.2; ethanol). The purity of each enantiomer was shown to be >98% by chiral HPLC analysis on a cyclodextrin (Cyclobond-1; Advanced Separations Technology, Whippany, New Jersey) column.'2
+
(2H,)Hexobarbital (1)
A solution of sodium ethoxide (from sodium (1.4 g, 61.4
mmol) in absolute ethanol (50 ml)) was cooled to 0°C and ethyl-1-cyclohexenylcyano acetate (10.0 g, 56.0 mmol) was added dropwise with stirring at 0°C. After stirring for 30 min, C2H,I in ether (8.10 g, 56 mmol) was added and the solution was heated at 60°C for 1.5 h. The reaction mixture was cooled, diluted with water, and the ester was extracted with ethyl acetate. The organic solvent was evaporated and the residue was distilled under reduced pressure to give 6 as a colorless oil. Nuclear magnetic resonance (NMR) (6) 5.99 (t, lH, olefink), 4 26) Qq,2H, C;OOa,CH,), 2.12 (m, 2H), 1.98 (m, 2H), 1.56 (m, 4H) and 1.28 (t, 3H, COOCH,=,).
5-(I - C y l o ~ g x e n y l ) ~ i m t i n o - f S ' K t , ) m e Q L y l acid
er>
A solution of sodium methoxide (from sodium (1.37 g, 57.14 miiof) and methanol (30 ml)) was added dropwise over 1 ' .0 rnm to a stirred solution of ester 6 (10.0 g, 47.61 mrnol a.nd N-methylurea (3.67 g, 47.61 mmoi) in anhydrous methanol (30 ml) at 60°C. The reaction mixture
Racemic unlabeled HB was resolved into its enantiomers in the same fashion as deuterated HB to provide [a]? + 12.30 S-(+)-(,H,)HB, m.p. 153-154"C, (c = 1.3; ethanol) and R-(-)-HB; m.p. 153-154"C, [a];' - 14.9 (c = 1.06; ethanol). The purity of each enantiomer was shown to be >98% by chiral HPLC." (1,5L2H,)Hexobarbital (1b)
This was prepared by the same procedure as described above for l a except that N-('H,)methylurea (isotopic purity > 99.5 atom%7) was used in place of unlabelled N-methylurea. 'H-NMR showed the absence of the N methyl group at 6 3.28 p.p.m.
3'-Ketohexobarbidal(3) Cr,O, (1.40 g) was suspended in a mixture of acetic anhydride (3.8 ml) and acetic acid (7.6 ml) at 0°C. To this solution was added a solution of HB (1.12 g) in
ANALYSIS OF HEXOBARBITAL ENANTIOMERS
benzene (7.6 ml) dropwise over 35 min at 18-20°C. After stirring the reaction mixture at room temperature for 10 min, benzene (7.6 ml) was added and the mixture was cooled in an ice bath. It was then cautiously neutralized with KOH solution (15.2 ml, 10 N). The biphasic solution was poured over ice-cold water and then extracted three times with ether (20 ml). The combined ether solution was washed twice with saturated aqueous NaHCO, solution (15 ml), water and dried (MgSO,). Crystallization of the residue gave a mixture of 3'-keto-HB and 6'-keto-HB. The mixture was reduced using aluminium isopropoxide as previously described." Under these conditions the 3'-keto-HB gave the corresponding 3'-OH-HB while the more hindered 6-keto-HB was unaffected. 3'-OH-HB was separated on a silica-gel column using 20% ethyl acetate in hexane and oxidized with MnO, to afford pure 3I-keto-HB; m.p. 159-161 "C. NMR (S) 6.03 (s, lH, olefinic), 3.30 (s, 3H, NB,), 2.40 (t, 2H), 2.26 (t, 2H), 1.99 (m, 2H) and 1.72 (s, 3H, CH3). (1,5-'H6)3'-Keto-HB was prepared in the same way starting from (1,5'H,)HB and it contained 5:f). Recovery of HB and metabolites ranged from 25% to 65% and intra- and inter-day variability was in the range 7.7% to 17.6% (Table 1). The assay was applied to the analysis of HB and metabolites in the plasma and urine of a subject given a
) .
c .-
In 0 c c
.-c
> 0
.L
-a L 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 time (mid Figure 3. Reconstructed total ion current chromatogram of HB and its putative metabalites: (a) 1,5-DMB-PFB; (b) HB-PFB; (c) fY-keto-HB-PFB; (d) 3’-OH-HB-TMS-PFB; (e) 3-keto-HB-PFB; ( f ) N-desmethyl-HB-bis-PF6. Injections were made in the splitless mode on a 20 m SPB-1 fused-silica capillary column (0.32 mm i.d., 0.25 pn coating thickness). The GC column was held at 140°C for 1 min then programmed to 260°C at 20°C min-’. Helium was used as carrier gas at a flow rate of 1 ml min-’.
2.0
3.0
4.0
5.0
6.0
7 .o
time (mid Figure 5. Selected ion current profile of plasma extract of HB and its metabolites: (a) (ZH,)HB-PFB; (b) (2H,)H6-PFB; (c) (ZH,)HB-PFB; (d) (2H,)1,5-DMB-PFB; (e) (2H,)l,5-DMBGC/MS conditions were as PFB; (f) (’He)1.5-DMB-PFB. described in the legend to Fig. 2, except that a 10 m column was used.
ANALYSIS OF HEXOBARBITAL ENANTIOMERS
563
0.8
z
-*a
0-HB A Y-ketO-HB
-
\
a
0.6
*-DW
2 c U a
2.0
3.0
4.0
5.0
6.0
1.0
time (minl
n -
J
0.4
0
z /
Figure 6. Selected ion current profile of a plasma extract of HB metabolites: (a) ('H0)3'-keto-HB-PFB; (b) ('H3)3'-keto-HBPFB; (c) ('H,)6-keto-HB-PFB; (d) ('H6)3-keto-HB-PFB; (e) ('Ho)3'-OH-HB-TMS-PFB; (f) ('H3)3'-OH-HB-TMS-PFB; (9) ('H,)Y-OH-HB-TMS-PFB; (h) ('H,)N-desmethyl-HB-bis-PFB; (i) ('H,)N-desmethyI-HB-bis-PFB. GC/MS conditions were as described in the legend to Fig. 2, except that a 10 m column was
0 0 4
3
0.2
5
a 0.0
2
0
100 mg oral dose of pseudoracemic HB containing equal amounts of S-(+)-(2H,)HB and R-(-)-(2H,)HB. This preliminary investigation confirmed the stereoselective disposition of HB in humans as reported previously. lS2 The two enantiomers demonstrated very rapid absorption which was completed within the first half hour after administration. R-(-)-HB was eliminated much more rapidly from the plasma than S-( +)HB, resulting in higher plasma concentrations of S-(+)HB over time. This difference in elimination also resulted in significantly higher plasma concentrations of the 3'-keto and DMB metabolites from the R-( -)-enantiomer. Only low plasma levels of N-desmethyl-HB were detected, while the 3'-OH metabolite was present only in trace to unmeasurable amounts from both enantiomers (Fig. 7). Pharmacokinetic parameters from these data revealed that R-(- )-HB had a tenfold greater oral clearance than S-(+)-HB (147.5 versus 16.8 1 h-I) and a threefold shorter terminal half-life (0.93 versus 3.1 1 h). Urinary excretion data (Table 2) showed 1,5-DMB as the major urinary metabolite of both enantiomers. More of the (-)-HB was excreted as the 3'-OH, 3'-keto and DMB metabolites than S-(+)-HB, while the reverse was the case for unchanged drug and N-desmethyl-HB. The observed stereoselectivity in the plasma disposition of HB was also apparent in the 0-8 h total urinary recovery, which was 14% of the administered dose for S-( +)-HB and 65% for R-( -)-HB.
6
4
lb)
a
0-HB A 3'-kct*HB
b-
*-DMB
-
n -
IECOVelV.
(%)
HB 3'-OH-HB 3'- Keto-HB DMB N-Desmethyl-HB
65.2 25.3 59.5 56.8 60.0
Coalficient of variation (%.)b Within-day Between-day n=8 n=10-13
10.5 15.2 7.7 15.3 15.2
11.1 16.1 10.5 11.3 17.6
Mean recovery over the entire linearity range ( n = 30). bAverage coefficient of variation found at concentrations of 10, 100and l o 0 0 ng ml-'.
Ndesmethvl-HB
5
a 0.0
2
0
4
6
8
TIME (hours)
Figure 7. Plasma concentration-time curves for HB enantiomers and their metabolites in an extensive metabolizer of mephenytoin after oral administration of 50 mg each of (a) S-(+)-('H,)HB and (b) R- (-)-('HJ HB.
Table2 Urinary recovery (0-8 h; % d w ) of HB and metabolites Excretion in 0 4 h urine
S-(+)
Table 1. Reproducibility of extraction of HB enantiomem and M i metabolites from human urine
0
TIME Ihourr)
Used.
Extraction
Ndesmethyl-HB
b-
fl-(-)
HB 3'-OH-HB 3I-Keto-HB DMB N-Desmethyl-HB
0.12 2.0 1.2 10.6 0.02
0.05 2.8 7.0 55.3
Total
14.0
65.1
0.004
Acknowledgements
a
This work was supported in part by US Public Health Service grants GM-31304, ES-00267 and RR00095.
C. PRAKASH, A. ADEDOYIN, G . R. WILKINSON A N D I. A. BLAIR
564
REFERENCES 1. M. van der Graaff, N. P. E. Vermeulen and D. D. Breimer, Drug. Mefab. Rev. 19,109(1988). 2. M. H. H. Chandler, S. R. Scott and R. A. Blouin, Clin. Pharmacol. Ther. 43.436 (1988). 3. G. R. Wilkinson, F. P. Guengerich and R. A. Branch, Pharmacol. Ther. 43,53 (1989). 4. R. G. Knodell, R. K. Dubey, G. R. Wilkinson and F. P. Guengerich, J. Pharmacol. Exp. Ther. 245,845(1988). 5. T. Yasumori, N. Murayama, Y. Yamazoe and R. Kato, Clin. Pharmacoi. Ther. 47.313 (1988). 6. T. A. Baillie, Pharmacol. Rev. 33, 81 (1 981 ). 7. M. van der Graaff, P. H. Hofman, D. D. Breimer, N. P. E. Vermeulen, J. Knabe and L. Schamber, Homed. Mass Spectrom. 12,464(1985).
8. I. A. Blair, in Methods of Enzymology, ed. by R. C. Murphy and F. A. Fitzpatrick, Vol. 187,p. 13. Academic Press, New York (1990). 9. N. P. E. Vermeulen, B. H. Bakker, J. Schultink, A. van der Gen and D. D. Breimer, Xenobiofica 9, 289 (1979). 10. H. Yoshimura, Chem. Pharm. Bull. 6, 13 (1958). 11. J. Knabe and R. Krauter, Arch. Pharm. 298,l (1965). 12. M. H. H. Chandler, R. J. Guttendorf, R. A. Blouin and P. J. Wedlund, J. Chromefogr.419,426(1987).
Enantiospecific Quantification of Hexobarbital and its Metabolites in Biological Fluids by Gas Chromatography/ElectronCapture Negative Ion Chemical Ionization Mass Spectrometry Cbandra Prakash, Adedayo Adedoyin, Grant R. Wilkinson and Ian A. Blair7 Department of Pharmacology, Vanderbilt University, Nashville,Tennessee 37232, USA
A highly sensitive and specak: assay based on gas cbromatography/electn capture negative ion chemicpI ionization mass spectrometry has been developed for the analysis of the aantiomers of hexobarbital and its mahr metabolites in human urine and plasmu. s-( )-(5-ZH,)hexobarbitaI aad R-( )-(SZH,)hexolbarbitd were syntbesized for clinical studies along with (fH1$-zH,)bxobarbital and the deuterated major metabolites for me as internal and reference standards. Hexobarbital enantismers and their metabolites were analyzed after pentaeoorob y 1 and trimethylsilyl derivatization, foilowing solid-plmse extraction from plasma and urine, Intense negative ion spectra were observed for all of the derivatives. Tbe base peak ia the spectra corresponded to the M - pentafluorobenzyl mion [ M PFBI- except for 1,5dimethylbnrbiturie acid, where MI' was the most abundant ion. Tbe applicability of the metbod was demonstrated by following the plasma concentration-time profiles and urinary excretion in a male extedve metabolizer of mephenytdn who was given II pseodorncemic oral dose of bexobuMtal containing equal 50 mg amounts of s-( +)-'(H,)hexohrbital a d R-( -)-(zH3)bexobPrbit.l. Muked stereowlective disposition was observed, with the R-(-)enautiomer king more effwiently met.bolizod, p r i d y by a k y c k O X h b t i Q O and M g ChVage.
-
+
-
-~
INTRODUCTION The metabolism of hexobarbital (HB; S-(cyclohexen-1'yl)-l,5-dimethylbarbitu~cacid, 1) has been well studied and the various findings have been recently reviewed.' In humans, alicyclic oxidation to 3'-hydroxy-HB (2) with its subsequent conversion to the 3'-keto (3) metabolite is a major pathway along with Wdimethylbarbituric acid (1,5-DMB, 4) resulting from formation of an intermediate epoxide-diol (Fig. 1). A number of additional minor metabolites (such as N-desmethyl-HB, 5) have also been reported. Because such metabolism is thought to be mediated by the cytochrome P-450-containing mixed-function rnonooxygenase system of the liver, hexobarbital has been widely used as an in uiuo probe to assess oxidative drug-matabolizing ability. Such use has, generally, been limited to the fate of the administered racemic drug despite evidence, particularly in the rat, of stereoselective metabolism. A recent study in humans has also demonstrated pronounced enantioselective disposition, with the oral clearance of R-( -)HB being fivefold higher than that of the S-( +)-enantiomer.2 However, the metabolic pathway(s) responsible for such differential disposition were not defined in this study because the high-performance liquid chromatography (HPLC)-based assay was unable to resolve the enantiomers of the various metabolites. In addition to stereoselective considerations, the metabolism of HB is also of interest because of a
nutative role for cvtochrome P45Qup. This specific knzyme is responsibie for the 4-hydroxylation of i 4 -)mephenytoin and its functional activity is bimodally distributed in the population because of genetic polyanorpl~iism.~ In uitto studies with human liver microsomes and a preliminary clinical study suggests that alicyclic formation of 3'-hydroxy-HJ3 is mediated by cytochrome P-450,, .**' Accordingly, HB disposition might be expected to be different in the extensive and poor metabolizer phenotypes, but the magnitude of such interphenotypic differences and the relative involvement of the two enantiomers is unknown.
5 f Author to whom correspondence should be addressed.
1052-9306/91fmsSu-06 W5.W by John Wiley 8r Sans, Ltd.
Q 1991
Figure 1. Metabolism of hexobarbital.
Received 15 Mmch 1y91 Revised IS May 1991
560
C. PRAKASH, A. ADEDOYIN, G. R. WILKINSON A N D I. A. BLAIR
A useful strategy for investigating stereoselective disposition in uioo is the administration of a pseudoracemate in which the enantiomers are differentially labeled using, for example, a stable isotope.6 Indeed, this approach has been used with HB in the rat;7 however, the sensitivity of the gas chromatography/electron impact mass spectrometry (GC/EIMS) methodology was insufficient for analogous studies to be performed in humans. We have shown previously, however, that electron capture negative ion chemical ionization (NICI) GC/MS of pentafluorobenzyl (PFB) derivatives of lipids can lead to an increase in detection sensitivity of two orders of magnitude.* Such derivatization was therefore applied to the analysis of HB and its metabolites in plasma and urine and the assay's suitability for clinical studies was investigated after administration of an oral dose of HB to an extensive metabolizer of mephenytoin. MATERIALS AND METHODS
Cdneral Bis(trimethylsily1)trifluoroacetamide (BSTFA) was obtained from Supelco (Bellefonte, Pennsylvania), ethyl-lcyclohexenylcyano acetate was from K &L K Laboratories (Cleveland, Ohio) and racemic HB was obtained from Sigma (St Louis, Missouri). C'H,I (isotopic purity > 99 atom%), other reagent-grade chemicals and solvents for synthetic procedures were obtained from Aldrich (Milwaukee, Wisconsin). HPLC-grade solvents were from Fisher (Atlanta, Georgia). Metabolites 3'-OH-HB (2) and 1,5-DMB (4) were synthesized as described previ~usly.~*~' Hexadeuterated analogs were prepared by similar methods using (1,5-'H6)HB (prepared as described below) in place of unlabeled HB. They both contained < O S % protium impurity. Ethyl-(3-2W,)-%cyian~2~l-cyclohexenyl)prop~onate (6)
was heated under reflux at 80°C for 5 h. After cooling the solution to O"C, 6.66 ml acetic acid in 167 ml icecold water was added and the mixture was kept at 0°C for 12h. The solid that separated was filtered, washed with water and dried in a vacuum desiccator overnight. It was crystallized from ethanol to give colorless prismatic needles (8.7 g, 79%), m.p. 105-108 "C.
(5-'H,)Hexobarbital (la) Imine 7 (8.0 g, 33.6 mmol) was mixed with dilute H,SO, (80 g, 20%, v/v) and stirred for 16 h at room temperature and at 60 "C for 2h. It was then cooled to 0 "C and left overnight. The separated solid was filtered and crystallized with ethanol to give colorless needles of racemic (5-'H,)HB (7.1 g, 88%). NMR (6) 8.16 (s, lH, NH), 5.74 (m, lH, olefinis), 3.28 (s, 3H, NCH,), 2.08 and 1.92 (m, 4H, H-3' and H-6) and 1.54 (m, 4H, H-4' and H-5'). Racemic (5-'H,)HB (7 g, 30 mmol) was stirred with a solution of N-methylquininium hydroxide in methanol (170 ml, 0.22 N) for 10 min." The methanol was evaporated under vacuum and the remaining oily residue was dissolved in absolute ethanol (70 ml). Ether was added to the solution until the turbidity persisted. The solution was kept at room temperature and crystallization allowed to proceed. The crystals were isolated and recrystalliied three times from ethanol to give R-( -)-('H,)HB as the N-methylquininium salt. The free HB acid was obtained by treatment of the salt with 2 N H,SO, in ethanol; m.p. 153-154"C, [a];' - 12.2 (c = 0.98; ethanol). The mother liquors were evaporated, the residue recrystallized three times from ethanol and treated with 2 N H'SO, to give S-(+)-('H,)HB; m.p. 153-154"C, [a];' 11.6 (c = 1.2; ethanol). The purity of each enantiomer was shown to be >98% by chiral HPLC analysis on a cyclodextrin (Cyclobond-1; Advanced Separations Technology, Whippany, New Jersey) column.'2
+
(2H,)Hexobarbital (1)
A solution of sodium ethoxide (from sodium (1.4 g, 61.4
mmol) in absolute ethanol (50 ml)) was cooled to 0°C and ethyl-1-cyclohexenylcyano acetate (10.0 g, 56.0 mmol) was added dropwise with stirring at 0°C. After stirring for 30 min, C2H,I in ether (8.10 g, 56 mmol) was added and the solution was heated at 60°C for 1.5 h. The reaction mixture was cooled, diluted with water, and the ester was extracted with ethyl acetate. The organic solvent was evaporated and the residue was distilled under reduced pressure to give 6 as a colorless oil. Nuclear magnetic resonance (NMR) (6) 5.99 (t, lH, olefink), 4 26) Qq,2H, C;OOa,CH,), 2.12 (m, 2H), 1.98 (m, 2H), 1.56 (m, 4H) and 1.28 (t, 3H, COOCH,=,).
5-(I - C y l o ~ g x e n y l ) ~ i m t i n o - f S ' K t , ) m e Q L y l acid
er>
A solution of sodium methoxide (from sodium (1.37 g, 57.14 miiof) and methanol (30 ml)) was added dropwise over 1 ' .0 rnm to a stirred solution of ester 6 (10.0 g, 47.61 mrnol a.nd N-methylurea (3.67 g, 47.61 mmoi) in anhydrous methanol (30 ml) at 60°C. The reaction mixture
Racemic unlabeled HB was resolved into its enantiomers in the same fashion as deuterated HB to provide [a]? + 12.30 S-(+)-(,H,)HB, m.p. 153-154"C, (c = 1.3; ethanol) and R-(-)-HB; m.p. 153-154"C, [a];' - 14.9 (c = 1.06; ethanol). The purity of each enantiomer was shown to be >98% by chiral HPLC." (1,5L2H,)Hexobarbital (1b)
This was prepared by the same procedure as described above for l a except that N-('H,)methylurea (isotopic purity > 99.5 atom%7) was used in place of unlabelled N-methylurea. 'H-NMR showed the absence of the N methyl group at 6 3.28 p.p.m.
3'-Ketohexobarbidal(3) Cr,O, (1.40 g) was suspended in a mixture of acetic anhydride (3.8 ml) and acetic acid (7.6 ml) at 0°C. To this solution was added a solution of HB (1.12 g) in
ANALYSIS OF HEXOBARBITAL ENANTIOMERS
benzene (7.6 ml) dropwise over 35 min at 18-20°C. After stirring the reaction mixture at room temperature for 10 min, benzene (7.6 ml) was added and the mixture was cooled in an ice bath. It was then cautiously neutralized with KOH solution (15.2 ml, 10 N). The biphasic solution was poured over ice-cold water and then extracted three times with ether (20 ml). The combined ether solution was washed twice with saturated aqueous NaHCO, solution (15 ml), water and dried (MgSO,). Crystallization of the residue gave a mixture of 3'-keto-HB and 6'-keto-HB. The mixture was reduced using aluminium isopropoxide as previously described." Under these conditions the 3'-keto-HB gave the corresponding 3'-OH-HB while the more hindered 6-keto-HB was unaffected. 3'-OH-HB was separated on a silica-gel column using 20% ethyl acetate in hexane and oxidized with MnO, to afford pure 3I-keto-HB; m.p. 159-161 "C. NMR (S) 6.03 (s, lH, olefinic), 3.30 (s, 3H, NB,), 2.40 (t, 2H), 2.26 (t, 2H), 1.99 (m, 2H) and 1.72 (s, 3H, CH3). (1,5-'H6)3'-Keto-HB was prepared in the same way starting from (1,5'H,)HB and it contained 5:f). Recovery of HB and metabolites ranged from 25% to 65% and intra- and inter-day variability was in the range 7.7% to 17.6% (Table 1). The assay was applied to the analysis of HB and metabolites in the plasma and urine of a subject given a
) .
c .-
In 0 c c
.-c
> 0
.L
-a L 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 time (mid Figure 3. Reconstructed total ion current chromatogram of HB and its putative metabalites: (a) 1,5-DMB-PFB; (b) HB-PFB; (c) fY-keto-HB-PFB; (d) 3’-OH-HB-TMS-PFB; (e) 3-keto-HB-PFB; ( f ) N-desmethyl-HB-bis-PF6. Injections were made in the splitless mode on a 20 m SPB-1 fused-silica capillary column (0.32 mm i.d., 0.25 pn coating thickness). The GC column was held at 140°C for 1 min then programmed to 260°C at 20°C min-’. Helium was used as carrier gas at a flow rate of 1 ml min-’.
2.0
3.0
4.0
5.0
6.0
7 .o
time (mid Figure 5. Selected ion current profile of plasma extract of HB and its metabolites: (a) (ZH,)HB-PFB; (b) (2H,)H6-PFB; (c) (ZH,)HB-PFB; (d) (2H,)1,5-DMB-PFB; (e) (2H,)l,5-DMBGC/MS conditions were as PFB; (f) (’He)1.5-DMB-PFB. described in the legend to Fig. 2, except that a 10 m column was used.
ANALYSIS OF HEXOBARBITAL ENANTIOMERS
563
0.8
z
-*a
0-HB A Y-ketO-HB
-
\
a
0.6
*-DW
2 c U a
2.0
3.0
4.0
5.0
6.0
1.0
time (minl
n -
J
0.4
0
z /
Figure 6. Selected ion current profile of a plasma extract of HB metabolites: (a) ('H0)3'-keto-HB-PFB; (b) ('H3)3'-keto-HBPFB; (c) ('H,)6-keto-HB-PFB; (d) ('H6)3-keto-HB-PFB; (e) ('Ho)3'-OH-HB-TMS-PFB; (f) ('H3)3'-OH-HB-TMS-PFB; (9) ('H,)Y-OH-HB-TMS-PFB; (h) ('H,)N-desmethyl-HB-bis-PFB; (i) ('H,)N-desmethyI-HB-bis-PFB. GC/MS conditions were as described in the legend to Fig. 2, except that a 10 m column was
0 0 4
3
0.2
5
a 0.0
2
0
100 mg oral dose of pseudoracemic HB containing equal amounts of S-(+)-(2H,)HB and R-(-)-(2H,)HB. This preliminary investigation confirmed the stereoselective disposition of HB in humans as reported previously. lS2 The two enantiomers demonstrated very rapid absorption which was completed within the first half hour after administration. R-(-)-HB was eliminated much more rapidly from the plasma than S-( +)HB, resulting in higher plasma concentrations of S-(+)HB over time. This difference in elimination also resulted in significantly higher plasma concentrations of the 3'-keto and DMB metabolites from the R-( -)-enantiomer. Only low plasma levels of N-desmethyl-HB were detected, while the 3'-OH metabolite was present only in trace to unmeasurable amounts from both enantiomers (Fig. 7). Pharmacokinetic parameters from these data revealed that R-(- )-HB had a tenfold greater oral clearance than S-(+)-HB (147.5 versus 16.8 1 h-I) and a threefold shorter terminal half-life (0.93 versus 3.1 1 h). Urinary excretion data (Table 2) showed 1,5-DMB as the major urinary metabolite of both enantiomers. More of the (-)-HB was excreted as the 3'-OH, 3'-keto and DMB metabolites than S-(+)-HB, while the reverse was the case for unchanged drug and N-desmethyl-HB. The observed stereoselectivity in the plasma disposition of HB was also apparent in the 0-8 h total urinary recovery, which was 14% of the administered dose for S-( +)-HB and 65% for R-( -)-HB.
6
4
lb)
a
0-HB A 3'-kct*HB
b-
*-DMB
-
n -
IECOVelV.
(%)
HB 3'-OH-HB 3'- Keto-HB DMB N-Desmethyl-HB
65.2 25.3 59.5 56.8 60.0
Coalficient of variation (%.)b Within-day Between-day n=8 n=10-13
10.5 15.2 7.7 15.3 15.2
11.1 16.1 10.5 11.3 17.6
Mean recovery over the entire linearity range ( n = 30). bAverage coefficient of variation found at concentrations of 10, 100and l o 0 0 ng ml-'.
Ndesmethvl-HB
5
a 0.0
2
0
4
6
8
TIME (hours)
Figure 7. Plasma concentration-time curves for HB enantiomers and their metabolites in an extensive metabolizer of mephenytoin after oral administration of 50 mg each of (a) S-(+)-('H,)HB and (b) R- (-)-('HJ HB.
Table2 Urinary recovery (0-8 h; % d w ) of HB and metabolites Excretion in 0 4 h urine
S-(+)
Table 1. Reproducibility of extraction of HB enantiomem and M i metabolites from human urine
0
TIME Ihourr)
Used.
Extraction
Ndesmethyl-HB
b-
fl-(-)
HB 3'-OH-HB 3I-Keto-HB DMB N-Desmethyl-HB
0.12 2.0 1.2 10.6 0.02
0.05 2.8 7.0 55.3
Total
14.0
65.1
0.004
Acknowledgements
a
This work was supported in part by US Public Health Service grants GM-31304, ES-00267 and RR00095.
C. PRAKASH, A. ADEDOYIN, G . R. WILKINSON A N D I. A. BLAIR
564
REFERENCES 1. M. van der Graaff, N. P. E. Vermeulen and D. D. Breimer, Drug. Mefab. Rev. 19,109(1988). 2. M. H. H. Chandler, S. R. Scott and R. A. Blouin, Clin. Pharmacol. Ther. 43.436 (1988). 3. G. R. Wilkinson, F. P. Guengerich and R. A. Branch, Pharmacol. Ther. 43,53 (1989). 4. R. G. Knodell, R. K. Dubey, G. R. Wilkinson and F. P. Guengerich, J. Pharmacol. Exp. Ther. 245,845(1988). 5. T. Yasumori, N. Murayama, Y. Yamazoe and R. Kato, Clin. Pharmacoi. Ther. 47.313 (1988). 6. T. A. Baillie, Pharmacol. Rev. 33, 81 (1 981 ). 7. M. van der Graaff, P. H. Hofman, D. D. Breimer, N. P. E. Vermeulen, J. Knabe and L. Schamber, Homed. Mass Spectrom. 12,464(1985).
8. I. A. Blair, in Methods of Enzymology, ed. by R. C. Murphy and F. A. Fitzpatrick, Vol. 187,p. 13. Academic Press, New York (1990). 9. N. P. E. Vermeulen, B. H. Bakker, J. Schultink, A. van der Gen and D. D. Breimer, Xenobiofica 9, 289 (1979). 10. H. Yoshimura, Chem. Pharm. Bull. 6, 13 (1958). 11. J. Knabe and R. Krauter, Arch. Pharm. 298,l (1965). 12. M. H. H. Chandler, R. J. Guttendorf, R. A. Blouin and P. J. Wedlund, J. Chromefogr.419,426(1987).
