Direct enantiomeric resolution of mephenytoin and its N-demethylated metabolite in plasma and blood using chiral capillary gas chromatography

Abstract

A gas chromatographic method was developed for the determination of the R- and S-enantiomers of the anticonvulsant, mephenytoin, and its N-demethylated metabolite, 5-phenyl-5-ethylhydantoin (PEH), in plasma and blood. Direct enantiomeric separation of mephenytoin and its internal standard was obtained using a chiral capillary column (Chirasil-Val®) followed by nitrogen specific detection. However, resolution of the enantiomers of PEH and its internal standard required propylation at the 3-position of the hydantoin ring prior to analysis. Similar linear and reproducible standard curves were obtained from both plasma and blood over the concentration range 50 ng/ml to 5 μg/ml, and above 100 ng/ml the reproducibility was less than 8% (coefficient of variation). Pronounced stereoselective differences in the plasma concentration—time curves for both mephenytoin and PEH were observed in a normal subject who received a single oral dose of 300 mg racemic mephenytoin. The peak plasma level of S-mephenytoin was only one-fifth that of the R-enantiomer and its elimination half-life was less than 3 h compared to over 70 h for R-mephenytoin. Similarly, S-PEH levels were barely detectable whereas concentrations of R-metabolite steadily increased over 4–6 days before slowly declining.