Determination of chlorzoxazone and 6-hydroxychlorzoxazone in human plasma and urine by high-performance liquid chromatography

A sensitive and reproducible method is described for the determination of the cytochrome P450 enzyme 2E1 substrate chlorzoxazone and its primary metabolite 6-hydroxychlorzoxazone in human plasma and urine. Plasma or diluted urine were acidified, incubated with β-glucuronidase and then were extracted with diethyl ether. Separation of the analytes was achieved on a C₁₈ column with UV detection set at 283 nm. Excellent linearity was observed over the concentration ranges of 100–3000 ng/ml and 4–400 μg/ml in plasma and urine, respectively. The intra-assay variability was 5.1% and the inter-assay variability was 8.2% for each compound in each matrix. The method presented is applicable to pharmacokinetic and pharmacogenetic studies utilizing chlorzoxazone.