Determination of diazepam and its major metabolites in man and in the cat by high-performance liquid chromatography

Abstract

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of diazepam, and its major metabolites, oxazepam, temazepam and nordiazepam in plasma, blood, and urine of humans and cats. The assay for the compounds involves extraction into benzene—methylene chloride (90:10) from plasma, blood or urine buffered to pH 9.0. In both species the overall recovery of diazepam and its major metabolites from plasma or blood ranged from 60 ± 3.2 to 89 ± 13% (S.D.) and for urine from 79 ± 7.9 to 93 ± 10.5% (S.D.). The sensitivity limit of the assay using UV detection at 254 nm was 50 ng/ml of plasma and blood in both species except for human urine (post-Glusulase) which was 200 ng/ml. The HPLC assay was used to monitor the plasma concentration—time profile in humans following a 10-mg oral dose of diazepam and the blood concentration time profile of diazepam and nordiazepam in cats following a 10 mg/kg intravenous dose of either diazepam or nordiazepam. The HPLC assay data correlated well with data generated by an electron-capture—gas—liquid chromatographic assay.