An improved HPLC method for rapid quantitation of diazepam and its major metabolites in human plasma

A rapid and specific HPLC method was developed and validated for simultaneous determination of diazepam and its main active metabolites, desmethyldiazepam, oxazepam and temazepam in human plasma. Plasma samples were extracted using toluene. HPLC system included a Chromolith™ Performance RP-18e 100 mm × 4.6 mm column, using 10 mM phosphate buffer (pH 2.5)–methanol–acetonitrile (63:10:27, v/v) as mobile phase running at 2 mL min−1. UV detector (λ = 230 nm) was used. The calibration curves were linear in the concentration range of 2–800 ng mL−1 for diazepam and 2–200 ng mL−1 for the three metabolites (r2 > 0.99). The lower limit of quantification was 2 ng mL−1 for all analytes. Within and between-day precisions in the measurement of QC samples were in the range of 1.8–18.0% for all analytes. The developed procedure was used to assess the pharmacokinetics of diazepam and its main metabolites following single dose administration of 10 mg diazepam orally to healthy subjects.