Analysis of oxazepam in urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection by post-column derivatization.

A reversed-phase high-performance liquid chromatographic method for oxazepam in human urine samples has been developed. The sample preparation consists of an enzymatic hydrolysis with beta-glucuronidase, followed by a solid-phase extraction process using Bond-Elut C2 cartridges. The mobile phase used was a methanol-water (60:40, v/v) mixture at a flow-rate of 0.50 ml/min. The column was a 3.5 cm x 4.6 mm I.D. C18 reversed-phase column. The detection system was based on a fluorescence post-column derivatization of oxazepam in mixtures of methanol and acetic acid. A linear range from 0.01 to 1 micrograms/ml of urine and a limit of detection of 4 ng/ml of urine were attained. Within-day recoveries and reproducibilities from urine samples spiked with 0.2 and 0.02 microgram/ml oxazepam were 97.9 and 95.0 and 2.1 and 9.4%, respectively.