High-performance liquid chromatographic determination of triazolam and its metabolites in human urine

A high-performance liquid chromatographic method for the determination of triazolam and its main metabolites (1-hydroxymethyltriazolam and 4-hydroxytriazolam) in human urine has been developed. After hydrolysis with beta-glucuronidase, the unchanged drug and its metabolites were extracted with a Sep-Pak C18 cartridge and further purified by a Sep-Pak silica cartridge. The extract was chromatographed on a reversed-phase column using a mobile phase consisting of methanol-10 mM phosphate buffer, pH 8 (65:35) and UV detection at 220 nm. The overall recoveries of triazolam, 1-hydroxymethyltriazolam and 4-hydroxytriazolam were ca. 88, 75 and 75%, respectively, and the detection limits of these compounds were 5 ng/ml, using a 10-ml specimen.