Determination of triazolam involving its hydroxy metabolites in hair shaftand hair root by reversed-phase liquid chromatography with electrospray ionization mass spectrometry and application to human hair analysis

A sensitive method using reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry has been developed for simultaneo us determination of triazolam and its hydroxy metabolites in hair. After th e addition of deuterium-labeled 1-hydroxymethyltriazolam as an internal sta ndard, the analytes in hair shaft and hair root samples were extracted with a basic medium, CH Cl,: MeOH:28% NH4OH (20:80:2) at room temperature overn ight. The chromatographic separation of the analytes was achieved using a s emimicro HPLC column (3-mum particle size; 100 x 2.0-mm i.d.) by gradient e lution with acetonitrile in water containing 1% acetic acid as eluent. The mass spectrometer was operated in selected-ion monitoring mode at quasi-mol ecular ions [M+H](+) of triazolam and its metabolites. The method has been applied to determine the incorporation of triazolam and its metabolites int o the hair shafts and hair roots of Dark Agouti rats administered 3 or 6 mg /kg triazolam intraperitoneally twice a day for 5 days. Triazolam, 1-hydrox ymethyltriazolam, and 4-hydroxytriazolam were incorporated into the hair sh afts and the hair roots. The concentration of 4-hydroxytriazolam was the hi ghest of all compounds detected. An unknown substance considered to be 1,4- dihydroxytriazolam also appeared in the hair samples. The structural elucid ation was performed with online HPLC-MS after acetylation of the substance with acetic anhydride and pyridine. The time course studies of triazolam an d the metabolites in both rat hair roots and plasma were carried out after single intraperitoneal administration of triazolam. The concentrations of t riazolam and the metabolites in the hair roots reflected those in the plasm a. The proposed method using selected-reaction monitoring was applied to th e determination of triazolam and the metabolites in human hairs of a triazo lam addict. Triazolam, 1-hydroxymethyltriazolam, and 4-hydroxytriazolam wer e identified in the black hair shafts, whereas only triazolam was detected in the hair roots and the white hair shafts. This is the first report on th e detection of triazolam and its metabolites in human hairs. (C) 2001 Acade mic Press.