Simultaneous determination of triazolam and its metabolites in human plasma by liquid chromatography–tandem mass spectrometry

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of triazolam and its metabolites, alpha-hydroxytriazolam (α-OHTRZ) and 4-hydroxytriazolam (4-OHTRZ), was developed and validated. Triazolam-D4 was used as the internal standard (IS). This analysis was carried out on a Thermo® C18 column and the mobile phase was composed of acetonitrile:H2O:formic acid (35:65:0.2, v/v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) and quantification was performed by multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 343.1 → 308.3, 359.0 → 308.3, 359.0 → 111.2 and 347.0 → 312.0 for triazolam, α-OHTRZ, 4-OHTRZ and triazolam-D4, respectively. LLOQ of the analytical method was 0.05 ng/mL for triazolam and 0.1 ng/mL for α-OHTRZ and 4-OHTRZ. The within- and between-run precisions were less than 15.26% and accuracy was −8.08% to 13.33%. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of triazolam in healthy Chinese volunteers.