Determination of Triazolam and Its Metabolites in Human Urine by EMIT, Gas Chromatography-Mass Spectrometry and Thermospray Liquid Chromatography-Mass Spectrometry.

Abstract

EMIT, gas chromatography-mass spectrometric (GC-MS) and thermospray high performance liquid chromatography-mass spectrometric (LC-MS) methods for the determination of triazolam (TRZ) and its metabolites (1-hydroxymethyltriazolam (α-HT) and 4-hydroxytriazolam (4-HT)) in the human urine were investigated. TRZ was analyzed by EMIT st Urine Benzodiazepine Assay. EMIT cutoff was 50 ng/ml of TRZ. TRZ and its metabolites were extracted from the human urine using Sep-Pak C18 cartridge with dichloromethane-methanol (90 : 10 v/v) as the eluent. The eluate was evaporated to dryness under stream of N2. The residue was dissolved in 1 ml of methanol, 50 μl was injected into the LC-MS. LC analyses were performed on a TSKgel Octyl-80Ts in the solvent system of 100 mM ammonium acetate-methanol (50 : 50 v/v) at a flow rate of 1.0 ml/min. For GC-MS, the extract was derivatized at 90°C for 30 min with 50 μl BTZ. GC analyses were performed on a fused silica column DB-1. The column temperature was 290°C. The detection limits of these compounds by scan mode were 800 ng/ml for α-HT and 4-HT with GC-MS, 50 ng/ml for TRZ and α-HT, 100 ng/ml for 4-HT with LC-MS and those by selected ion monitoring mode were 50 ng/ml for α-HT, 100 ng/ml for 4-HT with GC-MS, 5 ng/ml for TRZ and α-HT, 10 ng/ml for 4-HT with LC-MS.