DETERMINATION OF TRIAZOLAM AND ITS METABO LITES IN HUMAN URINE BY EMIT, GAS-CHROMATOGRAPHY MASS-SPECTROMETRY AND THERMOSPRAY LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY

EMIT, gas chromatography-mass spectrometric (GC-MS) and thermospray hi gh performance liquid chromatography-mass spectrometric (LC-MS) method s for the determination of triazolam (TRZ) and its metabolites (1-hydr oxymethyltriazolam (alpha-HT) and 4-hydroxytriazolam (4-HT)) in the hu man urine were investigated. TRZ was analyzed by EMIT st Urine Benzodi azepine Assay. EMIT cutoff was 50 ng/ml of TRZ. TRZ and its metabolite s were extracted from the human urine using Sep-Pak C-18 cartridge wit h dichloromethane-methanol (90:10 v/v) as the eluent. The eluate was e vaporated to dryness under stream of N-2. The residue was dissolved in 1 ml of methanol, 50 mu ul was injected into the LC-MS. LC analyses w ere performed on a TSKgel Octyl-80Ts in the solvent system of 100 mM a mmonium acetate-methanol (50:50 v/v) at a flow rate of 1.0 ml/min. For GC-MS, the extract was derivatized at 90 degrees C for 30 min with 50 mu l BTZ. GC analyses were performed on a fused silica column DB-1. T he column temperature was 290 degrees C. The detection limits of these compounds by scan mode were 800 ng/ml for alpha-HT and 4-HT with GC-M S, 50 ng/ml for TRZ and alpha-HT, 100 ng/ml for 4-HT with LC-MS and th ose by selected ion monitoring mode were 50 ng/ml for alpha-HT, 100 ng /ml for 4-HT with GC-MS, 5 ng/ml for TRZ and alpha-HT, 10 ng/ml for 4- HT with LC-MS.