Quantitation and confirmation of the diazolo- and triazolobenzodiazepines in human urine by gas chromatography/mass spectrometry.

We report a reliable, rapid, sensitive, and quantitative method for the N- and O-trimethylsilyl (TMS) derivatives of the diazolo- and triazolobenzodiazepines after enzymatic incubation (2 h) and liquid-liquid extraction of 0.5-mL urine volumes. Each analyte was identified by gas chromatography/mass spectrometry through the retention time for the total ion current (TIC) and selected ion monitoring (SIM) of four ion currents. Quantitation of the diazolobenzodiazepines was obtained by the ratio of the base peak ion of the TMS analyte to that of the internal standard bromazepam in the concentration range 50-500 ng/mL (1-10 ng injected into the GC/MS). The limit of detection (LOD) at p less than 0.01 was 50 ng/mL for all the diazolobenzodiazepines. The assay was not quite as sensitive for triazolobenzodiazepines (5-20 ng injected in the GC/MS). The extraction efficiency of the assay ranged from 75 to 92% for all the analytes. The coefficient of variation (CV) for the diazolobenzodiazepines ranged from 5.4 to 9.4% for within-day runs and from 11.1 to 13.9% for between-day runs. For the triazolobenzodiazepines the values were 3.8 to 18.9% for a single day and 3.4 to 19.9% between days. The selected ion current ratio for each analyte was determined for a single day and over a one-week period. There was no statistical difference in the ratios during this time. The confirmation of diazolobenzodiazepines in urine by this method was relatively easy after screening by the immunoassay technique. Identification of triazolobenzodiazepines appeared to be more difficult by both the screening technique and the GC/EI/MS analysis.