Benzodiazepine Analysis by Negative Chemical Ionization Gas Chromatography/Mass Spectrometry

The application of gas chromatography/mass spectrometry (GC/MS) for the simultaneous quantitation of seven commonly encountered urinary benzodiazepine metabolites is described. After comparison of the signal-to-noise ratios of high mass ions of benzodiazepines using electron impact (EI), positive chemical ionization (PCI), and negative chemical ionization (NCI), NCI was chosen because of its increased sensitivity, which ranged from four to several thousand times that of either PCI or EI. This method is novel because NCI spectra for many of these compounds have not been described. For quantitation of benzodiazepines in urine, sample preparation consisted of enzymatic hydrolysis, liquid-liquid extraction, and reaction with a silylating reagent to form trimethylsilyl derivatives. The extraction efficiency of the method was greater than 70% (range, 73–89%) for nordiazepam, oxazepam, temazepam, iorazepam, N-1-hydroxyethylflurazepam, α-hydroxyal-prazolam, and (α-hydroxytriazolam; the linear range for these compounds was from 50 to 2000 ng/mL. Within-run precision was less than 6% for all analytes in the range 50–2000 ng/mL; however, run-to-run precision ranged from 3 to 21%, depending on the analyte and concentration. Quantitation was based on area ratio of high mass ions relative to deuterated internal standards, acquired by scanning the mass range from m/z 250 to 450. Because these studies were performed in the scan mode, if desired, the sensitivity could be increased by using selected ion monitoring.