Determination of benzodiazepines in oral fluid using LC-MS-MS

A rapid, simple, highly sensitive procedure for the simultaneous analysis of bromazepam, alprazolam, clonazepam, lorazepam, oxazepam, diazepam, midazolam, flurazepam, flunitrazepam, nordiazepam, triazolam, temazepam, nitrazepam, and chlordiazepoxide in oral fluid is described, using liquid chromatography coupled to a triple quadrupole mass spectrometer in positive electrospray mode. Benzodiazepines in oral fluid samples (1 mL) were analyzed using the Quantisal collection device, quantified using solid-phase extraction, and detected using liquid chromatography with tandem mass spectrometric detection. For confirmation, two transitions were monitored and one ion ratio determined, which was within 20% of the ratio for known calibration standards. The limits of quantitation ranged from 0.5-5 ng/mL of neat oral fluid; the intraday precision of the assays (n=5) ranged from 2.8-7.29%; and the interday precision ranged from 1.42-6.8% (n=5). The percentage recovery of the drugs from the collection pads ranged from a low of 81.4% for midazolam to the highest of 90.17% for nitrazepam. The procedure can be applied to authentic oral fluid specimens.