Simultaneous analysis of 14 benzodiazepines in oral fluid by solid-phase extraction and LC-MS-MS

A simple and rapid procedure for the simultaneous screening of 14 benzodiazepines in oral fluid is presented. The procedure involves a solid-phase extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The target compounds include diazepam, oxazepam, temazepam, nordiazepam, lorazepam, chlordiazepoxide, alprazolam, alpha-hydroxyalprazolam, desalkylflurazepam, hydroxyethylflurazepam, clonazepam, 7-aminoclonazepam, flunitrazepam, and 7-aminoflunitrazepam. Oral fluid was obtained using a simple device that collects approximately 0.4 mL of oral fluid and dilutes it with 0.8 mL of preservative. The oral fluid sample preparation involves a solid-phase extraction on a Varian Bond Elut cartridge. Quantitation was performed by LC-MS-MS using nordiazepam-d(5) as the internal standard. The extraction efficiency exceeded 83% for all compounds except for 7-aminoclonazepam, which had a recovery of 55%. The limits of quantitation ranged from 0.1 ng/mL to 1.0 ng/mL in the multiple reaction monitoring mode. This method was used to confirm 41 patients that screened positive using the OraSure Technologies Benzodiazepine Intercept MICRO-PLATE Enzyme Immunoassay kit. All screened-positive patients were positive for at least one of the analyzed benzodiazepines, thus showing that this method is suitable for confirmation of the Intercept Benzodiazepine assay.