Simultaneous Analysis of 14 Benzodiazepines in Oral Fluid by Solid-Phase Extraction and LC–MS–MS

ISSN： 0146-4760

Source： Journal of Analytical Toxicology, Vol.31, Iss.7, 2007-09, pp. : 369-376

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Abstract

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, α-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-d₅ 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.