Published: Journal of Analytical Toxicology, ISSN 0146-4760, Volume 45, Number 7, September, pp.369-376

Simultaneous Analysis of 14 Benzodiazepines in Oral Fluid by Solid-Phase Extraction and LC–MS–MS
George Ngwa[1], Dean Fritch[2], Kristen Blum[2], Gregory Newland[3]
[1]Chemistry, Lehigh University, 6 E. Packer Ave., Seeley G. Mudd Building, Bethlehem, Pennsylvania 18015
[2]OraSure Technologies, Research and Development, Bethlehem, Pennsylvania
[3]Applied Biosystems, Applied Markets/Small Molecules, 21 S. 8th St., Bangor, Pennsylvania 18013

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, a-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.

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