Published: Journal of Analytical Toxicology, ISSN 0146-4760, Volume 30, Issue 7, September 2006, pp.441-448

Detection of Benzodiazepines in Hair Using ELISA and LC–ESI-MS–MS
Eleanor I. Miller, Fiona M. Wylie, and John S. Oliver
Forensic Medicine and Science Department, University of Glasgow, University Place, Glasgow, G12 8QQ, Scotland

This study was designed to validate an enzyme-linked immunosorbent assay (ELISA) and liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the detection of nine benzodiazepines in hair. Sixteen hair case samples were tested from drug-related deaths where a positive benzodiazepine blood result was obtained. The case samples were decontaminated with 0.1% sodium dodecyl sulfate, distilled water, and dichloromethane. For ELISA analysis, the samples were extracted by incubation in monobasic phosphate buffer for 1 h and then neutralized with dibasic phosphate buffer. They were diluted 1:5 with phosphate buffer saline (PBS) prior to analysis. For LC–MS–MS, the samples were incubated overnight in methanol/25% ammonium hydroxide (20:1). The benzodiazepines were extracted by solid phase. Thirteen samples were confirmed positive by LC–MS–MS. The benzodiazepines detected included diazepam, nordiazepam, temazepam, oxazepam, nitrazepam, and lorazepam. Using a cut-off concentration of 0.1 ng/mg oxazepam, the Immunalysis® Benzodiazepine Microplate ELISA demonstrated a sensitivity and specificity of 100% and 81%, respectively, compared with LC–MS–MS results.

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