Development of a rapid and sensitive method for the quantitation of benzodiazepines in Calliphora vicina larvae and puparia by LC-MS-MS

Abstract

Liquid chromatography-tandem mass spectrometry (LC-MS-MS) is emerging as the tool of choice for rapid analysis and the detection of biologically active compounds in complex mixtures. We describe the development of a sensitive method for the simultaneous quantitation of 10 benzodiazepines in Calliphora vicina (Diptera: Calliphoridae) larvae and puparia. The use of larvae for toxicological analyses offers some technical advantages over putrefied tissue. Four sample pretreatment methods for isolating the benzodiazepines out of larvae were evaluated. A simple homogenization, followed by acetonitrile precipitation yielded the highest recoveries. Puparia were pulverized and extracted by ultrasonification in methanol. All extracts were subsequently analyzed using reversed-phase LC-MS-MS. Larvae and puparia calibrators containing benzodiazepines at concentrations ranging from 25 to 750 pg/mg and 50 to 500 pg/mg, respectively, were prepared and analyzed. The method was demonstrated to be linear over the ranges investigated. Limits of detection were from 1.88 to 5.13 pg/mg larva and from 6.28 to 19.03 pg/mg puparium. The developed method was applied to the determination of nordiazepam and its metabolite oxazepam in larvae and puparia of the Calliphora vicina fly that had been reared on artificial foodstuff (beef heart) spiked with 1 μg/g nordiazepam. The larvae were harvested at day 5 for analysis of drug content. The method was sufficiently sensitive to allow the detection of nordiazepam and oxazepam in a single larva or puparium.