Detection of Nordiazepam and Oxazepam in Calliphora Vicina Larvae Using LC-MS/MS

This study details about detection of nordiazepam and oxazepam in Calliphora Vicina larvae using LC-MS/MS.

In addition to their use in the estimation of postmortem interval, insects may serve as reliable alternate source for toxicological analyses in the absence of tissues and fluids normally taken for such purpose. To date, a variety of compounds have been measured in fly larvae and pupae using different analytical procedures i.e. (Radio-Immunoassay (RIA), Gas Chromatography (GC) and Thin-Layer Chromatography (TLC)). In these studies a minimum of 1g (approximately 20 larvae) was needed to detect the toxic compound.

In this study we used LC-MS/MS (Liquid Chromatography-Tandem Mass Spectrometry) to detect the benzodiazepine Nordiazepam and its metabolite Oxazepam, in single larvae of the Calliphora vicina. Benzodiazepines are prescribed for the symptomatic treatment of anxiety and sleep disorders. They are frequently encountered in postmortem blood analysis (suicide or accidental deaths).

In addition, we compared the development of postfeeding larvae and pupae fed on different concentrations of Nordiazepam.

Study Design

Flies and larvae were from a stock colony of Calliphora vicina maintained in an environmental chamber at 18–24 °C and 60–70% humidity with cyclical artificial lighting simulating 16 hr daylight and 8 hr darkness.

Larvae were reared on artificial food (beef heart) spiked with a range of concentrations of Nordazepam (Table 1). Post-feeding larvae were harvested from day 4 till day 8. Thirty larvae were boiled and conserved (in a mixture of ethanol and acetic acid) prior to measurement of length. Another 30 were used for toxicological analysis. These were weighed and then killed, by freezing to -20 °C. The larvae were stored at -20 °C until analysis.

Sample Preparation

Individual larvae and pupae samples were prepared for LC-MS/MS as follows; the sample was transferred to a vial containing 500 μL water and vortex-mixed thoroughly. One millilitre of acetonitrile (containing deuterated internal standards) was then added and the samples mixed for a further minute. The mixture was evaporated to ~100 μL and then filtered. A 10 μL aliquot was analysed using LC-MS/MS.

All larvae, pupae and food spiked with Nordiazepam were positive for the drug, whereas all control samples were negative. 

Figures 1 and 2 show the larvae Nordiazepam and Oxazepam concentrations from days 4–8. Figure 3 shows the MRM chromatograms obtained following the LC-MS/MS analysis of a control larva and a Nordiazepam positive larva.