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An LC/MS/MS Method for the Simultaneous Determination of Diazepam and its Metabolites in Fly Larvae Sampled From Diazepam Spiked Porcine Liver
Analytical Unit St Georges - University of London, London, UK
Rebecca Beauchamp, Alan Beeby, Heidi Sanderson, Jennifer Button, Terry Lee and David W Holt

Forensic entomology is a recognised method of estimating post-mortem interval, butrelatively little research has examined the use of larvae in forensic toxicology. Forensic entomotoxicology includes the study of the effects of drugs and toxins on the development rate of carrion-feeding insects, and the use of these as alternative sample matrices in the absence of other samples. Analysis of living material, such as larvae, offers a number of technical advantages for drugdetection over putrefied human remains. The extraction of drugs from larvae is the same as that from tissue, however, no emulsion is formed, whereas this is not always the case with human tissue. There is also less contamination observed from endogenous substances, which is particularly problematic with putrefied human remains. Larvae are usually present in abundance on decomposed bodies and sampling isoften a relatively straight forward procedure. To date, most forensic entomotoxicological studies have concentrated on opiates rather than commonly prescribed drugs, such as benzodiazepines. LC/MS/MS is becoming widely used in toxicology laboratories and is an established technique for screening and quantifying benzodiazepines. A rapid, sensitive and selective LC/MS/MS method for theidentification of diazepam and its metabolites; desmethyldiazepam, oxazepam and temazepam in liver and fly larvae sampled from spiked porcine liver is described. Diazepam, first approved for usage in the early 1960s, is one of the most frequently prescribed drugs of the benzodiazepine group. Its uses include; treatment of anxiety and anxiety related insomnia, muscle relaxant, anti-epileptic and pre-operativesedative. The frequency with which it is prescribed, coupled with the fact that regular, long-term use can lead to physical and psychological dependence, means that diazepam and its metabolites are regularly detected in post-mortem samples, and often in conjunction with illicit drugs. Diazepam has a high oral bioavailability, with peak blood levels occurring within 1 to 2 hours of administration.It is metabolised to its principal active metabolite, desmethyldiazepam, by N-demethylation. Diazepam and desmethyldiazepam undergo 3-hydroxylation to form temazepam and oxazepam, respectively, which are also active. Whilst desmethyldiazepam accumulates during chronic dosing, temazepam and oxazepam do not accumulate to any significant extent.

Calliphora vicina larvae were reared onminced porcine liver containing 0, 4, 9, 10 and 21mg/kg diazepam. The temperature was maintained at 20oC and fresh food stuff was introduced as required. Larvae (n=10) were harvested at various stages of development (days 4, 5, 6, 8, 10 and 12) and analysed for drug content by LC/MS/MS. The harvested larvae were initially transferred to a piece of damp cotton wool and further incubated for 24hours after removal from the food source. This starvation period was to allow gut clearance. Drug levels detected are therefore representative of absorption into the larval tissues. The larvae were then sacrificed by immersion in boiling water and stored frozen at -20oC. Prior to analysis the larvae were washed with copious amounts of deionised water, to remove any remaining foodstuff and excretorywaste, and then dried on filter paper. They were individually weighed, ground down with a pestle and mortar and diluted one part in ten with deionised water. Materials Individual drug standards of diazepam, desmethyldiazepam, temazepam and oxazepam were obtained from Sigma-Aldrich (Poole, Dorset, England). Diazepam-D5 (1mg/mL in methanol) was obtained from LGC Promochem (Middlesex, U.K.). HPLC...
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