Use of necrophagous insects as evidence of cadaver relocation: myth or reality?

The use of insects as indicators of post-mortem displacement is discussed in many texts, courses and TV shows, and several studies addressing this issue have been published. Although the concept is widely cited, it is poorly understood, and only a few forensic cases have successfully applied such a method. The use of necrophagous insects as evidence of cadaver relocation actually involves a wide range of biological aspects. Distribution, microhabitat, phenology, behavioral ecology, and molecular analysis are among the research areas associated with this topic. This article provides the first review of the current knowledge and addresses the potential and limitations of different methods to evaluate their applicability. This work reveals numerous weaknesses and erroneous beliefs as well as many possibilities and research opportunities.

Methadone determination in puparia and its effect on the development of Lucilia sericata (Diptera, Calliphoridae).

This paper describes a sensitive UPLC-MS/MS method for quantification of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in single empty puparial case of Lucilia sericata. Larvae were reared on substrates spiked with different concentrations of methadone (0-4 µg/g). Methadone was quantified in puparia reared on high concentrated substrates (0.8-4 µg/g). The major metabolite of methadone (EDDP) was not detected, confirming rapid elimination of metabolites by the larvae before pupation. The effects of methadone on the development of L. sericata were also investigated. No effect on sex ratio was detected. A significant difference was calculated for emerged adults but no trends could be observed. Concerning the developmental curve, a significant difference was observed between control and high methadone concentrations using the Kolmogorov-Smirnov test.

Entomotoxicology, experimental set-up and interpretation for forensic toxicologists.

Forensic entomotoxicology studies the usefulness of insects as alternative toxicological samples. Use of insects as alternative matrix for drug detection is well documented and recommended when conventional matrices such as blood, urine or internal organs are no longer available. However, several limitations of entomotoxicology have been highlighted, especially concerning interpretation of the drug concentrations in insects on human forensic cases. In addition, the lack of knowledge in pharmacokinetic of drugs in insects, large variability of experimental set-up and toxicological analysis compromise the utility of this science. This review focuses on the current knowledge of factors influencing drug detection in insects. Reasons for the current limitations, but also recommendations for future research are discussed and proposed in this paper.

Quantification of methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in third instar larvae of Lucilia sericata (Diptera: Calliphoridae) using liquid chromatography-tandem mass spectrometry.

Entomotoxicology studies the application of toxicological analysis on necrophageous insects present on human remains. This paper describes the development and validation of a sensitive liquid chromatography tandem mass spectrometry method for quantification of methadone and its main metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in developmental stages of Lucilia sericata. One single larva was pulverized in a disposable vial and then extracted with 1-chlorobutane. After evaporation of the organic layer, samples were reconstituted in the mobile phase. Chromatographic separation was achieved on a NUCLEODUR Sphinx RP column with a liquid chromatographic gradient (0.1% formic acid and methanol), ensuring the elution of methadone and EDDP within 15 min. The method was fully validated according to international guidelines. The use of liquid liquid extraction was demonstrated to be effective (matrix effect < 27% and recovery > 66%). The method was linear over the dynamic range (10-400 pg/mg larva) with excellent within- and between-run precision and bias (CV% < 5%). The lower limit of quantification was fixed at 10 pg/mg larva. No instability of the extracted samples was observed in the autosampler after three freeze/thaw cycles and after two months at -20 degrees C. The validated method was applied to third instar larvae of Lucilia sericata reared on beef heart spiked with 4 microg/g methadone and on a postmortem methadone overdose case. The validation and actual sample analysis showed that the method is sensitive, rugged, precise, accurate, and well-suited for routine analysis of methadone and EDDP in a single larva obtained from forensic cases.

Analysis of amphetamines and metabolites in urine with ultra performance liquid chromatography tandem mass spectrometry.

A simple, rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry method was developed and fully validated for the quantitative determination of seven amphetamines and metabolites in urine. The method was validated for selectivity, linearity, LOQ, LOD, imprecision, bias, analyte and processed sample stability, matrix effect, recovery, carryover and dilution integrity. A classic liquid-liquid extraction with ethyl acetate was used as sample preparation procedure. The compounds were separated on an Acquity UPLC HSS C18 column in 6.8 min. The linear dynamic range was established from 25 to 500 ng/mL. The limit of quantification was fixed to the lowest calibrator level and the limit of detection ranged from 0.125 to 2.5 ng/mL. The method presented an excellent intra- and inter-assay imprecision and bias (<10.7%) at each measured concentration of two external quality controls (QC) and three "in house" QC. No matrix effects were observed and good recoveries (>70%) were obtained for all the compounds. No carryover was observed after the analysis of high concentrated samples (8000 ng/mL). The method was subsequently applied to authentic samples.

COI sequence variability between Chrysomyinae of forensic interest.

About 50 Chrysomyinae specimens belonging to three forensic relevant species (Chrysomia albiceps, Phormia regina and Protophormia terraenovae) were collected from different geographical locations in Belgium over the last 5 yr. A 304-bp fragment of their mitochondrial COI gene is sequenced. The monophyletic branches of the phylogenetic tree reveal that this marker is suitable for discrimination between these species. The intra versus interspecific variability marks clear threshold levels for DNA barcoding. Nineteen Chrysomyinae specimens, collected from four locations in France, show mitotypes that are identical or at least very similar to the Belgian mitotypes. Considering additional specimens from outside of Europe reveals no intraspecific geographical variation within C. albiceps and P. terraenovae, whereas P. regina is subbranched in a Belgian-French and a USA-Chinese population.