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

Abstract

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.

Simultaneous determination of prescription drugs, cocaine, aldicarb and metabolites in larvae from decomposed corpses by LC–MS–MS after solid–liquid extraction with low temperature partitioning

“…These values are within the same range as LOQs reported for different analytes in larvae [9,27,[41][42][43][44], and are below the concentrations usually found in larvae collected in intoxication cases, rendering the method applicable for forensic investigations.…”

“…Most likely, the two samples were collected from different sites of the corpse, a procedure that is recommended in forensic investigation [9,11,45]. Concentration variability could also be due to different physiological states of the larvae collected in different locations [9]. To the best of our knowledge, this is the first report worldwide of diazepam and carbamazepine in entomological samples collected from corpses.…”

“…The two samples collected from case G (51-year-old male) were positive for carbamazepine, with concentrations differing by two orders of magnitude. Most likely, the two samples were collected from different sites of the corpse, a procedure that is recommended in forensic investigation [9,11,45]. Concentration variability could also be due to different physiological states of the larvae collected in different locations [9].…”

“…In such forensic cases, entomological samples have been used as an alternative to investigate causa mortis [7,8]. Larvae are easily collected from a corpse, can be found for a long period after death, and are potentially easier to analyze due to fewer matrix effects [9]. Drug concentrations in these samples also appear to be more stable than in putrefied tissue [10].…”

“…The substances found include barbiturates [7], antidepressants and benzodiazepines [11], amphetamines [22], opiates [23], cocaine and its metabolite, benzoylecgonine [24,25], and pesticides [26]. The techniques used to determine these chemicals include gas chromatography coupled to a nitrogen-phosphorus detector [25] or mass spectrometry (MS) [24], and liquid chromatography coupled to a UV detector [23], MS [11], or MS-MS [9,27].…”