Validation and Quantification of Methamphetamine in Empty Puparia of Lucilia sericata (Diptera: Calliphoridae) Using Liquid-Liquid Extraction and Gas Chromatography-Mass Spectrometry

Entomotoxicology provides an alternative matrix for detecting drugs in decomposed human remains when conventional specimens are unavailable. This study aimed to develop and validate a quantitative method for detecting methamphetamine (MA) in different developmental stages of the blowfly Lucilia sericata.

Immature stages of L. sericata (larvae, pupae, empty puparia) were reared on chicken liver spiked with MA at concentrations of 45, 90 and 180 ng/mg. Samples underwent liquid-liquid extraction, derivatization via acetylation, and analysis using gas chromatography-mass spectrometry (GC-MS). The method was validated according to international guidelines, evaluating limit of detection (LOD), limit of quantification (LOQ), linearity, precision, accuracy and repeatability.

The method showed strong linearity across the studied concentration range (R²= 0.9817-0.9934). The limit of detection and LOQ were 15 ng/mg and 45 ng/mg, respectively, with a relative standard deviation (RSD)≤ 20% at the LOQ level. Intra- and inter-day precision showed RSD values< 10%, and accuracy ranged from 98.55% to 100.73%. Methamphetamine was detected in all immature stages and empty puparia. The highest observed concentrations were detected in third-instar larvae and pupae, whereas no drug residues were detected in adult flies. Methamphetamine retention in empty puparia supports their potential forensic relevance for retrospective drug detection.

This validated method demonstrated acceptable sensitivity, precision, accuracy and repeatability for quantitative entomotoxicological analysis. The detection of methamphetamine across insect developmental stages suggests the suitability of this approach as a supplementary tool for forensic investigations involving decomposed remains.