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



Parkhideh S Z ; Forouzesh M ; Akhgari M ; Akbarzadeh K ; Mohammadi A M A ; Rafizadeh S ; Rassi Y
Source: Journal of Arthropod-Borne Diseases Published:2026

Abstract

Background: 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. Methods: 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. Results: 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. Conclusion: 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. Copyright © 2026 The Authors.