Development of a standardized routine workflow for entomological and toxicological analysis using larvae of two forensically relevant fly species (Diptera, Calliphoridae).

For more than four decades, no standardized study design or workflow has been established for systematic entomotoxicological research in forensic casework. This study aimed to develop a universal rearing protocol for fly larvae grown on drug-spiked tissue surrogate and a standardized workflow for the qualitative analysis of pharmacologically active compounds (PACs) in insects of different developmental stages. Larvae of Protophormia terraenovae and Lucilia sericata were reared on minced meat serving as tissue surrogate. First-instar larvae were transferred to minced meat spiked with diazepam, amitriptyline, pipamperone, or lamotrigine, each triturated with placebo tablets. Actively feeding and post-feeding third-instar larvae were soaked for 24 h in ethanol (30 mL, 70:30, v:v), and both the larvae and the soaking solution were analysed using LC-MS/MS. The larval extraction method was optimized by replacing pure acetonitrile with an acetonitrile-methanol mixture and introducing centrifugation at -10°C. PAC detection was achieved in both larvae and soaking solution, confirming the sensitivity and applicability of the method. The workflow was further validated using authentic postmortem case samples. These findings demonstrate that ethanol-soaked larvae can be used effectively for both toxicological and entomological analyses. Overall, this study provides a universal and standardized workflow that simplifies and strengthens the application of entomotoxicology in forensic casework.