Chemical profiling of developmental stages in three major flies via direct infusion mass spectrometry: Improving estimates of minimum postmortem intervals in forensic entomology.

Forensic entomology has become an essential tool for estimating the minimum postmortem interval (mPMI), especially in scenarios where traditional methods are limited. This study aimed to characterize and differentiate the chemical profiles of three major forensically relevant fly species-Chrysomya albiceps, Cochliomyia macellaria, and Lucilia cuprina-across their developmental stages. Changes in chemical profiles during larval, pupal, and pharate adult stages were monitored via ethanol-based solvent extraction followed by direct infusion mass spectrometry (DIMS). Distinct chemical fingerprints were observed for each stage, with specific variations in ion compositions reflecting the dynamic nature of insect development. Chemometric analysis using PLS-DA models clearly distinguished the species and their developmental stages, especially during the larval phase, which is critical for mPMI estimation. These findings demonstrate that metabolomic profiling combined with chemometric tools offers, in forensic investigations, a precise and robust approach to estimate mPMI and to identify fly species in their larval stages.