Development of a multi-platform MS approach for the determination of organic micropollutants in terrestrial invertebrates

Terrestrial invertebrates occupy key ecosystem functions and accumulate organic micropollutants (OMPs) from agricultural, urban, and industrial sources, yet existing biomonitoring methods typically target narrow, class-specific chemical spaces. Here, four sample preparation methods, two based on bead beating (BB) and two on accelerated solvent extraction (ASE), were developed and compared through a smart validation scheme for gas and liquid chromatography coupled to high-resolution mass spectrometry (GC-HRMS, LC-HRMS). The two best-performing methods, BB-GC and BB-LC, were validated across three platforms: GC-HRMS, LC-HRMS, and GC tandem mass spectrometry (GC-MS/MS). A chemometric approach selected a validation set spanning plant protection products, pharmaceuticals, per- and polyfluoroalkyl substances, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), polychlorinated naphthalenes, and organochlorine pesticides, spanning the chemical space of in-house databases of more than 2800 unique compounds. In total, 303 LC-HRMS, 179 GC-HRMS, and 82 GC-MS/MS analytes were validated. Under the strict criteria of mean recovery of 70-120% and repeatability of ≤20% at every fortification level, 55%, 91%, and 95% of LC-HRMS, GC-HRMS, and GC-MS/MS analytes complied, respectively; the remainder met the expanded 30-140% recovery range permitted by SANTE/11312/2021 v2026 with consistent repeatability. Method quantification limits ranged from 3.4 to 15, 3.4 to 10, and 0.73 to 2.6 µg kg^-1 wet weight, for LC-HRMS, GC-HRMS, and GC-MS/MS, respectively. As proof of concept of the novel wide-scope screening approach, composite honeybee (Apis mellifera) and land snail (Eobania vermiculata) samples from Greece were analyzed, revealing co-occurring legacy contaminants (PCB 153, PAHs), current-use pesticides (pyrimethanil, cypermethrin), and industrial chemicals, thus compounds of distinct environmental origin that no single-class method would have captured.