Simultaneous determination of isoxazoline drugs in animal-derived foods using enhanced matrix removal–lipid solid-phase extraction combined with LC-MS/MS

Isoxazoline veterinary drugs, including afoxolaner, fluralaner, and lotilaner, are extensively used to control ectoparasite infestations, necessitating robust analytical methods for residue monitoring in animal-derived foods. In this study, a sensitive and robust liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of these compounds. Sample preparation involved extraction with acetonitrile, phase partitioning with anhydrous MgSO₄, and rapid clean-up using a Captiva EMR-Lipid pass-through cartridge. To ensure quantitative reliability, stable isotope-labeled internal standards were employed. The method exhibited excellent linearity (R² > 0.999) across a concentration range of 0.6–150 μg/kg. Limits of detection (LODs) and quantification (LOQs) were 0.2 and 0.6 μg/kg, respectively. Across five complex matrices (chicken muscle, egg, mutton, pork, and pork liver), the mean recoveries ranged from 90.8% to 111.5%, with relative standard deviations (RSDs) ≤ 5.19%. This validated protocol offers a highly efficient and robust analytical tool for routine isoxazoline residue surveillance in diverse animal-derived matrices.