Development and validation of a multianalyte method for quantification of isoxazolines in animal-derived matrix samples using SPE-HPLC–MS/MS

Isoxazoline drugs, including afoxolaner, fluralaner, lotilaner, and sarolaner, are widely used in veterinary medicine. However, the lack of available analytical methods has made regulatory monitoring of these drugs difficult. Therefore, in this study, we developed an optimized solid phase extraction process using hydrophilic–lipophilic cartridges, and subsequently analyzed the extracts using liquid chromatography–tandem mass spectrometry under optimized conditions. The detection limits for the four isoxazolines were 5.0 μg/kg, while the recoveries in six animal-derived matrices were 71.1–109.2 %, with intra-day and inter-day relative standard deviations below 11.9 % and 11.7 % respectively. Furthermore, an inter-laboratory comparison demonstrated that this method had good recovery (60.3–112.9 %) and acceptable precision (< 11.3 %). Additionally, the developed method exhibited excellent linearity, accuracy, precision, sensitivity, measurement uncertainty and robustness for isoxazoline residue determination in animal-derived samples. It was also successfully applied to determine incurred isoxazoline residues in medicated hens.