Novel LC-MS/MS strategies combining low-temperature partition extraction and DLLME for sensitive levamisole determination in fish

Sample preparation is a crucial and challenging step for determining veterinary drug residues (VDRs) in animal products. This study aimed to develop two novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the determination of levamisole in fish fillet (muscle plus skin in natural proportion): Low-Temperature Partition Extraction (LTPE) and a combination of LTPE with Dispersive Liquid-Liquid Microextraction (DLLME). Optimal extraction conditions were established through multivariate optimization. Both methods were validated, demonstrating selectivity, precision (CV < 20 %), accuracy (recoveries 98 %-111 %), low matrix effects, and linearity in the ranges of 2-200 and 0.2-20 μg kg<sup>-1</sup> for LTPE and LTPE-DLLME, respectively. The LTPE method was successfully applied in the analysis of real samples to evaluate levamisole residues in fish. To the best of our knowledge, this is the first study to combine LTPE and DLLME, representing a promising advance in sensitive and selective methods for routine monitoring of levamisole and potentially other VDRs in food-producing animals.