QuEChERS vs QuEChERS-DLLME: Toward an eco-friendly, selective, and sensitive method for levamisole determination in fish

Background The lack of regulated medicines for aquaculture challenges its expansion and its crucial role in global food security. Reliable bioanalytical methods are essential for monitoring drug residues in food, whether for research on new drugs or surveillance programs that ensure consumer safety. These methods face significant challenges in selectively detecting drugs at residual concentrations (often below 10 μg kg−1) in complex biological matrices such as muscle, fat, and skin. Since fish fillets consist of two distinct tissues (muscle and skin in natural proportions), the analytical challenges are even greater, requiring highly sensitive and selective approaches. Results We describe the development of eco-friendly, sensitive, and selective analytical methods for levamisole determination in fish fillets (tilapia, Oreochromis niloticus). The methods involved two sample preparation approaches: one based on a modified QuEChERS method and the other combining QuEChERS with DLLME (Dispersive Liquid-Liquid Microextraction). Both methods were coupled with LC-MS/MS and optimized using multivariate analysis to achieve the best extraction conditions. The proposed methods were validated and found to be selective, precise (CV = 0.6–13 %), and accurate (recovery 98.4–118 %), with linearity ranges of 2–200 μg kg−1 for QuEChERS and 0.2–20 μg kg−1 for QuEChERS-DLLME. The QuEChERS-DLLME approach enhanced detectability, achieving the lowest LOQ (0.2 μg kg−1) reported in the literature for levamisole quantification in fish fillets. The QuEChERS LC-MS/MS method was successfully applied to incurred tilapia samples treated with levamisole. Significance Despite the well-recognized advantages of QuEChERS-based methods, known for their versatility and robustness, and DLLME-based methods, valued for their high selectivity and sensitivity, to the best of our knowledge, the combination of QuEChERS and DLLME for levamisole determination in fish samples has not been reported. Therefore, this study may serve as a valuable reference to promoting the adoption of this combined approach for other applications.