Development of an LC-MS/MS method for analysis of levamisole in poultry and livestock products.

Background: Levamisole is an imidazothiazole anthelmintic agent widely used in poultry and livestock. In Taiwan, the Ministry of Health and Welfare has established MRLs ranging from 0.01 to 1 μg/g for eggs, milk, and various livestock and poultry tissues.

Objective: To support regulatory monitoring, a chiral LC-MS/MS method with simple sample preparation was developed for determining levamisole residues in chicken muscle, porcine muscle, liver, kidney, fat, poultry eggs, and milk from food-producing animals.

Method: Separation of levamisole and its enantiomer dexamisole was achieved using an Astec Cyclobond I 2000 DMP column with 100 mM ammonium acetate and acetonitrile as the mobile phase. The sample was extracted with acetonitrile/methanol (95:5, v/v) containing 1% formic acid, followed by clean-up with acetonitrile-saturated n-hexane.

Results: The method demonstrated good linearity (r > 0.995, 0.5-25 ng/mL) for three quantitative methods in all tested matrices. The method achieved a LOQ of 0.005 μg/g in all matrices. While the pre-spiked tissue calibration curve provided higher recoveries (96.3%-110.1%) by compensating for both matrix effects and analyte losses during extraction, it was more labor-intensive. In contrast, the matrix-matched calibration curve and ISTD-normalized solvent calibration curve offered slightly lower recoveries (84.8%-100.4%) but showed greater practicality for routine monitoring. All calibration strategies met the accuracy and precision criteria specified in European Commission Decision 2002/657/EC and the TFDA validation guidelines. Furthermore, levamisole residues were not detected in any of the 10 commercial livestock and poultry products analyzed, confirming the applicability of the method for food surveillance.

Conclusions: A simple and rapid LC-MS/MS method was developed for the determination of levamisole in poultry and livestock matrices, demonstrating satisfactory sensitivity, accuracy, and selectivity. The method is suitable for routine monitoring and large-scale surveillance of levamisole residues in food products.

Highlights: Development and validation of an LC-MS/MS method involving simple solvent extraction for chiral determination of levamisole in livestock and poultry products with satisfactory sensitivity, accuracy, and selectivity.