Validation of an LC–MS/MS multi-residue method for nine antibiotic classes in eggs according to EU regulation 2021/808 and assessment of their occurrence on Croatian poultry farms

An LC-MS/MS method was developed and validated to detect 68 antibiotics, including penicillins, cephalosporins, quinolones, macrolides, lincosamides, phenicols, pleuromutilins, sulphonamides, and tetracyclines, in accordance with CIR 2021/808. The extraction method used QUECHERS salts. Under Commission Regulation (EU) No 37/2010, only penicillin V, erythromycin A, lincomycin, tylosin A, tylvalosin, tiamulin, tetracycline, oxytetracycline and chlortetracycline are permitted for use in laying hens, with established maximum residue levels (MRL). To determine the detection capability (CCβ) for screening and the decision limit (CCα) for confirmatory testing, validation was conducted at concentrations of 0.1, 1, and 1.5 × MRL. For substances not authorised for use, the lowest calibration level was applied, ranging from 0.5 to 10 µg/kg. The estimated CCβ values ranged from 0.597 µg/kg for gamithromycin to 119 µg/kg for tylvalosin, while the CCα values for unauthorised substances ranged from 0.770 µg/kg for sulfacetamide to 36.7 µg/kg for nafcillin. Between 2022 and 2025, 200 egg samples were analysed. Three samples were found to be non-compliant, as they contained doxycycline and enrofloxacin residues above the decision limit. Compliance was confirmed for four samples in which residues of tiamulin and enrofloxacin were below the decision limit.