Multi-Analyte Method for Antibiotic Residue Determination in Honey Under EU Regulation 2021/808

Background/Objectives: Antibiotic detection in honey is challenging due to the complexity of this product, the typically low levels of residues, and the absence of Maximum Residue Levels (MRLs) for beehive products. The use of antibiotics in apiculture poses potential risks to human health, including antimicrobial resistance and toxic effects. Reliable, sensitive, and selective analytical methods are essential to ensure food safety and enable accurate monitoring of antibiotic contamination in honey. This study aimed to validate a multi-analyte procedure in accordance with the parameters established in Commission Implementing Regulation (EU) 2021/808 for the identification and quantification of antibiotics, including tetracyclines, lincosamides, quinolones, macrolides, β-lactams, sulfonamides, and diaminopyrimidines. Methods: An extraction protocol was developed using 0.1% formic acid in ACN:H2O (80:20, v/v), followed by a modified QuEChERS with the addition of 1 g NaCl and 2 g MgSO4. The extracts were analyzed by UHPLC-TOF-MS. Results: The method, validated under CIR (EU) 2021/808, demonstrated robust performance, with recoveries ranging from 80.1% to 117.6%, repeatability between 0.5% and 32.2%, reproducibility between 2.3% and 31.6%, and determination coefficients (R2) ranging from 0.9429 to 0.9982. Validation was achieved for 15 antibiotic residues, with CCβ from 3 to 15 μg·kg−1, LODs between 0.09 and 6.19 μg·kg−1, and LOQs between 0.29 and 18.77 μg·kg−1. Application to 10 commercial Portuguese honey revealed no detectable levels of the target antibiotics. Conclusions: The combination of a simplified extraction with UHPLC-TOF-MS provides a reliable approach for the determination of antibiotics in honey. This validated method represents a valuable tool for food safety monitoring and risk assessment of apiculture practices.