Antibiotics | Development and Validation of a Multi-Analyte Antibiotic Residue Detection Method in Honey under EU Regulation 2021/808

This study develops and validates a multi-class antibiotic residue detection method based on UHPLC-TOF-MS, compliant with EU Regulation (EU) 2021/808, applicable for complex food matrices like honey. The method demonstrates high sensitivity and accuracy, with detection limits as low as 0.09 µg·kg−1, providing a reliable tool for food safety monitoring.

 

Literature Overview
The article "Multi-Analyte Method for Antibiotic Residue Determination in Honey Under EU Regulation 2021/808", published in the journal Antibiotics, reviews and summarizes a multi-antibiotic residue detection method aligned with EU Regulation 2021/808. By optimizing QuEChERS pretreatment combined with UHPLC-TOF-MS analysis, the study successfully identified 15 antibiotics, offering scientific support for food safety assessments of antibiotic residues in honey.

Background Knowledge
Antibiotics are widely used in apiculture to prevent and treat bee infections, but their residues may trigger antimicrobial resistance and health risks. Monitoring antibiotic residues in honey is critical, yet no clear maximum residue limits (MRLs) exist for bee products, posing regulatory and risk assessment challenges. Honey, a complex matrix with high viscosity and sugar content, complicates antibiotic extraction and detection. Therefore, developing a universal, high-sensitivity detection method is essential. Although multi-residue methods (e.g., UHPLC-MS/MS and HRMS) are widely applied in food analysis, technical challenges remain in honey testing. This study validates a multi-class antibiotic detection method for honey under Commission Implementing Regulation (EU) 2021/808, providing a standardized approach for food safety regulation.

 

 

Research Methods and Experiments
Honey samples were extracted using 0.1% formic acid in acetonitrile-water (80:20, v/v), followed by modified QuEChERS purification (adding 1g NaCl and 2g MgSO4). Analysis was conducted using UHPLC-TOF-MS, with compound identification based on accurate mass deviation (≤5 ppm) and relative retention time deviation (≤1.0%). Method validation included linearity, recovery, repeatability, reproducibility, limit of detection (LOD), and limit of quantification (LOQ), followed by screening of 10 Portuguese commercial honey samples.

Key Conclusions and Perspectives

• The method demonstrated excellent performance for 15 antibiotics, with mass deviation ≤5 ppm and relative retention time deviation <1.0%, meeting regulatory requirements.
• Recoveries ranged from 80.1%–117.6%, repeatability 0.5%–32.2%, reproducibility 2.3%–31.6%, and R2 values between 0.9429–0.9982, indicating strong linearity.
• LOD values were 0.09–6.19 µg·kg−1, LOQ values 0.29–18.77 µg·kg−1, and CCβ values 3–15 µg·kg−1, confirming high sensitivity.
• No target antibiotic residues were detected in the 10 commercial Portuguese honey samples, suggesting minimal antibiotic contamination or usage in current honey production.

Research Significance and Prospects
This study provides an efficient and reliable method for antibiotic residue detection in honey, supporting food safety regulation and risk assessment. Future work should expand sample sizes and incorporate geographical and seasonal factors to comprehensively evaluate global antibiotic contamination in honey. Additionally, the method can be extended to other bee products like propolis and royal jelly.

 

 

Conclusion
This study successfully developed and validated a multi-class antibiotic residue detection method for honey under EU Regulation 2021/808. Through optimized extraction and purification protocols combined with high-resolution mass spectrometry, the method achieves high sensitivity, selectivity, and reproducibility, making it suitable for food safety testing. Results indicate no detectable antibiotic residues in Portuguese commercial honey samples, though broader investigations are needed to assess global antibiotic usage in bee products. This method offers scientific support for future food regulation, antimicrobial resistance research, and beekeeping management, particularly in the absence of established MRL standards.

 

Helena Rodrigues, Marta Leite, Maria Beatriz P P Oliveira, and Andreia Freitas. Multi-Analyte Method for Antibiotic Residue Determination in Honey Under EU Regulation 2021/808. Antibiotics.