Regulatory update and validation of a multi-matrix analytical method for the determination of aminoglycosides in food and feed products

Aminoglycosides are polar antibiotics used in veterinary medicine for the treatment of bacterial infections in food-producing animals. Due to their nephrotoxic and ototoxic risks, monitoring their residues in food and feed is essential. This study presents the validation and regulatory update of a multi-residue analytical method to determine eleven aminoglycosides (spectinomycin, streptomycin, dihydrostreptomycin, kanamycin A, apramycin, paromomycin, gentamicin C1, C2+C2A, C1A, neomycin B, and tobramycin) in muscle from different animal species, honey, feed, and drinking water. The method was updated to meet the requirements of Regulation (EU) 2021/808, replacing Decision 2002/657/EC, and optimized to omit pH adjustment while maintaining extraction efficiency and selectivity. Sample preparation included matrix-specific acidic extraction and solid-phase purification, followed by UHPLC-MS/MS analysis. Validation showed satisfactory selectivity (blank signal ≤30 % of the LCL), linearity (R 2 ≥ 0.98), accuracy (85–113 %), trueness (–15 to +13 %), precision (CV R , 3–24 %), and decision limits for confirmation (CCα), ranging from 11 to 1217 µg kg −1 ; for feed samples, from 582 to 6120 µg kg −1 ; and from 9 to 87 µg kg −1 for honey and water samples. Matrix effects were assessed, and appropriate internal standards were selected. The method is suitable for routine use in official control laboratories compliant with EU regulations. • Eleven aminoglycoside residues were determined in food and feed products. • Matrices included muscle, honey, animal feed, and drinking water. • The method used acidic extraction, SPE cleanup, and UHPLC-MS/MS. • Validation was performed according to Regulation (EU) 2021/808. • Simplified extraction avoided pH adjustment, improving throughput.