VALIDATION OF AN LC-MS/MS MULTI-RESIDUE ANALYTICAL METHOD FOR ANTIBIOTIC QUANTITATION IN DAIRY MILK

The use of antibiotics in dairy farming, particularly for the treatment and prevention of mastitis, may lead to the presence of antibiotic residues (ARs) in milk and dairy products, representing a significant risk to public health. Therefore, the availability of multi-residue methods for controlling and monitoring purposes is highly desirable to improve food safety. This work aimed to develop and validate a multi-residue analytical method for the simultaneous determination of 20 antibiotics belonging to four different classes (penicillins, sulfonamides, tetracyclines, and amphenicols) in milk. For sample preparation, a modified QuEChERS procedure, optimized by response surface methodology (RSM), was employed. The extraction was performed using acetonitrile acidified with 1% acetic acid and EDTA acidified with citric acid (2:1, m/m). The salting-out effect was induced by the addition of 1 g of NaCl and Na₂SO₄ mixture (1:4, w/w), with no clean-up step required. After centrifugation, the supernatant was filtered and analyzed by high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The chromatographic conditions were optimized to ensure a rapid separation, with a total run time of 11 minutes. Detection was performed in positive and negative electrospray ionization (ESI) modes, with data acquisition in dynamic Multiple Reaction Monitoring (dMRM) mode. Quantification was carried out by internal standard calibration (thiamphenicol, sulfamerazine, and roxithromycin). The method was validated according to the VICH GL49 (R)/2015 guidelines, evaluating the parameters of precision (intra- and inter-day), recovery, limit of detection (LOD), and limit of quantification (LOQ). Recoveries ranged from 74.4% to 117.2%, with relative standard deviations of 7.8% and 8.2%, respectively. The LODs and LOQs varied from 3.3 to 8.3 μg/kg and from 10 to 25 μg/kg, respectively. The intra-day precision (repeatability) and inter-day precision (intermediate precision) showed coefficients of variation between 1-14% and 3.7-19.8%, respectively. The developed method proved to be effective and can be successfully applied to the evaluation of ARs in milk samples.