Multiclass method for the determination of quinolones and β-lactams, in raw cow milk using dispersive liquid–liquid microextraction and ultra high performance liquid chromatography–tandem mass spectrometry

An analytical method based on a sample treatment by dispersive liquid–liquid microextraction (DLLME) followed by ultra high performance liquid chromatography–tandem mass spectrometry analysis (UHPLC–MS/MS) for the determination of 17 quinolones and 14 β-lactams (penicillins and cephalosporins) in raw cow milk, was validated according to the European Commission guidelines as cited in the Decision 2002/657/EC. The extraction efficiency of the DLLME depends on several parameters such as the nature and volumes of extractant and dispersive solvents, pH, concentration of salt, shaking time and time of centrifugation. These variables were accurately optimized using multivariate optimization strategies. A Plackett–Burman design to select the most influential parameters and a Doehlert design to obtain the optimum conditions have been applied. Two different pH values were used for the extraction of compounds (pH 3 for acidic quinolones and β-lactams and pH 8 for amphoteric quinolones). The method was validated using matrix-matched standard calibration followed by a recovery assay with spiked samples. The limits of quantification found ranged from 0.3 ng g−1 for amoxicillin to 6.6 ng g−1 for ciprofloxacin, and the precision was lower than 15% in all cases as is required by the European Regulation. The decision limits (CCα) ranged between 4.1 and 104.8 ng g−1, while detection capabilities (CCβ) from 4.2 to 109.7 ng g−1. These values were very close to the corresponding maximum residue limits (MLRs) for the studied antibiotics. Recoveries between 72 and 110% were also obtained. Finally, in order to evaluate the applicability of the method, 28 raw cow milk samples were analysed and it was observed that 28% of the samples were positive. However, only 11% were considered non-compliant with the current EU legislation (Commission Regulation 37/2010), due to some milk samples corresponded to treated cows with these antibiotics.