Efficient protein precipitation approach driven by design of experiment for rapid lc–ms/ms quantification of β-lactam and quinolone residues in milk

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This work presents the development and validation of a rapid and selective method for the quantification of residues of β-lactam and quinolone antibiotics (Penicillin G, Oxacillin, Cloxacillin, Ceftiofur, Enrofloxacin, and Ciprofloxacin, using Dicloxacillin and Sarafloxacin as internal standards) in raw bovine milk. Twenty distinct sample preparation methods were evaluated using design of experiments (DOE), with the most efficient option involving a single step of protein precipitation using a mixture of organic solvents acetone/acetonitrile at a ratio of 75:25 (v/v), assisted by ultrasonic bath. After centrifugation, the supernatant was injected into an Ultra-performance Liquid Chromatography system coupled with Electrospray Ionization Triple Quadrupole Mass Spectrometry (UPLC-ESI-QqQ-MS/MS). The separation occurred in 4.5 min using a reversed-phase Acquity UPLC BEH C18 column (1.7 µm, 50 × 2.1 mm), with the mobile phase consisting of water with 0.1% formic acid and acetonitrile. Multiple Reaction Monitoring (MRM) acquisition mode was employed in MS for high sensitivity and selectivity. The method was validated in accordance with the criteria established by Commission Implementing Regulation (EU) 2021/808, indicating recoveries from 81.1 to 107.8%, with coefficients of variation below 9% for both repeatability and intralaboratory reproducibility, and trueness in the maximum residue limit (MRL) concentration level ranging from 96,9 to 108,7%. Linearity, selectivity, matrix effect, CCα, robustness, detection limits, and quantification limits (LoQ) were also evaluated, achieving LoQ values as low as 0.1 × MRL. The method was successfully applied to eight milk samples collected from tanks on commercial farms.