A robust liquid chromatography-tandem mass spectrometry method was developed and comprehensively validated for the quantification of cefquinome considering the changing matrix composition from bovine colostrum to raw milk. Sample preparation consisted of addition of isotopically labeled cefquinome internal standard prior to protein precipitation of 2 g colostrum or milk followed by solid-phase extraction. A wide concentration range from 1 to 5000 ng cefquinome per gram of colostrum or milk was quantified using a 3200 QTRAP tandem mass spectrometer in positive ionization mode with electrospray ionization. Validation was performed according to the European Commission Decision 2002/657/EC guidelines. Matrix-comprehensive in-house validation included analytical limits CCα and CCβ, recovery, precision and calibration curves with prediction intervals, storage conditions, and evaluation of robustness based on factorial effect analysis. The detection limit was 0.2 ng cefquinome per gram of colostrum or milk. Recovery was between 98.4 and 99.4% for cefquinome concentrations from 4 to 240 ng/g. None of the investigated validation factors (matrix, storage of extracts, lot of SPE cartridges, and operators) exerted an influence higher than ± 3.2%, indicating that these factors make relatively low contributions to the respective combined measurement uncertainties. The comprehensively validated method enables routine residue control purposes and to monitor the pharmacokinetics of cefquinome in bovine colostrum and raw milk. In particular, residue depletion curves of cefquinome from high concentrations in first milking after treatment to concentrations far below the maximum residue limit can be measured.
Keywords: Cefquinome analysis; Colostrum; Dairy cattle; LC-MS/MS; Pharmacokinetics; Residue analysis.