Determination of flunixin and ketoprofen in milk by liquid chromatography–tandem mass spectrometry Original Research Article

A relatively fast, simple and very selective liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the detection of flunixin, its 5-hydroxymetabolite and ketoprofen in raw milk has been developed and validated. After a simple extraction with acetonitrile and partial evaporation of the organic phase, the extract was filtered and directly injected into the LC–MS–MS system on a Symmetry C18 column. The parent ions were selected for further fragmentation with argon. The method developed was partially validated according to Commission Decision 2002/657/EC [Commission Decision 2002/657/EC implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results]. The validation parameters were linearity, specificity, repeatability, recovery and decision limit (CCα). CCα varied from 0.5 μg kg−1 for flunixin and 5-hydroxyflunixin to 1 μg kg−1 for ketoprofen. Holstein–Friesian cows were given either Ketofen® or Finadyne® via an intravenous injection at the maximum dose as written in the instructions. Cows were milked twice a day and all samples were analysed by the method described. The highest concentrations found for ketoprofen, flunixin and 5-hydroxyflunixin were 2.5, 6.7 and 590 μg l−1, respectively. The concentration of 5-hydroxyflunixin declined rapidly to concentrations below the MRL value of 40 μg l−1. It can be concluded that the withdrawal time proposed by the pharmaceutical companies, 12 h after the last dosing, is acceptable for both compounds.