Determination of Flunixin and 5-hydroxyflunixin in Bovine Plasma with Hplc-uv-method Development, Validation and Verification

The method for quantitative determination of flunixin and its metabolite 5-hydroxyflunixin in bovine plasma has been developed and validated. Plasma samples were extracted with acetonitrile and analysed by liquid chromatography with ultraviolet detection. Extraction efficiency was tested and optimum pH conditions were established. Limit of the detection for both compounds was 0.03 μg/mL, limits of quantitation of flunixin and 5hydroxyflunixin were 0.06 and 0.05 μg/mL, respectively. Both substances were determined with an accuracy below 12% (CV, %) and overall recovery in the range of 63-85%. The method was verified by the analyses of plasma samples from cows treated with flunixin meglumine.