Comparison of ELISA and LC−MS/MS for the Measurement of Flunixin Plasma Concentrations in Beef Cattle after Intravenous and Subcutaneous Administration

Eight cattle (288 ± 22 kg) were treated with 2.2 mg/kg of body weight of flunixin free acid in a crossover design by subcutaneous (SC) and intravenous (IV) administration. After a minimum 1:10 dilution with 50 mM phosphate buffer, a commercial immunoassay was adapted to determine plasma concentrations of flunixin. The limit of detection was 0.42 ng/mL and the working range was 0.76–66.4 ng/mL when adjusted with the dilution factor. Plasma samples were extracted using mixed-mode cation exchange solid phase extraction prior to the LC–MS/MS analyses. The linear calibration curve for LC–MS/MS was 0.5–2000 ng/mL with a limit of detection of 0.1 ng/mL for flunixin and 0.3 ng/mL for 5-hydroxy flunixin. Flunixin concentrations determined using the ELISAs were compared to concentrations derived from the same samples using LC–MS/MS analyses. Pharmacokinetic parameters of time versus concentration data from each analysis were estimated and compared. Differences (P < 0.05) in estimates of area under the curve, volume...