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Comparison of ELISA and LC-MS/MS for the measurement of flunixin plasma concentrations in beef cattle after intravenous and subcutaneous administration

Weilin L. Shelver, Lisa A. Tell, Sarah Wagner, Scott E. Wetzlich, Ronald E. Baynes, Jim E. Riviere, David J. Smith
Journal of Agricultural and Food Chemistry 2013 v.61 no.11 pp. 2679-2686
absorbance, beef cattle, buffers, cation exchange, detection limit, enzyme-linked immunosorbent assay, equations, flunixin, intravenous injection, liquid chromatography, mass spectrometry, pharmacokinetics, phosphates, solid phase extraction, subcutaneous injection
Eight cattle (288 ± 22 kg) were treated with 2.2 mg/kg bw flunixin free acid in a cross-over design by subcutaneous (SC) and intravenous (IV) administration. After a minimum of 1:10 dilution with 50 mM phosphate buffer, a commercial immunoassay was adapted to measure plasma flunixin concentrations. A matrix matched, 1:10 diluted plasma calibration curve having points of 0 to 300 ng/mL was fitted to a four parameter logistic equation. The limit of detection, based on the inhibitor concentration required to produce 90% of the absorbance value where no flunixin is present (IC10), was 0.042 ng/mL. The working range (IC15 - IC85) was 0.076 to 6.64 ng/mL. Serum samples were cleaned up using mixed-mode cation exchange solid phase extraction prior to the LC-MS/MS analyses. The linear calibration curve for LC-MS/MS having concentration ranged from 0.5 – 2000 ng/mL with limit of detection 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. ELISA analysis typically gave higher flunixin plasma concentrations relative to LC-MS/MS with deviations between the two methods increasing as flunixin concentration increased. Pharmacokinetic parameters of time vs. concentration data from each analysis were estimated and compared. Differences (P < 0.05) in estimates of area under the curve, volume of distribution, and clearance were apparent between ELISA and LC-MS/MS analyses after IV dosing; after SC dosing, however, there were no differences among the estimated parameters between the two methods. This study indicates that quantitative immunoassay was a satisfactory method of flunixin analysis and that it would be difficult to differentiate routes of administration in healthy beef cattle based on the plasma elimination pattern of flunixin after IV and SC administration.