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

Authors: Shelver, Weilin; TELL, LISA - University Of California; WAGNER, SARAH - North Dakota State University; WETZLICH, SCOTT - University Of California; BAYNES, RONALD - North Carolina State University; RIVIERE, JIM - North Dakota State University; Smith, David

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/21/2013
Publication Date: 3/7/2013
Citation: Shelver, W.L., Tell, L.A., Wagner, S., Wetzlich, S.E., Baynes, R.E., Riviere, J.E., Smith, D.J. 2013. Comparison of ELISA and LC-MS/MS for the measurement of flunixin plasma concentrations in beef cattle after intravenous and subcutaneous administration. Journal of Agricultural and Food Chemistry. 61:2679-2686.

Interpretive Summary: Recently, regulators have measured illegal residue levels of the anti-inflammatory drug flunixin in cattle. The exact reason for these violations is not possible to determine, but the most likely causes would be too high of a dose, unapproved flunixin administration route, or too short of withdrawal time (the time from the last dose until the animal is marketed). This project investigated the effect that the dose route has on the time required for flunixin to deplete to levels acceptable for marketing; in addition, the usefulness of two very different analytical techniques for measuring flunixin was assessed. One method, known as ELISA, is very sensitive, easily performed, and is relatively inexpensive. The other method (HPLC-MS/MS) is precise and specific, but requires very expensive and sophisticated equipment; it is not typically cost effective for large numbers of samples. While both methods were useful for measuring relatively low flunixin concentrations, the ELISA assay tended to overestimate flunixin levels at times close to dosing. Nevertheless, it was convincingly shown that the ELISA assay would be a valuable, cost-effective tool for laboratories which do not have access to LC-MS/MS equipment.

Technical Abstract: The feasibility of using an enzyme linked immunosorbant assay (ELISA) with only simple preparation steps to determine flunixin plasma concentrations in healthy beef cattle was explored. 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.