Determination of flunixin in culled dairy cows using screening assays

Authors: Shelver, Weilin; Smith, David; SCHROEDER, J - North Dakota State University

Submitted to: International Association for Food Protection
Publication Type: Abstract Only
Publication Acceptance Date: 11/26/2014
Publication Date: 7/25/2015
Citation: Shelver, W.L., Smith, D.J., Schroeder, J.W. 2015. Determination of flunixin in culled dairy cows using screening assays [abstract]. International Association for Food Protection Annual Meeting. July 25-28, 2015. Portland, OR. P2-61.

Interpretive Summary: Introduction: Flunixin is a non-steroid anti-inflammatory agent used to treat serious inflammatory conditions in livestock; it is also a commonly detected violative residue in dairy cattle. Ante-mortem matrices such as milk, saliva, or urine could prove valuable for predicting possible violative tissue residues. Purpose: Using ELISA and lateral flow immunoassays to determine flunixin residues in tissues and ante mortem matrices Methods: After IACUC approval, 20 cows were given the labeled dose of flunixin by IV or IM administration (10 each) for 3 consecutive days. One-half of the cows were challenged with IV LPS. Milk, saliva, and urine were collected at timed intervals and cows were slaughtered with a 4-day withdrawal period (WP). USDA, FSIS methods (CLG-FLX3.01 and Bulletin 4246) were used to screen tissues for flunixin residues. Saliva flunixin was determined using a matrix matched calibration standards on a 96 well ELISA format. Lateral flow analyses were utilized to determine flunixin in milk, saliva, and urine. Milk was assayed directly while saliva and urine samples were diluted. Results: At a WP of 96 hours, no animals exceeded the muscle tolerance (25 ppb) while livers of two animals’ concentrations exceeded 100 ppb (tolerance 125 ppb). Saliva did not produced predictable flunixin elimination pattern using ELISA determination. Using lateral flow analysis, all 96-h urine samples were flunixin positive; 18 of 20 saliva samples were positive. Milk of 15 cows were positive at the 36-h milk WP. Significance: The FSIS’ ELISA screening method detected liver flunixin violative residues (confirmed by FSIS’ LC-MS method) with one false positive result. Based on lateral flow screening assay results, urine and saliva are not good ante-mortem predictors for tissue flunixin violations; rather, they potentially can be used to test flunixin exposure in off-label species.

Technical Abstract: Introduction: Flunixin is a non-steroid anti-inflammatory agent used to treat serious inflammatory conditions in livestock; it is also a commonly detected violative residue in dairy cattle. Ante-mortem matrices such as milk, saliva, or urine could prove valuable for predicting possible violative tissue residues. Purpose: Using ELISA and lateral flow immunoassays to determine flunixin residues in tissues and ante mortem matrices Methods: After IACUC approval, 20 cows were given the labeled dose of flunixin by IV or IM administration (10 each) for 3 consecutive days. One-half of the cows were challenged with IV LPS. Milk, saliva, and urine were collected at timed intervals and cows were slaughtered with a 4-day withdrawal period (WP). USDA, FSIS methods (CLG-FLX3.01 and Bulletin 4246) were used to screen tissues for flunixin residues. Saliva flunixin was determined using a matrix matched calibration standards on a 96 well ELISA format. Lateral flow analyses were utilized to determine flunixin in milk, saliva, and urine. Milk was assayed directly while saliva and urine samples were diluted. Results: At a WP of 96 hours, no animals exceeded the muscle tolerance (25 ppb) while livers of two animals’ concentrations exceeded 100 ppb (tolerance 125 ppb). Saliva did not produced predictable flunixin elimination pattern using ELISA determination. Using lateral flow analysis, all 96-h urine samples were flunixin positive; 18 of 20 saliva samples were positive. Milk of 15 cows were positive at the 36-h milk WP. Significance: The FSIS’ ELISA screening method detected liver flunixin violative residues (confirmed by FSIS’ LC-MS method) with one false positive result. Based on lateral flow screening assay results, urine and saliva are not good ante-mortem predictors for tissue flunixin violations; rather, they potentially can be used to test flunixin exposure in off-label species.