Determination and Confirmation of 5-Hydroxyflunixin in Raw Bovine Milk Using Liquid Chromatography Tandem Mass Spectrometry

Abstract

A method was developed and validated to determine 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry (LC/MS/MS). The mean recovery and percentage coefficient of variation (%CV) of 35 determinations for 5-hydroxyflunixin was 101% (5% CV). The theoretical limit of detection was 0.2 ppb with a validated lower limit of quantitation of 1 ppb and an upper limit of 150 ppb. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. A LC/MS/MS confirmatory method using the extraction steps of the determinative method was developed and validated for 5-hydroxyflunixin in milk from cattle. Briefly, the determinative and confirmatory methods were based on an initial solvent (acetone/ethyl acetate) precipitation/extraction of acidified whole milk. The solvent precipitation/extraction effectively removed incurred ((14)C) residues from milk samples. The organic extract was then purified by solid phase extraction (SPE) using a strong cation exchange cartridge (sulfonic acid). The final SPE-purified sample was analyzed using LC/MS/MS. The methods are rapid, sensitive, and selective and provide for the determination and confirmation of 5-hydroxyflunixin at the 1 and 2 ppb levels, respectively.

Quantification and confirmation of flunixin in equine plasma by liquid chromatography–quadrupole time-of-flight tandem mass spectrometry

“…Unlike GC/MS and HPLC, LC/Q-TOF/MS/MS provides enhancement in sensitivity, with LOD of 0.10 ng/ ml that is nearly 50 times more sensitive for flunixin in plasma than that by GC/MS or HPLC [24]. Other investigators [12][13][14] using LC/MS/MS have demonstrated increased sensitivity in the determination of flunixin and 5-hydroxyflunixin in milk and meat products. Although different matrices (milk versus plasma) were involved, the LOD (0.1 ng/ml) and LOQ (1.0 ng/ml) results of this study in plasma compared favorably with those in bovine milk [12], suggesting that the limiting factors in improved sensitivity are the extent of analyte recovery and instrumentation (LC/MS/MS versus GC/MS).…”

“…Other investigators [12][13][14] using LC/MS/MS have demonstrated increased sensitivity in the determination of flunixin and 5-hydroxyflunixin in milk and meat products. Although different matrices (milk versus plasma) were involved, the LOD (0.1 ng/ml) and LOQ (1.0 ng/ml) results of this study in plasma compared favorably with those in bovine milk [12], suggesting that the limiting factors in improved sensitivity are the extent of analyte recovery and instrumentation (LC/MS/MS versus GC/MS). Thus, the increased sensitivity resulting in the improved limit of detection and quantification of flunixin in plasma would help in the regulation of the use of flunixin in racehorses.…”

“…Methods available for the quantification of flunixin in plasma include high performance liquid chromatography (HPLC) [1][2][3][4][5][6]24,25], thin-layer chromatographic (TLC) densitometry and gas chromatography-mass spectrometry (GC/MS) [7][8][9][10][11]24,25]. The use of LC/MS/MS in the determination of flunixin and its metabolite in bovine milk and meat was recently reported [12][13][14]. Results of previous studies reported by the authors [3][4][5] and other investigators [15][16][17] indicated that intravenous administration of flunixin (1.10 mg/kg) 24 h prior to race time resulted in a plasma concentration of less than 10 ng/ml of flunixin.…”

Validation of a Rapid Lateral Flow Test for the Simultaneous Determination of β-Lactam Drugs and Flunixin in Raw Milk

“…In the spring of 2010, CVM collected incurred residue milk samples by treating three cows with flunixin after it was verified that the cows were healthy and had not been treated for the preceding 30 days. These incurred samples were provided to the IFSH for testing (1). Daily a.m. and p.m. milkings were collected until the prescribed drug clearance withhold time (4 days) and frozen.…”

“…Therefore, 5-hydroxyflunixin has been established as the marker residue to be used for monitoring of flunixin residues in bovine milk (5). More recently, an LC-tandem MS (LC-MS/MS) method was developed for the determi nation and confirmation of 5-hydroxyflunixin at 1-and 2ppb levels, respectively, in unpasteurized bovine milk (1).…”