Determination of flunixin in edible bovine tissues using liquid chromatography coupled with tandem mass spectrometry.

An accurate, reliable, and reproducible assay was developed and validated to determine flunixin in bovine liver, kidney, muscle, and fat. The overall recovery and percent coefficient of variation (%CV) of twenty-eight determinations in each tissue for flunixin free acid were 85.9% (5.9% CV) for liver, 94.6% (9.9% CV) for kidney, 87.4% (4.7% CV) for muscle, and 87.6% (4.4% CV) for fat. The theoretical limit of detection was 0.1 microg/kg (ppb, ng/g) for liver and kidney, and 0.2 ppb for muscle and fat. The theoretical limit of quantitation was 0.3, 0.2, 0.6, and 0.4 ppb for liver, kidney, muscle, and fat, respectively. The validated lower limit of quantitation was 1 ppb for edible tissues with the upper limit of 400 ppb for liver and kidney, 100 ppb for fat, and 40 ppb for muscle. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. Briefly, the method involves an initial acid hydrolysis, followed by pH adjustment ( approximately 9.5) and partitioning with ethyl acetate. A portion of the ethyl acetate extract was purified by solid-phase extraction using a strong cation exchange cartridge. The eluate was then evaporated to dryness, reconstituted, and analyzed using LC/MS/MS. The validated method is sensitive and specific for flunixin in edible bovine tissue.

Determination and confirmation of 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry.

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.

Flunixin residues in milk after intravenous treatment of dairy cattle with (14)C-flunixin.

Flunixin meglumine is used in veterinary medicine as an alternative to narcotic analgesics and as an antiinflammatory agent. Eight Holstein dairy cows were dosed intravenously once daily on three consecutive days with (14)C-flunixin meglumine at approximately 2.2 mg of flunixin free acid/kg of body weight. Milk was collected twice daily to determine the decline of the total radioactive residues (TRR) in milk and to identify or characterize residue components. TRR in milk declined rapidly and averaged 66, 20, and 14 ppb, respectively, for the first, second, and third milkings after administration of the last dose. Milk was extracted, and the extracts were examined for radioactive residues. Mean extractability of milk TRR was always greater than 80%. Flunixin and 5-hydroxyflunixin were identified by coelution with analytical standards using reverse phase HPLC. These two residues were the main radioactive residues found in milk and together accounted for 64, 37, and 44% of the extractable residues, for the first, second, and third milkings, respectively, after administration of the last dose. The presence of 5-OH flunixin in milk was confirmed by HPLC/MS/MS.