Screening, Quantification, and Confirmation of Phenylbutazone and Oxyphenbutazone in Equine Plasma by Liquid Chromatography-Tandem Mass Spectrometry

A sensitive liquid chromatographic-tandem mass spectrometric method was developed and validated for screening, quantification, and confirmation of phenylbutazone and oxyphenbutazone in equine plasma. Analytes were recovered from plasma by liquid-liquid extraction followed by separation in a reversed-phase column and identification by mass spectrometry with selected reaction monitoring in negative electrospray ionization mode. Extraction recovery for both analytes was > 80%. Limits of detection, quantification, and confirmation for both analytes were 0.01 µg/mL (S/N ≥ 3), 0.05 µg/mL, and 0.05 µg/mL, respectively. The assay with d9-labeled phenylbutazone as internal standard (IS) was linear over a range of 0.05–20 µg/mL (r2 > 0.995). Intra- and interday precision in terms of coefficient of variation was less than 15%. Intra- and interday accuracy (bias%) was within 80–120%. Hemolysis of red blood cells decreased analyte signal intensity but did not affect quantification results because an isotope-labeled IS was used. Analytes were stable in plasma for 24 h at room temperature, 9 days at 4°C, and 45 days at −20°C and −70°C. The method was successfully used in screening, quantification, and confirmation of phenylbutazone in post-competition plasma samples obtained from racehorses. The method is simple, rapid, and reliably reproducible.