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 d 9-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.