Quantification of phenylbutazone in equine sera by use of high-performance liquid chromatography with a nonevaporative extraction technique.

OBJECTIVE: To develop a sensitive, rugged high-performance liquid chromatography (HPLC) method for the measurement of phenylbutazone (PBZ) in equine sera, using a rapid, nonevaporative extraction technique. SAMPLE POPULATION: Sera from 5 nonexercising adult horses. PROCEDURE: After addition of sodium chloride and acetonitrile to serum samples, reverse-phase HPLC analysis for PBZ and oxyphenbutazone (OXY) was performed directly on extracts, using diode array UV spectrophotometric detection. Probenecid was used as an internal standard. Data were evaluated by standard means of statistical analysis. RESULTS: Recoveries of PBZ, OXY, and probenecid from spiked samples were acceptable (ie, > or = 80%) and within run retention times were reproducible. Chromatograms were free of interfering substances, and linearity of calibration curves was observed throughout operational ranges. Coefficients of variation at each fortified PBZ concentration were in the 5 to 10% range. The method was applicable to analysis of PBZ and OXY in serum extracts from horses dosed with PBZ (4.4 mg/kg of body weight, IV) in a controlled environment. Track samples analyzed by use of this method and a conventional liquid/liquid extraction method gave comparable results (mean deviation, 1.6%) for PBZ concentrations. CONCLUSION: The HPLC protocol described is suitable for measuring PBZ and OXY in equine sera to regulate PBZ administration in horses involved in pari-mutuel racing.