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 chrom atography (HPLC) method for the measurement of phenylbutazone (PBZ) in equine sera, using a rapid, nonevaporative extraction technique. Samp le 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 d irectly 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, greater than or equal to 80%) and within-run retention times were reproducible. Ch romatograms were free of interfering substances, and linearity of cali bra tion curves was observed throughout operational ranges. Coefficien ts of variation at each fortified PET concentration were in the 5 to 1 0% range. The method was applicable to analysis of PBZ and OXY in seru m extracts from horses dosed with PBZ (4.4 mg/kg of body weight, IV) i n a controlled environment, Track samples analyzed by use of this meth od 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 equin e sera to regulate PBZ administration in horses involved in pari-mutue l racing.