Simultaneous determination of hydrocortisone, dexamethasone, indomethacin, phenylbutazone and oxyphenbutazone in equine serum by high-performance liquid chromatography

Ethyl acetate extracts of equine serum, containing 0–5 μg/ml of hydrocortisone (HYD), dexamethasone (DEX), oxyphenbutazone (OPB), indomethacin (IND), phenylbutazone (PB) and probenecid as internal standard, were evaporated with nitrogen, resuspended in methanol and analyzed by HPLC, using a C-18 column equilibrated with 51:49 acetonitrile–water, 0.1% trifluoroacetic acid, at 1 ml/min. The eluate was monitored at 254 nm. The selectivity (inter-assay C.V.<4%), sensitivity (limits of quantitation of 0.25 μg/ml for HYD, DEX and IND, 0.5 μg/ml for PB and 1 μg/ml for OPB, despite the occurrence of significant degradation of OPB and PB during the analysis) and precision (intra-assay and inter-assay C.V.’s of about 3–6 and 9–15%, respectively) of the method appeared appropriate for anti-doping control of racehorses.