Quantitation of phenylbutazone and oxyphenbutazone in equine plasma by high-performance liquid chromatography with solid-phase extraction

Abstract

Abstract Phenylbutazone (bute) and oxyphenbutazone are non-steroidal anti-inflammatory drugs (NSAIDs) widely used in the equine world. Both substances are prohibited under the British Jockey Club Rules of Racing but are permitted up to a certain threshold level in blood plasma by some other equine governing bodies. There is therefore the requirement for accurate quantitation of these compounds in equine plasma samples. A quantitative analytical method utilising solid-phase extraction and reversed-phase HPLC was developed and a full validation exercise performed. Additional studies of analyte stability and development of a confirmatory analysis method were also carried out. A linear calibration over the plasma concentration range of 1–10 μg ml−1 for both analytes was achieved using fenclofenac as an internal marker. Inter-assay precision (n = 6) testing of plasma samples spiked at 2 μg ml−1 with both analytes produced results (R.S.D.) of 5.1% for phenylbutazone and 4.0% for oxyphenbutazone with standard error of the mean 0.0140 and 0.0138, respectively. The analytes were prone to oxidation during extraction and storage and preventative measures were incoporated into the methods. Confirmatory analysis was achieved by GC-MS with on-column derivatisation (methylation) of back exracted residues from the quantitative method.