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

This research study demonstrates a way to detect multiple drugs that can increase performance in racehorses, simultaneously in a single test. This is done using high-performance liquid chromatography (HPLC), and the drugs tested for include hydrocortisone, dexamethasone, oxyphenbutazone, indomethacin, and phenylbutazone.

Methodology

• The research was carried out using ethyl acetate extracts of horse serum containing hydrocortisone (HYD), dexamethasone (DEX), oxyphenbutazone (OPB), indomethacin (IND), phenylbutazone (PB).
• A control standard drug, probenecid, was also included.
• These extracts were evaporated using nitrogen and then resuspended with methanol for analysis.
• The HPLC analysis was performed with a C-18 column, which was prepared with a 51:49 acetonitrile-water solution and 0.1% trifluic acid at a rate of 1 ml per minute.
• The test output or “eluate” was then monitored at a wavelength of 254 nm.

Findings

• The researchers found that the method showed good selectivity, or ability to distinguish between the different drugs, which was apparent from an inter-assay C.V under 4%.
• The method also showed good sensitivity, the ability to accurately measure the minute quantities of each drug. This was recognized by the limits of quantitation, which were 0.25 microg/ml for HYD, DEX and IND, 0.5 microg/ml for PB, and 1 microg/ml for OPB.
• The precision of the method, reflected in its capacity to give consistent results over repeat tests, were also significant. The intra-assay and inter-assay values were reported at around 3-6% and 9-15% respectively.
• The researchers also noted significant degradation in some of the drugs (OPB and PB) during the testing process, indicating room for improvement in future tests.

Conclusion

• The researchers conclude that this testing process using HPLC could be useful to control doping in racehorses due to its selectivity, sensitivity, precision, and ability to simultaneously test for multiple performance-enhancing drugs.