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

The research focused on creating an efficient method for measuring levels of the drug phenylbutazone in horse serum using high-performance liquid chromatography coupled with a speedy, non-evaporative extraction technique.

Research Objectives and Methodology

The primary objective of the research was the development of a robust and effective method to measure the levels of phenylbutazone (PBZ) and oxyphenbutazone (OXY) in horse serum. Their focus was to make the process more sensitive, without compromising speed.

• Horse sera were prepared by adding sodium chloride and acetonitrile.
• Reverse-phase High Performance Liquid Chromatography (HPLC) was used to analyze the presence of PBZ and OXY directly from these extracts.
• To ensure accurate readings, probenecid was used as an internal standard for comparison.
• For analytical validation, measures like recoveries of PBZ, OXY, and probenecid from spiked samples, reproducibility, interference from other substances, linearity of calibration curves, and coefficients of variation were evaluated.

Results

The study concluded that the newly formulated HPLC method produced satisfactory recoveries (over 80%) for PBZ, OXY, and probenecid. The retention times were consistent, no additional substances interfered with the chromatograms, and the calibration curves showed linearity throughout different operational ranges.

• Compared to a conventional liquid/liquid extraction method, they found a small mean deviation of measurements (1.6%) for PBZ concentrations, indicating comparable accuracy.
• The coefficients of variation (a measure of precision) for the fortified PBZ concentration were between 5 to 10%, reinforcing the method’s reliability.

Conclusion

The newly developed HPLC method is a viable method to measure PBZ and OXY levels in equine serum to regulate PBZ use in competitive horse racing. The reliability, consistency, and speed of this method make it beneficial for drug regulation in the racing industry.