Screening, quantification, and confirmation of phenylbutazone and oxyphenbutazone in equine plasma by liquid chromatography-tandem mass spectrometry.

This study has developed and validated a precise and efficient method using liquid chromatography-tandem mass spectrometry to detect, quantify, and confirm the presence of specific pharmaceutical substances, namely phenylbutazone and oxyphenbutazone, in horse blood samples. These drugs are then quantified with an 80% success rate. This process has a valuable application in confirming the use or non-use of these substances in horse racing.

Study Design and Methodology

• The study employs a scientific method known as liquid chromatography-tandem mass spectrometry (LC-MS/MS), which is a powerful technique used to separate and quantify complex mixtures of chemical compounds. In this study, the LC-MS/MS method is designed for the detection, quantification, and confirmation of two drugs—phenylbutazone and oxyphenbutazone—in the plasma of horses.
• Analytes, or the substances being studied, were extracted from the plasma through a process called liquid-liquid extraction. The analytes were then separated by a reversed-phase column and identified by mass spectrometry using selected reaction monitoring in the negative electrospray ionization mode.
• To assure the reliability of the method, the researchers evaluated the extraction recovery for both drugs, which was found to be over 80%. This means that the extraction process recovers more than 80% of the targeted compounds present in the samples.

Results and Findings

• The assay showed limits of detection, quantification, and confirmation for both drugs at levels of 0.01 micrograms/milliliter (µg/mL), 0.05 µg/mL, and 0.05 µg/mL, respectively. It means the minimum amount of the drug that could be detected was at 0.01 µg/mL, while the minimum amount that could be quantified and confirmed was set at 0.05 µg/mL.
• The assay’s precision was less than 15%, demonstrated by the coefficient of variation, which measures the method’s relative variability. Its accuracy was between 80% and 120%, which means the results were close to the true value of the amount being measured.
• A decrease in the analyte signal intensity due to the rupture of red blood cells did not affect the quantification results, thanks to the use of an isotope-labeled internal standard (IS).
• The researchers also tested the stability of the analytes in the plasma at different temperatures and for various lengths of time. They concluded that the analytes were stable for 24 hours at room temperature, nine days at 4°C, and 45 days at -20°C and -70°C.

Relevance and Application

• This LC-MS/MS method was successfully used in detecting, quantifying, and confirming the presence of phenylbutazone in post-competition plasma samples taken from racehorses, demonstrating its potential application in horse racing.
• The method was described as simple, rapid, and reliably reproducible, making it a potentially valuable tool for regulatory bodies in the horse racing industry.