Development and validation of a quantitative UHPLC-HRMS bioanalytical method for equine anti-doping control.

The study develops and proves the effectiveness of a UHPLC-HRMS method to accurately identify and measure the concentration of prohibited substances in racehorse urine. These substances often exist in extremely low levels, making a highly sensitive detection method critical for the integrity of anti-doping measures.

Development and Validation of UHPLC-HRMS Method

• The research revolves around formulating a new quantitative analysis approach using Ultra High-Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS). This method aims to identify and quantify doping substances present in racehorse urine samples.
• The study emphasizes the need for superior analytical procedures as target substances generally appear in incredibly low amounts. Accuracy is crucial, especially for identifying substances with strict threshold limits whose breach could result to banning the horse from racing.
• Significant effort has been dedicated to minimizing the potential error that could result from minor variations in the testing process. This was achieved by validating the method at various concentration points, and applying a predefined acceptance limit of ±30%, as required for a biological matrix.

Substances of Interest and Threshold Limits

• The substances being tested include: diazepam and acepromazine that has a regulatory limit of 10 ng.mL; ketoprofen, flunixin, and caffeine with a permissible limit of 100 ng.mL; and meloxicam and lidocaine with a detection threshold of 25 ng.mL.
• These substances were validated at multiple concentration levels around these regulatory limits to ensure the robustness of the method across the entire range of potential substance concentrations.

Compliance with Standards

• The method’s development and validation process adhered strictly to the guidelines of the French Society of Pharmaceutical Sciences and Techniques (SFSTP) and the ISO 17025 standard to ensure credibility and reliability.
• Rigorous method validation was performed to confirm the method’s effectiveness and accuracy in detecting and quantifying the doping substances in the defined range.

Takeaways

• The research provides a validated bioanalytical method for equine anti-doping control, addressing the demand for robust analytical methods in a field where small variations can determine a racehorse’s career.
• The development of such methods not only supports fair competition in horse racing but also contributes to a wider research interest in drug detection and quantification in biological samples.