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

Abstract

The quantification of banned substances in equine antidoping control, especially in racehorse urine, necessitates robust analytical methods with high detection levels due to the extremely low concentrations of the target substances and the significant impact of minor variations on doping test results. Reliable quantification is important for substances near regulatory thresholds, which, if exceeded, are prohibited. This study presents the development and validation of a bioanalytical UHPLC-HRMS method for quantifying doping substances in equine urine, including diazepam and acepromazine with a regulatory limit level of 10 ng.mL^-1 (validated at 6, 8, 10, 12, and 14 ng.mL^-1); ketoprofen, flunixin, and caffeine with a permissible limit of 100 ng.mL^-1 (validated at 60, 80, 100, 120, and 140 ng.mL^-1); and meloxicam and lidocaine with a detection threshold of 25 ng.mL^-1 (validated at 15, 20, 25, 30, and 35 ng.mL^-1) using the accuracy profile. Different β values were applied to determine the proportion of future measurements that will fall within predefined acceptance limits, set at ±30 % (for a biological matrix). Method validation was carefully carried out and rigorously demonstrated in compliance with the French Society of Pharmaceutical Sciences and Techniques (SFSTP) commission in compliance with the ISO 17025 standard.

Keywords: Accuracy profile; Bio-analytical validation; Chromatography; Error total; Mass spectrometry; Quantification limit.