Development and Validation of Confirmatory Quantitative and Screening Methods for 63 Prohibited Substances in Horse Urine Using LC–MS/MS

The number of potentially abused substances in horse racing is very large and it is not easy to analyse the full range. To facilitate the analyses of substances of abuse, a method has been developed for 63 substances in horse urine using liquid chromatography–tandem mass spectrometry. The developed method has been validated according to the 2021/808/EC and AORC criteria. The horse urine samples were extracted after enzymatic hydrolysis via a C8-BCX SPE cartridge with an automated SPE and analyzed by LC–MS/MS. According to the validation study results, the within-laboratory reproducibility CV was no more than 13.9% and the average recoveries ranged from 90.71 to 117.94% for repeatability and reproducibility. The decision limits (CCα) for quantitative analysis and detection capabilities (CCβ) for qualitative screening were close to the targeted limit for each substance. The results of the present study showed that the method was able to perform quantitative analyses of 54 substances and screening analyses of 9 substances (total 63) in horse urine and that the results were valid and applicable. The validated method was successfully applied to five proficiency tests and three suspected samples.