Performance assessment of an equine metabolomics model for screening a range of anabolic agents

Abstract

Despite their ban, Anabolic Androgenic Steroids (AAS) are considered as the most important threat for equine doping purposes. In the context of controlling such practices in horse racing, metabolomics has emerged as a promising alternative strategy to study the effect of a substance on metabolism and to discover new relevant biomarkers of effect. Based on the monitoring of 4 metabolomics derived candidate biomarkers in urine, a prediction model to screen for testosterone esters abuse was previously developed. The present work now focuses on assessing the robustness of the associated method and de ne its scope of application. Several hundred urine samples were selected from 16 different horses of ethically approved animal experiments involving various doping agents' applications (AAS, SARMS, β-agonists, SAID, NSAID) (n = 349). In addition, 342 urine samples from untreated animals of general equine populations were included in the study. Samples were characterized with the previously described LC-HRMS/MS developed method, with the objective of assessing both its biological and analytical robustness's. The study concluded that the measurement of the 4 biomarkers involved in the model was t for purpose. Further, the classi cation model con rmed its effectiveness in screening for testosterone esters use; and it demonstrated its ability to screen for the administration of other anabolic agents, allowing the development of a global screening tool dedicated to this class of substances. Finally, the results were compared to a direct screening method targeting anabolic agent's residues, demonstrating complementarity performances of traditional and omics approaches in the screening of anabolic agents in horses.

Sample Preparation Techniques for Growth-Promoting Agents in Various Mammalian Specimen Preceding MS-Analytics

“…Equine urine analytics were conducted using C18 sorbents [ 51 , 52 ], or a specific reversed phase cartridge equipped with a polymeric phase with bimodal porosity and high surface area was applied [ 53 ]. Viljanto et al [ 54 ] analyzed equine tissue by combining a non-polar SPE with LLE.…”

“…To analyze equine urine, Cloteau et al [ 52 ] normalized their samples according to their determined specific gravity (SG). Subsequently, the mixture was spiked with an internal standard, diluted with an aqueous acetate buffer (pH 7.2), and extracted using C18 cartridges.…”

“…Subsequently, the mixture was spiked with an internal standard, diluted with an aqueous acetate buffer (pH 7.2), and extracted using C18 cartridges. The eluate was dried and reconstituted in water/MeOH (8/2, v/v ) and injected into the LC-MS [ 52 ]. One year earlier, the same group spiked urine with internal standard and diluted it with ammonium acetate buffer and water.…”

“…However, other sample preparation methods for the same sample type were also developed using C18-based columns. Depending on the number of implemented analytes, the SPE cartridges were modified [ 52 ]. Harding et al [ 51 ] implemented an additional LLE step after SPE.…”