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Drug testing and analysis2025; 17(12); 2411-2420; doi: 10.1002/dta.3951

Identification of Candidate Biomarkers Detected in the Urine of Racehorses After Anabolic Agent Administration: Use of Orthogonal Methods for Structural Elucidation.

Abstract: Biomarker identification by mass spectrometry represents a key step in the workflow of nontargeted metabolomic studies. Given the complexity of the data, this step, which must be carried out by a trained specialist, is time-consuming, and the biomarkers discovered are not always identified. While this stage is not an obstacle to the development of new screening and classification tools, it is nonetheless crucial to a better understanding of the results obtained. For this reason, the aim of this study was to perform structural elucidation of candidate biomarkers, which had previously been displayed to screen for the administration of anabolic agents in the urine of racehorses and whose robustness had been evaluated. The present study involved a combination of various analytical strategies, including enzymatic hydrolysis, high-resolution mass spectrometry and ion mobility (LC-HRMS, LC-IMS-HRMS), and in vitro experiments. Two candidate biomarkers were identified as phase II metabolites of tebuconazole, belonging to the equine exposome. This identification opens the way to further investigations into the relationship between the presence of this compound and its disruption in horse urine following anabolic agent administration. Overall, the use of orthogonal approaches provided better complementary information on the structure of the compound and ultimately enabled us to identify biomarkers with the highest possible level of confidence.
© 2025 John Wiley & Sons Ltd.
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Publication Date: 2025-09-18 PubMed ID: 40968575DOI: 10.1002/dta.3951Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study focused on identifying specific biomarkers in racehorse urine after anabolic agent administration using advanced mass spectrometry techniques.
  • The researchers aimed to structurally elucidate these biomarkers to deepen understanding and improve screening methods for doping control in horses.

Background

  • Biomarker identification is crucial in non-targeted metabolomic studies, especially in doping control for racehorses.
  • The process usually involves mass spectrometry and requires expert interpretation due to complex data sets.
  • Typically, not all discovered biomarkers get confidently identified, which can limit understanding despite being useful for screening.
  • Structural elucidation provides detailed knowledge about the chemical nature of biomarkers, supporting better validation and interpretation of screening results.

Objective

  • To perform detailed structural elucidation of candidate biomarkers previously recognized as screening indicators for anabolic agents in horse urine.
  • To use a combination of complementary analytical techniques (orthogonal methods) to improve confidence in biomarker identification.

Methods

  • Sample Collection: Urine samples were collected from racehorses exposed to anabolic agents.
  • Analytical Strategies:
    • Enzymatic Hydrolysis: Used to break down metabolites and understand conjugated compounds.
    • High-Resolution Mass Spectrometry (LC-HRMS): Provided precise mass data for metabolite identification.
    • Ion Mobility Spectrometry (LC-IMS-HRMS): Offered an additional dimension of separation to describe molecular structure and isomer differentiation.
    • In Vitro Experiments: Performed to replicate and confirm metabolic pathways outside the organism.
  • Data Analysis: Integration of these orthogonal approaches enabled comprehensive structural elucidation of biomarkers.

Findings

  • Two candidate biomarkers were successfully identified as phase II metabolites of tebuconazole, a fungicide, part of the equine exposome.
  • These metabolites likely indicate environmental exposure rather than direct anabolic substance administration.
  • The disruption or changes in levels of these biomarkers in urine post-anabolic agent administration suggest interplay between doping agents and exposure metabolites.
  • The structural identification supports the robustness of using these metabolites as indirect markers in doping control.

Significance

  • Combining orthogonal analytical techniques enabled a higher confidence level in biomarker identification than using single methods alone.
  • This structural elucidation aids the interpretation of metabolomic data, facilitating better understanding of doping effects and environmental factors in racehorses.
  • The study opens avenues for further research into the role of environmental chemicals like tebuconazole in doping tests and horse metabolism.
  • It contributes to improving screening strategies and supports regulatory agencies in maintaining fair competition through reliable biomarker detection.

Cite This Article

APA
Cloteau C, Delcourt V, Loup B, Chabot B, Pescher M, Susdorf E, Kaabia Z, Garcia P, Popot MA, Le Bizec B, Dervilly G, Bailly-Chouriberry L. (2025). Identification of Candidate Biomarkers Detected in the Urine of Racehorses After Anabolic Agent Administration: Use of Orthogonal Methods for Structural Elucidation. Drug Test Anal, 17(12), 2411-2420. https://doi.org/10.1002/dta.3951

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 17
Issue: 12
Pages: 2411-2420

Researcher Affiliations

Cloteau, C
  • Oniris, INRAE, LABERCA, Nantes, France.
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.
Delcourt, V
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.
Loup, B
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.
Chabot, B
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.
Pescher, M
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.
Susdorf, E
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.
Kaabia, Z
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.
Garcia, P
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.
Popot, M A
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.
Le Bizec, B
  • Oniris, INRAE, LABERCA, Nantes, France.
Dervilly, G
  • Oniris, INRAE, LABERCA, Nantes, France.
Bailly-Chouriberry, L
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, France.

MeSH Terms

  • Horses / urine
  • Animals
  • Biomarkers / urine
  • Anabolic Agents / urine
  • Anabolic Agents / administration & dosage
  • Anabolic Agents / metabolism
  • Doping in Sports
  • Substance Abuse Detection / methods
  • Substance Abuse Detection / veterinary
  • Mass Spectrometry / methods
  • Chromatography, Liquid / methods
  • Triazoles / urine
  • Triazoles / metabolism
  • Triazoles / administration & dosage

Grant Funding

  • 2019/1948 / Association Nationale de la Recherche et de la Technologie

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