From a non‐targeted metabolomics approach to a targeted biomarkers strategy to highlight testosterone abuse in equine. Illustration of a methodological transfer between platforms and laboratories

Abstract

In order to overcome the challenge associated with the screening of Anabolic‐Androgenic Steroids abuses in animal competitions, a non‐targeted liquid chromatography coupled to high resolution mass spectrometry based metabolomics approach was implemented on equine urine samples to highlight potential biomarkers associated with the administration of such compounds, using testosterone esters as model steroids. A statistical model relying on four potential biomarkers intensity could be defined to predict the status of the samples. With a routine application perspective, the monitoring of the highlighted potential biomarkers was first transferred into high‐throughput liquid chromatography‐selected reaction monitoring (LC‐SRM). The model's performances and robustness of the approach were preserved and providing a first demonstration of metabolomics‐based biomarkers integration within a targeted workflow using common benchtop MS instrumentation. In addition, with a view to the widespread implementation of such biomarker‐based tools, we have transferred the method to a second laboratory with similar instrumentation. This proof of concept allows the development and application of biomarker‐based strategies to meet current doping control needs.

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“…Metabonomics is an important part of systems biology [25]. Me tabonomics is a science that uses a variety of technical means to study the type, quantity and change law of small molecular metab olites in biological system [26]. Metabonomics can be divided into nontargeted and targeted metabonomics according to different re search purposes [27].…”

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

“…Afterwards, the samples were extracted using C18 cartridges. The eluate was dried, reconstituted in MeOH/water (8/2, v/v ), and injected into the LC-MS [ 55 ].…”

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

“…1). Quality control (QC) samples were prepared as described previously (Cloteau, 2022). All the samples of the study were stored at − 20 The general work ow to monitor the 4 candidate biomarkers of interest has previously been described (Cloteau, 2022).…”

“…Based on a previously published study reporting the discovery of 4 candidate biomarkers and their combination in a statistical model to suspect testosterone esters abuse in a gelding (Cloteau, 2022), the present work describes the rst steps of validation of the derived screening method based on the procedure reported by Dervilly-Pinel et al (Dervilly-Pinel, 2018). While the previous study focused on biomarkers nding and checking from an analytical point of view the transferability of their monitoring between laboratories and different analytical platforms, the aim of the present work was to assess the biological relevance of the biomarkers and to generate supplemental data dedicated to analytical robustness.…”