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

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.

Automated summary

A non-targeted metabolomics approach was used to identify potential biomarkers associated with the abuse of Anabolic-Androgenic Steroids in animal competitions using equine urine samples. The model's performances and robustness were successfully preserved in a high-throughput liquid chromatography-selected reaction monitoring method. The transferability of the method to a second laboratory demonstrated its applicability in developing biomarker-based strategies for doping control.