The use of in vitro technologies and high-resolution/accurate-mass LC-MS to screen for metabolites of ‘designer’ steroids in the equine.

This research focused on harnessing in vitro techniques and high-resolution LC-MS technology to detect ‘designer’ steroid abuse in equine sports by identifying associated metabolites. The study demonstrated the feasibility of identifying these metabolites using high-tech instrumentation and, importantly, provided an alternative approach in scenarios where in vivo studies would be unethical due to a lack of toxicity data.

Research Methodology

• The research investigated the metabolism of eight designer steroids, specifically sourced from the Internet. These were: androsta-1,4,6-triene-3,17-dione, 4-chloro,17α-methyl-androsta-1,4-diene-3,17β-diol, estra-4,9-diene-3,17-dione, 4-hydroxyandrostenedione, 20-hydroxyecdysone, 11-keto-androstenedione, 17α-methyldrostanolone, and tetrahydrogestrinone.
• In vitro metabolism took place by utilizing equine liver S9 and/or microsomes, with the exception of 20-hydroxyecdysone which was not successfully metabolized.
• The team employed a high-resolution Thermo LTQ-Orbitrap LC-MS instrument to identify metabolites from untreated sample extracts.
• The results gained from full scan LC-HRMS Orbitrap data were complemented by LC-HRMS/MS and GC-MS experiments to provide additional data on fragmentation, as well as to determine whether GC-MS could detect metabolites undetected by the LC-HRMS process.

Findings

• All steroids, except for 20-hydroxyecdysone, were successfully metabolized by the equine liver S9 and/or microsomes.
• Metabolites of all other compounds, except for 17α-methyldrostanolone (whose metabolites could only be identified by GC-MS), could be identified utilizing LC-HRMS.
• The findings suggest that it’s possible to conduct retrospective analysis of previously acquired full-scan data, resulting from routine equine drug testing.

Conclusion

• The viability and advantage of in vitro methodologies as an alternative to in vivo studies were underscored, especially in situations where administering untested drugs to live horses would be deemed unethical due to insufficient toxicity data.
• The ability to retroactively analyze data from routine equine drug testing can potentially aid in more robust detection and regulation of androgenic-anabolic steroid abuse in equine sports.