Oral Presentation 24th International Conference of Racing Analysts and Veterinarians 2026

Identifying urinary markers of doping agents with untargeted UPLC-MS/MS metabolomics. (129267)

Ryan C Kennedy 1 , Lance Brooker 2 , Sean Yamada 1 3 , Teresa Neeman 4 , Christopher Fitzgerald 5 6 , Adam Cawley 3 , Malcolm D McLeod 1
  1. Research School of Chemistry, Australian National University, Canberra, ACT, Australia
  2. Australian Racing Forensic Laboratory, Racing NSW, Sydney, NSW, Australia
  3. Racing Analytical Services LTD, Melbourne, VIC, Australia
  4. Research School of Biology, Australian National University, Canberra, ACT, Australia
  5. Max Planck Institute for Marine Microbiology, University of Bremen, Bremen, Germany
  6. Center for Marine Environmental Sciences, University of Bremen, Bremen, Germany

Among the biggest bottlenecks in implementing controls against doping agents in sport is identifying the incriminating metabolites to target. Targeted approaches to this issue are often slow and may miss the most relevant metabolites.1 Untargeted metabolomics presents unrivalled opportunity to characterise metabolic shifts and products from substance administration.1,2 However, while a wealth of programs are available to aid with data processing, the most daunting challenge remains; how to identify relevant compounds of interest. Manually sifting through tens of thousands of features is an unrealistic task and current programs are often prohibitively expensive, unreliable, or overly prescriptive.3

In this work, an efficient method has been developed to look through the noise that is generated when analysing whole metabolome data, using a simple, script-based workflow coupled to the untargeted LC-MS metabolomic data output from MS-DIAL. This workflow simultaneously, 1) performs statistical analysis on all high-quality features, to identify the most salient and characteristic indicators of drug administration; and 2) identifies putative sulfated and glucuronylated metabolites, the most likely phase II products of many potential doping agents, particularly steroids. The two pipelines are then combined to provide a list of the most statistically significant and chemically relevant features.

Oral Altrenogest is permitted in Australian Racing for oestrus control in mares and fillies but due to potentially elevated levels of illicit androgen contaminants, intramuscular administration is strongly discouraged.4 However, retrospectively distinguishing between administration routes remains an unsolved challenge. This method was tested on a 6-horse, cross-over study of oral and intramuscular administration of altrenogest to attempt to identify new and potentially distinguishing markers of prohibited intramuscular administration. Several metabolites of altrenogest were putatively identified, as well as a potential candidate feature capable of distinguishing between oral and intramuscular administration.

  1. 1. Botrè, F.; Georgakopoulos, C.; Elrayess, M. A. Metabolomics and Doping Analysis: Promises and Pitfalls. Bioanalysis 2020, 12 (11), 719–722. https://doi.org/10.4155/bio-2020-0137.
  2. 2. Heaney, L. M.; Deighton, K.; Suzuki, T. Non-Targeted Metabolomics in Sport and Exercise Science. J. Sports Sci. 2019, 37 (9), 959–967. https://doi.org/10.1080/02640414.2017.1305122.
  3. 3. Keen, B.; Cawley, A.; Reedy, B.; Fu, S. Metabolomics in Clinical and Forensic Toxicology, Sports Anti‐doping and Veterinary Residues. Drug Test. Anal. 2022, 14 (5), 794–807. https://doi.org/10.1002/dta.3245.
  4. 4. Elbourne, M.; Keledjian, J.; Cawley, A.; Fu, S. Administration Route Differentiation of Altrenogest via the Metabolomic LC-HRMS Analysis of Equine Urine. Molecules 2024, 29 (21), 4988. https://doi.org/10.3390/molecules29214988.