Synthesis of Designer Steroids to Improve Detection in Greyhounds

Abstract

Steroids occur naturally in different species and hold many important roles. Fascinated with their functions, chemists began synthesizing steroids in the laboratory last century. Some synthetic steroids later became approved as medications, but some were misused in the world of sports including greyhound racing. The presence of androgenic steroids or its metabolites in racing greyhounds has been banned by the authorities. However, with the existence of new "designer" steroids with potential for abuse, new detection methods must be developed to aid doping control efforts.

Chapter two presents the in vivo metabolism study of Furazadrol in greyhounds. Synthesised Furazadrol was given orally to two greyhounds and urinary phase II Furazadrol metabolites were detected as glucuronides. Following enzymatic hydrolysis, seven phase I urinary Furazadrol metabolites were confirmed with synthesised reference materials using liquid chromatography-mass spectrometry. Attempts to characterise an unknown hydroxyfurazadrol metabolite were performed by extensive tandem mass spectrometry analysis and preliminary results for synthesis towards this compound are reported.

Chapter three discusses the in vivo metabolism study of Jungle Warfare in greyhounds. Urinary phase I (unconjugated) and phase II (glucuronide) metabolites were detected following oral administration using liquid chromatography-mass spectrometry. The parent drug and one major phase I metabolite was confirmed by comparison with synthetically-derived reference materials, while glucuronide conjugated metabolites were found to be resistant to enzymatic hydrolysis. Early work on metabolites discovery using gas chromatography-mass spectrometry was also reported but proved inferior to liquid chromatography-mass spectrometry.

Chapter four describes the syntheses work on Superdrol and Ultradrol reference materials. The structure of these two "designer" steroids are very closely related, thus their syntheses were combined into a single route. Ten reference materials, including the parent compounds and mixtures of reduced compounds were reported and sent to the collaborator. These steroids have been given orally to two greyhounds separately, however the analysis of urine samples post-administration has not been performed and is the subject of future work.

The last chapter focuses on endogenous phase II glucuronide metabolites of testosterone in male greyhounds. A steroid monoglucuronide was synthesised chemically and characterised, while mixtures containing steroid diglucuronide were synthesised in vitro. Liquid chromatography-mass spectrometry methods were developed using these materials. Male greyhound urine samples were extracted, analysed, and the detected phase II metabolites were confirmed with the synthesised materials where available. An interesting fragmentation pattern that indicated the conjugation site of glucuronides was discovered. Finally, a study to assess the effectiveness of enzyme Escherichia coli beta-glucuronidase in deconjugating these metabolites was performed. Results were confirmed using both liquid chromatography- and gas chromatography-mass spectrometry.

Overall, this research provides new detection methods for Furazadrol and Jungle Warfare abuse in greyhounds suitable for routine screening conducted by anti-doping laboratories. Selection of Superdrol and Ultradrol reference materials were also synthesised to aid confirmation of in vivo urinary metabolites in the future. Furthermore, novel phase II testosterone metabolites have been discovered, advancing the knowledge of endogenous steroid metabolism in greyhounds. Show more