Strategies for the detection of designer steroids in greyhounds

Abstract

Steroids are naturally occurring organic compounds found in all eukaryotes, playing critical roles in biological processes such as hormone regulation and metabolism. Anabolic Androgenic Steroids (AAS), derived from the parent steroid testosterone, were initially developed to treat medical conditions but later gained notoriety as performance-enhancing drugs in human and animal sports. Despite established rules against AAS, including bans enforced by organisations like the World Anti-Doping Agency (WADA) and in greyhound racing, their misuse persists to gain an unfair advantage. The emergence of designer steroids, synthetic variants engineered to evade detection has further complicated doping control efforts. To address this, advanced analytical techniques such as high-resolution mass spectrometry, biomarker assays, and genetic analysis, along with long-term markers, are essential to detect these substances and pave the way for a future in sports free of AAS. Chapter two details an in vivo study on the metabolism of Prostanozol in greyhounds. Prostanozol was chemically synthesised and characterised before being administered orally to two greyhounds. In their urine, phase II Prostanozol metabolites were identified in the form of glucuronides. After enzymatic hydrolysis, four phase I urinary metabolites of Prostanozol were confirmed using synthesised reference materials and analysed through liquid chromatography-mass spectrometry. Chapter three focuses on the in vivo metabolism of Mechabol in greyhounds. After oral administration, both phase I (unconjugated) and phase II (glucuronide) metabolites were detected in urine using liquid chromatography-mass spectrometry. Enzymatic hydrolysis allowed the identification of the parent drug and three primary phase I metabolites, which were confirmed by comparison with synthetically prepared reference materials. Chapter four explores the in vivo metabolism of Trena in greyhounds. After oral administration, phase I (unconjugated) and phase II (glucuronide) metabolites were detected in urine using liquid chromatography-mass spectrometry. Through enzymatic hydrolysis, the parent drug was identified alongside one primary and one secondary phase I metabolite, verified by comparison with synthetically derived reference materials. Chapter four also details the synthesis of reference materials for Finaflex. In this work, four reference materials, including the parent compounds and mixtures of reduced and oxidised derivatives, were synthesised and provided to collaborators. While Finaflex was orally administered to two greyhounds individually, the analysis of post-administration urine samples has not yet been conducted and remains a focus for future research. Overall, this research introduces new detection methods for identifying Prostanozol and Mechabol abuse in greyhounds, making them suitable for routine screening by anti-doping laboratories. Additionally, reference materials for Finaflex and Trena were synthesised to support the future confirmation of in vivo urinary metabolites. Show more