Strategies for the detection of designer steroids in racehorses
Creators: name: Waller, Christopher Charles
Publisher: name: The Australian National University
Year: 2016
Subjects: steroid
anabolic steroid
designer steroid
anti-doping
drug testing
equine
horse
metabolism
review
sulfate ester
steroid sulfate
sulfation
solid phase extraction
SPE
bis-sulfate
constant ion loss
gas-chromatography
liquid chromatography
mass spectrometry
androgen bioassay
in vivo metabolism
in vitro metabolism
PAPS
synthesis
testosterone
furazadrol
[1’,2’]isoxazolo[4’,5’:2,3]-5α-androstan-17β-ol
3α-chloro-17α-methyl-5α-androstan-17β-ol
3β-chloro-17α-methyl-5α-androstan-17β-ol
hemapolin
2α
3α-epithio-17α-methyl-5α-androstan-17β-ol

description: Anabolic androgenic steroids (AAS) are a class of compounds which include the parent compound testosterone, and are well-known for their misuse as performance enhancing substances in sport. Over the last decade there has a push towards the illicit use of so-called “designer steroids” which are manufactured with the express aim of rendering these compounds undetectable by existing anti-doping methods. These compounds are readily accessible online in “dietary” or “nutritional” supplements containing compounds which have never been tested or approved as veterinary agents. Despite considerable focus in human sports, there has been limited investigation into these compounds in equine systems. As such, developing new tools to detect these compounds is of high priority. Chapter One presents a summary of the literature detailing the metabolism of designer steroid compounds in equine systems, with an aim to identify metabolites suitable for incorporation into screening protocols by anti-doping laboratories. Future directions for anti-doping laboratories are also proposed, highlighting the importance of alternate testing matrices, improved in vitro methodologies which can faithfully replicate in vivo metabolism, and the development of untargeted screening methods which can detect new instances of steroid misuse. Chapter Two presents new methodology for the synthesis of steroid sulfate compounds, suitable for use in analytical laboratories. Key to this synthetic methodology is the use of solid-phase extraction (SPE), a technique routinely used in anti-doping laboratories but with untapped potential in chemical synthesis. This methodology was applied to the synthesis of sixteen steroid mono-sulfate, and twelve steroid bis-sulfate reference materials, which encompass a representative range of steroid substitution patterns and configurations. The mass spectrometry behaviour of these bis-sulfate compounds has been studied, and used to develop a constant ion loss (CIL) scan method for the untargeted detection of all major classes of steroid bis-sulfate metabolites. Chapter Three presents a study detailing the discovery of a novel anabolic agent 3α/β-chloro-17α-methyl-5α-androstan-17β-ol, in samples seized by law-enforcement, and the subsequent investigations to elucidate its structure, identify the primary human and equine metabolites, and to incorporate these into routine anti-doping screening protocols. This study highlights a workflow suitable for adoption by anti-doping laboratories which allows for the development of suitable screening protocols in the event that new designer steroids are identified. Chapter Four presents in vivo and in vitro studies of the designer steroid furazadrol ([1’,2’]isoxazolo[4’,5’:2,3]-5α-androstan-17β-ol). Following a controlled equine in vivo administration, these investigations have identified the key urinary metabolites of furazadrol, many of which have been confirmed by comparison to synthetically-derived reference materials. These metabolites can be incorporated into anti-doping screening and confirmation procedures. Comparative in vitro studies were also undertaken alongside this work. A second study is presented in this chapter which investigates alternative conditions for in vitro phase II metabolism, and employs adenosine 5’-triphosphate (ATP) and Na2SO4 in place of the expensive phase II co-factor 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Although the ability to replicate in vivo metabolism using in vitro methods is currently limited, it is hoped that this work will allow for the prediction of in vivo metabolism of unknown compounds solely from in vitro results. Chapter Five presents an in vivo study of the designer steroid hemapolin (2α,3α-epithio-17α-methyl-5α-androstan-17β-ol). Following a controlled equine in vivo administration, these investigations have identified the key urinary metabolites of hemapolin, many of which have been confirmed by comparison to an extensive library of synthetically-derived reference materials. The excretion profiles of the major enone metabolites 17β-hydroxy-17α-methyl-5α-androst-3-en-2-one, and 17β-hydroxy-17α-methyl-5α-androst-2-en-4-one have been established through a MRM method. These metabolites can be incorporated into anti-doping screening and confirmation procedures, allowing for the future detection of hemapolin misuse.