Strategies for the detection of designer steroids in racehorses
Date
2016
Authors
Waller, Christopher Charles
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Abstract
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.
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Keywords
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
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