AbstractAnabolic-androgenic steroids (AAS) continue to be the drug class most abused amongst athletes, and despite the fact that adverse effects from pseudo-endogenous and synthetic AAS misuse are well known, the potential for irreversible effects on human health to be induced by AAS is still not sufficient to eliminate their abuse. Even as anti-doping laboratories have achieved significant advancements in the analytical performance of antidoping testing and established a deep knowledge of steroid profiles in humans, athletes continue to cheat and exploit the properties of emerging novel anabolic drug enhancers namely selective androgen receptor modulators (SARMs). SARMs were discovered in the late 1990s as promising alternatives to AAS due to their ability to separate anabolic effects on target tissues from unwanted androgenic effects. Nowadays, the potential therapeutic applications of SARM compounds has focussed on a range of human conditions including those involving muscle or bone wasting. Despite the fact that no SARM pharmacophore has yet received approval for clinical use in humans, they can still be easily accessed and sourced on the black market, being well recognised as drugs of abuse to enhance performance in both human and animal sports, and have also been suggested to potentially find use in foodproducing species to increase meat yield and production efficiency. In response to the illicit use of SARMs through doping and food fraud practices, SARMs are banned in human and equine sport by the World Anti-Doping Agency (WADA), the International Federation of Horseracing Authorities (IFHA) and the Fédération Equestre Internationale (International Equestrian Federation, FEI), as well as under EU Council Directive 88/146/EEC for animal husbandry use. The research presented here has sought to address the lack of multi-residue analytical methods enabling the effective detection of emerging SARM compounds in a range of biofluids collected from different animal species.
Considering that urine and blood are generally the biological matrices of choice regulated by both anti-doping testing and drug residue monitoring programmes, three semiquantitative ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) methods for the screening of 15 SARM compounds (equine, bovine, canine, rodent and human) with and without enzymatic hydrolysis (β-glucuronidase/arylsulfatase) in urine and in blood (equine plasma and bovine serum) were developed. Method validation was performed in all species of interest according to respective EU Commission Decision 2002/657/EC criteria and European Union Reference Laboratories for Residues (EU-RLs) guidelines, with detection capability (CCβ) values at 1-5 ng mL-1 for urine and at 0.5-5 ng mL-1 for blood, respectively.
Taking into account that stability studies are normally an integrated part of analytical method validation, and considering that information about SARM stability in peer reviewed scientific literature is exiguous, this research aimed also to determine the optimal storage conditions for various SARM compounds in solvent solutions (standard stock and intermediate mixed standard solutions), as well as in test matrices (urine and blood) and in solvent extracts under frozen storage and post freeze-thaw cycles. Findings have shown that most SARMs are stable in the corresponding reference solutions. A certain degree of instability was highlighted for some SARM analytes in both urine and blood matrices when stored at -20 °C as well as after repeated freeze-thaw cycles, whilst all compounds were found stable in respective urine and blood reconstitution solvent extracts.
Metabolites that could serve as additional marker residues of LGD-4033 and ostarine (S-22) were identified through analysis of biofluids obtained post-SARM administration in rodents. Whilst ostarine (S-22) was shown to be the principal marker residue in both urine and plasma, the bis-hydroxy metabolite of LGD-4033 was identified as the main marker residue in rodent urine with the methoxy metabolite most abundant in plasma. The various UHPLCMS/MS semi-quantitative screening assays developed in this research have potential to serve as approaches to monitor for SARM compounds in a range of biofluids enabling detection of their abuse in human and animal sport as well as in livestock animals. A screening survey using developed methods was performed with more than 600 samples (urine and blood) collected from abattoirs across Northern Ireland as well as donated by the Irish Equine Centre (IEC), the Irish Greyhound Board, the WADA Anti-Doping Laboratory of Rome and non-professional athletes based in Northern Ireland - to date none of the samples tested have been found to contain detectable quantities of SARM residues.
|Date of Award||Jul 2020|
|Supervisor||Mark Mooney (Supervisor) & Christopher Elliott (Supervisor)|
- selective androgen receptor modulators
- anti-doping control
- food safety monitoring
- residue analysis