Abstract
Selective androgen receptor modulators (SARMs) represent non-steroidal agents commonly abused in human and animal (i.e. equine, canine) sports, with potential for further misuse as growth promoting agents in livestock-based farming. As a direct response to the real and possible implications of illicit application in both sport as well as food production systems, this study incorporated enzymatic hydrolysis (β-glucuronidase/arylsulfatase) into a previously established protocol while maintaining the minimal volume (200 µL) of urine sample required to detect SARMs encompassing various pharmacophores in urine from a range of species (i.e. equine, bovine, human, canine and rodent). The newly presented semi-quantitative UHPLC-MS/MS-based assay is shown to be fit-for-purpose, being rapid and offering high-throughput, with validation findings fulfilling criteria stipulated within relevant doping and food control legislation.•CCβ values determined at 1 ng mL−1 for majority of analytes.•Deconjugation step included in the method led to significantly increased relative abundance of ostarine in analysed incurred urine samples demonstrating the requirement for hydrolysis to detect a total form of emerging SARMs.•Assay amenable for use within routine testing to ensure fair play in animal and human sports and that animal-derived food is free from contamination with SARM residues.
Highlights
Method ArticleAnna Gadaj a,b,∗, Emiliano Ventura a,∗, Jim Healy c,d, Francesco Botrè e, Saskia S
Assay amenable for use within routine testing to ensure fair play in animal and human sports and that animalderived food is free from contamination with SARM residues
SARM residues were extracted from urine (200 μL) with TBME without further clean-up and analysed by UHPLC-MS/MS
Summary
Anna Gadaj a,b,∗, Emiliano Ventura a,∗, Jim Healy c,d, Francesco Botrè e, Saskia S. As a direct response to the real and possible implications of illicit application in both sport as well as food production systems, this study incorporated enzymatic hydrolysis (β-glucuronidase/arylsulfatase) into a previously established protocol while maintaining the minimal volume (200 μL) of urine sample required to detect SARMs encompassing various pharmacophores in urine from a range of species (i.e. equine, bovine, human, canine and rodent). Deconjugation step included in the method led to significantly increased relative abundance of ostarine in analysed incurred urine samples demonstrating the requirement for hydrolysis to detect a total form of emerging SARMs. Chemistry Analytical chemistry UHPLC-MS/MS-based screening of SARMs following urine hydrolysis E. AC-262536 (P/N 96443-25MG, Sigma-Aldrich, Dublin, Ireland), andarine (S-4, P/N 78986-25MG, Sigma-Aldrich, Dublin, Ireland), bicalutamide (P/N PHR-1678-1G, Sigma-Aldrich, Dublin, Ireland), BMS-564929 (10 mM solution in DMSO, P/N HV-12111, MedChem Express, Sollentuna, Sweden), GLPG0492 (10 mM solution in DMSO, P/N HY-18102, MedChem Express, Sollentuna, Sweden), LGD-2226 (P/N 07682-25MG, Sigma-Aldrich, Dublin, Ireland), LGD-4033 (P/N CAY9002046-50mg, Cambridge Bioscience Ltd., Cambridge, UK), Ly2452473 (P/N CDS025139-50MG, Sigma-Aldrich, Dublin, Ireland), ostarine (S-22, P/N MK-2866, Cambridge Bioscience Ltd., Cambridge, UK), PF-06260414 (P/N PZ0343-5MG, Sigma-Aldrich, Dublin, Ireland), RAD140 (P/N CAY18773-1mg, Cambridge Bioscience Ltd., Cambridge, UK), S-1 (P/N 68114-25MG, Sigma-Aldrich, Dublin, Ireland), S-6 (P/N 79260-25MG, Sigma-Aldrich, Dublin, Ireland), S-9 (P/N D289535, Toronto Research Chemicals, Toronto, Canada), S-23 (P/N 55939-25MG, Sigma-Aldrich, Dublin, Ireland), bicalutamide-D4 (P/N B382002, Toronto Research Chemicals, Toronto, Canada), S-1-D4 (P/N D289532, Toronto Research Chemicals, Toronto, Canada); ultra-pure water (18.2 MOhm, generated in house using a Millipore (Cork, Ireland) water purification system), ethanol (EtOH) and dimethyl sulfoxide (DMSO) (both ACS reagent grade, Sigma-Aldrich, Dublin, Ireland), methanol (MeOH) and acetonitrile (MeCN) (both ChromasolvTM LC-MS grade, Honeywell, VWR International, Dublin, Ireland), acetonitrile-D (MeCN-D, 99.5%, Sigma-Aldrich, Dublin, Ireland), ammonium hydroxide solution, ≥25% (NH4OH) and acetic acid (CH3COOH) (both eluent additives for LC-MS, Honeywell, VWR International, Dublin, Ireland), tert-butyl methyl ether (TBME, LiChrosolv® LC grade, Sigma-Aldrich, Dublin, Ireland), sodium acetate (powder, BioReagent grade, Sigma-Aldrich, Dublin, Ireland), β-glucuronidase/arylsulfatase from Helix pomatia (stabilised saline solution, Roche, P/N 10127698001, Sigma-Aldrich, Dublin, Ireland); PAL-USG (CAT) pocket refractometer (Atago, Tokyo, Japan), SafeSeal polypropylene micro tubes (2 mL, Sarstedt, Nümbrecht, Germany), Hettich Micro 200R centrifuge (Davidson & Hardy, Belfast, UK), DVX-2500 multi-tube vortexer (VWR International, Dublin, Ireland), Grant GLS400 water bath with shaking (Davidson & Hardy, Belfast, UK), centrifuge filters 0.22 μm PTFE 750 μL centrifuge filters 0.22 μm PTFE (P/N F2517-9, Thermo Fisher Scientific, Hemel Hempstead, UK), Turbovap® LV evaporator (Caliper Life Sciences, Mountain View, USA); Waters Acquity I-Class UPLC® system (Milford, MA, USA) coupled to a Waters Xevo® TQ-MS triple quadrupole mass analyser (Manchester, UK) controlled by MassLynxTM software (TargetLynxTM software for data processing, Waters), Luna® Omega Polar C18 (100 × 2.1 mm, 100 A , 1.6 μm, P/N 00D-4748-AN, Phenomenex, Cheshire, UK), KrudKatcherTM Ultra HPLC in-line filter (P/N AF0-8497, Phenomenex, Cheshire, UK)
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