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Investigation of the selective androgen receptor modulators S1, S4 and S22 and their metabolites in equine plasma using high-resolution mass spectrometry
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
Univ Calif Davis, Sch Vet Med, KL Maddy Equine Analyt Chem Lab, Davis, CA 95616 USA.;Univ Calif Davis, Sch Vet Med, Dept Vet Mol Biosci, Davis, CA 95616 USA..
Univ Calif Davis, Sch Vet Med, KL Maddy Equine Analyt Chem Lab, Davis, CA 95616 USA..
German Sport Univ Cologne, Inst Biochem, Cologne, Germany.;German Sport Univ Cologne, Ctr Prevent Doping Res, Cologne, Germany..
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2016 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 30, no 7, p. 833-842Article in journal (Refereed) Published
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Text
Abstract [en]

RationaleSelective androgen receptor modulators (SARMs) are prohibited in sports due to their performance enhancing ability. It is important to investigate the metabolism to determine appropriate targets for doping control. This is the first study where the equine metabolites of SARMs S1, S4 (Andarine) and S22 (Ostarine) have been studied in plasma. MethodsEach SARM was administered to three horses as an intravenous bolus dose and plasma samples were collected. The samples were pretreated with protein precipitation using cold acetonitrile before separation by liquid chromatography. The mass spectrometric analysis was performed using negative electrospray, quadrupole time-of-flight mass spectrometry operated in MSE mode and triple-quadrupole mass spectrometry operated in selected reaction monitoring mode. For the quantification of SARM S1, a deuterated analogue was used as internal standard. ResultsThe numbers of observed metabolites were eight, nine and four for the SARMs S1, S4 and S22, respectively. The major metabolite was formed by the same metabolic reactions for all three SARMs, namely amide hydrolysis, hydroxylation and sulfonation. The values of the determined maximum plasma concentrations were in the range of 97-170 ng/mL for SARM S1, 95-115 ng/mL for SARM S4 and 92-147 ng/mL for SARM S22 and the compounds could be detected for 96 h, 12 h and 18 h, respectively. ConclusionsThe maximum plasma concentration of SARMs S1, S4 and S22 was measured in the first sample (5 min) after administration and they were eliminated fast from plasma. The proposed targets to be used in equine doping control are the parent compounds for all three SARMs, but with the metabolite yielding the highest response as a complementary target. 

Place, publisher, year, edition, pages
2016. Vol. 30, no 7, p. 833-842
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-286622DOI: 10.1002/rcm.7512ISI: 000372508100006PubMedID: 26969924OAI: oai:DiVA.org:uu-286622DiVA, id: diva2:924186
Available from: 2016-04-28 Created: 2016-04-21 Last updated: 2018-03-14Bibliographically approved
In thesis
1. Structural Determination of Drug Metabolites from Doping Classed Compounds Using Mass Spectrometry
Open this publication in new window or tab >>Structural Determination of Drug Metabolites from Doping Classed Compounds Using Mass Spectrometry
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Doping control in equine sports is important for a fair competition, but also to ensure the integrity of the betting system, as well as for animal welfare reasons. To detect the use of illicit compounds, screening for the parent compound is common. However, by using a metabolite as the analytical target instead, the detection time can be prolonged. For some compounds, the use of a metabolite is a necessity since the parent drug may not be detected at all.

The metabolites of the selective androgen receptor modulators (SARM) S1, S4 and S22 were investigated in horse urine and plasma. The unchanged parent compounds had the longest detection time in plasma, but were not detected at all in urine. Instead, the longest detection time was measured for the metabolites 2-amino-5-nitro-4-(trifluoromethyl)phenyl hydrogen sulfate (SARMs S1 and S4) and 2-amino-5-cyano-4-(trifluoromethyl)phenyl hydrogen sulfate (SARM S22). These metabolites were thus suggested as analytical targets for doping control in urine while the parent compounds were suggested for plasma samples. 2-amino-5-nitro-4-(trifluoromethyl)phenyl hydrogen sulfate could also be produced in large quantities by the fungus Cunninghamella elegans to potentially be used as reference compound.

The horse metabolites of the SARM LGD-4033 were also studied in urine and plasma. The formate adduct of LGD-4033 had the longest detection time in plasma and in urine after hydrolysis with β-glucuronidase. In non-hydrolyzed urine, the glucuronidated LGD-4033 was detected instead.

Different in vitro models were used to predict in vivo metabolites of roxadustat, a hypoxia-inducible factor stabilizer. Cunninghamella elegans was successful in producing more metabolites compared to human and equine liver microsomes and human hepatocytes.

The metabolite detection and identification in all experiments were accomplished using a UHPLC-Q-TOF MS instrument, where the high-resolution MS data was vital in determining which metabolites were formed.

The thesis shows the benefits of investigating the metabolites of doping substances to allow for a successful doping control method in horse urine and plasma by prolonging the detection time. It also highlights the usefulness of Cunninghamella elegans as an alternative to the more commonly used in vitro models for both predicting and producing metabolites.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 58
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 251
Keywords
mass spectrometry, UHPLC-MS/MS, doping control, Cunninghamella elegans, selective androgen receptor modulator, SARM, andarine, ostarine, LGD-4033, roxadustat, HIF stabilizer
National Category
Medicinal Chemistry
Research subject
Analytical Pharmaceutical Chemistry
Identifiers
urn:nbn:se:uu:diva-344310 (URN)978-91-513-0276-8 (ISBN)
Public defence
2018-05-04, B:42, BMC, Husargatan 3, Uppsala, 09:15 (English)
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Supervisors
Available from: 2018-04-12 Created: 2018-03-14 Last updated: 2018-04-24

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Hansson, AnnelieBondesson, UlfHedeland, Mikael

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