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Characterization of two major urinary metabolites of the PPARδ-agonist GW1516 and implementation of the drug in routine doping controls

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Abstract

Since January 2009, the list of prohibited substances and methods of doping as established by the World Anti-Doping Agency includes new therapeutics such as the peroxisome-proliferator-activated receptor (PPAR)-delta agonist GW1516, which is categorized as a gene doping substance. GW1516 has completed phase II and IV clinical trials regarding dyslipidemia and the regulation of the lipoprotein transport in metabolic syndrome conditions; however, its potential to also improve athletic performance due to the upregulation of genes associated with oxidative metabolism and a modified substrate preference that shifted from carbohydrate to lipid consumption has led to a ban of this compound in elite sport. In a recent report, two presumably mono-oxygenated and bisoxygenated urinary metabolites of GW1516 were presented, which could serve as target analytes for doping control purposes after full characterization. Hence, in the present study, phase I metabolism was simulated by in vitro assays employing human liver microsomal fractions yielding the same oxygenation products, followed by chemical synthesis of the assumed structures of the two abundant metabolic reaction products. These allowed the identification and characterization of mono-oxygenated and bisoxygenated metabolites (sulfoxide and sulfone, respectively) as supported by high-resolution/high-accuracy mass spectrometry with higher-energy collision-induced dissociation, tandem mass spectrometry, and nuclear magnetic resonance spectroscopy. Since urine samples have been the preferred matrix for doping control purposes, a method to detect the new target GW1516 in sports drug testing samples was developed in accordance to conventional screening procedures based on enzymatic hydrolysis and liquid–liquid extraction followed by liquid chromatography, electrospray ionization, and tandem mass spectrometry. Validation was performed for specificity, limit of detection (0.1 ng/ml), recovery (72%), intraday and interday precisions (7.7–15.1%), and ion suppression/enhancement effects (<10%).

The detection of GW1516, a drug candidate potentially enhancing athletic performance, and the identification of major urinary metabolites for doping control purposes is described using liquid chromatography-mass spectrometry.

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Acknowledgments

The study was carried out with support of the German Federal Ministry of the Interior, the Ministry of Sports, Tourism, and Youth Policy of the Russian Federation, and the Manfred-Donike Institute for Doping Analysis.

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Authors and Affiliations

  1. Institute of Biochemistry—Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany

    Mario Thevis, Ines Möller, Andreas Thomas, Simon Beuck, Hans Geyer & Wilhelm Schänzer

  2. Moscow Anti-Doping Center, Elizavetinsky per. 10, 105005, Moscow, Russia

    Grigory Rodchenkov

  3. Bayer CropScience AG, Alfred-Nobel-Str. 50, 40789, Monheim, Germany

    Wolfgang Bornatsch

Authors
  1. Mario Thevis
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  2. Ines Möller
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  3. Andreas Thomas
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  4. Simon Beuck
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  5. Grigory Rodchenkov
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  6. Wolfgang Bornatsch
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  7. Hans Geyer
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  8. Wilhelm Schänzer
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Correspondence to Mario Thevis.

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Thevis, M., Möller, I., Thomas, A. et al. Characterization of two major urinary metabolites of the PPARδ-agonist GW1516 and implementation of the drug in routine doping controls. Anal Bioanal Chem 396, 2479–2491 (2010). https://doi.org/10.1007/s00216-009-3283-x

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  • DOI: https://doi.org/10.1007/s00216-009-3283-x

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