Skip to main content

Advertisement

SpringerLink
Log in

Analytics of nonpeptidic erythropoietin mimetic agents in sports drug testing employing high-resolution/high-accuracy liquid chromatography-mass spectrometry

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Since its release as anti-anemic drug, recombinant erythropoietin (rEPO) gradually entered the illicit way to sports competitions as endurance-enhancing drug. Novel modifications biopharmaceutically introduced into the rEPO molecule in the form of carbohydrate or polyethylene glycol moieties made robust and sensitive test methods vital to doping controls in order to provide the necessary tools enabling the conviction of dishonest athletes. Modern protein analysis by means of gel electrophoretic separation and western blotting represents the status quo in rEPO anti-doping analysis. However, new therapeutically promising erythropoietin receptor activating compounds have been developed that exhibit cytokine hormone-mimicking properties but lack any protein structure. Progression to evade parenteral application and substitute for rEPO by low molecular mass and orally available compounds is still one of the major objectives in pharmaceutical research. In this approach, four promising in-house synthesized nonpeptidic erythropoietin mimetic agents, namely compound 129, compound 163, A1B10C1, and A5B10C4 were thoroughly evaluated by employing high-resolution/high-accuracy liquid chromatography tandem mass spectrometry experiments. Characteristic product ions were determined supporting the identification of these drugs and putative metabolites as well as related compounds in future doping controls. Test methods employing direct urine injection and receptor affinity purification strategies were assessed, which demonstrated that EPO receptor purification is of limited utility for nonpeptidic EPOR agonists while direct urine injection allowed for comprehensive method characterization. Thereby, achieved limits of detection were 1 ng/mL for compounds 129/163 and 5 ng/mL for A1B10C1/A5B10C4.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Leonard WJ, Lin JX. Cytokine receptor signaling pathways. J Allergy Clin Immunol. 2000;105(5):877–88.

    Article  CAS  Google Scholar 

  2. Cutler A, Brombacher F. Cytokine therapy. Ann N Y Acad Sci. 2005;1056:16–29.

    Article  CAS  Google Scholar 

  3. Wang H, Dong Q, Fu R, Qu W, Ruan E, Wang G. Recombinant human thrombopoietin treatment promotes hematopoiesis recovery in patients with severe aplastic anemia receiving immunosuppressive therapy. BioMed Res Int. 2015;2015:597293.

    Google Scholar 

  4. United States Anti-Doping Agency. U.S. Postal Service Pro Cycling Team Investigation. 2012. http://cyclinginvestigation.usada.org/. Accessed 28 June 2016.

  5. John MJ, Jaison V, Jain K, Kakkar N, Jacob JJ. Erythropoietin use and abuse. Indian J Endocrinol Metab. 2012;16(2):220–7.

    Article  CAS  Google Scholar 

  6. Lasne F, de Ceaurriz J. Recombinant erythropoietin in urine. Nature. 2000;405(6787):635.

    Article  CAS  Google Scholar 

  7. Reichel C, Abzieher F, Geisendorfer T. SARCOSYL-PAGE: a new method for the detection of MIRCERA- and EPO-doping in blood. Drug Test Anal. 2009;1(11–12):494–504.

    Article  CAS  Google Scholar 

  8. Thevis M, Piper T, Geyer H, Thomas A, Schaefer MS, Kienbaum P. Measuring xenon in human plasma and blood by gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom. 2014;28(13):1501–6.

    Article  CAS  Google Scholar 

  9. Brown A. French race walker admits positive test for FG-4592. 2015. http://www.sportsintegrityinitiative.com/french-race-walker-admits-positive-test-for-fg-4592/. Accessed 28 June 2016.

  10. Buisson C, Marchand A, Bailloux I, Lahaussois A, Martin L, Molina A. Detection by LC–MS/MS of HIF stabilizer FG-4592 used as a new doping agent: investigation on a positive case. J Pharm Biomed Anal. 2016;121:181–7.

    Article  CAS  Google Scholar 

  11. Wrighton NC, Balasubramanian P, Barbone FP, Kashyap AK, Farrell FX, Jolliffe LK. Increased potency of an erythropoietin peptide mimetic through covalent dimerization. Nat Biotechnol. 1997;15(12):1261–5.

    Article  CAS  Google Scholar 

  12. Stead RB, Lambert J, Wessels D, Iwashita JS, Leuther KK, Woodburn KW. Evaluation of the safety and pharmacodynamics of Hematide, a novel erythropoietic agent, in a phase 1, double-blind, placebo-controlled, dose-escalation study in healthy volunteers. Blood. 2006;108(6):1830–4.

    Article  CAS  Google Scholar 

  13. Guarnieri F. Designing a small molecule erythropoietin mimetic. Methods Mol Biol. 2015;1289:185–210.

    Article  CAS  Google Scholar 

  14. Goldberg J, Jin Q, Ambroise Y, Satoh S, Desharnais J, Capps K. Erythropoietin mimetics derived from solution phase combinatorial libraries. J Am Chem Soc. 2002;124(4):544–55.

    Article  CAS  Google Scholar 

  15. Qureshi SA, Kim RM, Konteatis Z, Biazzo DE, Motamedi H, Rodrigues R. Mimicry of erythropoietin by a nonpeptide molecule. Proc Natl Acad Sci U S A. 1999;96(21):12156–61.

    Article  CAS  Google Scholar 

  16. Ligand Pharmaceuticals. Ligand announces positive preclinical data on small molecule erythropoietin receptor agonists. 2010. http://investor.ligand.com/press-releases/detail/145/ligand-announces-positive-preclinical-data-on-small. Accessed 28 June 2016.

  17. Lin Zhi SD, CA (US), inventor; Ligand Pharmaceuticals, Inc., San Diego, CA (US). Small molecule hematopoietic growth factor mimetic compounds that activate hematopoietic growth factor receptors. 2011. Patent No. US 8680150 B2.

  18. Viking Therapeutics. EPOR Agonist program—overview and profile. 2016. http://www.vikingtherapeutics.com/pipeline/epor/. Accessed 28 June 2016.

  19. Novartis AG. A pilot study of a thrombopoietin-receptor agonist (TPO-R agonist), eltrombopag, in aplastic anemia patients with immunosuppressive-therapy refractory thrombocytopenia. 2009. https://clinicaltrials.gov/ct2/show/NCT00922883. Accessed 28 June 2016.

  20. Kuter DJ. New thrombopoietic growth factors. Blood. 2007;109(11):4607–16.

    Article  CAS  Google Scholar 

  21. Bussel JB, Kuter DJ, Aledort LM, Kessler CM, Cuker A, Pendergrass KB, et al. A randomized trial of avatrombopag, an investigational thrombopoietin-receptor agonist, in persistent and chronic immune thrombocytopenia. Blood. 2014;123(25):3887–94.

    Article  CAS  Google Scholar 

  22. Eisai Co. Ltd. Avatrombopag: phase III started. 2014. http://www.biocentury.com/weekinreview/clinicalstatus/2014-02-17/avatrombopag-phase-iii-started-349186. Accessed 28 June 2016.

  23. Boger DL, Chai W. Solution-phase combinatorial synthesis: convergent multiplication of diversity via the olefin metathesis reaction. Tetrahedron. 1998;54(16):3955–70.

    Article  CAS  Google Scholar 

  24. Heyl D, Fessner W-D. Facile direct synthesis of acetylenedicarboxamides. Synthesis. 2014;46(11):1463–8.

    Article  Google Scholar 

  25. Vogel M, Blobel M, Thomas A, Walpurgis K, Schänzer W, Reichel C, et al. Isolation, enrichment, and analysis of erythropoietins in anti-doping analysis by receptor-coated magnetic beads and liquid chromatography-mass spectrometry. Anal Chem. 2014;86(24):12014–21.

    Article  CAS  Google Scholar 

  26. Kuuranne T, Leinonen A, Schänzer W, Kamber M, Kostiainen R, Thevis M. Aryl-propionamide-derived selective androgen receptor modulators: liquid chromatography-tandem mass spectrometry characterization of the in vitro synthesized metabolites for doping control purposes. Drug Metab Dispos. 2008;36(3):571–81.

    Article  CAS  Google Scholar 

  27. Dib J, Thomas A, Delahaut P, Fichant E, Schänzer W, Thevis M. Identification and characterization of in vitro and in vivo generated metabolites of the adiponectin receptor agonists AdipoRon and 112254. J Pharm Biomed Anal. 2016;125:68–76.

    Article  CAS  Google Scholar 

  28. World Anti-Doping Agency. International standard for laboratories. 2012. https://wada-main-prod.s3.amazonaws.com/resources/files/WADA_Int_Standard_Laboratories_2012_EN.pdf. Accessed 28 June 2016.

  29. Van Eeckhaut A, Lanckmans K, Sarre S, Smolders I, Michotte Y. Validation of bioanalytical LC–MS/MS assays: evaluation of matrix effects. J Chromatogr B. 2009;877(23):2198–207.

    Article  Google Scholar 

  30. Güsten H, Klasinc L, Kramer V, Marsel J. Mass spectra of monosubstituted trans-stilbenes. Org Mass Spec. 1974;8(1):323–34.

    Article  Google Scholar 

  31. Thomas A, Görgens C, Guddat S, Thieme D, Dellanna F, Schänzer W, et al. Simplifying and expanding the screening for peptides <2 kDa by direct urine injection, liquid chromatography, and ion mobility mass spectrometry. J Sep Sci. 2016;39(2):333–41.

    Article  CAS  Google Scholar 

  32. Görgens C, Guddat S, Orlovius AK, Sigmund G, Thomas A, Thevis M, et al. “Dilute-and-inject” multi-target screening assay for highly polar doping agents using hydrophilic interaction liquid chromatography high resolution/high accuracy mass spectrometry for sports drug testing. Anal Bioanal Chem. 2015;407(18):5365–79.

    Article  Google Scholar 

  33. Mandel M. The statistical analyses of experimental data. 1st ed. New York: Wiley; 1964.

    Google Scholar 

Download references

Acknowledgments

The study was carried out with the support of the World Anti-Doping Agency, Montreal, Canada (#14B03MT); the German Sport University, Cologne, Germany; the Manfred Donike Institute for Doping Analysis, Cologne, Germany; and the Federal Ministry of the Interior of the Federal Republic of Germany.

Author information

Authors and Affiliations

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

    Matthias Vogel, Josef Dib, Laura Tretzel, Thomas Piper, Andreas Thomas, Wilhelm Schänzer & Mario Thevis

  2. European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany

    Mario Thevis

Authors
  1. Matthias Vogel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Josef Dib
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Laura Tretzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas Piper
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andreas Thomas
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wilhelm Schänzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mario Thevis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mario Thevis.

Ethics declarations

All studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee, and written informed consent was obtained from all volunteers.

Conflict of interest

The authors declare that they have no competing interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 353 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Vogel, M., Dib, J., Tretzel, L. et al. Analytics of nonpeptidic erythropoietin mimetic agents in sports drug testing employing high-resolution/high-accuracy liquid chromatography-mass spectrometry. Anal Bioanal Chem 408, 6431–6442 (2016). https://doi.org/10.1007/s00216-016-9761-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-016-9761-z

Keywords

Search

Navigation