APPROACHES TO CHORIONIC GONADOTROPIN QUANTITATIVE DETERMINATION IN ANTI-DOPING CONTROL
https://doi.org/10.32362/2410-6593-2017-12-1-64-75
Abstract
The article presents the results of the first stage of development of a new quantitative method for human chorionic gonadotropin (hCG) determination by means of ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) to uncover doping abuse by athletes. The identified tryptic peptides correspond to the most abundant hCG isoforms: the α- and β-subunits, the nicked and β-core fragment of the hormone. Identification and sequencing of specific fragments were performed with the use of nanoLC-MS/MS. A high resolution / high accuracy hybrid mass-spectrometer was applied. Optimization of mass-spectrometric determination of selected specific peptides was accomplished by UPLC-MS/MS. Quantitative evaluation of hCG using specific fragments determination by UPLC-MS/MS allows to detect corresponding hCG isoforms. This significantly increases the method specificity and decreases the probability of false-positive results.
About the Authors
I. O. Zvereva
Federal State Budgetary Institution “Antidoping Centre”
Russian Federation
Moscow, 105005 Russia
Russian Federation
Moscow, 105005 Russia
N. B. Savelieva
Federal State Budgetary Institution “Antidoping Centre”
Russian Federation
Moscow, 105005 Russia
Russian Federation
Moscow, 105005 Russia
P. V. Postnikov
Federal State Budgetary Institution “Antidoping Centre”
Russian Federation
Moscow, 105005 Russia
Russian Federation
Moscow, 105005 Russia
Yu. A. Efimova
Moscow Technological University (Institute of Fine Chemical Technologies)
Russian Federation
Moscow, 119571 Russia
Russian Federation
Moscow, 119571 Russia
M. A. Dikunets
Federal State Budgetary Institution “Antidoping Centre”
Russian Federation
Moscow, 105005 Russia
Russian Federation
Moscow, 105005 Russia
References
1. Kicman A.T., Brooks R.V., Cowan D.A. Human chorionic gonadotrophin and sport // Br. J. Sports Med. 1991. V. 25. № 2. P. 73–80.
2. 2014 Anti-Doping Testing Figures Report. [Электронный ресурс] – Access mode: https://wada-main-prod.s3.amazonaws.com/wada_2014_anti-doping-testingfigures_full-report_en.pdf (date of access 25.08.2016).
3. 2013 Anti-Doping Testing Figures Report. [Electronic resource] – Access mode: https://wadamain-prod.s3.amazonaws.com/wada_2013_antidoping_testing_figures_report_en.pdf (date of access 25.08.2016).
4. 2012 Anti-Doping Testing Figures Report. [Electronic resource] – Access mode: https://wada-mainprod.s3.amazonaws.com/resources/files/WADA-2012-Anti-Doping-Testing-Figures-Report-EN.pdf (date of access 25.08.2016).
5. Tsyrlina E.V., Poroshina T.E. Human chorionic gonadotropin as a marker of trophoblastic diseases // Prakticheskaya onkologiya (Practical Oncology). 2008. V. 9. № 3. P. 150–159. (in Russ.).
6. Bogerd J. Ligand-selective determinants in gonadotropin receptors // Mol. Cell Endocrinol. 2007. V. 2. № 260-262. P. 144–152.
7. The 2016 Prohibited list. [Electronic resource] – Access mode: https://www.wada-ama.org/en/resources/science-medicine/prohibited-list (date of access 25.08.2016).
8. Guidelines reporting & management of urinary human chorionic gonadotrophin (hCG) and luteinizing hormone (LH) findings in male athletes. [Electronic resource] – Access mode: https://wada-main-prod.s3.amazonaws.com/resources/files/wada-guidelineshcg-lh-findings-v2.0-2015-en.pdf (date of access 25.08.2016).
9. Lund H., Lovsletten K., Paus E., Halvorsen T.G., Reubsaet L. Immuno-MS based targeted proteomics: highly specific, sensitive, and reproducible human chorionic gonadotropin determination for clinical diagnostics and doping analysis // Anal. Chem. 2012. V. 84. № 18. P. 7926–7932.
10. Liu C.-L., Bowers L.D. Immunoaffinity trapping of urinary human chorionic gonadotropin and its highperformance liquid chromatographic-mass spectrometric confirmation // J. Chromatogr. B. Biomed. Sci. Appl. 1996. V. 687. P. 213–220.
11. Gam L.H. Tandem Mass Spectrometry – Applications and Principles / Ed. J. Prasain. Croatia: InTech, 2012. 788 p.
12. Gam L.H., Tham S.-Y., Latiff A. Immunoaffinity extraction and tandem mass spectrometric analysis of human chorionic gonadotropin in doping analysis // J. Chromatogr. B. 2003. V. 792. № 2. P. 187–196.
13. Lund H., Torsetnes S.B., Paus E., Nustad K., Reubsaet L., Halvorsen T.G. Exploring the complementary selectivity of immunocapture and MS detection for the differentiation between hCG isoforms in clinically relevant samples // J. Proteome Res. 2009. V. 8. № 11. P. 5241–5252.
14. Woldemariam G.A., Butch A.W. Immunoextraction-tandem mass spectrometry method for measuring intact human chorionic gonadotropin, free β-subunit, and β-subunit core fragment in urine // Clin.Chem. 2014. V. 60. № 8. P. 1089–1097.
15. Stenman U.H., Hotakainen K., Alfthan H. Gonadotropins in doping: pharmacological basis and detection of illicit use // Br. J. Pharmacol. 2008. V. 154. P. 569–583.
16. Alfthan H., Haglund C., Dabek J., Stenman U.H. Concentrations of human choriogonadotropin, its beta-subunit, and the core fragment of the beta-subunit in serum and urine of men and nonpregnant women // Clin. Chem. 1992. V. 38. № 10. P. 1981–1987.
17. Grenache D.G., Greene D.N., Dighe A.S., Fantz C.R., Hoefner D., McCudden C., Sokoll L., Wiley C.L., Gronowski A.M. Falsely Decreased Human Chorionic Gonadotropin (hCG) Results Due to Increased Concentrations of the Free β Subunit and the β Core Fragment in Quantitative hCG Assays // Clin. Chem. 2010. V. 56. № 12. P. 1839–1844.
18. Pan S., Aebersold R., Chen R., Goodlett D.R., McIntosh M.W., Zhang J., Brentnall T.A. Mass spectrometry based targeted protein quantification: methods and applications // J. Proteome Res. 2009. V. 8. № 2. P. 287–297.
Review
For citations:
Zvereva I.O., Savelieva N.B., Postnikov P.V., Efimova Yu.A., Dikunets M.A. APPROACHES TO CHORIONIC GONADOTROPIN QUANTITATIVE DETERMINATION IN ANTI-DOPING CONTROL. Fine Chemical Technologies. 2017;12(1):64-75. (In Russ.) https://doi.org/10.32362/2410-6593-2017-12-1-64-75
Views: 472
Email this article 
