Identification of hypoxene metabolites in urine samples using gas chromatography–tandem mass spectrometry for anti-doping control

Objectives. Hypoxen is a drug which possesses antioxidant and antihypoxic effects. It achieves this by increasing the utilization of oxygen by mitochondria, intensifying oxidative phosphorylation, and as a result, improving tissue respiration. Athletes take it during prolonged exercise, in order to increase efficiency and reduce physical overwork. Since 2023, the World Anti-Doping Agency has included the drug in the monitoring program, in the belief that it can be used to gain a competitive advantage. It is thus a candidate for inclusion in the Prohibited List as a potential regulator of the human metabolism. Currently, there are no studies or scientific publications focusing on the identification of hypoxene in biofluids for the purpose of anti-doping control. The aim of this study is to determine the possible metabolites of the drug and their chromato-mass spectrometric characteristics in urine samples using gas chromatography–tandem mass spectrometry (GC–MS/MS) for doping control screening purposes.
Methods. Sample preparation of urine samples was carried out using enzymatic hydrolysis, liquid–liquid extraction and derivatization. The GC–MS/MS method was used for analysis. Screening of hypoxene metabolites was carried out in the mode of total ion current after fragmentation of selected parent ions.
Results. Three specific metabolites of hypoxene (m/z 342, 300, and 346, including trimethylsilyl derivatives) were identified in urine samples of volunteers (n = 3). They can act as markers for taking the target antihypoxant, and their possible structural formulas are given. The excretion curves of two metabolites with an m/z of 300 and 346 respectively in urine were studied. The maximum concentration is reached after 8–14 and 1.5–6 h, respectively. It was established, that these metabolites are reliably identified in urine 90 h or more after a single dose of the drug.
Conclusions. Possible structures of hypoxene metabolites in urine samples from volunteers were determined for the first time and their chromato-mass spectrometric characteristics were established. The approach developed in this study can be used for screening analysis of hypoxene for the purpose of anti-doping control.

1. Levchenkova O.S., Novikov V.E., Pozhilova E.V. Pharmacodynamics of antihypoxants and their clinical use. Obzory po klinicheskoi farmakologii i lekarstvennoi terapii = Reviews on Clinical Pharmacology and Drug Therapy. 2012;10(3):3–12 (in Russ.). https://doi.org/10.17816/RCF1033-12

2. Shatalin Yu.V., Naumov A.A., Potselueva M.M. A comparison of antioxidant properties of hypoxen and duroquinone by the method of chemiluminescence. Biofizika. 2008;53(1):100–106 (in Russ.).

3. Murzaeva S.V., Belova S.P., Mironova G.D. Determination of the antioxidant properties of activators of mitochondrial ATP-dependent potassium channels with the Amplex Red fluorescent indicator. Appl. Biochem. Microbiol. 2013;49(4): 333–340. https://doi.org/10.1134/S0003683813040108 [Original Russian Text: Murzaeva S.V., Belova S.P., Mironova G.D. Determination of the antioxidant properties of activators of mitochondrial ATP-dependent potassium channels with the Amplex Red fluorescent indicator. Prikladnaya biokhimiya i mikrobiologiya. 2013;49(4):345–352 (in Russ.). https://doi.org/10.7868/s0555109913040107 ]

4. Semigolovskii N.Yu. Use of antihypoxants in the acute period of myocardial infarction. Anesteziol. Reanimatol. 1998;(2):56–59 (in Russ.).

5. Ivanova L.A., Pavlova M.V., Archakova L.I. Antioxidants in the combined therapy of patients with chronic destructive pulmonary tuberculosis. Therapeutic Archive. 1994;66(11):54–56 (in Russ.).

6. Ganapolskii V.P., Shabanov P.D. Meteoadaptogenic properties of antihypoxic drugs. Eksperimental’naya i Klinicheskaya Farmakologiya. 2009;72(6):36–41 (in Russ.).

7. Dolgareva S.A., Sorokin A.V., Konoplya N.A., Bushmina O.N., Bystrova N.A., Ovod A.I. The use of immunomodulators, antioxidants and hepatoprotectors for the correction of the liver, erythrocites and the immune system disorders in chronic ethanol intoxication. Biomed. Khim. 2018;64(4):360–367 (in Russ.). https://doi.org/10.18097/pbmc20186404360

8. Novikov V.E., Kriukova N.O., Novikov A.S. Gastroprotective properties of mexidol and hypoxen. Eksperimental’naya i Klinicheskaya Farmakologiya. 2010;73(5):15–18 (in Russ.).

9. Churganov O.A., Gavrilova E.A. Influence of gipoksen supplement on some values of psychological, immune, biochemical and functional status of athletes. Vestnik sportivnoi nauki = Sports Science Bulletin. 2009;(1):36–38 (in Russ.).

10. Zagorsky A.L., Kalninsh K.K., Toropov D.K. Mixture of poly-(1,4-dihydroxy)-phenylenes (polyhydroquinones): RF Pat. 2294918. Publ. 10.03.2007 URL: https://yandex.ru/patents/doc/RU2294918C1_20070310. Accessed January 29, 2024.

11. Zagorsky A.L., Kalninsh K.K., Toropov D.K. Mixtures of poly(1,4-dihydroxy)-phenylenes (polyhydroquinones): Pat. WO2007073236. Publ. 28.06.2007. https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2007073236. Accessed February 02, 2024.

12. Savelieva N.B., Ishutenko G.V., Polosin A.V., Radus F.V., Polyansky D.S., Kurbatkin S.A., Efimova Yu.A., Postnikov P.V. Validation of a method for the quantitative determination of narcotic and psychotropic substances in urine by UHPLC–MS/MS. Fine Chem. Technol. 2022;17(3):253–267. https://doi.org/10.32362/2410-6593-2022-17-3-253-267

13. Shoibonov B.B., Ontoboev A.N., Zinchenko A.A., Aleshkin V.A. Hypoxen is an Inhibitor of the c1q Subcomponent and c1 Component of the Human Complement System: RF Pat. 2205002. Publ. 27.05.2003. https://patents.google.com/patent/RU2205002C1/ru. Accessed February 05, 2024.

14. James A.M., Cochemé H.M., Smith R.A.J., Murphy M.P. Interactions of mitochondria-targeted and untargeted ubiquinones with the mitochondrial respiratory chain and reactive oxygen species. Implications for the use of exogenous ubiquinones as therapies and experimental tools. J. Biol. Chem. 2005;280(22):21295–21312. https://doi.org/10.1074/jbc.M501527200

15. Grishina E.V., Khaustova Ya.V., Pogorelova V.G., et al. Accelerated utilization of lactate under the effect of hypoxen after intensive exercise. Bull. Exp. Biol. Med. 2008;145(2): 198–201. https://doi.org/10.1007/s10517-008-0049-y [Original Russian Text: Grishina E.V., Khaustova Ya.V., Pogorelova V.G., Pogorelov A.G., Kuz’mich M.K., Maevskii E.I. Accelerated utilization of lactate under the effect of hypoxen after intensive exercise. Zhurnal eksperimental’noi biologii i meditsiny. 2008;145(2):158–161 (in Russ.).]

16. Murzaeva S.V., Abramova M.B., Popova I.I., et al. Effect of hypoxenum on bioenergetic processes in mitochondria and the activity of ATP-sensitive potassium channel. BIOPHYSICS. 2010;55(5):727–732. https://doi.org/10.1134/S0006350910050076 [Original Russian Text: Murzaeva S.V., Abramova M.B., Popova I.I., Gritsenko E.N., Mironova G.D., Lezhnev E.I. Effect of hypoxenum on bioenergetic processes in mitochondria and the activity of ATP-sensitive potassium channel. Biofizika. 2010;55(5):814–821 (in Russ.).]

17. Ignat’ev V.A., Petrova I.V., Tsvetkova L.N. Experience with the use of hypoxen (Olyphenum) in the treatment of patients with moderate and severe chronic obstructive pulmonary disease. Terra Medica. 2010;3(62):19–24 (in Russ.).