Introduction: Hypoxen, originally developed in the USSR, is recognized as an adaptogen with both anti-hypoxic and antioxidant activities. It’s been reported to enhance physical endurance by delaying muscle fatigue and improving physiological efficiency under stress. Due to its potential performance-enhancing effects, Hypoxen is included in the World Anti-Doping Agency (WADA) monitoring list. However, its polymeric nature creates significant challenges for detection, and limited research is available to guide reliable analytical approaches. To address this gap, a robust screening method is needed for effective anti-doping control.
Methods: This study focused on developing an advanced detection strategy for Hypoxen using liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS). Both in vitro (equine liver microsomes) and in vivo (Thoroughbred horse administration) models were employed to evaluate metabolic profiles and identify polymeric constituents of Hypoxen.
Results: The LC-ESI-MS method was developed for the detection of Hypoxen and its metabolites in biological samples. Analysis revealed that Hypoxen is composed of polymeric mixtures containing between one and six repeating 2,4-dihydroxyphenylene units. Each unit displayed structural diversity due to the sulfonic acid group being incorporated at both the –SH and –OH positions, resulting in multiple isomers. Both in vitro (equine liver microsomes) and in vivo (Thoroughbred horse administration) studies successfully detected relevant metabolites. These findings confirm the complexity of Hypoxen and its metabolic patterns, highlighting the necessity for specialized screening protocols.
Conclusion: The developed LC-ESI-MS method provides a reliable tool for detecting Hypoxen and its metabolites in biological samples. This work enhances the capacity of doping control laboratories to identify the misuse of Hypoxen in competitive sports and contributes to safeguarding the integrity of athletic performance.