Analysis of the polymeric mixture in hypoxen, using high-performance liquid chromatography coupled with high-resolution mass spectrometry

RationaleHypoxen is a medication known for providing individuals with a "second wind," by lowering the threshold for muscle fatigue and enhancing the body's efficiency under challenging conditions. Athletes who have used this medication report enhanced training outcomes and increased physical endurance. It is crucial to emphasize that hypoxen is not categorized as a prohibited substance as yet and is thus assumed safe for use in competitive sports. However, the polymeric nature of hypoxen presents challenges in detection and identification.MethodsTo prevent the potential misuse of this substance as a doping agent, doping control laboratories must therefore develop a detection method. This study aimed to address this gap by developing a comprehensive detection method for the polymeric mixtures within hypoxen, employing liquid chromatography-electrospray ionization-mass spectrometry.ResultsAmong the different columns tested, the Accucore and Syncronis HILIC columns demonstrated exceptional performance, yielding excellent separation with high-quality results. The study found that the hypoxen consisted of between 1 and 6 repetitions of 2,4-dihydroxyphenylene units. It was crucial to highlight that each unit contained multiple isomers due to the incorporation of the sulfonic acid group at both the -SH and -OH positions.ConclusionsThe results of this study will significantly contribute to the precise identification of hypoxen use, thereby aiding in the scrutiny of its abuse in competitive sports.