Urinary metabolites indicative of the administration of hypoxen monitored by liquid chromatography–high resolution/accurate mass tandem mass spectrometry

Hypoxen, a poly(dihydroxyphenylene) thiosulfonate‐based drug, has been investigated concerning its effect on mitochondrial respiration and the utilization of lactate, especially in the context of strenuous exercise. Since 2023, patterns of use regarding hypoxen amongst the athletic population are monitored by the World Anti‐Doping Agency (WADA) and its accredited anti‐doping laboratories, necessitating information on suitable urinary markers indicative of the administration of hypoxen. In this exploratory study, urine samples collected post‐administration of 1.5 and 2.0 g of hypoxen were analyzed by means of liquid chromatography‐high resolution/high mass accuracy (tandem) mass spectrometry, which allowed for the identification of eight analytes that were plausibly attributable to metabolites of hypoxen. The identified species were assigned to the unconjugated species of S‐(2,2′,5,5′‐tetrahydroxy‐[1,1′‐biphenyl]‐3‐yl) sulfurothioate and its glucuronide and additional tentatively identified analytes comprising a mercaptobenzene core structure. Including the identified markers into routine doping control analytical procedures enabled the detection of hypoxen use in athletes' doping control samples, thus contributing relevant information to WADA's monitoring program.