Detection of bemitil and its metabolite in urine by means of LC–MS/MS in view of doping control analysis

2‐(Ethylthio) benzimidazole is an active ingredient of Antihot, a dietary supplement sold in Ukraine. The substance, available also under names of Bemitil, Metaprot, and Bemaktor, was developed in the USSR in 1970s, and after tests on Soviet cosmonauts and soldiers, several studies on its influence on athletes' performances were conducted. The research showed that bemitil is a synthetic adaptogen which is capable to significantly increase physical performance and reduce the time of regeneration. Moreover, according to supplement's distributor, the substance improves both physical performance and resistance to stress. Taking into account these properties, it appears plausible that the World Anti‐Doping Agency (WADA) decided to include bemitil in its 2018 monitoring program. To select markers of bemitil use, six doses of the supplement (two per day, on three consecutive days) were administrated to six healthy volunteers (three men, three women, 26–49 years). Urine samples were collected before, during and up to 30 days after the first ingestion. Samples were analyzed by means of ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). The study revealed that bemitil can be traced in urine as either a parent compound or its glucuronide conjugate, which is more abundant and has a wider detection window. Application of a method involving enzymatic hydrolysis caused an increase in the peak area of bemitil, compared to the Dilute and Shoot method. The study revealed that bemitil can be traced in urine as either a parent compound or its glucuronide conjugate, which is more abundant and has wider detection window.