Determination of 2-(ethylthio)benzimidazole and its metabolites in rat urine by liquid chromatography−tandem high-resolution mass spectrometry
Research Institute of Hygiene, Occupational Pathology, and Human Ecology,
Federal Medical-Biological Agency
Bemithyl [2-(ethylthio)benzimidazole hydrochloride] is an actoprotector used for enhanced body's resistance to hypoxia, including better tolerance to physical stress. The inclusion of bemithyl in the WADA monitoring program necessitated the development of a method for the determination of 2-(ethylthio)benzimidazole in urine, identification of its metabolites and evaluation of the time window for detectable administration of this drug from the results of urine analysis. The goal of the present work was to identify urine metabolites of 2-(ethylthio)benzimidazole, to develop a procedure for the quantification of this drug and qualitative detection of its metabolites in urine and to determine their excretion time in preclinical study. A solution of bemithyl substance was intragastrically injected in rats for 3 days in a single daily dose of 25 mg/kg. Daily rat urine was collected for 29 days. The samples were analyzed by high-performance liquid chromatography hyphenated to tandem high-resolution mass spectrometry (HPLC−HRMS/MS). Bemithyl metabolites were identified in urine samples of rats exposed to an ultrahigh dose of bemithyl (100 mg/kg). A procedure for the quantification of 2-(ethylthio)benzimidazole in urine with the detection limit of 5 ng/mL was developed. Six metabolites of 2-(ethylthio)benzimidazole were identified in rat urine, of which two can be considered as retrospective biomarkers. It was established that 2-(ethylthio)benzimidazole and its main metabolites are excreted from the body within a few days and then only trace amounts of these analytes were detected in the urine samples throughout the entire experimental period (29 days). However, at the 29th day, the urine concentrations of bemithyl and its metabolites were still higher than the detection limit of the developed procedure.
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