Chlorthalidone in vitro metabolite identification for documenting exposure in doping

Diuretics are commonly used in doping because they can conceal the presence of performance-enhancing substances in an athlete's urine through dilution and promote rapid weight loss. As a result, these substances are prohibited in sports by the World Anti-Doping Agency (WADA) under the S5 category ("Diuretics and Masking Agents"). Chlorthalidone, a thiazide-like diuretic, is medically used as an antihypertensive agent and is prescribed for conditions such as heart failure and liver cirrhosis. However, it is also misused in doping. The detection of chlorthalidone or its metabolite markers in an athlete's urine is essential to prove consumption. Therefore, the aim of the study was to assess the metabolism of the substance in humans. For this purpose, chlorthalidone metabolites were predicted with GLORYx (Hamburg University, Germany) to identify the transformations that may occur with higher probability; the compound was incubated with 10-donor-pooled human hepatocytes to simulate hepatic metabolism; and the incubates were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) and software-aided data mining. In silico simulations predicted 11 Phase II metabolites, with N-acetylation at the sulfonamide group being the predominant transformation (88% probability score); other major reactions included O-glucuronidation, O-sulfation, and glutathione conjugation, with probability scores lower than 70%. Two metabolites were identified in in vitro hepatocyte incubates and presented a reduction or a hydroxylation at the phthalimidine moiety. To the best of the authors' knowledge, these metabolites are specific to chlorthalidone and can be targeted as markers for analytical screening in anti-doping controls.