Chlorthalidone in vitro metabolite identification for documenting exposure in doping

Area of Science:

• Pharmacology and Toxicology
• Anti-Doping Science
• Metabolomics

Background:

• Diuretics are prohibited by the World Anti-Doping Agency (WADA) under category S5 due to their masking and weight-loss effects in sports.
• Chlorthalidone, a thiazide-like diuretic, is medically prescribed but also misused for doping purposes.
• Accurate detection of chlorthalidone and its metabolites is crucial for anti-doping enforcement.

Purpose of the Study:

• To investigate the human metabolism of chlorthalidone.
• To identify specific metabolite markers for chlorthalidone consumption in athletes.
• To establish reliable analytical targets for anti-doping screening.

Main Methods:

• In silico prediction of chlorthalidone metabolites using GLORYx software.
• In vitro incubation of chlorthalidone with pooled human hepatocytes to simulate hepatic metabolism.
• Analysis of incubates using liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS).
• Software-aided data mining for metabolite identification.

Main Results:

• In silico analysis predicted 11 Phase II metabolites, with N-acetylation being the most probable transformation.
• Two novel chlorthalidone metabolites were identified in human hepatocyte incubates.
• These identified metabolites showed reduction or hydroxylation at the phthalimidine moiety.

Conclusions:

• The identified metabolites are specific to chlorthalidone.
• These novel metabolites serve as potential target markers for chlorthalidone detection in anti-doping controls.
• This research enhances the analytical capabilities for detecting diuretic misuse in sports.