Characterization of Novel Enarodustat Metabolites Using Liquid Chromatography–High Resolution Mass Spectrometry for Doping Control Purposes

ABSTRACT Hypoxia‐inducible factor prolyl hydroxylase inhibitors (HIF‐PHIs) represent a novel class of therapeutic substances that increase erythropoiesis. Due to their performance‐enhancing effects and potential risk of abuse, these agents were added to the World Anti‐Doping Agency (WADA) Prohibited List in 2011. Enarodustat is a novel HIF‐PHI and has been approved for clinical use in China in 2023. This study primarily aimed to characterize its major urinary metabolites for antidoping purposes. A single oral dose of 40‐mg enarodustat was administered to a volunteer. Urine samples were collected over 28 days and processed using solid‐phase extraction (SPE). Analytical methods included liquid chromatography‐high resolution mass spectrometry (LC‐HRMS) under both positive and negative electrospray ionization conditions, complemented by in vitro metabolism studies using human liver microsomes (HLMs) for the characterization and identification of metabolites. A total of eight metabolites were detected, including Phase I products such as parent compound (PC) isomer (M1), monohydroxylation (M2), dihydroxylation (M3), and dehydrogenation (M4) metabolites, as well as Phase II conjugates involving methylation (M5), glycosylation (M6), glucuronidation (M7), and monohydroxylation‐sulfation (M8) in vivo. Among these, M3–M5 are novel metabolites. In addition, compared with other metabolites, PC and M2 exhibited longer detection windows, suggesting they are valuable biomarkers for doping control purposes. The study elucidates enarodustat's metabolic pathways and provides a foundation for developing sensitive detection methods. Future work should focus on synthesizing reference materials to identify metabolite structures.