Comprehensive metabolic characterization of GBT1118 in humans using a combined approach via UHPLC-HRMS for doping...

Artificially enhancing muscular oxygen (O₂) delivery represents a major cheating method in competitive sports. GBT1118, an emerging hemoglobin S polymerization inhibitor (HbS-PI), has demonstrated considerable performance-enhancing effect by increasing the Hb affinity for O₂, raising concerns about potential abuse in athletics. However, the lack of metabolic knowledge and ethical restrictions on human subjects hinder the effective doping control of GBT1118. In this study, comprehensive metabolic profiling of GBT1118 was characterized by combining a human liver microsomes in vitro model and a multi-dimensional data mining strategy. Orbitrap-based ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) in data-dependent acquisition (DDA) mode was utilized to detect both phase-I and phase-II reaction products, together with in silico prediction and a home-made HbS-PI biotransformation database for in-depth metabolite screening. After duplicate exclusion, manual identification and verification with synthetic standards, extensive metabolism including 21 phase-I metabolites and 8 phase-II metabolites was discovered for GBT1118. Structure assignment revealed several key metabolic sites and diverse biotransformation pathways, including ester hydrolysis, aldehyde reduction, aldehyde oxidation, hydroxylation (monohydroxylation and dihydroxylation), glucuronidation, and the cross-combination of these pathways. Notably, the high proportion of glucuronide conjugates (> 60%) suggests the necessity of implementing enzymatic hydrolysis in routine anti-doping analyses. Furthermore, based on metabolite abundance and a comparative analysis with a recent in vivo metabolic study of an analogue, the drug prototype and two metabolites are proposed as promising urinary markers for abuse monitoring. This study provides the first comprehensive insight into the human-relevant metabolic profile of GBT1118, offering valuable guidance for doping control, which also serves as an effective tool for human metabolic research of other HbS-PIs.