Identification of related substances in baloxavir marboxil using 2D-LC-Q-TOF/MS

Baloxavir marboxil, a novel RNA polymerase inhibitor for the treatment of influenza, suppresses viral replication at the initiation stage of RNA transcription and has demonstrated significant clinical efficacy in reducing viral load and rapidly alleviating respiratory symptoms. Previous studies have primarily focused on its pharmacodynamics and clinical efficacy, whereas systematic investigations into its related substances (RSs) and their structural characteristics remain limited. In this study, two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry (2D-LC-Q-TOF/MS) was employed to detect and characterize process-related impurities in the baloxavir marboxil active pharmaceutical ingredient (API) and degradation products formed in its stressed samples, in accordance with the ICH Q1A(R2) guideline. Based on chromatographic retention behavior and high-resolution MS/MS fragmentation patterns, a total of 14 impurity-related peaks were characterized, including four process-related impurities and ten degradation products. Among them, six impurities, including IMP C and five degradation products (RSs 5, 6, 8, 9 and 12), were not found in the available literature and were characterized in this study. The major degradation pathways were proposed, involving side-chain hydrolysis, oxidation of the sulfur-containing aromatic ring, and the cleavage of heterocyclic linkages. Furthermore, a preliminary genotoxicity assessment of the characterized impurities was conducted in accordance with the ICH M7(R2) guideline using in-silico (Q)SAR prediction approaches, and revealed that these related substances did not exhibit genotoxic structural alerts. The determined impurity profile of baloxavir marboxil and the evaluated impurities' potential safety risks can provide a valuable reference for its impurity control, stability evaluation, and quality risk management.