Identification and characterization of related substances in EVT-401 by hyphenated LC–MS techniques

A sensitive and selective method was developed for the separation and characterization of related substances(RSs) in EVT-401 by hyphenated LC–MS techniques. Complete separation of the RSs was achieved with an Inertsil ODS-SP column (250 mm×4.6 mm, 5 μm) by linear gradient elution using a mobile phase consisting of0.2% formic acid solution, methanol and acetonitrile. EVT-401 was found to be susceptible to acid, alkaline and xidative stresses, while relatively stable under photolytic and thermal dry stress conditions. Fourteen RSs ncluding six process-related substances and eight degradation products were detected and identified in EVT-401 with positive ESI high-resolution TOF-MS analysis of their parent ions and the corresponding product mass pectra elucidation, and some of them were further verified by chemical synthesis and NMR spectroscopy. Thespecific LC–MS method developed for separation, identification and characterization of RSs is valuable for EVT-401 manufacturing process optimization and quality control.