Application of electro-activated dissociation fragmentation technique to identifying glucuronidation and oxidative metabolism sites of vepdegestrant by LC- HRMS

Drug metabolite identification is an integrated part of drug metabolism and pharmacokinetics (DMPK) studies in drug discovery and development. Definitive identification of metabolic modification sides of test compounds such as screening metabolic soft spots and supporting metabolite synthesis are often required. Currently, LC-HRMS is the dominant analytical platform for metabolite identification. However, the interpretation of product ion spectra generated by commonly used collision-induced disassociation (CID) and higher-energy collisional dissociation (HCD) often fails to identify locations of metabolic modifications, especially glucuronidation. Recently, a ZenoTOF 7600 mass spectrometer equipped with electron-activated dissociation (EAD-HRMS) was introduced. The primary objective of this study was to apply EAD-HRMS to identify metabolism sites of vepdegestrant (ARV-471), a model compound that consists of multiple functional groups. ARV-471 was incubated in dog liver microsomes and 12 phase I metabolites and glucuronides were detected. EAD generated unique product ions via orthogonal fragmentation, which allowed for accurately determining the metabolism sites of ARV-471, including phenol glucuronidation, piperazine N-dealkylation, glutarimide hydrolysis, piperidine oxidation, and piperidine lactam formation. In contrast, CID and HCD spectral interpretation failed to identify modification sites of three O-glucuronides and three phase I metabolites. The results demonstrated that EAD has significant advantages over CID and HCD in definitive structural elucidation of glucuronides and phase I metabolites although the utility of EAD-HRMS in identifying various types of drug metabolites remains to be further evaluated. Significance Statement Definitive identification of metabolic modification sites by LC-HRMS is highly needed in drug discovery and development, such as screening metabolic soft spots and supporting metabolite synthesis. However, commonly used CID and HCD spectra often fail to provide useful information for definitive structural elucidation. In this study, the EAD fragmentation technique was applied to identify glucuronidation and phase I metabolism sites of ARV-471, which generated significantly better results than CID and HCD.