Metabolite Profiling and Identification of Semaglutide in Liver S9 Across Species and Rat Plasma

Semaglutide, a glucagon-like peptide-1 receptor agonist, stands as a paradigm of successful peptide drug development. Although absorption, distribution, metabolism and excretion characteristics of semaglutide have been thoroughly studied in both animals and humans, a reliable in vitro screening model for evaluating metabolic behavior of semaglutide and other peptides remains an unmet need in drug development. This study established a novel ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry method to elucidate the species-specific metabolic pathways of semaglutide. Semaglutide was incubated with liver S9 fractions from human, monkey, dog, rat, and mouse for 1 h, respectively. For the in vivo study, rats were subcutaneously injected with semaglutide (10 mg/kg) for subsequent plasma collection. Data-dependent acquisition was performed to obtain the fragment ions, enabling identification of drug-related materials. Notably, a diagnostic fragment ion at m/z 469, formed from the cleavage of side chain, facilitated metabolite screening. Consequently, a total of 31 metabolites were detected and characterized, constituting a comprehensive metabolic profile of semaglutide in liver S9 fractions and rat plasma. Major metabolic pathways involved hydrolysis of peptide backbone. Our findings provided a robust methodological framework for the screening and metabolism prediction of peptide candidates, supporting the comprehensive safety and efficacy assessment.