Comprehensive Workflow For The Quantification Of Peptides And Proteins In Plasma: Semaglutide A Case Study

Over the past two decades, the pharmaceutical landscape has undergone a remarkable transformation with the rise of protein and peptide-based therapeutics. In 2023 alone, biologics represented 21 of the 55 newly approved medicines, which underscores their growing significance in modern medicine. Among these, semaglutide—a glucagon-like peptide-1 (GLP-1) receptor agonist—stood out for its clinical impact. These therapies are distinguished by their high potency and specificity, as well as their extended pharmacokinetic (PK) half-lives. As a result, they circulate in the bloodstream at extremely low concentrations, often in the nanomolar range, necessitating highly sensitive bioanalytical assays to accurately characterize their PK profiles.

However, developing robust LC-MS/MS methods for quantifying proteins and peptides in biofluids presents unique challenges. These include the formation of precursor ions with multiple charge states and the complexity of selecting from a vast array of potential product ions. To address these hurdles, we showcase an automated workflow that streamlines the selection, optimization, and comparison of peptide multiple reaction monitoring (MRM) transitions using the MassLynx™-Skyline Interface. This approach enables high-sensitivity quantification of semaglutide in human plasma, leveraging the advanced capabilities of the Xevo™ TQ Absolute Mass Spectrometer in conjunction with the ACQUITY™ Premier UPLC™ system.