In-Source Fragmentation of Pyrrolidine-Containing KRAS Scaffolds Leads to Enhanced Structure Elucidation and Tandem Mass Spectral Quality

The characterization of small-molecule therapeutics containing basic moieties, such as pyrrolidine groups common in KRAS G12C inhibitors, presents challenges for structure elucidation via tandem mass spectrometry (MS/MS). During fragmentation, the pyrrolidine preferentially sequesters the proton, leading to a dominant, uninformative fragment ion and a corresponding loss of structural detail. This hinders the efficient identification of related impurities and metabolites in complex mixtures. To circumvent this limitation, we developed an easily transferable analytical workflow that intentionally utilizes In-Source Fragmentation (ISF). Optimizing source parameters promotes the selective neutral loss of the pyrrolidine moiety prior to MS/MS, yielding core fragment ions (e.g., <i>m</i>/<i>z</i> 525 for GDC-6036). Tandem mass spectrometry on this ISF-generated precursor provides extensive fragmentation and structural coverage, outperforming traditional higher-energy collisional dissociation. We demonstrate the successful application of this optimized workflow to characterize GDC-6036, its synthetic intermediate, and structurally distinct KRAS inhibitors (Adagrasib and MRTX1133) across a chromatographic time scale. This approach offers a universal tool for enhancing the structure elucidation of challenging basic compounds, critical for supporting pharmaceutical process development.