Decoding MSn fragmentation rules of isoquinoline alkaloids: a comprehensive atlas.

Natural products constitute a valuable reservoir of lead compounds for drug discovery. Structural characterization and annotation of isoquinoline alkaloids, an important class of bioactive natural products, are prerequisites for the rapid discovery of novel molecular entities. Interpretation of fragmentation patterns is an indispensable step in structural confirmation of new compounds. However, structurally informative fragmentation behaviors of isoquinoline alkaloids remain insufficiently understood across diverse structural scaffolds. In this study, multistage fragmentation patterns of isoquinoline alkaloids were systematically investigated using high-resolution multistage mass spectrometry (HR-MSn). Through a comprehensive analysis of 139 compounds using HR-MSn, general guidelines and underlying principles were established for the first time. Characteristic skeletal fragmentation pathways, nitrogen-derived group losses, and common neutral losses were systematically elucidated, and several previously unreported fragmentation patterns were identified. In addition, the isomer-dependent fragmentation behavior was investigated. Collectively, these results provide a comprehensive reference framework for the mass spectrometric analysis of isoquinoline alkaloid, thereby laying a solid foundation for future investigations.