Molecular Networking-Based Annotation of Tuberostemonine Isomers Using MS/MS Spectral Similarity

Stemona alkaloids possess multiple stereogenic centers, giving rise to structurally diverse isomeric forms that remain challenging to differentiate by conventional mass spectrometric approaches. In this study, six tuberostemonine isomers were annotated and discriminated using LC-MS/MS in combination with molecular networking-guided spectral similarity analysis. Based on their MS/MS spectral features, these isomers were classified into two distinct groups. Gaussian fitting further revealed characteristic differences in fragmentation energy profiles and relative fragment-ion intensities, enabling refined differentiation of closely related isomeric alkaloids. The relative abundances of these isomers were subsequently compared across commercial "Bai-Bu" herbal materials. These results provide experimental evidence and an MS-based analytical strategy for the identification, discrimination, and quality evaluation of isomeric Stemona alkaloids.