Integrated HPLC and STOCSY reveal two new lanaroft type biflavonoids from Selaginella plants

Dereplicating by standalone LC-MS remains challenging for low-abundance biflavonoids (BFVs) in Selaginella extracts, as structural isomers share identical molecular weights, possess diverse interflavonoid linkages, and exhibit highly similar fragmentation patterns, thereby increasing the risk of misannotation. Here, an integrated high-performance liquid chromatography (HPLC)-statistical total correlation spectroscopy (STOCSY) workflow was introduced to enable cross-identification of BFVs across partially resolved fractions in two Selaginella extracts, namely Selaginella plana, and S. padangensis. In the workflow, initial screening of crude extracts was performed by matching HPLC retention time against an in-house BFV library. Subsequent STOCSY analysis of partially resolved fractions obtained from S. plana, enabled orthogonal validation of known BFVs. Crucially, unmatched correlated signals guided the selection of target fractions for purification and characterization, leading to the discovery of planaflavone A, an undescribed lanaroft-type BFV. A comparable HPLC RT observed in S. padangensis further directed the isolation of an additional lanaroft-type BFV, padangeflavone A. Collectively, this integrative workflow accelerated compound discovery by enabling discrimination of BFVs with diverse linkage types that are indistinguishable by LC–MS.