A comprehensive characterization strategy incorporating multiple filtration approaches and enhanced isomer identification: A case study of Eleutherococcus senticosus leaves.

Eleutherococcus senticosus (Rupr. & Maxim.) Maxim leaves (ESL) are consumed as tea or stir-fried dishes and exhibit neuroprotective activity. Their primary active components, triterpenoids, pose characterization challenges due to structural complexity and extensive isomerism. To address these challenges, we developed a novel integrated annotation strategy that incorporates polygonal mass defect filtering (p-MDF), molecular fingerprinting, molecular networks (MN) and ion mobility spectrometry (IMS). The EstMIL multidimensional database was established, encompassing 305 triterpenoids and addressing the challenge of insufficient database coverage. Finally, the integration of collision cross-section (CCS) with FBMN enhanced the characterization of unknown chemical compositions and improved the ability to identify isomers. This approach identified 13 triterpenoid isomer pairs, 6 of which were validated by ER-MS and quantum chemical calculations, confirming the reliability of the strategy. This integrated strategy ultimately identified 203 triterpenoids, comprising 64 newly identified compounds and 48 with novel potential.