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A simple, rapid method was developed using on-line high-performance liquid chromatography (HPLC)/electrospray ionization (ESI)-linear ion trap-Orbitrap hybrid mass spectrometry (LTQ-Orbitrap) to analyze bioactive constituents in the extract of Polygonum multiflorum Thunb., a traditional Chinese medicine (TCM). The qualitative analysis was carried out both in the negative and positive mode to acquire accurate mass data in full scan and data dependent MSn scan. Twenty eight new dianthrone glycosides were characterized and their fragmentation behaviors were first exploited by our LC-HR-MSn method. The identification and structure analysis of these chemicals provided essential data for further phytochemical studies of P. multiflorum. Moreover, the results of the present investigations indicated the relevance and usefulness of the combination of HPLC and LTQ-Orbitrap MS to detect and identify new natural …