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A method coupling high-performance liquid chromatography (HPLC) with quadrupole time-of-flight mass spectrometers (QTOF-MS) using an eletrospray ionization (ESI) source was firstly developed for detection,characterization and guiding target separation of variants of protopanaxdiol saponin from leaves of Panax notoginseng.Under the guidance of LC-QTOF-MS,a new trace saponin was probed according to the precise elemental compositions of molecular ions and the fragmentation behavior,and then separated from the ethanol extract of the plant by a set of chromatographic methods.It was further confirmed by NMR experiments as 3-O-β-D-glucopyranoside-3β,l2β,23β-triol-20-ene-dammar (Pn-1).The cytotoxic assay showed that Pn-1 had relatively stronger anti-tumor effects against three tumor cell lines (NCI-H460,HepG2 and SGC-7901) than Rg3,an approved clinic agent for cancer therapy.Meanwhile,based on accurate mass measurements within 5 ppm for each molecular ions and subsequent product ions,48 saponins,including 40 protopanaxadiol saponins,7 protopanaxatriol saponins and 1 oleanane saponin were identified.It is noted that the knowledge of the presence of abundant protopanaxadiol saponins in leaves of P.notoginseng may provide tools for a full understanding of the chemical diversity of secondary metabolites from the different parts of P.notoginseng.From the points of time consuming and accurate mass measurement capability,the LC-QTOF-MS is a highly powerful tool for screening and guiding target separation of new compounds in herbal extract,and thus benefit the speed of new drug discovery progress.