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Dioscoreapanthaicais a traditional Chinese medicinal herb which has been used to treat cardiovascular disease, gastropathy and hypertension.Steroidal saponins(SS) are the main active ingredients of this herb and have effects on myocardial ischemia, cancer, and parahormone function.Phytochemical analysis of SS, particularly isolation and structural determination, is time-consuming and laborious.Therefore, it is necessary to establish a method to speed up the process of identification, isolation and structure elucidation of SS.In this study, we developed an HPLC-IT/MS method for the identification of SS in D.panthaica.We used IT/MS to distinguish spirostanol and furostanol compounds.Spirostanol compounds were readily generated [M+HCOO]-ina(-) ESI-MS scan, while furostanol compounds with the exception of the ions [M+HCOO]-and[M+2HCOO]2-were indicators for the identification of this type of compound.The number, classes and the connected orders of the sugar moieties of aglucone were all studied in detail.Next, the established fragmentation features were applied for the analysis of the 65% ethanol fraction of the water extract from the dried rhizome of D.panthaica.As a result, 23 SS were identified, including eleven potential new compounds.Furthermore, two target compounds with high content were isolated by MSn analysis and their chemical structures were confirmed by 1H-, 13C-NMR.In this study, the LC-IT/MS technique was shown to be an efficient method for making the process of identification, isolation and structure determination of steroidal saponins more efficient.