Objective: To identify the steroidalsaponins in the tubers of Ophiopogonis Radix by ultra-performance liquid chromatography tandem with time-of-fight mass spectrometry (UPLC-Q-TOF-MSE)combined with the relative retention time (RRT) of furostanolsaponins in reverse chromatography. Method: The retention time of several furostanolsaponins was tested on two kinds of reversed-phase column

Acquity UPLC HSS T3 and Agelavenusil XBP C18-2

using water (0.1% formic acid)-acetonitrile (0.1% formic acid) and acetone-water

respectively. The mass spectrometric data of Ophiopogonis Radix were obtained by UPLC-Q-TOF-MSE. Result: The RRT of furostanolsaponins was consistently in three chromatographic conditions. When the sugar parts were identical

the RRT was shorter alone with the increase number of hydroxyl; location of hydroxyl affected the RRT

the orders were asfollow:1-hydroxyl< 17-hydroxyl< 14 -hydroxyl. When only the sugar chain at C-26 was different

the RRT was as follow:glucosyl-(1→6)-glucosylEcombined with RRT

a total of 21 compounds were identifiedincluding 14 furostanolsaponins and 7 spirostanolsaponins. Conclusion: The RRT of furostanolsaponins provide supportive data for rapidly identify isomers of steroidalsaponins from Ophiopogonis Radix by UPLC-Q-TOF-MSE.