Identification of chemical constituents in two traditional Chinese medicine formulae by liquid chromatography–mass spectrometry and off-line nuclear magnetic resonance

There have been increasing works on identification of the chemical constituents in traditional Chinese medicines (TCMs) by liquid chromatography–mass spectrometry (LC–MS). However, isomers cannot be distinguished generally only by MS data, and the structures of unknown compounds cannot be confirmed. In this study, semi-preparative LC guided by LC–MS was used to prepare the isomers in microscale followed by off-line NMR analysis to confirm their structures. This approach was applied to identifying the constituents in two TCM formulae, Zhi-Zi-Gan-Cao-Chi-Tang (ZZGCCT) and Zhi-Zi-Bai-Pi-Tang (ZZBPT). A total number of 119 constituents were identified tentatively or unambiguously by LC–IT–MS, LC–Q–TOF–MS, and NMR. Among them, 20 constituents were characterized unambiguously by comparing with the reference substances. In addition, 21 constituents without reference substances were prepared for the following 1D-NMR and/or 2D-NMR analysis, and their structures were unambiguously identified by MS, 1D-NMR, and 2D-NMR. Two triterpenoid glycosides (compounds 134 and 140) and one flavonoid glycoside (compound 62a or 62b) were showed to be novel compounds. Compounds 125 and 129, as well as 62a,b, were epimers.