Two Multidimensional Chromatography/High-Resolution Mass Spectrometry Approaches Enabling the In-Depth Metabolite Characterization Simultaneously from Three Glycyrrhiza Species: Method Development, Comparison, and Integration.

Two offline multidimensional chromatography/high-resolution mass spectrometry systems (method 1 : fractionation and online two-dimensional liquid chromatography, 2D-LC; method 2 : fractionation and offline 2D-LC) were established to characterize the metabolites simultaneously from three Glycyrrhiza species. Ion exchange chromatography in the first-dimensional ( 1 D) separation was well fractionated between the acidic (mainly triterpenoids) and weakly acidic components (flavonoids). These obtained subsamples got sophisticated separation by the second ( 2 D) and third dimension ( 3 D) of chromatography either by online reversed-phase chromatography × reversed-phase chromatography (RPC × RPC) or offline hydrophilic interaction chromatography × RPC (HILIC × RPC). Orthogonality for the 2 D/ 3 D separations reached 0.73 for method 1 and 0.81 for method 2, respectively. We could characterize 1097 compounds from three Glycyrrhiza species based on an in-house library and 33 reference standards, involving 618 by method 1 and 668 by method 2, respectively. They exhibited a differentiated performance and complementarity in identifying the multiple subclasses of Glycyrrhiza components.