A multidimensional chromatography/high-resolution mass spectrometry approach for the in-depth metabolites characterization of two Astragalus species

To address the chemical complexity is indispensable in a number of research fields. Herb metabolome is typically composed by more than one class of structure analogs produced via different biosynthetic pathways. Multidimensional chromatography (MDC), due to the greatly enhanced separation space, offers the potential solution to comprehensive characterization of herbal metabolites. Here, we presented a strategy, by integrating MDC and quadrupole time-of-flight mass spectrometry (QTOF-MS), to accomplish the in-depth herbal metabolites characterization. Using the metabolome of two Astragalus species (A. membranaceus var. mongholicus, AMM; A. membranaceus, AM) as the case, an off-line three-dimensional liquid chromatography (3D-LC) system was established: hydrophilic interaction chromatography using an XAmide column as the first dimension (1D) for fractionating the total extract, on-line reversed-phase × reversed-phase liquid chromatography separately configuring a CSH Fluoro-Phenyl column and a Cosmocore C18 column as the second dimension (2D) and the third dimension (3D) of chromatography to enable the explicit separation of three well fractionated samples. Moreover, the negative-mode collision-induced dissociation by QTOF-MS under the optimized condition could provide diversified fragments that were useful for the structural elucidation of AMM and AM. An in-house library (composed by 247 known compounds) and comparison with 43 reference standards were utilized to assist more reliable characterization. We could characterize 513 compounds from two Astragalus species (344 from AMM and 323 from AM), including 236 flavonoids, 150 triterpenoids, 18 organic acids, and 109 others. Conclusively, the established MDC approach gained excellent performance favoring the analogs-oriented in-depth characterization of herbal metabolites, but received uncompromising analytical efficiency.