Unveiling the complexity of Astragali Radix by comprehensive two-dimensional hydrophilic interaction liquid chromatography × reversed phase liquid chromatography system coupled to hybrid linear ion trap Orbitrap mass spectrometry

Comprehensive elucidation of the chemical constituents of Astragali Radix is crucial for its quality control and edible product development. Herein, online comprehensive two-dimensional hydrophilic interaction liquid chromatography × reversed phase liquid chromatography tandem high-resolution mass spectrometry was elaborately designed to separate, identify, and qualitatively compare various chemical components of Astragali Radix from two species. After selection and optimization of stationary phase, mobile phase, elution program and interface, an optimum method suitable for chemical components in Astragali Radix was established successfully. Under the optimized separation conditions, the orthogonality reached 99 %. A total of 212 compounds were identified and tentatively identified (198 from A. membranaceus var. mongholicus and 177 from A. membranaceus ). Additionally, the fragmentation pattern of flavonoids and saponins mass data in Astragali Radix have also been systematically studied. These findings not only unveiled multi-component complexity and diversity in two Astragalus species, but also proposed an innovative strategy for the rapid separation and identification of other complex systems.