A polarity-based two-dimensional LC-MS/MS distribution strategy combined with ion identity molecular networking for...

A polarity-based two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) distribution strategy was established by integrating hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) within a heart-cutting configuration. The resulting LC-MS data were further processed using an ion identity molecular networking (IIMN)-based annotation workflow, which systematically consolidates adducts, in-source fragments, and isotopes across positive and negative ionization modes, thereby facilitating coherent molecular network construction and structural inference. When applied to metabolomic profiling of two Astragalus Radix (AR) species, the proposed strategy enabled the characterization of 253 metabolites spanning multiple chemical classes and the identification of 45 discriminative metabolites associated with botanical origin. In addition, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) revealed distinct tissue-specific distribution patterns of representative differential flavonoids, with predominant localization in the phloem and xylem, providing complementary spatial evidence for species differentiation. Overall, the polarity-based 2DLC-MS/MS distribution strategy with IIMN workflow offers enhanced separation efficiency, improves annotation confidence, and supports structural interpretation of structurally related metabolites. This strategy provides a practical and extensible approach for in-depth characterization of complex plant metabolomes and quality evaluation of natural products.