Comprehensive Discovery and Characterization of Chemical Constituents in Huangqintang Decoction Using Off-Line Two-Dimensional Liquid Chromatography and High-Resolution Mass Spectrometry

Traditional Chinese prescriptions are characterized by complex chemical constituents and wide variations in constituent content, which pose a substantial challenge to their comprehensive characterization. As a classic traditional Chinese prescription known for its heat-clearing and detoxifying properties, Huangqintang Decoction (HQD) is composed of Scutellariae Radix, Paeoniae Radix Rubra, Glycyrrhizae Radix et Rhizoma, and Jujubae Fructus. In this study, we developed an off-line two-dimensional liquid chromatography that addressed the limitations of traditional analysis of unfractionated extracts, such as restricted peak capacity, which often obscured trace components. By coupling with ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS), this study successfully performed rapid identification or characterization of the complete chemical profile of HQD. Notably, beyond high-throughput identification, this approach leveraged characteristic fragment ions and reversed-phase chromatographic behaviors to differentiate some isomers of flavonoid glycosides and triterpenoid saponins, demonstrating its depth in structural identification. Flavonoid glycoside isomers were distinguished by diagnostic neutral losses, while flavanones and chalcones were characterized by retro-Diels–Alder (RDA) and β-rearrangement, respectively. Isomers of triterpenoid saponins were inferred from aglycone-specific pathways alongside RDA cleavages. Ultimately, a total of 192 compounds were identified, including 88 flavonoids, 80 triterpenoids, 7 monoterpene glycosides, 3 fatty acid amides, 3 phenylethanoid glycosides, 4 coumarins, 3 saccharides, 1 organic acid, and 3 others. This study demonstrated that the off-line two-dimensional liquid chromatography analysis strategy significantly enhanced chromatographic resolution and expanded the coverage of trace components. It presented an effective strategy for comprehensive compound identification in complex traditional Chinese medicine prescriptions.