Characterization and identification of the wide-polarity multicomponents from Prunella vulgaris by offline two-dimensional liquid chromatography and hydrophilic interaction chromatography coupled to ion mobility-quadrupole time-of-flight mass spectrometry

Metabolites identification is crucial to develop functional foods or perform quality control. Prunella vulgaris (Xia-Ku-Cao) is a medicinal and edible plant used as the herbal medicine or main additive in functional beverage. However, current analytical strategies can only on-line characterize tens of compounds, restricted by insufficient chromatographic resolution and low coverage of the mass spectrometric scan methods. This work was designed to characterize the wide-polarity components from the ear of P. vulgaris. The total extract was fractionated by semi-preparative high-performance liquid chromatography into the retained medium-polarity fraction and unretained polar fraction, which were further analyzed by offline two-dimensional liquid chromatography (2D-LC) and hydrophilic interaction chromatography, respectively. Data-independent high-definition MS^E of the Vion™ ion mobility time-of-flight mass spectrometer was utilized enabling the high-coverage acquisition of collision-induced dissociation-MS² data. The offline 2D-LC, configuring the XBridge Amide and HSS T3 columns, gave high orthogonality (0.81) and effective peak capacity (1555). Automatic peak annotation facilitated by the UNIFI™ bioinformatics platform and comparison with 62 reference compounds achieved the efficient and more reliable structural elucidation. We could characterize 255 compounds from P. vulgaris, with numerous phenylpropanoid phenolic acids and triterpenoid O-glycosides newly reported. Especially, collision cross section (CCS) prediction and targeted isolation of three compounds assisted in the identification of 39 groups of isomers. Additionally, 17 hydrophilic compounds, involving oligosaccharides and organic acids, were characterized from the unretained polar fraction. Conclusively, the in-depth metabolites identification of P. vulgaris was accomplished, and the results can benefit the development and better quality control of this valuable plant.