Offline three-dimensional chromatography coupled with high-definition data-independent acquisition of ion mobility quadrupole time-of-flight mass spectrometry enabling the multicomponent characterization of Centella asiatica

An offline three-dimensional liquid chromatography/high-resolution mass spectrometry system (3D-LC/HRMS) was developed for the in-depth metabolite profiling of Centella asiatica (Ji-Xue-Cao), an edible medicinal herb. The first-dimensional ion exchange chromatography (IEC) efficiently separated triterpenoids and flavonoids into distinct groups. Subsequent orthogonal separations, hydrophilic interaction chromatography (HILIC) on an XAmide column (second dimension) and reversed-phase chromatography (RPC) on a Luna Omega Polar C18 column (third dimension), significantly improved the resolution of fractionated triterpenoids and flavonoids. High-definition ion-mobility quadrupole time-of-flight mass spectrometry (IM-QTOF-MS) facilitated MS/MS data determination, while automatic peak annotation of UNIFI TM and Global Natural Products Social Molecular Network (GNPS) enabled the identification or tentative characterization of 170 compounds from C. asiatica , involving 23 potentially new triterpenoids. Collision-cross section (CCS) value determination and prediction further resolved seven groups of isomeric compounds. This study establish a robust methodological framework for the efficient discovery and reliable characterization of unknown components relevant to food development. • 3D-LC/IM-QTOF-HDMS E was used to identify multicomponents from Centella asiatica . • Automatic peak annotation, GNPS, CCS prediction, were used for data analysis. • 170 compounds were identified in C. asiatica , involving 23 potentially novel. • We provide a practical solution for efficient discovery of unknown components.