Identification and Characterization of the low-abundant constituents of Radix isatidis by off-line 2D-LC coupled with Q-TOF MS

Radix Isatidis (BanLanGen in Chinese) is the one of the most commonly used Traditional Chinese Medicine (TCMs) for its antiviral activity. But the constituents is unclear, especially the low-abundant ones. In this study, an orthogonal off-line two-dimensional high performance liquid chromatography (2D-LC) based on two different reversed-phase columns, a C18 column (XTerra MS C18) and a home-made polar-introduced C18 column (C18HC), provided with different molecular interaction was developed for the separation of Radix Isatidis. 1 The eluent was monitored by diode-array detection (DAD) and electrospray quadrupole time-of-flight tandem mass spectrometry (ESI-Q/TOF). Pure standards, accurate mass measurements and characteristic fragment ions were used to identify or tentatively characterize the structure of the separated compounds, including alkaloid, phenolic acid and phenyl propanoid. More than thirty compounds were identified or tentatively characterized based on their retention time, UV spectrometry and MS data. In this 2D-LC separation system, some low-abundant compounds covered by the major compounds were efficiently separated and identified for its highly separation resolution and sensitivity. For example, m/z 143 was identified as the molecular ion obviously in the 2D-LC analysis compared to the 1D-LC. As a result, the off-line 2D-LC-MS method in the present work was expected to be an effective technique to separate and identify the minor compounds for the advantage of reducing the influence of the signal suppression and providing more reliable structural information.