An ultra high performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry （UHPLC-quadrupole-orbitrap MS） method was developed to systematically analyze chemical constituents of Scutellaria barbata D. Don. The 70% methanol extract was separated on an Agilent Eclipse Plus Cl8 column （1.8μm, 150 mm×2.1 mm）, and eluted with a gradient of methanol-acetonitrile-water containing 0.1% formic acid at a flow rate of 0.25 mL/min. Constituents were identified by HRMS in the negative ion mode using both full scan and two-stage threshold-triggered mass modes. A total of 56 compounds, including 20 free flavonoids, 20 flavonoid O-glycosides, 2 flavonoid C-glycosides, 4 phenylethanoid glycosides, and 10 phenolic compounds were identified by analyzing their high resolution mass spectral data. Among them, 8 flavonoids and 2 phenylethanoid glycosides were unambiguously identified by comparing with reference standards. This study demonstrates that hybrid quadrupole-orbitrap mass spectrometry is a powerful tool in structural identification of unknown compounds in comolicated herbal extracts.
