Abstract: OBJECTIVE To identify the structure of berberine hydrochloride impurities by high performance liquid chromatography coupled to tandem quadrupole time-of-flight mass spectrometry(HPLC-Q-TOF-MS/MS). METHODS An Agilent ZORBAX SB-C₁₈ (4.6 mm×250 mm, 5 μm) column was used for the separation of berberine and its impurities. The impurities of berberine were separated by the 10 mmol·L^-1 ammonium acetate solution (adjusted pH to 3.0 with glacial acetic acid)-acetonitrile (75:25) as the mobile phase, the flow rate was 0.8 mL·min^-1, and the UV detection wavelength was 345 nm. The HPLC-Q-TOF-MS/MS was used to determine the precise first-order molecular weight and second-order fragment ions of berberine and its impurities, and the structure was elucidated. RESULTS Berberine and its impurities were well separated. There were 7 impurities in materials from plant extraction, and 5 impurities from chemical synthesis.CONCLUSION The LC-MS can effectively identify the structure of berberine and its impurities, providing a reference for quality control.