Abstract: AimTo establish a method for separation and identification of alkaloids in Stephania tetrandra S.Moore methanol extracts by using non-aqueous capillary electrophoresis interfaced with electrospray ionization ion trap mass spectrometry. MethodsThe molecular ions or adducts of alkaloids and fragments of specific parent ions were used for the identification. An uncoated capillary (86 cm×75 μm ID, on-line UV detection occurred at 21 cm from the inlet of the capillary) was used. Ammonium acetate (50 mmol·L^-1) containing 4% HAc in methanol was used as the running buffer; separation voltage was 25 kV. A coaxial sheath flow interface was used as the CE-MS interface; the electrospray voltage was 4.5 kV; the temperature of aluminium capillary was 170 ℃; 60% isopropanol-39% water-1% HAc was used as the sheath liquid with the flow rate of 5 μL·min^-1; the collision energy of MS-MS was set at 30% and the least ion counts was 1×10⁵. Results and conclusionThe alkaloids in Stephania tetrandra S. Moore methanol extracts were separated and identified by CE-ESI-MS/MS. The proposed method is of high accuracy and can be used for the investigation of traditional Chinese medicine.