Objective: To establish an HPLC method for simultaneous determination of polygalaxanthone Ⅺ, polygalaxanthone Ⅲ and 3,6’- disinapoylsucrose in Polygalae Radix, providing reference for the revision of quality standard of Polygalae Radix. Wherein,polygalanone Ⅺ is a newly discovered positional isomer from the peak of polygalanone Ⅲ. Methods: Chromatographic separation was achieved using a commercial Agilent InfinityLab Poroshell 120 EC-C18 column (150 mm×4.6 mm, 2.7 μm). Acetonitrile (A) and 0.1% phosphoric acid water (B) were used as the mobile phase with gradient elution (0-18 min,10%A→13%A;18-35 min,13%A→19%A;35-45 min,19%A→20%A), the flow rate was 1.0 mL·min-1, the column temperature was set at 30 ℃, the detection wavelength was 320 nm, and the injection volume was 10 μL. Results: The established method was verified by systematic methodology. The RSD values of precision, repeatability and stability experimental results were all below 1%, and the average recoveries for polygalaxanthone Ⅺ, polygalaxanthone Ⅲ and 3,6’- disinapoylsucrose were 90.6%, 94.4%, and 94.0%, respectively, with RSDs of 1.8%, 2.7%, and 3.6%, all of which met the requirements of Chinese Pharmacopoeia 2020. The RSD values of the contents from various parameters including different chromatographic columns, instruments, column temperatures, flow rates, and pH value of mobile phase, were all below 5%, indicating good tolerance of this method. As a result, the method was used for determining polygalaxanthone Ⅺ, polygalaxanthone Ⅲ and 3,6’- disinapoylsucrose in 21 bathes of Polygalae Radix samples, their contents were in range of 0.04%-0.11%, 0.06%-0.17% and 0.39%-0.82%, respectively. The content limit was finally recommended that the content sum of polygalaxanthone Ⅺ and polygalaxanthone Ⅲ should not less than 0.12% according to the data. Conclusion: The optimized method supplying good separation for 3,6’- disinapoylsucrose and two positional isomers polygalaxanthone Ⅲ and polygalaxanthone Ⅺ was more accurate for evaluation of the quality of Polygalae Radix than the method in Chinese Pharmacopoeia.
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