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Sennoside A and sennoside B are the major bioactive components in laxative herbs like rhubarb, senna, etc.The previously reported techniques for quantitative analysis of sennoside A and sennoside B have the disadvantages of defective peak purity and stability.This study investigates the influence of analytical parameters on efficiency of separation of sennoside A and sennoside B in rhubarb by ultra-high-performance liquid chromatography (UHPLC).The chromatographic parameters of column temperature and flow rate have a non-linear relationship with theoretical plate number and symmetry factor;the optimal column temperature was 30 ℃ and the optimal flow rate was 0.20 mL/min.A new UHPLC analytical method was developed based on these optimal parameters.The chromatographic peak purity of sennoside A and sennoside B in rhubarb was satisfactory.This UHPLC-based analytical method was successfully applied for quantitative determination of sennosides A and sennoside B in two sources of rhubarbs, Cassia angustifolia Vahl and Paidu Yangyan Capsule.There are remarkable content differences between sennoside A (more than 19 times) and sennoside B (more than 18 times) in the two species of rhubarb tested.In order to make the analytical method widely applicable, the analysis parameters of UHPLC have been converted into those of HPLC.In conclusion, the devised technique is suitable for quality control of laxative herbs and diet drugs containing sennoside A and sennoside B.