Application Research For Separation And Determination Of The Active Components In Radix Puerariae And Picriae Herba By HPCE

Objective:The first part of this article aims to study the influence on the separation of seven isoflavones including 3'-methoxypuerarin,puerarin,3'-hydroxypuerarin,ononin,daidzin,daidzein and genistin in Radix Puerariae by capillary electrophoresis based on binary cyclodextrin system.A rapid,sensitive and effective method of capillary electrophoresis were established and provide a method basis for the analysis of seven isoflavones in Radix Puerariae and related preparations.In the second part of this paper,the effect of organic solvent modified micellar electrokinetic capillary chromatography on the separation of four components(picfeltarraenins IA and IB,picfeltarraenin IV and acteoside)in Picriae Herba were studied.A rapid and practical new method of capillary electrophoresis has been developed.Additionally,the proposed method provides scientific evidences for quality control of Picriae Herba medicinal materials and its formulation.Methods:The experiments were performed through a P/ACE MDQ CE system(Beckman Coulter,Fullerton,America).The CE was equipped with a diode array detector(DAD),and uncoated capillary column(40.2 cm×75 m id,effective length 30 cm)was used as the separation channel.(1)The factors affecting the separation of seven isoflavones were studied: the concentration and pH of buffer solution,the concentration of sulfobutylether-?-cyclodextrin(SBE-?-CD)and the ?-cyclodextrin(?-CD),and the separation voltage and so on.The most optimum condition of simultaneous separation and determination of seven isoflavones were obtained: the electrolyte solution composed of 35 mM STB(pH 9.34),30 mM ?-CD and 9.0 mM SBE-?-CD,15 kV of applied voltage.Samples were injected into the capillary at a pressure of 0.5 psi for 5s.(2)The effects of buffer solution concentration and pH,the concentration of sodium cholate(SC),the content of methanol and 2-propanol,and the separation voltage on the separation of four active components in Picriae Herba were studied.Finally,the most optimum condition of simultaneous separation and determination of the four analytes(picfeltarraenins IA,picfeltarraenins IB,picfeltarraenin IV and acteoside)were as follows: the electrolyte solution consisting of 20 mM STB(pH 9.76),40 mM SC,10%(v/v)2-propanol and 10%(v/v)methanol,18 kV of applied voltage,with 25°C of capillary temperature and the injection condition was 0.5 psi for 5 s.Results:Under the optimum separation condition,3'-methoxypuerarin,puerarin,3'-hydroxypuerarin,genistin,ononin,daidzin and daidzein can reach baseline separation within 11 minutes,the linear relationships of seven isoflavones were good,and the correlation coefficient was in the range of 0.9978-0.9992.The seven analyte could be separated within 11 min.The intraday and interday precision ranged from 0.7% to 2.0%and from 0.8% to 1.9%,respectively.The recoveries of the actual samples were from97.7% to 103.1%.Under the optimum capillary electrophoresis condition,four active ingredients in Picriae Herba achieve baseline separation within 14 minutes,the linear relationships of four analytes were good and the correlation coefficients of the calibration curves were0.9984-0.9995 for the four analytes.Baseline separations of the four analytes were achieved within 14 min.The intraday and interday precision ranged from 1.2% to 2.4%and from 1.4% to 4.6%,respectively.The recoveries of analytes in actual samples varied from 98.0% to 102.5%.Conclusion:In this paper,a capillary electrophoresis(CE)method with a dual CDs system as the additive was established to simultaneous separate and determine the seven isoflavones(3'-methoxypuerarin,puerarin,3'-hydroxypuerarin,genistin,ononin,daidzin and daidzein)in the actual samples.The analytical method was simple and easy to operate,it provided a new method for the quality control of RP and its formulation.A simple and efficient organic solvent-modified micellar electrokinetic capillary electrophoresis of this paper was established to separate and determine the four active components in the Picriae Herba,and successfully used to determination of four active ingredients in the Picriae Herba and its preparations.The developed method of this paper provides a scientific basis and reference for the quality control of Picriae Herba and its preparations.