款冬花及蜜款冬花HPLC特征图谱和一测多评分析方法建立和应用

Establishment and application of HPLC characteristic chromatogram and determination by QAMS for Farfarae Flos and honeyed Farfarae Flos

目的:建立款冬花及蜜款冬花的HPLC特征图谱,采用一测多评方法测定10个指标性成分(新绿原酸、绿原酸、隐绿原酸、芦丁、异槲皮苷、异绿原酸B、异绿原酸A、异绿原酸C、款冬酮及甲基丁酰-3,14-Z-去氢款冬素酯)的含量,并验证方法的准确性,为其质量控制提供参考依据。方法:采用C18色谱柱(250 mm×4.6 mm,5μm),以乙腈(A)-0.1%磷酸水溶液(B)为流动相进行梯度洗脱,建立款冬花及蜜款冬花HPLC特征图谱;采用二极管阵列检测器按不同时间段变换波长(0~15 min,330 nm;15~20 min,254 nm;20~30 min,330 nm;30~45 min,220 nm),以芦丁为参照物,建立一测多评同时测定上述10个成分的方法。结果:建立款冬花与蜜款冬花的特征图谱,确定了14个特征峰,62批样品色谱图的相似度均大于0.90。新绿原酸、绿原酸、隐绿原酸、异槲皮苷、异绿原酸A、异绿原酸B、异绿原酸C、款冬酮、甲基丁酰-3,14-Z-去氢款冬素酯相对于芦丁的校正因子分别为0.694、0.690、0.876、0.744、0.585、0.529、0.571、0.896、1.778;且在不同实验条件下重现性良好(RSD<5.0%);一测多评法的计算结果与外标法实测值之间无显著差异。结论:建立的特征图谱可以提高款冬花与蜜款冬花的质量控制水平;一测多评同时测定10个成分的方法快速准确、简便高效,专属性良好。

Objective:To establish an HPLC characteristic chromatogram and quantitative analysis of multi-components by single-marker(QAMS)method for determining ten components(neochlorogenic acid,chlorogenic acid,cryptochlorogenic acid,rutin,isoquercitrin,isochlorogenic acid B,isochlorogenic acid A,isochlorogenic acid C,tussilagone and 7β-(3-ethyl-ciscrotonoyloxy)-1α-(2-methylbutyryloxy)-3(14)-dehydro-Z-notonipetranone)in Farfarae Flos and honeyed Farfarae Flos.And to validate the accuracy of the method.Methods:The analysis was performed on a C_(18)column(250 mm×4.6 mm,5μm)with gradient elution of acetonitrile(A)-0.1%phosphoric acid(B)at a flow rate of 1.0 mL·min^(-1).Variable wavelengths were set during different time periods(0-15 min,330 nm;15-20 min,254 nm;20-30 min,330 nm;30-45 min,220 nm).Rutin was used as reference substance.The relative correction factors(RCFs)of neochlorogenic acid,chlorogenic acid,cryptochlorogenic acid,isoquercitrin,isochlorogenic acid B,isochlorogenic acid A,isochlorogenic acid C,tussilagone and 7β-(3-ethyl-ciscrotonoyloxy)-1α-(2-methylbutyryloxy)-3(14)-dehydro-Z-notonipetranone to rutin were established.The RCFs were used to calculate the corresponding ingredients and achieve QAMS.Meanwhile,the ten components in Farfarae Flos and honeyed Farfarae Flos were determined by external standard method(ESM).The difference between the QAMS and ESM was compared to verify the accuracy and feasibility of the method.Results:HPLC characteristic chromatograms of Farfarae Flos and honeyed Farfarae Flos were established and 14 characteristic peaks were marked.The similarities of chromatograms of 62 batches of samples were above 0.90.RCFs of neochlorogenic acid,chlorogenic acid,cryptochlorogenic acid,isoquercitrin,isochlorogenic acid B,isochlorogenic acid A,isochlorogenic acid C,tussilagone and 7β-(3-ethyl-ciscrotonoyloxy)-1α-(2-methylbutyryloxy)-3(14)-dehydro-Z-notonipetranone to rutin were 0.694,0.690,0.876,0.744,0.585,0.529,0.571,0.896 and 1.778,respectively,and repeatability was good under different experimental conditions(RSD<5.0%).There was no significant difference between the quantitative results of the two methods.Conclusion:The established HPLC characteristic chromatograms and QAMS method can improve the quality control of Farfarae Flos and honeyed Farfarae Flos.