超高效液相色谱双内参法同时测定白茅根配方颗粒中六种有效成分

Determination of six effective ingredients in Imperatae Rhizoma concentrated granules by ultra-performance liquid chromatography using two internal references

建立超高效液相色谱方法并采用基于一测多评的双内参法同时测定白茅根配方颗粒中有效成分对香豆酸、绿原酸、5-咖啡酰奎宁酸、4-咖啡酰奎宁酸、咖啡酸、阿魏酸的含量。采用ZORBAX RRHD Eclipse Plus C18(2.1 mm×100 mm,1.8μm)色谱柱,流动相为1.7 mmol·L^(-1)草酸水-甲醇溶液梯度洗脱,流速0.4 mL·min^(-1),柱温35℃,进样量1μL。分别以对香豆酸为内参物,计算咖啡酸和阿魏酸的相对校正因子;以绿原酸为内参物,计算4-咖啡酰奎宁酸和5-咖啡酰奎宁酸的相对校正因子,考察其稳定性并利用相对保留时间法确定各化合物的色谱峰出峰时间。结果显示,5-咖啡酰奎宁酸、4-咖啡酰奎宁酸、咖啡酸和阿魏酸的相对校正因子分别为1.069、1.022、1.368和1.493,在不同条件下稳定性良好。使用外标法和双内参法对18批不同厂家的白茅根配方颗粒进行含量测定,两种方法所得的结果相对误差小于5.0%,表明建立的双内参法同时测定白茅根配方颗粒中6种有效成分可行,为白茅根配方颗粒质量标准建立提供依据。

To simultaneously determine the contents of p-coumaric acid,chlorogenic acid,5-caffeoylquinic acid,4-caffeoylquinic acid,caffeic acid and ferulic acid in Imperatae Rhizoma concentrated granules,an ultra-high performance liquid chromatography(UPLC)with two internal references method(TIRM)was established and validated.Chromatographic separation was achieved on a ZORBAX RRHD Eclipse Plus C18 column(2.1 mm×100 mm,1.8μm)using 1.7 mmol·L^(-1) oxalic acid in water and methanol as mobile phase.The flow rate was 0.4 mL·min^(-1) and the column temperature was set as 35℃.The relative correction factors(RCFs)of caffeic acid and ferulic acid using p-coumaric acid as internal reference were calculated and the RCFs of 4-caffeoylquinic acid and 5-caffeoylquinic acid were calculated using chlorogenic acid as the internal reference.The TIRM was fully validated for linearity,accuracy,repeatability,stability and recovery so that it could be compared with the external standard method(ESM).The RCFs of 5-caffeoylquinic acid,4-caffeoylquinic acid,caffeic acid,and ferulic acid were 1.069,1.022,1.368,and 1.493,respectively.The TIRM and ESM were used to determine the contents of six ingredients in Imperatae Rhizoma concentrated granules from different manufacturers and the variation between results was within acceptable limits.In conclusion,the newly established TIRM allowed simultaneous determination of six ingredients(p-coumaric acid,chlorogenic acid,5-caffeoylquinic acid,4-caffeoylquinic acid,caffeic acid,ferulic acid)in Imperatae Rhizoma concentrated granules,providing support for the quality control of this traditional Chinese medicine.