反相高效液相色谱法测定防己黄芪汤中6种活性成分的含量

Determination of six active constituents in Fangji Huangqi decoction by RP-HPLC

目的建立反相高效液相色谱法测定防己黄芪汤中粉防己碱、防己诺林碱、毛蕊异黄酮葡萄糖苷、甘草苷、甘草酸和白术内酯Ⅰ的含量。方法采用Diamonsil C18(2)色谱柱(250 mm×4.6 mm,5μm);流动相为0.2 mol/L乙酸铵0.1%甲酸溶液(A)-乙腈(B);流速为1.0 m L/min;柱温为30℃;进样量为20μL。测定粉防己碱、防己诺林碱、毛蕊异黄酮葡萄糖苷、甘草苷和甘草酸时采用梯度洗脱(0~5 min,15%~20%B;5~10 min,20%~30%B;10~15 min,30%~35%B;15~20 min,35%~45%B,V/V),平衡时间为10 min,检测波长为254 nm;测定白术内酯Ⅰ时采用等度洗脱(0~8 min,80%-80%B,V/V),检测波长为220 nm。结果粉防己碱、防己诺林碱、毛蕊异黄酮葡萄糖苷、甘草苷和甘草酸分别在5~250 mg/L(R=0.999 9)、1~50 mg/L(R=0.999 9)、1~50 mg/L(R=0.999 6)、5~250 mg/L(R=0.999 6)、5~250 mg/L(R=0.999 9)浓度范围内与峰面积呈良好的线性关系,平均回收率(n=9)分别为99.3%、98.7%、97.5%、99.1%和99.2%。白术内酯Ⅰ在0.05~2.5 mg/L(R=0.999 6)浓度范围内与峰面积呈良好的线性关系,平均回收率(n=9)为97.6%。结论方法结果准确,操作简便,具有良好的重现性和稳定性,可用于防己黄芪汤的质量控制。

Objective To develop a RP-HPLC method for determination of six active constituents （tetrandrine,fangchinoline,calycosin glucoside,liquiritin,glycyrrhizic acid and atractylenolideⅠ） in Fangji Huangqi decoction. Methods Chromatographic separation was performed on Diamonsil C 18 （2） （250 mm × 4.6 mm,5 μm）.The mobile phase consisted of 0.2 mol/L ammonium acetate （containing 0.1% formic acid） （A）-acetonitrile （B）.The flow rate was 1.0 mL/min and column temperature was maintained at 30 ℃.The injection volume was 20 μL.Tetrandrine,fangchinoline,calycosin glucoside,liquiritin and glycyrrhizic acid were determined at 254 nm with gradient elution （ 0- 5 min,15%-20%B;5-10 min,20%-30%B;10-15 min,30%-35%B;15-20 min,35%-45%B, V/V ） and the equilibration time was 10 min;AtractylenolideⅠ was determined at 220 nm with isocratic elution （0-8 min,80%-80%B, V/V ）. Results The linear ranges were 5-250 mg/L（ R =0.999 9） for tetrandrine,1-50 mg/L（ R = 0.999 6） for fangchinoline,1-50 mg/L（ R =0.999 6） for calycosin glucoside,5-250 mg/L（ R =0.999 6） for liquiritin,5-250 mg/L（ R =0.999 9） for glycyrrhizic acid;The average recoveries （n=9） of tetrandrine,fangchinoline,calycosin glucoside,liquiritin and glycyrrhizic acid were 99.3%,98.7%,97.5%,99.1% and 99.2%,respectively.The linear ranges were 0.05-2.5 mg/L（ R =0.999 6） for atractylenolideⅠ.The average recovery （n=9） of atractylenolide I was 97.6%. Conclusion The developed method is simple,accurate with high repeatability and stability,which is suitable for the quality control of Fangji Huangqi decoction.