优化的HPLC-DAD法同时测定罗布麻中4种黄酮类成分的含量

Optimized HPLC-DAD in simultaneous determination of 4 flavonoids in Apocynum venetum

目的建立优化的HPLC方法同时测定不同产地罗布麻药材中芦丁、金丝桃苷、异槲皮苷以及槲皮素含量。方法采用Shiseido C18（3.0μm,3.0 mm×100 mm）色谱柱;以乙腈：甲醇=10：1作为A相-0.1%甲酸水溶液作为B相,梯度洗脱（0~4 min,5%→16%A;4~15 min,16%→23%A;15~20 min,23%→35%A;20~22 min,35%→55%A）,流速0.6 ml.min-1;二极管阵列检测器检测波长：360 nm;柱温：30℃;进样量：5μl。结果芦丁、金丝桃苷、异槲皮苷、槲皮素在22 min内基线分离,线性范围分别为0.308 4~30.84μg.ml-1（r=0.999 9）,0.877 6~87.76μg.ml-1（r=0.999 9）,0.902 0~90.20μg.ml-1（r=0.999 9）,0.249 8~24.98μg.ml-1（r=0.999 9）。方法学考察表明,日内和日间精密度、最低检测限和定量限的范围均符合相关标准,加样回收率（n=3）分别为：芦丁102.0%、96.40%、103.8%;金丝桃苷98.50%、101.0%、102.9%;异槲皮苷98.40%、100.4%、101.4%;槲皮素104.4%、103.1%、103.5%。测定了9个产地的罗布麻中4个黄酮类成分的含量。结论本法快速,准确、简便,具有良好的重复性,可以为罗布麻药材质量控制提供依据。

Objective To establish an optimized HPLC method for simultaneous determination of rutin,hyperoside,isoquercitrin,and quercetin in Apocynum venetum obtained from different locations.Methods The separation was performed on a Shiseido C18（3.0 μm,30 mm×100 mm） column by gradient elution with acetonitrilemethanol=101 as the mobile phase A and 0.1%formic acid as the mobile phase B（0-4 min,5%→16%A;4-15 min,16%→23%A;15-20 min,23%→35%A;20-22 min,35%→55%A）at a flow rate of 0.6 ml·min-1.The detection wave length of DAD was set at 360 nm,and the column temperature was 30℃.The injection volume was 5 μl.Results Rutin,hyperoside,isoquercitrin,and quercetin were separated at base line within 22 min with good linearity（r=0.999 9）,with the linearity range being 0.308 4～30.84 μg·ml-1（r=0.999 9）,0.877 6-87.76 μg·ml-1（r=0.999 9）,0.902 0～90.20 μg·ml-1（r=0.999 9）,and 0.249 8～24.98 μg·ml-1（r=0.999 9）,respectively.The result of intra-day and inter-day precisions,limits of detection and quantitation were all within the normal ranges.The recovery rates（n=3） were 102.0%,96.40%,and 103.8% for rutin,98.50%,101.0%,and 102.9% for hyperoside,98.40%,100.4%,and 101.4% for isoquercitrin,and 104.4%,103.1%,and 103.5% for quercetin.The contents of the above 4 flavonoids were determined in Apocynum venetum from 9 different locations.Conclusion The method developed in this study is rapid,simple,accurate,reliable,and with good repeatability;the method provides evidence for the quality control of Apocynum venetum.