HPLC法同时测定怀牛膝中5个成分的含量

目的：建立高效液相色谱法同时测定怀牛膝中β-蜕皮甾酮、25R-牛膝甾酮、25S-牛膝甾酮、人参皂苷Ro和竹节参皂苷Ⅳa的含量。方法：采用ZORBAX SB-C₁₈色谱柱（4.6 mm×250 mm，5 μm），以乙腈（A）-0.5%磷酸溶液（B）为流动相，梯度洗脱，流速1.0 mL·min^-1，检测波长250 nm（β-蜕皮甾酮、25R-牛膝甾酮和25S-牛膝甾酮）和203 nm（人参皂苷Ro和竹节参皂苷Ⅳa）。结果：5个分析物分离良好，β-蜕皮甾酮、25R-牛膝甾酮、25S-牛膝甾酮、人参皂苷Ro和竹节参皂苷Ⅳa进样量分别在0.218~2.725 μg（r=0.999 8）、0.080~0.995 μg（r=0.999 9）、0.155~1.935 μg（r=0.999 9）、0.352~4.400 μg（r=0.999 8）和0.338~4.225 μg（r=0.999 8）范围内呈良好的线性关系，加样回收率分别为98.3%、101.5%、98.4%、99.3%和101.4%，相应的RSD分别为0.89%、0.93%、1.4%、0.69%和1.3%；16批不同产地的怀牛膝中β-蜕皮甾酮、25R-牛膝甾酮、25S-牛膝甾酮、人参皂苷Ro和竹节参皂苷Ⅳa的含量范围分别为0.032%~0.074%、0.008%~0.017%、0.008%~0.017%、0.044%~0.107%和0.047%~0.103%。结论：本方法可作为评价怀牛膝质量的方法。

Objective: To establish an HPLC method for simultaneous determination of β-ecdysterone,25R-inokosterone,25S-inokosterone,ginsenoside Ro and chikusetsusaponin Ⅳa in Achyranthis Bidentatae Radix. Methods: The chromatographic separation was performed on a ZORBAX SB-C₁₈ column(4.6 mm×250 mm, 5 μm)with the mobile phase consisting of acetonitrile(A)-0.5% phosphoric acid(B)in gradient elution mode at a flow rate of 1.0 mL·min^-1.The detection wavelength was set at 250 nm for β-ecdysterone,25R-inokosterone and 25S-inokosterone,and 203 nm for ginsenoside Ro and chikusetsusaponin Ⅳa.Results: The analytes were separated well.The linear ranges of β-ecdysterone,25R-inokosterone,25S-inokosterone,ginsenoside Ro and chikusetsusaponin Ⅳa were 0.218-2.725 μg(r=0.999 8),0.080-0.995 μg(r=0.999 9),0.155-1.935 μg (r=0.999 9),0.352-4.400 μg(r=0.999 8)and 0.338-4.225 μg(r=0.999 8),respectively.The average recovery rates were 98.3%,101.5%,98.4%,99.3% and 101.4%,with RSD of 0.89%,0.93%,1.4%,0.69% and 1.3%,respectively.In Achyranthis Bidentatae Radix from different habitats,the contents of β-ecdysterone,25R-inokosterone,25S-inokosterone,ginsenoside Ro and chikusetsusaponin Ⅳa were in ranges of 0.032%-0.074%,0.008%-0.017%,0.008%-0.017%,0.044%-0.107% and 0.047%-0.103%,respectively.Conclusion: The method can be used for quality evaluation of Achyranthis Bidentatae Radix.