HPLC-DAD-ELSD法同时测定牛膝中β-蜕皮甾酮、人参皂苷R0、竹节参皂苷Ⅳa的含量

Simultaneous Determination of β-ecdysterone, Ginsenoside R0 and Chikusetsusaponin Ⅳa in Radix Achyranthes Bidentatae by HPLC-DAD-ELSD

目的:采用高效液相色谱-二极管阵列检测器-蒸发光散射检测器(HPLC-DAD-ELSD)法同时测定牛膝中3种化合物β-蜕皮甾酮、人参皂苷R0、竹节参皂苷Ⅳa的含量。方法:采用Agilent 5 TC-C18(4.6 mm×250 mm,5μm)色谱柱,流动相为乙腈-0.08%甲酸(A),水-2.5%异丙醇-0.08%甲酸(B)不同比例进行梯度洗脱;流速为0.9 mL/min,运行时间80 min,β-蜕皮甾酮采用DAD进行检测,检测波长为248 nm;人参皂苷R0和竹节参皂苷Ⅳa采用ELSD进行检测,漂移管温度为110℃,载气流速3.2 L/min。结果:β-蜕皮甾酮在0.001 9~0.096 3 mg/mL浓度范围内浓度与峰面积间呈良好的线性关系(r^2=0.999 9);人参皂苷R0、竹节参皂苷Ⅳa分别在0.002 2~0.469 5 mg/mL和0.004 7~0.483 0 mg/mL浓度范围内浓度的对数与峰面积的对数间呈良好的线性关系(r^2=0.999 5,r^2=0.999 4)。牛膝中三种化合物(β-蜕皮甾酮、人参皂苷R0、竹节参皂苷Ⅳa)的平均加样回收率分别为99.29%(RSD为2.14%)、99.13%(RSD为2.04%)和98.30%(RSD为2.11%)。结论:本研究所建立的方法简便,快速,且稳定性、精密度、重复性良好,适用于牛膝中甾酮类和皂苷类成分的同时测定,也可用于牛膝药材的质量控制。

Objective: A method of high performance liquid chromatography-diode array detector- evaporative light scattering detection(HPLC-DAD-ELSD) was developed for simultaneous determination of β-ecdysterone(1), ginsenoside R 0(2) and chikusetsusaponin Ⅳa(3) in Radix Achyranthes Bidentatae . Methods: Optimum separations were obtained with a Agilent 5 TC-C 18 column(4.6 mm×250 mm, 5 μm), using a gradient elution with 0.08% aqueous formic acid(containing 2.5% isopropyl alcohol) and acetonitrile(containing 0.08% formic acid) as mobile phase.β-ecdysterone was detected by DAD at 248 nm, whereas ginsenoside R 0 and chikusetsusaponin Ⅳa were monitored by ELSD connected in series with DAD, temperature for the drift tube was 110 ℃ and the nitrogen flow rate was 3.2 L/min. Results: The calibration curve of β-ecdysterone showed good linear regression ( r^2=0.999 9). The regression equation showed a good liner relationship between logarithm of the peak area and logarithm of the concentration of ginsenoside R 0 and chikusetsusaponin Ⅳa ( r^2=0.999 5, r^2=0.999 4). The average recoveries of the three compounds were 99.29%(RSD: 2.14%), 99.13%(RSD: 2.04%) and 98.30%(RSD: 2.11%) respectively. Conclusion: The method is simple, fast, acceptable stability, precision and repeatability. The results demonstrated that the method is suitable for the simultaneous determination of ketosteroid and saponins in Radix Achyranthes Bidentatae , and the method is also can be used as the quality control of Radix Achyranthes Bidentatae .