HPLC法测定蟾酥中4种蟾毒内酯

Determination of four bufogenins in Bufonis Venenum by HPLC

目的建立同时测定蟾酥药材中蟾蜍他灵、蟾毒灵、华蟾酥毒基、脂蟾毒配基的方法。方法 10批蟾酥药材采用Agilent ZORBAX SB-C18(4.6 mm×250 mm,5μm)色谱柱;乙腈-0.04%三氟乙酸为流动相梯度洗脱;体积流量为1.0 mL/min;柱温为35℃;检测波长为296 nm。结果蟾蜍他灵、蟾毒灵、华蟾酥毒基、脂蟾毒配基分别在0.05～2.61μg(R2=1),0.10～4.96μg(R2=1),0.10～5.03μg(R2=1),0.10～5.18μg(R2=1)范围内线性关系良好;平均加样回收率分别为97.62%,97.34%,97.23%,97.34%,RSD值均<2.0%。结论此方法简便、准确。10批不同来源的蟾酥样品测定结果表明,药材中蟾蜍他灵、蟾毒灵、华蟾酥毒基含有量相对稳定,脂蟾毒配基含有量则波动较大。

AIM To establish a method for simultaneously determining bufotalin,bufalin,cinobufagin and resibufogenin in Bufonis Venenum.METHODS Agilent ZORBAX SB-C18 column（250 mm×4.6 mm,5 μm） was used for ten batches of Bufonis Venenum with the mobile phase consisted of acetonitrile-0.04% trifluoroacetic acid in a gradient elution manner.The flow rate was 1 mL/min.The column temperature was 35 ℃ and the detection wavelength was set at 296 nm.RESULTS The calibration curve of bufotalin was linear in the range of 0.05~2.61 μg（R2=1）,of bufalin was in the range of 0.10~4.96 μg（R2=1）,of cinobufagin was in the range of 0.10~5.03 μg（R2=1）,and of resibufogenin was in the range of 0.10~5.18 μg（R2=1）.The average recoveries were 97.62%,97.34%,97.23%,97.34%,respectively,with RSD less than 2.0%.CONCLUSION The method is simple and accurate.The determination of bufotalin,bufalin,cinobufagin are relatively stable,but the resibufogenin is fluctuated.