HPLC-MS法同时测定六神丸中9种蟾蜍二烯内酯类化合物

Simultaneous determination of nine bufadienolides in Liushen Pill by HPLC-MS

目的建立HPLC-MS法同时测定六神丸中9种蟾蜍二烯内酯类化合物(和蟾蜍他灵、沙蟾毒精、远华蟾毒精、去乙酰华蟾毒它灵、蟾毒它灵、华蟾毒它灵、蟾毒灵、华蟾酥毒基和脂蟾毒配基)的方法。方法采用ODS-2(250 mm×4.6mm,5μm)色谱柱,以乙腈-0.15%磷酸水溶液为流动相,梯度洗脱,体积流量1 mL/min,柱温40℃,检测波长296 nm。HPLC-MS联用的色谱条件与HPLC分离基本条件一致,流动相为乙腈-0.2%甲酸水溶液。通过电喷雾离子源,正离子模式扫描,对六神丸中9种蟾蜍二烯内酯类化合物进行分析鉴别。结果 9种蟾蜍二烯内酯类化合物均得到良好分离,线性关系良好(r≥0.999 6)。加样回收率均在92%~105%,RSD<5%。应用建立的HPLC-MS方法测定了10批六神丸中9种蟾蜍二烯内酯类化合物的量,分别为和蟾蜍他灵0.40~0.88 mg/g,沙蟾毒精0.32~0.83 mg/g,远华蟾毒精0.61~2.11 mg/g,去乙酰华蟾毒它灵0.15~0.32 mg/g,蟾毒它灵0.84~1.85 mg/g,华蟾毒它灵25.76~62.14 mg/g,蟾毒灵0.96~2.06 mg/g,华蟾酥毒基2.30~4.75 mg/g,脂蟾毒配基1.20~2.61 mg/g。结论所建立的定量测定方法简便、快速、准确可靠,专属性强,可用于六神丸的质量控制。

Objective To establish an HPLC-MS method for determining nine bufadienolides （gamabufotalin, arenobufagin, telocinobufagin, desacetylcinobufotalin, bufotalin, cinobufotalin, bufalin, cinobufagin, and resibufogenin） in Bufonis Venenum and Liushen Pill. Methods HPLC analysis was performed on ODS-2 column （250 mm×4.6 mm, 5 μm） with the mobile phase made up of acetonitrie and 0.15% phosphoric in water. Flow rate was set at 1 mL/min and column temperature was 40℃. The detection wavelength was 296 nm. Nine bufadienolides were determined by HPLC-ESI-TOF-MS with the mobile phase made up of acetonitrie and 0.2% formic acid in water. Eletrospray ionization source was applied and operated in the positive ion mode. Results Nine bufadienolides were separated at baseline with good linearity （r≥0.999 6）. The recovery rates were 92%-105% （RSD〈5%）. Conclusion The method established is rapaid, simple, and accurate, which is helpful to control the quality of Liushen Pill.