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目的建立RP-HPLC法同时测定女贞叶乙醇提取物中齐墩果酸与熊果酸的方法。方法通过系统地考察温度、pH值、甲醇-乙腈混合有机相中甲醇的量对齐墩果酸、熊果酸保留时间和分离度的影响,最终确定HPLC色谱条件为大连依利特KromasilC18(250mm×4.6mm,5μm)色谱柱;甲醇-乙腈-10mmol/L乙酸铵(55:27:18)为流动相,体积流量为1mL/min,检测波长为205nm,柱温20℃。结果齐墩果酸与熊果酸分别在0.300～3.00μg、0.692～6.92μg线性关系良好;加样平均回收率分别为101.4%、100.2%(n=9)。结论低温、中性pH、甲醇-乙腈混合有机相有利于齐墩果酸与熊果酸的分离。优化后的色谱方法分离度高,重现性好,可用于女贞叶乙醇提取物中齐墩果酸与熊果酸的测定。
OBJECTIVE To establish a method for the simultaneous determination of oleanolic acid and ursolic acid in ethanol extract of privet leaf by RP-HPLC. Methods The effects of temperature, pH value, amount of methanol in methanol-acetonitrile mixed organic phase on oleanolic acid and ursolic acid retention time and resolution were investigated systematically. The HPLC chromatographic conditions were determined to be Dalian Erolte Kromasil C18 (250 mm × 4.6 mm, 5μm). The mobile phase was methanol-acetonitrile-10mmol / L ammonium acetate (55:27:18), the volume flow rate was 1mL / min, the detection wavelength was 205nm and the column temperature was 20 ℃. Results The oleanolic acid and ursolic acid showed good linearity between 0.300 ~ 3.00μg and 0.692 ~ 6.92μg, respectively. The average recoveries were 101.4% and 100.2% (n = 9), respectively. Conclusion Low temperature, neutral pH and methanol-acetonitrile mixed organic phase are favorable for the separation of oleanolic acid and ursolic acid. The optimized chromatographic method has the advantages of high resolution and good reproducibility, and can be used for the determination of oleanolic acid and ursolic acid in the ethanol extract of privet leaf.