对照品替代法用于贯叶连翘提取物及其制剂中金丝桃素含量测定的研究

目的:研究对照品替代法在高效液相色谱法含量测定中的应用,建立以大黄素为替代物测定贯叶连翘提取物及其制剂中金丝桃素含量的方法。方法: 采用HPLC-DAD和HPLC-MS,通过定性和定量手段分别求得被测成分相对于替代物的计算因子,从而实现以大黄素为替代对照品,测定样品中金丝桃素的含量。色谱条件:色谱柱,Agilent TC-C₁₈(250 mm×4.6 mm,5 μm);流动相,10 mmol·L^-1醋酸铵缓冲液(以醋酸调pH 4)-乙腈梯度洗脱系统;流速,1.0 mL·min^-1;柱温,30℃;DAD检测器,检测波长284 nm。质谱条件:色谱柱,Agilent SB-C₁₈(2.1 mm×50 mm,1.8 μm);流动相,10 mmol·L^-1醋酸铵缓冲液(以醋酸调pH 4)-乙腈梯度洗脱系统;流速,0.4 mL·min^-1;柱温,30℃;MS-ESI正离子模式检测,去溶剂温度300℃,去溶剂气(N₂)流速10 L·min^-1,雾化气压力241 Pa,毛细管电压4 kV,离子扫描范围为 m/z 100~600,选定 m/z 269.1,503.2,519.2,535.5进行选择性离子扫描(SIM)。结果:对照品替代法与常规的含量测定法测得的结果基本一致。结论:本方法经济、实用、快速、高效,可用于贯叶连翘提取物及其制剂中金丝桃素的含量测定和产品质量控制,同时可解决因对照品缺乏而给含量测定带来的困难,大大降低了分析检测的成本。

Objective:To develop a method for determination of hypericin in the extracts of Hypericum perforatum L.and its preparations with one chemical reference as substitution substance. Methods:A series of calculator correct factors were obtained by HPLC-DAD and HPLC-MS,and the content of hypericin was quantified with emodin as standard reference.HPLC-DAD condition:column,Agilent TC-C₁₈(250 mm×4.6 mm,5 μm);column temperature,30℃;mobile phase,10 mmol·L^-1 ammonium acetate buffer solution(adjusted to pH 4 with acetic acid)-acetonitrile with a linear gradient elution;flow rate,1.0 mL·min^-1;DAD detector,detected wavelength 284 nm.HPLC-MS condition:column,Agilent SB-C₁₈(2.1 mm×50 mm,1.8 μm);column temperature,30℃;mobile phase,10 mmol·L^-1 ammonium acetate buffer solution(adjusted to pH 4 with acetic acid)-acetonitrile with a linear gradient elution;flow rate,0.4 mL·min^-1;MS-detection mode,electrospray positive ion;desolvation temperature,300℃;desolvation gas flow:10 L·min^-1;nenulizer 241 Pa;capillary voltage 4 kV;full scan,100-600 amu;selected ion monitoring(SIM, m/z) 269.1,503.2,519.2,and 535.5. Results: The quantitative results with new method were almost same to the results with conventional HPLC method. Conclusions:An economic,sensitive,simple and practicable HPLC-DAD and HPLC-MS method has been established for the determination of hypericin in the Hypericum perforatum L.extracts and its preparations with one standard chemical as replacement material.