HPLC-CAD一测多评法同时测定黄芪中6种成分含量

建立高效液相串联电雾式检测器(HPLC-CAD)同时测定黄芪药材中黄芪皂苷Ⅰ、黄芪皂苷Ⅱ、黄芪甲苷、毛蕊异黄酮葡萄糖苷、芒柄花素和7,2’-二羟基-3’,4’-二甲氧基异黄烷的一测多评方法。采用Agilent SB-C18(150 mm×4.6 mm,3.5μm)色谱柱,0.05%甲酸水-0.05%甲酸乙腈为流动相,梯度洗脱,流速1.0 mL·min-1,柱温35℃,进样量为20μL;CAD雾化室温度50℃。建立内参物黄芪皂苷Ⅱ与其他成分的相对校正因子,并通过相对校正因子计算其含量。同时采用外标法验证所建立的一测多评法的合理性、可行性和重复性。结果表明:各色谱峰分离度较高,黄芪皂苷Ⅰ、黄芪皂苷Ⅱ、黄芪甲苷、毛蕊异黄酮葡萄糖苷、芒柄花素和7,2’-二羟基-3’,4’-二甲氧基异黄烷分别在0.1132.250、0.0120.240、0.0040.080、0.0651.300、0.0050.100和0.0070.150 mg·mL^-1内线性关系良好;20批黄芪药材中黄芪皂苷Ⅰ、黄芪皂苷Ⅱ、黄芪甲苷、毛蕊异黄酮葡萄糖苷、芒柄花素、7,2’-二羟基-3’,4’-二甲氧基异黄烷的含量分别为0.3060.922、0.0530.183、0.0150.092、0.0690.823、00.098和0.0200.107 mg·g-1;内参物黄芪皂苷Ⅱ与黄芪皂苷Ⅰ、黄芪甲苷、毛蕊异黄酮葡萄糖苷、芒柄花素和7,2’-二羟基-3’,4’-二甲氧基异黄烷的相对校正因子分别为0.561、0.835、0.299、0.796和0.799;一测多评法含量测定结果与外标法测定结果无显著性差异。建立的HPLC-CAD一测多评法简便、准确,可用于黄芪中6种化学成分的含量测定,研究结果为黄芪药材的质量评价提供了科学依据。

Simultaneous quantitative analyses of six components in Astragalus membranaceus based on HPLC-CAD and quantitative analysis of multi-components with a single-marker

A quantitative analytical method based on HPLC coupled with the charged aerosol detector(CAD)for quantitative analysis of multi-components with a single marker(QAMS)was established for simultaneous determinations of astragalosideⅠ,astragalosideⅡ,astragalosideⅣ,calycosin-7-O-β-D-glucoside,formononetin and7,2’-dihydroxy-3’,4’-dimethoxyisoflavan in Astragalus membranaceus.The separation was performed on an Agilent SB-C18(150 mm×4.6 mm,3.5μm),with gradient elution using the mobile phase consisting of 0.05%formic acid solution and 0.05%formic acid acetonitrile at the flow rate of 1.0 mL·min-1.The column temperature was 35℃,and the injection volume was 20μL.For CAD,the drift tube temperature was at 50℃.The contents of six components in A.membranaceus were determined by both external standard method(ESM)and QAMS,and then were compared.The results showed that chromatographic peaks were separated well and the linear ranges of astragalosideⅠ,astragalosideⅡ,astragalosideⅣ,calycosin-7-glucoside,formononetin and 7,2’-dihydroxy-3’,4’-dimethoxyisoflavan were 0.113-2.250 mg·mL^-1,0.012-0.240 mg·mL^-1,0.004-0.080 mg·mL^-1,0.065-1.300 mg·mL^-1,0.005-0.100 mg·mL^-1and 0.007-0.150 mg·mL^-1,respectively.The content ranges of astragalosideⅠ,astragalosideⅡ,astragalosideⅣ,calycosin-7-glucoside,formononetin and 7,2’-dihydroxy-3’,4’-dimethoxyisoflavan were 0.306-0.922 mg·g-1,0.053-0.183 mg·g-1,0.015-0.092 mg·g-1,0.069-0.823 mg·g-1,0-0.098 mg·g-1 and 0.020-0.107 mg·g-1 in 20 batches of A.membranaceus,respectively.Using astragalosideⅡas an internal reference,the relative correlation factors of astragalosideⅠ,astragalosideⅣ,calycosin-7-O-β-D-glucoside,formononetin,and 7,2’-dihydroxy-3’,4’-dimethoxyisoflavan were calculated as 0.561,0.835,0.299,0.796,and 0.799,respectively.The results were compared with those obtained by the external standard method to verify the feasibility,rationality and repeatability of QAMS method,and there was no significant difference in assay results between the two methods.In conclusion,the QAMS method is accurate and feasible,and could be used to determine the contents such as astragalosideⅠ,astragalosideⅡ,astragalosideⅣ,calycosin-7-glucoside,formononetin and 7,2’-dihydroxy-3’,4’-dimethoxyisoflavan,and it can be used for quality control of A.membranaceus.