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

ABSTRACT

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 astragaloside Ⅰ, astragaloside Ⅱ, astragaloside Ⅳ, calycosin-7-O-β-D-glucoside, formononetin and 7,2'-dihydroxy-3',4'-dimethoxyisoflavan in Astragalus membranaceus. The separation was performed on an Agilent SB-C18 (150 mm×4.6 mm, 3.5 μm), 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 20 μL. 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 astragaloside Ⅰ, astragaloside Ⅱ, astragaloside Ⅳ, 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-1 and 0.007-0.150 mg·mL-1, respectively. The content ranges of astragaloside Ⅰ, astragaloside Ⅱ, astragaloside Ⅳ, 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 astragaloside Ⅱ as an internal reference, the relative correlation factors of astragaloside Ⅰ, astragaloside Ⅳ, 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 astragaloside Ⅰ, astragaloside Ⅱ, astragaloside Ⅳ, calycosin-7-glucoside, formononetin and 7,2'-dihydroxy-3',4'-dimethoxyisoflavan, and it can be used for quality control of A. membranaceus.