High performance liquid chromatography in determination of calycosin-7-O-β-D-glucoside and formononetin in Radix astragali / 第二军医大学学报

ABSTRACT

Objective: To determine the contents of calycosin-7-O-β-D-glucoside and formononetin in Astragalus membranaceus (Fisch.) Bge. by high performance liquid chromatography (HPLC). Methods: The HPLC condition was as follows column Hypersil ODS 2 column (4.6 mm×250 mm, 5 μm); mobile phase A was ACN-MeOH (9 1,V /V), B was H2O, with gradient elution; flow speed 1.0 ml/min; detection 260 nm; temperature of column room temperature; injection volume 20 μl. Astragalus membranaceus (Fisch.) Bge. was extracted with methanol solution twice, each time 20 min. Results: The theoretical plate numbers of calycosin-7-O-β-D-glucoside and formononetin were 50 134 and 25 258, respectively. The calibration curves were linear within the range of (2.022-101.1) μg/ml for calycosin-7-O-β-D-glucoside and (38.04-1522) μg/ml for formononetin, with their regression function being Y=58 924X - 12 352, r=0.999 9 and Y=9 237X - 124 447, r=0.999 9, respectively. The intra-day and inter-day precisions (RSD) at low, middle and high injection volume were all less than 2.0%. The limits of detection were 0.202 2 mg/ml for calycosin-7-O-β-D-glucoside and 1.522 mg/ml for formononetin. The recovery rates were 98.34% (RSD=1.33%, n=3) for calycosin-7-O-β-D-glucoside and 98.84% (RSD=0.12%, n=3) for formononetin. The contents of calycosin-7-O-β-D-glucoside and formononetin in 10 different batch of Astragalus membranaceus (Fisch.) Bge. were determined. Conclusion: HPLC is a simple and reliable method for determining the contents of calycosin-7-O-β-D-glucoside and formononetin in Astragalus membranaceus (Fisch.) Bge.