High performance liquid chromatography in determination of calycosin-7-O-βD-glucoside and formononetin in Radix astragali

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 H_2O,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-βD-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.