Objective: To establish an HPLC gradient elution method for simultaneous determination of stibene glucoside

physcione

calycosin-7-O-β-D-glucoside

ononin

calycosin and fermlononetin in Ejiao Yishou Jing. Method: An Agilent TC-C18 column (4.6 mm×250 mm

5 μm)

with acetonitrile-methanol(9:1)(A) and 0.1% phosphate acid solution (B) as the mobile phases for gradient elution. The column temperature was 30℃

and the flow rate was 1.0 mL·min-1

with an injection volume of 20 μL. Stibene glucoside and physcione were detected at 280 nm; calycosin-7-O-β-D-glucoside

ononin

calycosin and fermlononetin were detected at 254 nm. Result: Stibene glucoside

physcione

calycosin-7-O-β-D-glucoside

ononin

calycosin and fermlononetin showed good linearity within the concentration range of 5.23-104.60 mg·L-1 (r=0.999 5)

5.05-101.00 mg·L-1 (r=0.999 1)

5.92-118.40 mg·L-1 (r=0.999 3)

4.74-94.80 mg·L-1 (r=0.999 8)

4.85-97.00 mg·L-1 (r=0.999 7)

and 6.91-138.20 mg·L-1 (r=0.999 7) respectively. Their average recovery rate (n=6) and RSD were 98.52%(1.6%)

97.95%(1.4%)

99.47%(1.6%)

96.99%(1.0%)

99.24%(1.1%)

and 98.13%(1.4%) respectively. Conclusion: This HPLC gradient elution method could be used to simultaneously determine the contents of stibene glucoside

physcione

calycosin-7-O-β-D-glucoside

ononin

calycosin and fermlononetin in Ejiao Yishou Jing. This method is accurate

highly sensitive and repeatable

providing a reliable method for the quality control of Ejiao Yishou Jing.