Related substance determination of formononetin by HPLC

Abstract

To establish a method for the related substance determination of formononetin, HPLC is performed on the column of Agilent HC-C18 （250 mm × 4.6 mm,5 μm） with the mobile phase A of acetonitrile and mobile phase B of potassium dihydrogen phosphate solution at the flow rate of 0.8 mL/min. According to gradient elute, the sample size is 10 μL, the detection wavelength is 250 nm, and the column temperature is 30 ℃. The result shows that the main drug and related substances could be well separated and detected effectively, different impurities can be detected effectively, and there is a good linear relationship between the mass concentration of formononetin and impurity A, B, C, D, E, F and G and peak area in the range of 0.060~4.004 μg/mL(r=0.999 8), 0.056~3.756 μg/mL(r=0.999 3), 0.039~3.902 μg/mL(r=0.999 6), 0.060~4.026 μg/mL(r=0.999 5), 0.058~3.878 μg/mL(r=0.999 3), 0.058~3.869 μg/mL(r=0.999 5), 0.060~3.977 μg/mL(r=0.999 5) and 0.040~3.952 μg/mL(r=0.999 4), respectively. The RSD of instrument precision, intermediate precision and stability test is no more than 2.0%, and the average recovery of impurities is in the range of 98.49%~101.76% with the RSD in the range of 0.37%~1.37%(n=9). The method is simple, reliable and accurate, and can be used to detect related substance in sodium formononetin-3'-sulfonate.

Keywords

• chromatography
• formononetin
• HPLC
• gradient elute
• related substances