| Determination of Soybean Isoflavone by HPLC
a. Methanol (HPLC) b. Nanopure water
HPLC (SHIMADZU) Analytical balance (1/10000) Ultrasonic cleaner
0.45μm PTFE filter 50ml volumetric flask Screw cap vials
3) HPLC condition
Column: Shim-pack VP-ODS C18 5 m 4.6150mm
Detector: UV 260nm
Mobile Phase: A=water B= MeOH
Time（min）0.01 17 40 44 46 48 50
%A 69 69 43 43 69 69 stop
%B 31 31 57 57 31 31
Flow Rate: 1.1ml/min
Accurately weigh 25mg extraction into 50ml volumetric flask. Add MeOH to volume and sonicate to mix, filter through a 0.45um PTFE filter into a screw cap vial and proceed with the analysis.
Standard preparation /calibration
Prepare a mixed standard solution (approximately 0.5mg/ml in MeOH) using a commercial reference standard of Soybean Isoflavone，Sonicate for about 10 minutes and transfer to screw-cap vials and can be stored in the refrigeration.
Determine the peak areas of the Soybean Isoflavone on the sample chromatogram and compare to the corresponding peaks in the chromatogram for the reference standard solution. Calculate the weight percent.