Determination of Soybean Isoflavone by HPLC
1) Reagents a. Methanol (HPLC) b. Nanopure water 2) Apparatus 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 Gradient: 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 Injection: 10μl 4) Procedure Samples Preparation 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.
5) Calculation 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.
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