1 STANDARD PERATING PRTCL (SP) Subject: To determine the Contents of isoflavone in soybean products by HPLC Analysis Project/Core No.: Core B Total 9 Pages SP No.: CB0102 Modified Date: 05/15/01 BTANICAL CENTER FR AGE-RELATED DISEASES 1. Purpose: To determine the contents of isoflavones, Daidzein, Daidzin, Genistein, Genistin, Glycitein and Glycitin in soybean products using HPLC analysis. 2. Materials: The following materials are needed to carry out the analysis. 2.1 Standards: Daidzein, Sigma Chemical Co. Daidzin, Sigma Chemical Co. Genistein, Sigma Chemical Co. Genistin, Sigma Chemical Co. Glycitein, Sigma Chemical Co. Glycitin, Sigma Chemical Co. 2.2 Apparatus: HPLC instrument: Hewlett-Packard HP 1100 Series HPLC equipped with autosampler, DAD detector, and HP ChemStation Software or equivalent. HPLC column: waters symmetry C18, 5um 3.90*150 mm or Phenomenex Prodigy DS (3), 5 µ, 150 x 3.2 mm. Calibrated analytical balance accurate to ±0.01 mg
2 Sonicator, with temperature control Volumetric flasks, appropriate sizes Syringes, 3-cc disposable with Luer-lok tip Filters, 0.45-µm, glass PVDF or Cellulose Acetate Pipettes, class A, assorted sizes 2.3 Reagents: Water, deionized, HPLC grade Phosphoric acid, 86%, reagent grade Acetonitrile, HPLC grade 3. HPLC conditions: The following HPLC condition should be used when carrying out this analysis: 3.1 Project and location: Isoflavone HPLC program (soy01.m) for separating and checking and quantifying isoflavones is in the C:\HPCHEM\1\Methods\ directory of HP ChemStation software system. Sequence: soy01.s. 3.2 HPLC column: Water Symmetry RP C18, 5um, 3.90*150 mm or Phenomenex Prodigy DS (3), 5 µ, 150 x 3.2 mm. 3.3 Column temperature: 25 C. 3.4 Mobile phase: The mobile phase was consisted of solvent A (0.1% phosphoric acid solution) and solvent C (acetonitrile) with the following gradient solvent system (see table): Retention time (min.) %A %C 0.0 90 10 40 65 35 3.5. Retention times (see table) 3.6 Flow rate: 0.8 ml/min. 3.7 Injection volume: 10 µl. 3.8 Detection wavelength: 255 nm. 3.9 Running time: 40 minutes. 3.10 Post running time 15 minutes.
3 Analyte Waters Symmetry C18 Phenomenex Prodigy DS Daidzin 9.5 10.1 Glycitin 10.4 11.1 Genistin 14.7 15.4 Daidzein 23.5 24.1 Glycitein 24.7 25.4 Genisein 31.7 32.6 3.11 DAD detector option: Diode-array Detector (DAD) data were acquired on a mode 1100 series DAD detector over the wavelength range 200-400 nm at a rate of 1 pectrum/second and 1.2 nm resolution. 4. Preparation of Standard Weigh a known amount (8 mg) of Genistein standard into 25 ml volumetric flask. Add about 15 ml 70% MeH, shake and fill to full volume with 70% MeH. After using the standard solution, place new cap tightly and return the vial to refrigerator for next use. 5. Preparation of Samples 5.1 Weigh the appropriate amount of materials into 50 ml and record the weight. Sample Description Amount of sample 2% isoflavones 600 mg 20% isoflavones 100 mg 40% isoflavones 40 mg 5.2 Add appropriately 35 ml of 70% methanol and sonicate for 10 min and shake for 20 min. Allow the flask to cool to room temperature and fill to volume with 70% methanol. 5.3 Using a disposable syringe and 0.45 µm filter, filter the sample into an HPLC vial and cap the vial. 6. Calculation of Contents
4 Quantify each soy isoflavone against the Genistein standard using the following equation and factors. % w/w analyte = C x FV x d x F x 100% W Where: C = The sample's gallic acid concentration (mg/ml) from linear regression analysis FV = The final volume of the sample preparation, ml d = Dilution of the sample, if needed w = The weight of the sample in mg F = The correction factor for the individual isoflavone response vs. Genistein F=1.000 for Genistein F=1.458 for Genistin F=1.203 for Daidzein F=1.931 for Daidzin F=1.630 for Glycitein F=2.118 for Glycitin
5 APPENDIX I: Structures of the Standard Compounds: H Glu H H Daidzein (C 15 H 10 4, MW: 254) Daidzin (C 21 H 20 9, MW: 416) H Glu H H H H Genistein (C 15 H 10 5, MW: 270) Genistin (C 21 H 20 10, MW: 432) H Glu Me Me H H Glycitein (C 16 H 12 5, MW: 284) Glycitin (C 22 H 22 10, MW: 446
6 APPENDIX II: UV and MS Chromatographies, and MS spectra for each isoflavone standard in soy products: Fig. 1 UV Chromatography of Soy Products