AOAC Official Method Determination of Isoflavones in Soy and Selected Foods Containing Soy

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1 AOAC Officil Method Determintion of Isoflvones in Soy nd Selected Foods Contining Soy Extrction, Sponifiction, nd Liquid Chromtogrphy First Action 2001 (Applicble to the determintion of totl isoflvone content t 50 µg/g, individul isoflvone glucoside nd glycon content t 20 µg, nd isoflvone fmily subtotls t 20 µg/g in soy nd foods contining soy.) See Tbles A I for the results of the interlbortory study supporting cceptnce of the method. A. Principle Test smples re extrcted t 65 C for 2hinmethnol wter ( ), nd the extrcts re sponified t mbient temperture with NOH solution. The extrcts re cidified, filtered, nd diluted with wter to methnol wter ( ). The extrcts re then centrifuged to clrify them nd nlyzed by liquid chromtogrphy (LC). Isoflvone glucosides nd glycons re seprted on C18 reversed-phse column with methnol wter mobile phse nd determined by UV detection t 260 nm. Results re expressed in glycon units by summing the concentrtions of the glycon isoflvones (genistein, glycitein, nd didzein) nd the glycon equivlents of the corresponding glucoside forms (genistin, glycitin, nd didzin). B. Apprtus () LC system. With utomtic smpler nd 100 µl loop, binry grdient pumping system, UV detector t 260 nm, nd dt cquisition system. (b) Chromtogrphy column. C18 reversed-phse, mm id, or C18 reversed-phse, mm id. (c) Blnce. Anlyticl, weighing to g. (d) Dispenser. Dispensing 50 ± 0.5 ml methnol wter ( ). (e) Pipets. Dispensing 1 5 ml; with disposble tips. (f) Wter bth. Mintining 65 C, with shker. (g) Orbitl pltform shker. Holding 250 ml Erlenmeyer flsks. (h) Filter pper. 15 cm, quntittive grde, medium porosity, fn-folded. (i) Centrifuge. Centrifuging 1 ml fluid t 7000 g. (j) Microfuge tube. 1.5 ml, disposble. (k) Vils. Glss, for LC utosmpler, with Teflon-lined sept. C. Regents () Isoflvone stndrds. See Tble J. (b) Stock stndrd solutions. Using nlyticl blnce cpble of weighing to g, weigh 5 mg didzin, 5 mg genistin, 20 mg didzein, 20 mg genistein, nd 5 mg glycitein into 5 seprte 50 ml low-ctinic volumetric flsks. Quntittively trnsfer contents of 2 mg vil of glycitin into 50 ml low-ctinic volumetric flsk, rinsing vil repetedly with methnol nd dding rinsings to volumetric flsk. Dissolve contents of ech flsk in methnol nd dilute to volume. Stopper ech flsk nd mix well by repeted inversion. Store t room temperture in low-ctinic glss flsks for 6 months. (c) Working stndrd solutions. Prepre 5 levels of working stndrds by diluting the volume of ech stock stndrd shown in Tble K in corresponding volumetric flsk indicted. Add volume of wter shown in Tble K, nd dilute to volume with methnol wter (1 + 1). The pproximte concentrtion of ech isoflvone is shown in Tble L. For stndrds of <99+% purity, djust vlues for purity of stndrd ccordingly. Store solutions t room temperture in low-ctinic glss flsks for 6 months. (d) Methnol. LC grde. (e) Hexne. LC grde. (f) Acetic cid, glcil. (g) Extrction solution. Methnol wter ( ). Add 800 ml methnol to 1 L volumetric flsk. Add 200 ml wter (do not dilute to volume), stopper, nd mix well by inversion. (h) Methnol wter ( ). Combine 250 ml methnol with 250 ml wter, mix well, nd filter, using vcuum, through 0.45 µm filter. (i) Mobile phse A. Wter methnol cetic cid ( ). Combine 3520 ml wter, 400 ml methnol, nd 80 ml glcil cetic cid. Mix well nd filter, using vcuum, through 0.45 µm filter. (j) Mobile phse B. Methnol cetic cid (98 + 2). Add 3920 ml methnol to 6 L Erlenmeyer flsk. Add 80 ml glcil cetic cid, nd mix well. Filter through 0.45 µm filter disk with vcuum. (k) Sodium hydroxide solution. 2M. Weigh 80 g NOH into 1 L volumetric flsk, dissolve in wter, let cool to mbient temperture, nd dilute to volume with wter. D. Extrction nd Sponifiction Accurtely weigh mount of test smple tht contins c 1 g protein, but not >5 g test smple, into 250 ml Erlenmeyer flsk with ground-glss stopper. Add 40 ml extrction solution, nd stopper flsk. Cover stopper nd neck of flsk with luminum foil, nd shke flsk in 65 C wter bth for 2 h. Tble A. Interlbortory results for didzin in soy nd soy-contining foods Mtrix No. of lbs Men, µg/g RSD r, % RSD R, % HORRAT Soy isolte 10(2) Soy beverge 10(2) Soy flour 10(2) Vegetble burger 11(1) Soy molsses 12(0) Miso 10(2) No. of lbortories retined fter elimintion of outliers (in prentheses).

2 Tble B. Interlbortory results for glycitin in soy nd soy-contining foods Mtrix No. of lbs Men, µg/g RSD r, % RSD R, % HORRAT Soy isolte 12(0) Soy beverge 10(2) Soy flour 10(2) Vegetble burger 11(1) Soy molsses 11(1) Miso 10(2) No. of lbortories retined fter elimintion of outliers (in prentheses). Tble C. Interlbortory results for genistin in soy nd soy-contining foods Mtrix No. of lbs Men, µg/g RSD r, % RSD R, % HORRAT Soy isolte 12(0) Soy beverge 10(2) Soy flour 10(2) Vegetble burger 12(0) Soy molsses 11(1) Miso 11(1) No. of lbortories retined fter elimintion of outliers (in prentheses). Tble D. Interlbortory results for didzein in soy nd soy-contining foods Mtrix No. of lbs Men, µg/g RSD r, % RSD R, % HORRAT Soy isolte 10(2) Soy beverge 10(2) Soy flour 10(2) Vegetble burger 12(0) Soy molsses 10(2) Miso 10(2) No. of lbortories retined fter elimintion of outliers (in prentheses). Tble E. Interlbortory results for genistein in soy nd soy-contining foods Mtrix No. of lbs Men, µg/g RSD r, % RSD R, % HORRAT Soy isolte 12(0) Soy beverge 10(2) Soy flour 10(2) Vegetble burger 12(0) Soy molsses 11(1) Miso 11(1) No. of lbortories retined fter elimintion of outliers (in prentheses).

3 Tble F. Interlbortory results for didzin-didzein in soy nd soy-contining foods Mtrix No. of lbs Men, µg/g RSD r, % RSD R, % HORRAT Soy isolte 11(1) Soy beverge 10(2) Soy flour 10(2) Vegetble burger 11(1) Soy molsses 11(1) Miso 11(1) No. of lbortories retined fter elimintion of outliers (in prentheses). Tble G. Interlbortory results for glycitin-glycitein in soy nd soy-contining foods Mtrix No. of lbs Men, µg/g RSD r, % RSD R, % HORRAT Soy isolte 10(2) Soy beverge 11(1) Soy flour 10(2) Vegetble burger 11(1) Soy molsses 11(1) Miso 11(1) No. of lbortories retined fter elimintion of outliers (in prentheses). Tble H. Interlbortory results for genistin-genistein subtotl in soy nd soy-contining foods Mtrix No. of lbs Men, µg/g RSD r, % RSD R, % HORRAT Soy isolte 12(0) Soy beverge 10(2) Soy flour 10(2) Vegetble burger 12(0) Soy molsses 11(1) Miso 11(1) No. of lbortories retined fter elimintion of outliers (in prentheses). Tble I. Interlbortory results for totl isoflvones in soy nd soy-contining foods Mtrix No. of lbs Men, µg/g RSD r, % RSD R, % HORRAT Soy isolte 12(0) Soy beverge 10(2) Soy flour 10(2) Vegetble burger 12(0) Soy molsses 11(1) Miso 10(2) No. of lbortories retined fter elimintion of outliers (in prentheses).

4 Tble J. Isoflvone stndrds Stndrd Formul CAS Registry No. Indofine Ct. No. Didzin C 21 H 20 O Didzein C 15 H 10 O D-O101 Genistin C 21 H 20 O Genistein C 15 H 10 O G-103 Glycitin C 22 H 22 O GL-002 Glycitein C 16 H 12 O GL-001 Ct. Nos. from Indofine Chemicl Co., PO Box 473, Somerville, NJ 08876, USA; ; Fx Equivlent stndrds from other suppliers my be used. Tble K. Preprtion of working stndrds from dilutions of stock stndrd solutions Working stndrd Ech stock stndrd, ml Wter, ml Finl volume, ml Tble L. Approximte concentrtions of individul isoflvones in working stndrds Working stndrd Didzin, µg/ml Glycitin, µg/ml Genistin, µg/ml Didzein, µg/ml Glycitein, µg/ml Genistein, µg/ml Cool to room temperture, nd dd 3 ml 2M NOH. Replce luminum foil, nd shke flsk t room temperture on orbitl shker for 10 min. Remove flsk from shker, nd dd 1 ml glcil cetic cid. Swirl to suspend contents of flsk, nd pour into 50 ml grduted cylinder with ground glss stopper. Dilute to 50 ml with extrction solution nd mix well. Filter solution through quntittive-grde filter pper into 250 ml beker. Pipet 5 ml filtrte into 10 ml grduted cylinder with ground-glss stopper. Add 4.0 ml wter, nd dilute to 10 ml with methnol. Stopper grduted cylinder, nd invert cylinder repetedly to mix contents. Trnsfer c 1 ml extrct to 1.5 ml centrifuge tube, nd centrifuge for 5 min t 7000 g. Trnsfer cler superntnt to LC smple vil. Note: Do not filter superntnt through membrne filter. E. Determintion Set LC system to flow rte of 0.4 ml/min for 2.1 mm id column nd initil mobile phse composition shown in Tble M. For 4.6 mm id column, set flow rte to 1.5 ml/min, nd use sme grdient. Set detector wvelength to 260 nm. Let system equilibrte by running 1 complete grdient with no injection. Verify system performnce by injecting 20 µl working stndrd 3, using grdient conditions in Tble M. Verify bseline seprtion of didzein nd glycitein peks. The tiling fctor for ny pek should be 1.5. Adjust either %B or grdient times s needed to obtin required seprtion of ll 6 components. Typicl reltive retention times (in min) re s follows: didzin, 0.53; glycitin, 0.58; genistin, 0.66; didzein, 0.89; glycitein, 0.92; nd genistein, Retention times will vry with the ge nd condition of the column. Inject ll working stndrds nd ech test extrct. Determine re of ech isoflvone pek. F. Clcultion Determine response for ech isoflvone by clculting slope (m) nd intercept (b), using liner regression nlysis of re counts vs response for 5 levels of ech of the isoflvone stndrds. Clculte concentrtion of ech isoflvone in test smple, using following eqution: Isoflvone, µg/g = (( As m) b) Ws 5 where As = pek re of isoflvone in test solution; m = slope from liner regression for stndrd response; b = intercept from liner regression for stndrd response; Ws = weight of test portion, g; 50 = dilution volume in D; 10 = second dilution volume in D; 5 = liquot in D. Convert concentrtions of isoflvone glucosides genistin, glycitin, nd didzin to glycon equivlents, using following eqution: Tble M. LC pump grdient for ech run Mobile phse composition t end time Step Strt time, min End time, min %A %B Initil Stop run All grdients re liner.

5 Tble N. Aglycon conversion fctors Isoflvone glucoside MW MWg Genistin Glycitin Didzin of glucoside (Tble N); nd Cg = concentrtion of genistin, glycitin, or didzin, µg/g. Clculte totl isoflvones, µg/g glycon equivlents/g, by summing concentrtions of didzein, glycitein, nd genistein nd dding this totl to sum of glycon equivlent concentrtions of didzin, glycitin, nd genistin. T = C(didzein) + C(glycitein) + C(genistein) Ce = MW MWg Cg Te = Ce(didzin) + Ce(glycitin) + Ce(genistin) where T = sum of concentrtions of glycons, nd Te = sum of glycon equivlent concentrtions of glucosides. where Ce = isoflvone glycon equivlents, µg/g; MW = moleculr weight of glycon (Tble N); MWg = moleculr weight Totl isoflvones, µg glycon equivlents/g = T + Te Reference: J. AOAC Int. 84, 1865(2001).