Isoflavones are a class of chemical compounds found naturally

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1 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, FOOD COMPOSITION AND ADDITIVES Determintion of Isoflvones in Soy nd Selected Foods Contining Soy by Extrction, Sponifiction, nd Liquid Chromtogrphy: Collbortive Study STEPHEN P. KLUMP, MARYANN C. ALLRED, JOHN L. MACDONALD, nd JOAN M. BALLAM Rlston Anlyticl Lbortories, One Checkerbord Squre, 2RN, Sint Louis, MO Collbortors: A. Aror; S. Cole; M. Collison; W. Ellefson; J. Gensic; J. Hzebroek; P. Johns; S. Klump; S. Lewis; M. Moghddm; P. Murphy; D.C. Woollrd Isoflvones re biologiclly ctive compounds occurring nturlly in vriety of plnts, with reltively high levels found in soybens. Twelve lbortories prticipted in collbortive study to determine the glycon isoflvone content of 8 test smples of soy nd foods contining soy. The nlyticl method for the determintion of isoflvones incorportes mild sponifiction step tht reduces the number of nlytes mesured nd permits quntittion versus commercilly vilble, stble reference stndrds. Test smples were extrcted t 65 C with methnol wter ( ), sponified with dilute sodium hydroxide solution, nd nlyzed by reversed-phse liquid chromtogrphy with UV detection t 260 nm. Isoflvone results were reported s g/glycon/g or g glycon equivlents/g. The 8 test smples included 2 blind duplictes nd 4 single test smples with totl isoflvone concentrtions rnging from pproximtely 50 to 3000 g/g. Test smples of soy ingredients nd products mde with soy were distributed to collbortors with pproprite reference stndrds. Collbortors were sked to nlyze test smples in duplicte on 2 seprte dys. The dt were nlyzed for individul isoflvone components, subtotls of didzin didzein, glycitin glycitein, nd genistin genistein, nd totl isoflvones. The reltive stndrd devition (RSD) for repetbility ws %, nd the RSD for reproducibility ws % for totl isoflvone vlues of g/g. Submitted for publiction June The recommendtion ws pproved by the Methods Committee on Food Nutrition s First Action. See Officil Methods Progrm Actions, (2001) Inside Lbortory Mngement, November issue. Isoflvones re clss of chemicl compounds found nturlly in vriety of plnts such s soybens, which contin reltively high levels. Recent studies report tht isoflvones inhibit certin cncers, lessen symptoms of menopuse, nd contribute to improved bone density in tretments for osteoporosis (1 3). Three prent isoflvones re found in soy: didzein, glycitein, nd genistein (Figure 1). The isoflvones lso occur s the glucosides (didzin, glycitin, nd genistin; Figure 2) nd the glucoside esters of the prent isoflvones. The cetyl nd mlonyl isoflvone glucoside esters re the most bundnt forms found in soybens, with either n cetyl or mlonyl group ttched to the isoflvone glucoside t the 6 -O position, s shown in Figure 3. Severl methods reported for the determintion of isoflvones use vriety of extrction procedures followed by reversed-phse liquid chromtgrphy (LC) for seprtion nd UV or mss spectrometry for detection nd quntittion. Frnke et l. used cid hydrolysis, refluxing in ethnol wter (4), or enzymtic tretment of refluxed test smple t 37 C (5) to convert ll isoflvone compounds to the prent isoflvones (Figure 1). Wng nd Murphy (6) extrcted test smples with cetonitrile nd 0.1% HCl t room temperture nd nlyzed the extrcts by LC/UV. Brnes et l. (7) extrcted test smples by using either methnol wter ( ) or cetonitrile 0.1% HCl ( ), both t room temperture, with nlysis by LC/mss spectrometry. With the exception of methods employing enzymtic nd cid hydrolysis steps, these methods of nlysis determine levels of the prent isoflvones (Figure 1), the corresponding glucosides (Figure 2), nd the cetyl nd mlonyl esters (Figure 3). Becuse reference stndrds for the glucoside esters were not commercilly vilble until recently, estimtes of their concentrtions were commonly bsed on the ssumption tht the molr bsorptivities of the esters were equivlent to the bsorptivities of the corresponding glucosides. Reference stndrds for the cetyl nd mlonyl isoflvone esters (Figure 3) cn be purchsed, but they re reltively expensive nd re reported to be unstble in solution (8). The method in the collbortive study uses mild sponifiction step to convert the isoflvone glucoside ester

2 1866 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, 2001 Figure 1. Structures of prent isoflvones: () didzein, R=H,R = H; (b) glycitein, R=H,R = OCH 3 ; (c) genistein, R=OH,R =H. Figure 2. Structures of isoflvone glucosides: () didzin, R=H,R = H; (b) glycitin, R=H,R = OCH 3 ; (c) genistin, R=OH,R =H. forms (Figure 3) to the corresponding isoflvone glucoside forms (Figure 2), leving the 3 prent isoflvones (Figure 1) intct. This pproch llows direct comprison with stble, redily vilble, isoflvone prent nd glucoside reference stndrds. Becuse the method leves the isoflvone glucosides intct, it is possible to determine the reltive proportions of prent isoflvone nd glucoside present in test smples. Isoflvones re first extrcted from the test smple with methnol wter ( ) t 65 C for 2 h. Extrcts re then sponified for 15 min t mbient temperture with dilute (0.13M) sodium hydroxide solution. Extrcts re then filtered, centrifuged, nd nlyzed by using reversed-phse LC with C 18 column, followed by UV detection t 260 nm. Isoflvone results re reported s µg glycon/g or µg glycon equivlents/g. Collbortive Study Test smples of soy ingredients nd food products contining soy were distributed to 19 cdemic, industril, nd commercil testing lbortories. All 19 prticipting lbortories were provided with 1 prctice smple, 8 unidentified test smples, vil of stock reference stndrd solution 5, the method, nd instructions. The test smples included soy flour, soy protein isolte (blind duplictes), vegetble burger (freeze-dried), soy molsses, miso (freeze-dried), nd soy beverge (blind duplictes). Becuse of the cost of the isoflvone stndrds, mixed solution of stock reference stndrds ws provided to expedite completion of the study. Collbortors were required to mke ll necessry dilutions of the reference stndrds; they diluted n liquot of the stock stndrd solution provided to mke stndrd 4. Subsequent dilutions by collbortors provided ll 5 working stndrds required in this study. Isoflvone concentrtions were provided for the prctice smple nd ech collbortor ws sked to nlyze this smple to demonstrte method proficiency before proceeding with nlysis of the remining test smples. After successfully nlyzing the proficiency smple, collbortors were sked to nlyze ech test smple in duplicte on 2 seprte dys. These nlyses were in ddition to the nlyses of the blind duplictes nd provided further dt to estimte vribility within lbortory on given dy. Smple Preprtion nd Stndrds Five of the 8 test smples nd the prctice smple were homogenized by mixing nd then plced in test smple vils for distribution to the collbortors. Soy molsses ws llowed to settle for 7 dys nd the liquid lyer ws decnted to eliminte suspended mteril before the liquid portion ws plced in test smple vils. The vegetble burger nd miso were freeze-dried to enhnce stbility during both shipping nd storge. The freeze-dried miso ws mixed with dry ice before grinding. After grinding, the freeze-dried miso ws llowed to equilibrte t mbient temperture for 3 dys to vent CO 2 before it ws plced in test smple vils. The freeze-dried vegetble burger ws ground nd trnsferred directly to test smple vils. Eight test smples, lbeled A through H, were distributed to ech collbortor. The test smples were ssyed s received. Becuse the method specifies the weight of test smple to be used for extrction, bsed on the mount of soy protein in the test smple, collbortors were provided with recommended test smple sizes for the unidentified test smples. The recommended test smple sizes were s follows: test Figure 3. Structures of isoflvone glucoside esters: () 6 -O-[crbonyl group]-didzin, R=H,R =H,R = crbonyl group; (b) 6 -O-[crbonyl group]-glycitin, R=H, R = OCH 3,R = crbonyl group; (c) 6 -O-[crbonyl group]-genistin, R=OH,R =H,R = crbonyl group.

3 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, Tble 1. Didzin dt reported s glycon equivlents in g/g Lb Dy Smple Soy isolte smple Soy beverge smple A E D H Soy flour smple B Vegetble burger smple C Soy molsses smple F Miso smple G b 1290 b b b b 1214 b b b b 1346 b b b b 1346 b b b b b b b b c b c b c b c b 191 b b 190 b b 150 b b 240 b b 1297 b b 1357 b b 1217 b b 1247 b b 218 b b 222 b b 208 b b 210 b b c Smples A nd E were blind duplictes, nd smples D nd H were blind duplictes. Smples B, C, F, nd G were not run with blind duplictes. Outlier by Cochrn test. Outlier by Grubbs test.

4 1868 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, 2001 Tble 2. Interlbortory study results obtined for didzin in soy nd foods contining soy by extrction, sponifiction, nd LC Mtrix No. of lbs Men, µg/g s r RSD r, % s R RSD R, % r 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) Ech vlue is the number of lbortories retined fter elimintion of outliers; ech vlue in prentheses is the number of lbortories removed s outliers. smples A, B, E, nd the prctice smple, 1 g; test smples D nd H, 1 2 g; test smples F nd G, 2 3 g; nd test smple C, 4 5 g. A prctice smple of soy flour ws provided to ll collbortors. The expected concentrtion rnge ws provided for ech isoflvone level. Lbortories were sked to proceed with the study if the results of the nlysis of the prctice smple were within the given rnge. The concentrtion rnges were s follows: didzin didzein subtotl, µg/g; genistin genistein subtotl, µg/g; nd glycitin glycitein subtotl, µg/g. All concentrtions were in µg glycon equivlents/g. Preprtion of stndrds involved dilution of the stock stndrd solution before use. Prticipnts were instructed to trnsfer5mlliquot of the stock solution to 10 ml volumetric flsk, nd to dilute it to volume with methnol wter ( ) to mke stndrd 4. Subsequent dilutions of stndrd 4 were mde to prepre the remining working stndrds. Sttisticl Anlysis The dt nlysis for this summry follows the steps outlined by AOAC INTERNATIONAL (9). Ech isoflvone ssy ws treted s n independent mesurement. The method vribility ws determined seprtely for ech mesurement. Within-lbortory vrince ws estimted for ech mtrix nd lbortory by using the restricted mximum likelihood method (REML) in SAS s PROC MIXED (10). For those cses in which there ws no convergence, the estimte ws derived from method of moment estimtor (MIVQUE0). The REML nd MIVQUE0 methods were pproprite for estimting the vrince ttributed to within-dy, mong-dys, nd mong-smples components with unblnced dt. Repetbility for ech lbortory ws determined s the sum of these components. The Cochrn test ws pplied within ech mtrix to identify lbortories showing significntly greter vribility thn tht shown by other lbortories. These lbortories were removed from further nlysis. The Grubbs test for removl of lbortories with extreme verges ws performed fter the Cochrn test. The Grubbs test for single vlues ws followed by the Grubbs test for pirs of vlues (2 lowest, 2 highest, lowest nd highest). No more thn 2 of 12 lbortories were removed for ny test smple mtrix. After removl of outlying lbortories, the REML ws used to estimte the mong-lbortories, mong-smples within-lbortory (when pplicble), nd mong-dys within-smple nd within-dy components of vrince for ech mtrix. Repetbility vrince ws clculted s the sum of the mong-smples, mong-dys, nd within-dy components. Reproducibility vrince ws clculted s the sum of the repetbility vrince nd the mong-lbortories vrince. The collbortive dt nd results of sttisticl nlysis re shown in Tbles Lbortories tht were found to be outliers nd removed from the sttisticl nlysis re listed in Tbles 1, 3, 5, 7, 9, 11, nd 13. For didzein in test smple C (vegetble burger, Tble 7), the results rnged from 0 to 3 µg/g over ll lbortories. To retin s much dt s possible, no lbortories were removed s outliers from the sttisticl nlysis of didzein in this test smple, lthough 2 lbortories would hve qulified on the bsis of the sttisticl testing. A similr sitution ws found for test smples C (burger) nd F (soy molsses) in the glycitein ssy (Tble 9). After removl of lbortory 9 from the nlysis of results for test smple C (burger), the other vlues rnged from 0 to 3 µg/g in these 2 test smples. Sttisticl testing found other lbortories tht qulified for removl, but none were removed. Lbortories tht qulified for removl from the nlysis of results for genistein in test smple C (burger, Tble 11) lso were retined becuse of the low rnge of vlues (0 3 µg/g). There were mny zero vlues listed in the dt tbles, but they were minly for glycitin, glycitein, nd test smple C (burger). The glycitein fmily ws the lest bundnt soy isoflvone group found, nd this ws reflected by the plethor of zero vlues. Also, test smple C (burger) hd the lowest level of isoflvones by 1 order of mgnitude, nd therefore mny zero vlues ppered in the dt tbles. The HORRAT eqution gives vlues tht indicte whether the method is sufficiently precise for the level of nlyte being mesured nd is bsed on historicl collbortive study vlues. The HORRAT vlue is the rtio of the reproducibility reltive stndrd devition, in percent (RSD R, %), to the pre-

5 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, Tble 3. Glycitin dt reported s glycon equivlents in g/g Lb Dy Smple Soy isolte smple Soy beverge smple A E D H Soy flour smple B Vegetble Soy molsses burger smple C smple F Miso smple G b 0 55 b 10 b b 0 56 b 12 b b 0 14 b 0 b b 0 7 b 0 b b 79 b b 88 b b 85 b b 28 b b 27 b b 21 b b 39 b b b b b b b b b b b b b b c Smples A nd E were blind duplictes, nd smples D nd H were blind duplictes. Smples B, C, F, nd G were not run with blind duplictes. Outlier by Cochrn test. Outlier by Grubbs test.

6 1870 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, 2001 Tble 4. Interlbortory study results obtined for glycitin in soy nd foods contining soy by extrction, sponifiction, nd LC Mtrix No. of lbs Men, µg/g s r RSD r, % s R RSD R, % r 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) Ech vlue is the number of lbortories retined fter elimintion of outliers; ech vlue in prentheses is the number of lbortories removed s outliers. dicted reproducibility reltive stndrd devition, in percent (PRSD R, %): HORRAT = RSD, % R PRSD, % where PRSD R,%=2C nd C = the estimted men concentrtion. HORRAT vlues between 0.5 nd 2.0 indicte stisfctory method precision. 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 2 h in methnol 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). R 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, cpble of 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

7 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, Tble 5. Genistin dt reported s glycon equivlents in g/g Lb Dy Smple Soy isolte smple Soy beverge smple A E D H Soy flour smple B Vegetble burger smple C Soy molsses smple F Miso smple G b b 145 b b b 152 b b b 139 b b b 174 b b b b b b 390 b b 385 b b 298 b b 470 b b 425 b b 426 b b 403 b b 402 b b Smples A nd E were blind duplictes, nd smples D nd H were blind duplictes. Smples B, C, F, nd G were not run with blind duplictes. Outlier by Cochrn test.

8 1872 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, 2001 Tble 6. Interlbortory study results obtined for genistin in soy nd foods contining soy by extrction, sponifiction, nd LC Mtrix No. of lbs Men, µg/g s r RSD r, % s R RSD R, % r 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) Ech vlue is the number of lbortories retined fter elimintion of outliers; ech vlue in prentheses is the number of lbortories removed s outliers. (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 1000 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. 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

9 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, Tble 7. Didzein dt reported s glycon equivlents in g/g Lb Dy Smple Soy isolte smple Soy beverge smple A E D H Soy flour smple B Vegetble burger smple C Soy molsses smple F Miso smple G b 53 b 46 b 0 18 b 134 b b 49 b 48 b 0 20 b 139 b b 52 b 210 b 0 1 b 142 b b 61 b 149 b 0 1 b 169 b b 63 b b 65 b b 43 b b 0 b b 0 b b 0 b b 0 b c 0 c 0 c 0 c c 0 c 0 c 0 c c 0 c 0 c 0 c c 0 c 0 c 0 c c c c c b 1 16 b b 1 15 b b 1 9 b b 1 9 b b c Smples A nd E were blind duplictes, nd smples D nd H were blind duplictes. Smples B, C, F, nd G were not run with blind duplictes. Outlier by Cochrn test. Outlier by Grubbs test.

10 1874 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, 2001 Tble 8. Interlbortory study results obtined for didzein in soy nd foods contining soy by extrction, sponifiction, nd LC Mtrix No. of lbs Men, µg/g s r RSD r, % s R RSD R, % r 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) Ech vlue is the number of lbortories retined fter elimintion of outliers; ech vlue in prentheses is the number of lbortories removed s outliers. 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: Ce = MW Cg MWg where Ce = isoflvone glycon equivlents, µg/g; MW = moleculr weight of glycon (Tble N); MWg = moleculr weight 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) Te = Ce(didzin) + Ce(glycitin) + Ce(genistin) where T = sum of concentrtions of glycons, nd Te = sum of glycon equivlent concentrtions of glucosides. Totl isoflvones, µg glycon equivlents/g = T + Te Ref. J. AOAC Int. 84, (2001) Results nd Discussion The dt reported by collbortors re shown in Tbles 1, 3, 5, 7, 9, 11, nd 13, orgnized by isoflvone component nd isoflvone totl. Twelve prticipting lbortories nlyzed 8 test smples, including 2 blind duplictes, nd were sked to nlyze the test smples in duplicte on 2 seprte dys. Duplicte runs were dded to the collbortive protocol to better estimte within lbortory vribility on ny given dy. The sttisticl nlysis ws complicted becuse not ll of the lbortories rn duplictes ech dy. The method llows for distinction between bound isoflvone forms (i.e., glucosides nd glucoside esters) nd unbound (glycon) isoflvone forms. Six isoflvone components, didzin, glycitin, genistin, didzein, glycitein, nd genistein; subtotls of didzin didzein, glycitin glycitein, nd genistin genistein; nd totl isoflvones were ech sttisticlly nlyzed for method vlidity. This ws undertken becuse of the vriety of pproches used to report isoflvone dt. Typiclly, isoflvones re reported s totl isoflvones, or subtotls of isoflvone fmilies, or individul isoflvone components. Sttisticl summries for ech of the nlyzed isoflvone components, subtotls, nd totl isoflvones re listed in Tbles 2, 4, 6, 8, 10, 12, 14 16, nd I. The sttisticl summry tbles list the following: men, repetbility stndrd devition (s r ), reproducibility stndrd devition (s R ), repetbility reltive stndrd devition (RSD r ), reproducibility reltive stndrd devition (RSD R ), (r; 2.8 s r ), reproducibility (R; 2.8 s R ), nd HORRAT. The levels of isoflvone glucosides present re generlly higher thn those of the glycon isoflvone forms. As result, the isoflvone glucosides hd lower RSD vlues (Tbles 2, 4, nd 6). Of the 18 different test smple sets nlyzed for isoflvone glucosides, 16 hd HORRAT vlues of <2.0. The 2 smple sets with HORRAT vlues of >2.0 were for glycitin (Tble 4) with mens of 5.6 nd 2.8 µg/g. Clerly, isoflvone vlues t these low levels re below the limit of quntittion for individul components for this method. Men vlues for glucoside isoflvones tht gve HORRAT vlues of <2.0 were from 18 to 1449 µg/g. This result suggests tht the method is cpble of mesuring isoflvone glucosides down to 18 µg/g. For the glycon isoflvone forms, 18 smple sets lso were nlyzed (Tbles 7 12). Only 2 sets hd HORRAT vlues of <2.0. These were the didzein vlue (Tble 8) nd the

11 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, Tble 9. Glycitein dt reported s glycon equivlents in g/g Lb Dy Smple Soy isolte smple Soy beverge smple A E D H Soy flour smple B Vegetble burger smple C Soy molsses smple F Miso smple G b 0 b b b 0 b b b 0 b b b 0 b b b 7 b b 7 b b 5 b b 5 b b b b b b 17 b 0 c 3 c 13 b b 8 b 4 c 0 c 13 b b 15 b 7 c 7 c 14 b b 15 b 7 c 7 c 12 b b b b b b c Smples A nd E were blind duplictes, nd smples D nd H were blind duplictes. Smples B, C, F, nd G were not run with blind duplictes. Outlier by Cochrn test. Outlier by Grubbs test.

12 1876 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, 2001 Tble 10. Interlbortory study results obtined for glycitein in soy nd foods contining soy by extrction, sponifiction, nd LC Mtrix No. of lbs Men, µg/g s r RSD r, % s R RSD R, % r R HORRAT Soy isolte 10 (2) Soy beverge 10 (2) Soy flour 10 (2) Vegetble burger 11 (1) Soy molsses 12 (0) Miso 11 (1) Ech vlue is the number of lbortories retined fter elimintion of outliers; ech vlue in prentheses is the number of lbortories removed s outliers. genistein vlue (Tble 12) for smple G (miso). Miso is fermented soy product in which fermenttion pprently converts isoflvone glucosides nd glucoside esters to the glycon forms. Isoflvone glycons tht were present t <65.5 µg/g hd HORRAT vlues of >2.0. With the exception of miso, the test smples contined reltively little isoflvone glycon compred with the isoflvone glucoside. The mjority of vlues for didzein, glycitein, nd genistein were <25 mg/g. These smll peks were pprently difficult to identify nd mesure consistently. The mtrixes tested re nturl products or processed nturl products nd contin mterils tht cn produce interference peks. These interference peks cn occlude the pek of interest nd possibly be misidentified s the pek of interest. Low-level isoflvone glycon peks (<65 µg/g) were pprently more difficult to identify thn low-level isoflvone glucoside peks. In ddition, it is pprent tht some lbortories did not detect nlytes present t levels of <20 µg/g. Subtotls of isoflvone fmilies, didzin didzein, glycitin glycitein, nd genistin-genistein were lso sttisticlly nlyzed. These subtotls (Tbles 14 16) showed good reproducibility for high nd moderte vlues. Subtotls with low vlues were not s reproducible. Test smple C (vegetble burger) for didzin didzein (Tble 14) hd men of 18.8 µg/g nd good reproducibility (RSD R, 10.7%) with HORRAT of For test smple G (miso), glycitin glycitein hd men of 25.4 µg/g nd n RSD of 27.4% to give HORRAT of 2.79 (Tble 15). Agin for test smple C, the genistin genistein subtotl men ws 25.3 µg/g with n RSD R of 16.1% to give HORRAT of 1.64 (Tble 16). These results suggest tht 18 µg/g is the lower limit of quntittion for the subtotl vlues. The sttisticl results for totl isoflvone (Tble I) gve method reproducibility of %, nd ll HORRAT vlues were 1.80 for men vlues of µg/g. These results demonstrte tht the method is cpble of mesuring totl isoflvones in soy nd soy products down to 47 µg/g. Collbortors Comments Lbortory 4. The grdient used presented problems tht were trced to mobile phse out-gssing from mixing chmber locted prepump (i.e., low-pressure side). The use of different instrument tht hd 2 pumps nd post pump-mixing chmber solved the problem. Lbortory 11. The lbortory commented tht bringing the extrction solution to volume before filtrtion would produce high bis in the results. Studies undertken by the uthors lbortory showed tht bis of pproximtely 2 3% existed for test smples with totl isoflvone levels of 1100 µg glycon/g. Further evlution found tht bringing the extrction solution to volume fter filtrtion incresed the complexity of the procedure nd ws fr less repetble within the lbortory thn the method s written. Another lbortory declined to prticipte in the study becuse of concerns tht bse hydrolysis would destroy the isoflvones. A study undertken by the uthors lbortory evluted the bse hydrolysis step. Two test smples of stndrd solutions were tken through the method, but 1 test smple did not receive the NOH solution for sponifiction. Identicl recoveries were obtined for both stndrds, indicting tht the sponifiction solution does not destroy the isoflvone glycons or isoflvone glucons. It should be noted tht tretment of isoflvones with lkli t elevted tempertures does cuse loss of isoflvones. Recommendtion The results of this collbortive study show tht the method for determining isoflvones by using extrction nd sponifiction gives repetble performnce for ll components with limits of detection of 20 µg/g for glucoside nd subtotl components nd 47 µg/g for totl isoflvones. The Study Director recommends tht this method be dopted First Action.

13 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, Tble 11. Genistein dt reported s glycon equivlents in g/g Lb Dy Smple Soy isolte smple Soy beverge smple A E D H Soy flour smple B Vegetble burger smple C Soy molsses smple F Miso smple G b 58 b 41 b 0 15 b 191 b b 55 b 43 b 0 17 b 197 b b 47 b 61 b 0 63 b 201 b b 55 b 14 b 0 49 b 241 b b 42 b b 43 b b 29 b b 53 b b 0 26 b b 59 b b 0 26 b b 88 b b 3 31 b b 89 b b 3 31 b b Smples A nd E were blind duplictes, nd smples D nd H were blind duplictes. Smples B, C, F, nd G were not run with blind duplictes. Outlier by Cochrn test.

14 1878 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, 2001 Tble 12. Interlbortory study results obtined for genistein in soy nd foods contining soy by extrction, sponifiction, nd LC Mtrix No. of lbs Men, µg/g s r RSD r, % s R RSD R, % r R HORRAT Soy isolte 10 (2) Soy beverge 11 (1) Soy flour 10 (2) Vegetble burger 12 (0) Soy molsses 10 (2) Miso 11 (1) Ech vlue is the number of lbortories retined fter elimintion of outliers; ech vlue in prentheses is the number of lbortories removed s outliers. Tble 13. Totl Isoflvone dt reported s glycon equivlents in g/g Lb Dy Smple Soy isolte smple Soy beverge smple A E D H Soy flour smple B Vegetble burger smple C Soy molsses smple F Miso smple G b b 568 b b b 593 b b b 580 b b b 694 b b b b b b 663 b b 655 b b 508 b b 812 b

15 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, Tble 13. (continued) Lb Dy Smple Soy isolte smple Soy beverge smple A E D H Soy flour smple B Vegetble burger smple C Soy molsses smple F Miso smple G b 752 b b b 752 b b b 722 b b b 722 b b b Smples A nd E were blind duplictes, nd smples D nd H were blind duplictes. Smples B, C, F, nd G were not run with blind duplictes. Outlier by Cochrn test. Tble 14. Interlbortory study results obtined for didzin didzein subtotl in soy nd foods contining soy by extrction, sponifiction, nd LC Mtrix No. of lbs Men, µg/g s r RSD r, % s R RSD R, % r 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) Ech vlue is the number of lbortories retined fter elimintion of outliers; ech vlue in prentheses is the number of lbortories removed s outliers. Tble 15. Interlbortory study results obtined for glycitin glycitein subtotl in soy nd foods contining soy by extrction, sponifiction, nd LC Mtrix No. of lbs Men, µg/g s r RSD r, % s R RSD R, % r 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) Ech vlue is the number of lbortories retined fter elimintion of outliers; ech vlue in prentheses is the number of lbortories removed s outliers.

16 1880 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, 2001 Tble 16. Interlbortory study results obtined for genistin genistein subtotl in soy nd foods contining soy by extrction, sponifiction, nd LC Mtrix No. of lbs Men, µg/g s r RSD r, % s R RSD R, % r 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) Ech vlue is the number of lbortories retined fter elimintion of outliers; ech vlue in prentheses is the number of lbortories removed s outliers. 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). 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).

17 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, 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). 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).

18 1882 KLUMP ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 6, 2001 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). Tble J. Isoflvone stndrds Stndrd Formul CAS Registry No. Indofine Ct. No. Didzin C 21H 20O Didzein C 15H 10O D-O101 Genistin C 21H 20O Genistein C 15H 10O G-103 Glycitin C 22H 22O GL-002 Glycitein C 16H 12O 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