%REP]WMWSJ 2YXVEGIYXMGEP7S]-WSJPEZSRIW 9WMRK0'4(% .MQ/VSP1MOI=SYRKERH1EVO&IRZIRYXM ;EXIVW'SVTSVEXMSR 1EVGL 'ST]VMKLX;EXIVW'SVT -QTSVXERGISJ-WSJPEZSRIW # 4SXIRXREXYVEPERXMS\MHERXWJSYRHMRWS]FEWIH JSSHTVSHYGXW # -QTPMGEXIHMRVIHYGXMSRSJ0(0GLSPIWXIVSP # +IRMWXIMRERH(EMH^IMRMQTPMGEXIHMRVIHYGMRK GERGIVVMWO FVIEWXTVSWXVEXI GSPSR # ;IEOIWXVSKIRMGIJJIGXQE]VIPMIZIQIRSTEYWEP W]QTXSQW 'ST]VMKLX;EXIVW'SVT -QTSVXERGISJ-WSJPEZSRIW 2YXVEGIYXMGEP *IHIVEP6IKMWXEV3GX XLI*(%EPPS[WEPEFIPGPEMQXLEX l|KVEQWSJWS]TVSXIMREHE] EWTEVXSJEHMIXPS[MRWEXYVEXIHJEXERHGLSPIWXIVSP QE]VIHYGIXLIVMWOSJLIEVXHMWIEWIz 'ST]VMKLX;EXIVW'SVT %REP]WMWSJ-WSJPEZSRIW # +'17QIXLSHMWWIRWMXMZIERHEGGYVEXIFYXPEFSVMSYWERH I\TIRWMZI r WIZIVEP74)WXITWVIUYMVIHXSMWSPEXIHMJJIVIRXJSVQW r L]HVSP]WMWXSEKPYGSRI r HIVMZEXM^EXMSR # 0'4(%LEWEREHZERXEKIFIGEYWIEPPMWSJPEZSRIJSVQW GERFIWITEVEXIHERHUYERXMXEXIHYWMRK9:(IXIGXMSR r 7IRWMXMZMX] r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b¥ HOO E %KPYGSRI7XVYGXYVI (EMH^IMR 1;! O OH HOO H3CO +P]GMXIMR 1;! O OH T/Eb¥ HOO +IRMWXIMR 1;! OH O OH T/ b¥ E 'ST]VMKLX;EXIVW'SVT 'SQQSR7S]-WS*PEZSRI+P]GSWMHI 7XVYGXYVIERHT/E HO OH pKa ~ 11 O (EMH^MR O O 1;! HO (EMH^IMR+PYGSWMHI OH O OH %GIX]P(EMH^MR 1;! 1EPSR]P(EMH^MR (EMH^MR%GIX]P+PYGSWMHI O O 1;! O C (EMH^MR1EPSR]P+PYGSWMHI CH HO O 3 HO O OH pKa ~ 5.7 O O O O O O HO HO OH OH O O OH OH 'ST]VMKLX;EXIVW'SVT 4VMGISJ-WSJPEZSRI7XERHEVHW -WSJPEZSRI%REP]XIW 5X] QK 4VMGI (EMH^IMR (EMH^MR %GIX]P(EMH^MR 1EPSR]P(EMH^MR +P]GMXIMR +P]GMXMR %GIX]P+P]GMXMR 1EPSR]P+P]GMXMR +IRMWXIMR +IRMWXMR %GIX]P+IRMWXMR 1EPSR]P+IRMWXMR 8SXEPTVMGISJ7XERHEVHW 1EPSR]P)WXIVWSRFEGOSVHIVJSVWIZIVEP[IIOW 'ST]VMKLX;EXIVW'SVT 'LVSQEXSKVETLMG'LEVEGXIV 6IZIVWI4LEWI'SPYQRWEVI2SX%PMOI 7S]-WSJPEZSRIW *PS[6EXIQ0QMR &EGO4VIWW∼TWM -RNIGXMSR:SP®0 4 3 (IXIGXMSRRQ)\XVEGXIH 6 1SFMPI4LEWI%,3%GT, 5 <8IVVE¬17' ®Q 1SFMPI4LEWI&%G'2,3%G 7 89 \QQ 1 2 30% B 22% B 12% B ®KQ0 3 4 (EMH^MR Q%9 6 +P]GMXMR 2 5 7]QQIXV]' ®Q +IRMWXMR 7 8 9 %GIX]P(EMH^MR \QQ 30% B 1 %GIX]P+P]GMXMR 22% B (EMH^IMR +P]GMXIMR 12% B %GIX]P+IRMWXMR +IRMWXIMR 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 1MRYXIW 'ST]VMKLX;EXIVW'SVT -WSJPEZSRI7TIGXVE MR%G'2%GIXMG%GMHT, 259.4 TTQ)EGL-WSJPEZSRI 248.8 259.4 255.9 248.8 4SXIRXMEP7IGSRHEV] 255.9259.4 (IXIGXMSR;EZIPIRKXLW 248.8 Q%9*YPP7GEPI RQ %ZIλ1E\ RQ 200 220 240 260 280 300 320 340 360 380 RQ 'ST]VMKLX;EXIVW'SVT 7S]-WS*PEZSRI7IPIGXMZMX] 7]QQIXV]'®Q \QQ 30.00 Genistein pH of DI < 7, at ~ 5.5 25.00 AcetylGenistin Glycitein Aglucone IsoFlavones Daidzein 20.00 AcetylGlycitin Isoflavone AcetylGlucosides AcetylDaidzin k' Retention 15.00 Genistin Isoflavone Glycitin Glucosides 10.00 Daidzin pH 3.4 with 0.1% Acetic Acid pH 6.8 with 10 mM Na Acetate 5.00 345678pH T,FEWIHYTSR[SVOF] T,FEWIHYTSR[SVOF] 4EX1YVTL]IXEP-S[E7XEXI9RMZ 7XIZI&EVRIWIXEP9RMZSJ%PEFEQE 'ST]VMKLX;EXIVW'SVT 7S]-WS*PEZSRI7IPIGXMZMX] %POEPMRIT,[MXL%QQSRMYQ&M'EVFSREXI <8IVVE¬17' 30.00 Genistein pH of DI <7, ~5.5 25.00 AcetylGenistin Glycitein Daidzein 20.00 AcetylGlycitin Isoflavone AcetylGlucosides AcetylDaidzin 15.00 Genistin 6IXIRXMSR Ok Glycitin 10.00 Isoflavone Daidzin Glucosides 5.00 Aglucone IsoFlavones 0.00 345678910 T, pH 9.1 'ST]VMKLX;EXIVW'SVT 7S]-WS*PEZSRIW6IWSPYXMSR %POEPMRIT,[MXL%QQSRMYQ&M'EVFSREXI 7IPIGXMZMX]'LERKI ¬ 'SPYQR<8IVVE 17'®Q ®KQ0 \QQ (EMH^MR *PS[6EXIQ0QMR +P]GMXMR ∼ &EGO4VIWW TWM +IRMWXMR 9 -RNIGXMSR:SP®0 %GIX]P(EMH^MR 7 3 (IXIGXMSRRQ)\XVEGXIH %GIX]P+P]GMXMR 1SFMPI4LEWI%Q12,,'3T, (EMH^IMR 1SFMPI4LEWI&%G'2 +P]GMXIMR Q12,,'3 %GIX]P+IRMWXMR 1 T, 6 +IRMWXIMR 2 30% B Q%9 4 22% B 5 8 12% B 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 1MRYXIW 'ST]VMKLX;EXIVW'SVT -WSJPEZSRI7TIGXVE MR%G'22,,'3T, 259.4 255.9 TTQ)EGL-WSJPEZSRI 248.8 268.8 4SXIRXMEP7IGSRHEV] 253.5258.2 259.4 (IXIGXMSR;EZIPIRKXLW 258.2 248.8 RQ Q%9*YPP7GEPI %ZIλ1E\ RQ RQ9WIHJSV7MRKPI;EZIPIRKXL(IXIGXMSR RQ 'ST]VMKLX;EXIVW'SVT 7S]-WS*PEZSRIW6IWSPYXMSR %POEPMRIT,[MXL%QQSRMYQ&M'EVFSREXI 3TXMQM^IH6IWSPYXMSR ¬ 'SPYQR<8IVVE 17'®Q ®KQ0 \QQ (EMH^MR *PS[6EXIQ0QMR +P]GMXMR &EGO4VIWW∼TWM +IRMWXMR -RNIGXMSR:SP®0 %GIX]P(EMH^MR (IXIGXMSRRQ)\XVEGXIH %GIX]P+P]GMXMR 0S[IVMRMXMEPGSRHMXMSRWF] 1SFMPI4LEWI%Q12,,'3T, (EMH^IMR )\XIRHXMQIQMRJSVWXITERH 1SFMPI4LEWI&%G'2 +P]GMXIMR 9 3 Q12,,'3T, %GIX]P+IRMWXMR 7 +IRMWXIMR 4 5 30% B 6 1 2 22% B 8 Q%9 11% B 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 1MRYXIW 'ST]VMKLX;EXIVW'SVT 7S]-WS*PEZSRIW (IXIGXMSR)WXMQEXI %POEPMRIT,[MXL%QQSRMYQ&M'EVFSREXI 2 µg Each in 50% MeOH 4IEOXLVYEVIFVSEHIRIHHYIXS (EMH^IMR 1I3,MR7XERHEVH 6 7 +P]GMXIMR (EMH^MR -WSGVEXMG[MXL%G'2 8 +IRMWXIMR +VEHMIRXMRMXMEXMSREXQMRYXIW 9 +P]GMXMR +IRMWXMR %GIX]P(EMH^MR %GIX]P+P]GMXMR %GIX]P+IRMWXMR 2 3 5 Q%9 1 4 (IXIGXMSR0MQMX ®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¬17'®Q \QQ EW+P]GSWMHIW *PS[6EXIQ0QMR KQ0 1I3, &EGO4VIWW∼TWM -RNIGXMSR:SP®0 7 (EMH^IMR (IXIGXMSRRQ)\XVEGXIH +P]GMXIMR 1SFMPI4LEWI%Q12,,'3T, (EMH^MR 1SFMPI4LEWI&%G'2Q1T, +IRMWXIMR 3 +P]GMXMR +IRMWXMR 6 8 %GIX]P(EMH^MR 5 %GIX]P+P]GMXMR %GIX]P+IRMWXMR 9 1 2 3ZIVPEMH 4 Q%9 'LVSQEXSKVEQW 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 1MRYXIW 'ST]VMKLX;EXIVW'SVT 7S]-WSJPEZSRIW'SRJMVQEXMSR 'SRGIRXVEXIH)\XVEGX 'SPYQR7]QQIXV] '®Q 7EQTPI7S]0MJI -WSJPEZSRIW \QQ EW+P]GSWMHIW *PS[6EXIQ0QMR KQ0 1I3, &EGO4VIWW∼TWM -RNIGXMSR:SP®0 (EMH^IMR (IXIGXMSRRQ)\XVEGXIH +P]GMXIMR 1SFMPI4LEWI%,3%GT, (EMH^MR 1SFMPI4LEWI&%G'2,3%GT, +IRMWXIMR 7 +P]GMXMR 3 5 +IRMWXMR 6 %GIX]P(EMH^MR 8 %GIX]P+P]GMXMR %GIX]P+IRMWXMR 3ZIVPEMH 9 'LVSQEXSKVEQW 1 2 6 1500 mAU 1500 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 Minutes 'ST]VMKLX;EXIVW'SVT 7S]-WSJPEZSRIW (MIXEV]7YTTPIQIRX 'SPYQR<XIVVE¬17'®Q \QQ 2EXVSPJSV;SQIR¬ -RNIGXMSR:SP®0MR1I3, (IXIGXMSRRQ)\XVEGXIH 'ETWYPI!K 6 (MPYXIH 1SFMPI4LEWI%G'2Q12,,'3T, (EMH^IMR 'SRXEMRMRKQK +P]GMXIMR! 7S]-WSJPEZSRI)\XVEGX (EMH^MR! +IRMWXIMR! +P]GMXMR! Q%9 +IRMWXMR! %GIX]P(EMH^MR! %GIX]P+P]GMXMR 3 %GIX]P+IRMWXMR! 2 7 9 4 5 8 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 1MRYXIW 'ST]VMKLX;EXIVW'SVT 7S]-WSJPEZSRIW (MIXEV]7YTTPIQIRX =SYV0MJI 'SPYQR<XIVVE¬17'®Q \QQ 7XERHEVHM^I,IVFEP)\XVEGX -RNIGXMSR:SP®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