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12*21211000292100013AgilentZorbax300SB-C8250mm×4.6mm5μm--214nm45℃。κ-CNαs2-CNαs1-CNβ-CNWheyIgg0.99974.8%～132.5%。。9。4。2009-04-042009-06-23No.20775075No.2006BAF07B03*E-mailZhangqh@nim.ac.cn18。。80%κ-CNαs1-CNαs2-CNβ-CNα-Laβ-LgBSAIgg。、。、。1～5、67、8。、、。9。22.12LC-20AT、SIL-20A、CTO-20A、SPD-M20A、LCSolutionVer.1.21。SartoriusIKAMilliporeCEM。Bis-TrisBiotechnologyLotR1/83/03098%Inalcoκ-CNαS1-CNαS2-CNβ-CNWheyIggSigma-AldrichTedia。9。2.22mLA0.1mol/LBis-Tris、6mol/L、19.5mmol/L、5.37mmol/L46mL4.5mol/L-0.1%TFA0.22μm。β-CNαS1-CNαS2-CNκ-CNIggWhey5.3542.6370.4231.7522.1603.088g/L。37200911FENXIHUAXUEChineseJournalofAnalyticalChemistry111667～1670A1min1h4℃14800r/min20min34.5mol/L-0.1%TFA0.22μm。2.3AgilentZorbax300SB-C8250mm×4.6mm5μmA0.1%V/VTFAB0.1%V/VTFA0.8mL/min45℃214nm0→5.0min33%→35%B5.0→9.0min35%→37%B9.0→18.0min37%→40%B18.0→22.0min40%→41%B22.0→27.5min41%B27.50→28.0min41%→43%B28.0→36.0min43%→45%B36.0→40.0min45%→90%B40.0→45.0min90%B。33.1、、Bobe9pH7、。Bonfatti106mol/L、20mmol/L、5mmol/L1∶11h。4。100.1mol/LBis-Tris。3.23.2.1HPLC1030nmC1810nm。30nmC4、C8C18C8αs-CNβ-CNAgilentZorbax300SB-C8。200nm214nm280nm10214nm。3.2.2-TFA。B33%～45%。0.5～0.8mL/min。0.8mL/min45min。1abFig.1Chromatogramofcommerciallyavailablemilkaandsoymilkb1～5。κ-CNαs2-CNBSAβ-CN2、327～29min110BSA。1。3.3、3.3.1661yx6r0.999。86613716Table1LinearsrangesregressionequationsandcorrelationcoefficientofchromatogramsofsixproteinsstandardsProteinsInjectedamountμgRegressionequationsrCorrelationcoefficientκ-Caseinκ-CN0.88～35.04y=1.111×109x+9.903×1040.9999α-Caseinαs2-CN0.21～8.25y=5.324×108x+1.823×1050.9997αs1-CN1.32～51.39y=9.654×108x+4.562×1050.9999β-Caseinβ-CN2.68～107.09y=9.402×108x+4.130×1040.9995ImmunoglobulinsIgg1.08～43.20y=1.488×108x－2.815×1040.9999Whey10.29～102.93y=4.953×108x－9.077×1040.99973.3.22κ-CNαs1-CNαs2-CNβ-CNIggWhey2.1n=2。674.8%～132.5%2。26Table2RecoveriesofsixtargetproteinsspikedinmilkProteinsOriginalg/LAddedg/LFoundg/LRecovery%ProteinsOriginalg/LAddedg/LFoundg/LRecovery%αs1-CN7.8812.882.8420.0310.4394.389.6β-CN10.5710.641.7220.3511.8691.974.8αs2-CN3.511.566.841.307.5983.380.7Igg0.705.760.807.881.76124.7132.5κ-CN2.106.841.367.593.2380.783.1Whey4.6319.822.9125.997.30107.891.83.3.3100.5mL53。2.0%5.7%。3Table3RepeatabilityandreproducibilityoftheanalysismethodProteinRepeatabilityRetentiontimeRSD%PeakareaRSD%ReproducibilityRetentiontimeRSD%PeakareaRSD%αs1-CN0.30.71.32.1αs2-CN0.54.71.94.0κ-CN1.12.02.01.7β-CN0.32.00.51.5Igg0.82.20.25.7Whey0.13.90.70.23.494。61#～6#3.05%～3.56%37#～9#3.26%～3.38%。7.0%～9.2%。414.5%～20.0%11。4κ-CNαs2-CNαs1-CNβ-CN8%11%27%35%1.0∶1.3∶3.2∶4.211。34。96611149Table4Concentrationoftheproteinsin9kindsofcommerciallyavailablemilksMilkCTMPC%aCHPLCCCEMTE%dC%bκ-CNαs2-CNαs1-CNβ-CNWheyP%cκ-CNαs2-CNαs1-CNβ-CNWhey13.053.142.90.230.350.811.050.617.5511.426.634.520.023.053.07－0.70.250.340.841.100.528.2011.127.636.117.133.233.123.60.270.360.91.130.578.3611.227.935.017.643.243.152.80.260.360.881.180.568.0311.027.236.517.353.393.254.30.280.370.951.200.598.2610.928.035.417.463.563.269.20.280.420.991.220.657.8611.827.832.318.373.273.51－7.00.290.360.861.190.578.8810.926.336.517.583.263.49－6.70.290.360.861.150.608.9110.926.435.318.493.383.45－2.00.360.380.841.310.4910.6511.324.838.714.5a.TotalproteinsconcentrationTMPCb.Concentrationofindividualproteininmilkc.Proportionofindividualproteininmilkd.Relativeerror。References1DziubaJNaleczDMinkiewiczP.Anal.Chim.Acta20014491-2243～2522BobeGBeitzDCFreemenAELindbergGL.J.Agric.FoodChem.1998462458～4633ThomaCKrauseIKulozikU.InternationalDairyJoural2006164285～2934EnneGElezDFondriniFBonizziIFeliginiMAleandriR.J.Chromatogr.A200510941-2169～1745VisserSSlangenCJRollemaHS.J.Chromatogr.19915481-2361～3706Rehder-SilinskiMAMcGownLB.J.Chromatogr.A200310082233～2457TangPingTianJingSheZhen-BaoWengQian-FengXuGuo-Wang.J.Anal.Chem.20062215～88BraunFKrauseIKlostermeyerH.Milchwissenschaft199045113～99BobeGBeitzDCFreemenAELindbergGL.J.Agric.FoodChem.19984641321～132510BonfattiVGrigolettoLCecchinatoAGalloLCarnierP.J.Chromatogr.A200811951-2101～10611BordinGRaposoFCCalleBRodriguezAR.J.Chromatogr.A2001928263～76DeterminationofMajorBovineMilkProteinsbyReversedPhaseHighPerformanceLiquidChromatographyWANGJuan12ZHANGQing-He2WANGZhi-Hua1LIHong-Mei21StateKeyLaboratoryofChemicalResourceEngineeringBeijingUniversityofChemicalTechnologyBeijing1000292NationalInstituteofMetrologyBeijing100013AbstractARP-HPLCmethodforthequantificationofthesixmajorbovinemilkproteinsκ-caseinCNαs2-CNαs1-CNβ-CNWheyimmunoglobulinsIggisdescribed.SeparationandquantificationwereachievedbyareversedphaseanalyticalcolumnAgilentZorbax300SB-C8250mm×4.6mm5μmandthegradientelutionsolventsof0.1%trifluoroaceticacidTFAinwaterand0.1%TFAinacetonitrileataflowrateof0.8mL/min.Columntemperaturewassetat45℃andthesamplewasmonitoredwithphotodiodearraydetectorat214nm.Alinearrelationshipr0.999betweentheconcentrationsofproteinsandpeakareaswasobservedovertheconcentrationrange.Recoveriesofsixtargetproteinsspikedinmilkwereform74.8%to132.5%.Ninekindsofmilksofdifferentbrandswereanalyzedandthedifferenceoftheconcen-trationandrelativerationofκ-CNαs2-CNαs1-CNβ-CNandWhe