-发酵类抗生素中残留蛋白通用性测定方法的探讨

ChineseJournalofNewDrugs20152410117820152410“”2012BAK25B02。[e-mail]。。[e-mail]。··100050Bradford、。、Bradford。Sepha-dexTMG-15300mm×15mm40～120μmSephadexTMG-25100mm×26mm90μm280nm30℃500μL。97．4%Bradford2～40μg·mL－1r=0．9996BSA95．4%Bradford3．0%0．2μg·mL－1。0．001%0．002%。、。BradfordＲ917．795Ｒ978．1A1003－3734201510－1178－09AgeneralmethodforthedeterminationofresidualproteininfermentedantibioticsWANGYanYAOShang-chenCHANGYanCHONGXiao-mengMAYueHUChang-qinNationalInstituteforFoodandDrugControlBeijing100050ChinaAbstractObjectiveTodevelopagelfiltrationchromatographycombinedwithBradfordmethodforthedeterminationofresidualproteininantibiotics．MethodsThecolumnswereSephadexTMG-15300mm×15mm40～120μmandSephadexTMG-25100mm×26mm90μmrespectively．Themobilephasewaswater．Thedetectionwavelengthwas280nm．Thecolumntemperaturewas30℃andtheinjectionvolumewas500μL．Ｒe-sultsTherecoveryrateofgelfiltrationchromatographywasapproximately97．4%．Thelinearrangeofproteincon-centrationwas2～40μg·mL－1r=0．9996．TherecoveryrateofBSAwasapproximately95．4%．TheprecisionofBradfordmethodwaslessthan3．0%．TheLODwas0．2μg·mL－1．Thecombinedstandarduncertaintywas0.001%andtheexpandeduncertaintywas0．002%．ConclusionTheestablishedmethodisaccuratereliablerepeatableandsuitablefortheanalysisandcontrolofresidualproteininfermentedantibiotics．KeywordsfermentedantibioticsgelfiltrationchromatographyBradfordmethodresidualprotein、1－3。Streptomycesclavuligerusβ-41A。1B1CStreptomycesgriseusStreptomycescapreolus5。6－8ChineseJournalofNewDrugs20152410117920152410。1A、BCICH9EMA10。EDQM8．0“productsoffermenta-tion”11。FDA12。13、。3。1UltiMate3000Chromeleon6．8ThermoThermoSatoriousCP225DArium61196Corning201002001000μLEppendorf。BSAD0000255598．0%MerckBradfordSLBB8733VSigma06N1305001HSS1208201HSS1208203HSS1208205HCS1206202HCS1207201HCS1207202。2GEHealthcareSephadexTMG-15300mm×15mm40～120μmGEHealthcareSephadexTMG-25100mm×26mm90μm1.0mL·min－12．0mL·min－1280nm30℃500μL。33．1BSAIBSABSA5mg·mL－1。3．2BSAIII0．5mg·mL－1。V0。3．3BSA-III、IBSA1000．5mg·mL－1。3．4BSAIVBSABSA100μg·mL－1。3．5、100mg·mL－1。ChineseJournalofNewDrugs201524101180201524104SephadexSephadexG-10G-15G-255g350mL45mLSephadexG-155g250mL0.010．1mol·L－1pH7．045mL。1mL0．2mg·mL－1、200～400nmUV55．1、V0BSAII、BSA-III280nmA1A2A3。1。/%=A3－A2/A1×100%15．2BradfordBSAIV20．040．0μg·mL－11234、560Bradford595nmAAA12/%=A－A/A×100%21/μg·mL－1/μLIV/μL/μLBradford/μLan=300005005000．0811A203001001005000．19824030020005000．2763A030002005000．0804030002005000．0805A2001004005000．12364002003005000．208a0．00135．3IV1050100200μL1mL500μL2102040μg·mL－1。500μLBradford5min100μL96595nmACμg·mL－1。5．4LODLOQ10珔ASD珔A+3SD珔A+10SDLODLOQ。5．5“”2102040μg·mL－13ＲSD。6SephadexTMG-15300mm×15mm40～120μm500μL8．0～12．0min4mL624mL300mgSephadexTMG-25100mm×26mm90μm500μL7．0～11．0min8mL648mL300mg。48h1．0mL100μL400μLBradford500μL5min96595nmA3。X%=μg·mL－1×500Vx2μL×1Vx3ChineseJournalofNewDrugs20152410118120152410mL/100Vx4μL×300mg×1000×100%372。BradfordG250-595nm14－17。。21SephadexTMG-10SephadexTMG-15SephadexTMG-25。Sepha-dexTMG-10G-15G-257001500500018、237582751。318。0．010．1mol·L－1pH7．0。SephadexTMG-15、3ABC2SephadexTMG-15。。SephadexTMG-15SephadexTMG-25。SephadexTMG-15、SephadexTMG-25。ChineseJournalofNewDrugs201524101182201524101SephadexG-102SephadexG-153SephadexG-254SephadexG-150．01mol·L－1pH7．05Sepha-dexG-150．1mol·L－1pH7．03A、BC23BSAV045。V0。Sepha-dexTMG-15300mm×15mm40～120μmBSA9．8minV08．0～12．0min4mL4B1SephadexTMG-25100mm×26mm90μmBSA9．0minV07．0～11．0min8mL5BD1。8．0～12．0min、7．0～11．0min。1BSA23A100mg·mL－1B0．5mg·mL－1BSA100mg·mL－1SephadexTMG-154ABSA-BChineseJournalofNewDrugs201524101183201524101BSA23A100mg·mL－1B0．5mg·mL－1BSA100mg·mL－1C100mg·mL－1D0．5mg·mL－1BSA100mg·mL－1Sepha-dexTMG-255BSA-III3BradfordBradford。Bradford5∶11∶11∶21∶1500μL500μLBradford。、97%2。。3．1BradfordBradford95%95%3。Bradford。2BSA/mg·mL－1BSAIIA1A2IIIA3%n=30．50．0410．0080．04896．80．50．0210．0020．02498．10．50．0210．0060．02697．23、/μg·mL－1AAA%n=320．00．1980．0800．12395．540．00．2760．0800．20894．620．00．2020．0860．11799．740．00．2870．0860．21095．820．00．2130．1030．11794．140．00．2970．1030．21092．43．2logA=0．8626logC－1．7162r0．9996。2～40μg·mL－1。1。BradfordBSA19。3．3LODLOQLOD0．2μg·mL－133．3ppm300mgLOQ1．1μg·mL－1318．3ppm300mg。。3．4ＲSD≤1．6%。3．55～60minChineseJournalofNewDrugs201524101184201524105min60min10min。Bradford。Bradford5min60min5～60min10min2%20。4“6”、74。4/μg·mL－1/%UcP/%Uck=2/%06N130500122．1610．0370．0010．002HSS12082010aHSS12082030aHSS12082050aHSS12082010aHSS12082030aHSS12082050aaLOD=3．3ppm5。YX1X2…XnY=fX1X2…Xn4X5。UcY/Y2=UcX1/X12+UcX2/X22+…+UcXn/Xn221－224=SＲb1P+1n+()Cx－C2∑nj=1()Cj－C槡2225SＲ=∑nj=1Aj－()[]a+bCj2n－槡222P=3Cx3n=44。SＲ=0．01692μg·mL－1=0．01614μg·mL－1。BSA、、、643UcX6。UcX/X2=UcCx/Cx2+UcVx2/Vx22+UcVx3/Vx32+UcVx4/Vx42+UcWxi/Wxi2+UcVx1/Vx126CxUcCx/Cx=UcCsi/CsiBSA。、、《》JJG196—199023BA6ChineseJournalofNewDrugs20152410118520152410UcX/X。6BradfordUcX/X=UcCx/Cx2+UcVx2/Vx22+UcVx3/Vx32+UcVx4/Vx42+UcWxi/Wxi2+UcVx1/Vx121/2=0．011232+0．004162+0．002482+0．003372+0．0008172+0．0013521/2=0．01278。XXUcP/P=UcX/X2+UcX/X2+UcX/X21/2=0.012782+0．002002+0．0071321/2=0．01477。UcP=0．01477×0.037%=0．00001→0．001%。U=kUck295%U=2×0．001%=0．002