【doc】四环类抗生素的反相制备层析Ⅱ：应用反相LICHROPREP

【doc】四环类抗生素的反相制备层析Ⅱ：应用反相LICHROPREP 四环类抗生素的反相制备层析?:应用反相 LICHROPREP 《中国抗生素荣志~1993I18(3)}185~19I SEPARAT10NOFTETRACYCLINEANTIBIOTICSBY REVERSEPHASELICHR0PREPRP-18 HIGHPERFORMANCELOWPRESSURE LIQUIDCHR0MAT0GRAPHY ZouJing-yuan (SichuaaIndustrialInstituteofAntibiotics,Chendu610051) LestcrA.Mitschef (DepartmantofMedicinalChemistry,TheUniversityofKansas, Lawrence,KS66045U.S.A.) ABSTRACTThereversephasemodeofHPLC(RPLC)hasemergedasthemost popularandgeneralHPLCtechnique.Itwasanonpolarstationaryandapolarmo. bilephase.RPLCisideallysuitedforseparationsofnonpolarandmoderatepolar compounds.Inapreviousstudies,HPLCmethodsusingC,--andCIt-modifiedsilicagel colum?hayebeenusedforthe'determinationoftetracycllnes.TC,CTC,MTC,and DCcanbeseparatedrapidlyand~[icientlyfromeachotherusingLJchroprepRP—J8 columnconditionsinpreparativescale.Theresultsofwariousexperimentsshow thatMeOH}CH.CN,o.o1mel/Loxaficaeid(pH3)(5l0.5It0)wastheoptima/solvent systemfortheseparationofTC(orcTc,.OTC,MTCandDc,althoughthesepar- ationhetwee~TCandOTCwasnotachieved.TheLichroprepRP-tgcolumnused canherecoveredtouseagainafteritwastreatedwith0.01mol/LHCI,MeOH-H_io andMeOHrespectively. KEYWORDSTetracycllnesIChromatography;Reverse—phasechromatography;High performancelowpressurechromatography 1NTRODUCTION Thereverse-phasemodeofHPLC(RPLC)IRsesanonpolarstationaryanda polarmobilephase.IthasemergedasthemostpopularandgeneralHPLCte- chnique.RPLCisideallysuitedforseparationsofnonpolarandmoderately polarcompounds.Inapreviousstudies,HPLCmethodsusingCsandC18-me- difiedSilleagelcolumnhavebeenusedfordeterminationofTetracyclinesn". butitisalmosto/1a口analyticalscale.Tetracyclinesaretoopolartobe chro. matographedsuccessfullyonconventionalsupportssuchasalumina,silica gel,ionexchangeresin,SephadexLH-20issatisfactoryforsofa8purposesbut inourhandslacksresolvingpowef.RecentlyreversephasesilicagelLichrop? repRP一8andRP一18hasbeenusedwidelyforseparationandpurificationof ?185? naturalproducts.Forpreparativeseparation,tetracyclineandch1ortetracyc1inecan beseparatedrapidlyandeffiCientlyfromeach0therandfromthemixtureof methacyclineanddoxycyclineusingsillcagel60siI'anizedRP-2columncondi. 1ions.【-] Thispaperdescribesanothersimplerapidmethodtoseparatecommercially importanttetracyclineantibioticsthroughtheuseofreversephasesilicagel LichroprepRP一18columncondltions.Theresultsofva,rioUSexperimentsshow thatMeOH:CH.CN:0.01mol/Loxalicacid(pH3)(5;0.5:10)'wastheoptimalSOIre. ntsystemfortheseparationofTC(orOTC),CTC,MTCandDC.Although theseparationbetweenTCandOTCwasnotachieved. EXPERIMENTAL MaterlalsandMethods Apparatus ChromatoEraphicColumnlAnI.D.8x250mmglasscolnmn(MICHEL— PACKARD8540ADIODEARRAYSpectrophatometer. MILLER)JHEWLETT— M0DELFC-220Kfractionator. Reagents LichroprepRP一18(E.Merck13901),slllcagelHPTLCplate(E.Merck 5641)Jacetonltrile,chloroform,methanol,oxalicacid,hydrochloricacid.sod. iumhydroxideanddisodiamethylenedi8minetetraeetatewegcchemicalreagent grade.Tetracyclinehydroehloride(TC),chlortetracyc1inehydrochlorlde(CTC). 0xytetracyc1inehydrochloride(OTC).methacyclinehydrochloride(MTC)anddo. xycycllnehydrochlorlde(DC)wereprovidedbyLederleandRachellelaboratori. eS. PROCEDURE 1.Preparationofthesolventsystems. 0.01mo1]Loxalicacid(pH3.0),methanolandacetonitrileweremixedth. roughlyandallowedovernightatroomtemperature. '2.Preparationofthecolumnandchromat0gr8phy. LichroprepRP-18(8g)waspackedinteaspoonfulportionsintotheI.D.8× 250mmcolumn(MICHEL-MILLER)andcompactedbydroppingthecolumntwenty timesfromaheightofitabout0.5,linchesontoahardsurface.Addit.1onal LichroprepRP-18wasaddedinteaspoonfulandtampingprocesswasrepeated untilthecolumnwasful1.UsingtheFMILABpump(MODELRP—SY).theco. 1nmapackedLichroprepRP一18wasequilibratedwithMeOHIH2O(1l1)and MeOHfor2hrespectivelytotightenthecolumn.Thepressurewas20~70P.S. ItokeeptheflowrateOilO.5,0.7ml/min.Beforethechromatography.the columnprepareddescribedabovewasequilibratedwithdevelopingsolvents ?1R6? for1h.ThemixtureofTC,CTC,MTC,DCand0TC(30m1)wasdissolved in0.5,0.8mlofdevlopedsolventsandwasinjectedtothecolumn.Thenthe columnwasdevelopedwithfollowingsolventsystems. a.MeOHlCHsCN=oxalicacid(pH3)(O.01mol/L.0.Imol/Land0.2tool/L) (4f0.st10) b.MeOH:CHsCN:0.1mol/Loxalicacid(pH4,3and2)(4tO.5l10) c.MeOH:CHsCN:0.1mol/Loxalicacid(pH3)(4lO.5;10.5:O.5:10and6:O.5; lO) d.MeOH:CH3CN=0.1mol/Loxalicacid(pH3.O)(4l1.5tlO,4.5l1.OI10and 5.5l0l10) Theflowratewas0.7,O.8ml/min.Fractionofeach1.4,1.6mlwaseolle. atedusingthefractionator.Thecomponentsofeachfractionwereexaminedby HPTLCplateandbUVabsorbance. 3.Detectionoffractions a.Thin-layerchromat.graphy(- EachfractionwasanalyedusingsilicagelHPTLCplates.Asilicagel HPTLCplatewaspre-developedwithsaturatedNaIEDTAsolutionandthen driedinairatroomtemperatureforthandactivatedat13O?for2h.After applylngfraction,theplatewasdevelopedwit . hCHCle,MeOH:5Na:EDTA (65l20:5)(1owerlayer).ThedevelopedHPTLCplatedwassprayedevenlywith 1FastvioletBsaltsolutionandthenheatedat12O?todetectcoloredspo? ts. b.Determinationofabsorbanceoffraction5 Eachfractlon(O.2m1)waspipettedintoa14ramdiameter×150mmtesttube and5mlof0.01mol/LHCI—MeOHsolutionwasadded.Thentheabsorbance wasdeterminedat350or380um.E1utionsolvents(0.2m1)weredi1utedto5.2ml with5mlof0.01moi,LHC1一MeOHsolutionasablank. RESULTSANDDISCUSSlON 1.Selectionoftheappropirateconcentrationofoxalicacid UsingMeOHICH3CN:aqueousoxalicacidsolution(pH3)(4l0.5l10)asa mobilephase.theinfluenceofoxalicacidconcentrationwasexamined. Fig.1showstheelationdiagramseparationoftetracyclinesonLiehropr? epRP一18columnchromatographywithoxalicacidindifferentconcentrations. ItisclearthattheTC(orOTC),CTC,MTC(orDC)wereseparatedefficient? ly.buttheseparationbetweenTCandOTCwasnotachievedinallofthree concentrationofaqueousoxalicacid.Whe?O.01mol/Laqueousoxalicacid$o- lution@asused,theMTCandDCgavepoorseparation(Fig.1(a)].Asthe concentrationofoxalicacidincreased.MTCandDCgaveabetterresolution CFig.1(b,c)]althoughthepeakofTC,CTC,MTCandDCWerereta? ?187? rded.HoweyertheflowrateWaSdecreasedinthesamepressurewhen0.1mol/L andO.2mol/Loxlicacidsolutionwereusedduringchr0mat0graphy,theflow ratewasldeereusedto0.240.1ml/min.Sotheseparationtimeonthecolumn WaSincreasedwhenthec0ncentrationofoxalicacidincreased.ItseemSthat the0.1mol/LoxalicacidwatsuitableforseparationofTC(orOTC),CTC, MTCandDC. Tube~mber Fig?1SeparationoftetracyclinesbyLichroprepRP一18columnchromatography insolventofdifferentoxalicacidconcezltEat3o~.SamplesI30ragmixtureofTC. Oc.CTC,MTCandDC,elationsolventslMeOH,CH3CN:oxalicacid(pHs)(4,0.5? 10)}flowratet0.7,o.8mI/mf?lsml/fraction,detectionl380rim 2.pHoftheaqU~OnSoxalicacid. TheinfluenceofthepHoftheaqueousoxalicacidsolutionfnthemobile phusewasinvestigatedusingsolventsystemMe0H:CH3cN:0.1mol/Loxalicacid (4:0.5:10). Fig.2ShowstheelutiondiagramsseparationoftetracyclinesonLichroprep RP-18columnchromatographyusing0.1mol/LoxalicacidatdefferentpHs. AtpH4,althoughtheseparationofTCfromCTC,MTCandDCwasach- ieved,butonlythreeofpeaksweredlsplayedintheelutiondiagram(Fig.2 (a)]andCTC,MTCandDCgaveapoorresolution.AsthepHdecrease,itis clearthattheelationdiagramshowsfourpeaksofTC(+OTC),CTC,MTC andDCandCTC,MTCandDCwereseparatedwithhighresolution[Fig.2(b, c)3.HowevercomparedwiththepH4,theseparationtimesonthecolumnwas increasedinthepH3sadpH2.Asthetetracyclinesundergoreversiblespimeri— zationreadilyatC4atpH3—一5inaqueoussolutionandprogressi~edehydration atC5,atpHvalueslowerthan2.SothesolveatsystemMeOH:CHaCN*0.I ?188? :?2《 TubenumberTabe.utabcr Onli4 Tabenumber Fig.2SeparationoftetraeycllnesbyLiehroprepRP一18columnomatogchrarphy insolventsofdifferentpHoftheaqueousoxalicacid.Samples,30mgmixtureof TC.,OTC,CTC,MTCandDCIelutlonsolventsIMe0HIcH$CN,0.1mo1/Locxalicaid (410.5{10)}flowraterO.7,O.8ml/min)3ml/fraction)detectionl38Ohm mol/Loxalicacid(pH3)wastheoptimalsolventsystemforseparationofTC (orOTC),CTC,MTCandDC.. 3.Selectionofproportionsoforganicsolventsandaqueousoxalicacidsolu? tion VariousratiooftheorganicsolventZMeOH:CH3CN(4:O.5,510.5and6: 0.5)]andaqueousoxalicacidsolution(0.1mol/LtpH3)weretestedasmobile phase.Fig.3showstheelutiondiagramsseparatedoftetracyclineso11Lichroprep RP-18columnchr0mat0graphyusingvariousratiooftheorganicsolventand aqueousoxalicacidsolution.AsshowsinFig.3,TC(orOTC),CTC,MTC andDCweredisplayedagoodseparationinsolventsystemscontainingtheratio oftheorganicsolvent—aqueousoxalicacidat4.5:10and5.5:10CFig.3(atb)), butalowerresolutionbetweentheCTC,MTCandDCwasobtainedwithsolv? entsystemofratiooforganicsolventandaqueousoxalicacidat6.5:10,altho? ughitcanbeshortenedtheseparationtimeo11theColumn.Ontheotherhand, thepeaksofTC(+OTC),CTC,MTCandDCwereseparatedmoreconcentric. allyandseparationtimeonthecolumnwasshortenedwithincreasingtheratio 0forganicsolventsinthesolveatsystems.Thereforeitseemsthattheratiobe? tweenorganicsolventsandaqueousoxalicacidsolutionat5.5:10wasmoresuit? ableforseparationofTC(orOTC),CTC,MTCandDC. 4.Proportionofthemethanolandaeetonitrile Astheresultsoftheexperimentsofselectedsolventsystems,suggestingthat mixtureofMeOHandCH3CNisrequiredtoseparatetetracyelines.Sovarious fatio0fMeOHandofCH3CNweretestedusingtheorganicsolvents(MeOHl ? 189? r舄 ?:/}mm , m m? "0 "|J\一m厂.. , H,,,一. . . . 墓 g? T_k-mb虹 oz^ Fig.3Separationoftetracyc~nesbyLichrcprepRP一18columnchromatoggaphy iasolventsofdiifer~tpropor~onbetweentheorganicsolventandaqlleousoxalic ^cid.Samples*3OmgmixtureofTC,OTc'CTC,MTCandDCJdutlonsolvents? MeOH:CHsCNlImol/Loxalicacid(pH3)(4:0?5I10?5;0?5l10and8=0?5li0)lflow rate:O.7,O.8ml/mln,3ml/fractionjdetection=380nm CHCN):以1mol/Loxalicacidsolution(pH3)(5.5I10).Fig.4showsal1ofel? utiondiagraresoftheratiobetweentheMeOHandCH3CNin4:1.5,4.5:1.5and 5.5:OweredisplayedfourpeaksofTC(+OTC),CTC,MTCandDCandthe separationtimeonthecolumnwereshortenedwithCH3CNincreasedinsolvent systems.Howevertheseparationtimeonthecolumnwasincreasedwiththe mixtureofMeOHandoxalicacidandthesolventsofMeOHt0.1mol/Loxalic acidin4:1.5gaveapoorresolutionbetweenCTCtMTC,andDCCFig.4 (a)],the4.5;1mixturealsogavelowerresolutionbetweenMTCandDCthan didthe5:0.5mixture.Thusthe510.5mixturewasmoreoptimalforseparation ofTC(orOTC),CTC,MTCandDC. C0NCLUSl0N Basedontheoveral1resuits.itisdemonstratedthattheLichoprepRP一18 lowpressureliquidchromatographyisasimple,rapidmethodforseparation ofTC,CTC,MTCandDC.ThesolventsystemMeOH=CHaCNl0.1mol/Lox- alicacid(pH3)(5:O.5I10)wusthemostoptimalsolventsystemforseparation ofTC(0rOTC),CTC,MTCandDCalthoughitdidnotshowevidenceofse? parati0nbetweentheTCandOTC.Ontheotherhand,theLichroprepRP-18 columnusedcanberecoveredtouseagainaftertheyweretreatedwith0.01 mol/LHCI—MeOH,MeOH-Ht0andMeOHrespectlrely. REFERENCES [1)0h.H.etallJ.Chromatogr.,1987;393(2)l285~'298 ?190? 一 一 『 , 罨仙 C-一一 言j《 jjt 3 ?2 { 2 乏 - 0 3 2 { 喾 ,c Fig.4SeparationoftetracyclinesbyLichroprepRP_18columnchromatography insolventscontainingdifferentratioofMeOHandCHlCN.Samples,30ragTCt OTC,CTc.MTCandDC,elutionsolvents=MeOH,CHaCN,o.1mol/Loxalicacid(pH 3)(4}i.5l10,4.5t|l10,5}0.5ll0and5.5:0l19)Iflowrate=0.7,o.8,-1/mini3ml/ fraction~detectionl38Onm [2]Oka.H.etal:JChromatogr.1984314l303,31| [3]Oka,H.etallJChromatogr.1984}298(3)}435~45 []Ho丑,J,Y.C.etailJLjqChromatogrJ982.f5(1o)'】973,9D [5]Haso~,Tayyabaetal}JChromatogr.1985~8210)426 [6]Jiag—yuanZouetal}ChinJAnibiot,|992;iz(s)一3{1,347 f 环妻,航萄鳓 匹环类抗生素的反相制备层析I1.应用反相LICHROPREPRP-18 高效低压液相层析分离匹环类抗生素 (四川抗菌素工业研究所.成都610051) 米切尔 (堪萨斯太学药物化学系,劳伦斯美国,66045) 『双.弘 擒姜反相高压敢相层析是一种普遍应用的高压液相层桁技术.它使用一非投性的固定相和 一 极性的移动相,尤其适音分离非极性和中等扳性的化音钳.使用C和C..改造的硅腔柱的高压液 相来 分析 定性数据统计分析pdf销售业绩分析模板建筑结构震害分析销售进度分析表京东商城竞争战略分析 四环类抗生索的方法已有许多报道,但都是分折性的.对于四环类抗生索来说,要成功 地在一般常用的介质如氧化铝硅腔,离子交换树脂来进行制备性分离却显得极性太大.反相层 析硅腔LichroprepRP-18和RP一8的发展提供了一个新韵分离介质.应用四环索,土霉素,金霉 甲烯土霉索和强力霉索作材料,探索了应甩LJchroprepRP一|8高效低压柱层折来分离邀些抗生 索的条件.试验了瘩刺的配比.所用水相的pH,草酸疼液的浓度等.确定了使用溶剂景统MeOH CH,CN,9.1moI/Lozaficacid(pH3.0)(5I9.5I|0)艉成功地在这几种抗生素的混合物中有效的 分离出四环索,金霉索,甲烯土霉素和强力霉索.其方法简便,快速,且用过的LichrcprepRP一 |8柱经适当处理后可重复使用. 关t词四环共抗生索I层折,反耜层析|高效坻压层斩 ?191?