川芎嗪衍生物tbn的hplc含量测定方法

QUANTITATIVEHPLCANALYTICALMETHODFORTETRAMETHYLPYRAZINEDERIVATIVETBNSTUDENTNAMEMAKSAUHANGSTUDENTNOMAJORSUPERVISORPROFESSORWANG,YUQIANGDATEDD/MM/YYYY暨南大学本科生毕业论文论文题目川芎嗪衍生物TBN的HPLC含量测定方法学院国际学院学系药学系专业药学姓名学号指导教师STATEMENTOFORIGINALITYIHEREBYDECLARETHATTHETHESISPRESENTEDISTHERESULTOFRESEARCHPERFORMEDBYMEPERSONALLY,UNDERGUIDANCEFROMMYSUPERVISORTHISTHESISDOESNOTCONTAINANYCONTENTOTHERTHANTHOSECITEDWITHREFERENCESTHATHASBEENPREVIOUSLYPUBLISHEDORWRITTENBYOTHERS,NORDOESITCONTAINANYMATERIALPREVIOUSLYPRESENTEDTOOTHEREDUCATIONALINSTITUTIONSFORDEGREEORCERTIFICATEPURPOSETOTHEBESTOFMYKNOWLEDGEIPROMISETHATALLFACTSPRESENTEDINTHISTHESISARETRUEANDCREDITABLESIGNED_DATEQUANTITATIVEHPLCANALYTICALMETHODFORTETRAMETHYLPYRAZINEDERIVATIVETBNABSTRACTAHIGHPERFORMANCELIQUIDCHROMATOGRAPHYHPLCMETHODFORANEWTETRAMETHYLPYRAZINEDERIVATIVE,TBN,WASDEVELOPEDANALYTICALPERFORMANCEPARAMETERSSUCHASLINEARITY,PRECISION,ACCURACY,SPECIFICITY,LIMITOFDETECTIONLODANDLIMITOFQUANTIFICATIONLOQWEREDETERMINEDUNDERTHEGUIDELINEOFINTERNATIONALCONFERENCEONHARMONIZATIONQ2B1TBNWASANALYZEDBYRPHPLCWITHC18COLUMNUSINGMETHANOLWATER3565ASMOBILEPHASETHEFLOWRATEIS08ML/MINANDTHEDETECTORWASSETTO295NMTHELINEARITYOFCALIBRATIONCURVEISGOODR20999ANDTHELODANDLOQWERE12096NG/MLAND4032NG/MLRESPECTIVELYTHERELATIVESTANDARDDEVIATIONRSDOFPRECISIONANDACCURACYWERE01245AND06895,RESPECTIVELY,ANDTHESAMPLERECOVERYWAS9908RSD040THISMETHODISRELIABLEANDEASYFORTBNANALYSISKEYWORDSTETRAMETHYLPYRAZINEDERIVATIVE,TBN,HPLC川芎嗪衍生物TBN的HPLC含量测定方法摘要目的建立川芎嗪衍生物TBN高效液相含量测定方法。方法选用C18色谱柱；甲醇水3565；流速08ML/MIN；检测波长为295NM。结果TBN在12423105G/ML范围内线性关系良好R20999；定量限和检测限分别为12096NG/ML及4032NG/ML；精密度及准确度RSD分别为01245及06895；加样回收率结果为9908RSD040。结论本方法简便可靠，可作为TBN之定量分析方法。关键词川芎嗪衍生物，TBN，高效液相CONTENTS1INTRODUCTION32MATERIALS321CHEMICALS322APPARATUS33EXPERIMENTALMETHOD331TBNSTANDARDPREPARATION3311COLUMNCHROMATOGRAPHYFORTBNPURIFICATION3312SEMIPREPARATIVECOLUMNCHROMATOGRAPHY332DEVELOPMENTOFHPLCMETHODFORTBN3321MAXDETERMINATION3322OPTIMIZATIONOFHPLCCONDITIONSFORTBN333VALIDATIONOFHPLCMETHOD3331SPECIFICITY3332LINEARITY3333PRECISION3334LIMITOFQUANTIFICATIONANDLIMITOFDETECTION3335RANGE3336ACCURACY3337STABILITY3338RECOVERY3339CONTENTASSAY34RESULTSANDDISCUSSION35CONCLUSION3ACKNOWLEDGEMENT3REFERENCES31INTRODUCTIONTBNISANOVELCOMPOUNDDEVELOPEDBYTHEINSTITUTEOFNEWDRUGRESEARCHATTHEPHARMACYCOLLEGE,JINANUNIVERSITYTBNHASBEENSHOWNTOBEANTIOXIDATIVEANDTHROMBOLYTIC,ANDISUNDERDEVELOPMENTASATREATMENTFORISCHEMICSTROKE2TBNISADERIVATIVEOF2,3,5,6TETRAMETHYLPYRAZINETMP,WHICHISTHEMAINACTIVEINGREDIENTOFLIGUSTICUMWALLICHIIFRANCHATCHUANXIONG,FORMEDBYCONJUGATINGTMPANDANITRONEMOIETYSEEFIGURE1TMPISUSEDTOTREATISCHEMICSTROKEINCHINAFORMANYYEARSWHICHWASFOUNDBENEFICIALININHIBITINGPLATELETAGGREGATION3,LYSINGBLOODCLOTS4,BLOCKINGCALCIUMENTRY5ANDSCAVENGINGREACTIVEOXYGENSPECIESROS6WHILERETAININGTHETHROMBOLYTICACTIVITYOFTMP,THENITRONEADDEDTOTMPIETBNHADBEENPROVEDTOPROVIDEASTRONGANTIOXIDATIVEACTIVITYNITRONESAREUSEFULASTHERAPEUTICAGENTSFORNEURALANDSYSTEMICDISEASESSUCHASATHEROSCLEROSIS,SEPTICEMIA,STROKE,ANDALZHEIMERSDISEASE712NNH3CCH3H3CCH3NCOR3R2R13NNH3CCH3CHH3CNOCCH3CH3CH3FIGURE1STRUCTUREOFTMP1NITRONE2TBN3ASAPOTENTIALNEWDRUG,SERIESOFRESEARCHESLIKEPHARMACOLOGY,TOXICOLOGY,PHARMACOKINETICS,PHARMACODYNAMICSARENEEDEDTHEREFORE,QUALITYANALYSISANDCONTROLONTBNISNECESARYFIRSTOFALL,AQUANTITATIVEMETHODOFTBNHADTOBEDEVELOPEDHPLCMETHODFORANALYZINGCHEMICALCOMPOUNDISQUICK,SIMPLEANDRELIABLETHEREFOREWECHOSEITASTHEANALYZINGMETHODFORTBNARELIABLEHPLCMETHODSHOULDBEABLETOSEPARATETHESAMPLEFROMITSIMPURITIESCOMPLETELYANDCANBEVALIDATEDPROPERLY,SOCALLEDTHEMETHODOLOGYTHEVALIDATIONOFDEVELOPEDHPLCMETHOD1,8INCLUDEDSEVERALPARTSWHICHARELINEARITY,PRECISION,RANGE,LIMITOFQUANTIFICATIONANDLIMITOFDETECTION,ACCURACY,STABILITY,SPECIFICITY,ANDRECOVERYTHECONTENTASSAYWOULDBEDONEAFTERTHEVALIDATIONOFMETHODHOWEVER,REFERENCESTANDARDOFTHESUBSTANCETOBETESTEDISNEEDEDINSOMEITEMSOFTHEHPLCMETHODVALIDATIONFORTHISCASE,TBNISANEWCOMPOUNDANDITSREFERENCESTANDARDISUNAVAILABLETHEREFORE,INTHISPAPER,ARELATIVELYPURESAMPLEWASUSEDASAREFERENCESTANDARDHENCE,TOOBTAINARELATIVELYPURETBNSAMPLE,APURIFICATIONOFTBNWASALSODONEINTHISRESEARCHTHETARGETOFTHISRESEARCHISTOESTABLISHANEASYANDRELIABLEHPLCMETHODFORTBNANALYSIS,USEDINROUTINEQUALITYCONTROLOFITSRELATEDSTUDIESAFTERTHEESTABLISHMENTOFTBNREFERENCESTANDARD,THISHPLCMETHODCANBEAPPLIEDDIRECTLYTOQUANTIFYTBNCONTENTINTHISRESEARCH,THEREWERETHREEMAINPARTSTBNSTANDARDPREPARATION,HPLCMETHODESTABLISHMENT,ANDMETHODVALIDATION2MATERIALS21CHEMICALSTBN,PREPAREDBYTHEINSTITUTEOFNEWDRUGRESEARCHATPHARMACYCOLLEGE,JINANUNIVERSITYPETROLEUMETHER,ETHYLACETATE,ACETONEANDDICHLOROMETHANEANALYTICALREAGENTSPURCHASEDFROMFUYUREFINEDCHEMICALPRODUCTSLTDREAGENTGRADEDSILICAGEL200300MESHPURCHASEDFROMBRANCHOFQINGDAOHAIYANGCHEMICALPLANTMETHANOLHPLCGRADEPURCHASEDFROMJIANGSUHANBONSCIDOUBLEDISTILLEDWATERPROVIDEDBYTHEINSTITUTESODIUMDIHYDROGENPHOSPHATEANDDISODIUMHYDROGENPHOSPHATEANALYTICALREAGENTS,PURCHASEDFROMGUANGZHOUCHEMICALREAGENTFACTORY22APPARATUSGLASSCHROMATOGRAPHICCOLUMNTHINLAYERCHROMATOGRAPHICSILICAGELPLATESROTARYEVAPORATOREYELAN1001ANDDIGITALWATERBATHEYELASB1000VACUUMDRYINGOVENDZF6050ULTRASONICCLEANERSKQ250EMELTINGPOINTMEASURINGINSTRUMENTSGWX4SHIMADZUUVVISSPECTROPHOTOMETERSUV2450SHIMADZU10ATHPLCANDSPD10AVPUVVISDETECTORLUBEXKROMASILC18COLUMN5100250MM46MMSEMIPREPARATIVEHPLCCOLUMNVYDACRPC1890APHARMACEUTICALELECTRONICBALANCEACCULABALC1104OTHERAPPARATUSVOLUMETRICFLASKS,CONICALFLASKS,PIPETTE,MICROFILTERSANDMEMBRANES045M3EXPERIMENTALMETHOD31TBNSTANDARDPREPARATIONTBNISAWHITECRYSTALWHICHISWATERSOLUBLE,WITHMOLECULARWEIGHTOF221G/MOLDUETOTHEDIFFERENCEINPURITYBETWEENEACHBATCHESOFPRODUCTION,THEPHYSICALPROPERTIESOFTBNSAMPLESAREVARIEDFROMONEANOTHERINTHISPART,RELATIVELYPURETBNSAMPLEWOULDBEPREPAREDANDTHENUSEDINTHEVALIDATIONOFHPLCMETHODASREFERENCESTANDARD311COLUMNCHROMATOGRAPHYFORTBNPURIFICATION1TLCMETHODUSEETHYLACETATEASTHESOLVENTTODISSOLVETBNSAMPLE,SEPARATETHESAMPLECOMPONENTSWITHDIFFERENTMOBILEPHASESATHECONTINUOUSSEPARATINGCONDITIONPETROLEUMETHERETHYLACETATE11BMODIFIEDCONDITION1PETROLEUMETHERETHYLACETATE41CMODIFIEDCONDITION2PETROLEUMETHERACETONE51FIGURE2TLCOFTBNWITHDIFFERENTMOBILEPHASEFROMLEFTTORIGHTA,B,C2COLUMNCHROMATOGRAPHYFROMTHERESULTSOFTLC,METHODBANDCWEREIMPLEMENTEDINCOLUMNABOUT300MGOFTBNWEREUSEDINEACHMETHODFORMETHODB,YELLOWSEMISOLIDWASOBTAINEDVERYPALEYELLOWSOLIDWASOBTAINEDINMETHODCHENCE,METHODCWASCHOSENBATCH20081011TBN426GWASPUTINCOLUMNCHROMATOGRAPHYWITHTHEMOBILEPHASEPETROLEUMETHERACETONE51AND288GOFPURIFIEDTBNWASYIELDEDMELTINGPOINTWASTESTED77312SEMIPREPARATIVECOLUMNCHROMATOGRAPHYSINCETHEREWERETRACEAMOUNTOFIMPURITIESINTHEPURIFIEDTBNFROM311,SEMIPREPARATIVECOLUMNCHROMATOGRAPHYWASPERFORMEDFORFURTHERPURIFICATIONONEHUNDREDMGOFTBNWASUSEDTHISTIME,ANDAMETHANOLWATER3565SYSTEMWASUSEDASAMOBILEPHASEHOWEVER,SHIMADZU10ATHPLCISNOTDESIGNEDFORSEMIPREPARATIVECOLUMN,TOOMUCHSOLVENTWASUSEDANDTOOFEWTBNHAVEBEENPURIFIEDTHISMETHODISNOTFEASIBLEFORPRODUCINGSUFFICIENTPURIFIEDTBNANDFORTHETBNPURIFIED,SINCETHEREWASTOOMUCHWATERINTHEMOBILEPHASETHATWASDIFFICULTTODRY,ONLYTRACEAMOUNTOFTBNWASOBTAINEDWHICHWASNOTENOUGHFOREXPERIMENTALUSEWITHTHERESULT,THETBNPURIFIEDFROM311WASUSEDASREFERENCESTANDARD32DEVELOPMENTOFHPLCMETHODFORTBN321MAXDETERMINATIONBEFORETHEPROCEDUREOFHPLC,THEMAXOFTBNSHOULDBEDETERMINEDWITHTHEUVVISSPECTROPHOTOMETER,THETBNSAMPLESOLUTIONWASSCANNEDIN200400NMFORTHEDETERMINATIONOFMAXMETHODTBN001GFROM311WASWEIGHTEDANDWASDISSOLVEDIN10MLVOLUMETRICFLASKWITHMETHANOLDILUTETHESOLUTIONTO50G/MLBYUSINGPIPETTEANDVOLUMETRICFLASKSSCANTHESAMPLESOLUTIONWITHTHEUVVISSPECTROPHOTOMETERANDRECORDTHERESULTFIGURE3UVSPECTROGRAMFORTBNFROMTHEABOVERESULT,295NMWASCHOSEFORTBNDETECTION322OPTIMIZATIONOFHPLCCONDITIONSFORTBNLUBEXKROMASILC18COLUMN5100250MM46MMWASUSEDINTHISRESEARCHWHICHISSUITABLEFORMOSTORGANICCOMPOUNDDIFFERENTRATIOOFMOBILEPHASE,FLOWRATE,PH,HAVEBEENUSEDTODETERMINETHEMOSTSUITABLEHPLCMETHODFORTBNWITHTHEAIMOFSEPARATINGTHETBNFROMITSIMPURITIES,TBNSAMPLEWITHOUTPURIFICATIONBATCH20081019WEREUSEDTHEMOBILEPHASEWEREPREPAREDDAILYANDDEGASSEDBYTHEULTRASONICCLEANSERFORNOTLESSTHAN20MINUTESTABLE1DIFFERENTHPLCCONDITIONSTESTEDFORTBNMETHANOLWATERDETECTORFLOWRATEPH70300910ML/MINNOADDED60400808ML/MINNOADDED50500810ML/MINNOADDED,707645550810ML/MINNOADDED,7240600810ML/MINNOADDED3565295NM08ML/MINNOADDED,697233VALIDATIONOFHPLCMETHODTHEREFERENCESTANDARDUSEDINTHISPARTWASPURIFIEDIN31,WHICHWASASSUMED985PURETHEHPLCCHROMATOGRAMOFTBNREFERENCESTANDARDSHOWEDABOUT99PURITYOFTBN,BUTSINCETHEUVDETECTORINHPLCCANNOTDETECTOREVERYCOMPONENTINTHESAMPLE,THEREFORETHEPERCENTAGECONTENTOFTBNISSTILLUNKNOWN331SPECIFICITYTWOMETHODSHAVEBEENUSEDTOEVALUATETHESPECIFICITYOFTHEESTABLISHEDHPLCMETHOD1RAWMATERIAL2DEGRADEDSAMPLE1METHOD1WEIGHTPRECISELYABOUT10MGOFTMPRAWMATERIAL,DILUTEDIN10MLVOLUMETRICFLASK,TAKES2MLINTOA5MLVOLUMETRICFLASKANDDILUTEDTOTHEMARK,TAKE1MLOFTHESOLUTIONINTOA10MLVOLUMETRICFLASK,ADD05MLOFTHETBNSTANDARDSOLUTIONANDDILUTEDTOMARKTHEDILUTIONSWEREDONEWITHMOBILEPHASEINJECTANDRECORDTHECHROMATOGRAMMETHOD2USETHETBNSTANDARDSOLUTIONSTAYEDFOR48HOURSINJECTANDRECORDTHECHROMATOGRAM332LINEARITYTHEMETHODUSEDINLINEARITYHEREHADBEENUSEDINLOTSOFLITERATURE917WEIGHT00630GOFTBNIN50MLVOLUMETRICFLASKANDDILUTETOMARKPURITYOFTBNWASASSUMED985,THUSTHEREWERE00621GOFTBNINTHESTANDARDCONCENTRATIONWASEQUALTO1242MG/ML,THUS1242G/MLTHESTANDARDSOLUTIONOFTBNMEASUREPRECISELY01ML,05ML,10ML,15ML,AND20MLINDIVIDUALLYINTOFIVE10MLVOLUMETRICFLASKSANDDILUTETOMARKSALLOFDILUTIONSWEREDONEBYMOBILEPHASEINJECTTHESOLUTIONANDRECORDHEPEAKAREASPLOTASTANDARDCURVEANDCALCULATETHER2333PRECISIONMEASURE22MLFROMTHETBNSTANDARDSOLUTION1242G/MLINTOA10MLVOLUMETRICFLASK,DILUTEDTOTHEMARKWITHMOBILEPHASEINJECTTHETBNSOLUTIONFOR6TIMESCONTINUALLYANDRECORDTHEPEAKAREASFOREVALUATINGTHEPRECISIONANDREPEATABILITY1,818334LIMITOFQUANTIFICATIONANDLIMITOFDETECTIONDILUTETHETBNSTANDARDSOLUTION1242G/MLGRADUALLYUNTILTHEPEAKHEIGHTEQUALTO101AND31TOTHEBASELINENOISERESPECTIVELY1335RANGETHESTANDARDCURVEOBTAINEDFROMLINEARITYWOULDGIVETHERANGE1,818336ACCURACYSIXPORTIONSOF15MLTBNSTANDARDSOLUTION1242G/MLWEREMEASUREDANDDILUTEDINSIX10MLVOLUMETRICFLASKS,NAMED16INJECTTHESOLUTIONSANDRECORDTHEPEAKAREAS1518337STABILITY15MLTBNSTANDARDSOLUTION1242G/MLWASMEASUREDANDDILUTEDIN10MLVOLUMETRICFLASKTHESOLUTIONWASINJECTEDAT0,2,4,6,8HOURSRESPECTIVELYAFTERPREPARATION,RECORDTHEPEAKAREAS1518338RECOVERY1CONTENTASSAYFORSAMPLERECOVERY1,917USETHEBATCH20081107ASSAMPLEABOUT10MGTBNWASPRECISELYWEIGHTEDANDDISSOLVEDIN10MLVOLUMETRICFLASKWITHMOBILEPHASESOLUTIONTOTHEMARKMEASURE1MLFROMTHESAMPLESOLUTIONANDDILUTEDIN10MLVOLUMETRICFLASKWITHMOBILEPHASEINJECTTHESOLUTIONFOR3TIMESANDRECORDTHEPEAKAREAAPPLYTOTHESTANDARDCURVEANDCALCULATETHEPERCENTAGECONTENTOFTHETBNSAMPLE2RECOVERYTESTTBNBATCH20081107WASUSEDINTHISPARTASSAMPLEMETHODMEASURE3MLFROMTHETBNSAMPLESOLUTIONCONCENTRATION109G/ML,8615TBNCONTENTTONINE10MLVOLUMETRICFLASKSRESPECTIVELYMEASURETHESTANDARDSOLUTIONCONCENTRATION1242G/MLWHICHWASEQUIVALENTTO80,100AND120OFTHETBNCONTENTINTHESAMPLESOLUTIONRESPECTIVELY,ANDTRANSFERTOTHEVOLUMETRICFLASKSWITHTBNSAMPLESOLUTION,3PORTIONSFOREACHWEREPREPAREDDILUTETHESOLUTIONTOTHEMARKSWITHMOBILEPHASEINJECTANDRECORDTHEPEAKAREAS339CONTENTASSAYABOUT10MGTBNWASWEIGHTEDPRECISELYANDDISSOLVEDIN10MLVOLUMETRICFLASKWITHMOBILEPHASESOLUTIONTOTHEMARKREPEATSTEP1FORTOTAL4BATCHESOFTBNMEASURE1MLTBNSOLUTIONFROMEACHOFFLASKSANDDILUTEINANOTHER4VOLUMETRICFLASKSWITHMOBILEPHASESOLUTIONTOTHEMARKSINJECTTHE4BATCHESOFDILUTEDSOLUTIONTOHPLCFOR3TIMESEACHANDRECORDTHEPEAKAREAAPPLYTHERESULTTOTHESTANDARDCURVEANDCALCULATETHETBNCONTENTINEACHBATCH15184RESULTSANDDISCUSSIONOPTIMIZATIONOFHPLCCONDITIONSFORTBNFIGURE4METHANOLWATER3565FLOWRATE08ML/MINSOLUTIONSTAYEDFOR48HOURSTHISHPLCMETHODWASPERFORMEDINTWOSHIMADZU10ATHPLCMACHINESOFPHARMACYCOLLEGEANDHADSIMILARRESULTSIDECIDEDTOSTARTTHEMETHODOLOGYEXPERIMENTWITHTHISCONDITIONTOSEEIFTHERESULTWOULDBEVALIDTHEHPLCMETHODDEVELOPEDINTHISARTICLEWASEVALUATEDTHATISSUITABLEFORTBNANALYSISSINCETHEPRESENCEOFIMPURITYWITHSIMILARTBNPOLARITY,NOTONLYTHERATIOOFMOBILEPHASE,BUTTHEFLOWRATEHASTOBEADJUSTED,HENCETHELONGERANALYZINGTIMEINTHEMETHODFROMTHECHROMATOGRAMSHAVEBEENDONE,VARIATIONINTHERETENTIONTIMEOFTBNWASPRESENCEDUETOTHESMALLDIFFERENCEINTHEVOLUMERATIOOFDAILYPREPAREDMOBILEPHASEANDTHEVARIATIONISBETWEEN2427MINVALIDATIONOFHPLCMETHODSPECIFICITYMETHOD1FIGURE5CHROMATOGRAMOFTBNTMPMETHOD2FIGURE6CHROMATOGRAMOFTBNSTANDARDSOLUTIONSTAYEDFOR48HOURSMETHOD2WASMORESIGNIFICANTHERE,ASLONGASTHESTUDY,THEREWERENOOBSERVABLETMPINTHETBN,BUTTHEHPLCMETHODSTILLPROVEDFEASIBLEWHENTMPISPRESENTFROMTHERESULTSABOVE,THEHPLCMETHODESTABLISHEDWASWELLSEPARATEDWITHTHEIMPURITIESINTBNSAMPLELINEARITYTABLE2RESULTSOFLINEARITYCONCENTRATIONG/MLPEAKAREAUVS124287310462104298987186301350701627324200228853105023350922FIGURE7THESTANDARDCURVEWASRELIABLE,R20999BESIDESTHEABOVERESULT,THEREARETWOINVALIDRESULTSHAVEBEENDONEDURINGTHERESEARCHMETHOD1THISMETHODWASUSEDBECAUSEITWASUSEDINANARTICLEPUBLISHEDFROMINSTITUTEOFDRUGCONTROL18ABOUT10MGTBNWASWEIGHTEDFROMTBNSTANDARDPRECISELY,DILUTESIN50MLVOLUMETRICFLASKTOTHEMARKINJECT2,4,6,8,10,12,14,16LPRECISELYINTOHPLC,RECORDTHEPEAKAREASTABLE3RESULTSOFMETHOD1INJECTIONVOLUMELPEAKAREAUVS217131154407336466280787887568301010976877121315832914151845861617755992FIGURE8STANDARDCURVEOFMETHOD1THERESULTOFTHISMETHODWASGOODINCORRELATIONCOEFFICIENTHOWEVER,THERANGEOFTHISCURVEWASTOONARROWALSO,ITISNOTACCURATETOUSETHEMICROINJECTORFORSAMPLINGASAMPLERSHOULDBEUSEDMETHOD2THISMETHODWASUSEDCOMMONLYINTHEESTABLISHMENTOFSTANDARDCURVEWHICHHAVEBEENUSEDINMOSTOFARTICLESIFOUND917THEADVANTAGESOFTHISMETHODAREACCURATE,WIDERANGEINTHECURVE,BUTITISSOMEWHATMOREDIFFICULTTOOPERATEBECAUSETHEERRORCOMESFROMTHESOLUTIONSPREPARATIONWOULDAFFECTTHECORRELATIONCOEFFICIENTOFTHESTANDARDCURVEDIRECTLY00630GOFTBNWASWEIGHTEDIN50MLVOLUMETRICFLASKANDDILUTEDTOMARKASSUMEDPURITYOFTBNWAS985,THUSTHEREWAS00621GOFTBNINTHESTANDARDCONCENTRATIONWAS1242G/MLMEASUREPRECISELY01ML,05ML,10ML,15ML,AND20MLINDIVIDUALLYINTOFIVE10MLVOLUMETRICFLASKSANDDILUTETOMARKSALLOFDILUTIONSWEREDONEBYMOBILEPHASETABLE4RESULTSOFMETHOD21STTIMECONCENTRATIONG/MLPEAKAREAUVS124111061562620554848641124119518987186165133669442482217509484,FIGURE11THESTANDARDCURVEOFMETHOD2WHICHWASUNRELIABLEBECAUSER20999INTHEEVALUATIONOFLINEARITY,2METHODSHAVEBEENUSED,ANDONLYMETHOD2ISVALIDTHEINVALIDOFMETHOD1WEREDUETO1THERANGEOFEVALUATIONWASTOONARROWIE8FOLDS2THEERRORCOMESFROMMANUALINJECTIONOFDIFFERENTVOLUMEWERETOOLARGETHEREFORE,METHOD2WASUSEDAFTERHAVINGDISCUSSIONWITHPROFESSORBUTTHERESULTOFMETHOD2WASNOTGOODINTHEFIRSTTIMEANDTHEOPERATIONTECHNIQUEPROBABLYTHESOURCEOFTHEERRORBYINSPECTINGTHEPROCEDUREANDTECHNIQUEIUSED,SOMEIMPROPERANDCARELESSMISTAKESHADBEENFOUND1USEMETHANOLINSTEADOFMOBILEPHASETORINSETHEMICROINJECTOR2INJECTTHENEXTSAMPLEWITHOUTRINSETHEMICROINJECTORWITHTHATSAMPLE3BOTHOFTHESAMPLERANDTHEMICROINJECTORSHOULDBECLEANEDWITHMOBILEPHASECOMPLETELYBEFOREUSEANDBETWEENEACHOFINJECTIONS,INSIDEANDOUTSIDEBYCORRECTINGTHEIMPROPERTECHNIQUE,THESTANDARDCURVEWASDONEWITHR20999SEEFIGURE7STANDARDCURVEPROVIDEDARELIABLEQUANTITATIVEMEASUREMENTFORASSAYINGTHEUNKNOWNSUBSTANCEPRECISIONTABLE5RESULTOFTHE6INJECTIONS,THEREWERENOSIGNIFICANTDIFFERENCEBETWEENINJECTIONSSAMPLINGNOPEAKAREAUVS120080153220067338320094488420022885520051016620071876SUM120387756MEAN20064626S2498114248RSD01245BYTHERESULTABOVE,THEDEVELOPEDHPLCMETHODHADAGOODPRECISION,THERSDBETWEENEACHINJECTIONSWERENEGLIGIBLETHISHPLCMETHODHASAGOODREPEATABILITYTHEGOODREPEATABILITYOFHPLCMETHODENSUREDAGOODPRECISIONINTHECONTENTASSAY,THELESSTHERSD,THEHIGHERTHEPRECISIONLIMITOFQUANTIFICATIONANDLIMITOFDETECTIONFIGURE10HPLCCHROMATOGRAMOFTBN4032NG/MLLOQFIGURE11HPLCCHROMATOGRAMOFTBN12096NG/MLLODFIGURE12BLANKTHEREWERESOMEDIFFICULTIESINTHISPARTBECAUSEEVENTRACEAMOUNTOFRESIDUEWOULDAFFECTTHERESULTAFTERSOMEFAILURE,IFOUNDTHATHARSHREQUIREMENTSWERENEEDEDINTHISEXPERIMENT,HEREISMYCONCLUSION,1RINSETHEMICROINJECTORWITHMOBILEPHASECOMPLETELYBEFOREANDAFTERUSE,ANDWIPETODRY2RINSETHESAMPLERWITHMOBILEPHASEINSIDEANDOUTSIDE3RINSETHEMICROINJECTORWITHTHESAMPLENOTLESSTHANTENTIMEBEFOREINJECTION4INJECTTHEMOBILEPHASEWITHOUTBINDINGTHEINJECTIONVALVETOCLEANTHESAMPLERFINALLY,THEVALIDRESULTSWEREOBTAINEDWITHIMPROVINGOFMYSKILLSTHELOQOFTBNIS4032NG/MLANDTHELODOFTBNIS12096NG/MLFROMTHOSEMAGNIFIEDCHROMATOGRAMS,THEPEAKSINTHEVERYBEGINNINGOFTHECHROMATOGRAMSARENOTBELONGTOTBN,THEYSHOULDBEELECTRONICSIGNALSRANGETHECALIBRATIONCURVEGAVEANACCURATE,PRECISERANGEFROM12423105G/MLFIGURE11THERANGEDONEINTHESTANDARDCURVEWASWIDEENOUGHMORETHAN20FOLDSBUTITCANBESTILLIMPROVEDBYCOVERINGTHECONCENTRATIONOFTHELOQ4032NG/MLIFTHISPOINTISADDEDINTOTHESTANDARDCURVESEEFIGURE7,THEVALUEOFR2ISTHESAMEFIGURE15STANDARDCURVEWITHLOQBUTTHISCURVEISNOTVALIDSINCEITISNOTACONTINUALLYINJECTION,SOTHEHPLCCONDITIONHAVESLIGHTLYDIFFERENCEACCURACYTABLE7RESULTSOFACCURACYTEST,THEREWERENOSIGNIFICANTDIFFERENCEBETWEENSAMPLESSOLUTIONNOPEAKAREAUVS113545598213505194313712559413693187513591888613725935SUM81774361MEAN1362906017RSD06895FROMTHERESULTS,THEDEVELOPEDHPLCMETHODWASPROVEDAGOODACCURACYWITHLOWRSDVALUEFROMTHERESULTSOFPRECISIONANDACCURACY,THEHPLCMETHODOFTBNISHIGHINBOTHACCURACYANDPRECISIONSTABILITYTABLE8RESULTSOFSTABILITYTEST,SHOWSTHATTHESAMPLESOLUTIONWASSTABLEWITHIN8HOURSINJECTINGTIMEPEAKAREAUVS0HOUR135775112HOUR136297784HOUR135425786HOUR136087888HOUR13609209SUM67967864MEAN135935728RSD02507THERESULTSHOWEDABOVEPROVEDTHATTBNSOLUTIONWASSTABLEWITHIN8HOURSFROMTHEPROGRESSOFRESEARCH,TBNINSOLUTIONISNOTSTABLESODAILYPREPARATIONISNECESSARYDURINGTHEEXPERIMENT,ITISFOUNDTHATTBNISUNSTABLEINSOLUTIONFORM,ANDTHEPEAKAT18509MINOFTHECHROMATOGRAMSHOWEDINFIGURE6WASAPPEAREDAFTER24HOURSANDBECOMEMUCHLARGERASTIMEGOESIFTHESOLUTIONWASSTOREDINREFRIGERATOR,THISPEAKWOULDBESMALLERANDUNEASYTOOBSERVERECOVERYTHEPERCENTAGECONTENTOFBATCH20081107OFTBNWASTESTED8615TABLE10RESULTSOFRECOVERYTESTTBNCONTENTSAMPLEGTBNCONTENTSTANDARDGPEAKAREAUVSRECOVERYMEANRSD356114810078354038010051802817122340354576510083392998599023923723987210028171279253899721975743318249850433475498621202817133510429672497119908040THEFLUCTUATIONINTHEDIFFERENTCONCENTRATIONBATCHESMAYDUETOTHEERRORINPREPARINGSOLUTIONSTHERECOVERYPERCENTAGESHOULDBETWEEN95105,SOTHERESULTWASVALID,ANDTHERSDVALUESWERELOWTHATTHERESULTSHAVENOSIGNIFICANTERRORWITHINEACHPERCENTAGESAMPLEWITHINTEGRATINGTHERESULTFROMRECOVERYTESTANDACCURACYTEST,THEHPLCMETHODESTABLISHEDWASPROVEDAGOODACCURACYCONTE