川芎嗪衍生物TBN的HPLC跟量测定方法.docx

川芎嗪衍生物TBN的HPLC跟量测定方法.docx

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川芎嗪衍生物TBN的HPLC跟量测定方法

QuantitativeHPLCAnalyticalMethodforTetramethylpyrazineDerivativeTBN

StudentName

MAKSAUHANG

StudentNo.

2005055160

Major

Pharmacy

Supervisor

ProfessorWang,Yuqiang

Date（dd/mm/yyyy）

25/05/2009

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

2008年5月25日

StatementofOriginality

Iherebydeclarethatthethesispresentedistheresultofresearchperformedbymepersonally,underguidancefrommysupervisor.Thisthesisdoesnotcontainanycontent（otherthanthosecitedwithreferences）thathasbeenpreviouslypublishedorwrittenbyothers,nordoesitcontainanymaterialpreviouslypresentedtoothereducationalinstitutionsfordegreeorcertificatepurposetothebestofmyknowledge.Ipromisethatallfactspresentedinthisthesisaretrueandcreditable.

Signed:

_________________Date：

25-05-2009

QuantitativeHPLCanalyticalmethodfortetramethylpyrazine

derivativeTBN

Abstract:

Ahigh-performanceliquidchromatography（HPLC）methodforanewtetramethylpyrazinederivative,TBN,wasdeveloped.Analyticalperformanceparameterssuchaslinearity,precision,accuracy,specificity,limitofdetection（LOD）andlimitofquantification（LOQ）weredeterminedundertheguidelineofInternationalConferenceonHarmonizationQ2B[1].TBNwasanalyzedbyRP-HPLCwithC18columnusingmethanol-water（35:

65）asmobilephase.Theflowrateis0.8ml/minandthedetectorwassetto295nm.Thelinearityofcalibrationcurveisgood（r2>0.999）andtheLODandLOQwere12.096ng/mland40.32ng/mlrespectively.Therelativestandarddeviation（RSD）ofprecisionandaccuracywere0.1245%and0.6895%,respectively,andthesamplerecoverywas99.08%（RSD:

0.40%）.ThismethodisreliableandeasyforTBNanalysis.

KeyWords:

tetramethylpyrazinederivative,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C-18色谱柱；甲醇-水（35:

65）；流速：

0.8ml/min；检测波长为295nm。

结果：

TBN在12.42-310.5g/ml范围内线性关系良好（r2>0.999）；定量限和检测限分别为12.096ng/ml及40.32ng/ml；精密度及准确度RSD分别为0.1245%及0.6895%；加样回收率结果为99.08%（RSD:

0.40%）。

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

Contents

1.Introduction1

2.Materials2

2.1.Chemicals2

2.2.Apparatus3

3.ExperimentalMethod3

3.1.TBNstandardpreparation3

3.1.1.ColumnchromatographyforTBNpurification4

3.1.2.Semi-preparativecolumnchromatography5

3.2.DevelopmentofHPLCmethodforTBN5

3.2.1.maxdetermination5

3.2.2.OptimizationofHPLCconditionsforTBN6

3.3.ValidationofHPLCmethod7

3.3.1.Specificity7

3.3.2.Linearity7

3.3.3.Precision7

3.3.4.LimitofQuantificationandLimitofDetection7

3.3.5.Range7

3.3.6.Accuracy7

3.3.7.Stability7

3.3.8.Recovery7

3.3.9.ContentAssay7

4.ResultsandDiscussion7

5.Conclusion7

Acknowledgement7

References7

1.Introduction

TBNisanovelcompounddevelopedbytheInstituteofNewDrugResearchatthePharmacyCollege,JinanUniversity.TBNhasbeenshowntobeantioxidativeandthrombolytic,andisunderdevelopmentasatreatmentforischemicstroke[2].

TBNisaderivativeof2,3,5,6-tetramethylpyrazine（TMP）,whichisthemainactiveingredientofLigusticumwallichiiFranchat（ChuanXiong）,formedbyconjugatingTMPandanitronemoiety（seeFigure1）.

TMPisusedtotreatischemicstrokeinChinaformanyyearswhichwasfoundbeneficialininhibitingplateletaggregation[3],lysingbloodclots[4],blockingcalciumentry[5]andscavengingreactiveoxygenspecies（ROS）[6].WhileretainingthethrombolyticactivityofTMP,thenitroneaddedtoTMP（i.e.TBN）hadbeenprovedtoprovideastrongantioxidativeactivity.Nitronesareusefulastherapeuticagentsforneuralandsystemicdiseasessuchasatherosclerosis,septicemia,stroke,andAlzheimer’sdisease[7].

（1）

（2）

（3）

Figure1StructureofTMP

（1）;Nitrone

（2）;TBN（3）

Asapotentialnewdrug,seriesofresearcheslikepharmacology,toxicology,pharmacokinetics,pharmacodynamicsareneeded.Therefore,qualityanalysisandcontrolonTBNisnecesary.Firstofall,aquantitativemethodofTBNhadtobedeveloped.

HPLCmethodforanalyzingchemicalcompoundisquick,simpleandreliable.ThereforewechoseitastheanalyzingmethodforTBN.AreliableHPLCmethodshouldbeabletoseparatethesamplefromitsimpuritiescompletelyandcanbevalidatedproperly,socalledtheMethodology.

ThevalidationofdevelopedHPLCmethod[1,8]includedseveralpartswhicharelinearity,precision,range,limitofquantificationandlimitofdetection,accuracy,stability,specificity,andrecovery.Thecontentassaywouldbedoneafterthevalidationofmethod.However,referencestandardofthesubstancetobetestedisneededinsomeitemsoftheHPLCmethodvalidation.

Forthiscase,TBNisanewcompoundanditsreferencestandardisunavailable.Therefore,inthispaper,arelativelypuresamplewasusedasareferencestandard.Hence,toobtainarelativelypureTBNsample,apurificationofTBNwasalsodoneinthisresearch.

ThetargetofthisresearchistoestablishaneasyandreliableHPLCmethodforTBNanalysis,usedinroutinequalitycontrolofitsrelatedstudies.AftertheestablishmentofTBNreferencestandard,thisHPLCmethodcanbeapplieddirectlytoquantifyTBNcontent.

Inthisresearch,therewerethreemainparts:

TBNstandardpreparation,HPLCmethodestablishment,andMethodvalidation.

2.Materials

2.1.Chemicals

TBN,preparedbytheInstituteofNewDrugResearchatPharmacyCollege,JinanUniversity;

Petroleumether,ethylacetate,acetoneanddichloromethane（Analyticalreagents）purchasedfromFUYURefinedChemicalProductsLtd.;

Reagentgradedsilicagel（200-300mesh）purchasedfromBranchofQingdaoHaiyangChemicalPlant;

Methanol（HPLCgrade）purchasedfromJiangsuHanbonSci.&Tech.Co.,Ltd.;

Double-distilledwaterprovidedbytheInstitute;

Sodiumdihydrogenphosphateanddisodiumhydrogenphosphate（Analyticalreagents）,purchasedfromGuangzhouChemicalReagentFactory.

2.2.Apparatus

GlassChromatographiccolumn;

Thinlayerchromatographicsilicagelplates;

RotaryEvaporator（EYELAN-1001）andDigitalWaterBath（EYELASB1000）;

Vacuumdryingoven（DZF-6050）;

UltrasonicCleaners（KQ-250E）;

MeltingPointMeasuringInstrument（SGWX-4）;

ShimadzuUV-VISSpectrophotometers（UV-2450）

Shimadzu-10ATHPLCandSPD-10AVPUV-VISDetector;

LUBEXKromasilC18column（5100Å250mm4.6mm）;

semipreparativeHPLCcolumn（VYDACRPC1890APHARMACEUTICAL）;

Electronicbalance（ACCULABALC110.4）;

Otherapparatus:

volumetricflasks,conicalflasks,pipette,microfiltersandmembranes（0.45m）.

3.ExperimentalMethod

3.1.TBNstandardpreparation

TBNisawhitecrystalwhichiswatersoluble,withmolecularweightof221g/mol.Duetothedifferenceinpuritybetweeneachbatchesofproduction,thephysicalpropertiesofTBNsamplesarevariedfromoneanother.

Inthispart,relativelypureTBNsamplewouldbepreparedandthenusedinthevalidationofHPLCmethodasreferencestandard.

3.1.1.ColumnchromatographyforTBNpurification

（1）TLCmethod

UseethylacetateasthesolventtodissolveTBNsample,separatethesamplecomponentswithdifferentmobilephases.

A.Thecontinuousseparatingcondition:

petroleumether-ethylacetate（1:

1）

B.Modifiedcondition1:

petroleumether-ethylacetate（4:

1）

C.Modifiedcondition2:

petroleumether-acetone（5:

1）

Figure2TLCofTBNwithdifferentmobilephase（fromlefttoright:

A,B,C）

（2）Columnchromatography

FromtheresultsofTLC,methodBandCwereimplementedincolumn.About300mgofTBNwereusedineachmethod.FormethodB,yellowsemisolidwasobtained.VerypaleyellowsolidwasobtainedinmethodC.Hence,methodCwaschosen.

Batch2008.10.11TBN（4.26g）wasputincolumnchromatographywiththemobilephasepetroleumether-acetone（5:

1）and2.88gofpurifiedTBNwasyielded.Meltingpointwastested77℃.

3.1.2.Semi-preparativecolumnchromatography

SincethereweretraceamountofimpuritiesinthepurifiedTBNfrom3.1.1,semi-preparativecolumnchromatographywasperformedforfurtherpurification.

OnehundredmgofTBNwasusedthistime,andamethanol-water（35:

65）systemwasusedasamobilephase.However,Shimadzu-10ATHPLCisnotdesignedforsemi-preparativecolumn,toomuchsolventwasusedandtoofewTBNhavebeenpurified.ThismethodisnotfeasibleforproducingsufficientpurifiedTBN.AndfortheTBNpurified,sincetherewastoomuchwaterinthemobilephasethatwasdifficulttodry,onlytraceamountofTBNwasobtainedwhichwasnotenoughforexperimentaluse.

Withtheresult,theTBNpurifiedfrom3.1.1wasusedasreferencestandard.

3.2.DevelopmentofHPLCmethodforTBN

3.2.1.maxdetermination

BeforetheprocedureofHPLC,themaxofTBNshouldbedetermined.WiththeUV-VISSpectrophotometer,theTBNsamplesolutionwasscannedin200-400nmforthedeterminationofmax.

Method:

TBN（0.01g）from3.1.1wasweightedandwasdissolvedin10mlvolumetricflaskwithmethanol.Dilutethesolutionto50g/mlbyusingpipetteandvolumetricflasks.

ScanthesamplesolutionwiththeUV-VISSpectrophotometerandrecordtheresult.

Figure3UVspectrogramforTBN

Fromtheaboveresult,295nmwaschoseforTBNdetection.

3.2.2.OptimizationofHPLCconditionsforTBN

LUBEXKromasilC18column（5100Å250mm4.6mm）wasusedinthisresearchwhichissuitableformostorganiccompound.

Differentratioofmobilephase,flowrate,pH,havebeenusedtodeterminethemostsuitableHPLCmethodforTBN.WiththeaimofseparatingtheTBNfromitsimpurities,TBNsamplewithoutpurification（Batch200