高效液相方法验证课件

AnalyticalMethodDevelopmentandValidation分析方法的建立和验证28March2008AnalyticalMethodDevelopment1Outline总纲

HPLCMethodDevelopment高效液相方法建立HPLCTrouble-shootingGuideHPLC问题解答MethodOptimization方法优化MethodValidation方法验证SystemSuitabilityRequirements系统适应性要求GMP“Mind-set”GMP理念Outline总纲HPLCMethodDevelopm2MethodDevelopment

–MethodClassification

方法开发－分析方法分类

AssayMethod含量方法ImpurityProfile杂质情况-RelatedSubstances有关物质ChiralPurity手性纯度ContentUniformity含量均一性DissolutionCleaningValidation清洁验证Identity–Fingerprint鉴别－指纹法MethodDevelopment

–Method3MethodDevelopment方法开发

–UsefulInformation有用的信息

ChemicalstructureandMW化学结构和分子量Solventsolubility溶剂中的溶解度pKaandpHsolubility酸性解离常数和PH溶解度UVspectrum紫外光谱Processimpurities工艺杂质Possibledegradationimpurities可能的降解杂质LiteratureReferences参考文献MSDS–Materialsafetydatasheets安全数据表MethodDevelopment方法开发

–Us4MethodDevelopment方法开发

–ResourcePlanning&Gathering资源计划收集Solventsandreagents溶剂和试剂Placebomaterials安慰剂Individualformulationcomponents各个处方成分Relatedsubstancestandards有关物质标准品APIReferenceStandards活性成分标准品Acceleratedstresssample强降解样品MethodDevelopment方法开发

–Reso5MethodDevelopment方法开发

–HPLCColumn

色谱柱Reversedphase:C18(USPL1),C8(L7)反相：C18(USPL1),C8(L7)Normalphase:Silica,CN,andNH2Silica,CN,andNH2Ion-Exchange:Dionexcolumn离子交换：DionexcolumnIonpairChromatography:C18/C8/CN离子对色谱法：C18/C8/CNSizeExclusionChromatography排阻色谱法Chiralseparation手性分离MethodDevelopment方法开发

–HPLC6MethodDevelopment

–HPLCColumn(cont.)

色谱柱ColumnDimension:柱子的尺寸4.6mmx25cm4.6mmx15cm4.6mmx10cmParticleDiameter:粒径10µm,5µm,3.5µm,1.8µmPoresize,Surfacearea,Endcapping孔径，表面积，封尾

MethodDevelopment

–HPLCCol7MethodDevelopment方法开发

–HPLCColumn色谱柱：考考你

???

ParticleSize粒径Length长度ExpectedN要求 3.5µm 5cm 4200 3.5µm 10cm ??? 5µm 25cm 12000 10µm 25cm ???

ParticleSize粒径Length长度Resolution分离度 3.5µm 5cm 1.5 3.5µm 10cm ???MethodDevelopment方法开发

–HP8MethodDevelopment方法开发

–HPLCColumn(cont.)色谱柱

GuardColumn预柱ColumnWash柱子冲洗ColumnTemperature(35˚C-60˚C)柱温（35˚C-60˚C）ColumnStorage柱子的储存MethodDevelopment方法开发

–HP9MethodDevelopment方法开发

–HPLCcolumnselectionsummary

色谱柱选择：总结

C18Column:firstchoiceC18柱：首选Shortlength:10cmor15cm短柱：10cmor15cmSmalldiameter:3.5µmor5µm

小的粒径：3.5µmor5µm

End-capped封尾MethodDevelopment方法开发

–HPL10MethodDevelopment方法开发

–HPLCMobilePhase流动相

Strongsolventcomponent:强溶剂组份 Methanol甲醇 Acetonitrile乙腈 Tetrahydrofuran四氢呋喃Weaksolventcomponent:弱溶剂组份 Water水 Buffer缓冲液 Dilutedacid(0.1%H3PO4)MethodDevelopment方法开发

–HP11MethodDevelopment

–HPLCMobilePhase:Buffer

流动相：缓冲液

BufferSelectionPrinciple:

缓冲液选择原则 ThepHofthemobilephasebuffershouldNOTbewithin±2pHunitsofthepKavaluesoftheanalytes.

流动相缓冲液的pH值不应在样品酸性解离常数值pH单位的±2范围内

Thisensuresreproducibleretentiontimesandgoodpeakshapes.这保证了保留时间的重复性和良好的峰形。

MethodDevelopment

–HPLCMo12MethodDevelopment

–HPLCMobilePhase:Buffer

流动相：缓冲液

Henderson-HasselbalchEquation公式:

pH=pKa+log([A-]/[HA])

BuffersforuseinHPLCseparations:色谱分离使用的缓冲液

Buffer pKa BufferRange UVCutoff Phosphate 2.11.1–3.1 <200nm磷酸盐 Acetate 4.75 3.75–5.75 210nm醋酸盐 Citrate 3.1 2.1–4.1 230nm柠檬酸盐 MethodDevelopment

–HPLCMo13MethodDevelopment方法开发

–HPLCDetectors：HPLC检测器

UVabsorption紫外吸收 Fixed-wavelengthUVdetector固定波长紫外检测器 Variable-wavelengthUVdetector可变波长紫外检测器 Photodiodearraydetector二极管阵列检测器Fluorescencedetector荧光检测器Massspectrometer(LC/MS)质谱仪（液质联用）Electrochemicaldetector电化学检测器Conductivitydetector电导率检测器Evaporativelightscatteringdetector蒸发光散射检测器MethodDevelopment方法开发

–HPL14MethodDevelopment方法开发

–HPLCUVDetectors紫外检测器MethodDevelopment方法开发

–HPL15MethodDevelopment

–OtherChromatographicConditions

其它色谱条件

Flowrate流速:1.0±0.5mL/minInjectionVolume进样量:10µL–100µLSampletemperature样品温度:5˚C–roomtemperature室温Isocraticvs.Gradient等度VS.梯度

MethodDevelopment

–OtherC16MethodDevelopment

方法开发

–InitialChromatographicConditions

Initial色谱条件

Column柱:WatersSymmetryC18 4.6mmx15cm,3.5µmColumntemperature柱温:35˚C

Sampletemperature样品温度:RoomTemperature室温

流动相MobilephaseA:0.1%H3PO4inwater

MobilephaseB:0.1%H3PO4inacetonitrile

流速FlowRate:1.0mL/min

进样量InjectionVolume:20µL

检测器Detector:Photodiodearraydetector

梯度GradientProfile:10%B-80%Bin60min.MethodDevelopment

方法开发

–In17HPLCTrouble-shooting：高效液相问题解答

–Sourcesofpeaktailing：拖尾的原因pH问题pHissue

问题柱子（柱床坏掉）Badcolumn(packedbeddamaged)

化学效应（与硅烷醇结合）Chemicaleffects(interactwithsilanol)

柱过载Columnoverloaded

不正确的稀释液Wrongdiluent(100%ACN)

死体积Deadvolume

温度问题Temperatureissue

等等etc.

HPLCTrouble-shooting：高效液相问题解18HPLCTrouble-shooting

–Sourcesofghostpeak：鬼峰的原因Contaminatedsolution溶液受到污染Badreagents不合格的试剂Carry-over残留物DetectorCell检测池

HPLCTrouble-shooting

–Sourc19MethodOptimization方法优化

RegulatoryRequirements法规要求

TechnicalRequirements技术要求

PracticalRequirements实践要求ValidationRequirements验证要求MethodOptimization方法优化Regul20MethodOptimization-Four“S”

方法优化－4S

Simple简，不复杂

Short短（时）

Sensitive灵敏Suitable适合MethodOptimization-Four“S”21MethodDevelopment

方法开发

–methodoptimizationsample方法优化举例

柱Column:WatersSymmetryC18 4.6mmx

25cm,5µm

柱温Columntemperature:20˚C

流动相MobilephaseA:0.01%

HAC

inwater

流动相MobilephaseB:0.01%

HAC

inACN

流速FlowRate:0.8mL/min

进样量InjectionVolume:5µL

检测器Detector:UVat203nmMethodDevelopment

方法开发

–met22MethodDevelopment

方法开发

–methodoptimizationsample(cont.)方法优化举例梯度GradientProfile:Time时间 %MobilePhaseA %B0 80 2015 65 3525 65 3540 57 4350 57 4365 42 5875 25 7580 80 2085 80 20

MethodDevelopment

方法开发

–me23MethodValidationProtocol

方法验证方案

Objective目的Responsibilities职责Reagents/Standards试剂/标准物质Samples样品Procedures程序Acceptancecriteria可接受标准MethodValidationProtocol

方法24MethodValidation方法验证

Accuracy(Spikerecovery)准确度（加标回收率）Precision(RSD%)精密度（RSD％）Repeatability–injectionprecision重复性－进样精密度IntermediatePrecision–within-Lab中间精密度－同一实验室范围内Reproducibility–betweenLabs重现性－不同实验室之间MethodValidation方法验证Accurac25MethodValidation(continued)

方法验证

Linearity(fivelevels)线性（五个浓度水平）Range(50%to150%)范围(50%to150%)Robustness耐用性

MethodValidation(continued)

26MethodValidation(continued)

方法验证

Specificity专属性LimitofDetection(LOD)检测限LimitofQuantitation(LOQ)定量限SolutionStability,etc.

溶液稳定性，等等MethodValidation(continued)

27MethodValidation:APIAssaymethod

方法验证：原料药含量测定

–Day1Experiment第一天的试验Accuracy,Precision,LinearityandRangeforAssayofDrugSubstance(API):原料药含量分析的准确度、精密度、线性和范围 5levelsintherangeof50%to150%ofthenominalconcentration(forexample:0.1mg/mL)规定浓度（例如：0.1mg/mL）50％至150％之间的5个浓度水平 ByweighingthesoliddrugsubstanceorusingtheAPIStocksolution(1.0mg/mL)通过称量固体原料药或使用其储备液(1.0mg/mL)Repeattheaboveprocedure(byweighingthesoliddrugsubstanceorusingasecondseparatelypreparedAPIStocksolution)重复以上程序（通过称量固体原料药或使用第二次另外制备的储备液）Analyzeaspermethod(preparetheworkingstandardsolutions,etc)按照方法进行分析（制备标准品溶液，等等）

MethodValidation:APIAssaym28MethodValidation:API方法验证：原料药

–Day2Experiment第二天的试验RepeattheDay1experiment(twomoreindependentpreparations)重复第一天的试验（重新进行两次独立的配制）Calculatethe%Recoveryforeachsample计算每一个样品的回收率Calculatethe%RSDforeachlevel计算每个水平的RSDAssesstwolinearitycurvesforeachday (%Addedvs.%Found)评价每天的共两个线性曲线（加标VS.测得值）

MethodValidation:API方法验证：原料29MethodValidation:APIAssay

方法验证：原料药含量分析

–AcceptanceCriteria可接受标准Accuracy:The%Recoverymustbewithin98.0%to102.0%准确度：回收率98.0%to102.0%Precision:%RSDis≤1.0%精密度：RSD≤1.0%Linearity:r≥0.995foreachcurve线性：相关系数r≥0.995（对每一个曲线）

MethodValidation:APIAssay

30MethodValidation:ImpuritiesofAPI

方法验证：原料药的杂质

–Day1Experiment第一天试验Accuracy,Precision,LinearityandRangeforImpurityEstimation:准确度、精密度、线性和范围 5levelsintherangeof0.05%w/wto150%oftheknownimpurityspecification(forexample:0.3%)已知杂质限度（例如0.3%)） UsingtheAPIStocksolutionandtheimpuritystocksolutionforeachknowprocessimpurityanddegradates对每一个已知工艺杂质和降解杂质，使用原料药储备液和杂质储备液 Repeattheaboveprocedure(usingasecondseparatelypreparedimpuritystocksolution)重复以上程序（使用另外一个单独配制的杂质储备液）Analyzeaspermethod(preparetheworkingstandardsolutions,etc)按照方法进行分析（制备工作标准品溶液，等等）MethodValidation:Impurities31MethodValidation:APIImpurity

方法验证：原料药的杂质

–Day2Experiment第二天的试验RepeattheDay1experiment(twomoreindependentpreparations)重复第一天的试验（重新进行两次独立的配制）Calculatethe%Recoveryforeachsample对于每个样品，计算回收率Calculatethe%RSDforeachlevel计算每个浓度水平的RSDAssesstwolinearitycurvesforeachday (%Addedvs.%Found)评价每一天共两个线性曲线（加标VS.测得值）

MethodValidation:APIImpurit32MethodValidation:APIImpurity

方法验证：原料药的杂质

–AcceptanceCriteria可接受标准Accuracy:The%Recoverymustbewithin70%to130%foreachimpurity准确度：对每个杂质，回收率应为70%to130%

Precision:%RSDis≤20%精密度：RSD≤20%Linearity:r≥0.99

foreachcurve线性：r≥0.99

（对每个曲线）

MethodValidation:APIImpurit33MethodValidation:DrugProduct

方法验证：制剂

–Day1Experiment第一天的试验Accuracy,Precision,LinearityandRangeforAssayofDrugProduct:制剂含量分析的准确度、精密度和线性及范围 5levelsintherangeof50%to150%ofthenominalconcentration(forexample:0.1mg/mL)规定浓度（例如0.1mg/mL）50%to150%范围内5个水平 ByweighingthesoliddrugsubstanceorusingtheAPIStocksolution(1.0mg/mL)通过称量固体原料药或使用原料药储备液（1.0mg/mL）

Addtheplaceboproductortheplacebostocksolutionaspertheformulation根据配方加安慰剂或安慰剂储备液Repeattheaboveprocedure(byweighingthesoliddrugsubstanceorusingasecondseparatelypreparedAPIStocksolution)重复以上程序（通过称量固体原料药或使用另外单独配制的原料药储备液）Analyzeaspermethod(preparetheworkingstandardsolutions,etc)安装方法分析（配制工作标准品溶液，等等）MethodValidation:DrugProduc34MethodValidation:DrugProduct

方法验证：制剂

–Day2Experiment第二天的试验RepeattheDay1experiment(twomoreindependentpreparations)重复第一天的试验（重新进行两次独立的配制）Calculatethe%Recoveryforeachsample对每个样品计算回收率Calculatethe%RSDforeachlevel计算每个浓度水平的RSDAssesstwolinearitycurvesforeachday (%Addedvs.%Found)评价每天共两个线性曲线（加标VS.测得值）

MethodValidation:DrugProduc35MethodValidation:DrugProduct

方法验证：制剂

–AcceptanceCriteria可接受标准Accuracy:The%Recoverymustbewithin97.0%to103.0%准确度：回收率应在97.0%to103.0%Precision:%RSDis≤2.0%精密度：RSD≤2.0%Linearity:r≥0.990

foreachcurve线性：r≥0.990

（对每个曲线）

MethodValidation:DrugProduc36MethodValidation:

方法验证

–PurposefulStressing

强降解Light(UV)stressing(controldarksample)紫外光降解(controldarksample)Heatstressing(80˚Cinanovenandanalyzedonaweeklybasisforuptoonemonth)加热降解（置于80˚C烘箱中，每周分析一次，共一个月的时间）Acid(1NHCl)andbasehydrolysis(1NNaOH)酸、碱降解(1NHCl)

和(1NNaOH)Oxidationstressing氧化降解Desireddegradationby5%to15%level.降解5%to15%

MethodValidation:

方法验证

–Pu37MethodValidation:

方法验证

–RobustnessStudies耐用性研究Mobilephasecomposition流动相组份FlowRate(±10%)流速(±10%)UVdetectorwavelength(±5nm)紫外检测波长(±5nm)Column(sametype,3differentlots)柱（同型号，3个不同批号）ColumnTemperature(±10˚C)柱温(±10˚C)

MethodValidation:

方法验证

–Ro38MethodValidation:

–SamplePreparation样品制备Threelotsandduplicatepreparationforeachlot(forbothAPIorproduct)三个批号的样品，每批重复制备两份（对原料药和制剂）Sampleextraction/mixingtime样品提取/混合时间Filteradsorptionstudies过滤吸附研究Methodprecision:sixduplicatepreparationsforthesamesample

方法精密度：对同一个样品，重复制备六份溶液

MethodValidation:

–SampleP39MethodValidation:

–RelativeResponseFactor(RRF)

相对响应因子（RRF）RRF=(AREAimpurity/CONCimpurity)/ (AREAParent/CONCParent)RRF=(杂质峰面积/杂质浓度)/(主峰面积/主成分浓度)

MethodValidation:

–Relativ40SystemSuitability系统适应性

Resolution(Rs>2.0)分离度(Rs>2.0)InjectionPrecision(RSD%<2.0%forHPLC)进样精密度RSD%<2.0%TailingFactor(<2.5)拖尾因子(<2.5)RRT(RelativeRetentionTime)相对保留时间SystemSuitability系统适应性Resol41SystemSuitability(continued)

系统适应性

LOQorReportingLimit (0.1%forProductor0.05%forAPI)定量限或报告限（制剂0.1%,原料药:0.05%）ReferenceStandardCheck(98.0%to102.0%)标准品检查(98.0%to102.0%)TheoreticalPlates,etc.理论塔板数，等等SystemSuitability(continued)42SystemSuitability:Practicalguide

系统适应性：实用指南

–Samplesetmethod/sequencetable

样品分析安排/进样顺序表ForAssayandImpuritiesMethods针对含量和杂质检查SampleID样品号

SampleName样品名称 #Injections进样次数12107001:01A Blank(Diluent) 212107001:03A ReportLimitSolution 312107001:03B Resolutionsolution 212107001:06A CheckStandard 5

12107001:06B WorkingStandard 212107001:07A SampleLot07058 212107001:07A SampleLot07059 212107001:07A SampleLot07060 212107001:06B WorkingStandard 2

SystemSuitability:Practical43SystemSuitability:Practicalguide

–Samplesetmethod/sequencetable

ForHPLCIdentity–FingerprintHPLC鉴别－指纹SampleID SampleName #Injections12107001:01A Blank(Diluent) 212107001:03A ControlSamplesolution

212107001:03B ReferenceSTDsolution 212107001:07A SampleLot07058 212107001:07A SampleLot07058 212107001:07A SampleLot07058 212107001:01A Blank(Diluent) 112107001:03A ControlSamplesolution

2

SystemSuitability:Practical44CalculationTips“Asis”Basis

“Dried”Basis干燥的“Anhydrous”Basis无水的

CalculationTips“Asis”Basi45GMP“Mind-set”

GMP理念

Rawdata(ontime,completed,accurate,reliable,etc.)原始数据（及时、完整、准确、可靠，等等）Qualifiedchemist/staff/reviewer合格的分析人员/职员/审核者Calibratedinstruments/equipments经过校正的仪器/设备Validatedmethod,etc.经过验证的方法，等等Systemsuitabilityrequirements系统适应性要求

GMP“Mind-set”

GMP理念Rawdat46GMP“Mind-set”

–Date,initial,andtime

日期、姓名缩写和时间记录的格式Date: 14–2–2007(14.2.2007) 8–2–2007(8.2.2007) 08FEB2007Initial:SWorSQWTime:13:30or1:30PM

GMP“Mind-set”

–Date,initi47Acknowledgements(致谢)TogetherWewillmakeadifference!!!Thankyouverymuch!!!Acknowledgements(致谢)Together48AnalyticalMethodDevelopmentandValidation分析方法的建立和验证28March2008AnalyticalMethodDevelopment49Outline总纲

HPLCMethodDevelopment高效液相方法建立HPLCTrouble-shootingGuideHPLC问题解答MethodOptimization方法优化MethodValidation方法验证SystemSuitabilityRequirements系统适应性要求GMP“Mind-set”GMP理念Outline总纲HPLCMethodDevelopm50MethodDevelopment

–MethodClassification

方法开发－分析方法分类

AssayMethod含量方法ImpurityProfile杂质情况-RelatedSubstances有关物质ChiralPurity手性纯度ContentUniformity含量均一性DissolutionCleaningValidation清洁验证Identity–Fingerprint鉴别－指纹法MethodDevelopment

–Method51MethodDevelopment方法开发

–UsefulInformation有用的信息

ChemicalstructureandMW化学结构和分子量Solventsolubility溶剂中的溶解度pKaandpHsolubility酸性解离常数和PH溶解度UVspectrum紫外光谱Processimpurities工艺杂质Possibledegradationimpurities可能的降解杂质LiteratureReferences参考文献MSDS–Materialsafetydatasheets安全数据表MethodDevelopment方法开发

–Us52MethodDevelopment方法开发

–ResourcePlanning&Gathering资源计划收集Solventsandreagents溶剂和试剂Placebomaterials安慰剂Individualformulationcomponents各个处方成分Relatedsubstancestandards有关物质标准品APIReferenceStandards活性成分标准品Acceleratedstresssample强降解样品MethodDevelopment方法开发

–Reso53MethodDevelopment方法开发

–HPLCColumn

色谱柱Reversedphase:C18(USPL1),C8(L7)反相：C18(USPL1),C8(L7)Normalphase:Silica,CN,andNH2Silica,CN,andNH2Ion-Exchange:Dionexcolumn离子交换：DionexcolumnIonpairChromatography:C18/C8/CN离子对色谱法：C18/C8/CNSizeExclusionChromatography排阻色谱法Chiralseparation手性分离MethodDevelopment方法开发

–HPLC54MethodDevelopment

–HPLCColumn(cont.)

色谱柱ColumnDimension:柱子的尺寸4.6mmx25cm4.6mmx15cm4.6mmx10cmParticleDiameter:粒径10µm,5µm,3.5µm,1.8µmPoresize,Surfacearea,Endcapping孔径，表面积，封尾

MethodDevelopment

–HPLCCol55MethodDevelopment方法开发

–HPLCColumn色谱柱：考考你

???

ParticleSize粒径Length长度ExpectedN要求 3.5µm 5cm 4200 3.5µm 10cm ??? 5µm 25cm 12000 10µm 25cm ???

ParticleSize粒径Length长度Resolution分离度 3.5µm 5cm 1.5 3.5µm 10cm ???MethodDevelopment方法开发

–HP56MethodDevelopment方法开发

–HPLCColumn(cont.)色谱柱

GuardColumn预柱ColumnWash柱子冲洗ColumnTemperature(35˚C-60˚C)柱温（35˚C-60˚C）ColumnStorage柱子的储存MethodDevelopment方法开发

–HP57MethodDevelopment方法开发

–HPLCcolumnselectionsummary

色谱柱选择：总结

C18Column:firstchoiceC18柱：首选Shortlength:10cmor15cm短柱：10cmor15cmSmalldiameter:3.5µmor5µm

小的粒径：3.5µmor5µm

End-capped封尾MethodDevelopment方法开发

–HPL58MethodDevelopment方法开发

–HPLCMobilePhase流动相

Strongsolventcomponent:强溶剂组份 Methanol甲醇 Acetonitrile乙腈 Tetrahydrofuran四氢呋喃Weaksolventcomponent:弱溶剂组份 Water水 Buffer缓冲液 Dilutedacid(0.1%H3PO4)MethodDevelopment方法开发

–HP59MethodDevelopment

–HPLCMobilePhase:Buffer

流动相：缓冲液

BufferSelectionPrinciple:

缓冲液选择原则 ThepHofthemobilephasebuffershouldNOTbewithin±2pHunitsofthepKavaluesoftheanalytes.

流动相缓冲液的pH值不应在样品酸性解离常数值pH单位的±2范围内

Thisensuresreproducibleretentiontimesandgoodpeakshapes.这保证了保留时间的重复性和良好的峰形。

MethodDevelopment

–HPLCMo60MethodDevelopment

–HPLCMobilePhase:Buffer

流动相：缓冲液

Henderson-HasselbalchEquation公式:

pH=pKa+log([A-]/[HA])

BuffersforuseinHPLCseparations:色谱分离使用的缓冲液

Buffer pKa BufferRange UVCutoff Phosphate 2.11.1–3.1 <200nm磷酸盐 Acetate 4.75 3.75–5.75 210nm醋酸盐 Citrate 3.1 2.1–4.1 230nm柠檬酸盐 MethodDevelopment

–HPLCMo61MethodDevelopment方法开发

–HPLCDetectors：HPLC检测器

UVabsorption紫外吸收 Fixed-wavelengthUVdetector固定波长紫外检测器 Variable-wavelengthUVdetector可变波长紫外检测器 Photodiodearraydetector二极管阵列检测器Fluorescencedetector荧光检测器Massspectrometer(LC/MS)质谱仪（液质联用）Electrochemicaldetector电化学检测器Conductivitydetector电导率检测器Evaporativelightscatteringdetector蒸发光散射检测器MethodDevelopment方法开发

–HPL62MethodDevelopment方法开发

–HPLCUVDetectors紫外检测器MethodDevelopment方法开发

–HPL63MethodDevelopment

–OtherChromatographicConditions

其它色谱条件

Flowrate流速:1.0±0.5mL/minInjectionVolume进样量:10µL–100µLSampletemperature样品温度:5˚C–roomtemperature室温Isocraticvs.Gradient等度VS.梯度

MethodDevelopment

–OtherC64MethodDevelopment

方法开发

–InitialChromatographicConditions

Initial色谱条件

Column柱:WatersSymmetryC18 4.6mmx15cm,3.5µmColumntemperature柱温:35˚C

Sampletemperature样品温度:RoomTemperature室温

流动相MobilephaseA:0.1%H3PO4inwater

MobilephaseB:0.1%H3PO4inacetonitrile

流速FlowRate:1.0mL/min

进样量InjectionVolume:20µL

检测器Detector:Photodiodearraydetector

梯度GradientProfile:10%B-80%Bin60min.MethodDevelopment

方法开发

–In65HPLCTrouble-shooting：高效液相问题解答

–Sourcesofpeaktailing：拖尾的原因pH问题pHissue

问题柱子（柱床坏掉）Badcolumn(packedbeddamaged)

化学效应（与硅烷醇结合）Chemicaleffects(interactwithsilanol)

柱过载Columnoverloaded

不正确的稀释液Wrongdiluent(100%ACN)

死体积Deadvolume

温度问题Temperatureissue

等等etc.

HPLCTrouble-shooting：高效液相问题解66HPLCTrouble-shooting

–Sourcesofghostpeak：鬼峰的原因Contaminatedsolution溶液受到污染Badreagents不合格的试剂Carry-over残留物DetectorCell检测池

HPLCTrouble-shooting

–Sourc67MethodOptimization方法优化

RegulatoryRequirements法规要求

TechnicalRequirements技术要求

PracticalRequirements实践要求ValidationRequirements验证要求MethodOptimization方法优化Regul68MethodOptimization-Four“S”

方法优化－4S

Simple简，不复杂

Short短（时）

Sensitive灵敏Suitable适合MethodOptimization-Four“S”69MethodDevelopment

方法开发

–methodoptimizationsample方法优化举例

柱Column:WatersSymmetryC18 4.6mmx

25cm,5µm

柱温Columntemperature:20˚C

流动相MobilephaseA:0.01%

HAC

inwater

流动相MobilephaseB:0.01%

HAC

inACN

流速FlowRate:0.8mL/min

进样量InjectionVolume:5µL

检测器Detector:UVat203nmMethodDevelopment

方法开发

–met70MethodDevelopment

方法开发

–methodoptimizationsample(cont.)方法优化举例梯度GradientProfile:Time时间 %MobilePhaseA %B0 80 2015 65 3525 65 3540 57 4350 57 4365 42 5875 25 7580 80 2085 80 20

MethodDevelopment

方法开发

–me71MethodValidationProtocol

方法验证方案

Objective目的Responsibilities职责Reagents/Standards试剂/标准物质Samples样品Procedures程序Acceptancecriteria可接受标准MethodValidationProtocol

方法72MethodValidation方法验证

Accuracy(Spikerecovery)准确度（加标回收率）Precision(RSD%)精密度（RSD％）Repeatability–injectionprecision重复性－进样精密度IntermediatePrecision–within-Lab中间精密度－同一实验室范围内Reproducibility–betweenLabs重现性－不同实验室之间MethodValidation方法验证Accurac73MethodValidation(continued)

方法验证

Linearity(fivelevels)线性（五个浓度水平）Range(50%to150%)范围(50%to150%)Robustness耐用性

MethodValidation(continued)

74MethodValidation(continued)

方法验证

Specificity专属性LimitofDetection(LOD)检测限LimitofQuantitation(LOQ)定量限SolutionStability,etc.

溶液稳定性，等等MethodValidation(continued)

75MethodValidation:APIAssaymethod

方法验证：原料药含量测定

–Day1Experiment第一天的试验Accuracy,Precision,LinearityandRangeforAssayofDrugSubstance(API):原料药含量分析的准确度、精密度、线性和范围 5levelsintherangeof50%to150%ofthenominalconcentration(forexample:0.1mg/mL)规定浓度（例如：0.1mg/mL）50％至150％之间的5个浓度水平 ByweighingthesoliddrugsubstanceorusingtheAPIStocksolution(1.0mg/mL)通过称量固体原料药或使用其储备液(1.0mg/mL)Repeattheaboveprocedure(byweighingthesoliddrugsubstanceorusingasecondseparatelypreparedAPIStocksolution)重复以上程序（通过称量固体原料药或使用第二次另外制备的储备液）Analyzeaspermethod(preparetheworkingstandardsolutions,etc)按照方法进行分析（制备标准品溶液，等等）

MethodValidation:APIAssaym76MethodValidation:API方法验证：原料药

–Day2Experiment第二天的试验RepeattheDay1experiment(twomoreindependentpreparations)重复第一天的试验（重新进行两次独立的配制）Calculatethe%Recoveryforeachsample计算每一个样品的回收率Calculatethe%RSDforeachlevel计算每个水平的RSDAssesstwolinearitycurvesforeachday (%Addedvs.%Found)评价每天的共两个线性曲线（加标VS.测得值）

MethodValidation:API方法验证：原料77MethodValidation:APIAssay

方法验证：原料药含量分析

–AcceptanceCriteria可接受标准Accuracy:The%Recoverymustbewithin98.0%to102.0%准确度：回收率98.0%to102.0%Precision:%RSDis≤1.0%精密度：RSD≤1.0%Linearity:r≥0.995foreachcurve线性：相关系数r≥0.995（对每一个曲线）

MethodValidation:APIAssay

78MethodValidation:ImpuritiesofAPI

方法验证：原料药的杂质

–Day1Experiment第一天试验Accuracy,Precision,LinearityandRangeforImpurityEstimation:准确度、精密度、线性和范围 5levelsintherangeof0.05%w/wto150%oftheknownimpurityspecification(forexample:0.3%)已知杂质限度（例如0.3%)） UsingtheAPIStocksolutionandtheimpuritystocksolutionforeachknowprocessimpurityanddegradates对每一个已知工艺杂质和降解杂质，使用原料药储备液和杂质储备液 Repeattheaboveprocedure(usingasecondseparatelypreparedimpuritystocksolution)重复以上程序（使用另外一个单独配制的杂质储备液）Analyzeaspermethod(preparetheworkingstandardsolutions,etc)按照方法进行分析（制备工作标准品溶液，等等）MethodValidation:Impurities79MethodValidation:APIImpurity

方法验证：原料药的杂质

–Day2Experiment第二天的试验RepeattheDay1experiment(twomoreindependentpreparations)重复第一天的试验（重新进行两次独立的配制）Calculatethe%Recoveryforeachsample对于每个样品，计算回收率Calculatethe%RSDforeachlevel计算每个浓度水平的RSDAssesstwolinearitycurvesforeachday (%Addedvs.%Found)评价每一天共两个线性曲线（加标VS.测得值）

MethodValidation:APIImpurit80MethodValidation:APIImpurity

方法验证：原料药的杂质

–AcceptanceCriteria可接受标准Accuracy:The%Recoverymustbewithin70%to130%foreachimpurity准确度：对每个杂质，回收率应为70%to130%

Precision:%RSDis≤20%精密度：RSD≤20%Linearity:r≥0.99

foreachcurve线性：r≥0.99

（对每个曲线）

MethodValidation:APIImpurit81MethodValidation:DrugProduct

方法验证：制剂

–Day1Experiment第一天的试验Accuracy,Precision,LinearityandRangeforAssayofDrugProduct:制剂含量分析的准确度、精密度和线性及范围 5levelsintherangeof50%to150%ofthenominalconcentration(forexample:0.1mg/mL)规定浓度（例如0.1mg/mL）50%to150%范围内5个水平 ByweighingthesoliddrugsubstanceorusingtheAPIStocksolution(1.0mg/mL)通过称量固体原料药或使用原料药储备液（1.0mg/mL）

Addtheplaceboproductortheplacebostocksolutionaspertheformulation根据配方加安慰剂或安慰剂储备液Repeattheaboveprocedure(byweighingthesoliddrugsubstanceorusingasecondseparatelypreparedAPIStocksolution)重复以上程序（通过称量固体原料药或使用另外单独配制的原料药储备液）Analyzeaspermethod(preparetheworkingstandardsolutions,etc)安装方法分析（配制工作标准品溶液，等等）MethodValidation:DrugProduc82MethodValidation:DrugProduct

方法验证：制剂

–Day2Exp