AnalyticalMethodDevelopmentandValidation分析方法的建立和驗證28March2008Outline總綱

HPLCMethodDevelopment高效液相方法建立HPLCTrouble-shootingGuideHPLC問題解答MethodOptimization方法優化MethodValidation方法驗證SystemSuitabilityRequirements系統適應性要求GMP“Mind-set”GMP理念MethodDevelopment

–MethodClassification

方法開發－分析方法分類

AssayMethod含量方法ImpurityProfile雜質情況-RelatedSubstances有關物質ChiralPurity手性純度ContentUniformity含量均一性DissolutionCleaningValidation清潔驗證Identity–Fingerprint鑒別－指紋法MethodDevelopment方法開發

–UsefulInformation有用的信息

ChemicalstructureandMW化學結構和分子量Solventsolubility溶劑中的溶解度pKaandpHsolubility酸性解離常數和PH溶解度UVspectrum紫外光譜Processimpurities工藝雜質Possibledegradationimpurities可能的降解雜質LiteratureReferences參考文獻MSDS–Materialsafetydatasheets安全數據表MethodDevelopment方法開發

–ResourcePlanning&Gathering資源計劃收集Solventsandreagents溶劑和試劑Placebomaterials安慰劑Individualformulationcomponents各個處方成分Relatedsubstancestandards有關物質標準品APIReferenceStandards活性成分標準品Acceleratedstresssample強降解樣品MethodDevelopment方法開發

–HPLCColumn

色譜柱Reversedphase:C18(USPL1),C8(L7)反相：C18(USPL1),C8(L7)Normalphase:Silica,CN,andNH2Silica,CN,andNH2Ion-Exchange:Dionexcolumn離子交換：DionexcolumnIonpairChromatography:C18/C8/CN離子對色譜法：C18/C8/CNSizeExclusionChromatography排阻色譜法Chiralseparation手性分離MethodDevelopment

–HPLCColumn(cont.)

色譜柱ColumnDimension:柱子的尺寸4.6mmx25cm4.6mmx15cm4.6mmx10cmParticleDiameter:粒徑10μm,5μm,3.5μm,1.8μmPoresize,Surfacearea,Endcapping孔徑，表面積，封尾

MethodDevelopment方法開發

–HPLCColumn色譜柱：考考你

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ParticleSize粒徑Length長度ExpectedN要求 3.5μm 5cm 4200 3.5μm 10cm ??? 5μm 25cm 12000 10μm 25cm ???

ParticleSize粒徑Length長度Resolution分離度 3.5μm 5cm 1.5 3.5μm 10cm ???MethodDevelopment方法開發

–HPLCColumn(cont.)色譜柱

GuardColumn預柱ColumnWash柱子沖洗ColumnTemperature(35?C-60?C)柱溫（35?C-60?C）ColumnStorage柱子的儲存MethodDevelopment方法開發

–HPLCcolumnselectionsummary

色譜柱選擇：總結

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

小的粒徑：3.5μmor5μm

End-capped封尾MethodDevelopment方法開發

–HPLCMobilePhase流動相

Strongsolventcomponent:強溶劑組份 Methanol甲醇 Acetonitrile乙腈 Tetrahydrofuran四氫呋喃Weaksolventcomponent:弱溶劑組份 Water水 Buffer緩沖液 Dilutedacid(0.1%H3PO4)MethodDevelopment

–HPLCMobilePhase:Buffer

流動相：緩沖液

BufferSelectionPrinciple:

緩沖液選擇原則 ThepHofthemobilephasebuffershouldNOTbewithin±2pHunitsofthepKavaluesoftheanalytes.

流動相緩沖液的pH值不應在樣品酸性解離常數值pH單位的±2范圍內

Thisensuresreproducibleretentiontimesandgoodpeakshapes.這保證了保留時間的重復性和良好的峰形。

MethodDevelopment

–HPLCMobilePhase:Buffer

流動相：緩沖液

Henderson-HasselbalchEquation公式:

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

BuffersforuseinHPLCseparations:色譜分離使用的緩沖液

Buffer pKa BufferRange UVCutoff Phosphate 2.11.1–3.1 <200nm磷酸鹽 Acetate 4.75 3.75–5.75 210nm醋酸鹽 Citrate 3.1 2.1–4.1 230nm檸檬酸鹽 MethodDevelopment方法開發

–HPLCDetectors：HPLC檢測器

UVabsorption紫外吸收 Fixed-wavelengthUVdetector固定波長紫外檢測器 Variable-wavelengthUVdetector可變波長紫外檢測器 Photodiodearraydetector二極管陣列檢測器Fluorescencedetector熒光檢測器Massspectrometer(LC/MS)質譜儀（液質聯用）Electrochemicaldetector電化學檢測器Conductivitydetector電導率檢測器Evaporativelightscatteringdetector蒸發光散射檢測器MethodDevelopment方法開發

–HPLCUVDetectors紫外檢測器MethodDevelopment

–OtherChromatographicConditions

其它色譜條件

Flowrate流速:1.0±0.5mL/minInjectionVolume進樣量:10μL–100μLSampletemperature樣品溫度:5?C–roomtemperature室溫Isocraticvs.Gradient等度VS.梯度

MethodDevelopment

方法開發

–InitialChromatographicConditions

Initial色譜條件

Column柱:WatersSymmetryC18 4.6mmx15cm,3.5μmColumntemperature柱溫:35?C

Sampletemperature樣品溫度:RoomTemperature室溫

流動相MobilephaseA:0.1%H3PO4inwater

MobilephaseB:0.1%H3PO4inacetonitrile

流速FlowRate:1.0mL/min

進樣量InjectionVolume:20μL

檢測器Detector:Photodiodearraydetector

梯度GradientProfile:10%B-80%Bin60min.HPLCTrouble-shooting：高效液相問題解答

–Sourcesofpeaktailing：拖尾的原因pH問題pHissue

問題柱子（柱床壞掉）Badcolumn(packedbeddamaged)

化學效應（與硅烷醇結合）Chemicaleffects(interactwithsilanol)

柱過載Columnoverloaded

不正確的稀釋液Wrongdiluent(100%ACN)

死體積Deadvolume

溫度問題Temperatureissue

等等etc.

HPLCTrouble-shooting

–Sourcesofghostpeak：鬼峰的原因Contaminatedsolution溶液受到污染Badreagents不合格的試劑Carry-over殘留物DetectorCell檢測池

MethodOptimization方法優化

RegulatoryRequirements法規要求

TechnicalRequirements技術要求

PracticalRequirements實踐要求ValidationRequirements驗證要求MethodOptimization-Four“S”

方法優化－4S

Simple簡，不復雜

Short短（時）

Sensitive靈敏Suitable適合MethodDevelopment

方法開發

–methodoptimizationsample方法優化舉例

柱Column:WatersSymmetryC18 4.6mmx

25cm,5μm

柱溫Columntemperature:20?C

流動相MobilephaseA:0.01%

HAC

inwater

流動相MobilephaseB:0.01%

HAC

inACN

流速FlowRate:0.8mL/min

進樣量InjectionVolume:5μL

檢測器Detector:UVat203nmMethodDevelopment

方法開發

–methodoptimizationsample(cont.)方法優化舉例梯度GradientProfile:Time時間 %MobilePhaseA %B0 80 2015 65 3525 65 3540 57 4350 57 4365 42 5875 25 7580 80 2085 80 20

MethodValidationProtocol

方法驗證方案

Objective目的Responsibilities職責Reagents/Standards試劑/標準物質Samples樣品Procedures程序Acceptancecriteria可接受標準MethodValidation方法驗證

Accuracy(Spikerecovery)準確度（加標回收率）Precision(RSD%)精密度（RSD％）Repeatability–injectionprecision重復性－進樣精密度IntermediatePrecision–within-Lab中間精密度－同一實驗室范圍內Reproducibility–betweenLabs重現性－不同實驗室之間MethodValidation(continued)

方法驗證

Linearity(fivelevels)線性（五個濃度水平）Range(50%to150%)范圍(50%to150%)Robustness耐用性

MethodValidation(continued)

方法驗證

Specificity專屬性LimitofDetection(LOD)檢測限LimitofQuantitation(LOQ)定量限SolutionStability,etc.

溶液穩定性，等等MethodValidation:APIAssaymethod

方法驗證：原料藥含量測定

–Day1Experiment第一天的試驗Accuracy,Precision,LinearityandRangeforAssayofDrugSubstance(API):原料藥含量分析的準確度、精密度、線性和范圍 5levelsintherangeof50%to150%ofthenominalconcentration(forexample:0.1mg/mL)規定濃度（例如：0.1mg/mL）50％至150％之間的5個濃度水平 ByweighingthesoliddrugsubstanceorusingtheAPIStocksolution(1.0mg/mL)通過稱量固體原料藥或使用其儲備液(1.0mg/mL)Repeattheaboveprocedure(byweighingthesoliddrugsubstanceorusingasecondseparatelypreparedAPIStocksolution)重復以上程序（通過稱量固體原料藥或使用第二次另外制備的儲備液）Analyzeaspermethod(preparetheworkingstandardsolutions,etc)按照方法進行分析（制備標準品溶液，等等）

MethodValidation:API方法驗證：原料藥

–Day2Experiment第二天的試驗RepeattheDay1experiment(twomoreindependentpreparations)重復第一天的試驗（重新進行兩次獨立的配制）Calculatethe%Recoveryforeachsample計算每一個樣品的回收率Calculatethe%RSDforeachlevel計算每個水平的RSDAssesstwolinearitycurvesforeachday (%Addedvs.%Found)評價每天的共兩個線性曲線（加標VS.測得值）

MethodValidation:APIAssay

方法驗證：原料藥含量分析

–AcceptanceCriteria可接受標準Accuracy:The%Recoverymustbewithin98.0%to102.0%準確度：回收率98.0%to102.0%Precision:%RSDis≤1.0%精密度：RSD≤1.0%Linearity:r≥0.995foreachcurve線性：相關系數r≥0.995（對每一個曲線）

MethodValidation:ImpuritiesofAPI

方法驗證：原料藥的雜質

–Day1Experiment第一天試驗Accuracy,Precision,LinearityandRangeforImpurityEstimation:準確度、精密度、線性和范圍 5levelsintherangeof0.05%w/wto150%oftheknownimpurityspecification(forexample:0.3%)已知雜質限度（例如0.3%)） UsingtheAPIStocksolutionandtheimpuritystocksolutionforeachknowprocessimpurityanddegradates對每一個已知工藝雜質和降解雜質，使用原料藥儲備液和雜質儲備液 Repeattheaboveprocedure(usingasecondseparatelypreparedimpuritystocksolution)重復以上程序（使用另外一個單獨配制的雜質儲備液）Analyzeaspermethod(preparetheworkingstandardsolutions,etc)按照方法進行分析（制備工作標準品溶液，等等）MethodValidation:APIImpurity

方法驗證：原料藥的雜質

–Day2Experiment第二天的試驗RepeattheDay1experiment(twomoreindependentpreparations)重復第一天的試驗（重新進行兩次獨立的配制）Calculatethe%Recoveryforeachsample對于每個樣品，計算回收率Calculatethe%RSDforeachlevel計算每個濃度水平的RSDAssesstwolinearitycurvesforeachday (%Addedvs.%Found)評價每一天共兩個線性曲線（加標VS.測得值）

MethodValidation:APIImpurity

方法驗證：原料藥的雜質

–AcceptanceCriteria可接受標準Accuracy:The%Recoverymustbewithin70%to130%foreachimpurity準確度：對每個雜質，回收率應為70%to130%

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

foreachcurve線性：r≥0.99

（對每個曲線）

MethodValidation:DrugProduct

方法驗證：制劑

–Day1Experiment第一天的試驗Accuracy,Precision,LinearityandRangeforAssayofDrugProduct:制劑含量分析的準確度、精密度和線性及范圍 5levelsintherangeof50%to150%ofthenominalconcentration(forexample:0.1mg/mL)規定濃度（例如0.1mg/mL）50%to150%范圍內5個水平 ByweighingthesoliddrugsubstanceorusingtheAPIStocksolution(1.0mg/mL)通過稱量固體原料藥或使用原料藥儲備液（1.0mg/mL）

Addtheplaceboproductortheplacebostocksolutionaspertheformulation根據配方加安慰劑或安慰劑儲備液Repeattheaboveprocedure(byweighingthesoliddrugsubstanceorusingasecondseparatelypreparedAPIStocksolution)重復以上程序（通過稱量固體原料藥或使用另外單獨配制的原料藥儲備液）Analyzeaspermethod(preparetheworkingstandardsolutions,etc)安裝方法分析（配制工作標準品溶液，等等）MethodValidation:DrugProduct

方法驗證：制劑

–Day2Experiment第二天的試驗RepeattheDay1experiment(twomoreindependentpreparations)重復第一天的試驗（重新進行兩次獨立的配制）Calculatethe%Recoveryforeachsample對每個樣品計算回收率Calculatethe