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1、分析方法的建立-课件,分析方法的建立-课件,Method Development Method Classification方法开发分析方法分类,Assay Method 含量方法 Impurity Profile 杂质情况 - Related Substances 有关物质 Chiral Purity 手性纯度 Content Uniformity 含量均一性 Dissolution Cleaning Validation 清洁验证 Identity Fingerprint 鉴别指纹法,Method Development Method,Method Development 方法开发 Use
2、ful Information 有用的信息,Chemical structure and MW化学结构和分子量 Solvent solubility 溶剂中的溶解度 pKa and pH solubility酸性解离常数和PH溶解度 UV spectrum紫外光谱 Process impurities工艺杂质 Possible degradation impurities可能的降解杂质 Literature References参考文献 MSDS Material safety data sheets 安全数据表,Method Development 方法开发 Us,Method Develo
3、pment 方法开发 Resource Planning & Gathering 资源计划收集,Solvents and reagents溶剂和试剂 Placebo materials安慰剂 Individual formulation components各个处方成分 Related substance standards有关物质标准品 API Reference Standards活性成分标准品 Accelerated stress sample强降解样品,Method Development 方法开发 Reso,Method Development 方法开发 HPLC Column 色谱
4、柱,Reversed phase: C18(USP L1), C8(L7)反相：C18(USP L1), C8(L7) Normal phase: Silica, CN, and NH2Silica, CN, and NH2Ion-Exchange: Dionex column离子交换： Dionex column Ion pair Chromatography: C18/C8/CN离子对色谱法： C18/C8/CN Size Exclusion Chromatography排阻色谱法 Chiral separation手性分离,Method Development 方法开发 HPLC,Met
5、hod Development HPLC Column (cont.) 色谱柱,Column Dimension: 柱子的尺寸 4.6 mm x 25 cm 4.6 mm x 15 cm 4.6 mm x 10 cm Particle Diameter: 粒径 10 m, 5 m, 3.5 m, 1.8 m Pore size, Surface area, End capping孔径，表面积，封尾,Method Development HPLC Col,Method Development 方法开发 HPLC Column色谱柱：考考你 ?,Particle Size 粒径Length长度 E
6、xpected N 要求3.5 m 5 cm42003.5 m 10 cm ?5 m 25 cm1200010 m 25 cm ? Particle Size粒径 Length长度 Resolution分离度3.5 m 5 cm 1.53.5 m 10 cm ?,Method Development 方法开发 HP,Method Development 方法开发 HPLC Column (cont.)色谱柱,Guard Column预柱 Column Wash柱子冲洗 Column Temperature (35 C - 60 C)柱温 （ 35 C - 60 C ） Column Stora
7、ge柱子的储存,Method Development 方法开发 HP,Method Development 方法开发 HPLC column selection summary色谱柱选择：总结,C18 Column: first choiceC18柱：首选 Short length: 10 cm or 15 cm短柱： 10 cm or 15 cm Small diameter: 3.5 m or 5 m 小的粒径： 3.5 m or 5 m End-capped封尾,Method Development 方法开发 HPL,Method Development 方法开发 HPLC Mobile
8、 Phase 流动相,Strong solvent component: 强溶剂组份Methanol 甲醇Acetonitrile 乙腈Tetrahydrofuran 四氢呋喃 Weak solvent component: 弱溶剂组份Water 水Buffer 缓冲液 Diluted acid (0.1% H3PO4),Method Development 方法开发 HP,Method Development HPLC Mobile Phase: Buffer流动相：缓冲液,Buffer Selection Principle:缓冲液选择原则The pH of the mobile phas
9、e buffer should NOT be within 2 pH units of the pKa values of the analytes. 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 This ensures reproducible retention times and good peak shapes.这保证了保留时间的重复性和良好的峰形。,Method Development HPLC Mo,Method Development HPLC Mobile Phase: Buffer 流动相：缓冲液,Henderson-Hasselbalch Equatio
10、n 公式: pH = pKa + log (A- / HA) Buffers for use in HPLC separations: 色谱分离使用的缓冲液BufferpKaBuffer RangeUV Cutoff Phosphate 2.1 1.1 3.1 200 nm磷酸盐Acetate4.75 3.75 5.75 210 nm醋酸盐 Citrate3.1 2.1 4.1 230 nm 柠檬酸盐,Method Development HPLC Mo,Method Development 方法开发 HPLC Detectors：HPLC 检测器,UV absorption 紫外吸收Fixe
11、d-wavelength UV detector 固定波长紫外检测器Variable-wavelength UV detector 可变波长紫外检测器Photodiode array detector 二极管阵列检测器 Fluorescence detector 荧光检测器 Mass spectrometer (LC/MS) 质谱仪 （液质联用） Electrochemical detector 电化学检测器 Conductivity detector 电导率检测器 Evaporative light scattering detector 蒸发光散射检测器,Method Developmen
12、t 方法开发 HPL,Method Development 方法开发 HPLC UV Detectors 紫外检测器,Method Development 方法开发 HPL,Method Development Other Chromatographic Conditions其它色谱条件,Flow rate 流速: 1.0 0.5 mL/min Injection Volume 进样量: 10 L 100 L Sample temperature 样品温度: 5C room temperature室温 Isocratic vs. Gradient 等度 VS. 梯度,Method Develo
13、pment Other C,Method Development 方法开发 Initial Chromatographic Conditions Initial 色谱条件,Column 柱: Waters Symmetry C184.6 mm x 15 cm, 3.5 m Column temperature 柱温: 35C Sample temperature 样品温度: Room Temperature室温 流动相 Mobile phase A: 0.1% H3PO4 in water Mobile phase B: 0.1% H3PO4 in acetonitrile 流速 Flow R
14、ate: 1.0 mL/min 进样量 Injection Volume: 20 L 检测器 Detector: Photodiode array detector 梯度 Gradient Profile: 10% B - 80% B in 60 min.,Method Development 方法开发 In,HPLC Trouble-shooting ：高效液相问题解答 Sources of peak tailing：拖尾的原因,H问题 pH issue 问题柱子 （柱床坏掉） Bad column (packed bed damaged) 化学效应 （与硅烷醇结合）Chemical eff
15、ects (interact with silanol) 柱过载 Column overloaded 不正确的稀释液 Wrong diluent (100% ACN) 死体积 Dead volume 温度问题Temperature issue 等等 etc.,HPLC Trouble-shooting ：高效液相问题解,HPLC Trouble-shooting Sources of ghost peak：鬼峰的原因,Contaminated solution溶液受到污染 Bad reagents不合格的试剂 Carry-over残留物 Detector Cell检测池,HPLC Troubl
16、e-shooting Sourc,Method Optimization 方法优化,Regulatory Requirements法规要求 Technical Requirements技术要求 Practical Requirements实践要求 Validation Requirements验证要求,Method Optimization 方法优化 Regul,Method Optimization - Four “S”方法优化 4S,Simple简，不复杂 Short 短（时） Sensitive灵敏 Suitable适合,Method Optimization - Four “S”,Me
17、thod Development 方法开发 method optimization sample 方法优化举例,柱 Column: Waters Symmetry C184.6 mm x 25 cm, 5 m 柱温 Column temperature: 20C 流动相 Mobile phase A: 0.01% HAC in water 流动相 Mobile phase B: 0.01% HAC in ACN 流速 Flow Rate: 0.8 mL/min 进样量 Injection Volume: 5 L 检测器 Detector: UV at 203 nm,Method Develop
18、ment 方法开发 met,Method Development 方法开发 method optimization sample (cont.) 方法优化举例,梯度 Gradient Profile:Time 时间% Mobile Phase A % B08020156535256535405743505743654258752575808020858020,Method Development 方法开发 me,Method Validation Protocol 方法验证方案,Objective 目的 Responsibilities 职责 Reagents/Standards 试剂/标准物
19、质 Samples 样品 Procedures 程序 Acceptance criteria 可接受标准,Method Validation Protocol 方法,Method Validation 方法验证,Accuracy (Spike recovery)准确度 （加标回收率） Precision (RSD%)精密度 （RSD） Repeatability injection precision重复性进样精密度 Intermediate Precision within-Lab中间精密度同一实验室范围内 Reproducibility between Labs重现性不同实验室之间,Met
20、hod Validation 方法验证 Accurac,Method Validation (continued)方法验证,Linearity (five levels)线性 （五个浓度水平） Range (50% to 150%)范围 (50% to 150%) Robustness耐用性,Method Validation (continued),Method Validation (continued)方法验证,Specificity专属性 Limit of Detection (LOD)检测限 Limit of Quantitation (LOQ)定量限 Solution Stabil
21、ity, etc. 溶液稳定性，等等,Method Validation (continued),Method Validation: API Assay method 方法验证：原料药 含量测定 Day 1 Experiment 第一天的试验,Accuracy, Precision, Linearity and Range for Assay of Drug Substance (API):原料药含量分析的准确度、精密度、线性和范围5 levels in the range of 50% to 150% of the nominal concentration (for example: 0
22、.1 mg/mL)规定浓度（例如： 0.1 mg/mL ） 50至150之间的5个浓度水平By weighing the solid drug substance or using the API Stock solution (1.0 mg/mL) 通过称量固体原料药或使用其储备液 (1.0 mg/mL) Repeat the above procedure (by weighing the solid drug substance or using a second separately prepared API Stock solution)重复以上程序（通过称量固体原料药或使用第二次另
23、外制备的储备液）Analyze as per method (prepare the working standard solutions, etc)按照方法进行分析（制备标准品溶液，等等）,Method Validation: API Assay m,Method Validation: API 方法验证：原料药 Day 2 Experiment 第二天的试验,Repeat the Day 1 experiment (two more independent preparations)重复第一天的试验 （重新进行两次独立的配制） Calculate the % Recovery for ea
24、ch sample计算每一个样品的回收率 Calculate the % RSD for each level计算每个水平的RSD Assess two linearity curves for each day (%Added vs. %Found) 评价每天的共两个线性曲线 （加标 VS.测得值）,Method Validation: API 方法验证：原料,Method Validation: API Assay 方法验证：原料药含量分析 Acceptance Criteria 可接受标准,Accuracy: The % Recovery must be within 98.0% to
25、102.0%准确度：回收率 98.0% to 102.0% Precision: %RSD is 1.0 %精密度：RSD 1.0 % Linearity: r 0.995 for each curve线性：相关系数r 0.995 （对每一个曲线）,Method Validation: API Assay,Method Validation: Impurities of API 方法验证：原料药的杂质 Day 1 Experiment 第一天试验,Accuracy, Precision, Linearity and Range for Impurity Estimation:准确度、精密度、线
26、性和范围5 levels in the range of 0.05% w/w to 150% of the known impurity specification (for example: 0.3%)已知杂质限度（例如0.3%)）Using the API Stock solution and the impurity stock solution for each know process impurity and degradates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 Repeat the above procedure (using a second sep
27、arately prepared impurity stock solution)重复以上程序（使用另外一个单独配制的杂质储备液）Analyze as per method (prepare the working standard solutions, etc)按照方法进行分析（制备工作标准品溶液，等等）,Method Validation: Impurities,Method Validation: API Impurity 方法验证：原料药的杂质 Day 2 Experiment 第二天的试验,Repeat the Day 1 experiment (two more independe
28、nt preparations) 重复第一天的试验（重新进行两次独立的配制）Calculate the % Recovery for each sample对于每个样品，计算回收率Calculate the % RSD for each level计算每个浓度水平的RSDAssess two linearity curves for each day (%Added vs. %Found) 评价每一天共两个线性曲线 （加标VS.测得值）,Method Validation: API Impurit,Method Validation: API Impurity 方法验证：原料药的杂质 Acce
29、ptance Criteria 可接受标准,Accuracy: The % Recovery must be within 70% to 130% for each impurity准确度：对每个杂质，回收率应为70% to 130% Precision: %RSD is 20 %精密度：RSD 20 %Linearity: r 0.99 for each curve线性： r 0.99 （对每个曲线）,Method Validation: API Impurit,Method Validation: Drug Product 方法验证：制剂 Day 1 Experiment 第一天的试验,A
30、ccuracy, Precision, Linearity and Range for Assay of Drug Product:制剂含量分析的准确度、精密度和线性及范围5 levels in the range of 50% to 150% of the nominal concentration (for example: 0.1 mg/mL)规定浓度（例如0.1 mg/mL ） 50% to 150% 范围内5个水平By weighing the solid drug substance or using the API Stock solution (1.0 mg/mL)通过称量固体
31、原料药或使用原料药储备液（ 1.0 mg/mL ） Add the placebo product or the placebo stock solution as per the formulation根据配方加安慰剂或安慰剂储备液Repeat the above procedure (by weighing the solid drug substance or using a second separately prepared API Stock solution)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）Analyze as per method (prepa
32、re the working standard solutions, etc)安装方法分析（配制工作标准品溶液，等等）,Method Validation: Drug Produc,Method Validation: Drug Product 方法验证：制剂 Day 2 Experiment 第二天的试验,Repeat the Day 1 experiment (two more independent preparations) 重复第一天的试验 （重新进行两次独立的配制）Calculate the % Recovery for each sample对每个样品计算回收率 Calculat
33、e the % RSD for each level计算每个浓度水平的RSD Assess two linearity curves for each day (%Added vs. %Found)评价每天共两个线性曲线 （加标VS.测得值）,Method Validation: Drug Produc,Method Validation: Drug Product 方法验证：制剂 Acceptance Criteria 可接受标准,Accuracy: The % Recovery must be within 97.0% to 103.0%准确度：回收率应在97.0% to 103.0% P
34、recision: %RSD is 2.0 %精密度：RSD 2.0 % Linearity: r 0.990 for each curve线性： r 0.990 （对每个曲线）,Method Validation: Drug Produc,Method Validation: 方法验证 Purposeful Stressing 强降解,Light (UV) stressing (control dark sample)紫外光降解(control dark sample)Heat stressing (80C in an oven and analyzed on a weekly basis
35、for up to one month)加热降解（置于80C 烘箱中，每周分析一次，共一个月的时间） Acid (1 N HCl) and base hydrolysis (1 N NaOH)酸、碱降解(1 N HCl) 和(1 N NaOH) Oxidation stressing氧化降解 Desired degradation by 5% to 15% level. 降解5% to 15%,Method Validation: 方法验证 Pu,Method Validation: 方法验证 Robustness Studies 耐用性研究,Mobile phase composition流
36、动相组份 Flow Rate ( 10%)流速( 10%) UV detector wavelength ( 5 nm)紫外检测波长( 5 nm) Column (same type, 3 different lots)柱（同型号，3个不同批号）Column Temperature ( 10C )柱温( 10C ),Method Validation: 方法验证 Ro,Method Validation: Sample Preparation 样品制备,Three lots and duplicate preparation for each lot (for both API or prod
37、uct)三个批号的样品，每批重复制备两份 （对原料药和制剂） Sample extraction/mixing time样品提取/混合时间 Filter adsorption studies过滤吸附研究 Method precision: six duplicate preparations for the same sample 方法精密度：对同一个样品，重复制备六份溶液,Method Validation: Sample P,Method Validation: Relative Response Factor (RRF)相对响应因子（RRF）,RRF = (AREAimpurity/CO
38、NCimpurity) / (AREAParent/CONCParent)RRF=(杂质峰面积/杂质浓度)/(主峰面积/主成分浓度),Method Validation: Relativ,System Suitability 系统适应性,Resolution ( Rs 2.0)分离度( Rs 2.0) Injection Precision (RSD% 2.0% for HPLC)进样精密度 RSD% 2.0% Tailing Factor ( 2.5)拖尾因子 ( 2.5) RRT (Relative Retention Time) 相对保留时间,System Suitability 系统适
39、应性 Resol,System Suitability (continued)系统适应性,LOQ or Reporting Limit(0.1% for Product or 0.05% for API)定量限或报告限 （制剂0.1%, 原料药:0.05% ） Reference Standard Check (98.0% to 102.0%)标准品检查 (98.0% to 102.0%) Theoretical Plates, etc.理论塔板数，等等,System Suitability (continued),System Suitability: Practical guide 系统适
40、应性：实用指南 Sample set method/sequence table样品分析安排/进样顺序表,For Assay and Impurities Methods针对含量和杂质检查Sample ID 样品号 Sample Name 样品名称# Injections 进样次数12107001:01A Blank (Diluent)212107001:03A Report Limit Solution312107001:03B Resolution solution2 12107001:06A Check Standard5 12107001:06B Working Standard212
41、107001:07A Sample Lot 07058212107001:07A Sample Lot 07059212107001:07A Sample Lot 07060212107001:06B Working Standard2,System Suitability: Practical,System Suitability: Practical guide Sample set method/sequence table,For HPLC Identity FingerprintHPLC 鉴别指纹Sample ID Sample Name# Injections12107001:01
42、A Blank (Diluent)212107001:03A Control Sample solution212107001:03B Reference STD solution2 12107001:07A Sample Lot 07058212107001:07A Sample Lot 07058212107001:07A Sample Lot 07058212107001:01A Blank (Diluent)112107001:03A Control Sample solution2,System Suitability: Practical,Calculation Tips,“As
43、is” Basis “Dried” Basis干燥的 “Anhydrous” Basis无水的,Calculation Tips “As is” Basi,GMP “Mind-set” GMP 理念,Raw data (on time, completed, accurate, reliable, etc.)原始数据 （及时、完整、准确、可靠，等等） Qualified chemist/staff/reviewer合格的分析人员/职员/审核者 Calibrated instruments/equipments经过校正的仪器/设备 Validated method, etc. 经过验证的方法，等
44、等 System suitability requirements系统适应性要求,GMP “Mind-set” GMP 理念 Raw dat,GMP “Mind-set” Date, initial, and time 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)08 FEB 2007 Initial: SW or SQW Time: 13:30 or 1:30 PM,GMP “Mind-set” Date, initi,Acknowledgements (致谢),Together We will make a difference !Thank you very much !,Acknowledgements (致谢)Together,
