1、Analytical Method Analytical Method Development and ValidationDevelopment and Validation分析方法的建立和验证 28 March 2008Outline总纲z HPLC Method Development 高效液相方法建立高效液相方法建立z HPLC Trouble-shooting GuideHPLC问题解答问题解答z Method Optimization方法优化方法优化z Method Validation方法验证方法验证z System Suitability Requirements系统适应性要求
2、系统适应性要求z GMP “Mind-set”GMP理念理念Method Development Method Classification方法开发分析方法分类z Assay Method 含量方法z Impurity Profile 杂质情况 - Related Substances 有关物质z Chiral Purity 手性纯度z Content Uniformity 含量均一性z Dissolution z Cleaning Validation 清洁验证z Identity Fingerprint 鉴别指纹法Method Development 方法开发 Useful Informa
3、tion 有用的信息z Chemical structure and MW化学结构和分子量z Solvent solubility 溶剂中的溶解度z pKa and pH solubility酸性解离常数和PH溶解度z UV spectrum紫外光谱z Process impurities工艺杂质z Possible degradation impurities可能的降解杂质z Literature References参考文献z MSDS Material safety data sheets 安全数据表Method Development 方法开发 Resource Planning &
4、Gathering 资源计划收集z Solvents and reagents溶剂和试剂z Placebo materials安慰剂z Individual formulation components各个处方成分z Related substance standards有关物质标准品z API Reference Standards活性成分标准品z Accelerated stress sample强降解样品Method Development 方法开发 HPLC Column 色谱柱z Reversed phase: C18(USP L1), C8(L7)反相：C18(USP L1), C
5、8(L7)z Normal phase: Silica, CN, and NH2Silica, CN, and NH2z Ion-Exchange: Dionex column离子交换： Dionex columnz Ion pair Chromatography: C18/C8/CN离子对色谱法： C18/C8/CNz Size Exclusion Chromatography排阻色谱法z Chiral separation手性分离Method Development HPLC Column (cont.) 色谱柱z Column Dimension: 柱子的尺寸 4.6 mm x 25 c
6、m 4.6 mm x 15 cm 4.6 mm x 10 cmz Particle Diameter: 粒径 10 m, 5 m, 3.5 m, 1.8 m3.5 m, 1.8 mz Pore size, Surface area, End capping孔径，表面积，封尾 Method Development 方法开发 HPLC Column色谱柱：考考你考考你 ?z Particle Size 粒径Length长度 Expected N 要求3.5 m 5 cm42003.5 m 10 cm ?5 m 25 cm1200010 m 25 cm ?z Particle Size粒径 Leng
7、th长度 Resolution分离度3.5 m 5 cm 1.53.5 m 10 cm ?Method Development 方法开发 HPLC Column (cont.)色谱柱z Guard Column预柱预柱z Column Wash柱子冲洗柱子冲洗z Column Temperature (35 C - 60 C)柱温柱温 （ 35 C - 60 C ）z Column Storage柱子的储存柱子的储存Method Development 方法开发 HPLC column selection summary色谱柱选择：总结色谱柱选择：总结z C18 Column: first c
8、hoiceC18柱：首选柱：首选z Short length: 10 cm or 15 cm短柱：短柱： 10 cm or 15 cmz Small diameter: 3.5 m or 5 m 小的粒径：小的粒径： 3.5 m or 5 m z End-capped封尾封尾Method Development 方法开发 HPLC Mobile Phase 流动相z Strong solvent component: 强溶剂组份强溶剂组份Methanol 甲醇甲醇Acetonitrile 乙腈乙腈Tetrahydrofuran 四氢呋喃四氢呋喃z Weak solvent component:
9、 弱溶剂组份弱溶剂组份Water 水水Buffer 缓冲液缓冲液 Diluted acid (0.1% H3PO4) Method Development HPLC Mobile Phase: Buffer流动相：缓冲液 Buffer Selection Principle:缓冲液选择原则缓冲液选择原则The pH of the mobile phase buffer should NOT be within 2 pH units of the pKa values of the analytes. 流动相缓冲液的流动相缓冲液的pH值不应在样品酸性解离常数值值不应在样品酸性解离常数值pH单位的
10、单位的2范围内范围内 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。这保证了保留时间的重复性和良好的峰形。Method Development HPLC Mobile Phase: Buffer 流动相：缓冲液z Henderson-Hasselbalch Equation 公式公式: pH = pKa + log (A- / HA) z Buffers for use in HPLC separations: 色谱分离使用的缓冲液色谱分离使用的缓冲液BufferpKaBuff
11、er Range UV Cutoff Phosphate 2.1 1.1 3.1 2.0)分离度( Rs 2.0)z Injection Precision (RSD% 2.0% for HPLC)进样精密度 RSD% 2.0% z Tailing Factor ( 2.5)拖尾因子 ( 2.5)z RRT (Relative Retention Time) 相对保留时间 System Suitability (continued)系统适应性z 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
12、料药:0.05% ）z Reference Standard Check (98.0% to 102.0%)标准品检查 (98.0% to 102.0%)z Theoretical Plates, etc.理论塔板数，等等System Suitability: Practical guide 系统适应性：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:
13、01A Blank (Diluent)212107001:03A Report Limit Solution312107001:03B Resolution solution2 12107001:06A Check Standard5 12107001:06B Working Standard212107001:07A Sample Lot 07058212107001:07A Sample Lot 07059212107001:07A Sample Lot 07060212107001:06B Working Standard2 System Suitability: Practical g
14、uide Sample set method/sequence table For HPLC Identity FingerprintHPLC 鉴别指纹Sample ID Sample Name# Injections12107001:01A Blank (Diluent)212107001:03A Control Sample solution212107001:03B Reference STD solution2 12107001:07A Sample Lot 07058212107001:07A Sample Lot 07058212107001:07A Sample Lot 0705
15、8212107001:01A Blank (Diluent)112107001:03A Control Sample solution2 Calculation Tips z “As is” Basisz “Dried” Basis干燥的z “Anhydrous” Basis无水的 GMP “Mind-set” GMP 理念 zRaw data (on time, completed, accurate, reliable, etc.)原始数据 （及时、完整、准确、可靠，等等）z Qualified chemist/staff/reviewer合格的分析人员/职员/审核者z Calibrate
16、d instruments/equipments经过校正的仪器/设备z Validated method, etc. 经过验证的方法，等等z System suitability requirements系统适应性要求 GMP “Mind-set” Date, initial, and time 日期、姓名缩写和时间记录的格式z Date:14 2 2007 (14.2.2007)8 2 2007 (8.2.2007)08 FEB 2007z Initial: SW or SQWz Time: 13:30 or 1:30 PM Acknowledgements (致谢)Together Together We will make a difference ! We will make a difference !Thank you very much !Thank you very much !
