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1、Analytical Method Development and Validation,分析方法的建立和验证28 March 2008,Outline总纲,HPLC Method Development 高效液相方法建立HPLC Trouble-shooting Guide HPLC问题解答Method Optimization 方法优化Method Validation 方法验证System Suitability Requirements 系统适应性要求GMP “Mind-set” GMP理念,Method Development Method Classification 方法开发分析
2、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 方法开发 Useful 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 solubil
3、ity 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 Resource Planning & Gathering 资源计划收集,Solvents and reagents 溶剂和试剂Placebo materials 安慰剂Individual formulation component
4、s 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 HPLC Column 色谱柱,Reversed phase: C18(USP L1), C8(L7) 反相：C18(USP L1), C8(L7)Normal phase: Silica, CN, and NH2 Silica, CN, and NH2 Ion-Exchange: Dionex column 离子交换： Dionex c
5、olumnIon pair Chromatography: C18/C8/CN 离子对色谱法： C18/C8/CNSize Exclusion Chromatography 排阻色谱法Chiral separation 手性分离,Method Development HPLC Column (cont.) 色谱柱,Column Dimension: 柱子的尺寸4.6 mm x 25 cm4.6 mm x 15 cm4.6 mm x 10 cmParticle Diameter: 粒径10 m, 5 m, 3.5 m, 1.8 mPore size, Surface area, End capp
6、ing 孔径，表面积，封尾,Method Development 方法开发 HPLC Column色谱柱：考考你 ?,Particle Size 粒径Length长度 Expected N 要求3.5 m 5 cm 42003.5 m 10 cm ?5 m 25 cm 1200010 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 HPLC Column (cont.)色谱柱,Guard Column 预柱Column Wash 柱子冲洗Colu
7、mn Temperature (35 C - 60 C) 柱温 （ 35 C - 60 C ）Column Storage 柱子的储存,Method Development 方法开发 HPLC column selection summary 色谱柱选择：总结,C18 Column: first choice C18柱：首选Short length: 10 cm or 15 cm 短柱： 10 cm or 15 cmSmall 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 HPLC
8、 Mobile 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 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 phase buffer should NOT b
9、e 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 Mobile Phase: Buffer 流动相：缓冲液,Henderson-Hasselbalch Equation 公式:pH = pKa + log (A- / HA) Buffers for use i
10、n HPLC separations: 色谱分离使用的缓冲液Buffer pKa Buffer Range UV Cutoff Phosphate 2.1 1.1 3.1 200 nm 磷酸盐Acetate 4.75 3.75 5.75 210 nm 醋酸盐 Citrate 3.1 2.1 4.1 230 nm 柠檬酸盐,Method Development 方法开发 HPLC Detectors：HPLC 检测器,UV absorption 紫外吸收Fixed-wavelength UV detector 固定波长紫外检测器Variable-wavelength UV detector 可变
11、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 Development 方法开发 HPLC UV Detectors 紫外检测器,Method Development Other Chromatographic C
12、onditions 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 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 mColumn temperature 柱
13、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 Rate: 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.,HPLC Trouble-shooting ：高效液相问题解答
14、Sources of peak tailing：拖尾的原因,pH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 effects (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 Sources of ghost peak：鬼峰的原因,Contaminated
15、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,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 - Four “S” 方法优化 4S,Simple 简，不复杂Short 短（时）Sensitive 灵敏Suitable 适合,Method Develo
16、pment 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 Development 方法开发 method optimization sample (cont.) 方法优化举例,
