Method Development – HPLC Column (cont.) 色谱柱
Col mm x 15 cm 4.6 mm x 10 cm
Particle Diameter: 粒径 10 µm, 5 µm, 3.5 µm, 1.8 µm Pore size, Surface area, End capping 孔径，表面积，封尾
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 Storage 柱子的储存
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 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
封尾
Method Development 方法开发
– HPLC 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 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 – 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 cm Small diameter: 3.5 µ m or 5 µ m 小的粒径： 3.5 µ m or 5 µ m End-capped
Method Development – HPLC Mobile Phase: Buffer 流动相：缓冲液
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 µm 5 cm 4200 3.5 µm 10 cm ??? 5 µm 25 cm 12000 10 µm 25 cm ??? Particle Size粒径 Length长度 Resolution分离度 3.5 µm 5 cm 1.5 3.5 µm 10 cm ???
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 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 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 – 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