药品质量控制中的 现代分析方法与技术,Modern analytical methods & techniques in quality control of drugs,第十六章,现代分析方法与技术，为药学的发展提供了适时而有效的手段与动力。,色谱及其联用技术： 药学研究分子水平。 手性分析： 毛细管电泳及手性色谱技术药物研究与质量控制提供了保障。 现代光谱技术： 药物结构鉴定， 微量杂质检定。,第一节 概况,Capillary electrophoresis,CE,Modern chromatogr & its application,药物现代色谱法及其应用,UPLC,UltraPerformance LC (UPLC ) technology starts with unique 1.7 m small-particle chemistries. Chromatographers no longer need to choose between speed and resolutionwith UPLC you get both.,Mass spectroscopy MS Nuclear magnetic resonance spectrometry NMR X-ray diffraction method Near infrared spectrometry NIRS,现代光谱法及其应用,Modern spectroscopy & its application in pharmaceutical analysis,Hyphenated Techniques in Chromatography,现代联用技术及其应用,HPLC-MS,CE-MS,第二节 液质联用技术与应用,2.1 离子化方式,2.2 离子分离与测定模式,Full-Scan Mass Spectrometry,Advantage Provides MW Information,Full-Scan MS of Buspirone,Buspirone (丁螺环酮) C21H31N5O2 MW = 385,(M+H)+,(M+Na)+,Single Ion Monitoring (SIM),Advantages Targeted Analyte Monitoring High Duty Cycle Simple,Disadvantages Can suffer from interferences Not as sensitive or selective as SRM (see below),Fixed m/z,Pass All,Pass All,Product Ion Scanning: A Tandem MS Method,Advantage Provides Structural Information,Disadvantage Low duty cycle,Fixed m/z,Pass All,Scanning,Product Ion Spectrum,Q3,Q2,Q1,Product Ion Spectrum of Buspirone,(M+H)+,Precursor Ion Scanning,Advantage ID compounds producing specific fragment ion (e.g., PO3 for phosphopeptides),Disadvantage Low duty cycle,Fixed m/z,Pass All,Scanning,Precursor Ion Spectrum,Q3,Q2,Q1,Precursor Ion Scan Mode for Buspirone Metabolites,Precursor Ion Scan: Q3 set to m/z 122,Neutral Loss Scanning,Advantage Screen for compounds producing specific neutral loss (e.g., loss of 176 for glucuronide conjugates),Disadvantage Low duty cycle,Scanning,Pass All,Scanning,Neutral Loss Spectrum,Linked,Q3,Q2,Q1,Neutral Loss Scan of Buspirone Metabolites,Neutral Loss Scan: Q1/Q3 difference set to 121 Da,Selected Reaction Monitoring (SRM),Advantages Targeted Analyte Monitoring High Duty Cycle “Simultaneous” Monitoring of Multiple Transitions,Disadvantage No “advanced” structural information,Q1,Q2,Q3,MS/MS Selectivity in Complex Matrices,息斯敏阿斯咪唑(astemizole),Chlroamphenicol(氯霉素，CAP)残留测定 黄杨生物碱成分鉴定 苯甲酸利扎曲普坦人体药代动力学研究,2.3药物分析中的典型应用,C11H12Cl2N2O5 FMW=323.13,【类别】 酰胺醇类抗生素 【适应症】本品是治疗伤寒、副伤寒的首选药物，外用可治疗沙眼。因脑脊液浓度高，故常用于治疗细菌性脑膜炎和脑脓肿。此外，尚可外用治疗痤疮、酒糟鼻、脂溢性皮炎等。,被农业养殖滥用！ 肉食品中严格检查。,2.3.1 Chlroamphenicol(氯霉素，CAP)残留测定,HPLC analysis was performed on the Finnigan Surveyor HPLC module with MS Pump and Autosampler Column: Thermo Hypersil Gold C18 (1002.1 mm, 5) Mobile Phase: A: Water; B: Acetonitrile Column Temperature: 40 oC Gradient Program: 0.25 mL/min Injection: 20 uL with loop,Operation Conditions for CAP,Q1 peak width and H-SRM experiment,Enabling the H-SRM experiment Highly Selective Selected Reaction Monitoring (H-SRM) Reduces “isobaric” chemical noise Increases confidence of analysis & improved LOQ,CAP SRM Result: CAP Standard Q1 peak width = 0.7 Da,CAP,Peak Area Counts = 2.4E4,CAP SRM Result: Kidney Blank,CAP SRM Result: Kidney Spiked (0.5ng/g),CAP,Not accurate for confirmation,CAP detected,CAP H-SRM Result: CAP Standard Q1 peak width = 0.2 Da,Peak Area Counts = 7.3E3,CAP H-SRM Result: Kidney Blank,No CAP detected,CAP H-SRM Result: Kidney Spiked (0.5ng/g),CAP,2.3.2 黄杨宁生物碱HPLC-MS联用鉴定,黄杨科植物小叶黄杨Buxus microphlla Sieb. et. Zucc. var. sinica Rehd.et Wils中含有具有较强心血管疾病治疗活性的孕甾烷生物碱，主要含环维黄杨星D、环黄杨碱D和环常绿黄杨碱C等生物碱成分。,黄杨生物碱HPLC-ELSD色谱图,色谱条件 色谱柱：Lichrospher SiO2 (250mm4.6mm,5 m) 流动相：四氢呋喃-甲醇-乙腈-氨水 ( 32:50:13:3) 流速：1mLmin -1 柱温：30 ELSD参数：漂移管温度70 雾化气体（N2） 流速：1.5 Lmin -1,环维黄杨星D和有关生物碱含量测定结果,环维黄杨星D及其有关生物碱的鉴别 质谱条件 电喷雾离子化正离子检测 喷口电压5000V 雾化气压35psi 辅助气压力5psi 毛细管温度350 碰撞气氩气压力1.5mTorr 色谱条件 色谱柱：Lichrospher SiO2 (250mm4.6mm,5 m) 流动相：四氢呋喃-甲醇-乙腈-氨水 ( 32:50:13:3) 流速：1mLmin -1 柱温：30,黄杨宁LC-MS/MS全扫描色谱图,黄杨宁LC-MS/MS全扫描色谱放大图,1,2,3,4,5,6,7,8,9,峰1母离子质荷比,峰1二级质谱图,M+H+=370,可能为峰1的黄杨宁有关生物碱,Cyclobuxomicreine K,Cyclobuxosuffrine K,Buxenone M,Cyclobuxoviridine B,峰2母离子质荷比,峰2二级质谱图,M+H+= 431,可能为峰2的黄杨宁有关生物碱,Cyclomicrophylline B,Buxazidine B,Cyclobuxoxazine C,Cyclomicrophylline C,峰3母离子质荷比,峰3二级质谱图,M+H+= 415,可能为峰3的黄杨宁有关生物碱,Cycloprotobuxine A,Cyclokreanine B,Cyclovirobuxeine B,16-deoxybuxidienine C,峰4母离子质荷比,峰4二级质谱图,环常绿黄杨碱C,M+H+= 417,峰5母离子质荷比,峰5二级质谱图,M+H+= 401,383.34,可能为峰5的黄杨宁有关生物碱,Cycloprotobuxine C,Buxaminol E,Buxocyclamine A,Cyclobuxine B,峰6母离子质荷比,峰6二级质谱图,环黄杨碱D,M+H+= 387,峰7母离子质荷比,峰7二级质谱图,环维黄杨星D,M+H+= 403,峰8母离子质荷比,M+H+= 375,357.15,峰8二级质谱图,峰9二级质谱图,371.17,m/z=375,m/z=357,m/z=344,m/z=326,m/z=309,357.15,峰9母离子质荷比,M+H+= 389,峰9二级质谱图,371.17,m/z=389,m/z=371,m/z=358,m/z=340,m/z=309,371.17,HPLC-ELSD法黄杨宁有关生物碱归属表,苯甲酸利扎曲普坦 (Rizatriptan Benzoate) MW: 391.47 分子式：C15H19O5C7H6O2,5-HT受体拮抗剂,2.3.3 苯甲酸利扎曲普坦人体药代动力学研究,药理作用,刺激大脑血管壁的后接点5-HT1B受体收缩血管，降低颅内血管通透性； 刺激三叉神经前突触5-HT1D受体，调节神经递质的释放，抑制硬膜的神经原性炎症反应和血浆外渗； 阻止血管肽的释放，使血管口径正常化，通过收缩颅内血管并抑制神经炎症； 刺激脑干5-HT1B或5-HT1D受体，抑制三叉神经核兴奋；