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1、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,UltraPerfo
2、rmance 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
3、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
4、,(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 In
5、formation,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,Scanni
6、ng,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
7、,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 “ad
8、vanced” 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
9、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
10、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
11、 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),
12、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,
13、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,C
14、yclobuxomicreine 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
15、 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=
16、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；
