药品质量控制中的现代分析方法与技术文档资料.ppt

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 MSNuclear magnetic resonance spectrometry NMRX-ray diffraction methodNear 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,AdvantageProvides MW Information,Full-Scan MS of Buspirone,Buspirone(丁螺环酮)C21H31N5O2MW=385,(M+H)+,(M+Na)+,Single Ion Monitoring(SIM),AdvantagesTargeted Analyte MonitoringHigh Duty CycleSimple,DisadvantagesCan suffer from interferencesNot as sensitive or selective as SRM(see below),Fixed m/z,Pass All,Pass All,Product Ion Scanning:A Tandem MS Method,AdvantageProvides Structural Information,DisadvantageLow duty cycle,Fixed m/z,Pass All,Scanning,Product Ion Spectrum,Q3,Q2,Q1,Product Ion Spectrum of Buspirone,(M+H)+,Precursor Ion Scanning,AdvantageID compounds producing specific fragment ion(e.g.,PO3 for phosphopeptides),DisadvantageLow 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,AdvantageScreen for compounds producing specific neutral loss(e.g.,loss of 176 for glucuronide conjugates),DisadvantageLow 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),AdvantagesTargeted Analyte MonitoringHigh Duty Cycle“Simultaneous”Monitoring of Multiple Transitions,DisadvantageNo“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,C11H12Cl2N2O5FMW=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 AutosamplerColumn:Thermo Hypersil Gold C18(1002.1 mm,5)Mobile Phase:A:Water;B:AcetonitrileColumn Temperature:40 oCGradient Program:0.25 mL/minInjection: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：30ELSD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；减少颈动脉血流；透过血脑屏障，增加脑血流量。,实验内容,建立苯甲酸利扎曲普坦在血浆、尿样浓度的LC-MS/MS测定方法苯甲酸利扎曲普坦分散片和胶囊进行生物等效性试验苯甲酸利扎曲普坦片体内药代动力学研究 单剂量(5,10,15 mg)多次给药(10 mg)稳态(10 mg),LC-MS/MS测定方法建立,测定方法选择质谱条件优化色谱条件选择 色谱柱选择 缓冲盐选择血浆处理方法 液液萃取法 蛋白沉淀法内标选择,LC-MS/MS,m/z 270m/z 158,Phenomenx PFP,1%冰醋酸,0.2%醋酸铵,蛋白沉淀法,盐酸曲马多,色谱条件,流动相A：醋酸盐缓冲液(1%冰醋酸，2%醋酸铵;pH 3.5)流动相B：甲醇(0.1%甲酸)梯度条件过程：0 min(B50%)1.0 min(B95%)4.5 min(B95%)4.6 min(B50%)6.5 min(B50%),空白溶剂色谱图 预处理的空白血浆色谱图,质谱条件,离子检测方式：ESI+SRM检测对象：利扎曲普坦 m/z 269.9158.1 曲马多 m/z 263.958.0喷口电压：5000 V雾化气压：35 psi辅助气压力：5 psi毛细管温度：350碰撞气氩气压力：1.3mTorr碰撞能量：20 eV,质谱条件优化,利扎曲普坦LC-MS质谱扫描图（m/z=270）,利扎曲普坦LC-MS/MS质谱扫描图（m/z=158）,利扎曲普坦质谱裂解图 m/z 270m/z 158（碰撞能量：20ev）,色谱条件选择,色谱柱,Intersil-ODS C18Lichrospher-C18 Zorbak-ODS C18 Lichrospher-CN Phenomenx Curosil-PFP,缓冲盐条件(0.1%HCOOH,v/v;0.2%醋酸铵,w/v;pH3.0）,采用PFP（五氟苯基）柱，样品的保留适当，峰型良好，有机相和水相的比例也较为适当。,醋酸铵(w/v),0.05%0.1%0.2%,甲醇:水=(50:50),0.2%(w/v)醋酸铵样品峰响应高，拖尾得到一定程度的改善。,0.1%HCOOH0.1%HAC0.2%HAC 0.5%HAC 1.0%HAC,1%HAC水相pH值为3.5，样品保留时间合适，峰形良好。,血浆处理方法,苯甲酸利扎曲普坦血浆提取条件试验,内标选择,佐米曲普坦(Zolmitriptan)MW：287 分子式：C16H21N3O2,盐酸曲马多(Tramadol hydrochloric)MW：299.8 分子式：C16H25NO2,曲马多LC-MS质谱扫描图（m/z=264）,曲马多LC-MS/MS质谱扫描图（m/z=58）,曲马多质谱裂解图 m/z 264 m/z 58,单次给药,受试者单次口服苯甲酸利扎曲普坦片后，苯甲酸利扎曲普坦的Cmax和AUC与服药剂量成正比关系。,CmaxDose,AUCDose,数据分析,受试者单剂量口服低中高剂量后的平均血药浓度-时间曲线,受试者单剂量口服低中高剂量后的平均累积排泄量-时间曲线,受试者多次服药第36天时平均谷时血药浓度-时间曲线,受试者多次口服达稳态后的平均药时曲线,2.4 其他质谱技术与特点,成功来源于,创新意识,不断探索,勇于实践,