药品质量控制中的现代分析方法与技术.ppt

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,*,资料仅供参考,不当之处，请联系改正。,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,资料仅供参考,不当之处，请联系改正。,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,资料仅供参考,不当之处，请联系改正。,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 NMRX-ray diffraction method,Near infrared spectrometry NIRS,现代光谱法及其应用,Modern spectroscopy&its application in pharmaceutical analysis,GC-FTIR,GC-MS,UPLC-MS,HPLC-NMR,H,yphenated,T,echniques in,C,hromatography,现代联用技术及其应用,HPLC-MS,CE-MS,+,+,样品与溶剂脱,离及电离,EI,ESI,APCI,LC/MS,接口,离子源,质量分析器,检测离子,HPLC,数据系统,质谱,离子识别,Quadrapole,Time of Flight,Fourier Transform,+,+,+,+,+,+,+,离子检测,+,+,-,+,+,+,+,+,+,-,+,+,+,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),C,21,H,31,N,5,O,2,MW=385,150,200,250,300,350,400,450,500,m/z,25,50,75,100,Relative Abundance,386,408,(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,100,150,200,250,300,350,400,m/z,25,50,75,100,Relative Abundance,122,386,222,150,265,180,(M+H),+,Precursor Ion Scanning,Advantage,ID compounds producing specific fragment ion(e.g.,PO,3,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 122,100,200,300,400,500,m/z,Relative Abundance,402,386,9,10,11,12,13,14,15,16,Time(min),25,50,75,100,Relative Abundance,11.62,13.84,13.16,14.40,12.13,10.45,15.45,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,100,200,300,400,500,m/z,Relative Abundance,402,386,9,10,11,12,13,14,15,16,Time(min),25,50,75,100,Relative Abundance,13.92,11.69,13.21,15.50,10.58,Selected Reaction Monitoring(SRM),Advantages,Targeted Analyte Monitoring,High Duty Cycle,“Simultaneous”Monitoring of Multiple Transitions,Disadvantage,No“advanced”structural information,Fixed m/z,Pass All,Fixed m/z,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,C,11,H,12,Cl,2,N,2,O,5,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(100,2.1 mm,5),Mobile Phase:,A:,Water,;B:Acetonitrile,Column Temperature:,40,o,C,Gradient Program:0.25 mL/min,Injection:20 uL with loop,Time(min),%A,%B,0,80,20,2.5,20,80,3.0,20,80,3.1,80,20,5.0,80,20,Operation Conditions for CAP,Ion source:,ESI,Ion polarity:,Negative,Spray voltage:,4000 V,Sheath gas pressure:,45,Auxiliary gas pressure:,15,Ion transfer capillary temperature:,300,o,C,Source CID:,8 V,Scan Type:,SRM,3 transitions of M-H,-,(m/z:321),(321,152,321,194 and 321,257),Q1 peak width,0.7 Da in SRM,or,0.2 Da in H-SRM,Q3 peak width,0.7 Da,Collision Pressure:,Ar at 1.3 mTorr,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,Q1 peak width,0.7 Da in SRM,or,0.2 Da in H-SRM,Q3 peak width,0.7 Da,CAP SRM Result:CAP Standard,Q1 peak width=0.7 Da,TIC,321-152,321-194,321-257,CAP,Peak Area Counts=2.4E4,CAP SRM Result:Kidney Blank,TIC,321-152,321-194,321-257,CAP SRM Result:Kidney Spiked(0.5ng/g),TIC,321-152,321-194,321-257,CAP,Not accurate for confirmation,CAP detected,CAP H-SRM Result:CAP Standard,Q1 peak width=0.2 Da,TIC,321-152,321-194,321-257,Peak Area Counts=7.3E3,CAP H-SRM Result:Kidney Blank,TIC,321-152,321-194,321-257,No CAP detected,CAP H-SRM Result:Kidney Spiked(0.5ng/g),TIC,321-152,321-194,321-257,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 SiO,2,(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,次数,环维黄杨星D含量%,峰1生物碱含量%,峰2生物碱含量%,峰4生物碱含量%,峰5生物碱含量%,有关生物碱总含量%,1,86.85,3.57,8.09,0.79,0.92,13.37,2,87.08,3.84,8.54,0.80,0.98,14.15,3,85.83,3.46,8.82,0.81,0.95,14.03,4,85.70,3.54,8.76,0.87,0.93,14.10,5,85.05,3.52,8.64,0.74,0.85,13.75,6,84.84,3.74,9.03,0.79,0.97,14.53,Mean,85.89,3.61,8.65,0.80,0.93,13.99,RSD%,1.07,3.65,3.38,4.62,4.63,2.8,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 SiO,2,(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,峰位号,t,R,(min),M+H+,(m/z),特征碎片离子,(,m/z),可能生物碱名称,1,3.92,370,339，325，283，,135，70，58,Cyclobuxomicreine K,Cyclobuxosuffrine K,Buxenone M,Cyclobuxoviridine B,2,4.03,431,413，382，323，,86，70，58,Buxazidine B,Cyclomicrophylline B,Cyclobuxoxazine C,Cyclomicrophylline C,3,5.35,415,384，84，58,Cycloprotobuxine A,Cyclokreanine B,Cyclovirobuxeine B,16-deoxybuxidienine C,4,5.92,417,399，386，368，,84，58,Cylcyclovirobuxine C,5,6.11,401,370，352，326，,171，58,Cycloprotobuxine C,Buxaminol E,Buxocyclamine A,Cyclobuxine B,6,7.03,387,369，356，338，,171，58,Cyclobuxine D,7,7.63,403,385，372，354，,70，58,Cyclovirobuxine D,8,8.28,375,357，344，326，,309，58,未有相关文献报道,9,9.77,389,371，358，340，,173，70，58,可能为,Cyclobuxine D,双键加氢还原产物,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,：,C,15,H,19,O,5,C,7,H,6,O,2,5-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-HT,1B,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-HT,1D,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-HT,1B,或5-HT,1D,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 270,m/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 270,m/z 158,（碰撞能量：,20ev,）,色谱条件选择,色谱柱,Intersil-ODS C18,Lichrospher-C18,Zorbak-ODS C18,Lichrospher-CN,P,henomenx 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%HCOOH,0.1%HAC,0.2%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：,C,16,H,21,N,3,O,2,盐酸曲马多,(Tramadol hydrochloric),MW,：,299.8,分子式：,C,16,H,25,NO,2,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。,Cmax,Dose,AUC,Dose,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,3,6,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,