药物分析 药品质量控制中的现代分析方法与技术PPT学习教案

1、会计学112/16/20212 现代分析方法与技术，为药学的发展提供了适时而有效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。的手段与动力。色谱及其联用技术：色谱及其联用技术： 药学研究分子水平。药学研究分子水平。手性分析：手性分析： 毛细管电泳及手性色谱技术药物研究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。与质量控制提供了保障。现代光谱技术：现代光谱技术： 药物结构鉴定，药物结构鉴定， 微量杂质检定。微量杂质检定。第一节 概况第1页/共89页12/16/20213Modern chromatogr & its application药物现代色谱法及其应用药

2、物现代色谱法及其应用第2页/共89页12/16/20214第3页/共89页12/16/20215现代光谱法及其应用现代光谱法及其应用Modern spectroscopy & its application in pharmaceutical analysis第4页/共89页12/16/20216GC-FTIRGC-MSUPLC-MSHPLC-NMRHyphenated Techniques in Chromatography 现代联用技术及其应用现代联用技术及其应用HPLC-MSCE-MS第5页/共89页+样品与溶剂脱离及电离 EI ESI APCILC/MS接口离子源质量分析器检测

3、离子HPLC数据系统离子识别 Quadrapole Time of Flight Fourier Transform +离子检测+-+-+第二节第二节 液质联用技术与应用液质联用技术与应用第6页/共89页2.1 离子化方式离子化方式第7页/共89页12/16/20219第8页/共89页12/16/202110第9页/共89页12/16/202111第10页/共89页12/16/202112第11页/共89页12/16/2021132.2 离子分离与测定模式离子分离与测定模式第12页/共89页12/16/202114第13页/共89页12/16/202115Full-Scan Mass Spec

4、trometryAdvantage Provides MW Information第14页/共89页12/16/202116NNNNNOOBuspirone (丁螺环酮)C21H31N5O2MW = 385150200250300350400450500m/z255075100Relative Abundance386408(M+H)+(M+Na)+第15页/共89页12/16/202117Advantages Targeted Analyte Monitoring High Duty Cycle SimpleDisadvantages Can suffer from interference

5、s Not as sensitive or selective as SRM (see below)Fixed m/zPass AllPass All第16页/共89页12/16/202118Product Ion Scanning: A Tandem MS MethodAdvantage Provides Structural InformationDisadvantage Low duty cycleFixed m/zPass AllScanningProduct Ion SpectrumQ3Q2Q1第17页/共89页12/16/202119NHNNNOONHNOO100150200250

6、300350400m/z255075100Relative Abundance122386222150265180NNNNHNOO(M+H)+第18页/共89页12/16/202120Precursor Ion ScanningAdvantageID compounds producing specific fragment ion (e.g., PO3 for phosphopeptides)DisadvantageLow duty cycleFixed m/zPass AllScanningPrecursor Ion SpectrumQ3Q2Q1第19页/共89页12/16/202121P

7、recursor Ion Scan: Q3 set to m/z 122NNNNNOOOH100200300400500m/zRelative Abundance402386910111213141516Time (min)255075100Relative Abundance11.6213.8413.1614.4012.1310.4515.45NNNNNOONHNN第20页/共89页12/16/202122Neutral Loss ScanningAdvantageScreen for compounds producing specific neutral loss (e.g., loss

8、 of 176 for glucuronide conjugates)Disadvantage Low duty cycleScanningPass AllScanningNeutral Loss SpectrumLinkedQ3Q2Q1第21页/共89页12/16/202123Neutral Loss Scan: Q1/Q3 difference set to 121 Da100200300400500m/zRelative Abundance402386910111213141516Time (min)255075100Relative Abundance13.9211.6913.2115

9、.5010.58NNNNNOONNNNNOOOH第22页/共89页12/16/202124Selected Reaction Monitoring (SRM)AdvantagesTargeted Analyte MonitoringHigh Duty Cycle“Simultaneous” Monitoring of Multiple TransitionsDisadvantage No “advanced” structural informationFixed m/zPass AllFixed m/zQ1Q2Q3第23页/共89页12/16/202125MS/MS Selectivity

10、in Complex Matrices息斯敏阿斯咪唑(astemizole)第24页/共89页12/16/202126第25页/共89页12/16/202127OHOHNHHHOClClO2NC11H12Cl2N2O5FMW=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，氯霉素，CAP)残留测定残留测定第26页/共89页12/16/202128HPLC analy

11、sis 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 loopTime (min)% A% B080202.520803.020803.180205.08020第2

12、7页/共89页12/16/202129Operation Conditions for CAPIon source:ESIIon polarity:NegativeSpray voltage:4000 VSheath gas pressure:45Auxiliary gas pressure:15Ion transfer capillary temperature:300 oCSource CID:8 VScan Type:SRM, 3 transitions of M-H-(m/z: 321)(321152, 321 194 and 321 257)Q1 peak width0.7 Da i

13、n SRM or 0.2 Da in H-SRMQ3 peak width0.7 DaCollision Pressure:Ar at 1.3 mTorrOHOHNHHHOClClO2N第28页/共89页12/16/202130Q1 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

14、& improved LOQQ1 peak width0.7 Da in SRM or 0.2 Da in H-SRMQ3 peak width0.7 Da第29页/共89页12/16/202131OHOHNHHOClClO2NONHHNOClClOHONHHHOClClO2NOHO2NOHCO2NOHHm/z 321m/z 257m/z 321m/z 194m/z 152第30页/共89页12/16/202132RT: 0.00 - 5.000.00.20.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.6

15、4.8Time (min)020406080100020406080100020406080100Relative Abundance020406080100RT: 2.83AA: 60868SN: 15803.843.254.652.341.030.802.473.561.530.324.523.113.431.731.880.172.064.872.644.170.644.311.230.471.402.20RT: 2.83AA: 23577SN: 16694.653.201.032.133.793.574.213.392.373.970.114.421.400.834.901.692.5

16、41.890.680.310.461.241.53RT: 2.83AA: 13035SN: 1644RT: 2.83AA: 24218SN: 6393.843.252.341.030.802.474.593.564.761.530.323.113.431.734.431.880.172.062.644.174.311.230.541.402.20NL:1.82E4TIC F: MS ICIS 1221C03NL:7.07E3Base Peak m/z= 151.50-152.50 F: MS ICIS 1221C03NL:4.48E3Base Peak m/z= 193.50-194.50 F

17、: MS ICIS 1221C03NL:6.77E3Base Peak m/z= 256.50-257.50 F: MS ICIS 1221C03TIC321-152321-194321-257CAPPeak Area Counts = 2.4E4第31页/共89页12/16/202133RT: 0.00 - 5.000.00.20.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.8Time (min)020406080100020406080100020406080100Relative Abundance0

18、204060801001.281.481.751.892.052.612.231.032.832.462.953.073.223.783.480.694.603.624.324.060.364.870.184.454.720.512.772.052.231.851.641.522.421.772.572.953.253.351.313.544.321.034.163.974.870.420.930.224.723.824.463.690.811.180.662.152.682.991.651.852.293.212.831.372.392.033.524.504.013.673.451.124

19、.610.194.924.324.200.940.420.820.691.281.481.751.892.612.052.231.032.462.833.073.293.783.490.694.603.624.060.364.374.214.830.180.51NL:5.30E4TIC F: MS 1221D05NL:1.79E3Base Peak m/z= 151.50-152.50 F: MS 1221D05NL:4.69E2Base Peak m/z= 193.50-194.50 F: MS 1221D05NL:5.27E4Base Peak m/z= 256.50-257.50 F:

20、MS 1221D05TIC321-152321-194321-257第32页/共89页12/16/202134RT: 0.00 - 5.000.00.20.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.8Time (min)020406080100020406080100020406080100Relative Abundance0204060801001.292.061.482.881.842.262.421.082.762.623.033.343.483.973.624.943.784.814.390.5

21、80.130.464.240.280.774.104.66RT: 2.88AA: 13715SN: 30RMS2.231.572.111.452.451.821.283.342.593.483.933.191.124.163.710.694.390.434.710.190.870.554.864.54RT: 2.88MA: 6787SN: 17RMS2.511.882.661.661.081.261.972.261.473.820.950.724.123.033.204.673.654.403.374.854.543.980.550.330.134.26RT: 2.87MA: 14653SN:

22、 INF1.292.061.481.842.291.002.422.613.033.253.393.973.624.943.784.814.390.580.130.464.240.280.774.104.66NL:4.68E4TIC F: MS 1221D08NL:3.47E3Base Peak m/z= 151.50-152.50 F: MS ICIS 1221D08NL:2.26E3Base Peak m/z= 193.50-194.50 F: MS 1221D08NL:4.63E4Base Peak m/z= 256.50-257.50 F: MS 1221D08TIC321-15232

23、1-194321-257CAPNot accurate for confirmationCAP detected第33页/共89页12/16/202135RT: 0.00 - 5.000.00.20.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.8Time (min)020406080100020406080100020406080100Relative Abundance020406080100RT: 2.89AA: 16306SN: 1762RMS1.661.780.221.153.864.324.893

24、.462.031.010.551.512.302.614.593.311.320.773.083.680.363.994.202.732.444.724.442.15RT: 2.89AA: 7313SN: 24498RMS3.423.083.854.384.831.073.980.151.331.532.061.763.673.301.932.560.362.200.492.694.120.690.884.592.36RT: 2.91AA: 1841SN: 273RMSRT: 2.89AA: 7129SN: 768RMS1.661.780.221.154.323.863.461.012.030

25、.552.301.512.614.593.311.324.870.773.680.364.202.444.003.074.724.46NL:5.46E3TIC F: MS ICIS 1221G11NL:2.39E3Base Peak m/z= 151.50-152.50 F: MS ICIS 1221G11NL:7.50E2Base Peak m/z= 193.50-194.50 F: MS ICIS 1221G11NL:2.46E3Base Peak m/z= 256.50-257.50 F: MS ICIS 1221G11TIC321-152321-194321-257Peak Area

26、Counts = 7.3E3第34页/共89页12/16/202136RT: 0.00 - 5.000.00.20.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.8Time (min)020406080100020406080100020406080100Relative Abundance0204060801001.301.782.051.952.591.471.012.412.201.672.853.220.290.520.733.504.643.864.320.863.330.114.114.894.7

27、74.443.691.521.623.111.871.432.264.641.774.320.953.332.832.550.751.214.843.560.454.150.580.193.854.544.013.731.133.862.732.922.580.222.461.621.502.060.991.292.351.724.373.371.420.670.802.224.690.481.850.144.093.103.244.943.563.731.301.782.051.952.591.471.012.412.202.853.220.290.520.733.503.110.860.1

28、14.114.624.894.233.863.694.41NL:2.08E3TIC F: MS 1221G04NL:2.14E2Base Peak m/z= 151.50-152.50 F: MS 1221G04NL:2.70E1Base Peak m/z= 193.50-194.50 F: MS 1221G04NL:2.06E3Base Peak m/z= 256.50-257.50 F: MS 1221G04TIC321-152321-194321-257No CAP detected第35页/共89页12/16/202137RT: 0.00 - 5.000.00.20.40.60.81.

29、01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.8Time (min)020406080100020406080100020406080100Relative Abundance020406080100RT: 2.86MA: 7799SN: 23841.302.052.581.581.832.202.340.991.172.990.813.144.893.680.313.293.960.094.490.573.414.264.703.554.13RT: 2.85AA: 2757SN: 274RMS2.061.461.651.92

30、1.292.241.112.641.773.293.964.492.404.934.700.983.783.093.554.130.530.800.300.144.330.67RT: 2.85AA: 1107SN: 240RMS1.564.891.661.382.362.191.05 1.172.042.604.430.163.333.493.103.992.724.133.861.863.640.794.260.534.780.390.674.60RT: 2.86MA: 3554SN: 1NL:2.45E3TIC F: MS 1221G07NL:8.97E2Base Peak m/z= 15

31、1.50-152.50 F: MS ICIS 1221G07NL:3.82E2Base Peak m/z= 193.50-194.50 F: MS ICIS 1221G07NL:2.39E3Base Peak m/z= 256.50-257.50 F: MS 1221G07TIC321-152321-194321-257CAP第36页/共89页12/16/202138 第37页/共89页12/16/202139黄杨生物碱HPLC-ELSD色谱图 色谱条件色谱条件色谱柱：Lichrospher SiO2 (250mm4.6mm,5 m)流动相：四氢呋喃-甲醇-乙腈-氨水 ( 32:50:13:3

32、)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第38页/共89页12/16/202140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% 186.853.578.090.790.9213.37287.083.848.540.800.9814.15385.833.468.820.810.9514.03485.703.548.760.870.9314.10585.053.528.640.740.8513.75684.843.749.030.

33、790.9714.53Mean85.893.618.650.800.9313.99RSD%1.073.653.384.624.632.8第39页/共89页12/16/202141第40页/共89页12/16/202142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 123456789第41页/共89页12/16/202143峰1母离子质荷比峰1二级质谱图M+H+=370第42页/共89页12/16/202144可能为峰1的黄杨宁有关生物碱NOCH3CH3NCH3CH3HOCH3HONHNHOCH3Cyclobuxomicreine KCyclobuxosuffri

34、ne KBuxenone MCyclobuxoviridine B第43页/共89页12/16/202145峰2母离子质荷比峰2二级质谱图M+H+= 431第44页/共89页12/16/202146可能为峰2的黄杨宁有关生物碱NNHCH3CH2OHCH3CH3OHNONHCH3CH2OHCH3CH3NOHNOCH3CH3CH3NCH3CH2OHCH3OHNHCH3Cyclomicrophylline BBuxazidine BCyclobuxoxazine CCyclomicrophylline C第45页/共89页12/16/202147峰3母离子质荷比峰3二级质谱图M+H+= 415第46

35、页/共89页12/16/202148可能为峰3的黄杨宁有关生物碱NNCH3CH3CH3CH3NNHCH3CH3OHCH3NNHCH3CH3OHCH3NHNCH3CH2OHCH3CH3Cycloprotobuxine ACyclokreanine BCyclovirobuxeine B16-deoxybuxidienine C第47页/共89页12/16/202149峰4母离子质荷比峰4二级质谱图CH3CH3NHCH3CH3OHNHCH3HCH3HCH3CH3环常绿黄杨碱CM+H+= 417第48页/共89页12/16/202150峰5母离子质荷比峰5二级质谱图M+H+= 401383.34第4

36、9页/共89页12/16/202151可能为峰5的黄杨宁有关生物碱NCH3CH3CH3NHNH2CH3OHNCH3NCH3CH3CH3NCH3CH3CH3NCH3CH2NHOHCycloprotobuxine CBuxaminol EBuxocyclamine ACyclobuxine B第50页/共89页12/16/202152峰6母离子质荷比峰6二级质谱图NHCH3CH3OHNHCH3HCH3HCH3CH2环黄杨碱DM+H+= 387第51页/共89页12/16/202153峰7母离子质荷比峰7二级质谱图CH3CH3NHCH3CH3OHNHCH3HCH3HCH3环维黄杨星DM+H+= 40

37、3第52页/共89页12/16/202154峰8母离子质荷比M+H+= 375第53页/共89页12/16/202155 357.15峰8二级质谱图峰9二级质谱图 371.17第54页/共89页12/16/202156CH3OHNH2CH3HCH3HCH3CH3NH2H2OCH3NH2CH3HCH3HCH3CH3NH2CH3OHHCH3HCH3CH3NH2CH3HCH3HCH3CH3NH2CH3HCH3HCH3CH3NH2CH3H2ONH3m/z=375m/z=357m/z=344m/z=326m/z=309 357.15第55页/共89页12/16/202157NHCH3CH3OHNHCH3

38、HCH3HCH3CH2峰9母离子质荷比M+H+= 389NHCH3CH3OHNHCH3HCH3HCH3CH3第56页/共89页12/16/202158峰9二级质谱图 371.17第57页/共89页12/16/202159NHCH3CH3OHNH2CH3HCH3HCH3CH3H2ONHCH3NH2CH3HCH3HCH3CH3NH2CH3NHCH3OHHCH3HCH3CH3H2ONHCH3CH3HCH3HCH3CH3NH2CH3CH3HCH3HCH3CH3m/z=389m/z=371m/z=358m/z=340m/z=309 371.17第58页/共89页12/16/202160峰位号tR(min

39、)M+H+(m/z)特征碎片离子(m/z)可能生物碱名称13.92370339，325，283，135，70，58Cyclobuxomicreine K, Cyclobuxosuffrine K,Buxenone M, Cyclobuxoviridine B24.03431413，382，323，86，70，58Buxazidine B, Cyclomicrophylline B,Cyclobuxoxazine C, Cyclomicrophylline C35.35415384，84，58Cycloprotobuxine A,Cyclokreanine B,Cyclovirobuxeine

40、B, 16-deoxybuxidienine C45.92417399，386，368，84，58Cylcyclovirobuxine C56.11401370，352，326，171，58Cycloprotobuxine C, Buxaminol E,Buxocyclamine A, Cyclobuxine B67.03387369，356，338，171，58Cyclobuxine D77.63403385，372，354，70，58Cyclovirobuxine D88.28375357，344，326，309，58未有相关文献报道99.77389371，358，340，173，70，5

41、8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第59页/共89页12/16/202161NNNNHN*.COOH苯甲酸利扎曲普坦苯甲酸利扎曲普坦 (Rizatriptan Benzoate) MW: 391.47 分子式分子式：C15H19O5C7H6O25-HT受体拮抗剂受体拮抗剂2.3.3 苯甲酸利扎曲普坦人体药代动力学研究苯甲酸利扎曲普坦人体药代动力学研究第60页/共89页12/16/202162第61页/共89页12/16/202163第62页/共89页12/16/202164LC-MS/MSm/z 270m/z 158Phenomenx

42、PFP1%冰醋酸冰醋酸, 0.2%醋酸铵醋酸铵蛋白沉淀法蛋白沉淀法盐酸曲马多盐酸曲马多第63页/共89页12/16/202165色谱条件色谱条件流动相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%)第64页/共89页12/16/202166空白溶剂色谱图 预处理的空白血浆色谱图 RT: 0.00 - 6.500246Time (min)020406080100020406080100Relative Abu

43、ndanceNL:1.00E5TIC F: + c sid=-12.00 SRM ms2 263.90-20.00 57.99-58.00 MS washNL:1.00E3TIC F: + c sid=-12.00 SRM ms2 269.90-20.00 158.10 MS washRT: 0.00 - 6.500246Time (min)020406080100020406080100Relative AbundanceNL: 1.00E5TIC F: + c sid=-12.00 SRM ms2 263.90-20.00 57.99-58.00 MS M-Cline-4-01NL: 1.

44、00E3TIC F: + c sid=-12.00 SRM ms2 269.90-20.00 158.10 MS M-Cline-4-01第65页/共89页12/16/202167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第66页/共89页12/16/202168质谱条件优化质谱条件优化LZQPT #2385 RT: 20.83 AV: 1 NL: 1.95E5T: +

45、c sid=-8.00 Q3MS 170.00-350.00180200220240260280300320340m/z0102030405060708090100Relative Abundance270.14187.02274.25340.23241.02318.24286.14228.01301.03346.26247.05182.08222.04262.24312.25205.97288.09328.17利扎曲普坦LC-MS质谱扫描图（m/z =270） 第67页/共89页12/16/202169LZQPT #2994 RT: 26.34 AV: 1 NL: 3.60E5T: + c

46、sid=-8.00 Full ms2 270.00-25.00 50.00-350.0050100150200250300350m/z0102030405060708090100Relative Abundance158.0358.10201.08155.92172.00225.03129.9490.86270.5159.24332.51314.94利扎曲普坦LC-MS/MS质谱扫描图（m/z=158） 第68页/共89页12/16/202170NNNNHN6844NHm/z 270m/z 158利扎曲普坦质谱裂解图 m/z 270m/z 158 （碰撞能量：20ev）第69页/共89页12/16/202171色谱条件选择色谱条件选择色谱柱色谱柱Intersil-ODS C18Lichr