1、藥質量量控制中的藥質量量控制中的現代分析方法與技術現代分析方法與技術Modern analytical methods & techniquesin quality control of drugs第十六章第十六章 現代分析方法與技術，為藥學的開展提供了適時而有效的現代分析方法與技術，為藥學的開展提供了適時而有效的手段與動力。手段與動力。色譜及其聯用技術：色譜及其聯用技術： 藥學研討分子程度。藥學研討分子程度。手性分析：手性分析： 毛細管電泳及手性色譜技術藥物研討毛細管電泳及手性色譜技術藥物研討與質量控制提供了保證。與質量控制提供了保證。現代光譜技術：現代光譜技術： 藥物構造鑒定，藥物

2、構造鑒定， 微量雜質檢定。微量雜質檢定。第一節 概略Capillary electrophoresis,CEModern chromatogr & its application藥物現代色譜法及其運用藥物現代色譜法及其運用UPLCUltraPerformance 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 b

3、oth.Mass spectroscopy MSNuclear magnetic resonance spectrometry NMRX-ray diffraction methodNear infrared spectrometry NIRS現代光譜法及其運用現代光譜法及其運用Modern spectroscopy & its application in pharmaceutical analysisGC-FTIRGC-MSUPLC-MSHPLC-NMRHyphenated Techniques in Chromatography 現代聯用技術及其運用現代聯用技術及其運用HPLC-

4、MSCE-MS+樣品與溶劑脫離及電離 EI ESI APCILC/MS接口離子源質量分析器檢測離子HPLC數據系統離子識別 Quadrapole Time of Flight Fourier Transform +離子檢測+-+-+第二節第二節 液質聯用技術與運用液質聯用技術與運用2.1 離子化方式離子化方式2.2 離子分別與測定方式離子分別與測定方式Full-Scan Mass Spectrometry Advantage Provides MW InformationFull-Scan MS of BuspironeNNNNNOOBuspirone (丁螺環酮丁螺環酮)C21H31N5O2

5、MW = 385150200250300350400450500m/z255075100Relative Abundance386408(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/zPass AllPass AllProduct Ion

6、Scanning: A Tandem MS Method Advantage Provides Structural Information Disadvantage Low duty cycleFixed m/zPass AllScanningProduct Ion SpectrumQ3Q2Q1Product Ion Spectrum of BuspironeNHNNNOONHNOO100150200250300350400m/z255075100Relative Abundance122386222150265180NNNNHNOO(M+H)+Precursor Ion ScanningA

7、dvantageID compounds producing specific fragment ion (e.g., PO3 for phosphopeptides)DisadvantageLow duty cycleFixed m/zPass AllScanningPrecursor Ion SpectrumQ3Q2Q1Precursor Ion Scan Mode for Buspirone MetabolitesPrecursor Ion Scan: Q3 set to m/z 122NNNNNOOOH100200300400500m/zRelative Abundance402386

8、910111213141516Time (min)255075100Relative Abundance11.6213.8413.1614.4012.1310.4515.45NNNNNOONHNNNeutral Loss Scanning Advantage Screen for compounds producing specific neutral loss (e.g., loss of 176 for glucuronide conjugates) Disadvantage Low duty cycleScanningPass AllScanningNeutral Loss Spectr

9、umLinkedQ3Q2Q1Neutral Loss Scan of Buspirone MetabolitesNeutral Loss Scan: Q1/Q3 difference set to 121 Da100200300400500m/zRelative Abundance402386910111213141516Time (min)255075100Relative Abundance13.9211.6913.2115.5010.58NNNNNOONNNNNOOOHSelected Reaction Monitoring (SRM) Advantages Targeted Analy

10、te Monitoring High Duty Cycle “Simultaneous Monitoring of Multiple Transitions Disadvantage No “advanced structural informationFixed m/zPass AllFixed m/zQ1Q2Q3MS/MS Selectivity in Complex Matrices息斯敏阿斯咪唑(astemizole) Chlroamphenicol(氯霉素，氯霉素，CAP)殘留測定殘留測定 黃楊生物堿成分鑒定黃楊生物堿成分鑒定 苯甲酸利扎曲普坦人體藥代動力學研討苯甲酸利扎曲普坦人體藥

11、代動力學研討2.3藥物分析中的典型運用藥物分析中的典型運用OHOHNHHHOClClO2NC11H12Cl2N2O5FMW=323.13【類別】酰胺醇類抗生素【順應癥】本品是治療傷寒、副傷寒的首選藥物，外用可治療沙眼。因腦脊液濃度高，故常用于治療細菌性腦膜炎和腦膿腫。此外，尚可外用治療痤瘡、酒糟鼻、脂溢性皮炎等。 被農業養殖濫用！ 肉食品中嚴厲檢查。2.3.1 Chlroamphenicol(氯霉素，氯霉素，CAP)殘留測定殘留測定HPLC analysis was performed on the Finnigan Surveyor HPLC module with MS Pump and

12、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% B080202.520803.020803.180205.08020Operation Conditions for CAPIon source:ESIIon polarity:NegativeSpray volta

13、ge: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 in SRM or 0.2 Da in H-SRMQ3 peak width0.7 DaCollision Pressure:Ar at 1.3 mTorrOHOHNHHHOClClO2N

14、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 LOQQ1 peak width0.7 Da in SRM or 0.2 Da in H-SRMQ3 peak width0.7 DaOHOHNHHOClClO2NONHHNOClClOHONHHHO

15、ClClO2NOHO2NOHCO2NOHHm/z 321m/z 257m/z 321m/z 194m/z 152CAP SRM Result: CAP Standard Q1 peak width = 0.7 DaRT: 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: 2.83AA: 60868SN: 15803.

16、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: 16694.653.201.032.133.793.574.213.392.373.970.114.421.400.834.901.692.541.890.680.310.461.241.53RT: 2.83AA: 13035SN: 1644RT: 2.83AA: 24218SN: 6393.843.252.341.030.802.474.593.56

17、4.761.530.323.113.431.734.431.880.172.062.644.174.311.230.541.402.20NL: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: MS ICIS 1221C03NL:6.77E3Base Peak m/z= 256.50-257.50 F: MS ICIS 1221C03TIC321-152321-194321-257CAPPeak A

18、rea Counts = 2.4E4CAP SRM Result: Kidney Blank RT: 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 Abundance0204060801001.281.481.751.892.052.612.231.032.832.462.953.073.223.783.480.694.603.624.324.060.364.8

19、70.184.454.720.512.772.052.231.851.641.522.421.772.572.953.253.351.313.544.321.034.163.974.870.420.930.224.723.824.463.690.811.180.662.152.682.991.651.852.293.212.831.372.392.033.524.504.013.673.451.124.610.194.924.324.200.940.420.820.691.281.481.751.892.612.052.231.032.462.833.073.293.783.490.694.6

20、03.624.060.364.374.214.830.180.51NL: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: MS 1221D05TIC321-152321-194321-257CAP SRM Result: Kidney Spiked (0.5ng/g)RT: 0.00 - 5.000.00.20.40

21、.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.8Time (min)020406080100020406080100020406080100Relative Abundance0204060801001.292.061.482.881.842.262.421.082.762.623.033.343.483.973.624.943.784.814.390.580.130.464.240.280.774.104.66RT: 2.88AA: 13715SN: 30RMS2.231.572.111.452.451.821

22、.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: INF1.292.061.481.842.291.002.422.613.033.253.393.973.624.943.784.814.390.580.13

23、0.464.240.280.774.104.66NL: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 1221D08TIC321-152321-194321-257CAPNot accurate for confirmationCAP detectedCAP H-SRM Result: CAP St

24、andard Q1 peak width = 0.2 DaRT: 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: 1762RMS1.661.780.221.153.864.324.893.462.031.010.551.512.302.614.593.311.320.773.083

25、.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: 768RMS1.661.780.221.154.323.863.461.012.030.552.301.512.614.593.311.324.870.773.680.364.202

26、.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 Counts = 7.3E3CAP H-SRM Result: Kidney BlankRT:

27、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 Abundance0204060801001.301.782.051.952.591.471.012.412.201.672.853.220.290.520.733.504.643.864.320.863.330.114.114.894.774.443.691.521.623.111.871.432.264.641.

28、774.320.953.332.832.550.751.214.843.560.454.150.580.193.854.544.013.731.133.862.732.922.580.222.461.621.502.060.991.292.351.724.373.371.420.670.802.224.690.481.850.144.093.103.244.943.563.731.301.782.051.952.591.471.012.412.202.853.220.290.520.733.503.110.860.114.114.624.894.233.863.694.41NL:2.08E3T

29、IC 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 detectedCAP H-SRM Result: Kidney Spiked (0.5ng/g)RT: 0.00 - 5.000.00.20.40.60.81.01.21.41.61.82.02.2

30、2.42.62.83.03.23.43.63.84.04.24.44.64.8Time (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: 274RMS2.061.461.651.921.292.241.112.641.7

31、73.293.964.492.404.934.700.983.783.093.554.130.530.800.300.144.330.67RT: 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= 151.50-152.50 F: MS I

32、CIS 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 1221G07TIC321-152321-194321-257CAP2.3.2 黃楊寧生物堿黃楊寧生物堿HPLC-MS聯用鑒定聯用鑒定黃楊科植物小葉黃楊Buxus microphlla Sieb. et. Zucc. var. sinica Rehd.et Wils中含有具有較強心血管疾病治療活性的孕甾烷生物堿，主要含環維黃楊星D、環黃楊堿D和環常綠黃楊堿C等生物堿成分。 黃

33、楊生物堿HPLC-ELSD色譜圖 色譜條件色譜條件色譜柱：色譜柱：Lichrospher SiO2 (250mm4.6mm,5 m)流動相：四氫呋喃流動相：四氫呋喃-甲醇甲醇-乙腈乙腈-氨水氨水 ( 32:50:13:3)流速：流速：1mLmin -1柱溫：柱溫：30ELSD參數：漂移管溫度參數：漂移管溫度70 霧化氣體霧化氣體N2 流速：流速：1.5 Lmin -1環維黃楊星D和有關生物堿含量測定結果次數環維黃楊星D含量% 峰1生物堿含量%峰2生物堿含量%峰4生物堿含量%峰5生物堿含量%有關生物堿總含量% 186.853.578.090.790.9213.37287.083.848.540.

34、800.9814.15385.833.468.820.810.9514.03485.703.548.760.870.9314.10585.053.528.640.740.8513.75684.843.749.030.790.9714.53Mean85.893.618.650.800.9313.99RSD%1.073.653.384.624.632.8環維黃楊星環維黃楊星D及其有關生物堿的鑒別及其有關生物堿的鑒別 質譜條件質譜條件 電噴霧離子化正離子檢測電噴霧離子化正離子檢測 噴口電壓噴口電壓5000V 霧化氣壓霧化氣壓35psi 輔助氣壓力輔助氣壓力5psi 毛細管溫度毛細管溫度350 碰撞氣

35、氬氣壓力碰撞氣氬氣壓力1.5mTorr 色譜條件色譜條件 色譜柱：色譜柱：Lichrospher SiO2 (250mm4.6mm,5 m) 流動相：四氫呋喃流動相：四氫呋喃-甲醇甲醇-乙腈乙腈-氨氨水水 ( 32:50:13:3) 流速：流速：1mLmin -1 柱溫：柱溫：30黃楊寧LC-MS/MS全掃描色譜圖 黃楊寧LC-MS/MS全掃描色譜放大圖 123456789峰1母離子質荷比峰1二級質譜圖M+H+=370能夠為峰1的黃楊寧有關生物堿NOCH3CH3NCH3CH3HOCH3HONHNHOCH3Cyclobuxomicreine KCyclobuxosuffrine KBuxenon

36、e MCyclobuxoviridine B峰2母離子質荷比峰2二級質譜圖M+H+= 431能夠為峰2的黃楊寧有關生物堿NNHCH3CH2OHCH3CH3OHNONHCH3CH2OHCH3CH3NOHNOCH3CH3CH3NCH3CH2OHCH3OHNHCH3Cyclomicrophylline BBuxazidine BCyclobuxoxazine CCyclomicrophylline C峰3母離子質荷比峰3二級質譜圖M+H+= 415能夠為峰3的黃楊寧有關生物堿NNCH3CH3CH3CH3NNHCH3CH3OHCH3NNHCH3CH3OHCH3NHNCH3CH2OHCH3CH3Cycl

37、oprotobuxine ACyclokreanine BCyclovirobuxeine B16-deoxybuxidienine C峰4母離子質荷比峰4二級質譜圖CH3CH3NHCH3CH3OHNHCH3HCH3HCH3CH3環常綠黃楊堿CM+H+= 417峰5母離子質荷比峰5二級質譜圖M+H+= 401383.34能夠為峰5的黃楊寧有關生物堿NCH3CH3CH3NHNH2CH3OHNCH3NCH3CH3CH3NCH3CH3CH3NCH3CH2NHOHCycloprotobuxine CBuxaminol EBuxocyclamine ACyclobuxine B峰6母離子質荷比峰6二級質

38、譜圖NHCH3CH3OHNHCH3HCH3HCH3CH2環黃楊堿DM+H+= 387峰7母離子質荷比峰7二級質譜圖CH3CH3NHCH3CH3OHNHCH3HCH3HCH3環維黃楊星DM+H+= 403峰8母離子質荷比M+H+= 375 357.15峰8二級質譜圖峰9二級質譜圖 371.17CH3OHNH2CH3HCH3HCH3CH3NH2H2OCH3NH2CH3HCH3HCH3CH3NH2CH3OHHCH3HCH3CH3NH2CH3HCH3HCH3CH3NH2CH3HCH3HCH3CH3NH2CH3H2ONH3m/z=375m/z=357m/z=344m/z=326m/z=309 357.1

39、5NHCH3CH3OHNHCH3HCH3HCH3CH2峰9母離子質荷比M+H+= 389NHCH3CH3OHNHCH3HCH3HCH3CH3峰9二級質譜圖 371.17NHCH3CH3OHNH2CH3HCH3HCH3CH3H2ONHCH3NH2CH3HCH3HCH3CH3NH2CH3NHCH3OHHCH3HCH3CH3H2ONHCH3CH3HCH3HCH3CH3NH2CH3CH3HCH3HCH3CH3m/z=389m/z=371m/z=358m/z=340m/z=309 371.17峰位號tR(min)M+H+(m/z)特征碎片離子(m/z)可能生物堿名稱13.92370339，325，283

40、，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 B, 16-deoxybuxidienine C45.92417399，386，368，84，

41、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未有相關文獻報道99.77389371，358，340，173，70，58可能為Cyclobuxine D雙鍵加氫還原產物HPLC-ELSD法黃楊寧有關生物堿歸屬表N

42、NNNHN*.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受體拮抗劑受體拮抗劑2.3.3 苯甲酸利扎曲普坦人體藥代動力學研討苯甲酸利扎曲普坦人體藥代動力學研討藥理作用v 刺激大腦血管壁的后接點5-HT1B受體收縮血管，降低顱內血管通透性；v 刺激三叉神經前突觸5-HT1D受體，調理神經遞質的釋放，抑制硬膜的神經原性炎癥反響和血漿外滲；v 阻止血管肽的釋放，使血管口徑正?；涍^收縮顱內血管并抑制神經炎癥； v 刺激腦干5-HT1B或5-HT1D受體，抑制三叉神經核興奮； v 減

43、少頸動脈血流； v 透過血腦屏障，添加腦血流量。 實驗內容v 建立苯甲酸利扎曲普坦在血漿、尿樣濃度的LC-MS/MS測定方法v 苯甲酸利扎曲普坦分散片和膠囊進展生物等效性實驗v 苯甲酸利扎曲普坦片體內藥代動力學研討v 單劑量 (5,10,15 mg)v 多次給藥(10 mg)v 穩態(10 mg)LC-MS/MS測定方法建立測定方法建立v 測定方法選擇測定方法選擇v 質譜條件優化質譜條件優化v 色譜條件選擇色譜條件選擇v 色譜柱選擇色譜柱選擇v 緩沖鹽選擇緩沖鹽選擇v 血漿處置方法血漿處置方法v 液液萃取法液液萃取法v 蛋白沉淀法蛋白沉淀法v 內標選擇內標選擇LC-MS/MSm/z 270m/

44、z 158Phenomenx PFP1%冰醋酸, 0.2%醋酸銨蛋白沉淀法蛋白沉淀法鹽酸曲馬多鹽酸曲馬多色譜條件色譜條件流動相A：醋酸鹽緩沖液(1%冰醋酸，2%醋酸銨;pH 3.5)流動相B：甲醇(0.1%甲酸)梯度條件過程：0 min (B50%)1.0 min (B95%)4.5 min (B95%)4.6 min (B50%) 6.5 min (B50%)空白溶劑色譜圖 預處置的空白血漿色譜圖 RT: 0.00 - 6.500246Time (min)020406080100020406080100Relative AbundanceNL:1.00E5TIC F: + c sid=-12

45、.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: 1.00E3TIC F: + c sid=-12.00 SRM ms2

46、 269.90-20.00 158.10 MS M-Cline-4-01質譜條件質譜條件離子檢測方式： ESI+ SRM檢測對象： 利扎曲普坦 m/z 269.9158.1 曲馬多 m/z 263.958.0噴口電壓： 5000 V霧化氣壓： 35 psi輔助氣壓力： 5 psi毛細管溫度： 350碰撞氣氬氣壓力： 1.3mTorr碰撞能量： 20 eV質譜條件優化質譜條件優化LZQPT #2385 RT: 20.83 AV: 1 NL: 1.95E5T: + 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質譜掃描圖m/z =270 LZQPT #2994 RT: 26.34 AV: 1 NL: 3.60E5T: + c sid=-8.00 Full ms2 270.00-25.00 50.00-350.0050