藥品質(zhì)量控制中的現(xiàn)代分析方法與技術(shù)

1、藥品質(zhì)量控制中的現(xiàn)代分析方法與技術(shù),Modern analytical methods & techniquesin quality control of drugs,第十六章,現(xiàn)代分析方法與技術(shù)，為藥學(xué)的發(fā)展提供了適時(shí)而有效的手段與動(dòng)力。,色譜及其聯(lián)用技術(shù)： 藥學(xué)研究－－分子水平。手性分析： 毛細(xì)管電泳及手性色譜技術(shù)－－藥物研究與質(zhì)量控制提供了保障?，F(xiàn)代光譜技術(shù)： 藥物結(jié)構(gòu)鑒定， 微量雜質(zhì)檢定。,,第一節(jié)

2、 概況,Capillary electrophoresis,CE,Modern chromatogr & its application,藥物現(xiàn)代色譜法及其應(yīng)用,,UPLC,UltraPerformance LC® (UPLC® ) technology starts with unique 1.7 µm small-particle chemistries. Chromatographers n

3、o longer need to choose between speed and resolution—with UPLC you get both.,Mass spectroscopy MSNuclear magnetic resonance spectrometry NMRX-ray diffraction methodNear infrared spectrometry NIRS,現(xiàn)代光譜法及其應(yīng)用,Modern s

4、pectroscopy & its application in pharmaceutical analysis,,Hyphenated Techniques in Chromatography,現(xiàn)代聯(lián)用技術(shù)及其應(yīng)用,HPLC-MS,CE-MS,第二節(jié) 液質(zhì)聯(lián)用技術(shù)與應(yīng)用,2.1 離子化方式,2.2 離子分離與測(cè)定模式,Full-Scan Mass Spectrometry,AdvantageProvides MW Info

5、rmation,Full-Scan MS of Buspirone,Buspirone (丁螺環(huán)酮)C21H31N5O2MW = 385,(M+H)+,(M+Na)+,Single Ion Monitoring (SIM),AdvantagesTargeted Analyte MonitoringHigh Duty CycleSimple,DisadvantagesCan suffer from interferences

6、Not 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,Pr

7、oduct 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,Fixe

8、d 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 specifi

9、c 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:

10、Q1/Q3 difference set to 121 Da,,,,Selected Reaction Monitoring (SRM),AdvantagesTargeted Analyte MonitoringHigh Duty Cycle“Simultaneous” Monitoring of Multiple Transitions,DisadvantageNo “advanced” structural informat

11、ion,Q1,Q2,Q3,MS/MS Selectivity in Complex Matrices,,息斯敏——阿斯咪唑(astemizole),Chlroamphenicol(氯霉素，CAP)殘留測(cè)定黃楊生物堿成分鑒定苯甲酸利扎曲普坦人體藥代動(dòng)力學(xué)研究,2.3藥物分析中的典型應(yīng)用,C11H12Cl2N2O5FMW=323.13,【類別】　酰胺醇類抗生素　【適應(yīng)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

12、脊液濃度高，故常用于治療細(xì)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(nóng)業(yè)養(yǎng)殖濫用！ 肉食品中嚴(yán)格檢查。,2.3.1 Chlroamphenicol(氯霉素，CAP)殘留測(cè)定,HPLC analysis was performed on the Finnigan Surveyor HPLC module with MS Pump and AutosamplerColumn: Thermo Hypersil G

13、old C18 (100×2.1 mm, 5µ)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

14、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 pea

15、k 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

16、,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 黃楊寧生物堿HPLC-MS聯(lián)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(qiáng)心血

17、管疾病治療活性的孕甾烷生物堿，主要含環(huán)維黃楊星D、環(huán)黃楊堿D和環(huán)常綠黃楊堿C等生物堿成分。,,黃楊生物堿HPLC-ELSD色譜圖,色譜條件色譜柱：Lichrospher SiO2 (250mm×4.6mm,5 µm)流動(dòng)相：四氫呋喃-甲醇-乙腈-氨水 ( 32:50:13:3)流速：1mL·min -1柱溫：30℃ELSD參數(shù)

18、：漂移管溫度70℃ 霧化氣體（N2） 流速：1.5 L·min -1,環(huán)維黃楊星D和有關(guān)生物堿含量測(cè)定結(jié)果,環(huán)維黃楊星D及其有關(guān)生物堿的鑒別 質(zhì)譜條件 電噴霧離子化正離子檢測(cè) 噴口電壓5000V 霧化氣壓35psi

19、 輔助氣壓力5psi 毛細(xì)管溫度350℃ 碰撞氣氬氣壓力1.5mTorr 色譜條件 色譜柱：Lichrospher SiO2 (250mm×4.6mm,5 µm) 流動(dòng)相：四氫呋喃-甲醇-乙腈-氨

20、水 ( 32:50:13:3) 流速：1mL·min -1 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,1,2,3,4,5,6,7,8,9,峰1母離子質(zhì)荷比,峰1二級(jí)質(zhì)譜圖,[M+H]+=370,可能為峰1的黃楊寧有關(guān)生物堿,,Cyclobuxomicreine K,Cyclobuxosuffrin

21、e K,Buxenone M,Cyclobuxoviridine B,峰2母離子質(zhì)荷比,峰2二級(jí)質(zhì)譜圖,[M+H]+= 431,可能為峰2的黃楊寧有關(guān)生物堿,,Cyclomicrophylline B,Buxazidine B,Cyclobuxoxazine C,Cyclomicrophylline C,峰3母離子質(zhì)荷比,峰3二級(jí)質(zhì)譜圖,[M+H]+= 415,可能為峰3的黃楊寧有關(guān)生物堿,,Cycloprotobuxine A,Cyc

22、lokreanine B,Cyclovirobuxeine B,16-deoxybuxidienine C,峰4母離子質(zhì)荷比,峰4二級(jí)質(zhì)譜圖,環(huán)常綠黃楊堿C,,[M+H]+= 417,峰5母離子質(zhì)荷比,峰5二級(jí)質(zhì)譜圖,[M+H]+= 401,383.34,,,,可能為峰5的黃楊寧有關(guān)生物堿,,Cycloprotobuxine C,Buxaminol E,Buxocyclamine A,Cyclobuxine B,峰6母離子質(zhì)荷比,峰6二

23、級(jí)質(zhì)譜圖,環(huán)黃楊堿D,,[M+H]+= 387,峰7母離子質(zhì)荷比,峰7二級(jí)質(zhì)譜圖,環(huán)維黃楊星D,,[M+H]+= 403,峰8母離子質(zhì)荷比,[M+H]+= 375,357.15,峰8二級(jí)質(zhì)譜圖,峰9二級(jí)質(zhì)譜圖,371.17,,,,,,,,,m/z=375,m/z=357,m/z=344,m/z=326,m/z=309,,,357.15,,,,,峰9母離子質(zhì)荷比,[M+H]+= 389,,,,,峰9二級(jí)質(zhì)譜圖,371.17,,,,,,,m

24、/z=389,m/z=371,m/z=358,m/z=340,m/z=309,371.17,,,,,,HPLC-ELSD法黃楊寧有關(guān)生物堿歸屬表,苯甲酸利扎曲普坦 (Rizatriptan Benzoate) MW: 391.47 分子式：C15H19O5·C7H6O2,5-HT受體拮抗劑,2.3.3 苯甲酸利扎曲普坦人體藥代動(dòng)力學(xué)研究,藥理作用,刺激大腦血管壁的后接點(diǎn)5-HT1B受體收縮血管，降低顱內(nèi)血管通透性

25、；刺激三叉神經(jīng)前突觸5-HT1D受體，調(diào)節(jié)神經(jīng)遞質(zhì)的釋放，抑制硬膜的神經(jīng)原性炎癥反應(yīng)和血漿外滲；阻止血管肽的釋放，使血管口徑正?；ㄟ^收縮顱內(nèi)血管并抑制神經(jīng)炎癥； 刺激腦干5-HT1B或5-HT1D受體，抑制三叉神經(jīng)核興奮； 減少頸動(dòng)脈血流； 透過血腦屏障，增加腦血流量。,實(shí)驗(yàn)內(nèi)容,建立苯甲酸利扎曲普坦在血漿、尿樣濃度的LC-MS/MS測(cè)定方法苯甲酸利扎曲普坦分散片和膠囊進(jìn)行生物等效性試驗(yàn)苯甲酸利扎曲普坦片體內(nèi)藥代動(dòng)力學(xué)

26、研究 ? 單劑量 (5,10,15 mg) ? 多次給藥(10 mg) ? 穩(wěn)態(tài)(10 mg),LC-MS/MS測(cè)定方法建立,測(cè)定方法選擇質(zhì)譜條件優(yōu)化色譜條件選擇 ? 色譜柱選擇 ? 緩沖鹽選擇血漿處理方法 ? 液液萃取法 ? 蛋白沉淀法內(nèi)標(biāo)選擇,LC-MS/MS,m/z 270?m/z 158,Phenomenx PFP,1%冰醋酸

27、, 0.2%醋酸銨,蛋白沉淀法,鹽酸曲馬多,色譜條件,流動(dòng)相A：醋酸鹽緩沖液(1%冰醋酸，2%醋酸銨;pH 3.5)流動(dòng)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(yù)處理的空白血漿色譜圖,質(zhì)譜條件,離子檢測(cè)方式： ESI+ SRM檢測(cè)

28、對(duì)象： 利扎曲普坦 m/z 269.9→158.1 曲馬多 m/z 263.9→58.0噴口電壓： 5000 V霧化氣壓： 35 psi輔助氣壓力： 5 psi毛細(xì)管溫度： 350℃碰撞氣氬氣壓力： 1.3mTorr碰撞能量： 20 eV,質(zhì)譜條件優(yōu)化,利扎曲普坦LC-MS質(zhì)譜掃描圖（m/z =

29、270）,,利扎曲普坦LC-MS/MS質(zhì)譜掃描圖（m/z=158）,,利扎曲普坦質(zhì)譜裂解圖 m/z 270?m/z 158 （碰撞能量：20ev）,色譜條件選擇,,色譜柱,Intersil-ODS C18Lichrospher-C18 Zorbak-ODS C18 Lichrospher-CN Phenomenx Curosil-PFP,緩沖鹽條件(0.1%HCOOH,v/v; 0.2%醋酸銨,w/v; pH3.0）,采用PF

30、P（五氟苯基）柱，樣品的保留適當(dāng)，峰型良好，有機(jī)相和水相的比例也較為適當(dāng)。,,醋酸銨(w/v),0.05%0.1%0.2%,甲醇:水=(50:50),,0.2% (w/v)醋酸銨樣品峰響應(yīng)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值為3.5，樣品保留時(shí)間合適，峰形良好。,血漿處理方法,苯甲酸利扎曲普坦血漿提取條件試驗(yàn),,內(nèi)標(biāo)

31、選擇,佐米曲普坦 (Zolmitriptan) MW：287 分子式：C16H21N3O2,,鹽酸曲馬多 (Tramadol hydrochloric) MW：299.8 分子式：C16H25NO2,曲馬多LC-MS質(zhì)譜掃描圖（m/z=264）,,曲馬多LC-MS/MS質(zhì)譜掃描圖（m/z=58）,,曲馬多質(zhì)譜裂解圖 m/z 264 ?m/z 58,單次給藥,受試者單次口服苯甲酸利扎曲普坦片后，苯甲酸利扎曲普坦的Cmax和