藥物分析藥物現(xiàn)代儀器分析法課件

1、第十六章第十六章 藥物現(xiàn)代儀器分析法藥物現(xiàn)代儀器分析法藥物現(xiàn)代儀器分析法1.色譜法2.光譜法3.質(zhì)譜法(MS)4.毛細(xì)管電泳法 5.聯(lián)用技術(shù)6.微型全分析系統(tǒng)(Lab-on-a-chip)7.場(chǎng)流分離技術(shù)(FFF)1.色譜法色譜法色譜法1. 薄層色譜法(TLC)：定性鑒別、雜質(zhì)檢查2. 毛細(xì)管氣相色譜法(GC)殘留溶劑的檢測(cè)，藥物的定量分析。3. 高效液相色譜法 (HPLC)藥物含量測(cè)定，手性藥物拆分。色譜系統(tǒng)適色譜系統(tǒng)適用性試驗(yàn)用性試驗(yàn):a.柱效 (即理論塔板數(shù) n)b.分離度 R (一般R1.5)c.拖尾因子T ( 0.95 1.05)d.重復(fù)性 (一般RSD2.0%)2. 光譜法1.紫外

2、可見(jiàn)光譜法 (UV) , A=ECL 靈敏度達(dá) 10-4 10-6 g/ml 波長(zhǎng)（200 400 760 nm）2. 熒光光度法 (檢測(cè)光與入射光成直角), F=KC 靈敏度達(dá) 10-7 10-10 g/ml 一般采用激發(fā)光波長(zhǎng) 250 700 nm4. 紅外光譜法(IR)主要用于有機(jī)藥物的定性鑒別 波長(zhǎng) 400 760 nm5. 核磁共振法 (NMR)，主要用于有機(jī)藥物的定性鑒別6. X射線粉末衍射法，主要用于藥物晶型的定性鑒別3. 原子吸收光度法主要用于金屬元素的分析，靈敏度達(dá) 10-6 10-9 g/ml現(xiàn)代光譜法3. 質(zhì)譜法(MS)原理：將化合物形成離子和碎片離子，按其質(zhì)荷比的不同進(jìn)

3、行分離，可做成分和結(jié)構(gòu)分析。特點(diǎn)：應(yīng)用范圍廣、靈敏度高(10-11g)、分析速度快(小于1秒)。樣品導(dǎo)入系統(tǒng)離子源質(zhì)量分析器檢測(cè)器記錄儀用途：測(cè)定分子量鑒定化合物推測(cè)未知物結(jié)構(gòu)測(cè)定分子中Cl 和Br等的原子數(shù)如與色譜聯(lián)用可進(jìn)行多組分的定性定量分析。4. 高效毛細(xì)管電泳法 (High Performance Capillary Electrophoresis, HPCE)原理：依據(jù)樣品中各個(gè)組分之間淌度和分配行為上的差異實(shí)現(xiàn)分離分析。以石英毛細(xì)管為分離通道、高壓直流電場(chǎng)為驅(qū)動(dòng)力。優(yōu)點(diǎn)：高效(n可達(dá)幾十萬(wàn)至幾千萬(wàn))高速 (最快可在1分鐘內(nèi)完成，一般幾分鐘即可)微量(nL級(jí)，即10-9L)低消耗 (

4、流動(dòng)相 幾mL即可)分離模式：毛細(xì)管區(qū)帶電泳 (CZE)膠束電動(dòng)毛細(xì)管色譜 (MECC)毛細(xì)管凝膠電泳 (CGE) 毛細(xì)管等電聚焦 (CIEF)02468101214161805101520minmAU 01020304050607005101520minmAU 051015202505101520mAUmin 05101520253005101520minmjAU A D L B DL C D L A D L B D L C D L Fi gure 2.Fi gure 2. Chi ral separat i on of D - and L- t rypt ophan i n avi di

5、n and phosphol i pi ds-avi di n w i t hout PS coat ed capi l l ari es. (A ) D PPC (B) Bi ot i nyl -cap-D PPE and (C) Bi ot i nyl -PE coat i ng capi l l ari es. Runni ng condi t i ons: 50m ID fused si l i ca capi l l ary; t ot al l egt h 48. 5 cm ; l engt h t o t he det ect i on w i ndow 40 cm ; capi

6、 l l ary t em perat ure 25 0C; appl i ed vol t age -20kV ; sam pl e i nj ect i on 10s at 50 m br; U V det ect i on 214 nm ; runni ng buffer 20 m M (Ioni c st rengt h) Tri s at pH 7. 4.Fi gure 3.Fi gure 3. Chi ral separat i on of D - and L-t rypt ophan i n Phosphol i pi d/ PS (90: 10 m ol % )-avi di

7、n coat ed capi l l ary. (A ) D PPC/ PS, (B) Bi ot i nyl -cap-D PPE/ PS, (C) Bi ot i nyl -PE/ PS. Runni ng condi t i ons as i n Fi g. 2.05101520253035024681012minmAU 0246810121416182005101520minmAU 5. 聯(lián)用技術(shù)1. GC IR：中成藥復(fù)方制劑、 藥用揮發(fā)油、香精香料分析、毒物檢測(cè)、廢水和農(nóng)藥分析等 2. GC MS ：合成產(chǎn)物的確證、副產(chǎn)物的鑒定、中藥未知成分的鑒定、藥物代謝的研究。 LOQ可達(dá) 1

8、0-9 10-12 g 3. HPLC MS：藥品質(zhì)量控制(雜質(zhì)、降解產(chǎn)物、生物轉(zhuǎn)化產(chǎn)物等)體內(nèi)藥物分析藥物代謝研究 4. HPLC NMR ：藥物雜質(zhì)鑒定、藥物體內(nèi)外代謝產(chǎn)物的結(jié)構(gòu)鑒定、天然產(chǎn)物化學(xué)篩選等5. HPCE MS ：合成產(chǎn)物的確證、副產(chǎn)物的鑒定、中藥未知成分的鑒定couple6. 微型全分析系統(tǒng)(Miniaturized Total Chemical Analysis System, -TAS) 1990年左右， Manz 和Widmer 率先提出微全分析系統(tǒng) (Miniaturized total analysis system, TAS) ，全世界分析化學(xué)家高度重視，2001

9、年新的學(xué)術(shù)季刊 “Lab-on-a-chip” 創(chuàng)立，美國(guó) Aanalytical Chemistry 期刊每?jī)赡甑木C述中列有專門的文章綜述 TAS領(lǐng)域的發(fā)展。 TAS領(lǐng)域中微加工方法，大致以最簡(jiǎn)易方法為主。 TAS檢測(cè)器：激光誘導(dǎo)螢光檢測(cè)器；吸收光度檢測(cè)器；化學(xué)發(fā)光檢測(cè)器；折光率檢測(cè)器；發(fā)射光譜檢測(cè)器；電化學(xué)檢測(cè)器；質(zhì)譜檢測(cè)器等。 TAS卡脖子難點(diǎn)： 1) 進(jìn)樣系統(tǒng) 2) 前處理不容易 3) 重復(fù)性差 TAS的巨大潛力：1) 多重平行檢測(cè) 2) 大規(guī)模集成化 未來(lái)的研究領(lǐng)域： 與生命科學(xué)相關(guān)，如生物醫(yī)學(xué)、高通量的藥物合成篩選、衛(wèi)生檢疫等。隨著人類基因組計(jì)劃的初步完成，已步入后基因組時(shí)代，單核

10、甘酸多態(tài)性分析、基因表達(dá)分析、基因變異分析以及蛋白組分析等， 由于TAS有大規(guī)模平行處理能力，可望成為后基因組時(shí)代的支撐性技術(shù)。 7. 場(chǎng)流分離技術(shù) Field-flow fractionation (FFF) is the most versatile separation technique of all macro molecular and particle separation methodologies in terms of separation range, selectivity and resolution. FFF comprises a family of techni

11、ques that have demonstrated, over more than two decades, the ability to characterize supra molecular species in a size range spanning many order of magnitude, from macromolecules to micron-sized particles. The separation occurs by differential retention in a stream of liquid flowing through a thin,

12、empty channel. The separated components are eluted one at a time into the detector.As in classical chromatography, sample components elute at given retention times which are related, and often rigorously predictable, to various physicochemical properties of the retained species. Measurements of rete

13、ntion times can yield these properties for each fractionated component.This is the main distinguishing feature of FFF with respect to classical chromatography that can neither directly predict retention for a given component nor extract chemical information straight from retention parameters.The FFF

14、 mechanism combines elements of chromatography and field-driven techniques such a electrophoresis and ultracentrifugation. Like chromatography, FFF is an elution technique; like field-driven techniques, FFF requires a field or gradient. The field in FFF is applied at right angles to flow and serves

15、to drive components into different stream laminae in a capillary channel. The different velocities of the fluid laminae across the channel develops the separation induced by the action of field. The FFF FamilySedimentation FFFFlowFFFThermalFFFSteric FFF What can you play with FFF?SOME APPLICATIONS P

16、roteins, protein aggregates, lipoproteins DNA, viruses, chromosomes Pharmaceutical emulsions, liposomes Bacterial cells, blood cellsWhat do you need to work with FFFFFF differs from a standard HPLC in the column. Here the column is the channel. The channel is capillary with rectangular cross-section. There is no stationary phase inside