中南大學(xué)中藥現(xiàn)代化研究中心

1、Chemometrics and Multivariate Resolution and its Application in analysis of TCM1Chemometrics Chemometrics is a new chemical discipline that uses the theory and methods from mathematics, statistics, computer science and other related disciplines to optimize the procedure of chemical measurement, and

2、to extract chemical information as much as possible from chemical data. Chemometrics could be defined as a discipline of fundamental theory and methodology of chemical measuring. 化學(xué)計(jì)量學(xué)運(yùn)用數(shù)學(xué)、統(tǒng)計(jì)學(xué)、計(jì)算機(jī)科學(xué)、以及其他相關(guān)學(xué)科的理論與方法，優(yōu)化化學(xué)量測(cè)過程，并從化學(xué)量測(cè)數(shù)據(jù)中最大限度地獲取有用的化學(xué)信息，可以說是一門化學(xué)量測(cè)的基礎(chǔ)理論與方法學(xué)。2Brief History of ChemometricsSva

3、nte Wold used it firstly for applying scientific project in 1970 in Sweden;Wold and Kowalski founded the International Chemometric Society in 1974;Analytical Chemistry published special review on “Chemometrics” every two year since 1978;Two special chemometric international journals named “J.Chemom.

4、” and “ChemLab” appeared in 1987 from both American and Europe.3Contents in chemometrics4Chemometrics consists of fundamental and methodology of chemical measurements.5Necessary fundamental knowledge of statistics and linear algebraVector and Matrix6Is Mathematics really useful for chemists?Data exp

5、loding;Extract chemical information from the data;Revolution of Information technique;Progress in Computer ability; 7Vector in analytical chemistryAll the spectra, chromatograms and etc. can be numerated into a group of numbers, which is called a vector in mathematics. 8Hyphenated Instruments, such

6、as HPLC-DAD, GC-MS, GC-IR, HPLC-MSTwo-way data containing both chromatography and spectra;Data matrix with more than 10 Megabytes;Data base of lots of chemical standards9The mixture spectrum of two different chemical compounds a and b according to the Lambert-Beer law10Geometric sense of vector addi

7、tion11Geometric sense of vector subtraction12Direction and length of vectorThe direction of a vector is decided by all its elements, since the different ratios between them can defines different directions in linear subspace;The length of a vector is also decided by magnitudes of its elements, since

8、 we have, |a|=(a12+.+an2)1/213different ratios between them can defines different directions in linear subspace14Subtraction of two vectors defines the distance between the two points in n dimensional space15Numerical multiplication of vectors16The spectra of different concentrations17Inner product

9、and outer product between the vectors 18Inner or dot product between two vectors producing a number19Geometric sense of inner product between two vectors20Inner product and projection between vectors21Outer product between two vectors producing a bilinear matrix, which is of special importance in mu

10、ltivariate resolution22What is the chemical meaning of rank of a matrix?Linearly independent?Rank of a matrix= the number of the chemical components in the mixture?Mixture number and compound number?23A part of a real two-way data measured for Cortex Cinnamomi (肉桂) 24Lambert-Beer Law25The problem he

11、re for chemists to solve is that, with the measurement matrix at hand, one needs to find out :The number of absorbing chemical components: AThe spectrum of each chemical component: si (i=1, 2, , A) (first step of qualifications) The concentration pro each chemical component: ci (i=1, 2, , A) (first

12、step for quantification)Is it possible? Yes, if we have the two-way data!26Mixture spectra and compound spectraThis is a two compound system;Two red vectors are the compound spectra;Seven blue vectors are mixture spectra; 27Linearly independentThe two spectra are from two different chemical compound

13、s, so they are independent with each otherSeven spectra are all from the spectra of the two chemical compounds, so they are dependent upon the concentrations of the two compounds in the mixture28Mixture number and compound number29Rank of a matrix= the number of the chemical components in the mixtur

14、e?Unchanged are chemical compounds;Rank of a matrix is also certain, which is not changed;Thus, if we collect all the spectra to form a matrix, the rank of which should be 2.30How can we find the rank of a matrix with measurement noise?What are noises and how do they influence the data analysis?Thus

15、, we need statistics and also algebra;Useful technique: Principal component analysis!31This is really a difficult problem!Now, lets go through this problem step by step. 32Single value decomposition33Question 2We have a data matrix at hand, we need to know there are how many chemical components in i

16、t. What technique do you want to use?Principal component analysis? Can we use singular value decomposition to solve the problem?34Multivariate calibration and multivariate resolutionThis is the major topic in analytical chemometrics, we will discuss it in some detail.35Single value decomposition and

17、 number of chemical component in the system36Local factor analysis and Zero component regionsEvolving factor analysis (EFA)Fix-sized moving window evolving factor analysis(FSMWEFA);Eigenstructure tracking analysis (ETA)Evolving latent projections37Evolving Factor Analysis38Evolving factor analysis39

18、Fix-sized Moving Window Evolving Factor Analysis(FSMWEFA)40Fix-sized Moving Window Evolving Factor Analysis(FSMWEFA)41Some problems with local factor analysisLocal rank Zero component regions, Window size Eigenstructure tracking analysisRank map and local data structureEvolving latent projections42P

19、rincipal Component analysis and Latent projections43Evolving latent projections44Selective information and Evolving latent projections 45Zero component region and noise level46A simple example47Resolution into pure chromatograms and spectraBecause A=CStIf we have pure spectra S, then we can use the

20、following equation: C=AS(StS)-148Resolution into pure chromatograms and spectra49美國(guó)標(biāo)準(zhǔn)局16組分PAH混合物標(biāo)樣(Sulpeco)已知峰9為苯并a蒽和屈，峰14為苯并芘和二苯并蒽的二組分重疊峰50Peaks 5 and 6 in the plot51峰5、峰6的演進(jìn)特征投影圖峰5的前5個(gè)特征值依次為16382，2436，1294，22，1152分辨所得的芴、苊、菲、蒽的色譜與光譜53J.C Giddings in “Statistical theory of component overlap in multi

21、component chromatograms” claimed thatIt is shown that, relative to the maximum peak content or peak capacity for closely spaced peaks, a random chromatogram will never contain more than about 37% of its potential peaks and, worst of all from an analytical point of view, 18% of its potential single-c

22、omponent peaks.The number of observed peaks is not, then, the same as the number of distinct chemical components. The loss of analytical information resulting from this overlap is by itself serious, but the severity of the problem is greatly magnified if we do not have a good estimate of the magnitu

23、de of the loss. Anal. Chem. 1983, 55, 418-424 54Application to environmental chemistryPAHs represent a class of organic pollutants that are widely found in the environment. They have been shown to exert a very strong carcinogenic action on animals. This is an example for qualitative and quantitative

24、 analysis of PAHs in air-borne particulates in Hong Kong city by the chemometric resolution method. 551.8.2 HELP 分辨 直觀推導(dǎo)式演進(jìn)特征投影法充分利用了二維數(shù)據(jù)的色譜信息和波譜化學(xué)信息，以逐步推導(dǎo)的方式分辨各組分的純色譜和波譜 。56具體步驟簡(jiǎn)述如下： 1） 將響應(yīng)矩陣X沿色譜方向分解為一系列相對(duì)簡(jiǎn)單的子 體系Xi，檢測(cè)各個(gè)子體系Xi存在的背景與噪聲，扣 除背景及噪聲 2）利用演進(jìn)特征投影圖和特征結(jié)構(gòu)跟蹤分析與秩圖確定 其組分?jǐn)?shù)以及各組分的選擇性區(qū)域和零濃度區(qū)域 3）利用各組分的選

25、擇性區(qū)域和零濃度區(qū)域分辨出各組 分的純色譜和波譜 4）將解析所得的各組分的純色譜和波譜乘積線性加和與 實(shí)際數(shù)據(jù)比較，驗(yàn)證解 析結(jié)果的可靠性 57Figure 2.6 The amplified total ioncurrent chromatogram of fraction N6obtained from GC-MS. 58596061621.9 定性分析 用質(zhì)譜庫(kù)相似檢索定性，結(jié)合色譜保留指數(shù)確正質(zhì)譜的定性結(jié)果 色譜峰B分辨所得六種物質(zhì)定性，它們依次是 ：6-methoxy-2-methyl-beta-carboline(C13H12N2O)、3,3-dimethyl-1,1-biphen

26、yl-4,4- diamine(C14H16N2)、2,5-dimethyl-4-3-amino-4-methylphenylpyridine(C14H16N2)5-acetyl-4-amino-2-methylthio-thiophene(C8H8N2OS2)、4,9-dimethyl-naphtho-2,3-b-thiophene(C14H12S)、1,2,5,6-tetramethylacenaphthylene(C16H16) 依此原理，分辨七個(gè)餾分的所有色譜峰，對(duì)各個(gè)組分進(jìn)行定性分析。在1200多組分中鑒別出130多種含氮化合物 63化學(xué)計(jì)量學(xué)與中藥現(xiàn)代化研究64中草藥的現(xiàn)代化與國(guó)際

27、化是勢(shì)不可擋的世界潮流美國(guó)自30到60年代，幾乎停止植物藥的研究（Interest in medical plants progressing significantly diminished in the 1930s and botancals fell into almost complete disuse until 1960s, Roy Upton, Herbalist Executive Director, American Herbal Pharmacopoeia），到現(xiàn)在（2000） Global Herbal Market : US$ 19.6 billion !（196個(gè)億美

28、金）；65德國(guó)和法國(guó)聯(lián)合開發(fā)銀杏葉提取物EG6761，找到最佳銀杏提取物的指紋圖譜，導(dǎo)致植物藥的在歐美重新流行66Health Products (Western Countries)Ginggo Biloba (銀杏)St. Jones Wort(貫頁(yè)連翹)67 Health Products (Western Countries)Green tea(綠茶)Echinacea(紫錐花)68 Traditional Chinese Medicine (TCM) Health ProductsDong Quai(當(dāng)歸)Ginseng(人參)69 Traditional Chinese Medic

29、ine (TCM) Health ProductsCordyceps(冬蟲夏草)Galic(大蒜)70 Natural Product Market Global Herbal Market 2000 : US$ 19.6 billion ! 71Natural Product Market72缺乏適當(dāng)或可接受的評(píng)價(jià)傳統(tǒng)草藥的研究方法Despite its existence and continued use over many centuries, and its popularity and extensive use during the last decade, traditiona

30、l medicine has not been officially recognized in most countries. The quantity and quality of the safety and efficacy data on traditional medicine are far from sufficient to meet the criteria needed to support its use worldwide. The reasons for the lack of research data are due not only to health car

31、e policies, but also to a lack of adequate or accepted research methodology for evaluating traditional medicine.73傳統(tǒng)植物化學(xué)方法研究中藥的窘境每個(gè)化學(xué)物質(zhì)都分離了（耗時(shí)），但藥效似乎都不強(qiáng)，找到活性單體藥無法與同類西藥相比；中藥的君臣佐使，協(xié)同作用難以體現(xiàn)；日本人、德國(guó)人（銀杏）的發(fā)現(xiàn)，指紋圖譜的提出；中藥現(xiàn)代化需要新思路，新方法。74中草藥現(xiàn)代化與國(guó)際化研究中的幾個(gè)難點(diǎn)問題中草藥的化學(xué)基礎(chǔ);(多組分復(fù)雜混合體系)中藥復(fù)方制劑;(“膏丹丸散,神仙難辯”);中藥國(guó)際化的主要難點(diǎn)：中

32、藥基地軌范化(GAP 和GEP)，中藥生產(chǎn)標(biāo)準(zhǔn)化(GMP)，基于化學(xué)指紋圖譜的中藥質(zhì)量控制;中藥作用機(jī)制研究（君臣佐使,協(xié)同作用）中藥藥效及藥代動(dòng)力學(xué)方法的建立;中藥譜（化學(xué)指紋圖譜）效（藥理藥效）學(xué)的創(chuàng)立;中醫(yī)藥理論科學(xué)化的艱巨任務(wù)75廣西玉林廣東肇慶越南云南76中藥指紋圖譜在中藥現(xiàn)代化研究中的核心地位77Essential Constituents of Ramulus Cinnamomi(桂枝） 78中藥現(xiàn)代化的機(jī)遇與挑戰(zhàn)基于中藥指紋圖譜的質(zhì)量控制、基于中藥指紋圖譜的藥效研究、基于中藥指紋圖譜的藥代動(dòng)力學(xué)研究、餾分組合（雞尾酒方法），新型更有效復(fù)方制劑的發(fā)現(xiàn)與開發(fā)、全依賴于中藥指紋圖譜的

33、快速定性定量解析！79中藥色譜指紋圖譜的測(cè)定方法以解決中藥長(zhǎng)期處于“丸散膏丹，神仙難辨”的被動(dòng)局面80Instruments Commonly used for Chemical Analysis of Chinese MedicineThin Layer Chromatography (TLC)(薄層色譜) qualitative analysis & semi-quantitative analysis High Performance Liquid Chromatography (HPLC)(高效液相色譜) Both qualitative analysis & quantitative

34、 analysis Gas Chromatography (GC)(氣相色譜) Both qualitative analysis & quantitative analysis DNA Analysis( DNA分析) Capillary Electrophoresis (CE) (毛細(xì)管電泳)81中藥指紋圖譜剖析的新工具聯(lián)用色譜儀器82Hyphenated Instrument (聯(lián)用儀器)Most modern and advanced analytical instrument.e.g. HPLC-DAD, GC-MS, CE-DAD, etc.Combination of two o

35、r more instrumentse.g. HPLC DAD (diode-array detector)GC MS (Mass-spectrometer)Separation InstrumentSignalInstrument83Get more data/informationHPLC -DAD3D chromatogramHPLC chromatogram of nuclueside of Cordyceps Sinensis (冬蟲草) at one wavelengthHyphenated Instrument (聯(lián)用儀器)84Hyphenated Instrument (聯(lián)用儀

36、器)GC instrumentGC chromatogram of peptic powder (平胃散)Mass spectrum taken at retention time 10.2 minutes85Hyphenated Instrument (聯(lián)用儀器)Advantages :More data is obtained at a retention time.More spectral information acquired to give three dimensional (3D) data.Much more information available to analyze

37、 complicated system like TCM.86Hyphenated Instrument (聯(lián)用儀器)Disadvantages :Huge amount of 3D data is obtained.Need data processing methods for information extraction, pattern recognition, etc.Computer power was poor before. Usual practice : Use a few data obtained from HPLC-DAD/GC-MS to find marker c

38、omponents or active ingredients. Now, everything becomes possible with high tech.! 87國(guó)家藥品監(jiān)督管理局付局長(zhǎng)任德權(quán)先生在現(xiàn)代化中藥產(chǎn)業(yè)關(guān)鍵技術(shù)系列研討會(huì)的發(fā)言中指出： 實(shí)際上，從分析化學(xué)學(xué)科自身發(fā)展看，純品的分析現(xiàn)已無特殊困難，而對(duì)復(fù)雜有機(jī)混合物體系給出快速定性定量分析，.。從現(xiàn)代化學(xué)計(jì)量學(xué)的觀點(diǎn)看，儀器分析數(shù)據(jù)的傳統(tǒng)處理方法“如光譜、波譜只取其峰值，對(duì)色譜只計(jì)算其面積等，這樣做的后果只能造成大量有用信息的浪費(fèi)?！泵鎸?duì)形形色色的多組分體系，在充分利用化學(xué)量測(cè)儀器所產(chǎn)生的化學(xué)信號(hào)特點(diǎn)的基礎(chǔ)上，化學(xué)計(jì)量學(xué)運(yùn)用統(tǒng)計(jì)

39、學(xué)和應(yīng)用數(shù)學(xué)及計(jì)算機(jī)，最大限度地從中抽取不同樣本中的定性、定量化學(xué)信息。在這方面我國(guó)學(xué)者梁逸曾先生按對(duì)被分析的多組分體系的定性組成知悉程度，提出了白色、灰色和黑色多組分體系三種分析體系。現(xiàn)代分析化學(xué)學(xué)科，特別是化學(xué)計(jì)量學(xué)的發(fā)展，為中成藥指紋圖譜用于定性定量分析，提供了現(xiàn)代科學(xué)技術(shù)基礎(chǔ)。歷史的挑戰(zhàn)與機(jī)遇并存,我們則無旁貸,充分利用時(shí)代的條件,去解決時(shí)代的問題.開創(chuàng)打破“神仙難辯”,逐步實(shí)現(xiàn)中藥定性定量分析的新時(shí)代.88中藥指紋圖譜的定性定量剖析聯(lián)用色譜與化學(xué)計(jì)量學(xué)89Traditional Chinese medicines我們對(duì)二十幾種單味藥和復(fù)方制劑進(jìn)行了HPLC-DAD和GC-MS分析;對(duì)

40、某些單味藥的道地性、季節(jié)性影響也進(jìn)行了系統(tǒng)研究，以得到它們的mono-graphs；對(duì)我省正清制藥集團(tuán)的魚腥草和風(fēng)痛寧注射液指紋圖譜的建立進(jìn)行了系統(tǒng)的方法學(xué)研究。90Chemometrics : Cordyceps sinensisChromatograms of the fungal part of Cordyceps sinensis at wavelength(a) 234 nm, (b) 260nm and the larva part of Cordyceps sinensis atwavelength (c) 234 nm, (d) 260 nm.91A simple exampl

41、e92Chemometrics : Cordyceps sinensis(a) The selective chromatograms of the fungus part from 11.0 to 14.0 minutes with an interval of5 nm and (b) shows the spectra of the fungus part in the range of 195 to 312 nm with an intervalof 0.1 minute. (c) and (d) show the corresponding eigenvalues plot and t

42、he latent project graph of .(e) and (f) show the resolved chromatograms and spectra for components f1 and f2 inthe fungal part. 93Chemometrics : Cordyceps sinensisThe HELP method was used for resolving these chromatograms. The overall resolved chromatogram of the fungal partThe overall resolved chro

43、matogramof the larval part94Fan Gong, Yizeng liang, F.t. Chau, Anal. Lett., 33（2000）2105-2128Gan Feng and Yizeng Liang , Analytical Science, 16 (2000) 603-607 Chengjian Xu, Yizeng Liang and Jianhui Jiang, Analytical Letters, 33 (2000) 2105-2128 Hailin Shen, Yizeng Liang, O.K. Kvalheim and R. Manne,

44、Chemometrics and Intelligent Laboratory Systems, 51 (2000) 49-59 (in English)Hailin Shen, L. Stordrange, R. Manne, O.M. Kvalheim and Yizeng Liang, Chemometrics and Intelligent Laboratory Systems, 51 (2000) 37-47 (in English)Hailin Shen, Xiaoning Li, Yizeng Liang, Chinese Science Bulltin（科學(xué)通報(bào)）, 45 (2

45、000) 587-592Hailin Shen, Youqun song, Hui Cui, Yizeng Liang, Acta Chimica Sinica （化學(xué)學(xué)報(bào)）, 58 (2000) 438-442Qingsong Xu, Yizeng Liang and Kaitai Fang, Chemometrics and Intelligent Laboratory Systems, 52 (2000) 155-166.95Peptic powderPeptic powder is an ancient concentrated preparation in China. It can

46、 clear away pathogenic dampness, moisturize spleen, promote the circulation of qi and regulate the stomach. The formulation is composed of four single herbs. They are Rhizoma Atractylodis (atractylodes rhizome), Pericarpium Citri Reticulatae (tangerine peel), Cortex Magnoliae Officinalis (magnolia b

47、ark) and Radix Glycyrrhizae (licorice root). The volatile constituents of peptic powder are pharmacological active. 96Gong Fan, Y.Z. Liang, Hui Cui, F.T. Chau, Benny T.P. Chan, Determination of volatile components in peptic powder by gas chromatography-mass spectrometry and chemometric resolution, J

48、. Chromatography A, 905（2001）193-205 (in English)Gong Fan, Y.Z. Liang, Qing-Song Xu, F.T. Chau, Gas chromatography-mass spectrometry and chemometric resolution applied to determination of essential oils in Cortex Cinamomi, J. Chromatography A, 909（2001）237-247 (in English)龔范, 梁逸曾, 宋又群，彭源貴, 崔卉, A.K.M

49、. Leung, Foo-tim Chau，高等學(xué)校化學(xué)學(xué)報(bào), 2001 (in press).97Gan Feng, Jia-hong Yang and Y.Z. Liang, Liberary search of mass spectra with a new matching algorithm based on substructure similarity, Analytical Science, 17 (2001) 635-638 (in English)Y.Z. Liang and O.M. Kvalheim, Resolution of two-way data: theore

50、tical background and practical problem solving Part 1: theoretical background and methodology, Frenenius Anal. Chem., 370 (2001) 694-704. (in English)Minghao Zhang and Yizeng Liang, Analyst, (2001) (in press).Gong Fan, Y.Z. Liang, Qing-song Xu，F(xiàn).T. Chau, King-man Ng, Anal. Chim. Acta, 2001 (in press

51、). (in English)Y.Z. Liang, K.T. Fang, Q. S. Xu, Chemometrics and Intelligent Laboratory Systems, 2001 (in press). Chengjian Xu, Y.Z. Liang, You-Qun Song and Ji-shan Li, Frenenius Anal. Chem., (2001) (in press) 98 fingerprints for some herbal medicinest Top part: (蒼術(shù)揮發(fā)油); Lower part: (厚樸揮發(fā)油)99Finding

52、 Fingerprint : HELPGC-MS of peptic powder (平胃散)100peptic powder(平胃散)There are four herbs in peptic powder: say Rhizoma Atractylodis (atractylodes rhizome), Pericarpium Citri Reticulatae (tangerine peel), Cortex Magnoliae Officinalis (magnolia bark), and Radix Glycyrrhizae (licorice root).The results

53、 obtained so far showed that the volatile oil in peptic powder are almost the mixture of the above four herbs proportional to their relative amounts taken in the preparation. 101Finding Fingerprint:Chemical Composition ApproachA TCM preparation: Si-wu decoction (四物湯)100g Radix Angelicae Sinensis ,當(dāng)歸

54、80g Rhizoma Chuanxiong, 川芎120g Radix Paeoniae Alba,白芍 120g Radix Rehmanniae Praeparata,熟地Volatile oil extractionAccording to the standard extraction method in Chinese Pharmacopoeia 102Finding Fingerprint:Chemical Composition ApproachRhizoma Chuanxiong 川芎Radix Angelicae Sinensis當(dāng)歸103Finding Fingerprint:Chemical Composition ApproachPreliminary resultsComponentsfoundComponents identifiedRelativecontentRadix Angelicae Sinensis 當(dāng)歸805092.09%Rhizoma Chuanxiong川芎1278284.09%Si-wu d