ChemometricsandMultivariateResolutionanditsApplicationinanalysisofTCM中南大學中藥現代化研究中心梁逸曾Chemometrics

Chemometricsisanewchemicaldisciplinethatusesthetheoryandmethodsfrommathematics,statistics,computerscienceandotherrelateddisciplinestooptimizetheprocedureofchemicalmeasurement,andtoextractchemicalinformationasmuchaspossiblefromchemicaldata.Chemometricscouldbedefinedasadisciplineoffundamentaltheoryandmethodologyofchemicalmeasuring.化學計量學運用數學、統計學、計算機科學、以及其他相關學科的理論與方法，優化化學量測過程，并從化學量測數據中最大限度地獲取有用的化學信息，可以說是一門化學量測的基礎理論與方法學。BriefHistoryofChemometricsSvanteWolduseditfirstlyforapplyingscientificprojectin1970inSweden;WoldandKowalskifoundedtheInternationalChemometricSocietyin1974;AnalyticalChemistrypublishedspecialreviewon“Chemometrics”everytwoyearsince1978;Twospecialchemometricinternationaljournalsnamed“J.Chemom.”and“ChemLab”appearedin1987frombothAmericanandEurope.ContentsinchemometricsChemometricsconsistsoffundamentalandmethodologyofchemicalmeasurements.NecessaryfundamentalknowledgeofstatisticsandlinearalgebraVectorandMatrixIsMathematicsreallyusefulforchemists?Dataexploding;Extractchemicalinformationfromthedata;RevolutionofInformationtechnique;ProgressinComputerability;VectorinanalyticalchemistryAllthespectra,chromatogramsandetc.canbenumeratedintoagroupofnumbers,whichiscalledavectorinmathematics.

HyphenatedInstruments,suchasHPLC-DAD,GC-MS,GC-IR,HPLC-MSTwo-waydatacontainingbothchromatographyandspectra;Datamatrixwithmorethan10Megabytes;DatabaseoflotsofchemicalstandardsThemixturespectrumoftwodifferentchemicalcompoundsaandbaccordingtotheLambert-BeerlawGeometricsenseofvectoradditionGeometricsenseofvectorsubtractionDirectionandlengthofvectorThedirectionofavectorisdecidedbyallitselements,sincethedifferentratiosbetweenthemcandefinesdifferentdirectionsinlinearsubspace;Thelengthofavectorisalsodecidedbymagnitudesofitselements,sincewehave,||a||=(a12+….+an2)1/2differentratiosbetweenthemcandefinesdifferentdirectionsinlinearsubspaceSubtractionoftwovectorsdefinesthedistancebetweenthetwopointsinndimensionalspaceNumericalmultiplicationofvectorsThespectraofdifferentconcentrationsInnerproductandouterproductbetweenthevectors

InnerordotproductbetweentwovectorsproducinganumberGeometricsenseofinnerproductbetweentwovectorsInnerproductandprojectionbetweenvectorsOuterproductbetweentwovectorsproducingabilinearmatrix,whichisofspecialimportanceinmultivariateresolutionWhatisthechemicalmeaningofrankofamatrix?Linearlyindependent?Rankofamatrix=thenumberofthechemicalcomponentsinthemixture?Mixturenumberandcompoundnumber?Apartofarealtwo-waydatameasuredfor

CortexCinnamomi(肉桂)

Lambert-BeerLawTheproblemhereforchemiststosolveisthat,withthemeasurementmatrixathand,oneneedstofindout:Thenumberofabsorbingchemicalcomponents:AThespectrumofeachchemicalcomponent:si(i=1,2,…,A)(firststepof

qualifications)Theconcentrationprofileofeachchemicalcomponent:ci(i=1,2,…,A)(firststepfor

quantification)Isitpossible?Yes,ifwehavethetwo-waydata!!MixturespectraandcompoundspectraThisisatwocompoundsystem;Tworedvectorsarethecompoundspectra;Sevenbluevectorsaremixturespectra;

LinearlyindependentThetwospectraarefromtwodifferentchemicalcompounds,sotheyareindependentwitheachotherSevenspectraareallfromthespectraofthetwochemicalcompounds,sotheyaredependentupontheconcentrationsofthetwocompoundsinthemixtureMixturenumberandcompoundnumber

Rankofamatrix=thenumberofthechemicalcomponentsinthemixture?

Unchangedarechemicalcompounds;Rankofamatrixisalsocertain,whichisnotchanged;Thus,ifwecollectallthespectratoformamatrix,therankofwhichshouldbe2.Howcanwefindtherankofamatrixwithmeasurementnoise?Whatarenoisesandhowdotheyinfluencethedataanalysis?Thus,weneedstatisticsandalsoalgebra;Usefultechnique:Principalcomponentanalysis!!Thisisreallyadifficultproblem!Now,let’sgothroughthisproblemstepbystep.SinglevaluedecompositionQuestion2Wehaveadatamatrixathand,weneedtoknowtherearehowmanychemicalcomponentsinit.Whattechniquedoyouwanttouse?Principalcomponentanalysis?Canweusesingularvaluedecompositiontosolvetheproblem?MultivariatecalibrationandmultivariateresolutionThisisthemajortopicinanalyticalchemometrics,wewilldiscussitinsomedetail.SinglevaluedecompositionandnumberofchemicalcomponentinthesystemLocalfactoranalysisandZerocomponentregionsEvolvingfactoranalysis(EFA)Fix-sizedmovingwindowevolvingfactoranalysis(FSMWEFA);Eigenstructuretrackinganalysis(ETA)EvolvinglatentprojectionsEvolvingFactorAnalysisEvolvingfactoranalysisFix-sizedMovingWindowEvolvingFactorAnalysis(FSMWEFA)Fix-sizedMovingWindowEvolvingFactorAnalysis(FSMWEFA)SomeproblemswithlocalfactoranalysisLocalrankZerocomponentregions,WindowsizeEigenstructuretrackinganalysisRankmapandlocaldatastructureEvolvinglatentprojectionsPrincipalComponentanalysisandLatentprojectionsEvolvinglatentprojectionsSelectiveinformationandEvolvinglatentprojections

ZerocomponentregionandnoiselevelAsimpleexampleResolutionintopurechromatogramsandspectraBecauseA=CStIfwehavepurespectraS,thenwecanusethefollowingequation:

C=AS(StS)-1Resolutionintopurechromatogramsandspectra美國標準局16組分PAH混合物標樣(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Peaks5and6intheplot峰5、峰6的演進特征投影圖

峰5的前5個特征值依次為16382，2436，1294，22，11分辨所得的芴、苊、菲、蒽的色譜與光譜J.CGiddingsin“Statisticaltheoryofcomponentoverlapinmulticomponentchromatograms”claimedthatItisshownthat,relativetothemaximumpeakcontentorpeakcapacityforcloselyspacedpeaks,arandomchromatogramwillnevercontainmorethanabout37%ofitspotentialpeaksand,worstofallfromananalyticalpointofview,18%ofitspotentialsingle-componentpeaks.Thenumberofobservedpeaksisnot,then,thesameasthenumberofdistinctchemicalcomponents.Thelossofanalyticalinformationresultingfromthisoverlapisbyitselfserious,buttheseverityoftheproblemisgreatlymagnifiedifwedonothaveagoodestimateofthemagnitudeoftheloss.

Anal.Chem.1983,55,418-424

ApplicationtoenvironmentalchemistryPAHsrepresentaclassoforganicpollutantsthatarewidelyfoundintheenvironment.Theyhavebeenshowntoexertaverystrongcarcinogenicactiononanimals.ThisisanexampleforqualitativeandquantitativeanalysisofPAHsinair-borneparticulatesinHongKongcitybythechemometricresolutionmethod.

1.8.2

HELP分辨

直觀推導式演進特征投影法充分利用了二維數據的色譜信息和波譜化學信息，以逐步推導的方式分辨各組分的純色譜和波譜。具體步驟簡述如下：

1）將響應矩陣X沿色譜方向分解為一系列相對簡單的子體系Xi，檢測各個子體系Xi存在的背景與噪聲，扣除背景及噪聲

2）利用演進特征投影圖和特征結構跟蹤分析與秩圖確定其組分數以及各組分的選擇性區域和零濃度區域

3）利用各組分的選擇性區域和零濃度區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際數據比較，驗證解析結果的可靠性

Figure2.6’TheamplifiedtotalioncurrentchromatogramoffractionN6obtainedfromGC-MS.

1.9定性分析

用質譜庫相似檢索定性，結合色譜保留指數確正質譜的定性結果

色譜峰B分辨所得六種物質定性，它們依次是：6-methoxy-2-methyl-beta-carboline(C13H12N2O)、3,3’-dimethyl-[1,1’-biphenyl]-4,4’-diamine(C14H16N2)、2,5-dimethyl-4-[3-amino-4-methylphenyl]pyridine(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，分辨七個餾分的所有色譜峰，對各個組分進行定性分析。在1200多組分中鑒別出130多種含氮化合物

化學計量學與中藥現代化研究中草藥的現代化與國際化是勢不可擋的世界潮流美國自30到60年代，幾乎停止植物藥的研究（Interestinmedicalplantsprogressingsignificantlydiminishedinthe1930sandbotancalsfellintoalmostcompletedisuseuntil1960s,RoyUpton,HerbalistExecutiveDirector,AmericanHerbalPharmacopoeia），到現在（2000）

GlobalHerbalMarket:US$19.6billion!!（196個億美金）；德國和法國聯合開發銀杏葉提取物EG6761，找到最佳銀杏提取物的指紋圖譜，導致植物藥的在歐美重新流行HealthProducts(WesternCountries)GinggoBiloba

(銀杏)St.Jone’sWort(貫頁連翹)HealthProducts(WesternCountries)Greentea(綠茶)Echinacea(紫錐花)TraditionalChineseMedicine(TCM)HealthProductsDongQuai(當歸)Ginseng(人參)TraditionalChineseMedicine(TCM)HealthProductsCordyceps(冬蟲夏草)Galic(大蒜)NaturalProductMarketGlobalHerbalMarket2000:US$19.6billion!!

NaturalProductMarket

缺乏適當或可接受的評價傳統草藥的研究方法Despiteitsexistenceandcontinueduseovermanycenturies,anditspopularityandextensiveuseduringthelastdecade,traditionalmedicinehasnotbeenofficiallyrecognizedinmostcountries.…

Thequantityandqualityofthesafetyandefficacydataontraditionalmedicinearefarfromsufficienttomeetthecriterianeededtosupportitsuseworldwide.

Thereasonsforthelackofresearchdataareduenotonlytohealthcarepolicies,

butalsotoalackofadequateoracceptedresearchmethodologyforevaluatingtraditionalmedicine.傳統植物化學方法研究中藥的窘境每個化學物質都分離了（耗時），但藥效似乎都不強，找到活性單體藥無法與同類西藥相比；中藥的君臣佐使，協同作用難以體現；日本人、德國人（銀杏）的發現，指紋圖譜的提出；中藥現代化需要新思路，新方法。中草藥現代化與國際化研究中的幾個難點問題中草藥的化學基礎;(多組分復雜混合體系)中藥復方制劑;(“膏丹丸散,神仙難辯”);中藥國際化的主要難點：中藥基地軌范化(GAP和GEP)，中藥生產標準化(GMP)，基于化學指紋圖譜的中藥質量控制;中藥作用機制研究（君臣佐使,協同作用）中藥藥效及藥代動力學方法的建立;中藥譜（化學指紋圖譜）效（藥理藥效）學的創立;中醫藥理論科學化的艱巨任務廣西玉林廣東肇慶越南云南中藥指紋圖譜在中藥現代化研究中的核心地位EssentialConstituentsofRamulusCinnamomi(桂枝）

中藥現代化的機遇與挑戰基于中藥指紋圖譜的質量控制、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力學研究、餾分組合（雞尾酒方法），新型更有效復方制劑的發現與開發、全依賴于中藥指紋圖譜的快速定性定量解析！中藥色譜指紋圖譜的測定方法以解決中藥長期處于“丸散膏丹，神仙難辨”的被動局面InstrumentsCommonlyusedforChemicalAnalysisofChineseMedicineThinLayerChromatography(TLC)(薄層色譜)

qualitativeanalysis&semi-quantitativeanalysisHighPerformanceLiquidChromatography(HPLC)(高效液相色譜)

Bothqualitativeanalysis&quantitativeanalysisGasChromatography(GC)(氣相色譜) Bothqualitativeanalysis&quantitativeanalysisDNAAnalysis(DNA分析)CapillaryElectrophoresis(CE)(毛細管電泳)中藥指紋圖譜剖析的新工具聯用色譜儀器HyphenatedInstrument(聯用儀器)Mostmodernandadvancedanalyticalinstrument.

e.g.HPLC-DAD,GC-MS,CE-DAD,etc.Combinationoftwoormoreinstrumentse.g. HPLC DAD(diode-arraydetector)

GC MS(Mass-spectrometer)SeparationInstrumentSignal

InstrumentGetmoredata/informationHPLC-DAD3DchromatogramHPLCchromatogramofnucluesideofCordycepsSinensis(冬蟲草)atonewavelengthHyphenatedInstrument(聯用儀器)HyphenatedInstrument(聯用儀器)GCinstrumentGCchromatogramof

pepticpowder(平胃散)Massspectrumtakenatretentiontime10.2minutesHyphenatedInstrument(聯用儀器)Advantages:Moredataisobtainedataretentiontime.Morespectralinformationacquiredtogivethreedimensional(3D)data.MuchmoreinformationavailabletoanalyzecomplicatedsystemlikeTCM.HyphenatedInstrument(聯用儀器)Disadvantages:Hugeamountof3Ddataisobtained.Needdataprocessingmethodsforinformationextraction,patternrecognition,etc.Computerpowerwaspoorbefore.Usualpractice:UseafewdataobtainedfromHPLC-DAD/GC-MSto findmarkercomponentsoractiveingredients.

Now,everythingbecomespossiblewithhightech.!

國家藥品監督管理局付局長任德權先生在現代化中藥產業關鍵技術系列研討會的發言中指出：

實際上，從分析化學學科自身發展看，純品的分析現已無特殊困難，而對復雜有機混合物體系給出快速定性定量分析，...。從現代化學計量學的觀點看，儀器分析數據的傳統處理方法“如光譜、波譜只取其峰值，對色譜只計算其面積等，這樣做的后果只能造成大量有用信息的浪費。”面對形形色色的多組分體系，在充分利用化學量測儀器所產生的化學信號特點的基礎上，化學計量學運用統計學和應用數學及計算機，最大限度地從中抽取不同樣本中的定性、定量化學信息。在這方面我國學者梁逸曾先生按對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。現代分析化學學科，特別是化學計量學的發展，為中成藥指紋圖譜用于定性定量分析，提供了現代科學技術基礎。……歷史的挑戰與機遇并存,我們則無旁貸,充分利用時代的條件,去解決時代的問題.開創打破“神仙難辯”,逐步實現中藥定性定量分析的新時代.中藥指紋圖譜的定性定量剖析聯用色譜與化學計量學TraditionalChinesemedicines我們對二十幾種單味藥和復方制劑進行了HPLC-DAD和GC-MS分析;對某些單味藥的道地性、季節性影響也進行了系統研究，以得到它們的mono-graphs；對我省正清制藥集團的魚腥草和風痛寧注射液指紋圖譜的建立進行了系統的方法學研究。Chemometrics:CordycepssinensisChromatogramsofthefungalpartofCordycepssinensisatwavelength(a)234nm,(b)260nmandthelarvapartofCordycepssinensisatwavelength(c)234nm,(d)260nm.AsimpleexampleChemometrics:Cordycepssinensis(a)Theselectivechromatogramsofthefunguspartfrom11.0to14.0minuteswithanintervalof5nmand(b)showsthespectraofthefunguspartintherangeof195to312nmwithanintervalof0.1minute.(c)and(d)showthecorrespondingeigenvaluesplotandthelatentprojectgraphof.(e)and(f)showtheresolvedchromatogramsandspectraforcomponentsf1andf2inthefungalpart.Chemometrics:CordycepssinensisTheHELPmethodwasusedforresolvingthesechromatograms.TheoverallresolvedchromatogramofthefungalpartTheoverallresolvedchromatogramofthelarvalpartFanGong,Yizengliang,F.t.Chau,Anal.Lett.,33（2000）2105-2128GanFengandYizengLiang,AnalyticalScience,16(2000)603-607ChengjianXu,YizengLiangandJianhuiJiang,AnalyticalLetters,33(2000)2105-2128HailinShen,YizengLiang,O.K.KvalheimandR.Manne,ChemometricsandIntelligentLaboratorySystems,51(2000)49-59(inEnglish)HailinShen,L.Stordrange,R.Manne,O.M.KvalheimandYizengLiang,ChemometricsandIntelligentLaboratorySystems,51(2000)37-47(inEnglish)HailinShen,XiaoningLi,YizengLiang,ChineseScienceBulltin（科學通報）,45(2000)587-592HailinShen,Youqunsong,HuiCui,YizengLiang,ActaChimicaSinica（化學學報）,58(2000)438-442QingsongXu,YizengLiangandKaitaiFang,ChemometricsandIntelligentLaboratorySystems,52(2000)155-166.Pepticpowder PepticpowderisanancientconcentratedpreparationinChina.

Itcanclearawaypathogenicdampness,moisturizespleen,promotethecirculationofqiandregulatethestomach.Theformulationiscomposedoffoursingleherbs.TheyareRhizomaAtractylodis(atractylodesrhizome),PericarpiumCitriReticulatae(tangerinepeel),CortexMagnoliaeOfficinalis(magnoliabark)andRadixGlycyrrhizae(licoriceroot).Thevolatileconstituentsofpepticpowderarepharmacologicalactive.GongFan,Y.Z.Liang,HuiCui,F.T.Chau,BennyT.P.Chan,Determinationofvolatilecomponentsinpepticpowderbygaschromatography-massspectrometryandchemometricresolution,J.ChromatographyA,905（2001）193-205(inEnglish)GongFan,Y.Z.Liang,Qing-SongXu,F.T.Chau,Gaschromatography-massspectrometryandchemometricresolutionappliedtodeterminationofessentialoilsinCortexCinamomi,J.ChromatographyA,909（2001）237-247(inEnglish)龔范,梁逸曾,宋又群，彭源貴,崔卉,A.K.M.Leung,Foo-timChau，高等學校化學學報,2001(inpress).GanFeng,Jia-hongYangandY.Z.Liang,Liberarysearchofmassspectrawithanewmatchingalgorithmbasedonsubstructuresimilarity,AnalyticalScience,17(2001)635-638(inEnglish)Y.Z.LiangandO.M.Kvalheim,Resolutionoftwo-waydata:theoreticalbackgroundandpracticalproblemsolvingPart1:theoreticalbackgroundandmethodology,FreneniusAnal.Chem.,370(2001)694-704.(inEnglish)MinghaoZhangandYizengLiang,Analyst,

(2001)(inpress).GongFan,Y.Z.Liang,Qing-songXu，F.T.Chau,King-manNg,Anal.Chim.Acta,2001(inpress).(inEnglish)Y.Z.Liang,K.T.Fang,Q.S.Xu,ChemometricsandIntelligentLaboratorySystems,2001(inpress).

ChengjianXu,Y.Z.Liang,You-QunSongandJi-shanLi,FreneniusAnal.Chem.,(2001)(inpress)

fingerprintsforsomeherbalmedicinestToppart:(蒼術揮發油);Lowerpart:(厚樸揮發油)FindingFingerprint:HELPGC-MSofpepticpowder(平胃散)pepticpowder(平胃散)Therearefourherbsinpepticpowder:sayRhizomaAtractylodis(atractylodesrhizome),PericarpiumCitriReticulatae(tangerinepeel),CortexMagnoliaeOfficinalis(magnoliabark),andRadixGlycyrrhizae(licoriceroot).Theresultsobtainedsofarshowedthatthevolatileoilinpepticpowderarealmostthemixtureoftheabovefourherbsproportionaltotheirrelativeamountstakeninthepreparation.

FindingFingerprint:

ChemicalCompositionApproachATCMpreparation:Si-wudecoction(四物湯)100gRadixAngelicaeSinensis,當歸80gRhizomaChuanxiong,川芎120gRadixPaeoniaeAlba,白芍

120gRadixRehmanniaePraeparata,熟地VolatileoilextractionAccordingtothestandardextractionmethodinChinesePharmacopoeiaFindingFingerprint:

ChemicalCompositionApproachRhizomaChuanxiong川芎RadixAngelicaeSinensis當歸FindingFingerprint:

ChemicalCompositionApproachPreliminaryresults

ComponentsfoundComponentsidentifiedRelativecontentRadixAngelicaeSinensis當歸805092.09%RhizomaChuanxiong

川芎1278284.09%Si-wudecoction

四物湯975785.27%當歸、川芎與復方四物湯揮發油化學組分的定性定量結果鑒定了當歸、川芎及四物湯揮發油的50、82和57個化學組分，約占總量的92.02%、84.09%和85.27%。結果表明,在16個具有藥理活性的主要化學組分中，有6個組分含量明顯增加，4個組分基本不變，1個組分減少，另有5個組分在四物湯揮發油中檢測不到。反式藁本內酯的相對含量增加得很多，而它正是四物湯復方中抗膽堿、抑制子宮收縮最強的組分；丁基酞內酯、丁烯基酞內酯相對含量也是成倍增加，這2個組分均是復方具有解痙作用的主要成分，同時兼有抑制子宮收縮與平喘的作用；4-松油醇、

-松油醇、香茅醇具有平喘、消毒與抑制病菌的功能，其相對含量比理論計算值也略有增加。為詳盡地研究由單味藥配伍成復方四物湯時各化學成分在質和量上的變化，以更好地說明該復方配伍的科學性和各單味藥之間的協同作用，我們將對復方與各單味藥提取揮發油后的水相部分進行更進一步的定性定量檢測。利用化學計量學方法分析遼五味子揮發性成份的GC/MS數據遼五味子是木蘭科植物五味子的干燥成熟果實，主產我國東北，認為有益氣、明目、補不足、養五臟、壯筋骨等作用，中醫作為收斂、滋補強壯劑。遼五味子粉碎后有愉快的香氣。

六種不同提取方法得到樣品的TIC圖兩個來自不同樣品的