中南大学中药现代化研究中心

ChemometricsandMultivariateResolutionanditsApplicationinanalysisofTCM中南大学中药现代化研究中心梁逸曾Chemometrics

Chemometricsisanewchemicaldisciplinethatusesthetheoryandmethodsfrommathematics,statistics,computerscienceandotherrelateddisciplinestooptimizetheprocedureofchemicalmeasurement,andtoextractchemicalinformationasmuchaspossiblefromchemicaldata.Chemometricscouldbedefinedasadisciplineoffundamentaltheoryandmethodologyofchemicalmeasuring.化学计量学运用数学、统计学、计算机科学、以及其他相关学科的理论与方法，优化化学量测过程，并从化学量测数据中最大限度地获取有用的化学信息，可以说是一门化学量测的基础理论与方法学。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美国标准局16组分PAH混合物标样(Sulpeco)

已知峰9为苯并[a]蒽和屈，峰14为苯并芘和二苯并蒽的二组分重叠峰Peaks5and6intheplot峰5、峰6的演进特征投影图

峰5的前5个特征值依次为16382，2436，1294，22，11分辨所得的芴、苊、菲、蒽的色谱与光谱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

直观推导式演进特征投影法充分利用了二维数据的色谱信息和波谱化学信息，以逐步推导的方式分辨各组分的纯色谱和波谱。具体步骤简述如下：

1）将响应矩阵X沿色谱方向分解为一系列相对简单的子体系Xi，检测各个子体系Xi存在的背景与噪声，扣除背景及噪声

2）利用演进特征投影图和特征结构跟踪分析与秩图确定其组分数以及各组分的选择性区域和零浓度区域

3）利用各组分的选择性区域和零浓度区域分辨出各组分的纯色谱和波谱

4）将解析所得的各组分的纯色谱和波谱乘积线性加和与实际数据比较，验证解析结果的可靠性

Figure2.6’TheamplifiedtotalioncurrentchromatogramoffractionN6obtainedfromGC-MS.

1.9定性分析

用质谱库相似检索定性，结合色谱保留指数确正质谱的定性结果

色谱峰B分辨所得六种物质定性，它们依次是：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，分辨七个馏分的所有色谱峰，对各个组分进行定性分析。在1200多组分中鉴别出130多种含氮化合物

化学计量学与中药现代化研究中草药的现代化与国际化是势不可挡的世界潮流美国自30到60年代，几乎停止植物药的研究（Interestinmedicalplantsprogressingsignificantlydiminishedinthe1930sandbotancalsfellintoalmostcompletedisuseuntil1960s,RoyUpton,HerbalistExecutiveDirector,AmericanHerbalPharmacopoeia），到现在（2000）

GlobalHerbalMarket:US$19.6billion!!（196个亿美金）；德国和法国联合开发银杏叶提取物EG6761，找到最佳银杏提取物的指纹图谱，导致植物药的在欧美重新流行HealthProducts(WesternCountries)GinggoBiloba

(银杏)St.Jone’sWort(贯页连翘)HealthProducts(WesternCountries)Greentea(绿茶)Echinacea(紫锥花)TraditionalChineseMedicine(TCM)HealthProductsDongQuai(当归)Ginseng(人参)TraditionalChineseMedicine(TCM)HealthProductsCordyceps(冬虫夏草)Galic(大蒜)NaturalProductMarketGlobalHerbalMarket2000:US$19.6billion!!

NaturalProductMarket

缺乏适当或可接受的评价传统草药的研究方法Despiteitsexistenceandcontinueduseovermanycenturies,anditspopularityandextensiveuseduringthelastdecade,traditionalmedicinehasnotbeenofficiallyrecognizedinmostcountries.…

Thequantityandqualityofthesafetyandefficacydataontraditionalmedicinearefarfromsufficienttomeetthecriterianeededtosupportitsuseworldwide.

Thereasonsforthelackofresearchdataareduenotonlytohealthcarepolicies,

butalsotoalackofadequateoracceptedresearchmethodologyforevaluatingtraditionalmedicine.传统植物化学方法研究中药的窘境每个化学物质都分离了（耗时），但药效似乎都不强，找到活性单体药无法与同类西药相比；中药的君臣佐使，协同作用难以体现；日本人、德国人（银杏）的发现，指纹图谱的提出；中药现代化需要新思路，新方法。中草药现代化与国际化研究中的几个难点问题中草药的化学基础;(多组分复杂混合体系)中药复方制剂;(“膏丹丸散,神仙难辩”);中药国际化的主要难点：中药基地轨范化(GAP和GEP)，中药生产标准化(GMP)，基于化学指纹图谱的中药质量控制;中药作用机制研究（君臣佐使,协同作用）中药药效及药代动力学方法的建立;中药谱（化学指纹图谱）效（药理药效）学的创立;中医药理论科学化的艰巨任务广西玉林广东肇庆越南云南中药指纹图谱在中药现代化研究中的核心地位EssentialConstituentsofRamulusCinnamomi(桂枝）

中药现代化的机遇与挑战基于中药指纹图谱的质量控制、基于中药指纹图谱的药效研究、基于中药指纹图谱的药代动力学研究、馏分组合（鸡尾酒方法），新型更有效复方制剂的发现与开发、全依赖于中药指纹图谱的快速定性定量解析！中药色谱指纹图谱的测定方法以解决中药长期处于“丸散膏丹，神仙难辨”的被动局面InstrumentsCommonlyusedforChemicalAnalysisofChineseMedicineThinLayerChromatography(TLC)(薄层色谱)

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

Bothqualitativeanalysis&quantitativeanalysisGasChromatography(GC)(气相色谱) Bothqualitativeanalysis&quantitativeanalysisDNAAnalysis(DNA分析)CapillaryElectrophoresis(CE)(毛细管电泳)中药指纹图谱剖析的新工具联用色谱仪器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.!

国家药品监督管理局付局长任德权先生在现代化中药产业关键技术系列研讨会的发言中指出：

实际上，从分析化学学科自身发展看，纯品的分析现已无特殊困难，而对复杂有机混合物体系给出快速定性定量分析，...。从现代化学计量学的观点看，仪器分析数据的传统处理方法“如光谱、波谱只取其峰值，对色谱只计算其面积等，这样做的后果只能造成大量有用信息的浪费。”面对形形色色的多组分体系，在充分利用化学量测仪器所产生的化学信号特点的基础上，化学计量学运用统计学和应用数学及计算机，最大限度地从中抽取不同样本中的定性、定量化学信息。在这方面我国学者梁逸曾先生按对被分析的多组分体系的定性组成知悉程度，提出了白色、灰色和黑色多组分体系三种分析体系。现代分析化学学科，特别是化学计量学的发展，为中成药指纹图谱用于定性定量分析，提供了现代科学技术基础。……历史的挑战与机遇并存,我们则无旁贷,充分利用时代的条件,去解决时代的问题.开创打破“神仙难辩”,逐步实现中药定性定量分析的新时代.中药指纹图谱的定性定量剖析联用色谱与化学计量学TraditionalChinesemedicines我们对二十几种单味药和复方制剂进行了HPLC-DAD和GC-MS分析;对某些单味药的道地性、季节性影响也进行了系统研究，以得到它们的mono-graphs；对我省正清制药集团的鱼腥草和风痛宁注射液指纹图谱的建立进行了系统的方法学研究。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（科学通报）,45(2000)587-592HailinShen,Youqunsong,HuiCui,YizengLiang,ActaChimicaSinica（化学学报）,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)龚范,梁逸曾,宋又群，彭源贵,崔卉,A.K.M.Leung,Foo-timChau，高等学校化学学报,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:(苍术挥发油);Lowerpart:(厚朴挥发油)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%当归、川芎与复方四物汤挥发油化学组分的定性定量结果鉴定了当归、川芎及四物汤挥发油的50、82和57个化学组分，约占总量的92.02%、84.09%和85.27%。结果表明,在16个具有药理活性的主要化学组分中，有6个组分含量明显增加，4个组分基本不变，1个组分减少，另有5个组分在四物汤挥发油中检测不到。反式藁本内酯的相对含量增加得很多，而它正是四物汤复方中抗胆碱、抑制子宫收缩最强的组分；丁基酞内酯、丁烯基酞内酯相对含量也是成倍增加，这2个组分均是复方具有解痉作用的主要成分，同时兼有抑制子宫收缩与平喘的作用；4-松油醇、

-松油醇、香茅醇具有平喘、消毒与抑制病菌的功能，其相对含量比理论计算值也略有增加。为详尽地研究由单味药配伍成复方四物汤时各化学成分在质和量上的变化，以更好地说明该复方配伍的科学性和各单味药之间的协同作用，我们将对复方与各单味药提取挥发油后的水相部分进行更进一步的定性定量检测。利用化学计量学方法分析辽五味子挥发性成份的GC/MS数据辽五味子是木兰科植物五味子的干燥成熟果实，主产我国东北，认为有益气、明目、补不足、养五脏、壮筋骨等作用，中医作为收敛、滋补强壮剂。辽五味子粉碎后有愉快的香气。

六种不同提取方法得到样品的TIC图两个来自不同样品的