h第八章 生物碱类药物分析

第八章生物碱类药物分析

Chapter8TheAnalysisofAlkaloids

药物分析课件系列SpecialidentificationtestAssayAlkaloidsPropertyImpuritytestClassificationcontentsfundamentalrequirementsreviewContents

ThestructuresandpropertiesofAlkaloids(6groups);TheprimaryidentificationtestsofAlkaloids;SpecialimpuritytestsfortheAlkaloids;AssaysofAlkaloids.Fundamentalrequirements

Masteringthetheories,proceduresandconditions:non-aqueoustitration,extractivetitration,andaciddyecolorimetry;UnderstandingthestructuresandpropertiesofAlkaloids;UnderstandingthespecificimpuritytestsofAlkaloids;UnderstandingtheapplicationsofhighperformanceliquidchromatographicandgaschromatographicmethodsinassaysofAlkaloids.FamiliarizingwiththeprimaryidentificationtestsofAlkaloids;Fundamentalrequirements

Benzoalkylamines

TropanesQuinolines

IsoquinolinesIndoles

XanthinesClassificationPressheretoseethedetailedclassificationGotoreviewephedrinehydrochloridepseudoephedrinehydrochloridecolchicinThestructuralcharacteristicofthistypealkaloidsisthattheNatomisinthealphaticchainratherthaninaromaticring.ThetypicaldrugsareEphedrinehydrochloride,Pseudoephedrinehydrochloride,Colchicin.Benzoalkylamineatropinesulfateanisodaminehydrobromide

Tropanes

Thistypeofalkaloidsarethecondensationproductsofalkaloidderivedfromtropanesanddifferentkindsofesters.Thetypicaldrugsareatropineandanisodamine.Quinolinesquininesulfate

quinidinesulfate

Thecommonlyuseddrugsarequininesulfate,quinidinesulfateetc.··Isoquinolines

morphinehydrochloridecodeinephosphateThetypicaldrugsaremorphine,codeine,narcotine.Indoles

reserpinestrychninenitrateThenumberofthistypeofalkaloidislarge.Manyofthemhavecomplexstructuresandobviousphysiologicalactivity.Suchasstrychninenitrate,reserpineetc.

Property：Basicity:Generally,alkaloidsshowweakbasicity.Nearlyallofalkaloidscanformsaltwithacids.Solubility:Mostofalkaloidsarelipophilicandtheirsaltscanbedissolvedinwater.UV:Becauseoftheirhighlyconjugatedsystem,mostofthealkaloidshaveUVabsorption.d.ChiralityGotoreview生物碱来源于氨基酸来源于异戊烯来源于萜类来源于甾体来源于鸟氨酸吡咯类吡咯里西丁类托品烷类哌啶类吲哚里西丁类喹诺里西丁类喹啉类吖啶酮类简单苯丙胺类四氢异喹啉类苄基四氢异喹啉类生物碱苯乙基四氢异喹啉类生物碱苄基苯乙胺类生物碱吐根碱类生物碱简单吲哚碱类简单β–卡波林类半萜吲哚碱类单萜吲哚碱类单萜生物碱倍半萜生物碱二萜生物碱三萜生物碱孕甾烷生物碱环孕甾烷生物碱胆甾烷生物碱来源于赖氨酸来源于色胺酸来源于苯丙氨酸/酪氨酸来源于邻氨基苯甲酸Xanthines

caffeinetheophyllineXanthinesincludemanyalkaloids,suchascaffeineandtheophylline.Instructure,theyhave4Natoms,butallofthemshowweakbasicitybecauseoftheinfluencefromtheortho-positioncarbonylgroupelectronabsorbingeffect.Theyaredifficulttoformsalt,So,inclinic,weusecaffeinefreebase.IdentificationTestsGeneralidentificationtestBiuretreactionVitailireactionThakkeioquinreactionMarquisreaction;Frohdereaction;MurexidereactionMeasurementofmeltingpointColorreactionPrecipitationreactionUltravioletabsorptionspectroscopyInfraredspectrometryChromatographySpecificidentificationtestGeneralidentificationtestsMeasurementofmeltingpointtheophylline,codeinephosphateinChP(2000)andtheophyllineinBP(2002)areidentifiedbythemeasurementofmeltingpoint.

ColorreactionConcentratedsulfuricacid,concentratednitricacid,molybdenumsulfuricacid,vanadiumsulfuricacid,seleniumsulfuricacidetc.arecommonlyusedasthereactionagents.Themechanismmaybeinvolvedindehydration,oxidation,condensationetc.GeneralidentificationtestsPrecipitationreactionAlkaloidscanforminsolubleorundissolvedsalts,compoundsorcomplexsaltsbyreactwithheavymetallicsalt(bismuthpotassiumiodide,potassiummercuriciodide,etc.)ormacro-moleculeacidprecipitatingagents(phosphomolybdicacid,silicotungsticacidetc.)inacidicwatersolution.GeneralidentificationtestsUltravioletabsorptionspectroscopyMostofalkaloidshavearomaticringandconjugatedoublebondstructure,sotheyhaveoneormorecharacteristicpeaksinultravioletbandforidentification.Generalidentificationtests

Pressheretoseethe

commonlyusedmethodsCommonlyusedmethodsComparingwithmaximumabsorbancewavelength,minimumabsorbancewavelengthandcorrespondingvarianceofabsorbance.e.gIdentificationofcolchicine[ChP(2000)秋水仙碱]2.Comparinguniformityofsampleabsorptionspectrumwiththatofstandard.e.gIdentificationofemetine[USP(24)吐根碱]3.Compareuniformityofabsorbanceindex.e.gReserpine[JP(14)利血平].Infraredspectrometry

Infraredabsorptionspectralmethodisalsoregardedasoneoftheprimaryidentificationmethod.ThealkaloidsidentifiedbyinfraredabsorptionspectralmethodandnotedinChP(2000)arereserpine,morphine,atropine,colchicine,etc.GeneralidentificationtestsChromatography

TLCisthemajormethodforidentificationofalkaloids,andHPLCisalsoused.Presently,silicagelisthecommonlyusedsorbent.Foreffectiveseparation,alkaloidsshouldbeintheirfreebaseform.Onthecontrary,ifthealkaloidsinsaltform,theywouldbeheldsotightlythatthespotsshowserioustailing.Thefollowingmethodsarecommonlyusedtoovercometheproblem.

Generalidentificationtests1.Addingalittlebaseintodevelopingsolvent,suchasammonia,diethylamine,etc.2.Basifyingthesilicagelplatewithbase.Usually,bymixthesorbentwithsomesodiumhydroxideorPBSintheplatepreparationprocedure.method:Specificidentificationtests

双缩脲反应(Biuretreaction):芳环侧链具有氨基醇结构生物碱药物的特征反应。

Vitailireaction:托烷类生物碱莨菪酸结构的特征反应，与发烟硝酸得黄色三硝基衍生物，遇醇制氢氧化钾即显深紫色。

绿奎宁反应(Thakkeioquinreaction):6位含氧喹啉衍生物的反应。可在其盐类的微酸性水溶液中，滴加溴水和氯水，至微过量时，再加入过量的氨水，应呈翠绿色反应。Gotoreview

Murexidereaction黄嘌啉生物碱的特征反应，即样品加盐酸和氯酸钾，在水溶液上共热蒸干，此残渣遇氨气呈紫色，再加氢氧化钠溶液颜色即消失。

Marquisreaction含酚羟基异喹啉类生物碱的特征反应。该类药物遇甲醛硫酸可形成具有醌式结构的有色化合物。

Frohdereaction系吗啡生物碱的特征反应。吗啡加钼酸试液即显紫色，继而蓝色，再为棕绿色。SpecialImpurityTestsMostofthealkaloidsaregottenfromherbs,thoughsomeofthemaresynthesized.Forthecomplexstructureandlongproductionprocess,theotheralkaloidsmaypresentinthefinalproducts.Ontheotherhand,alkaloidsmaybedecomposedduringstorage.Becauseofthetoxicityandactivityofalkaloids,theimpuritiesmustbecontrolledstrictlyforsafety.TheSpecialImpuritiesintheAlkaloidsDrugsImpurityatropinesulfatetropine,otheralkaloidsquininesulfateundissolvedinchloroform-ethanol，otherchininesmorphinehydrochlorideapomorphine，narceine，otheralkaloidscodeinephosphatemorphinestrychninenitratestrychninereserpineoxidationproductCommonlyusedexaminationmethodsPhysicaltestsDifferenceinColorDifferenceinopticalactivityDifferenceinspectrum

ChemicalreactiontestsColordifferencewhilereactwithsomereagentsChromatographyTLCHPLC

DifferenceinabsorptionSpecialImpurityTestsGotoreviewAssayCommonlyusedmethods：

Non-aqueoustitration

Extractivetitration

Aciddyecolorimetry

Ultravioletspectroscopy

ChromatographyNon-aqueoustitration：

Theory

Non-aqueoustitrationisthemostcommonvolumetricmethodusedinassays.Itissuitableforthetitrationofveryweakacidsorbasesandprovidesasolventinwhichorganiccompoundsaresoluble.Themostcommonlyusedprocedureisthetitrationoforganicbaseswithperchloricacidinaceticacid.Theseassayssometimestakesomeperfectingintermsofbeingabletojudgepreciselytheend-point.TheoryWaterbehavesbothasaweakacidandaweakbasethusinanaqueousenvironmentitcancompeteeffectivelywithveryweakacidandbaseswithregardtoprotondonationandacceptanceasshowninFigure1.TheeffectofthisisthattheinflectioninthetitrationcurvesforveryweakacidsandveryweakbasesissmallbecausetheyapproachthepHlimitsinwaterof14and0respectivelythusmakingend-pointdetectionmoredifficult.AgeneralruleisthatH2O+H+H3O+

CompeteswithRNH2+H+RNH3+

H2O+BOH-+BH+

CompeteswithROH+BRO-+BH+Fig.1CompetitionofwaterwithweakacidandbasesforprotonbaseswithpKa<7oracidwithpKa>7cannotbedeterminedaccuratelyinaqueoussolution.Variousorganicsolventsmaybeusedtoreplacewatersincetheycompetelesseffectivelywiththeanalyteforprotondonationoracceptance.Non-aqueoustitrationofweakbasesAceticacidisaveryweakprotonacceptorandthusdoesnotcompeteeffectivelywithweakbasesforprotons.Onlyverystrongacidswillprotonateaceticacidappreciablyaccordingtotheequationshownbelow:CH3COOH+HACH3COOH2++A-Perchloricacidisthestrongofthecommonacidsinaceticacidsolutionandthetitrationmediumusuallyusedfornon-aqueoustitrationofbasesisperchloricacidinaceticacid.Additionofaceticanhydride,whichhydrolysestoaceticacid,isusedtoremovewaterfromaqueousperchloricacid.Weakbasescompeteveryeffectivelywithaceticacidforprotons.Crystalviolet(veryweakbases)orquinalidineredisusedastheindicatorinthistypeoftitration.AtypicalanalysisisshowninFigure2forL-DOPA.＋

CH3COOH2+

＋CH3COOH

Fig.2AnalysisofDOPAbynon-aqueous

Whenthebasesisintheformofasaltofaweakacid,removalofananioniccounterionpriortotitrationisnotnecessary,e.g.forsaltsofbaseswithweakacidssuchastartrate,acetateorsuccinate.However,whenabaseisintheformofachlorideorbromidesalt,thecounterionhastoberemovedpriortotitration.Thisisachievedbyadditionofmercuricacetate;theliberatedacetateisthentitratedwithacetousperchloricacid.ThisisillustratedinFigurefortheexampleofphenylephrine.HCl.Non-aqueoustitrationwithacetousperchloricacidisusedinthepharmacopoeialassaysof:adrenaline,codeine,chlorpromazine.HCl,amitriptyline.HCl,propranolol.HCl,andquaternaryaminesaltssuchasneostigminebromide.＋Cl－

Hg(CH3COO)2+2Cl－HgCl2+2CH2COO－2CH3COOH2++2CH3COO－4CH3COOH

Fig.3TheanalysisofphenylephrinehydrochlorideNon-aqueoustitrationofweakacidsForthenon-aqueoustitrationofweakacidsasolventsuchasanalcoholoranaprotic,solventisusedthatdoesnotcompetestronglywiththeweakacidforprotondonation.Typicaltitrantsarelithiummethoxideinmethanolortetrabutylammoniumhydroxideindimethylformamide.End-pointdetectionmaybecarriedoutwiththymolblueasanindicatororpotentiometrically.Non-aqueoustitrationofacidicgroupsiscarriedoutinpharmacopoeialassaysof:barbiturates,uracilsandsulphonamides.Condition:

Alkaloidsusuallyhaveweakbacisity,andthereisnosatisfactoryresultwhentitratedwithaciddirectlyinaqueousbecauseofunobviousrising.Butinnon-aqueousacidconditions,ifKbisabove10-10,thebasicitycouldberaisedtothelevelofsolventAc-withglacialaceticacid,andtherefore,thebasicitystrengthensobviously.Thismethodisusuallytodeterminethecontentsofalkaloidsinrawmaterials.

Theoreticsofnon-aqueoustitrationNon-aqueoustitrationPrinciple:Thetitrationofalkaloidsisareplacementprocess,infact,thatis,strongacidreplacestheweakacidcombinedwithalkaloids.BH+·A-+HClO4BH+·ClO4-+HABH+·A-standsforsaltsofalkaloids,HAstandsforreplacedweakacid.ThedifferentacidityofreplacedHAmakesthedifferentinfluencestothetitration,sothecorrespondingconditionsshouldbeadoptedaccordingtothedifferentinstance.Non-aqueoustitrationGotoreviewMethod：

Addingglacialaceticacid10~30mltosamplewhichisdriedproperlytilldissolution.Ifitissaltofhalogenacid,put5％glacialaceticacidsolutionofmercuricacetate3~5ml,titratewith5%perchloricreagenttoendpoint,andcorrecttheresultwithblanktest.Non-aqueoustitrationDiscussion:

Therangeofapplication：Kb<10-8organicbasicsalts；Influenceofacidradicals:生物碱盐中被置换出的无机酸类在醋酸中的酸性依下列排序递减：HClO4>HBr>H2SO4>HCl>HSO4->HNO3Methodofindicatingendpoint:Electricpotentialmethodandindicatormethod;crystalvioletiscommonlyusedastheindicator.Non-aqueoustitrationApplication：氢卤酸盐的测定：由于氢卤酸在冰醋酸中酸性较强，对测定有干扰，必须先加入过量的醋酸汞冰醋酸溶液，使形成难以电离的卤化汞，而氢卤酸盐则转变为可测定的醋酸盐，然后用高卤酸滴定可得满意结果。

Inordertoovercomethedisturbanceofhalogenacid,putexcessmercuricacetatetoformmercurichalidewhichisdifficulttoionizeinthesolvent,thenthealkaloidsaltofhalogenacidtransformstoameasurableacetateandtheacceptableresultcouldbeattainedfortitrationbyperhalogenacid.Non-aqueoustitrationB.Theassayofsulfate：硫酸为二元酸，在水溶液中能完成二级电离，生成SO42-,但在冰醋酸介质中，只能解离为HSO4-，不再发生二级解离。因此，生物碱的硫酸盐，在冰醋酸的介质中只能被滴定至生物碱的硫酸氢盐(BH+)2·SO42-+HClO4BH+·ClO4-+BH+·HSO4-

测定生物碱硫酸盐时，应注意分子中氮原子的碱性强弱，方能正确地进行结果计算；并能正确地理解各测定方法项下滴定度的由来。如：硫酸阿托品[USP(24)]。Non-aqueoustitrationASSAY-Dissolveabout1gofAtropineSulfate,accuratelyweighed,in50mLofglacialaceticacid,andtitratewith0.1Mol/LperchloricacidVS,determiningtheendpointpotentiometrically.Performablankdetermination,andmakeanynecessarycorrection.EachmLof0.1Mol/Lperchloricacidisequivalentto67.68mgof(C17H23NO3)2·H2SO4.Theassayofnitrate:

硝酸在冰醋酸中是弱酸，但它具有氧化性使指示剂变色，所以用非水溶液测定生物碱硝酸盐时，一般不采用指示剂而用电位法指示终点。

Nitricacidshowsweakacidityinglacialaceticacid,butitsoxygenationwouldcolorizetheindicator.So,weusuallyusepotentiometricmethodratherthanindicatorwhentitratingalkaloidsnitrateinnon-aqueoussolution.Non-aqueoustitration

Theassayofpreparations:Injections：Takesomesampleintowaterbathandmakeittodryness,thendryitinacertaintemperatureandtreatitaccordingtotheitemofrawmaterial.Tablets：Inmostoftablets,theexcipientsuchasstearate,carboxymethylcellulosesodiumetcalwaysconsumeperchloricacid.So,thesamplesshouldbeextracted,separatedanddriedbeforeassay.Non-aqueoustitrationExtractivetitrationCondition:Somealkaloidswithstrongbasicity(pKb6~9)canbedetermineddirectlybyacid/basetitrationafterbasifyingandextractingbyorganicsolvent.Principle:Alkaloidsaltsaredissolvedinwater,whilefreealkaloidsaredissolvedinorganicsolvent.Withtheseproperties,alkaloidsareextractedandtitrated.samplewaterordil.mineralacidsolutionalkalinereagentdissociativealkaloidsorganicsolventextractalkaloidswateralkaloidsalkalinereagentwatersolubleimpuritywashanhydroussodiumsulfatedehydratepurealkaloidswaterMethod:directtitrationbacktitrationbacktitrationwithbaseafterextractedwithacidExtractivetitrationGotoreviewDiscussion:

AlkalinizingreagentCommonlyusedreagent：ammonia,sodiumbicarbonate,sodiumhydroxide,calciumhydroxideandmagnesiumoxide.Strongbasesarenotsuitableforthedissociationofthefollowingalkaloids:

[1] Drugswiththestructureofester.e.g.atropineandreserpine,astheyareeasilydecomposed.[2] Drugswithphenolstructure.e.g.morphine,areeasilytoformsaltwithalkalinizingreagentanddissolvedinwater.[3] Drugscontainingfattyexcipientsareeasilytobeemulsifiedandmaketheextractiondifficultandincomplete.ExtractivetitrationB.Extractionsolvent:Selectionrules：[1]Alkaloidsareeasilydissolvedinthesolvent,andthesolventisundissolvablewithwater；[2]Itisinerttoalkaloidsandbasifiers；

Accordingtotheaboverules,exceptforfewalkaloidssuchasmorphinewhichisundissolvedinchloroform,themostefficientandcommonlyusedextractionsolventischloroform.Ethyletherisanothercommonlyusedextractionsolvent.Discussion:

ExtractivetitrationC.IndicatorselectionThebasicityofalkaloidsisnotstrongenough.Ifitistitratedwithstrongacid,theformedsaltshowsacidity.Sotheindicatorselectedshouldchangecolorinacidrange.D.Preventionofemulsificationchenshi:chenshi:Discussion:

ExtractivetitrationPreventionofemulsification：1.Usingweakbasifier;2.Usingproperextractionsolventwhichdoesnotemulsifyeasily;3.Avoidingshakingfiercely.Application：

Thismethodisusedtodeterminmorphineinopium.

Therearemorethanthirtykindsofalkaloidsinopium.Themosteffectivecomponentsismorphine,anditscontentis10%.Theninsequence,narcotine(about2％～9％),codeine(about0.3％～2％),papaverine(about0.8％).ExtractivetitrationMethod：

供试品滤液醚层水层精密称定，加水研磨，加入Ca(OH)2，定溶过滤除去可待因，罂粟碱吗啡，罂粟酸，硫酸等形成可溶性钙盐加入乙醇，乙醚，氯化胺，振摇使分层那可汀等易溶于有机溶剂的阿片生物碱吗啡结晶醚液洗涤，中性甲醇重结晶，用硫酸滴定液溶解溶液硫酸吗啡溶于水中，过量硫酸黄色溶液甲基红试剂，NaOH滴定液滴定ExtractivetitrationAcid-dyecolorimetry:Conditions：Thismethodiscommonlyusedinsmalldosagesamples(especiallyinjections)orquantitativeanalysisofalkaloidsinorganism.Principle：Inpropermedium,alkaloidscancombinewithhydrogeniontoformpositiveion(BH+).Someacid-dyes,suchasbromophenolblue,bromcresolgreenandsoon，candisassociatetobenegativeion(In-).Thepositiveandnegativeionscanquantitativelycombinetoformionpair,whichcanbequantitativelyextractedbyorganicsolventandhaveaabsorptionatcertainwavelength.Wecancalculatethecontentofalkaloidbydeterminingthesampleabsorbance.

BH++In-(BH+·In-)waterphase(BH+·In-)organicphase

Aciddyecolorimetry:[BH+·In-]organicphase

[BH+]waterphase[In-]waterphase

E=BH+waterphase＋In-waterphase(BH+