第八章生物堿類藥物分析

Chapter8TheAnalysisofAlkaloids

藥物分析課件系列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生物堿來源于氨基酸來源于異戊烯來源于萜類來源于甾體來源于鳥氨酸吡咯類吡咯里西丁類托品烷類哌啶類吲哚里西丁類喹諾里西丁類喹啉類吖啶酮類簡單苯丙胺類四氫異喹啉類芐基四氫異喹啉類生物堿苯乙基四氫異喹啉類生物堿芐基苯乙胺類生物堿吐根堿類生物堿簡單吲哚堿類簡單β–卡波林類半萜吲哚堿類單萜吲哚堿類單萜生物堿倍半萜生物堿二萜生物堿三萜生物堿孕甾烷生物堿環孕甾烷生物堿膽甾烷生物堿來源于賴氨酸來源于色胺酸來源于苯丙氨酸/酪氨酸來源于鄰氨基苯甲酸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)秋水仙堿]2.Comparinguniformityofsampleabsorptionspectrumwiththatofstandard.e.gIdentificationofemetine[USP(24)吐根堿]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

雙縮脲反應(Biuretreaction):芳環側鏈具有氨基醇結構生物堿藥物的特征反應。

Vitailireaction:托烷類生物堿莨菪酸結構的特征反應，與發煙硝酸得黃色三硝基衍生物，遇醇制氫氧化鉀即顯深紫色。

綠奎寧反應(Thakkeioquinreaction):6位含氧喹啉衍生物的反應。可在其鹽類的微酸性水溶液中，滴加溴水和氯水，至微過量時，再加入過量的氨水，應呈翠綠色反應。Gotoreview

Murexidereaction黃嘌啉生物堿的特征反應，即樣品加鹽酸和氯酸鉀，在水溶液上共熱蒸干，此殘渣遇氨氣呈紫色，再加氫氧化鈉溶液顏色即消失。

Marquisreaction含酚羥基異喹啉類生物堿的特征反應。該類藥物遇甲醛硫酸可形成具有醌式結構的有色化合物。

Frohdereaction系嗎啡生物堿的特征反應。嗎啡加鉬酸試液即顯紫色，繼而藍色，再為棕綠色。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:生物堿鹽中被置換出的無機酸類在醋酸中的酸性依下列排序遞減：HClO4>HBr>H2SO4>HCl>HSO4->HNO3Methodofindicatingendpoint:Electricpotentialmethodandindicatormethod;crystalvioletiscommonlyusedastheindicator.Non-aqueoustitrationApplication：氫鹵酸鹽的測定：由于氫鹵酸在冰醋酸中酸性較強，對測定有干擾，必須先加入過量的醋酸汞冰醋酸溶液，使形成難以電離的鹵化汞，而氫鹵酸鹽則轉變為可測定的醋酸鹽，然后用高鹵酸滴定可得滿意結果。

Inordertoovercomethedisturbanceofhalogenacid,putexcessmercuricacetatetoformmercurichalidewhichisdifficulttoionizeinthesolvent,thenthealkaloidsaltofhalogenacidtransformstoameasurableacetateandtheacceptableresultcouldbeattainedfortitrationbyperhalogenacid.Non-aqueoustitrationB.Theassayofsulfate：硫酸為二元酸，在水溶液中能完成二級電離，生成SO42-,但在冰醋酸介質中，只能解離為HSO4-，不再發生二級解離。因此，生物堿的硫酸鹽，在冰醋酸的介質中只能被滴定至生物堿的硫酸氫鹽(BH+)2·SO42-+HClO4BH+·ClO4-+BH+·HSO4-

測定生物堿硫酸鹽時，應注意分子中氮原子的堿性強弱，方能正確地進行結果計算；并能正確地理解各測定方法項下滴定度的由來。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劑變色，所以用非水溶液測定生物堿硝酸鹽時，一般不采用指示劑而用電位法指示終點。

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：

供試品濾液醚層水層精密稱定，加水研磨，加入Ca(OH)2，定溶過濾除去可待因，罌粟堿嗎啡，罌粟酸，硫酸等形成可溶性鈣鹽加入乙醇，乙醚，氯化胺，振搖使分層那可汀等易溶于有機溶劑的阿片生物堿嗎啡結晶醚液洗滌，中性甲醇重結晶，用硫酸滴定液溶解溶液硫酸嗎啡溶于水中，過量硫酸黃色溶液甲基紅試劑，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+