Quantitative determination of major alkaloids in Cinchona bark by Supercritical Fluid Chromatography

Chinoline alkaloids found in Cinchona bark still play an important role in medicine, for example as antimalarial and antiarrhythmic drugs. For thefirsttime Supercritical Fluid Chromatography has been utilized for their separation.

Journal of Chromatography A, 1554 (2018) 117–122

jou rn al h om ep a g e : w w w e l s e v i e r c o m / l o c a t e / c h r o m a

Adele Murauer, Markus Ganzera∗

a r t i c l e i n f o

Keywords:

Quinine

Quantification

a b s t r a c t

(http://creativecommons.org/licenses/by/4.0/)

1 Introduction

Cinchonaecortex,whichoriginatesfromseveralrelatedspecies

ofthegenusCinchona(C.pubescens,C.calisaya,C.ledgerianaand

hybrids)accordingtotheEuropeanPharmacopeia, wasusedas

antimalarialdrugbytheindigenouspopulationofSouthAmerica

forcenturies.Itbecametheprimaryremedyagainstthisdisease

worldwide,and only afterWorldWar 2 syntheticantimalarials

likechloroquinereplacedthenaturalproduct[1].However,dueto

increasingresistancesandalsoavailabilityissuesquinineisstill

rel-evantformalariatreatmenttoday[2];besidesthatthecompound

isaddedtobeveragesasbitteragent[3],servesacatalystin

asym-metricorganicsynthesis[4],oractsaschiralselectorinstationary

phases[5].Thealkaloid patterninCinchonabarkisrather

com-plexwithmorethan30knownrepresentatives[6].Theymainly

arechinolinederivatives,includingthediastereomericpairs

qui-nine/quinidineandcinchonine/cinchonidine.Additionalalkaloids

are,amongothers,theirdihydro-derivatives

Notonlyduetothemedicinalandcommercialimportanceof

Cinchonabarkbutalsothenarrowtherapeuticwindowofquinine

many analytical studies focused on the determination of

alka-loidsinthecrudedrug.Thecompendialmethodinthe9thedition

of the Ph.Eu is based ona photometric determination of qui-nine(348nm)andcinchonine-type(316nm)alkaloids.Research papersmainlyemphasizedontheseparationofthedominant rep-resentativesutilizingTLC[7],isotachophoresis[8],aqueous[9]and non-aqueousCE[10],vibrationalspectroscopy[11],NMR[12]and HPLC[3,6,13–16].Forexample,Hoffmannetal.utilized achiral strongcationexchangematerialtoexcellentlyresolveeight Cin-chonaalkaloidsin15min,yetanapplicationtoplantmaterialis missing[16].Thelatterwaspresentedinthemostrecentstudy,

inwhich Holmfredetal reportedontheseparation ofthefour mainisomerson2.6␮mC-18coreshellmaterial(KinetexXB-C18)

in25min[17].WhetherSupercriticalFluidChromatography(SFC)

is apossibleand equivalentalternativehasnever been investi-gated.Inthepastthetechniquewaspredominantlyusedforthe analysisofnon-polarcompoundslikefattyacids[18],triglycerides [19]orcarotenoids[20].Recentpublicationspointtoamuchwider rangeofpossibleapplicationsalsoincludingpolarnaturalproducts [21,22]andalkaloids[23–25].Therefore,weattemptedtoseparate andquantifythealkaloidsinCinchonabarkbySFC

2 Materials and methods

2.1 Standardsandreagents SixCinchonaalkaloids(compounds1-6,seeFig.1forstructures) withapurity≥98%wereavailableasstandards;theywere pur-https://doi.org/10.1016/j.chroma.2018.04.038

Fig 1.Chemical structure of the assayed Cinchona alkaloids.

chasedfrom Phytolab(Vestenbergsreuth, Germany;compounds

1and 2)and SigmaAldrich(St.Louis,MO,USA; compounds

3-6).Plant samples(CC-2017-1 toCC-2017-4)were bought2017

indifferentpharmaciesinInnsbruck,Austria;voucherspecimens

aredepositedattheInstituteofPharmacy,Pharmacognosy,

Uni-versityofInnsbruck.CompressedcarbondioxideforSFCanalysis

had a purity of ≥99.995% (4.5 grade) and came from Messer

(Gumpoldskirchen,Austria).Allsolventsandreagents(methanol,

acetonitrile, diethylamine, trimethylamine, sodium hydroxide,

aceticacid,ammoniumacetate,phosphoricacid)utilizedin this

studywereofanalyticalgradeandpurchasedfromMerck

(Darm-stadt,Germany).AnArium 611water purificationsystemfrom

Sartorius(Göttingen,Germany)producedtherequiredHPLCgrade

water

2.2 Samplepreparation

Theplantmaterial(CinchonaecortexPh.Eu.)wasfinely

pulver-izedin amilland150mgwereextractedfollowingapublished

protocol[6].Extractionsolventwasamethanol/0.1MNaOH

mix-turein theratio 49/1; thesampleswere extracted three-times

with10mlofthismixturebysonication(BandelinSonorex,Berlin,

Germany)for20mineach.Aftereachsteptheywerecentrifuged

for 10min at 1500g, and the clear supernatant combined in a

50mlvolumetricflask.Thenthelatterwasfilledtovolumewith

theextractionsolvent.Samplesolutionsweremembranefiltered

rightbeforeanalysis(0.45␮mcelluloseacetatemembrane,VWR,

Vienna,Austria)andinjectedintriplicate.Ifstoredat4◦Csample

andstandardsolutionsarestableforatleast2weeks

2.3 Analyticalmethod

For all experiments an Acquity UPC2-SFC instrument from

Waters(Milford,MA,USA),equippedwithconvergencemanager,

columnoven,samplemanager,binarysolventmanagerandPDA

detectorwasused;theoperatingsoftwarewasEmpower3

Opti-mumseparationofthesixstandardswasachievedonanAcquity

UPC2TorusDEAcolumn(3.0×100mm,1.7␮m)fromWaters,

pro-tectedbyaguardfilter(criticalclean;Waters).Themobilephase

comprisedCO (A)and0.8%diethylamineinamixtureof10%

ace-tonitrileand90%methanol(B).Isocraticseparationwasachieved

by maintaininga concentration of 97.7A/2.3Bover 10min.The injectedsamplevolumewas1␮l,whileflowrate,column temper-atureandABPRpressureweresetto1.8ml/min,15◦Cand150bar (2175psi).Thecompoundsofinterestweredetectedat275nm Thesamplemanagerwasmaintainedat10◦C, andamixtureof methanol/2-propanol(1:1) andmethanolservedasa weakand strongwash,respectively

2.4 Methodvalidation

ToassurethatthedevelopedSFCmethodconformsto regula-torystandardsitwasvalidatedaccordingtoICHguidelines[26] Fortheconstructionofcalibrationcurvesaswellastodetermine thelinearrangeapproximately1mgofeachstandardwas accu-ratelyweightedanddissolvedin1mlmethanol(stocksolution) Thissolution wasused to preparefurthercalibration levelsby serialdilutionintheratioof1:1withthesamesolvent.LOD(limit

ofdetection)andLOQ(limitofquantification)valueswere calcu-latedasdescribedintheguidelinesbasedonstandarddeviation

oftheresponseandslopeofthecalibrationcurve.Selectivitywas confirmedby utilizing PDA data and thepeak purity option of theoperatingsoftware.Precisionwasassuredbypreparingand analyzing five solutions of sample CC-2017-2 oneach of three consecutivedays.Variationswithinoneday(intra-dayprecision) andwithinthreedays(inter-dayprecision)werecalculatedbased

onthepeakarea Accuracywasinvestigated byspikingsample CC-2017-2 withdifferent concentrations of allstandards (high, mediumandlowspike).Spikedsampleswerethenextractedand analyzedasproposed.Recoveryrateswerecalculatedby compar-ingtheactuallyfoundconcentrationswiththetheoreticallypresent ones.Allresultsofthevalidationexperimentsaresummarizedin Table1

3 Results and discussion

Sinceitsbeginningsinthe1960sSFChasevolvedintoawidely utilizedandefficientseparationtechnique.Abetter understand-ingoftheunderlyingtheory,togetherwithsignificantlyimproved instrumentsand stationaryphaseshave ledtomanysuccessful

A Murauer, M Ganzera / J Chromatogr A 1554 (2018) 117–122 119

Table 1

y = 297.1 x +848.7 y = 315.1 x +182.1 y = 267.6 x −189.4 y = 273.4 x −34.1 y = 239.1 x −679.9 y = 252.8 x −724.0

Precision

Accuracyd

separationsandabroadfield ofapplications.However,relevant

medicinalplants,whoseingredientsseemtobenotsuitableforSFC

becauseoftheirpolarity,have neverbeeninvestigatedtilldate

OneofthemisCinchonabark,adrugwhichisanalytically

chal-lengingasitcontainsdiastereomericchinolinealkaloidsasactive

constituents

3.1 Methoddevelopment

TheoptimumSFCseparationofsixmajorCinchonaalkaloids,

namelydihydroquinidine(1), dihydroquinine (2),quinidine (3),

quinine(4),cinchonine(5)andcinchonidine(6),withinlessthan

7min is shown in Fig.2A During methoddevelopmentit was

observed that this result is only feasible by one specific

com-binationof mobileand stationary phase Concerning thelatter,

eightdifferentSFCcolumnsfromWaterswithidenticaldimensions

(3.0×100mm)andaparticlesize≤2␮mweretested:fourfromthe

Torusseries,i.e.2-PIC,Diol,1-AAandDEA,andfourViridiscolumns

(BEH,BEH2-EP,CSHFluoro-PhenylandHSSC18SB).According

toWestand colleagues,who classifiedmore than30ultra-high

performanceSFCstationaryphasesusingamodifiedLSER(linear

solvationenergyrelationship)model,fromallthestationaryphases

availableinthisstudyTorusDEA(diethylamine)materialhasthe

highestbasiccharacter[27].Forthismaterialtherelevanta-term

(basicity)ishigherthan2.6,whereasforexampleforViridisphases

itrangesfrom0.3(CSHFluoro-Phenyl)to1.4(BEH2-EP)

Accord-ingly,thismaterialisdesignedtoprovidesuperiorpeakshapefor

bases[28].WithpKavalues around8.5[29] thetargetanalytes

aresuchcompounds,andthereforeitseemedlogicthatthis

sta-tionaryphasewasselectedforfurtherexperiments.OnlyonTorus

DEAmaterialthecompoundscouldbeseparatedwithacceptable

resolutionandpeakshape,onothersincludingallViridiscolumns

thecompoundselutedasbroadandoverlappingsignalsonly(see

supportinginformation)

Concerningthemobilephase itwasrequiredtoadd organic

solventsanddiethylamineasmodifiers.ThepolarityofpureCO2

is similar to hexane [30], and therefore a small percentage of

methanol wasrequired;the combination withacetonitrilewas

advantageousintermsofresolution(Fig.2B),thusaMeOH/ACN

mixtureintheratioof9:1wasemployed.However,withoutan

alkalineeluentnoacceptableresultwaspossible.Thisobservation

wasin agreementtoliterature,whereanenhancedSFC

separa-tionofbasicsubstanceswithanalkalinemobilephaseisreported

[31].Theauthorsattributedthiseffecttoreducedsecondaryionic

interactionswithresidualsilanols.Forthecurrentapplicationthe

additionof0.8%diethylamine(DEA)tothemodifier(i.e.the

afore-mentionedmixtureofMeOHandACN)showedtobetheoptimum

Intermsofelutionmodeconditionshadtobefine-tunedaswell Evenwithaveryflatgradientthefirstfoursignalsmerged,sothat isocraticconditionshadtobeselected;97.7%phaseA(CO2)and 2.3%B(MeOH,ACNandDEA)providedthebestresolution.Itis note-worthytosaythatalreadyaslightchange(e.g.to2.5%B;Fig.2C) hadanegativeimpactontheseparation Loweringthemodifier concentrationto2.0%resultedinprolongedretentiontimes,yet compounds2and3graduallyoverlapped

Anotherparameterwithsignificantinfluence onthe separa-tionofthesixalkaloidswascolumntemperature(Fig.2D).Rather surprisingly,byloweringcolumntemperaturedownto15◦C reten-tion times steadily increased.The opposite would beexpected becauseatlowertemperaturesfluiddensityincreases,resultingin reducedretention.Apossibleexplanationfortheobservedeffects mightbechangesinthepolarityofthestationaryphaseduetoa temperature-dependentadsorptionofmobilephasecomponents [32].Itisobviousthatcarbondioxidewasnotpresentinthe super-critical stateanymore,becauseitscritical temperatureis 31◦C; however,workinginthesubcriticalstagehasnosignificant dis-advantages and it is described (butnot necessarily mentioned) quiteoften[33].Furtherchromatogramsindicatingtherelevance

ofindividualmethodparametersarecompiledassupplementary material.Aninterestingfactshownthereistheinfluenceofapplied backpressure(ABPR).Thissettingisusuallyofminorimportance, yetinthecurrentapplicationitmodifiedresolution,particularly betweencompounds2and3.Thelattercouldberesolvedbestat

anappliedABPRof150bar

3.2 Methodvalidation Assaydevelopmentwasfollowedbymethodvalidation;data presentedinTable1confirmsthatallrequirementswere satisfac-torilymetinthisrespect.Selectivitywasdeducedbyseveralfacts First,structurallycloselyrelatedcompounds(including diastere-omers)couldberesolved,second,nosignsofco-elutions(e.g.peak shoulders)werevisible,andthird,thePDAdatawasvery consis-tentwithinindividualpeaks.Afinalconfirmationofpeakpurity

by SFC-MSwasnotpossible,because thistechnical option was notavailable.Forallstandardscalibrationcurveswerelinearfrom approx 1000–30␮g/ml, withdetermination coefficients always beinghigherthan0.999.LODvaluesshowedtobeintherangefrom 0.6(5)to2.4(2)␮g/ml,LOQvaluesvariedfrom1.9–7.3␮g/ml.They naturallycannotcompetewiththoseachievablebyfluorescence detection(e.g.LODforquinineis2fmol,[6]);however,theyare comparabletoconventionalHPLC-UVasLOQvaluesof5␮g/gare statedinreference[17].Precisionwasinvestigatedbyrepeatedly assayingsampleCC-2017-2underoptimizedextractionand

sepa-Fig 2. Separation of Cinchona alkaloids by SFC; optimum conditions(A;column: Acquity UPC 2 Torus DEA 1.7 ␮m, 3.0 × 100 mm; mobile phase: CO 2 (A) and 0.8% diethylamine

rationconditions.Intra-day(≤2.2%)aswellasinter-dayvariations

(≤3.0%)wereacceptableandtypicalforinvestigatingplant

mate-rial,whichusuallyshowssomedegreeofinhomogeneity.Lastbut

notleast,accuracywasdeterminedinspikingexperiments(high

spike:200␮g/ml,mediumspike:100␮g/ml,lowspike:50␮g/ml)

Recoveryrateswerenotlowerthan97.2%(3,lowspike)andnot

higherthan103.7%(6,highspike),indicatingvalidityofthis

param-etertoo

3.3 Analysisofthesamples FoursamplesofdriedandmilledCinchonabark,allofthemwith Ph.Eu.quality,wereavailablefor quantification.Concerningthe optimumextractionprotocolaproceduredescribedbyGattietal wasadopted[6].Itutilizesalkalinemethanolandsonication,and showedtobeadvantageousoverotherslikesoxhletextractionin theirworkduetothemildconditionsapplied;theobserved

quan-A Murauer, M Ganzera / J Chromatogr A 1554 (2018) 117–122 121

Table 2

cinchonidine(6) 0.90 (0.91) 1.15 (1.47) 1.26 (1.14) 1.05 (0.89)

inFig.3.ThecompiledquantitativeresultspresentedinTable2

indicate that all of the investigated specimens were of similar

composition.Threeof thesix standardswereclearly assignable

by matching retention times and UV-spectra; if these criteria

werenotmet,e.g.peaksweretoosmallforprovidingmeaningful

spectra,respectivesignalswerenotconsideredforquantitation

Theassignedcompoundswerequinine,cinchonineand

cinchoni-dine, with the latter always being the least abundant alkaloid

(0.90%–1.26%).Mostdominantwascinchonine(1.87%–2.30%),

fol-lowedbyquinine,whichrangedfrom1.59%to1.89%;anexcellent

repeatabilitywasobservedwhile performingtheseexperiments

(␴rel≤1.55, n=3).The total alkaloid content variedfrom 4.75%

(sampleCC-2017-3)to5.20%(sampleCC-2017-2)

4 Conclusion

Thisstudyisanotherprooffortheexcellentseparationefficiency

andversatilityofSFC,especiallyinthefieldofnaturalproducts.The

determinationofalkaloidsinCinchonabarkisachallengingtask,

becausethetargetanalytesarestructurallyverysimilarandthe

investigatedmatrixiscomplexlikemostbiologicalsamples.Due

tothepersistingpracticalrelevance ofthedrugmany attempts

havebeenmadetodeterminethesecompounds,mostlybyusing

conventionalRP-HPLCincombinationwithfluorescencedetection

Thisassuredanexcellentsensitivity;however,therequired

anal-ysistimewasintherangefrom15[16]to50min[6],whenonly

recentpublicationsareconsidered.Thatacomparableseparation

isalsofeasibleinlessthan7minbyusinga“greentechnology”has beenshowninthecurrentstudy.Thiswasonlypossibleafter metic-ulousmethodoptimization,butoncecompleted,areproducible, accurateandruggedsystemwasavailableforroutineuse;method validationconfirmedthisestimation.Inthesamplesanalyzedthree outofsixstandardscouldbeassigned.Thisislessthaninprevious reports,butexplainablebythedifferentdetectiontechniquesused However,ifsuitableinstrumentationispresent(e.g.fluorescence detectorsforSFCareavailable)therewillbeprobablyno differ-enceinthenumberofidentifiedcompounds.Withtheavailable instrumentationquinine,cinchonineandcinchonidinecould eas-ilybeassignedincrudeCinchonabarkextracts.Thequantitative resultswerewellcomparabletopublisheddata,whichfor exam-plereportthefollowingvaluesforadrugwithPh.EU.quality:1.80% quinine,1.65%cinchonine,and1.25%cinchonidine[17].This suc-cessfulapplicationofSFC,onfortheutilizedtechnique“untypical” compounds,shouldraisefurtherinteresttofullyexplorethe poten-tialofthisseparationtechnique,whichdefinitelyisnotlimitedto the“classics”likecarotenoids,fattyacidsorterpenes.Thisandother studiesonnaturalproductslikeanthraquinones[34],kavalactones [35]orfurocoumarins[36]aregoodindicatorsactually

Conflict of interest

Theauthorsdeclarenoconflictofinterest

Acknowledgement

TheauthorswouldliketothanktheAustrianScienceFund(FWF, projectP269170)forfinancialsupport

Appendix A Supplementary data

Supplementarydataassociatedwiththisarticlecanbefound,

intheonlineversion,athttps://doi.org/10.1016/j.chroma.2018.04

038

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