Proceedings of the 14th International Students Conference “Modern...

ISBN 978-80-7444-059-5

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Prague, 2 —2 September 2010 1 8

Edited by Karel Nesměrák

Prague 2018

Proceedings of the

14 ath Intern tional Students Conference

“Modern Analytical Chemistry”

Proceedingsofthe

14thInternationalStudentsConference

“ModernAnalyticalChemistry”

Prague, 20—21 September 2018


Prague 2018

Proceedings of the

14th International Students Conference


CATALOGUING-IN-PUBLICATION–NATIONALLIBRARYOFTHECZECHREPUBLIC

KATALOGIZACEVKNIZE–NA RODNIKNIHOVNACR

ModernAnalyticalChemistry(konference)(14.:2018:Praha,Cesko)

Proceedingsofthe14thInternationalStudentsConference“Modern

AnalyticalChemistry”:Prague,20-21September2018/editedbyKarel

Nesmerak.--1stedition.--Prague:FacultyofScience,Charles

University,2018.--x,298stran

ISBN978-80-7444-059-5(brozovano)

543*(062.534)

analytickachemie

sbornık ykonferencı

analyticalchemistry

proceedingsofconferences

543– Analytickachemie[10]

543–Analyticalchemistry[10]

TheelectronicversionoftheProceedingsisavailableattheconferencewebpage:

http://www.natur.cuni.cz/isc-mac/

©CharlesUniversity,FacultyofScience,2018.

ISBN978-80-7444-059-5

Preface

Forthefourteenthtimewewelcometheparticipantsoftheinternationalstudent

conference “ModernAnalytical Chemistry” inPrague at – as it canbe already

called – the traditional meeting of Ph.D. students of analytical chemistry.

Participants from six countries (Belarus, Czech Republic, Germany, Poland,

Russia,andSlovakia)arecomingtopresenttheresultsoftheirresearch,tomaster

theirpresentationandlanguageskillsandtoenjoyinternationalcommunityof

analyticalchemists.Webelievethat, likeallpreviousones,thisyearwillbean

interesting,beneficialandenjoyableencounter.

Forty eight contributions are presented in this volume of the conference

proceedings,assortedbythesequenceoftheirdelivery,accompaniedwiththe

indexesattheendoftheproceedingsenablingeasynavigationthroughitspages.

Let’sexpressourhopesthatallcontributionswillbefoundinterestingandwill

prove that analytical chemistry is trendy, multifaceted, steadily developing

sciencewithnew,unsuspectedwaysofitsinnovationandapplication.Andthisis

whatmakestheorganizationofthismeetingveryfulfillingandsatisfactory.

Weareverygrateful to theDivisionof

Analytical Chemistry of EuCheMS for its

long-lasting auspices of our conference.

Also,wearethankfultooursponsors,not

only for their kind financial sponsorship

making the conference possible, but also

for all their support and cooperation in

manyofourotheractivities.

prof.RNDr.VeraPacakova,CSc. doc.RNDr.KarelNesmerak,Ph.D.

Proceedingsofthe14thISCModernAnalyticalChemistry Prague2018 iii

Sponsors

The organizersof14th International Students Conference “ModernAnalytical

Chemistry” gratefully acknowledge the generous sponsorship of following

companies:

iv Proceedingsofthe14thISCModernAnalyticalChemistryPrague2018

www.waters.com

www.thermofisher.czwww.ecomsro.com

www.lach-ner.com

www.watrex.com

Proceedingsofthe14thISCModernAnalyticalChemistry Prague2018 v

Contents

MilanowskiM.,RudnickaJ.,LigorT.,BuszewskiB.:Determinationofvolatileorganiccom- poundsinheadspaceabovesalivaspecimensusingSPME-GC/MStechnique............................................1DurnerB.,EhmannT.,MatysikF.N.:Separationoflinearandcyclicpoly(dimethylsiloxanes) withinteractivechromatography ............................................................................................................................7DęboszM.,WieczorekM.,KoscielniakP.:Simplecalibrationapproachtoelimination oftheadditiveinterferenceeffect ..........................................................................................................................14Nguyen-MarcinczykC.T.,KarasinskiJ.,WojciechowskiM.,KrataA.A.,HaliczL.,BulskaE.: Separationofchromium(III)andchromium(VI)byreversed-phaseion-pairing chromatography .........................................................................................................................................................19GaworA.,KonopkaA.,TorresElgueraJ.C.,RuszczynskaA.,CzaudernaM.,BulskaE.: Label-freeproteomicapproachtoidentificationandquantificationofproteinsinanimal tissuesamples ...............................................................................................................................................................25GranicaM.:Distance-basedmeasurementsusingmicrofluidicpaper-basedanalyticaldevices modifiedwithPrussianBlue ........................................................................................................................................31MadejM.,KochanaJ.,BasB.:Cyclicvoltammetryandstaircasevoltammetryincitalopram determination ..............................................................................................................................................................37JagielskaA.,WagnerB.,RuszczynskaA.,GaworA.,BulskaE.,ZieminskaE.,ToczyłowskaB., JakuczunW.,SzostekM.:ElementalanalysisofatheroscleroticplaquebyICP-MSand LA-ICP-MS ......................................................................................................................................................................44GajdarJ.,BarekJ.,FischerJ.:Determinationofdifenzoquatatamercurymeniscusmodified silversolidamalgamelectrodebydifferentialpulsevoltammetry ............................................................51BraunP.,RablH.P.,MatysikF.M.:Investigationsontheelectrochemicallyinduceddecomposition ofAdBlue-urea..............................................................................................................................................................55NikolaevaA.A.,BulychevaE.V.,KorotkovaE.I.,LinertW.:Simultaneousdetermination ofsyntheticdyesPonceau4R(E124)andSunsetYellow(E110)byfluorimetryinsoftdrinks ..........61KrolA.,PomastowskiP.,Railean-PlugaruV.,BuszewskiB.:AnalysisofLactococcuslactis modifiedwithzincionsbycapillaryelectrophoresis.......................................................................................68FestingerN.,MorawskaK.,SmarzewskaS.,CiesielskiW.:Voltammetricstudies ofacemetacin................................................................................................................................................................77Makrlık ovaA.,DejmkovaH.,NavratilT.,BarekJ.,VyskocilV.:HPLC-ED/UVfordetermination ofvanillylmandelicacidinhumanurineaftersolidphaseextraction .......................................................82PatockaJ.,KrejcovaA.,KlausovaK.:ICP-MSanalysisasatoolformonitoringoftheefficiency ofthesorptionbasedremovalofiodinatedcontrastagents .........................................................................87RogowskaA.,PomastowskiP.,ZłochM.,Railean-PlugaruV.,KrolA.,RafinskaK., Szultka-MłynskaM.,BuszewskiB.:TheinfluenceofpHontheelectrophoreticbehaviour ofyeastmodifiedbycalciumions ...........................................................................................................................93SoukalJ.,MusilS.:Optimizationofphotochemicalvaporgenerationofmolybdenumas asampleintroductionforICP-MS .......................................................................................................................100MaleckovaM.,VrzalT.,OlsovskaJ.:Developmentofminiaturizedextractionmethodusedfor GC-NCDscreeningofnon-volatilenitrosocompoundsinmalt..................................................................105PlatonovI.A.,KolesnichenkoI.N.,KarapetianD.D.,IgitkhanianA.E.:Chromato-desorption methodforproducinggasmixturesofvolatileorganiccompounds .......................................................114GalbavaP.,SzaboovaZ .,GabrisovaĽ.,MachoO.,KubinecR.,BlaskoJ.,MikulecJ.:MS/MSana- lysisoffattyacidmethylestersindiesel.............................................................................................................120StarzecK.,KochanaJ.:Newbiosensormatricesbasedoncarbonnanomaterials fortyrosinaseimmobilization ..............................................................................................................................126

vi Proceedingsofthe14thISCModernAnalyticalChemistryPrague2018

GusarA.,GashevskyaA.,DorozhkoE.,DerinaK.:Carboncontainingelectrodesmodified withtheiodatesaltsofaryldiazoniumforelectroanalysis .........................................................................134BaluchovaS.,TaylorA.,MortetV.,Schwarzova-PeckovaK.:Boron-dopeddiamondelectrode fabricatedbymicrowaveplasmaenhancedchemicalvapourdepositionprocesswith linearantennadeliveryforneurotransmitterssensing...............................................................................140LewinskaI.,MichalecM.,TymeckiŁ.:Fluorometricmethodofcreatininedetermination employing3,5-dinitrobenzoicacid.....................................................................................................................145ZusťakovaV.,DusekM.,OlsovskaJ.:Screeningofpesticideinapplecidersbyliquidchromato- graphy-highresolutionmassspectrometry ....................................................................................................152PlatonovI.A.,KolesnichenkoI.N.,IgitkhanianA.E.,KarapetianD.D.:Chromato-desorption microsystemsfordeterminationofbiomarkersintheexhaledbreath...................................................158KorbanA.:Anovelwayofestablishingthequantitativecompositionofgravimetrically preparedstandardsolutionsofvolatilecompoundsinwater-ethanolmatrix ....................................162ShikunM.,VrublevskayaO.:MethodofcyclicvoltammogramsinthedeterminationofSn(II) instronglyacidelectrolytesfortinelectrodeposition ..................................................................................169PegierM.,PyrzynskaK.,KilianK.:AdsorptionofSc(III)onoxidizedcarbonnanotubesfor separationandpreconcentrationfromaqueoussolutions–studyofmechanism .............................175JandovskaV.,DusekM.:Behaviorandfateofpesticides duringbeerbrewing ................................................................................................................................................181BakhytkyzyI.,Hewelt-BelkaW.,Kot-WasikA.:DesignofExperimentapproachforlipid extractionoptimisationinlipidomics................................................................................................................188BorowskaM.,Kot-WasikA.,Kucinska-LipkaJ.:Releaseofactivesubstancesfrompolymeric coatingsinmedicalapplications .........................................................................................................................194BystrzanowskaM.,TobiszewskiM.:Multi-criteriadecisionanalysisforselectionofthebest procedureforPAHsdeterminationinsmokedfood.......................................................................................200FabjanowiczM.,Płotka-WasylkaJ.:Metalcontentinwines ofPolishorigin ...........................................................................................................................................................205GarwolinskaD.,Hewelt-BelkaW.,NamiesnikJ.,Kot-WasikA.:Newsamplepreparation strategiesforcomprehensivelipidomicsofhumanbreastmilk...............................................................211GlinkaM.,Kucinska-LipkaJ.,WasikA.:Determinationofamikacinandciprofloxacin byliquidchromatographywithpre-columnderivatizationtoevaluatesustaineddelivery ofantibioticsfromDrug-ElutingBiopsyNeedle.............................................................................................218KalinowskaK.,WojnowskiW.,Płotka-WasylkaJ.,NamiesnikJ.:Poultrymeatfreshness assessmentbasedonthebiogenicaminesindex.............................................................................................224KempinskaD.,Kot-WasikA.:Highresolutionliquidchromatographyandtimeofflight massspectrometryinperfumeanalysis ............................................................................................................230Lubinska-SzczygełM.,RozanskaA.,DymerskiT.,NamiesnikJ.:Studyoftheeffectofthe hybridisationprocessonthecontentofterpenesinoroblancofruit (Citrusparadisi×Citrusgrandis) ......................................................................................................................236PawlakF.,JankowskaK.,PolkowskaZ .:Correlationbetweenchemicalcompositionand thepresenceofselectedgroupsofbacteriainfreshwatersamplescollected fromIsfjordenandBillefjorde...............................................................................................................................240PytelK.,MarcinkowskaR.,ZabiegałaB.:Influenceofterpenes onindoorairquality ................................................................................................................................................246RozanskaA.,Lubinska-SzczygełM.,DymerskiT.,NamiesnikJ.:Classificationofadulterated raspberryjuiceusingultra-fastgaschromatography .................................................................................253SwierczekL.,CieslikB.,KonieczkaP.:Thepotentialofrawsewagesludge inconstructionindustry .........................................................................................................................................258SzulczynskiB.,RybarczykP.,GębickiJ.:Estimationoftheodourintensityofairsamples undergoingbiofiltrationprocessusingelectronicnoseandartificialneuralnetwork ....................263WronaO.,RafinskaK.,MozenskiC.,BuszewskiB.:Supercriticalcarbondioxideextraction asacrucialstepintheenrichingsampleindesiredgroupofbioactivecompounds...........................269

Proceedingsofthe14thISCModernAnalyticalChemistry Prague2018 vii

PopovaV.,KrivosheinaA.,KorotkovaE.:Developmentofavoltammetricmethodfor detectionofethylnitrite .........................................................................................................................................276KhristunovaY.,BarekJ.,KratochvılB.,VyskocilV.,KorotkovaE.,DorozhkoE.:Control ofelectrochemicalsignalfromsilvernanoparticlesatdifferentmodificationsteps forelectrochemicalimmunosensordevelopment .........................................................................................280SmolejovaJ.,DousaM.:Effectofchaotropicsaltsadditionintomobilephases onseparationofmodelanalytesonpolarstationaryphases inhydrophilicinteractionchromatography ...................................................................................................285

Authorindex .......................................................................................................................................................................293

Keywordindex ...................................................................................................................................................................295

Contributions

1.Introduction

Volatileorganiccompoundscanemanatefrommanydifferentbiologicalspeci-mensincludingblood,urine,feces,salivaandskinsecretions.Theycontainvola-tileorganiccompoundsofvariousfunctionalgroupssuchasaldehydes,alcohols,alkanes, esters, fatty acids, and ketones [1]. Analyses of volatile profiles andsubsequentcomparisonofnormalandpathologicalstatesmayprovideimportantinformationastotheetiology,pathogenesisordiagnosisofcertaindiseases.Salivacanbecollectednoninvasivelybyindividualswithmodesttraining,anditoffersacost-effectiveapproachforscreeningoflargepopulations[1,2].Salivaprovidesalargenumberofvolatilebiomarkerssuchasvolatilesulphurcompoundsres-

ponsibleforhalitosis[3],nonanalforceliacdisease[4]andbenzophenoneforlungcancer[5].Thetypicalmethodforenrichmentofvolatileorganiccompounds

Determination of volatile organic compounds in headspace above saliva specimens using SPME-GC/MS technique

MACIEJMILANOWSKI*,JOANNARUDNICKA,TOMASZLIGOR,BOGUSŁAWBUSZEWSKI

NicolausCopernicusUniversityinToruń,FacultyofChemistry,DepartmentofEnvironmentalChemistryandBioanalytics,Gagarina7,87-100Toruń,Poland*[email protected]

Proceedingsofthe14thISCModernAnalyticalChemistryPrague2018 1

AbstractTheaimofthisstudywastoapplyheadspace-solidphasemicroextra-ction-gaschromatography/massspectrometry(HS-SPME-GC/MS)toevaluateprofilesofvolatileorganiccompoundsfromsalivaofnon-smokersandsmokers.AlsodifferenttypesofSPMEfibreswereusedand influence of such factors as sample volume, incubation, andadsorption time were evaluated. We found that 75 µm Carbo-xen/PDMS fibre gave the highest abundances and the greatestnumberofextractedvolatileorganiccompoundsfromsaliva.Volumeof2mLandincubationtime20minwerechosenasthebestparame-ters of conducted experiments. In the typical profile of salivaryheadspacewefoundatleasttencompoundssuchas:acetaldehyde,propanal,2,3-butanedione,ethylether,dimethylsulphide,andpyr-role.Dailyvariationsofsalivaryconstituentswereinvestigated.Thegreat diversity of compounds were observed in the afternoon.Comparisonof smokersandnonsmokers revealed thepresenceofpyridineinsalivarysamplesofpersonssmokingcigarettes.

KeywordsHS-SPME-GC/MSpreconcentrationsalivavolatileorganic

compounds

insalivaryheadspacearesolventextraction,stir-barextractionandsolidphasemicroextraction(SPME)[1].ThepurposeofourstudywastoapplySPME-GC/MStechnique toevaluatevolatileprofiles in theheadspaceabove saliva.WehavetesteddifferenttypesofpolymersforSPMEpreconcentration.Influenceofsto-rageconditions,samplevolume,incubationandadsorptiontimewasevaluated.Volatileorganiccompoundswereextractedusing75μmCarboxen/PDMSfibreandanalysedbyGC/MS.Dailyvariationofsalivaryprofilesandcomparisonofsmokersandnonsmokerswerealsoinvestigated.

2.Experimental

2.1Reagentsandchemicals

The15mLsterilepolypropylenetubes(ISOLAB,Wertheim,Germany)wereused

for collection of saliva specimens. 22 mL headspace crimp top vials andPTFE/butyl septa for HS-SPME-GC/MS experiments were purchased from

PerkinElmer(Waltham,MA,USA).SPMEfibres:75µmCarboxen/PDMS,100µmPDMSand65µmPDMS/DVBwerefromSupelco,Bellefonte,PA,USA.

2.2Instrumentation

Gaschromatograph7890A(Agilent)coupledwithaspectrometerTruTOF(Leco).ColumnCP-Porabond-Q(Varian)25m×0.25m×3μm.Theoventemperatureprogramme:theinitial40°Cwerekeptfor2min,andrampedat10°C/minto140°C and then again ramped at 5°C/min to 270°C andkept for 5min. Thetemperature of the split-splitless injector was 235°C. The acquisition wasperformedatthemassrange30–350m/z,theacquisitionratewas50spectra/sec.Theionsourceandlinetransfertemperaturesweresetat250°C.ThecollectionofchromatographicdatawasperformedbymeansofChromaTOFsoftware(Leco).Compounds were identified by comparing their mass spectra with thosecontained in spectral library; each peak was searched manually (includingbaseline subtraction and averaging over a peak). Forward and reversematchqualityofatleast800/1000wasusedasthelowermatchthreshold,otherwiseacompoundwaslabelledunknown.

2.3SelectionofSPMEfibre

ThreeSPMEfibres:75µmCarboxen/PDMS,100µmPDMSand65µmPDMS/DVBweretestedtomaximizethepeaksresponses.Selectionwascarriedoutwithfixedextractionandadsorptionconditions forall three fibres.A fibreproviding thehighestabundancesandthegreatestnumberofextractedvolatileorganiccom-poundswaschosen.

2 Proceedingsofthe14thISCModernAnalyticalChemistryPrague2018

2.4Collectionofsaliva

Salivasampleswerecollectedinanon-stimulatedfashionfromelevenhealthyvolunteers(8males,3females),includingtwoactivesmokers.Theywereaskedtorefrainfromeatinganddrinkingatleast1hbeforecollectionofsalivasamples.Participantswereinstructedtorinsetheirmouthswithtapwaterpriortosam-pling,withoutbrushingtheirteethorusinganymouthwashes.After10min,salivasamplesweretakeninglassvialbyspitting.

2.5Incubationandextractionconditions

Salivasampleswereincubatedfinallyfor20minat40°C,extractedfor20minat40°Cwithpreconditioned75µmCarboxen/PDMSfibreandanalysedwithGC/MSsystem.

3.Resultsanddiscussion

3.1ChoosingofSPMEfibre

AmongthreetestedSPMEfibreswechose75µmCarboxen/PDMSfibreasadeviceused for conducted experiments. This fibre was characterized by the highestabundancesandthegreatestnumberofextractedvolatilesfromsalivasamples.

3.2Influenceofsamplevolumeandincubationtime

InfluenceofsamplevolumeisdemonstratedinFig.1(A).Theresultsfor2mLofsaliva shows the highest abundances and comparable number of extracted


Fig. 1Influenceoftwofactorsaffectingvolatileorganiccompoundsanalysis:(A)effectofsamplevolumeonthenumbervolatileorganiccompoundsreleasedfrom1mL,2mL,and3mLofsalivasamples. (B) influence of incubation time and the following extractions on the level of volatileorganiccompoundsfromsinglesalivasample.

compounds. An effect of subsequent volatile organic compounds adsorptionsfromone samplewith increasing incubation time from20min to 195min ispresentedinFig.1(B).Itcanbefoundthatafter195minofincubationthemostnoticeableobservation isadecreaseof2,3-butadieoneandsignificantraiseoflacticacid levelafter145minof incubation.For furtherexperimentsweused20min of incubation because excessive extension of maintaining time led toappearanceofcompoundsfrombacterialmetabolisminobtainedsalivarypro-files,likeethanol.Ourgoalwastoanalyseprofilesfrom“fresh”saliva,hencebothincubationandadsorptionstepswerecarriedoutat40°Cfor20minboth.Wetriedtoreducethecontributionofvolatileorganiccompoundsfromputrefactiveactivityofmicroorganismsinmouthbyshorteningthetimeofpreconcentration,butleavingsufficienttimetodifferentiatethedistinctvolatileorganiccompoundprofilesofindividualsubjects.

3.3Reproducibilityofmethodandcharacteristicoftypicalprofile

Thereproducibilityofthemethodfortriplicatesamplesof1mLisshowninFig.2.UsageofSPMEprovidedsatisfactorylevelofreproducibility.Typicalchromato-gramofvolatileorganiccompoundsobtainedfromsinglefemalesubjectispre-sentedinFig.3.Wewereabletodetecttenvolatileorganiccompoundsinobtainedprofile.Theywere: acetaldehyde, ethanol, propanal, dimethyl sulphide,1-pro-panol, ethyl ether, 2,3-butanedione, 1-propen-2-ol, acetate, ethyl acetate andpyrrole.

3.4Dailyvariationsofsalivaryconstituentsandcomparisonofsmokersandnonsmokers

FluctuationsindailysalivarycompositionisdemonstratedinFig.4(A).Samplesfromsinglemanweretakenthreetimesperday,e.g.,inthemorning,afternoonand evening. Subject did not have any dietary restrictions that day and was


Fig. 2ThereproducibilityofSPME-GC/MSmethodfor1mLofsalivasample.

allowedtoperformordinarydailyroutine.Thegreatdiversityofcompoundswereobservedintheafternoon.However,thepeaksof2,3-butadienonewerehigherinthemorningandevening.Possibleoriginofthesefluctuationsmaybemealsandinterferencesofenvironment. Nineteenvolatileorganiccompoundswere identified inaltogethersamplesfromhealthynon-smokingsubjects.Theclassesofvolatileorganiccompoundswere: three alcohols and phenols, three volatile sulphur compounds, twoaldehydes,twoketones,twoesters,twoacids,twovolatilenitrogencompounds,onehydrocarbon,oneether,andanoxide(Fig.4(B)).2,3-butadienonewasthemost frequently observed substance found in the samples. Ethyl acetate wasdetectedinsevensamples.Propanalanddimethylsulphidewerethethirdmostseencompoundsinvolatileorganiccompoundprofiles.Fig.5demonstratesthatinchromatogramsobtainedfromactivesmokers(M8andF3),onevolatilefromcigarettesmokewasdetectedanditwaspyridine.


Fig. 3SPME-GC/MSchromatogramofsalivasamplefromsinglefemalesubject.

Fig. 4 (A)daily variations of volatile organic compounds from single subject. (B) frequency ofoccurrenceofvolatileorganiccompoundscalculatedforelevenhealthysubjects.

4.Conclusions

Applicationof75µmCarboxen/PDMSfibreallowedtheextractionofthegreatest

numberofcompoundswith thehighestabundances, incontrast to theresults

from100µmPDMSand65µmPDMS/DVBfibres.Moreover,theproposedSPME-

GC/MSmethodallowstoobtainreproduciblesalivaryvolatileorganiccompound

profilesthatcanbeusedfordifferentiationofindividuals.Prolongationoftimeof

incubation can influence the composition of salivary headspace by increasing

ofabundances of volatiles.Comparison of volatile organic compound profiles

fromelevenhealthysubjectsrevealedsignificantdifferencesintheirsalivarycom-

positionsaswellasthediversityofdistributionofcertainvolatiles.Pyridinewasfound in chromatograms of two participants in this study that were active

smokers.Thepossibleoriginofthiscompoundistobaccosmoke.Dailyvariations

insalivaryprofileswereobserved.Thenumberofcompoundsandtheirdifferent

abundancescanbeassociatedwithmealseatenbeforethecollectionofsaliva.

References[1] deLacyCostelloB.,AmannA.,Al-KatebH.,FlynnC.,FilipiakW.,KhalidT.,OsborneD,RatcliffeN.

M.:Areviewofthevolatilesfromthehealthyhumanbody.J.BreathRes.8(2014),014001.[2] PfaffeT.,Cooper-WhiteJ.,BeyerleinP.,KostnerK.,PunyadeeraC.:Diagnosticpotentialofsaliva:

Currentstateandfutureapplications.Clin.Chem.57(2011),675–687.[3] delN.SanchezM.,GarcıaE.H.,PavonJ.L.,CorderoB.M.:Fastanalyticalmethodologybasedon

mass spectrometry for the determination of volatile biomarkers in saliva.Anal. Chem.84(2012),379–385.

[4] Francavilla R., Ercolini D., PiccoloM., Vannini L., Siragusa S., De Filippis F., De Pasquale I.,DiCagno R., Di Toma M., Gozzi G., Serrazanetti D. I., De Angelis M., Gobbetti M.: Salivarymicrobiotaandmetabolomeassociatedwithceliacdisease.Appl.Environ.Microbiol.80(2014),3416–3425.

[5] SoiniH.A.,KlouckovaI.,WieslerD.,OberzaucherE.,GrammerK.,DixonS.J.,XuY.,BreretonR.G.,PennD.J.,NovotnyM.V.:Analysisofvolatileorganiccompoundsinhumansalivabyastaticsorptive extractionmethod and gas chromatography-mass spectrometry. J. Chem. Ecol.36(2010),1035–1042.


Fig. 5Distributionofvolatileorganiccompoundsacrossthesalivaryprofilesfromelevenindividuals,includingtwosmokers(M8andF3).

1.Introduction

Siloxanesareusedinabroadvarietyfordifferentapplicationareas.Ingeneral,siloxanesconsistofalternatingsilicone-oxygenbondsinthebackboneanddiffe-renttypesoffunctionalgroups.Animportantclassofsiloxanesispoly(dimethyl-siloxane)containingonlymethylandmethylenegroupsboundedtothepolymerbackbone.Thebasicnotationofpoly(dimethylsiloxane)dependsonthenominalnumber of oxygen bonded to silicon: the basic building blocksM,D, T andQpresent one, two, three or four oxygen(s) bonded to silicone, respectively.Therefore, themolecular architecture is clearly defined by the nomenclature,e.g.D4standsforthecyclictetramer[1–4]. Theuniquecharacteristicsofsiloxanes,likehighflexibilityintheirbackbone,lowintermolecularforcesbetweenmethylgroupsorlowsurfaceenergiesmakeapplications in cosmetics,medicine aswell as in construction industries veryattractive. Especially in case of poly(dimethylsiloxane), the usage in releaseagents,antifoams,heattransferliquidsorcoatingsdemonstratetheimportanceof

Separation of linear and cyclic poly(dimethylsiloxanes) with interactive chromatography

a,b, a bBERNHARDDURNER *,THOMASEHMANN ,FRANK-MICHAELMATYSIK

a WackerChemieAG, Johannes-Hess-Straße24,84489,Burghausen,Germany*[email protected] DepartmentofChemistryandPharmacy,UniversityofRegensburg, Universitätsstraße31,93040,Regensburg,Germany


AbstractDuetotheirattractiveproperties,siloxaneshavefoundmanyappli-cationsinvariousindustrialareas,e.g.cosmetics,healthcareorcon-structionindustriesareinrecentyears.Therefore,amethodforsepa-ration of linear and cyclic poly(dimethylsiloxane), applying liquidchromatographictechniqueswasdevelopedandoptimized.Byinter-activechromatography,oligomerresolutionandseparationoflinearfrom cyclic poly(dimethylsiloxane) could be achieved for poly(di-methylsiloxane)withupto30monomericunits.Resultsofinvestiga-tions of the underlying separation mechanism pointed out thatacombinationof fractionated-re-dissolutionandadsorptioneffectsprimarilydependingontheadequatechoiceoftheeluentsystemwasessential.

Keywordsinteractivechromato-

graphylinearandcyclicpoly(di-

methylsiloxane)precipitation-re-disso-

lutionmechanismpolymerHPLC

thistypeofpolymer[5,6].Concerningapplicationsinpharmaceuticalsormedicalcare products, comprehensive analytical methods are necessary. Therefore,investigationsoflowmolecularweightoligomer,linearandcyclicpoly(dimethyl-siloxane)aremainlydonewithgaschromatography[7,8].Moreover,linearandcyclicpoly(dimethylsiloxane)canalsobeseparatedwithliquidchromatographyatcriticalconditions,wheretheseparationonlydependsonchemicalfunctiona-lities[9].Amajordrawbackofliquidchromatographyatcriticalconditionsisthehighsusceptibility tosmall changes inanalytical conditions,e.g.mobilephasecomposition,temperaturechangesorsmallvariationsoftheinvestigatedpolymersample[10].Apartfromliquidchromatographyatcriticalconditions,interactivechromatography,focusingondifferencesinthechemicalstructureofmacromole-cules,isanappropriatealternative.ComparedtoconventionalHPLC,peculiaritieslikesmalldiffusioncoefficientsinsolution,reducedsolubilityoramorecomplexretentionmechanismon the stationaryphase, occur.Thus, polymer elution iscontrolledbydifferenttypesofinteractionsofvariousseparationmechanisms,caused by adsorption, partition or solubility effects. Consequently, optimizingvariousparametersinmethoddevelopment,e.g.choiceofmobileandstationaryphase,LCflowrate,temperature,arenecessaryforexplainingthemainseparationmechanism[11–13].Thepresentcontributionisconcernedwithcorrespondingmethoddevelopments.

2.Experimental


AllsolventsusedwereHPLCgrade.Acetonitrile,acetone,methanol,ethanol,iso-propanol, and non-stabilized tetrahydrofuran were purchased from Merck(Darmstadt,Germany)andusedwithoutfurtherpurification.WaterofaMilli-Q-Advantage A10water system (MerckMillipore)was used. All used analyticalstationaryphasesappliedinthisstudyaresummarizedinTable1.ForfractioncollectionofsinglelinearandcyclicoligomersaThermo-Fisher(Waltham,USA)Accucore C30 (150×4.6 mm, 2.6 µm) was used. The used linear and cyclicpoly(dimethylsiloxane) samples were obtained from Wacker Chemie AG(Burghausen,Germany).Asreferencematerialforlinearpoly(dimethylsiloxane)asiliconeoilwithaviscosityof10mPasandforcyclicpoly(dimethylsiloxane)amixtureofD8–D17wasused.

2.2Instrumentation

Theinvestigationswereperformedona1100seriesLCsystemofAgilent(Wald-bronn, Germany) with a tetrahydrofuran-resistant 3215α degasser from ERC(Riemerling,Germany)anda385ELSDofAgilentequippedwithanenhanced

®parallel-path MiraMist poly(tetrafluoroethylene) nebulizer from Burgener


Research(Mississauga,Ontario,Canada)at40°Cevaporatortemperature,90°Cnebulizertemperatureand1.2SLM(standardliterperminute)gasflow.Alltestmeasurementswere donewith a linear gradient from 100%A to 100%B in40min,unlessotherwisestated.Changingcolumndimensions,thegradientpara-meters were adapted to obtain the same effective linear gradient. The finalmethoddevelopmentwasdoneonanAccucoreC30(50×4.6mm,2.6µm)ataLC

–1flowrateof2mL∙min startingat(methanol:water(75:25,v/v)):acetone50:50and ending at 100% acetone in 160min. Applying silica beads the stepwisegradientwasperformedwith5%stepheight,5minsteplengthwithwaterandacetoneaseluentsystem.


3.1Optimizationofstationaryphase

Accordingtocommonliteratureforpoly(dimethylsiloxane)separation[9]withRP-Polymer-HPLC,acetonitrileasadsorptionpromotingsolventandtetrahydro-furanasdesorptionpromotingsolventwerechoseninpreliminaryexperiments.Thus,aC8stationaryphasewasselectedseparatinglinearandcyclicpoly(dimet-hylsiloxane),Fig.1.Theseparationperformanceofthissystemislimitedbyrepea-


Number Manufacturer Name Particletype Dimensions/mm

1 Thermo-Fisher AccucoreC18 2.6µm,80Å 100×4.6 2 Thermo-Fisher AccucoreC8 2.6µm,80Å 100×4.6 3 Thermo-Fisher AccucoreC30 2.6µm,150Å 50×4.6 4 Phenomenex KinetexPFP 2.6µm,100Å 100×4.6 5 YMC CarotenoidC30 3µm,80Å 100×4.6 6 Thermo-Fisher AccucoreC18aQ 2.6µm 100×4.6 7 Agilent EclipseC18 5µm,80Å 150×4.6 8 Phenomenex EVOC18 2.6µm,100Å 100×4.6 9 MicroSolvTechnology CogentBidentateC18 4.2µm,100Å 150×4.6 10 Macherey-Nagel Nucleosil100C18 5µm,100Å 125×4 11 Macherey-Nagel NucleodurPyramidC18 5µm,110Å 150×4.6 12 Thermo-Fisher HypersilBDSC18 2.4µm,120Å 100×4.6 13 Phenomenex HyperCloneBDSC18 5µm,130Å 150×4.6 14 Thermo-Fisher HyPurityC18 5µm,190Å 150×4.6 15 Macherey-Nagel NucleosilC18EC 5µm,50Å 100×4.6 16 Macherey-Nagel NucleosilC18EC 5µm,100Å 100×4.6 17 Macherey-Nagel NucleosilC18EC 5µm,300Å 150×4.6 18 Macherey-Nagel NucleosilC18EC 7µm,1000Å 150×4.6 19 Self-prepared Silicabeads 75µm 50×7.0

Table 1Overviewofinvestigatedstationaryphasesfortheseparationofpoly(dimethylsiloxane);columnswerepurchasedbyAgilent(Waldbronn,Germany),Macherey-Nagel(Düren,Germany),MicroSolvTechnologyCorporation(Leland,USA),Thermo-Fisher(Waltham,USA),Phenomenex(Aschaffen-burg,Germany),andYMC(Dinslaken,Germany).

Fig. 1Separationoflinearandcyclicpoly(dimethylsiloxane)with(a)acetonitrile/tetrahydrofuranonanAccucoreC8column(100×4.6mm,2.6µm)andwith(b)methanol:water(75:25)/acetoneona Kinetex pentafluorophenyl column (100×4.6 mm, 2.6 µm); cyclic poly(dimethylsiloxane) isannotatedasDplusmonomoricnumber,andlinearpoly(dimethylsiloxane)isannotatedasSiplusmonomericnumber.

ted peak overlap of linear and cyclic siloxanes. Following a classical HPLCapproach,different stationaryphases (Table1)were tested for improving theseparationperformance.Withapentafluorophenyl (PFP) columnan improve-mentoftheseparationcouldbeachievedbyreplacingtheadsorptionpromotingsolventfromacetonitriletoanadequatemixtureofmethanol:water(75:25)–thetriple bond of acetonitrile prevents the interaction of analyte and stationaryphase.Finally,thedeterminationcouldconsiderablybeimprovedwhenusinganAccucoreC30stationaryphaseincombinationwiththeeluentsystemmethanol:water/acetone(Fig.2).


Fig. 2Optimized separation of poly(dimethylsiloxane) applying an Accucore C30 (50×4.6 mm,–12.6µm)aLCflowrateof2.0mLmin ,methanol:water(75:25)asadsorptionpromotingsolventand

acetoneasdesorptionpromoting solvent, the chromatogram indetailhighlights theoligomericseparationoflinearandcyclicoligomersupto30repetitionunits.

3.2Optimizationofmobilephasecomposition

Using acetonitrile, methanol or water as adsorption promoting solvent andacetone,ethanol, isopropanolortetrahydrofuranasdesorptionpromotingsol-vent and mixtures thereof, allowed the investigation of various solventcombinationswhileoptimizingthestationaryphaseforseparatingpoly(dimeth-ylsiloxane).Thechoiceofanappropriatemobilephasecompositioninterferedwithseparation improvement in termsofpolymersolubility instationaryandmobile phase. Consequently, the originally used eluent system for the penta-fluorophenylcolumnconsiderablyimprovedtheanalysismethodonothermorerobuststationaryphases,too,e.g.AccucoreC30(Fig.2).Thisparticularcombi-nationofstationaryandmobilephasesenabledanextendedseparationrange,mainlycausedbyprecipitation-re-dissolutionandadsorptionofthepolymeratthecolumn.

3.3Explanationofseparationmechanism

According to the aforementioned findings, amore detailed description of thepredominant separation mechanism was possible. Particularly, when

–1investigatinglowmolecularweightpoly(dimethylsiloxane)(upto3000gmol )liquid adsorption chromatography is theprominent separationmodebecauseseparationefficiencywashighlydependingontheappliedstationaryphase.Apartfromthis,thesignificanceofwell-definedmobilephasecompositionsuggestedthatanadsorptionmechanismissuperimposedbyamechanismofprecipitation


Fig. 3Separationofasiliconeoilwithaviscosityof10mPas,containingonlylinearpoly(dimethyl-siloxane)oligomers,whenapplyingastepwisegradient(steplength5minandstepheight5%)usingwaterandacetoneasmobilephaseonasilicabeads(75µm)columnexcludingHPLCadsor-ptioneffectsofthestationaryphase.

and re-dissolving. Furthermeasurementswere performedwith a silica beadscolumn(Fig.3),whichshowednousefulHPLCseparationduetoabsenceofstatio-nary phase modifications. Applying a stepwise gradient, the poly(dimethyl-siloxane)(viscosityof10mPas)polymericdistributionwasmeasuredprimarilyto fraction re-dissolutionmechanism. Fractionated re-dissolving elution over-laying HPLC adsorption effects indicated the significance of mobile phasecomposition.Withidealsettings,resolvingvariousmolecularweightoligomersbecomepossible.

4.Conclusion

Aninteractivechromatographymethodwasdevelopedforseparatinglinearandcyclic poly(dimethylsiloxane) up to 30 repetition units (D30). Therefore, themobilephasecompositionandselectionof stationaryphaseswereoptimized.ApplyingpreparativeHPLC,puresingleoligomericstandardsoflinearandcyclicpoly(dimethylsiloxane)canbeobtainedfor improvingquantitativeanalyses ininteractive chromatography as well as in gas chromatography. Based on thecombinationofprecipitation-re-dissolutionandadsorptionmechanismsvariousothertypesofpolymersmightbeanalyzedwithinteractivechromatography.

Acknowledgments

TheauthorsthankthegroupofProcessChemistryPolymerandFluidsoftheBusinessUnitBasicsandIntermediatesatWackerChemieAGBurghausenforsupportwithsiliconeoils.


References

[1] FendingerN.J.,LehmannR.G.:Polydimethylsiloxanes.In:OrganosiliconMaterials.G.Chandra(ed.).Berlin,Springer1997.

[2] Liu,Y.:Siliconedispersions.In:SurfactantScienceSeries,Vol.159.London,CRCPress2017.[3] NollW.:ChemieundTechnologiederSilicone.2nded.Weinheim,Chemie1968.[4] KoernerG.,Schulze,M.,WeisJ.:Silicone.ChemieundTechnologie.Symposiumam28.April1989.

Essen,Vulkan-Verlag1989.[5] LambrechtJ.,BrunnigM.:AdvantagesofsiliconesandfuturechallengesintheworldofT&D.In:

SiliconeElastomers.Berlin,SmithersRapraTechnology2012.[6] SiliconeElastomersShawbury,SmithersRapraTechnology2013.[7] BletsouA.A.,AsimakopoulosA.G.,StasinakisA.S.,ThomaidisN.S.,KannanK.:Massloadingand

fate of linear and cyclic siloxanes in awastewater treatment plant inGreece.Environ. Sci.Technol.47(2013),1824–1832.

[8] BrothersH.M.,BoehmerT.,CampbellR.A.,DornS.,KerbleskiJ.J.,LewisS.,MundC.,PeroD.,SaitoK.,WieserM.,ZollerW.:Determinationofcyclicvolatilemethylsiloxanesinpersonalcareproductsbygaschromatography.Int.J.CosmeticSci.39(2017),580–588.

[9] MackoT.,HunkelerD.:Liquidchromatographyundercriticalandlimitingconditions:Asurveyof experimental systems for synthetic polymers. In: Liquid Chromatography / FTIRMicrospectroscopy / Microwave Assisted Synthesis. A. Abe, A.C. Albertsson (edits.). Berlin,Springer2003.

[10] BerekD.:Criticalassessmentof“critical”liquidchromatographyofblockcopolymers.J.Sep.Sci.39(2016),93–101.

[11] GlocknerG.:GradientHPLCofCopolymersandChromatographicCross-Fractionation.Berlin,Springer1991.

[12] PaschH.,TrathniggB.:HPLCofPolymers.SpringerLaboratory.Berlin,Springer1998.[13] BerekD.:PolymerHPLC.In:HandbookofHPLC.D.Corradini(edit.),2nded.,BocaRaton,Taylor

&Francis2010,p.447–504.


1.IntroductionOverthelastfewdecadesvariouscalibrationmethodswereproposedwiththeaim of obtaining the result that is accurate, meaning that it reflects the trueamountoftheanalyteinthesample.Unfortunately,mostofthosemethodsisnotused in laboratorypractice and the calibration curvemethod is still themostpopularone.However,whenthismethodisemployed,thefinalresultisaffectedbyaserioussystematicerrorsincethecompositionofthesample’smatrixisnottaken into considerations. Moreover the interference effects might cause thecalibrationrelationshiptobechanged.Thestandardadditionmethodisalsowell-knowncalibrationapproachandmightbeusedforeliminationoftheinterferenceeffectwhen the change of the analytical signal is proportional to the analyteconcentration(itiscausedbecauseallcalibrationsolutionscontaintheanalyteinthe environment of all sample components). The standard addition method,however,doesn'talloweliminationoftheadditiveinterferenceeffect,whichcauseaconstantchangeofameasuredsignalregardlessoftheanalyteconcentration.Theotherdrawbackisthatthecalibrationcurvehastobeconstructedforeach

Simple Calibration Approach to Elimination of the Additive Interference Effect

MAREKDĘBOSZ*,MARCINWIECZOREK,PAWEŁKOSCIELNIAK

DepartmentofAnalyticalChemistry,FacultyofChemistry,JagiellonianUniversityinKrakow,Gronostajowa2,30-387,Krakow,Poland*[email protected]

AbstractThedeterminationofanalytesusuallyrequiresthecalibrationpro-ceduretobecarriedout.Todothisthecalibrationcurvemethodiscommonlymetinlaboratorypractice.However,itsuseisfacedwithseriousproblems.Forinstance,thefinalresultmightbeaffectedbyserioussystematicerrorinthecasewhentheinterferenceeffectsarepresent.Inthisworkitisshownhowthecalibrationcurvemethodcanbemodifiedinordertoeliminatetheadditiveinterferenceeffect.Theconceptisbasedonthemeasurementsofboththestandardsolutionsandthesamplesattwovariouswavelengthsselectedsotokeepthesignalsmeasuredforaninterferentconstant.Astheresult,theanalyteinasampleisdeterminedwiththeuseoftwocalibrationcurvesmoreaccuratelythanintheconventionalway.IncontrasttothealternativeH-pointstandardadditionmethodtheproposedapproachallowsthecalibrationcurvestobeusedforanalysisofaseriesofsamples.Themethod was verified on the example of the spectrophotometricdeterminationofFe(II)inthepresenceofFe(III)astheinterferent.

Keywordsanalyticalcalibrationflowanalysisironinterferenceeffectsspectrophotometry


analysedsampleseparatelymakingthecalibrationprocedurelaboriousandtime-consuming[1–2]. Theoneofthecalibrationmethodwhichisnotpopularinlaboratorypractice,but compensates both the multiplicative and additive interference effect isH-pointstandardadditionmethod[3].Thisapproachisbasedontheanalyticalmeasurements carried out according to the standard additionmethod at twodifferentwavelengths.Theconditionshastobeselectedtomeasuretheanalyticalsignal with different sensitivities but to keep the signals produced by theinterferentsconstant.UndersuchconditionscalibrationgraphsarecrossedattheH-point indicating both the additive effect and the analyte concentration.However,as for thecaseof thestandardadditionmethod, the requirementofpreparingseveralworkingsolutionsforeachsampleneedstobemet. Toovercomethisissueanewapproachispresentedinthiswork.Namely,itisproposedtoprepare twocalibrationcurvesaccording to thecalibrationcurvemethodattwowavelengths(selectedasabove)andtomeasurethesignalsforthesampleinthesameconditionsseparatelyfromthestandardsolutions.Whenbothcalibrationgraphsarelinear(R =A +B c ,i=1, 2)andtheadditiveeffectisconstanti i i x atbothwavelengths(A =A =A),theconcentrationc ofananalyteinasampleand1 2 xthevalueAoftheadditiveinterferenceeffectarecalculatedfromtheequations:

c =(R –R )/(B –B ) (1)x 1 1 22

A=(R B –R B )/(B –B ) (2)2 1 1 2 1 2

whereR andB (i=1,2)are thesignals for thesampleandtheslopesof thei i

calibrationgraphs,respectively. Thesuitabilityofthisapproachwasverifiedontheexampleofthespectro-photometricdeterminationofFe(II)inthepresenceofFe(III),whichplayedtheroleoftheinterferent(asin[4]).

2.Experimental

2.1Reagentsandsamples

Thereagents,allofwhichwereanalyticalgradechemicals,wereusedtopreparetheappropriatesolutions:phenantrolinemonohydrate(Lachner,CzechRepub-lic),salicylicacid(FabrykaOdczynnikowChemicznych,Gliwice,Poland),iron(III)nitrate nonahydrate (Sigma Aldrich, Germany), ammonium iron(II) sulphatehexahydrate (Chempur, Poland), 37% fuming hydrochloric acid (Merck, Ger-many)andpotassiumhydrogenphthalate(FabrykaOdczynnikowChemicznych,Gliwice,Poland).

–1 Stockironsolutionscontaining1000mgL Fe(II)andFe(III)werepreparedbywater dissolving of an adequate amount of Fe(NH ) (SO ) ·6H O and4 2 4 2 2

Fe(NO ) ·9H O, respectively. Stock solution of mixture of 1,10-phenantroline3 3 2


monohydrateandsalicylicacidwaspreparedbydissolving0.843gand0.575gofthesereagents,respectively,in10,0mLofethanolandadjustingthevolumeto100mL with distilled water. The use of ethanol was utilized to increase thesolubility of salicylic acid. Buffer solution (pH=3.0) was prepared by mixing

–3appropriatevolumeof0.2moldm solutionsofpotassiumhydrogenphthalateand hydrochloric acid [5]. All stock solutions were prepared fresh daily. Thesolutionswereprepared indistilledwater.Thedistilledwater fromanHLP5system(Hydrolab,Poland)wasusedthroughoutthework.

2.2Instrumentation

Theinstrumentalflow-injectionmanifolddedicatedtotheproposedcalibrationmethodispresentedinFig.1.Itconsistedofaneight-portinjectionvalveequippedwithahomemade,electricswitchingsystem,twoperistalticpumps(Minipuls3,Gilson,France)and16–channelcontrollerUVCTR-16(KSPElectronicsLabora-tory,Poland).Lambda25spectrometer(PerkinElmer,USA)equippedwithaglassflowcellwithpathlengthequal10mm,wasutilizedasthedetector.TheworkofpumpsandinjectionvalvewascontrolledbyValveandPumpControllerSoftware(KSPElectronicsLaboratory,Poland).

2.3.Procedure

Samplesorstandardsolutionswerepreparedbyadding1mLofbuffersolutionand7mLofsamplesorappropriatevolumeofstocksolutionsofFe(II)andFe(III)to10mLvolumetricflaskandmadeuptomarkwithdeionizedwater.Theconcen-

–1trationofFe(II)inthecalibrationsolutionswasintherangeof0–5mgL (with–11mgL step).Thesampleorstandardsolutionwasinjectedintoastreamofwater,


Fig. 1 Scheme of the constructed flow-injection manifold: (S) sample, (ST) standard solution,(P1)and(P2)peristalticpumps,(IV)injectionvalve(MC)mixingcoil,(W)waste,(r ,r ,r )flowrates.1 2 3

whichwasconnectedwithastreamofmixtureofphenantrolineandsalicylicacid,resulting in formationofanorangeorpurplederivativecomplexofFe(II)andFe(III),respectively.Theformedproductwasdirectedtowardsthedetectorwhereabsorbancewasrecordedat twoselectedsetwavelengths.Heightof recordedcharacteristicpeakswastreatedasananalyticalsignal.Eachdeterminationwasrepeatedthreetimesinthesameinstrumentalconditions.


Workingparametersoftheflow-injectionmanifold,suchas:flowrate,reactionloop lengthandthevolumeof injectedsample,wereoptimized.Thefollowing

–1parameterswerechosen:flowrates2.0mLmin ,thelengthofthereactioncoil

100cmand thevolumeof injected sample 200µL.The linearity rangeof the–1proposedmethodis0–25mgL forbothanalytes.

Basedonthespectraof1,10-phenantrolineandsalicylicacidcomplexeswithFe(II)andFe(III)(Fig.2)threepairsofwavelengthwerechoseninaccordancewithprincipleofthemethod:530and573nm,542and549nm,542and551nm. Table 1 presents the results of analysis of several synthetic samples withdifferentconcentrationsofFe(II)(analyte)andFe(III)(interferent).Theresultsobtained by the proposed method are compared with these obtainedconventionally,i.e.usingthecalibrationcurvemethod.Theresultsobtainedwiththecalibrationcurvemethodintermsoftherelativeerrorareunacceptable.ThisisduetothepresenceofFe(III)thatactsasaninterferent.Thecalibrationcurvemethodevidentlycannotdealwiththeinterferenceeffect.However,theuseofthe


–1 –1Fig. 2Absorptionspectraof(a)5mgL Fe(II),and(b)10mgL Fe(III)withthemixedreagentsatpH=3.

proposedmethodisabletotakeintoaccountsucheffectand,asitcanbeseeninTable1,theresultsarequitesatisfactory.TheexceptionaretheresultsobtainedforFe(II)insampleIattwopairsofwavelengths.ItcanbecausedbythefactthatselectedconcentrationofFe(II)isnearbythelimitofdetectionandtheinfluenceofinterferentisespeciallystronginthiscase.Inaddition,thevaluesoftheadditiveeffect caused by Fe(III) were calculated. The validity of these parameters isevidentwhencomparedwiththeincreasingvalueofinterferent’sconcentration–thehighertheconcentrationthehighertheadditiveeffect.

4.Conclusions

Thepresentedstudyshowsthatthepresentedcalibrationmethodisaneffectiveandhelpfulanalyticaltool.Itoffersdeterminationofananalytewithimprovedaccuracyduetoeliminationoftheadditiveinterferenceeffect.Furthermore, itallowsthevalueofthiseffecttobeestimated.AsthemethodisfasterandsimplerincomparisonwithH-pointstandardadditionmethod,itcanberecommendedtobeusedinroutineanalyticalpractice.

References

[1]K oscielniakP.,WieczorekM.,KozakJ.:Calibrationproblemsintraceanalysis.In:HandbookofTraceAnalysis.FundamentalsandApplications.I.Baranowska(Ed.).Springer2016.

[2]K oscielniak P., Wieczorek M.: Univariate analytical calibration methods and procedures:Areview.AnalyticaChimicaActa944(2016),14–28.

[3]B oschR.F.,CampinsF.P.:H-pointstandardadditionmethod:Part1fundamentalsandapplicationtoanalyticalspectroscopy.Analyst113(1988),1011–1016.

[4]S afaviA.,AbdollahiH.:ApplicationoftheH-pointstandardadditionmethodtothespeciationoftheFe(II)andFe(III)withchromogenicmixedreagents.Talanta54(2001),727–734.

[5]h ttp://www.ochemonline.com/Buffer_solutions(accessed5thMay2018)


–1Sample λ/nm Concentration/mgL Relativeerror/% A

Expected CCM PM CCM PM

Fe(II)/Fe(III) Fe(II) I 530/573 1.00/5.00 6.20 1.10 520.0 10.0 0.0498 542/549 3.19 1.13 219.0 13.0 0.0498 542/551 3.62 1.01 262.0 1.0 0.0529II 530/573 5.00/10.00 13.92 4.88 178.4 –2.4 0.0935 542/549 8.79 4.89 75.8 –2.2 0.0947 542/551 9.02 4.86 80.4 –2.6 0.0953III 530/573 5.00/20.00 22.36 4.58 347.2 –8.4 0.1861 542/549 12.30 4.66 146.0 –6.8 0.1910 542/551 12.71 4.71 154.2 –5.8 0.1895

Table 1Resultsobtainedbythecalibrationcurvemethod(CCM)andtheproposedmethod(PM)inthreesyntheticsamples(Aisadditiveeffectinabsorbance).

1.Introduction

Chromiumwithdifferentoxidationstatesexhibitswidelydifferentbehavioursintermsofpotential toxiceffectsonenvironmentalandbiological system.Chro-mium(VI) is toxicandknownas carcinogenicwhileCr(III) isa traceessentialelementfortheproperfunctioningoflivingorganism[1,2].Becausechromiumisused widely in industrial activates such as electrical plating, Cr(VI) is easilyreleasedintotheenvironment,especiallyinsurfacewaterandgroundwaterandcouldposeahealthrisk.Therefore,extractionandchemicaltreatmentofcontami-nated groundwater in order to remove Cr(VI) become an important issue.Chromium(VI)canbereduced into immobileCr(III) formwhich ismuch lesstoxic.ItwasreportedthatCr(VI)reductionisoftenassociatedanisotopicfractio-

53 52nationandisthedominantprocesscausingthechangeof Cr/ Crratio[3,4].Asaresultofwhich,CrstableisotopicratioscanbeservedasindicatorstoquantifytheextentofCr(VI)reductioninenvironmentalremediationefforts.

Separation of chromium(III) and chromium(VI) by reversed-phaseion-pairing chromatography

a, a aCUCTHINGUYEN-MARCINCZYK *,JAKUBKARASINSKI ,MARCINWOJCIECHOWSKI ,a b aAGNIESZKAANNAKRATA ,LUDWIKHALICZ ,EWABULSKA

a BiologicalandChemicalResearchCenter,FacultyofChemistry,UniversityofWarsaw, ŻwirkiIWigury101,02-089,Warsaw,Poland*[email protected] GeologicalSurveyofIsrael,30MalkheiIsraelStreet,Jerusalem95501,Israel


AbstractTheseparationofCr(III)andCr(VI)byreversed-phase ionpairingliquidchromatographyforaspecificpurposehasbeeninvestigated.Chromium(III)waschelatedwithEDTAatpH=6.0priortoanalysis.

–1Themobilephase(pH=6.0)consistedofa0.32mmolL EDTAand–10.83mmolL tetrabutylammoniumhydroxide.Themethodshowed

thatCr(III)andCr(VI)wereeffectivelyseparatedatlowmobilephaseflowratewhichissuitableforafutureexperimentdesignofisotopicratiomeasurements.Themostdifficulttaskwasnotonlytoensuretheeffectiveseparationbutalsotoavoidanypossibleinter-conversionbetweentwochromiumforms.Forabetterdetectionofchromiumspecies, the chromatographic system was coupled with a typicalquadrupoleinductivelycoupledplasmamassspectrometry.

KeywordschelationchromiumRPIPHPLCreduction

Accordingtoourbestknowledge,therehasnotbeenanypublishedarticleyetwhichstudiestheisotopicratioofeachchromiumformCr(III)andCr(VI)existingsimultaneously in an analytical sample. Therefore, the aim of this work is todevelopamethodtoseparatetwoformsofchromium:Cr(III)andCr(VI)byusingreversed-phase ion pairing liquid chromatography. The separation methodshouldbecapableforcouplingwithamulticollectorinductivelycoupledplasmamass spectrometry (MC ICP-MS) in a future experiment designwhich allowschromiumisotopicratiomeasurement.

2.Experimental


Reagentswere analytical grade chemicals andwereusedwithout any furtherpurification. Standards and other solutions including a mobile phase werepreparedwithdeionizedwater(18,2MΩcm)obtainedbyMilli-QSystem(Merck

–1Millipore, Germany). Standard solutions of 10mg L Cr(III) and Cr(VI)wereprepared by diluting the standard stock solution of Cr(NO ) and K CrO at3 3 2 4 –11000mgL (Merk,Germany).Themobilephasewasobtainedbydissolutionofanappropriate amount of ethylenediaminetetraacetic acid disodium dehydrate(EDTA, EDM Millipore, Germany) in deionized water and by an addition oftetrabutylammoniumhydroxide(40%inwater,Sigma-Aldrich,USA).Foradjust-ing pH sodium hydroxide (NaOH 50%, Fisher Scientific, USA) and acid nitric

®(HNO 65%Suprapur ,Merck,Germany)wereused.3

2.2Instrumentation

AnAgilent1200Infinityserieshigh-performanceliquidchromatography(AgilentTechnologies,USA)equippedwithapeltier-cooledautosamplertray,quaternarypump, a peltier-heated column oven, and an UV/ VIS detector with variablewavelengthwasused.Areversed-phaseAgilentZorbaxC8(2.1×150mm,1.8µm)wasutilizedastheseparationcolumn,itstemperaturewassetat35°C.Mobile

–1 –1phase in isocraticmode consisted of 0.32mmol L EDTA and 0.83mmol L tetrabutylammoniumhydroxide; itspHwasmaintained to6.0.Theseparationwascarriedoutwithintotalanalysistimeof16minwiththeflowrateofmobile

–1phase0.25mlmin andsampleinjectionvolume20µL.TheICP-MSdetectorwasamodelofElan6100DRC(Perkin-ElmerSCIEX,Canada).Theoperationalcondi-

–1tionsforICP-MSwere:RFpower1200W,nebulizergas(argon)flow0.92Lmin ,52 +lensvoltage15V.Monitoredionwas Cr .

2.3Procedure

WorkingstandardswithdifferentconcentrationscontainingCr(III)and/orCr(VI)werepreparedbymixinganappropriatevolumeofstandardssolutionsofCr(III)


–1andCr(VI)withEDTA0.05or0.1molL atpH=6.0(adjustedbyNaOH)inglassvials(volumeratioof1/1).Theobtainedvolumethenwasbroughtupto500µLbyaddingthemobilephase.Thesolutionswerekeptatanambienttemperaturefor1hour prior to analysis orwere incubated at 40°C/60°C in awater bath for15–60minutestoallowformationofCr(III)-EDTA. For the optimization of chromatographic conditions, several factorswhichaffecttheseparationsuchastheconcentrationofEDTAandtetrabutylammoniumhydroxide in the mobile phase as well as the pH (adjusted by HNO ) were3

investigatedtogetthebestseparation.


Inthiswork,reversed-phaseion-pairingchromatographywasusedforthesepa-2–rationofCr(III)andCrO .Firstly,EDTAwasusedtochelateandstabilizeCr(III)4– 2–intheformof[Cr-EDTA] ,whichisalsoananionicionasCrO .Theseanionicions4

interactedwithtetrabutylammoniumasthecationion-pairingreagent,whichinturninteractedwithreversedphaseC8column. Itwasreportedthattheinter-conversionbetweenchromiumspeciesoccursatacidic pH, and Cr(VI) is much more stable in neutral and basic pH [5]. TheCr(III)-EDTA chelation is pH dependent. A high pH favours the complexation,however, Cr(III) can be precipitated in a form of chromium hydroxide [6].Thereforeinthiswork,thechelationofCr(III)withEDTApriortoanalysiswascarried out at pH=6.0 which allows to keep Cr(VI) stable and avoid Cr(III)precipitation.Toexaminetheeffectoftemperatureonthechelationefficiency,thecomplexationwasperformedatambient roomtemperatureandat60 °C.Theresultsshowedthat,afterseveralhoursCr(III)-EDTAwasnot formedatroomtemperaturewhileanintensivevioletcolourwasobtainedwhenthesolutionwasheated at 60 °C for 30 min, which indicated the formation of Cr(III)-EDTA.Therefore,unlikeotherworks[7,8]heatingwasrequiredbecauseofCr(III)-EDTAcomplexationreactionkinetic.

–1 Themobilephase flowrateof0.25mlmin wasuseddue to theplanningutilizationofadesolvatingnebulizersystemdesignedfortheMCICP-MS.Thisvalueisalmostmaximumforthesampleflowrateatwhichthenebulizercanstilloperate. The future experiment design for isotopic ratio measurements bycouplingHPLCwithMCICP-MSwillnotdiscussedinthiswork.However,tobecapable forbeingcoupledwithMSICP-MS, theseparationbyusingreversed-phaseionpairingliquidchromatographyshouldfulfilsomecriteriasuchas:goodresolutionandacceptablelongretentiontimes.Thebestseparationwasobtained

–1 –1withmobilephase(pH=6.0)containing0.32mmolL EDTAand0.83mmolL tetrabutylammoniumhydroxide.AtypicalobtainedchromatogramisshowninFig.1. Themostdifficulttaskinthisworkwastoavoidinter-conversionbetweentwochromiumformsduringtheexperiment.AsaforementionedtheCr(VI)reduction


is often associated with an isotopic fractionation, hence the inter-conversionbetweentwoformsisnotbeneficialforthefurtherisotopicratioanalysis.TheoxidationofCr(III)toCr(VI)didnotoccurattheoptimalchromatographiccondi-tions.Unfortunately,Cr(III)-EDTApeakwasobservedinthechromatogramoftheCr(VI)standardsolutions.ThiscouldbecausedbythereductionofCr(VI).Otherinvestigationswere carried out in order to confirmwhether the reduction ofCr(VI)toCr(III)occurredbeforeorduringtheexperiments.TheeffectofEDTAconcentration, temperatureand time in the chelation stepwasexamined (seeprocedure). It was observed that after heating the Cr(VI) working standard

–1solutionscontainingequalchromiumconcentrationswithEDTA0.05molL or–10.1molL at40°Cor60°Cfor15–60min,inallcasesthesamepeakareasfor

Cr(VI)wereobtained.Interestingly,Cr(III)-EDTApeakwasalsoobservedwhenexperimentwasperformedwithoutanypre-treatment(Fig.2),andthesamepeakareawasalsoobtainedforCr(VI).TheseresultsimpliedthattheEDTAconcen-tration,temperatureandtimewerenotthereasonsfortheobservationofCr(III)inCr(VI)standardsolutions.TheeffectofthepHofmobilephasewasalsotested.The results showed that the more acidic the mobile phase was, the biggerCr(III)-EDTApeakareawasobtained.However,forpHbiggerthan5.5(6.0,6.5and7.0) Cr(III)-EDTApeak areaswere the same. Calibration curves (Fig. 3) show

2avery good regression coefficient (R >0.99) for both Cr(VI) and Cr(III). OnepossibilityexplainingthisphenomenacouldbethatCr(III)alreadyexistsinthe


Fig. 1TypicalchromatogramobtaniedfortheseparationofCr(III)-EDTAandCr(VI).Concentration–1 –1of chromium was 500 µg L . Mobile phase (pH = 6.0) contained 0.32 mmol L EDTA and

–10.83mmolL tetrabutylammoniumhydroxide.DetectorUV/VIS,detectionwavelength540nm(continuousline)and370nm(dottedline).


52Fig. 2Chromatogramof Cr(VI)standardsolutionobtainedwhennosamplepre-treatmentwas–1 –1carriedout.ConcentrationofCr(VI)was100µgL .Mobilephase(pH=6.0)contained0.32mmolL

–1EDTAand0.83mmolL tetrabutylammoniumhydroxide.DetectorICP-MS.

Fig. 3CalibrationcurvesofchromiumstandardsolutionsobtainedbyusingtheoptimizedHPLCprocedure:Cr(VI)=dottedline,Cr(III)-EDTA=continuousline.

–1initialstandardstocksolutionofchromate(1000mgL )duetothereduction(about25%).Thereductionmightoccurduringtransportationand/orstorage.

4.Conclusions

Inthiswork,reversed-phaseionpairingliquidchromatographywasshowntobeagoodtechniquewitheasysamplepreparation foreffectiveseparationof twodifferentchromiumformsCr(III)andCr(VI).Themobilephase(pH=6.0)con-

–1 –1taining0.32mmolL EDTAand0.83mmolL tetrabutylammoniumhydroxide–1andtheusedlowflowrate0.25mlmin ensuredthegoodseparationresolution

andacceptablelongretentiontimes,whicharerequiredforthefutureexperimentdesignofisotopicratiomeasurements.However,25%ofCr(VI)wasfoundtobereduced into Cr(III) evenwhen therewas no pre-treatment prior to analysiscarriedout.TheexperimentsshowedthatthereductionofCr(VI)mightoccurinthe initial standard stock solution due to an inappropriate transportation orstorage.However,furtherworkneedstobedoneinordertofindtheexactproblemcausingthistransformation.

Acknowledgments

This research is financial supported by the Nation Center of Science (NCN, Poland), project501-D312-66-0004878.AspecialthanktoJulioTorresforyourtechnicalsupport.

References

[1]GomezV.,CallaoMP.:Chromiumdeterminationandspeciationsince2000.TrendsAnal.Chem.25(2006),1006–1015.

[2]KotasJ.,StasickaZ.:Chromiumoccurrenceintheenvironmentandmethodsofitsspeciation.Environ.Pollut.107(2000),263–283.

[3]EllisAndreS.,JohnsonThomasM.,BullenThomasD.:UsingchromiumstableisotoperatiostoquantifyCr(VI)reduction:lackofsorptioneffects.Environ.Sci.Technol.38(2004),3604–3607.

[4]ColeD.B.,WangX.,QinL.,PlanavskyN.J.,ReinhardC.T.:Chromiumisotopes.In:EncyclopediaofGeochemistry.Springer2018.

[5]SunJ.,MaL.,YangZ.,WangL.:Optimizationofspeciesstabilityandinterconversionduringthecomplexingreactionforchromiumspeciationbyhigh-performanceliquidchromatographywithinductivelycoupledplasmamassspectrometry.J.Sep.Sci.37(2014),1944–1950.

[6]JenJ.F.,Ou-YangG.L.,ChenC.S.,YangS.M.:Simultaneousdeterminationofchromium(III)andchromium(VI)withreversed-phaseion-pairhigh-performanceliquidchromatography.Analyst118(1993),1281–1284.

[7]BarałkiewiczD., PikoszB., BelterM.,MarcinkowskaM.: Speciation analysis of chromium indrinkingwatersamplesbyion-pairreversed-phaseHPLC–ICP-MS:validationoftheanalyticalmethodandevaluationoftheuncertaintybudget.Accredit.Qual.Assur.18(2013),391–401.

[8]WolfR.E.,MorrisonJ.M.,GoldhaberM.B.:SimultaneousdeterminationofCr(III)andCr(VI)usingreversed-phased ion-pairing liquid chromatographywith dynamic reaction cell induc-tivelycoupledplasmamassspectrometry.J.Anal.At.Spectrom.22(2007),1051–1060.


1.Introduction

Seleniumisanessentialelementfortheproperfunctionofhumanandanimalorganisms.Seleniumisacomponentofmanyproteins,thuscontributingtobio-chemical processes. For many years, research has continued on the role ofselenium inbothphysiological andpathologicalprocessesof livingorganisms[1,2].Itissuggestedthatthebeneficialeffectsofseleniumonlivingorganismsismainlycausedbytheactivityofa lowmolecularcompoundSe-methyl-Seleno-cysteineandselenoproteins(containingseleniumintheformofselenocysteine),enzymesthatprotectthebodyfromoxidativestressbyreducingfreeradicals[3].Dietarysupplementationwithseleniumcompoundsmayleadtoanincreaseinthe

Label-free proteomic approach to identifi-cation and quantification of proteins in animal tissue samples

a, a aANDRZEJGAWOR *,ANNAKONOPKA ,JULIOCESARTORRESELGUERA ,a b aANNARUSZCZYNSKA ,MARIANCZAUDERNA ,EWABULSKA

a BiologicalandChemicalResearchCentre,FacultyofChemistry,UniversityofWarsaw, ZwirkiiWigury101,02-093Warsaw,Poland*[email protected] TheKielanowskiInstituteofAnimalPhysiologyandNutrition,PolishAcademyofSciences, Instytucka3,05-110Jabłonna,Poland


AbstractMassspectrometry–basedproteomicsisrecogniseasanusefulpro-cedureforlarge-scaleidentificationandquantificationofproteinsinnaturalsamples.Here,wepresentthelabel-freeproteomicapproachfor liver tissue samples obtained from lambs fed with the dietenrichedwithinorganiccompoundsofSe(VI)andorganicseleniumcompounds(selenomethionineinseleniumyeast).Thestudyaimedto examine how the presence of inorganic and organic forms ofseleniuminthelambs’forageaffectstheexpressionoftheproteins,inparticular, those containing selenium. In the course of the study,protein tissue homogenates were in-solution digested by trypsin.Peptideanalysiswasperformedusinghigh-resolutionelectrospraymassspectrometerequippedwithOrbitrapmassanalysercoupledtoultra-high performance liquid chromatography (nano-UHPLC-ESI-(ORBITRAP)-MS/MS).Basedonregistereddata,usingtheappropri-ate proteomic software (Mascot, MaxQuant) with access to theSwissProtproteindatabase,qualitativeandquantitativeanalysisofproteinswasachieved.

Keywordslabel-freeproteomicsmassspectrometryselenoproteins

concentrationoftheseenzymesandaffectstheexpressionoftheproteins.Identi-fyinga specificprotein in tissues, then comparing its concentrationsbetweenphysiologicalandpathologicalstatesoftissuescansupportthedetectionofthoseassociatedwiththespecificdiseaseandcontributetothedevelopmentofnewdrugsandtherapies. Thestudyaimedtoexaminehowthepresenceofinorganicandorganicformsofseleniuminthelambs’forageaffectstheexpressionoftheproteins,inparti-cular, those containing selenium. Here, we present the complete label-freeproteomics methodology for identification and quantification of the proteinsusingnano-UHPLC-ESI-(ORBITRAP)-MS/MSandtheappropriateproteomicsoft-ware(Mascot,MaxQuant)withaccesstotheSwissProtproteindatabase[4].

2.Experimental

2.1Examinedobjects

Thesubjectof thestudywas lambliver fromanimalsonanexperimentaldietsupplemented with selenium. Breeding of animals and tissue collection wascarriedoutbytheRegulationoftheCounciloftheEuropeanUnion(EC)No.1301099/2009of24September2009ontheprotectionofanimalsatthetimeoftheirkilling [5]. Thirty Corriedale male lambs of similar and age after a 3-weekstandarddietweredividedintocontrolandtwodietgroupsenrichedinseleniumcompounds(0.35mgofSe(VI)/1kgstandarddietand0.35mgofselenomethio-nine/1kgstandarddiet).After35daysofexperimentaldietaryenrichmentanddecapitationofanimals, thelivertissuewerecollected.Thesamplesoftissueswerefreeze-dried(below–40°C)andstoredat4°Cbeforeconductingpropersamplepreparation.


Analytical reagent grade chemicalswere purchased from SigmaAldrich (Ger-many), Baker (USA), Promega (USA) and Bio-Rad (USA). Solutions includingamobilephase forUHPLC-ESI-MS/MSwerepreparedwithLCMSgradewater(Baker,USA).

2.2Instrumentation

Theseparationofsupernatantsfromlivertissueresiduewasachievedusingthecentrifuge5804/5804R(Eppendorf,USA).Thevacuumcentrifuge(Eppendorf,USA)wasusedforconcentrationoftheextractssolution.Themeasurementswere

®carried out using the nano-UHPLC system equipped with Accucore column(75μm×500mm;C-18;2.6μm)purchasedfromThermoScientificcoupledtotheESI-MS/MSmass spectrometer (Orbitrap Fusion Tribrid™Mass Spectrometer,DionexUltimateSeriesUHPLC,ThermoScientific,USA).


Fig. 1Schemeoftheanalyticalprocedure.

2.3Procedure

Around100mgoftissueswereextractedwith1mLoflysisbuffer(1%SDS,0.05%–1lipase, 1 mmolL phenylmethylsulfonyl) in room temperature over 6 hours

supportedbymixingandultrasonicbaths.Thesupernatantwasseparatedfromtheresiduebycentrifugationfor30minat20000×g.TotalproteinconcertationwasdeterminedusingtheBradfordmethod[6].Allextractswerekeptat−80°Cbefore theproteinsdigestion.The5µLofproteinextractswerereducedwith

–175µLof5mmolL 1,4-dithiothreitolfor45minat56 °C,thenalkylatedwith75µL–1of55mmolL iodoacetamidefor30mininthedark.Thesamplesweresubjected

–1toenzymaticdigestionfor18hoursat37 °Cwith75µLof20ngL trypsinand –1100µLof50mmolL ammoniumbicarbonate.Thereactionwasquenchedwith

theadditionof150µL5%formicacid.Thesolutionscontainingtheelutedpepti-des were purified by the Zip Tip C18 clean-up procedure (EMD Millipore,

Germany)andconcentratedinavacuumcentrifugeat20 °Candresuspendedin0.1%formicacid.Thesampleswereinjected(1µL)intoUHPLCsysteminarever-

–1sephaseataflowrateof300nLmin usinggradientelutionconsistedof0.1%formicacidinwater(solventA)and0.1%formicacidinacetonitrile(solventB)for360min.SchemeoftheprocedureispresentedinFig.1.


Figure2AshowsanexampleofTotalIonCurrent(TIC)chromatogramfromthegroupsupplementedinseleniumorganiccompounds.RegisteredMS/MSspectraofpeptideswereanalysedtoidentifyselenium-containingproteinsusingMascotsoftware(MatrixScience,USA).Mascotisapowerfulsearchenginewhichusesmassspectrometrydatatoidentifyproteinsfromprimarysequencedatabases.The programsearchesall possiblepeptides from the theoreticaldigestionof


Fig. 2 (A)TIC chromatogram forpeptide extracts from lambs supplemented inorganic formofselenium.(B)EvaluationofMS/MSspectrabyMascotforpeptideLKQPAMPR.

availableproteinsequencesinthedatabaseandthen,indicatesthose,whichmassandfragmentationspectracorrespondtotheresultofexperimentalMSanalysis.Search parameters used during research were followed: SwissProt database,mammalian taxonomy, fixed modification: cysteine carbamidomethylation,variablemodifications:Se-cysteine,Se-methionineandoxidationofSe-cysteine,Se-methionine and carbamidomethylation of Se-cysteine. About 30 proteinscontaining selenium in the form of selenomethionine or selenocysteine withvarious modifications were found in the studied liver lambs’ samples (scoreparameterabove23).InordertoconfirmtheproteinidentificationproposedbytheMascot,theregisteredisotopicpatternfortheidentifiedselenium-containingpeptidewascomparedmanually to the theoreticalone.Manualverificationofexperimentalisotopicpatternallowedtheconfirmationofthepresenceofsele-niuminonlyoneofthem:dipeptidylpeptidase.Figure2BshowstheevaluationoftheMS/MS spectrum for the peptide LKQPAMPRwith Se-methioninemodifi-cation fromdipeptidylpeptidaseprotein.Thedifferences inexperimentalandtheoretical isotopicpatternsobservedforothersmayberelatedtoavery lowconcentration of those selenium-containing proteins in the examined liversamples,whichhavebeenalreadyreportedbyothers[7,8]. Quantitativeanalysiswasperformedmanuallybycomparingtheintensitiesofagiven unique peptide, which correlates with a given protein, on the TIC


Fig. 3Volcanoplotchartshowingdifferingintheproteinsexpressionbetweenthecontrolgroupandgroupswithseleniumsupplementation.

chromatograms usingMaxQuant. For determining the variations between theproteomeexpressedinvariousconditions,itisnecessarynotonlytoidentifytheproteinspresentbutalsotoperformstatisticalteststodetermineiftheobservedexpressionchangesarestatisticallysignificant (PrincipleComponentAnalysis,PCA).Perseuswasusedforstatisticalanalysisandvisualisationofthereceiveddata.Fig.3showsthequantitativeanalysisrevealedthattheseleniumsupplemen-tationoflambs’diet,bothinorganicandinorganicforms,changestheexpressionofsomeproteins(comparisonsofgroupsAandBvsgroup0). Incontrast,noexpressionchangewasobservedwhentwogroupswithseleniumsupplemen-tationwerecomparedtoeachother(comparisonofgroupAvsgroupB).Itcanbeconcludedthatwhereasseleniumpresenceitselfinthediethassomeinfluenceontheproteinexpressioninthelivercellsoflambs,theseleniumform(organicorinorganic)isnotsocrucial.

4.Conclusions

Theappliedanalyticalprocedureallowsautomaticidentificationofthousandsofproteins.Nearly30proteinscontainingseleniumintheformofselenomethionineor selenocysteine with variousmodifications were found in the studied liverlambs'samples(scoreparameterabove23).Manualverificationofexperimentalisotopicpatternallowedtheconfirmationofthepresenceofseleniuminonlyoneof them. Quantitative analysis revealed that the selenium supplementation oflambs'diet,bothinorganicandinorganicforms,changestheexpressionofsomeproteins,butnoexpressionchangewasobservedwhentwogroupswithseleniumsupplementationwerecomparedtoeachother.Furtherstudiesofproteinexpre-ssioninothertissuesoflambs(muscles,brain,serum,etc.)areinprogress.


Acknowledgments

ThestudywascarriedoutattheBiologicalandChemicalResearchCentre,UniversityofWarsawestablished within the project co-financed by European Union from the European RegionalDevelopmentFundundertheOperationalProgrammeInnovativeEconomy2007–2013.

References

[1] NavarroA.,Lopez-MartinezM.C.:Essentialityofseleniuminthehumanbody:relationshipwithdifferentdiseases.Sci.TotalEnviron.249(2000),347–371.

[2] WierzbickaA.,BulskaE.,PyrzynskaK.,WysockaI.,ZacharaB.A:Selen.Pierwiastekważnydlazdrowia,fascynującydlabadacza.Warszawa,WydawnictwoMalamut2007.(InPolish.)

[3]Ruszczynska A., Konopka A., Kurek E., Torres Elguera J C., Bulska E.: Investigation ofbiotransformationofseleniuminplantsusingspectrometricmethods.Spectrochim.ActaB130(2017),7–16.

[4]CoxJ.,MannM.:MaxQuantenableshighpeptideidentificationrates,individualizedp.p.b.-rangemass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26 (2008),1367–1372.

[5]RuszczynskaA.,BulskaE.,CzaudernaM.,Krajewska-BieniasL.:Dietaryselenium,carnosicacidandfishoilaffecttheconcentrationsofselectedelements,fattyacids,tocopherols,cholesterolandoxidativestress in the liverandselectedmusclesof lambs. Ital. J.Anim.Sci.21 (2017),285–301.

[6]BradfordM.:Rapidandsensitivemethodforthequantitationofmicrogramquantitiesofproteinutilizingtheprincipleofprotein-dyebinding.Anal.Biochem.72(1976),248–254.

[7] GuillaumeB.,LisaE.,EricL,ClayW.:Detectionandcharacterizationofselenoproteinsbytandemmassspectrometryassistedbylaserablationinductivelycoupledplasmamassspectrometry:applicationtohumanplasmaselenoproteins.J.Anal.At.Spectrom.26(2011),383–394.

[8]GammelgaardB.:Complementaryuseofmolecularandelement-specificmassspectrometryforidentificationofseleniumcompoundsrelatedtohumanseleniummetabolism.Anal.Bioanal.Chem.390(2008),1691–1706.


1.Introduction

Modernanalytic trends are focusedon theminiaturization, cost-effectiveness,andportabilityofthecreatedsystemsormethodologies[1].Theabilitytoperformqualitativeandquantitativeanalysisinsourcelimitedconditionsorinthefieldisoneofthesignificantfactorsthatcausedthegrowthoftheideaofquickpaper-madetests.Microfluidicpaper-basedanalyticaldevices(µPADs),wereintroducedasfullyutilizeplatformscapableofperformingcompleteanalyticalprocedures,providingresultscomparabletotheseoneobtainedusingstandardlaboratoryequipment. Minimal, user-friendly operation and versatility stay in excellentagreementtothechallengestheyforced,andforthisreason,paperbased-systemareintensivelyinvestigated. Withyears,simpletestsevolvetomorecomplicatedplanaroreventhree-di-mensionalsystems,wheresamplecouldproceedwithmanyindividualoperationsproviding the opportunity for simultaneous testing or analyte multistepderivatizationwithinoneµPAD[2].Developmentofthisdeviceswasconnectedwiththepossibilitytocoverthepapermatrixwithhydrophobicsubstances.Thepaperpatterningallowsforremotesamplesplittingormixingwithinonemicro-fluidicsystemwithoutuser intervention.Wax-printing technique isoneof the

Distance-based measurements using microfluidic paper-based analytical devices modified with Prussian blue

MATEUSZGRANICA*

FacultyofChemistry,UniversityofWarsaw,Pasteur1St.,02-093,Warsaw,Poland*[email protected]


AbstractInthecourseofthisresearchthedistance-basedmicrofluidicpaperanalyticaldevices(µPADs)wereinvestigated.Prussianbluewasusedasasensingsubstrate,depositedintheworkingzoneofthesystem.Asaresultofthecapillaryaction,thesampleintroducedonthedrypapersurface was transported along the channel, and the analyte wasreactingwithPrussianblue.Aseffectthediscolorizationofthesen-singzone,proportionaltotheanalyteconcentrationwasobserved.using created µPADs the calibration curves for ascorbic acid wasrecordedwithintheexperimentandoptimizationofthedyedeposi-tionwasprovided.

Keywordsdistance-baseddetectionmicrofluidicpaper

analyticaldevicesPrussianblue

mostusedforµPADpreparation[3]duetocomputerdesigning,simpleprepara-tionwithsolidinkprintersandgoodreproducibility. ThemostproblematicinamatterofµPADusefulnessisthesignaldetectionstep.Colorimetricschemesshowthegreatestpopularitybecauseofthefastsignalreadout and a plurality of available colorimetric procedures dedicated to thedeterminationofcommonanalytes.However,thebiggestconcernisstillfocusedoncolorintensityevaluationbecausethiskindofmeasurementsrequiresreliablephotosensitivedevices.Theunaidedeyecolorintensitydetection,whichistheunquestionablymostconvenienttoperforminthesource-limitedlocalizationisdisruptedwithuser individualattributesand impressions[4]and isnotreco-mmended inquantitativeanalysis.Tosolvethisproblemthedifferent idea forcolorimetricsignalquantificationwiththenakedeyewasproposedbyZuketal.[5].Thisideaappliedinflowpaper-basedsystemutilizetheeffectoftheanalyteconsumptionduringsampleflowthroughthesystem.Whentheflowchanneliscoveredwiththereagentinadvance,theanalytefromthesampleisreactingwiththereagentintherestrictedzone.Ifthereactionproductiscolored,acolorbandproportionaltotheanalyteconcentrationpresentinthesampleisgenerated.Thequantificationofthesignalisaccomplishedbysimplymeasuringthelength/sizeofthecoloredzone.Extremelyfacilereadoutofthesignalwiththeabsenceoftheelectronicequipmentisdescribedwithproperreproducibilityandaccuracy[6].This kind of detection has been already used in the aspect of environmentalpollutionanalysis[7]andpointofcaretesting[8]whereitsapplicationpotentialinrealscenarioswasconfirmed.

2.Experimental


All used solutions were prepared using deionized water (Mili-Q PurificationSystem)andanalyticalgradereagents.ThePrussianbluedepositionwascarriedfromthe1-Mhydrochloricacidsolutionsoftheiron(III)chlorideandpotassiumhexacyanoferrate(III) (Sigma-Aldrich).Theconcentrationsof theapplied solu-

–3tionswereexperimentallyadjusted.Thestock0.1moldm standardofascorbicacid was prepared daily form the pure reagents of the Ascorbic Acid (PoCH,Poland)tolimitthestoragetimeandavoidbreakdownoftheanalyte.Working

–3standardsoftheascorbicacidin2–10mmoldm waspreparedwithanappro-priatedilutionofthestockstandardwithwater.

2.2Instrumentationandresearchmethodology

Paper-based systemswere prepared usingWhatman Qualitative paper GradeNo.1asasupport.Usingsolidinkprinter(Xerox8580DN)thepatterns,whichdesign is presented on the Fig.1, were printed and then heated in standard


laboratorydrierfor2minin120°C.Theheattreatmentensurethepaperpene-tration with wax, causing that obtained barriers were fully impermeable foraqueoussolutions.Inthenextstep,patternswerecoveredwithPrussianblue.Thesolutions used for Prussianblue depositionweremixed together equally justbefore the introduction into the paper surface. The 25µL of themixturewasspreadalongthechannelusingthepipette,andsoakedsystemswereleftforthedepositionfor24h.Afterthattime,systemswerewashedwithdeionizedwateranddriedintheopenair.ThesamplewasplacedintheSpartofthesystemusingthe pipette. For signal readout, the colored band lengthwasmeasured usingconventionalruler.


ThefirstpartoftheworkwasdevotedtoexaminingtheinfluenceofthePrussianblueamountdepositedonthesensitivityandresolutionofthecreatedsystems.Because the distance-based detection uses the analyte consumption effect, asaprimaryreasonwhythesignalsfordifferentstandardconcentrationareobser-ved,theamountofthedyedepositedinthechannelhassignificantimportance.Thedecolorizationoccursonlyinthezonewherethesensingdyewasabletoreactwiththereductant(inthiscontributionasamodelreductantanascorbicacidwaschosen).TheamountofthePrussianbluedepositedintothepapercanbeeasilycontrolled by the concentration of the iron(III) chloride and potassium hexa-cyanoferrate(III)solutionsusedforitsfabrication.HigherconcentrationsprovideenhancedPrussianbluedeposition,whatismanifestedwithlowersensitivitybutthewiderlinearityofthecalibrationcurves,aspresentedinFig.2. AscanbenoticedfromtheFig.2,whenthechannelismodifiedwithahighamountofPrussianblue,thesignals(measureddistance)obtainedforlowcon-centrationsofanalytearelow,duetothehighrateoftheascorbicacidoxidationatthebeginningofthechannel.Fordilutedstandards,asmallamount(amixtureofthe10mMstandards)ofthedyeispreferable,becauseitprovideshighsensitivity


Fig. 1 The system design used during theexperiments.ThePrussianbluewasdepositedalong the channel described with S = 5 mmdiameter,W=3mm,andL=50mm.

Fig. 2ThedifferentsensitivityofthesystemsobtainedusingdifferentconcentrationsofFeCl and3

K [Fe(CN) ]appliedforPrussianBluedeposition.3 6

andhighmeasurementresolution.Formtheotherhand,forhighlyconcentratedsamples,asignificantamountofthedye(amixtureof25mMsolutions)depositedon the systems provide constant decolorization during the sample flow, thusexpandedcalibrationcurveslinearitycanbeobserved,butalsolowersensitivity

–4 –3 3+isnoticeable.Basedontheresultsthemixtureofthe2.5×10 moldm ofFe and3–[Fe(CN) ] solutionswaschosenasoptimalfordistance-basedpaperanalytical6

systems preparation. All further investigations will be conducted with thePrussianbluedepositedinthisdescription. Thesystemspreparedusingoptimizedconditionswereused to recordcali-brationcurveforascorbicacid.Theobtainedcalibrationcurve ispresented inFig.3.Thecalibrationshowsgoodlinearityinallrespectedrangeofascorbicacidapplied.Thesystemalsoischaracterizedbyhighreproducibilityandfacilesignalreadout.Theerrorbarsrepresentthestandarddeviationforthree-timerepeatedmeasurementsforthesamestandardsolution.

4.Conclusions

Inthecourseofthesepreliminaryinvestigations,thefullyworkingdistance-basedmicrofluidic sensoric systemwas developed. The system allows ascorbic aciddetermination in the millimolar range of concentrations with good reprodu-cibilityandaccuracy.Moreover,duetoitschemicalproperties,Prussianbluecanbeusedashydrogenperoxidesensor.AfterthereductionintoPrussianwhiteformitissensitivetoanyoxidizingagentpresentinthesample.Usingthisproperty,presentedplatformwill be in the future reducedwith ascorbic acid and thenmodified with enzymes to provide high-selectively devices dedicated to thespecified analyte recognition. The oxidases generate the hydrogen peroxide


Fig. 3Calibrationgraphforascorbicacidobtainedunderoptimizedconditions.

duringitscatalyticactivityandshowgreatpotentialtobeinvolvedindistance-basedsensorsdevelopment. Distance-baseddetectionshowsexcellentapplicationpossibilityintheaspectof the paper-basedmicrofluidic systems due to its simplicity and device-freereadout.However,thiskindofdetectionstillrequiresanevaluationofthefactorsinfluencing the signal generation, e.g., the geometry and deposited reagentconcentration.

Acknowledgments

HelpfulcommentstothispaperfromDrŁukaszTymecki(UniversityofWarsaw)arekindlyacknow-ledged.TheauthorthanksthesupportfromtheUniversityofWarsaw(DSM501-D112-86-DSM-115100project).TheseinvestigationsweresupportedbythePolishNationalScienceCentre(projectOPUSNCNno.2014/13/B/ST4/04528).

References

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[2] YetisenA.K.,AkramM.S.,LoweC.R.:Paper-basedmicrofluidicpoint-of-carediagnosticdevices.Lab.Chip.13(2013),2210–2251.

[3] CarrilhoE.,MartinezA.W.,WhitesidesG.M.:Waxprinting–asimplemicropatterningprocessforpaper-basedmicrofluidics.Anal.Chem.81(2009),1–5.

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[5] ZukR.F.,GinsbergV.K.,HoutsT.,RabbieJ.,MerrickH.,UllmanE.F.,FischerM.M.,SiztoC.C.,StisoS.N.,LitmanD.J.:Enzymeimmunochromatography:Aquantitativeimmunoassayrequinngnoinstrumentation.Clin.Chem.31(1985),1144–1150.

[6] Cate D.M., Dungchai W., Cunningham J.C., Volckens J., Henry C.S.: Simple, distance-basedmeasurementforpaperanalyticaldevices.Lab.Chip.13(2013),2397–2404.


[7] Quinn C.W., Cate D.M., Miller-Lionberg D.D., Reilly T., Volckens J., Henry C.S.: Solid-phaseextractioncoupledtoapaper-basedtechniquefortracecopperdetectionindrinkingwater.Environ.Sci.Technol.52(2018),3567–3573.

[8] WeiX.,TianTianT.,JiaS.,ZhuZ.,MaY.,SunJ.,LinZ.,YangC.J.:Microfluidicdistancereadoutsweethydrogelintegratedpaper-basedanalyticaldevice(μDiSH-PAD)forvisualquantitativepoint-of-caretesting.Anal.Chem.88(2016),2345–2352.


1.Introduction

Citalopram (Fig. 1) is known chemically as(±)-1-(3-dimethylaminopropyl)-1-(4-fluoro-phenyl)-1,3-dihydroisobenzofuran-5-carbo-nitrile. It is a selective serotonin-reuptakeinhibitor and exhibits broad spectrum oftherapeutic activity against depressivedisorders [1].Citalopram is one of themostcommonlyusedantidepressants,hencethere

Cyclic voltammetry and staircase voltammetry in citalopram determination

a, a bMARIAMADEJ *,JOLANTAKOCHANA ,BOGUSŁAWBAS

a DepartmentofAnalyticalChemistry,FacultyofChemistry,JagiellonianUniversityinKraków, Gronostajowa2,30-387,Kraków,Poland*[email protected] DepartmentofAnalyticalChemistry,FacultyofMaterialsandCeramics,AGHUniversityofScienceandTechnologyAdamaMickiewicza30,30-059,Kraków,Poland


AbstractThis work deals with electrochemical detection of citalopram,commonly used antidepressant. the comparison of two electro-chemicaltechniques:cyclicvoltammetry(CV)andstaircasevoltam-metry(SCV)arepresented.themeasurementparameters,influencingpeakcurrentsrecordedusingbothstudiedtechnics,i.e.scanrateforCV, and potential step and step time for SCV were verified andexamined. The research has shown that employingSCV techniqueallowedtoobtainlargercalibrationslopeincomparisontoCV,andconsequentlybettersensitivityoftheanalyticalmethod.EmployingSCVinsteadofstandardCVseemstoprovidebetteropportunitiesformoresensitiveelectrochemicaldetectionoforganiccompounds.

Keywordscitalopramcyclicvoltammetryelectrochemistrystaircasevoltammetry

is a great need to develop fast and reliablemethods for its determination intablets, biological fluids or even in the environment. The application ofvoltammetrictechniquesforthispurposecouldbeverypromisingduetothelowcostoftheapparatus,thesimplicity,rapidityandhighsensitivityofvoltammetricmeasurements[2]. Cyclic voltammetry (CV) is one of themost commonly used voltammetrictechniqueforanalyticalandmechanismofelectrochemicalreactionsstudiesoforganic and inorganic compounds. Its effectiveness is limited by the charging

Fig. 1Chemicalstructureofcitalopram.

current,alwaysoccurringduetothecontinuouslychangingpotential.Staircasevoltammetry (SCV), the equivalent technique in the pulse version, allows toseparatetheFaradaiccurrentfromthenon-Faradaicandthustoeliminatethechargingcurrent[3]. AccordingtoRandles-Sevcikequation(1)peakcurrentdependsonlyononemeasuringparameter: thespeedof changingpotentialapplied to theworkingelectrode,commonlyknownasascanrate

3/2 1/2 1/2 I =kz AD v c (1)p

where:I ispeakcurrent,kconstantvalue,znumberofelectronsinvolvedinthep

redoxprocess,Aelectrodesurface,Ddiffusioncoefficient,vscanrate,andc isconcentrationofelectroactivecompound. Incyclicvoltammetry,potentialischangedlinearly,thereforescanratedoesnotdependonanyotherparameters.Duringstaircasevoltammetricmeasure-ments,potentialofworkingelectrodeischangedintheformofintervals,so-calledstairs,withacertainpotentialvalue(steppotential)andthespecifiedduration(steptime).Therefore,thescanrateinSCVtechniqueisdeterminedbythesetwoparameters[4]. Theaimofthisworkwastocomparetwoelectrochemicaltechnics:SCVandCVincitalopramdetermination.Forthispurpose,theCVandSCVmeasurementsatdifferent experimental conditionswere carried out.The influence of experi-mentalparametersonrecordedsignalswascompared,particularlyintermsofanalyticalmethodsensitivity.

2.Experimental


Thefollowingreagentswereused:MicroPolishAluminasuspensionwithgrainsize0.5µm(Buechler,USA);potassiumchloride99.5%(Poch,Poland);sodiumnitrate99.5%(Merck,Germany);nitricacid65%(Merck,Germany);disodiumhydrogenphosphate99%(Poch,Poland);sodiumdihydrogenphosphate 99.5%(Chempur,Poland);citalopramhydrobromide(LGCStandards,Canada);distilledwaterderivedfromaHLP5system(Hydrolab,Poland).

2.2Instrumentation

ThevoltammetricmeasurementswereconductedwiththeM161electrochemicalanalyzer(Mtm-Anko,Poland).TheEALab2.1softwarewasusedfordataacqui-sition.Themeasurementswerecarriedinthree-electrodesysteminquartzvesselof5mLvolumecoveredwithaplasticcoverwithfourholesthatmatchedthesizeofeachelectrodeandanadditionalholeforaddingstandardsolution.Theroleof


–1Fig. 2Voltammogramsrecordedfor10µmolL citalopraminphosphatebuffersolution(pH=8.00;–1100mmolL )usingcyclic(solidline)andstaircasevoltammetry(dottedline)techniques.

workingelectrodeplayedglassycarbonelectrodeconsistingof3mmsilverwirecoveredbyglassycarbonlayerplacedinteflonholder(MTMAnkoM10X1).Silver

–1chlorideelectrodewithdoublecoat(Ag/AgCl,saturatedKCl/2molL NaNO ;3

MTMAnkoM6)wasusedasareferenceelectrodeandplatinumplateplacedinteflonholderwasused as an auxiliary electrode.Alsoultrasonicbath Sonic-3(Polsonic,Poland)andmagneticstirrerMS11(Wigo,Poland)wereused.


According toRandles-Sevcikequationpeak current recorded in cyclic voltam-metric measurements depends on square root of scan rate. To confirm thisrelationship cyclic voltammogramswere registeredusingdifferent scan rates:

–112.5,25,50,100,200,250and500mVs .Themeasurementswereconductedin–1phosphate buffer solution (pH=8.00; 100mmol L ) containing citalopramat

–1concentrationof10µmolL inpotentialrangefrom300to1100mV.TheexampleofrecordedCVvoltammogramispresentedinFig.2.Ascanbeseen,citalopramundergoesan irreversibleoxidationprocess, thereforeoxidationpeak currentwastakenasanalyticalsignalinfurtherstagesofresearch.Theobtaineddepen-denceofpeakcurrentinfunctionofscanrateispresentedinFig.3.Asshowninthischart,thecorrelationofpeakcurrentandsquarerootofthescanrateassumedthe linear function,which is consistentwith theRandles-Sevcik equation andprovesthatthecitalopramoxidationisaprocesscontrolledbydiffusion[5].


Fig. 4Dependenceofscanratefromsteptime(blackpointswithsolidline)orpotentialstep(greypointswithdottedline)achievedinstaircasevoltammetricmeasurements.

Fig. 3Dependenceofpeakcurrentinafunctionofsquarerootofthescanrateobtainedincyclicvoltammetrymeasurements.

TostudytheimpactofthechangeinthepotentialstepandsteptimeonrecordedsignalSCVvoltammogramswereregisteredusingvariousvaluesofthepotentialstep (1, 2, 3, 4, 6, 8, and 10 mV) and constant value of step time (40 ms).Subsequently, one value of the potential step (8 mV) was selected andvoltammogramsusingdifferentvaluesofsteptime(8,16,20,40,80,160and320ms) were recorded (Fig. 4). Themeasurements were conducted in the same


Fig. 5Influenceofpotentialstep(greypointswithdottedline)andsteptime(blackpointswithsolidline)onregisteredpeakcurrentinstaircasevoltammetricmeasurements.

electrolytesolutionasCVmeasurementswerecarriedoutwiththecorrespondingpotentialrangeandcitalopramhydrobromideconcentration.Thegraphpresen-tedinFig.4provesthegrowinglinearrelationshipbetweenthepotentialstepandscanrate.Withtheincreaseofthepotentialstep,thenumberofregisteredpointsdecreased,thusthescanrategrewup.Therefore,duetodecreasingmeasurementresolution,itisrecommendedtoapplypotentialstepnothigherthan10mV.Ifthesteppotentialisnothigherthan8mV,theSCVmeasurementscorrespondtotheCVtechnique[4].Forsteptimethereverserelationshipwasobserved:thescanratedecreasedwiththegrowthoftheeachstepduration,whichcanbedescribedas inversely proportional relationship. Registered values of peak current fordifferentpotentialstepsandsteptimesareshowninFig.5.Itcanbeseenthatpeakcurrentincreasedwiththegrowthofpotentialstepanddecreasedwithgrowingofstep time. According to obtained correlations between scan rate and stepparametersitwasobservedthatdependencesofpeakcurrentonpotentialstepandsteptimewereconsistentwiththeRandles-Sevcikequation(1). Inordertoverifytheinfluenceofappliedvoltammetrictechniqueoncitalo-pramcalibrationgraphsmeasurementswereperformedusingbothCVandSCV

–1methods.Forthispurpose,theappropriatevolumesof1.0mmolL citalopramsolution were gradually added to the measuring vessel filled with 5 mL of

–1phosphate buffer solution (pH=8.00; 100 mmol L ), obtaining citalopram–1concentrations of 0, 10, 20, 30, 40 and 50 µmol L . For both techniques,

measurementswereconductedinpotentialrangefrom300to1100mVandscan–1rateequal100mVs .ForSCV,potentialstepequal8mVandsteptime40ms,was

applied, which correspond to mentioned scan rate value. For each analyteconcentration voltammograms were registered three times and peak current


Fig. 6Calibrationgraphsobtainedfromcyclicvoltammetric(blackpointswithsolidline)andstair-casevoltammetric (greypointswithdotted line)measurementsconducted inphosphatebuffer

–1solution(pH=8.00;100mmolL )containingcitalopramwithpotentialrange300–1100mVand–1scanrateequal100mVs .

valuewascalculatedastheaverageofperformedrepetitions.Thepeakcurrentwasreadfromthemaximumoftheoxidationpeakandthenthevaluefrombasevoltammogram(registeredforbuffersolution)wassubtracted.Theresultsofthisexperiment are presented in the Fig. 6. Both calibration graphs exhibit goodlinearity,howeverestimatedlinearfunctionachievedforSCVmeasurementsischaracterized by a greater slope value than calibration graph obtained by CVtechnique. The phenomenon may be related to the fact that SCV methodeliminatesthechargingcurrentwhichisresponsibleforthehighbackgroundinCVmethod,especiallydisturbingathighpotentialvalues:thecitalopramoxida-tionpeakisrecordednearto900mV,werenoticeableincreasingofbackgroundcurrentinCVmeasurementswasobserved(Fig.2).

4.Conclusions

The conducted preliminary studies proved that staircase voltammetry can beappliedforelectrochemicaldeterminationofcitalopraminsteadofcyclicvoltam-metry.InSCVmeasurementsscanrate,soalsopeakcurrent,dependsonpotentialstepvalueandstepduration.Appropriateselectionoftheseparametersallowsforthe modification of the measurement resolution: higher potential step valuedecreasesnumberofrecordedpointsandtheresolutionisdescending.Thus,thevalues of the step potential greater than 10mV are not recommended in thequantitative analysis. It was observed that SCV technique allowed to obtain


calibrationgraphwithalargerslopethanusingCVandsoimprovedsensitivityofdetermination.Accordingtoliterature,alsothepointinthestepduration,whenthecurrentstartstobemeasured,seemstohavesignificantimpactonregisteredvalueofpeakcurrent[3].Theinvestigationofthisparameter’simpactonthepeakcurrentwouldbeanimportantpartoffurtherresearch.

Acknowledgments

ThisresearchhasbeenpartlysupportedbytheEUProjectPOWR.03.02.00-00-I004/16.

References

[1] AtmacaM.,KulogluM.,TezcanE.,SemerciozA.:Theefficacyofcitalopraminthetreatmentofprematureejaculation:aplacebo-controlledstudy.Int.J.Impot.Res.14(2002),502–505.

[2] KeypourH.,SaremiS.G.,VeisiH.,NorooziM.:ElectrochemicaldeterminationofcitalopramonnewSchiffbasefunctionalizedmagneticFe3O4nanoparticle/MWCNTsmodifiedglassycarbonelectrode.J.Electroanal.Chem.780(2016),160–168.

[3] SeralathanM.,OsteryoungR.,OsteryoungJ.:Generalequivalenceoflinearscanandstaircasevoltammetry.J.Electroanal.Chem.222(1987),69–100.

[4] CyganskiA.:Podstawymetodelektroanalitycznych.3rded.Warszawa,WNT1999.[5] GosserD.K:CyclicVoltammetry:SimulationandAnalysisofReactionMechanisms.2nded.New

York,VCH1993.


1.Introduction

AccordingtostatisticalreportsvasculardiseasesremainthefirstcauseofdeathinPoland,responsiblefornearly46%ofdeceasesin2013[1].Thatkindofdeceaseisusuallyassociatedwithatherosclerosis,definedasnarrowing the lumenofanarteryduetothebuild-upofplaque[2].Atheroscleroticplaqueiscomposedofglycolipoprotein part and inorganic part,mainly calcium compounds [3]. Themostclinicallyimportantdifferentiationofatheroscleroticplaquedistinguishestwotypes:stableandunstableplaque[4].Stableplaquehassmalllipidpoolandthickfibrouscap.Itcangrowinmanyyearswithoutanysymptoms.Inturn,bigger

Elemental analysis of atheroscleroticplaque by ICP-MS and LA-ICP-MS

a, a a aAGATAJAGIELSKA *,BARBARAWAGNER ,ANNARUSZCZYN SKA ,ANDRZEJGAWOR ,a b c dEWABULSKA ,ELZBIETAZIEMIN SKA ,BEATATOCZYŁOWSKA ,WAWRZYNIECJAKUCZUN ,

dMAŁGORZATASZOSTEK

a BiologicalandChemicalResearchCentre,FacultyofChemistry,UniversityofWarsaw, ul.ŻwirkiiWigury101,02-093Warsaw,Poland*[email protected] MossakowskiMedicalResearchCentre,PolishAcademyofSciences, ul.Pawińskiego5,02-106Warsaw,Polandc NałęczInstituteofBiocyberneticsandBiomedicalEngineering,PolishAcademyofSciences, ul.ks.Trojdena4,02-109Warsaw,Polandd DepartmentofGeneralandEndocrineSurgery,MedicalUniversityofWarsaw, ul.Banacha1a,02-097Warsaw,Poland

AbstractAtherosclerosiscanbedescribedasnarrowingthelumenofanarteryduetothebuild-upofplaque.Chemicalcompositionofanatheros-cleroticplaquestronglydependsonitsclinicallyimportantstability.Theaimofastudywaselementalanalysisofatheroscleroticplaquebyusinginductivelycoupledplasmamassspectrometry(ICP-MS)andcreating the elemental distribution maps by using laser ablationinductively coupled plasma mass spectrometry (LA-ICP-MS). Ele-mentssuchasLi,Mg,Ca,V,Mn,Fe,Co,Cu,As,Se,Rb,Sr,Ag,Cd,Pbweresuccessfullyquantified,with thegreatest contentof typical calcifi-cationcomponents:CaandMg.LA-ICP-MSanalysiswasperformedwithnoneedofsamplemineralization.Inhomogeneousdistributionofseveralelementswasobserved,againmainlyincaseofcalcification(Ca,Mg, Sr,P).Moreover, the influenceof samplepreparationwasstated. Lyophilisation enhanced analytes concentration, but alsocausedthelossofseveralelements,suchasPborS.

Keywordsatherosclerosislaserablationmassspectrometry


lipid pool and thinner fibrous cap in unstable plaque make it much morevulnerable and easier to rupture. Chemical composition of a plaque can varydepending on its stable or unstable character. Currently, there are no drugsavailabletostabilizeunstableatheroscleroticplaque[5].However,itisimportanttoquicklydiagnosethestabilityor instabilityof theplaque inordertodecideaboutsurgeryandtoplanfurthertreatment.

2.Experimental


StocksolutionswerepreparedbydilutingICPmulti-elementstandardMerckVI(Merck, Germany). CertifiedReferenceMaterials (CRM)were used:MODAS-4,cormoranttissue(MODAS,Poland),MODAS-3,herringtissue(MODAS,Poland),NIST1577c,bovinelivertissue(NIST,USA).Forsamplemineralization65%nitricacid,analyticalgrade,wasused(Merck,Germany).Samplesandstandardswerediluted with deionized water obtained by Milli-Q System (Merck, Millipore,Germany).

2.2Instrumentation

The measurement were carried out using inductively coupled plasma massspectrometerNexION300D(PerkinElmer,USA).LaserablationwasperformedwithLSX-213(CetacTechnologies,USA).InstrumentsconditionsarepresentedinTable1.Sample lyophilisationwasexecutedwithLiofilizatorAlphamodel1-2LDplus(Donserv,Germany).


LA-ICP-MS ICP-MS

Sampleintroductionsystem LSX-213(CETAC) MeinhardnebulizerNumberofablationlines 5 –Laserenergy/mJ 2.5 –Lasershotfrequency/Hz 20 –Spotsize/μm 100 –

–1Scanrate/μms 50 –Plasmapower/W 1400 1350

3 –1Carryinggas(Ar)flow/dm min 0.9 0.8Dwelltime/ms 5 50Shutterdelay/s 30 –

Table 1Laserablationinductivelycoupledplasmamassspectrometryandinductivelycoupledplasmamassspectrometryinstrumentationconditions.

Fig. 1Schemeofusedanalyticalprocedure.

2.3Analyticalprocedure

SchemeoftheanalyticalprocedureispresentedinFig.1.Atheroscleroticplaquetaken intraoperatively from thepatient’s femoral arterywasdivided into twopieces(Plaque1andPlaque2).Thefirstpieceremainedunchanged(Plaque1)andthesecond(Plaque2)waslyophilized(temperature:–15°C,pressure:1mbar,time:6h).Ahalfoflyophilizedmaterial(Plaque2a)wastreatedwithmicrowave-assistedmineralizationintheclosedsystem(65%HNO ,220°C)andanalysedby3

usinginductivelycoupledplasmamassspectrometry(ICP-MS).Theunchangedpiece (Plaque 1) and the second half of lyophilized sample (Plaque 2b)wereanalysedbyusinglaserablationinductivelycoupledplasmamassspectrometry(LA-ICP-MS).AfterlaserablationitisstillpossibletomineralisethesampleandthatiswhyPlaque2bcouldbetransferredtothesolutionandalsoanalysedbyICP-MS.


In order to estimate total elemental content in atherosclerotic plaque, thescreeningICP-MSmethodwasused.Preliminaryscreeninganalysisreliesonmassspectrumregistrationinawiderangemasstochargeratio.Then,theresultsareinterpreted by using mathematical algorithm with automatic correction fortypical isobaricandmultielemental interferences.Registeredsignal intensities


Fig. 2Laserablationinductivelycoupledplasmamassspectrometryscreeningmethodresultsfortwosamplesofatheroscleroticplaque.

arecomparedwithavailabletableofresultsforeachelementandpresentedasapproximateconcentrations. ResultsobtainedbyusingICP-MSscreeningmethodarepresentedinFig.2.Preliminary testingallowed tonarrowdown the listof elementsdesigned forfurtherquantitativeanalysisbyexternalstandardcalibration.TheresultswerereferredtoCRMinordertoprovethereliabilityofthemethod.ChosenelementalcontentinatheroscleroticplaqueispresentedinTable2(nextpage).Elementssuchascalcium(above10%ofplaquedrymatter)ormagnesium(above0.1%)canbeconsideredastypicalcomponentsofcalcifiedplaqueareas.Significantironcontent(above0.01%)comesprobablyfromtherestofmorphoticcomponentoftheblood.Apartfromtheseelements,quantitativeresultsforcobalt,copper,leadandcadmiumwereachievedthatmaybeastartingpointforinvestigationaboutmetalaccumulationinatheroscleroticplaque. Directanalysisofelementaldistributioninatheroscleroticplaquewasperfor-medusingLA-ICP-MS.Linear laserablationwaschosen inordertoobtain theinformation of elemental content on sample surface. Signals reconstruction

2allowedvisualisingelementaldistributioninchosenarea(1.35mm )ofaplaque(Fig. 3, page 49). For each element, signal intensities were normalised andpresented as percent of maximum signal intensity. Most of the elements areinhomogeneouslydistributed,particularlycharacteristiccomponentsofcalcifi-cation(Ca,Mg,Sr,P).Acorrelationbetweenhighcontentoftheseelementscanbeobserved in themaps. It isworthmentioning thatphosphorusnotonly is thecomponentof calcifiedparts,butalso itoccurs inphospholipids.Additionally,phosphatesmaybenottheonlyanionsincalcification.



–1Sample

Quantification/mgkg

drymatter

Li

Mg

Ca

V

Mn

Fe

Co

Cu

As

Se

Rb

Sr

Ag

Cd

Pb

Plaque2a

<LOD3454230361

4.86

1.39

1179

0.206

5.65

1.01

0.9860.668

53.50.097

0.3972.088

CV/%

644

0.6

3.8

5.2

2.0

2.0

4.8

4.3

6.4

14.5

18.2

1.5

112.6

3.6

1.8

Plaque2b0.0241819126672

<LOD<LOD

485.30.287

<LOQ<LOD0.2510.206

39.8<LOD0.0590.928

CV/%

3.1

0.8

0.9

2.6

0.7

0.7

1.4

1.1

1.0

5.0

1.2

1.5

4.8

0.5

0.9

LOD

0.01215

46

0.02

0.09

4.7

0.003

1.07

0.21

0.0180.196

0.2

0.024

0.0030.062

LOQ

0.01615

55

0.02

0.13

7.6

0.005

1.76

0.25

0.0290.210

0.2

0.037

0.0040.079

Tab

le 2

Chosenelementalcontentinatheroscleroticplaquequantifiedbyusingexternalstandardm

ethod(CV–coefficientofvariation,

LOD–limitofdetection,LOQ–limitofquantitation).

MultielementalcompositionofanatheroscleroticplaquefromheartvesselswasperformedforthefirsttimebyZhuravskayaetal.in2016[6].Authorsusedsynchrotron radiation–induced X-ray fluorescence (SRXRF) method and alsostated the correlation between calcium and strontium distribution, as inpresented research.Moreover, in the cited study [6], the association betweencalcium,zincandironwasobserved. SamplepreparationwasconsideredasanimportantfactorinfluencingLA-ICP-MS measurements. Lyophilisation has many advantages, such as increasingstabilityandinhibitionofmetabolicprocesses[7].Therearereportsthatlyophili-sationpreserves tissue cellular structure [8,9]. Concentrationof the analytesincreasesafterremovingwaterthatmakeitpossibletodeterminetracecontents.However, there are also some limitations of the process, among others beingaconsequence of sample specificity. Atherosclerotic plaque is composed ofcellularrestsrichinwater,butitcontainsalsolipidpoolsandcalcificationwithmuch lower water contribution. For such inhomogeneous samples, resultscalculatedaspercentofdrymattermaynotbethebestwayofmeasurementdatapresentation. The second considerable drawback of lyophilisation observedduring thepreliminaryresearchwas losing the informationaboutseveralele-ments(mainlysulphurandlead).Averagesignalfortheseelementswasmuchlowerforlyophilizedsampleincomparisonwithunpreparedsamplesthatisinopposition to normally observed effect of increasing content after removingwater.


Fig. 3Elementaldistributionmapsofchosenelementsinatheroscleroticplaquewithnopreparation(Plaque1)and in lyophilizedsample(Plaque2b)performedbyusing laserablation inductivelycoupledplasmamassspectrometry

4.Conclusions

Atheroscleroticplaqueanalysisbyusingmassspectrometrytechniquesiscom-plementary in relation to histological examination that provides only theinformation about cellular composition of the sample. ICP-MS method givesapossibilitytoanalyseelementalcontentanditscombinationwithlaserablationallows visualizing superficial distribution of many elements. Inhomogeneousdistributionofseveralelementswasobservedmainlyincaseofcalcification(Ca,Mg,Sr,P)thatcanbecomeanimportanttoolinatheroscleroticplaquedifferen-tiation.Theinfluenceofsamplepreparationwasstated,seeingthatlyophilisationenhancesanalytesconcentration,butalsocauses the lossof severalelements,suchasPborS.Presentedstudycanbeagoodstartingpointforfurtherresearchleadingtochemicaldifferentiationofatheroscleroticplaqueinclinicalpractice.

Acknowledgments

ThestudywascarriedoutattheBiologicalandChemicalResearchCentre,UniversityofWarsawestablishedwithintheprojectco-financedbyEuropeanUnionfromtheEuropeanRegionalDevelop-mentFundundertheOperationalProgrammeInnovativeEconomy2007–2013.

References

[1] StrzeleckiZ.,SzymborskiJ.:ZachorowalnośćiumieralnośćnachorobyukładukrążeniaasytuacjademograficznaPolski.RządowaRadaLudnosciowa2015.

[2] HerringtonW.,LaceyB.,SherlikerP.,ArmitageJ.,LewingtonS.:Epidemiologyofatherosclerosisandthepotentialtoreducetheglobalburdenofatherothromboticdisease.Circ.Res.118(2016),535–546.

[3] LaraM.J.,RosE.,SierraM.,DorronsoroC.,AguilarJ.:Compositionandgenesisofcalciumdepositsinatheromaplaques.Ultrastruct.Pathol.38(2014),167–177.

[4] Liu J.,WangZ.,WangW.M.,LiQ.,MaY.L.,LiuC.F.,LuM.Y.,ZhaoH.:FeasibilityofdiagnosingunstableplaqueinpatientswithacutecoronarysyndromeusingiMap-IVUS.J.Zhejiang.Univ.Sci.B16(2015),924–930.

[5] Thompson P.L., Nidorf S.M., Eikelboom J.: Targeting the unstable plaque in acute coronarysyndromes.Clin.Ther.35(2013),1099–1107.

[6]ZhuravskayaE.Y.,SavchenkobT.I.,ChankinabO.V.,.PolonskayaaY.V,ChernyavskiiA.M.,RaginoY.I,ShcherbakovaL.V.:SRXRFstudyofchemicalelementscontentintheatheroscleroticplaqueofheartvessels.Phys.Procedia84(2016),270–274.

[7] ShuklaS.:Freezedryingprocess:areview.Int.J.Pharm.Sci.Res.2(2011),3061–3068.[8]TangM.,WolkersW.F.,Crowe J.H.,TablinF.:Freeze-driedrehydratedhumanbloodplatelets

regulateintracellularpH.Transfusion46(2006),1029–1037.[9]WolkersW.F.,WalkerN.J.,TablinF.,CroweJ.H.:Humanplateletsloadedwithtrehalosesurvive

freeze-drying.Cryobiology42(2001),79–87.


1.Introduction

Recently,therehasbeenadecreaseintheuseofmercuryelectrodescausedbythefearofitstoxicitythatpromptedresearchofnovelelectrodematerialswithsimi-larproperties.Mercuryelectrodesaregenerallyconsideredasthebestforstudiesof reducible substances.Non-toxic amalgamelectrodesproved to be aworthyreplacementofmercuryelectrodeswithsimilarlywidepotentialwindow,compa-rablesensitivityandtheyalsoofferadvantageofbettermechanicalstability[1].TheywereintroducedbyNovotnyandYosypchuk[2]andsincethentheyhavebeenwidelyusedforvoltammetricdeterminationofvariousorganiccompounds,environmental pollutants and pesticides [3–7]. Amercurymeniscusmodified

Determination of difenzoquat at a mercury meniscus modified silver solid amalgam electrode by differential pulse voltammetry

JU LIUSGAJDA R*,JIR IBAREK,JANFISCHER

UNESCOLaboratoryofEnvironmentalElectrochemistry,DepartmentofAnalyticalChemistry,FacultyofScience,CharlesUniversity,Hlavova8,12843Prague2,CzechRepublic*[email protected]


AbstractInthisstudyherbicidedifenzoquatisdeterminedbydifferentialpulsevoltammetryatamercurymeniscusmodifiedsilversolidamalgamelectrode. The optimal medium for the determination is Britton-RobinsonbufferpH=11.0.AtthispHdifenzoquatgivesonecathodicsignalat–1.33V(vs.Ag|AgCl|3MKClreferenceelectrode).Anadditionofafewdropsofgelatinasasurface-activecompoundgreatlyimpro-vesrepeatabilityofdeterminationofdifenzoquatandremovessharpmaximainvoltammograms.Undertheseconditionsitispossibletoachievelimitsofquantitationinsubmicromolarrange.

Keywordsdifenzoquatherbicidemercurymeniscus

modifiedsilversolidamalgamelectrode

voltammetry

Fig. 1Structuralformulaofdifenzoquat

silversolidamalgamelectrode(m-AgSAE) isconsideredtobethemostsimilartoamercuryelectrodewithitswidepotentialwindowandhighsensitivity[8]. The analyte in this study is a herbicidedifenzoquat(Fig.1),aquaternaryammoniumsalt, usually distributed as1,2-dimethyl-3,5-diphenyl-pyrazolium methyl sulfate. This

herbicideisusedforwildoat(Avenafatua)controlinbarleyandwheat.Itbearsalmostnoacuteorchronicthreattoavian,waterormammalianspecies[9].Thiscompoundwasstudiedbyelectrochemicalmethodsatmercuryelectrodeswitheitherpureorganicormixedsolventsandthedeterminationwasunsuccessfulinaqueousmedia[10,11].Inthiswork,astudiedherbicidedifenzoquathasbeendeterminedbydifferentialpulsevoltammetryatm-AgSAEinaqueousmedium.

2.Experimental


–1Astocksolutionofdifenzoquat(1mmolL ,1,2-dimethyl-3,5-diphenylpyrazo-liummethylsulfate,CASnumber:43222-48-6,99.9%fromSigmaAldrich)waspreparedindeionizedwater.Britton-Robinsonbufferswerepreparedbymixing

–1 –10.04molL phosphoric,acetic,andboricacidwith0.02molL sodiumhydroxide(allp.a.,Lach-Ner).

2.2Instrumentation

All voltammetric measurements were carried out on Eco-Tribo Polarographcontrolled by Polar Pro 5.1 software (both Polaro-Sensors, Czech Republic).Voltammetricmeasurementswerecarriedoutinathree-electrodesystemwithworking m-AgSAE (0.5 mm diameter, Eco-Trend Plus, Czech Republic),

–1Ag|AgCl|3molL KClreferenceelectrode(Elektrochemickedetektory,Turnov,CzechRepublic)andaplatinumwireauxiliaryelectrode(Eco-TrendPlus,CzechRepublic).Samplesforvoltammetricmeasurementswerepreparedbymeasuringan appropriate amount of the stock solution of difenzoquat into a 10.0 mLvolumetric flask, twodropsof0.5%gelatinsolutionwere thenaddedand thevolumetricflaskwasfilledwithBritton-RobinsonbufferpH=11.0tothemark.Thesolutionwaspurgedwithnitrogenfor5minandcorrespondingvoltammo-gram was recorded. The purging was repeated for 30 s before every singlemeasurement. Working m-AgSAE was pre-treated as described in [12]. Them-AgSAE was activated before every series of measurements by applying

–1potential–2.2Vfor300sin0.2molL KCl.differentialpulsevoltammetrypara-–1meterswere:scanrate20mVs ,pulsewidthof100ms,pulseamplitude–50mV,

andsamplingtime20ms.


VoltammetricresponseofdifenzoquatwasinvestigatedintheBritton-Robinsonbuffer.Reductionatm-AgSAEwasobservedonlyatpH=8.0andmorealkalinebuffersolutions;inneutralandacidicpHthereductionresponsecoincidedwiththehydrogenevolution.Thepotentialofreductionwasconstantat–1.33Vinthe


pHrange8.0–12.0.ThepeakcurrentresponsedidnotsignificantlychangeinthepHrange10.0–12.0andthevalueinthemiddleofthisrangepH=11.0waschosenasoptimalforthevoltammetricdeterminationofthecompound.Afewdropsofgelatinwereaddedtothemeasuredsolutionbecauseiteliminatedsomesharpmaximathatwereobservedinvoltammogramsatmorenegativepotentialsthanthereductionpeak.Undertheseconditionsitwaspossibletomeasure20curveswithgoodreproducibilityaftertheactivationofm-AgSAEwithoutanyregene-ratingstepin-betweenmeasurements. Difenzoquat was at first successfully determined in the Britton-Robinsonbufferwith the addition of two drops of 0.5% gelatin solution. Voltammetric

–1curvesandacalibrationdependencyfortheconcentrationrange2–10μmolL areshowninFig.2.Difenzoquatwasalsodeterminedinrealmatrices(drinkingandriverwater)spikedwithconcentrateddifenzoquatsolutionandinthepre-sence of 10% Britton-Robinson buffer of pH=11.0 and the surface-activecompoundgelatin.Parametersofcalibrationcurvesforthelineardynamicrangeof the method are summarized in Table 1. At higher concentrations of the


Fig. 2Differentialpulsevoltammogramsofdifenzoquat(0(1);2(2);4(3);6(4);8(5);and10(6)–1μmolL ) atm-AgSAE inBritton-RobinsonbufferpH=11.0medium in thepresenceof gelatin

–1(25µgmL ).Thecalibrationcurveisshownintheinset.

–1 –1Matrix Slope/mALmol Intercept/nA R LOQ/µmolL

Deionizedwater –1.78±0.05 +0.13±0.15 –0.9978 0.46Drinkingwater –2.14±0.04 –0.36±0.14 –0.9983 0.34Riverwater –1.59±0.06 –0.55±0.09 –0.9946 0.47

Table 1Calibrationstraightlineparametersfordifferentialpulsevoltammetrydeterminationofdifenzo-

–1quat in lineardynamicrangeof0.2–10µmolL atm-AgSAE invariousmatricesusingBritton-–1RobinsonbufferpH=11.0asasupportingelectrolyteandaftertheadditionofgelatin(25µgmL )to

suppressobservedmaxima.

–1herbicide(morethan10µmolL )slopesoftheconcentrationdependenciesweresignificantlydifferent(lower)incaseofallmatrices.

4.Conclusions

The herbicide difenzoquat was successfully determined by differential pulsevoltammetry at them-AgSAE in the Britton-Robinson buffer buffer solution(pH=11.0)andinrealmatricesofdrinkingandriverwaterwithlimitsofquanti-tation in submicromolar range. An addition of gelatin eliminated some sharpmaximathatwerepresentinaqueousmedium.

Acknowledgments

FinancialsupportoftheCzechScienceFoundation(project17-03868S)isgratefullyacknowledged.

References

[1] GajdarJ.,HorakovaE.,BarekJ.,FischerJ.,VyskocilV.:Recentapplicationsofmercuryelectrodesformonitoringofpesticides:Acriticalreview.Electroanalysis28(2016),2659–2671.

[2] Novotny L., Yosypchuk B.: Pevne strıbrne amalgamove elektrody. Chem. Listy 94 (2000),1118–1120.

[3] Danhel A., Barek J.: Amalgam electrodes in organic electrochemistry. Curr. Org. Chem. 15(2011),2957–2969.

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[6] Janikova-BandzuchovaL.,SelesovskaR.,ChylkovaJ.,NesnidalovaV.:Voltammetricanalysisofherbicidepicloramonthesilversolidamalgamelectrode.Anal.Lett.49(2016),19–36.

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[10]Pospıs ilL.,ColombiniM.P.,FuocoR.,StreletsV.V.:Electrochemicalpropertiesofdifenzoquatherbicide (1,2-dimethyl-3,5-diphenyl-pyrazolium). J. Electroanal. Chem. Interfacial Electro-chem.310(1991),169–178.

[11]RuhlingI.,SchaferH.,TernesW.:HPLConlinereductivescanningvoltammetricdetectionofdiquat,paraquatanddifenzoquatwithmercuryelectrodes.FreseniusJ.Anal.Chem.364(1999),565–569.

[12]YosypchukB.,BarekJ.:Analyticalapplicationsofsolidandpasteamalgamelectrodes.Crit.Rev.Anal.Chem.39(2009),189–203.


1.Introduction

Inordertocounteractenvironmentalairpollution,legislationworldwideincrea-singlylimitsthepermittedamountsofexhaustgasesfrominternalcombustionengines.Nitrogenoxides(NO ),meaningthesumofnitrogenmonoxideNOandx

nitrogendioxideNO haveattractedattentioninrecentyearsinconnectionwith2

thediscussionsaboutthediesel-engine’sNO emissions.Incontrasttogasolinex

engines,dieselenginesareingeneraloperatedwithaleancombustionmixture,meaningthatastoichiometricexcessofoxygencomparedtotheamountoffuelispresentinsidethecombustionchamber[1].Forthiscase,theon-boardchemicalreductionofNO isacomplexprocessduetoundesiredreactionsofexhaust’sx

potentialreducingagents(unburnedhydrocarbons,carbonmonoxide)withtheoxygenpresent in the leanexhaustgas.Therefore,vehiclesoperatedbydieselenginesrequireanadditionaltechnologyforreductionofNO emissionssincethex

Investigations on the electrochemically induced decomposition of AdBlue-urea

a,b, b aPETERBRAUN *,HANS-PETERRABL ,FRANK-MICHAELMATYSIK

a InstituteofAnalyticalChemistry,Chemo-andBiosensors,FacultyofChemistryandPharmacy,UniversityofRegensburg,Universitätsstraße31,93053,Regensburg,Germany*[email protected]

b LaboratoryCombustionEnginesandEmissionControl,FacultyofMechanicalEngineering,OstbayerischeTechnischeHochschule,Galgenbergstraße30,93053,Regensburg,Germany


AbstractAmmoniabasedselectivecatalytic reductionsystemsare themostwidely used technology for reduction of nitrogen oxide emissionsfrom lean-burn engines such as diesel engines. However, at lowexhaust temperatures the selective catalytic reduction process islimitedbydifficulties indecompositionof the ammoniaprecursorurea,whichiscarriedon-boardusinganaqueoussolution“AdBlue”.InpreviousworkthenickelspeciesNiOOHwasshowntobecatalyticallyactiveindecomposingureaatlowtemperatures,forthecaseofhighlyconcentratedpotassiumhydroxideinAdBlue.Sincethisapproachisdifficulttoapplyinpractice,inthepresentstudytheelectrochemicalbehaviourofanickelsurfaceinammoniumcarbonatewascomparedtothatinsodiumhydroxideusingcyclicvoltammetry.Itwasfoundthat the electrochemical behaviour changes significantly whenchangingtheelectrolyte.

KeywordsDeNO systemsx

dieselengineNiOOHselectivecatalytic

reductionureadecomposition

introductionoftheEUROVIstandard.BesidestheleanNO -trapcatalyst,whichisx

mainlyusedforlightdutyapplications,theselectivecatalyticreductionprocessisthemostcommonlyusedtechnologyforeliminationofNO emissionsfromleanx

exhaustgas[2].Theselectivecatalyticreductionprocessrequiresammoniaasexternalreducingagentandmustbecarriedon-boardasadditionaloperatingmaterial.However,asammoniaisatoxicgasatambientconditions,thereducingagentiscarriedon-boardinformoftheprecursorurea.Therefore,ureaas32.5%bymasssolutioninwater(“AdBlue”or“DEF”)isusedasexternalreducingagent.TheprocessofNO reductionisthusprecededbytheprocessofureadecompo-x

sitiontoammonia.WhilemodernSCRcatalyticcoatingsreach90%NO reductionx

at exhaust temperaturesof 165 °C [2], for the caseof available ammonia, thedecompositionofureatoammoniarequirestemperaturesofatleast180–190°Cevenwhen applying a hydrolysis catalyst (e.g., TiO ) [3]. Sincemodern diesel2

engineshavebecomemoreandmoreefficientoverthe lastyears,adeclineofexhausttemperaturecouldbeobservedconcurrently[4].Hence,incaseoflow-loaddriving like incitytraffic, thedecompositionprocessofureatoammonialimitstheapplicabilityoftheoverallselectivecatalyticreductionsystem.Conse-quently,methodsenablingpreparationofammoniaatlowertemperatureswouldreduceNO emissionsofdieselvehiclesatlowexhausttemperatures[5].x

Oneelectrochemicalapproachfordecomposingureaintheliquidphaseatlowtemperatureswas presented by Lu et al. [6]. In their study they showed thatanickel-basedelectrodewasabletoincreasetherateofammoniagenerationbyafactorof~28incomparisontothethermalhydrolysisofureaatatemperatureaslowas70°C.TheinvestigationswerecarriedoutinAdBluecontaining7MKOH.Ina furtherpublication the same authorspresentedmechanistic studies on thiselectrochemically inducedconversionofureatoammonia[7].Theyconcludedthatnickeloxyhydroxide,NiOOHisthespeciescatalyticallyactiveinthedecompo-sition of urea on thenickel surface [8].However, since all these studieswerecarriedoutinureasolutionscontainingpotassiumhydroxideatrelativelyhighconcentrations, the experiments represent an approach which is difficult toimplement inpractice.Themostdesirableapproach in termsofpracticabilitywouldbetouseAdBluewithoutanyadditivesoringeneralwithoutchangingitscomposition. Along with the decomposition of urea in aqueous solution, theevolutionofcarbondioxidetakesplaceinadditiontotheformationofammonia.Ifthereactioniscarriedoutinaqueoussolution,anammoniumcarbonatesolutionisformedbydissolution. Because of that, in this study the electrochemical behaviour of a nickelelectrode in ammonium carbonate solutions was investigated by cyclicvoltammetrystudiesandcomparedtothebehaviourofthenickelelectrodeinsodiumhydroxidesolution.


2.Experimental


Sodium hydroxide, purity “p.a.”was purchased from Merck. Am-monium carbonate, purity “purefood grade” was purchased fromAppliChem. Millipore water wasusedthroughouttheexperiments.

2.2Instrumentation

A laboratory-made nickel-discelectrodewasused in the experi-ments. A nickel wire (diameter0.5mm)wasembeddedintothetipofaglasspipette,usingepoxyresin.Forthecyclicvoltammetrystudiesa three electrode configurationwasappliedconsistingofthework-


Fig. 1 Schematic setup of the electrochemical cellconfiguration.

ingelectrode(nickeldisc)whichwaspolishedbeforeeachexperiment,theauxi-liaryelectrode(platinumwire)andthereferenceelectrode(Ag/AgCl/3MKCl).TheschematicsetupisshowninFig.1. A797VAComputraceofMetrohmwasusedaspotentiostat.Toenablecyclicvoltammetry experiments at elevated temperatures, a reaction vessel withathermostatjacketandaHaakeD8thermostatwereused.Tokeepthereferenceelectrode at a constant temperature and thereby enable comparability of thepotentialsinthedifferentexperiments,thereferenceelectrodewasplacedinanexternalcompartment,connectedtothereactionvesselviaasaltbridge filledwith3MKCl.Theexperimentswereperformedwiththefollowingvoltammetric

–1parameters:sweeprate:50mVs ,voltagestep:0.001V,numberofsweeps:30.


Sincesignalsinthecyclicvoltammogramsofammoniumcarbonateweregrowingduringtheexperiments,cyclicvoltamogramsinbothelectrolyteswererepeated30timesandthe30thsweepofeachexperimentisshowninFigs.2and3.First,thebehaviour of the nickel electrode in sodium hydroxide solutions of differentconcentrations(0.1M,0.5M,1.0M,2.0Mand4.0M)wasinvestigatedat25°Cand40°C.TheresultsofeachcyclicvoltammetryexperimentareshowninFig.2.

Ascanbeseenforallinvestigatedconcentrations,at25°Cand40°Cthereisasignalofoxidationandreductionforthenickelsurface.Accordingtoliterature

2+ 3+theprocesscanbeattributedtotheoxidationofNi toNi [9]by

– – Ni(OH) +OH →NiOOH+H O+e (1)2 2

therebyformingNiOOHwhichwasshowntobecatalyticallyactiveconcerningthedecompositionofurea[7]. Inthenextstepthecyclicvoltammetryexperimentswerecarriedoutinammo-niumcarbonatesolutionsofdifferentconcentrations(0.1M,0.5M,1.0M,and2.0M).Theexperimentswereagaincarriedoutat25°Cand40°C.TheresultsoftheseexperimentsareshowninFig.3. Regardingthe0.1Mammoniumcarbonatesolution,twooxidationsignalsatpotentialsofabout0.9Vand1.1Vandonereductionsignalatabout0.65Vwere


Fig. 2Cyclicvoltammogramsofanickel-discelectrode(d=0.5mm)insodiumhydroxidesolutionsofvariousconcentrations,left:at25°C,right:at40°C.

Fig. 3Cyclicvoltammogramsofanickel-discelectrode(d=0.5mm)inammoniumcarbonatesolu-tionsofvariousconcentrations,left:at25°C,right:at40°C.

foundinthecyclicvoltammetryexperimentsforbothtemperatures.Thereduc-tionsignalwasofsimilarsizeastheoxidationpeakat0.9V.Theoxidation,aswellas thereduction, signalsweregrowingwiththenumberofsweepsduringtheexperiment, meaning the signals did not reach a steady state even after therecordingof30cyclicvoltammograms.Ascanbeseen,theintensityofthesignalsat0.65Vand0.9Vwasmostpronouncedintheexperimentswithlowammoniumcarbonate concentrations. In the experiments with 0.5 M, 1.0 M and 2.0 Mammoniumcarbonateat25°Cand40°C,thesesignalsweremuchweaker.Thisfindingwasadditionallyconfirmedbyanexperimentwitha0.05Mammoniumcarbonatesolution.Literatureindicatesthatthesesignalscouldberelatedtothe

2+ 3+transitionNi /Ni [10].However,asvisibleinFig.3theintensityofthesecondoxidationpeakincreasedbyincreasingtheammoniumcarbonateconcentration,whichcanbeanindicationthattheoxidationpeakatabout1.1Visduetotheoxidationofammoniaonthenickelsurface[10].BesidestheexperimentsshowninFig.3,thecyclicvoltammetryexperimentwith0.1Mammoniumcarbonatewasalsocarriedoutwiththeuppervertexpotentialbeing1.0Vinsteadof1.4V.Inthiscasethesignalsat0.65Vand0.9Vcouldnotbedetected.Thisobservationshowsthat this process cannot proceed independently of processes occurring atpotentials>1.0V.OnepossibleexplanationmightbealocalacidicpHshiftatthenickel electrode, which is associated with the formation of oxygen at highpotentialswithprogressingexperiment.

4.Conclusions

Thepresentedresultsshowthatthebehaviourofthenickelelectrodestudiedbycyclicvoltammetryexperimentschangessignificantlybychangingtheelectrolytefromsodiumhydroxidetoammoniumcarbonate.Oxidationofthenickelsurfacemightalsooccurintheammoniumcarbonatesolutions.However,furtherdetailedstudiesarenecessary.Experimentswithureadissolvedinsodiumhydroxideaswellasammoniumcarbonatesolutionsareplannedinordertoevaluatepossibleinteractionsbetweenthespeciesformedonthenickelsurfaceandurea.

References

[1] TschokeH.,MollenhauerK.,MaierR.:HandbuchDieselmotoren,4thed.Wiesbaden,Springer2018.

[2] BraunP.,GebhardJ.,MatysikF.M.,RablH.P.:Potentialtechnicalapproachesforimprovinglow-temperatureNO conversionofexhaustaftertreatmentsystems.Chem.Ing.Tech.90(2018),x

762–773.[3] BernhardA.M.,PeitzD.,ElsenerM.,SchildhauerT,KrocherO.:Catalyticureahydrolysisinthe

selectivecatalyticreductionofNO :catalystscreeningandkineticsonanataseTiO andZrO .x 2 2

Catal.Sci.Technol.3(2013),942–951.[4] BraunP.,GebhardJ.,RablH.P.:LowtemperatureDeNO .FinalReportFVVproject1155,2017.x

[5] RoppertzA.,FugerS.,KuretiS.:Investigationofurea-SCRatlowtemperatures.Top.Catal.60(2017),199–203.


[6] LuF.,BotteG.G.:Electrochemicallyinducedconversionofureatoammonia.ECSElectrochem.Lett.4(2015),E5–E7.

[7] LuF.,BotteG.G.:Understandingtheelectrochemicallyinducedconversionofureatoammoniausingnickelbasedcatalysts.Electrochim.Acta246(2017),564–571.

[8] DaramolaD.A.,SinghD.,BotteG.G.:DissociationratesofureainthepresenceofNiOOHcatalyst:ADFTanalysis.J.PhysChem.A114(2010),11513–11521.

[9] HahnF.,FlonerD.,BedenB.,LamyC.:InsituinvestigationofthebehaviourofanickelelectrodeinalkalinesolutionbyUV-VisandIRreflectancespectoscopies.Electrochim.Acta32(1987),1631–1636.

[10]ZhengG.,CaoH.,ZhengL.:Influenceofammoniaconcentrationonanodicdepositionofnickeloxide.J.Appl.Electrochem.37(2007),799–803.


1.Introduction

Themostfrequentlyusedofsyntheticfoodcoloursisthegroupofazodyes[1],whichischaracterizedbythepresenceofoneormoreazogroups–N=N–inthemolecule.Unfortunately,azodyesareoftenpotentialcarcinogens[2]. “Ponceau”, identical to “Crimson” (food additive E124) is a dye of syntheticorigin,whichexhibitingaredcolour[3].SunsetYellow(foodadditiveE110)isusedtocolorationofmanyproductsinorangecolours[4].AmixtureofsyntheticdyesE110andE124isoftenusedtoproducecaramelshadesintheproductionoffruitfillings“Rowanwithcognac”andsoftdrinks“pear”. Feketeaetal.[5]commissionedbytheFoodStandardsAgencyoftheUnitedKingdom(FSA)conductedstudiesthatshowedthattheuseofproductscontainingazodyesleadstoincreasedhyperactivityandreducedattentionconcentrationinchildren.

Simultaneous determination of synthetic dyes Ponceau 4R (E124) and Sunset Yellow (E110) by fluorimetry in soft drinks

a, a aALENAA.NIKOLAEVA *,ELIZABETHV.BULYCHEVA ,ELENAI.KOROTKOVA ,bWOLFGANGLINERT

a DepartmentofChemicalEngineering,EngineeringSchoolofNaturalResources,NationalResearchTomskPolytechnicUniversity,Leninavenue30,634050,Tomsk,Russia*[email protected]

b InstituteofAppliedChemistry,TechnicalUniversityofVienna, Getreidemarkt9,1060,Vienna,Austria


AbstractAfluorimetricmethodforsimultaneousdeterminationandquanti-ficationforofthesyntheticfoodazodyesSunsetYellow(E110)andPonceau4R(E124)insoftdrinksisproposed,whichischaracterizedbyhighsensitivityandselectivity.Thesedyesarewidelyusedinsoftdrinks,astheyhaveabrightandattractiveorangecolor.Fordetermi-nationofthedyesinbeveragestheworkingconditionsfortheanalysiswereselected:excitation wavelength is330nm, logging interval isfrom 350 to 500 nm for Ponceau 4R; the excitation wavelength is250nm,loggingintervalisfrom280to450nmforSunsetYellow.Thepossibilityofsimultaneousdeterminationoftwodyesofaredshadeinamixtureisproved,aswellastherepeatabilityofmeasurementsoffood azo dyes determination in non-alcoholic beverages by fluori-metricandspectrophotometricmethodsofanalysis.

Keywordsfluorimetrysimultaneous

determinationsyntheticdyes

This encouraged us to develop rapid, inexpensive and accurate methods todeterminationofdyesinfoodasanurgenttaskforsolvingproblemsofqualitycontrolandfoodsafety.Todate,manyanalyticalmethodsareusedtoanalyzethesynthetic dyes [6]: chromatographic, spectrophotometric, electrochemical andcapillary electrophoresis. But among the listed methods of determination ofseveraldyes inamixture ispossibleonlywiththehelpofexpensiveandtimeconsumingchromatographicmethods. Intheliterature,therearealsoreferencesconcerningtheapplicationofthefluorimetric method for the determination of dyes [7, 8]. However, despiteanumberofmeritsoffluorimetricanalysis,suchashighsensitivity,awiderangeofdetectableconcentrations,simplicityofinstrumentation,theapplicationofthismethodtodyestudieshasnotbeenpaidenoughattention. Theaimofthisresearchwastodevelopafluorimetrictechniqueforthesimul-taneous determination of synthetic food dyes Ponceau 4R (E124) and SunsetYellow(E110)inamixtureinmodelsolutionsandnon-alcoholicbeverages.Asacomparisonmethod,aspectrophotometricanalysismethodwasused.

2.Experimental


–3Work solutions of the dyes Ponceau 4R and Sunset Yellow (c=10.00 mgdm )

werepreparedusingareferencesubstanceofdyewith95%purity. Softdrinkswerechosenasthemaintargetoftheresearchforthedeterminationofsyntheticfood colors of Ponceau 4R (E124) andSunsetYellow(E110):“Japanese pear”,manufacturer of TM Irbis, Russia, Novokuznetsk; “Mirinda”, manufacturer ofPepsiCo,Spain;“Irn-Bru”(Ironbrew),manufacturerofA.G.Barrp.l.c.,Scotland.

2.2Instrumentation

The investigations were carried out on a Fluorat-02-Panorama Fluid Analyzer.Theprincipleoftheanalyzerisbasedonmeasuringtheintensitiesofthelightfluxesfromtheobjectunderinvestigation,whichariseundertheactionofexcitingoptical radiation of the selected spectral range and recorded by the opticalreceiversofthedevice. Spectrophotometric determination of Ponceau 4R and Sunset Yellow inbeverageswascarriedoutusingtheAgilentTechnologyCary60UV-Visspectro-photometer.ThequantitativeconcentrationofE124andE110dyesinthebevera-gesstudiedwasdeterminedusingacalibrationcurveofthedependenceoftheoptical density of beverage solutions on the concentration of standard dyesolutions.Samplepreparationofthebeveragesforspectrophotometricdetermi-nationwascarriedoutinaccordancewithGOSTP52470-2005“Foodproducts.Methods for identification and determination of the mass concentration of


synthetic dyes in alcohol products” and GOST P 52671-2006 “Food products.Methods for identification and determination of the mass concentration ofsyntheticdyesincaramel”.

2.3Samplepreparation

Thesamplepreparationofthebeveragesstudiedconsistedofdilutingtheinitialsample1:100withdistilledwater,whichmadeitpossibletogetridoftheharmfulinfluenceofothercomponentsinthebeverages.Thequantitativeconcentrationofthetestdyes inthesamplesofsoftdrinkswasdeterminedusingacalibrationcurveofthedependenceoffluorescencesignalintensityontheconcentrationofthedye,whichwasconstructedusingstandardsolutions.


At the first stage of the work, the dyes Sunset Yellow (E110) and Ponceau 4R(E124)weresynchronouslyscannedinordertosearchforallpotentialexcitationwavelengthsatwhichthedyeluminescenceprocessispossible[9].Theinvesti-gation showed that the highest signal intensity is observed at an excitationwavelengthof330nmforPonceau4Rand250nmfortheSunsetYellow.Inthisregard, the synchronous scanning mode was subsequently used as the majormode for simultaneous identification of two dyes E110 and E124 in themixture(Fig.1). Withthebenefitoftheadditionoptionoffluoride-02-Panoramais“Analysisofamulticomponentmixture”,mixturesoftwodyesPonceau4RandaSunsetYellowindifferentsplitswereinvestigated(Table1).AstheTable1shows,usingfluori-metricanalysis,itispossibletoquantitativelydeterminesyntheticfooddyesinamixturewithoutconstructingacalibrationcurvewithasinglestandardsolutionofeachdyeinthemixture.


–3Fig. 1Synchronous scanning of a mixture of azo dyes (c=10 mgdm ,V = 3 ml) with a mono-chromatordisplacing60nm:E110–SunsetYellow,E124–Ponceau4R,andwater.

ItcanbeseenfromFig.2thatamaximumoffluorescenceisobservedatadetec-tionwavelengthof420nmforPonceau4Rand347nmforaSunsetYellow.Theobtained wavelengths are used as operating fluorescence wavelengths todeterminethedyesinthetestbeverages. Todeterminethedyestobestudiedinbeveragesamples,aseriesofstandarddyesolutionsofvariousconcentrationswerepreparedusingacalibrationplotandthefluorescenceintensityofdyesunderthesameconditionswasmeasured.According to the calibration graph in the concentration range from 0.10 to

–31.00mgdm withregressionequationforSunsetYellow(E110)

I=0.0442x+0.1478 (1)2 R =0.9919

andforPonceau4R(E124)

I=0.0355x+0.0081 (2)2 R =0.9922


–3Fig. 2Luminescentspectrumsofaqueoussolutionsofdyestandards(c=10mgdm ):E110–SunsetYellow,E124–Ponceau4R,andwater.

Table 1Fluorimetric analysis of the simultaneous determination of dyes E110 – Sunset Yellow and

–3E124–Ponceau4R(c =10mgdm ).total

–3 –3Dyesratio(Е110:Е124) c /mgdm c /mgdm added assay

3:1 7.50:2.50 8.00:2.201:3 2.50:7.50 3.58:7.223:3 5.00:5.00 5.45:4.43

a quantitative determination of the dyes in non-alcoholic beverages has beencarried out. As a comparison method, a spectrophotometric method for theanalysisofsyntheticfooddyeswasused. Todeterminethewavelengthcorrespondingtothemaximumofabsorptivity,thespectrumsofstandardsolutionsoftheSunsetYellowandPonceau4Randtheresearchbeverageswererecordedinthewavelengthrange400–650nm(Fig.3).From the figure becomes obvious that the absorption maximum, both for thestandardsolutionandforthetestbeverage,correspondstotheSunsetYellowis482nm,andforthePonceau4Ris505nm,whichcorrespondstoGOSTRandliterature[10]. For the quantitative determination of food dyes in the research samples ofbeverages,thecalibrationcurveforthedependenceoftheopticaldensityofthesolutionontheconcentrationofstandardsolutionsE110andE124atawave-lengthof482nmand505nm,respectively,wasconstructed.Thecalibrationgraph

–3is linear in the concentration range 1.00–10.00 mgdm with the regressionequationforSunsetYellow(E110)

A=0.0579x+0.0923 (3)2 R =0.9972

andforPonceau4R

A=0.0391x+0.0827 (4)2 R =0.9995

TheresultsofthedeterminationofthesyntheticfooddyesoftheSunsetYellowandPonceau4RinsoftdrinksbytwomethodsofanalysisarepresentedinTable2.


–3Fig. 3Absorbancespectrumsofaqueoussolutionsofdyestandards(c=10mgdm ):E110–Sunsetyellow,E124–Ponceau4R,andwater.

Fromthetableitisseenthattherepeatabilitymeasurementsoffluorimetricandspectrophotometricanalysisisappeared.Besides,itwasfoundthattheconcen-trationofdyesinallsamplesofbeveragesstudieddoesnotexceedthepermissible

–3normof50mgdm .

4.Conclusions

Theinvestigationscarriedoutshowthepossibilityandusefulnessoffluorimetricmethod for the simultaneous analysis of qualitative and quantitative determi-nationofsyntheticfooddyesSunsetYellow(E110)andPonceau4R(E124)inamixture in soft drinks without using complex and prolonged samplepreparation.Comparedtothespectrophotometricanalysisforthedeterminationof dyes in food products, the fluorimetricanalysis are characterized by highersensitivity,selectivityandsimplesamplepreparation,andalsoallowsthesimul-taneousdetectionoftwodyesofredshadesinthemixture.

Acknowledgments

The work is supported by grant of Ministry ofEducation andScienceofRussianFederation forprogram“Science”.

References

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Table 2ResultsofthedeterminationoftheE110–SunsetYellowandE124–Ponceau4R insoftdrinksbyfluorimetricandspectrophotometricmethodsofanalysis(n=3,p=0.95,t =4.3).table

Softdrink Dye Fluorimetricmethod Spectrophotometricmethod tcalc.

–3 –3 c /mgdm s c /mgdm sdye r dye r

“Mirinda” E110 45.29±1.42 0.02 44.78±1.86 0.03 1.37“Japanesepear” E124 30.05±0.09 0.01 30.03±0.03 0.02 1.41“Irn-Bru” E110 21.18±0.07 0.03 – – – E124 17.08±1.06 0.02 – – –

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[10]Benvindi A., Abbasi S., Gharaghani S., Dehghan M.D., Masoum S.: SpectrophotometricdeterminationofsyntheticcolorantsusingPSO-GA-ANN.J.FoodChem.220(2017),377–384.


1.Introduction

Nowadays,theuseofcapillaryelectrophoresis(CE)fortheanalysis,identification,and characterization of different types ofmicroorganisms has receivedmuchattention.Thistechniquehasbeenfoundtoprovidemanyadvantagessuchashighseparationefficiency,shortanalysistimeorthepossibilityofdirectanalysisofbiologicalsamples[1,2].ThereisafewexamplesoftheCEuseforthedetermi-nationofbacterialpathogens[3],yeastcells[4]orvarioustypesofviruses[5].Unfortunately, this analytical method has limitations such as uncontrolledaggregation of bacterial cells and their adhesion to the capillary surface [6].Microorganismsareoftenconsiderasabiocolloids,mainlyduetothecomplexstructureoftheircellwall,andtheunderstandingoftheelectrophoreticprocessismorecomplicatedforsuchaparticles.Thecellwallcompositionischaracteristicfor various types of bacterial species, they have different content of proteins,phospholipids, polysaccharides or another organic components [7]. All com-poundspresent in thebacterial cellwall structurestronglyaffects thesurface

Analysis of Lactococcus lactis modified with zinc ions by capillary electrophoresis

a,b, b a,bANNAKRO L *,PAWEŁPOMASTOWSKI ,VIORICARAILEAN-PLUGARU ,a,bBOGUSŁAWBUSZEWSKI

a DepartmentofEnvironmentalChemistryandBioanalytics,FacultyofChemistry,NicolausCopernicusUniversity,GagarinaStreet7,87-100Toruń,Poland*[email protected]

b InterdisciplinaryCentreforModernTechnologies,NicolausCopernicusUniversity,Toruń,Poland

AbstractAdhesion to the capillary surface and uncontrolled aggregation ofbacterialcellsisasignificantdrawbackofcapillaryelectrophoresis.Inourstudy,theinfluenceoftheLactococcuslactissurfacemodificationbyzincionsatdifferentconcentrationwasdetermined.Resultsofthe

2+studyindicatedthatusingaZn causedtheuncontrolledaggregationof bacterial cells and new peaks of microbial agglomerates wereobservedintheelectropherogram.Capillaryelectrophoresiswasper-formedinanisotachophoreticmodeusingTris+boricacid+hydro-chloricacid(inlet)andTris+boricacid(outlet)buffersatpH=7.3and8.0, respectively. In addition, data from fluorescence microscopypointedoutifcapillaryelectrophoresisdoesnotcausethedeathofmicrobialcellsbutonlytheirdamage.Therefore,CEmaybeapoten-tialmethodforevaluatingtheaggregationactivityofdifferenttypesofmetalionsagainstmicrobialcells.

KeywordsbacterialaggregationcapillaryelectrophoresisLactococcuslactiszincions


chargeofmicroorganisms.Itcanbeexplainedbythepresenceofmanyfunctionalgroupsundergoingtheprotonationprocess.Therefore,itisimportanttodevelopamethod allowing to eliminate the aggregation and adhesion problem. Someresearchgroupshaveproposedtheadditionofpoly(ethyleneoxide)tothebuffersolution[8,9]whichfunctionedasafocussingagent.Anotherapproachiscapi-llarysurfacemodificationbythechemicalssuchasdivinylbenzeneortrimethyl-chlorosilaneacrylamide[10,11].Recently,thenewapproachwassuggested;itisbasedonthemodificationofthebacterialsurfacebyspecificdivalentmetalionssuchas,e.g.calcium[12].Applicationofthismethodmayresultinthecontrolledaggregationofmicroorganismscells.Althoughseveralsurfacemodificationsbycalciumionshavebeenevaluatedfortheseparationofmicroorganisms[12],thereisalackofpapersdescribedbacteriasurfacemodificationviaanothertypesofdivalentmetalions,e.g.zincions.Therefore,understandingtheinfluenceofdiffe-rent ions on microorganisms surfaces and their potential use for furthermodifications,ispivotalforanalyticalchemistry.Suchaproblemledourresearchgrouptothemaintaskoftheconductedexperiment.ThemainaimofthisstudywastoinvestigatetheelectrophoreticbehavioroflacticacidstrainofGram(+)

2+LactococcuslactisduringCEanalysisandtoexaminetheinfluenceofZn ionsatdifferent concentration on the bacterial cells aggregation. Additionally, theviabilityofLactococcuslactisbeforeandafterCEanalysisbyusingfluorescencemicroscopywasperformed.2.Experimental

2.1.SamplepreparationforCEanalysis

Therequirednumberofcellsin1mLofthesuspensionwasachievedbytheserialdilution.Thebacterialpelletwerethensuspendedinthesolutionofzincnitrateat1,3or10mMconcentrationandincubatedfor1hourattheroomtemperature.Aftertheincubation,thesuspensionwascentrifuged(20°C,9000rpm,15min)and the obtained precipitate was washed twice with distilled water andtransferredtotheoutletTBbuffer(Trisandboricacid;pH=8.0).Asacontrol,unmodifiedLactococcuslactiscellswereused.

2.2Capillaryelectrophoresisanalysis

CapillaryelectrophoresisnalysiswereperformedusingPA800plus(BeckmanCoutner system,Brea,USA) equippedwith aDADwith theuseof fused silicacapillary (i.d.=75μm;L =33.5cm;L =25cm;CompositeMetalServices,tot eff

Shipley,UK).Thebacterialsampleswereinjectedintothecapillarywithapres-suremode(10psi,8s)andtheanalysiswereperformedataconstantvoltage(20kV)andthetemperatureat23°C.Astheinletbuffer,TBH(Tris,boricacidandhydrochloric acid; pH = 7.3) were choose. The samples were detected atλ=214nm.


2.3.Fluorescencemicroscopyanalysis

Determination of Lactococcus lactis cells viability after the CE analysis wasperformed by using fluorescencemicroscopy approach according to the [13].DuringtheCEanalysis,fractionsofbacterialcellsnotandmodifiedwithzincionswere collected. Obtained bacterial samples were then stained using acridineorange (0.12 μg/mL) and ethidium bromide (0.4 μg/mL) and analyzed usingaZeissAxiocomD1 (Germany) fluorescencemicroscopewith the setof filters(43Heand38).RecordedimageswereanalyzedwithAxioVision4.8.software.


Inthisstudy,Gram(+)Lactococcuslactismodifiedwiththedifferentconcentrationof zinc ions (1,3, and10mM)wereexamined inanappliedvoltageof20kV.Bacterialstrainwithoutanysurfacemodificationwastestedasacontrolsample(Fig.1A).Theelectromigrationtimeoftheprobioticstrainmodifiedbythezincnitratesolutionatconcentrationof1,3and10mMwas2.026(RSD=0.784%),2.064(RSD=1.287%)and2.083(RSD=0.512%)minrespectively.Applicationofelectrophoretic buffers with different ionic strength (TB and TBH) allowedfocusingthezoneofcontrolsampleattheelectromigrationtimeof1.670min(RSD=1.794%).ItisinagoodcorrelationwiththeresultsobtainedbyPomastowskietal. [14], who have observed the peak of Lactococcus lactis ATCC 11454 atmigrationtimeofabout2minutes.Allelectrophoreticanalysiswerereproduciblebecauseofthemigrationtimeslowstandarddeviationvalues(Table1).Moreover,thevalueofLactococcuslactiselectrophoreticmobilitynon-andmodifiedwithzinc ions, were decreasing with the increasing CE duration time (Table 1).OutcomesofourstudyshowthataftertheLactococcuslactissurfacemodification

2+byZn ions,theformationofbacterialaggregateswithdifferentsizeandsurfacecharge occurred; it was observed by an increasing number of signals in theelectropherograms(Fig.1B,CandD).Thenumberofbacterialagglomeratesriseswith an increasing concentration of modifier. In the case of 1 and 3mMmodification,thenumberofCEzonesoccurredat18,whereasusingof10mMzincnitrateresultedintheobservationof23signals.Theobserveddifferencesintheintensity,numberandtimeofsomesignals,canbeaconsequenceofthevarioussorptionand interactionof zincatdifferent concentration levelwithbacterialproteins.Kroletal.[15]haveobservedthatadditionof3mMzincnitratetotheprobioticstrain(LactobacillusparacaseiLB3)haveresultedintheintracellularformationofzincoxidenanocompositeswhichcouldalsoformaggregatesandbeobservedasamultipliedsignalsontheCEelectropherogram.AccordingtotheirdatafromFT-IRanalysis,themaingroupsinvolvedintheZnObiosynthesiswerecarboxylandamidgroupsderivedfrombacterialproteins.Itisstronglyrelatedwithresultsobtainedinourstudy.



Fig. 1Electropherogramof(A)non-modifiedLactoccocuslactis,andmodifiedLactoccocuslactisby(B)1mM,(C)3mM,and(D)10mMofZn(NO ) .Conditions:inletbuffer:TBH(pH=7.3),outlet3 2buffer:TB(pH=8.0),suspensivebuffer:TB(pH=8.0);I=100μA,U=20kV,t=23°C,λ=214nm,L=33.5cm,L =25cm,φ=100μm,injection:10psi,t=8s.eff

Fig.2showstheFT-IRspectraofLactococcuslactisunmodifiedandmodifiedby2+Zn ions. It indicated that the main groups involved in the bacterial cells

aggregation process are deprotonated carboxyl groups (spectral bands at–11530–1560cm )whichcanderivefrombothamino-acidsofbacterialproteins

–1andpeptidoglycanoftheircellwall[16].Thespectrabandat1610–1670cm correspondswithamidegroupsofbacterialproteins.Anothercharacteristicband

–1appearsat1440cm mayderivesfromthestretchingvibrationofC–NandN–Hbond from surfacemicrobial proteins. Furthermore, as aresult of themodifi-

–1cation, the increaseof the signalat1720cm intensity canbeobserved. It isrelatedwiththestretchingvibrationofcarbonylgroups(C=O).Additionally,itcan


2+

2+

2+

1mMZn

3mMZn

10mMZn

2–1

–1

2–1

–1

2–1

–1

t/min

μ/c

mV

sRSD

/%

t/min

μ/c

mV

sRSD

/%

t/min

μ/c

mV

sRSD

/%

–2–2

–22.026

2.07×10

0.784

2.064

2.03×10

1.287

2.083

2.01×10

0.512

–2–2

–22.173

1.93×10

0.981

2.241

1.87×10

1.427

2.143

1.95×10

0.498

–2–2

–22.230

1.88×10

0.713

2.372

1.77×10

1.490

2.407

1.74×10

0.443

–2–2

–23.093

1.35×10

1.397

3.024

1.38×10

0.705

2.896

1.45×10

1.473

–2–2

–23.427

1.22×10

0.802

3.182

1.32×10

0.670

3.306

1.27×10

0.164

–3–2

–24.464

9.38×10

0.899

3.455

1.21×10

2.063

3.430

1.22×10

0.927

–3–2

–24.598

9.11×10

1.494

3.539

1.18×10

1.664

3.642

1.15×10

0.246

–3–2

–24.631

9.04×10

1.494

3.656

1.15×10

1.744

3.920

1.07×10

0.272

–3–2

–25.309

7.89×10

0.878

3.908

1.07×10

0.412

4.062

1.03×10

1.008

–3–3

–35.410

7.74×10

0.197

4.451

9.41×10

1.315

4.198

9.98×10

1.519

–3–3

–35.580

7.51×10

2.005

5.546

7.55×10

1.554

4.496

9.32×10

0.828

–3–3

–35.862

7.15×10

2.725

5.685

7.37×10

0.842

4.725

8.86×10

0.451

–3–3

–36.299

6.65×10

0.169

5.859

7.15×10

1.342

5.166

8.11×10

0.883

–3–3

–36.773

6.18×10

1.886

6.524

6.42×10

2.135

5.542

7.56×10

1.056

–3–3

–38.082

5.18×10

1.430

6.788

6.17×10

1.245

5.704

7.34×10

0.935

–3–3

–38.591

4.87×10

0.557

7.812

5.36×10

1.499

5.945

7.04×10

0.717

–3–3

–39.400

4.46×10

1.359

8.188

5.11×10

1.430

6.059

6.91×10

1.252

–3–3

–39.712

4.31×10

0.930

8.635

4.85×10

1.286

6.212

6.74×10

0.087

–3

6.310

6.64×10

0.252

–3

6.709

6.24×10

1.508

–3

7.560

5.54×10

1.195

–3

8.752

4.78×10

0.113

–3

9.639

4.34×10

1.019

Tab

le 1

Mig

rati

on

tim

esa

nde

lect

rophore

tic

mobilit

yo

fLactoccocuslactis

modif

iedw

ithd

iffe

ren

tco

nce

n-

trat

ion

ofz

inc

ion

s.

be observed that the most significant changes occur in the spectrum of theLactococcus lactismodifiedby10mMzincnitrate.Amongthecommonuseofcapillaryelectrophoresisintheidentificationofpathogens,yeastcellsandvarioustypesofviruses[3–5],aCEapproachhasalsobeenappliedforthedirectdetectofthe cell viability determination. Szumski et al. [17] have performed theassessment of the viability ofStaphylococcus aureus andEscherichia coli cellsduringcapillaryelectrophoreticprocess.Theyhavetestedtwodifferentappliedvoltage(20kVand30kV)andcollectedbacterialzonesafterCEwerecultivatedontheBrothAgar.Incomparisontothecontrolsample(withoutelectrictreatment),


Fig. 2FT-IRspectra for(A)non-modifiedLactoccocus lactis, andmodifiedLactoccocus lactisby−1(B) 1 mM, (C) 3mM, and (D) 10 mM of Zn(NO ) . Spectral bands: (1) 1420–1480 cm ,3 2

−1 −1 −1(2)1530–1560cm ,(3)1610–1670cm ,and(4)1710–1740cm .

only3.4%and0.7%oftheEscherichiacolicellssurvivedtheappliedvoltagetreat-mentof20kVand30kV,respectively.Staphylococcusaureusstrainswerefoundtobemoreresistanttotheelectricfield,resultsoftheexperimenthaveshownthatabout35%and20%Staphylococcusaureuscellssurvivedvoltageof20kVand30kV, respectively. Szumski et al. [17] have emphasized such a significantdifferencesisrelatedtothedifferentcellwallcompositionoftreatedstrains.Thebigger peptidoglycan layer ofGram(+) (Staphylococcus aureus) cellwallmakethemmore toughandresistant tocapillaryelectrophoresisconditions. It is inagoodcorrelationwithdatareceivedinourstudy.InthefluorescencemicroscopyassaytheamountoftotalcellsbeforeCEwerecomparedtothecontrolaftertheCEanalysis(Fig.3).Theresultsofthefluorescentmicroscopyanalysisindicatedthat


Fig. 3Fluorescentmicroscopyanalysisafterthecapillaryelectrophoresis(A)Lactoccocuslactisnon-modified,andLactoccocuslactismodifiedby(B)1mM,(C)3mM,and(D)10mMofZn(NO ) .3 2

capillaryelectrophoresisdonotcausethedeathofmicrobialcellsbutonlytheirdamage.ItcanbeexplainedbythefactthatLactococcuslactisistheGram(+)strainwith the thickpeptidoglycan layer in its bacterial cellwall.Applicationof thefluorescencemicroscopyconfirmedalsothatthemodificationofthebacterialcellsurfacewith 1, 3 and 10mM zinc ions causes their uncontrolled aggregation(Fig.3B,CandD).Theresultsobtainedduringtheexperimentalsoindicatethatzincionswiththehighestconcentration(10mM)causedthegreatestaggregationoftheLactococcuslactis.

4.Conclusions

In this work, CE was used to evaluate the effect of zinc ions at differentconcentrationlevel(1,3and10mM)ontheLactococcuslactiscellsaggregation.Thisstudyconfirmstheoccurrencetheformationofbacterialcellsagglomeratesunder thespecific surfacemodification.According to theFT-IRdata, themaingroupsinvolvedinthoseprocessarecarboxylandamidgroupswhichmayderivefrom surface bacterial proteins. The results of the fluorescence microscopyshowed that Lactococcus lactis cells stay alive after surface modification andduringcapillaryelectrophoresis.Incomparisonwithpreviouspapersfocussedon

2+themicrobialsurfacemodificationbycalciumions,itcanbeconcludedthatZn isnot sufficient for the Lactococcus lactis controlled clumping in CE approach.However, the described work shed new light on several important issues,includinganinterpretationofprobioticbacteriaaggregationprocessandunder-standingtheinfluenceofzincionsonmicroorganismssurfacesandtheirpotentialuseforfurthermodifications,whichseemstobecrucialforseparationsciencefield.

Acknowledgments

This work was supported by the Opus 11 No. 2016/21/B/ST4/02130 (2017–2020) from theNationalScienceCentre,Poland.

References

[1] Klodzinska E., Kupczyk W., Jackowski M., Buszewski B.: Capillary electrophoresis in thediagnosisofsurgicalsiteinfections.Electrophoresis34(2013),3206–3213.

[2] DesaiM.J.,ArmstrongD.W.:Separation,identification,andcharacterizationofmicroorganismsbycapillaryelectrophoresis.MicrobiolMolBiolRev.67(2003),38–51.

[3] HorkaM.,TesarovaM.,KarasekP.,RuzickaF.,HolaV.,SittovaM.,RothM.:Determinationofmethicillin-resistantandmethicillin-susceptibleStaphylococcusaureusbacteriainbloodbycapillaryzoneelectrophoresis.Anal.Chim.Acta.868(2015),67–72.

[4] ShenY.,Berger S.J., SmithR.D.: Capillary isoelectric focusingof yeast cells.Anal. Chem.72(2000),4603–4607.

[5] OkunV.M.,MoserR.,BlaasD.,KenndlerE.:Complexesbetweenmonoclonalantibodiesandreceptorfragmentswithacommoncoldvirus:determinationofstoichiometrybycapillaryelectrophoresis.Anal.Chem.73(2001),3900–3906.

[6] DziubakiewiczE.,BuszewskiB.: Capillary electrophoresisofmicrobial aggregates.Electro-phoresis35(2014),1160–1164.


[7] SaltonM.R.J.:Studiesofthebacterialcellwall:IV.Thecompositionofthecellwallsofsomegram-positiveandgram-negativebacteria.Biochim.Biophys.Acta10(1953),512–523.

[8] SchneiderheinzeJ.M.,ArmstrongD.W.,SchulteG.,WestenbergD.J.:Highefficiencyseparationofmicrobialaggregatesusingcapillaryelectrophoresis.FEMSMicrobiol.Lett.189(2000)39–44.

[9] KlodzinskaE.,DahmH.,RozyckiH.,SzeligaJ.,JackowskiM.,BuszewskiB.:RapididentificationofEscherichiacoliandHelicobacterpyloriinbiologicalsamplesbycapillaryzoneelectrophoresis.J.Sep.Sci.29(2006),1180–1187.

[10]BuszewskiB.,SzumskiM.,KłodzinskaE.,DahmH.:Separationofbacteriabycapillaryelectro-phoresis.J.Sep.Sci.26(2003),1045–1049.

[11]BuszewskiB.,KłodzinskaE.:DeterminationofpathogenicbacteriabyCZEwithsurface-mo-difiedcapillaries.Electrophoresis29(2008),4177–4184.

[12]RogowskaA.,PomastowskiP.,ZłochM.,Railean-PlugaruV.,KrolA.,RafinskaK.,Szultka-MłynskaM., Buszewski B.: The influence of different pH on the electrophoretic behaviour ofSaccharomycescerevisiaemodifiedbycalciumions.Sci.Rep.8(2018),DOI:10.1038/s41598-018-25024-4.

[13]Railean-PlugaruV.,PomastowskiP.,RafinskaK.,WypijM.,KupczykW.,DahmH.,BuszewskiB.:Antimicrobialpropertiesofbiosynthesizedsilvernanoparticlesstudiedbyflowcytometryandrelatedtechniques.Electrophoresis37(2016),752–761.

[14]PomastowskiP.,Szultka-MłynskaM.,KupczykW.,JackowskiM.,BuszewskiB.:EvaluationofintactcellMatrix-AssistedLaserDesorption/IonizationTimeof-FlightMassSpectrometryforCapillaryElectrophoresisdetectionofcontrolledbacterialclumping.J.Anal.Bioanal.Tech.S13(2015),008.DOI:10.4172/2155-9872.S13-008.

[15]Krol A., Railean-Plugaru V., Pomastowski P., Złoch M., Buszewski B.: Mechanism study ofintracellularzincoxidenanocompositesformation.ColloidsSurf.A553(2018),349–358.

[16]Naumann D., Keller S., HelmD., Schultz C., SchraderB.: FT-IR spectroscopy and FT-Ramanspectroscopy are powerful analytical tools for the non-invasive characterization of intactmicrobialcells.J.Mol.Struct.347(1995),399–405.

[17]SzumskiM.,KłodzinskaE.,DziubakiewiczE.,HrynkiewiczK.,BuszewskiB.:Effectofappliedvoltage on viability of bacteria during separation under electrophoretic conditions. J. Liq.Chromatogr.Relat.Technol.34(2011),2689–2698.


1.Introduction

Since thedate of their invention in 1958, carbonpaste electrodesunderwentavery impressive development, pursuing the progress in electrochemistry,electroanalysis,andinstrumentalanalysisassuch.Thisyear,60yearshavepassedsincethedesignofthecarbonpasteelectrodesbyR.N.Adams[1].Overthepastsixdecades, carbon paste – amixture of graphite powder and a binder (pastingliquid), hasbecomeoneof themostpopular electrodematerials used for thepreparationofvariouselectrodesandsensors[2].Graphiteiscommonlyusedintheelectrochemicalstudiesduetoitslowbackgroundcurrentandwidepotentialwindow.Moreover,thegraphitepasteelectrodeshavehighsensitivityandlowcostwhichmakesthemmoreandmorepopular[3,4].Carbonpasteelectrodesareused as working electrodes in voltammetry for the determination of electro-chemicalactivecompounds,suchaspharmaceuticals[5]orpesticides[6].Typicalpropertiesoftherespectivecarbonpastemixturedependsonthetypeandqualityofusedgraphite,aswellasitsamountinthemixture.Theparaffinoilsarethemostpopularbindingagentsusedforpreparationofcarbonpastemixtures[2]. Acemetacin, the glycolic acid ester of indometacin, is non-steroidal anti-inflammatorydrug,whichiscommonlyusedinrheumatoidarthritistreatment.Acemetacinmayinhibitprostaglandinsynthesisandproduceanti-inflammatory,analgesic,andantipyreticeffects[7].Thedrugispracticallyinsolubleinwater. In this paper, the voltammetric method of acemetacin determination inpharmaceuticalswasdeveloped.

Voltammetric studies of acemetacin

NATALIAFESTINGER*,KAMILAMORAWSKA,SYLWIASMARZEWSKA,WITOLDCIESIELSKI

DepartmentofInorganicandAnalyticalChemistry,FacultyofChemistry,UniversityofLodz,12TamkaStreet,91-403,Lodz,Poland*[email protected]


AbstractAvoltammetricmethodforthesensitivedeterminationofacemetacinusingcarbonpasteelectrodeisproposed.Undertheoptimumcondi-tions,thecalibrationcurvewaslinearintheconcentrationrangefrom

–8 –6 –13.0×10 to1.0×10 molL .Thelimitofdetectionandlimitofquanti-–9 –1fication were calculated and were equal to 6.45×10 molL and

–8 –12.15×10 molL , respectively. The developed method wassuccessfully applied for the determination of acemetacin in thepharmaceuticalformulations.

Keywordsacemetacincarbonpasteelectrodedeterminationvoltammetry

2.Experimental


Allchemicalswereprovidedfromcommercialsources.Thesematerialswereusedwithout furtherpurification.Thestandardsolutionsofacemetacinweremade

–3 –1dailybyappropriatedilutionofthestocksolution(c=1.0×10 molL ),whichwasmadebydissolvingappropriateamountof thecompoundinacetone.The0.04M Britton–Robinson buffer was used in experiments as the supportingelectrolyte. The pharmaceutical formulation (Rantudil Retard, 90 mg) wasobtained from local pharmacy store and used as received. The content of sixcapsuleswasmixedwithproperamountofacetoneandthendilutedtoreceiveappropriateacemetacinconcentration.2.2Instrumentation

ElectrochemicalexperimentswereconductedusingMultiAutolabpotentiostat/-galvanostatwithNova1.10softwareandelectrodestandM164type(mtm-anko)withthree-electrodesystemconsistingofcarbonpasteelectrodeasaworking

–1electrode, Ag/AgCl (3molL KCl) as a reference electrode and Ptwire as anauxiliaryelectrode.Thecarbonpasteelectrodewasmadebyplacingthepaste(300 μL of paraffin oil and 1.0 g of graphite) in a piston-driven carbon pasteelectrodeholderwithinnerdiameter6mm.Beforeeachsetofmeasurements,thesurface of electrode was refreshed by polishing on wet filter paper. Allmeasurementswerecarriedoutatroomtemperature.


TheBritton-RobinsonbufferinwidepHrange(1.5–9.0)wastestedassupportingelectrolyteforacemetacindetermination.ThehighestsignalsofacemetacinwererecordedinBritton-RobinsonbufferpH=2.1(Fig.1),thereforeBritton-RobinsonbufferpH=2.1wasselectedforfurtherstudiesassupportingelectrolyte.Next,parameters of squarewave voltammetrywere chosen (Table 1) according toacemetacinsignals’shapeandheight.

Usingtheoptimizedparametersofsquarewavevoltammetrytechnique,thecalibrationcurveofacemetacindeterminationwasfoundintherangefrom0.03to

–11.0μmolL (Fig.2).Thelimitofdetermination(LOD)andlimitofquantification(LOQ) for acemetacin were calculated from the calibration curve using thefollowingequations:

LOD=3SD/b (1)

LOQ=10SD/b (2)

whereSD isstandarddeviationandb isaslopeofthelinearcalibrationcurve.


Fig. 1RelationshipbetweentheacemetacinpeakcurrentandthepHofBritton-Robinsonbuffersin(A)pHrange1.5–9.0,and(B)pHrange1.5–2.5.


Parameter Examinedrange Optimalvalue

Steppotential/mV 1–15 7Amplitude/mV 10–100 20Frequency/Hz 2–100 20

Table 1Optimumparametersofsquarewavevoltammetrytechniqueforacemetacindetermination

Fig. 2Squarewavevoltammogramsforaceme-tacincalibrationcurve.Acemetacinconcentra-tions:0.03,0.05,0.07,0.10,0.30,0.50,0.70,and

–11.00 μmol L . Voltammograms recorded inBritton-RobinsonbufferpH=2.1.

Limit of determination and limit of quantification were equal to 6.45 and –121.5nmolL ,respectively(Table2).Tocheckthecorrectnessofthedeveloped

method,theaccuracyandprecisionofsquarewavevoltammetrywerecalculatedforincreasingconcentrationsofacemetacininthelinearrange.TheCVwasequalintherangeof4.5–9.0%.Fromtheobtainedresults,itcanbeconcludedthatthe

proposedmethodprovidesgoodsensitivityandrepeatabilityfortheacemetacindetermination. Because the acemetacin is frequently used as anti-inflammatory drug inrheumatoidarthritis, itsdetermination in real samples isvery important.Thedetermination of acemetacin in pharmaceutical formulations was conductedusing triple standard addition method under the optimum conditions ofexperiment.Foranalysedpharmaceuticalformulationwithdeclaredamountof90.00mg of acemetacin, the found amountwas 90.09mg of acemetacin; therecoverywas100.11%andCV1.70%.Developedmethodgavesatisfyingresultsofrecovery.Itwasobservedthatpharmaceuticalformulationmatrixdonotproduceadditionalsignalsanddonotinterfereinacemetacindetermination.

4.Conclusions

Aninnovative,simpleandlow-costmethodfortheacemetacindeterminationwasdeveloped.Thecalibrationcurvewasobtainedforacemetacinconcentrationin

–1range0.03to1.0μmolL .Thecarbonpasteelectrodewasappliedforacemetacindeterminationinrealsampleswithgoodrecovery(100.1%forRantudilRetard).To our knowledge, this is the first study to demonstrate the possibility ofelectrochemicaldeterminationofacemetacinbasedonoxidationprocess.

Acknowledgments

ThisworkwassupportedbytheUniversityofLodz,PolandunderGrantforyounginvestigatorsB1711100001602.02.


Table 2RegressiondataofthecalibrationcurveforthedeterminationofacemetacininBritton-RobinsonbufferpH=2.1.

Parameter Value

–1 –8 –6Linearrange/molL 3.0×10 –1.0×10 –1 –7Linearregressionequation I[A]=5.66c[molL ]+3.55×10

2R 0.9993–1 –9LOD/molL 6.45×10 –1 –8LOQ/molL 2.15×10

References

[1] AdamsR.N.:Carbonpasteelectrodes.Anal.Chem.30(1958),1576–1576.[2] Svancara I., Vytras K., Kalcher K.,Walcarius A.,Wang J.: Carbon paste electrodes in facts,

numbers,andnotes:areviewontheoccasionofthe50-yearsjubileeofcarbonpasteinelectro-chemistryandelectroanalysis.Electroanalysis21(2009),7–28.

[3] SvancaraI.,WalcariusA.,KalcherK.,VytrasK.:Carbonpasteelectrodesinthenewmillennium.Cent.Eur.J.Chem.7(2009),598–656.

[4] AlAqadK.M.,SuleimanR.,Al-HamouzO.C.,SalehT.A.:Novelgraphenemodifiedcarbon-pasteelectrodeforpromazinedetectionbysquarewavevoltammetry.J.Mol.Liq.252(2018),75–82.

[5] Smarzewska, S., Pokora, J., Leniart,A., Festinger,N.,Ciesielski,W.:Carbonpasteelectrodesmodified with graphene oxides – comparative electrochemical studies of thioguanine.Electroanalysis28(2016),1562–1569.

[6] PappI.,SvancaraI.,GuzsvanyV.,VytrasK.,GaalF.:Voltammetricdeterminationofimidaclopridinsecticideinselectedsamplesusingacarbonpasteelectrode.Microchim.Acta166(2009),169–175.

[7] hehataT.M.,AbdallahM.H.,IbrahimM.M.:Proniosomaloraltabletsforcontrolleddeliveryandenhanced pharmacokinetic properties of acemetacin. AAPS PharmSciTech 16 (2015),375–383.


1.Introduction

Vanillylmandelic acid (DL-4-hydroxy-3-methoxybenzeneacetic acid) is found inurinewithothercatecholaminemetabolites(homovanillicacid,metanephrine,andnormetanephrine).Normalconcentrationsofvanillylmandelicacidinurine

–1describedinthebibliographyarefrom11.6to28.7µmolL [1].Increasedurinaryvanillylmandelicacidlevelisfoundinpatientswithtumors,pheochromocytomaandneuroblastoma[2].Themostcommonmethodsfordeterminationofvanillyl-mandelicacidareHPLCandELISA(enzyme-linkedimmunosorbentassay)[3].Duetohydroxylgrouponaromaticsystem,vanillylmandelicacidiselectrochemi-callyoxidizableandthussuitableforelectroanalyticaldetermination.

HPLC-ED/UV for determination of vanillylmandelic acid in human urine after solid phase extraction

a,b, a b, aANNAMAKRLIKOVA *,HANADEJMKOVA ,TOMA S NAVRATIL ,JIR IBAREK ,aVLASTIMILVYSKOCIL

a UNESCOLaboratoryofEnvironmentalElectrochemistry,DepartmentofAnalyticalChemistry,FacultyofScience,CharlesUniversity,Hlavova2030/8,12843Prague2,CzechRepublic

*[email protected] J.HeyrovskýInstituteofPhysicalChemistryoftheASCR,v.v.i., Dolejškova3,18223Prague8,CzechRepublic

AbstractHPLCwithelectrochemicalandspectrophotometricdetection(ED/-UV)aftersolidphaseextraction(SPE)wasusedfordeterminationofvanillylmandelic acid in human urine. HPLC-ED was performed ataglassycarbonelectrodeina“wall-jet”arrangementinacetate-phos-phatebufferatpH=2.5andgradientelution(increasingcontentofacetonitrilefrom5to25%in10minutes)wasused.Optimizedpara-

−1meters were following: flow rate of mobile phase 1mLmin ,detection potential +1.1V, detection wavelength 279nm, injectedvolume 20µL. Dependence of the peak current on the analyteconcentration was linear in the concentration range from 10 to

−1 −1150µmolL , with obtained limits of detection2.6µmolL (calcu-−1latedfrompeakheight)and1.9µmolL (calculatedfrompeakarea)

−1for HPLC-ED, and 11.0 µmolL (calculated from peak height) and−19.8µmolL (calculatedfrompeakarea)forHPLC-UV.

KeywordsHPLCsolidphaseextractiontumorbiomarkervanillylmandelicacid


In this work, determination of vanillylmandelic acid in human urine usingHPLCwithelectrochemicalandspectrophotometricdetection(ED/UV)withsolidphaseextraction(SPE)willbepresented.Theaimofthisstudywastodeterminevanillylmandelic acid in human urine without difficult sample pre-treatment.Solid phase extraction was used for filtration of samples to protect the HPLCcolumnfromclogging.

2.Experimental


–1Fortestingexperiments,thestocksolution(1000µmolL )ofvanillylmandelicacid was prepared by dissolving 4.95mg of the pure substance in 25mL of

–1deionized water and then diluted to 100 µmolL solution containing 25% ofacetonitrile.ToobtainhighrecoveryduringtheSPEprocedure,10%aceticacid

–1was added to each sample of urine. Acetate-phosphate buffer (0.05molL in–1phosphoricacidand0.05molL inaceticacidwiththeappropriateamountof

–10.2molL sodiumhydroxidesolution)atpH=2.5wasusedasamobilephase.

2.2Instrumentation

HPLC-ED measurements were performed in a “wall-jet” arrangement withaglassy carbon working electrode (3 mm, Metrohm, Switzerland), a reference

–1argentochloride electrode (3 molL KCl, Monokrystaly, Czech Republic), andaplatinum counter electrode (Monokrystaly, Czech Republic). Apparatus forHPLC-ED/UVconsistsofhighpressurepumpBeta10(Ecom,CzechRepublic),injection valve with a 20 µL loop (Ecom, Czech Republic), degasser DG 4014(Ecom,CzechRepublic),UV/VISdetectorSapphire800(Ecom,CzechRepublic),and amperometric detector ADLC 2 (Laboratornı prıstroje, Czech Republic)connectedinseries.AKromasilEternity-5-PhenylHexyl4.6×150mm(AkzoNobel,Netherlands)HPLCcolumnwasusedforseparations.Apartfromvanillylmandelicacid,othertumorbiomarkers(5-hydroxyindole-3-aceticacidandhomovanillicacid) were added into human urine for separation and detection. For simulta-neousseparation,agradientelutionprogramwasused,linearlyincreasingthecontentofacetonitrileinthemobilephasefrom5to25%in10minutes.Optimumconditionsforobtainingbestresultswereasfollows:flowrateofmobilephase

–1was 1mLmin , detection potential for ED was set up to +1.1 V, and for UVdetection,wavelength279nmwasused. Samplepre-treatmentincludedonlySPEasafiltrationtechniquewithcom-merciallyavailableSPEcolumns(LiChrolutEN200mg3mLstandardPP-tubes,MerckMillipore,Germany),wheremethanolwasusedasaSPEeluent. SoftwareClarity2.3(DataApex,CzechRepublic)wasusedforrecordingHPLCchromatograms,MicrosoftOfficeExcel2010(Microsoft,USA)andOriginPro8.0


(Origin Lab, USA) were used for calculating calibration curve parameters andgraphic expressions of results. The limit of detection (LOD) was calculatedaccordingequation

LOD=3s/a (1)

wheresisthestandarddeviationofthreerepetitivemeasurementsofthelowestmeasurableconcentrationandaistheslopeofthecalibrationcurve[4].


Firstly,pilotexperimentswithbuffer(compositionoftheusedbufferwasadoptedfrom paper [5]) were done. Mobile phase was a mixture of acetate-phosphatebuffer at pH=2.5 and acetonitrile, where the content of acetonitrile linearlyincreasedfrom5to25%in10minutes.Calibrationdependencesforbiomarkers

–1inbufferwerelinearinthewholetestedconcentrationrange(0.5to10µmolL ).Relative standard deviations for 10 measurements (concentration of vanillyl-

–1mandelicacidwas100µmolL )werenothigherthan16%forHPLC-EDandnothigherthan3.5%forHPLC-UV. NextstepofthisresearchwastodeveloptheoptimumSPEprocedure.Methanolprovidedbetterextractionrecovery(almost100%)incomparisonwithaceto-nitrile.TheSPEcolumnwasusedonlyforfiltrationtoavoiddestructionoftheHPLCcolumn.ActivationoftheSPEcolumnandelutionwereperformedwith5mLofmethanol,andalsoinjectionofsamples(20µL)wasperformedfrom5mLofeachsample. Last part of this research was devoted to application of this method fordetermination of vanillylmandelic acid in human urine. Fig. 1 shows HPLC-EDrecordings of urine and urine with addition of 1 mL of the stock solution ofvanillylmandelicacid,5-hydroxyindole-3-aceticacid,andhomovanillicacid(each

–1of1000µmolL )intoa7mLurinesample.Fig.2thendepictsHPLC-UVrecor-dingsofthesameanalysisasinFig.1.Therewerenoproblemswithinterferences

–1inHPLC-ED;foundnativeconcentrations(⁓13µmolL )inurinefullycorrespond–1withpublishedconcentrations(from11.6to28.7µmolL )[1].Ontheotherhand,

thereweresomeinterferencesobservedinHPLC-UV,andfoundnativeconcen-trations are thus not corresponding with the published values. For thedeterminationoftheconcentrationoftheanalyte,thestandardadditionmethodwasused.Dependenceswerelinearinthetestedconcentrationrangefrom10to

–1 –1150µmolL ;obtainedLODswere2.6µmolL (calculatedfrompeakheight)and–1 –11.9µmolL (calculatedfrompeakarea)forHPLC-ED,and11.0µmolL (calcu-

–1latedfrompeakheight)and9.8µmolL (calculatedfrompeakarea)forHPLC-UV.



Fig. 1 HPLC-ED recordings of urine and urine with addition of vanillylmandelic acid (VMA),–15-hydroxyindole-3-aceticacid(5-HIAA),andhomovanillicacid(HVA),each100µmolL .Glassy

carbon electrode, acetate-phosphate buffer at pH =2.5, gradient elution (increasing content of–1acetonitrilefrom5to25%in10minutes),flowrateofmobilephase1mLmin ,detectionpotential

+1.1V,injectedvolume20µL.

Fig. 2HPLC-UV recordings of urine and urinewith addition ofof vanillylmandelic acid (VMA),–15-hydroxyindole-3-aceticacid(5-HIAA),andhomovanillicacid(HVA),each100µmolL .Acetate-

phosphatebufferatpH=2.5,gradientelution(increasingcontentofacetonitrilefrom5to25%in–110minutes),flowrateofmobilephase1mLmin ,detectionwavelength279nm,injectedvolume

20µL.

4.Conclusions

HPLC-ED/UV after SPE was successfully used for determination of vanillyl-mandelicacidinhumanurine.Solidphaseextractionprocedurereplacedfiltra-tionofsamplesofhumanurinetoavoidproblemswithclogginganHPLCcolumn;anyothersamplepre-treatmentwasnotused.Presentedmethodcouldbeusedfor screening of human urine, especially of infants, because HPLC-ED is verysensitive method and allows simultaneous determination of all three tumorbiomarkers, vanillylmandelic acid, 5-hydroxyindole-3-acetic acid, and homo-vanillicacid.

Acknowledgments

This research was carried out within the framework of the Specific University Research(SVV260440).A.M.thankstheGrantAgencyoftheCharlesUniversity(ProjectGAUK734216)forthefinancialsupport.J.B.andT.N.thanktheGrantAgencyoftheCzechRepublic(project17-03868S).

References

[1] Garcıa A., Heinanen M., Jimenez L. M., Barbas C.: Direct measurement of homovanillic,vanillylmandelic and 5-hydroxyindoleacetic acids in urine by capillary electrophoresis.J.Chromatogr.A871(2000),341–350.

[2] Magera M. J., Thompson A. L., Matern D., Rinaldo P.: Liquid chromatography-tandem massspectrometrymethodforthedeterminationofvanillylmandelicacidinurine.Clin.Chem.49(2003),825–826.

[3] Shirao M. K., Suzuki S., Kobayashi J., Nakazawa H., Mochizuki E.: Analysis of creatinine,vanilmandelic acid, homovanillic acid and uric acid in urine by micellar electrokineticchromatography.J.Chromatogr.B693(1997),463–467.

[4] InczedyJ.,LengyelT.,UreA.M.:CompendiumofAnalyticalNomenclature:DefinitiveRules1997.3rdEd.BlackwellScience,Malden1998,p.106–127.

[5] DejmkovaH.,AdamkovaH.,BarekJ.,ZimaJ.:Voltammetricandamperometricdeterminationofselectedcatecholaminemetabolitesusingglassycarbonpasteelectrode.Monatsch.Chem.148(2017),511–515.


1.Introduction

The presence and ecotoxicological effects ofmedicinal products in thewaterenvironmentareoneofcurrentproblemsinenvironmentalchemistry.Morethaneightypharmaceuticallyactivesubstances,includingiodineX-raycontrastagents,

–1werefounduptothelevelofμgL insewage,surface,andgroundwater.Theirincreasedabundanceinthewaterenvironmentisconnectedtotheimprovementof health care over the past fifty years. From tens up to hundreds grams ofiodinatedcontrastagentsarerequired foronediagnostic test.Then iodinatedcontrastagentsareeliminatedfromhumanbodyintheurineinnon-metabolizedform,whichisnotcompletelyremovedinwatertreatmentplantsandleadstotheenhancedadsorbableorganichalidesformation[1]. Analytical techniques suitable for the determination of single iodinatedcontrastagentsare,e.g.,highperformanceliquidchromatographyorcapillaryelectrophoresiswithsuitabledetectionasUV-Vis,photometricandmassspectro-metry.Liquidchromatographyconnected tomass spectrometryusingelectro-

ICP-MS analysis as a tool for monitoring of the efficiency of the sorption based removal of iodinated contrast agents

JANPATOCKA*,ANNAKREJCOVA ,KATER INAKLAUSOVA

DepartmentofEnvironmentalandChemicalEngineering,UniversityofPardubice,Studentská95,53210Pardubice,CzechRepublic*[email protected]


AbstractTheinductivelycoupledplasmamassspectrometry(ICP-MS)methodfortheiodinedeterminationwasdeveloped.Theinstrumentallimitof

127 –1detection(3σ)for Iwas0.28ngl ,recoveries100–118%,repeat-abilities1.23–4.99%.IodineinthecertifiedreferencematerialNCS

–1ZC81002b (human hair, the certified value 0.96±0.2μgkg ) was–1found1.15±0.07μgkg . Themethodwasusedfortheevaluationof

thesorptionbasedremovalofiodinatedcontrastagentsfromwaterand artificial urine using activated carbon, humic acids and drybiomass of green algae Chlorella kessleri. The tested iodinatedcontrast agentswere Iomeron andXenetix. Potassium iodidewasusedtoevaluatethesorptionofinorganiciodineinordertoassessfullytheapplicabilityofproposedapproachesinrealhospitalwaste-waterscontainingiodineinvariousforms.

KeywordsICP-MSiodinatedcontrastagentsiodinesorption

sprayionization,inthecomparisonwiththeothers,allowsperformingnotonlyquantificationbut also identificationof iodinated contrast agents inunknownsamples[2–4].Totaliodinecanbedeterminedusingspectraltechniquessuchareinductivelycoupledplasmaopticalemissionspectrometryandinductivelycou-pledplasmamassspectrometry(ICP-MS)[5]. Incaseofourlaboratoryresearchexperiments,iodinatedcontrastagentscanbemonitoredasiodine.

2.Experimental


ThedeionizedwaterpurifiedusingthesystemMilli-Q(Merck,Germany)wasusedinthiswork.Allreagentswereofanalytical-reagentgrade.Nitricacid65%(v/v),analyticalgrade(LachNer,CzechRepublic)wasdistilledinsub-boilingdistillationequipment(BSB939IR,Berghof,Germany).Forstabilizationofiodinesolutions,25%tetramethylammoniumhydroxide(analyticalgrade,Sigma-Aldrich,Germa-ny)wasused. The stock artificial urine was prepared according to Dawson [6]: 3.9 gNH H PO ,5.08gNaCl,2.86gKCl,0.312gCaCO ,0.418gMgCl .6H O,18.1gof4 2 4 3 2 2

urea,8.7mL35%HCl,and0.67mL96%H SO tothefinalvolumeof100mL(all2 4

analyticalgradefromLach-Ner,CzechRepublic).Fornextexperiments,thestockartificialurinewastentimesdiluted. Theiodinecalibrationstandardsolutionsofconcentrations0.1,0.5,1.0,5.0,

–1and10.0μgL werepreparedfrompotassiumiodide(analyticalgrade,Lach-Ner,CzechRepublic)in1%tetramethylammoniumhydroxide.However,tetramethyl-ammoniumhydroxidewasusedonlyat themethoddevelopingandvalidatingstage.Fortheiodinedeterminationinsamplesfromsorptionexperiments,tetra-methylammonium hydroxide was not used and calibration standards werepreparedfreshrightbeforeanalysis.

–1 The single element standard solution of tellurium 1.000±0.002gL (SCPScience,Canada)wasusedfortheinternalstandardpreparation. ThecertifiedreferencematerialNCSZC81002b(humanhair,China)withthe

–1certifiedvalueofiodine0.96±0.2μgkg wasusedforthemethodvalidation. The evaluated sorbentswere activated carbonHydraffin CC 8X30 (Donau-chem,CzechRepublic),theChlorellakesslerialgaebiomass(InstituteofBotanyoftheCzechAcademyofSciences,CzechRepublic)andhumicacids(sodiumhumate,Humatex,CzechRepublic).


Thetestediodinatedcontrastagents(structuresinFig.1;[7,8])wereIomeron–1containingjomeprol(400gL ofiodine,BraccoImagingDeutschland,Germany)

–1andXenetixcontainingjobritridol(350gL ofiodine,Guerbert,France).


Samples of liquid phase from sorption experiments were taken after thecentrifugation(5minutes)andsubsequently1000folddilutedforanalysis. Theoriginaliodinatedcontrastagentsweremineralizedusingthemicrowaveoven:1mLof1000folddilutedsamplein6mLof65%HNO ,(i)160°Cat50bars,3

(ii)200°Cat75bars,(iii)50°Cat75bars,filledupto100mLandsubsequently1000folddilutedforanalysis. About0.3goftheCRMNCSZC81002bwasmineralizedunderthesamecondi-tionsasweretheiodinatedcontrastagents,filledupto25mLanddilutedtoget

–1 –1thetheoreticaliodineconcentration4µgL forIomeronand3.5µgL forXenetix. Wastewaters obtained from the Pardubice Hospital were stabilized in 1%tetramethylammonium hydroxide and then: (i) filtrated and hundred timesdiluted,(ii)filtratedandfivehundredtimesdilutedforaseriesofspikedsamples

–1withpotassiumiodide(1,2and4μgL )forstandardadditionapproach,and(iii)digested (1 ml of raw wastewater, the same conditions as for the iodinatedcontrastagents,filledupto50mL,fourtimesdiluted).

2.3Instrumentation

The inductively coupled plasma orthogonal acceleration time-of-flight massspectrometer (oaTOF-ICP-MS) Optimass 9500 (GBC Scientifc Equipment,

–1Australia)equippedwiththeconcentricnebuliserMicroMist(0.4mLmin )anda70mLthermostatted(10°C)cyclonicspraychamber(bothGlassexpansion,Australia)wasusedforthedeterminationofiodine. ThemicrowavesystemSpeedwaveXpert(Berghoff,Germany)wasusedformineralizationofsamples. ThesorptionexperimentswererealizedusingthelaboratoryshakerHeidolphVibramax 100 (Heidolph Instruments GmbH & CO. KG, Germany) and theEppendorfCentrifuge5804R(EppendorfAG,Germany).


Fig. 1Chemicalstructuresof(a)Iomeprol,and(b)Iobitridol.

(a) (b)


3.1TheICP-MSmethodvalidation

127 125Iodinewasmeasuredontheisotope I.Basedontheliterature[9], Tewas–1addedasaninternalstandard(10µgL )incalibrationstandardsandmineralized

samplesofcertifiedreferencematerialNCSZC81002b. ThereliabilityoftheICP-MSmethodwasprovenbyrecoveriesandrepeatabi-litiesobtainedfromtherepetitiveanalysisof iodinestandardsolutions,which

125were101–112%and1.08–4.91%whenusing the internal standard Te and100–118%and1.23–4.99%withouttheinternalstandard. The determined iodine concentration in certified reference material NCS

–1ZC81002b was 1.15±0.07 mg kg (n = 4) complied with the certified value–10.96±0.2mgkg .

Thelimitsofdetectioncalculatedastheconcentrationrelatedtothreetimesthestandarddeviationoftheintegratedpeakareameasurednearthemonitoredion

–1peakforthestandardsolutioncontaining0.1µgL ofIinthreereplicateswere–1 –10.28ngL (instrumental)and0.28µgL (proceduralforsorptionexperiments).

3.2Theanalysisofiodinatedcontrastagents

Thedilutedormineralizediodinatedcontrastagentswereanalysedinordertoassess the ICP-MSmethodapplicability for themonitoringof sorptionexperi-ments.Tetramethylammoniumhydroxidewasnotused for thestabilizationofthesesamplesbecausetheyareconsideredtobeverystable.

–1 The iodine concentration for Iomeron was 371±18.2gL (diluted) and–1 –1385±44.7gL (digested) and for Xenetix 287±12.9gL (diluted) and

–1294±39.1gL (digested). The lower recoveries were probably caused by thesamplematrixincaseofonlydilutediodinatedcontrastagentsandbyanalytelossesduringdigestionincaseofdigestedsamples.Possiblesystematicerrorsinsorptionexperimentswereovercomebyrelatingofactualtooriginconcentration.Forthesorptionmonitoring,thesampleswereonlydilutedandnotetramethyl-ammoniumhydroxideortechnetiumwasadded.

3.3Sorptionexperiments

Thesorptionexperimentswererealizedusingactivatedcarbon,biomassofalgaeChlorella kessleri and humic acids as sorbents for iodinated contrast agentsIomeronandXenetixandpotassiumiodideremovalfromwaterandtheartificialurine.Thesorptionexperimentswereperformedin15mLcentrifugetubeswith0.1;0.2;0.5;1.0gofsorbentsandtestedsubstanceswithiodineconcentration

–1 –120mgL for Iomeron and potassium iodide and 17.5mgL for Xenetix. Thetesting tubeswereplacedon laboratory shaker for2hours.Then, samplesofliquidphaseweretakenforiodineanalysis.


Thesorptionexperimentswerecarriedoutforeverycombinationoftestediodi-natedcontrastagentswithsorbentsusedinwaterandartificialurine.TheresultsarepresentedinTable1.Fortheexperimentsshowingverylowiodineremovalefficiency,theiodineremovalpercentagecouldnothavebeencalculatedbecausethedeterminediodineconcentrationattheendoftheexperimentwasslightlyhigher than the starting one. This situation may have occurred from several

–1reasonsandtheircombinations:(i)memoryeffectsforiodineatsubµgL levelswerenoticedduring ICP-MSmeasurements, (ii) severe iodinememoryeffectsoccurredduringsamplepreparationandmanipulation,whichwasdiminishedbutnotcompletelyavoidedusingdisposablelaboratoryequipment,and(iii)theabsence of internal standardization, which may have led to the short timenebulizingefficiencychangesandtheinstrumentsignalinstability.

3.4Analysisofrealhospitalwastewater

The iodine concentrations in the real hospital wastewater were:–1 –1(i)0.85±0.02mgL for filtered and diluted, (ii) 1.07±0.04mgL for filtered,

diluted and quantified using the standard addition approach, and –1(iii)0.71±0.03mgL formineralizedsamples.Theobtainedresultssupportthe


m /g Removalefficiency/%s

Water Artificialurine

Activated Biomassof Humicacid Activated Biomassof Humicacid carbon Chlorellakessleri carbon Chlorellakessleri

Iomeron0.1 88 5 7 69 6 4

a a0.2 98 16 3 73 – –a0.5 99 10 8 89 – 1a1.0 98 11 11 89 – 9

Xenetixa a a a0.1 62 2 – – – –

a a a a0.2 95 – 2 – – –a a a0.5 96 4 2 – – –

a a a a a1.0 97 – – – – –Potassiumiodide

a a a a0.1 46 – – 67 – –a a a0.2 31 – 1 79 – –

a a a0.5 75 1 – 89 – –a a a a1.0 90 – – 89 – –

aImpossibletoevaluate;nosorptionhasoccurred.

Table 1Thepercentageefficiencyofiodinatedcontrastagentsremovalfromchosenmediausingevaluatedsorbents(m –massofusedsorbent).s

ideaofanalytelosesduringmicrowavedigestionandmildmatrixinterferencewhenexternalcalibrationstandardsusedforquantification.

4.Conclusions

The developed ICP-MS method provided sufficient sensitivity (LOD =procedure–1=0.28µgL )foriodinemonitoringinthesorptionexperimentsandrealsamples

ofhospitalwastewater.Theresultsrevealedthatthebestofsorbentsusedwasactivated carbonwith the removal efficiencies 99, 97, and 90% for Iomeron,Xenetixandpotassiumiodideinwaterand89%forbothIomeronandpotassiumiodideintheartificialurine.TheChlorellakesslerialgaebiomassandhumicacidsrevealed significantly lower removal efficiencies: 11, 2, and 1% for Iomeron,Xenetixandpotassiumiodideinwaterand9%forIomeronintheartificialurinewhen the difference between these two sorbents was insignificant. Activatedcarbonseemstobeasuitablesorbentfortheremovalofiodineinbothorganicandinorganicformseveninthepresenceofcomplexmatrix.

Acknowledgments

AuthorsacknowledgethesupportfromtheUniversityofPardubice,FacultyofChemicalTechnologyprojectsSGFCHT05/17andSGFCHT05/18.

References

[1] Steger-HartmannR,LangeR.,SchweinfurthH.:EnvironmentalriskassessmentforthewidelyusediodinatedX-raycontrastagentiopromide(Ultravist).Ecotoxicol.Environ.Saf.42(1999),274–281.

[2] ArbughiT.,BertaniF.,CelesteR.,GrottiA.,TironeP.:High-performanceliquidchromatographicdeterminationoftheX-rayimagingcontrastagent,iofratol,inplasmaandurine.J.Chromatogr.B701(1997),103–103.

[3] VanHouckeS.K.,SeauxL.,CavalierE.,SpeeckaertM.M.,DumoulinE.,LecocqE.,DelangheJ.R.:Determinationof iohexoland iothalamate inserumandurinebycapillaryelectrophoresis.Electrophoresis37(2016),2363–2367.

[4] Hirsch R., Ternes T.A., Lindart A., Haberer K., Wilken R.D.: A sensitive method for thedeterminationofiodinecontainingdiagnosticagentsinaqueousmatricesusingLC-electro-spray-tandem-MSdetection.FreseniusJ.Anal.Chem.366(2000),835–841.

[5] OliveiraA.A.,TrevizanL.C.,NobregaJ.A.:Iodinedeterminationbyinductivelycoupledplasmaspectrometry.ApplSpectroscRev.45(2010),447–473.

[6] DawsonJ.B.,EllisD.J.,Newton-JohnH.:Directestimationofcopperinserumandurinebyatomicabsorptionspectroscopy.ClinChimActa.21(1968),33–42.

[7] WhitehouseG.H.:Foreword.Eur.J.Radiol.18(1994),vii.[8] ContiM.,MottaR.,PuggioliC.,BrambillaP.:Surface-activatedchemicalionization-electrospray

ionization mass spectrometry combined with two-dimensional serial chromatography isapowerful tool for drug stability studies. Rapid. Commun. Mass. Spectrom. 27 (2013),1231–1236.

[9] JerseA.,JacimovicR.,MarsicN.K.,GermM.,S irceljH.,StibiljV.:DeterminationofiodineinplantsbyICP-MSafteralkalinemicrowaveextraction.Microchem.J.137(2018),355–362.


1.Introduction

Capillaryzoneelectrophoresis(CZE)isawellknownandwidelyusedtechniqueenablingtheseparation,andidentificationofmicroorganisms[1].DespitesuchwidespreaduseofCZE,thistechniqueisintheprocessofevolutionandrequiresthedevelopmentofnewmethodsforsamplepreparation.Inrecentyearsalotofinterest has been focused on the possibility of applying this technique toelectroanalysistoprovidetheidentificationofwildstainsofmicroorganismsindiagnosticslaboratory.However,theelectrophoreticanalysisofsuchcomplicatedsystemsasmicroorganismscancausemanydifficultiesassociatedwithuncon-trolledcellaggregationandadhesiontotheinnersurfaceofthecapillary.Thenewapproachtoeliminatethisprobleminvolveschangesoffunctionalgroupsonthemicrobialsurfacebydivalentmetalionsresultingincontrolledcellsclumping[2]. Saccharomyces cerevisiae canbe calledamodelorganismbecause theyaresmallwithashortgenerationtimeandcanbeeasilycultured[3].Theyeastscellwallstructureiscomposedofpolysaccharidesandproteins[4].Suchaconstru-ctionoftheyeastcellwallresultsintherebeingmanyfunctionalgroupsonits

The influence of pH on the electrophoretic behaviour of yeast modified by calcium ions

a, b a bAGNIESZKAROGOWSKA ,PAWEŁPOMASTOWSKI ,MICHAŁZŁOCH ,a, b a, b a, bVIORICARAILEAN-PLUGARU ,ANNAKRO L ,KATARZYNARAFIN SKA ,

b a, b,MAŁGORZATASZULTKA-MŁYN SKA ,BOGUSŁAWBUSZEWSKI *

a CentreforModernInterdisciplinaryTechnologies,NicolausCopernicusUniversity, Wileńska4,87-100Torun,Polandb DepartmentofEnvironmentalChemistryandBioanalytics,FacultyofChemistry,NicolausCopernicusUniversity,Gagarina7,87-100Torun,Poland*[email protected]


AbstractTheinfluenceofadifferentpHonSaccharomycescerevisiaeyeastcellsmodifiedwith calcium ionswas investigatedby the capillary zoneelectrophoresis.Theobtainedresultsindicatethatthemodificationofsurfacefunctionalgroupsbycalciumionssignificantlyaffectedtheefficiencyofelectrophoreticmobility.Moreover,themicroscopicandspectrometricanalysisshowsthatthepHvalueofthecalciumionssolutionhasasignificanteffectontheintensityyeastcellsclumping.However,thesechangesdidnotaffectontheaccuracyofS.cerevisiaeidentificationbyMALDIequipmentwithBioTyperplatform.Theseresults formtheanalytical solution forcouplingofelectrophoresisandMALDI-TOFMStechnique.

Keywordscapillaryzone

electrophoresiscellsclumpingMALDI-TOFMSyeast

surface such as phosphate, carboxyl and amino groups. Under appropriateconditionsofpH,thesegroupsaredeprotonated,whichallowstheirinteractionwithpositivechargedmetalionsresultsincellsflocculation[5].Manymetalions

2+ 2+such asMg or Zn have been described as yeast cell aggregation inducers.However, calcium ions are known as the most effective in flocculation pro-motion[6]. The aim of this study was to investigate the impact of the cells surfacemodification by calcium ions on its clumping and on the effectiveness of theelectrophoreticmobility.ThenovelapproachofthemicrobialsamplepreparationfortheCZEanalysismayconstituteasignificantcontributiontothefutureuseofthistechniqueindiagnosticslaboratory.Moreover,thespectrometricanalysisofyeastmodifiedbycalciumionsmaybeafoundationforthecouplingofcapillaryelectrophoresisandMALDI-TOFMSanalysistoeliminatethepreconcentrationproblemofmicrobiologicalsamples.

2.Experimental

AllsolventsandmaterialswerepurchasedfromSigmaAldrich(Poznan,Poland).Ultra-purewaterwaspurifiedusingtheMilli-QRGsystem(Bedford,USA). InordertopreparationofyeastcellsmodifiedbycalciumionsatdifferentpHS.cerevisiaesamplesweresuspendedin5mMCa(NO ) adjustedtothedesiredpH3 2

bytheadditionofCa(OH) .After60minutesofincubation,thesuspensionwas2

centrifuged.Theresultingprecipitatewaswashedtwicewithwatertoremoveunboundcalciumions. In order to performedmicroscopic analysis, sample ofmodified yeastwasdropped on the watch glass and observed using stereoscopic fluorescentmicroscopewithCellsoftware(Olympus,SZX16,Shinjuku,Tokyo,Japan). Forthespectrometricanalysisthesampleofmodifiedandnon-modifiedyeastwasanalyzedaccordingtothepreviouslydescribedmethodology[7]. To performed capillary zone electrophoresis analysis modified and non-modifiedS.cerevisiaecellsweretransferredtotheoutletTBbufferofpH=7.98(4.5mMtris(hydroxymethyl)aminomethane,50mMboricacid)astheinletbufferwasusedTBHofpH=7.31(4.5mMtris(hydroxymethyl)aminomethane,50mMboric acid, 3.31 mM hydrochloric acid). Directly before the CZE analysis theobtainedyeastsamplewassonificated.TheCZEanalysiswasperformedusingPA800plus(BeckmanCoutnersystem,USA)equippedwithaDADdetectorwiththeuseoffusedsilicacapillaries(id=75μm;L =33.5cm;L =25cm;Compositetot eff

MetalServices,UK).


To determine the effect of the surface modification of S. cerevisiae on theelectrophoreticmobility,theelectrophoreticanalysiswasconducted.Fig.1shows


theelectropherogramsoftheyeastunmodifiedandmodifiedbycalciumionsatdifferentpHconditions.Theelectromigrationtimeoftheyeastmodifiedbythecalcium solution of pH = 6.0, 8.0 and 9.0 was 3.099, 4.013 and 4.099 min,respectively.Theresultsindicatethatthesurfacemodificationof(bio)colloidhasasignificantimpactonitselectrophoreticmobility.ThisphenomenonwasalsoobservedbyPomastowskietal. [7] inthecaseofbacteria.Moreover,afterthemodification,thesharpeningofthepeaksandimprovementoftheshapeofthebase line on the electropherogram aswell as the reduction in the number ofaggregatesandtheimprovementofthereproducibilitycanbeobserved.Anotherinteresting observation is the effect of pH on modified (bio)colloid behaviorduring theelectrophoreticanalysis.With the increase in thepHofmediuminwhich sorption was conducted, the increase in the electromigration time ofmodifiedyeastcellsandasadecreaseintheelectrophoreticmobility.Thehighestreproducibility (RSD < 5%) of the electrophoretic analysis was obtained forS.cerevisiae which was modified at pH=9.0. This phenomenon is probablyconnectedwiththetotaldeprotonationofsurfacefunctionalgroups.


Fig. 1 Electropherogram of (A) non-modified Saccharomyces cerevisiae, and (B) and modifiedSaccharomycescerevisiaebyCa(NO ) atpH=6.0,7.0,8.0and9.0.3 2

Inordertohighlightthechangesonmolecularprofileof(bio)colloidsunderdifferentpHconditions,theinfluencetheelectrophoreticproperties,microscopicand spectrometric studies ofmodified yeastswere performed.On theMALDIspectra (Fig. 2) for calcium-modified yeast, can be observedmany significantchangesincomparisontospectraofnativecells.However,clusteranalysisofMSP


Fig. 3MSP dendrogram for MALDI-TOF MS spectra of Saccharomycescerevisiae modified with Ca(NO ) 3 2

solution at pH = 6.0, 8.0, 9.0 andnativeS.cerevisiae.

Fig. 2MALDI-TOF MS spectra of Saccharomyces cerevisiae suspended in Ca(NO ) solution at3 2

(A)pH=6.0,(B)pH=8.0,(C)pH=9.0,and(D)nativeS.cerevisiae.

(MainSpectrumProfile)spectra(Fig.3)ofcontrolandyeastsincubatedincalciumsolution at pH = 6.0, 8.0 and 9.0 show that the greatest change inmolecularprofiles,ascomparedtothecontrolsample,wasobservedfortheyeastincubatedincalciumsolutionatpH=9.0.Thechangeswereobservedatmolecularlevelof

2+ribosomal,cytosolandmembraneproteins.ItisknownthatpresenceoftheCa inthe culture medium may strongly influence the expression of proteins inS.cerevisiaecells,mainlyribosomalorribosome-associatedproteinsaswellas

2+proteinsrelatedtothecellwall.HighconcentrationsofCa leadstooverexpress-ionofcellwallintegrityregulatorsasaresponsetotheosmoticandionicchangesthat occur in themedia [8] which can explain the observations in this workchangesofproteinprofilesduringMALDITOF/MSanalysis. Themicroscopicobservationshowsthatthehighestaggregationoftheyeastcellswasachievedforthesystem,wheresolutionatpH=9.0wasused.Inthecaseof thecalciumsolutionatpH=6.0, cluster formationwasn'tobserved (Fig.4).Moreover,microscopicresultsprovethattheyeastsurfacechangeswithacalciumion affect the aggregates formation. A similar phenomenon was observed byDziubakiewiczetal.forthebacterialcells[2].Asaresultofthebacteriasurface


Fig. 4Microscopic imagesofSaccharomyces cerevisiae suspended in5mMCa(NO ) solutionat3 2

(A)pH=6.0,(B)pH=8.0,and(C)pH=9.0.

charge modification by calcium ions, the cells created a compact aggregates,whereby,fewerhigh-intensitysignalsontheelectropherogramswereobserved.Theyattributedthisscoretothebridgingeffectofcalciumionsbetweenbacterialcells. According to thewell-known theory of dicationic bridges, carboxyl andamine surface groups are involved in the formation of aggregates. Partiallydeprotonatedcarboxylgroupsandprotonatedaminogroupsarenotabletoformbridges between (bio)colloids. After yeast centrifugation from calcium saltsolution and suspended in TB buffer, probably borate ions associate and/orcomplex adsorbed calcium ions. The created ions of TB buffer respectivelyassociatecomponentsofthemicroorganismssurface.Inturn,yeastsuspendedina calcium solution at pH = 8.0 form aggregates with part-compact structure(Fig.4).

4.Conclusion

Capillary zone electrophoresis allows for the determination of a variety ofbiological systems. However, the complexity of the microorganism surfacemorphologyforcedustocarryoutaseriesofresearchonthephysicochemicalproperties in order to interpret the phenomena occurring at the interface of(bio)colloids. Themicroscopic and spectrometric studies performed for non-modifiedandmodifiedS.cerevisiaehaveshownthatunderspecifiedconditionsthetestedsystemtendstoaggregate.Thisphenomenonexplainsthestabilityof(bio)colloidsubjectedtothebiosorptionprocessinelectrophoreticseparationconditions. In addition, it was observed that alongwith the increasing pH ofmodifiertheweightandsurfaceofparticlesofcolloidalsystemincreases,whichresults in the reduction of the (bio)colloid electrophoreticmobility. Obtainedresults indicate that the proposed new sample preparation approach of wildmicroorganisms strains may be a foundation for the application of capillaryelectrophoresisindiagnosticslaboratoryinthefuture.Moreover,theresultsshow

2+thatthedifferentpHandmodificationofthecellsbyCa influencethemolecularprofilesofyeastcellsbutdonotaffecttheidentificationqualitybytheMALDI-TOFMS equipmentwith the BioTyper database. Such resultsmay provide a basisenablingforcouplingofcapillaryelectrophoresisandtheMALDI-TOFMSanalysis.

Acknowledgments

ThisstudywasfinanciallysupportedbyOpus11No.2016/21/B/ST4/02130(2017–2020)fromtheNationalScienceCentre,Poland.

References

[1] Buszewski B., Kłodzinska E.: Rapidmicrobiological diagnostics inmedicine using electro-migrationtechniques.TrendsAnal.Chem.78(2016),95–108.

[2] Dziubakiewicz E., Buszewski B.: Capillary electrophoresis ofmicrobial aggregates.Electro-phoresis35(2014),1160–1164.

[3] BlackwellM.:TheFungi:1,2,3…5.1millionspecies?Am.J.Bot.98(2011),426–438.


[4] SomervilleC.,BauerS.,BrininstoolG.,FacetteM.,HamannT.,MilneJ.,OsborneE.,ParedezA.,PerssonS.,RaabT.,VorwerkS.,YoungsH.:Towardasystemsapproachtounderstandingplantcellwalls.Science306(2004),2206-2211.

[5] MillP.J.:ThenatureoftheinteractionsbetweenflocculentcellsintheflocculationofSaccharo-mycescerevisiae.J.Gen.Microbiol.35(1964),61–68.

[6] MikiB.L.A.,PoonN.H.,JamesA.P.,SeligyV.L.:PossiblemechanismforflocculationinteractionsgovernedbygeneFLO1inSaccharomycescerevisiae.J.Bacteriol.150(1982),878–889.

[7] PomastowskiP.,Szultka-MłynskaM.,KupczykW.,JackowskiM.,BuszewskiB.:EvaluationofintactcellMatrix-AssistedLaserDesorption/IonizationTimeof-FlightMassSpectrometryforcapillaryElectrophoresisdetectionofcontrolledbacterialclumping.J.Anal.Bioanal.Tech.S13(2015),008.

[8] Haramati O., Brodov A., Yelin I., Atir-Lande A., Samra N., Arava Y.: Identification andcharacterizationofrolesforPuf1andPuf2proteinsintheyeastresponsetohighcalcium.Sci.Rep.7(2017)3037.


1.Introduction

Photochemicalvaporgeneration isanexpandingandpromisingsample intro-duction technique for analytical atomic spectrometry. Inphotochemical vaporgeneration,ananalyteisconvertedtothevolatilespeciesthroughtheactionofUV-radiation. The presence of a photochemical agent in the liquid phase isrequired (e.g., formic acid). The photochemical generator usually consists ofasourceofUV-radiation,mostoftenamercuryUVtubelamp(emittingmainlyat254nm),andareactioncoilthatistightlywrappedaroundandwherethesampleisconvertedtothevolatilespecies.ThematerialofthereactioncoilmustbemadeofamaterialtransparentforUV(quartzorteflon).Theadvancedphotoreactors(high-efficiencyflow-throughphotoreactors)utilizeamodifiedmercuryUVlampwhere thesample is irradiated in the innerchannel (quartz tube) thatpassesthroughthedischargeoftheUVlamp.SinceUVlighthastotransmitonlyaquartzwalloftheinnerchannel,thesephotoreactorswereshowntoirradiatethesample

Optimization of photochemical vapor generation of molybdenum as a sample introduction for ICP-MS

a,b,* aJAKUBSOUKAL ,STANISLAVMUSIL

a DepartmentofTraceElementAnalysis,InstituteofAnalyticalChemistryoftheCzechAcademyofSciences,Veveří97,60200Brno,CzechRepublic

b DepartmentofAnalyticalChemistry,FacultyofScience,CharlesUniversity, Hlavova8,12843Prague,CzechRepublic*[email protected]

AbstractThisarticledealswithoptimizationofconditionsofphotochemicalvaporgenerationofmolybdenum.Thevolatilespeciesofmolybde-num were generated in the flow arrangement, when sample wasinjectedtoastreamofareactionmedium.Efficientgenerationwasaccomplishedusinga19Whigh-efficiencyflow-throughphotoreactorusing formic acid as the reactionmedium. The generated volatileproduct(mostprobablymolybdenumhexacarbonyl)wasintroducedto an inductively coupled plasmamass spectrometer for sensitivedetection. Irradiation time, formicacid concentrationandeffectofadditiveswere carefully studiedwith theaim to reach thehighest

– –generationefficiency.Interferencesfrominorganicanions(NO , Cl ,32– – –SO , NO , andClO ) werealsoindetailinvestigated.Thelimitof4 2 4

–1detectionachievedatoptimalconditionswas1.2pgmL .

KeywordsICP-MSmolybdenumphotochemicalvapor

generationUV


withphotochemical agentmoreefficiently.Photochemicalvaporgeneration isapplicablenotonlytomercuryandhydrideformingelements(Se,As,Sb,Te,Pb,Bi)butalsononmetals(I,Br,Cl,S)andsometransitionmetals(Ni,Fe,Co,Cu,Cd,Os) [1–4]. In this work, we present the photochemical vapor generation ofmolybdenum,whichhasneverbeenearlierreported.

2.Experimental

AschematicdiagramoftheUV-photochemicalvaporgenerationsysteminaflow-injectionmodehyphenatedtoICP-MSisdepictedinFig.1.Photochemicalvaporgenerationwasaccomplishedusinga19Whigh-efficiencyflow-throughphoto-reactor(BeijingTitanInstruments,China).Thegeneratedvolatileproductwasdirectedbyanargoncarriergas toaplasticgas-liquidseparator(50mL)andintroducedintoaspraychamberofanAgilent7700xinductivelycoupledplasmamassspectrometer(ICP-MS).Asolutionof1%nitricacidmixedwithasolutionofrhodiuminternalstandardwasnebulizedintothespraychambercreatingrobustwetplasmaconditions.ThetypicalconditionsofICP-MSusedfordetectionare:RF

–1 –1power 1600W, nebulizer Ar 1.02Lmin , dilution Ar (via HMI) 0Lmin ,Ar–1chemifold for photochemical vapor generation 100mLmin , ICP-MS pump

–1 –10.35mLmin carrierliquidand0.06mLmin internalstandard,spraychamber–1temperature2 °C,Hecollisioncellgas4.1mLmin ,measurementmode: time

95 98resolved analysis, measured isotopes (dwell time): Mo (0.1 s), Mo (0.1 s),103Rh(internalstandard,0.05s).


Fig. 1Schematicdiagramoftheflowinjection-photochemicalvaporgenerationsystemhyphenatedtoICP-MSforvaporintroductionofvolatilemolybdenumspecies


Firstly, a flow rate ofAr (chemifold) for photochemical vapor generationwasoptimizedbutithadnosignificantinfluenceonsensitivityintherangebetween

–1100and500mLmin .Theimportantaspectforphotochemicalvaporgenerationisanirradiationtime.Theinfluenceofirradiationtimeofanalytewasinvestigatedbychangingtheflowrateofthereactionmedium(30%formicacid).Thehighest

–1sensitivitywasfoundat1.25mlmin .Thisflowrateofthereactionmediumis–1equaltoirradiationtimeof39sinthephotoreactor.Theflowrate1.25mlmin of

thereactionmediumisreflectedinatotaltimeofthemeasurementof350speronesampleinjection.Afterward,aconcentrationofformicacidasthereactionmediumwasoptimized.TheworkspublishedonphotochemicalvaporgenerationofNi so far suggest that thehigher generating efficiency is achievedbyusingformicacidasareactionmediumforphotochemicalvaporgeneration[5–6].Inthiswork, themaximumsensitivity at50% formic acidwas found formolyb-denum.However,thisconcentrationwasnotusedfurtherduetoaninevitabledilutionofsample,instabilityofthesignalbaseline,increaseinsignalbaselinelevelduetocontaminationandformicacidvaporsloadintoplasma.Hence,30%formicacidwaschosenasacompromiseforfurtherexperiments.Aninfluenceof

3+ 2+ 2+variousadditives(transitionmetalsFe ,Ni ,Cu ionsoraceticacid,etc.)aswellas adjustment inpHof the reactionmediumwith respect topotential enhan-

3+cementingenerationefficiencywasalsoinvestigated.OnlyFe ionsincreasedsignificantlygenerationefficiencybyafactorofaround1.4.Apartoftheresearchwastargetedoninterferencefromcommoninorganicacidsandtheirsalts(Fig.2).Especially nitric acid and nitrates were found serious interferents duringphotochemicalvaporgenerationofmolybdenum.


Fig. 2Relative effects of added inorganic acids and salts onphotochemical vapor generationof–11ngmL molybdenumin30%formicacid.


–1 Theachievedlimitofdetectionatchosenoptimalconditionswas1.2pgmL .–1Repeatabilityofthemeasurementat1ngmL ofmolybdenumwasbetterthan

3%.Theaccuracyandfeasibilityofthissensitivemethodologywassuccessfullyverifiedbyanalysisofcertifiedreferencematerials:onefreshwater(SRM1643e)andtwoseawatermaterials(NASS-7andCASS-6).Duetonitricacidpresenceusedfor stabilization of the certified referencematerials, the materials had to bediluted andmeasured using standard addition technique (Table 1). The flowinjectionphotochemicalvaporgenerationICP-MSwasalsosuccessfullyappliedontwosamplesofdietarysupplements.Thetabletspurchasedatalocalpharmacyweredissolvedin30%formicacid,sonicatedat50°Candfurtherdilutedwith30%formicacid(forphotochemicalvaporgenerationICP-MS)orwith1%nitricacid(nebulization-ICP-MS).AlltheresultsarecomparedinTable2.

4.Conclusion

Photochemical vaporgenerationofmolybdenum hasbeen thoroughly investi-gatedusingICP-MSasadetector.Themethodisextremelysensitivewithaverygoodrepeatability.Molybdenumhexacarbonylissupposedtobethegeneratedvolatileproduct.Veryhighefficiencyofphotochemicalvaporgenerationhasbeenreached using 30% formic acid. Further enhancement can be achieved with

3+additionof Fe ions.Accuracyof thedevelopedmethodhasbeenverifiedonwatercertifiedreferencematerialsandontwosamplesofdietarysupplements.Photochemicalvaporgenerationispronetoseriousinterferencesfromnitrates

–1Certifiedreferencematerial Concentrationofmolybdenum/ngmL

Certified Found

SRM1643e(Freshwater) 121.4±1.3 127.7±18.9NASS-7(Seawater) 9.29±0.4 9.51±0.37CASS-6(Nearshoreseawater) 9.15±0.52 8.98±0.07

Table 1ConcentrationsofmolybdenuminwatercertifiedreferencematerialsdeterminedbyflowinjectionphotochemicalvaporgenerationICP-MSusingstandardadditiontechnique.

Dietarysupplement Concentrationofmolybdenum/µgpertablet

declared FI-PVG-ICP-MS nebulization-ICP-MS CentrumAZ 50 56.8±5.1 56.7±1.9SupradynCoQ10energy 50 45.7±2.9 46.8±1.6

Table 2Concentrationsofmolybdeumindietarysupplementsamples(µgper1tablet)determinedbyflowinjectionphotochemicalvaporgenerationICP-MS(FI-PVG-ICP-MS)usingstandardadditiontech-niqueandbynebulization-ICP-MSusingexternalcalibration.

butalsochlorideswhichhavetobetakenintoaccountwithrespecttorealsamplepreparation.

Acknowledgments

The support of the Grant Agency of the CzechRepublic (P206/17-04329S), CzechAcademy ofSciences (Institutional supportRVO:68081715)andCharlesUniversity (projectSVV260440) isgratefullyacknowledged.

References

[1] RybınovaM., CervenyV.,Hranıcek J.,RychlovskyP.: UV-fotochemicke generovanı tekavychsloucenin pro potreby atomovych spektrometrickych metod. Chem. Listy 109 (2015),930–937.

[2] YinY.G.,LiuJ.F.,JiangG.B.:Photo-inducedchemical-vaporgenerationforsampleintroductioninatomicspectrometry.TrACTrendsAnal.Chem.30(2011),1672–1684.

[3] HeY.H.,HouX.D.,ZhengR.E.,SturgeonR.E.:Criticalevaluationof theapplicationofphoto-chemicalvaporgenerationinanalyticalatomicspectrometry.Anal.Bioanal.Chem.30(2007),769–774.

[4] SturgeonR.E.:Photochemicalvaporgeneration:aradicalapproachtoanalyteintroductionforatomicspectrometry.J.Anal.Atom.Spectrom.32(2017),2319–2340.

[5] GuoX.,SturgeonR.E.,MesterZ.,GardnerG.:UVphotosynthesisofnickelcarbonyl.Appl.Organo-metal.Chem.18(2004),205–211.

[6] LiuL.,DengH.,WuL.,ZhengC.,HouX.:UV-inducedcarbonylgenerationwithformicacidforsensitivedeterminationofnickelbyatomic fluorescencespectrometry.Talanta80 (2010),1239–1244.


1.Introduction

VolatileN-nitrosamines are well-known carcinogens, which were detected infood,cosmeticproducts,beer,andmalt.ThedeterminationofvolatileN-nitros-aminesinamaltandbeerareroutinelymeasuredbyagaschromatographywithmassspectrometric(GC-MS)oranitrosospecificchemiluminescencedetection(GC-NCD).Thesumofallnitrosogroupsinthesampleischaracterizedbyappa-rent total nitroso compounds. VolatileN-nitrosamines forms less than 5% ofapparenttotalnitrosocompoundsinmalt[1].Therestofapparenttotalnitrosocompoundsconsistedofnon-volatilenitrosocompoundsofunknownstructure.Studyofthesecompoundsmoredeeplywouldrevealtheirhealtheffect. Theaimof thisstudywas todevelopaminiaturizedextractionmethod forscreeningofnon-volatilenitroso compoundsusing theGC-NCD.Anextractionmethodconsistsofthepreparationofamaltsample,namelyanextractionand

Development of miniaturized extraction method used for GC-NCD screening of non-volatile nitroso compounds in malt

a,b, a,b aMICHAELAMALECKOVA *,TOMA S VRZAL ,JANAOLSOVSKA

a ResearchInstituteofBrewingandMalting,Inc.,Lípová15,12044Prague2,CzechRepublicb DepartmentofAnalyticalChemistry,FacultyofScience,CharlesUniversity, Hlavova8,12843Prague2,CzechRepublic*[email protected]


AbstractThe aim of this study was to develop a miniaturized extractionmethodforafastscreeningofnon-volatilenitrosocompoundsusinggaschromatographywithanitrosospecificchemiluminescencedete-ction.Accordingtoafinalmethodology,thesampleswerepreparedbyextractionofgrindedmaltusingamixtureofpyridineandacetonitrileinratio60:40(v/v).Toenhancevolatilityofthedeterminedanalytes,the two-step derivatization using hexamethyldisalazane andN,O-bis(trimethylsilyl)trifluoroacetamidewasused.The total volu-meofthesamplewas200μlandthepreparationtimeafteroptimi-zationwas80min.Theextractionmethodwasconnectedtoaclassifi-cation method, which can divide chromatographic peaks into thegroupsofN-nitrosoandC-nitrosocompounds,andinterferingsub-stances.Afterapplicationof themethodsmentionedabove to realmaltsamples, thespecificchromatographicpeaksofC-nitrosoandN-nitrosocompoundswereselected.

Keywordschemiluminescence

detectorderivatizationextractiongaschromatographynitrosocompounds

aderivatization by hexamethyldisilazane and N,O-bis(trimethylsilyl)trifluoro-acetamide[2]. ForatotalGC-NCDscreeningofthenon-volatilenitrosocompoundsinmalt,used extractionmethod is coupled with a classificationmethod [3], that candistinguishchromatographicpeaksintothegroupsofN-nitroso,C-nitroso,C-nit-roso/nitrocompoundsandinterferingsubstances.

2.Experimental


Used chemicals: pyridine (≥99.8%), acetonitrile (≥99.9%), ethyl acetate(≥99.5%),N,O-bis(trimethylsilyl)trifluoroacetamidewith trimethylchlorosilane(99:1,v/v),hexamethyldisilazane(≥99.0%),andtrifluoroaceticacid(≥99.0%),allfromSigma-Aldrich,chloroform(≥99.8%,Merck),diethylether(99.9%),acetone(99.97%),andtoluene(≥99%)allfromLach-Ner.

2.2Instrumentation

ExtractionandderivatizationstepswereperformedonaheatingblockPierceReacti-ThermI.ThesampleswereanalysedonthechromatographThermoTrace1310,equippedwithcapillarycolumnTG-200MS(30m,0.25mmIDand0.25μmdf,trifluoropropylmethylpolysiloxane).Sampleinjectionsof2.0µlwereperfor-medat210°Cbyasplittechnique(1:10).Aconstantflowofargon,asacarriergas,

–1wasmaintainedat0.6mlmin .Atemperatureprogrammewaschosenasfollows:–1 –1 –150°C(1min);20°Cmin ;150°C(5min);10°Cmin ;210°C(3min);10°Cmin ;

320°C(6min).DetectionwascarriedoutbyEllutia820TEANCDatdifferenttemperaturesofapyrolytictube(500,650,700,750and800°C)toobtainrelated

–1pyrolyticprofiles.Anoxygenflowforozonegenerationwas3.2mlmin .


Usedmaltswerepilsner,munich,caramel,wheatandcolouredtype.Barleygrainswerealsoused.Groundmaltorhuskormaltflour(50mg)wasextractedby50μlofamixtureofpyridine:acetonitrile(60:40,v/v)for10minat65°C.Hexamethyl-disilazane(100μl)andtrifluoroaceticacid(1μl)wasadded,andafter30minat65°C,N,O-bis(trimethylsilyl)trifluoroacetamide(50μl)wasadded.Sampleswereestablished for 30min at 65°C and subsequently were cooled for 10min atlaboratory temperature. The extractwas transferred to a new vial insert andanalysedbytheGC-NCD.


2.4Dataprocessing

Experimental designs were evaluated by the ANOVAwith a back eliminationstrategy. Data were analysed by the principal component analysis (PCA), theANOVAandhierarchicalclusteringwithEuclideandistanceandWardmethod.Datawereevaluatedatasignificancelevelof0.05.


3.1Developmentoftheextractionmethod

Inprimaryexperiment,groundmalthuskswereextractedby100µlofsolventandderivatizedby100µlofN,O-bis(trimethylsilyl)trifluoroacetamidefor60minutesat 65 °C. In the first three samples, the pyridine, chloroform, andN,O-bis(tri-methylsilyl)trifluoroacetamidewereusedassolvents.Resultingchromatogramsshowed satisfying peak intensities of the extracted analytes, therefore, otheraprotic solvents were tested in the next experiment. Causentively, pyridine,chloroform,acetone,acetonitrile,diethylether,ethylacetateandtoluenewereusedandtestedassolvents,separately.Aftercomparingintensitiesofthechro-matographicpeaks,thesamplesextractedusingpyridine,acetonitrile,andethylacetatehadthehighestanalytes’abundance.Tofindoutthebestmixtureofthesethree solvents, the 3-factor mixture design was applied. The samples werepreparedusing100μlofthemixtureofthedifferentsolventsratioand100μloftheN,O-bis(trimethylsilyl)trifluoroacetamideandanalysedbyGC-NCD.Becauseofthedifferenceofthechromatographicpeaks’intensities,thedataweredividedintotwoblocksasfollows:Block1includespeakselutingintherangeofretentiontimes4–16minandBlock2intherangeofretentiontimes23–27min.Thesumofthechromatographicpeakareaoftheblockswasusedasaresponse(y)inthemixturedesignexperimentsdescribedbyalinearmathematicalmodel

y=Σβ (±ε )x +Σβ (±ε )x x (1)i i i ij ij i j

whereβ is the regression coefficient of the factor x , andβ is the regressioni i ij

coefficientoftheijinteractionbetweenthefactorsx andx ,andε ,ε arestandardi j i ij

errorsoftheregressioncoefficientsβ ,β ,respectively.Thelinearmathematicali ij

model with evaluated regression coefficients and their standard errors aredescribedbytheequation(2)forBlock1,andequation(3)forBlock2

y=18.66(±5.86)x +10.11(±5.86)x +4.91(±5.86)x +p a e

+73.66(±27.00)x x +24.19(±27.00)x x +10.90(±27.00)x x (2)p a p e a e

y=321.40(±89.42)x +157.00(±89.42)x +223.70(±89.42)x +p a e

+1024.60(±412.14)x x +184.20(±412.14)x x –p a p e

–255.10(±412.14)x x (3)a e


Fig. 1Thecontourplotofthe3-factormixturedesign.

wherefactorsx ,x andx representsthevolumeratioofthepyridine,acetonitrilep a e

andethylacetate,respectively.Thecoefficientsofdeterminationofthesemodelswere81.04%and76.20%,respectively.ThemodelsprojectedintothecontourplotsareinFig.1.Accordingtobothlinearmathematicalmodels,theoptimummixtureofpyridine:acetonitrile:ethylacetateis60:40:0(v/v/v). In the next step of themethod development, acetone and chloroformweretested,inadditiontotheprevioussolventsbya5-factormixturedesign.Thesumof classified chromatographic peak area was used as a response. The linearmathematicalmodelwiththeevaluatedregressioncoefficientsandtheirstandarderrorsareinequation

y= 191.00(±51.51)x +283.00(±56.00)x +42.00(±56.00)x +ac a ch

+448.00(±56.00)x +322.00(±56.00)x –p e

–2690.00(±821.87)x x +1344.00(±821.87)x x (4)a e ch p

where factors x , x , x , x and x represents volume ratio of the acetone,ac a ch p e

acetonitrile,chloroform,pyridineandethylacetate,respectively.Thecoefficientof determination for this model was 89.46%. The model projected into thecontourplotisinFig.2,wherefactorsofacetoneandacetonitrilewereconstant.Accordingtothe linearmathematicalmodel, theoptimummixtureofacetone:acetonitrile:chloroform:pyridine:ethylacetateis0:0:35:65:0(v/v/v/v/v). Two optimum mixtures were obtained and compared in the followingexperiment(simultaneouslytestedtheeffectofultrasoundontheextraction).Thesamplesweretreatedintheultrasoundbathfor0,1,5and15minbeforethederivatization.Thesumofallchromatographicpeakareaswasusedasresponsevalues,whichwereevaluatedbythetwo-wayanalysisofvariance(ANOVA)forthesix repeats. According to two-way ANOVA, the effect of ultrasound on theextractionwasstatisticallysignificant(α=0.05).Thesamplesextractedbythe


mixtureofpyridine:acetonitrile(60:40,v/v)showeddecreasingresponsewithincreasing ultrasound treatment time. Samples extracted by the mixture ofpyridine:chloroform(65:35,v/v)showedconstantresponsesinthewholetimerangeoftheultrasoundtreatment.Themixtureofpyridine:acetonitrileshowsatleasttwo-timeshigherresponseareathanthemixtureofpyridine:chloroform.Thisobservationleadstoaconclusion,thatthemixtureofpyridine:acetonitrilehadahigherextractivityfortargetanalytes. AccordingtoWainwright[4],themostabundanceofnitrosocompoundsarelocated in the husk. The next experiment tested the differences among thesamplespreparedfromtheextractionofmalthusk,maltflour,andmaltgrain.Thechromatographicpeakareasofselected14peakselutinginallsampleswereusedasresponsevaluesandwereevaluatedbyPCA.Fig.3depictsthebiplotofPCAanalysis, where 14 variables are represented by a vector. The samples weredividedintothreeclustersaccordingtoausedmatrix.Pointsofmalthuskandmalt


Fig. 2Theresponseareaplotofthe5-factormixturedesign.

Fig. 3ThebiplotofPCAofsamplespreparedby extraction of malt grain, malt husk andmaltflour.Vectorsrepresentretentiontimeofselectedpeaks.

Fig. 4Thechromatogramsofthesamplespreparedbyone-step(dashedline)andtwo-stepderiva-tization(solidline).

grainmatricesareintheareaofhigherpeakareas.Thesamplesfrommaltgrainswerechosenastheoptimalmatrix,duetothesimplerpreparationincomparisonwithmalthusk. Sofar,onlyN,O-bis(trimethylsilyl)trifluoroacetamidewasusedforthederivati-zation.Anewderivatizingagenthexamethyldisilazanewasaddedandrevealedsomenewanalytes(Fig4).Theratioofextractionsolution:hexamethyldisilazane:N,O-bis(trimethylsilyl)trifluoroacetamide(50:100:50,v/v/v)werechooseaccor-dingtopreviouslypublishedmethod[2].Thewholeprocessincludingthetwo-stepderivatizationwasoptimizedusingthePlackett-Burmandesign.Thesumofstandardized chromatographic peak areawasused as the response (y) in thePlackett-Burmandesignexperimentsdescribedbyalinearmathematicalmodel

y=β (±ε )+Σβ (±ε )x (5)0 0 i i i

whereβ isanintercept,andβ isaregressioncoefficientofthefactorx andε is0 i i i

standarderroroftheregressioncoefficients.Linearmathematicalmodel,withevaluated regression coefficients and their standard errors, is described inequation

y= 3.67(±0.63)+0.33(±0.63)x –0.67(±0.63)x +0.83(±0.63)x +ex H B +0.00(±0.63)x (6)st


st ndwhere factors x , x , x , and x represent times of extraction, 1 and 2 ex H B st

derivatization and time to the stabilization of the solution. This model was2evaluatedasverypoor(R =0.3082),thereforethedecisionaboutsignificance

level of the factorswould be inaccurate. All prior factorsweremaintained at30min(intotal120min)andaftertheoptimization,onlyfactorx andx werecutex stdownfor10min(intotal80min).

3.2Realsampleapplication

Thedevelopedextractionmethodtogetherwiththeclassificationmethodwasappliedtoninepilsner,threemunich,threecaramel,onewheat,onecoloured,andtwo artificially nitrosated malts, and three samples of barley grains. Resultclassified peaks into the groups ofN-nitroso, C-nitroso, C-nitroso/nitro com-poundsand interferencesaredepictedbyaheatmap inFig.5(onpage112).According to the similar classification, the sampleswere clustered into threegroups (A,B, C). There was observed an association between theN-nitroso-dimethylamine concentrations inmaltsofparticulargroupsand charactersofclusters:thegroupAincludesmaltswithN-nitrosodimethylamineconcentration≥0.9ppband it is characterizedbyahighnumberofdetectedC-nitrosocom-pounds,theclusterBcontainsmaltsofN-nitrosodimethylamineconcentration≤0.9ppbanditischaracterizedbythelowernumberofC-nitrosocompoundsbutthehighernumberofC-nitroso/nitrocompounds.TheclusterCcontainsmostlythebarleysamplesandthespecialmalt,inwhichtheconcentrationofN-nitroso-dimethylamine is ≤0.2 ppb, this cluster is characteristic with no or very lownumberofthenitrosocompoundsdetected.

4.Conclusions

Miniaturizes extraction method for the screening of the non-volatile nitrosocompoundsinmaltsamples,usingtheGC-NCD,wasdeveloped.Forthesamplepreparation, thegrainedmaltwas chosen tobeappropriate for the screeninganalysis.Themethoduses50μlofthepyridine:acetonitrilemixtureinratio60:40(v/v)andnoneultrasonicationtreatmentarerequired.Two-stepderivatizationisalso suitable.Theoptimumratioof extraction solution:hexamethyldisilazane:N,O-bis(trimethylsilyl)trifluoroacetamideis50:100:50(v/v/v).Thetotaltimeofsamplepreparationis80min(including10minoftheextractionat65°C,two-times30minofthetwo-stepderivatizationat65°Cand10minofstabilizingat20°C).TherealsampleapplicationrevealstheassociationbetweenN-nitroso-dimethylamineconcentrationandthecharacteristicclassificationofthenitrosocompoundsamongthesampleswithinthesameclusters.



Fig.

5Theheatm

apofpeakclassificationinmaltsam

ples.

Acknowledgments

ThisstudywassupportedbytheprojectofMinistryofEducationYouthandSportsoftheCzechRepublicNo.LO1312.

References

[1] Johnson P., Pfab J., Tricker A., Key P., Massey R.: An investigation into the apparent totalN-nitrosocompoundsinmalts.J.Inst.Brew.93(1987),319–321.

[2] GabrisovaL.,KotoraP.,Peciar,P.:CharacterizationofnutritionalsupplementwithcontentofvacciniummacrocarponbasedoncomparisonofchosenflavonolglycosidesbyHTGC-MS.In:Proceedingsof the13thISCModernAnalyticalChemistry.Prague,FacultyofScience,ChalesUniversity2017,p.126–130.

[3] VrzalT.,MatoulkovaD.,Olsovska, J.:Anewmethod fordetectionandclassificationofnon-volatilenitrosocompoundsinbeercombininggaschromatographywithchemiluminescencedetection and discriminant analysis. In: Proceedings of the 13th ISC Modern AnalyticalChemistry.Prague,FacultyofScience,ChalesUniversity2017,p.12–19.

[4] WainwrightT.:Nitrosaminesinmaltandbeer.J.Inst.Brew.92(1986),73–80.


1.Introduction

Todate,theenvironmentalsituationintheworldcanbecharacterizedbyahighlevelofanthropogenicimpactonthenaturalenvironment.Inaddition,recently,humanhabitathasdramaticallychanged,includinginconnectionwithpollutionwithmutagenicandteratogenic factorsofvariousorigins,whichputsthevastmajority of the population in other conditions of existence than in previousgenerations[1]. Worldoverwidechemical,metallurgical,oilrefiningandgasprocessingindus-triesarewidespread,whichleadtoanincreaseinthereleaseofindustrialwaste.Oneofthemostcommonproduction-relatedairpollutantsisacetone,whosehighcontentintheaircancauseseriousharmtothehumanbody. Despite the fact that acetone is a natural metabolite of the human body,exceeding itspermissibleconcentrations inthebloodwithprolongedexternalactionisaccompaniedbyadisturbanceofmetabolicprocesses,amanifestationofsignsofliverdysfunction[2].Theliveristhecentralbodyofneutralizationandutilizationofforeigncompoundsofexogenousoriginandfurthertheseverityofdamage to itsmetabolic systemsmaydependon the subsequent toxic effectsofecotoxicantsonallorgansandsystemsofthebody[3].

Chromato-desorption method for producing gas mixtures of volatile organic compounds

a aIGORARTEMYEVITCHPLATONOV ,IRINANIKOLAEVNAKOLESNICHENKO ,b, bDIANADAVIDOVNAKARAPETIAN *,ASTKHIKEDIKOVNAIGITKHANIAN

a DepartmentofChemistry,SamaraNationalResearchUniversity, Moskovskoyeshosse,443086Samara,Russia*[email protected] InstituteofSpaceRocketEngineering,SamaraNationalResearchUniversity, Moskovskoyeshosse,443086Samara,Russia*[email protected]

AbstractAnewsimple chromato-desorptionmethod for thepreparationofcalibrationgasmixturescontainingmicroconcentrationsofvolatileorganiccompoundsisproposed.Variousmethodsforthepreparationofcalibrationmixturesareconsidered.Thepossibilityofobtaining

–3calibration gas mixtures in the concentration range 3–10mgm (δ =15%)isshown.Acomparativeevaluationoftheproposedandmax

standardizedmethodsiscarriedout.Itisprovedthatthechromato-graphy-desorptionmethodallowstoincreasetheaccuracyofprepa-rationofcalibrationmixtures,andalsotoreducethetotalerroroftheanalysisby13–15%.

Keywordscalibrationmixtureschromato-desorption

microsystemsecologicalcontrolgaschromatography


Fig. 1Preparationofgasmixturesbyachromato-desorptionmethod.

Atpresent, gasanalyzersandgas chromatographsareused to reliablyandsystematicallycontrolthelevelofenvironmentallyhazardoussubstancesinairenvironments[4].Atthesametime,theaccuracyofthemeasurementsdependsontheefficiencyofcalibrationandcalibrationofgasanalyzingequipment[5],which is associated with the use of liquid, gas and vapor-gas mixtures withastandardizedcontentofvolatileorganiccompounds. Thepurposeofthisworkisthedevelopmentofnewmethodsanddevicestoimprovetheaccuracyandadequacyofthepreparationofcalibrationmixturesforthesubsequentquantificationofvolatileorganiccompoundsinairenvironments.

2.Experimental

2.1.Preparationofliquidandgas-vaporgradingmixtures

Liquidacetonecalibrationmixturesintherangeofmeasurableconcentrations–3from0.05to5.00mgm werepreparedbythevolumetricmethod,bysucces-

sivelydilutingthepuresubstance. Vapor-gascalibrationacetonemixturesintherangeofmeasuredconcentra-

–3tionsfrom0.0005to5.0000mgm wereobtainedusingstaticgasextraction[6].

2.2Gasmixturesobtainedbydynamicchromatography-desorptionmethod

Tobuildthecalibrationcharacteristics,alsousedgasmixturesofacetone,obtai-nedbythechromato-desorptionmethod.Thismethodisbasedontheequilibriumsaturationoftheinertgasflowofvolatileorganiccompoundsasitpassesthroughthechromato-desorptionmicrosystem[7].Thesystemisamedicalneedle(length32mm,internaldiameter0.5mm)filledwithasorbentwithaknownamountofvolatile organic compounds. In this study, the following sorbents: ChromatonN-AW-DMCS/25% CaCl , Chromaton N-AW-DMCS/25% CoCl and Fiber-2 2

glass/50%PEG. Toobtaingasmixturesofacetonebyachromatographic-desorptionmethod,aoutlinewasimplemented,thealgorithmofwhichisshowninFig.1.


Theprocessofobtaininggasmixtureswascarriedoutintwostages[7]:1.T hechromato-sorptionstageincludesthepreparationofasorbent,thefillingof

a column,and theequilibriumsaturationof the sorbentwith impuritiesofvolatilesubstances.Inthiscase,thereisamultipleredistributionoftheanalyteinthevolumeofthesorbent,sothataninsignificantconcentrationgradientofthe analyte canbe achievedduring theuseof chromato-desorptionmicro-system. Thus, by changing the temperature, by adjusting the value of thedistributionconstantofsubstancesinthesystem,itispossibletoobtaingasflowscontainingmicro-quantitiesofvolatilecomponents.

2.T he chromato-desorption stage consists in the equilibrium desorption ofvolatilesubstancesattemperaturesequaltothesorptiontemperature.

2.3Calibrationofanalyticalequipment

Theexperimentwas carriedouton a gas chromatographCrystal 5000.1withaflameionizationdetector.Toseparatethesamplecomponents,aquartzcapillarycolumn50mlong,0.32mminnerdiameterand0.5μmthicknon-polarstationaryphase OV-351 filmwas used. Themeasurementswere carried out under theconditionsofprogrammingthetemperaturefrom600°С(for7minutes)to200°С

–1with a gradient of 20°Сmin , temperature of thedetector220°С, evaporatortemperatureof100°С.Asthecarriergas,nitrogenwasused(osm.,GOST9093-

3 –1 3 –174),consumption20.9cm min ,hydrogenandairconsumption20cm min and3 –1200cm min ,respectively.Inthecaseofchromato-desorptionmicrosystem,the

columntemperaturerangedfrom50,70or100°Ctoprovidegasmixtureswithdifferentconcentrationsofthetargetcomponent.


Estimation of convergence limit and reproducibility limit of the calibrationmixturespreparationarepresentedinTable1.Itshouldbenotedthattheuseofliquidcalibrationmixturesislimitedbythelevelofpurityofthesolvent,whichhasmatrixmicroimpuritiesinitscomposition,whichmakesitimpossibletodirectlydeterminetheanalyteatthelevelofmicroconcentrationsandatvaluescloseto


Calibrationmixtures r/% R/% K

Liquid 6.1 7.7 0.90Vapor-gas 10.2 15.8 0.93Gas,obtainedbyadiscretechromato- 12.4 17.6 0.95desorptionsystemmethod

Table 1Estimationofconvergencelimit(r),reproducibilitylimit(R),andcorrelationcoefficient(K)ofthecalibrationmixturespreparation.

Fig. 2Dependenceofacetoneconcentrationondurationofusechromato-desorptionmicrosystem(ChromatonN-AW-DMCS/25%CoCl )attemperatures(1)50°С,(2)70°С,and(3)100°С.2

thethresholdofthesensitivityofthedetector.Toreducethemeasurementerror,itispossibleusingthesubtractionmethodofthesolventbackground,buteveninthiscaseitisnotpossibletoachievetherequiredaccuracy. The use of chromato-desorption microsystem makes it possible to obtain

–3calibrationgasmixtureswithanacetonecontentof3to10mgm whenoperatinginthetemperaturerange50-100°С. Itwasexperimentallyestablishedthatwhendiscretedosingofagasmixturecontainingastandardizedmicrocumulativeamountofacetone,theoperatinglifeofchromato-desorptionmicrosystemisatleast6cycleswithastandarddeviationofδ=15%.Atthesametime,therenewableresourceofthesystemwasatleast3cycles.Fig.2–4showsrelationbetweenacetoneconcentrationandthenumberof consecutively obtained gas mixtures with the use of chromato-desorptionmicrosystemfilledwithdifferenttypeofsorbents. An important advantage of chromato-desorption microsystem is thepossibilityofobtainingmulti-pointcalibrations,withoutadditionaldilutionoftheflow,byvaryingthedesorptiontemperature. Comparison of data on calibrations obtained using standardmethods andusing chromato-desorption microsystem shows that the application of thedeveloped systems makes it possible to improve the accuracy of the lineardependencedescriptionbyreducingthetotalerroroftheanalysiswhenusingchromato-desorptionmicrosystemby13–15%.


Fig. 4Dependenceofacetoneconcentrationondurationofusechromato-desorptionmicrosystem(Fiberglass/50%PEG)attemperatures(1)50°С,(2)70°С,and(3)100°С.

Fig. 3Dependenceofacetoneconcentrationondurationofusechromato-desorptionmicrosystem(ChromatonN-AW-DMCS/25%CaCl )attemperatures(1)50°С,(2)70°С,and(3)100°С.2

4.Conclusions

Anewsimplemethodforobtainingcalibrationgasmixturescontainingknownconstantmicroconcentrationsof volatileorganic compounds isproposed.Theexperimentalverificationoftheproposedmethodshowedtheexpediencyofitspracticaluse.Gasmixturesobtainedbythechromatographic-desorptionmethodareapplicableforcalibrationandcalibrationofgasanalyticalequipmentforthequantitative analysis of organic and inorganic contaminants in ambient air,workplaceair,andalsoforotherpurposes,forexample,intheanalysisofbiomar-kersinexhaledair.Itshouldbenotedthattheuseofdevelopedmicroanalytical


systemshasseveraladvantages, themainofwhicharesimplicityofhardwaredesign,universality,efficiency,exponentialityandthepossibilityofautomationofanalysis.

Acknowledgments

The reported study was funded by the Ministry of the Education and Science of the RussianFederationunderprojectNo.4.6875.2017/8.9.

References

[1] Delgado-SaboritJ.M.,AquilinaN.J,MeddingsC.,BakerS.,HarrisonR.M.:Modeldevelopmentandvalidationofpersonalexposuretovolatileorganiccompoundconcentrations.Environ.HealthPerspect.117(2009),1571–1579.

[2] Фоменко С.Е., Кушнерова Н.Ф.: Экспериментальная оценка токсического влиянияацетона на метаболические реакции печени в условиях повышеннои влажностивоздуха.Токсикологическийвестник2(2013),9–14.

[3] МышкинВ.А.,ЕникеевД.А.,СрубилинД.В..ГимадиеваА.Р.:Экспериментальнаяоценкапроизводныхпиримидинанамоделяхтоксическогопораженияпечени:обзор.Научноеобозрение.Медицинскиенауки3(2016),88–98.

[4] BellarT.,SigsbyJ.E.,ClemonsC.A.,AltshullerA.P.:Directapplicationofgaschromatographytoatmosphericpollutants.Anal.Chem.34(1962),763–765.

[5] McKinley J., Majors R.E.: The preparation of calibration standards for volatile organiccompounds−aquestionoftraceability.LC-GCEur.13(2000),892−895.

[6] Vitenberg, A. G., Konopelko L.A.: Gas chromatographic headspace analysis: metrologicalaspects.J.Anal.Chem.66(2011),438–457.

[7] Platonov I.A., Kolesnichenko I.N., Lange P.K.: Chromatographic-desorption method forpreparing calibration gasmixtures of volatile organic compounds.Meas. Tech.59 (2017),1330–1333.


1.Introduction

Interestinbiodiesel,asanalternativetofossilfuels,iscontinuingtoincrease,whatisaresultofpopulationgrowing,exhaustionoffossilfuels,globalwarmingandoilpricefluctuations[1].Biodieselisanacceptablealternativefuelfordieselengine,duetoitstechnical,environmentalandstrategicadvantages.Biodiesel,definedasthemonoalkylestersoflongchainfattyacids,isproducedfromseveraltypesofconventionalandnon-conventionalvegetableoilsandanimalfats[2]. Inadditiontoitsrenewability,otheradvantagesofbiodieselincludenon-car-cinogenic and non-mutagenic properties, biodegradability, miscibility withpetroleumdiesel,lubricity,highflashpointandcetanenumberandabsenceofaromaticcompounds[1].BiodieselcanbeusedpureormixedwithpetroleumdistillatestoattainblendsdefinedwiththeabbreviationBXX,whereXXstandsforthebiodieselpercentage (v/v) [3]. It canbeused for industrialprocessesandtransportengines. InSlovakiaatpresent, theminimumcontentofbio-compo-

MS/MS analysis of fatty acid methyl esters in diesel

a, a b b,PAULINAGALBAVA *,ZOFIANIZNANSKA ,ĽUDMILAGABRISOVA ,OLIVERMACHO ,a a cRO BERTKUBINEC ,JAROSLAVBLASKO ,JOZEFMIKULEC

a InstituteofChemistry,FacultyofNaturalSciences,ComeniusUniversityinBratislava, Ilkovičova6,84215Bratislava,Slovakia*[email protected] InstituteofProcessEngineering,FacultyofMechanicalEngineering.SlovakUniversityof TechnologyinBratislava,Námestieslobody17,82143Bratislava,Slovakiac VÚRUP,a.s.,Vlčiehrdlo,82003Bratislava,Slovakia

AbstractTheworkisfocusedonthedevelopmentofanewmethodforfattyacidmethylesteranalysisindiesel.Themethodallowsrapidanalysisof individual fatty acidmethyl ester in diesel. The linearity of themethod is in the range of 0.5% to 40% for biodiesel, providingcomparableresultswiththeGC-FIDmethodwitha10-foldshorteranalysistime.Unlikethestandardmethod(STNEN14078),withthisnewmethoditispossibletoidentifyindividualmethylestersindieseland due to high selectivity, the analysis is not burdened by theinterferenceofothercomponentspresent indiesel.Thecontentoffattyacidmethylestersindieselobtainedfromthelocalgasstationatthelevel6.1%witharelativestandarddeviationof3%wasdeter-mined.

KeywordsbiodieseldieselGC-FIDfattyacidmethylesterMS/MSrestrictor


nentsindieselis6%,withthepercentageofbiodieselbeingincreasedasamendedbytheEUregulation[3]. Thecontentofbiodieselinfuelispossibletodetermineusingvariousmethods,wherebytothemostpreferredbelonggaschromatographywithflameionizationdetector(GC-FID)ormassspectrometricdetector(GC-MS).Thesemethodsareable todetermine thecontentof the individual fattyacidmethylesters in thesamples,whatmakesitpossibletodeterminetheoriginoftheoilusedtopreparefattyacidmethylester[4].Thedisadvantageofthesemethodsistherelativelylonganalysistime,whichinsomecasestakesalmostanhour.Amorerapidandinexpensive method for determining bio-components in diesel is the use ofinfraredspectroscopy,wherethepresenceoftheC=Ogroupismonitored.Thelimitation of infrared spectroscopy method is that for correct bio-compoundcontentdeterminationindieselisnecessarytoknowtheoriginorthecompo-sitionoftheindividualfattyacidmethylesters.Furthermore,othercomponentscontainingC=Ogrouppresentindieselcanprovideapositivesignal,notneces-sarilybelongingtothebio-components.Bydefault,thebiodieselcontentindieselisdeterminedaccordingtoSTNEN14078byinfraredspectroscopy. Theaimofthisworkistodevelopanewmethod,whichcombinesthebenefitsof thepreviouslyproposedmethods,asrapidanalysis(infraredspectroscopy)andhighreliability(asGC-FID;GC-MSmethods),wherethepossibilityofinter-ferencewithothersubstancespresentinfuelisminimized.

2.Experimental

2.1Chemicalsandsamplepreparation

Samplesofplantoils(rapeseed,sunflower,palmoils)wereboughtonlocalmarketandbiodieselwaspurchased from localgasstation.Sodiummethoxide,oxalicacid, methanol, hexane were purchased from Sigma-Aldrich (Steinheim,Germany).Themethylestersoffattyacidswerepreparedusingbasictransesteri-

3 –3ficationbyadding100mm ofa0.5moldm solutionofsodiummethoxideindry3methanoltothesamplesin1000mm hexane.Samplesweremixedandreactedat

3 340°Cfor15minutes.Inthenextstep60mm ofoxalicacid(0.5gin15cm ofdiethylether)wasaddedtothesolution,itwasmixedthoroughlyandcentrifuged

–1at 2000 rmin for 3 minutes to settle the precipitated sodium oxalate. The–3samplesofbiodieselwerepreparedattheconcentration100µgmm inhexane

–3withdiethylphtalateasinternalstandardwithconcentration10mgmm .

2.2Instrumentation

GC-MS/MSanalyseswereperformedusingaTraceGCUltragaschromatographwithaTriPlusautosamplerandaTSQQuantumXLSmassspectrometer(ThermoFisher,Austin,TX,USA).Theinjectortemperaturewassetto350°C.Sampleswith


3injectionvolumeof1mm wereinjectedina2mlongfused-silicacapillarywith50μmI.D.,withadeactivatedsurface(restrictor)(AgilentTechnologies,PaloAlto,

3 –1CA,USA).Heliumwasusedasacarriergaswithconstantflowof0.01cm min insplitlessmode.Thetemperatureofthechromatographicovenandtransferlinewas350°C.Themainparametersrelatedtothemassspectrometersetupwere:theionsourcetemperaturewas230°C,thecollisiongaswasargonwithpressureof1.5Painthecollisionchamber,electronenergy70eV,emissioncurrent50µA.ThemassspectrainSCANmodewereobtainedintherangem/z=33–350,MS/MSdetectionwasperformedusingSelectedReactionMonitoring(SRM)transitionsexperimentally optimized for the selected analytes. The used SRM transitionswerem/z270→101and270→73forC16:0,m/z292→121and292→33forC18:3,m/z 294→81 and 294→95 for C18:2,m/z 296→101 and 296→81 for C18:1,m/z 298→101and298→73 forC18:0,m/z326→121 forC20:0with collisionenergysetto15V,andfordiethylphthalateasinternalstandard177→149withcollisionenergy15V,withascantimeof20mseceach. ComparativeanalyzeswereperformedusingGC-FID6890N(AgilentTechno-logies,PaloAlto,CA,USA).Theinjectortemperaturewassetto300°C.Volumeof

31mm ofsampleswereinjectedinsplitmode(70:1).Heliumwasusedasacarriergasatconstantpressure255.1kPa.ChromatographicseparationwascarriedoutonaDB-2360m×0.25mm×0.25μmcapillarycolumn.Theoventemperature

–1wassetto70°Cfor2minandgraduallyincreasedto150°Catarateof25°Cmin , –1thanthetemperaturewasincreasedto240°Catarateof5 °Cmin andheld5min.

Thedetectortemperaturewas280°C.Totalanalysistimewas28.2min.


Tostudythesuitabilityofthenewlydevelopedmethodforthefattyacidmethylestersdeterminationindiesel,methylestersofmostusedoilsintheproductionofbiofuelswereprepared.Table1showsthepercentagesofindividualfattyacidsin


Fattyacid Palmoil Rapeseedoil Sunfloweroil

Name Abb. t /min meas. publ. meas. publ. meas. publ.r

Myristicacid 14:0 12.0 1.1 1.1 0.1 0 0.1 0.1Palmiticacid 16:0 14.5 44.3 42.5 4.8 4.2 6.2 6.4Palmitoleicacid 16:1 14.9 0.5 0.2 0.5 0.1 0.1 0.1Stearicacid 18:0 17.2 4.4 4.1 1.9 1.6 3.7 3.6Oleicacid 18:1 17.6 39.9 41.3 63.3 59.5 23.8 21.7Linoleicacid 18:2 18.3 9.9 9.5 19.2 21.5 64.5 66.3Linolenicacid 18:3 19.2 0.0 0.3 7.7 8.4 0.3 1.5Arachidicacid 20:0 20.0 0.0 0.3 0.6 0.4 0.4 0.3Gondoicacid 20:1 20.3 0.0 0.1 1.5 2.1 0.2 0.2Behenicacid 22:0 22.6 0.0 0.1 0.3 0.3 0.7 0.6Erucicacid 22:1 23.0 0.0 0 0.3 0.5 0.0 0.1

Table 1Fattyacidcompositionalprofilesoffatsandoils(asfattyacidmethylester)inpercentages.

oilsobtainedbyGC-FIDanalysisafterbasic transesterification.The individualfattyacidcontents(percentages)varysignificantlydependingontheoilused.InCentralEurope,rapeseedoilismostoftenusedforbiodieselproduction.Forthisreason,weobservedamixtureofmethylestersobtainedmainlyfromrapeseedoil.Fig.1showstheGC-FIDchromatographicrecordofdieselpurchasedonalocalgasstation. Using a given separation system, bio-components (biodiesel componentsoriginatingfromplantoils)canbereliablydistinguishedfromthosederivedfromcrudeoil(Fig.1).TotalGC-FIDanalysistimeis25minutesplusseveralminutestocoolandequilibratethechromatographicsystem. Fig. 2 shows a GC-MS/MS chromotographic record of oleic acid in dieselcontainingbio-componentandwithoutbio-component.FromFig.2isapparent,thatitispossibletoclearlydistinguishdieselwithfattyacidmethylesteraddition(biodiesel)fromdieselwithoutfattyacidmethylesteraddition.Theindividual


Fig. 1GC-FIDchromatogramofdieselpurchasedonlocalgasstation.

Fig. 2GC-MS/MSchromatographicrecordofoleicacidinbiodieselanddieselwithoutbio-compo-nents.ThemethylestersofrapeseedoilaremarkedwithabbreviationMERO.

SRMtransitions,characteristicsofthecalibrationcurvesfortheindividualfattyacidmethylesteroriginatingfromrapeseedoilareshowninTable2. ThenewlydevelopedmethodbasedonMS/MSdetectionwithoutchromato-graphicseparationallowstomonitorthecontentofindividualfattyacidmethylesterindieselataconcentrationrangeof0.5–40.0%(v/v)withahighlinearityof

2themethod(R intherange0.992–0.996).FromthecalibrationcurveslistedinTable2wecalculatedthecontentofbio-componentsindieselpurchasedatthelocalgasstation.Thedeterminedbiodieselcontentswere5.9%(v/v)usingGC-FIDmethodand6.1%(v/v)using theGC-MS/MSmethod.TheaverageRSDof theindividualfattyacidmethylesterinbiodieselis3.1%.

4.Conclusion

Thenewlydevelopedanalyticalmethodmakesitpossibletodeterminethefattyacidmethylesterscontentintherangeof0.5–40.0%(v/v)indiesel.Theadvantageofthismethodcomparedtotheofficialmethodbasedoninfraredspectroscopyisthe possibility to determine the content of individual fatty acidmethyl esterindiesel,whatmeans that the typeofplantoilusedasbio-componentcanbedetermined.Also,thehighselectivityofthemethodmakesitpossibletofilteroutvarious impurities that give positive response using infrared spectroscopy.ComparedtotheclassicGC-FIDorGC-MSmethod,ourmethodprovidescompa-rabledataaboutthecompositionoffattyacidmethylester,howeveris10timesfaster.

Acknowledgments

Thisworkwas supportedby theSlovakResearchandDevelopmentAgencyunder the contractnumberAPVV-15-0466.


Table 2Characteristicsofthecalibrationcurvesforrapeseedfattyacidmethylester.

2Fattyacidmethylester SRM a(slope) b(intercept) R

C16:0 270-101 925 18900 0.996 270-73 9600 7970 0.992C18:3 292-121 9320 8030 0.993 292-93 1510 4730 0.995C18:2 294-81 42900 21300 0.994 294-95 22900 10350 0.994C18:1 296-101 2460 20480 0.995 296-81 9730 17800 0.996C18:0 298-101 2220 10700 0.995 298-73 460 4780 0.993C20:0 326-101 –1500 3740 0.995

References

[1] HoekmanS.K.,BrochA.,RobbinsC.,CenicerosE.,NatarajanM.:Reviewofbiodieselcompo-sition,properties,andspecifications.Renew.Sust.Energ.Rev.16(2012),143–169.

[2] KnotheG.:Analyzingbiodiesel:Standardsandothermethods.J.Am.Oil.Chem.Soc.83(2006),823–833.

[3] MogollonN.G.S.,RibeiroA.L.F.,LopezM.M.,HantaoL.W.,PoppiR.J.,AugustoF.:Quantitativeanalysisofbiodiesel inblendsofbiodieselandconventionaldieselbycomprehensivetwo-dimensional gas chromatography andmultivariate curve resolution.Anal. Chim. Acta.796(2013),130–136.

[4] RagoneseC.,TranchidaP.Q.,SciarroneD.,MondelloL.:Conventionalandfastgaschromato-graphyanalysisofbiodieselblendsusingan ionic liquidstationaryphase. J.Chromatogr.A1216(2009),8992–8997.


Fig. 1Schemeofelectrochemicalbiosensor.

1.Introduction

Abiosensorisasmallmeasuringdevicewhichallowsthebiologicalresponse tobe transformed intoanalytically useful signal, mostcommonly electrical [1]. TheschemeofbiosensorisshowninFig.1.Biosensorsbasedontyro-sine are the most sensitivesensors used for the determi-nation of phenolic compounds.Tyrosinaseisacoppercontainingenzymebelongingtotheclassofoxidoreductases (EC 1.14.18.1).Tyrosinase catalyzes the oxida-

New biosensor matrices based on carbon nanomaterials for tyrosinase immobilization

KAROLINASTARZEC*,JOLANTAKOCHANA

DepartmentofAnalyticalChemistry,FacultyofChemistry,JagiellonianUniversityinKraków,Gronostajowa2,30-387Kraków,Poland*[email protected]

AbstractBiosensormatricesbasedontitaniagelmodifiedwithcarbonnano-materials: multi-walled carbon nanotubes (MWCNTs) and meso-porous carbon CMK-3 were examined. Matrix composites wereadditionally enriched by poly(diallyldimethylammonium), gold

®nanoparticles,Nafion andglutaraldehyde.Tyrosinasewasusedasmodelactivebiologicalcomponent.Tochoosetheoptimummatrixcomposite, cyclic voltammetrymeasurementswere carried out inacatecholsolution.SensitivityofeachbiosensortowardcatecholandcorrespondingvalueofMichaelis-Mentenconstants,anindicatorofbiologicalaffinityofbiosensortosubstrate,wereestimated.Themor-phologyofmatrixnanocompositeswascharacterizedbySEMimages.

Keywordsbiosensorstitaniageltyrosinasevoltammetry


tionofmonophenolsbymolecularoxygentoformo-diphenols,whicharethentransformedbydehydrogenationtoo-quinones.Theresultingo-quinonescanbereducedat theelectrodesurfacebygeneratingasignal (current)enabling theelectrochemicaldeterminationofphenoliccompounds[2].

Crucialstepinthedevelopmentofnewbiosensorsistheimmobilizationofbioelementinthereceptorlayerinthewaytoretainitscatalyticactivity[3,4].Additionally,thematricesshouldprovideeasyaccessofanalytetobioreceptor,resulting from developed surface area, be characterized by high mechanicalstrengthand,forelectrochemicalbiosensors,theexcellentelectrontransfer[5].Thereareseveralmethodsofimmobilizingbiocatalyticelementsonthetrans-ducersurfacelikephysicaladsorption,covalentbinding,matrixentrapment,intermolecularcross-linkingandencapsulation[6]. Inthescientificreportsvariouskindsofbiosensorsusingtyrosinase-enzymeas a biocatalytic element have been reported, like screen-printed carbonelectrodesmodifiedbygoldnanoparticles[7],graphenemodifiedcarbonworkingelectrodesbycross-linkingwithglutaraldehyde[8],orgraphene-goldnanopar-ticles[9]. Currently, carbon materials including multiwalled carbon nanotubes(MWCNTs)andmesoporouscarbonCMK-3areverypromisingmaterialsinbio-sensingresearch[10,11].Bothmentionedcarbonmaterialsareveryattractivebecauseofitsapplicationsandproperties.Carbonmaterialsarecharacterizedbyhighspecificporevolume(CMK-3),largesurfacearea,hydrophobicity,thermalstabilityandchemical inertness[6,12].Moreover, theuseofcarbonnanoandmesoporousmaterialshavebecomeimportantduetotheirexcellentconductivityresultingintheimprovementofelectrontransferbetweentheenzymesandtheelectrodesurfaces[10,13]. Thegoalofthepresentedresearchwastodevelop,studyandcomparevariousmatricesoftyrosinasebiosensorsbasedontitaniumdioxidegelmodifiedwithtwotypeofnanostructuredcarbonmaterials:multiwalledcarbonnanotubesandmesoporouscarbonCMK-3.Matrixcompositeswereadditionallyenrichedwithgoldnanoparticles(AuNPs),Nafion®,glutaraldehyde(GA)orpolycationit,poly-(diallyldimethylammonium)chloride(PDDA).Forverificationpurposes,multi-componentmatricesdepositedonthegraphiteelectrodesurfacewereexaminedwithrespecttothemechanicaldurability.Sensitivityofeachbiosensortowardcatechol,themodelsubstrateofenzyme,andcorrespondingvalueofMichaelis-Menten constants, an indicatorofbiological affinityofbiosensor to substrate,wereestimated.2.Experimental

2.1.Reagentsandchemicals

–1Catechol (≥99%), tyrosinase from mushroom (EC1.14.18.1; 1881Umg and–13130Umg ), poly(diallyldimethylammonium chloride) PDDA (average

M <100,000,35wt%inwater),glutaraldehyde(70%),Nafion(5%,w/v,solutionw

inmixtureoflowaliphaticalcoholandwater)andmulti-walledcarbonnanotubeswere purchased from Sigma-Aldrich (USA); mesoporous carbon CMK-3 was


synthesizedinDepartmentofChemicalTechnologyattheJagiellonianUniversityinKrakow,ethanol(96%),HNO (65%),2-propanol,L-(+)-ascorbicacid,KH PO 3 2 4

andNa HPO .2H OwereobtainedfromAvantorPerformanceMaterialsPoland2 4 2

S.A.(Poland);HCl(35%),NH (25%aqueoussolution),andacetonewerepur-3

chased from Lach-Ner (Czech Republic); 0.3 mm alumina powder was fromBuehler MicroPolish (USA); acetic acid (100%) was from Merck (Germany).PhosphatebuffersolutionsofpH=6.0inaconcentrationof0.1Mwerepreparedbymixing appropriate volumesofKH PO andNa HPO solutions.Tyrosinase2 4 2 4

(0.048mg/10 μL) solutionwas prepared in 0.1M phosphate buffer solutionpH=7.0.Ultrapurewaterwasused throughout.All chemicalswere analytical-gradereagents.

2.2Instrumentation

MeasurementsbymeansofcyclicvoltammetrywererealizedwithusingofM161electrochemicalanalyzer(mtm-anko,Poland)inthermostaticcabinet(Pol-Eko-Aparatura,Poland).Allexperimentswerecarriedoutwithusingaconventionalthree-electrode electrochemical cell equippedwith the saturated silver/silverchloridereferenceelectrode,aplatinumwireasacounterelectrodeandagra-phiteworkingelectrodecoatedwithenzymaticmatrixcomposite.AsasupportingelectrolytephosphatebuffersolutionofpH=6(0.1M)wasused.Thestudieswereperformedinpotentialrangefrom–0.3to0.5V(vs.saturatedAg/AgCl)atascan

–1rangeof62.5mVs andattemperatureof25°C.

2.3Biosensorpreparation

Topreparealltyrosinase-basedbiosensors,titaniasolwassynthesizedbyacidhydrolysisandthenpolycondensationoftitanium(IV)isopropoxide[14].Inorderto prepare the Tyr/TiO /CNTs/Nafion compositeMWCNTswere dispersed in2

TiO solution. Then, the mixture of tyrosinase was added to the remaining2

suspensionandshaken.Inthelaststep,Nafionwasaddedtothecompositeandthenallmixturewasshaken.Toreceivehomogenouscomposite,themixturewassonicated. For the others of prepared matrix composites the method of pre-parationwassimilar.However,alsothemodificationoftheorderofadditionthetitaniasolandtyrosinasehavebeenexamed.TheamountsofeachcomponentsofthestudiedmatrixcompositesarepresentedinTable1. Topreparebiosensortwo10μLportionsofnanocompositeweredepositedonthesurfaceofthegraphiteelectrode,dryingaftereachportioninairfor10min.Afterwards,theelectrodeswerelefttodryoversaturateddisodiumphosphatesolutionfor20hat4°C.Biosensorswerestoredat4°Cin0.1Mphosphatebuffersolution(pH=6.0).



Matrices

Com

pon

ents

T

yro

sinase/μ

L

TiO

/μL

CM

K-3

/mg

MW

CN

Ts/m

gN

afion

/μL

AuN

Ps/μ

L

PD

DA

/μL

GA

/μL

2

Tyr/T

iO/C

NTs/N

afion

7.1

5

7.1

5

–0.0

4

5.7

–

––

2a

7.1

5

7.1

5–

0.0

4

5.7

–

––

Tyr/T

iO/C

NTs/N

afion

/PD

DA

7.1

5

6.1

2

–0.0

4

5.7

–

1.0

–

2a

7.1

5

6.1

2–

0.0

4

5.7

–

1.0

–

7.1

5

5.7

0

–0.0

4

5.7

–

1.4

–

Tyr/T

iO/C

NTs/A

uN

Ps/N

afion

7.1

5

8.1

6

–0.0

4

5.7

2.0

–

–2

Tyr/T

iO/C

NTs/N

afion

/GA

7.1

5

7.1

5

–0.0

4

5.7

–

–5.0

2

Tyr/T

iO/C

MK

-3/N

afion

7.1

5

7.1

5

0.0

4

–5.7

–

––

2

Tyr/T

iO/C

MK

-3/P

DD

A

9.5

9.5

0.0

6

––

–0.9

5

–2

Tyr/T

iO/C

MK

-3/N

afion

/GA

7.1

5

7.1

5

0.0

4

–5.7

–

–1.0

2

Tyr/T

iO/C

MK

-3/N

afion

/PD

DA

7.1

5

6.5

0.0

4

–5.7

–

0.6

–

2

7.1

5

6.1

2

0.0

4

–5.7

–

1.0

–

a

7.1

5

6.1

2

0.0

4

–5.7

–

1.0

–

7.1

5

6.1

2

0.0

4

–5.7

–

1.4

–

aModificatio

no

ftheo

rdero

fadditio

nth

etitaniaso

landty

rosin

ase(MW

CN

Tso

rCM

K-3

wered

ispersed

inT

iOo

rtyro

sinaseso

lutio

n).

2

Table

1T

heco

mp

ositio

no

fthestu

died

bio

senso

rmatricesw

ithth

ecorresp

on

din

gquan

titiesofeach

com

pon

entfo

ron

eelectrode(C

MK

–meso

poro

us

carbon

CM

K-3

,MW

CN

Ts–m

ultiw

alledcarb

on

nan

otu

bes,A

uN

PS–go

ldn

anoparticles,P

DD

A–p

oly

(diallyld

imeth

ylamm

on

ium

),GA

–glutar-

aldehyde.


3.1SEMimagesofmatricescomposites

To characterize the morphology of developed matrix composites scanningelectronmicroscopyhavebeenemployed.TheobtainedimagesarepresentedintheFig.2.Eachmodificationofthematrixcompositenoticeablychangethemor-phology of biosensor surface. Three-dimensional porous structure for eachbiosensorlayerwithvisibleconvexitiesinsidethecrackscanbeobserved.

3.2Analyticalcharacteristicsofbiosensors

TheanalyticalcharacterizationofthepreparedbiosensorsincludedcalculationtheMichaelis-Mentenconstantandsensitivitytothecatecholusedastherefe-rence substance. The aim of the above-mentioned tests was to examine andcomparebothparameters forbiosensorsbasedondifferentmulti-componentcompositesimmobilizingtheenzymeandselectingamatrixcharacterizedbythehighestsensitivityandthelowestvalueoftheMichaelis-Mentenconstant. AlltheconstructedbiologicalsensorsbasedonMWCNTswerecharacterizedbygoodmechanicalstrength.Whereas,themechanicalstrengthoftheonebiosensorbasedonmesoporouscarbonCMK-3waspoor(TYR/TiO /CMK-3/PDDA).2


Fig. 2 SEM images of (A) TYR/TiO /CNTs/Nafion, (B) TYR/TiO /CNTs/Nafion/PDDA,2 2

(C)TYR/TiO /CMK-3/Nafion,(D)TYR/TiO /CMK-3/PDDA,(E)TYR/TiO /CMK-3/Nafion/GA,and2 2 2

(F)TYR/TiO /CMK-3/Nafion/GAcompositessupportedonthegraphiteelectrodesurface.2

To estimated sensitivity of prepared biosensors the cyclic voltammetrymeasurementswere carried out, first in a supporting electrolyte, and then inacatecholsolutionofdifferentconcentrationsintherangeof8to80μM.Basedontheobtainedresultsthecalibrationsgraphsweredraftedandthesensitivityofeachbiosensorasaslopeofthelinearcalibrationcurvewerereadout. For the biosensors containing in thematrix compositemultiwalled carbonnanotubesthebestresultofthesensitivitywasobtainedforthebiosensorbasedon Tyr/TiO /CNTs/Nafion/PDDA (1.4 µL for one electrode) in a value of2

–1 –2817.0μAmM cm .AnalysingtheresultsofsensitivityofthebiosensorsbasedonmesoporouscarbonCMK-3,definitelyhighervaluesofsensitivity,incomparisonto the samematrices based onMWCNTs,were observed. Biosensor based onTyr/TiO /CMK-3/Nafion/PDDAsolutioninvolumeof1.0µL(foroneelectrode)2 –1 –2wascharacterizedbythehighestvalueofsensitivity1247.0μAmM cm towardcatechol.AttheFig.3acomparisonoffivevoltammogramsisshown.Eachofthevoltammogram registered in the catechol solution corresponds to differentbiosensor.Itcanbenoticedthatthecompositionofthematrixlayerhadnotonlyasignificantimpactontheanalyticalparametersofthetestedbiosensors,butalsoontheshapeofvoltammetriccurves. The dependence of the enzymatic electrode response as a function of theanalyte concentration (catechol), by the Michaelis-Menten equation was alsodescribed,whereastheenzymeactivityischaracterizedbytheMichaelis-Menten


Fig. 3Comparisonofvoltammogramsrecordedforbiosensorswithdifferentcompositionofmatrixcompositesatthecatecholsolutionof78μM.

constant(K ).Michaelis-Mentenconstantvaluedeterminesthesubstrateconcen-M

trationatwhichhalfof theactivesiteofmolecules in theenzymestructure isoccupied.LowvalueoftheMichaelis-Mentenconstantmeansthestrongaffinityoftheenzymetothesubstrate,whileahighvalueindicatesareversetendency.ForallconstructedbiosensorstheMichaelis-MentenconstantwascalculatedusingtheLineweaver-Burkequation,whichconvertstheMichaelis-Mentenequationtolinearform[15]

(1)

wherethec istheconcentrationofcatechol,I –I isacurrentcorrectedbythecat cat buf

value registered in the supporting electrolyte, I is the maximum currentmax

measuredatthemomentofcompletesaturationoftheenzymesubstrate.Thefirststep of theK value estimation was based on the relationship of the inverseM

current1/(I –I )asafunctionoftheinversecatecholconcentration1/c .Tocat buf cat

thedesignatedmeasurementpointstrendlineswereadjustedintheformoftheequation

(2)

Then,knowingthatb=1/I ,anda=K /I ,Michaelis-Mentenconstantsweremax M max

calculated.IncaseofCNTs-basedbiosensorsthebestresultsofK constantwereM

obtainedforbiosensorbasedonTYR/TiO /CNTs/Nafion/PDDA(1.0µL),thatwas2

notcorrelatedtothebestsensitivity.ForthebiosensorsbasedonCMK-3,thelovervalue of Michaelis-Menten constant was received for biosensor based onTYR/TiO /CNTs/Nafion/GA did not agreeing with the highest sensitivity.2

ConsideringallobtainedresultsthatthebestbiosensorwasbasedoncompositeofTyr/TiO /CMK-3/Nafion/PDDA(1.0µLforoneelectrode).Thebiosensorwas2

–1 –2characterized by the highest value of sensitivity 1247.0μAmM cm towardcatechol and relatively low Michaelis-Menten constant, 105.2 µM, as well asexhibitedgoodmechanicalstrength.

4.Conclusions

Thebiosensormatricesfortyrosinaseimmobilizingbasedontitaniumdioxidegelmodifiedwithnanostructuredcarbonmaterials,additionallyenrichedwithgoldnanoparticles, Nafion®, glutaraldehyde or polycationit PDDA (poly(diallyldi-methylammonium)chloride)wereexamined.Theperformedexperimentsprovedthatthecarbonmaterialswereanessentialcomponentsofthematrixcomposites


influencing the electrochemical biosensor response. Some of the constructedbiosensorswerecharacterizedbygoodmechanical strength,highsensitivitiesandrelativelylowMichaelis-Mentenconstantvaluesindicatingthehighaffinityofcatecholtothetyrosinaseimmobilizedinthesematrixcomposites.SensitivitiesofbiosensorsandMichaelise-Mentenconstantvaluescouldsuggestentrappedofa largeramountof tyrosinase in its catalyticallyactive formsuggesting largersurfaceareaofmesoporouscarbonCMK-3incomparisontoMWCNTs.

References

[1] http://www1.lsbu.ac.uk/water/enztech/biosensors.html(accessed22ndJune,2018)[2] TangL.,YangG.D.,ZengG.M.,CaiY.,LiS.S.,ZhouY.Y.,PangY.,LiuY.Y.ZhangY.,LunaB.:Synergistic

effectofirondopedorderedmesoporouscarbononadsorption-coupledreductionofhexa-valentchromiumandtherelativemechanismstudy.Chem.Eng.J.239(2014),114–122.

[3] ZhaoH.,CuiQ.,ShahV.,XuJ.,WangT.:Enhancementofglucoseisomeraseactivitybyimmo-bilizingonsilica/chitosanhybridmicrospheres.J.Mol.Catal.B126(2016),18–23.

[4] Yu J., Du W., Zhao F., Zeng B.: High sensitive simultaneous determination of catechol andhydroquinone at mesoporous carbon CMK-3 electrode in comparison with multi-walledcarbonnanotubesandVulcanXC-72carbonelectrodes.Electrochim.Acta54(2009),984–988.

[5] BhardwajT.:Areviewonimmobilizationtechniquesofbiosensors.Int.J.Adv.Res.Technol.3(2014),294–298.

[6] MonosıkaR.,StreďanskybM.,S turdıkaE.:Biosensors-classification,characterizationandnewtrends.ActaChim.Slov.5(2012),109–120.

[7] Del Torno-De Roman L., Asuncion Alonso-Lomillo M., Domın guez-Renedo O., Julia Arcos-Martın ez M.: Tyrosinase based biosensor for the electrochemical determination of sulfa-methoxazole.SensorActuat.B227(2016),48–53.

[8] ApetreiI.M.,PopaC.V.,ApetreiC.,TutunaruD.:Biosensorsbasedongraphenemodifiedscreen-printed electrodes for the detection of catecholamines. Rom. Biotech. Lett. 19 (2014),9801–9809.

[9] Pan D., Gu Y., Lan H., Sun Y., Gao H.: Functional graphene-gold nano-composite fabricatedelectrochemicalbiosensorfordirectandrapiddetectionofbisphenolA.Anal.Chim.Acta853(2015),297–302.

[10]PerezLopezB.,MerkoçiA.:Improvementoftheelectrochemicaldetectionofcatecholbytheuseofacarbonnanotubebasedbiosensor.Analyst134(2009),60–64.

[11]ParkJ.A.,KimB.K.,ChoiH.N.,LeeW.Y.:Electrochemicaldeterminationofdopaminebasedoncarbonnanotube-sol-geltitania-nafioncompositefilmmodifiedelectrode.Bull.KoreanChem.Soc.31(2010),3123–3127.

[12]KumarS.,ZhengD.,Al-RubeaanK.,LuongJ.H.T.,SheuF.S.:Advancesincarbonnanotubebasedelectrochemicalsensorsforbioanalyticalapplications.Biotechnol.Adv.29(2011),169–188.

[13]ParkE.J.,JinJ.H.,KimJ.H.,MinN.K.:Surfaceactivationofplasma-patternedcarbonnanotubebasedDNAsensingelectrodes.Microchim.Acta174(2011),231–238.

[14]KochanaJ.,NowakP.,Jarosz-WilkołazkaA.,BieronM.:Tyrosinase/laccasebienzymebiosensorforamperometricdeterminationofphenoliccompounds.Microchem.J.89(2008),171–174.

[15]ApetreiC.,Rodrıg uez-MendezM.L.,DeSaja J.A.:Amperometric tyrosinasebasedbiosensorusinganelectropolymerizedphosphate-dopedpolypyrrolefilmasanimmobilizationsupport.Applicationfordetectionofphenoliccompounds.Electrochim.Acta56(2011)8919–8925.


1.Introduction

Chemicallymodifiedelectrodesareeffectiveforelectrochemicalanalysisduetoitsutilityinawiderangeofinvestigations,suchas,electrostaticphenomenaatelectrode surfaces, the relationship of heterogeneous electron transfer andchemical reactivity to electrode surface chemistry, and electron and ionictransportphenomenainpolymers.Moreover,chemicallymodifiedelectrodesarebeneficial for the design of electrochemical sensing systems, electro-organicsyntheses,energystorage,molecularelectronics,corrosionprotection.Comparedwithotherelectrodeconceptsinelectrochemistry,thedistinguishingfeatureofachemicallymodifiedelectrodeisthatagenerallyquitethinfilm(fromamole-cularmonolayer toperhapsa fewmicrometers-thickmultilayer)of a selectedchemicalisbondedtoorcoatedontheelectrodesurfacetoendowtheelectrodewith the chemical, electrochemical, optical, electrical, transport, and otherdesirablepropertiesof the film ina rational, chemicallydesignedmanner [1].Therefore, the search of novel electrodemodifiers is extremely necessary forelectroanalysis. Theiodatearyldiazoniumsaltsareprospectiveaselectrodesurfacemodifiers.The salts allows the covalent bonding of aryl functional groups [2] with theelectrodesurfaceunderelectrolysis.ThefirstapplicationofdiazoniumaromaticsaltsforsurfacemodificationwascarriedoutbyDelamaretal.[3].Traditionally

Carbon containing electrodes modifiedwith the iodate salts of aryldiazonium for electroanalysis

ANNAGUSAR*,ANNAGASHEVSKYA,ELENADOROZHKO,KSENIADERINA

DivisionforChemicalEngineering,SchoolofEarthSciences&Engineering,TomskPolytechnicUniversity,Leninav.,30,634050Tomsk,Russia*[email protected]

AbstractThispaperdealswiththemodificationofaglassycarbonelectrodewitharyldiazoniumiodatesalts(withdifferentfunctionalgroups)bythe spontaneous formation of organic layers for different electro-analyticalpurposes.Theresultswereobtainedbycyclicvoltammetry,scanning (raster) electron microscopy and IR spectroscopy. Theoptimal conditions of modification were established. Obtainedsurfacescouldbeusedforthefurtherdevelopmentofbiosensors.

Keywordsaryldiazoniumsaltsglassycarbonelectrodevoltammetry


aryldiazoniumsaltsareusedforthemodificationofsolidelectrodes.Neverthe-less,thecontrolofmodifierlayeronelectrodesurfaceisdifficultwhileelectro-chemicalmodification.Therefore,themodifierlayercouldessentiallygrowandblocktheconductiveelectrodesurface[4].Thenoveltyofpresentworkisobtain-ingofoptimalconditionsfornonelectrochemicalmodification.

2.Experimental


The following iodate salts of aryldiazoniumwere chosen as modifiers of theelectrode surfaces of the glassy carbon electrodes: [HOOCC H N ]IO ,6 4 2 3

[NCC H N ]IO ,[O NC H N ]IO ,[C H N ]IO ,[H C C H N ]IO .Theirstructures6 4 2 2 6 4 2 6 5 2 33 16 6 4 23 3 3 3

are depicted in Fig. 1. All reagents were of analytical grade. The water wasobtained by water purification system Milli-Q Direct; water resistivity was18.2MΩcm. Silver chloride electrodes were used as auxiliary electrode andreference electrode. To select the optimal conditions for spontaneousmodifi-cationofglassycarbonelectrodewithiodatesaltsofaryldiazonium,theconcen-

–1trationofthemodifier(mgl )andthetimeofagingoftheglassycarbonelectrodeinthesolutionsofthecorrespondingmodifiers(sec)werevaried.Theworking

–1concentrationsofdiazoniumsaltsolutionsformodificationwere10,30,60mgl .Thetimeforthemaintenanceofglassycarbonelectrodeinthesolutionsofthemodifiers was 2, 5, 10, 30, 60, 120 seconds. To evaluate the reversibility ofelectrode processes on the glassy carbon electrode, cyclic voltammograms of

3–/4–hexacyanoferratesalts[Fe(CN) ] ofconcentration0.25M(background0.5M6

KCl)wererecordedbeforeandafterchemicalmodification.


Fig. 1Structuralformulasofaryldiazoniumsalts:(A)4-carboxybenzodiazoniumiodate,(B)iodatearyldiazonium; (C) 4-cyanobenzodiazonium iodate, (D) 4-hexadecylbenzodiazonium iodate,(E)4-nitrobenzodiazoniumiodate,(F)4-(phenyldiazenyl)benzodiazoniumiodate.

2.2Instrumentation

A universal electrochemical workstation TA-2 (Tomanalyt, Tomsk, RussianFederation)withathreeelectrodecellwasused.SilverchlorideelectrodesandglassycarbonelectrodesformodificationwerepurchasedfromLLCTomanalyt(Tomsk,RussianFederation).Investigationsofthecutoffsoftheelectrodewerecarriedoutusingascanning(raster)electronmicroscopeJEOLJSM-7500FA.Tofurtherconfirmthepresenceoforganicfunctionalgroupsontheglassycarbonelectrodesurface,IRreflectionspectrawereobtained.TheinvestigationswerecarriedoutusingtheCary660IRspectrometer(manufacturedbyAgilent).


Toassesstheeffectivenessofmodifyingtheglassycarbonelectrodewithdifferentmodifiersundertheconditionsofchangingtheconcentrationofthemodifierandthetimeofagingoftheglassycarbonelectrode,thevalueofΔI(%)wascalculated

(1)

3–/4– 3–/4–whereI iscurrentof[Fe(CN) ] withoutmodifier,I iscurrentof[Fe(CN) ] 0 6 1 6

afteraginginthesolutionofthemodifier. In thecourseof thestudy(Fig.2), itwasestablishedthat theoxidationand

3–/4–reductioncurrentsof[Fe(CN) ] aremaximalfor4-carboxybenzodiazonium6

iodateoftheglassycarbonelectrodemodifieratthetimeofholdingtheelectrode–1initssolutionfor5secondsandthemodifierconcentration10mgl (ΔI=230%

cathodescanandΔI=185%anodescan).


3–/4–Fig. 2Dependenceofthechangeinthecurrentsof[Fe(CN) ] (ΔI,%)onthetimeofagingofSEMin6

the solution of the 4-carboxybenzodiazonium iodate modifier at different concentrations:–1 –1 –11−10mgl ;2−30mgl ;3−60mgl :(A)cathodepotentialsweep,(B)anodepotentialsweep.The

–1backgroundelectrolyteisKCl0.5M,v=80mVs .

(A) (B)

Foramoreaccuratedescriptionofthemechanismoftheprocessesoccurringattheelectrode,astudyofthemorphologyoftheelectrodesurfacewasrequired(Fig.3).Thefirstsampleisthesurfaceofpureglassycarbonelectrode,beforethe

3–/4–reversible[Fe(CN) ] pairisaddedtothecell.Onthesamplesurface,selective6

microporosityisobserved(Fig.3A).Theporesizedoesnotexceed10μm.Onthesurfacethereisaslightcontaminationwithanextraneousphase,whichhastheformofglobularparticles.Presumably,thisphaseisaparticleofsalt,whichispartof the background electrolyte.The second sample is the surface of the glassy

3–/4–carbonelectrodeafterintroducingareversible[Fe(CN) ] pairintothecell.On63–/4–amicroscopicphotograph,[Fe(CN) ] aggregatesareobservedonthesurface6

oftheelectrode,coveringthemicroporesofglassycarbon.Areaswithinclusionsofasphericalshapeareobserved(Fig.3B).Presumably,thisphasecorrespondstoiron-containing hexacyanoferat.The third sample is the surface of the glassycarbon electrode after modification of 4-carboxybenzodiazonium iodate

–1(10mgl )for5seconds.O namicroscopicimage(Fig.3C),laminatedaggregatesofirregularshapeareobservedconfirmingtheflowofadsorptiononthesurfaceoftheelectrodesubstrate,whichprovesthefactofthechemicalreactionontheglassycarbonelectrodesurfacebetweenthecarbonandthediazoniummodifier.Itisobviousthatacovalentmodificationoftheglassycarbonelectrodeispossiblewithoutimposingapotentialinaveryshortperiodoftime.Inaddition,afterthemodi-fication,thecurrent-conductingpropertiesoftheSEMareincreaseddueto

3–/4–anincreaseinthecurrentsofthereversible[Fe(CN) ] pairatthepotentialsof6

0.15Vand0.35Vundertheoptimummodificationconditions.


Fig. 3Scanningelectronmicroscopyofglassycarbonelectrodesurfacesandcorrespondingcyclicvoltamograms:(A)glassycarbonelectrodewithoutamodifier;(B)glassycarbonelectrodeafterthe

3–/4–addition of the reversible [Fe(CN) ] pair to the cell; (С) glassy carbon electrode after6–1modificationofthesolutionofthe4-carboxybenzodiazoniumiodatemodifier(10mgl )for5sec.

To further confirm the presence of organic functional groups on the glassycarbonelectrodesurface,IRreflectionspectra(Fig.4)wereobtained:1)4-carbo-

–1xybenzodiazoniumiodatemodifierwithaconcentrationof10mgl ;2)theinitialsurface of the glassy carbon electrode; 3) the surface of the glassy carbon

–1electrodeaftermodification.Theabsorptionbandsat3659,1685,1590,786cm ,correspondingtothecarboxylgroupandthephenylnucleusareobservedinthespectrum.

4.Conclusions

Thus, it has been established that themost suitablematerial of the workingelectrodeistheglassycarbonelectrode,andthemaximumvaluesofthecurrentsareachievedusingthe4-carboxybenzodiazoniumiodatemodifier.Theoptimummodificationconditionswere:concentrationof4-carboxybenzodiazoniumiodate

–110mgl ,electrodeholdingtime5sec.

Acknowledgments

TheworkissupportedbygrantofMinistryofEducationandScienceRFforprogram“Science”.

References

[1] DurstR.A.,BaumnerA.J.,MurrayR.W.,BuckR.P.,AndrieuxC.P.:Chemicallymodifiedelectrodes:Recommendedterminologyanddefinitions.PureAppl.Chem.69(1997),1317–1323.

[2] BergerF.,DelhalleJ.,MekhalifZ.:Hybridcoatingonsteel:ZnNielectrodepositionandsurfacemodificationwithorganothiolsanddiazoniumsalts.Electrochim.Acta53(2008),2852–2861.


Fig. 4The transmissionspectrumof the IR surfaceof theglassy carbonelectrode: (1)modifier–14-carboxybenzodiazonium iodate (c = 10 mg l ); (2) the initial surface of the glassy carbon

electrode;(3)surfaceoftheglassycarbonelectrodeaftermodification.

[3] DelamarM.,HitmiR.,PinsonJ.,SaveantJ.M.:Covalentmodificationofcarbonsurfacebygraftingoffunctionalizedarylradicalsproducedfromelectrochemicalreductionofdiazoniumsalts.J.Am.Chem.Soc.114(1992),5883–5884.

[4] BelangerD.,PinsonJ.:Electrografting:apowerfulmethodforsurfacemodification.Chem.Soc.Rev.40(2011),3995–4048.


1.Introduction

Boron-dopeddiamond(BDD)hasbecomeawell-establishedelectrodematerialforneurotransmittersdetection,especiallybiogenicamines,duetoitsexceptionalcharacteristicsincludingbiocompatibility,resistancetofouling,andsufficiently

Boron-doped diamond electrode fabricated by microwave plasma enhanced chemical vapour deposition process with linear antenna delivery for neurotransmitters sensing

a,b, c b,cSIMONABALUCHOVA *,ANDREWTAYLOR ,VINCENTMORTET ,a,bKAROLINASCHWARZOVA -PECKOVA

a UNESCOLaboratoryofEnvironmentalElectrochemistry,DepartmentofAnalyticalChemistry, FacultyofScience,CharlesUniversity,Hlavova8,12843Prague2,CzechRepublic *[email protected],CzechTechnicalUniversityinPrague, SítnáSq.3105,27201Kladno,CzechRepublicc InstituteofPhysicsoftheCzechAcademyofSciences, NaSlovance2,18221Prague,CzechRepublic

AbstractMorphological, spectral and electrochemical characterization ofboron-doped diamond (BDD) electrode deposited by microwaveplasma enhanced chemical vapour deposition process with linearantenna delivery apparatus was performed by scanning electronmicroscopy, Raman spectroscopy and cyclic voltammetry. Fastelectrontransferkineticswasconfirmedforstudiedredoxmarkersatas-deposited hydrogen-terminated BDD surface (H-BDD) and alsoafter applying anodic activation resulting in oxidized surface.Moreover, the voltammetric behaviour of dopamine, an essentialbiologically active compound of which abnormal levels in physio-logical fluids are associated with neurological disorders, wasexamined.Due to thesubstantial foulingof theH-BDDfilmduringdopamine oxidation process, anodically activated BDD film waschosentoperformfurtherexperimentswiththisneurotransmitter.Concentration dependency for dopamine in phosphate buffer

−1pH=7.4 was constructed and detection limit of 1.22μmolL wasachieved.

Keywordsboron-dopeddiamondcyclicvoltammetrydopaminelinearantennaRamanspectroscopy


wide potential window in the region of positive potentials for sensitiveelectrochemicalsensingofdopamine,(nor)adrenaline,andserotonine[1]. Boron-dopeddiamondthinfilmscanbegrownbyusingoneofseveralenergy-assisted chemical vapour deposition methods, the most popular being hot-filament andmicrowave plasma enhanced (MW-PECVD) [2]. However, typicalcavitybasedMW-PECVDarerestrictedtoanareaofdiameterof15cmandhighgrowthtemperaturesabove600°C.Conversely,MW-PECVDsystemwithlinearantennadelivery(MW-LA-PECVD)enablesgrowthwithgoodhomogeneityoverlargeareasatlowtemperatures(<600°C)[3],thereforemakingitmoreattractiveformanyapplications,includingcoverageofglassplatformsformicroelectrodearrays (MEA) used favourably for neurotransmitters sensing.Nevertheless, insuchlinearantennasystemstheadditionofoxygenspecies,typicallyintheformofCO , is necessary which leads to, on one hand, enhanced growth rates and2

diamond quality, but on the other hand, reduced active boron incorporationmanifestedbydecreasedelectricalconductivity[4]. Hence,withinthisstudy,nanocrystallineBDDsamplesdepositedbyMW-LA-PECVD system were characterized by scanning electron microscopy (SEM),Raman spectroscopy and electrochemically by cyclic voltammetry (CV) usingdifferent redox probes. The possibility of dopamine determination was alsoverified.

2.Experimental


Analyticalgradereagentswereusedas-receivedwithoutanyfurtherpurification:dopaminehydrochloride,hexaamminerutheniumchloride(bothSigma-Aldrich,Darmstadt, Germany), potassium chloride, potassium hexacyanoferrate tri-hydrate,sodiumdihydrogenphosphatedihydrate(allLach-Ner,Neratovice,CzechRepublic), sodium hydroxide and sulfuric acid (both Penta, Chrudim, CzechRepublic).Allaqueoussolutionswerepreparedwithdeionizedwater(MilliporeMiliplusQsystem,Billerica,USA)withresistanceofnotlessthan18.2MΩ.

2.2Instrumentation

AMW-LA-PECVDsystemwasusedforthepreparationofBDDsamples,furtherdetails are reported in [3]. The surface morphology of deposited layers wasobservedbySEMusingaTescanFERA3tool.RamanspectroscopywascarriedoutatroomtemperatureusingaRenishawInViaRamanMicroscopeatawavelengthof488nmanda laserpowerof6mWatthesampletoassessthequalityanddiamondlayercomposition.

−1 Cyclicvoltammetrymeasurementswithascanrateof100mVs werecarriedoutbyacomputer-drivenEco-TriboPolarographwithPolarPro5.1software(Eco-TrendPlus,Prague,CzechRepublic).Athree-electrodeset-upwasusedinwhich


linearantennaBDDsamplewasplaced ina laboratory-madeTeflonelectrodebody [5] to form a working electrode. A silver chloride electrode

−1(AgAgCl3molL KCl)andaplatinumwire(bothElektrochemickedetektory,Turnov,CzechRepublic)servedasareferenceandcounterelectrodes,respecti-vely. pH measurements were performed using a digital pH meter 3510 withacombinedglasselectrode(Jenway,Essen,UK).Allelectroanalyticalexperimentswerecarriedoutatlaboratorytemperature(23±1°C). The concentration dependency was constructed from the average of fourreplicatemeasurementsforeachdopaminestandardsolutionandevaluatedbytheleastsquareslinearregressionmethod.Limitofdetection(LOD)wascalcu-latedasathreefoldandlimitofquantification(LOQ)asatenfoldofthestandarddeviationofthepeakcurrents(tenruns)ofthelowestmeasurableconcentration,dividedbytheslopeoftheconcentrationcurvek.


SEMimagesofinvestigatedlayerswerefoundtobeclosedandfreeofpinholes.Layersexhibitedadistinctcrystallinestructurewithgrainsconsistingofamixture

3 −1oforientations.Ramanspectrashowedpeaksrelatedtosp carbonat1332cm aswellasbroadfeaturesrelatingtotranspolyacetylenelyingingrainboundaries

−1 −1at1150cm and1490cm .Furthermore,additionalpeakswereobservedat−1 −1 2 −11360cm and1585cm belongingtosp carbon,andtwopeaksat500cm and−11230cm relatedtoboronincorporation.

Electrochemical characterization of the linear antenna BDD electrode was3−/4−performedby recording CVs of the inner-sphere [Fe(CN) ] and the outer-6

3+/2+ −1 −1sphere[Ru(NH ) ] redoxmarkers(bothof1mmolL in1molL KCl).BDD3 6

filmwastestedas-receivedwithhydrogen-terminatedsurface(H-BDD)directlyafterthedepositionprocedure,andalsoafteritsanodicactivation(performedin

−1acidicmediumof0.5mol L H SO by applyinghighlypositivepotentialE 2 4 ACT

+2.4Vfor20min;intheregionofwaterdecompositionleadingtothehydroxylradical evolution and subsequently the oxygen formation) resulting to theintroductionofvariousoxygen-functionalitiesattheBDDsurface(O-BDD)[6].Evaluated∆E valueswere81mVand66mVatH-BDD,and87mVand64mVatp

3−/4− 3+/2+O-BDDfor[Fe(CN) ] and[Ru(NH ) ] ,respectively,indicatingthatconver-6 3 6

sion to oxidized surface did not significantly altered the redox behaviour ofstudiedprobes.Hence,fastelectrontransferkineticswasconfirmedatH-BDDaswellasatO-BDDsurface.

−1 −1 Theelectrochemicalbehaviourof1mmolL dopaminein0.1molL phos-phatebufferpH=7.4wasinvestigatedbycyclicvoltammetry.DopamineoxidationonH-BDDoccurredat+0.35Vandthepeakheightscontinuouslydecreasedby57% within the set of five consecutive scans, confirming electrode fouling.Noticeablyhigherpositiveoxidationpotentialof+0.44VwasobservedatO-BDDsurface, and repeatable signals were obtained because anodic activation wasappliedbetweentheindividualscans(E +2.4Vfor30s)ensuringresistanceACT


towardselectrodesurfacepassivation.ThereversibilityofdopamineoxidationprocesswassuppressedatO-BDDwhichwasmanifestedbyindistinctivecathodicpeakinthereversescanoftherecordedCVs.Clearly,theoxidationofdopamineisstrongly influenced by surface termination of the BDD sample. Consecutively,onanodicallyactivatedBDDsampletheconcentrationdependenceofdopamine

−1in 0.1mol L phosphate buffer pH=7.4was recorded by cyclic voltammetry−1(Fig.1)withinthelinearrangefrom4to100μmolL anditcanbedescribedby

thefollowingequation

−1 −1 I (nA)=29.9±22.5(nA)+23.6±0.5(nAμmol L)×c(μmolL ) (1)p

R=0.9985

−1 −1ThecalculatedLODandLOQvaluesare1.22μmolL and4.06μmolL ,respec-tively.

4.Conclusions

Withinthisstudy,BDDelectrodeproducedbyMW-LA-PECVDprocesswascharac-terizedbyusingarangeoftechniquesandpreliminaryresultswithneurotrans-mitter dopamine were obtained, which suggest that linear antenna BDD isaperspectivematerialfordopaminesensinganditmaybeusedadvantageously


−1Fig. 1Cyclicvoltammogramsofdopamine in0.1molL phosphatebufferpH=7.4recordedatanodically activated BDD film. Following dopamine concentration levels were measured:

−1 −1 −1 −1 −1 −1(1)4μmolL ,(2)6μmolL ,(3)8μmolL ,(4)10μmolL ,(5)20μmolL ,(6)40μmolL ,−1 −1 −1(7)60μmolL ,(8)80μmolL ,and(9)100μmolL .Dottedlinerepresentssupportingelectrolyte.

for many applications such as fabrication of BDD based MEA device. ThecomparisonoflinearantennaBDDsampleswithothertypesofBDD,includingconventional planar and nanostructured samples prepared by MW-PECVDapparatuswillbeperformed.

Acknowledgments

TheresearchwascarriedoutwithintheframeworkofSpecificUniversityResearch(SVV260440).ItwasfinanciallysupportedbytheCzechScienceFoundation(project17-15319S)andtheJ.E.Pur-kynefellowshipawardedtoV.MortetbytheCzechAcademyofSciences.

References

[1] GarrettD.J.,TongW.,SimpsonD.A.,MeffinH.:Diamondforneuralinterfacing:Areview.Carbon102(2016),437–454.

[2] XuJ.S.,GrangerM.C.,ChenQ.Y.,StrojekJ.W.,ListerT.E.,SwainG.M.:Boron-dopeddiamondthin-filmelectrodes.Anal.Chem.69(1997),591–597.

[3] Taylor A., Fekete L., Hubik P., Jager A., Janicek P., Mortet V., Mistrik J., Vacik J.: Large areadeposition of boron doped nano-crystalline diamond films at low temperatures usingmicrowaveplasmaenhancedchemicalvapourdepositionwithlinearantennadelivery.Diam.Relat.Mat.47(2014),27–34.

[4] TaylorA.,AshcheulovP.,CadaM.,FeketeL.,HubikP.,KlimsaL.,OlejnicekJ.,RemesZ.,JirkaI.,JanicekP.,Bedel-PereiraE.,KopecekJ.,MistrikJ.,MortetV.:Effectofplasmacompositiononnanocrystallinediamondlayersdepositedbyamicrowavelinearantennaplasma-enhancedchemicalvapourdepositionsystem.Phys.StatusSolidiA-Appl.Mat.212(2015),2418–2423.

[5] Cizek K., Barek J., Fischer J., Peckova K., Zima J.: Voltammetric determination of 3-nitro-fluoranthene and 3-aminofluoranthene at boron doped diamond thin-film electrode.Electroanalysis19(2007),1295–1299.

[6] HuttonL.A.,IacobiniJ.G.,BitziouE.,ChannonR.B.,NewtonM.E.,MacphersonJ.V.:ExaminationoftheFactorsAffectingtheElectrochemicalPerformanceofOxygen-TerminatedPolycrysta-llineBoron-DopedDiamondElectrodes.Anal.Chem.85(2013),7230–7240.


1.Introduction

Creatinineisoneofmostcommonanalytesintheclinicalanalysis[1].Itisformedinmusclesinoneoftwopossibleprocesses:eitherinnonenzymaticdehydrationanddephosphorylationofcreatinephosphateorinenzymaticcreatinedehydra-tion.Creatinineservesnobiologicalfunctionsintheorganismandisfiltratedbykidneys and removed with urine. However, due to the fact that creatinine issynthesized inmuscles, its level is related to themusclemass aswell as it isregardedtobearenalfunctionmarker[2].Thephysiologicalrangeofcreatinineis

–10.6–1.3mgdL inserumand800–2000mgin24-hoururine. AcommonlyusedmethodforcreatininedeterminationinbiologicalsamplesisJaffemethod,proposedbyMaxJaffein1886[3]andadaptedforclinicalanalysispurposesbyOttoFolin[4].Itbasesonanorangecomplexformationwhencrea-tininereactswithpicricacidinalkalinesolution.AlthoughJaffemethoddomi-natesinclinicallaboratories,itishighlynonspecific.Compoundssuchasglucose,pyruvate,bilirubin,albumin,cephalosporinsandmanyothersarecausingabiasindeterminedcreatininelevel[5].Toovercometheseobstacles,akineticvariant

Fluorometric method of creatinine determination employing 3,5-dinitrobenzoic acid

a, a, b aIZABELALEWIN SKA *,MICHAŁMICHALEC ,ŁUKASZTYMECKI

a FacultyofChemistry,UniversityofWarsaw,Pasteura1,02-093Warsaw,Poland *[email protected] CollegeofInter-FacultyIndividualStudiesinMathematicsandNaturalSciences, UniversityofWarsaw,Banacha2C,02-097Warsaw,Poland


AbstractAfluorometricmethodofcreatininedeterminationwasinvestigated.Asareagent3,5-dinitrobenzoicacid isemployed.Thefluorometric3,5-dinitrobenzoicacid-creatininecomplexisformedinalkalinecon-ditionswithexcitationandemissionmaximaat410nmand480nm,respectively.Theconditionsoftheassayslikesolventfor3,5-dinitro-benzoicacid,baseconcentrationandreactiontimewereoptimized.

–1Alinear calibration curve in the range from 10 to 500µmolL ofcreatininewasobtained.However,thesubstrate(3,5-dinitrobenzoicacidinorganicsolvent)isunstable,whichlimitsapplicationofpropo-sedmethod.

Keywords3,5-dinitrobenzoicacidcreatininefluorimetry

ofJaffemethodiswidelyemployed,whichbasesonatwo-pointabsorbancemea-surement. The difference between obtained values is treated as an analyticalsignal.Thisapproachallowstoeliminatetheinfluenceonthemeasurementofcompoundsreactingwithpicricacidwithadifferentratethancreatinine. Asanothersolutiontotheproblemofmanyinterferences,in1936threegroups–Bollinger[6],LangleyandEvans[7],andBenedictandBahre[8]independentlyproposedanalternativemethodforthedeterminationofcreatinine.Insteadofpicric acid, 3,5-dinitrobenzoic acidwas employed as achromophoric residue.Since thenonlya fewpapershavebeenpublishedon thismatter [9–13].The3,5-dinitrobenzoicacid-basedmethodisreportedtoexhibitfewerinterferencesthanJaffeassay.However,asystematicandcomprehensiveresearchinthisareaisstillmissingfromtheliterature.Itwillbeasubjectoffurtherinvestigationinourgroupandwillbepublishedelsewhere. Furthermore,thereactionbetweencreatinineand3,5-dinitrobenzoicacidissuitable not only for colorimetric but also for fluorometric determination of

–1creatinine[14].Inorganicbaseinaconcentrationabove0.25molL promotestheformation of fluorescent product with the excitation and emission maxima

–1410nm and 475 nm, respectively. 3,5-dinitrobenzoic acid (0.05molL ) wasdissolvedinanorganicsolvent.Accordingto[14],solventslike1,4-butanediol,isopropanol,dimethylsulfoxide(DMSO),dimethylformamide(DMF)andethanolcan be employed to prepare 3,5-dinitrobenzoic acid solution and facilitatefluorescence.Thegreatest sensitivitywasachieved in1,4-butanediol solution.However,thegreatestfluorescenceintensitywasobservedinDMSOsolution.The

–1linear range of the assay is up to 800µmolL of creatinine and the limit of–1detectionisbelow1µmolL .Thismethodofcreatininedeterminationwaspaten-

ted[15].Inthiscontribution,weverifyresultsobtainedbyBlassandoptimizetheconditions to potentially apply fluorometric creatinine assay to a single-pointcreatininedetermination.

2.Experimental


Thecreatinineand3,5-dinitrobenzoicacidwerepuregradeandobtainedfromSigma Aldrich (USA). Other reagents like 1,4-butanediol, sodium hydroxide,ethanol, methanol, DMSO of analytical grade were obtained from AvantorPerformance(Poland).Waterusedinallexperimentswasthefirstlevelofpurity.2.2Instrumentation

For fluorometricmeasurements, a Scinco FS-2 (SouthKorea) fluorimeterwasemployed. In all experiments disposable, polystyrene fluorometric cuvettesobtainedfromSarstedt(Germany)wereused.


–1Fig. 1Kineticsoffluorescentproductformationafterreactionof0.05molL 3,5-dinitrobenzoicacid–1 –1dissolvedin1,4-butanediol,ethanol,methanol,DMSOor0.45molL NaOHwith500µmolL of

–1creatininestandardand1molL NaOHrecordedat410nmexcitationwavelengthand480nmemissionwavelength.


–1A solution of 0.05molL 3,5-dinitrobenzoic acid was prepared in various–1solvents:1,4-butanediol,DMSO,ethanol,methanol,and0.45molL NaOH.The

kineticswererecordedfor410nmand480nmexcitationandemissionwave-lengths, respectively. Themeasurementswere conducted in a volume ratio of3,5-dinitrobenzoicacidsolution:NaOH:sampleequalto1:1:2.AsshowninFig.1outofallfivesolvents,afluorescentsignalafteradditionofcreatininewasdevelo-ped only in 1,4-butanediol, contrary to Blass’ findings. In case of 3,5-dinitro-benzoicaciddissolvedinDMSO,apurplecolourwasdevelopedandariseinthesolutiontemperaturewasnoticedaftercreatinineaddition.However,theproductwas not fluorescent. For further experiments, 1,4-butanediol was selected asasolventfor3,5-dinitrobenzoicacid. Next,theeffectofNaOHconcentrationon3,5-dinitrobenzoicacid-creatininecomplexfluorescencewasinvestigated.Accordingtooriginalpublication[13],theincreaseofbaseconcentrationresultedinfasterdevelopmentofsignal.KineticsshowninFig.2confirmtheseobservations.AhighersignalisobtainedwiththeincreaseofNaOHconcentration.However,afasterdecayinfluorescenceintensity

–1isalsoobserved.For3molL NaOHasteady-stateisreachedinstantly.Forfurther–1measurements,1molL waschosenduetothehighestfluorescenceintensityof

formedproduct.Thehighestemissionoftheproductisreachedafter9minutesafterreactioninitiationbymixing3,5-dinitrobenzoicacidin1,4-butanediolwithNaOHandcreatininestandard.


–1Fig. 2Kineticsoffluorescentproductformationafterreactionof0.05molL 3,5-dinitrobenzoicacid–1dissolvedin1,4-butanediolwith500µmolL ofcreatininestandardindifferentconcentrationsof

–1NaOH:0.5,1.0,2.0and3.0molL recordedat410nmexcitationwavelengthand480nmemissionwavelength.

Acalibrationcurveforcreatininewaspreparedintheconcentrationrangefrom–110to500µmolL andisshowninFig.3withtheemissionspectraofconsecutive

creatininestandards. However, a significant decay in obtained signal was noticed after storing3,5-dinitrobenzoicacidin1,4-butanediolforseveralhoursinaclosedvolumetricflask.A50%decreaseinsignalwasachievedafter48hoursofstoringthereagentandan80%decreaseafter72hoursofstorageinaclosedvolumetricflask,as


–1Fig. 3Emissionspectra(left)ofconsecutivecreatininestandards(10,50,100,250and500µmolL )

andacorrespondingcalibrationcurve (right).

–1Fig. 5Kineticsoffluorescentproductformationafterreactionof0.05molL 3,5-dinitrobenzoicacid–1dissolvedin75%,50%or25%1,4-butanediolsolutioninwaterwith500µmolL ofcreatinine

–1standardand1molL NaOHrecordedat410nmexcitationwavelengthand480nmemissionwavelength.Thereagentswerefreshlypreparedorstoredfor120hours.

shown inFig.4.This is amajorobstacle to implementing thismethod in realsamplesanalysis.AnattempttostabilizethesubstratebyadditionofwaterwasundertakenTheresultsarepresentedinFig.5.Eventhen,notonlyasmallersignalwasdevelopedthaninpure1,4-butanediol,butalsosimilardecayinsignalwas


–1Fig. 4Kineticsoffluorescentproductformationafterreactionof0.05molL 3,5-dinitrobenzoicacid–1 –1dissolvedin1,4-butanediolwith500µmolL ofcreatininestandardand1molL NaOHrecordedat

410nmexcitationwavelengthand480nmemissionwavelength.Thereagentwasfreshlyprepared,storedfor48hoursorstoredfor72hours.

observed in case of 75% and 50% 1,4-butanediol solution in water used asasolventfor3,5-dinitrobenzoicacid.Anoppositetrendisnoticedincaseof25%1,4-butanediolsolutioninwater.Apossibleexplanationofthisisthat3,5-dinitro-benzoicacidwasnotcompletelysolublein25%butanediolanditsstoragefor120hours resulted in the increase of the amount of 3,5-dinitrobenzoic aciddissolved.

4.Conclusions

Despite the fact, that the conditionswere successfully optimized and a linearcalibrationcurvewasobtained,thisfluorometriccreatinineassayisnotsuitableforrealsamplesanalysisandfoundashighlyunpractical.Anewbatchof3,5-di-nitrobenzoicacid in1,4-butanediol is required foreverysetofmeasurementsbecausethesubstrateisnotstableinsuchasolution.Whatismore,areactionthatlasts9minutesisnotanattractivealternativeforcreatininedeterminationaccor-dingtoJaffeprotocol,whichlastsupto1minute.

Acknowledgments

TheseinvestigationsweresupportedbythePolishNationalScienceCentre,projects:PreludiumNCNno.2017/25/N/NZ5/01556andOpusNCNno.2014/13/B/ST4/04528.HelpfulcommentstothispaperfromProf.RobertKoncki(UniversityofWarsaw)arekindlyacknowledged.

References

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[2] NarayananS.,AppletonH.D.:Creatinine:Areview.Clin.Chem.26(1980)1119–1126.[3] JaffeM.:UeberdenNiederschlag,welchenPikrinsaureinnormalemHarnerzeugtundubereine

neueReactiondesKreatinins.Z.Physiol.Chem.10(1886),391–400.[4] OttoF.,WuH.:Asystemofbloodanalysis.J.Biol.Chem.38(1919),81–110.[5] PeakeM.,WhitingM.:Measurementofserumcreatinine–currentstatusandfuturegoals.Clin.

Biochem.Rev.27(2006),173–184.[6] BollingerA.:Thecolorimetricdeterminationofcreatinineinurineandbloodwith3,5-dinitro-

benzoicacid.Med.J.Aust.2(1936),818–821.[7] LangleyW.,EvansM.:Thedeterminationofcreatininewithsodium3,5-dinitrobenzoate.J.Biol.

Chem.4(1936),333–341.[8] BehreJ.A.,BenedictS.:Studiesincreatineandcreatininemetabolism.IV.Onthequestiononthe

occurrenceofcreatinineandcreatineinbloodcreatinineandcreatineinblood.J.Biol.Chem.52(1922),11–33.

[9] CarrJ.:Reactionsofaromaticnitrocompoundswithactivemethyl,methylene,methinegroupsinpresenceofbase.Anal.Chem.25(1953),1859–1863.

[10]SabbaghM., RickW., Schneider S.: Eine kinetischeMethode zur direktenBestimmung desKreatinins im Serum mit 3,5-Dinitrobenzoesaure ohne Enteiweiβung. J. Clin. Chem. Clin.Biochem.26(1988),15–24.

[11]ParekhA.C.,CookS.,SimsC.,JungD.:Anewmethodforthedeterminationofserumcreatininebasedonreactionwith3,5-dinitrobenzoylchlorideinanorganicmedium.Clin.Chim.Acta80(1976),221–231.

[12]SimsC.,ParekhA.C.:Determinationofserumcreatininebyreactionwithmethyl-3,5-dinitro-benzoateinmethylsulfoxide.Ann.Clin.Biochem.14(1977),227–232.


[13] Cocco C., SchinellaM., Lippi U., Clin C., Maggiore O.C.: New kineticmethod for creatininemeasurementnowautomatedinthecobasfaracentrifugalanalyzer.Clin.Chem.34(1988),2577.

[14]BlassK.G.:Reactivityofcreatininewithalkaline3,5-dinitrobenzoate:Anewfluorescentkidneyfunctiontest.Clin.Biochem.28(1955),107–111.

[15]Blass K.G.: Sensitive and highly specific quantitative fluorometric assay for creatinine inbiologicalfluids.USPat.5,507,708(1996).


1.Introduction

Thepesticideshaveveryimportanttheroleintheagriculture[1–2].Theyhelptoincreaseefficiencyprofitabilityandqualityproducts.On theotherhand, theirapplicationtofarmlandincreasingrapidlyovertheworld,andthecontaminationoccursofagriculturalproduce,water,air,andsoil.Theapplesbelongtocommo-ditiestreatedwidescaleofvariousagrochemicals.Themostusedpesticidesfortreatmentofapplesare insecticides,suchasacetamiprid,chlorpyrifos-methyl,methoxyfenozide, pirimicarb and fungicides, such as boscalid, carbendazim,cyprodinil,dithiocarbamate,fluopyram,andmandipropamid[3–4]. Basedon the trendof increasingconsumptionofappleciders in theCzechRepublic,especiallycidersproducedindomesticcraftciderhouses,wedesignedanexperimentwhichprovidesinformationaboutlevelsofpesticidesresiduesinthesekindsofbeverages.Thesampleswereobtainedmainlyfromlocalcidersandseveralforeignciderswereinvolvedintothestudy.Severalorganicciderswere

Screening of pesticide in apple ciders by liquid chromatography-high resolution mass spectrometry

a, b, a aVERONIKAZUSTA KOVA *,MARTINDUSEK ,JANAOLSOVSKA

a ResearchInstituteofBrewingandMalting,Inc.,Lípová15,12044Prague2,CzechRepublicb D epartmentofAnalyticalChemistry,FacultyofScience,CharlesUniversity, Hlavova8,12843Prague2,CzechRepublic*[email protected]

AbstractThepresentstudyisfocusedonseparationanddetectionofpesticidesresiduesinappleciders.Themaintargetofthisexperimentwasnotonlytodeterminetheconcentrationsoftheseresiduescarryingoverfrom apple peels into the beverages but also to evaluate theirpotentialrisktoconsumer’shealth.FortheextractiontheQuEChERSmethodwasusedincombinationwithadditionalcolumnSPEsampleclean-up to achieve the lowestdetection limitpossibledue to lowconcentrations of pesticide residues. The samples were analysedusing a high-resolution accurate-mass (HPLC-HR/AM) instrumentand this applied method assured the quantification of pesticideresiduesintheciderspossibleat0.2ppblevelformostoffiftytargetpesticides.Additionally,non-targetscreeningmodeenabledtoverifypresence of over 300 pesticides using comprehensive libraryincludingretentiontimes,empiricalformulas,andverifiedfragments.

KeywordsappleciderHPLC-HR/MSpesticidesresiduesQuEChERS


alsoanalysedinordertoconfirmthepesticidefreedeclaration.Inthisstudy,theQuEChERSbasedextractionmethodincombinationwithcolumnSPEadditionsampleclean-uptoachievethelowestdetectionlimitofpesticideresiduesinthesamples[5].TheQ-Exactiveinstrumentandthisappliedmethodmadequanti-ficationofpesticideresiduesinciderspossibleat0.2ppblevelformostoffiftytargetpesticides.

2.Experimental


Acetonitrile,methanol, formicacidandammoniumformate(allLC-MSgrade),sodium citrate tribasic dihydrate and sodium hydrogencitrate sesquihydratewereandpurchasedfromSigma-Aldrich(Steinheim,Germany).Sodiumchloride(anal.grade)wasobtainedfromLach-Ner(Neratovice,CzechRepublic).Magnesi-umsulfate(anal.grade,>98%)wasobtainedfromPenta(Prague,CzechRepublic).PurewaterwasobtainedfromaMilli-Qpurificationsystem(Merck-Millipore). Pesticidestandardsabamectin,acephate,acetamiprid,ametoctradin,azoxy-strobin,bifenthrin,boscalid,bupirimate,carbendazim,chlorantraniliprole,chlor-pyrifos, clothianidin, cyazofamid, cymoxanil, diflubenzuron, dimethomorph,etoxazole, fenpropimorph, fenpyroximate, flonicamid, fludioxonil, fluopicolide,fluopyram,hexythiazox,imazalil,imidacloprid,indoxacarb,malaoxon,malathion,mandipropamid, mepanipyrim, metalaxyl, methoxyfenozide, metrafenone,myclobutanil,oxadiazon,penconazol,pendimethalin,pirimicarb,propamocarb,propargite,propiconazol,pyraclostrobin,pyridaben,quinoxyfen, spirodiclofen,spirotetramat,spiroxamine,tebuconazole,tebufenozide,tebufenpyrad,thiaben-dazole, thiacloprid, thiamethoxam, triadimefon, triadimenol, trifloxystrobin,triflumizole, and internal standards azoxystrobin-d4, thiamethoxam-d3,triphenylphosphatewerepurchasedfromSigmaAldrich(St.Louis,USA).

–1 Standardand internalstandardstockssolutions(1.0mgmL forallexcept –10.2mgmL forametoctradin, carbendazimandchlorantraniliprole)werepre-

paredinacetonitrileor,incaseofsolubilityproblem,inmethanoloracetoneandstoredat–20°C.Astandardmixturesolution,withall58pesticides,wasprepared

–1inacetonitrileat1µgmL ofeachpesticide.

2.2Instrumentation

HPLC-MSwascarriedoutusingaDionexUltiMate3000UHPLCsystem(ThermoScientific,Germering,Germany)consistedofabinarypump(HPG-3400RS),anautosampler(WPS-3000TRS),adegasser(SRD-3400)andacolumnoven(TCC-3000RS).DetectionwascarriedoutbyaQExactivehybridquadrupole-orbitrapmass spectrometer (Thermo Scientific, Waltham, MA, USA). Analytes wereseparatedonareversed-phaseC18AtlantisT3column(2.1×100mm,3µm)fromWaters(Milford,MA,USA)withacorrespondingguardC18column(Security-


GuardULTRA)fromPhenomenex(Aschaffenburg,Germany).TheLC-MSsystemwasequippedwithaheatedelectrosprayionizationsource(HESI-II)andTrace-Finder software version 4.1. Chromatographic separation was realized usinggradientelutionwith2mMammoniumformatecontaining0.1%formicacidinwaterassolventAandmethanolassolventB;LCgradient:0min:85%ofsolventA+15%ofsolventB,0.5min:85%A+15%B,9min:5%A+95%B,15min:95%A+5%Bwithaflowrateof340µLperminutewasused.Thecolumnovenwasheatedto40°Candinjectionvolumewas2µL. Inthepositiveelectrosprayionization(ESI)mode,theionsprayvoltagewassetat2.8kV,thesheathgasflowwasat32arbitraryunits,theauxiliarygasflowratewaskeptat7arbitraryunits,thecapillarytemperaturewassetat295°Candtheauxiliarygasheatertemperaturewassetat295°C.InthenegativeESImode,theion spray voltage was set at –2.5 kV. Nitrogen was used as both sheath andauxiliarygas. Themassspectrometerwasgenerallyoperatedinparallelreactionmonitoring(PRM).Theprecursorionsintheinclusionlistwere,withintheretentiontimewindow±0.3min,filteredinthequadrupoleatisolationwindow(targetm/z±0.7amu),fragmentedinHCDcollisioncell,productionswerecollectedintheC-trapat17,500resolution(FWHM,fullwidthathalfmaximum,atm/z200),AGCtargetvalueof2e5,andmaximumioninjectiontimeof40msandfinallytwospecificpairsofprecursor-productiontransitionsweremonitoredforeachcompoundofinterest. Amass tolerance of 5 ppmwas employed. The normalized collisionenergy(NCE)wasoptimizedforeachcompound.Theinstrumentwasexternallycalibratedpriortoeachmeasurementusingthemixtureofmasscalibrants.

2.3Standardpreparation

–1Stock solutions (1mgmL ) of each compound of interest were prepared byadding10mg(correctedforpurity)oftheanalyticalgradecompoundtoseparate10mLvolumetricflasksandbringinguptovolumewithacetonitrile(exceptcar-bendazim,whichwasdilutedwithmethanol).Thestocksolutionswerestoredgenerally at–20 °C.Ahigh level fortification solutionwaspreparedby taking0.1mLaliquotsof each stock solutionanddilutingup tovolume in a100mL

–1volumetricflaskwithacetonitrile,resultingina1μgmL mixedsolution.

2.4Cidersamplepreparation

Fifteen samplesof theapple cidersoriginally fromCzechRepublic (CZ)were:CiderHopHop;CiderMagneticApple–chmeleny;Tatuvsad–chmeleny;BBcidre;CiderBohemia;CiderMagneticApple–original;12PragCider;CiderDRY;Tatuvsadcider;Prvnı Prajzskejablko;Ceskorajskycider;Redbrookcider;Rychnovskycider;Johannescyder;Cidre99dry,andfiveforeignciders:ValdeRance(FR);Dunkertons(GB);AspollSuffolk(GB);SidraBere(ESP);Opre’Cidery(SK)were


involved in the experiment. The samples were prepared using a modifiedQuEChERSmethod:(1)10mLofdegassedsamplewasaddedintoa50mLPFTEcentrifugation tube, 50µLof internal standard solution (triphenylphosphate,

–1c=10mgL )wasaddedandthetubesweremixedthoroughly;(2)10mLofaceto-nitrilewasadded(thetubesweretightlyclosed)and1minutemixedontheVortexshaker;(3)thenwasaddedthemixofsalts(4gMgSO ,1gNaCl,1gsodiumcitrate4

tribasicdihydrate,0.5gsodiumhydrogencitratesesquihydrate)and(4)thesam-plesweremanuallyshakenfor1minute;(5)Thesampleswerecentrifuge7minu-tesat4500rpm;(6)6mLoforganicphasefromsamplewastookintothecentrifu-gationtubes(15mL)with0.9MgSO andthetubeswereagaincentrifugationfor4

7minutesat4500rpm.Inthesecondstep,thesampleswerecleaned-upusingSPEtechnique.ASPEcolumnswithPSAsorbent(200mg,3mLtube;Supelco)wasconditionedwith2mLacetonitrilepriorsclean-up.Thenwasadded2mLsample(from15mLcentrifugationtube).TheeluentwascollectintotheheartshapedflaskandsubsequentlytheSPEcolumnswerewashedwith10mLofacetonitrile.Thenthesampleswereevaporatedtodrynessontheevaporator(40min/35°C).Atthelast,thesamplesweredissolvedwith1mL0.1%formicacidinmixtureof50%methanolinwater.


Inthisstudy,12pesticidesresidueswerefoundintwentysamplesofapplecidersthatwerereliablyquantified.Atleasttwopesticidesresiduesweredetectedineach sample. Themost occurring pesticidewas pirimicarb, an insecticide foraphidcontrolinawiderangeofcrops.Itwasdetectedinseventeensamples,infour samples was quantified, and the highest concentration was 3.6 ppb in“JohannesCyder”(CZ).Thesecondmostoccurredpesticidewasboscalid,afungi-cideactiveagainstawiderangeoffungalpathogens.Itwasdetectedinfourteensamples,ineightsampleswasquantification,andthehighestconcentrationwas14.1ppbin“Bohemiacider”(CZ).Thefrequentlyfoundpesticideresiduewasalsomethoxyfenozide,aninsecticideusedtocontrolvariousinsectmoths,butterflies.Itwasdetectedinelevensamples,insevenwasquantification,andthehighestconcentration1.2ppbwas in apple ciderwithhops addition “CiderMagneticApple–chmeleny”(CZ).Acetamiprid(aninsecticideusedforthecontrolaphid)waspesticideresiduefoundquiteoftenincidersanditwasquantificationinfivesamplesatthehighestconcentrationwas4.5ppbindrycider“CiderMagneticApple–original”(CZ),andinotherfivesampleswereonlydetected.Fluopyram,abroad-spectrumfungicideforuseasafoliarapplicationandasaseedtreatmenttocontrolvariousdiseases,wasdetectedineightsampleswiththehighestcon-centration1ppbin“CiderBohemia”(CZ)andmandipropamidweredetectedinfoursamples,intwosamplesatthehighestconcentration2.1ppb.Theremainingpesticide residues (for example abamectin B1A, carbendazim, imazalil,myclobutanil,pyraclostrobin, thiacloprid)wereonlydetecteddue to their lowconcentration.


Wehad in our experiment also three organic ciders (“Cider hop hop” (CZ);“Appleciderdry”(CZ);“Dunkertons”(GB)). In“Ciderhophop”weredetectionboscalid(0.5ppb)andmandipropamid(<0,5ppb),inthe“Appleciderdry”wereboscalid (1 ppb) and myclobutanil (<0.5 ppb), and in “Dunkertons” wereidentifiedabamectinB1A(0.6ppb)andpirimicarb(<0.5ppb). Fivesamplesofapplescidersweremadewithhopsadditionforbittertasteandaroma.Unfortunately,hopsbelongbetweencommoditiestreatedbywidescaleoftheagrochemicals.Thus,hopsasarawmaterialcouldbeapotentialdonorofpesticidesresiduescarriedoverintociders.Thecider“Opre’dryhoppedcider”originate from Slovakia contained a quite high concentration of imidacloprid(5.3ppb)and imazalil (2.6ppb).Presenceof imazalil suggests that theapplescould be determined for post harvest storage originally. The sample “CiderMagneticApple–chmeleny”(CZ),whichcontainedacetamipridat1.2ppb,bos-calid at 2.6 ppb, methoxyfenozide at 1.2 ppb, fluopyram at <0.5 ppb,mandipropamidat2.1ppb,andpirimicarbat<0.5ppb.Insamples“Tatuvsad–chmeleny”wereacetamipridat<0.5ppb,boscalidat1.1ppb,methoxyfenozideat0.9ppb, fluopyramat<0.5ppb,pirimicarbat1.1ppb,andmandipropamidat


Fig. 1Selectedionchromatogramsofpesticidesresiduesinsampleapplecider“Tátůvsad–chme-lený”:(a)acetamiprid(m/z=223.0745,t = 4.69min),(b)boscalid(m/z=343.0399,t = 7.88min),r r (c)methoxyfenozide(m/z=369.173,t =8.09min),(d)fluorpyram(m/z=397.0537,t = 8.23min),r r (e)pirimicarb (m/z = 239.1503, t =5.51min), (f) triphenyl phosphate, i.e., internal standardr

(m/z=327.0780,t =8.83min).r

<0.5ppb.TheselectedionchromatogramsofpesticidesresiduesinsampleforthissampleisinFig.1.Inthesample“CiderHopHop”weredetectedonlyboscalid(0.5ppb) andmandipropamid (<0.5ppb), and in the sample “BB cidre”weredetectedboscalid(<0.5ppb)tooandpirimicarb(<0.5).Theoccurrenceofmandi-propamidwasobservedonlyincaseofciderswithhop,thusthispesticideresiduecomingfromthehopmostprobably.Theapplesqualityisnottheonlyinfluencingfactorfortheoccurrenceofpesticidesresidues.

4.Conclusions

Thefoundconcentrationsofpesticideresiduesinciderasaprocessedfoodwere–1 –1evaluated base on acceptable daily intake (ADI,mgkg bwday ) for each

pesticides.Theresultshowedthattheevenhighestdeterminedconcentrationofboscalidat14.1ppbrepresentonlysmallpartofADIfor80kgweighman.Thelevelsoffoundpesticideresiduesthuscouldnotberiskforconsumershealth.Nevertheless, the monitoring of pesticide residues in this type of beveragesproducts is important because they contain various pesticide residues,whichlong-termconsumptioncouldbepotentiallyhazardousforconsumer’shealth.

Acknowledgments

ThestudywassupportedbytheprojectofMinistryofEducationYouthandSportsoftheCzechRepublicNo.LO1312.

References

[1] SimonS.,BrunL.,GuinaudeauJ.,SauphanorB.:PesticideuseincurrentandinnovativeappleorchardSystemsAgronomy.Agron.Sustain.Dev.31(2011),541–555.

[2] www.food.gov.uk(accessed15thMay2018)[3] www.sitem.herts.ac.uk(aceessed10thJune2018)[4] LozowickaB.:Healthrickforchildrenandadultsconsumingappleswithresidue.Sci.Total.

Environ.502(2015),184–198.[5] LehatayJ.S,AnastassiadesM.,MajersE.R.:TheQuEChERSrevolution.LC-GCEurope23(2010),

418–429.


1.Introduction

Analysisofexhaledairisanattractiveareaofnon-invasivemedicaldiagnostics,because thismethodexcludes invasive interventionsandcanbe implementedrepeatedly. It provides an opportunity to study thoroughly the dynamics ofphysiologicalprocesses.Also,theanalysisofexhaledairallowstorevealapatho-logy at those stages of development, when othermethods of diagnostics areinsensitive,nonspecificanduninformative. Theexhaledaircontainsaboutthreethousandvolatileorganiccompounds[1],which are the products of physiological and biochemical processes in theorganism.Manyofthemarebiomarkersoffunctionaldisordersofhumanbody,about20ofthemareusedaspredictorsofsomediseases.Theacetoneisoneoftheselectivebiomarkers.It isformedasaresultoftheoxidationoffats.Increasedconcentrationofacetoneintheexhaledairsignalsaboutexcesslevelofglucoseintheblood[2,3]. Thedevelopmentofnon-invasivediagnosticsishamperedbythelackofanoptimizedmethodforthequantitativedeterminationofmicroquantitiesofbio-markersintheexhaledair.Thelimitingfactorswhichdeterminetheaccuracyandtherateofmeasurementsareappropriatesamplingandsamplepreparationthateliminatethepossibilityofadditionalsamplecontamination.

Chromato-desorption microsystems for determination of biomarkers in the exhaled breath

a aIGORARTEMYEVITCHPLATONOV ,IRINANIKOLAEVNAKOLESNICHENKO ,b, bASTKHIKEDIKOVNAIGITKHANIAN *,DIANADAVIDOVNAKARAPETIAN

a DepartmentofChemistry,SamaraUniversity, 34,Moskovskoyeshosse,443086Samara,Russia*[email protected] InstituteofSpaceRocketEngineering,SamaraUniversity, 34,Moskovskoyeshosse,443086Samara,Russia*[email protected]

AbstractChromato-desorption microsystems have been developed in thisresearch work. They can be used to increase the accuracy ofquantitative determination of biomarkers in the exhaled air. Theevaluation of the accuracy determination of acetone inmodel gasmixturesshowsanadvantageincomparisonwithstandardmethods.

Keywordsbiomarkersbreathacetonechromato-desorption

microsystemsdiabetesnon-invasivediagnostics


Thepurposeoftheresearchisthedevelopmentofmethodologicaltechniquesandthequantitativedeterminationofacetoneintheexhaledair.

2.Experimental


Wehave chosen four types of sorbents formicro systems: ChromatonN-AW-MCS+25%CaCl , Chromaton N-AW-MCS+25%CoCl , Al O , fiberglass+50%2 2 2 3

polyethyleneglycolandfilledwiththemdevelopedchromato-desorptionmicro-systems.Wehaveusedacetoneasatargetcomponenttoconductexperiment.

2.2Instrumentation

Developed chromato-desorptionmicrosystems andmethods allow to concen-tratetracecontaminantsofaliphaticvolatileorganiccompoundsfromexhaledairsamplesbysolidphasemicroextractiontechnique.Chromato-desorptionmicro-systemshavebeenmadefrommedicalneedles(innerdiameter0.5mm)andfilledwithsorbents.Fig.1showsstagesofchromato-desorptionmicrosystemprepar-ation.WehaveusedscanningelectionmicroscopeTescanVEGAforanalysisofsorbentsurfacemicrostructures(Fig.2).


Fig. 1Stagesofchromato-desorptionmicrosystempreparation:(1)fillingwithasorbent,(2)satura-tionchromato-desorptionsystemwithatargetcomponent,(3)preparationprocedure.

(a) (b) (c) (d)

Fig. 2Microphotograph of sorbent surfaces: (a) Al O , (b) Chromaton N-AW-MCS+25% CaCl ,2 3 2

(c)ChromatonN-AW-MCS+25%CoCl ,(d)fiberglass+50%polyethyleneglycol.2


Itisknownthatsurface-layersorbents,modifiedwithsorption-activeinorganicsalts,havealargeadsorptioncapacity,chemicalinactivityandthermalstability,allowtheprocessofconcentratingasamplewithdirectthermaldesorptionofimpuritiestotransferthemtoagaschromatograph,therebyshorteningthetime,increasingthesensitivityanalysis.Inthisconnection,itisexpedienttostudythepotentialuseof sorbentsof this type for themanufactureof chromatographicdesorptionmicrosystemsdesigned toproducegasmixturescontainingmicro-quantitiesofacetone.ChromatonN-AW-DMCSwithmodificationof25%CaCl 2andChromatonN-AW-DMCSwithmodificationof25%CoCl acquireastrongly2

developedsurfacestructure,whichgreatlyincreasesitssurfacearea,moreoversorbent particles are small and have irregular geometric form, whereby thepackingdensityofchromato-desorptionmicrosystemincreases.Thecumulativeeffectofthesefactorsmakesitpossibletopredictanincreaseinsorptioncapacityofthesystem.Asimilareffectofincreasingthesorptivecapacityofthesystemdueto the largesurfaceareaand isshowntoAl2O3, thecapacityreduction isnotobserved. Thesystemwithfiberglass+50%polyethyleneglycolshowedhighthroughput.The salt is planted partially on the fiberglass. This sorbent has a specialconfiguration,sothedeadvolumeisalmostabsent.Itwasestablishedexperimen-tally that chromato-desorption microsystem life of dispensing discrete gasmixtureofatleastsixcycleswithastandarddeviationδ=15%(Fig.3).

4.Conclusions

Analytical micro concentrating systems and methods for concentrating tracealiphatic hydrocarbons from exhaled air samples have been developed.Characteristicsofchromato-desorptionmicrosystemsfilledwithChromatonN-AW-DMCS+25%CaCl , Chromaton N-AW-DMCS+25%CoCl , Al O , fiber-2 2 2 3

glass+50%polyethylene glycol have been determined. It is reasonable to use


(a) (b)

Fig. 3Acetoneconcentrationindependenceonusechromato-desorptionmicrosystemfilledwith(a)CromatonN-AW-DMCS+25%CaCl ,(b)fiberglass+50%polyethyleneglycol.2

chromato-desorption microsystem filled with Chromaton modified withsorption-active inorganicsaltsandwith fiberglass+50%polyethyleneglycol toconcentrate volatile organic compounds from breath air samples. Chromato-desorptionmicrosystemmethodmeetsgreenchemistryprinciplesasitreducesdramaticallychemicalagentconsumption.

Acknowledgments

ThestudywassupportedbytheMinistryofEducationandScienceoftheRussianFederationunderprojectnumber4.6875.2017/8.9.

References

[1] КопыловФ.Ю.,СыркинА.Л.,ЧохамидзеП.Ш.,БыковаА.А.,ШалтаеваЮ.Р.,БеляковВ.В.,ПершенковВ.С., СамотаевН.Н., ГоловинА.В., ВасильевВ.К.,МалкинЕ.К., ГромовЕ.А.,ИвановИ.А.,ЛипатовД.Ю.,ЯковлевД.Ю.:Перспективыдиагностикиразличныхзаболе-вании посоставувыдыхаемоговоздуха.Клиническаямедицина10(2013),16–21.

[2] ZhouM.G.,LiuY.,LiW.W.,YuanX.,ZhanX.F.,LiJ.,DuanY.X.,LiuY.,GaoZ.H.,ChengY.,ChengS.Q.,LiH.,LiangY.:Investigationandidentificationofbreathacetoneasapotentialbiomarkerfortype2diabetesdiagnosis.Chin.Sci.Bull.59(2014),1992–1998.

[3] GalassettiP.R.,NovakB.,NemetD.,Rose-GottronC.,CooperD.M.,MeinardiS.,NewcombR.,ZaldivarF.,BlakeD.R.:Breathethanolandacetoneasindicatorsofserumglucoselevels:aninitialreport.DiabetesTechnol.Ther.7(2005),115–123.


1.Introduction

Each and every quantification analysis undoubtedly cannot proceed correctlywithoutpreliminarycalibrationprocess.Speakingaboutwater-ethanolmixturesexcellentexponentsoftheirapplicationspherearealcoholicbeveragesandthematerialsof theirorigin,suchasdistillates.Amonglotsofcontrol testsof thisoutputvolatilecompoundsquantification is the topmostqualityparameter,aschemicalcontentofalcoholicbeverageinfluencebothorganolepticparameters(flavourandtaste)andsafety.Consequentlyanalyticalchemistschallengeistoestablish the concentrations of volatile compounds in the testing sample asaccuratelyaspossible.

A novel way of establishing the quantitative composition of gravimetrically prepared standard solutions of volatile compounds in water-ethanol matrix

a, bANTONKORBAN

a DepartmentofAnalyticalChemistry,ChemistryFaculty,BelarusianStateUniversity,LeningradskayaStr.14,220030Minsk,Belarus*[email protected]

b InstituteforNuclearProblemsofBelarusianStateUniversity, BobruyskayaStr.11,POB220030,Minsk,Belarus

AbstractTheiterationmethodofvolatilecompoundsconcentrationsandcali-brationcoefficientscalculationinwater-ethanolstandardsolutionswas proposed. It lies in the gravimetric preparation of a mixturewhichcontainsimpuritiesatmuchhigherconcentrationsthanthoseininitialethylalcoholappliedasablendingagent.The“EthanolasInternalStandard”methodisthenusedforthecalculationofRelativeResponse Factors of zero-order approximation for each analysedvolatile.Ethylalcoholusedforpreparationisthenmeasuredbygaschromatographyandtheadulterantsconcentrationsinitaredeter-mined.FurtherthevolatilesconcentrationsinstandardsolutionandRelativeResponseFactorsarespecified.Finallytheprocessoftakingintoconsiderationtheadulterationofinitialethanolallowscorrectdeterminationoflowvolatilesconcentrationsinpreparedstandardssolutions. The efficiency of themethodwas demonstrated experi-mentally.

Keywordsethanolgravimetricpreparationstandardsolutionsuccessiveiterations,volatileimpurities


Separation and detection of components in ethanol-containing products ismainlyperformedwithgas chromatography (GC)methodas themost robust,rapidandmodern.Thus,lotsofinternationallegislativedocumentsestablishtheGCwayofquantitativedeterminationofvolatilecompoundsinalcoholicproducts[1–5].Butthestandardsolutionspreparationalgorithmdiffersfromdocumenttodocument. European regulation [1] concerns the GCmethod of volatile com-pounds quantification in spirit drinks and describes the whole procedure ofstandard solutions gravimetric preparation fromwater and ethyl alcohol freefrom volatile impurities. Oppositely, the European Pharmacopeia (EP)mono-graphsconcerningethanol(96%)andanhydrousethanolanalysisestablishthepreparation of reference solutions on the basis of testing substance [5]. Inaddition,theauthorsofamajorpartofresearcharticlesconcerningthedetermi-nationofcontaminantsinalcoholicproductspreparestandardsolutionsintheirown way, applying methods different from those described in the legislativedocuments.InthiscasebothExternalandInternalstandardmethodsareappliedalthoughthelatterispredominant. Eventually let us describe the problem which unavoidably exists duringstandardsolutionspreparation.Thefactisthatethylalcoholcannotbe100%pureasitalwayscontainsvolatileimpurities.Andthechallengeistotaketheethanoladulterantsconcentrationsintoconsiderationduringfinalcalculationofstandardsolutionscomposition.Table1includestheinformationaboutlimitsofvolatilesconcentrations in ethyl alcohol found in both legislative documents and openofficialcommercialsources,suchasSigma-Aldrich.Finallyinsomecasesthecon-centrationsofvolatilesinstandardsolutionsarecomparablewiththoseintheinitialethylalcohol.Thisphenomenoncausesaproblemofincorrectestablish-mentofcertifiedvalues.Theproblemcanbesolvedbytheproposediterationmethod. Firstly,astandardsolutionswhichcontainsanalysedvolatilecompoundsat

–1the concentrationsnear to500mgl absolute alcohol (AA) is tobepreparedgravimetrically according toASTM recommendations [6]. Zero-order approxi-

–1matedconcentration(mgl AA)ofi-thvolatileinthepreparedstandardsolutionstc (0)isdeterminedaccordingtotheformulai

(1)


–1Component(s) Concentrationlimitofabsolutealcohol/mgL

EPmonograph EC110/2008 Opencommercialsources

acetaldehyde(+acetal) 8 50 8–40methanol 165 300 80–300higheralcohols/othervolatiles 240 50 80–240

Table 1Limitsofvolatileconcentrationsinethylalcohol.

–1 i stwhereρ =789300mgl istheethanoldensity;c andm arethepurityofi-theth i i–1compound in the i-th substance (mgmg ) and recorded mass of added i-th

st –1substance; c is the mass concentration (mgmg ) of ethanol in preparedethststandardsolutionandm isthemass(mg)ofstandardsolution.Furtherthezero-

orderapproximatedRelativeResponseFactors(RRF)aredeterminedaccordingtothe“EthanolasInternalStandard”method[7–8]bythefollowingequation

(2)

st swhereA andA arethevaluesofGCresponsetoi-thvolatileandethanol,fori eth

instance,peakarea.Hereafteritisnecessarytoestablishvolatilesconcentrationsin the initial ethyl alcohol, that is why it should be measured by GC. Theconcentrations of impurities are then calculated according to the followingformula

(3)

–1Theobtainedmgl AAconcentrationunitsfullycomplywiththeinternationalregulatory documents [1–5]. Most common volatiles presented in pure ethylalcoholareacetaldehyde,ethylacetate,methanolandhigheralcohols(fuseloils).ThenthevolatilesconcentrationsinstandardsolutionsmustbeclarifiedbytakingintoconsiderationtheamountofimpuritiesestablishedaccordingtotheEq.(3).Itisdoneaccordingtothefollowingformula

(4)

Thisishowtheiterationcircleisclosed.FurtheritisnecessarytomakeoneortwoadditionaliterationsbysubsequentusageofEqs.(2)–(4).Afterthisthequantita-tiveanalysisofatestsampleproceedsaccordingtotheEq.(3)butwithclarified

ethRRF values.i

Inordertoshowtheinfluenceofethanolspoilageontheestablishmentofcerti-fiedconcentrationsvaluestherelativebiasisapplied

(5)


2.Experimental


Initialhigh-purityethanolwaspurchasedfromJSC“DyatlovoWineandDistilleryPlantAlgon”(Belarus).

2.2Instrumentation

ThesampleswereanalysedwithaChromateс-Kristall5000gaschromatographequippedwithFIDandanautosampler.InstrumentcontrolanddataanalysiswereperformedwithUniChromsoftware(NewAnalyticalSystems,Minsk,Belarus).ThegaschromatographwasfittedwithcapillarycolumnRt-Wax,60m×0.53mmwith1μmphasethickness.Theoventemperaturewasthefollowing:theinitial

–1isothermat75°Cfor9minwasraisedto130°Catarateof5°Cmin thenraisedto–1180°Catarateof10°Cmin withfinalisothermof155°Cfor5min.Thecarrier

–1gaswasnitrogen(≥99.99%purity);thegasflowwas6.9mlmin ;theinjectortemperature was 160°C; the detector temperature was 200°C; the injectorvolumewas1μL;thesplitratiowas1:7.AnalyticbalanceOHAUSPA-214Cwithaprecisionof0.2mgwasusedforgravimetricpreparations.

2.3Standardsolutionspreparationandanalysis

Sixstandardwater-ethanolsolutionswith40%ethanolbyvolumewerepreparedgravimetrically according to [6] recommendations by subsequent addition ofindividualchemicalsubstancesorsolutions into initialwater-ethanolmixture.Theaddedindividualchemicalsubstanceswere:acetaldehyde,methylacetate,ethylacetate,methanol,2-propanol,1-propanol,isobutylalcohol,1-butanol,andisoamylalcohol.


Theconcentrationsofvolatilesinthepreparedstandardsolutionswerenearlythe–1following:3,10,50,200,250and500mgl AA.Eachsamplewasmeasuredtriply

in repeatability conditions. The standard solutions “WES-B”, which contained–1nearly500mgl AAofeachcomponentwasusedasacalibrationsolution.Then

theRRF(0)valueswerecalculatedaccordingtotheEq.(2)andinitialethylalcoholwasalsotriplymeasuredbyGC.Itwasfoundthatinitialethanolcontainedonlythree impurities,whichwere acetaldehyde,methanol and2-propanol (Fig.1).ThentheabovementionediterationsweredoneinaccordancewithEqs.(2)–(4).The detailed results of iterations are given in Table 2. Then the biases werecalculatedaccordingtotheEq.(5)andtransferredintographicalform(Fig.2).Astherewereonlythreeimpuritiesintheinitialethylalcohol,therearethreelineson


thecorrespondingdiagram.Itcanbeseen,thatincaseofstandardsolutionswith–1volatilesatverylowconcentrations,e.g.solutionswith3and10mgl AAvola-

tiles contamination, biases reach extremely high values. Asmethanol concen-tration inethylalcohol is thebiggestamongothervolatiles, itscorrespondinggraphisthemostrapidofall.Itisclearthattherelativebiasconstantlydecreaseswhenspeakingaboutmoresaturatedsolutionsbutstilltakesrathergreatvaluesformethanol.


Fig. 1Thechromatogramofethylalcoholusedforstandardsolutionspreparation.

–1Component Concentrationofabsolutealcohol/mgL Average RRF(0) RRF(I) RRF(II) detector Zero-order Intheinitial Specifiedin response approxima- ethylalcohol SSWES-B /nAmin tioninstan- dardsolu- tionsWES-B

acetaldehyde 435.9 1.85 437.8 4.80 1.290 1.295 1.295methylacetate 487.2 – 487.2 4.54 1.524 1.524 1.524ethylacetate 476.1 – 476.1 5.04 1.342 1.342 1.342methanol 526.2 18.16 544.3 6.32 1.182 1.222 1.2232-propanol 512.2 1.15 513.3 8.26 0.881 0.883 0.883ethanol 789300 789300 789300 11210 1.000 1.000 1.0001-propanol 532.4 – 532.4 11.32 0.668 0.668 0.668isobutylalcohol 553.0 – 553.0 12.34 0.636 0.636 0.6361-butanol 531.8 – 531.8 11.96 0.631 0.631 0.631isoamylalcohol 555.7 – 555.7 12.64 0.624 0.624 0.624

Table 2TheestablishmentofvolatilesconcentrationsinstandardsolutionsandthespecificationofRelativeResponseFactors(RRF).

4.Conclusions

Thecurrentstudyrevealedtheproblemofthecorrectestablishmentofstandardsolutionscertifiedvaluesasethylalcoholinitiallycontainsanalysedvolatiles.Thesuccessiveiterationsmethodofvolatilecompoundsconcentrationsandcalibra-tioncoefficientscalculationinwater-ethanolstandardsolutionswasproposedandexperimentallytested.Theanalysisofobtainedresultsshowsthatthemethodallowstheaccuracyofvolatilecompoundscertifiedvaluesincreasegreatly.Themost intelligent application of themethod seems to be duringpreparation ofstandardsolutionswithcongenersatlowconcentrations.Thereisnonecessityinanyexpensivematerials,additionalinstrumentsorlabour-costoperations,thatiswhytheproposedmethodcanbeappliedasaneasy,accurateandcheapproblemsolution.

Acknowledgments

AuthorthankshisscientificadvisersDr.SiarheiCharapitsaandDr.SvetlanaSytovafromInstituteforNuclearProblemsofBelarusianStateUniversity.

References

[1] CommissionRegulation(EC)No2870/2000of19December2000layingdownCommunityreference methods for the analysis of spirits drinks. Official Journal of the EuropeanCommunitiesL333/20.

[2] AOACOfficialMethods 972.10 and972.11.Alcohol (higher), methanol and ethyl acetate indistilledliquors(2005).Alternativegaschromatographicmethod.

[3] InternationalOrganizationofVineandWine(OIV).Compendiumofinternationalmethodsofanalysis of spirituous beverages of viti-vinicultural origin. Determination of the principalvolatilesubstancesofspiritdrinksofviti-viniculturalorigin.OIV-MA-BS-14:R2009.

[4] EuropeanStandardEN15721.Ethanol asa blendingcomponent forpetrol– Determinationhigheralcohols,methanolandvolatileimpurities–Gaschromatographicmethod,2009.


Fig. 2Thediagramofrelativebiasescausedbydisregardingofethanolspoilage.

[5] E Strasbourg,CouncilofEurope2017,p.2417–2420.uropeanPharmacopoeia.9thEd.[6] ASTMD 4307-99. Standard Practice for Preparation of Liquid Blends for Use as Analytical

Standards.[7] CharapitsaS.V.,KavalenkaA.N.,Kulevich N.V., MakoedN.M.,MazanikA.L.,SytovaS.N.,Zayats

N.I., Kotov Y.N.: Direct determination of volatile compounds in spirit drinks by gaschromatography.J.Agric.FoodChem.61(2013),2950–2956.

[8] CharapitsaS.,SytovaS.,KorbanA.,BoyarinN.,ShestakovichI.,CabalaR.:Theestablishmentofmetrological characteristics of the method “Ethanol as Internal Standard” for the directdeterminationofvolatilecompoundsinalcoholicproducts.JournalofChemicalMetrology12(2018),59–69.


1.Introduction

Tincoatingsarewidelyusedasafinishingcoatingforfoodindustryequipmentworkingsurfaces,forstoragepacksfromsteel,inelectronics,intheproductionofprintedcircuitboard,intheautomotiveindustryandtheproductionofdetailsusedinfriction. Stronglyacidicelectrolytesforindustrialelectrochemicaldepositionoftinaredemandedmostlyduetothepossibilitytoobtainshinydecorativecoatingsandduetohighstabilityanddepositionrateincomparisonwiththeknownweaklyacidic and alkaline electrolytes. Strongly acidic electrolytes consist of: Sn(II)sulphateorchlorideasametalsources;citrate-,tartrate-ionsasligandsfortheincreasingofsolutionstabilityowingtheformationofSn(II)bidentatecomplexes.pHofsolutionsisregulatedbyusingasulphuric,methanesulphonicorhydrochlo-ricacid[1].Differentsurfactantssuchas2-naphthol,gelatin,polyethyleneglycol,peptone[1,2]areusedintheelectrolytesforfine-crystallineclosely-packedcoa-tingselectroplating.Sn(IV)isformedduringtheelectrolytesstorageandexploi-tation both by reaction of Sn(II) oxidation by dissolved oxygen and anodicoxidationofSn(II).Inordertopreventpartlytheseprocessestheusageofantioxi-dants,suchasquinones,ascorbicacid,isneeded. Sn(II)ionscouldexistinhighmentionedsulphuricelectrolytesinnextforms:

2+ 2– +Sn , SnSO , Sn(SO ) , SnL , SnL ,which arepresented simultaneously at the4 4 2 2pH=0.5–2.0 (ref. [3]), aswellasSn(IV)compounds.Thepresenceofdifferent

Method of cyclic voltammograms in the determination of Sn(II) in strongly acid electrolytes for tin electrodeposition

a, bMARINASHIKUN *,OLGAVRUBLEVSKAYA

a InorganicChemistryDepartment,ChemistryFaculty,BelarusianStateUniversity, LeningradskayaStr.14,220030Minsk,RepublicofBelarus*[email protected] ResearchInstituteforPhysicalChemicalProblemsoftheBelarusianStateUniversity,LeningradskayaStr.14,220030Minsk,RepublicofBelarus


AbstractThewayofSn(II)quantitativedeterminationinthepresenceofSn(IV)in acid electrolyteswhichareused for the tin electrodeposition isproposed.ThemethodisbasedontheanalysisofcurrentdensityinthemaximumofthefirststageofSn(II)reduction.

KeywordsSn(II)Sn(IV)cyclicvoltammetrycurrentdensity

Sn(II)formsinthesolutioncausesthecomplicate(forexampletwostage)processofitsreduction. DuringtheelectrolyteexploitationSn(II)concentrationreduceswithcoatingdepositionandtheusageofsolubletinanodesdoesnotcompensatethedecreaseinSn(II)concentration.TheadjustmentofSn(II)concentrationandothersolutioncomponentsisneededforthelong-termelectrolytesexploitation.CorrectionofthesolutioncompositionisrequirediftheresidualconcentrationofSn(II)closeto30–40%oftheinitialconcentration. QuantitativedeterminationofSn(II)compoundsinaqueoussolutionscanbeproceededbychemicalmethodssuchas:iodometrictitration[4];precipitationwithinsolubleSn(II)sulphideformation;complexometrytitration,basedontheformationofasufficientlystableSn(II)complexwithethylenediaminetetraaceticacid. Physicochemical methods such as atomic absorption spectroscopy andinductivelycoupledplasmaatomicemissionspectroscopy[5]arealsousedforquantitative tin analysis. Chemicalmethods require a lot of time andmay beinaccurate if there are many components in the solution. Physicochemicalmethods are two or three orders of magnitude more accurate than chemicalmethods,butrequirespecialequipmentandspeciallytrainedpersonnel. Thepurposeoftheworkwastoanalysethepossibilityofquantitativeanalysisof Sn(II) compounds in amulticomponent solution in the presence of Sn(IV)compoundsbycyclicvoltammetry(CV).

2.Experimental


ModelsolutionsforSn-coatingselectrodepositionarepresentedinTable1.AllchemicalswerepurchasedfromSigma-Aldrich.


–3Component Concentration/moldm Sulphatesolution Chloridesolution Chloridesolution withSn(IV)

SnSO 0.020–0.100 – –4

SnCl .2H O – 0.020–0.100 0.020–0.0752 2

SnCl .5H O – – 0.020–0.0304 2

Thiourea 0.053 0.053 0.053Hydroquinone 0.005 0.005 0.005

Table 1Compositionsofmodelsolutionsfortindeposition.

2.2Instrumentation

Voltammetricmeasurementswerecarriedoutinthree-electrodecellwithcarbon2workingelectrode(withworkingsquare0.38cm ).Ag/AgCl/KCl(sat)electrode

andaplatinumwirewereusedasreferenceandcounter-electrodescorrespon-dingly. All potentials are referred to the Ag/AgCl/KCl(sat) electrode. Cyclic

–1voltammetrywascarriedoutatscanrate10mVs withtheusageofprogrammerPR-8andpotentiostatPI-50-1.1(Russia)controlledbysoftware.Thesurfacelayerofcarbonworkingelectrodewasdelayedmechanicallybeforeeachexperiment.


Fortheanalysisofthepossibilityofusagecyclicvoltammogramsforthequanti-tative determination of Sn(II) concentration CV curves were received for thesulphateandchloridesolutions(Fig.1а).ItispossibletoallotthreeareasofCVcurvesperspective for thedata comparison forquantitativeanalysisof Sn(II).Suchareasarethemaximumcurrentdensityofanodicpeakofthevolt-amperecurves(areaA)characterizingthedissolutionof tindepositedontheworkingelectrode;thepotentialsoftheSn(II)reductionbeginning(areaB);themaximumcurrentdensityof thepeakon the cathodicbranchof thevolt-ampere curvescharacterizingSn(II)reductiononthefirststagewhichisintherangefrom–0.50Vto–0.52Vforsulphatesolutionsandintherangefrom–0.53Vto–0.57V forchloridesolution(areaC).


Fig. 1–3(a)CV curves for chloride solutions with Sn(II) concentration 0.050mol dm (curve 1) and

–3 –30.100moldm (curve)andsulphatesolutionswithSn(II)concentration0.05moldm (curve2)–3and0.10moldm (curve4)

(b)The dependences between maximum anodic current density and Sn(II) concentration inchloride(curve5)andsulphate(curve6)solutions.

ThedependenceofSn(II)concentrationinsulphateandchloridesolutionsoncurrentdensityatthemaximumoftheanodicbranchofvolt-amperecurvesisshowninFig.1b.Theabovementioneddependenceswerenotlinearandcouldnotbeusedasacalibrator.TherewasnoanydependenceofSn(II)concentrationintheelectrolyteonthepotentialsoftheSn(II)reductionbeginning(areaB).AreaCissuitableforthequantitativeanalysisofSn(II).InsertinFig1aisthecathodicscansofvolt-amperecurvesintherangeofpotentialsfrom–0.48Vto–0.67Vfor

–3sulphateandchloridecontaining0.050and0.100moldm Sn(II).ItwasfoundthatthedependencesbetweenmaximumcathodiccurrentandtheSn(II)concen-tration for sulphate and chloride solutions are linear. Calibration curves arepresentedinFig.2. Mixed chloride solutions containing SnCl .5H O in concentrations4 2

–3 –30.010–0.030moldm andSn(II)inconcentrationequalto0.100moldm werepreparedinordertoanalysethepossibilityofquantitativeSn(II)determinationinthesolutionswithSn(IV).CathodicbranchesofCVcurvesarepresentedinFig.3.AccordingtotheanalysisofthecathodicbranchofCVcurveforchloridesolutioncontainingonlySn(IV)(Fig.3,curve3)itisobviousthatreductionpotentialoftheSn(IV)isequalto–0.63V.Itisnecessarytonotethatincaseofhighestconcen-

–3trationofSn(II)(0.100moldm )inchlorideelectrolytethemaximumofcurrentdensityisobservedat–0.57VthatismorepositiveincomparisonwithSn(IV)reduction. IncaseofmixedchloridesolutionsthemaximumofthecurrentdensityofSn(II)reductionisobservedatthe–0.65VpotentialandtheprocessofSn(IV)reductionisstartedat–0.63V.So,inthepresenceofSn(IV)itisimpossibletoobservethe


Fig. 2ThedependenceofSn(II)concentrationonthecurrentdensityinthemaximumofSn(II)reduc-tion(areaC)forsulphate(1)andchloride(2)solutions.

currentdensitypeakofSn(II)reduction.ThecomparisonofthecurrentdensitiescorrespondingtothemaximumofcathodecurrentsdensitiesinsolutionswithoutSn(IV)allowstodetermineSn(II)concentrationsinmixedchlorideelectrolyte.TheresultsofquantitativedeterminationofSn(II)inthepresenceofSn(IV)aregiveninTable2.ItisnecessarytonotethatconcentrationofSn(IV)ionsincaseofelectrolyteexploitationforelectrochemicaltindepositioninmanufactoryisoneorderofmagnitudelessthanforSn(II). TheresultsoftheexperimentalworkshowthatincaseofSn(II):Sn(IV)moleratioequalto3.75:1therelativeerrorisminimal.WiththedecreasingofSn(II)concentrationwhenSn(II):Sn(IV)=1.3:1therelativeerrorreaches6.9%.


Fig. 3CathodicbranchesofCVcurves forsulphate(curve1)andchloride(curve2)electrolytes–3containingSn(II)inconcentration0.100moldm ;thesolutioncontainingSn(IV)inconcentration

–30.020moldm (curve3).

–2 ac(Sn(IV)) c(Sn(II)) moleratio j/mAcm c Relativeerrordefined–3 –3 –3/moldm /moldm Sn(II):Sn(IV) /moldm /%

0.02 0.040 2:1 0.696 0.042 4.80.02 0.050 2.5:1 0.840 0.051 1.90.02 0.075 3.75:1 1.251 0.075 –0.03 0.040 1.3:1 0.712 0.043 6.90.03 0.050 1.7:1 0.872 0.053 5.70.03 0.075 2.5:1 1.257 0.075 –

acalculatedbymean-squireerrormethod

Table 2TheresultsofquantitativedeterminationofSn(II)inthepresenceofSn(IV)inthechloridemodelsolutions.

4.Conclusions

TheexpresswayofSn(II)concentrationdeterminationinsulphateandchloridesolutionsforelectrochemicaltindepositionwasfound.ItisbasedontheanalysisofthecurrentdensityinthemaximumofthefirststageofSn(II)reduction.TheanalysiscouldbeheldinthepresenceofSn(IV)compoundsaccumulatedinthesolutionduringitsexploitation.Therelativeerrorreachesthehighestvalueof6.9%inthecaseofSn(II):Sn(IV)moleratioequalto1.3:1andthelowestoneof1.9%at2.5:1moleratio.TheSn(II):Sn(IV)ratiois10:1intheconditionsofelectrolytesexploitationinmanufactory.Thatiswhyitispossibletoconcludethatproposed method is applicable for the high accuracy tin (II) quantitativedetermination.

Acknowledgments

The studywas supported by the Belarusian Republican Foundation for Fundamental ResearchunderProjectNo.X18М-060.

References

[1] WalshF.C.,LowC.T.J.:A reviewofdevelopments in the electrodepositionof tin.Surf. Coat.Technol.288(2016),79–94.

[2] ZaikoskaS.P.,MuloneA.,HansalW.E.G.,KlementU.,MannR.,KautekW.:Alkoxylatedβ-naphtholasanadditivefortinplatingfromchlorideandmethanesulfonicacidelectrolytes.Coatings8(2018),79–96.

[3] SurvilaA.,MockusZ.,KanapeckaiteS.,StalnionisG.:KineticsofSn(II)reductioninacidsulphatesolutionscontaininggluconicacid.J.Electroanal.Chem.667(2012),59–65.

[4] http://www.ecitechnology.com/articles/control-tinlead-solutions-electrodeposition-bumps(accessed22thJune,2018)

[5] RoncevicS.,BenuticA.,NemetI.,GabelicaB.:Tincontentdeterminationincannedfruitsandvegetablesbyhydridegenerationinductivelycoupledplasmaopticalemissionspectrometry.Int.J.Anal.Chem.2012,ArticleID376381.


1.Introduction

Scandiumtogetherwithyttriumandlanthanidesarecountedamongrareearthelements.Scandiumanditscompounds,duetoitsinterestingpropertiesismoreandmorewidelyappliedamongothersinoptical,chemical,laser,superconductor,medical treatment industries [1].Growinguseofscandiuminhigh-techposesathreatforitsreleaseandaccumulationinenvironment.Duetothesefactsthereisaneedforsimpleandaccurateanalyticalmethodsfordeterminationofscan-diuminenvironmentalsamples,suchaswater.Themainobstacleforapplicationof methods that are used for metal determination in water samples, namelyinductively coupled plasma optical emission spectrometry andmass spectro-metry(ICPOESandICPMS,respectively)islowconcentrationofscandium.Forthisreasonapplicationofpreconcentrationtechniquesthatallowforseparationof analytes frommatrix,with solid phase extraction as themost popular, are

Adsorption of Sc(III) on oxidized carbon nanotubes for separation and preconcen-tration from aqueous solutions – study of mechanism

a,b, a bMATEUSZPEGIER *,KRYSTYNAPYRZYNSKA ,KRZYSZTOFKILIAN

a FacultyofChemistry,UniversityofWarsaw, Pasteur1st.,02-093Warsaw,Poland*[email protected] HeavyIonLaboratory,UniversityofWarsaw,Pasteur5ast.,02-093Warsaw,Poland


AbstractOxidized multiwalled carbon nanotubes (CNT-COOH) has beenrecentlyproposedforseparationandpreconcentrationofscandiumionsfromwatersamples.InthepresentstudyadsorptionbehaviourofscandiumcationsonCNT-COOHhavebeeninvastigated.TheresultsshowthatatpH>4removalofSc(III) formsolutioncanbemainlyattributed to precipitation of insoluble scandiumhydroxide,whiledominatingmechanisminthepHrange1–3isadsorption.PropertiesofCNT-COOHasexcellentsorbentareconfirmedbysorptioncapacity

–1of40.1mgg atpH=3.0and25°C.Asfarastheadsorptionparame-ters is concerned, equilibrium data are in good agreement withFreundlich isotherm. Adsorption kinetics is best represented bypseudo-secondordermodelwithfilmdiffusionasmaincontrollingfactor.

Keywordsadsorptioncarbonnanotubesscandium

essential for obtaining accurate results. Carbon nanotubes (CNTs) have beenproventobeeffectivesorbentsfortheremovalofawidevarietyoforganicandinorganic pollutants dissolved in aqueousmedia [2].OxidizedCNTs, obtainedafterintroductionofoxygencontainingfunctionalgroups(suchas–OH,–C=Oand–COOH)ontheirsurfacewithseveralshowexceptionallyhighsorptioncapacityandefficiencyforremovalofheavymetalions[3].Theyhavebeenappliedforpreconcentrationofscandiumformwatersamples[4,5].

2.Experimental


–1Solutionsofscandiumwerepreparedfrom1000mgL stocksolutionfromMerckbystepwisedilution.Multiwalledcarbonnanotubescarboxylicacidfunctionali-zed (purity > 95%, length 1–5 μm and average diameter of 9.5 nm) werepurchasedfromSigmaAldrichandwereusedwithoutfurtherpurification.

2.2Instrumentation

AThermoScientific iCAP6000 ICPOES spectrometerwasused for scandiumdetermination.ThepHvaluesweremeasuredusingHannaInstrumentsHI2210-01 pH-meter supplied with glass-combined electrode. Boehm titrations weremadeusingMetrohmTitrandoautomatictitrator.PorousstructureofCNT-COOHwasevaluatedusingtheMicrometricsASAP2010analyzer.

2.3CharacterizationofCNT-COOH

ContentofsurfacecarboxylicgroupswasevaluatedusingBoehmtitration.Thespecific surface area was calculated by the Brunauer-Emmer-Teller (BET)method.TheporesizedistributionwasobtainedusingtheBarrett-Joyner-Halen-damethod.TheporevolumeswereobtainedfromthevolumeofN adsorbedator2

nearP/P =0.99.TheporousstructureofCNT-COOHwasevaluatedbynitrogen0

adsorptionisothermsat77K.

2.4Kineticstudy

Theadsorptionkineticsexperimentswerecarriedoutinordertoestablishthe–1kineticsoftheadsorptionprocessforaninitialSc(III)concentrationof2mgL

andapH=3.0asafunctionofcontacttimeintherangeof1–50min.

2.5Adsorptionstudies

Thesorptionstudieswereconductedbyequilibrating50mgofCNT-COOHwith–110mLof 5mgL 1 of Sc(III) solution having specified acidity for 4 h at room


temperature.Thesupernatantswere thendecantedand theconcentrationsofSc(III)wasmeasured.Theamountofretainedmetalionswerecalculatedasthedifferencebetweentheinitialandfinalconcentrationatequilibrium.TheresultsarebasedonatleastthreereplicateexperimentsforeachpHvalue.Simultane-ously,tocheckthepossibilityofscandiumremovalbyprecipitationinthestudiedpH range, similar experiments but in the absence of carbon nanotubes were

–1conducted.Thesolutionscontaining5mgL ofSc(III)andappropriatepHwerepassed through a 0.2 μm filter (Whatmann) to trap a possible precipitate.Subsequently,scandiumfromthefilterwaselutedbyHNO3anddeterminedbyICPOES. Forestimationofthesorptioncapacity,50mgofCNTswasmixedwith10mLof

–1Sc(III)solutionatpH=3.0andconcentrationrangeof1–300mgL .Aftershakingthe solution for 4 h, the metal concentration was determined. The sorption

–1capacityq (mgg )werecalculatedusingthefollowingequatione

q =[(c –c )V]/m (1)e o e

–1wherec andc arethe initialandequilibriumSc(III)concentrations(mgL ),o e

respectively,Vthevolumeofsolutionandmistheweightofansorbent.


3.1CharacterizationofCNT-COOH

TheN adsorption-desorption isothermofCNT-COOH isof type IV, typical for22 –1mesoporousmaterials.Thespecificsurfaceareawascalculatedtobe352m g

3 –1andtotalporevolume0.72cm g .Thecontentofsurfaceacidicgroupsevaluated–1bytheBoehmtitrationmethodwas2.41mmolg .

3.2EffectofsolutionpH

Astheacidityinfluencesnotonlytheadsorbentsurfacechargebutalsothemetalspecies present in solution it is one of themost important parameters in theadsorptionofmetalions.TheadsorptionofSc(III)ionsonCNT-COOHwasinvesti-gatedatpHintherangeof1–7.Theexperimentalresults,showninFig.1(nextpage),indicatethatadsorptionquicklyincreasesatpHrangingfrom1to4andremainsalmostconstantathigherpHvaluesandremovalofSc(III)isquantitative. TheresultsobtainedforexperimentswithoutCNT-COOHwerepresentedalsoinFig.1.ForpHvaluesupto3,therewasalittleornoprecipitationbutsignificantincreaseofscandiumremovalwasobservedinthepHrangeof3–7.Precipitation

–1ofscandiumhydroxidefromoursolution(0.11mmolL )iseffectiveatpH=4.77–31basedonitsK valueof2.22×10 .UntilpH=3Sc(III)isthepredominantspeciessp

present in thesolutionand in thepHrangeof4–9 itexists simultaneouslyas+ 4+Sc(OH) , Sc(OH) andSc (OH) .2 3 2 2


TheresultsofbothseriesindicatedthatremovalofSc(III)inthepHrangeof1–3couldbemainlyattributedtoitssorptiononcarbonnanotubes,whileatpH>4theresults obtained in the presence of CNT-COOHoverlapwith the precipitation,indicating insignificance of adsorption. Therefore, further experiments wereperformedatpH=3.0.ItistheadvantageintheviewofapplicationofCNT-COOHforseparationofSc(III)fromheavymetalionsastheyexhibitverylowaffinityforcarbonnanotubesundertheseconditions[6].

3.3Kineticstudy

Resultsforkineticsshowedthattheadsorptionraterapidlyincreasedduringthefirst10minandthen,asthenumberofsurfacesitesforsorptioncomesdown,graduallytendedtoequilibriumandadsorptionofSc(III)wasover95%duringthefirst2min,whichindicatedthatkineticsadsorptionequilibriumwasveryfast. InordertoinvestigatemechanismofSc(III)adsorptionfourdifferentkineticmodelswereappliedtotesttheexperimentaldata.EstimatedmodelsandkineticparametersarelistedinTable1(nextpage).Itcanbeconcluded,thatadsorptionofSc(III)followspseudo-secondorderkineticmodelandthemechanismofthatprocessmightbechemisorption.Furthermore,intra-particlediffusionmodelwasused to investigate the contribution of intraparticle and film diffusions intoadsorption process. The linear plot of experimental data to Weber-Morrisequationshowsearlyadsorptionstepupto10mintheprocessisgenerallycon-trolledbybothfactors.Thesubsequentadsorptionstep,characterizedbylowerslope,ismainlycontrolledbythefilmdiffusion.


–1Fig. 1EffectofpHonthesorptionandprecipitationofSc(III).Metalconcentration5mgL ;50mgofCNT-COOH.

2 3 4 5 6 70

20

40

60

80

100

Re

co

ve

ry,

%

pH

% ( sorption % ( precipitation))

3.4Adsorptionstudies

InordertodescribeSc(III)adsorptionbehaviouroncarbonnanotubes,theexpe-rimental datawas analyzed by three isothermmodels. The Freundlichmodeldescribesadsorptiononheterogeneoussurfacesandisrepresentedbyequation

–1/n q =K C (2)e F e

whereK andnaretheFreundlichisothermconstantsrelatedtoadsorptioncapa-F

cityandintensity,respectively. TheLangmuirmodel,whichassumesmonolayercoverageisdescribedbytheequation

–1 q =q K C (1+K C ) (3)e max L e L e

whereq andK are themaximummonolayers adsorption capacity and themax L

adsorptionenergyrelatedconstant,respectively. TheTempkinisothermassumesthatsorptionenergydecreaseslinearlywithsurfacecoverageandhasbeengenerallyappliedinthefollowingform

q =RT/bln(A C ) (4)e T e

whereA istheTempkinisothermequilibriumconstant,bistheTempkinconstantT

relatedtoheatofsorptionandTistheabsolutetemperature. The adsorption data for Sc(III) a little better fit the Freudlich equation

2 2(R =0.9974)thanforLangmuirisothermmodel(R =0.9881)aswellasTempkin2model(R =0.7476)reflectingmultilayeradsorption.Freudlichisothermisoften

usedforcasesofheavymetaladsorptionontocarbonmaterials[3].


Kineticmodel Equation Parameters

–1Pseudo-firstorderkinetic ln(q –q )=lnq –k t k =0.0643mine t e 1 12 R =0.8488

2 –1 –1Pseudo-secondorderkinetic t/q =1/k q +(1/q )t k =0.032gmg mint 2 e e 22 R =1.000

92 –1 –1Elovichequation q =βln(αβ)+βlnt α =4.4×10 mgg mint–1 β =0.0019gmg

2 R =0.97850.5 –1Intra-particlediffusion q =θ+k t k =0.393mint i i

θ =0.39302 R =0.8845

Table 1Parametersofvariouskineticmodelsfittedtoexperimentaldata.

4.Conclusions

CNT-COOHhashighadsorptionabilitytowardsscandiumionsfortheirprecon-centration and removal fromaqueous solutions. Themultilayer adsorption ofSc(III)wasobservedatpH=3.0.TheadsorptionkineticsontocarbonnanotubeswasfastandtheremovalofSc(III)wasover95%duringthefirst2min.Theobtain-eddatawere fittedusing thepseudo-secondorderkineticmodeland the filmdiffusionisthemaincontrollingfactor.AscomparedwithotherreportedmethodsregardingtheadsorptionofSc(III)ondifferentsolidmaterials,carbonnanotubesofferhighadsorptioncapacityandfastsorptionkinetics.

References

[1] PyrzynskaK.,KilianK.,PegierM.:Separationandpurificationofscandium:Fromindustrytomedicine.Sep.Pur.Rev.,inpress,DOI:10.1080/15422119.2018.1430589.

[2] PyrzynskaK.: Use of nanomaterials in sample preparation.Trends Anal. Chem.43 (2013),100–108.

[3] AbbasI.A.,Al-AmerA.M.,LaouiT.,Al-MarriM.J.,NasserM.S.,KhraishehM.,AtiehM.A.;Heavymetal removal from aqueous solution by advanced carbon nanotubes: Critical review ofadsorptionapplications.Sep.Purif.Technol.157(2016),141–161.

[4] JerezJ.,IsaguirreA.C.,BazanC.,MartinezL.D.,CeruttiS.:DeterminationofscandiuminacidminedrainagebyICPOESwithflowinjectionon-linepreconcentrationusingoxidizedmulti-walledcarbonnanotubes.Talanta124(2014),89–94.

[5] PegierM.,KilianK.,PyrzynskaK.:Enrichmentofscandiumbycarbonnanotubesinthepresenceofcalciummatrix.Microchem.J.137(2018)371–375.

[6] Stafiej A., Pyrzynska K.: Solid phase extraction of metal ions using carbon nanotubes.Microchem.J.89(2008),29–33.


1. Introduction

Rawmaterialsusedforbeerbrewingarewater,malt,hopsandyeast.Thequalityofbeerbasesonthequalityoftheserawmaterials[1].Thehop,Humuluslupulus,isapoplinplantbelongtothefamilyCannabaceae,whosefemalegenderproducescones,strobiles.Thishopconescontaincompoundsthatareusedinbrewingfortheiraromaticandflavoringproperties,especiallyasabitteringagent.Thecostofhops is related to theyieldperplant,becausegrowersmustcombatbacterialdiseases,fungusandmildew,virusdiseases,aswellaspestsandparasiticinvasiontoproduceacommoditythatisbothhighinyieldandqualitywithusingpesti-cides[2]. And themain sourcesofpesticides inbeerarehopsandbarleyaswell as.Pesticides are applied at many stages of barley cultivation and during post-harveststorageespeciallyagainstmicrobialpathogensandinsects,fungiandforweedcontrol[1].Maltingprocessincludesseveralstages,duringsomeofthemmayoccurtoreduceofsomepesticideresidues,especiallyinthecaseofwater-so-lubleresiduesduringthesteeping[3].Thehopbelongstoagriculturalcropswith

Behavior and fate of pesticides during beer brewing

a,b, bVLADIMIRAJANDOVSKA *,MARTINDUSEK

a DepartmentofAnalyticalChemistry,FacultyofScience,CharlesUniversity, Hlavova8/2030,12843Prague2,CzechRepublic*[email protected],PraguePLC, Lípová15,12044Prague2,CzechRepublic


AbstractThearticledealswithstudyingofthefateof16pesticides,commonlyusedtotreathopsinCzechRepublicduringbeer,brewingtoestimatetheirriskof theircarryover intobeer.Organichopswerecontami-natedusingpesticidesinthelevelof20ppmandthebeerwasbrewedwiththusspikedhopsonlaboratoryscale.ThesamplesweretakenaftereachstageofbrewingfordeterminationofpesticideresiduesandtheanalysiswascarriedoutusingbyLC/HR-MSinpositiveandnegativemode.Thepercentagesofresiduescarryoverintohoppedwortandthepercentagesofdecayreductionrelativetotheamountspikedonhopswerecalculated.Thepesticideresiduesweredividedintothreegroups,thatcharacterizedbehaviorofpesticides,amongother things, basedon their partition coefficientsn-octanol–water(logPvalues).

Keywordsbeerfatehopspesticide

intensive protection by pesticides. These compounds have prominent orpotentiallynegativehealtheffect[4]andtheycanpersistinthecropforalongtime.Basedonthatthemaximumresiduelevels(MRLs)ofpesticideresidueswereestablishedfordriedhops(butnotforbeer)andthecontrolofthesechemicalagentandtheirresiduesbecamenecessity.Theaimofthisworkisanswerthequestion: what is the fate of some commonly used pesticides during beerbrewing?

2.Experimental

2.1Chemicalandmaterial

Pesticide standards of abamectin, azoxystrobin, boscalid, cymoxanil, fenpyro-ximate, flonicamid, hexythiazox, imidacloprid, mandipropamid, metalaxyl,pyraclostrobin,quinoxyfen, spirotetramat, tebuconazole, thiamethoxam, triflu-mizole and internal standard azoxystrobin-d4, thiamethoxam-d3, triphenylphosphate(TPP)werepurchasedfromSigmaAldrich(St.Louis,MO,USA).

–1 Standard and internal standard stocks solutions (1.0 gL for all) werepreparedinacetonitrileor,incaseofsolubilityproblem,inmethanoloracetoneand stored at–20°C.A standardmixture solution,with all 16pesticides,was

–1preparedinacetonitrileat1mgL ofeachpesticide. Acetone (99.9% purity), acetonitrile, methanol, formic acid, amonniumformate (LC-MS grade) were purchased from Sigma Aldrich (USA). Sodiumchloride(analyticalgrade)wasobtainfromLach-ner(Neratovice,CzechRepub-lic) andmagnesiumsulfate (analytical grade)wasobtain fromPenta (Prague,Czech Republic). Sodium citrate tribasic dihydrate (99% purity), disodiumhydrogencitratesesquihydrate(99%purity)werepurchasedfromSigma-Aldrich(Germany).PurewaterwasobtainusingbyMilli-Qpurificationsystem(Merck-Millipore). Forbeerbrewinginlaboratoryscaleamalt,organichops(Saazvariety,CzechRepublic,TheHopResearchInstituteCo.)andyeast(Saccharomycespastorianus,RIMB95,TheResearchInstituteofBrewingandMalting,PraguePLC)wereused.

2.2Preparationhopsspikedwithpesticides

For spiking the organically grown, pesticide-free, dried hop coneswere used.A10gportionofthesegroundhopconeswereappliedtoPetridishinthinlayerandsubsequentlyspikedbypesticidemixturecontains200μgoftheeachabovepesticide. For spiking was used 100ml glass bottle with spray head. Afterapplicationofpesticidesthebottlewasadded3mlofacetonitriletorinsetherestof pesticides. Thus prepared hop sample was dried at room temperatureovernight.Nextdaythehopsamplewasgroundagain.


2.3Brewingbeerwithexperimentalpreparedhops

Infusionmashwaspreparedusingmashingdevice.Intoeachof5mashingbeakerswasmixed73.5gofgroundmaltwith400mLofbrewingwaterheatedon44°C.The mashing device was controlled by a computer software with setup oftemperaturegradient showed inTable1.Duringwholeprocess themashwasmixing.Thuspreparedmashesinbeakerswerecooleddownon60°Candfilteredbyusingafolderpaperfilter(type),whichwasinadvancerinsedbyhotwater.Thewort from the beakers were combined, the total volume of wort wasapproximately1700mLandtheoriginalgravitywasestablishedto12.25°P.Afteraddition of 380mL of brewingwater, the original gravity of sweetwortwasadjustedat10.04°P.Hoppedwortwas transferred intoboiling flask(4L)andheatedunderreflux,whenthewortstartedtoboilthen5g(2.5gperliterofsweetwort)ofthespikedhopwasadded.Hoppingofwortwascarriedoutfor90min.Subsequently, the hop wort was left at room temperature for 30 min withoccasionalroundedmixingtoprecipitatingsolidparticularincooleddownwort.Thecoldwortwasfilteredbyusingfolderfiltrationpaperrinsedinadvancewithhotwater.Thespenthopswerekeptforfurtheranalysis.Clearhoppedwortwascooledrapidlyto15°C.Then10gofactivatedyeastinwortwasaddedandtheprimaryfermentationwasconcludedinafermentationvesselwithairaccessat12°Cfor7days.Theprocessofmaturingcarriedoutin2Ltightlyclosedbottleat3°Cfor6weeks.

2.4Samplepreparationandextraction

Preparedsamplesforanalysiscanbedividedtotwogroups:hopsamplesandliquid samples.Groupofhop samples include spikedhopsand spenthops, toliquidsamplesbelongtohoppedwort,greenbeerandfinishbeer.ThespenthopsweretransferredonoriginalfiltrationpaperontoPetridishandspreadinthinlayer.Thedishwascoveredbyafiltrationpaperandletdryatroomtemperaturefortwodays.Driedspenthopsweregentlymincedtocrushlumps. A1gportionofhopsorspenthopssampleswasplacedintoa50mLcentrifugetube,10mLofwaterwasaddedandthesamplewasmixedusingvortexfor1minandlettosoakfornext30min.Then10mLofacetonitrileand50µLofinternal


t/°C Heatingtimeatthe Process temperature/min

50 20 Proteinrest62 30 Lowersaccharificationrest71 20 Highersaccharificationrest78 20 Mash-offrest

Table 1–1Temperatureprogramforinfusionmashing(therateofheating1°Cmin ).

–1standard(1mgL )wereaddedandthecontentwasmixedbyvortexfor1min.Subsequently,themixtureof4gofanhydrousmagnesiumsulfate,1gofsodiumchloride,1g trisodiumcitratedehydrate,and0.5gdisodiumhydrogencitratesesquihydrate were added, the tube was thoroughly capped and shakenvigorouslybyhandfor1min.After7minofcentrifugationat4500rpm6mLofupperacetonitrilelayerwastransferredtoa15mLcentrifugetubewith900mg

ofmagnesiumsulfate.Thetubewasmixedfor30susingvortexandcentrifuged

for7minat4500rpm.Finally,a2mLportionof theQuEChERSextractsweredilutedwithacetonitrilein20mLvolumetricflasks. Avolumeof10mLofliquidsampleswerepippetedinto50mlcentrifugetube,10mLofacetonitrilewereadded,andtheprocessofextractionwasthesamelikeincaseofhopssamples.Liquidsampleswereanalyzedundiluted.

2.6Standardadditionmethod

The method of standard addition was used for quantification of pesticides.Dilutionextract(hopsamples)ofvolumeof200µLwaspipettedinfour2mLvials

–1foreachsample.Thestandardsolution(1mgL )wasaddedtothreeofthesevialsatvolumecorresponding50,100and150%ororiginalamount.Theacetonitrileoftheappropriatevolumewasaddedtomakeafinalvolumeof600µLineachvial.Foranalysisofliquidundilutedsamples,thevolumeof400µLwaspipettedinfour2mLvialsforeachsampleandthestandardsolutionwasaddedinthesamerationasthepreviouscase.

2.6AnalysisusingLC-MS/MS

LC/HR-MS consisted of the chromatographic system Dionex UltiMate 3000UHPLC(ThermoScientific,Germany)includedabinarypumpHPG-3400RS,anautosampler WPS-3000TRS, a degasser SRD-3400 and a column over TCC-3000RS,andmassspectrometerQExactivehybridquadrupole-orbitrap(ThermoFisherScientificInc.Waltham,USA)withaheatedelectrosprayionizationsource(HESIII).TraceFindersoftwareversion4.1wasusedforevaluationofresults. Analyteswere separated on a reverse-phaseC18Atlantis T3 column (2.1 ×100mm, 3 µm) (WatersMilford, USA)with corresponding guard C18 columnSecurityGuardULTRA(Phenomenex,Aschaffenburg,Germany).Separationwasrealizedusingbygradientelution:0min:85%ofsolventA+15%ofsolventB,0.5min:85%A+15%B,9min:5%A+95%B,15min:95%A+5%Bwithaflow

–1rateof340µLmin ,whenthesolution2mMammoniumformatecontaining0.1%formicacidinwaterwasusedassolventAandmethanolwasusedassolventB.

Thecolumntemperaturewas40°Candinjectionvolumewas2µL. Ionswereionizedinpositiveandnegativeelectrosprayionizationmodeandtheionsprayvoltagewassetat2.8kVand–2.5kV,respectively.Thesheathgasflowwasat32arbitraryunits,theauxiliarygasflowratewaskeptat7arbitraryunits,


thecapillarytemperaturewas295°Candtheauxiliarygasheatertemperaturewassetat295°C.Nitrogenwasusedassheathandauxiliarygas. Themassspectrometerwasgenerallyoperatedinparallelreactionmonitoring(PRM). The precursor ions from scheduled inclusion list were, within theretentiontimewindow±0.3min,filteredinthequadrupoleatisolationwindow(targetm/z ± 0.7 amu), fragmented in HCD collision cell, product ions werecollectedintheC-trapat17.500resolution(FWHM,fullwidthathalfmaximum,atm/z=200),AGCtargetvalueof2e5,andmaximumioninjectiontimeof40msandfinallytwospecificpairsofprecursor-productiontransitionsweremonitoredforeach compound of interest. A mass tolerance of 5 ppm was employed. Theinstrument was externally calibrated prior to each measurement using themixtureofmasscalibrants.

3. Resultsanddiscussion

TheQuEChERSmethodused forextractionofpesticides fromhopmatrixwasmodifiedandvalidated[5].Theconcentrationofspikedpesticideswaschosento

–1be20mgkg toallowdilutiontoovercomematrixeffectsthataremassiveforthistypeofmatrix.Theamountofhopforhoppingwasmodifiedtosetupthepesticide

–1residueconcentrationto35–40μgL ofwort. Analyteswerequantifiedusingbystandardadditionmethod. Thetransferrates(inpercentages)ofeachpesticidedeterminedinspenthopsandhoppedworttotheoriginalresidueconcentrationinthepesticideenrichedhopswerecalculated(Table2).Allanalyteswerethensortedinfothreegroupsbased on their behavior during the beer brewing: (group A) the carryoverpercentagesintohoppedwortagainsttheamountincontaminatedhopwereatleast55%;(groupB)pesticidesremainedinspenthoporthatwereextractedlessthan from 45%; (group C) pesticideswhichwere not detected at all orweredetectedattracelevel. The results clearly showed that 90min boil has a significant influence onamountofafewpesticidessortedintogroupAandBaswell.Thesecarryoversofpesticidescouldberelatingtotheirpartitioncoefficientsbetweenn-octanolandwater(logPvalues)[1,6]andsolubilityinwater.Theresultsshowthatwatersolublepesticides(logP<3)wereextractedat>70%andpesticidesthathavelowlogP value, being<2,were almost fully extracted fromhops to hoppedwort.

–1PesticidesingroupBwerenotextractedatalloronlyminimally(0.001mgL forhexythiazoxandquinoxyfeninhoppedwort),correspondingtotheirlogPvalue.Totalamountofpesticidessuchastriflumizole(71%),wasreducedmorethanabout 50% due to unspecified thermal decomposition, pyrolysis, hydrolysisor/andadsorptionontoinsolublecomponentswhichrepresentthedominantandcommonreason for losespesticidesduringhoppingworth [7].PercentagesoftheselosseswerecalculatedandarelistedinTable2foreachpesticide.Theabilityofpesticidestobecarriedoverintohoppedwortwasexpressedasresidualratio


(R ;Table 2) and calculatedon the basin of pesticide amount in hoppedwortw

comparedtothesumofamountsofthepesticideinspenthopsandhoppedwort.Fromthebrewingtrialconductedwithpesticidespikedhops,theconcentrationsofpesticideresiduesweredeterminedinhoppedwortpriortheadditionofyeast,after seven days of fermentation and finally after 4weeks of fermentation intightenplasticbottle.All8pesticidescarriedoverintohoppedwort(groupA)werefoundingreenbeerandremainedinbeeratvariousratesofinitialtofinalconcentration(R values;Table2),whichseemedtobealsorelatedtologPvaluesb

ofthesepesticides.ResultsshowsthatpesticideswithalogPvalues(<3)tendedtoremaininthefinalbeer.

4.Conclusions

Inthestudy,abrewingtrialsimulatedthepreparationofabeerfrompesticidespiked hops as inmass production scale. The results showed that half of theanalyzed pesticides (group A)were carried over at an appreciable level. Thepesticideresiduetransferrateswerecalculatedfromhopstofinalbeerformostanalytes.Theaboveresultsconfirmedthatthetransferrateofpesticideresiduesmainly depends on its octanol-water partition coefficient (log P) and watersolubility.IfthepesticideslogPvalueslessthan3.75,theytendedtobecarriedovertofinalbeer.Otherwise,thehighlogPvalues(>4)ofpesticidesindicatethatthesepesticidessuchasfenpyroximate(logP=5.01),quinoxyfen(logP=4.66),largelyremainedinspenthops.Thelistof16LC-amenablepesticidesinvolvedin


Table 2Concentrationforeachpesticideinspikedhops,spenthops,hoppedwort,greenbeerandbeer,andwort(R )andresidualratioofpesticideinbeertotheoriginalresidueconcentrationinhoppedwortw

Group Compound Concentration Concentration Transferrate Concentration–1 inhops/mgkg inspenthops tospenthops inhopped

–1 /mgkg /% wort/%

A Azoxystrobin 15.54 2.17 14 0.028 Boscalid 15.16 4.28 28 0.025 Flonicamid 12.35 n.d. – 0.032 Imidacloprid 15.86 0.51 3 0.037 Mandipropamid 16.67 4.11 25 0.024 Metalaxyl 16.31 0.54 3 0.043 Tebuconazole 14.41 n.d. – 0.024 Thiamethoxam 13.70 n.d. – 0.036B AbamectinB1A 12.54 5.81 46 n.d. Fenpyroximate 16.18 9.98 62 n.d. Hexythiazox 14.21 11.91 84 0.001 Quinoxyfen 14.41 9.47 66 0.001C Cymoxanil 13.04 n.d. – n.d. Pyraclostrobin 15.53 n.d. – n.d. Spirotetramat 18.76 n.d. – n.d. Triflumizole 6.65 1.11 17 0.002

thisstudycovermajorityofcommonlyusedpesticidesfortreatmenthopplantsinCzechRepublic.BasedonthedatagatheredfromthemeasurementsshowninTable2,thecarryoverofpesticideresiduesfoundoncommerciallytreatedhopscanbeeasilyestimated.

Acknowledgments

ThisstudywassupportedbytheprojectofMinistryofEducationYouthandSportsoftheCzechRepublicNo.LO1312.

References

[1] NavarroS.,PerezG.,NavarroG.,VelaN.:Declineofpesticideresiduesfrombarleytomalt.FoodAddit.Contam.28(2007),851–859.

[2] HengelM.J.,MillerM.:Analysisofpesticidesindriedhopsbyliquidchromatography-tandemmassspectrometry.J.Agric.FoodChem.56(2008),6851–6856.

[3] Miyake, Y., Hashimoto, K.,Matsuki, H., OnoM., TajimaR: Fate of insecticide and fungicideresiduesonbarleyduringstorageandmalting.J.Am.Soc.Brew.Chem.60(2002),110–115.

[4] FantkeP.,FriedrichR.,JollietO.:HealthimpactanddamagecostassessmentofpesticidesinEurope.Environ.Int.49(2012),9–17.

[5] Dusek M., Jandovska V., Olsovska J.: Analysis of multiresidue pesticides in dried hops byLC–MS/MSusingQuEChERSextractiontogetherwithdSPEclean-up.J.I.Brewing,inpress,DOI:10.1002/jib.490.

[6] MiyakeY.,TajimaR.:Fateofpesticidemetabolitesonmaltduringbrewing.J.Am.Soc.Brew.Chem.61(2003),33–36.

[7] InoueT.,NagatomiY.,SugaK.,UyamaA.,MochizukiN.:Fateofpesticidesduringbeerbrewing.J.Agric.FoodChem.59(2011),3857–3868.


Group Compound Concentration Concentration Transferrate Concentration Transferrate Decomposition Concentration Concentration R /% R /% logP Solubilityw b–1 inhops/mgkg inspenthops tospenthops inhopped tohopped rate/% ingreenbeer inbeer inwater

–1 –1 –1 –1 /mgkg /% wort/% wort/% /mgL /mgL /mgL

A Azoxystrobin 15.54 2.17 14 0.028 71 15 0.029 0.024 0.84 0.88 2.50 6.7 Boscalid 15.16 4.28 28 0.025 65 7 0.022 0.018 0.70 0.72 2.96 4.6 Flonicamid 12.35 n.d. – 0.032 103 0 0.028 0.027 1 0.86 –0.24 5200 Imidacloprid 15.86 0.51 3 0.037 93 4 0.033 0.033 0.97 0.89 0.57 610 Mandipropamid 16.67 4.11 25 0.024 57 18 0.024 0.019 0.70 0.81 3.20 4.2 Metalaxyl 16.31 0.54 3 0.043 105 0 0.035 0.036 0.97 0.84 1.65 8400 Tebuconazole 14.41 n.d. – 0.024 68 32 0.017 0.018 1 0.75 3.70 36 Thiamethoxam 13.70 n.d. – 0.036 105 0 0.033 0.032 1 0.89 –0.13 4100B AbamectinB1A 12.54 5.81 46 n.d. – 54 n.d. n.d. – – 4.40 n.a. Fenpyroximate 16.18 9.98 62 n.d. – 38 n.d. n.d. – – 5.01 0.023 Hexythiazox 14.21 11.91 84 0.001 3 13 n.d. n.d. 0.03 – 2.67 0.1 Quinoxyfen 14.41 9.47 66 0.001 4 31 n.d. n.d. 0.05 – 4.66 0.047C Cymoxanil 13.04 n.d. – n.d. – 100 n.d. n.d. – – 0.67 780 Pyraclostrobin 15.53 n.d. – n.d. – 100 n.d. n.d. – – 3.99 1.9 Spirotetramat 18.76 n.d. – n.d. – 100 n.d. n.d. – – 2.51 29.9 Triflumizole 6.65 1.11 17 0.002 13 71 n.d. n.d. 0.43 – 4.77 10.5

theirlogPandwatersolubilityvalues.Thecalculatedresidualratioofpesticideafterhoppingofafterfermentation(R ),transferrates(carryovers)anddecompositionrateduringworthopping.b

1.Introduction

Lipidomicsisapartof“omics”sciencethatallowstostudythebiochemicalandmolecularcharacterisationoflipidspresentinagivenbiologicalsystem,fluid,cellortissue[1],andlipidchangesthatwereinductedbysomefactors[2–3].Lipidsarealargenumberofstructurallyandfunctionallydiversemolecularspeciesthatcoverabroadrangeofpolarity,fromnon-polar(e.g.,triacylglycerides)topolar(e.g.,phospholipids)[4–5].Inabiologicalsample,lipidssometimesarepresentinsignificantlyranginglevels,fromfemtomoleleveluptomicromolelevel[6].Thesedifferencesinlipidstructuresandlevelsofpresenceinbiologicalsampleintro-duceconsiderablechallengestocompleteefficientlipidomeextraction.Carefulsamplepreparationisacriticalstepinanalyticalchemistrytogeneratesampleforachemicalmeasurementaccurately.Itiscriticaltodevelopsamplepreparationmethods for lipidomics that are reproducible, fast, and enables extraction ofawiderangeofanalyteswithdifferentpolaritiesandconcentration[7]. Conventionalmethodsofoptimisingaprocessrequirevaryingoneparameterpertrial[8].Thisapproachisverytime-consuming[9–10].Takingintoaccountthevastnumberofparametersthatcanaffectasuccessfullipidextraction,itis

Design of Experiment approach for lipid extraction optimisation in lipidomics

INALBAKHYTKYZY*,WERONIKAHEWELT-BELKA,AGATAKOT-WASIK

DepartmentofAnalyticalChemistry,FacultyofChemistry,GdanskUniversityofTechnology,11/12NarutowiczaSt.,80-233Gdansk,Poland*[email protected]

AbstractLipidomics isa lipid targetedmetabolomicsapproach thataimsatcomprehensiveanalysisoflipidsingivensample.Lipidsarestructu-rally and functionally diverse group of small molecules that playmultipleimportantrolesinbiologicalsystems.Thelargediversityinstructureandfunctionoflipidsmakesitahugechallengetodevelopacomprehensivelipidextractionmethod.Inthestudy,anoptimiza-tionoflipidextractionmethodbasedonDesignofExperimentshasbeendescribed.A two level full-factorial experimental designwasused as amultivariate strategy for the evaluation of the effects ofvarrying several variables at once. The effects of four differentvariables elution solvent, ratio of stationary phase, extraction andelutionvortexingtime,onthenumberofextractedmolecularfeatureshavebeeninvestigated.Fromthesestudies,certainvariablesshowedupassignificant.

KeywordsDesignofExperimentlipidomicslipidextraction


clearthattheimplementationofDesignofExperimentapproachisneededtobeeconomical,bysavingtimeandmoneyonchemicals. Theimportantfeaturesofdesigninganexperimentusingstatisticaltoolsaretobetimeeffective,enhancecapability,andincreaseprocessfeasibility.ThemainintentofusingDesignofExperimentinthisworkistosimultaneouslyexaminethevarious factorsaffecting the lipidextractionefficiency.Thisapproachhelps toeliminate the less significant factors and focus on optimising only the mostimportantones.Thefullfactorialdesignisacommonlyusedtwo-leveldesign.Itis

kdescribedas2 designwhere2standsforthenumberoffactorlevelsandkisthenumberoffactors,eachwithalowandhighvalue[12]. Thegoalof this research is tooptimisehumanbreastmilk lipidextractionprocessusingFactorialDesign.Humanbreastmilkisusedasamodelbiologicalmatrix because it is complex and contains lipid classes with considerabledifferenceinabundanceandchemicalstructure.

2.Experimental


LC–MSgrademethanol,HPLCgradehexanewerepurchasedfromMerck(Ger-many),2-propanolandammoniumformate(99.9%purity)werepurchasedfromSigma–Aldrich(USA).DeionizedwaterwaspurifiedbyanHLP5system(Hydro-lab,Poland).

2.2Experimentaldesign

Four variables affecting the extraction efficiency were selected to define theexperimentaldomain.Thesevariableswere:1.elutionsolvent,2.stationaryphaseratio(HybridSPE-PhospholipidandC18bothfrom(Sigma-

Aldrich,USA)),3.extractiontime,4.elutiontime.

4Fourvariablesat2levelsgive2 fullfactorialdesign.Thetotalnumberofexperi-ments including replicates and one control sample for each set was 48. Thevariables considered, the code used, the low and high levels studied, andresponsesareshowninTable1(nextpage).

2.3Samplesandsampletreatment

Thepooledsample,whichwaspreparedbymixing150μLofpreviouslycollected(n=71) human breast milk samples, was used for lipid extraction methoddevelopmentandoptimizationusingfactorialdesign.Milksampleswerestoredinpolypropylenetubeat–80°Cpriortoanalysis.


Samplepreparationwasbasedontheextractionoflipidscontainedinhumanbreastmilkwith theuseofSolidPhaseDispersiveExtraction technique.First,30mg of an apropriate stationary phase was weighed in 1.7 mL Eppendorfmicrotubes (VWR International, Poland). Protein precipitation was done asfollows:100µLofhumanbreastmilksamplewastransferredto15mLpolypro-pylenetubeandmixedwith900µLof1%formicacidinmethanol.Aftervortexingfor30s,samplewascentrifugedfor5minat10000rpm.900µLofsupernatantwastransferredtotheEppendorfmicrotubesandvortexed.Afterthestationaryphase was precipitated, the supernatant was carefully discarded using GlassPasteurPipette(150mm,VWRInternational,Gdansk,Poland).Then,1000µLofelutionsolventwasaddedtothestationaryphaseandvortexed.Thesupernatantwas carefully collected using a syringe with needle (1 mL, Terumo, LagunaTechnopark,Binan,Laguna,Philippines),filtratedandanalysed.

2.4Instrumentation

TheHPLCsystemusedwasanAgilent1290LCsystemequippedwithabinarypump, an online degasser, an autosampler and thermostated column


Run X X X X Numberofmolecularfeatures1 2 3 4

1 – – – – 4152 + – – – 5313 – + – – 4394 + + – – 7325 – – + – 3886 + – + – 6047 – + + – 4318 + + + – 7779 – – – + 41510 + – – + 57911 – + – + 41312 + + – + 74913 – – + + 36814 + – + + 57815 – + + + 42216 + + + + 782

Table 14Experimentalvariables,levels,designmatrix,andresponsesinthe2 fractionalfactorialdesignfor

lipidextractionfromhumanbreastmilk.

Variable Coded Level Low(–) High(+)

elutionsolvent,methanol:2-propanol:NH X 81:14:5(v/v/v) 14:81:5(v/v/v)3 1

stationaryphaseratio X 9:1(w/w) 1:9(w/w)2

extractionvortextime X 1min 5min3

elutionvortextime X 1min 5min4

compartmentcoupledtoa6540Q-TOF-MSwithadualelectrosprayionization(ESI)source(AgilentTechnologies,USA). The chromatographic separationmethodwas previously developed in ourgroupanddescribedinanarticlewhichisstillinpress.Briefly,theseparationwascarriedoutbyusinganAgilentPoroshell120EC-C8,(150×2.1mmI.D.,1.9μmparticlesize)columnwith0.2µmin-linefilter.Theelutionprogramwasgeneratedwithamixtureof5mMammoniumformate inwaterandmethanol(1:4,v/v)(componentA)andamixtureof5mMammoniumformateinwater,n-hexaneand2-propanol(1:20:79,v/v/v)(componentB)asfollows:0to15min,B(%)10to50(linealincrease);15to20min,B(%)50to100(linealincrease).Subsequently,columnwaswashedfor0.5minat100%Bandthegradientreturnedtostartingconditions and system was re-equilibrated for 10 min. The flow rate was

–10.5mLmin and the injection volume was 0.5 μL. The column was kept atconstanttemperatureof45°C.DatawereacquiredinESI+(SCAN)modeintherangefrom200to1700m/zinthehigh-resolutionmode(4GHz).TheESIsourceconditionappliedwasoptimizedearlieranddescribedindetailelsewhere[11].

2.5DataAnalysis

TheTICchromatogramswerefirstvisuallyexaminedtodetectanyretentiontimeshift.Then,dataprocessingwasdoneusingmolecularfeatureextractionoptioninMassHunterWorkstationSoftwareQualitativeAnalysis,B.03.01version(AgilentTechnologies,USA).Parametersforthemolecularfeatureextractwereasfollows:extractionalgorithm,smallmolecule;inputdatarange,restrictedretentiontime0.90−20.00min,restrictedm/z=200−1700;peakfilters;peakwithhight≥1000,ionspecies,+H,–H;peakspacingtolerance0.0025m/zplus7.0ppm; isotopemodel, common organic molecules charge state, 2.molecular feature extractresultedinalistofallmolecularentities,whichincludedthefullTOFmassspectraldataforeachsample. Identification of lipids compoundswas performed by comparing themassaccuracyofobtainedMFagainstonlinedatabase-LipidMAPS.Masserrorwassetto5ppm.


We applied screening factorial design to evaluate the factors (variables)influencinglipidextractionefficiency.Thefactorsconsideredinthisstudywere:elutionsolvent,stationaryphaseratio,extractiontime,elutiontime.Thevariableswere chosen according to their importance in extraction process. Number ofmolecularfeaturewasusedasresponse. Obtained results of molecular features for each experimental setup arepresentedinthelastcolumnofTable1.Thehighestnumberofmolecularfeaturesswereobservedinexperimentalruns4,8,12and16.Intheseexperimentalsetups


X andX variableswereatthesamelevel,whereasX andX weredifferent.Itcan1 2 3 4

be assumed that vortexing time (X and X ) did not influence the number of3 4

extractedmolecular features. To check the effect of individual factors on thenumberofextractedmolecularfeatures,themaineffect(ME)ofeachfactorwascalculatedusingequation

(1)

Fig.1displaysthemaineffectsplot.Theslopeofthelineisproportionaltothesizeoftheeffect,anditconfirmsthatvortexingtimedidnotinfluencethenumberofextractednumberofmolecularfeatures.Elutionsolventandstationaryphaseratiopositivelyinfluencedthenumberofmolecularfeatures.Theseparameterscanbefurtherconsideredintheoptimizationdesign,toobtainoptimumcondi-tions.

4.Conclusions

Factorial design is a fast and universal tool for evaluating the significance ofagivenparametertowardsobservedresponse.Ascouldbeshown,elutionsolventandstationaryphaseratiohadmoreimpactonthenumberofextractedmolecularfeaturesinthisextractionmethodandshouldbeconsideredduringthefurtheroptimizationdesigns.


Fig. 1MainEffectsPlot.

References

[1] Navas-IglesiasN.,Carrasco-PancorboA.,Cuadros-Rodrıg uezL.:Fromlipidsanalysistowardslipidomics,anewchallengefortheanalyticalchemistryofthe21stcentury.PartII:Analyticallipidomics.TrACTrendsAnal.Chem.28(2009),393–403.

[2] ZhaoY.Y.,VaziriN.D.,LinR.C.:Lipidomics:Newinsight intokidneydisease. In:Advances inClinicalChemistry.MakowskiG.S.(edit.).Elsevier2015,p.153–175.

[3] HuC.,vanderHeijdenR.,WangM.,vanderGreefJ.,HankemeierT.,XuG.:Analyticalstrategiesinlipidomics and applications in disease biomarker discovery. J. Chromatogr. B877 (2009),2836–2846.

[4] LamS.M.,ShuiG.:Lipidomicsasaprincipaltoolforadvancingbiomedicalresearch.J.Genet.Genomics40(2013),375–390.

[5] SandraK., Sandra P.: Lipidomics froman analytical perspective.Curr. Opin. Chem.Biol.17(2013),847–853.

[6] QuehenbergerO., ArmandoA.M., BrownA.H.,Milne S.B.,MyersD.S.,Merrill A.H., Bandyo-padhyayS.,JonesK.N.,KellyS.,ShanerR.L.,SullardsC.M.,WangE.,MurphyR.C.,BarkleyR.M.,LeikerT.J.,RaetzC.R.,GuanZ.,LairdG.M.,SixD.A.,RussellD.W.,McDonaldJ.G.,SubramaniamS.,FahyE.,DennisE.A..:Lipidomicsrevealsaremarkablediversityof lipids inhumanplasma.J.LipidRes.51(2010),3299–3305.

[7] CajkaT.,FiehnO.:Comprehensiveanalysisoflipidsinbiologicalsystemsbyliquidchromato-graphy-massspectrometry.TrACTrendsAnal.Chem.61(2014),192–206.

[8] DejaegherB.,HeydenY.Vander:Experimentaldesignsandtheirrecentadvancesinset-up,datainterpretation,andanalyticalapplications.J.Pharm.Biomed.Anal.56(2011),141–158.

[9] KalilS.J.,MaugeriF.,RodriguesM.I.:Responsesurfaceanalysisandsimulationasatool forbioprocessdesignandoptimization.ProcessBiochem.35(2000),539–550.

[10] VanderHeydenY.,PerrinC.,MassartD.L.:OptimizationstrategiesforHPLCandCZE.In:Hand-bookofAnalyticalSeparations.ValkoK.(edit.).Elsevier2000,p.163–212.

[11] GarwolinskaD.,Hewelt-BelkaW.,Namiesnik J.,Kot-WasikA.:Rapidcharacterizationof thehuman breast milk lipidome using a solid-phase microextraction and liquid chromato-graphy–massspectrometry-basedapproach.J.ProteomeRes.16(2017),3200–3208.

[12] Quinn G.P., Keough M.J.: Experimental Design and Data Analysis for Biologists. New York,CambridgeUniversityPress2002.


1.Introduction

Urinarycathetersareusedwhenthenaturalurinaryoutputishindered.Oneoftheindicationforcatheterizationoftheurinarybladderisanaccurateassessmentoftheamountofurineoutput.Catheterizationisgiventopatientsundergoingsomesurgicalprocedures.Anotherindicationforcatheterizationisthestateofurinaryretention occurring involve in bladder inflammation. Catheters are used afterurologicalprocedurestohealtheurinarytract,whenit isnecessary.Themostfrequently complication reported after catheterization are urinary tractinfections[1].Urinarytractinfectionsarethemostcommoninfectionsassociatedwith healthcare and constitute about 40% of all infections in hospitalizedpatients.Catheter-associatedurinarytractinfections)are80%ofurinarytractinfectionsandareresultfromthepresenceofcathetersintheurinarytract[2]. Catheterizationmaycontributetothedisruptionofthenaturaldefensesystemoftheurinarytractandcanallowingthecolonizationofbacteria,leadingtothecreationofbiofilmsandthedevelopmentofinfections.Microbeshavetheabilityto adhere and create a biofilm on the surface of the cathetersmaterials. It is

Release of active substances from polymeric coatings in medical applications

a, a bMAŁGORZATABOROWSKA *,AGATAKOT-WASIK ,JUSTYNAKUCIN SKA-LIPKA

a DepartmentofAnalyticalChemistry,FacultyofChemistry,GdańskUniversityofTechnology, 11/12GabrielaNarutowiczaStreet,80-233Gdańsk,Poland*[email protected] DepartmentofPolymerTechnology,FacultyofChemistry,GdańskUniversityofTechnology, 11/12GabrielaNarutowiczaStreet,80-233Gdańsk,Poland

AbstractCatheter-associatedurinarytractinfectionsaretheresultofcathe-terization of the bladder. The risk of infection increases withlengtheningtimerequiredforcatheterization.Bacterialcellscantoadhereandcreatethebiofilmonthesurfaceofcathetermaterials.Microbes that are the integral part of the biofilm show greaterresistance to agents used for their degradation. The treatment ofurinarytractinfectionsareassociatedwithrequiretheoraladmini-strationoflargeamountsofantimicrobialdrugs.Analternativemaybe the use of antimicrobial release coatings on the surface ofcatheters. These coatings can to allow target drug delivery andcontribute to reducing thedose and improving adrug availability.HPLC-MSsystemisaverygoodsolutiontoanalysisofreleasethedrugfromantimicrobialcoatingsanditisusedinthisstudies.

Keywordsantimicrobialcoatingscatheterizationurinarycathetersurinarytractinfections


importancewhenthecathetersareplacedinbodyforalongtime[3].Theriskofdevelopingurinarytractinfectionsisdirectlyproportionaltothelengthofstayofthecatheterintheurinarytract.Short-termcatheterization(lessthan7days)isthecauseofinfectionsinalmost50%ofpatients.Forpatientsundergoinglong-termcatheterization(upto28days),theriskofinfectionincreasesto100%[2]. Bacteria’sbiofilmprovidesprotectionforthebacteriaagainstantimicrobialagents, antibodies and defences of the human body. It is a serious problembecausebacterialcellsthatareanintegralpartofbiofilmareupto1000-foldmoreresistanttoantimicrobialagentscomparedtoplanktonicformofbacteria[4].Inadditioncathetercolonizedbymicrobesmustbereplacedresultinginincreasedmorbidityforthepatientandincreasedcosttothehealthcaresystem[3]. Froma preventionurinary tract infections standpoint, themost importantaspect are develop catheters with materials that prevent microorganismattachmentandbiofilmformation[3].Themosteffectivechoiceseemstocoatingofcathetersurfacewithantimicrobialagentsorpolymercoatingloadedwithanti-microbialagents[4].Thepolymercoatingshouldbedegradable,sothatduringtheimplementationgraduallyreleasethedrugsubstances. Theaimofthisstudywastoprepareantimicrobialcoatingsfrompolyvinyl-pyrrolidone(PVP)withclindamycinwhichwerechooselikepolymertopreparethecoatingsandantimicrobialagent,respectively.AntimicrobialactivityofPVP-clindamycincoatingsweretestedagainstStaphylococcusaureus(Gram-positivebacteria) which is the microorganism most frequently involved in catheter-relatedinfections[3].

2.Experimental

2.1Chemicalsandreagents

PolyvinylpyrrolidonewaspurchasedfromSigma-Aldrich(USA).ClindamycinwaspurchasedfromPfizer(USA)informoftabletslabeledtocontain300mgclinda-mycinpertablet.Salinesolution(0.9%)waspurchasedfromPolpharma(Poland).Acetonitrile(HPLCgrade)andformicacid(>98%)werepurchasedfromMerck(Germany). Acetonitrile (LC-MS grade) was purchased from VWR Chemicals(USA).UltrapurewaterwaspreparedusingHPL5systemfromHydrolab(Poland).


PVPcoatingswerepreparedbydissolvingtheappropriateamountofpolymerindeionizedwater.Thesesolutionsconsisted1,3,5and7%ofPVP,respectively.AfterthedissolutionoftheentirePVP(usingmixingandheating),thesolutionswerepouredintoPetridishesfordrying.Thecoatingwiththebestpropertieswasthen selected and shells were prepared with the addition of clindamycin,respectively,1,3,5%fortheselectedconcentrationofPVP.


2.3CharacterizationofPVPcoatings

CoatingswithdifferentPVPconcentrationswereobservedusingOpticalMicro-scopy fromcompanyBresser toassess theirhomogeneity.Thedegradationofcoatingsaftertheimpactof0.9%NaClwasalsotested.

2.4Chromatographyandmassspectrometry

AnAgilentG1379BLCsystemconsistedwithbinarypump,anon-linedegasser,anautosamplerandathermostatedcolumncompartmentcoupledwith1100SeriesLC/MSD.Purospher®STARRP-C18e(125×3mm,5µm;Merck,Germany)column

–1wasusedduringanalysis.Theflowratewas1mLmin ,injectionvolumewas5µL,columntemperaturewas30°C.Themobilephasegradientwas0–2min:15.0%B,2–4min:increaseofeluentBto30.0%,4–6,5min:30.0%Bandbetween6.5and6.6min change to start conditions (15.0%B),whereAwaswater andBwasacetonitrile,bothofsolventsincludedof0.1%formicacidaddition.TheESIsourcewasoperatedwithpositiveionmode.Thefragmentatorvoltagewassetat80eV.Nebulizergaswassetat35psianddryinggastemperaturewassetat300°C.

2.5Clindamycinreleasestudies

Drugreleasestudieswereconductedfortheclindamycinmodifiedcoatings(5%additionofclindamycinincoatings).Thepreparedcoatingswerecutintosquares(5×5mm)andwereweighted.Inthenextstage,thepreparedsampleswereplacedin0.9%NaClsolutionfor15,30and60minutes,respectively.Afterapredeter-minedtime,asamplesolutionwastakenandtestedbyHPLC-MS(procedure2.4).

2.6Microbiology

Antibacterialactivitiesofclindamycin-PVPbasedcoatings(3%PVPcoatingswith1, 3 and 5% addition of clindamycin) were evaluated against Staphylococcusaureus,Gram-positivebacteria,usingdiskagardiffusionmethod.ThebacterialcellswererefreshedbygrowinginLuria-Bertanibrothmedium(10 gNaCl,10 gpeptone,and5 gyeastextractperliterofdistilledwater),andincubatedat37 °Cfor24  h.Then, thebacterialcultureswerediluted10-foldusingLuria-Bertanimedium,and0.1  mLof thebacterialsuspensionswerespreadovertheLuria-Bertaniagarplatesanincubatedat37°Cfor24 h.Thepolymericcoatingswerecut,sterilizedwith70%ethanolandfollowedbydryingunderUVlamp(30min).Thecoatingsweregentlyplacedontheagarplatesusingforceps,andtheplateswereincubatedat37°Cfor24 h.Afterincubation,zonesofinhibitionofbacterialgrowthwereobserved.


Fig. 1 The growth inhibition zonesof Staphylococcus aureus subjected tointeraction with antimicrobial coatingcontaining1,3and5%ofclindamycin.


3.1CharacterizationofPVPcoatings

Polymericcoatingscontaining1,3,5and7%ofPVPwereprepared.Inthecaseof1and3%ofPVPcoatings,ahomogeneousstructurewasobservedwhilefor5and7% amount of PVP in coatings, homogeneity was not obtained. The coatingcontaining1%PVPturnedouttobebrittleandunstableincontactwith0.9%NaCl(too fast solubility),whichmeans that it cannot be used in next studies. Theremainingcoatings(3,5,7%)showedgoodflexibilityandsufficientdurability.Degradation of these three coatings were slower. In this step coating whichinclude3%ofPVPwaschoosetopreparecoatingswithadditionofclindamycin.

3.2Microbiologytest

Polymericcoatingscontaining3%ofPVPanddifferentamountofclindamycin(1,3,5%)subjectedtointeractionwiththeStaphylococcusaureusbacterialstrain.ThegrowthinhibitionzonesofthisbacteriaareshowninFig.1. Basedonthedrawing,itiseasytonoticethatthehighertheconcentrationofantibiotic in the coating, that the growth inhibition of Staphylococcus aureusincreases.Butfor5%ofdrugamounttheinhibitionzoneisnotverymuchbiggerthanto3%containingofclindamycin.

3.3Clindamycinreleasefromantimicrobialcoatings

Theanalysisoftheclindamycinreleaseratefromcoatingscontaining3%ofPVPwascarriedoutatvarioustimeintervals(15,30,60min).Thisisthetimefromplacingtheantimicrobialcoatingfragmentinsalinesolutiontotakeasampleofthe solution foranalysisbyHPLC-MS.Thereleaseprofileof clindamycin fromcoatingswasdefinedasthedissolutionofPVPwiththesimultaneousreleaseof


clindamycinat threedifferent time intervals (15,30,60min).The results areshowninFig.2andarepresentedastheratioofthepeakareatothemasscoatingimmersedinthesolutionasafunctionoftime. ReleaseofclindamycinfromPVPcoatingswasoccurredimmediately.Afterthefirsttimeinterval(15min)waselutedapproximately80%ofclindamycin.After30minuteswas observedmaximally concentration of drug in saline solution.After the thirdpointof time(60min)approximately90%ofclindamycinwasreleasedfromcoating. At the beginning, release of clindamycin from PVP-based coatings happensquicklyandreachesmaximumconcentrationafterjust30minutes.Thenthereisaslowdecreaseintheconcentrationofdruginsalinesolution.Thatmechanismreleasingofclindamycincanprovideaquickcounteractingthegrowthofbacteria.Itmayhelptoreducedtheriskofinfection.Inaddition,theuseofantimicrobialcoatings should contribute to the immediate release of antimicrobial agentsthroughtargeteddeliveryandmaintenanceofhighconcentrationsforalongtime.

4.Conclusions

TheconductedstudyprovedthatthePVPcanbeusedasdrugelutingcoating.Thispolymer facilitates quick release of the antimicrobial agent, like clindamycin.Coatingsmadebasedonthe3%contentofPVPcontainingantimicrobialdrugcanbeusedinmedicalapplications.PVP-clindamycinbasedcoatingspreparedinthisstudiesshowgrowthinhibitionforStaphylococcusaureus.ThepossibilityofusingPVPasacatheter’scoatingwillbetestedinthenextstudies.


Fig. 2 Release of clindamycin frompolyvinylpyrrolidone-basedcoatings.

References

[1] WarrenJ.W.:Catheter-associatedurinarytractinfections.Int.J.Antimicrob.Agents17(2001),299–303.

[2] DohntK.,SauerM.,MullerM.,AtallahK.,WeidemannM.,GronemeyerP.,RaschD.,TielenP.,KrullR.: An in vitro urinary tract catheter system to investigate biofilm development incatheter-associatedurinarytractinfections.J.Microbial.Methods87(2011),302–308.

[3] KowalczukD.,GinalskaG.,GolusJ.:Characterizationofthedevelopedantimicrobialurologicalcatheters.Int.J.Pharm.402(2010),175–183.

[4] Dayyoub E., FrantM., Pinnapireddy S. R., LiefeithK., BakowskyU.: Antibacterial and anti-encrustationbiodegradablepolymercoatingforurinarycatheter.Int.J.Pharm.531(2017),205–214.


1.Introduction

Themostcommonanalysts’considerationsinvolvetheselectionofappropriatemethodsofsamplepreparation,reagents,analyticaltechniques,andconditionsforanalyticaldetermination.Unfortunately, thereisahugenumberofalterna-tives,thusmakingaproperdecisionisnotaneasytask.Itisnecessarytoknowthedecisionproblem,theneedandpurposeoftheanalysis,aswellasthecriteriaofthedecisionandtheavailablealternatives.It isadifficulttasktojudgeclearly,whichoftheanalyticalproceduresisthebestinagivencase.Inthissituationtheapplication of Multi-Criteria Decision Analysis methods may be a useful anddesirablesolution.Thesetoolsallowdescribingagivenproblemusingnumericalvalues,andenabletoobtainfinalresultsalsoasnumericalvalues.Thescoresarepresentedinaformofafullrankingofavailableoptions,whichallowsselectingobjectivelythebestalternative.Moreover,thedecisionismadeinasystematicway.DetailedinformationaboutMulti-CriteriaDecisionAnalysisusageinareaofchemicalsciences,especiallyanalyticalchemistrymaybefoundin[1]. OneofthemostpopulartoolsisPROMETHEE(PreferenceRankingOrganiza-tionMethod for Enrichment Evaluations). In this work selection of themostpreferable analytical procedure for polycyclic aromatic hydrocarbons (PAHs)determination in smoked products using PROMETHEE is presented and dis-cussed.

Multi-criteria decision analysis for selection of the best procedure for PAHs determination in smoked food

MARTABYSTRZANOWSKA*,MAREKTOBISZEWSKI

DepartmentofAnalyticalChemistry,ChemicalFaculty,GdańskUniversityofTechnology,G.NarutowiczaSt.,80-233Gdańsk,Poland*[email protected]

AbstractMakingaproperdecisioninmultifacitatedsituationisverychallen-gingtask.Especially,iftherearemanyalternativesandcriteria,evencontradictoryones.ThesupporttoolsmaybeapplicationofMulti-CriteriaDecisionAnalysismethods.InthisstudytheapplicationofPROMETHEE(PreferenceRankingOrganizationMethodforEnrich-mentEvaluations)asoneofMulti-CriteriaDecisionAnalysismethodinselectionofthemostpreferableanalyticalprocedureforpolycyclicaromatichydrocarbonsdeterminationinsmokedproductsispresen-ted.

Keywordsgreenanalyticalchemistry

Multi-CriteriaDecisionAnalysis

PROMETHEEsmokedmeat


2.Experimental

2.1Polycyclicaromatichydrocarbonsinsmokedproducts

Polycyclicaromatichydrocarbonsarealargeclassoforganiccompoundsthatarecomposed of two or more fused aromatic rings [2]. Mainly they are formedthroughincompletecombustionorpyrolysisoforganicmatterandduringvariousindustrialprocesses.AdditionallyPAHsarealsoformedduringfoodpreparationmethods such as grilling, roasting and smoking. In Europe about 15%of fishproducts for consumption are prepared using smoking process [3]. In foodindustrymostlybenzo[a]pyrene is controlled as amarkerof the carcinogenicPAHs in foodwithmaximum limits in certain foods in the EU [4]. Analyticalproceduresmayinvolvevarietyofsamplepreparationtechniques,forinstanceSoxhletextraction,solid-phaseextraction,andliquid–liquidextraction,pressu-rizedliquidextractionandQuEChERS,etc.[5].Therefore,whichoftheanalyticalproceduresisthebestforthisgivenpurpose?

2.2ComponentsofMulti-CriteriaDecisionAnalysis

2.2.1Maingoalofanalysis

Mainaimof theanalysis is findingthegreenestanalyticalprocedure forPAHsdetermination in smoked products such as meat and fish. Analysis includesassessmentonlyforbenzo[a]pyrene,asamarkerofcarcinogenicPAHsinfood.Incaseofanalyticalprocedureconsideration,alsometrologicalfactorshavetobesatisfactorybutmainlyenvironmentalfactorsareconsidered.

2.2.2Criteriaofassessment

InMulti-CriteriaDecisionAnalysismethodscriteriaarefactorsthatareallowtomake an evaluation of a given problem, and describe alternatives. Technicalevaluationofanalyticalprocedureinvolvelimitofdetection(LOD)andprecision,expressedasrelativestandarddeviation(RSD).Criteriaasamountofsample,totaltimeneededtoperformanalysisandnumberofproceduralstepsareinvolved.Theinformation on reagents are designate in a reference to Analytical Eco-Scaleapproach [6].On the other hand, solvents evaluation is based on calculationsproposedbyTobiszewskiandNamiesnik[7].CriteriawithpreferencesfunctionsareLOD,RSD,Amountofsample,Timeofanalysis,Scoreforsolvents,Scoreforother reagents,Numberofprocedural steps, allwithpreference function “thelower the better”. It is possible to differentiate the importance of criteria byassessingappropriateweightvaluestoallcriteria.Inthisparticularcasestudyweassumedthatallcriteriainfluencesimilarlyonthemaingoal.


2.2.3Alternatives

Alternativesarethesubjectofconsiderations.Theyrepresentpossibleanalyticalproceduresthatmayreachthestatedgoal.ProposedanalyticalproceduresforPAHsdeterminationinsmokedproductsaresummarizedinTable1.

2.3PROMETHEEanalysisAllthedatavaluesaretakendirectlyorindirectlyfromindicatedabovescientificpapers(Table1.).Indirectlymeans,thatsomeofthemarecalculatedintonumeri-cal values. The set of data prepared for PROMETHEE analysis consists ofalternativesdescribedbycriteria.InthisworkPROMETHEEalgorithmisusedascommercialcomputersoftware-VisualPROMETHEEsoftware.


ForPAHsdeterminationinsmokedfishandmeat,allintroducedcriteriaaredefineasbeingequallyimportant.Withsuchassumptions,itispossibletoobtainresultasacompleterankingofalternatives,whatispresentedinTable2.PhipresentedinTable2isabalancebetweenthepositiveandnegativepreferenceflowsanditincludesbothofthemandpresentsasasinglescore.Asitispresented,thebest


Rank Alternatives Number(cf.Table1) Phi

1 MAE-RP-HPLC-FLD 5 0.61902 ASE-GC-MS 1 0.19053 MAE-DLLME-GC-MS 6 0.07144 LLE-HPLC-FLD 3 0.00005 LLE-GC-MS 2 –0.07146 SPE-GC-FID 4 –0.35717 Soxhlet-GC-MS 7 –0.4524

Table 2FinalresultsofPROMETHEEanalysis.

Table 1Analyticalproceduresofbenzo[a]pyrenedeterminationinsmokedmeatandfish.

Number Matrix Abbreviation Ref.

1 Smokedfish ASE-GC-MS [8]2 Cold-smokedfish(mackerel) LLE-GC-MS [9]3 Cold-smokedfish(salmon) LLE-HPLC-FLD [10]4 Smokedmeat SPE-GC-FID [11]5 Smokedmeat MAE-RP-HPLC-FLD [12]6 Smokedfish MAE-DLLME-GC-MS [13]7 Smoke-curedfishproducts Sox.-GC-MS [14]

analyticalprocedureforPAHsdeterminationinsmokedmeatandfishistechniquebased on high performance liquid chromatography with spectrofluorometricdetection,precededbymicrowave-assistedextraction.MAE-RP-HPLC-FLDproce-dureischaracterizedbythemostdesiredcriteria'svaluesinresponsetootheralternatives.Ontheotherhand,theworstanalyticalproceduresareSoxhlet-GC-MSandSPE-GC-FID.Theirlowpositionsintherankingareduetohighscoreforsolvents.Thus,highlytoxicandhazardoussolventsareused,involvingtheirhugeamounts.InprocedurewithSoxhletextractionasapre-treatmentover300mLofdichloromethane is used. Moreover, Soxhlet-GC-MS is characterized by thehighestvalueforlimitofdetection,whatisnotdesired.

4.Conclusions

Manychemicaldecisionproblemsarecomplexandarecharacterizedbyinterdis-ciplinarynature.Thusthereisaneedofcomprehensiveassessmentthatincludesenvironmental,economicandmetrologicalpointofview.Multi-CriteriaDecisionAnalysismethodscombinemultioutputinformationintosinglevalue,thatiseasytobecomparedotherpossibilities.Theyallowsolvingcomplexproblems(withmanycriteriaandalternatives)inatechnicallyvalidandpracticallyusefulway.ItwasfoundthatthebestprocedureforPAHsdeterminationinsmokedmeatandfishisMAE-RP-HPLC-FLD.

References

[1] BystrzanowskaM., Tobiszewski M.: How can analysts usemulticriteria decision analysis?TrendsAnal.Chem.105(2018),98–105.

[2] EuropeanFoodSafetyAuthority(EFSA):Polycyclicaromatichydrocarbonsinfood-scientificopinionofthepaneloncontaminantsinthefoodchain.EFSAJ.724(2008),1–114.

[3] StołyhwoA.,SikorskiZ.E.:Polycyclicaromatichydrocarbonsinsmokedfish–acriticalreview.FoodChem.91(2005),303–311.

[4] EuropeanCommission:CommissionRegulation(EC)No.1881/2006of19December2006settingmaximumlevelsforcertaincontaminantsinfoodstuffs.O.J.EUL364/5(2006).

[5] PissinattiR.,deSouzaS.V.:HC-0A-02:Analysisofpolycyclicaromatichydrocarbonsfromfood.In: Biodegradation and Bioconversion of Hydrocarbons – Analysis of Polycyclic AromaticHydrocarbons from Food. K.Heimann, O.P.Karthikeyan, S.S. Muthu (Eds.). Springer 2017,p.67–104.

[6] GałuszkaA.,MigaszewskiZ.M.,KonieczkaP.,NamiesnikJ.:AnalyticalEco-Scaleforassessingthegreennessofanalyticalprocedures.TrendsAnal.Chem.37(2012),61–72.

[7] Tobiszewski M., Namiesnik J.: Scoring of solvents used in analytical laboratories by theirtoxicologicalandexposurehazards.Ecotox.Environ.Safe.120(2015),169–173.

[8] Duedahl-OlesenL.,ChristensenJ.H.,HøjgardA.,GranbyK.,Timm-HeinrichM.:Influenceofsmoking parameters on the concentration of polycyclic aromatic hydrocarbons (PAHs) inDanishsmokedfish.FoodAddit.Contam.27(2010),1294–1305.

[9] YurchenkoS.,MolderU.:Thedeterminationofpolycyclicaromatichydrocarbonsinsmokedfishbygaschromatographymassspectrometrywithpositive-ionchemicalionization.J.FoodCompos.Anal.18(2005),857–869.

[10]ViscianoP.,PeruginiM.,AmorenaM.,IanieriA.:Polycyclicaromatichydrocarbonsinfreshandcold-smokedAtlanticsalmonfillets.J.FoodProtect.69(2006),1134–1138.


[11]Olatunji O.S., Fatoki O.S., Opeolu B.O., Ximba B.J.: Determination of polycyclic aromatichydrocarbons[PAHs]inprocessedmeatproductsusinggaschromatography–flameionizationdetector.FoodChem.156(2014),296–300.

[12]PurcaroG.,Moret S., Conte L. S.: Optimisation ofmicrowave assisted extraction (MAE) forpolycyclicaromatichydrocarbon(PAH)determinationinsmokedmeat.MeatSci.81(2009),275–280.

[13]Ghasemzadeh-MohammadiV.,MohammadiA., HashemiM., KhaksarR.,Haratian P.:Micro-wave-assisted extraction and dispersive liquid–liquid microextraction followed by gaschromatography–massspectrometryforisolationanddeterminationofpolycyclicaromatichydrocarbonsinsmokedfish.J.Chromatogr.A1237(2012),30–36.

[14]EssumangD.K.,DodooD.K.,AdjeiJ.K.:Polycyclicaromatichydrocarbon(PAH)contaminationinsmoke-curedfishproducts.J.FoodCompos.Anal.27(2012),128–138.


1.Introduction

Metalsareimportantinhumandiethowever,theexcessiveintakeofmetalsmaybetoxicandveryharmful forhumanhealth[1].Thus,monitoringofmetals iscrucialinthecontrolofthequalityoffoodproductsandbeverages.Duetotheincreasing consumption of wines from year to year the monitoring of metalcontent in given alcoholic beverages is of high importance. The law alreadyestablishedtheacceptablelevelsofparticularmetalconcentrationsandinmostoenologicallaboratoriesitisroutinelyperformed.Theacceptedlimitsofmetalscontent inwine ispresented inTable1,onnextpage, [2].Metalsarestronglyimpactingthequalityofwinesduetotasteandorganolepticpropertieschanging,whatalsoencouragetheircontrolling[3].Moreover,thecontentofsomemetalsmightbeusedtoidentifytheoriginofwine(vineyardandregionallevels)duetoitscorrelationwiththesoiltype[4].Therewasalotofresearchperformedinthisfield.However,thereisstillalottostudy.Forthepracticalissuesoflegalfinger-printing ofwines themulti-element dataset is needed,multivariate statisticaltechniques are required for data analysis.What is more, inductively coupledplasma–massspectrometry(ICP-MS)ispromisinganalyticalmethodsfortraceandultra-traceelementfingerprintingofwines,whichisinexpensive,fast,routine

Metal content in wines of Polish origin

MAGDALENAFABJANOWICZ*,JUSTYNAPŁOTKA-WASYLKA

DepartmentofAnalyticalChemistry,FacultyofChemistry,GdańskUniversityofTechnology,11/12NarutowiczaStreet,80-233Gdańsk,Poland*[email protected]


AbstractMetalsareimportantinthehumandiet.However,theexcessiveintakemaybetoxictohumanbody.Thatiswaymonitoringofmetalsinfoodproductsandbeverages,includingwinesisofhighimportance.Theaim of given study is to characterizewines coming fromdifferentregioninPoland,intermsofmetalcontent.Duetodesiredfeatureslike:sensitivity,lowlimitofdetectionaswellasspeedofanalysis,theinductively coupled plasma-mass spectrometry (ICP-MS) and theinductivelycoupledplasma-opticalemissionspectrometry(ICP-OES)techniqueswereusedtodeterminethemetalconcentrationinwinesamples.Resultsweresatisfiedshowingallowableconcentrationsofexaminedmetalsaccordingtothetoxiclevelsformetalsreportedintheliterature.Additionally,chemometricanalysiswasperformedinorder to find possible correlation between the wine samples orbetweenchemicalvariables.Thechemometricanalysisfoundspecificcorrelation.

KeywordsICP-MSICP-OSmetalwine

andaccurate.FollowingworkisfocusedonthedeterminationofselectedmetalsinwinesfromdifferentPolishvineyards.Twoanalyticaltechniqueswereapplied:ICP-MS and ICP-OES. Moreover, chemometric analysis were performed usingclusteranalysis(CA,hierarchicalandnon-hierarchicalwithK-meansalgorithm)andprincipalcomponentsanalysis(PCA)tosearchforthespecificrelationshipsbetweenthewinesamplesorbetweenthechemicalvariables.

2.Experimental


MetalswereanalyzedwiththeuseofCertifiedReferenceMaterialERMCA713(sample125)traceelementsinwastewater(IRMM–InstituteforReferenceMate-rialsandMeasurements).CalibrationinvolvedtheICPIVmultielementstandardusage(Merc,USA)andsinglestandards:As,Sb,Se,MoandV(Sigma-Aldrich,USA),Hg(Merc,USA)andinternalstandards:Sc,Rh,TbandGestandardsinsuprapure1% HNO (Merc, USA) and deionized water from the Milli-Q Direct 8 Water3

PurificationSystem(MercMillipore)forthesample(pre)treatmentandsampledilution. To prepare the calibration standard the Sigma Aldrich (USA) stock

–1solutioncontainingCa,MgandK(1000mgL foreachelement)wereused.

2.2Instrumentation

44bottlesofwinefrom9differentvineyardsformPolandwereanalyzedusingICP-OES(Shimadzu ICPE-9820, Japan)bywhichCa,K,Mgconcentrationwereinvestigated. However, ICP-MS analytical technique (ICP-MS 2030 Shimadzu,Japan)wasusedtodetermineconcentrationof:Ag,Al,As,B,Ba,Bi,Cd,Co,Cr,Cu,Fe,Hg,Li,Mn,Mo,Na,Ni,Pb,Sb,Se,Sn,Sr,Ti,Tl,V,Zn,Zr.Morever,thechemometrictoolsofmultivariatedata interpretation, clusteranalysis (CA,hierarchicalandnon-hierarchicalclustering)wereused[5].


–1Country Concentrationofmetals(mgL )

Al As Cd Cu Na Pb Ti Zn

Australia – 0.10 0.05 5.00 – 0.20 – 5.00Germany 8.00 0.10 0.01 5.00 – 0.30 1.00 5.00Italy – – – 10.00 – 0.30 – 5.00Poland – 0.20 0.03 – – 0.30 – –OIV – 0.20 0.01 1.00 60 0.15 – 5.00

Table 1TheacceptedlimitsofthemetalscontentinwineindifferentcountriesandgivenbyInternationalOrganizationofVineandWine(OIV).


InordertocharacterisePolishwines,comingfromdifferentregion,intermsofmetalcontent,30elementswereselectedtobeanalyzed.Almostallchosenmetalswere determined in Polishwine samples, excluding Ag, Co, Cu, Sn and V, notdetectedinsomesamples.Theamountofparticularelementwasdependentonthetypeofspecificwinesample.Thehighestconcentrationswereobservedfor:

–1 –1 –1K(97to3250mgL ),Mg(42.7to161mgL ),Ca(32to137mgL ),B(0.333to–1 –1 –112.1 mgL ) and Mn (0.329 to 9.219 mgL ). Moreover: Mn (18.61 µgL to

–1 –1 –1 –19.219mgL ), Fe (0.1558 to2.775mgL ),Na (5.33µgL to3.823mgL ),Al–1 –1 –1 –1(64.12µgL to2.729mgL )andZn(74.71µgL to2.339mgL )wereathigh

concentrationlevels.Thelowestconcentrationswerenotedforsuchmetalsas:Hg–1 –1 –1(0.31to0.51µgL ),Ag(<0.001to4.92µgL ),Co(<0.002to6.98µgL ),Cd–1 –1(0.023to2.54µgL ),andTi(0.54to2.37µgL ).

There isobserveda correlationbetween the colorofwine sampleand theconcentrationlevelofmarkedmetals.Thisiswellvisibleontheexampleofsilver,whichtheconcentrationishigherinthewhitewinethanintheredwinesamples.Thesamerelationshipisobservedincaseof:As,Bi,Co,Sb,Se,Sn,VandZn.Andoppositefor:B,Ba,Fe,KandMn,whichwerepresentathigherconcentrationinsamplesofredwines.Chemometryanalysiswasappliedtofindmorecomplexcorrelations.Itwasusedtoexaminespecificcorrelationsbetween44wine(redandwhite)samples(objectofthestudy),comingfromdifferentregionsofPolandlike West Pomerania, Kyuavian Pomerania, Subcarpathia, Lesser Poland andMasovia)andbetween30metalconcentrations,variablesinordertoclassifythewinesaccordingtotheirmetalcontentinawaybeingdiscriminatingchemicalindicatorsforeachclustergroupofwinetype. Thehierarchicalclusteranalysis(HCA)wasusedtogroupchemicalvariables,Fig.1(nextpage).Theunsupervisedclusteringleadtotheformationof4clusters:∎ K1:Zn,Pb,Cu,Co∎ K2:Cd,Mg,Ca,K,Mn,Ba∎ K3:Ni,Fe,Li,Cr,Sr,B,V,Na,Zr,Al∎ K4:Tl,Ti,Se,Sb,Mo,Sn,Bi,As,Hg,Ag.Later,theprincipalcomponentswasusedtoexplainover80%ofthetotalvarianceofthesystem:∎ PC1(27%)–indicateshighcorrelationbetweenAg,As,Bi,Hg,Mo,Sb,Se,Sn,Tl

andTi–onecannoticeditisentireK4fromHCA;∎ PC2(>10%)showinghighfactorloadingforAl,VandZr.∎ PC3(~10%)pointingoutsignificantloadingfactorofBa,K,Mg,Ti.∎ PC4(~7%)showingtherelationshipbetweenB,NaandSr.∎ PC5(9%)indicatingcorrelationbetweenCa,Co,CuandZn.∎ PC6(7%)showinghighloadingfactorofelementslike:Cr,Fe,Li,Ni.∎ PC7andPC8 (explaining together >10%of total variance) pointing out the

simpleimpactofCdandPb.



20 40 60 80 100 120

(Dlink/Dmax)*100

ZnPbCuCoCdMgCa

KMnBaNiFeLiCrSrBV

NaZrAlTlTi

SeSbMoSnBi

AsHgAg

Fig. 1Hierarchicaldendrogramfor30chemicalvariablesanalyzedinwinesamples.

20 40 60 80 100 120

(Dlink/Dmax)*100

20R9R8R7R

12W11W10W17R18R16R14R13R11R15R10R20W24W19W18W

13 W23W22W

6R5R

12R4R3R

21W17W16W15W

2R1R

14W19R9W7W6W8W5W4W3W2W1W

Fig. 2Hierarchicaldendrogramfor44winesamples,comingfromdifferentregioninPoland.

PCA broaden the information about the relationship of analyzed variables,showingsimilartrendasinHCAdemonstratedbefore,atthesametime. Nextstepincludedthemoreimportanttaskwhichwastodetectthesimilaritygroupsofwinesamples.TheresultsarepresentonFig.2,where3significantclusterswerecreated:∎ K1:10w,11w,12w,7r,8r,9r,20r∎ K2:18w,19w,20w,24w,10r,11r,13r,14r,15r,16r,17r,18r∎ K3:2w,3w,4w,5w,6w,7w,8w,9w,13w,14w,15w,16w,17w,21w,22w,23w,

1r,2r,3r,4r,5r,6r,12r,19r1wisanoutlier. Itshows,thatK1thesmallestclusterconsistsofequalnumberofwhiteandredwines, includingtheSubcarpathiawines.K2 isdominatedbyredwines intherelationof2:1tothewhitewines,fromMasovia.K3,thebiggestcluster,consistsmostlyofwhitewinesinrelationof2:1totheredwines,fromWestPomerania,PomeraniaandKyuavianPomerania. Furthermore, the supervised classification by the use of K-means non-hierarchical clustermethodwas used todetermine thediscriminant chemicalvariables.TheanalysisentirelyconfirnedtheresultsfromtheHCA.Oneoutlierwasobtainedandthreeclustershavingthesamenumberofwinesamples. Outlier(1w)isawinesampleofaspecificcharacteristic:highestlevelofAg,As,Bi,Hg,Mo,Sb,Se,Sn,Ti,Tl(thosearemetalshavinghighfactorscoresinPC1whichcouldbeconditionallynamed“toxicated”bysoilconditions)andlowestlevelsofAl,V,Zr(elementsfromPC2),B,Ba,K,Mg,Mn(PC3),i.e.earthconstituentsor“specific”soilconditions).ItismuchdifferentwinesamplefromWestPomeraniathanothersfromthesameregion. Cluster1isofthesamecharacteristicasK3inHCAandincludesalldifferentPomeranianwines.Nodiscriminatingspeciesweredetected.Itcharacterizedby“background”levelsofallmetals.Itcanbeconcluded,thatthesoilconditionsareveryappropriateforthegrapesensuringhighwinequality. Cluster 2 is the same as K2 in HCA and includes Masovian wines and ischaracterizedbysimilarqualityasPomeranianwines.Thesoilconditionsmaybeconsideredasgood(no“toxic”or“specific”soilconditionsimpactingthewinequality). Cluster4isthesameasK1inHCAandconsistsofSubcarpathianwines.Theircharacterization is closer to the outlier wine sample than Masovian orPomeranianwinesamples.TherewereenhancedlevelsofAl,B,Co,Fe,Li,Na,Ni,Sr,Zr observed, thus becoming subject to soil specificity (of natural, noranthropogenicorigin)intheregion.

4.Conclusions

Almostall30selectedmetalsweredeterminedinPolishwinesamples,excludingAg,Co,Cu,SnandV.However,thetypeofanalyteandquantitydependsonthe


specific wine sample. K, Mg, Ca, B and Mn were present at highest amount,whereasHg,Ag,Co,CdandTiwerepresentatlowestconcentrations.Thecontentofmetalsinallanalysedwinesampleswaswithintheacceptablelimitsaccordingto the toxic levels of metals stated in the literature. Polish wines may beconsideredassafeintermsofmetalcontent.Thelevelofselectedmetalsstronglydependson thecolourofwine, specificelementscontent inaparticularwinesample.Therewerehigherconcentrationsofmetalslike:Ag,As,Bi,Co,Sb,Se,Sn,VandZninwhitewinesamplesthanredones.However,B,Ba,Fe,KandMnwerepresentathigherconcentrationsinredwinesamplesthanwhite.Inordertoseemore complex correlations the chemometric analysis were performed whichhelpedtofind,thatwinequalitystronglydependsonsoilnaturalcompositionaswell as soil toxic metals content. Wine would be successfully separated ongeographicobjectivesandtodiscriminatemetalvariablesforeachregion.Furtherresearchwould be helpful to find possible sources of themetal content bothnaturalandanthropogenic.

Acknowledgments

JustynaPłotka-WasylkawouldliketothankFacultyofChemistry,GdanskUniversityofTechnologyforfinancialsupportwithintheminigrantprogram(Decisionno.4914/E-359/M/2017).Theau-thors would like to thank Zodiak Vineyard, PrzyTalerzyku Vineyard, Kozielec Vineyard, PodOrzechemVienyard,StokVineyard,SpotkaniowkaVineyard,andDworKomborniaVineyardforthesamplesofwine.

References

[1] Płotka-WasylkaJ.,RutkowskaM.,CieslikB.,TyburcyA.,NamiesnikJ.:Determinationofselectedmetals in fruit wines by spectroscopic techniques. J. Anal. Meth. Chem. (2017), ArticleID5283917.

[3] Pyrzynska K.: Chemical speciation and fractionation of metals in wine. Chem. SpeciationBioavailability19(2007),1–8.

[4] GreenoughJ.D.,Mallory-GreenoughL.M.,FryerB.J.:Geologyandwine:regionaltraceelementfingerprintingofCanadianwines.Geosci.Can.32(2005),229–137.

[5] MassartD.L.,KaufmanL.:TheInterpretationofAnalyticalChemicalDataBytheUseofClusterAnalysis.Amsterdam,Elsevier1983.


1.Introduction

Humanbreastmilkisconsideredasthegoldstandardinnutritionofthenew-born[1].Despite a huge contributionof humanbreastmilk lipids to the totalamount of humanbreastmilk nutrients and their impact on theproper childdevelopment,theyremaintheleastunderstoodpartofmilk.Duetotheineffici-encyand time-consumingofprior available traditional analyticalmethods forlipidanalysis,theextensionofknowledgeabouthumanbreastmilklipidswaslimited.Theapplicationofuntargetedlipidomicsthatoffersinvestigationoflipidsina fast andprecisewayallows fordetailed characterizationof anenormousnumberofhumanbreastmilklipidsspecies,eventhoseunidentifiedpreviously,inoneanalyticalrun. One of themain concerns in untargeted lipidimic study is to achieve highlipidomecoverageusingsimple,reproduciblesamplepreparationstrategyandlimitednumberofanalytical techniques. Inhumanbreastmilk, thesignificantvariety in concentration level of lipid species occurs. Some lipid classes aredominantwithconcentration(μMtomMrange)incontrasttootherone,whicharemuchlessabundant(pM–nMrange).Thisappliesprimarilyforlowabundant

New sample preparation strategies for comprehensive lipidomics of human breast milk

DOROTAGARWOLIN SKA*,WERONIKAHEWELT-BELKA,JACEKNAMIESNIK,

AGATAKOT-WASIK

DepartmentofAnalyticalChemistry,FacultyofChemistry,GdanskUniversityofTechnology,11/12NarutowiczaSt.,80-233Gdansk,Poland*[email protected]


AbstractGlobalprofilinganalysisofhumanbreastmilklipidsishinderedbycomplex composition of humanbreastmilk. This problem is hugefromanalyticalpointofview,sinceduetothatdetectionofallhumanbreastmilklipidsduringoneanalyticalrunislimited.Thus,samplepreparationstepconstitutesacrucial step inuntargeted lipidomicanalysis of human breast milk, especially when semi-quantitativeanalysisisassumed.Herein,wepresentacomparisonofpublishedandproposedbyussamplepreparationproceduresusedinlipidomicstudy of human breast milk, including indication of advantages,drawbacksandpossibleapplication.

Keywordshumanbreastmilkuntargetedlipidomics

glycerophospholipids and sphingophospholipids [2], and compared withglycerophospholipidsandsphingophospholipidshighabundantglycerolipids[3].This problem is important from analytical point of view, since due to thatdetectionofallhumanbreastmilklipidsduringoneanalyticalrunislimited.Lowabundantlipidsrequireenrichingsteptobreakthelimitofdetection,whereaslipids at high concentration levels frequently require dilution step to avoidsaturationofMSsignal.Therefore,thepropersamplepreparationisthecrucialstepinlipidomicsanalysisofhumanbreastmilk.

2.Experimental


LC-MS-grademethanolandHPLC-gradechloroformandhexanewerepurchasedfromMerck(Germany),andHPLC-grade2-propanol,ammoniumformate(99.9%purity)formicacidandammoniap.awerepurchasedfromSigma-Aldrich(USA).DeionizedwaterwaspurifiedonanHLP5system(Hydrolab,Wislina,Poland).

2.2Samplepreparationprocedure

For the global lipidomics of human breast milk we developed two samplepreparationprotocols.Firstonehasbeenbasedonsolid-phasemicroextraction(SPME)technique[4]andthesecondoneinvolvescombinationofsolid-phaseextraction(SPE)andliquid-liquidextraction(LLE)techniques[5].TheflowchartsofsamplepreparationstrategiesforgloballipidomicofhumanbreastmilkarepresentedonFig.1and2respectively. The first samplepreparationprotocolwasdescribed indetails inpreviousresearch[4].Inshort, priortoadsorption,theSPMEtipthatconsistedofafibercoatedwithasilica-basedsorbentmodifiedwithC18groups(Supelco,Sigma-Aldrich,USA)waspreconditionedinamixtureofsolvents(MeOH/H O).Then,it2


Fig. 1OptimizedprotocolforhumanbreastmilklipidextractionusingSPMEtechnique[4].

wasimmersedin1mLofhumanbreastmilk.Afterlipidadsorption,theSPMEtipwastransferredtoaLCvialcontainingaglassinsertfilledwith2-propanolforlipiddesorption.Afterdesorptionwithshaking,theSPMEtipwasremoved.TheobtainedlipidextractwassubsequentlyanalyzedusingLC-Q-TOF-MS. The second developed sample preparation strategy for comprehensivecharacterizationofhumanbreastmilklipidswithusedLLEandSPEtechnique[5]includedtwobasissteps:1. SPEbasedphospholipidsenrichment:100μLofhumanbreastmilksamplehas

tobemixedwiththesolutionof1%formicacidinmethanolandsubsequentlyvigorous vortexing and centrifuged in order to precipitate proteins. Nextsupernatant has to be loading to the HybridSPE-Phospholipid cartridge(Supelco,SigmaAldrich,USA).Afterstationaryphasewashing,phospholipidscanbeelutedwith5%ammoniainmethanol.Theobtainedextracthastobeevaporatedtodrynessanddissolvedwiththeuseoflipidextractobtainedinthesecondstepofsamplepreparationprocedure.

2. LLEbasedlipidextraction,wherejust225μLofhumanbreastmilksamplehasto be mixed with the chloroform/methanol mixture and vortexed. Next,appropriatevolumeofchloroformandwaterhastobeaddedandaftervigo-rousvortexingsamplehastobecentrifugedtoseparateaqueousandorganic


Fig. 2LipidextractionbySPEandLLEbasedapproach[5].

phase.Thelowerorganicphasecontaininglipidshastobedilutedandnext,100μLofdilutedextracthastobeusedasadissolvingsolutionforenrichedphospholipidfraction(obtainedinthefirststep).

Finally,thepreparedhumanbreastmilklipidextractcanbetransferredtothechromatographicvialandanalysedbyLC-Q-TOF-MS

2.3Instrumentation

TheRP-LC-Q-TOF-MSanalysisofobtainedextractwasperformedusingAgilent1290LCsystemequippedwithabinarypomp,anonlinedegasser,anautosamplerand thermostated column compartment coupledwith a 6540Q-TOF-MSwithaDualelectrosprayionization(ESI)source(AgilentTechnologies,USA). Lipid extracts obtained by SPME based strategy were chromatographicallyseparatedonanAgilentZORBAXSB-C18column(50×2.1mm,1.8μmparticlesize)intheconditiondescribedindetailsinpreviousresearch[4]. ToreducetimeofanalysislipidextractsobtainedbySPEandLLEbasedstrategywerechromatographicallyseparatedonareversed-phasecolumn(Poroshell120EC-C8,150×2.1mm,1.9µmparticlesize,Agilent)witha0.2µmin-linefilter.Thecolumnandautosamplertemperaturethroughouttheanalysisweremaintainedat45°Cand4°C,respectively. Inbothcasestheinjectionvolumewas0.5µLandmobilephaseconsistedofmixtureof5mMammoniumformateinwaterandmethanol(1:4,v/v)ascompo-nentAandamixtureof5mMammoniumformateinwater,hexaneand2-propanol

–1(1:20:79,v/v/v)ascomponentB).Themobilephasewaspumpedat0.5mLmin withinatotalruntimeof30.5min.Thegradientelutionprogramwasinitiatedwith10%eluentBduringthefirst10minutesandwasthenrampedfrom10%to50%Bfrom10to15minutesand50%to100%Bfrom15to20minutes.Then,after 0.5 minute, the gradient was switched to 10% eluent B for 10min forequilibrationpriortothenextrun. The ESI source condition and data analysis parameters were described indetailselsewhere[8].


Humanbreastmilklipidanalysisishinderedbyhugediversityofthiscompoundsandtheirwiderangeofconcentration.Duetothatthesamplepreparationstepiscrucial.Accordingtoavailableliteraturedata,previouslyreportedmethodsforhumanbreastmilk lipidanalysisbyHPLC-MSapproach includingLLE[6]andsinglephaseextraction[6], [7]assamplepreparationstep.Wedevelopedtwoanothermethodsforlipidextraction,onebasedonSPMEtechnique,andsecondonebasedonSPEandLLEtechniques. Samplepreparationmethod,basedonSPMEtechniqueallows forrapidandsimplecomprehensivecharacterizationoflipidsinhumanbreastmilksamples.


Our sample preparation method offers significant improvements over otherpublishedmethods forhumanbreastmilk lipidextraction. Itdoesnotrequireproteinprecipitation(extractionisperformeddirectlyfromhumanbreastmilksamples),whatminimizessamplepreparationsteptimeandsignificantlyreducesthe organic solvents use. Moreover, the development of a SPME method incombinationwithLC-Q-TOF-MSenablesdetectionofbroadrangeofhumanbreastmilk lipids. Comparison of the individual lipid classes extracted from humanbreast milk using different extraction procedures revealed that all extractionprocedures provide similar lipidome coverage [4]. However, due to the lowprecision it can be only use for qualitative purpose. The relative standarddeviationofthe69%ofmolecularfeaturevolumes(detectedinthreeextractionreplicatesofthesamehumanbreastmilksample)washigherthan20%,whichdoesnotmeetthecriteriaforsemi-quantitativeanalysis.Weassumedthatmainreason of low precision of develop extraction method, may be high level ofglycerolipidsconcentration,whichcancausenonlinearisotherm.Sampledilutionmayimproveprecisionofextractionprocedure,butlowlevelofconcentrationofsome other lipids, limits it. Moreover, due to the wide concentration rangeofdifferentlipidclassesinthehumanbreastmilksamples,theMSsignalobtainedforsomeoflipidswassaturatedandrequireddilutionforquantification,andtheMS signals for other lipid classeswere very low and limit dilution. Thus, thesimultaneousquantificationofalldetectedlipidclassesinoneanalyticalrunwaslimited also for this reason. However, in our studywe focused on developedmethod for rapid qualitative analysis and for this purpose, developed samplepreparationmethod,evenwithlowrepeatability,issufficient. Toovercomedrawbacksofpreviouslyevaluatedmethod,wehavedevelopedsamplepreparationmethod,basedonSPEandLLEtechniques.Thecombinationof these two extraction techniques enables the enrichment of low abundanthumanbreastmilklipidscontainingthephosphatemoiety(glycerophospholipidsandsphingomyelins),anddilutionofhumanbreastmilklipidsthatareatthehighconcentration level in human milk (glycerolipids). To confirm usefulness ofdevelopedlipidextractionprotocolthehumanbreastmilklipidchromatogramsobtainedwiththepreviousanddevelopedmethodarepresentedonFig.3. The lipidome coverage obtained by implementation of this enrich-dilutestrategywashigherthanthosedescribedinpreviouslyreports[8,10,11],parti-cularlyinthetermofphospholipidscontent(approximatelyfourfoldmorehumanbreastmilklipidscontainingphosphatemoietywereidentified.Thisextractionprocedure enables both highly effective separation of phospholipids andglycerolipids. Thus, the developed sample preparation strategy representsvaluabletoolforcomprehensiveanalysisofhumanbreastmilklipids.


4.Conclusions

Preparationofhumanbreastmilksampletolipidanalysisperformedinuntar-getedmannerisachallenge.Manyofavailablelipidextractionsarenotsuitableforisolationofhumanbreastmilklipids,duetothehugediversityofthiscompoundsand their wide range of concentration. We have developed two samplepreparation procedures and compared them with other sample preparationstrategyusedinanalysisofhumanbreastmilklipids.Oneofthemensuressimilarlipidomcoveragetothosedescribedinpreviousreports,butisnotsuitableforquantitative analysis. However, second one provides higher lipidom coveragethanthosedescribedinpreviousreportsandissuitableforbothqualitativeandsemi-quantitativelipidanalysisperformedinuntargetedmanner.

References

[1] WorldHealthOrganization:Globalstrategyforinfantandyoungchildfeeding.UNICEF2003.[2] Tavazzi I., Fontannaz P., Lee L.Y., Giuffrida F.: Quantification of glycerophospholipids and

sphingomyelininhumanmilkandinfantformulabyhighperformanceliquidchromatographycoupledwithmassspectrometerdetector.J.Chromatogr.B1072(2018),235–243.

[3] SokolE.,UlvenT.,FærgemanN.J.,EjsingC.S.:Comprehensiveandquantitativeprofilingoflipidspecies in human milk, cow milk and a phospholipid-enriched milk formula by GC andMS/MS(ALL).Eur.J.LipidSci.Technol.117(2015),751–759.


Fig. 3Lipidchromatogramsobtainedwith the previous and developedmethodbasedonSPEandLLEandLC-MStechniques.


[4] GarwolinskaD.,Hewelt-BelkaW.,NamiesnikJ.,Kot-WasikA.:RapidCharacterizationofthehumanbreastmilklipidomeusingasolid-phasemicroextractionandliquidchromatography-massspectrometry-basedapproach.J.ProteomeRes.16(2017),3200–3208.

[5] Hewelt-BelkaW.,GarwolinskaD.,BelkaM.,BączekT.,NamiesnikJ.,Kot-WasikA.:unpublisheddata.

[6] Andreas N.J. Hyde M.J., Gomez-Romero M., Lopez-Gonzalvez M.A., Villasenor A., Wijeye-sekeraA., Barbas C.,ModiN., Holmes E., Garcia-Perez I.:Multiplatform characterization ofdynamicchangesinbreastmilkduringlactation.Electrophoresis36(2015),2269–2285.

[7] VillasenorA.,Garcia-PerezI.,GarciaA.,PosmaJ.M.,Fernandez-LopezM.,NicholasA.J.,ModiN.,HolmesE.,BarbasC.:Breastmilkmetabolomecharacterizationinasingle-phaseextraction,multiplatformanalyticalapproach.Anal.Chem.86(2014),8245–8252.

1.Introduction

AmikacinandciprofloxacinarethemostcommonlyusedantibioticsusedtofightbacterialinfectionsmainlycausedbyEscherichiacoli[1].Thankstotheireffici-encytheseantibioticsareusedtopreventpossibleinfectionsinthecaseofatrans-rectalprostatebiopsy.Nowadays,forthispurpose,medicinewillinglyusesthe“controlled drug delivery systems”, i.e., polymer-based drug-eluting biopsyneedles. This approach allows administrationof antibiotics during thebiopsydirectlyintothetissue,eliminationofonerousantibiotictherapyanddecreasepossibilityofinfectioncomplications. Amikacin is anaminoglycosideantibiotic especially effective againstGram-Negativebacteria.Chemicallyamikacinconsistofaminosugars(D-glucosamine,D-kanosamine)whichareconnectedtoaminocyclitolbyglyosidicbonds. Ciprofloxacinisasecondgenerationquinolonederivative.Incomparisontoamikacin, ciprofloxacin is characterizedby completely different chemical pro-pertiessuchaslowerpolarityandwatersolubility(Table1).

Determination of amikacin and ciprofloxacin by liquid chromatography with pre-column derivatization to evaluate sustained delivery of antibiotics from Drug-Eluting Biopsy Needle

a, b aMARTAGLINKA *,JUSTYNAKUCIN SKA-LIPKA ,ANDRZEJWASIK

a DepartmentofAnalyticalChemistry,FacultyofChemistry,GdańskUniversityofTechnology, 11/12GabrielaNarutowiczaStreet,80-233Gdańsk,Poland*[email protected],FacultyofChemistry,GdańskUniversityofTechnology, 11/12GabrielaNarutowiczaStreet,80-233Gdańsk,Poland

AbstractDetermination of chosen antibacterial antibiotics: amikacin andciprofloxacin was carried out by hplc-uv after derivatization with9-fluorenylmethylchloroformateandintheirnativeformbyHPLC-MS/MS.Developedmethodshavebeenappliedtocontrolthekineticsof antibiotic release from polymer-based controlled drug deliverysystem.

Keywordsantibacterialantibioticsderivatizationdrugdeliverysystemshighperformanceliquid

chromatography


Asaresultoftheaforementioneddifferences,thechromatographicdetermi-nationofamikacinandciprofloxacinmaycauseproblemsduetotheircompletelydifferent retention behaviour during separation. In addition, due the lack ofchromophoresandhighpolarity,directanalysisofamikacininitsnativeform,especiallyunderreversedphaseconditionsisdifficultandthederivatizationstepisrequired[2].Derivatizationofantibioticswithamino-groupsisrealizedmainlywith:9-fluorenylmethylchloroformate,6-aminoquinolyl-N-hydroxysuccinimidylcarbamate, ortho-phthaldialdehyde with 3-mercaptopropionic acid, etc. How-ever,derivatizationoftenresultsinthedecreaseofprecisionandincreaseofcostsandtimeofanalysis[2–4].Theprocedureforprecolumnderivatizationofami-kacin and ciprofloxacin with 9-fluorenylmethyl chloroformate was proposed.Additionally obtained resultswere comparedwith direct analysis of the anti-biotics in their native forms using high performance liquid chromatographycoupledwith tandemmassspectrometry(HPLC-MS/MS).Proposedprocedurewas applied to control the kinetic of antibiotics release from polymer-basedcontrolleddrugdeliverysystem,namelythetrans-rectalbiopsyneedle.

2.Experimental


AmikacinandCiprofloxacinwerepurchasedfromInterquim(CuautitlanIzcalli,Mexico)andAartiDrugsLimited(Maharashtra,India)respectively.9-fluorenyl-methyl chloroformate (≥ 99%), acetonitrile (LC-MS grade) and glycine werepurchasedfromSigmaAldrich.UltrapurewaterwaspreparedusingHLP5systemfromHydrolab(Wislina,Poland).FormicacidwaspurchasedfromMerck.Boricacid,phosphoricacid,sodiumhydroxideandpotassiumchloridewerepurchasedfromPOCH(Gliwice,Poland).Boratebufferwaspreparedbytitratingthewatersolutionofboricacid(0.2M)andpotassiumchloride(0.2M)withsodiumhydro-xidetopH=7.3.Polymer-coatedbiopsyneedleswithdifferentconcentrationsofhydrophilicpolymersandantibioticswerepreparedincooperationwithDepart-mentofPolymerTechnology(GdanskUniversityofTechnology,Poland).


–1 Name Molecularformula M/gmol Log P Watersolubility pK pKa b

–1amikacin C H N O 585.6 –7.40 185 gL (25 °C) 12.1 9.7922 43 5 13 –1ciprofloxacin C H FN O 331.3 0.28 30 gL (20 °C) 5.76 8.6817 18 3 3

Table 1Selectedpropertiesofamikacinandciprofloxacin(dissotiationexponentspredictedfromChem-Axon).

2.2Instrumentation

HPLC-UVdeterminationofantibioticwasperformedusinganAgilent1200LCsystem consisting of degasser, binary pump, ALS autosampler, thermostatedcolumncompartmentanddiodearraydetector(DAD)detector.TheseparationofantibioticswascarriedoutusingKinetexC18,1.7µm(50×2.1mm)chromato-graphiccolumnworkingat40°C.Asamobilephasewater(componentA)andacetonitrile(componentB)bothwith0.5%(v/v)ofdilutedphosphoricacid(inamassratio1:1withdeionizedwater)wereusedinfollowinggradientelution:0min–20%B,15min–95%B,20min–95%B,20.1min–20%B,24.5min–20%

–1B.Flowrateof0.3mLmin wasusedandtheinjectionvolumewas2µL. HPLC-MS/MS determination of antibiotics in multiple reaction monitoringmode(Table2)wasperformedusinganAgilent1200SeriesRapidResolutionLCsystem(USA)consistingofanonlinedegasser,abinarypump,ahigh-performanceSL autosampler and a thermostated column compartment. The system wascoupledtotheQ-Trap4000triplequadrupolemassspectrometer(ABSCIEX,USA)withelectrospray ionization(ESI)sourceworking inpositive ionmode.OtherparametersofESIsourcewereasfollows:curtaingaspressure:20psi,sourcetemperature:550°C,nebulizergaspressure:30psi,heatergaspressure:30psiandcapillaryvoltage:4000V.ForseparationZORBAXEclipseXDB-C181.8µm(50×4.6mm) chromatographic columnwas used. Temperature of columnwasmaintained at 35 °C. Separation was carried out in isocratic conditions withmixtureofacetonitrileandwater(85:15,v/v)with0.1%offormicacidasamobile

–1phase.Flowrateof0.8mLmin wasusedandtheinjectionvolumewas2µL.Thetimeofanalysiswas3minutes.


Twosetsofbiopsyneedleswerepreparedwithtwodifferentcompositionsandthicknessofcoatings(8needlesforeachset)andinjectionsimulationtestwasperformed.Forthispurpose,porkprostatesobtainedfromalocalslaughterhousewereused.Prostateswere frozenandheatedtoapprox.37°Conthetestday.Additionally,inordertosimulatetherealbiopsyprocedure,thespecialbiopsygunwasused.Foreachof8needles fromagivenseries (with thesamecoatings),


Analyte Multiplereaction Declustering Collision monitoring potential/V energy/V

amikacin 586.4–163.1 81 47 586.4–425.3 81 27ciprofloxacin 332.3–288.1 91 27 322.3–231.0 91 49

Table 2Multiplereactionmonitoringmodeparameters.

adifferentnumberof tissue injectionswereperformed:0 (without injection–referencesample),1,3,5,7,9,11and12injections.Subsequently,eachneedlewasimmersedin5.5mLofdeionizedwaterindedicatedtest-tubefor40min.Afterthat,thesolutionwastransferredintoEppendorfvialsandvortexfor5minutes.TheclarifiedsolutionwastransferredtothevialsandanalysedbyHPLC-MS/MS. In the case of HPLC-UV analysis, the modified Chang’s [4] procedure wasapplied.Theclarifiedsolutionwasmixedwithacetonitrileinvolumeratio1:1.The200µLofthismixturewasintroducedintothevialwith200µLboratebuffer(0.2M,pH=7.3).Forderivatization,200µLof9-fluorenylmethylchloroformateacetonitrilesolution(2.5mM)wasaddedandmixed.After20minutes,the50µLofglycine(0.1M)wasaddedandmixedforstoppingthederivatizationreaction.Afterall,sampleswereanalysedbyHPLC-UV.

2.4Calibrationcurves

–1 –1Stock solution of amikacin (10 mgmL ) and ciprofloxacin (1 mgmL ) werepreparedindeionizedwater.Workingstandardsolutionswerepreparedfreshlybymixingvolumesofstocksolutionswithwateringraduatedflasks.AmikacinandciprofloxacinLC-MS/MScalibration standard solutionsof0.5, 1, 5, 15,25,50,

–1100µgmL wereprepared.InthecaseofLC-UVmethod,calibrationstandardsof–1 –15,20,40,75,150µgmL foramikacinand0.5,2,4,7.5,15,30,60,100µgmL for

ciprofloxacin after derivatization with 9-fluorenylmethyl chloroformate were.used


Inthecaseofderivatizationprocedure,thebufferpH(7.3and8.5),concentrationof9-fluorenylmethylchloroformate(1,2.5,3.5,5and25mM)andtimeofreaction(1,3,5,15,20and45minutes)wereoptimized(datanotshown).Theoptimalconditionsweresummarizedinsection2.3. Validationofpre-columnderivatizationHPLC-UVmethodwasperformed.Itconsisted of estimation of linearity, limits of detection (LOD) and limits of


(a) (b)

–1 –1Fig. 1Calibrationcurveof(a)amikacin(5–150µgml ),and(b)ciprofloxacin(0.5–100µgml ).

quantification(LOQ).Bothcalibrationcurves(Fig.1)werelinearwithinthestu-diedconcentrationranges.Determinationcoefficientsweresatisfactory. Alsothelimitsofdetection(LOD=calibrationcurveinterceptstandarddevi-ationmultipliedby3.3anddividedbytheslopeofthecalibrationcurve)andlimitsof quantitation (LOQ = 3×LOD) were established. For amikacin the LOD=

–1 –1=3.2µgmL and LOQ = 9.6 µgmL , whereas for ciprofloxacin 0.65 and–11.95µgmL respectively.

Resultsofreal-worldsampleanalysis(withandwithoutpre-columnderiva-tization)aresummarizedinTable3.DevelopedHPLC-UVmethodcanbeappliedfor determination of antibiotics in polymermatrix. BothHPLC-UV andHPLC-MS/MSmethodsproduces similar results.For ciprofloxacin,HPLC-UVmethodinvolvingthederivatizationstepgivesmorepreciseresultswhiletheaccuracyremainssimilar.IncaseofamikacinresultsobtainedwiththehelpofHPLC-UVmethodarelessprecisethanthoseprovidedbydirectHPLC-MS/MSmethod.Thisismostprobablycausedbyinconsistentderivatizationreactionefficiency.Ontheother hand we can observe that the results obtained with the HPLC-MS/MSmethodareslightlylowerthanthoseproducedbyHPLC-UV.ThisphenomenoncanbeexplainedbythefactthatamikacinwasnotretainedonC18stationaryphaseandelutedinadeadtimetogetherwithothersignalsuppressioncausingcomponents.


–1 –1Coating Injection c(amikacin)±SD/μgmL c(ciprofloxacin)±SD/μgmLnumber number HPLC-UV HPLC-MS/MS HPLC-UV HPLC-MS/MS

1 0 68.4±6.2 60.9±1.8 48.63±0.51 45.1±1.6 1 44.2±7.1 39.2±1.3 37.60±0.46 35.0±1.7 3 28.7±4.8 26.23±0.67 23.85±0.47 24.0±1.6 5 17.1±1.3 13.27±0.55 15.61±0.57 16.8±1.4 7 20.9±2.1 17.50±0.92 25.2±2.2 23.5±1.9 9 6.86±0.88 3.96±0.52 4.53±0.33 4.82±0.65 11 n.a. 1.60±0.63 0.55±0.13 n.a. 12 5.0±1.1 2.84±0.58 1.56±0.22 1.46±0.35

2 0 59.9±4.1 44.92±0.32 39.42±0.72 42.1±1.3 1 44.5±8.3 38.9±1.5 40.83±0.84 40.0±1.9 3 35.6±1.1 25.50±0.54 36.3±2.2 33.7±2.1 5 42.0±4.1 34.75±0.97 35.52±0.73 37.3±2.4 7 16.24±0.37 14.08±0.31 20.15±0.17 19.0±1.5 9 n.a. 1.051±0.051 0.96±0.21 0.92±0.11 11 9.69±0.71 11.19±0.75 13.99±0.51 16.4±1.9 12 6.86±0.24 7.24±0.61 8.95±0.28 10.79±0.56

Table 3Comparison of amikacin and ciprofloxacin content in polymer-based coatings after injectionsimulation test obtained by pre-column derivatization HPLC-UV and derivatization-less HPLC-MS/MSmethods(n=3).

4.Conclusions

TheresultsobtainedwiththedevelopedHPLC-UVmethodaresimilartothoseobtainedbyHPLC-MS/MSanalysisoftheantibioticsintheirnativeforms.Itcanbeusedtostudythekineticsofamino-antibioticdrugreleasefromdrug'scontrolled-deliverysystems.InrelationtoamikacinitisslightlylesssensitivethanHPLC-MS/MSmethodbutseeminglytheaccuracyisbetter.Withregardtociprofloxacinthe developedmethod seems to be superior over theHPLC-MS/MS since theprecision of the results is higher while accuracy stays the same. Also theinstrumentationissimpler,cheaperandwidelyavailable.

References

[1] SieczkowskiM.,GibasA.,WasikA.,Kot-WasikA.,PiechowiczL.NamiesnikJ.MatuszewskiM.:Drug-Elutingbiopsyneedleasanovelstrategy forantimicrobialprophylaxis in transrectalprostatebiopsy.Technol.CancerRes.Treat.16(2017),1038–1043.

[2] FaroukF.,AzzayH.M.E.,NiessenW.M.A.:Challengesinthedeterminationofaminoglycosidedetermination,areview.Anal.ChimicaActa890(2015),21–43.

[3] Baranowska I., Markowski P., Baranowski J.: Simultaneous determination of 11 drugsbelonging to four different groups in human urine samples by reversed-phase high-performanceliquidchromatographymethod.Anal.ChimicaActa570(2006),46–58.

[4] ChangX.-J.,PengJ.-D.,LiuS.-P.:Asimpleandrapidhighperformanceliquidchromatographicmethodwith fluorescencedetection for estimationof amikacin inplasma– application topreclinicalpharmacokinetics.J.Chin.Chem.Soc.57(2010),34–39.


1.Introduction

Poultrymeat is an important part ofmost diets due to its being a source ofawholesomeproteinessentialforproperfunctioningofthehumanbody.Intermsofthenutritionalvalue,poultrymeatsurpassesporkandbeef,asitcontainsmoreproteins and less fat. It is an easily digestible source of minerals, such aspotassium,calcium,phosphorusandiron[1].Asaresult,poultryisoneofthemostpopulartypesofmeatanditsconsumptionissteadilyincreasing.Itisestablishedthat it constitutes 40%of the overallmeat consumed in EuropeanUnion [2].Moreover,poultrymeatisbecominganimportantcomponentofthedietofPoles.Sincetheyear2000,itsannualconsumptionincreasedbyasmuchas63.3%andatthepresentmomentreaches29kgpercapita[3,4]. Thegrowingdemandforsafemeatproductshascausedanincreasedinterestinnewmethodsofpoultry'sspoilageassessment,bothattheindustrialandretaillevels.Thequalityevaluationisnecessarytoensuretheconsumers’satisfaction,aswellastheirsafetysincetheconsumptionofspoiledmeat,canbeacauseofserioushealthhazards[5]. It was reported that the deterioration of meat’s quality due to chemicalchangesaccompanyingbacterialgrowthcanbequantifiedbythemeasurementoftotalvolatilebasicnitrogen.Thismethodisusedmostlyfortheevaluationoffish

Poultry meat freshness assessment based on the biogenic amines index

KAJAKALINOWSKA*,WOJCIECHWOJNOWSKI,JUSTYNAPŁOTKA-WASYLKA,

JACEKNAMIESNIK

DepartmentofAnalyticalChemistry,FacultyofChemistry,GdanskUniversityofTechnology,11/12NarutowiczaSt.,80-233Gdańsk,Poland*[email protected]

AbstractInordertosafeguardthewell-beingoftheconsumers,itisimportanttoaccuratelydetermine theshelf lifeofpoultryandpoultry-basedproducts.Inthiswork,itwasevaluatedwhetherthemeasurementoftheconcentrationofcadaverine,putrescine,histamineandtyraminecan be used to assess the shelf-life of poultrymeat stored in thedifferent types of the containers. Based on the results it can beconcludedthatthecollectivemeasurementofthebiogenicaminescanbesuccessfullyusedinthepoultrymeatfreshnessassessmentandcould potentially supplementmore traditionalmethods of qualityandshelf-lifeevaluation.

Keywordsbiogenicaminesfoodanalysismeatfreshness


freshnesssincethevolatilenitrogenousbasesthatareprimarilyformedintheprocess of the enzymatic decarboxylation of certain amino acids are oftenassociatedwiththearomaofspoiledfish[6,7].However,itwasdeterminedthatthevalueoftotalvolatilebasicnitrogenincreaseswiththepoultrymeat’sspoilageandthusmaybeusedasoneofthemeatfreshnessindicators[8–10]. Thealternativeapproachtomeatqualityevaluation is themeasurementofconcentrationvaluesofbiogenicamines.Asvolatilenitrogenousbases,theyarepredominantlyformedbythedecarboxylationofaminoacidsduetotheactivityofvariousmicroorganisms.Eventhoughthereareseveralpolyaminesoccurringinfreshmeat,biogenicaminessuchascadaverineandhistaminearemainlyformedduringprocessingandstorageofmeatandthusmayserveasqualityindicatorsofpoultryandpoultry-basedproduce[11–14].Moreover,thereisagrowinginterestintheirdeterminationnotonlybecauseoftheirpotentialuseinmeatspoilageassessment but also due to the toxicological effect that can be related to theingestionofbiogenicamines.Itwasreportedthattheconsumptionofproductswithhighlevelsofcertainamines(e.g.,tyramineandhistamine)maycausefoodpoisoningandmigraines[15–16]. Sincetheconcentrationofindividualbiogenicaminesmaydifferdependingonthenumerousfactors,meatqualityassessmentmayyieldabetterresultwiththeapplicationofmethodsbasedonthemeasurementofthelevelsofseveralamines.Veciana-Noguesetal.[17]proposedtheuseofbiologicalaminesindexinwhichthevaluesofhistamine,cadaverine,putrescineandtyramineareaddedup.Thismethodisemployedprimarilyinfishfreshnessevaluation;however,ithasfoundan application in the spoilage assessment of poultry and poultry-based pro-ducts[18].

2.Experimental

2.1Samples

Freshchickenbreastmuscle,porkloinandbeefloinweresourcedfromalocaldistributioncentreinGdansk,Poland.Animalswereslaughteredontheeveningprior to the first day of the analysis and then transported to the distributioncentres,inwhichthecarcasseswereprocessed.Onthefirstdayoftheexperiment,meat was transported in a portable cooler to the laboratory, where it wasrefrigerated at 4°C. Samples of each meat were stored in three differentcontainers:polypropyleneco-polymervacuumfoodbox,aerobicallyinastandardpolypropyleneco-polymerfoodboxandaerobicallyinastandardhigh-densitypolyethylenerefrigeratorbag.Onthe1st,3rd,and5thdayoftheexperiment,theconcentrationof the fourbiogenic amineswasdeterminedusingapreviouslydescribedmethod[14].


2.2Reagents

Standardsofcadaverine,histamine,putrescine,tyramine,aswellastheinternalstandard(hexylamine)weresuppliedbySigmaAldrich.Forderivatization,iso-butylchloroformate(SigmaAldrich)wasused.Asextractivesolvent,chloroformofhighpurityHPLCanalysisgrade,alsosuppliedbySigmaAldrich,wasused.Bothhydrochloric acid and high-purity grade dispersive solvent methanol wereobtained from Fluka. In order to prepare the solution of alkaline methanol,potassiumhydroxidewasdissolvedinmethanoluntilsaturation.

2.3Biogenicaminesdetermination

Insituderivatizationwithisobutylchloroformatecoupledtodispersiveliquid-liquidmicroextractionwasusedassamplepreparationmethodologypriortothefinaldeterminationwithgaschromatography-massspectrometry.

2.4Multivariatedataanalysismethod

DataanalysiswasperformedusingadedicatedPythontoolboxOrangev.3.13[19].Theconcentrationvaluesofthefourbiogenicamineswerenormalisedandusedasinputsforbothprincipalcomponentanalysisandforsupervisedmachinelearningalgorithms.Theperformanceofthelatterwasassessedusingastratified10-foldcross-validation.HierarchicalclusteranalysiswasperformedbasedonEuclideandistancesbetweenthedatapointswithWardlinkage.


Theaimoftheexperimentwastodeterminewhetheritispossibletoassessthefreshnessofpoultrymeat stored indifferent containersbasedon theconcen-trationofcadaverine,tyramine,putrescineandhistamine.Duringthecourseoftheexperiment,theoverallconcentrationofbiogenicaminesinallsamples(listedinTable1)hasincreasedsignificantly.Thedifferencesbetweentheresultsobtain-ed for the samples stored in different containers were noticeable, but notprominent. Theaveragedconcentrationofbiogenicamineshasbeenusedasinputvaluesfor chemometricanalysis.Fristof all, principal componentanalysis (PCA)hasbeen performed. First two principal components covered 99% of variance.AscatterplotobtainedwiththeuseofPCAispresentedinFig.1.Asitcanbeseen,datapointscorrespondingtosamplesanalysedondifferentdaysofstorageformdistinctlyseparatedgroups.Moreover,thewithin-groupvarianceincreasedafterthefirstdayofstorage,asthedifferencesbetweentheconcentrationsofbiogenicamines in thesamplesstored invariouscontainersbecomemorepronouncedovertime.


InFig.2,thedendrogramofhierarchicalclusteringisshown.Datapointsaregrouped primarily based on the time of storage. However, samples stored indifferenttypesofcontainersformseparateclusterswithinthegroups.Itisalsoworthnotingthattheresultforthemeatstoredinafoodboxandinabagformagroupseparatedfromtheresultsofpoultrymeatkept inavacuumfoodbox(adistinctgroupatthe0.33clusteranalysiscut-off).Thisislikelyduetothefactthatlimitedoxygenaccessinvacuumfoodboxaffectsthemicrobialdevelopment.


–1Biogenicamine Storage Concentration/mgg

Day1 Day3 Day5

cadaverine I n.d. 870.6±4.4 1041.4±4.8 II n.d. 781.8±1.2 980.6±4.7 III n.d. 915.0±1.9 1104.2±3.2tyramine I n.d. 311.6±1.3 410.0±2.1 II n.d. 241.8±1.5 305.0±3.8 III n.d. 413.0±1.1 531.4±3.6putrescine I 98.84±0.19 111.60±0.17 179.58±0.58 II 98.86±0.17 103.3±1.7 153.78±0.52 III 99.140±0.040 115.0±0.2 197.7±1.4histamine I 148.14±0.46 433.2±3.2 380.6±4.7 II 148.00±0.47 411.4±3.0 365.4±3.7 III 148.46±0.05 507.8±4.8 338.0±1.9

Table 1Informationonconcentrationofbiogenicaminesinsamplesondifferentdaysofstorage:(I)foodbox,(II)vacuumfoodbox,(III)high-densitypolyethylenerefrigeratorbag.Averagedconcentration

–1100mgg .

Fig. 1ResultsofPCAanalysisrefrige-ratedovertheperiodof5days.

Method AUC CA Precision

k-nearestneighbours 1.000 1.000 1.000classificationTree 0.981 0.977 0.398supportvectormachines 1.000 1.000 1.000randomforest 1.000 1.000 1.000NaiveBayes 1.000 0.955 0.960

Table 2Classificationparameters.

Furthermore, theclassificationaccuracyof fivedifferentalgorithms,namelyk-nearestneighbours,classificationtree,supportvectormachines,randomforestandNaive-Bayes,wasassessed.AsitcanbeseeninTable2, itwaspossibletoclassifythesamplesbasedonthestoragetimewithsatisfactoryresults.Itisworthnotingthattheperfectclassification(AUC1.000,CA1.000,precision1.000)wasachievedwiththeuseofthreeoutoffivealgorithms.

4.Conclusions

Basedontheresultsoftheexperiment,itispossibletoconcludethatthecollectivemeasurement of the concentration of cadaverine, putrescine, histamine andtyraminecanbeusedtoassesstheshelf-lifeofmeat.ThebiologicalaminesindexproposedbyVeciana-Noguesetal.[17]enablestheclassificationofmeatbasedonthedurationofstorageevenwhenthesamplesarestoredindifferenttypesofcontainers.Thissuggeststhatthistechniquecouldbeusedasasupplementarymethodinproductionordistributioncentresusingmoretraditionalmethodsofqualityassessment,suchasTotalViableCount.


Fig. 2Thedendrogramofhierarchicalclusteringwithacut-offlineat33%oftherange.

References

[1] RachwałA.:Cechychemicznemięsadrobiowego.Hod.Drobiu2(2006),28–33.(InPolish)[2] OECD-FAOAgriculturalOutlook2015.Paris,OECDPublishing2016.[3] www.stat.gov.pl/obszary-tematyczne/roczniki-statystyczne/roczniki-statystyczne/rocznik-

statystyczny-rzeczypospolitej-polskiej-2016,2,16.html.[accessed21stMay,2018].(InPolish)[4] NowakM.,TrziszkaT.:Zachowaniakonsumentownarynkumięsadrobiowego.Żywność.Nauk.

Technol.Jakość¨1(2010),114–120.(InPolish.)[5] SaucierL.,Microbialspoilage,qualityandsafetywithinthecontextofmeatsustainability.Meat

Sci.120(2016),78–84.[6] BalamatsiaC.C.,PatsiasA.,KontominasM.G.,SavvaidisI.N.:Possibleroleofvolatileaminesas

quality-indicatingmetabolitesinmodifiedatmosphere-packagedchickenfillets:Correlationwithmicrobiologicalandsensoryattributes.FoodChem.104(2007),1622–1628.

[7] WojnowskiW.,MajchrzakT.,DymerskiT.,GębickiJ.,NamiesnikJ.:Electronicnoses:Powerfultoolsinmeatqualityassessment.MeatSci.131(2017),119–131.

[8] WangY.,LiY.,YangJ.,RuanJ.,SunC.,Microbialvolatileorganiccompoundsandtheirapplicationinmicroorganismidentificationinfoodstuff.TrendsAnal.Chem.78(2016),1–16.

[9] KhulalU.,ZhaoJ.,HuW.,ChenQ.:Intelligentevaluationoftotalvolatilebasicnitrogen(TVB-N)contentinchickenmeatbyanimprovedmultipleleveldatafusionmodel.Sens.ActuatorsB238(2017),337–345.

[10] Huang L., Zhao J., Chen Q., Zhang Y.: Nondestructive measurement of total volatile basicnitrogen(TVB-N)inporkmeatbyintegratingnearinfraredspectroscopy,computervisionandelectronicnosetechniques.FoodChem.145(2014),228–236.

[11] FrankeC.,BeauchampJ.:Real-timedetectionofvolatilesreleasedduringmeatspoilage:acasestudy of modified atmosphere-packaged chicken breast fillets inoculated with Br.thermosphacta.FoodAnal.Methods10(2017),310–319.

[12] Balamatsia C.C., Paleologos E.K., Kontominas M.G., Savvaidis I.N.: Correlation betweenmicrobialflora,sensorychangesandbiogenicaminesformationinfreshchickenmeatstoredaerobicallyorundermodifiedatmospherepackagingat4°C:possibleroleofbiogenicaminesasspoilageindicators.AntonieVanLeeuwenhoek89(2006),9–17.

[13]WojnowskiW.,MajchrzakT.,SzwedaP.,DymerskiT.,GębickiJ.,NamiesnikJ.:RapidevaluationofpoultrymeatshelflifeusingPTR-MS.FoodAnal.Methods11(2018),2085–2092.

[14]Wojnowski W., Płotka-Wasylka J., Kalinowska K., Majchrzak T., Dymerski T. Szweda P.,NamiesnikJ.:Directdeterminationofcadaverineinthevolatilefractionofaerobicallystoredchickenbreastsamples.Monatsh.Chem.,inpress,doi:10.1007/s00706-01802218-7.

[15] LehaneL.,OlleyJ.:Histaminefishpoisoningrevisited.Int.J.FoodMicrobiol.58(2000),1–37.[16] CrookM.:Migraine:Abiochemicalheadache.Biochem.Soc.Trans.9(1981),351–357.[17] Veciana-NoguesM.T.,Marine-FontA.,Vidal-CarouM.C.:Biogenicaminesashygienicquality

indicators of tuna. Relationships with microbial counts, ATP-related compounds, volatileamines,andorganolepticchanges.J.Agric.FoodChem.45(1997),2036–2041.

[18] Silva C.M., GloriaM.B.A.: Bioactive amines in chickenbreast and thigh after slaughter andduringstorageat4±1°Candinchicken-basedmeatproducts.FoodChem.78(2002),241–248.

[19] DemsarJ.,CurkT.,ErjavecA.,HocevarT.,MilutinovicM.,MozinaM.,PolajnarM.,ToplakM.,Staric,A.,StajdoharM.,UmekL.,ZagarL.,ZbontarJ.,ZitnikM.,ZupanB.:Orange:DataminingtoolboxinPython.J.Mach.Learn.Res.14(2013),2349–2353.


1.Introduction

Perfumeshavebeenusedforthousandsofyearsandnowadaystheyareconsi-deredasanessentialpartofhumanlife[1,2].Onaverage,every43hoursanewfemalefragranceappears,whilethemaleoneappearsonceevery96hours.Asaresult,itisassessedthattheperfumebusinessisabillion-dollarindustry[3]. Perfumeshavecomplexmatricesthatconsistofawiderangeofnaturalandsyntheticcompoundsbelongingtodifferentchemicalclasses.Hence,theriskofcontactallergyinducedbytheiringredientsisstillbeingtheobjectofscientificdebate [1]. Furthermore, due to the adverse effects of some of perfumescomponentsonhumanhealthandtheirpotentialbioaccumulation,theypresentaclearlygrowingthreattohealthandenvironment.Besides,thehighpricesofessentialoilscausethatfragrancedealersmoreandmoreoftendecidetofalsifytheirproductsbyaddingcheapermaterials,butstillaskingforthesamepriceforthemixture.Accordingtothesereasons,theuseofanalyticaltechniquestoassessallergenicpropertiesofperfumecomponents,environmentalcontaminationoradulterationofperfumesisinevitable[4].Duetothefactthatthemostperfumeingredientshaveapolarand(semi-)volatilecharacter,gaschromatography(GC)

High resolution liquid chromatography and time of flight mass spectrometry in perfume analysis

DAGMARAKEMPIN SKA*,AGATAKOT-WASIK

DepartmentofAnalyticalChemistry,FacultyofChemistry,GdańskUniversityofTechnology,11/12GabrielaNarutowiczaStreet,80-233Gdańsk,Poland*[email protected]

AbstractPerfumesconsistofawiderangeofnaturalandsyntheticcompoundsthatbelongstodifferentchemicalclasses.MostofthesecompoundsaregenerallydeterminedbyGC.However,inthisstudyRP-HPLC-Q-TOF-MS and HILIC-Q-TOF-MS technique was applied for thedetermination of ingredients of original perfumes and their imi-tations.Antioxidantsandcompoundsspecifictofragrancesofanimaloriginwerefoundinoriginalperfumesamples,whereascarrieroilscomponents were generally determined in their imitations.Furthermore,somecomponentsofessentialoilswerealsodetected.This research confirmed the theory that results obtained in theanalysisofperfumeusingHPLCcanbecomplementarytothoseoneobtainedduringGCanalysis.

KeywordsHILIC-Q-TOF-MSperfumesRP-HPLC-Q-TOF-MS


isthemostpopulartechniqueusedforperfumeapplication.However,reversed-phasehighperformanceliquidchromatography(RP-HPLC)istechniquethancanbeappliedforthedeterminationofnon-volatileperfumesingredientsthathavelowthermostability.Betweenthevarietyofdetectors,massspectrometer(MS)issuperiorforeitherGCorHPLC,becauseofitshighsensitivityandoutstandingidentificationpossibilities. Electronicnoseisanotherpopulardeviceusedfordeterminingtheperfumeingredients[3,4]. TheaimofthisstudywastoshowthepotentialofHPLC-MStechniqueindirectanalysisoforiginalperfumesandtheirimitations.Twodifferenttypesofliquidchromatographywerepresentedandcompared.Furthermore,theidentificationofseveralperfumesingredientshasbeendone.

2.Experimental


Acetonitrile (HPLCgrade)and formicacid (>98%)wereobtained fromMerck(Germany). Acetonitrile (LC-MS grade) was purchased from VWR Chemicals(USA) and ultrapure water was prepared using HPL5 system from Hydrolab(Wislina,Poland).

2.2Instrumentation

BothHILIC-Q-TOF-MSandRP-HPLC-Q-TOF-MSanalyseswereperformedusingtheAgilent1290LCsystemequippedwithabinarypump,anonlinedegasser,anautosamplerandathermostatedcolumncompartmentcoupledwiththe6540Q-TOF-MSwithaDualESIionsource(AgilentTechnologies,SantaClara,USA).TheESI sourcewas operatedwithpositive andnegative ion ionizationmode.Thefragmentorvoltagewassetat100Vandthemassrangewassetat100–1500m/zinMS.Furthermore,nebulizergaswassetat35psig,capillaryvoltagewassetat

–13500V, anddryinggas flow rate and temperaturewere set at10Lmin and

300°C,respectively.TheTOF-MSsystemwascalibratedonadailybasis. In case of RP-HPLC, LiChrospher 100 RP-18e (125×4 mm, 5 μm; Merck,Germany)columnwasusedinordertoseparateanalytes.Twodifferentsolventmixtureswereexaminedandappliedasamobilephase:onemixturewasbasedonacetonitrileandwatermixturewithformicacid(0.05%,v/v)andthesecondonewasbasedonacetonitrileacidifiedwithformicacid(0.05%,v/v).Inbothcase,theisocraticelutionwasperformed(100%B).The flowrateofmobilephasewas

–10.8mLmin and the injection volume was 20 µL. The column temperaturethroughouttheseparationprocesswaskeptat25°C. In case of hydrophilic interaction liquid chromatography (HILIC), KinetexHILIC100A(150×4.6mm,2.6μm;Phenomenex,USA)columnwasusedinordertoseparateanalytes.Themixtureofacetonitrileandwatermixturewithformicacid


(0.05%,v/v)wasusedasamobilephase.Theotherparametershavebeensetasabove.

2.3Sampleandsamplepreparation

Incaseofthisresearch,twosamplesofperfumesformen(A,B),twosamplesofperfumesforwomen(C,D)andfoursamplesofperfumeimitationswereana-lyzed.TheoriginaloneswereboughtinpopularperfumeryinGdansk,whereastheircheaperversionswereboughtinChineseshop.ThescentcompositionsoforiginalperfumesareshownatFig.1. Bothoriginalandcheaperperfumesamples(250μL)weredilutedin250μLofacetonitrilecontaining3%ofwater.Suchpreparedsampleswereinjected(20μL)directlyintotheHPLC-Q-TOF-MSsystem.


Inthispresentedstudy,differentchromatographicsystemhasbeenusedinordertodetermineperfumescomponents.ThesamplesoforiginalperfumesandtheirimitationswereanalyzedbySCANmode.EachobtainedchromatogramLC-HRMS


Fig. 1FragrancepyramidsoforiginalperfumesanalyzedbyHPLC-MStechnique.

were processed with Molecular Feature Extraction (MFE) mode. The resultsachievedforsampleA(bothESImodes)areshowninFig.2.IncaseofRP-HPLC-Q-TOF-MS,lowmolecularcompounds(200–500Da)andmediummolecularweightcompounds(500Da<)weregenerallydetected.IncaseofHILIC-Q-TOF-MS,thesamesituationwasobserved.However,theuseofHILICenabledtodeterminemorecompoundswithmasshigherthan700Da. The next stepwas to identify the perfumes ingredients. Perfumes containvariouschemicalsthatcanbeclassifiedinsixcategories:solvents,essentialoils,dyes,modifiers,blenders,andfixatives[5].Itwasdecidedthattheidentificationwouldbebasedoninformationaboutperfumescomposition,whichispartiallyavailable,andthelistofcompoundsusedinperfumerypublishedbyInternationalFragranceAssociation(IFRA).SomecompoundsdetectedintheperfumesampleswerepresentedinTable1.TheresultsfortwotypesofperfumesareshowninTable2. Compoundsbelongtofattyacidsweredetectedinallsamples.Theyarethecomponentsofcarrieroils thatareusedtodiluteessentialoilsandabsolutes.Compoundsspecifictofragrancesofanimalorigin(musk,ambergris)werefoundinbothoriginalperfumesamples.Thesesubstancesarenotonlyusedasbasenotes inperfumery,butalsoas fixatives.Duetothe limitedamountofnaturalmuskandambergrisavailableonthemarket,theyareveryexpensive.Further-more,antioxidants(avobenzone,diethylaminohydroxybenzoylhexylbenzoate)


Fig. 2Therelationshipbetweenthemassofchemicalspeciesdetectedinperfumessamplesandtheretentiontime.

thatareaddedtoperfumestoprotect theircolorandscentcompositionweredetected in original perfumes. In case of perfumeA, atranol, dimethyl benzylcarbinylbutyrateandvanillinweredetected.Thesecompoundsarecharacteristicforsomeessentialoils.Thefirstonecanbedeterminedinoakmossessentialoils,thesecondoneinplumessentialoils,whereasthethirdoneisspecifictovanillaessentialoils.Incaseofitsimitation,atranolwasonlydetected.Thiscompoundhasbeenidentifiedastheallergen,soitsconcentrationinperfumesshouldberegulated.MintlactonewasidentifiedinbothsamplesCandC’.Itisafragrancecompound that can be found in Tonka bean oils. Coumarin is the secondcompoundthatcanbedetectedinthisessentialoil.

4.Conclusions

Thepriceofperfumesisaffectedbythecostoftheirproduction.Becauseoftheusageofsmallavailabilitycomponents,someoftheperfumesareexpensiveandstillverydesirable.Forthisreason,manyperfumeimitationsarereachingthemarkets. Most of compounds that vary perfumes and their imitations arecommonlydetectedduringGCanalyses.However,thedeterminationofessentialoilcomponents,muskandotherfixativesconfirmedthattheLC-MScanbeusedasa complementary technique to GC or GC-MS. Two different chromatographicsystemswereappliedforperfumeanalysis.Inbothcaseslowmolecularweightcompounds(from200Dato500Da)weregenerallydetected.Nonetheless,mostidentifiedperfumesingredientswereonlydeterminedinsamplesanalyzedwithRP-HPLC-Q-TOF-MSsystem.


Compound Molecular Monoisotopic Ionizationmode Theoreticalm/z formula mass/Da

ambroxide C H O 236.2140 Positive 237.221316 28 2

atranol C H O 152.0473 Negative 151.04018 8 3

avobenzone C H O 310.1569 Positive 311.164220 22 3

dimethylbenzyl C H O 220.1463 Positive 221.153614 20 2

carbinylbutyratediethylaminohydroxy- C H NO 397.2253 Positive 398.232624 31 4

benzoylhexylbenzoatemintlactone C H O 166.0994 Positive 167.106610 14 2

muscone C H O 238.2296 Positive 239.236916 30

oleicacid C H O 282.2559 negative 281.248618 34 2

palmiticacid C H O 256.2402 Negative 255.232916 32 2

pentadecanoicacid C H O 242.2246 Negative 241.217315 30 2

stearicacid C H O 284.2715 Negative 283.264218 36 2

vanillin C H O 152.0473 Positive 153.05468 8 3

Table 1Basicinformationaboutcompoundsdetectedinperfumessamples.

References

[1] MondelloL.,CasilliA.,TranchidaP.Q.:Comprehensivetwo-dimensionalgaschromatographyincombination with rapid scanning quadrupole mass spectrometry in perfume analysis.J.Chromatogr,A1067(2005),235–243.

[2] GhergelS.,MorganR.M.,BlackmanC.S.,KaruK.,ParkinI.P.:Analysisoftransferredfragranceanditsforensicimplications.Sci.Justice56(2016),413–420.

[3] vanAstenA.:TheimportanceofGCandGC-MSinperfumeanalysis.TrendsAnal.Chem.21(2002),698–708.

[4] Abedi G., Telebpour Z., Jamechenarboo F.: The survey of analytical methods for samplepreparationandanalysisoffragrancesincosmeticsandpersonalcareproducts.TrendsAnal.Chem.102(2018),41–59.

[5] PalmerI.:Perfumemaking-Anoverview.In:Perfume,SoapandCandleMaking.TheBeginner’sGuide.Omaha,WowEnterprises2013,p.11–28.


Sample Compound Experimental Massaccuracy RP-HPLC HILIC m/z /ppm

A ambroxide 237.2217 1.81 + – atranol 151.0410 –5.96 + + dimethylbenzyl carbinylbutyrate 221.1537 0.45 + + diethylaminohydroxybenzoylhexylbenzoate 398.2331 1.26 + + oleicacid 281.2480 –2.13 + – palmiticacid 255.2332 1.76 + + pentadecanoicacid 241.2178 –2.07 + + stearicacid 283.2639 –1.06 + + vanilin 153.0539 –4.57 + –A' atranol 151.0406 3.31 + – palmiticacid 255.2339 3.92 + – C ambroxide 237.2214 –0.42 + – avobenzone 311.1644 0.64 + – mintlactone 167.1067 0.56 + – muscone 239.2372 –1.25 + – oleicacid 281.2494 2.84 + – palmiticacid 255.2336 2.74 + + stearicacid 283.2647 1.77 + +C’ coumarin 147.0441 0.00 + – mintlactone 167.1064 –1.20 + – oleicacid 281.2491 1.78 + – palmiticacid 255.2338 3.53 + + pentadecanoicacid 241.2180 2.90 + + stearicacid 283.2648 2.12 + +

Table 2Compoundsdetected inoriginalperfumesamples (A,C) and their imitations (A’, C’)using twodifferentchromatographicsystem(+=detectedunderusedconditions,–=notdetectedunderusedconditions).

1.Introduction

Inrecentyears,therehasbeenanincreasinginterestinhealthfood,whichiswhynew varieties of plantswith a high content of pro-health substances such asvitamins,mineralsandbioactivesubstancesarebeingsoughtfor.Fromthepointof view of fruit producers, the new varieties should have specific functionalproperties,suchasthoseassociatedwithgreaterresistancetoclimaticfactors,higheryieldsorlackofseeds[1].Oneofthepopularsolutionsinrecentyearsisthecreationofhybridplants.Fruithybridization,orcross-breeding,istheprocessofbotanicalmatingoftwodifferentplantspeciesinordertocreatehybridsthathaveallthebestqualitiesofmotherplantsandnoneofitsdisadvantages[2]. Oneofthecommonlyavailablehybridfruitinrecentyearsisoroblancocalledsweetie(Citrusparadisi×Citrusgrandis),hybridbetweengrapefruit(Citruspara-disi)andthegiantorange,calledpummelo(Citrusgrandis).Itisseedlessfruits,lessacidicandlessbitterthanthegrapefruit[3],whichisrelatedtothedifferent

Study of the effect of the hybridisation process on the content of terpenes in oroblanco fruit (Citrus paradisi × Citrus grandis)

MARTYNALUBINSKA-SZCZYGEŁ*,ANNARO Z AN SKA,TOMASZDYMERSKI,JACEKNAMIESNIK

DepartmentofAnalyticalChemistry,ChemicalFaculty,GdańskUniversityofTechnology,NarutowiczaStreet11/12,80952,GdańskPoland*[email protected]

AbstractFruithybridizationisaprocessthathasbeenusedformanyyearsandresults in formation of fruits with new organoleptic and health-promotingproperties.Thepurposeofhybridizationisalsotoimprovethefruit’sfunctionalproperties.Thepaperpresentsthecomparisonofthecontentofterpenesofhybridfruitnamelyoroblancoanditsparentfruits,grapefruitandpummelo.Bytheuseoftwo-dimensionalgaschromatographycoupledwithmassspectrometryitwaspossibletodeterminethemostabundantterpenesinthevolatilefractionofsweetie, grapefruit, and pummelo.Α-terpineol and limonenewereselectedasmainterpenescompoundsdeterminedintheheadspaceofsweetieandgrapefruit.Inthecaseofpummelo,itwaspossibletodetermineonlyonechemicalcompound,namelylimonene.

Keywordsaromapropertiesfruithybridizationsweetieterpenes


contentofterpenes,aldehydes,esters,ketonesandcarboxylicacids.Accordingtotheliterature,terpenesarethemostabundantcompoundinmostofcitrusfruits[4].Thesearecompoundsbelongingtothegroupofbioactivecompounds,show-ing antioxidant, antibacterial activity, etc. Their presence also determines thetaste and aroma of newly-created fruits, which affects their consumption byconsumers. Duetothecomplexcompositionofthematrixforthedeterminationofvolatilecompounds,ananalyticaltechniquetogiverisetoimprovedresolutionandpeakcapacityisrequired.Inlinewiththeprinciplesofgreenanalyticalchemistry,theaimisalsotousesolvent-freeextractiontechniques,thereforeHS-SPME-GC×GC-TOFMSispureforeignperformed[5].Theaimoftheconductedresearchwastocomparethecontentofterpenesinsamplesoforoblancoanditsparentsfruitsusingtwo-dimensionalgaschromatography.

2.Experimental


Standards of terpenes: α-pinene, limonene, ocimene, myrcene, γ-terpinene,α-terpineol(Sigma-Aldrich,USA)weredilutedinmethanol(AvantorPerformanceMaterials Poland). The fruits for testingwere purchased at local distributionpoints in the PomeranianVoivodship.Due to the complex composition of thematrix,whicharefruitsandvariedstructureandpropertiesofthecompoundsidentifiedtoeliminatethematrixeffect,thestandardadditionmethodwasused.

2.2Instrumentation

AnAgilent6890Agaschromatograph(AgilentTechnologies,USA)equippedwithasplit/splitlessinjectorandaliquidnitrogen-baseddualstagecryogenicmodu-lator, coupled with Pegasus IV time-of-flight mass spectrometer. In case ofisolationandenrichmentofanalytessolidphasemicroextractionwasused.


Theidentifiedcompoundsweredividedintoeightchemicalclasses.AsitcanbeseenatFig.1.thedominantclassinthefruitsamplestestedareterpenes.Thehighcontentofterpenescandeterminethebittertasteofcitrus[6].Asignificantche-micalclass,regardingtotheamountofidentifiedsubstances,wasthegroupofalcohols,esters,aldehydesandketones.Thesearechemicalswithaspecific,oftenintenseodour.Theirpresenceandsynergisticinteractionscauseintensearomaoffruits. Table1presentsallterpenesidentifiedandquantifiedinsweetie,pummeloandgrapefruitsamples.Basedontheresultsitcanbestatedthatpummeloisthe


leastaromaticone.DespitetheuseofGC×GCtechnique,itwaspossibletoquantifyonly one substance, namely limonene, in the fruit pulp. In contrast, in thegrapefruit and sweetie samples, six chemical compounds, belonging to theterpenes group, were identified and determined. In both cases, the highestcontentofα-terpineolwasindicated.Thefourfoldmoreamountofthissubstancewasnotedingrapefruitthaninsweetiesamples.Theearthyodordescriptionofα-terpineol is one of the reasons for the bitter taste of the fruit. The highest

–1content of limonene was noted in the grapefruit, namely 15.79±0.30μgg .Moreover,sweetiecontainsmoremyrceneandγ-terpinenewithpleasantcitrusaromawhichmayexplainitssweetandlessbittertastecomparedtograpefruit.Consideringthecontentofindividualterpenesinthesamplesofthesweetiepulp


Fig. 1Contentofselectedclassesofchemicalsinsamplesofsweetie,pummeloandgrapefruit.

2 –1 –1 –1Compound R Concentration/μgg LOQ/μgg LOD/μgg

sweetie pummelo grapefruit

α-Pinene 0.999 0.8241±0.0096 <LOQ 2.851±0.015 0.664 0.219Limonene 0.996 5.298±0.058 2.057±0.092 15.79±0.30 1.431 0.472Ocimene 0.995 1.600±0.097 <LOQ 2.057±0.078 1.519 0.501β-Myrcene 0.991 4.1±0.14 <LOQ 3.224±0.0293 2.098 0.692γ-Terpinene 0.997 7.27±0.34 <LOQ 2.566±0.026 1.163 0.384α-Terpineol 0.992 20.96±0.70 <LOQ 87.9±2.0 1.947 0.643

Table 1Quantitationofselectedterpenespresentinthevolatilefractionofsweetie,pummelo,andgrape-fruit.

anditsparentfruit,itcanbeconcludedthatthehybridfruitcorrelatestogrape-fruit. Due to the high content of chemical compounds in this chemical class,sweetieisarichsourceofhealth-promotingingredients.

4.Conclusions

Usingtheanalyticalprocedureduringthepresentedstudies,itwaspossibletodeterminethecontentofselectedterpenesinthesweetieanditsparentfruitssamples and to find the reasons for the less bitter and acidic sweetie tastecomparedtothegrapefruit.Itwasalsoshownthatthehighcontentofterpeneswith bioactive activity is a feature that sweetie inherited from the grapefruitduringthehybridizationprocess.

References

[1] SansaviniS.,DonatiF.,CostaF.,TartariniS.:Advances inapplebreeding forenhancedfruitqualityandresistancetobioticstresses:newvarietiesfortheeuropeanmarket.J.FruitOrnam.PlantRes.12(2004),13–52.

[2] BurkeJ.M.,ArnoldB.J.:Geneticsandthefitnessofhybrids.Annu.Rev.Genet.35(2001),31–52.[3] Gazit Y., Kaspi R.: An additional phytosanitary cold treatment against Ceratitis capitata

(diptera:tephritidae)in'oroblanco'citrusfruit.J.Econ.Entomol.110(2017)790–792.[4] DharmawanJ.,KasapisS.,CurranP.,JohnsonJ.R.:Characterizationofvolatilecompoundsin

selected citrus fruits fromAsia. Part I: freshly-squeezed juice.FlavourFragr. J.22 (2007),228–232.

[5] Lubinska-SzczygełM.,RozanskaA.,DymerskiT.,NamiesnikJ.,KatrichE.,GorinsteinS.:AnovelanalyticalapproachintheassessmentofunprocessedKaffirlimepeelandpulpaspotentialrawmaterialsforcosmeticapplications.Ind.CropsProd.120(2018),313–321.

[6] RenJ.N.,TaiY.N.,DongM.,ShaoJ.H.,YangS.Z.,PanS.Y.,FanG.:Characterisationoffreeandboundvolatilecompoundsfromsixdifferentvarietiesofcitrusfruits.FoodChem.185(2015),25–32.


1.Introduction

The interest in thesubjectofpollution in theArctichas increasedwhencom-pounds, whose nearest potential source of emission was several thousandkilometersaway,weredetectedthere.Sincethen,themainfocushasbeenontheimpactofpollutantsonthehigher-orderorganismsfoundinthisregion,andlittleattentionhasbeenpaidtomicroorganisms.[1]Itisassumedthatthecontamina-tioninthepolarregionsarederivedmainlyfromtheemissionsourceslocatedinregionswithtemperateclimates.Itisbelievedthatthetransportofthechemicalsover largedistances is causedby thegrasshoppereffect.Thisphenomenon isconcerning mainly the light organic compounds characterized by low vapor

Correlation between chemical composition and the presence of selected groups of bacteria in freshwater samples collected from Isfjorden and Billefjorde

a, b aFILIPPAWLAK *,KATRZYNAJANKOWSKA ,ZANETAPOLKOWSKA

a DepartmentofAnalyticalChemistry,FacultyofChemistry,GdanskUniversityofTechnology, 11/12NarutowiczaSt.,80-233Gdańsk,Poland*[email protected] DepartmentofWaterandWaste-WaterTechnology,FacultyofCivilandEnvironmentalEngineer-ing,GdanskUniversityofTechnology,11/12NarutowiczaSt.,80-233Gdańsk,Poland

AbstractTheaverageconcentrationsofpollutantsinthearcticwater,snowandtheatmospherearemuchlowerthanthoseobservedinthetemperateclimate. Specific conditions occurring in the polar regions haveapotentialtoaccumulatethepollutantstransportedfromotherpartsoftheworld.Inthisstudy,attemptsweremadetofindacorrelationbetweenselectedchemicalcomponentsandthebacterialpopulation.Theanalysisinvolved11samplesofwatercollectedfromriversandstreams flowing into Isfjorden and Billefjorde in summer 2017(Spitsbergen,Svalbard).Water sampleswere analysed in order todetermine the concentration of various substances such as PAHs,metals,maincations,andanions.Additionally,parameterssuchaspH,SEC,TOC,number,averagesizeandbiomassofbacterioplanktonwerealsomeasured.Onthebasisoftheobtainedresults,itwasimpossibletodeterminetherelationshipbetweenthemeasuredsubstancesandthebacterialcommunity.

KeywordsArcticBacteriapollutantsSpitsbergen


pressure.Themechanismofthiseffectisasfollows:volatilecompoundsevapo-rate intensely into the atmosphere and then they are transportedwithwarmmassesofairtoArctic.Inpolarregions,thetemperaturedecreasessignificantly,whichleadstothecondensationanddepositionofpollutants.Solidparticles,onwhichnonvolatilesubstancescanbeabsorbed,arealsocarriedwithwarmmassesofair.Moreover,pollutantscanbe transportedbyseaandriver,aswellas iceformingonthecoastofSiberia[2].Inaddition,insomepartsoftheArcticlocalsourcesofpollutionsuchashumansettlements,hardcoalmines,powerplants,and means of transport may be located [3]. However, concentration of thepollutantsisnottheonlyfactorthataffectsthebacterialcommunity.Inordertoassess its condition, concentration of naturally occurring compounds andelementsshouldalsobemeasured[4].Moreover,specificclimaticconditionssuchaslowaverageannualtemperature,theoccurrenceofpolarnightandday,lowinsolation,presenceoficeandsnowmaycontributetotheincreaseofdurabilityoforganiccompounds.Theglobalwarmingand,ineffect,rapidlychangingclimaticconditionsintheArcticcausedbyglobalwarmingcancauseremissionsofthecompoundsaccumulatedinthisregion[5].Itmaycreateathreattothefaunaandfloraoftheregion.Inaddition,organismsandmicroorganismsadaptedtolifeinacolderclimatecanbesupplantedbyorganismsbetteradjustedtohighertempe-ratures[6].

2.Experimental

2.1Studyarea

ThesampleswerecollectedfromriversandstreamsflowingintoIsfjordenandBillefjorde in summer 2017 (Spitsbergen, Svalbard). Water samples can bedividedintotwocategoriesbasedonthedifferentresearchareas:threewatersampleswerecollectedfromriversandlakesthatgoouttotheBillefjordenfjord,andremainingeightwerecollectedfromtheIsfjordenfjord.BothofthesefjordsarelocatedinthewesternpartofSpitsbergen.TheexactgeographicallocationsarepresentedontheFig.1.

2.2Sampling

Inordertoobtainsamplesrepresentativeof thewholewatercourse,samplingplaceswerechosencarefully,withmorphologicalandhydrologicalcharacteristicsofthewatercourse,aswellastheproximityofthelocalpollutionsourcestakenunderconsideration.Sampleswerecollectedtohermeticallyclean1Lcontainerswiththeuseofmanualsamplingtechnique.


2.3Instrumentation

The following methods were used to determine the analytes: PAHs – gaschromatography (GCShimadzu2010plus) coupledwithamassspectrometry(MSShimadzuTQ8050),fittedwithadetectorwiththeelectronionizationIonchromatographycoupledwithaconductivitydetector(DIONEXICS-3000)wasused in order to analyse the ions, while – while metals were analysed withInductivelyCoupledPlasmaMass Spectrometry (ThermoScientificXSERIES2ICP-MS).SumofphenolsandformaldehydewasmeasuredwithSpectroquant-Pharo100Spectrophotometer,TOC–catalyticoxidationwithoxygenat680°Cwithnon-dispersiveinfraredspectroscopy(TotalOrganicCarbonAnalyserTOC-VCSH/CSN).MeasurementsofpHandelectricalconductivity(EC)wereperfor-medwiththeuseofmicrocomputerpH-meterandconductivitymeterCPC-411(Elmetron)equippedwithanEC60conductivitysensor.Microbiologicalanalysisconsistedintheestimationofthenumber,averagesizeandbiomassofbacterio-plankton. The direct countingmethodwas used to perform the analysis. Themeasuring apparatus consisted of a Nikon 80i epifluorescence microscope,aNikonDS-5Mc-U2colordigitalcamera.Pre-countingofbacteriawascarriedoutusing theNisElements andMultiscan programs. Then the preliminary resultswere entered into Microsoft Excel and converted into a suitably configuredmacrodefinition.


Fig. 1Themapofsamplingareawiththelocationofsamplingpoints.


Duetothepresenceofthesuspensionobscuringthebacterialcells,thetotalviablecountcouldnotbemeasuredinseveralsamples.Themeasurementwascarriedoutfor6outof11testedsamples.Thelackofresultspreventedameaningfulinterpretationoftherelationshipbetweenthechemicalcompositionofwaterandbacterialcommunities. However, itwaspossible to find correlationsbetween the concentrationofsomechemicalcompounds.InFig.2,theinterconnectionbetweenthecontentofmagnesiumions,calcium,elementalstrontium,andsulphateionsinsamplesfromriversandstreamsflowingintoIsfiorden,canbeseen.Bysettingthetrendlineforcalciumcontent,thesampletakenatpointL1wasnottakenintoaccountbecauseof the possibility of toomuch sewage from Barentsburg. The L1 samplewasmarkedon thegraphas -Rejectedpoint.The linear relationshipbetween theabove-mentionedanalytessuggestthattheymaycomefromthesameorigin.Itismostlikelythattheycomefromthedissolutionoflocalrocks. The Principal components analysis was performed for selected variables.Variablestakenintoaccountare:pH,electricalconductivity,totalorganiccarbon,sumofanionconcentrations,sumofcationconcentrations,sumofconcentrationsofpolycyclicaromatichydrocarbons,andsumofselectedmetals(Ag,As,Bi,Cd,Co,Cr,Cu,Fe,Hg,Mo,Ni,Pb,Sb,Se,V).Somevariableshavebeenlogarithmizedtobettershowdistributions.Firsttwoprincipalcomponentscovered82%ofthevariance.AscatterplotobtainedusingPCAispresentedinFig.3.Asitcanbeseen,samplesfromtwogroups:thefirstofthemincludessamplesL6,L8,Pi1,Pi2,Pi3takenfromriversandstreamsoccurringnearthecoalmine,whilethesecondcluster includes samplesL2,L3,L5, andL7collected fromriversandstreamsaroundwhichtherearenoidentifiedlocalsourcesofpollution.


Fig. 2Therelationbetweenthecontentofmagnesiumions,calciumandelementalstrontium,andsulphateionsforsamplesfromriversandstreamsflowingintoIsfiorden.

4.Conclusions

Duringthecourseoftheexperiment,severalanalysishavebeenperformed:deter-minationofPAHs,ions,formaldehyde,sumofphenolTOC,andselectedmetals,aswellasthemeasurementofpH,EC,totalcountandaveragevolumeofbacterialcellsandbiomass.However,nosignificantcorrelationbetweentheconcentrationoftheanalytesandtheconditionofbacterialcommunityhasbeenfound.Lackofdependencecanbecausedby thecomplexityofbacterialmetabolism,speciesdiversityofmicroorganisms,andtheirenvironmentaladaptation.Thelackoffullresultscouldmakeitimpossibletoobservedependence.However,theanalysisofthe composition of the tested samples made it possible to find the linearrelationshipbetweenelementssuchascalcium,magnesiumandstrontium,andsulphateions,whichmaysuggestthattheyoriginatefromthesamesource,e.g.,localrocks.Theanalysisofthemaincomponentsmadeitpossibletovisualizethesimilaritybetweenthesamplestakenfromriversandstreams.

References

[1] KosekK.,KozakK.,KoziołK.,JankowskaK.,ChmielS.,PolkowskaZ .:Theinteractionbetweenbacterial abundance and selected pollutants concentration levels in an arctic catchment(southwestSpitsbergen,Svalbard).Sci.Total.Environ.622–623(2018),913–923.

[2] MaJ.,HungH.,MacdonaldR.:Theinfluenceofglobalclimatechangeontheenvironmentalfateofpersistentorganicpollutants:AreviewwithemphasisontheNorthernHemisphereandtheArcticasareceptor.Glob.Planet.Change.146(2016),89–108.

[3] Zaborska A., Beszczynska-Moller A., Włodarska-Kowalczuk M.: History of heavy metalaccumulationintheSvalbardarea:Distribution,originandtransportpathways.Environ.Pollut.231(2017),437–450.

[4] GrebmeierJ.,OverlandJ.,MooreS.,FarleyE.,CarmackE.CooperL.,FreyK.,HelleJ.,McLaugh-linF.,McNutt S.:Amajor ecosystem shift in theNorthernBering Sea.Science311 (2006),1461–1464.

[5] Kozak K., Polkowska Z ., Ruman M., Kozioł K., Namiesnik J.: Analytical studies on theenvironmentalstateoftheSvalbardArchipelagoprovideacriticalsourceofinformationaboutanthropogenicglobalimpact.TrendsAnal.Chem.50(2013),107–126.


Fig. 3TheresultofthePCAanalysis.

[6] KallenbornR.,HungH.,Brorstrom-LundenE.:Chapter13–Atmosphericlong-rangetransportof persistent organic pollutants (POPs) into Polar Regions In.: Comprehensive AnalyticalChemistry.Vol.64.E.Y.Zeng(ed.).Elsevier2015,p.411–432.


1.Introduction

According toUnitedStatesEnvironmentalProtectionAgency90%of the timepeoplespentinclosedspaces,whichmakesindoorairanimportantfactorthatinfluenceshumanhealth[1–3].Theproblemofindoorairqualityaroseinlate1960sand1970s,whentheterm“sickbuildingsyndrome”appeared[4].Thistermreferstosymptomslike:irritationofupperairway,eyes,mucousmembranesandskin[5].Itisbelievedthatsickbuildingsyndromeiscausedbyvolatileorganiccompounds[6]withterpenesasoneofthecontributors.Terpenesareproducedmostlybyconiferplants[7–8],howevertheyarealsoemittedintoindoorairfromcleaningagents,perfumes,airfreshenersandcosmeticproducts[9–11].Terpenesmostcommonlypresent in indoorairare:d-limonene,α-pineneandβ-pinene[12]. Products formed due to the series of reactions initiated by terpenesoxidationmaycausehumanhealthdeterioration[6;13–15].Thereasonwhythepresenceofterpenesinindoorairmaybeconsideredasathreattohumanhealthis because products of their transformations undergo condensation and formsecondaryorganicaerosol[17].Secondaryorganicaerosolnanosizedparticlesare able to penetrate deeply human respiratory track and even reach thebloodstream[18]. Therewerealreadyalotofstudiesonterpenespresenceinindoorair,buttothebestofourknowledge,therewasnoresearchconcerningterpenesdeterminationinhairdressers salonspublishedyet. In thisworkwepresent thepreliminary

Influence of terpenes on indoor air quality

KLAUDIAPYTEL*,RENATAMARCINKOWSKA,BOZENAZABIEGAŁA

DepartmentofAnalyticalChemistry,FacultyofChemistry,GdańskUniversityofTechnology,G.Narutowicza11/12,80-233GdańskPoland*[email protected]

AbstractThe aimof this studywas to investigate air quality in hairdressersalons, focusing on terpenes determination. Terpenes are knownreactive volatile organic compounds that contribute to secondaryorganicaerosolformation.Thosecompoundsarefrequentlyfoundincosmeticproductsasfragranceagents.Hairdressersalonsarespecialkindofenvironment,wheresecondaryorganicaerosolconcentrationmaybeelevated.OnehairdressersaloninGdyniawaschosentocarryoutpreliminary research. Indoor air samples from the salonwerecollected using diffusive samplers and sorbent tubes, whichwerelaterdesorbedandanalyzedbyTD-GC-FIDandTD-GC-MS.Determi-

–3nedlimoneneconcentrationvariedbetween6–74µgm .

Keywordshairdresserindooraird-limonenesecondaryorganic

aerosolterpenes


researchconcerningindoorairqualityofthistypeofpublicservicespace.Theaimistodeterminechemicalcompositionofindoorair,withaspecialattentionpaidtoterpenesconcentration,anditsvariabilityinhairdressersalon.

2.Experimental


Methanol(gradientgradeforLC,Sigma-Aldrich),(R)-(+)-limonene(Sigma-Ald-rich),calibrationsolutionswereoffollowingconcentrations:10;100;200and

–1500ngµl .

2.2Instrumentation

® ®Radiello diffusivepassivesamplerswithCarbograph sorbentandtubesfilled®withTenax wereappliedtosampletheindoorair.Passivesamplersandsorbent

®tubes were desorbed using thermal desorption (TD) unit Markes . Gaseoussampleswereanalyzedbygaschromatographycoupledtomassspectrometry(GCAgilentTechnologies6890,MSAgilentTechnologies5973)andflame-ionizationdetector(GC-FIDAgilentTechnologies7820A).ParametersofthermaldesorptionunitandtwochromatographicunitsarepresentedinTable1.

2.3Methodologyofanalyticalprocedure

®AllofanalyticalproceedingstagesarepresentedinFig.1.LocationofRadiello andactivesamplingwithsamplingtubesispresentedinFig.2.


GC-MS GC-FID

Column DB5MS60m×0.25mm×1µm DB130m×0.32mm×5µm (AgilentJ&W) (AgilentTechnologies)

–1 –1Columnflow 0.5mlmin 2.2mlminTemperatureprogram 70°Cheldfor1min,rampedat 40°Cheldfor1min,rampedat

–1 –1 15°Cmin to120°Candheldfor 10°Cmin to125 °C,rampedat –1 –1 1min,rampedat10°Cmin to 15°Cmin to240 °Candheldfor

280°Candheldfor5min 5minDetectortemperature Ionsource:250°C 250°C Quadrupole:150°CThermaldesorptionprepurge 1min(splitON) 1min(splitON)tubedesorption 10minat300°C(splitOFF) 10minat300°C(splitOFF)trapdesorption Traplow:1°C Traplow:1°C Traphigh:300°C(splitOFF) Traphigh:300°C(splitOFF)

Table 1Thermaldesorption,GC-MSandGC-FIDparametersappliedduringanalysis.


Fig. 2 Location of passive samplers and sorbent tubessamplingpointsathairdressersalon.

Fig. 1Analyticalproceedingstepsappliedinthisresearch.


®SamplingwithRadiello samplerslast5hours(ca.since10a.m.to3p.m.)eachdayofsampling,whereassamplingwithsamplingtubeswasperformedaround2–3p.m.eachday.Samplingwascarriedoutduringtheworkingdaysandattheweekend.LimoneneconcentrationscalculatedonthebasisofthemassadsorbedonsamplingtubesanddeterminedbyTD-GC-FIDarepresentedinTable2. Accordingtoobtainedresults,limoneneconcentrationsdeterminedinhair-dresser salonwere sometimes higher in comparison to an exemplary studiescarried out in Germany,which indicated limonene concentration at homes at

–3alevelof15–20µgm [19–22].Alldifferencesinconcentrationcouldbecausedbyactivitiesperformedinthesalonduringtheday.Forexampleat09.06.2018limoneneconcentrationwasthelowestandsamplingwascarriedoutveryclose(1meter)fromtheclientwithahairdye.Thetypeofhairdyesusedinchosensalondoes not have terpenes indicated in International Nomenclature of CosmeticIngredientsandnonoticeablescent.Thehighestconcentrationdeterminedonthe16.06.2018mayberelatedtothefactthatsamplingoccurredwhilehairdresserwasusinghairsprayandothercosmeticsintheformofaerosols,withrelativelylarge amount of scented chemicals included. Chromatograms obtained by

®desorptionofRadiello samplerspresentmoreprecisepictureofthecomplicityofindooraircomposition,whichcanbeseenonanexemplarychromatogramsobtainedbyTD-GC-FIDandTD-GC-MS(Fig.3)analysis. MS detector was applied in order to carry out qualitative analysis anddeterminecomponentsoftheindoorair.OntheGC-MSchromatogramlimonenepeakintensityisrelativelylowincomparisontosiloxanes(Fig.3B).Therearealsoalcoholspeaksofhigh intensityvisibleat thebeginningofchromatogramandtheirpresenceisjustifiedduetothefactthattheyarecommoncosmeticingre-dients.FIDdetectorwasappliedmainly togetquantitative informationaboutanalytespresentinthesample.ChromatogramobtainedbyGC-FIDprovesthatindoorairfromhairdressersalonisacomplexsamplecontainingasetofdifferentchemicals(alotofchromatographicpeaksonFig.3A).SiloxanesarenotvisibleonFIDchromatogram,becauseFIDisselectivetowardscompoundsthatpossessC–Hbondinginastructure. Suchhighconcentrationofsiloxanesinindoorairofhairdressersalonmaybeexplained by the fact that siloxanes are common ingredients of hair cosmetic


Dayofsampling Limonenecon- Totalconcentration–3 –3 centration/µgm oforganiccompounds/µgm

09.06.2018 6 32216.06.2018 74 224620.06.2018 69 5122

Table 2Limoneneconcentrationdeterminedinachosenhairdressersalon.

products.Suchhighconcentrationofsiloxanesmayalsobeconsideredasathreatforworkersandclientshealth,becauseaccordingtotheliterature,siloxanesweredeterminedascomponentsofatmosphericaerosolandmayirritatehumanrespi-ratorysystem[23].

4.Conclusions

Inthispaperweprovedthatindoorairofhairdressersalonscontainsprecursorsforsecondaryorganicaerosolformation.Itisnecessarytocontinuethisresearchinordertoimproveanalyticalapproachandobtainmoredata.ItwouldbeveryhelpfultouseinthefutureparticlecounterslikeSMPS(ScanningMobilityParticleSizers)todeterminetheexactdependencebetweentheamountofterpenesinindoorairandtheamountandspatialdistributionofcreatedsecondaryorganicaerosolparticles.


Fig. 3Chromatogramobtainedby(A)GC-FID,and(B)GC-MSanalysisofthesamplecollectedonthefourthdayofsampling.

References

[1] USEPA:AirQualityCriteriaforParticulateMatter.NationalCenterforEnvironmentalAssess-mentOfficeofResearchandDevelopment.1.2001

[2] USEPA:AirQualityCriteriaforParticulateMatter.NationalCenterforEnvironmentalAssess-mentOfficeofResearchandDevelopment.2.2004.

[3] USEPA:AirQualityCriteriaforParticulateMatter.NationalCenterforEnvironmentalAssess-mentOfficeofResearchandDevelopment.3.1996.

[4] SundellJ.:Onthehistoryofindoorairqualityandhealth.IndoorAir14(2004),51–58.[5] Spengler.JD.,SametJ.M.,McCarthyJ.F.:IndoorAirQualityHandbook.NewYork,McGraw-Hill

2001.[6] MissiaD.A.,DemetriouE.,MichaelN.,TolisE.I.,Bartzis J.G.: Indoorexposure frombuilding

materials:Afieldstudy.AtmosphericEnviron.44(2010),4388–4395.[7] CurciG.,BeekmanM.,VautardR.,SmiatekG.,SteinbrecherR.,ThelokeJ.,FriedrichR.:Modeling

studyof the impact of isopreneand terpenebiogenic emissiononEuropeanozone levels.AtmosphericEnviron.43(2009),1444–1455.

[8] SchrippT., LangerS., SalthammerT.: Interactionofozonewithwoodenbuildingproducts,treatedwoodsamplesandexoticwoodsamples.AtmosphericEnviron.54(2012),365–372.

[9] NazaroffW.W.,WeschlerJ.W.:Cleaningproductsandairfresheners:exposuretoprimaryandsecondaryairpollutants.AtmosphericEnviron.38(2004),2841–2865.

[10]Wolkoff P., Clausen P., Wilkins C., Nielsen G.: Formation of strong airway irritants interpene/ozonemixtures.IndoorAir10(2000),82–91.

[11]TsigoniaA.,LagoudiA.,ChandrinouS.,LinosA.,EylogiasN.,AlexopoulosEC.: Indoorair inbeautysalonsandoccupationalhealthexposureofcosmetologiststochemicalsubstances.Int.J.Environ.Res.PublicHealth7(2010),314–324.

[12]Hodgson A., Beal D.: Sources of formaldehyde, other aldehydes and terpenes in a newmanufacturedhouse.IndoorAir12(2001),235–242.

[13]Holcomb L.C., Seabrook B.S.: Indoor concentrations of volatile organic compounds:implicationsforcomfort,healthandregulation.IndoorEnvironment.4(1995),7–26.

[14]KotziasD.,GeissO.,TirendiS.,JosefaBM.,ReinaV.,GottiA.,GraziellaCR.,CasatiB.,MarafanteE.,Sargiannis D.: Exposure to Multiple air contaminants in public buildings, schools,kindergartens–theeuropeanindoorairmonitoringandexposureassessment(AIRMEX)study.FreseniusEnviron.Bull.18(2009),670–681.

[15]Eriksson K.A., Levin J.O., Sandstrom T., Lindstrom–Espeling K., Linden G., Stjernberg N.L.:TerpeneexposureandrespiratoryeffectsamongworkersinSwedishjoineryshops.Scand.J.WorkEnviron.Health23(1997),114–120.

[16]Wolkoff P., Larsen ST.,HammerM.,Kofoed–SørensenV., ClausenP.A.,NielsenG.D.:Humanreferencevalues for acute airwayeffectsof five commonozone–initiated terpene reactionproductsinindoorair.Toxicol.Lett.216(2013),54–64.

[17]ItoK.,HarashimaH.:CoupledCFDanalysisofsizedistributiononindoorsecondaryorganicaerosolderivedfromozone/limonenereaction.Build.Environ.46(2011),711–718.

[18]Borduas N., Lin V,S.: Researsh highlights: Laboratory studies of the formation andtransformation of atmospheric organic aerosols.Environ. Sci. Process. Impacts18 (2016),425–428.

[19]Schlink U., Rehwagn M., Damm M., Richter M., Borte M., Herbarth O.: Seasonal cycle ofindoor–VOCs: comparison of apartments and cities. Atmospheric Environ. 38 (2004),1181–1190.

[20]Schlink U., Roder S., Kohajda T.,Wissenbach D.K., Franck U., Lehmann I.: A framework tointerpretpassivelysampledindoor–airVOCconcentrationsinhealthstudies.Build.Environ.105(2016),198–209.

[21]MatysikS.,RamadanA.B.,SchlinkU.:SpatialandyemporalvariationofoutdoorandindoorexposureofvolatileorganiccompoundsinGreaterCairo.Atmos.Pollut.Res.1(2010),94–101.


[22]RoschC.,KohajdaT.,RoderS.,BergenM.,SchlinkU.:RelationshipbetweensourcesandpatternsofVOCsinindoorAir.Atmos.Pollut.Res.5(2014),129–137.

[23]ChandramouliB.,KamensM.R.:Thephotochemicalformationandgas–particlepartitioningofoxidation products of decamethyl cyclopentasiloxane and decamethyl tetrasiloxane in theatmosphere.AtmosphericEnviron.35(2001),87–95.


1.Introduction

Consumer awareness is increasing in recent years. They paymore andmoreattentiontothehealth-promotingpropertiesoffoodproducts.Consumersdrinkmoreandmorepureandnaturallycloudyfruitjuices,whichbelongtothegroupofNotFromConcentratejuices.AccordingtotheEuropeanFruitJuiceAssociation,thedemandforNotFromConcentratejuiceshasincreasedofabout14.0%(overthepast fiveyears)[1].ThemostcommonlyconsumedNotFromConcentratejuiceisorangejuice[2],however,itcanbenoticedthatthepopularityofjuicesproduced from berries, such as raspberries, blueberries, blackberries andchokeberry increased. These juices are characterized by a high content ofbioactivecompounds,suchaspolyphenols,carotenoidsoranthocyanins[3–5].Therefore,theingestionofthesejuicesasapartofthedietcanhaveapositiveeffect on health and the human body. Luo et. al. [6] exhibit that consumingraspberry juicemayresult in improvedmetabolism.Hatcher [7],on theotherhand,hasproventhatraspberryjuicehasapositiveeffectonbonehealthandprotects it against osteoporosis. In Poland, there was a problem with theutilizationofchokeberryfruit.Therefore,accordingtoexperts’opinions,thesefruitscanbeusedasanadditiontojuicesproducedfromotherberries.

Classification of adulterated raspberry juice using ultra-fast gas chromatography

ANNARO Z AN SKA*,MARTYNALUBINSKA-SZCZYGEŁ,TOMASZDYMERSKI,JACEKNAMIESNIK



AbstractInordertoensuretheproperqualityoffoodproducts,itisimportanttodetectfoodcontaminations.Theaimofthisworkwastopresentthepossibilityofusingultra-fastgaschromatographytechniquetodetectthe adulteration of raspberry juice. The subjects were Not FromConcentrateraspberryandchokeberryjuices,aswellasmixturesofthese juices. Classification of juice samples was carried out usingunsupervised statistical method – Hierarchical Cluster Analysis.Basedontheresultsitcanbeconcludedthattheusingultra-fastgaschromatography technique coupled with Hierarchical ClusterAnalysismethodallowedtodistinguishadulteratedandunadulter-atedraspberryjuice.

Keywordselectronicnosefruitjuicesgaschromatographyhierarchicalcluster

analysis

Inordertoprovideconsumerswithsafeandhigh-qualityfoodproducts,itisextremely important that fooddoes not contain additives. In the case of fruitjuices,impuritiesmaybewater,dyesortheadditionofothercheaperjuices[8,9].Averyimportantelementofthistypeofproductsistheiraroma,whichdirectlyaffectstheflavour.Forthisreason,thegaschromatographytechniqueisusedtoevaluate the aromaprofile of raspberry juices [10].On theotherhand, liquidchromatographictechniquesarethemostcommonlyusedtodetectadulterationof these juices [11,12]. The use of thesemethods is often labour- and time-consuming.Onthisaccount,newrapidprocedurestodetectadulterationoffruitjuicesamplesaresought.

2.Experimental

2.1Samples

RaspberryandchokeberryNotFromConcentratejuiceswereobtainedatlocaldistributioncentresinGdansk.Thejuicemixtures(5/10/30/50%v/vadditionof chokeberry) were prepared immediately after their purchase. Samples of

35.0±0.1gramswerepouredinto20cm glassvialsandsealedwithacapwithasilicone-PTFEmembrane.Sampleswererefrigeratedat4 Cfor24hours.Foreachtypeofsamples,theanalyseswereperformedintenreplicates.

2.2Instrumentation

Headspaceanalysisoffruitjuicesampleswasperformedusinganultra-fastgaschromatographHeraclesII(AlphaMOS,Toulouse,France)equippedwiththeHS100autosampler(Gerstel,Mulheim,Germany).Theanalytesaretransferredinacarriergasstream(hydrogen)toasorptiontrapfilledwith10mgofTenaxTAsorbent.Then, theanalytesaredesorbedandput into twoparallel chromato-graphiccolumnswhosestationaryphasesarecharacterizedbydifferentpolarity(non-polarMTX-5andmedium-polarMXT-1701).After theelutionofanalytesfromchromatographic columns, theywere transferred to the flame ionizationdetectorsmeasuringcells(μFID).Parametersofthechromatographicsystemthatwere used are summarized in Table 1. AlphaSoft 12.4 software was used toprocessthedata.

2.3MultivariatedataanalysisDataanalysiswasperformedusingOrangeCanvasDataMiningv.3.3.9software(Bioinformatics Lab, University of Ljubljana, Slovenia). The chromatographicpeakareavalueswerenormalisedandusedasinputdataforHierarchicalClusterAnalysis.



Element Operation Parameter

AutosamplerHS-100 Incubation Incubationtemperature:40°C Incubationtime:120s Agitation Agitationspeed:500rpm

3HeraclesII Injection Samplevolume:2.5cm Injectortemperature:200°C Adsorption/desorption Trappingtemperature:40°C ofanalytes Trappingduration:30s Analysis Temperatureprogram:from40°Cwith

–1 speed4°Cmin to200°C Detectionofanalytes Detectortemperature:270°C

Table 1Ultra-fastGCparametersusedduringanalysis.

Fig. 1Thedendrogramofhierarchicalclustering(cut-offlineat7.0%oftherange).


Theaimoftheresearchwastodetermine,whetheritispossibletodistinguishbetweenadulteratedandunadulteratedsamplesofraspberryjuicebasedonitsheadspace.Thechangesinthevolatilefractionofsamplesweremonitoredusingan electronic nose device which based on the ultrafast gas chromatographytechnique.Thechromatographicpeakareasfromthedetectorsweretreatedassignalsofgassensors.Theseresultswereusedas inputdata forchemometricanalysis,namelyHierarchicalClusterAnalysis.Itisamethodofcombininggroupsofthemostcloselyrelatedsamples.Inthestudy,theEuclideandistancewasusedto determine the similarity between the objects. In addition, Ward's linkagemethodwasapplied.TheresultofHierarchicalClusterAnalysisisthedendrogram(Fig.1),whichtakesintoaccountthecutoffpoint(meandistancelessthan3.0).Inthisway,5clusterswereformed. Thecompositionofthearomafor100.0%chokeberryjuicesamples(markedas100.0)and100.0%raspberryjuicesamples(markedas0.0)formingtwosingleclusters. Subsequently two separated clusters were obtained for samples ofmixtures of raspberry and chokeberry juice containing 50.0% and 30.0% ofchokeberry juice (samples 50.0 and 30.0). However, for data for samplescontaining between 5.0% and 10.0% of chokeberry juice, the distinction isdifficultbecauseoneclusterhasbeencreatedforthisdata.ThismeansthattheHierarchicalClusterAnalysismethodmakesitpossibletodistinguishsamplesofunadulteratedraspberryjuicefromsamplesadulteratedwithchokeberryjuice,however, for some samples this method is insufficient to determine the per-centageofchokeberryjuice.

4.Conclusions

Basedontheobtainedresults,itcanbeconcluded,thatitispossibletoclassifybetween adulterated and unadulterated raspberry juice samples based on itsvolatile fraction. This suggests that the use of Hierarchical Cluster Analysismethodcouldbeused todetermine theauthenticityofNotFromConcentrateraspberryjuice.However,inordertoindicatethepercentageofthechokeberryjuice in raspberry juicewith greaterprecision, supervised statisticalmethodsshouldbeused.

References

[1] AIJNEuropeanFruitJuiceAssociation:FruitJuiceMatters2017Report.http://www.aijn.org/-files/default/aijn-fjm-report-final-digital.pdf

[2] LiuY.,HeyingE.,TanumihardjoS.A.:History,GlobalDistribution,andNutritionalImportanceofCitrusFruits.Compr.Rev.FoodSci.FoodSaf.11(2012),530–545.

[3] BorgesG.,MullenW.,CrozierA.:ComparisonofthepolyphenoliccompositionandantioxidantactivityofEuropeancommercialfruitjuices.FoodFunct.1(2010),73–81.

[4] BobinaitR.,ViskelisP.,VenskutonisP.R.:Variationoftotalphenolics,anthocyanins,ellagicacidandradicalscavengingcapacityinvariousraspberry(Rubusspp.)cultivars.FoodChem.132(2012),1495–1501.


[5] CarvalhoE.,FraserP.D.,MartensS.:Carotenoidsandtocopherolsinyellowandredraspberries.FoodChem.139(2013),744–752.

[6] Luo T., Miranda-Garcia O., Sasaki G., Shay N.F.: Consumption of a single serving of redraspberriesperdayreducesmetabolicsyndromeparametersinhigh-fatfedmice.FoodFunct.8(2017),4081–4088.

[7] HatcherK.:Theeffectofwholeredraspberryjuiceonbonedensityandbiomarkersofboneinpostmenopausalosteopenicwomen.MasterThesisatTheGraduateSchoolofTexasWoman’sUniversity2017.https://twu-ir.tdl.org/twu-ir/handle/11274/9756

[8] FryJ.,MartinG.,LeesM.:Authenticationoforangejuice.In:ProductionandPackagingofNon-CarbonatedFruitJuicesandFruitBeverages.AshurstP.(edits.).Boston,Springer1994,p.1–52.

[9] ElkinsA.,HeuserJ.,ChinH.:Detectionofadulterationinselectedfruitjuices.In:AdulterationofFruitJuiceBeverages.NagyS.,AttawayJ.,RhodesM.(edits.).NewYork,MarcelDekker1988,p.317–341.

[10] DuarteW.F.,DragoneG.,DiasD.R.,OliveiraJ.M.,TeixeiraJ.A.,SilvaJ.B.,SchwanR.F.:Fermentativebehavior of Saccharomyces strains during microvinification of raspberry juice (RubusidaeusL.).Int.J.FoodMicrobiol.143(2010),173–182.

[11] ObonJ.M.,Dıa z-GarcıaM.C.,CastellarM.R.:RedfruitjuicequalityandauthenticitycontrolbyHPLC.J.FoodCompos.Anal.24(2011),760–771.

[12] VersariA.,BiesenbruchS.,BarbantiD.,FarnellP.,GalassiS.:Effectsofpectolyticenzymesonselectedphenoliccompoundsinstrawberryandraspberryjuices.FoodRes.Int.30(1997),811–817.


1.Introduction

Sewagesludgemanagementisachallengeformunicipalwastewatertreatmentplantsintermsofsocial,ecologicalandeconomicaspects.Theirproductioninrecentyearsshowsanupwardtrend.Itisestimatedthatby2020inEurope,theamount of generated sewage sludge on a drymatter basiswill reach approx.13milliontonesDMofsludge[1].Therearemanymethodsfortheirmanagementandthequalityofstabilizedfinalproductsdependsmainlyonthecharacteristicsofthesewagesludgeproducedinthetreatmentplant.Themostcommonlyusedmethods of management include direct soil application, biological-chemicalstabilization (methane fermentation, composting) or thermal neutralization(mono-orco-incineration,gasification,pyrolysis)[2].Inthecaseofusingcompostobtained through biological stabilization, concerning legal requirements, thelimiting aspect is the content of heavy metals. Therefore, other methods ofutilizationaresought.OneofBestAvailableTechnologiesisthermalstabilization,whichallowsthesludgetobehygienizedwhilereducingtheirvolume.Theendproduct isash,whichduetoheavymetalconcentrationmaybeconsideredas

The potential of raw sewage sludge in construction industry

LESŁAWS WIERCZEK*,BARTŁOMIEJCIESLIK,PIOTRKONIECZKA


AbstractExcesssewagesludgeproducedinany,municipalorindustrialwaste-water treatment plant becomes a serious problem due to itsincreasingamount.Thisincreaseisrelated,e.g.,totheimprovementoftreatmenttechnologies.Theuseofsewagesludgeinbuildingmate-rialseliminatessomeoftheexpensiveandenergy-intensivestagesofutilization, therefore the finalproductobtained isoftenstableandsafe.Heavymetalsarenotleachoutfromobtainedmaterialswhilestrangepropertiesareconsideredsatisfying.Duetotheoccurrenceofheavymetalsinsewagesludge,theirstabilizationwithmineralandhydraulicbindersbecomesapromisingmethodofdevelopment.Thispaperpresents the resultsof researchon the contentofmetals insewage sludge from various steps of the technological processprovidedinsewagetreatmentplant.Thisisoneofthekeyparametersresponsibleforthedurabilityandstrengthofcementedproducts.

Keywordsbuildingindustryheavymetalsewagesludgesewagesludgeashstabilization


ahazardouswaste.Inaddition,theprocesscanbeenergy-consumingduetothedryingofsludgebeforebeingincinerated[3]. Indevelopedcountries,theconstructionindustryplaysasignificantrole.Theuseofrawsludgeasanadditiveformortars,concretesorceramicproducts isapro-environmental approach.On theonehand, theenvironmentallyharmfulwaste is disposed of, on the other hand, a good quality building material isproduced,savingrawmaterialatthesametime.Inaddition,suchanapproachpromotesacirculareconomy[4,5]. Theadditionofexcesssludgetorawcementandmortarproductsmaybeanalternativetotheexistingmethodsofitsmanagement.Intheliterature,onecan


Fig. 1Exampleofsewagesludgetreatmenttechnologicalline.

findapproachesusingofunhydrated sewage sludge (1–2%DM) tomortarorconcreteasasubstituteforwater.Ontheotherhand,dehydratedsludgecanbeusedasasubstituteforfineaggregates[6].However,significantpresenceofheavymetalsinsewagesludgemaynegativelyaffectthebondingreactionsofcementproducts. It is possible to use raw sewage sludge from various steps of thetechnologicalprocess.AnexampleofasewagetreatmentplantwithemergingsludgeispresentedinFig.1.Thepurposeofthisworkwastodetermineheavymetals(Cd,Cu,Ni,Pb,Zn,Cr,Mg,Mn,Fe)intheprimarysludge,dehydratedsludge,digestedsludgeandexcesssludgetocheckandcompareheavymetalcontent.Theobtaineddatacanbeusedtooptimizetheproductionprocessofcementproductsthat contain raw sewage sludge, and confirm the environmental safety ofproposedapproaches.

2.Experimental


SewagesludgewascollectedfromoneofthemunicipalsewagetreatmentplantinGdansk.Inordertodeterminethecontentofheavymetalsinvariousobtainedsludge,drymasseswereinitiallydetermined.Forthispurpose,about2gofeachsludgewasweighed,placedinaporcelaincrucibleanddriedat105°Ctoconstantweight.Eachanalysiswasdonewith3repetitions.Determinationofpseudototalmetalconcentrationconsistedinweighingabout1gofeachtypeofsedimentintoaspecialvessel(Teflonbomb)andsubjectingthemtowetpressuremineralizationusingmicrowaveenergy.TheDigestionmixturewasconcentratedwithHNO and3

HClina1:2byvolumeratio.Themineralizationwascarriedoutfor1.5hoursatamaximumtemperatureof150°C.Theobtainedsolutionswerequantitatively

3transferredinto25cm graduatedflasksandmadeuptovolumewithdeionizedwater. AnalysisofheavymetalsinobtainedsolutionswascarriedoutusingAtomicAbsorption Spectrometry with flame atomization. Calibration solutions were

–3prepared from stock solution (1000mgdm ). Depending on the determinedelement (Cd, Cu,Ni, Pb, Zn, Cr,Mg,Mn, Fe) proper calibration solutionswere

–3prepared(from0.1to5.0mgdm ).Iftheconcentrationoftheelementwasabovethe range of the calibration curves, the sample solutions were appropriatelydiluted.Eachmeasurementwasrepeated3to4timesforuncertaintycalculation.Onthebasisoftheresultsofdrymatterandtheconcentrationofheavymetals,thecontent of heavy metals in particular sewage sludge from various steps oftechnologicallinewascalculated.

2.2Instrumentation

Microwave assisted mineralization was carried out using the Anton PaarMineralizationsystem,modelMultiwaveGO.


TheAtomicAbsorptionSpectrometerfromGBCSCIENTIFICEQUIPMENTmodelSensAAwasusedtoanalyzeheavymetalsinthesamples.Hollowcathodelampsfor every element determination was also supplied by GBC SCIENTIFICEQUIPMENT.


In Table 1 the results of determination of selected metals are presented.ConcentrationsofmetalssuchasCu,Cr,PborNidonotdiffersignificantly.Theconcentrations of the remaining elements vary depending on the purificationstage.Differencesincontentmayberelatedwiththeformationofstablemetal-organiccompounds,whichcanbebuilt-insewagesludgebiotatissues.Mentionedcompoundsaredegradedduringmineralization.Inaddition,unitprocessesandoperationsprecededindifferentstepsofthetreatmentplanttechnologicallinecausesthesludgetodiffersignificantly,whichcanalsoaffectthedifferencesinmetalcontent.ThisismostevidentinthecaseofFeandMg.FeandAlsaltsareoftenusedascoagulants,thankstowhichdewateringofsewagesludgeiseasier.They are also used to remove phosphates fromwastewater, as well as otheralkaline salts (Ca(OH) ), which simultaneously promotes the precipitation of2

Mg(OH) [6].Moreover,mentioned reagentsmay be addedduring, previously2

mentioned,differentstepsofsludgetreatment. ThepresenceofFeintheendproductisundesirable,duetothefactitmayadverselyaffectthepozzolanicactivityofmortarorconcreatewithadditives(e.g.,coalburnash)[7].Thebiggestproblemofsewagesludgeinbuildingindustryuseisthefact,thattheFebasedsaltaremostcommonlyusedprecipitatingreagent.Alternatively used Al based reagents are used seldom because of economicaspects (Al based reagents prices are significantly higher). Concentrations ofmoretoxicmetals(Cd,Ni,Pb,CrandMn)arerelativelylowandatthesametime


Element Preliminaly Dehydrated Fermented Excesssludge Limitof sludge sludge sludge sludge detection

–1 –1 –1 –1 –1 /mgkg DM /mgkg DM /mgkg DM /mgkg DM /mgkg DM

Cd 6.189±0.093 10.88±0.15 <LOD <LOD 1.8Cu 186.0±9.4 269±12 230±11 257±13 –Ni <LOD <LOD <LOD <LOD 5.0Pb <LOD <LOD <LOD <LOD 6.8Zn 129.0±3.5 1216±47 110.4±3.2 188.9±5.9 1.9Cr <LOD <LOD <LOD <LOD 24Mg 660±179 8147±1947 853±190 2395±565 –Mn <LOD 208±24 <LOD 14.3±1.2 5.8Fe 2069±224 27113±2932 2230±244 6649±938 –

Table 1Heavymetals concentration in sewage sludge collected at different step of treatment and pro-cessing.

comparablewith literature values.However, since those could cause environ-mentalhazardandpollution,theirconcentrationshavetobemonitored[8–10].

4.Conclusions

Sewagesludgecanbeconsideredasapotentiallyattractiveadditiontobuildingmaterialsmainly due to, their physicochemical properties. Themainmineralcomponentsof sewagesludge include: calcium, ironandaluminumandphos-phoruscompounds,which,intheformofoxides,areincludedincementmortarsand other commercially used building materials. Thanks to this, there is thepossibilityofpro-ecologicaluseofsewagesludge,whichisverybeneficialinthecontextofthecirculareconomy.Whendesigninganewmethodofmanagement,itis important to determine broad range of heavymetals due to their possibleimpactonbondingofcementbindersandalsoforecologicalreasons.Inaddition,suchproductsmustmeetthestrengthandleachingcriteriaincludedinlegislativestandards.Theeffectoftheadditionofrawsewagesludgeonthedurabilityandtheriskofleachingofheavymetalsfromthefinalproductsarethetargetoffutureresearch.

Acknowledgments

TheresearchwassupportedbyGdanskUniversityofTechnology,FacultyofChemistryfunds.Nospecificgrantsandfundingwereavailable.

References

[1] Mininni G., BlanchA. R., Lucena F., S. Berselli: EU policy on sewage sludge utilization andperspectivesonnewapproachesofsludgemanagement.Environ.Sci.Pollut.Res.22(2015),7361–7374.

[2] CieslikB.M.,Namiesnik J., KonieczkaP.: Reviewof sewage sludgemanagement: Standards,regulationsandanalyticalmethods.J.Clean.Prod.90(2015),1–15.

[3] LiJ.,XueQ.,FangL.,PoonC.S.:CharacteristicsandmetalleachabilityofincineratedsewagesludgeashandairpollutioncontrolresiduesfromHongKongevaluatedbydifferentmethods.WasteManag.64(2017),161–170.

[4] SmolM.,KulczyckaJ.,HenclikA.,GorazdaK.,WzorekZ.:Thepossibleuseofsewagesludgeash(SSA) in theconstruction industryasaway towardsacirculareconomy. J.Clean.Prod.95(2015),45–54

[5] SupinoS.,MalandrinoO.,TestaM.,SicaD.:SustainabilityintheEUcementindustry:TheItalianandGermanexperiences.J.Clean.Prod.112(2016),430–442.

[6] Klaczynski E.: Oczyszczalnia sciekow-chemiczne usuwanie fosforu.Wodociągi-Kanalizacja(2013),26–28.(InPolish.)

[7] Giergiczny Z.,Małolepszy J., Szwabowski J., S liwinski J.:Cementy z dodatkamimineralnymiwtechnologiibetonównowejgeneracji.Opole,GorazdzeCement2002.(InPolish.)

[8] TellaM.,DoelschE.,LetourmyP.,ChataingS.,CuoqF.,BravinM.N.,SaintMacaryH.:Investigationofpotentiallytoxicheavymetalsindifferentorganicwastesusedtofertilizemarketgardencrops.WasteManag.33(2013),184–192.

[9] S cancarJ.,MilacicR.,StrazarM.,BuricaO.:TotalmetalconcentrationsandpartitioningofCd,Cr,Cu,Fe,NiandZninsewagesludge.Sci.TotalEnviron.250(2000),9–19.

[10]AntunesE.,SchumannJ.,BrodieG.,JacobM.V.,SchneiderP.A.,Biocharproducedfrombiosolidsusingasingle-modemicrowave:Characterisationanditspotentialforphosphorusremoval.J.Environ.Manage.196(2017),119–126.


1.Introduction

Municipal services aswell as the industrial activities are themain sources ofemissionstotheatmosphereofodorouscompounds.Thedominantgroupofsuchcompoundsarevolatileorganiccompounds.Suchcompoundsarebothunplea-sant and dangerous for people. Therefore, odorous substances should beeffectivelyremovedfromair. Thereareseveralaircleaningtechnologiesdevotedtotheremovalofodorouscompounds, i.e., catalytic combustion or adsorptive and absorptive methods.Biological methods, including biofiltration, are interesting group of odorabatementtechnologies.Biofiltrationconsistsinthedecompositionofpollutantsbymeansofbacteriaandothermicrobesinhabitingtheporouspackingofafilter.Themechanismoftheprocesstakesadvantageofthediffusionofpollutantsfromgaseous phase to a liquid phase of a biofilm covering the elements of a filterpacking. The purified stream of air leaves the biofilter whilst the adsorbedpollutants undergo the process of biodegradation. As a result, pollutants

Estimation of the odour intensity of air samples undergoing biofiltration process using electronic nose and artificial neural network

BARTOSZSZULCZYN SKI*,PIOTRRYBARCZYK,JACEKGĘBICKI

DepartmentofChemicalandProcessEngineering,FacultyofChemistry,GdanskUniversityofTechnology,NarutowiczaStreet11/12,80-233Gdansk,Poland*[email protected]


AbstractBiofiltrationisoneofthetechniquesusedtoreduceodorantsintheair. It is based on the aerobic degradation of pollutants bymicroorganismslocatedinthefilterbed.Theresearchpresentsthepossibility of using the electronic nose prototype combined withartificial neural network for estimation of the odour intensity oftoluenecontaminatedairsamples.Thestudywasconductedusing3-section biotrickling filter settled with selected environmentalisolatesofCandidafungiduring21days.Asaresultofthestudies,itwasfoundthattheelectronicnoseprototypealongwiththeproposedartificialneuralnetworkcanbesuccessfullyusedtoestimateoftheodour intensity of toluene contaminated air samples undergoingbiofiltrationprocess.

Keywordsbiofiltrationbiotricklingelectronicnoseodourintensitytoluene

previouslyharmfultohumanhealtharetransformedmainlyintoCO ,waterand2

biomass,dependingonthepollutedaircomposition[1,2].Biofiltrationisahighlyefficientmethod for the treatment of large volumes of air polluted with lowconcentrations of odorants. Themost frequently used techniques to evaluateeffectivenessofbiofiltrationprocessaregaschromatographytechniques.Theyenableseparationanddeterminationofconcentrationsofindividualcomponentsofthemixture.Intermsoftheevaluationoftheodourqualityofpurification,suchinformationisnotdirectlyuseful.Therefore,electronicnoses–devicesenablingholisticanalysisofgassamples–areincreasinglyusedtoassesstheeffectivenessofbiofiltrationintermsofodourintensityreduction[3].Odourintensityisoneofthemostcommondeterminedfeatureofthesmell.Itisdefinedastheperceivedstrengthofodorsensationthatwillbetriggeredbyaspecificstimulus.Typically,intensityisassessedbysensoryanalysisusingpointscales.AnexampleofascaleaccordingtotheGermanstandardisshowninTable1. Theevaluationoftheodourintensityisalsopossiblewiththeuseofelectronicnoses – devices that are supposed to imitate the human sense of smell. Theelectronicnosesystemconsistsoffourmaincomponents: Samplingsystemwhichprovidesstableandreproduciblemeasuringcondi-

tions(temperature,humidity,gasflowvelocity)andeliminatesallundesirablefactorsthatcanaffectthesensorresponse;

Detectionsystemwhichisbuilt fromthesetofsensors locatedinthemea-suringchamber.Themostcommonlyusedtypeofsensorsarecommerciallyavailablesensorsfordetectionofvolatileorganiccompounds,e.g.,MetalOxideSensors [4].Theyshowdifferent selectivityandsensitivity,butasawhole,produceacharacteristicchemicalimageofthegasmixture(“fingerprint”);

Dataprocessingsystem; Patternrecognitionsystemwhichassignsthereceivedsetofsignalstooneof

thepatternclasses.As pattern recognition system various chemometric algorithms are used, e.g.,Principal Component Analysis, Linear Discriminant Analysis, Support VectorMachineorPartialLeastSquare.Butthemostvaluablemethodusedinthee-nosesysteminArtificialNeuralNetwork(ANN).Artificialneuralnetworksarenowconsideredthebestmethodofanalyzingdatafromartificialsenses,mainlyduetothe fact thatANNs in theirarchitectureand functioningresemble thenervoussystem in humans. The simplest, having only one neuron, ANN is called theperceptron. The main and most important element of the perceptron is the


Odourstrength OdourIntensitylevel

Notperceptible 0Veryweak 1Weak 2Distinct 3Strong 4Verystrong 5Extremelystrong 6

Table 1Odour intensity scale described in German Stan-dardVDI3940.

McCulloch-Pittsneuron,whichisasimplifiedmodelofthebiologicalnervecell.ThesimilarityintheconstructionofbothneuronsispresentedinFig.1. TheuseofANNfordataanalysisispossibleonlyafterpriorcollectionofthetrainingdataset–examplesofinputsalongwithdefined,correspondingoutputvalues.Neuralnetworklearningprocessinvolveschangingitsinternalparame-ters(weightcoefficientsandneuronactivationthresholds).Thisisdoneusingtheappropriatealgorithm,usuallylearningundersupervision.Themostfrequentlyusedalgorithmforthispurposeisthebackerrorpropagationalgorithm[5].Itsoperationconsistsinthemodificationofweightsandthresholdvaluesbasedontrainingdatainsuchawayastominimizetheerrormadebythenetworkwhileperformingitsassignedtasksforalldataincludedinthetrainingset. Thearticlepresentsestimationoftheodourintensityofairsamples(conta-minatedwithtoluene)undergoingbiofiltrationprocessusingelectronicnoseandartificialneuralnetwork.Theobtainedresultswerecomparedwiththeresultsofsensoryanalysis.

2.Experimental

2.1Biofiltrationunit

Thestudiesuseda three-sectionbiotrickling filter.The installationdiagram isshowninFig.2.Thebiofilterwas filledwith10×2.4mmRashigceramicrings(sectionA)and6×1.5mm(sectionsBandC).Thebiofilterbed,aftersterilization,


Fig. 1SimilarityofneuroncellandArtificialNeuralNetworkconstruction.

wassettledwithselectedenvironmentalisolatesofCandidafungi.Themediumwithyeast,containingK HPO ,MgSO .7H O,KH PO ,NH Clandmicroelements,in2 4 4 2 2 4 4

3avolumeof1.5dm waspouredintothebioreactorandthencirculatedfor5days3 –1(ataflowrateof50cm min ).Afterthistime,thesupplyofaircontaminatedwith

-toluenewas started.The tolueneconcentration in the inlet air streamcorresponded to theodour intensityequal to3.Every thirdday,approx.20%of thetricklingliquidvolumewaschangedtofresh.Sixgassampleswerecollectedattheinletandoutletof the installation foreachday.Sampleswerecollected to the

3Tedlarbags(withavolumeof1.5dm ).Threeofthemwereanalyzedbyanelec-tronicnose,whiletheotherthreewereanalyzedbysensoryanalysis.

2.2Sensoryanalysis

Sensoryevaluationofodorintensitywascarriedoutby4persons,selectedaccor-dingtotheproceduredescribedin[6].Eachmemberofthepanelwasresponsiblefor assigning the appropriate odour intensity value to a given sampleusing a7-stepscaledescribedinGermanStandardVDI3940(Table1).

2.3Electronicnoseanalysis

The prepared samples were analyzed using a constructed electronic noseprototype.Thedevicewasequippedwitheightmetaloxidesensorsmanufacturedby Figaro Engineering: TGS2104, TGS2106, TGS2180, TGS2600, TGS2602,TGS2201A,TGS2201BandTGS2611.


Fig. 2Biotricklingfiltersystemusedduringtheresearch.

The collected sampleswere sucked by amembrane pump into the e-nosechamberfor15seconds.Thesamplewasthenkeptinthechamberfor30seconds.The purified air was then directed into the chamber for regeneration of thesensors.Fordataanalysisthemaximumsignalvalueofeachsensorwasused. TheodourintensityofthesamplewasdeterminedusingpreviouslydesignedArtificial Neural Network (topology: 8-7-1). Architecture of the network ispresentedintheFig.3.Threelayerneuralnetworkwasdesigned.Theweightsweremodifieduntiltheerrorbetweenthemeasuredandpredictedvaluesareminimized.RStudioDesktop(v.1.0.143)softwarewasusedasthecomputationalsoftware.


Theresultsofsensoryanalysiscomparedto theelectronicnosewithartificialneuralnetworkanalysisobtainedduring21daysofbiofilter’sworkarepresentedintheFig.4.Onthebasisoftheobtainedresults,theremovalefficiency(RE)ofthetestedsampleswasdetermined,accordingtothedependence


Fig. 3ArchitectureoftheArtificialNeuralNetwork.

Fig. 4Changesintheremovalefficiencyoftolueneasafunctionoftheduration.

(1)

where:OI areodourintensitiesattheinlet,andOI areodourintensitiesatthein out

outletofthebiofiltrationsystem. TheresultsobtainedusingtheelectronicnosecombinedwithArtificialNeuralNetworkarecharacterizedbya largeconvergencewith theresultsof sensoryanalysis.Inmostcases,theyareoverestimated,soremovalefficiencyislower.

4.Conclusions

Asaresultofthestudies,itwasfoundthattheelectronicnoseprototypealongwiththeproposedArtificialNeuralNetworkcanbesuccessfullyusedtoestimateof the odour intensity of toluene contaminated air samples undergoingbiofiltrationprocess.Theresearchhasshownthattheuseofe-nosesinsteadofsensoryanalysisispossiblewhichisadvantageousduetosignificantlyshortertimeandcostsofasingleanalysis.Inaddition,itisalsopossibletoworkthistypeofdevicesinon-linemode,whichcanalsobeusedforcontinuousmonitoringandcontrol of the air biofiltration process, which is now increasingly useddeodorizationmethod.

Acknowledgments

TheinvestigationswerefinanciallysupportedbytheGrantNo.UMO-2015/19/B/ST4/02722fromtheNationalScienceCentre(Poland).

References

[1]ChengY.,HeH.,YangCh.,ZengG.,LiX.,ChenH.,YuG.:Challengesandsolutionsforbiofiltrationofhydrophobicvolatilecompounds.Biotechnol.Adv.34(2016),1091–1102.

[2]SchiavonM.,RagazziM.,RadaE.C.,TorrettaV.:Airpollutioncontrolthroughbiotricklingfilters:areviewconsideringoperationalaspectsandexpectedperformance.Crit.Rev.Biotechnol.36(2016),1143–1155.

[3]SzulczynskiB.,NamiesnikJ.,GębickiJ.:Monitoringandefficiencyofbiofilterairdeodorizationusingelectronicnoseprototype.Chem.Pap.72(2018),527–532.

[4]Szulczynski B., Gębicki J.: Currently commercially available chemical sensors employed fordetectionofvolatileorganiccompoundsinoutdoorandindoorair.Environments4(2017),21.

[5]RumelhartD.E.,HintonG.E.,WilliamsR.J.:Learningrepresentationsbyback-propagatingerrors.Nature323(1986),533–536.

[6]GębickiJ.,DymerskiT.,RutkowskiSz.:Identificationofodorofvolatileorganiccompoundsusingclassical sensory analysis and electronic nose technique. Environ. Prot. Eng. 40 (2014),103–116.


1.Introduction

Solidagogigantea(commonlyknownasgoldenrod)iswidelyspreadinPolandandhastraditionalusageinthedietandasamedicinalplant.GenusofSolidagoincludesover100speciesmainlyinhabitingAmerica,AsiaandEurope.Twoofthemostpopularspecies:S.canadensisandS.giganteaareveryinvasiveandnowconsidered among the most aggressive plant in Europe. Therefore, it is veryreasonabletofindadditionalapplicationforthoseplants.Alsoliteraturestudy

Supercritical carbon dioxide extraction as a crucial step in the enriching sample in desired group of bioactive compounds

a,b, b,c aOLGAWRONA *,KATARZYNARAFIN SKA ,CEZARYMOZEN SKI ,b,cBOGUSŁAWBUSZEWSKI

a NewChemicalSynthesesInstitute,Al.TysiącleciaPaństwaPolskiego,24-110Puławy,Poland *[email protected] InterdisciplinaryCentreofModernTechnologies,NicolausCopernicusUniversity,Wilenska4, 87-100Toruń,Poland

c DepartmentofEnvironmentalChemistryandBioanalytics,FacultyofChemistry, NicolausCopernicusUniversity,Gagarina7,87-100Toruń,Poland


AbstractThemaingoalofthisstudywastoobtaintheoptimalconditionsofsupercriticalcarbondioxideextractionofSolidagogigantea(golden-rod)atthequarter-technicalplant.Criterionfortheselectionofthoseconditionswas the highest amount of fatty acidsmethyl esters inobtainedextract.Fattyacids,especiallyunsaturatedfattyacids,arevaluablecompoundsduetotheirhealth-promotingproperties.Fattyacids methyl esters was determined by GC-MS. For optimizationpurpose,Box-Behnkendesignwasusedtoanalyzetheeffectsofthreeindependentprocessparameters (pressure, temperature,and flowrate of CO ) on selected criterion. Box-Behnken design allows to2analyze of obtained results by Response Surface Methodology.Asecond-order quadratic polynomial model was suitable for the

2experimental data and obtained results (R for fatty acidsmethylesterswas0.90).Therefore,theresponsesurfacemethodologycanbeapplied to optimize the supercritical carbon dioxide extraction ofSolidagogigantea.ResponseSurfaceMethodologyresultsandANOVAindicate that the highest amount of desired group is achieved by

–1extractingat318K,35MPaandtheflowofCO 6.3kgh .2

KeywordscarbondioxidefattyacidsGC-MSgoldenrod(Solidago

giganteaL.)isolationresponsesurface

methodologysupercriticalfluids

extraction

had shown that preparations obtained from goldenrods have a diuretic,spasmolytic, hypotensive, anti-inflammatory, bacteriostatic and analgesicproperties[1–4].Allofthepropertiesofthegoldenrodpreparationarearesultsoftheir composition. Goldenrods are rich in secondary metabolites: flavonoids,monoterpenes, diterpenes (clerodane-type), saponins and different nitrogen-containingcompound[5,6]. Supercriticalfluidextraction(SFE)isagreentechnology,providingefficientisolation of valuable components from plant materials. Supercritical fluidextractionoffersseveraladvantagesoverconventionalsolventextraction,renderhigherselectivityandshorterextractiontime.Thequalitativeandquantitativecompositionofthefinalextractisdeterminedbythephysicochemicalpropertiesofthesolventandparametersoftheprocess.Hencethenecessitytooptimizetheprocessconditionsforindividualplantmaterials. Samplepreparationisacrucialfirststepintheanalyticalchemistrywhichmaycauseanerrorsinfurtheranalysis.Attheisolationstage(SFE)itispossibletocontrol thecompositionof thesampleandenrich itwiththedesiredgroupofcompounds.Asa resultofSFE, freeof contaminationandenrichedproduct isobtainedandsamplecanbeanalyzeddirectlybydissolutionoftheextract.

2.Experimental

2.1Chemicalsandreagents

Allchemicalsandreagentswereofanalyticalgradeandwerepurchased fromSigmaAldrich,Germany.

2.2Plantmaterial

SolidagogiganteausedinthisstudywereharvestedinChocen,Poland.Golden-rodsweredriedandgroundinto2–3cmpieces.

2.3Experimentalprogram

In our case, Box-Behnken design was used to analyze the effects of threeindependent variables on selected criterion. Complete design consisted of 15experimental steps at the different conditions (Table 1). Three independent

–1variableswere:temperature(K)pressure(MPa)andsolventflowrate(kgh ).Toevaluate the effect of those factors, fatty acids methyl esters (FAME) weredetermined. All the results and statistical analysis were accomplished usingDesignExpert9.0.Optimalextractionconditionsweredeterminedbasedontheconcentrationonselectedcompoundsasaresponse.


2.4Extractionoftheplantmaterial

Extractions were carried out at the quarter-technical plant placed in NewChemicalSynthesesInstituteinPuławy,accordingtotheBox-Benhkendesign(theprocessparametersareincludedintheTable1).Briefly,150gofplantmaterialwere loaded into the 1L extraction basket (vessel).When equilibrium wasreached,CO wasfedtotheextractorthroughahighpressurepump.Theextract2

ladenCO was sent to a separator.At reducedT andP conditions, the extract2

precipitatedintheseparator,whileCO wasrecycledtotheextractor.Attheend,2

afterfinishingtheextraction,theextractcontainedintheseparatorwascarefullycollectedinacontainerandtightly-closed.

2.5Determinationoffattyacidsmethylesters

Qualitativeandquantitativeidentificationoffattyacidsmethylesters(FAME)inobtainedextractofSolidagogiganteawascarriedoutusingaTraceGCUltragaschromatograph coupled with Thermo Scientific TSQ Quantum XLS massspectrometer.10mgofextractwasweighed,500μLoft-butylmethyletherand250μLofTMSHwereadded,andthemixerwasplacedinmagneticstirrerfor15minutesat40°C.Thepreparedsamplewasleftfor30minutestoestablishtheequilibrium.Afterthesettime,thesamplewasanalyzed.TheanalysiswascarriedoutontheTR-FAMEpolarcolumn(30m×0.25mm×0.25um).Otheroperatingconditions of the gas chromatograph: column temperature control: 90°C for

–11min,thenheatedto140°Catarate4°Cmin ,holdupat140°Cfor5min.,then –1heated to180°Cata rate2°Cmin , isothermalat180°C fora5minutes,and –1heatedto220°Catarate10°Cmin andmaintainedat220°Cfor2min;carrier


–1 –1 T/K P/MPa S/kgh FAME/mgg DM

NwOE1 333.15 80.00 7.00 22.37NwOE2 353.15 80.00 5.00 41.31NwOE3 353.15 20.00 5.00 36.23NwOE4 313.15 50.00 3.00 114.13NwOE5 333.15 50.00 5.00 102.43NwOE6 353.15 50.00 3.00 95.77NwOE7 333.15 50.00 5.00 48.42NwOE8 333.15 50.00 5.00 72.11NwOE9 313.15 80.00 5.00 157.25NwOE10 333.15 20.00 3.00 101.21NwOE11 313.15 20.00 5.00 211.94NwOE12 313.15 50.00 7.00 217.16NwOE13 333.15 80.00 3.00 134.24NwOE14 333.15 20.00 7.00 157.22NwOE15 353.15 50.00 7.00 56.91

Table 1ResultsofBox–BehnkendesignforthesupercriticalcarbondioxideextractionofSolidagogigantea.

–1gasflow1.1mLmin ; split 100:1; injectionvolume 1μL;electronenergy70eV;ionsourcetemperature230°C;temperatureofsampleinlet230°C. ThesamplecomponentswereidentifiedbycomparingobtainedresultstomassspectrafromNISTmassdatabase.Thequantitativeanalysiswasmadeonthebasisofthepreviouslypreparedcurvesforthefivemethylestersfattyacidsstandard.


GC-MSisveryusefultechniqueforseparationandidentificationofcompoundsextractedfromcomplexmatrix.Asaresultofqualitativeanalysis,bycomparingobtainedresultstomassspectrafromNISTmassdatabase,palmitic,oleic,stearic,linoleic and α-linolenic acidsmethyl esters were identified (with the highestaccuracy,over99.9%)(Fig.1). Thequantitativeanalysiswasmadeon thebasisof thepreviouslypreparedcurvesforthosefivemethylestersfattyacidsstandard.TheresultsarelistedontheTable1.Theconcentrationofthosecompoundishighdespitethefactthatgoldenrodisnon-oilyplantmaterial).Thisisduetothefactthatfattyacidsarenon-polar compounds,which are very easily extractable by non-polar carbondioxide. Box-Behnkendesign allows analyzing obtained results byResponse SurfaceMethodology(RSM).RSMisastatisticaltoolthatcanbeusedtoevaluatetheeffect(correlation) between responses and independent variables as well as theirinteractions which allows finding the levels of input variables (P, T, S) thatoptimize a particular response of a extraction process. The obtained results


Fig. 1MSmassspectrumofSolidagogiganteaextract:palmiticacidmethylester(t = 15.56min),r stearicacidmethylester(t =20.56min),oleicacidmethylester(t =21.23min),linoleicacidmethylr r

ester(t =22.79min),andα-linolenicacidmethylester(t =24.78min).r r

showedtheinfluenceoftheprocessparametersonconcentrationoffattyacidsmethylesters(Fig.2–4).Alloftheprocessparametershaveagreatimpactontheresponse;theslopesoftheresponsesurfaceatP,T,Saresignificant. Regressioncoefficientof0.90indicatesthattheadoptedmodelexplains96%the dependence of responses on input variables. High values of regressioncoefficientandtheadjustedcoefficientprovetheaccuracyoftheadoptedmodel.Non-statistically significant lack of fit and statistically significant p-value testadmitthatthemodeldescribeswellthedependenceofinputandoutputvariables(Table2).


Fig. 2DependenceontheconcentrationofFAMEinextractonthepressureandtemperature.

Fig. 3 DependenceontheconcentrationofFAMEinextractontheflowrateofcarbondioxideandtemperature.

Fig. 4 DependenceontheconcentrationofFAMEinextractontheflowrateofcarbondioxideandpressure.

As a result of process optimization, by using the analysis of variation, forindividual input variables the following optimal parameters were obtained:

–1318K,35MPaandtheflow6.3kgh .Inordertoverifythecorrectnessoftheadoptedmodel,extractionsinoptimalparameterswerecarriedout.Asresults,we

–1obtained the concentration of 218 mg FAMEg DM, which was the highestobtainedvalueandwhichwaswithintheconfidenceinterval.

4.Conclusion

Ssupercritical fluid extraction canbe considered as an analyticalmethod thatprovides a very pure and rich sample for further determinations. To produceahighqualityextractforfurtherapplication,supercriticalcarbondioxideextr-action of Solidago gigantea was developed. Our studies showed that the bestconditionsoftheextractionoffattyacidsmethylesterswere318K,35MPaand

–1theflow6.3kgh whichindicatedthehighestamountofFAME.

Acknowledgments

ThisstudywassupportedbyPLANTARUMprojectNo.BIOSTRATEG2/298205/9/NCBR/2016fromNationalCentreforResearchandDevelopment,Poland.


2RegressioncoefficientR 0.902AdjustedR 0.72

Lackoffit(LOF) 1.70p-value 0.0443

Table 2Valuesofimportantstatisticalparametersoftheadopted

2model for obtained results (critical values: R > 0.8;p<0.0001veryhighlysignificant,p<0.01verysignificant,p < 0.05 significant, p > 0.1 not statistically significant;LOF>0.05).

References

[1] Radusiene J.,MarskaM., IvanauskasL., JakstasV.,KarpavicieneB.:Assessmentofphenoliccompoundaccumulationintwowidespreadgoldenrods.Ind.CropsProd.63(2015),158–166.

[2] PaunG.,NeaguE.,AlbuC.,RaduG.L.:VerbascumphlomoidesandSolidagovirgaureaeherbsasnaturalsourceforpreventingneurodegenerativediseases.J.Herb.Med.6(2016),180–186.

[3] AmtmannM.:Thechemicalrelationshipbetweenthescentfeaturesofgoldenrod(SolidagocanadensisL.)floweranditsunifloralhoney.J.FoodCompost.Anal.23(2010),122–129.

[4] KundelD.,vanKleunenM.,DawsonW.:InvasionbySolidagospecieshaslimitedimpactsonsoilseedbankcommunities.BasicAppl.Ecol.15(2014),573–580.

[5] WeberE., JakobsG.:Biological flora of central Europe: Solidago giganteaAiton.Flora200(2005),109–118.

[6] HendersonM.S.,McCrindleR.,McMasterD.:ConstituentsofSolidagospecies.PartV.Non-acidicditerpenoidsformSolidagogiganteanvar.serotina.Can.J.Chem.51(1973),1346–1358.


1.Introduction

Despitetheemergenceofnewdrugsandtherapeuticapproachesinthefieldofoncology, theperformance indicatorsofantitumortreatmentofnon-smallcelllungcancerremainlow.Oneofthereasonsthatnon-smallcelllungcancerissohardtotreatisthatinthelatestagesmalignantcellsdevelopnovelproperties,suchastheavoidanceofimmunologicalsurveillance[1]. Increasedproductionofnitricoxidehasbeenimplicatedinthedevelopmentofmalignancy[2].Thedevelopmentofasensitiveandselectivemethodologyforthedeterminationnitricoxideconcentrationsdirectlyinbiologicalsystemsrequiresisrequiredtounderstanditsroleinthepathogenesisofmalignanttumors.Manypapersinthisfieldhavebeenpublished,yet,thereisstillnodevelopedtestsystemfordeterminingnitricoxide(II)inbiologicalfluidsandtheproblemisstillrele-vant[3].

2.Experimental


All solutions were prepared with nanopure water. Britton-Robinson buffer–1solutionwaspreparedbymixingof0.2molL sodiumhydroxidewiththemixture

–1of0.04molL ofboric,aceticandphosphoricacid.

Development of a voltammetric method for detection of ethyl nitrite

VALENTINAPOPOVA*,ANNAKRIVOSHEINA,ELENAKOROTKOVA

ChemicalEngineeringDepartment,NationalResearchTomskPolytechnicUniversity,30LeninAvenue,634050Tomsk,Russia*[email protected]

AbstractA simple and sensitive voltammetric method was developed todetermineofethylnitriteatgraphiteelectrodeinBritton-Robinsonbuffer solutionwithapH=4.02. Surfaceof graphiteelectrodewasmodifiedwithcarbonink.Theethylnitritewaspre-accumulatedontheelectrodesurfaceat+0.4Vfor4s.Awell-definedoxidationpeakwasobtainedat0.9V.Anodicvoltammetryindifferentialmodewas

−7 −1appliedforthecalibrationplotanddetectionlimit(3.8×10 molL ).

Keywordscarboninkethylnitritegraphiteelectrodevoltammetry


Formodification of the electrode surface the carbon inkwas prepared bymixing0.01gofpolystyrene,0.09gofcarbonpowder(particlesizeof3.5–5.5μmAldrich)and0.5mLof1,2-dichloroethane(99.97%,Aldrich).

2.2.Instrumentation

Allmeasurementswere carriedout using analyzerTA-2 (Tomsk,Russia). Thethree-electrode electrochemical cell was equipped with the: Ag/AgCl 1M KClreference and auxiliary electrode, graphite electrode and carbon-containingworking electrode. The pH was measured with a pH-meter/ionomer ITAN,Tomanalyt,Russia.


Firstly, theoptimalpH forethylnitritedeterminationwas found.TheBritton-RobinsonbuffersolutioninthepHrangefrom2.4to9.1werecontrolled.Thehighest and the best developed peak was obtained in the pH=4.02. Britton-RobinsonbufferpH=4.02waschosenastheoptimalmedium. Moreover, two types of material of working electrodes for ethyl nitritedetermination were studied: graphite electrode; carbon-containing workingelectrode with a renewable surface. Measurements was carried out in thepotentialrangefrom+0.4to+1.4V. Ethylnitritegiveswelldevelopedpeakinpotentialrangebetween1.15Vand1.4 V at graphite electrode (E = 1.2 V). On the carbon-containing workingp

electrodecarbon-containingworkingelectrodeelectrochemicalsignalwasnotobserved(Fig.1). Toincreasethesensitivitythesurfaceofelectrodesweremodifiedbycarbonink.Themodifierwaspreparedaccordingtotheproceduredescribedabove.Theefficiencyofthemodificationwasevaluatedonastandardoxidation-reduction

3– 4–pair [Fe(CN) ] /[Fe(CN) ] .Thehighestandthebestdevelopedpeakofethyl6 6


Fig. 1Anodic voltammograms of C H ONO at (1) graphite electrode, or (2) carbon-containing2 5

workingelectrodeinBritton-Robinsonbuffersolution(pH=4.02)(––––).Backgroundelectrolyte–4 –1 –1(---).Conditions:c(C H ONO)=7×10 molL ,scanrate90mVs .2 5

nitritewasobtainedonelectrodesaftertheirmodificationbycarbonink(Fig.2).PeakofethylnitriteoxidationwasobtainedattheE =1.0Vongraphiteelectrode,p

and at the E = 1.13 V on carbon-containing working electrode. For futurep

measurementsgraphiteelectrodewaschosenasworkingelectrode. Theinfluenceofpotentialandtimepre-accumulationhavebeentested.Optimalworking conditions are: potential pre-accumulation: +0.4 V; time pre-accumulation:4s. Thedependenceofthepeakcurrentontheconcentrationofethylnitritewas

–1linearintherangefrom1to10μmolL withtheregressionequation

–1 I [µA]=0.0786 с[mgL ]–0.0537 (1)2 R =0.9959

Thedetection limitcalculatedbyequationLOD=3s/b,wheres is thestandarddeviationinthemeasurementofthesignalofblanksample;bistheinstrumentalsensitivityfactoroftheslopeofthestraightsectionofthecalibrationcurve,was

–7 –13.8×10 molL .

4.Conclusions

Inthisresearch,electrochemicalsignalfromethylnitriteatdifferentelectrodesurfaceswascontrolled.Optimalworkingconditionswerefound.ThesestudieswillbeusedtoevaluateNOmetabolitesinbiologicalobjectstodeterminetheiractivityinhumanandanimalcancercells.

Acknowledgments

TheauthorsthankTomskPolytechnicUniversityforfinancialsupportofthiswork(RussianFundforBasicResearch,researchprojectNo.4.5752.2017/BP,RussianStateassignment“Science”).


Fig. 2Anodic voltammograms of C H ONO on (1) graphite electrode, or (2) carbon-containing2 5

working electrode in Britton-Robinson buffer solution (pH = 4.02) after modification (––––).–4 –1 –1Backgroundelectrolyte(---).Conditions:c(C H ONO)=7×10 molL ,scanrate90mVs .2 5

References

[1] Janakiram N.B., Rao C.V.: iNOS-selective inhibitors for cancer prevention: promise andprogress.FutureMedChem.17(2012),2193–2204.

[2] GowA.J.,DavisC.W.,MunsonD.:ImmunohistochemicaldetectionofS-nitrosylatedproteins.MethodsMol.Biol.279(2004),167–172.

[3] Ischiropoulos H., Gow A.: Pathophysiological functions of nitric oxide-mediated proteinmodifications.Toxicology208(2005),299–303.


1.Introduction

In the past decade, electrochemical immunoassays have become an attractiveoptionforhigh-throughputanalysiscombinedwithadvantagesofeasyhandling,enhancedsensitivity,highselectivity,andrapidityofdatacollection[1–3].Themainideaofthisresearchistodevelopanelectrochemicalimmunosensorforthequantitativedetectionofantibodiesagainsttick-borneencephalitis.Tick-borneencephalitisvirusisoneoftheendemicflavivirusesinRussia,whichcancauseseriousinfectionsinhumansthatmayresultinencephalitis/meningoencephalitis[4].Inthiswork,silvernanoparticles(SNPs)wereusedasdirectsignalingmar-kersfortheantibodydetection,andtheirsignalwasrecordedbyvoltammetry.Such types of electrochemical immunosensors based on a signal from metalnanoparticlesrepresentanupcomingtrendinanalyticalchemistry[5,6].

Control of electrochemical signal from silver nanoparticles at different modification steps for electrochemical immunosensor development

a,b,c, a bYEKATERINAKHRISTUNOVA *,JIR IBAREK ,BOHUMILKRATOCHVIL ,a c cVLASTIMILVYSKOCIL ,ELENAKOROTKOVA ,ELENADOROZHKO

a UNESCOLaboratoryofEnvironmentalElectrochemistry,DepartmentofAnalyticalChemistry,FacultyofScience,CharlesUniversity,Hlavova8,12843Prague2,CzechRepublic*[email protected]

b DepartmentofSolidStateChemistry,UniversityofChemistryandTechnology,Prague, Technická5,16628Prague6,CzechRepublicc NationalResearchTomskPolytechnicUniversity,LeninAvenue30,634050Tomsk,Russia

AbstractDevelopmentofelectrochemical immunosensorstowardsantibodydetection consists of several steps. Specific attention was paid toexclude the non-specific electrochemical signal from silver nano-particles at different modification steps. A tick-borne encephalitisvirus antigen was attached to glutaraldehyde on a glassy carbonelectrode modified with gold nanoparticles and cysteamine. Cyclicvoltammetricstudiesdemonstratetheelectrochemicalsituationon

II/IIIthe electrode surface by electron transfer of Fe as a probe.Detection of the SNPs was performed by anodic stripping voltam-

+metryofAg attheglassycarbonelectrode.

Keywordselectrochemical

immunosensorsilvernanoparticlestick-borneencephalitis


Preparation of the electrochemical immunosensor is performed in severalsteps:i)immobilizationoftick-borneencephalitisvirusantigenontheelectrodesurface; ii) production of silver nanoparticle–antibody (against tick-borneencephalitisvirus)conjugates;iii)incubationoftheelectrodewiththeantigeninthe silver nanoparticle–antibody bioconjugate solution; iv) silver dissolutionfromthesurfaceoftheelectrochemicalimmunosensor;v)recordingofvoltam-mogramscorrespondingtotheoxidationofcathodicallypre-accumulatedsilveronthebareglassycarbonelectrode(GCE).Immobilizationoftheantigenontheelectrode surface is one of the most significant preparation steps, and it iscritically importanttocontrol theelectrochemicalsignal fromSNPsaftereachmodificationstep.Resultsofthisresearchareimportantforunderstandingthenature of SNP signals and could help avoiding questions about non-specificinteractionsofSNPs.

2.Experimental


HAuCl ·3H O (99.99%), NaBH (99%), AgNO (99.99%), cysteamine (95%),4 2 4 3

glutaraldehyde solution (25% in H O), K [Fe(CN) ]·3H O (99%), KNO , HNO 2 4 2 2 3 3

(65%)wereobtainedinanalyticalgradepurityfromSigma-Aldrich,Germany.Anantigenagainsttick-borneencephalitisviruswassuppliedbyVector-Best,Novo-sibirsk, Russia. All solutions were prepared with nanopure (deionized) water(18MΩcm).

2.2Instrumentation

Voltammetricmeasurementswerecarriedoutinathree-electrodesystemwithaGCEasaworkingelectrode(3mmdiameter,Metrohm,Switzerland),anauxiliaryplatinumwireelectrode(Eco-TrendPlus,CzechRepublic),andaAg|AgCl(3MKCl,Elektrochemicke detektory, Czech Republic) reference electrode. The GCE waspolishedpriortomeasurementswithaqueousslurryofaluminapowder(1.1μm)tomirror-likeappearance.Linear-sweepASVwascarriedoutusingscanrateof

–10.1Vs ,potentialscanrangefrom–0.2to+0.6V,accumulationpotentialof–0.8V,andaccumulationtimeof60s.CVmeasurementswerecarriedoutfrom–0.9to

–1+1V at scan rate of 0.05 V s . ASV and CV were carried out onμAutolab III(Metrohm)controlledbyNova1.11software(Metrohm).


Intheinitialresearchstage,sphericallyshapedSNPs(5.3±1.2nminsize)weresynthesized by the method of Mulfinger and Solomon [7]. In the UV/Visabsorptionspectra(Fig.1),themaximumabsorptionofSNPsisintherangeof


395–400nm, which is in accordance with the average SNP size of 5.3±1.2nmcalculatedfromthetransmissionelectronmicroscopic(TEM)observations. Afterwards, an aliquot (2 mL) of silver colloid solution was centrifuged at8,000rpm for 10 min. The pellet was collected and resuspended in deionizedwater. Preparation of electrochemical immunosensor was as follows. The GCE waschosenasaplatformforantigenimmobilization.Firstly,goldnanoparticlesweredeposited electrochemically on the surface of the GCE [8]. During the secondstage,thethiolationofAu-GCEsurfacewasperformedbydippingitinto2mLof

–1acysteamine solution (0.05 mol L ) for 2 hours at room temperature. Afterrinsingtheelectrodewithdeionizedwater,theelectrodewasplacedintoaglutar-aldehydesolution(2.5%)for45minutesatroomtemperature.Afterwards,theelectrode was rinsed with a phosphate buffer (pH=7.4) three times, and theantigenwasthenimmobilizedontheelectrodesurface.Theelectrodeincubationtimewas1houratroomtemperature. ElectrochemicalsignalsfromSNPswerecheckedaftereachmodificationstep.The electrode was immersed into the SNP solution for 30 min. Chemical

–1dissolutionofsilverfromtheelectrodesurfacewith1molL HNO followedby3

ASVattheGCEwasfoundasasuitabletechniqueforthedeterminationoftheSNPs.Infurtherinvestigations,boththeelectrodesurfaceandthesolutionwerecontrolledbecauseofthepossibilityoftheSNPresiduesremainingonthesurface.Electrochemical situation on the surface of variously modified electrodes

II/IIIdemonstratedbyCVofFe asaprobeisshowninFig.2.Theobtainedresultsshow that the surface of the electrode was successfully modified. This can beobservedviaincreasingthepotentialdifferencebetweentheanodicandcathodicpeak,whichindicatesformationofthenextlayer,leadingtotheinhibitionoftheelectrontransfer.

+ DetectionoftheSNPswasperformedbyASVofAg attheGCE.OurstudieshaveshownthatafterthemodificationoftheAu-GCEwithcysteamine,signalsfrom


Fig. 1UV/Vis absorption spectra of clearyellow colloidal Ag (SNPs), optical pathlengthof1.0cm,blank–deionizedwater.

SNPswererecorded.Themoleculeofcysteaminecontainsan–NH groupwhich2

canreactwiththeSNPs.Uponthenextmodificationstagewithglutaraldehyde,nosignalsfromSNPswereobserved.ThisexcludesthepossibilityofSNPpenetrationtothecysteaminelayerandavoidsquestionsaboutnon-specificinteractionsofSNPs. Moreover, in the case of the subsequent modification with the antigen,signalsfromSNPswererecordedagain.Thisproblemcouldbesolvedwithintheconstructionofthefutureimmunosensor,whereantigenfirstlybindstounlabeledantibodiesandthewholeelectrodesurfaceshould,moreover,beblockedwithaprotein–thus,therewouldbenopossibilityofnon-specificbindingoffreeSNPs.

4.Conclusions

Inthisresearch,electrochemicalsignalfromSNPsatdifferentmodificationstepswas controlled. The obtained data showed that after all steps of modification,there is no possible binding of free SNPs through the individual modificationlayers.ResultofthisresearchareequallyimportantforunderstandingthenatureofSNPsignalandforconfirmationthatthefinalsignalisreceivedasaresultoftheantigen–antibody(againsttick-borneencephalitisvirus)interaction.


4− –1Fig. 2 Cyclic voltammograms of [Fe(CN) ] (c= 10 mmol L ) at different electrode surfaces in6–1 –10.1molL KClsolution,Evs.Ag|AgCl,scanrate0.05Vs .

Acknowledgments

This work was financially supported by the Grant Agency of the Czech Republic (ProjectP206/12/G151).TheauthorsalsothankTomskPolytechnicUniversityforfinancialsupportofthiswork(RussianFundforBasicResearch;ResearchProjectNo.4.5752.2017/BP).Thisresearchwasalso supported by the project „Research in Pharmaceuticals and Biomaterials“ (grant No.A1_FCHT_2017_004).

References

[1] ZhangK.,LvS.,LinZ.,LiM.,TangD.:Bio-bar-code-basedphotoelectrochemicalimmunoassayfor sensitive detection of prostate-specific antigen using rolling circle amplification andenzymaticbiocatalyticprecipitation.Biosens.Bioelectron.101(2018),159–166.

[2] Wang R., Liu W.D., Wang A.J., Xue Y., Wu L., Feng J.J.: A new label-free electrochemicalimmunosensor based on dendriticcore-shell AuPd@Au nanocrystals for highly sensitivedetectionofprostatespecificantigen.Biosens.Bioelectron.99(2018),458–463.

[3] Wan Y., ZhouY.G., PoudinehM.,Safaei T.S., MohamadiR.M., Sargent E.H., Kelley S.O.: Highlyspecific electrochemical analysis of cancer cells using multi-nanoparticle labelling.Angew.Chem.Int.Ed.53(2014),13145–13149.

[4] KhasnatinovM.,DanchinovaG.,LiapunovA.,ManzarovaE.,PetrovaI.,LiapunovаN.,SolovarovI.: Prevalence of tick-borne pathogens in hard ticks that attacked human hosts in EasternSiberia.Int.J.Biomed.7(2017),307–309.

[5] HaoaN.,LiH.,LongY.,ZhangbL.,ZhaoaX.,XuaD.,ChenaH.Y.:Anelectrochemicalimmuno-sensingmethodbasedonsilvernanoparticles.J.Electroanal.Chem.656(2011),50–54.

[6] Perez-Lopez B., Merkoci A.: Nanoparticles for the development of improved (bio)sensingsystems.Anal.Bioanal.Chem.399(2011),1577–1590.

[7] MulfingerL.,SolomonS.D.,BahadoryM.:Synthesisandstudyofsilvernanoparticle.J.Chem.Educ.84(2007),322–325.

[8] NoskovaG.N.,ZakharovaE.A.,ChernovV.I.:Fabricationandapplicationgoldmicroelectrodeensemblebasedoncarbonblack-polyethylenecompositeelectrode.Anal.Methods3(2011),1130–1135.


1.Introduction

Hydrophilicinteractionchromatographybelongstomorerecentchromatographymethods.CurrentlyitiscommonlyusedforseparationofverypolarcompoundswhichprovidesagoodalternativetoNP-HPLCandRP-HPLC.StationaryphasesaresimilartothoseusedinusinginRP-HPLCandmobilephasesaresimilartothoseusedinNP-HPLC.Amechanismofseparationisverycomplexconsistingofhydrophilic partitioning, adsorbtion, ionic interactions and hydrophobicinteraction. [1–6]Theprocessofseparationalsodependsonamountofwatercontent in mobile phase. Lesser the water content in mobile phase, lesserhydrophilicinteractionsandotherphenomenonparticipatesonseparation. Chaotropicsaltsaresubstances(Hofmeisterserieofsalts)whichareusingforpeptide analysis and have “salting in” properties. There are many theoriesexplainingthemechanismbehindeffectofchaotropicsalts inmobilephaseofseparationinRP-mode.Firstoneisanon-specific“ionassociation”model[7,8].Chaotropic additives are less polar thenwater and destroy hydrogen bridgeswhile thehydrophobicity increases. Second theory says thatmechanism is ananalogyof“dynamicion-exchange”[9].Large,poorlyhydratedanionsofchao-tropic salts penetrate deeper into the non-polar stationary phase and createchargedsurfaceofion-exchangeproperties.

Effect of chaotropic salts addition into mobile phases on separation of model analytes on polar stationary phases in hydrophilic interaction chromatography

a, bJANASMOLEJOVA *,MICHALDOUSA

a DepartmentofAnalyticalChemistry,FacultyofScience,CharlesUniversity, Hlavova8,12843Prague2,CzechRepublic*[email protected] ZentivaGroupa.s.,UKabelovny130,10237,Prague10,CzechRepublic


AbstractEffectofadditionchaotropicsalts into themobilephasesonsepa-rationinhydrophilicinteractionchromatographywasstudied.Fourcolumns,twelvemodellinganalytesandthirteenmobilephaseswereused.Interestingphenomenonwereobserved.Ammoniumformatebufferwithhexafluorophosphoricacidadditionhaspositiveeffectonretention behaviour of p-toluenesulfonic acid, 4-hydroxybenzene-sulfonicacid,nicotinicacidandascorbicacid.

Keywordschaotropicsaltsformicacidhexafluorophosphoric

acidHydrophilicinteraction

chromatographyTSK-amidecolumn

2.Experimental


Thiaminehydrochloride(95.7%,SigmaAldrich,USA),amproliumhydrochloride(99.4%,SigmaAldrich,USA),adenine(≥99%,SigmaAldrich,USA),guanine(98%,SigmaAldrich,USA),cytosine(≥99%,Sigma,USA),uracil(≥99%,SigmaAldrich,USA),melamine (99%, Sigma, USA), ascorbic acid (99% Sigma Aldrich, USA),nicotinicacid(99%,SigmaAldrich,USA),p-toulenesulfonicacid(p-TSA)(≥99%,SigmaAldrich,USA)and4-hydroxybenzenesulfonicacid(4-OH-BSA)(98%,Sigma

–1Aldrich,USA)wereusedassampleswhichwerepreparedas1mgmL solutiondissolvedin50%acetonitrileandthendilutedbypureacetonitrile(UltraGradient

–1HPLCGrade,J.T.Baker,Poland)to0.1mgmL solution. Thefollowingreagentswereusedforbuffers:aceticacid(100%Sigma,USA),formicacid(100%,Merck,Germany),phosphoricacid(85%,Merck,Germany),citricacid(99%,Sigma,USA),malonicacid(99%,Sigma,USA),methansulfonicacid(100%,Sigma,USA),perchloricacid(70%,Aldrich,USA),trifluoroaceticacid(≥99.5%,Sigma,USA),hexafluorophosphoricacid(55%,Aldrich,USA),ammo-niumhydroxide (25%, J.T.Baker,Poland), triethylmine (≥99.5%,Sigma,USA),tert-butylamine(≥99.5%,Sigma,USA),potassiumhydroxide(45%,Merck,Ger-

–1many).Therewerethreetypesofpreparation.First0.5Lof25mmolL aceticacid,formicacid,phosphoricacid,citricacidormalonicacidwerepreparedandthentitratedby25%ammoniumhydroxidetopH=3.5.Secondwerebufferswith

–1chaotropic salt: 0.25L of 25mmolL methansulfonic acid, perchloric acid,trifluoroacetic acidorhexafluorophosphoric acidwere titratedbyammonium

–1hydroxidetopH=3.5or6.6then0.25Lof25mmolL formicacidwasaddedandtitratedby25%ammoniumhydroxidetopH=3.5or6.6.Thirdwerebasicbuffers:

–10.5L of 25mmolL ammonium hydroxide, triethylamine, tert-butylamine orpotassiumhydroxidewere prepared and then titrated by 25% formic acid topH=3.5.

2.2Instrumentation

Allexperimentswereperformedon theWaters (USA)Aliance2695withPDA2996asadetector.Thefollowingcolumnswereused:TSKgel®Amide-803µm4.6×150mm, (Sigma Aldrich, USA), Atlantis® HILIC Silica 5µm 4.6×150mm(Waters,USA),Luna®3µmHILIC200A 4.6×150mm(Phenomenex,USA)andX-BridgeTHHILIC3.5µm4.6×150mm(Waters,USA).Flowrateofmobilephase

–1was1mLmin ,rationoforganicandinorganicpartwas80:20(v/v),injectionvolumewas5µL,columnwasthermostatedon30°Canddetectionwasat230nmformelamine,4-OH-BSA,p-TSAand260nmforthiamine,amprolium,adenine,cytosine,guanine,uracil,ascorbicacid,nicotinicacid.



TherearenotmanyworksdealingwitheffectonadditionofchaotropicsaltintothemobilephaseinconnectionwithHILICcolumns.Thereforeascreeningwasmade.Fourcolumnswereused for separationof12modellinganalytes (fromstrongacidstostrongbasis)using13differentmobilephases.Interestingresultsweresearched.Retentionofacidsoncolumnwasimportantaspectbecauseoftheirdifficultseparationonreversephases. RetentionofanalytesoncolumnTSK-amidewithcarbamoylasastationaryphasewithHCOONH andHPF asmobilephaseseemedtobemostinteresting.4 6

Changes in concentration of formic acid and HPF and in pH were made.6

Significant drift in retention times were observed only by acids therefore4-OH-BSA,p-TSA,ascorbicacidandnicotinicacidwereexamined.

–1 Experimentstookplacebyfollowingway:5,10,20and30mmolL .HPF was6–1addedto10mmolL HCOOHandtitratedby25%NH OHtopH=3.5and6.6.And4

–1 –1then5,10,20and30mmolL HCOOHwasmixedwith25mmolL HPF and6

titratedby25%NH OHtopH3.5and6.6.4

Therearetwogroupsofanalytes:strongacids(4-OH-BSA,p-TSA)andweakacids(ascorbicacid,nicotinicacid).Groupofweakacidsaremoreretainedonthecolumnandstrongeracidofonegroupismoreretainedonthecolumn. AscorbicacidisretainedmorewhenmobilephasehaspH=6.6thanwhenpHis3.5. While opposite trend was observed for all other acids. Analyte with thesmallestretentiontimesisp-TSA.Thebiggestretentionofp-TSAwasachievedby

–1 –1using10mmolL HCCONH with25mmolL HPF andpH=3.5.4 6

In case of pH=3.5 (Fig.1) increasing ionic strength of buffer decreasesretentionofascorbicandnicotinicacidbuthasnotaninfluenceonretentiontime


Fig. 1Effectofretentionfactorof4-OH-BSA,p-TSA,ascorbicacid,andnicoticicacidonconcentration–1ofHCOONH .Unchangingconditions:pH=3.5,25mmolL HPF .4 6

of4-OH-BSAandp-TSA.WhenpH=6.6(Fig.2)retentionofacidisnotchangingbecause of their pK . Interesting effect on retention was observed whena

concentration of HPF was changed. Increasing concentration of HPF causes6 6

increaseofretentiontimesforallof4acidsincaseofbothpH=3.5(Fig.3)and6.6(Fig.4).Furtherexperimentsarenecessaryandareinprogress.


Fig. 3 Effectofretentionfactorof4-OH-BSA,p-TSA,ascorbicacid,andnicoticicacidonconcentration–1ofHPF .Unchangingconditions:pH=3.5,10mmolL HCOONH .6 4

Fig. 2 Effectofretentionfactorof4-OH-BSA,p-TSA,ascorbicacid,andnicoticicacidonconcentration–1ofHCOONH .Unchangingconditions:pH=6.6,25mmolL HPF .4 6

4.Conclusions

TheeffectofpresenceofchaotropicsaltsinHILICmobilephaseswasinvestigated.AfterascreeningstudycombinationofTSK-amidecolumnwithcarbamoylasastationaryphaseandHCOONH withHPF asamobilephasewasselected for4 6

detailed study. Increasing ionic strength of buffer with pH=3.5 decreasesretention of ascorbic acid and nicotinic acid and doesn't have an impact on4-OH-BSAandp-TSA.IncreasingconcentrationofHPF6increasesretentiontimesofall4acids.

References

[1] AlpertA.J:Hydrophilic interaction chromatography for the separation of peptides, nucleicacidsandotherpolarcompounds.J.Chromatogr.A499(1990),177–196.

[2] McCalleyD.V.:Studyoftheselectivity,retentionmechanismandperformanceofalternativesilica-based stationary phases for separation of ionized solutes in hydrophilic interactionchromatography.J.Chromagr.A1217(2010),3408–3417.

[3] JinG.,GuoZ.,ZhangF.,XueX.,JinY.,LiangX.:Studyontheretentionequationinhydrophilicinteractionchromatography.Talanta76(2008),522–527.

[4] KarazapanisA.E.,FiamegosY.C.,StalikasC.D.:Studyofthebehaviorofwater-solublevitaminsinHILIConadiolcolumn.Chromatographia71(2010),751–759.

[5] McCalleyD.V.,NeueU.D.:Estimationoftheextentofthewater-richlayerassociatedwithsilicasurfaceinhydrophilicinteractionchromatography.J.Chromatogr.A1192(2008),225–229.

[6] Alpert A.J.: Electrostatic repulsion hydrophilic interaction chromatography for isocraticseparation of charged solutes and selective isolation of phosphopeptides.Anal. Chem.80(2008),62–75.

[7] LoBruttoR.,JonesA.,KazakevichY.V.:Effectofcounter-anionconcentrationonretentioninhighperformancechromatographyofprotonatedbasicanalytes.J.Chromatogr.A913(2001),189–196.

[8] Jones A., LoBrutto R., Kazakevich Y.V.: Effect of counter-anion type and on the liquidchromatographyretentionofβ-blockers.J.Chromatogr.A964(2002),179–188.


Fig. 4 Effectofretentionfactorof4-OH-BSA,p-TSA,ascorbicacid,andnicoticicacidonconcentration–1ofHPF .Unchangingconditions:pH=6.6,10mmolL HCOONH .6 4

[9]HorvathC.,MelanderW.,MolnarI.,MolnarP.:Enhancementofretentionbyion-pairformationinliquidchromatographywithnonpolarstationaryphases.Anal.Chem.49(1977),2295–2305.


Indexes

Author Index

BakhytkyzyI.188

BaluchovaS.140

BarekJ.51,82,280

BasB.37

BlaskoJ.120

BorowskaM.194

BraunP.55

BulskaE.19,25,44

BulychevaE.V.61

BuszewskiB.1,68,93,

269

BystrzanowskaM.200

CiesielskiW.77

CieslikB.258

CzaudernaM.25

DęboszM.14

DejmkovaH.82

DerinaK.134

DorozhkoE.134,280

DousaM.285

DurnerB.7

DusekM.152,181

DymerskiT.236,253

EhmannF.7

FabjanowiczM.205

FestingerN.77

FischerJ.51

GabrisovaĽ.120

GajdarJ.51

GalbavaP.120

GarwolinskaD.211

GashevskyaA.134

GaworA.25

GaworA.44

GębickiJ.:263

GlinkaM.218

GranicaM.31

GusarA.134

HaliczL.19

Hewelt-BelkaW.188,

211

IgitkhanianA.E.114,

158

JagielskaA.44

JakuczunW.44

JandovskaV.181

JankowskaK.240

KalinowskaK.224

KarapetianD.D.114,

158

KarasinskiJ.19

KempinskaD.230

KhristunovaY.280

KilianK175

KlausovaK.87

KochanaJ.37,126

KolesnichenkoI.N.114,

158

KonieczkaP.258

KonopkaA.25

KorbanA.162

KorotkovaE.61,276,

280

KoscielniakP.14

Kot-WasikA.188,194,

211,230

KrataA.A.19

KratochvılB.280

KrejcovaA.87

KrivosheinaA.276

KrolA.68,93

KubinecR.120

Kucinska-LipkaJ.194,

218

LewinskaI.145

LigorT.1

LinertW.61

Lubinska-SzczygełM.

236,253

MadejM.37

MachoO.120

Makrlık ovaA.82

MaleckovaM.105

MarcinkowskaR.246

MatysikF.M.7,55

MichalecM.145

MikulecJ.120

MilanowskiM.1

MorawskaK.77

MortetV.140

MozenskiC.269

MusilS.100

NamiesnikJ.211,224,

236,253

NavratilT.82

Nguyen-MarcinczykC.T.

19

NikolaevaA.A.61

NiznanskaZ .120

OlsovskaJ.105,152

PatockaJ.87

PawlakF.240

Proceedingsofthe14thISCModernAnalyticalChemistry Prague2018 293


PegierM.175

PlatonovI.A.114,158

Płotka-WasylkaJ.205,

224

PolkowskaZ .240

PomastowskiP.68,93

PopovaV.276

PyrzynskaK.175

PytelK.246

RablH.P.55

RafinskaK.93,269

Railean-PlugaruV.68,

93

RogowskaA.93

RozanskaA.236,253

RudnickaJ.1

RuszczynskaA.25,44

RybarczykP.263

ShikunM.169

Schwarzova-PeckovaK.

140

SmarzewskaS.77

SmolejovaJ.285

SoukalJ.100

StarzecK.126

SwierczekL.258

SzostekM.44

SzulczynskiB.263

Szultka-MłynskaM.93

TaylorA.140

TobiszewskiM.200

ToczyłowskaB.44

TorresElgueraJ.C.25

TymeckiŁ.145

VrublevskayaO.169

VrzalT.105

VyskocilV.82,280

WagnerB.44

WasikA.218

WieczorekM.14

WojciechowskiM.19

WojnowskiW.224

WronaO.269

ZabiegałaB.246

ZieminskaE.44

ZłochM.93

ZusťakovaV.152

Keyword Index

acemetacin77

adsorption175

analyticalcalibration14

antibacterialantibiotics

218

antimicrobialcoatings

194

applecider152

Arctic240

aromaproperties236

aryldiazoniumsalts134

atherosclerosis44

Bacteria240

bacterialaggregation68

beer181

biodiesel120

biofiltration263

biogenicamines224

biomarkers158

biosensors126

biotrickling263

boron-dopeddiamond

140

breathacetone158

buildingindustry258

calibrationmixtures114

capillaryelectrophoresis

68,93

carbondioxide269

carbonink276

carbonnanotubes175

carbonpasteelectrode

77

catheterization194

cellsclumping93

chaotropicsalts285

chelation19

chemiluminescence

detector105

chromato-desorption

microsystems114,

158

chromium19

citalopram37

creatinine145

currentdensity169

cyclicvoltammetry37,

140,169

DeNOxsystems55

derivatization105,218

DesignofExperiment

188

determination77

diabetes158

diesel120

dieselengine55

difenzoquat51

dinitrobenzoicacid,3,5-

145

distance-baseddetection

31

d-limonene246

dopamine140

drugdeliverysystems

218

ecologicalcontrol114

electrochemical

immunosensor280

electrochemistry37

electronicnose253,263

ethanol162

ethylnitrite276

extraction105

fate181

fattyacids269

fattyacidmethylester

120

flowanalysis14

fluorimetry61,145

foodanalysis224

formicacid285

fruithybridization236

fruitjuices253

gaschromatography

105,114,253

GC-FID120

GC-MS269

glassycarbonelectrode

134

goldenrod(Solidago

giganteaL.)269

graphiteelectrode276

gravimetricpreparation

162

greenanalytical

chemistry200

hairdresser246

heavymetal258

herbicide51

hexafluorophosphoric

acid285

hierarchicalcluster

analysis253

HILIC-Q-TOF-MS230

hops181

HPLC82,218

HPLC-HR/MS152

HS-SPME-GC/MS1

Proceedingsofthe14thISCModernAnalyticalChemistry Prague2018 295

humanbreastmilk211

hydrophilicinteraction

chromatography285

ICP-MS87,100,205

ICP-OS205

indoorair246

interactive

chromatography7

interferenceeffects14

iodinatedcontrast

agents87

iodine87

iron14

isolation269

label-freeproteomics25

Lactococcuslactis68

laserablation44

linearandcyclic

poly(dimethyl-

siloxane)7

linearantenna140

lipidextraction188

lipidomics188

MALDI-TOFMS93

massspectrometry25,

44

meatfreshness224

mercurymeniscus

modifiedsilversolid

amalgamelectrode

51

metal205

molybdenum100

MS/MS120

Multi-CriteriaDecision

Analysis200

NiOOH55

nitrosocompounds105

non-invasivediagnostics

158

odourintensity263

µPAD31

perfumes230

pesticide181

pesticidesresidues152

photochemicalvapor

generation100

pollutants240

polymerHPLC7

precipitation-re-

dissolution

mechanism7

preconcentration1

PROMETHEE200

Prussianblue31

QuEChERS152

Ramanspectroscopy

140

reduction19

responsesurface

methodology269

restrictor120

RP-HPLC-Q-TOF-MS230

RPIPHPLC19

saliva1

scandium175

secondaryorganic

aerosol246

selectivecatalytic

reduction55

selenoproteins25

sewagesludge258

sewagesludgeash258

silvernanoparticles280

simultaneous

determination61

smokedmeat200

Sn(II)169

Sn(IV)169

solidphaseextraction

82

sorption87

spectrophotometry14

Spitsbergen240

stabilization258

staircasevoltammetry

37

standardsolution162

successiveiterations162

supercriticalfluids

extraction269

sweetie236

syntheticdyes61

terpenes236,246

tick-borneencephalitis

280

titaniagel126

toluene263

TSK-amidecolumn285

tumorbiomarker82

tyrosinase126

untargetedlipidomics

211

ureadecomposition55

urinarycatheters194

urinarytractinfections

194

UV100

vanillylmandelicacid82

volatileimpurities162

volatileorganic

compounds1

voltammetry51,77,

126,134,276

wine205

yeast93

zincions68


Proceedings of the 14th International Students Conference “Modern Analytical Chemistry”

EditedbyKarelNesmerak.

PublishedbyCharlesUniversity,FacultyofScience.

Prague2018.

1stedition–x,298pages

ISBN978-80-7444-059-5

ISBN 978-80-7444-059-5

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Prague 2018

Proceedings of the

14 ath Intern tional Students Conference


Proceedings of the 14th International Students Conference “Modern...

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