Development of sol-gel silica-based mixed-mode zwitterionic sorbents for determining drugs in environmental water samples

Four novel mixed-mode zwitterionic silica-based functionalized with strong moieties sorbents were synthesized and evaluated through solid-phase extraction (SPE) to determine acidic and basic drugs in environmental water samples.

Contentslistsavailableat ScienceDirect

journalhomepage: www.elsevier.com/locate/chroma

Alberto Morala,∗, Francesc Borrulla, Kenneth G Furtonb, Abuzar Kabirb, Núria Fontanalsa,∗,

Rosa Maria Marcéa

a Department of Analytical Chemistry and Organic Chemistry, Universitat Rovira i Virgili, Sescelades Campus, Marcel ·lí Domingo 1, Tarragona 43007, Spain

b Department of Chemistry and Biochemistry, Florida International University, International Forensic Research Institute, Miami, FL 33199, USA

a r t i c l e i n f o

Article history:

Received 31 March 2022

Revised 10 June 2022

Accepted 10 June 2022

Available online 12 June 2022

Keywords:

Mixed-mode zwitterionic sorbents

Silica sorbents

Environmental samples

Solid-phase extraction

Basic compounds

a b s t r a c t

Fournovelmixed-modezwitterionicsilica-basedfunctionalizedwithstrongmoietiessorbentswere syn-thesizedand evaluatedthroughsolid-phaseextraction(SPE)todetermineacidicandbasicdrugsin en-vironmentalwatersamples.Allsorbentshadthesamefunctionalization:quaternaryamineandsulfonic groupsand C18 chainssothathydrophobic andstrong cationicexchange(SCX) andstrong anionic ex-change(SAX)interactionscouldbeexploited,inaddition,twoofthemhadcarbonmicroparticles embed-ded

Allsorbentsretainedbothacidicand basic compoundsinthepreliminaryassays butonlythe ba-siccompoundswereretainedselectivelythroughionicexchangeinteractionswhenaclean-upstepwas introduced.TheSPEmethodwasthereforeoptimizedtopromotetheselectiveretentionofthebasic com-pounds,initiallywiththetwobest-performingsorbents

Afteroptimizationof theSPE protocol,thesesorbentswereevaluated forthe analysis of environ-mentalwatersamplesusingliquidchromatography-tandemmassspectrometry(LC-MS/MS).Themethod withthebest-performingsorbentwasthenvalidatedwith100mLofriversamplesand50mLof efflu-entwastewatersamplesintermsofapparentrecoveries (%Rapp)spikingsamples at50ng/L(river)and

200ng/L(riverandeffluent),matrixeffect,linearrange,methodquantificationanddetectionlimits, re-peatability,and reproducibility.Itshould behighlightedthat%Rappranged from40to85%and matrix effectsrangedfrom-17to-4%forspikedriversamples.Whenthemethodwasappliedtoriverand efflu-entwastewatersamples,mostcompoundswerefoundintherangefrom24to1233ng/Lwithdetection limitsfrom1to5ng/L

© 2022TheAuthors.PublishedbyElsevierB.V ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/)

1 Introduction

Complex samplesrequireselectivesampletreatments to

sepa-ratetheanalytesfromtheinterferencesthatmaycausematrix

ef-fect,mainlyinliquidchromatography-massspectrometry(LC-MS)

Onewaytoachievethisistouseselectivematerialsinsorptive

ex-tractiontechniques,themostrepresentativeofwhichissolidphase

extraction (SPE) [1,2] Variants of this technique are also used,

such as microsolid-phase extraction (μSPE) [3], dispersive

solid-phase extraction(dSPE)[4],on-lineSPE[5]andpipette tip

solid-phase extraction(PT-SPE) [6],aswell asother sorptive extraction

∗ Corresponding authors

E-mail addresses: alberto.moral@urv.cat (A Moral), nuria.fontanals@urv.cat (N

Fontanals)

techniquessuchasstirbarsorptiveextraction(SBSE)[7]orfabric phasesorptiveextraction(FPSE)[8]

In recent years,research has focused on developing new sor-bents [9] that can improve the sensitivity and selectivity of the methods in which they are applied, through the decrease of the interferencesandthematrixeffect

Mixed-mode ion-exchange sorbents are an example of these new types of sorbents [10,11] These sorbents can retain non-chargedcompoundsthroughhydrophobicinteractionsandcharged compoundsthroughion-exchangeinteractions,thusenablingthem

tointeract withawide rangeof compounds.The compounds re-tainedbyhydrophobicinteractionsareelutedwithanorganic elu-ent Those retained by ion-exchange interactions, on the other hand,requireanacidic orbasiceluenttodisrupttheinteractions withthesorbent.Thisdualityaffordsgreatflexibility.Forinstance,

ifthetargetcompounds areintheionicstate(e.g.acidicorbasic

https://doi.org/10.1016/j.chroma.2022.463237

0021-9673/© 2022 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

compounds),a clean-upstep withanorganicsolvent canremove

thehydrophobiccompoundsattachedtothesorbent.Theacidicor

basiccompounds canthen beelutedselectivelywithan acidicor

basic solvent [10–12], which neutralizes the compounds and

en-ablestheionicinteractionstobedisrupted

These sorbents can beclassified accordingto thetype of

ion-exchange interaction established On the one hand, the sorbents

are anionic exchangers if they retain anionic compounds, being

strongexchangers(SAX)orweakexchangers(WAX)dependingon

thefunctionalization.Ontheotherhand,thesorbentsarecationic

exchangersiftheyretaincationiccompounds.Inthiscase,theycan

alsobestrong(SCX)orweak(WCX)dependingonthe

functional-ization

ThepHinthedifferentstepsoftheextractionprotocolis

there-forethekeyparameterwhenthiskindofsorbentisused.Toselect

thepHtopromotehighretentionoftheanalytes, thepKa ofeach

compoundmustbe takenintoaccounttoensurethat,atthat pH,

theanalytesarechargedandcaninteractwiththesorbent,which

isalsochargedattheworkingpH

The most common mixed-mode ion-exchange sorbents are

polymericsorbents,andtheyareavailablecommerciallyas,for

ex-ample, Oasis (from Waters) or Strata X (from Phenomenex)

An-other interestinggrouparesilica-basedsorbents,thoughtheseare

lessstableatextremepHthanpolymericsorbents.Theyalso

usu-ally present low retention of polarcompounds, though this may

be beneficial since they have fewer unspecific interactions than

polymeric-basedsorbents[12].However,silica-basedsorbentshave

high organic resistance and good mechanical stability Moreover,

the silanolgroupspresentinthesilica networkare easyto

mod-ify, which enables a wide range of functionalization [13–19] Liu

et al [14], for example, developed two sorbents when

function-alizing mesoporous silica with octadecylsilane or octylsilane and

sulfonic acidtoobtain amixed-modesorbent basedon

reversed-phaseandSCXinteractions.Thesesorbentsweresatisfactorily

eval-uatedfordeterminingveterinarydrugresidues

Oneofthemainproblemswithmixed-modeion-exchange

sor-bents is that most ofthem are only based on one type of ionic

interaction (as occurs withthe commercialsorbents [12]), which

meansthatthey areselectiveforonlyonetype ofcompound

(ba-sicoracidic).Oneapproachtoextractboth acidicandbasic

com-poundscouldbethecombinationofcommercialpolymericanionic

and cationic mixed-mode ion-exchange sorbents in a single

car-tridge[20]orinseries[21,22]todetermineacidicandbasic

com-pounds in one extraction For instance, commercial anionic and

cationic Oasissorbents werecombinedinasinglecartridgeto

se-lectively extract acidicand basiccompounds fromwatersamples

[20].Anotherapproachisthe developmentofsorbentsthat

com-bineanionicandcationicinteractions,i.e.zwitterionicexchangers

Oneofthedevelopmentsinthefieldofnewsorbentsisthestudy

of materials that can simultaneously retain cationic and anionic

compoundsthroughzwitterionic-exchangeinteractions.One

exam-ple is the microporous polymer developed by Nadal et al [23],

which was used to determine a mixture of drugs,

pharmaceuti-cals and sweeteners with acidic and basiccharacter in water.In

thisstudy,polymeric-based microspheres weredeveloped forSPE

based on weak anionic and cationic interactions that were

con-trolled using the pH ofthe loading solution By loading samples

at pH6, itwaspossible to retainacidic andbasiccompounds to

determinethosecompoundsinriverandeffluentwastewater

sam-plesthroughliquidchromatography-massspectrometryintandem

LC-MS/MS

Some silica-based [16,18] and polymer-based [23–25]

zwitte-rionic sorbents have already been developed, though research

is still needed The silica-based sorbents reported [16,18] are

based on weak ionic interactions since they are functionalized

with carboxylic groups and primary amines, in both cases the

chargeability of the sorbents dependedon the pH along the SPE protocol

In our study, we present a series of zwitterionic silica-based sorbents based on the functionalization of a silica network Two

of thesesorbents were based onsilica without modification and two were based on silica with carbon microparticles embedded Allsorbentswere functionalizedwithquaternaryaminesand sul-fonic acid groups, therefore the novelty of the sorbents arise in the functionalizationof silicawith strongionic moieties, so that, thesorbent willbe always chargedatanypH.Oncethe sorbents were synthesized, they were evaluated using SPE and the best-performingsorbentwasusedtoselectivelydeterminebasicdrugs

inriverandeffluentwastewaterwatersamplesthroughLC-MS/MS

2 Experimental

2.1 Reagents and standards

Chemicals and reagents for sol-gel mixed-mode zwitterionic sorbents include methyl trimethoxysilane (MTMS), tetramethyl orthosilicate (TMOS), activated carbon, trifluoroacetic acid (TFA), isopropanol(IPA),methylenechloride,methanol(MeOH),and am-monium hydroxide purchased fromSigma-Aldrich (St Louis,MO, USA) Octadecyl trimethoxysilane (C18-TMS), 3-mercaptopropyl trimethoxysilane (3-MPTMS), N-Trimethoxysilylpropyl-N,N,N-trimethyl ammonium chloride and 4-(Trimethoxysilylethyl) ben-zyltrimethyl ammonium chloride were obtained from Gelest Inc (Morrisville,WI,USA)

Thirteendrugs wereselectedforthesorbentevaluation.Sixof these were basic, atenolol (ATE), trimethoprim (TRI), metoprolol (MTO),venlafaxine(VEN),ranitidine(RAN)andpropranolol,while sevenwereacidic,bezafibrate(BEZ),clofibricacid(CLO),diclofenac (DICLO),fenoprofen(FEN), flurbiprofen(FLB),naproxen(NPX)and valsartan(VAL).Allthesedrugswerepurchasedaspurestandards fromSigma-Aldrich(purity>96%)

Stock solutions of individual standards were prepared in methanol(MeOH)at aconcentration of 1000 mg/Landstoredat

−20 °C Working solutions of a mixture of all compounds were preparedweeklyinamixtureofultrapurewaterandMeOH(80/20 v/v) and stored at 4 °C in brown bottles in the dark Ultrapure water was provided by a water purification system (Millipore, Burlington,UnitedStates),while“HPLCgrade” MeOHand acetoni-trile(ACN)werepurchasedfromJ.T.Baker(Deventer,The Nether-lands).“MSgrade” ACNandwaterwere purchasedfromScharlab (Barcelona, Spain) Formic acid (HCOOH), acetic acid(AcOH) and HClwereacquiredfromSigma-Aldrich

2.2 Synthesis of sol-gel mixed-mode zwitterionic sorbents

Sol solutions to create the sol-gel mixed-mode zwitteri-onic sorbents were obtained by sequential addition and sub-sequent vortexing of methyl trimethoxysilane (MTMS), tetram-ethyl orthosilicate (TMOS), octadecyl trimethoxysilane (C18-TMS), 3-mercaptopropyl trimethoxysilane (3-MPTMS), N-trimethoxysilyl propyl N,N,N-trimethyl ammonium chloride (N-TMPTMAC), iso-propanol(IPA)andtrifluoroaceticacid(TFA,0.1M)ina50mL cen-trifugetube.Therelativeratiosofthevarious ingredients(MTMS, TMOS, C18-TMS, 3-MPTMS,N-TMPTMAC, IPA,and TFA were 1: 1: 0.1: 0.1: 0.2: 3.8: 3, respectively To introduce phenylethyl linker connected to trimethyl ammonium chloride, N-trimethoxysilyl propylN,N,N-trimethylammonium chloride wasreplaced with 4-(trimethoxysilylethyl)benzyltrimethyl ammoniumchloridein an-othersetofsol-gelsorbents.The mixturewasvortexedfor5min and then sonicated for 15 min to remove any trapped air bub-blesfromthesolsolution.Thesolsolutionwaskeptatroom tem-peraturefor8h toallow thesol-gelprecursorsto behydrolysed

2

Freshly prepared ammoniumhydroxide (1 M)wasthen added in

droplets to the sol solution under continuous stirring in a

mag-netic stirrer The solution slowly became viscous before turning

intosolidgel.Toproduceactivatedcarbonimpregnatedsol-gel

sor-bent,0.5gofactivatedcarbonwasaddedtothesolsolutionbefore

ammoniumhydroxidesolutionwasadded

The solidgelwasthermallyconditionedandagedat60°Cfor

48h.Themonolithicbedofthesol-gelnetworkwasthencrushed

anddried at80 °Cfor24h andthe sol-gelsorbent wascrushed

intofineparticlesinaballmillandrinsedwithMeOH:methylene

chloride (50:50, v/v) under sonication for 30 min The particles

were air-dried andtreatedwith 30% H2O2 (with0.1 M sulphuric

acid)for4h.Theparticleswererinsedwithdeionizedwater

sev-eral times and then dried at 80 °C for 12 h The sol-gel

mixed-mode zwitterionic sorbents were then ready forloading intothe

SPEcartridges

2.3 Structure of sol-gel mixed-mode zwitterionic sorbents

ThecharacterizationofthesorbentswasperformedwithaCary

670 FTIR,AgilentTechnologiesCary 600SeriesFTIRSpectrometer

(AgilentTechnologies,Santa Clara,CA,USA)fortheFourier

Trans-form Infrared Spectroscopy (FT-IR) and with a JEOL JSM 5900LV

ScanningElectronMicroscope(SEM)equippedwithEDS-UTW

de-tector, JEOLUSA, Inc (Peabody, MA, USA) forrecording SEM

im-ages

The four sorbents (Fig 1) tested inthis studywere basedon

a silica skeleton functionalized with C18 to perform hydrophobic

interactions; quaternaryaminestoperform SAX interactions;and

sulfonicgroupstoperformSCXinteractions

All sorbentswere functionalizedwiththesamegroupsto

per-formSAXandSCXinteractions.Twoofthem(SiO2-SAX/SCX SiO2

SAX/SCX(Ph)) were based on a silica network (S-type) and two

(SiO2-C-SAX/SCX SiO2-C-SAX/SCX(Ph))werebasedonasilica

net-work withactivated carbonembedded(C-type) Fig 1showsthe

structure of the four sorbents tested SiO2-SAX/SCX and SiO2

-C-SAX/SCX had the same functionalization,with propylgroups

be-tween the network and the quaternaryamine SiO2-SAX/SCX(Ph)

and SiO2-C-SAX/SCX(Ph) also had the same functionalization,

though in this case witha phenylethyl group in the anionic ex-changechain

2.4 Solid-phase extraction procedure

An empty 6mL SPE cartridge(Symta, Madrid,Spain) was fit-tedwitha10μmpolyethylenefrit(Symta)andfilledwith200mg

ofsorbents.A10μmpolyethylenefritwasthenplaced abovethe sorbentbed

The SPE procedure was performed in an SPE manifold (Teknokroma,Barcelona,Spain)connectedtoavacuumpump.The firststep wastoconditionthe sorbents with5mL ofMeOHand

5mL ofultrapure wateradjustedatpH 3.100mL ofsample ad-justedatpH3withHClwereloadedintothecartridge.Forthe ef-fluentwastewatersamples,thevolumewas50mL.Afterthe load-ing step, the washing step was performed with 5 mL of MeOH Finally, the elution step involved 5 mL of MeOH containing 5%

ofNH4OH Theelutedvolume wasevaporated withamiVacDuo centrifugeevaporator (Genevac,Ipswich,UK)to completedryness andthen reconstitutedwith1mLofinitial mobilephasesolution (H2O/ACN, 95/5,v/v) The reconstitutedextracts were filtered us-ing 0.45 μm polytetrafluoroethylene (PTFE) syringe filters (Schar-lab) before analysis To reuse the SPE cartridges a washing step withMeOHwasperformedandthen, itwascompletely dried by applyingvacuumfor10min

Samples from river and effluent wastewater treatment plants werefilteredthrougha0.45μmNylonmembranefilter(Scharlab) Theeffluentsampleswerepreviouslyfilteredusinga1.2μm glass-fibremembranefilter(Fisherbrand,Loughborough,UK)

2.5 Instrumentation and chromatographic conditions

The initial tests and the optimization of the SPE conditions wereperformedwithanAgilent1200UHPLCequippedwitha bi-narypump,anautosampler,anautomaticinjector,andadiode ar-raydetector(DAD) (Agilent,Waldbronn, Germany).The chromato-graphic column used was a Luna® Omega 5 μm Polar C18 100 (150× 3.0mm,5μmparticlesize)suppliedbyPhenomenex (Tor-rance,CA,UnitedStates).Themobilephasewasamixtureof

ultra-Fig 1 Structure of the sol-gel mixed mode zwitterionic sorbents

3

B) The gradientprofilebegan with5%ofB The% ofB wasthen

increased to 40% within 10 min,then to 45% within 4 min, and

finally to100% within1min.It wasthenheld at100% for3 min

before returning to the initial conditions in1 min,where it was

held for 3 min tostabilize the column The column temperature

was 30 °Cand the flow ratewas 0.4mL/min The injection

vol-umewas20μL.ATE,TRI,MTO,PRO, BEZ,VAL,FEN,FLB andCLO

weremeasured at210nm,whileRAN,VEN,DICLO,andNPXwere

measuredat230nm

OncetheSPEconditionswere optimized,themethodwas

val-idated for the basic compounds analysing real samples with

LC-MS/MS using an Agilent 1260 Infinity 2 connected to a triple

quadrupole mass detector Agilent 6460 and electrospray

ioniza-tion(ESI)interface.Thechromatographicconditionswerethesame

as in LC-DAD, except that the injection volume was 10 μL and

the pH ofsolvent A was adjusted with HCOOH rather than HCl

The optimizedparameters inthe (ESI)MS/MSwere gas

tempera-ture 320 °C, gas flow rate 10 mL/min, nebulizer pressure 35psi

andthecapillaryvoltage3000 V.The fragmentorpotentialforall

transitionswas100V.Foreachcompound,thediagnostic ionwas

[M+H]+.Oneofthetransitionswasusedasquantifierandatleast

one morewasused asqualifier.Table S1showsthe MRM

transi-tionsselectedandtheircollisionenergies

2.6 Validation parameters

The method wasvalidatedinterms ofrecovery,matrix effect,

linear range,methodquantification anddetectionlimits,

repeata-bilityandreproducibility

Recovery(%R)andapparentrecovery(%Rapp)wereusedto

eval-uate the yield of the extraction %R was obtained with LC-DAD,

being the ratio of the concentration obtained after the SPE of a

spikedsampleandtheconcentrationexpected.%Rappwasobtained

inthesamewaythat%Rbuttheanalysiswasperformedwith

LC-MS/MS,anditconsiderstheextractionrecoveryandthematrix

ef-fect

The matrix effect (%ME) was calculated from the formula:

%ME = (CExp/CTheo × 100)– 100, where“CExp” isthe

concentra-tion obtained by spiking a blank sampleafter SPEand “Ctheo” is

theexpectedconcentration.Anegativevalueindicatessuppression

ofthesignal,whileapositivevalueindicatesenhancement

Theinstrumentallinearrangewasevaluatedwithexternal

cali-brationcurvesanalysingintriplicatesevensolutionswithdifferent

concentrations Matrix matched calibration curves were obtained

spikingriversamplesatsevendifferentconcentrations

Method quantification limit (MQL) was obtained from the

matrix-matchedcalibrationcurves,beingthelowestconcentration

fromthe curve andmethoddetectionlimit (MDL)wascalculated

astheconcentrationthatprovidedasignal-to-noiseratioof3

Repeatability was obtainedasthe % relative standar deviation

(%RSD) intra-day (n = 3) analysing by triplicate samples spiked

at the same concentration the same day The reproducibility

be-tween days wasobtainedasthe %RSDinter-day (n=3) analysing

samples(n=3) spikedatthesameconcentration duringdifferent

days(n=3)

3 Results and discussion

3.1 Synthesis of the sol-gel mixed-mode zwitterionic sorbents

Many environmental and biological samples simultaneously

containneutral, acidicandbasicanalytes.Ifalltheanalytesareof

interest, theseparationandpreconcentrationofthesecompounds

pose serious analytical challenges One wayto solve this

analyt-ical challenge isto create a mixed-modezwitterionic sorbent by

incorporating a neutralcarbon chain, a cation exchanger, andan anion exchanger into a single sorbent To maintain the cations andanions intheir chargedstate atfullpH range,thecation ex-changerandanionexchangershouldbestrongsothat they main-taintheir ionicstateatall pHlevels.Octadecylsilaneisthemost prevalent sorbent in SPE Octadecyl trimethoxysilane was there-fore chosen as the neutralsorbent To includea SCX in the sor-bents,3-mercaptopropyl trimethoxysilane,whichgeneratespropyl sulfonic acid after oxidation, was used N-trimethoxysilyl propyl N,N,N-trimethyl ammonium chloride and 4-(trimethoxysilylethyl) benzyl trimethyl ammonium chloride were used as SAX To in-corporate thesefunctional groups intothe silica network, sol-gel synthesis,whichisconsideredapopular,environment-friendlyand facilesynthesis approach,wasused.Sol-gelsynthesis can be per-formedunderacidicorbasiccatalysisoracidichydrolysisfollowed

by condensationin basicenvironment Acidic hydrolysis followed

by basic condensation renders the sol-gel network stronger and moreporous[26].Moreover,tofacilitatesynthesis,thesol-gel pro-cess enables the creation of sol-gel sorbent particles or surface coating insituatroom temperature Propylsulfonic acidwas ob-tained afterpost-gelation treatment of thesorbent with 30% hy-drogenperoxide(impregnatedwith0.1Msulphuricacid).The cre-ation ofsol-gelmixed-modezwitterionicsorbents isanew mile-stoneinseparationscience

3.2 Characterization of sol-gel silica based mixed mode zwitterionic sorbents

All the sorbents were subjected to characterization using FourierTransformInfraredSpectroscopy(FT-IR)andScanning Elec-tron Microscopy (SEM) However, as the results provided were quite similar, we only present the resultsof the testsperformed with SiO2-SAX/SCX FT-IR spectrareveal valuable information re-gardingthefunctionalcompositionofthebuildingblocksandtheir successfulintegration into thefinal compositematerial SEM im-ages,ontheother hand,shedlight onthesurfacemorphologyof thecompositematerial

3.2.1 Fourier Transform Infrared Spectroscopy (FT-IR)

The FT-IR spectra of the individual building blocks, methyl trimethoxysilane (MTMS), octadecyl trimethoxysilane (C18-TMS), 3-mercaptopropyl trimethoxysilane (3-MPTMS), N-trimethoxysilyl N,N,N-trimethyl ammonium chloride (TMTAMC) and the sol-gel mixedmodezwitterionicsorbentare presentedinFig.2(a–e), re-spectively AllFT-IR spectra were collectedover a rangebetween

3000and700cm−1 ataresolution4cm−1 FT-IRspectraofMTMS(Fig.2a)displaysseveralsignaturebands

at 1266 and 789 cm−1 which are attributed to the vibration of

CH3 group connected toSion theprecursor molecule.The peaks

at 1077 and 1189 cm−1 are attributed to C-O stretching vibra-tion Si-O-CH3.The peaks at2842 and 1464 cm−1 are attributed

toC-H stretching andbendingvibration ofSi-O-CH3,respectively [27] The noteworthy peaks in the C18-TMS spectra 2922 cm−1 and2852cm−1 whichcanbeassignedtoantisymmetric[va(CH2)] andsymmetric[vs (CH2)]bandsforthealkenechainsofC18-TMS The FT-IR spectra of 3-mercaptopropyl trimethoxysilane demon-strate signature band at 2560cm−1 that can be attributed to

S-H stretching [28] The bands at 1187 and 1080cm−1 are related

to–CH3OCH3 [28]. ThesignaturebandinN-trimethoxysilyl N,N,N-trimethyl ammonium chloride FT-IR spectra includes 1480 cm−1 that can be attributedto N-CH3 bending vibration [29] It is im-portanttonotethat allprecursorshaveacommonendconsisting

of-Si (CH3)3.As a result, manyspectral bands are common.The FT-IRspectraofsol-gelSiO2-SAX/SCXincludemanybandssuchas

1505,1441,1314,1061,and778cm−1thatalsoappearedinthe

FT-4

Fig 2 FT-IR spectra of (a) methyl trimethoxysilane; (b) octadecyl trimethoxysilane; (c) 3-mercaptopropyl trimethoxysilane; (d) N-trimethoxysilyl N,N,N-trimethyl ammonium

chloride; (e) sol-gel mixed-mode zwitterionic sorbent

Fig 3 Scanning electron microscopy of sol-gel SiO 2 -SAX/SCX sorbent at (a) 100x magnifications ; (b) 10 0 0x magnifications

IRspectraofindividualbuildingblockswhichmanifestssuccessful

integrationofthebuildingblocksintosol-gelSiO2-SAX/SCX

3.2.2 Scanning Electron Microscopy (SEM)

Thesurfacemorphologyofthesol-gelSiO2-SAX/SCXwas

inves-tigatedusingaScanningElectronMicroscope.TheSEMimagesare

presentedinFig.3(a,b)at100xand1,000xmagnifications,

respec-tively.TheSEMimagesrevealedthattheparticlesizesarenot

ho-mogeneouslydistributedandpossessirregularshapes.Some

parti-cles oftheSiO2-SAX/SCXare insub-micronsizewhile othersare

bigger, inthe range of50-60micron (gross estimation) The

sur-faceoftheparticlesapparentlylookroughthatshouldenhancethe

interaction betweentheparticles andthe analytesduringthe

ex-traction process.Thebroadrangeofparticlesizedistributionalso

helps reducing the void volume due to the close packing of the sorbents

3.3 Optimization of the SPE procedure

The SPEprocedure wasoptimizedusinga mixture solutionof standardspreparedinultrapurewater.Theanalysiswasperformed usingLC-DAD

3.3.1 Extraction performance evaluation of the sorbents

Sincethefunctionalizationofthesorbentsevaluatedwasbased

onstrongionicinteractions,theywillalwaysbechargedatanypH

Toselectthe initialpH, thepKa ofthecompounds wastherefore considered (Table 1) andit wasset at5(pH atwhich theacidic

5

Table 1

pK a of the compounds and recoveries performed by each sorbent at initial conditions (see text)

SiO 2 -SAX/SCX SiO 2 -SAX/SCX(Ph) SiO 2 -C-SAX/SCX SiO 2 -C-SAX/SCX(Ph)

∗ RSD (%) < 10% ( n = 3)

a pK a values obtained from PubChem for all compounds except for BEZ, FLB and CLO (values obtained from Drugbank)

andbasiccompoundswerecharged).Theconditionsofloading

vol-umeandelutionwerebasedonaprevious studyreportedby our

group[23]thatanalyzedacidicandbasiccompoundsusingaweak

zwitterionicsorbent.Theseconditionswere:25mLofloading

vol-umeandanelutionstepwith5mLof5%AcOHinMeOHtoelute

the acidiccompounds;and5mLof5% NH4OH inMeOH toelute

thebasiccompounds

The foursorbents were initially testedto discern which ones

provided the highest recoveries.The four sorbents had thesame

functionalization,withsulfonicgroupstoperformSCXinteractions

andquaternaryaminestoperformSAXinteractions.Thedifference

between thesesorbents wasthe support since some were based

onthesilicanetwork (S-type),whileforothersthesilicanetwork

wasembeddedwithactivatedcarbonmicroparticles(C-type).Each

grouphadtwovariants:oneinwhichtheSAXgroupswerebonded

through a propylgroup to the silica network (SiO2-SAX/SCX and

SiO2-C-SAX/SCX),andanotherwhichhada phenylethylgroup

be-tween the silica network andthe SAX groups (SiO2-SAX/SCX(Ph)

andSiO2-C-SAX/SCX(Ph))

As Table 1 shows, the sorbents that provided the greatest

re-coveries were SiO2-SAX/SCX and SiO2-C-SAX/SCX The recoveries

of sorbents SiO2-SAX/SCX(Ph) and SiO2-C-SAX/SCX(Ph) were

sig-nificantly lower thanthose ofSiO2-SAX/SCXandSiO2-C-SAX/SCX

Adding the aromatic ring seemed to hamper interactions

be-tween the compounds and the ionic exchange groups, thus

re-sulting in lower recoveries.Sorbents SiO2-SAX/SCX(Ph) andSiO2

C-SAX/SCX(Ph)werethereforediscarded,andthesubsequenttests

wereperformedwithSiO2-SAX/SCXandSiO2-C-SAX/SCX

Moreover, bycomparingtheS-typeandC-typesorbentsitcan

be observed thatthe S-typesorbents presentedhigherrecoveries

than the C-type sorbents Forexample, DICLO presented a %R of

82%withSiO2-SAX/SCXand53%withSiO2-C-SAX/SCX

3.3.2 Optimization of the loading pH

As we explained inthe Introduction,the control of pHis

im-portant whenevaluating thesesorbents, thus,the firstparameter

tobeevaluatedwasthepHoftheloadingsolution,whichgoverns

theretentionofthecompounds.Sincethesorbentswerebasedon

strongion-exchangeinteractions,theywerechargedatanypH.The

loading pHwasthereforeusedto controlthe chargeabilityofthe

analytes

AshasbeenhighlightedinSection3.3.1.,pH5wasinitially

se-lected since in thisrange all compounds were charged

consider-ing the pKa of the analytes Moreover, a cleaning step of 2 mL

was also introduced to check whether the compounds were

be-ing retained through ionic interactions As can be observed in

Fig 4, whereresults of SiO2-SAX/SCX are presented,good

recov-eries are obtainedfor basic compounds, only ATE and RAN pro-vided%Rbelow80%.However,theacidiccompoundsprovidedlow recoveries

Then,pH3and9wereevaluatedtopromotethespecificionic interactions ineach range; atpH 3,the cationic interactions dis-playedbythe basiccompounds andatpH 9,theanionic interac-tions by theacidic compounds.Attending to Fig.4,it can be ob-servedthat therecoveriesofthebasiccompoundsimprovedwith

pH3,achievingrecoverieshigherthan80%forthesixcompounds However,atpH9,therecoveriesoftheacidiccompoundsdidnot improve.pH4wasevaluated sincethebestresultswereobtained

atpH3 andweconsidered interesting totest thispH.As can be observed inFig 4,the resultsfor basiccompounds were slightly better than pH5 andslightly worse than pH3 The good recov-erieswere explainedsinceatthesepHs,theanalyteswere proto-natedandthereforeabletointeractwiththesorbentthroughionic interactions.Thelowrecoveriesobtainedfortheacidiccompounds suggested that retention occurred only via hydrophobic interac-tions sincethesecompounds wereelutedfromthesorbent when MeOH was applied, meaning that the SAX interactions did not work

TheoptimizationofthepHwasperformedwithSiO2-SAX/SCX and SiO2-C-SAX/SCX Although Fig 4 shows the results obtained fromthepHevaluationwithSiO2-SAX/SCXbothsorbentsprovided similarresults,beingthe%RofSiO2-SAX/SCXslightlyhigher Jin etal [16] also observed that only basic compounds were retained via ionic interactions These authors evaluated a home-made mixed-mode zwitterionic sorbent based on weak interac-tionsgroundedincarboxylicacidsandsecondaryaminesto deter-mineagroupofacidic,basicandneutralcompoundswitha load-ingpHof6

Giventhe zwitterionic nature ofthe sorbents, the loading pH shouldhavebeencloserto theneutralpHusedby Jinetal.[16], whochosealoadingpHof6todeterminebasicantidepressantsin aquaticproductsusingahomemadezwitterionicmixed-mode sor-bent functionalized withcarboxylic acids and secondary amines The above authorsobserved that theacidic compounds were not retainedthroughionicexchangeinteractions[16].Asimilar expla-nationcan beadapted inour study,inwhich all theacidic com-poundspresentedaromaticringsthattended tointeractwiththe

C18chainsthroughhydrophobicinteractions

Whentheclean-upstepwasincluded,thebehaviorofthe sor-bentswasthereforeclosertoacationicexchangerthantoa zwitte-rionicexchanger.Asoccurredinpreviousstudies[30,31]that eval-uatedSCXsorbentstoselectivelydeterminebasiccompoundsfrom aqueoussamplesandselectedapHintheacidicrange,theloading

pHforourstudywasacidic

6

Fig 4 Comparison of the recoveries obtained at pH 3, 4, 5 and 9 with the SiO 2 - SAX/SCX sorbent

3.3.3 Optimization of the clean-up step

A clean-up stepis neededto remove theinterferences andto

increase theselectivityofthemethod.Intheprevioussection,we

introduced a clean-up with2 mL of MeOH.We then used 5mL

ofMeOHto testwhetherthecleaningvolume couldbe increased

without the recoveries being affected,thereby enhancing the

se-lectivityoftheextraction

Table2showsresultswhen25mLofsamplewasloadedatpH

3withorwithoutaclean-upstep(2or5mL).Whenthisclean-up

step(2or5mLofMeOH)wasapplied,bothsorbentsshowedthe

sameperformance,withrecoveriesforthebasiccompoundsabove

80%andthosefortheacidiccompoundsbelow10%

As we mentioned earlier, the results for acidic compounds

proved that these compounds were retained through

hydropho-bic interactions since they were removed from the sorbent with

MeOH On the other hand, the basic compounds were retained

through ionic exchange interactions since they were not eluted

duringtheclean-upstep

Table 2

Recoveries obtained when 100 mL of ultrapure water were loaded without cleaning

and cleaning with 2 and 5 mL of MeOH clean

SiO 2 -SAX/SCX SiO 2 -C-SAX/SCX

No clean 2 mL 5 mL

No clean 2 mL 5 mL

∗ RSD (%) < 10% ( n = 3)

Theclean-upstepwassetat5mLofMeOHsincetherewasno evidentdecreaseintherecoverieswhenthevolumewasincreased from2mLto5mL.Moreover,thisincreasewouldhelptoincrease selectivity.ItiscommontouseMeOHtoperformtheclean-upstep whenworkingwithmixed-modeion-exchangesorbentstodisrupt thehydrophobic interactions andpromoteselectivity Using5 mL hasbeenreportedinastudywitha homemademixed-modeSCX sorbent [32] Inanother study[23],the volume wasset at1 mL

toreducethelossofanalytesinthedeterminationofillicitdrugs, sweeteners andpharmaceuticals usinga homemade mixed-mode ion-exchange zwitterionic sorbent based on weak ionic interac-tions

Otherstudies,ontheotherhand,havereportedaclean-upstep not fullybased onMeOH Hu etal.[33], forexample,performed thisstepwith2mLofamixtureofwater/MeOH(95/5,v/v)when using a modified silica sorbent with a triazine to determine an-thraquinonesinurine,whichcouldnotbeenoughtoproducea re-markableclean-upeffect.Therefore,an aqueousclean-upwasnot evaluated in thisstudyanda clean-up step with5 mL ofMeOH wasselected

3.3.4 Optimization of the elution

Initially,theelutionwasconductedintwosteps:anacidicstep (5% AcOH in MeOH) to elute the acidic compounds and a basic step (5% NH4OH in MeOH) to elute the basic compounds Since the acidiccompounds are elutedjust withMeOH,the AcOHwas notneeded,andtheacidicstepwasthenremoved

Aftertesting5%NH4OHinMeOHinaprevioussection,thetwo optionstestedwere5mLof10%NH4OHinMeOHand10mLof5%

NH4OH in MeOH All three options provided similar results: 85-100% forthe SiO2-C-SAX/SCX sorbent and90–105% for the SiO2 SAX/SCXsorbent.Thefirstoptionwasthereforechosen sinceitis greenerandgeneratesalowervolumetoevaporate

Thiselutionhaspreviously beenusedinsomestudiestoelute basic compounds [20,23,31] from mixed-mode ion-exchange sor-bents Moreover, when Salas et al [20] studied combinations of commercialcationandanionic exchangers,theauthorsalsobegan withelutionintwosteps,i.e.an acidicstepbasedon5%AcOHin MeOHandabasicstepwith5%NH4OHinMeOH.However,during

7

elutedinthebasicstep

3.3.5 Optimization of the loading volume

Thefinal stepintheoptimizationprocesswastheloading

vol-ume Thelargertheloadingvolume,thehigherthe

preconcentra-tionfactor,though thebreakthroughvolumeshould alsobe taken

intoaccount

Theinitialvolumewas25mL,while100,250and500mLwere

alsotestedwithstandardsolutions.Everyvolumeshowedgood

re-coveries forboth sorbents (80–100% forSiO2-C-SAX/SCXand 85–

105%forSiO2-SAX/SCX).Theresultswerethereforegoodevenwith

500mLwithstandardsolutions

Then, 100 and250mLwere evaluated withspiked river

sam-ples to select the loading volume with river samples As Fig S1

shows, asignificantdecreaseintherecoveries occurredwhenthe

volume wasincreased from100 to 250mL Thevolume selected

with riversamples wastherefore 100 mL Klan etal.[30] tested

100,200,500and 1000mL and obtainedsatisfactory results for

mostoftheir analyteswhenanalysing riverwatersamples

How-ever,a significantdecrease in%Rwasobserved inthemostpolar

compounds Theauthorsconsideredtheincrease intimeinherent

totheincreaseinvolume.Theyalsoconsideredthepossibilitythat

the cartridgewouldgetclogged anddecidedto select200mL as

theloadingvolume

When working with effluent wastewater samples, recoveries

were low with 100 mL The loading volume was therefore

re-ducedto50mL,whichledtosatisfactoryrecoveries(44–78%)

Gi-lart etal [32] alsoevaluated the loadingvolume andfound that

500 mL presented good recoveries in standard solutions For

ef-fluent wastewatersamples,however,they also hadtoreduce the

volumeto50mL

3.4 Validation of the method

The method wasvalidated for river water samples and

efflu-entwastewaterfromtreatmentplantsusingLC-ESI-MS/MSto

im-provesensitivityandselectivity.Thechromatographicmethodwas

transferredtoLC-MS/MS,whichenabledworkatlower

concentra-tions Theparametersofgastemperature, gasflowrate,nebulizer

pressureandcapillaryvoltagewereoptimizedexperimentally.Gas

temperaturewasevaluatedbetween200and400°C;gasflowrate

between 6 and 14 mL/min; nebulizer pressure between 20 and

60psi; andcapillaryvoltagebetween2500and5000V.Foreach

compound, the fragmentor potential was alsoevaluated between

50and200V.Thecollisionenergy(CE)wasevaluatedbetween0

and30 eV.The conditionsselected areshown inSection 2.5and

TableS1.Forallfragments,theCErangedfrom15to25eV,except

forVEN,whichrangedfrom7to5eV

Theinstrumental linearrangewas0.5–250 μg/Lformost

com-pounds The R2 wasabove 0.995 for all compounds exceptMTO,

whoseR2was0.992.TheinstrumentalLODandLOQwere0.1μg/L

and 0.5 μg/L, respectively for all compounds except VEN, whose

limitswere0.05and0.1μg/L,respectively

Beforevalidatingthemethod,thebestperformingsorbentwas

selected.Sincebothsorbentsprovidedgoodresultsduringthe

op-timizationoftheSPEprocedure,toselectoneofthem,theywere

testedin termsofapparent recovery (%Rapp), whenriver samples

were spiked at 200ng/L Tocalculate theapparent recovery

cor-rectly,ablankwasmeasuredtosubtractthesignaloftheanalytes

naturallypresentfromthesignalofthespikedsamples.AsTable3

shows(andaswehighlightedduringtheoptimizationprocedure),

theSiO2-SAX/SCXsorbentshowedhigher%Rappforthespikedriver

samples (with values ranging from60 to 78%), while the results

for the SiO2-C-SAX/SCX sorbent ranged from 47 to 62 % (except

Table 3

%R app obtained with each sorbent when 100 mL of river samples spiked at 200 ng/L was extracted

Compound SiO 2 -SAX/SCX SiO 2 -C-SAX/SCX

∗ RSD (%) < 10% ( n = 3)

forATE,whose%Rapp were40and34%,respectively).Thesorbent chosentovalidatethemethodwasthereforeSiO2-SAX/SCX Theadditionofcarbonaceousparticlesintothesol-gel compos-itesorbentincreasedtheoverallsurfaceareaofthecomposite sor-bent butdecreased the absoluteloading of thesol-gel silica sor-bent,and consequently, the overall interactionsites It isevident fromthe recovery data that the sorption feature ofthe carbona-ceousparticles inthecompositesol-gelsorbentplayedno rolein theextractionprocess.Inafutureproject,weintendtoinvestigate theimpact ofcarbonaceous particles on other type ofmolecules After selecting the best sorbent, river samples were analyzed to perform the validation,according to theparameters describedin Section 2.6, in terms ofrecovery at two concentrations (50 ng/L and 200 ng/L), matrix effect,linear range, method quantification anddetectionlimits(MQLandMDL),repeatability(%RSD,n= 3) andreproducibilitybetweendays(%RSD,n=3)

AsTable4shows,%Rapp spikingat50ng/Lweregood,i.e.60– 85% for all compounds except ATE, whose %Rapp were 40%, be-ing similar results tothe %Rapp spiking at200 ng/Lpresented in Table 3 These recoveries were comparableto those obtained by Nadaletal.[23] (58%–87%) whendeterminingTRI,MTO andPRO

in100mLofriversamplesusingahomemademixed-mode zwit-terionic sorbentbasedon weakionicinteractions Zhuetal.[13], whosevaluesrangedfrom75to98%,alsoobtainedslightlyhigher recoverieswhenanalysing aromaticaminesinenvironmental wa-ter sampleswitha WCXmixed-modesilica-based sorbent More-over, Afonso-Olivareset al.[34] obtainedrecoveries rangingfrom

78to98% whendeterminingpharmaceuticals(ATEamongothers)

inseawaterwithacommercialsorbent(OasisHLB)

The%MEs(Table 4)were remarkablylow (rangingfrom-17to -4%),whichindicateslowionsuppressionduetotheinclusionof

aclean-upstepwith5mLofMeOH.Theseresultsare lowerthan thosefound inother studies,e.g.Krizmanetal.[31],who useda commercial sorbent (Oasis MCX) and obtainedmatrix effects for opioids and their metabolites ranging from -38to -7% Fortheir part,Nadaletal.[24]obtainedmatrixeffectsrangingfrom-30to +5whenusingamixed-modeSAX/WCXsorbenttodetermine,for example,TRI,MTO,RAN,ATEandPRO

Thelinearrangewasobtainedfrommatrix-matchedcalibration curvesand riversamples were spiked from 1to 500 ng/L In all cases,determinationcoefficient(R2)wasabove0.99

Attending to the methodquantification andmethod detection limits(Table 4),inboth cases,thevalueswere intheng/Lrange, which were comparable to those found in developed methods based on determining those analytesin riversamples [23,30,32], whoselimitswerealsointheng/Lrange

The values for repeatability (intra-day precision, n=3) and reproducibility (inter-day precision, n=3) were acceptable (as Table4shows,inallcasestheywerebelow16%)

The method was also evaluated in terms of %Rapp, %ME and

%RSD witheffluent wastewatersamples Since the analyteswere presentathighconcentrationsintheeffluentwastewatersamples,

nomatrix-matched calibrationcurves weredone Toquantify the

8

Fig 5 Chromatogram of an effluent wastewater sample when it was analyzed using the developed method

Table 4

Validation parameters for SiO 2 -SAX/SCX sorbent with river samples

Compound %R app (50 ng/L) %ME Linear range (ng/L) MQL (ng/L) MDL (ng/L) % RSD intra-day ( n = 3) % RSD inter-day ( n = 3)

analytes in real samples, external calibration curves and

appar-ent recoverieswereused.The%Rapp obtainedspikingat200ng/L

ranged from40to 71%.Lowerconcentrationswere not evaluated

due to the presence ofthe compounds in the sample Moreover,

spiking at higherconcentration was neitherevaluated since

sim-ilar recoveries in river samples were obtained when spiking the

samples bothat50and200ng/L.Theseresultsare comparableto

othersreported,e.g.acombinationofcommercialcationicand

an-ionic exchangers (where the %Rapp ranged from 50 to 73% [20]),

andwithanovelSCXsorbent(wherethe%Rapp rangedfrom39to

84%[32]).Inboth casessimilarcompounds weredetermined.The

%MEobtainedrangedfrom-25%to-18%.Theseresultsare compa-rabletothoseobtainedbyGilartetal.[32](rangingfrom-12and +21%), who used a novel SCX sorbent when determining similar compounds (ATE, PRO, MET, RAN andTRI among others) More-over, Jaukovic et al [35] determined cardiovascular drugs (MTO amongothers)ineffluentwastewatersamplesusingacommercial sorbent(Oasis HLB), andobtainedhigherrecoveriesrangingfrom

84to106%andhigher%MErangingfrom-28to+23%.Inallcases,

%RSDintraday(n=3)wasbelow14%

9

Table 5

Range of concentrations (ng/L) obtained after the analysis of river and effluent

wastewater samples through SPE-LC-MS/MS method based on the SiO 2 -SAX/SCX

sorbent

Compound River samples Effluent wastewater samples

∗ RSD (%) < 15% ( n = 3)

3.5 Analysis of real samples

SamplesfromEbroriverandsamplesfromeffluentwastewater

treatmentplantsfromReusandTarragona,Spainwereanalyzed

Table5showstheoccurrenceofthecompoundsinriverand

ef-fluentwastewatersamples.Ascanbeobserved,theconcentrations

of ATEandPROwere belowthe MQLintheriversamples,while

those of RAN,TRI,MTO andVEN were found atsimilar levels of

concentrationrangingfrom24to72ng/L.Nadaletal.[36]also

an-alyzedEbroriversamplesandfoundthattheconcentrationsofATE

andPRO(rangingfrom0.7to9.5ng/L)wereabovetheirMQL.On

the other hand,the concentration ofTRIwasbelowitsMQL.The

concentrationofMTOfoundinthepresentstudywasoneorderof

magnitudehigherthanthatfoundinthestudybyNadaletal.:29–

67ng/L comparedto3–7ng/L.When Klanetal.[30] determined

MTO,PRO,RANandVENinriverwatersamplesfromSlovenia,all

compounds were below theMQLexcept forVEN, whose

concen-trationrangedfrom0.08to3.01ng/L

Regarding the effluent wastewater samples (Fig 5), all

com-poundswerequantified.RANandVEN presentedthehighest

con-centrations(653–1233 ng/L),whiletheconcentrationsofPRO and

MTO were lower (29–113 ng/L) These levels were comparable

to those quantified previously with similar samples.When Gilart

et al [32] measured ATE, RAN, TRI,MTO and PRO from effluent

wastewatertreatmentplantsamples,obtainingconcentrations

val-ues similar to the ones presented in Table 5 PRO, for example,

was found between 50 and 100 ng/L while RAN was found at

around1000ng/L.Nadaletal.[36],fortheirpart,measuredMTO,

ATE,PROandTRI(amongothers)ineffluentwastewatertreatment

plants andalso found similar concentrations tothose in Table 5

Moreover, whenIancu etal.[37] quantifiedATE andPRO (among

others)ineffluentwastewatersamplesfromRomania,the

concen-trationsofPROrangedfrom5to40ng/L,whichareclosetothose

found in our study (48–82 ng/L) On the other hand, the

aver-age concentrationsofATE(94.6 ng/L)waslower than ours(234–

282ng/L)

4 Conclusions

In this study, we developed andevaluated four novel

mixed-mode zwitterionic-exchange sorbents to determine

pharmaceuti-calsinenvironmentalwatersamplesthroughSPEfollowedby

LC-MS/MS It hasbeen shownthat the sol-gelapproach successfully

worksforthe preparationofaseriesof mixed-modezwitterionic

silica-basedsorbents

When applying a clean-up step to achieve selective

extrac-tion, only basic compounds were retained through ion-exchange

interactions, whereas acidic compounds were mainly retained

by reversed-phase interactions A method based on the

best-performing sorbent wassuccessfully developedto selectively

de-terminethebasicdrugsinenvironmentalsamples.Theapplication

of this methodin environmental samplesis therefore interesting

duetothelowmatrixeffectachievedthankstotheclean-upstep

Theresultsobtainedinthisstudy,especiallywhenitcomesto thelowmatrixeffect,areencouragingforthedeterminationof ba-siccompounds in complex samples.Moreover, the sorbents used couldbe interestingforextractingothercompounds inthefuture andapplyingthemtoothermatrices

Declaration of Competing Interest

Theauthorsdeclarethattheyhavenoknowncompeting finan-cialinterestsorpersonalrelationshipsthatcouldhaveappearedto influencetheworkreportedinthispaper

CRediT authorship contribution statement Alberto Moral: Investigation, Resources, Validation, Writing – original draft Francesc Borrull: Supervision, Funding acquisi-tion Kenneth G Furton: Supervision, Conceptualization Abuzar Kabir: Project administration, Conceptualization, Methodology

Núria Fontanals: Project administration, Conceptualization, Writ-ing – original draft Rosa Maria Marcé: Methodology, Writing – originaldraft,Writing– review&editing

Acknowledgments

The authors would like to thank the Spanish Ministry of Economy, Industry and Competitivity, the Spanish State Research Agency, and the European Regional Development Fund (ERDF) (PID2020-114587GB-I00andRED2018-102522-T)fortheirfinancial support.A.MoralwouldalsoliketothankUniversitatRovirai Vir-gili(URV)forhisPhDgrant(2020PMF-PIPF-33)

Supplementary materials

Supplementary material associated with this article can be found,intheonlineversion,atdoi:10.1016/j.chroma.2022.463237

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