Membrane-based liquid-phase microextraction of basic pharmaceuticals – A study on the optimal extraction window

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ContentslistsavailableatScienceDirect

Journal of Chromatography A

journalhomepage:www.elsevier.com/locate/chroma

Membrane-based liquid-phase microextraction of basic

pharmaceuticals – A study on the optimal extraction window

Maria Schüller

^a

, Kim Tu Thi Tran

^a

, Elisabeth Leere Øiestad

^a^,^b

, Stig Pedersen-Bjergaard

^a^,^c^,^∗

aDepartment of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, 0316 Oslo, Norway

bOslo University Hospital, Division of Laboratory Medicine, Department of Forensic Sciences, P.O. Box 4459 Nydalen, 0424, Oslo, Norway

cDepartment of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark

a rt i c l e i nf o

Article history:

Received 5 July 2021 Revised 10 December 2021 Accepted 20 December 2021 Available online 23 December 2021 Keywords:

Sample preparation Liquid-phase microextraction Hollow ﬁber

Pharmaceuticals Deep eutectic solvents

a b s t r a c t

Thepresentpaperdeﬁnestheoptimalextractionwindow(OEW)forthree-phasemembrane-basedliquid- phasemicroextraction(MP-LPME)intermsofanalytepolarity(logP),and anchorsthistoexistingthe- ories forequilibrium partitioningand kinetics.Using deepeutectic solvents(DES) assupported liquid membranes(SLM),weinvestigatedhowtheOEWwasaffectedbyionic-,hydrogenbondandπ^-π^inter- actionsbetweentheSLMandanalyte.Elevenbasicmodelanalytesintherange-0.4<logP<5.0were extracted byMB-LPMEin a96-wellformat. Extractionwas performedfrom 250μLstandard solution in25 mM phosphatebuffer (pH 7.0)into50 μLof10 mMHClacceptor solution (pH 2.0)with mix- turesofcoumarin,camphor,DL-menthol,andthymol,withandwithouttheioniccarrierdi(2-ethylhexyl) phosphate(DEHP),astheSLM.TheOEWwithpureDESwasintherange2<logP<5,andlowSLM aromaticitywasfavorablefortheextractionofnon-polaranalytes.Here,extractionrecoveriesupto98%

wereobtained.UponadditionofDEHPtotheSLMs,theOEWshiftedtotherange-0.5<logP<2,and acombinationof5%DEHPandmoderatearomaticityresultedinextractionrecoveriesupto80%forthe polaranalytes.Extractionwithioniccarrierwasineﬃcientforthenon-polaranalytes,duetoexcessive trappingintheSLM.TheresultsfromourstudyshowthatLPMEperformsoptimallyinarelativelynar- rowlogP-windowof≈2–3unitsandthattheOEWisprimarilyaffectedbyioniccarrierandaromaticity.

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

1. Introduction

Microextraction techniques like liquid-phase microextraction (LPME) arepopularchoicesfortheextractionoftargetedanalytes frombiologicalmatrices.Offeringlowconsumptionoforganicsol- vents and the potential for automation, they are favorable with regards to greenness and eﬃciency in high-throughput applications [1].Current efforts focusontheimplementationofLPMEin routinelaboratories,includingthecommercializationofequipment andbetterunderstandingoftheoptimalareaofuse.Thisworkwill focusonthelatter.

LPMEcanbeperformedwithbothtwoandthreephaseswhere the formerisbased onthe partitionof asubstancebetweentwo immiscible liquids, like an aqueous sample and organicacceptor solution. In athree-phase system, asecond aqueousphase is in- troducedastheacceptor,allowingfortheyieldofcleanerextracts andbetterHPLCcompatibility[2].Theprincipleisbasedonliquid-

∗Corresponding author.

E-mail address: [email protected] (S. Pedersen-Bjergaard).

liquidextractionwithbackextraction,withaconﬁgurationallow- ing the extraction to take place in a single step [3]. Fig. 1B il- lustrates thebasicprinciple withtwo aqueousphases,the donor, and acceptor, separated by an immiscible organic supported liq- uidmembrane (SLM). Ina typical schemefor basicanalytes, like manypharmaceuticaldrugs,the donorisalkaline,andtheaccep- tor is acidic.A highpH in the donorpromotes theextraction of theanalyte asa neutralspeciesintothe SLM, whilea low pHin theacceptorpromotes extractionandcollectionofthesameana- lyteastheprotonatedspecies.Extractionrecoverywilldependon thepartitionbetweenthesethreephases[3].Extractioninathree- phasesystemistypicallyusedfororganicanalyteswithweakbase oracidproperties.

An array of conﬁgurations for LPME has been developed through the years,each with speciﬁc advantagesandlimitations.

Beginning in 1996, pioneering work for two-phase LPME was conducted by Dasgupta [4] and Cantwell [5] with the introduction of single-drop microextraction (SDME), where a drop of organic liquid is immersed into an aqueous sample. Another im- portant milestone in the ﬁeld of microextraction, pioneered by Pedersen-Bjergaard and Rasmussen, was the introduction of the

https://doi.org/10.1016/j.chroma.2021.462769

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Fig. 1. A) Membrane-based liquid phase microextraction in 96-well format. B) The principle of three-phase liquid-phase microextraction. For the extraction of basic analytes the donor is alkalized to deprotonate the base. The neutral analyte diffuses across the SLM and is protonated in the acidic acceptor solution.

membrane-basedtechniquehollow-fiberliquid-phasemicroextraction (HF-LPME), which initiated the development of three-phase microextraction systems [6]. In later years, several alternatives have been developed, including dispersive liquid-liquid microextraction(DLLME)[7],dispersiveliquid-liquidmicroextractionbased on solidified floating organic droplets (DLLME-SFO) [8], solvent bar microextraction(SBME)[9],andother membrane-basedtech- niquessuchasmembrane-basedLPME(MB-LPME)ina96-wellformat[10,11] andelectromembraneextraction(EME) [12,13]. Inthe presentstudy,MB-LPMEina96-wellformatwasused(Fig.1A).

LPME has been applied in various fields of health and life science, such aspharmacology [14], forensics[15],environmental chemistry [16], clinical chemistry [17], toxicology [18], and anti- doping control[19]withscientificpublicationssteadilyincreasing from the mid-’90s until today. Although LPME has kept its rele- vance, implementationintoroutine laboratorieshasstill notbeen facilitated.Onereasonistheneedforcommercializedequipment, such asseenforsolid-phasemicroextraction(SPME).While LPME equipment isstill not commerciallyavailable,equipment forEME isveryclosetomarket,andwhenusedwithoutvoltage,itoperates asaMB-LPMEsystem[20].Anotherreasonistheneedforgeneric methods,wheremolecular descriptorscanbeusedto predictand selectappropriatestandardizedextractionconditionsforgivenan- alytes. Although a large number of validated applications have beenpublished[21–25],developmentofgenericmethodsfromthis material isdifficult.Extractionshavebeendone usingverydiffer- entexperimental conditions,andperformanceshavenotbeenan- chored sufficientlyinfundamentalunderstandingaboutpartition- ingandmolecularinteractions.

Therefore,inthepresentworkwehavelookedintoLPMEagain, nowfromahighlyfundamentalangle.Wehavestudiedtheextrac- tionofselectedbasicpharmaceuticalsasmodelanalytesinthelog Prangefrom−0.4to5.0, usingselecteddeepeutecticsolventsas theSLM.Withthelatter,hydrogenbonds,

π

^-

π

^-,ândîonicînterac-

tions werecontrolledandvariedsystematically.Foreach SLM,we investigated(1)whichmodelanalytessufferedfrompoorpartition into theSLM,(2)whichmodelanalyteswere extractedefficiently across theSLM,and(3)whichmodelanalytessufferedfrompoor partition fromSLMandintotheacceptor.Modelanalytesbelong- ing to (2) were within the optimal extraction window (OEW) of each SLM, andOEWs were assigned fordifferent SLMs basedon molecular interactionsandanalytelogPasasinglemolecularde- scriptor. The purpose of this wasto develop a starting point for the development ofgeneric methods.The work istherefore fundamental andgeneral, andextractionswere conductedonly from pure standard solutions.Applications, quantifications inbiological fluids, methoddevelopment,andvalidation,whicharerequiredto developthefinalgenericmethods,willfollowinfuturepapers.The

intentionisthatthecurrentpaperwillserveasfundamentalrefer- ence.

2. Experimental

2.1. Chemicalsandreagents

Coumarin,thymol,camphor,DL-menthol,di(2-ethylhexyl)phosphate,hydrochloride acid37%,sotalolhydrochloride, metaraminol bitartrate, atenolol, tyramine, ephedrine hydrochloride, metopro- loltartrate, pethidinehydrochloride,haloperidol,nortriptyline hydrochloride,loperamidehydrochloride,andmethadonehydrochlo- ride were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Sodiumhydroxide, acetonitrile(HPLCgrade), andformic acid99%

(LC-MS grade) were purchased from VWR (Radnor, PA, USA).

DeionizedwaterwasobtainedwithaMilipak® (0.22μmﬁlter)pu- riﬁcationsystemfromMilli-Q(Molsheim,France).

Individualstocksolutionsofeachanalytewerepreparedatcon- centrationsof1–4mgmL⁻¹anddissolvedinpuredeionizedwater or30%v/vmethanolindeionizedwater.Fromthese, apolarana- lytemix(sotalol,metaraminol, atenolol,tyramine,ephedrine,and metoprolol) and a non-polar analyte mix (pethidine, haloperidol, nortriptyline,loperamide,andmethadone)werepreparedwithan- alyte concentrations of 10 μg mL⁻¹ in 25 mM phosphatebuffer.

Theseanalytemixeswere pipettedintothedonorwells.Theﬁnal methanolconcentrationneverexceeded0.3%andwasnotassumed to affectextraction recoveries. Tocalculate therecovery, a 50μg mL⁻¹ standardpolarmixand50μg mL⁻¹standardnon-polarmix werepreparedin10mMHCl.Allsolutionswerestoredat4°Cand protectedfromlight.

DES were prepared by weighing and mixing appropriate amounts of HBA components (coumarin or camphor) and HBD components(thymolorDL-menthol)inmolarratiosof1:1and1:2.

The mixtures were heated in an oven (80 °C)for approximately 10minandvortexeduntilhomogenous.InSLMswithioniccarrier, DEHPwasaddedtotheDESmixturesinvolumeratiosof0.5,2,5, or10%.

2.2. Liquid-phasemicroextractionprocedure

The equipment used is previously described in [22]. The ex- tractionwasperformedwitha96-wellpolypropylenedonorplate with 0.5 mL wells from Agilent (Santa Clara, CA, USA). The ac- ceptorplatewasa96-well MultiScreen-IPﬁlterplatefromMerck Millipore (Carrigtwohill, Ireland). The membrane material was polyvinylidene ﬂuoride (PVDF) with a pore size of 0.45

μ

^m. ^A

Platemax Pierceable Aluminum Sealing Film (Axygen, Union City,

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CA,USA)wasusedtosealtheacceptorplate.Agitationduringex- traction wasaccomplishedwitha Vibramax 100agitation system fromHeidolph(Kellheim,Germany).

The extractionwasperformedby pipetting250μLofstandard solutionintothewellofthedonorplate.Theﬁlterontheacceptor platewasimpregnatedwith4μLofDES,creatingtheSLM.Accep- tor solution (50 μL) waspipetted into the wells of the acceptor plate. The acceptor and donor plate were clamped together and sealed with adhesive foil. The whole set-up wasplaced into the agitationdeviceforextractionfor60minat900rpm.Afterextrac- tion,thedonorandacceptorsolutionwerecollectedandanalyzed with HPLC-UV. Details to theuse of the extractionunit are pro- videdinSupplementarySection2.

2.3. HighperformanceliquidchromatographywithUV-detection

HPLC-UV analysis was performed on a 3000 Ultimate HPLC- UV (ThermoFisherScientiﬁc,Waltham,MA,USA)withanAcquity UPLC HSST3(2.1 mm I.D × 150mm, 1.8

μ

^m ^particle ^size)^pur-

chased fromWaters(Wexford, Ireland).Mobile phaseA consisted of0.1%formicacidin95:5deionizedwater/methanol(v/v).Mobile phase Bconsistedof0.1%formicacidin95:5methanol/deionized water(v/v).Furtherdetails ontheelutiongradients, detectionpa- rameters andchromatogramsareprovided inSupplementarySec- tion1.

2.4. Calculations

TheextractionrecoverywascalculatedbyEq.(1):

R= C_{a,f inal} Cd,initial ×Va

Vd × 100% (1)

Here,C_a_,_{f inal} andC_d_,_initialaretheconcentrationsintheacceptor aftertheextractionandtheconcentration ofanalyteinthedonor beforetheextraction,respectively.ThetermsV_aandV_ddenotethe volumeoftheacceptoranddonor,respectively.

3. Resultsanddiscussion

Eleven drugcompounds wereselected asmodelanalyteswith

−0.4<logP<5.0.Thisrangewaschosen,asitrepresentsacom- monrangeforsmall-moleculedrugs[26].Polaranalyteswerede- ﬁned ashaving logP < 2.0andnon-polar analyteswere deﬁned ashavinglogP>2.0.AnalytelogP,logDatpH2,andpKavalues areshowninTable1.

Table 1

Physical-chemical properties of the studied drugs.

Drug compound Log P Log D (pH 2) pK a

Sotalol −0.4 −3.2 9.43

Metaraminol −0.1 −2.5 9.68

Atenolol 0.4 −2.8 9.67

Tyramine 0.7 −2.0 9.66

Ephedrine 1.3 −1.9 9.52

Metoprolol 1.8 −1.5 9.67

Pethidine 2.5 −1.1 8.16

Haloperidol 3.7 0.2 8.05

Nortriptyline 4.4 1.2 10.47

Loperamide 4.7 1.3 9.41

Methadone 5.0 1.5 9.12

Chemicalize was used to generate structure properties (retrieved 27.02.2021, https://chemicalize.com ) developed by ChemAxon ( https://www.chemaxon.

com ) [28] .

3.1. Extractiontheoryanddeﬁnitionofoptimalextractionwindow

ThepartitioncoeﬃcientK₁betweenthedonorandtheSLMcan bewrittenas:

K1=Ceq,SLM

C_eq_,_d (2)

Here,C_eq,d andC_eq_,_SLMaretheequilibriumconcentrationsinthe donorandSLM,respectively.Correspondingly,thepartition coeﬃ- cientK₂betweentheSLMandacceptorcanbewrittenas:

K₂= Ceq,a

C_eq,SLM (3)

Here,Ceq,aistheequilibriumconcentrationintheacceptor.The overallpartitioncoeﬃcientKbetweenthedonorandacceptorcan beexpressedastheproductofK₁andK₂:

K= Ceq,a

C_eq_,_d = K1×K2 (4)

AssumingthatK isunaffectedby theorganicphase, K₂ isde- creasingwhenK₁isincreasing,andviceversa.

Basedonthepartitioncoeﬃcients,thetheoreticalextractionre- coveryinthree-phaseLPMEmaybecalculatedby:

R

(

^%

)

⁼ _K_×_V ^K^×^V^a

a+K1×VSLM+V_d ×100% (5)

Here, Va, V_d, and V_SLM denote the volumes of the acceptor, donor,andSLM,respectively.

Eq. (4) is valid forestimating extraction recoveries when the systemhasenteredequilibrium. The timerequiredto reachequi- librium is compound-dependent. Previous work has shown that partition into the SLM often is the rate-limiting factor in three- phase LPME[27].Thus, kinetics arecontrolled by K₁, andcan be modelledusingthefollowingequation:

C_d

(

^t

)

⁼^Cd⁰·exp

−ASLMDSLMK1

V_dh t

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Here,C_d(^t) ^is ^theconcentration inthe donorasa function of time,C_d⁰ is theinitial concentration in thedonor (t = 0), A_SLM is the surface area of the SLM, h is the thickness of the SLM, and D_SLMisthediffusioncoeﬃcientoftheanalyteintheSLM.

ExactvaluesofK₁andK₂ aregenerallynotavailable,unless1- octanol is used as the SLM. With 1-octanol, computer-generated logPandlogDvaluescanbeusedtodescribe theeﬃciencyofa three-phaseLPMEsystem. K₁isequaltoPfortheneutralspecies, whileK₂ issetto1/DforthespeciesatpH2.

SelectingthreecompoundswithdifferentlogPandlogDvalues fromoursetofmodelanalytes,namelymethadone,pethidine,and sotalol,thephasedistributionfort=60minwascalculatedbased onEq.(5) andEq.(6)usingcomputer-generated log PandlogD values[28].DetailsofthecalculationsarefoundinSupplementary Section3.Formethadone,logPis5.0, whilelogDatpH2is1.5.

ThecalculateddistributionisillustratedinFig.2.Thedonorisde- pleted,70%istrappedinthe SLM,andonly30%ofthe analyteis extractedintotheacceptor.Becausemethadoneisnon-polar,K₁is large,andthedonoristhereforerapidlydepleted.Correspondingly, K₂ is small,causing methadoneto suffer fromserious membrane trapping.Similar behavior isexpected forother analyteswithlog Phigherthan3.5,includingloperamideandnortriptylinefromour setofmodelanalytes,with1-octanolastheSLM.After60min,the extraction system has entered equilibrium, and membrane trap- pingremainsunaffecteduponextensionoftheextractiontime.

Forsotalol,logPandlogDare−0.4and−3.2,respectively.So- talolis a polarsubstance; K1 islow, while K2 is high. Therefore, partition intotheSLM ishighlyunfavorable,andmass transferis correspondinglyslow.After 60minofextraction,99%ofsotalolis

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Fig. 2. Calculated relative amounts of sotalol, pethidine, and methadone found in the donor (blue), SLM (yellow), and recovered in the acceptor (green) with t = 60 min.

still left in the donor, and extraction is limited by slow kinetics. With1-octanolastheSLM, slowkineticsistobe expectedforall substances withlog Pbelow 2.0. Recoveries can be improved by increasing extractiontime,andtheoretically, exhaustiveextraction isachievedattheend.However,thisrequiresextractionfarbeyond 60min,whichisoflittlerelevanceforanalyticalapplications.

For pethidine, logP and log Dare 2.5 and −1.0, respectively.

Comparedtomethadone,K₁islowerandK₂ishigher.Duetothis, thebalancebetweenK₁andK₂ismoreappropriate,andtheequi- librium distribution is much more in favor of high recovery for pethidine(Fig.2).WithK₁suﬃciently hightoprevent slowkinet- ics,withoutcausingmembranetrapping,theanalytesareextracted underidealconditions.With1-octanolastheSLM,similarbehavior isexpectedforotheranalyteswithlogPintherangeof2.0to3.5.

Thisistheoptimalextractionwindow (OEW)for1-octanol, where, intheory,exhaustiveextractionistobeexpected.

Although the partition coeﬃcients change if 1-octanol is re- placed byanothersolvent,theprinciplesdiscussedabove arestill valid. The here described theoretical trends have also been ob- servedinpreviousliterature[29,30].Thus,agivenSLMsolventhas an OEW,whereK₁ andK₂ arebalancedandexhaustiveextraction canbeexpected.Morepolaranalytes(lowK₁)areexpectedtosuf- ferfromslowkinetics,whilemorenon-polaranalytes(lowK₂)are pronetomembranetrapping.

In the following, OEWs were established for different SLMs, withreferencetoanalytelogP.

3.2. TuningSLMpropertieswithdeepeutecticsolvents

OEWs were investigated with different deepeutectic solvents (DES), also including combinations with DEHP as the ionic carrier.This enabledindividualassessmentofhydrogenbond-,

π

^-

π

^-

, and ionic interactions, and their impact on the OEW. Four eutectic components, two HBA components, and two HBD components were mixed inmolar ratios of 1:1and 1:2. Coumarin and camphorwereselectedasHBAcomponents,whilethymolandDL- mentholwereselectedasHBDcomponents.Coumarinandthymol, provided aromaticcharactertothedeepeutecticsolvents.Table2 gives an overview ofthe tested membrane compositions,including the number of HBA and HBD sites, and aromatic properties

(aromatic ring count).Mixtures ofcoumarin andDL-menthol did notformstabledeepeutecticmixtures,seenasprecipitationafter heating,andwere thereforenottested. DEScompositionswithor withoutioniccarrierwereselectedbasedon literatureandprevi- ousexperience[31–33].

3.3. Pureeutecticsolvents

Intheﬁrstsetofexperiments,theeffectofhydrogenbondin- teractionswasinvestigatedusingSLMswithdifferentHBA/HBDra- tios and an aromatic ring count equal to zero. Two SLMs were compared, namely camphor:DL-menthol in molar ratios 1:1 and 1:2 (HBA/HBD ratio 2.0 and 1.5, respectively). To establish mass balancedata,boththeacceptorandthedonorwereanalyzed.

Experimentaldataobtainedwithcamphor:DL-menthol(1:1)are summarized in Fig.3A. The resultswere in accordancewith the theoreticaldiscussioninSection3.1.Thepolarmodelanalytesso- talol, metaraminol,atenolol,tyramine,ephedrine,metoprolol,and pethidinelargelyremainedinthedonor.Thesecompounds,repre- sentingthe logP rangefrom −0.4to 2.5, suffered fromslow kinetics. Forhaloperidol,nortriptyline, loperamide,andmethadone, withlogPvalues between3.7and5.0, recoverieswere generally high, and this indicated that the extraction was under ideal or near-idealconditions inthe OEW.When the molarratio ofcam- phorandDL-mentholwaschangedfrom1:1to1:2(resultsshown inFig.3B),theHBA/HBDratiodecreasedfrom2.0to1.5.TheOEW wasseeminglyunaffected, buttheoverall membranetrappingin- creasedfor themodel analyteswithin thisregion. Since thearo- maticring count waszerowithboth SLMs,theslightincrease in membranetrappingwascausedbyincreasedHBDactivity.

Inanext setofexperiments,theeffectofaromaticring count was investigated. Three different SLMs were compared, namely coumarin:thymol, camphor:thymol, and camphor:DL-menthol, all inmolarratiosof1:1.TheseSLMsrepresentedthree,one,andzero aromatic ring counts, respectively, while the HBA/HBD ratio was 2.0inallcases.

Experimental data obtained with coumarin:thymol (1:1) are summarizedinFig.3C.ThisSLMwashighlyaromatic,witharing countofthree.Sotalol,metaraminol,atenolol,tyramine,ephedrine, andmetoprolol(−0.4< logP<1.8) wereproneto slowkinetics.

Pethidine(logP=2.5),however,wasextractedwith65%recovery andwaswithin theOEWforthisSLM.Forhaloperidol,nortriptyline,loperamide,andmethadone,withlogPbetween3.7and5.0, mass balance data veriﬁed signiﬁcant membrane trapping in the rangebetween64and100%.Withcamphor:thymol(1:1)(Fig.3D), thearomaticringcount wasone.Again,thepolaranalytesinthe range −0.4 < log P < 1.8 suffered from slow kinetics and were notextractedfromthedonor.Here,theOEWwasshiftedtowards lowerlogPvaluescomparedtopreviouslydiscussedSLMs.Thean- alytesin thelog Prangefrom2.5to 5.0were extractedto some extent, but membrane trapping still dominated. This was espe- cially evident for loperamide, possibly due to strong

π

^-

π

^inter-

actions between the SLM (ring count one) and loperamide (ring countthree).

With camphor:DL-menthol (1:1), the SLM was non-aromatic.

Pethidine wasnolonger within theOEW, whilehaloperidol, nortriptyline,loperamide,andmethadonewereallextractedwithhigh recoveries(Fig.3A).Sinceallthenon-polarmodelanalytesarearo- matic, thechange to a non-aromatic SLM increased their extraction into the acceptor. Therefore, the optimal extraction window wasshiftedslightlytowardshigherlogP.Loperamidewasnowex- tractedwithhighrecovery,mostprobablybecausethe

π

^-

π

^inter-

actions were absent. No signiﬁcant membrane trapping was ob- servedforthisSLM,butisassumedtobepresentabovethetested logPrange.

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Fig. 3. Relative amounts of model analyte found in the donor (blue), SLM (yellow), and recovered in the acceptor (green) after 60 min of extraction for A) camphor:DL- menthol (1:1) and B) camphor:DL-menthol (1:2), C) coumarin:thymol (1:1), D) camphor:thymol (1:1) The error bars represent the standard deviation (SD) of the acceptor with n = 4.

3.4. EutecticsolventswithDEHP

In thenext set ofexperiments,the effectofionic interactions was investigated by adding 0.5, 2, 5,and 10% DEHP to the non- aromaticSLMcamphor:DL-menthol(1:1).

Experimental data obtained with 0.5% DEHP are summarized in Fig.4A.Thepolaranalytessotalol, metaraminol,atenolol,tyra-

mine,andephedrineandthenon-polaranalyteshaloperidol,nortriptyline, andloperamidewere all extracted withlow recoveries (<30%).Asobservedinthemassbalancedatathemajorityofan- alyte was trapped in the SLM. Satisfactory extraction recoveries (> 60%),were measuredforthenon-polaranalytespethidineand methadone. The data show no apparent correlation with analyte log P values. When increasing the DEHP percentage to 2% (See

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Fig. 4. Relative amounts of model analyte found in the donor (blue), SLM (yellow), and recovered in the acceptor (green) after 60 min of extraction for A) camphor:DL- menthol (1:1) + 0.5% DEHP, B) camphor:DL-menthol (1:1) + 2% DEHP, C) camphor:DL-menthol (1:1) + 5% DEHP, and D) camphor:DL-menthol (1:1) + 10% DEHP, E) camphor:DL-menthol (1:1) + 5% DEHP, F) camphor:thymol (1:1) + 5% DEHP, and G) coumarin:thymol (1:1) + 5% DEHP. Sample size n = 4. The error bars represent the standard deviation (SD) of the acceptor with n = 4.

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Table 2

Overview of tested membranes, including computer-generated HBA sites, HBD sites, aromatic ring count, and molecular structures of the eutectic components.

SLM composition HBA sites ^a HBD sites ^a

Aromatic ring count ^a

Ionic carrier (Y/N)

Coumarin:thymol (1:1) 2 1 3 N

Camphor:thymol (1:1) 2 1 1 N

Camphor: DL-menthol (1:1) 2 1 0 N

Camphor: DL-menthol (1:2) 3 2 0 N

Camphor: DL-menthol (1:1) + 0.5% DEHP 2 1 0 Y

Camphor: DL-menthol (1:1) + 2% DEHP 2 1 0 Y

Coumarin:thymol (1:1) + 5% DEHP 2 1 3 Y

Camphor:thymol (1:1) + 5% DEHP 2 1 1 Y

Coumarin Thymol Camphor DL-menthol DEHP

aChemicalize was used to generate structure properties (assessed by 01.2020, https://chemicalize.com ) developed by ChemAxon ( https://www.chemaxon.com ) [28] .

Fig. 4B), theextraction recoveries of the polaranalytes generally increased. The increase was both due to increased donor deple- tion anddecreased membrane trapping. Extraction recoveries all decreasedforthenon-polaranalytes.Fromthemassbalancedata, it isevident that thiswasdueto an increasein membranetrapping.

ForhigherDEPHconcentrations,namely5and10%,similarob- servationsweremade(Fig.4Cand4D).Theextractionrecoveriesof thepolaranalyteswerearound50%,exceptforatenolol,whichsuf- fered frommembranetrapping.Forthe majorityofthe non-polar analytes,therecoverieswerebelow10%,andmassbalancedatare- vealed serious membranetrapping.Pethidine wasextracted close to ideal conditions when DEPH concentrations were high, while the extraction recovery of methadoneﬂuctuated with the higher DEPHconcentrations.

From theobtainedmassbalancedata,additionofDEHPgener- ally increasedthe extraction ofpolar analytesfrom 5-to a 50%- level. This showsthat ionic interactions are essential for the ex- tractionoftheseanalytes,evenwithdeepeutecticsolvents.DEHP increases the partition into the SLM, and the donor is depleted.

For thenon-polar analytes, exceptpethidine andmethadone, the addition of an ionic carriersigniﬁcantly increasedthe amount of membrane trapping. For nortriptyline, asan example,membrane trapping increased from 20 to 93% upon addition of 10% DEHP.

It is suspected that the ionexchange at the membrane-acceptor interface is too weak to accommodate for the strong ionic and hydrophobic interactions betweenthenon-polaranalytesandthe SLM.Pethidineandmethadonearebehavingsimilarlytothepolar analytes, wherehigher DEHP concentrations increaseddonor de- pletion.

Inthelastsetofexperiments,thecombinedeffectofaromatic- ityandionicinteractionswereinvestigated.Hydrogenbondingwas not investigatedfurther, as previous experiments revealedan in- signiﬁcant effect on the OEW. For this last set of experiments, 5% DEHP was added to coumarin:thymol, camphor:thymol, and camphor:DL-menthol, all inmolar ratios of1:1. These SLMsrep- resented zero, one, and three aromatic ring counts, respectively;

while theHBA/HBDratio wasequal to2.0in allSLMs.Mass balance data are shownin Fig. 4. Forthe polaranalytes, the high-

estextractionrecoverieswere obtainedforcamphor:thymol +5%

DEHP(Fig.4F).ThisSLM hasan aromaticringcountequaltoone.

Furtherincreasingordecreasingthearomaticringcountincreased theamountofmembranetrapping.ThisSLMwastheoverallbest for the polar analytes. For the non-polar analytes, the increase inaromaticringcount decreasedthe extractionrecoveriesdueto membrane trapping.This showsthat aromatic SLMs incombina- tionwith DEHPare highlyunfavorablefor theextractionof non- polaranalytes.

4. Conclusion

In the present study, we proposed the terms optimal extraction window (OEW), slow kinetics, and membrane trapping, to express the log P range where a given three-phase membrane- basedliquid-phasemicroextraction(MB-LPME)systemcan beex- pectedtobeoptimal.Weinvestigatedaselectionofsupportedliq- uidmembranes(SLM)basedondeepeutecticsolvents(DES)with varying HBA/HBD andaromatic ring count, and established their OEWs using a set ofpharmaceutical drugs (−0.4< logP < 5.0) as model analytes. With pure DES, extraction was primarily facilitated by hydrogen bond and

π

^-

π

interactions, and the OEWs were typically within the range 2 < log P < 5. Model analytes withlogP<∼2sufferedfromslowkinetics,whilemodelanalytes withlogP>∼3.5werepronetomembranetrapping.OEWswere slightlyaffectedby theHBA/HBDratioandshiftedtowardshigher logPvalueswithincreasingaromaticringcount.WithintheOEWs, themodelanalyteswereextractedwithhighrecoveries,exceptfor highlyaromaticones,whichwerepronetostrong

π

^-

π

interactions andmembranetrapping.Althoughthe pureDESwere strongsol- vents regardinghydrogen bond and

π

^-

π

interactions, they were insuﬃcientfor theextraction ofpolaranalytes (logP < 2). With the addition of ionic carrier (DEHP) to the SLMs, polar analytes wereeﬃcientlyextractedandtheOEWsshiftedtotherange−0.5

<logP<2.TheSLMwith5%DEHPandmoderatearomaticityre- sultedinextractionrecoveriesofupto80%forthepolaranalytes.

Forthenon-polaranalytes,however,thesameSLM sufferedfrom membranetrapping.Theresultsfromourstudyshowthatagiven three-phase liquid-phase microextraction systemis eﬃcient only

(8)

in a relatively narrow log P range within the optimal extraction window(OEW).MinorshiftsoftheOEWcanbeexpectedfromal- terationsofHBA,HBD,andaromaticringcount,whilemajorshifts canbeexpectedwhenintroducingioniccarriers.

Identifying OEWs for different SLMs is a step towards a bet- ter understanding of LPME. In the future, this can help users in routine laboratoriestodeﬁne thepossibilitiesandlimitationsofa given LPME system. More research on SLMs with an added carrier is plannedto increase knowledge on carrier-mediated LPME.

Thiswill helpdevelopingbettertheoretical modelstopredictop- erationalconditionsandperformance.

Supplementaryinformation

HPLC-UVmethod– Additionaldetails;MB-LPMEin96-wellformat– Equipmentandhandling;Calculationsfortheoreticalextrac- tionmodel.

DeclarationofCompetingInterest

Theauthorsdeclarethattheyhavenoknowncompetingﬁnan- cial interests that could have appeared toinﬂuence the workre- portedinthispaper.

Supplementarymaterials

Supplementary material associated with this article can be found,intheonlineversion,atdoi:10.1016/j.chroma.2021.462769. CRediTauthorshipcontributionstatement

MariaSchüller:Conceptualization,Methodology, Formalanaly- sis,Investigation,Writing– originaldraft,Writing– review&editing,Visualization.KimTuThiTran:Methodology,Formalanalysis, Investigation,Writing– review&editing.ElisabethLeereØiestad:

Supervision,Writing– review&editing.StigPedersen-Bjergaard:

Conceptualization, Methodology, Formal analysis, Writing – originaldraft,Writing– review&editing,Supervision.

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