Solvent-assisted stir bar sorptive extraction by using swollen polydimethylsiloxane for enhanced recovery of polar solutes in aqueous samples: Application to aroma compounds in beer and pesticides in wine

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ContentslistsavailableatScienceDirect

Journal

of

Chromatography

A

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

Solvent-assisted

stir

bar

sorptive

extraction

by

using

swollen

polydimethylsiloxane

for

enhanced

recovery

of

polar

solutes

in

aqueous

samples:

Application

to

aroma

compounds

in

beer

and

pesticides

in

wine

Nobuo

Ochiai

a,∗

,

Kikuo

Sasamoto

a

,

Frank

David

b

,

Pat

Sandra

b a_GERSTEL_K.K._1-3-1_Nakane,_Meguro-ku,_Tokyo_152-0031,_Japan

b_Research_Institute_for_{Chromatography,}_President_Kennedypark_26,₈₅₀₀_Kortrijk,_Belgium

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received12April2016

Receivedinrevisedform24May2016

Accepted25May2016

Availableonline26May2016

Keywords:

Solvent-assistedstirbarsorptiveextraction

(SA-SBSE)

SolventswollenPDMS

Polarsolutes

Aromacompoundsinbeer

Pesticidesinwine

a

b

s

t

r

a

c

t

Anovelsolvent-assistedstirbarsorptiveextraction(SA-SBSE)techniquewasdevelopedforenhanced recoveryofpolarsolutesinaqueoussamples.AconventionalPDMSstirbarwasswolleninseveral sol-ventswithlogKowrangingfrom1.0to3.5whilestirringfor30minpriortoextraction.Afterextraction,

thermaldesorption–gaschromatography–(tandem)massspectrometry(TD-GC-(MS/)MS)orliquid desorption–largevolumeinjection(LD-LVI)-GC–MSwereperformed.Aninitialstudyinvolved investi-gationofpotentialsolventsforSA-SBSEbyweighingoftheresidualsolventintheswollenPDMSstirbar beforeandafterextraction.ComparedtoconventionalSBSE,SA-SBSEusingdiethylether,methylisobutyl ketone,dichloromethane,diisopropyletherandtolueneprovidedhigherrecoveriesfromwatersamples fortestsoluteswithlogKow<2.5.ForSA-SBSEusingdichloromethane,recoverieswereimprovedby

fac-torsof1.4–4.1,whilemaintainingorevenimprovingtherecoveriesfortestsoluteswithlogKow>2.5.

TheperformanceoftheSA-SBSEmethodusingdichloromethane,diisopropylether,andcyclohexaneis illustratedwithanalysesofaromacompoundsinbeerandofpesticidesinwine.

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

1. Introduction

Severalminiaturized and solventless (orsolvent minimized) samplepreparationtechniqueshavebeendescribedforisolation and extractionof traceorganiccompounds in various matrices prior to chromatographic analysis. These include liquid phase microextraction (LPME) [1], dispersive liquid–liquid microex-traction (DLLME) [2], single drop microextraction (SDME) [3], headspace(HS)[4],solidphase microextraction(SPME)[5],etc. Successfulapplicationofthedifferenttechniquesdependson suit-ablematchingtoanalyteandmatrixcharacteristics.

Stirbarsorptiveextraction(SBSE)wasintroducedin1999as aminiaturized andsolventless extractiontechnique initiallyfor aqueoussamples[6].SBSEallowsextractionandconcentrationin asinglestepprovidingveryhighsensitivityespeciallywith ther-maldesorptionon-linecoupledtogaschromatography(TD-GC). SBSEhasbeensuccessfullyappliedindifferentﬁeldsincludingfood, ﬂavor,environmental,lifeandbiomedicalscience[7–10].Several

∗Correspondingauthor.

E-mailaddress:[email protected](N.Ochiai).

authorsindicatedthatSBSEusingpolydimethylsiloxane(PDMS)as extractionphaseallowshighrecoveryandextremelylowlimitsof detection(LOD)downtothesub-ngL−1 _level_for_extraction_and

enrichmentofrelatively apolarsolutescharacterizedby a loga-rithmofoctanol-waterpartitioningcoefﬁcient(logKow)>3.0.For

morepolarsolutes(logKow<3.0),SBSEwithin-situderivatization

wasdeveloped[7].Derivatizationreactionsarefunctionoftheclass ofcompoundstargeted(e.g.phenolic solutes)andaretherefore notgenerallyapplicable.Severalalternativestirbarcoatingswith different polarities (e.g. alkyl-diol-silica (ADS) restricted access material(RAM)[11],monolithicmaterials[12],polyurethane[13], PDMS/polypyrrole[14],andPDMS/␤-cyclodextrin[15])havebeen developed to extendthe applicability of SBSE to polar solutes

[16].However,theseextractionphasesaremostlyonly compati-blewithliquiddesorption(LD)and/orhaveinferiorperformance characteristicsrelatedtorobustness,bleeding,stability,etc. com-paredtoPDMS[10].Acommerciallyavailablestirbarcoatedwith polyethyleneglycol-modiﬁedsilicone(EG SiliconefromGERSTEL GmbH&Co.KG,MulheimanRuhr,Germany)canbeusedwith TD-GCanalysisbutthestirbartendstoencounterphysicalchangeon thecoatingwhenre-usedseveraltimes.Toovercomethis, multi-SBSE(mSBSE)incorporatingaspecial samplingmodefortheEG

http://dx.doi.org/10.1016/j.chroma.2016.05.085

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Siliconestirbar(aswellasforthePDMSstirbar)wasdevelopedin 2013[17].TheEGSiliconestirbarisattachedontheinnersidewall ofthevialbyusingamagneticclipplacedontheoutersidewall ofthevial,whilearobustPDMSstirbarisstirringatthebottomof thevialforagitationofthesampleaswellasforextractionofthe solutes.Theseresearchworkshighlightthechallengesin develop-ingnewphasesforSBSE.Duringresearchitbecameclearthatthe followingcriteriashouldbemet:(1)acoatingshouldbethermally stabletoallowthermaldesorption,(2)thecoatingshouldberobust enoughtoavoidphysicaldamages(e.g.scratchesand/orcracks) duringstirringinthevial,(3)thecoatingshouldgiveasigniﬁcant improvementversusPDMS,and(4)productionofthecoatedstir barshouldbepossibleinaneasyandreproducibleway(asinthe caseofPDMScoatedstirbar).

Xuand LeedevelopedanalternativeLPMEapproach usinga

silicamonolithasan extractionphase holder insteadofhollow ﬁber.ThisLPMEmethodtermedsolvent-barmicroextractionusing asilicamonolith(SBME/SM)with1-octanolastheextraction sol-ventfollowedbyLD-liquidchromatography(LC)wasdemonstrated foranalysisofpolycyclicaromatichydrocarbons(PAHs) inriver water(spikedat50ng/mL)[18].Spangenbergetal.demonstrated asimilarapproachusingamonolithicstirbarwithimmobilized 1-butanolforextractionof17␣-ethinylestradiolinwater[19].

Interesting techniques were introduced by Bicchi et al. The authorsdevelopedtwokindsofPDMS-tubingdevicesin combina-tionwithcarbonmaterialorsolventinside.Theformerdevice(with carbonmaterial)termedDualPhaseTwisterwasdemonstratedfor headspacesorptiveextraction(HSSE)ofcoffeeandsage,andSBSEof whisky[20].Thelatterdevice(withsolvent)wasdemonstratedfor HSSEofsage,thyme,andcoffee.ThemethodusingPDMS-tubing andsolventwastermedsolvent-enhancedHSSE (SE-HSSE)[21]. IncontrastwithotherapproachesusingPDMS-tubingandsolvent

[22,23],theinnersolventinSE-HSSEisnotusedasanacceptor oftheanalytes,butactsasamodiﬁerofthepolarityofthePDMS inwhichitdiffuses.SE-HSSEusingethylacetateandcyclohexane improvedboththeanalyterangeenrichedandthesensitivityfor theabovementionedapplications.

Inthisstudy,weextendedtheconceptsofbothSBMEand SE-HSSEtoSBSEusingasolventswollenPDMSstirbar.Wecallthe techniquesolvent-assistedstirbarsorptiveextraction(SA-SBSE). Afterextraction,TD-GCcoupledto(tandem)massspectrometry (MS/)MSorliquiddesorption(LD)followedbylargevolume injec-tion(LVI)-GC–MSwere performed.Thesolventabsorbed inthe swollenPDMSphasepartitionsintotheaqueousphaseandreaches anequilibriumbetweenPDMSandaqueousphaseduring extrac-tion.Hereby,thesolventactsnotonlyasamodiﬁerofthePDMS (increasingdiffusion),butalsoasanadditionalextractionmedium, resultinginenhancedrecoveryofsolutesfromtheaqueousphase. EspeciallyforrelativelypolarsoluteswithlogKow<3.0,recoveries

aresigniﬁcantlyimproved.TwelvesolventswithlogKowranging

from−0.24(acetone)to3.90(hexane)wereinitiallyexaminedon theirusefulnessforSA-SBSE.EightwithlogKow:0.86–3.90were

selectedforSA-SBSEofspikedwaterwithawiderangeoftest com-pounds(logKow:0.56–4.21).TheperformanceofSA-SBSEisfurther

illustratedwithtworealworldapplicationsnamelytheanalysisof aromacompoundsinbeerandthedeterminationofpesticidesin wine.

2. Experimental

2.1. Reagentsandmaterials

Acetone, acetonitrile, cyclohexane, dichloromethane, diethyl ether, diisopropyl ether, ethyl acetate, hexane, methyl acetate,

methyl isobutyl ketone, tetrahydrofuran, and toluene were

obtainedfromWakoPureChemicalindustriesLtd.(Osaka,Japan). 2-acetylpyrrole,2-acetylthiazole,benzylalcohol,butanoicacid, carbaryl, citronellol, coumarin, ␤-damascenone, decanoic acid, ethiofencarb,ethylhexanoate,ethyloctanoate,furaneol,guaiacol, hexanoicacid, cis-3-hexenol, 1-hexanol,indole, linalool,maltol, methionol,2-methylpropanoicacid,3-methyl butanoicacid,␥ -nonalactone,octanoicacid,phenethylacetate,phenethylalcohol, and vanillin were obtained from Kanto Kagaku (Tokyo, Japan). Azoxystrobin, benalaxyl, bitertanolI/II, chlorpyrifos, cyprodinil, dichlobenil, diethofencarb, difenoconazole I/II, dimethomorph Z/E, diphenylamine, epoxiconazole, fenbuconazole, flucythri-nateI/II,fludioxonil,flusilazole,iprodione,iprodionemetabolite, kresoxim-methyl,metalaxyl,metolachlor,myclobutanil, paclobu-trazol,penconazol,o-phenylphenol,procymidone,pyraclostrobin, pyrimethanil,tebuconazole,tetraconazole,triadimefon,and triadi-menolI/II wereobtainedfromHayashiPure ChemicalIND.Ltd. (Tokyo,Japan).For aromacompounds(including12test solutes usedintheSection3.1.2),astockstandardsolutioncontainingeach solutewaspreparedat1000␮g/mLor10,000␮g/mLinethanoland keptat4◦C.Thestockstandard solutionswerethenmixedand dilutedwithethanoltoprepareseverallevelsofworkingstandard mixtures.Onetofortymicrolitersoftheworkingstandardmixtures werefinallyaddedtoasampleaccordingtothecalibrationlevel. Forpesticides,astockstandardsolutioncontainingeachsolutewas preparedat1000␮g/mLinacetoneandkeptat−20◦C.Thestock standardsolutionswerethenmixedanddilutedwithacetoneto prepareseverallevelsofworkingstandardmixtures.Onetoten microlitersoftheworkingstandardmixtureswerefinallyaddedto asampleaccordingtothecalibrationlevel.Pilsnertypebeerand whitewinewerepurchasedfromlocalstoresinTokyo,Japan. 2.2. Instrumentation

Thethermaldesorption(TD)-GC–MSanalysiswasperformed

witha thermal desorption unit (TDU) equipped witha MPS 2

auto-samplerandaPeltiercooledCIS4programmedtemperature vaporization(PTV)inlet(GERSTEL,MülheimanderRuhr,Germany) installedonanAgilent7890Agaschromatographwitha 5975C singlequadrupoleMS(QMS)(AgilentTechnologies,CA,USA)or a7000BtriplequadrupoleMS(QQQ-MS)(Agilent).TheGC-QMS wasequippedwithacapillaryﬂow technology(CFT)platewith make-upgasforback-ﬂushcapability.

2.3. Samplepreparation

Stir bars (Twister) coated with 63␮L PDMS (10mm

length×1.0mm thickness)were obtainedfromGERSTEL.PDMS

stirbarsfromthesamebox(having thesameproductionbatch andlotnumber)wereusedasonebatchforSA-SBSE.ForSA-SBSE, 10mLheadspace(HS)vialswithscrewcapcontainingPTFE-coated siliconsepta(GERSTEL),andamultiplepositionmagneticstirrer (20 positions) fromGlobal Change (Tokyo, Japan) was applied. Priortouse,thestirbarswereconditionedfor30minat280◦Cina ﬂowofhelium.Fivemillilitersofsamplewastransferredtoa10mL

HSvial and30% NaCl wasdissolvedin thesample. To prepare

swollen PDMS stir bars, the conditioned PDMS stir bars were

initiallystirredatroomtemperature(25◦C)with1–2mLofsolvent inthesealed10mLHSvialsfor30minwhilestirringat800rpm. Then,theswollenPDMSstirbarwasaddedtothesamplevialand thevialwassealed.SA-SBSEwasperformedatroomtemperature (25◦C)for60minwhilestirringat800rpm.Afterextraction,the stirbarswereremovedwithamagneticrod(Twistertakingtool, GERSTEL)andforceps,rinsedbrieﬂyinultrapurewater,anddried withalint-freetissue.ForTD-GC-(MS/)MSanalysis,thestirbars wereplaced ina glassthermal desorptionliner.Theglass liner wasplacedintheTDUtray.ForLD-LVI-GC–MSanalysis,thestir

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barswereplacedinthesealed10mLHSvialcontaining0.5mLof acetone.Thestirbarswerestirred atroomtemperature(25◦C) for30minwhilestirringat800rpm.Aftersolventbackextraction, theacetoneextractwastransferredtoa2mLvial.Thesealed2mL vialwasplacedintheMPS2tray.Reconditioningofstirbarsafter usewasdonebysoakinginultrapurewater andacetonitrilefor 1–2h each;stir bars werethen removed fromthesolvent and driedonacleansurfaceatroomtemperaturefor1h.Finally,the stirbarswerethermallyconditionedfor30minat280◦Cinaﬂow ofhelium.Typically,thesamePDMSstirbarcouldbeusedmore than50timesforSA-SBSE.

2.4. Thermaldesorption(TD)

It is very important to use a two-step thermal desorption

(TD)programwithmoderatetemperatureprogrammingrate(e.g. 10–40◦C/min)whichenablesintheﬁrststepsolventventingandin thesecondstepanalytedesorption.TheuseofasinglestepTD pro-gramwithfasttemperatureprogrammingrate(e.g.>100◦C/min) forsolventswollenPDMSstirbarsmaycausecrackingofthePDMS phase.

For the analysis of test compounds in spiked water and

pesticidesinwine,thestirbarswerethermallydesorbedby pro-grammingtheTDUfrom30◦C(heldfor0.5min)at10◦C/minto 60◦C or100◦C(heldfor5min),at35◦C/minto250◦Cor280◦C (heldfor3min)with100mL/mindesorptionﬂow.Desorbed com-poundswerefocusedeitheronaTenaxTApackedlinerat20◦C or ona quartzwoolpackedliner at10◦C inthe Peltiercooled PTVinletforsubsequentGC–MS(/MS)analysis.Afterdesorption, thePTVinletwasprogrammedfrom20◦Cto240◦C(heldduring thetotalGCruntime)or10◦Cto280◦C(heldforGCruntime)at 720◦C/mintoinjecttrappedcompoundsontotheanalytical col-umn.Theinjectionwasperformedeitherinthesplitmodewitha splitratioof1-1usingthelowsplitoption(GERSTELK.K.,Tokyo, Japan)orinthesplitlessmodewiththesplitvalveclosedfor3min. 2.5. Largevolumeinjection(LVI)usingathermaldesorptionunit andmicro-vialinsert

For the analysis of aroma compounds in beer, 100␮L large volumeinjection(LVI)oftheacetoneextract(obtainedfromthe sol-ventbackextraction)wasperformedwiththeTDUsystemthatacts asatwo-stageinlet.Thissystemallowsoptimizationofinlet con-ditionsforsolventventing,analyterefocusingandtransfertothe columnindependentofthepresenceofmatrixcomponents[24]. Afterautomatedinjectionintoaglassmicro-vialthatcanbeheated intheTDU,non-volatilematrixcompoundsareleftinthemicro-vial anddonotcontaminatestheinlet.Volatilesaresplitlesstransferred totheinletwheretheyarerefocusedbeforeintroductionintothe GCcolumn.Finally,theTDUlinercontainingthemicro-vialinsertis returnedtotheauto-samplertray.TheTDUwasprogrammedfrom 30◦C(heldfor0.5min)at140◦C/minto80◦C(heldfor7min)with

100mL/mindesorptionﬂow.Desorbedcompoundswerefocused

onaTenaxTApackedlinerat20◦CinthePeltiercooledPTVinlet forsubsequentGC–MSanalysis.Afterdesorption,thePTVinletwas programmedfrom20◦Cto240◦C(heldfortotalGCruntime)to injecttrappedcompoundsontotheanalyticalcolumn.The injec-tionwasperformedinthepulsedsplitmodewithasplitratioof 1–3.

2.6. GC-QMSanalysis

Foranalysisoftestcompoundsinspikedwaterandaroma com-poundsinbeer,separationswereperformedona20m_×0.18mm i.d.×0.30␮mﬁlmthicknessDB-Waxcolumn(Agilent).The col-umntemperaturewasprogrammedfrom40◦C(heldfor3min)at

Fig.1.Acomparisonbetweenasolvent(diisopropylether)swollenPDMSstirbar

(a)andaconventional(conditioned)PDMSstirbar.

5◦C/minto240◦C(heldfor17min).After50min,thecapillary col-umnwasback-ﬂushed.Heliumwasusedascarriergasataﬂowof 1.0mL/min.TheMSwasoperatedinscanmodeusingelectron ion-izationat70eV.Scanrangewassetfromm/z29–300andasampling rateofthree,resultinginscanrateof2.68scan/s.

2.7. GC-QQQ-MSanalysis

Foranalysisofpesticidesinwine,separationswereperformed ona 30m×0.25mm i.d.×0.25␮mﬁlm thicknessDB–5ms

col-umn(Agilent).Thecolumn temperaturewasprogrammedfrom

50◦C(heldfor1min)at25◦C/minto125◦C,at10◦C/minto300◦C (heldfor10min).Postrun wasperformedat 320◦C for10min. Thisisthetemperatureprogramthatcanbeusedincombination withtheintelligentMRMpesticidedatabase(AgilentTechnologies Japan,Ltd.).Heliumwasusedascarriergas.Theheadpressurewas adjustedtoelutechlorpyrifosmethylataconstantretentiontime of13.443min[25].TheQQQ-MSwasoperatedinselectedreaction monitoring(SRM)modewiththeselectedtransitions(precursor toproduction)[26].Transitionsofthedetected32pesticidesin thewinesamplearelistedinTable3.Theelectronaccelerating voltageoftheEIwas70eV.Nitrogenwasusedascollisiongasat 1.5mL/min,andacollisionenergyof0–40VwasusedforMS/MS experiments.

2.8. Dataanalysis

MSDChemStationversionE.02.02.1431(Agilent),MassHunter qualitative analysis version B.06.00633 (Agilent), MassHunter Quantitative Analysisversion B.07.00.457(Agilent), and Aroma Ofﬁce 2D data base version 4.01.00 (Gerstel KK, Tokyo, Japan) wereusedfor dataanalysis.AromaOfﬁce2Dcontainsthemost comprehensivedatabaseofaromacompoundsavailable(>101,000 entries).Thissoftwareisasearchabledatabasewhichcontains lin-earretentionindices(LRI)informationforawiderangeofaroma compoundsfrommanyliteraturereferences.ThelogKow values

werecalculatedwithanSRC-KOWWINversion1.68software pack-age(SyracuseResearch,Syracuse,NY).

3. Resultsanddiscussion 3.1. EvaluationofSA-SBSE

3.1.1. SelectionofpotentialsolventsforSA-SBSE

Fig.1showsacomparisonbetweenasolventswollenPDMSstir bar(30minstirringindiisopropylether)andaconventional (con-ditioned)PDMSstirbar.Swellingandde-swellingofPDMSwith

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Table1

TheamountoftheabsorbedsolventintheswollenPDMSsirbarbeforeandafterSA-SBSE.

No. Compound logKow Sratioa BeforeSA-SBSE AfterSA-SBSE Rratiob

Solventabsorbed(mg) RSD%(n=3) Solventresidual(mg) RSD%(n=3)

1 Acetone −0.24 1.06 21 0.10 0.50 0.26 0.024 2 Acetonitrile −0.15 1.01 20 5.0 0.90 0.20 0.046 3 Methylacetate 0.37 – 37 0.30 2.4 0.058 0.064 4 Ethylacetate 0.86 1.18 67 1.3 13 0.95 0.20 5 Tetrahydrofuran 0.94 1.38 100 0.64 5.6 0.32 0.056 6 Diethylether 1.05 1.38 84 1.6 35 1.7 0.41

7 Methylisobutylketone 1.16 – 59 0.70 47 0.85 0.79

8 Dichrolomethane 1.34 1.22 110 0.31 62 1.1 0.56

9 Diisopropylether 1.88 – 88 2.5 72 3.9 0.81

10 Toluene 2.54 1.31 82 2.1 77 1.6 0.94

11 Cyclohexane 3.18 1.33 79 2.9 71 2.2 0.90

12 Hexane 3.90 1.35 77 3.1 66 4.9 0.85

a_Swelling_ratio:_the_difference_of_the_length_between_PDMS_in_the_solvent_and_the_dry_PDMS_[Ref._[27]].

b _Residual_ratio:_the_ratio_of_the_solvent_amount_in_the_PDMS_stir_bar_between_before_SA-SBSE_and_after_SA-SBSE.

Table2

Selectedaromacompounds,logKow,selectedions,relativepeakratioofSBSEandSA-SBSE,linearity,concentration,andrepeatabilityobtainedforSA-SBSE-LD-LVI-GC–MS

ofbeer.

No. Compound logKow m/zd Relativepeakratioe QuantitationbySA-SBSEusingDIPE

SBSE SA-SBSE r2i _{Concentration}

(ng/mL) RSD(%)j (n=6) DCMf _DIPEg _CyHxh 1 2,3-Butanediol −0.36 45 1.0 11 27 1.0 – – 6.2 2 Maltol −0.19 126 1.0 22 5.4 0.63 0.9919b ₄₁₀ _6.9 3 Methionol 0.44 106 1.0 16 17 1.7 0.9929b ₁₁₀₀ _6.3 4 Furfurylalcohol 0.45 98 1.0 12 21 1.8 – – 6.3 5 2-Acetylpyrrole 0.56 109 1.0 13 9.2 1.5 – – 4.8 6 2-Acetylfuran 0.80 110 1.0 10 3.9 1.9 – – 7.3 7 Furaneol 0.82 128 1.0 33 22 1.4 0.9959b ₁₅₀ _6.2

8 2-Methylpropanoicacid 1.00 73 1.0 6.8 25 2.0 0.9907c ₁₇₀₀ _5.1

9 Vanillin 1.05 152 1.0 8.2 1.4 1.5 0.9941a _3.7 _2.5

10 Butanoicacid 1.07 60 1.0 3.8 12 2.7 0.9962c ₁₁₀₀ _5.3

11 Guaiacol 1.34 124 1.0 4.6 9.6 2.0 0.9985a _1.6 _8.0

12 3-Methylbutanoicacid 1.56 60 1.0 9.1 25 2.1 0.9863b ₁₅₀₀ _3.8

13 Phenethylalcohol 1.57 122 1.0 6.8 6.8 1.8 0.9920c ₅₀₀₀ _3.7 14 1-Hexanol 1.82 56 1.0 2.3 3.6 2.4 0.9958a ₁₅ _2.6 15 Hexanoicacid 2.05 60 1.0 5.3 6.5 1.9 0.9994c ₁₄₀₀ _3.6 16 Indole 2.05 117 1.0 1.7 2.1 1.2 0.9930a _1.2 _5.4 17 ␥-Nonalactone 2.08 85 1.0 1.8 2.2 1.2 0.9917a ₃₂ _3.4 18 4-Vinylguaiacol 2.24 150 1.0 2.3 2.6 1.5 – – 2.1 19 4-Vinylphenol 2.41 120 1.0 4.8 7.8 1.4 – – 2.3 20 Phenethylacetate 2.57 104 1.0 0.90 1.0 1.0 0.9970c ₁₀₀₀ _1.8 21 Ethylhexanoate 2.83 88 1.0 0.55 0.87 1.0 0.9986b ₁₇₀ _3.2 22 Octanoicacid 3.03 60 1.0 2.1 3.2 1.0 0.9901b ₂₀₀₀ _3.2 23 Linalool 3.38 93 1.0 0.71 0.89 0.95 0.9951a _1.1 _4.6 24 Nonanoicacid 3.52 60 1.0 1.8 2.4 1.2 – – 4.0 25 Citronellol 3.56 95 1.0 0.63 0.85 1.0 0.9939a _1.4 _8.7 26 Ethyloctanoate 3.81 88 1.0 0.27 0.67 0.75 0.9953b ₄₄₀ _3.7 27 Decanoicacid 4.02 60 1.0 2.3 4.1 3.9 0.9891b ₃₉₀ _3.7 28 ␤-Damascenone 4.21 190 1.0 0.42 0.72 0.79 0.9959a _1.4 _8.0

a_Linearity_range_was_1–40_ng/mL.

b _Linearity_range_was_200–4000_ng/mL.

c _Linearity_range_was_1000–10000_ng/mL.

d _Selected_ions_for_relative_peak_ratio,_{quantitation,}_and_{repeatability.}

e_Relative_peak_ratio_of_SBSE_and_SA-SBSE_(normalized_by_SBSE_peak_area).

f _SA-SBSE_using_{dichloromethane.}

g_SA-SBSE_using_diisopropyl_ether.

h _SA-SBSE_using_cyclohexane.

i _Linearity_of_the_standard_addition_calibration_method.

j _{Repeatability.}

solventdonotinﬂuencetheabilityofthepolymerforextraction. LiquiddesorptionofaPDMSstirbarfollowedbyreconditioningand reusehasbeenintensivelyusedandshowsthatthePDMS charac-teristicsarenotalteredbythisprocedure.Toinvestigatepotential solventsforSA-SBSE,theamountofabsorbedsolventintheswollen PDMSstirbarbeforeandafterSA-SBSEwasmeasuredby weigh-ing.TwelvesolventscoveringalogKowrangefrom−0.24to3.90

wereselected,includingnamelyacetone(logKow:−0.24),

acetoni-trile(logKow:−0.15),methylacetate(logKow:0.37),ethylacetate

(logKow:0.86),tetrahydrofuran(THF)(logKow:0.94),diethylether

(logKow: 1.05), methyl isobutyl ketone (MIBK) (logKow: 1.16),

dichloromethane(logKow:1.34),diisopropylether(logKow:1.88),

toluene(logKow: 2.54),cyclohexane(logKow:3.18),and hexane

(logKow: 3.90). Triplicate measurements wereperformed using

differentstirbarsbuthavingthesameproductionlotandbatch number. In SBSE, besides the log Kow of the solute, there are

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Table3

Detectedpesticides,logKow,retentiontime,selectedtransitions,relativepeakratioofSBSEandSA-SBSE,linearity,concentration,andrepeatabilityobtainedfor

SA-SBSE-TD-GC–MS/MSofwine.

No. Compound logKow RT(min) Target transitione

(m/z→m/z)

Relativepeakratiof _Quantitation_by_SA-SBSE_using_DCM

SBSE SA-SBSE r2j _{Concentration}

(pg/mL) RSD(%) k_(n₌₆₎ DCMg _DIPEh _CyHxi 1 Azoxystrobin 1.58 23.44 344.0→329.0 1.0 1.8 1.8 1.8 0.9994c ₄₆₀ _7.7 2 Metalaxyl 1.70 13.73 234.0→146.0 1.0 4.6 2.9 2.4 0.9984c ₆₇₀ _2.6 3 Ethiofencarb 2.04 13.16 168.0→77.0 1.0 1.7 1.3 1.3 0.9998a _6.7 _6.1 4 Carbaryl 2.35 13.69 144.0→115.0 1.0 1.8 1.8 1.5 0.9971b ₂₁ _7.9 5 DimethomorphZ/E 2.36 23.62/24.07 301.0→165.0 1.0 2.7 2.3 2.3 0.9939c ₉₆₀₀d _8.2 6 Procymidone 2.59 15.41 283.0→96.0 1.0 1.0 1.0 1.2 0.9935b ₂₆ _1.9 7 Dichlobenil 2.70 7.66 171.0→100.0 1.0 1.2 2.2 1.7 0.9951a _2.1 _8.5 8 Iprodione 2.85 18.54 314.0→245.0 1.0 1.4 1.4 1.4 0.9948c ₁₇₀₀₀d _8.9 9 Triadimefon 2.94 14.59 208.0→181.0 1.0 1.1 1.1 1.2 0.9987b ₃₁ _4.2 10 TriadimenolI/II 2.95 15.41/15.56 168.0→70.0 1.0 2.0 1.9 1.7 0.9995c ₁₆₀₀ _3.0 11 Pyrimethanil 3.19 12.58 199.0→198.0 1.0 4.4 2.8 2.7 0.9971c ₁₇₀₀₀d _2.5 12 Metolachlor 3.24 14.34 238.0→162.0 1.0 1.3 1.7 1.2 0.9981a _3.8 ₁₁ 13 Iprodione(metabolite) 3.24 19.23 329.0→142.0 1.0 1.8 2.2 1.5 0.9965c ₁₅₀₀ _6.2 14 o-phenylphenol 3.28 9.61 170.0→169.0 1.0 1.0 1.3 1.4 0.9913b ₁₄ _9.9 15 Diphenylamine 3.29 10.79 168.0→167.0 1.0 0.93 1.2 1.3 0.9979b ₁₇ _9.2 16 Diethofencarb 3.29 14.39 267.0→225.0 1.0 1.2 1.1 1.3 0.9998b ₈₈ _2.8 17 Paclobutrazol 3.36 15.79 236.0→125.0 1.0 3.2 4.5 3.4 0.9847a _4.2 ₁₂ 18 Fludioxonil 3.36 16.13 248.0→127.0 1.0 1.3 1.3 1.2 0.9991c ₁₂₀₀ _3.2 19 Epoxiconazole 3.47 18.36 192.0→138.0 1.0 1.1 0.96 1.2 0.9990a _6.8 _4.4 20 Myclobutanil 3.50 16.4 179.0→125.0 1.0 1.5 1.3 1.4 0.9991c ₂₉₀ _3.5 21 Benalaxyl 3.69 17.58 266.0→148.0 1.0 0.98 0.98 1.2 0.9985c ₂₇₀ _3.9 22 Tebuconazole 3.89 18.1 250.0→125.0 1.0 1.2 1.2 1.3 0.9994c ₁₁₀ _3.6 23 Cyprodinil 3.99 15.05 225.0→224.0 1.0 1.7 1.4 1.7 0.9997c ₂₂₀ _2.8 24 BitertanolI/II 4.07 20.44/20.55 170.0→115.0 1.0 5.5 7.3 4.6 0.9986a _6.3 ₁₁ 25 Fenbuconazole 4.23 21.03 198.0→129.0 1.0 1.4 1.2 1.5 0.9994b ₁₀ _5.3 26 Tetraconazole 4.25 14.58 336.0→218.0 1.0 1.4 1.2 1.2 0.9952b ₂₂ _8.4 27 Chlorpyrifos 4.66 14.37 314.0→258.0 1.0 1.8 2.8 1.8 0.9941a _1.4 ₁₁ 28 Penconazol 4.67 15.15 248.0→157.0 1.0 1.2 1.1 1.2 0.9995a _4.3 _8.2 29 Flusilazole 4.89 16.44 233.0→165.0 1.0 1.1 1.0 1.2 0.9983b ₄₇ _6.9 30 DifenoconazoleI/II 5.20 22.85 323.0→265.0 1.0 1.8 1.5 1.6 0.9918b ₃₁ _9.1 31 Pyraclostrobin 5.45 22.441 164.0→132.0 1.0 2.7 4.3 2.7 0.9948b ₆₇ _2.6 32 Kresoxim-methyl 5.88 16.46 206.0→116.0 1.0 0.92 0.99 1.2 0.9962c ₂₆₀ _4.0 33 FlucythrinateI/II 6.56 21.52/21.72 199.0→107.0 n.d. – – – 0.9945b ₃₆ ₁₆

a_Linearity_range_was_1–50_pg/mL.

b_Linearity_range_was_10–200_pg/mL.

c _Linearity_range_was_200–2000_ng/mL.

d _50-fold_diluted_sample_was_used_for_{qunatitation.}

e_Selected_transitions_for_relative_peak_ratio,_{quantitation,}_and_{repeatability.}

f _Relative_peak_ratio_of_SBSE_and_SA-SBSE_(normalized_by_SBSE_peak_area).

g_SA-SBSE_using_{dichloromethane.}

h _SA-SBSE_using_diisopropyl_ether.

i _SA-SBSE_using_cyclohexane.

j _Linearity_of_the_standard_addition_calibration_method.

k_{Repeatability.}

severalimportantparametersthat influencetheextraction effi-ciency,includingphase ratio(␤:samplevolume/PDMSvolume), extractiontime,stirringspeed,saltaddition,organicmodifier addi-tion,pHadjustment,etc.Forhighextractionefficienciesofpolar solutes(e.g.logKow<3.0),smallerphaseratioand saltaddition

are often required [7,8]. Therefore, a 63␮L PDMS stir bar and 5mLofwater(correspondingto␤=79)including30%NaClwere used.SA-SBSEwasperformedfor1hwhilestirringat800rpm.The initialsolventswellingprocessofthePDMSstirbarandthe SA-SBSEprocedureweredescribedintheSection2.3.Aconditioned PDMSstirbarinasealedHSvialwasinitiallyweighedas refer-ence.Then,thesolventswollenPDMSstirbarinthesealedHSvial wasweighedjustafterthesolventswellingprocess.Finally,the solventswollenPDMSstirbarinthesealedHSvialwasweighed justafterSA-SBSE.ThisallowedustomeasuretheSolventabsorbed

(PDMS absorbedsolvent−PDMS initial)and theSolvent residual (PDMS residualsolvent−PDMSinitial).

Table1showstheamountoftheabsorbedsolventintheswollen PDMSstirbarbeforeandafterSA-SBSEandresidualratio(R_ratio:

the ratioof the solventamount in the PDMSstir bar between

beforeSA-SBSE andafterSA-SBSE).TheR _ratiowascalculatedas Solventresidual/Solventabsorbed.Swellingratio(Sratio)asdescribed inRef.[27]isalsoshowninTable1.Triplicatemeasurementsof thesolventswollenPDMSstirbarusingweighingshowedgood repeatabilitywithrelativestandarddeviation(RSD)valuesinthe rangeof0.06–4.90%foralltestsolvents.Asexpected,theamountof absorbedsolventintheswollenPDMSstirbarbeforeSA-SBSE gen-erallyincreasedwithanincreaseofswellingratio.Solventswith swellingratiorangingfrom1.18to1.38gavetherelativelyhigher amountsofSolvent_absorbed rangingfrom59to110mg,while sol-ventswithlowswellingratiosuchasacetonitrile(1.01)andacetone (1.06)gaveloweramountsof20mgand21mg,respectively.After SA-SBSE,theamountoftheSolventresidual and theRratioin the swollenPDMSstirbargenerallydecreasedwithdecreaseofthe logKowvalue.AlthoughTHF(S_ratio:1.38)gavethesecond

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high-estamount(100mg)oftheabsorbedsolventbeforeSA-SBSE,the

R_ratiowasquitelowwith0.056duetoitslowlogKow valueof

0.940.SolventswithlogKow>1.0showedtheR_ratiorangingfrom

0.41to0.94,whilesolventslogKow<1.0showedtheR_ratio<0.070

(exceptforethylacetateat0.20).Therefore,eightsolvents(ethyl acetate,diethylether,methylisobutylketone,dichloromethane, diisopropylether,toluene,cyclohexane,andhexane)withaR_ratio >0.20wereselectedaspotentialsolventsforfurtherstudy. 3.1.2. Comparisonofextractionefﬁciencyanduptakerate betweenSBSEandSA-SBSE

Toinvestigate extractionefﬁciencyof SA-SBSE, therecovery obtainedbySA-SBSEusingtheeightsolventswithlogKow

rang-ing from 0.86 (ethyl acetate) to 3.90 (hexane) for twelve test soluteswithlogKowrangingfrom0.56(2-acetylpyrrole)to4.21

(␤-damascenone)inwaterwerecomparedwiththoseobtainedby conventionalSBSE.AllSBSEconditionswereperformedfor1hin duplicate.Theconcentrationofthetestsoluteswas5ng/mLeach. Therecoverywascalculatedbycomparingpeakareaswiththose ofacalibrationcurvepreparedbydirectliquidinjectionofa stan-dardsolutioninjectedintotheTDUthroughaseptumhead.Fig.2

showstherecoveriesbetweenconventionalSBSEandSA-SBSEwith theeightselectedsolventsforeachtestsolute.Deviationbetween duplicate analyses were less than 10%. For conventional SBSE, soluteswithlogKow>2.5(phenethylacetate,linalool,citronellol,

and␤-damascenone)showedrecoverieshigher than80%,while thesoluteswithlogKow<2.5showedlowrecoveries,especiallyfor

2-acetylpyrrole(logKow:0.56,recovery:6.8%),2-acetylthiazole

(logKow:0.67,recovery:18%),benzylalcohol(logKow:1.08,

recov-ery:11%),guaiacol(logKow:1.34,recovery:21%),andphenethyl

alcohol(logKow:1.57,recovery:15%).SA-SBSEusingseveral

sol-ventssigniﬁcantlyimprovedtherecoveriesfor thesoluteswith logKow<2.5.Especiallydichloromethane(logKow:1.34)provided

enhancedrecoveriesforallsoluteswithlogKow<2.5byfactorsof

1.4–4.1,whilemaintainingorevenimprovingtherecoveriesforthe soluteswithlogKow>2.5.Theserecoveriesarethehighestor

sec-ondhighestrecoveriesforalltestconditions.SA-SBSEusingmethyl isobutylketone(logKow:1.16),diisopropylether(logKow:1.88),

andtoluene(logKow:2.54)alsoimprovedtherecoveriesfor

sev-eralsoluteswithlogKow<2.5,e.g.2-acetylpyrrole(from6.8%to

20–21%),guaiacol(from21%to50–62%),phenethylalcohol(from 15%to35–50%),andindole(from51%to77–86%).However,the recoveriesofcis-3-hexenolandlinaloolweredecreasedfrom31% to8.1–24%,andfrom82%to52–68%,respectively.Thisdeviating behaviorofcis-3-hexanolandlinalool,twounsaturatedaliphatic alcohols,cannotbeexplained.SA-SBSEusingdiethylether(logKow:

1.05)showedasimilartrendbutwithoutanydecreasedrecoveries ofthetestsolutes.SA-SBSEusingethylacetatewhichhasthe low-estlogKowof0.86showeddecreasedrecoveriesforseveralsolutes

withlogKow<2.5suchas2-acetylpyrrole(from6.8%to3.6%),

2-acetylthiazole(from18%to12%),benzylalcohol(from11%to5.2%), coumarin(from38%to20%),andcis-3-hexenol(from31%to16%), whilemaintainingtherecoveriesfortherestofthesolutes. SA-SBSEusingrelativelyapolarsolventssuchascyclohexane(logKow:

3.18)showedsimilarrecoverieswiththoseofconventionalSBSE orslightlyincreasedrecoveries(byafactorof1.1–1.3)forninetest solutes,whiledecreasingtherecoveriesforthreesolutesnamely coumarin,cis-3-hexenol,andlinalool.SA-SBSEusinghexane(log Kow:3.90)showedasimilartrend.Consequently,SA-SBSEusing

relativelypolarsolventswithlogKow rangingfrom1.05(diethyl

ether)to2.54(toluene)enhancebesttheextractionefﬁcienciesof polarsoluteswithlogKow<2.5inaqueoussample.Thesesolvents

actnotonlyasamodiﬁerofthePDMSphase(increasingdiffusion), butalsoasanadditionalextractionmediumfromtheswollenPDMS phase.Fig.3showsanextractionmodelofSA-SBSE.ForSA-SBSE usingasolventwithlogKow<1.0(Fig.3a),e.g.ethylacetate,the

residualsolventintheswollenPDMSphaselargelydiffusesintothe samplesolutionandtheequilibrium(ofthesolventbetweenPDMS phaseandsamplesolution)isbiasedtowardsamplesolution,while increasingthesolubilityofsoluteswithlogKow<2.5intheaqueous

sample.Thisexplainsthelowgaininextractionyieldusingthese solvents.ForSA-SBSEusingasolventwithlogKowintherangeof

1.0–3.0(Fig.3b),e.g.dichloromethane,moresolventisretainedin thePDMSphase,resultinginhighrecoveriesforsoluteswithlog Kow<2.5.Bothextractionphasepolarityandvolume(phaseratio)

arechanged,resultinginthehighestgaininextractionefﬁciency. ForSA-SBSEusingasolventwithlogKow>3(Fig.3c),e.g.

cyclohex-ane,theresidualsolventintheswollenPDMSphasepartitionless intothesamplesolution.Therecoveryofpolarsolutesismainly basedontheincreaseofextractionmediumvolume,butnotonthe polarityofthephase.

SincetheextractionefﬁciencydependsonthelogKowofthe

sol-ventinSA-SBSE,theuptakerateofSA-SBSEusingthreesolvents, dichloromethane(logKow:1.34),diisopropylether(logKow:1.88),

andcyclohexane(logKow:3.18)andconventionalSBSEwerealso

compared.Sixextractiontimesbetween1and60minwere per-formedinduplicate.Fig.4illustratesacomparisonoftheuptake rate(extractiontimeproﬁles)ofsixsoluteswithlogKowranging

from0.67 (2-acetyl thiazole) to3.56(citronellol).It is interest-ingtoobservethattheuptakerates ofSA-SBSE clearlydiffered fromthose ofconventional SBSEfor all test solutes. Generally, SA-SBSE reachedtheequilibrium earlier (30–45min) than con-ventionalSBSE(45–60min), whilerecoveries weresigniﬁcantly higher amounts for solutes withlog Kow<2.5 (especially using

dichloromethaneanddiisopropylether).AlthoughSA-SBSEusing diisopropyletherandcyclohexanereachedtheequilibriumearlier forlinalool,conventionalSBSEﬁnallyreachedhigherrecoveryat 60min.Consequently,SA-SBSEusingrelativelypolarsolventsnot onlyprovideshigherpartitioningcoefﬁcients(fromaqueous sam-pletoswollenPDMS)butalsohigherdiffusionconstantsofthe solutes(inswollenPDMSandaqueoussample).

3.2. ApplicationofSA-SBSEtorealsamples

TooutlinetheimprovedperformanceofSA-SBSEforpolar com-poundsinrealsamples,beerﬂavorandwhitewinecontaminants wereanalyzedtodemonstratedetectionofawidevarietyof com-poundsinthepg/mL(ppt)to␮g/mL(ppm)range.Conventional SBSEwasalsoperformedascomparison.

3.2.1. Analysisofaromacompoundsinbeerby SA-SBSE-LD-LVI-GC–MS

OneoftheadvantagesofSBSEusingPDMSisthetargeted extrac-tionofrelativelyapolarand GCamenablesolutesfromaqueous foodmatriceswithoutenrichmentofnon-volatilesolutessuchas aminoacids,sugars,polyphenols,etc.Therefore,thermal desorp-tion (TD)can be used for SBSEwithout a risk of heat-induced artifactformation(e.g.Maillardreactions).SBSEhasbeenapplied toanalysisofaroma/off-ﬂavorcompoundsinbeer[28–30]. How-ever, SA-SBSE mayhave the potential to extract precursors of heat-inducedartifacts(e.g.polyphenols),whichcanbeextracted withtraditionalliquid–liquidextraction(LLE).Fromthe prelimi-narytestofbeerwithSA-SBSEusingdichloromethanefollowedby TD-GC–MSanalysis,severalphenoliccompoundsincludinga typ-icalaromacompoundsuchasguaiacolwereclearlydetectedand guaiacolshowedabout20timeshigher“concentration”by stan-dardadditioncalibration, compared tothatofSBSE.Thismight beduetothermaldegradationofpolyphenolswhichareextracted inthesolventswollenPDMSstirbar.Tominimizeariskof heat-inducedartifactformation,liquiddesorption(LD)followedbylarge volumeinjection(LVI)atmoderatetemperature(80◦C)wasused forSA-SBSEanalysisofbeer.

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30 20 10 0 Hx (log Kow: 3.90) CyHx (log Kow: 3.18) Tol (log Kow: 2.54) DIPE (log Kow: 1.88) DCM (log Kow: 1.34) MIBK (log Kow: 1.16) DEE (log Kow: 1.05) EtAc (log Kow: 0.86) SBSE

2-Acetylpyrrole

(log Kow: 0.56)

80 60 40 20 0 Hx (log Kow: 3.90) CyHx (log Kow: 3.18) Tol (log Kow: 2.54) DIPE (log Kow: 1.88) DCM (log Kow: 1.34) MIBK (log Kow: 1.16) DEE (log Kow: 1.05) EtAc (log Kow: 0.86) SBSE

2-Acetylthiazole

(log Kow: 0.67)

50 40 30 20 10 0 Hx (log Kow: 3.90) CyHx (log Kow: 3.18) Tol (log Kow: 2.54) DIPE (log Kow: 1.88) DCM (log Kow: 1.34) MIBK (log Kow: 1.16) DEE (log Kow: 1.05) EtAc (log Kow: 0.86) SBSE

Benzyl

alcohol

(log Kow: 1.08)

80 60 40 20 0 Hx (log Kow: 3.90) CyHx (log Kow: 3.18) Tol (log Kow: 2.54) DIPE (log Kow: 1.88) DCM (log Kow: 1.34) MIBK (log Kow: 1.16) DEE (log Kow: 1.05) EtAc (log Kow: 0.86) SBSE

Guaiacol (log Kow: 1.34)

Coumarin (log Kow: 1.51)

60 40 20 0 Hx (log Kow: 3.90) CyHx (log Kow: 3.18) Tol (log Kow: 2.54) DIPE (log Kow: 1.88) DCM (log Kow: 1.34) MIBK (log Kow: 1.16) DEE (log Kow: 1.05) EtAc (log Kow: 0.86) SBSE

Phenethyl alcohol (log Kow: 1.57)

cis-3-Hexenol

(log Kow: 1.81)

Indole

(log Kow: 2.05)

Pheneth

yl acetat

e (log Kow: 2.57)

Linalool (log Kow: 3.38)

Citronellol (log Kow: 3.56)

ß-Damascenone (log Kow: 4.21)

Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery (%)

n/a

Fig.2.AcomparisonofrecoverybetweenconventionalSBSEandSA-SBSE.SBSE:conventionalSBSE,EtAc:SA-SBSEusingethylacetate,DEE:SA-SBSEusingdiethylether,

MIBK:SA-SBSEusingmethylisobutylketone,DCM:SA-SBSEusingdichloromethane,DIPE:SA-SBSEusingdiisopropylether,Tol:SA-SBSEusingtoluene,CyHx:SA-SBSEusing

cyclohexane,Hx:SA-SBSEusinghexane.n/a:notavailableduetoblankvalueinthesolvent.

Thepresenceofarelativelyhighlevelofethanolinbeer(e.g. 4.5–5%)mayinﬂuencetheresidualratio(R_ratio)ofthesolventin

aswollenPDMSstirbar.SA-SBSEusingdichloromethane(logKow:

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Fig.3.ExtractionmodelofSA-SBSEforsoluteswithlogKow<2.0inaqueoussample.

Fig.4.Acomparisonoftheuptakerate(extractiontimeproﬁle)ofsixtestsolutesbetweenSA-SBSEandconventionalSBSE.DCM:dichloromethane,DIPE:diisopropylether,

CyHx:cyclohexane.

3.18)wereﬁrstperformedfora5%ethanol-watersampleto inves-tigatetheR_ratiovalues.SA-SBSE usingdichloromethaneshowed a slightly lowerR _ratio of 0.51 than that of 100% water sample (R _ratio: 0.56)duetoitsrelativelyhigher watersolubilityinthe selectedsolvents;howeverSA-SBSEusingdiisopropylether and

cyclohexane showed the same R _ratio values of 0.81 and 0.90, respectively.Therefore,SA-SBSEusingthesethreesolventswere performedforthebeersample.Fig.5illustratesacomparisonofa majorpartofthetotalionchromatogram(TIC)betweenSA-SBSE usingdichloromethane(a),SA-SBSEusingdiisopropylether(b),

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SA-0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 28.0 26.0 24.0 22.0 20.0 30.0 32.0 34.0 36.0 0 0.5 1.0 1.5 2.0 38.0 Int en sity (x 10 5a.u.) Int en sity (x 10 5a.u.) Int en sity (x 10 5a.u.) Int en sity (x 10 5a.u.) Retention time 6 1 8 10 4 12 3 20 15 13 2+5 7 22 24 18 27 19 11 25 28 17 23

(a) SA-SBSE (DCM)

(b)

SA-SBSE

(DIPE)

(min) 28.0 26.0 24.0 22.0 20.0 30.0 32.0 34.0 36.0 38.0

(c)

SA-SBSE (C

yHx

)

(min) 28.0 26.0 24.0 22.0 20.0 30.0 32.0 34.0 36.0 38.0

(d)

SBSE

(min) 28.0 26.0 24.0 22.0 20.0 30.0 32.0 34.0 36.0 38.0 (min)

Fig.5.Acomparisonofthetotalionchromatogram(TIC)betweenSA-SBSEandconventionalSBSE.(a)SA-SBSEusingdichloromethane,(b)SA-SBSEusingdiisopropylether,

(c)SA-SBSEusingcyclohexane,(d)ConventionalSBSE.

SBSEusingcyclohexane(c),andconventionalSBSE(d).Therelative peakratioofSBSEandSA-SBSE(normalizedtoSBSEpeakarea)of 28selectedaromacompounds(logKow:−0.45–4.21)arelistedin

Table2.ThecompositionsofTICsbetweenSA-SBSEand conven-tionalSBSEclearlydifferedbothqualitativelyandquantitatively. It isinteresting toobservethat severalaromacompounds with logKow<2.0,e.g.2,3-butanediol(1;butter;logKow:−0.36),

mal-tol(2;caramel;logKow:−0.19),2-acetylpyrrole(5;nutty;logKow:

0.56),methionol(3;bakedvegetable;logKow:0.44),furfuryl

alco-hol(4;burntsugar;logKow:0.45),2-acetylfuran(6;toasty;log

Kow:0.80),3-methylbutanoicacid(12;cheese;logKow:1.56),and

phenethylalcohol (13;rose;logKow:1.57),aremoreintensein

chromatograms(a)and(b)comparedtochromatograms(c)and

(d).Therelativepeakratioofthesepeaksinthechromatogram(a) and(b)are3.9–27timesgreaterthanthoseinthechromatogram (d).Itisalsoclearthatsomeofthesecompoundssuchas2-acetyl pyrrole,guaiacol,and phenethylalcohol, whichhave beenused astestsolutesforthespikedwaterintheSection3.1.2,showed greater differences of therelative peak ratio between SA-SBSE andconventionalSBSE,resultinginhigherextractionefﬁciencies

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15.0 14.0 13.0 0 5.0 0 5.0 0 5.0 0 5.0 Int en sity (x 10 5a.u.)

Metalaxyl

(log

K

ow

: 1.70)

Dimethomorph

Z

/

E

(log

K

ow

: 2.36)

25.0 24.0 23.0 Int en sity (x 10 6a.u.) 0 2.0 4.0 0 2.0 4.0 0 2.0 4.0 2.0 4.0 0 13.0 12.0 Int en sity (x 10 8a.u.) 0 1.0 0 1.0 0 1.0 0 1.0

Pyrimethanil

(log

K

ow

: 3.19)

Paclobutrazol

(log

K

ow

: 3.36)

17.0 16.0 15.0 Int en sity (x 10 4a.u.) 0 1.0 0 1.0 0 1.0 0 1.0

Biter

tano

l I/I

I

(log

K

ow

: 4.07)

Int en sity (x 10 4a.u.) 20.0 0 5.0 0 5.0 0 5.0 0 5.0

Chlorpyrifos

(log

K

ow

: 4.66)

Int en sity (x 10 3a.u.) 15.0 14.0 0 2.0 4.0 0 2.0 4.0 0 2.0 4.0 0 2.0 4.0

Pyraclo

strobi

n

(log

K

ow

: 5.45)

Flucythrina

te I/I

I

(log

K

ow

: 6.56)

Int en sity (x 10 4a.u.) 23.0 22.0 0 5.0 0 5.0 0 5.0 0 5.0 Int en sity (x 10 3a.u.) 22.0 21.0 0 5.0 0 5.0 0 5.0 0 5.0 Retention time (min)

Retention time (min)

Retention time (min) Retention time (min)

I I _II Z E II SA-SBSE (DCM) SA-SBSE (DIPE) SA-SBSE (CyHx) SBSE SA-SBSE (DCM) SA-SBSE (DIPE) SA-SBSE (CyHx) SBSE SA-SBSE (DCM) SA-SBSE (DIPE) SA-SBSE (CyHx) SBSE SA-SBSE (DCM) SA-SBSE (DIPE) SA-SBSE (CyHx) SBSE SA-SBSE (DCM) SA-SBSE (DIPE) SA-SBSE (CyHx) SBSE SA-SBSE (DCM) SA-SBSE (DIPE) SA-SBSE (CyHx) SBSE SA-SBSE (DCM) SA-SBSE (DIPE) SA-SBSE (CyHx) SBSE SA-SBSE (DCM) SA-SBSE (DIPE) SA-SBSE (CyHx) SBSE 21.0

Fig.6. Acomparisonoftheselectedreactionmonitoring(SRM)chromatogrambetweenSA-SBSEandconventionalSBSE.DCM:dichloromethane,DIPE:diisopropylether,

CyHx:cyclohexane.

comparedtothosewiththespikedwatersample(seetheSection

3.1.2andFig.2).Consequently,SA-SBSEusingdichloromethaneand diisopropylethersignificantlyimprovetheextractionefficiencies ofpolarsolutesinbeer matrices,whilecompensatingthe nega-tivematrixeffectfrom5%ethanolobservedinconventionalSBSE. Althoughall SA-SBSE conditionsshowed higherextraction effi-ciencyforC8–C10fattyacidshavingpolarcarboxylicfunctioneven

withlogKowrangingfrom3.03to4.02,conventionalSBSEshowed

higherextractionefﬁcienciesforseveralapolararomacompounds suchas linalool(23; ﬂoral; logKow: 3.38),ethyl octanoate (26;

fruity;logKow:3.81),and ␤-damascenone(28; honey;logKow:

4.21).

Repeatabilitytestsofthe28aromacompoundsandquantitation of21compoundswerecarriedoutwithSA-SBSEusingdiisopropyl etherinsixreplicateanalyses.Fivepointsofstandardaddition cal-ibrationcurvesbetween1and40ng/mLor200and4000ng/mL

or1000and10000ng/mL wereusedforthequantitation.Good repeatabilitywithRSDssmallerthan8.0%wasobtainedforall com-pounds.Thelinearitywasalsogoodwithr2_higher_than_0.99_for₁₉

compounds.Ther2_values_for_3-methyl_butanoic_acid_and_decanoic

acidwere0.9863and0.9891,respectively.Thedeterminedvalues wereintherangeof1.1–5000ng/mL.Selectedaromacompounds, logKow, selectedions,relative peakratio ofSBSEand SA-SBSE,

linearity,concentration,and repeatabilityareallsummarizedin

Table2.

3.2.2. AnalysisofpesticidesinwinebySA-SBSE-TD-GC–MS/MS SBSEhasbeenusedforseveraltypesofwineanalyses includ-ingpesticideanalysis[31,32].SBSEcanbeappliedtonon-diluted winesamplesincluding10–15%ethanol,andallowsthermal des-orption(TD)intoGCsystem.SA-SBSEhasthepotentialtoextract

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higheramountsofpolarvolatilematricessuchasfuselalcohols, fattyacids,and oxygen-containingaromatics, which maycause overloadingofthecolumnandretentiontimeshiftwhencombined withTDfollowedbyPTVsplitlessinjectionintoGCsystem.These “excess”matricesmayalsoreducecleanlinessofTDsystem.To pre-ventthesematrixeffects,winewas5-folddilutedwithwaterbefore SA-SBSE,resultingin2.5%ethanol.SA-SBSEwasperformedwith dichloromethane,diisopropylether,andcyclohexane.Thirtythree pesticides(including27fungicides,4insecticides,and2herbicides) withlogKow intherangeof1.58–6.56werepositivelydetected

withallSA-SBSEconditionsusingtheintelligentMRMpesticide database(Agilent).Although32pesticideswerealsodetectedwith conventionalSBSE,therelativepeakratioofthosepesticideswere mostlylowerthanthoseofSA-SBSE(Table3).Fig.6illustratesa

comparisonoftheSRMchromatogrambetweenSA-SBSEand

con-ventionalSBSEforrepresentativepesticides.AllSA-SBSEconditions generallyenhancedtheextractionefﬁcienciesnotonlyfor rela-tivelypolarpesticideswithlogKow<3.0suchasmetalaxyl(logKow:

1.70)anddimethomorphisomers(logKow:2.36)butalsoforapolar

pesticideswithlogKow>3.0suchaspaclobutrazol(logKow:3.36),

chlorpyrifos (logKow: 4.66), and pyraclostrobin (logKow: 5.45).

AlthoughsaltadditioninconventionalSBSEdecreasesthe extrac-tionefﬁcienciesofmorehydrophobic(apolar)solutes[33,34],all SA-SBSEconditionscompensatethenegativeeffectofsalt addi-tion.Consequently,SA-SBSE usingdichloromethane,diisopropyl ether,andcyclohexaneprovidesbetteropportunitiestodetecta widerangeofpesticidesinwinecomparedtoconventionalSBSE. Quantitationofthedetected33pesticideswascarriedoutwith SA-SBSEusingdichloromethaneinsixreplicateanalyses.Fivepoints ofstandardadditioncalibrationcurvesbetween1and 50pg/mL

or10 and200pg/mLor 200and 2000pg/mL wereusedforthe

quantitation.Good repeatabilitywithRSDofless than10%was obtainedfor27compounds.TheRSDvaluesfortheother5 pesti-cideswereintherangeof11–16%.Thelinearitywasalsogoodwith r2_higher_than_0.9913_for₃₂_pesticides._The_linearity_for

pacrobu-trazolwasr2_of_0.9847._The_determined_values_were_in_the_range_of

1.4–17000pgmL−1_._Detected_pesticides,_log_K

ow,relativepeakratio

ofSBSEandSA-SBSE,selectedtransitions,linearity,concentration, andrepeatabilityaresummarizedinTable3.

4. Conclusion

AnewSBSEmethodreferredtoassolvent-assisted(SA)-SBSE hasbeendeveloped.SA-SBSEusingasolventswollenPDMSstir barwithsolventswithinthelogKow:1.0–3.0rangecanenhancethe

extractionefﬁcienciesofpolarsoluteswithlogKow<3.0in

aque-oussample.Theperformance ofthemethodwasdemonstrated

bySA-SBSE-LD-LVI-GC–MSanalysisofaromacompoundsinbeer

andbySA-SBSE-TD-GC–MS/MSanalysisofpesticidesinwine.For aromacompoundanalysisinbeer,SA-SBSEusingdichloromethane anddiisopropyletherprovidedimprovedsensitivitiesespecially forcompoundswithlogKow<2.0atng/mLto␮g/mL,while

com-pensatingthenegativeeffectof5%ethanol.Forpesticideanalysis ofwine, SA-SBSEusingdichloromethane,diisopropylether, and cyclohexaneofferedimprovedsensitivitiesnotonlyforpolar pes-ticideswithlogKow<3.0butalsoforapolarpesticideswithlogKow

upto6.56atpg/mLtong/mL,whilecompensatingthenegative effectofsaltadditiontoapolarpesticides.

Acknowledgement

Dr.RyoOgasawaraofAgilentTechnologiesJapan,Ltd.isthanked forprovidingtheintelligentMRMpesticidedatabase.

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