Atmospheric pressure gas chromatography coupled to quadrupole-time of flight mass spectrometry as a...

Atm osphericpressure gaschrom atographycoupled to quadrupole-tim e offlightm assspectrom etry asa toolforidenti cation ofvolatile m igrantsfrom autoadhesive labelsused fordirectfood contactElena Canellas,a,b Paula Veraa and Cristina Nerína*

Pressure-sensitive adhesives(PSA)are used to m anufacture labelsthatare applied directly on the food.These adhesivescouldcontainnotonlyintentionallyaddedcom pounds(IAS)totheadhesiveform ulabutalsonon-intentionallyaddedsubstances(NIAS),duetotheim puritiesfrom theraw m aterialsused,decom position oftheinitialcom ponentsorfrom chem icalinteractionsbetweenthem .These com poundscould m igrate to the food and contam inate it.In thisstudy,gaschrom atography coupled with m assspectrom etry(GC-M S/Q)and atm osphericpressuregaschrom atography coupled to aquadrupole hyphenated to a tim e of ightm assspectrom eter(APGC-M S/Q-TOF)have been used foridenti cation ofunknown com poundsand NIAS com ing from aPSA.Seven com poundswereidenti ed byGC-M S/Q,and othereightcom poundsrem ained initiallyunknown.Thestructureofthese

eightnew com poundswaselucidated byworking with thespectraobtained byAPGC-M S/Q-TOF.Finally,two differentm igrationstudieswerecarried out.The rstonewith Tenaxassolid food sim ulantin contactwith thepaperlabelcontaining theadhesiveand thesecond one with isooctane lled in a naturalpork intestine wherethe labelcontaining the adhesive wasapplied on theexternalside.The resultsareshown and discussed.Copyright© 2014 John W iley & Sons,Ltd.

Keywords:APGC-Q-TOF;NIAS;pressure-sensitiveadhesives;food contactm aterial;riskexposure

Introduction

Food packaging labelshave the purpose ofinform ing abouttheproductandm otivatetheconsum ertobuyit.Insom ecases,wheretheproductissold withoutapackaging,labelsarestickdirectlyonthefood with apressure-sensitiveadhesive(PSA).Som eexam plesarefruits,cured cheesesand cold cured m eats.Adhesivesare com plex m ixturesthatinclude a high variety of

com pounds.The m ain com ponents ofan adhesive are a resin,catalysts, hardeners, accelerators, inhibitors, retarders, solvents,thickners,diluents,extenders, llers,carriers,plasticizers, exibilizers,tackiers, lm form ers, antioxidants, antihydrolisis, antifungals,soaps,surfactantsand wetting agents.[1]W ith the exception oftheresin,m ostofthese additiveshave m olecularweightlowerthan1000am u,and consequently,theycould m igrate from the labeltothe food,as they are in direct contact with it.As a result,them igration study iscom pulsory before launching the autoadhesivelabels to the m arket.However,notonly the intentionally addedsubstances(IAS)butalso the non-intentionally added substances(NIAS)from the adhesive form ula could end in the food.In theadhesives form ula,NIAS could appearfrom im purities from theraw m aterials used,decom position ofthe initialcom ponents orbecause of chem ical interactions between them .[2,3] Thesecom poundscouldalsom igrateandaffectthefoodsafety.Therefore,itisveryim portantto identifythem .Identicationofunknowncom poundsisachallengingwork.Gas

chrom atographycoupled to m assspectrom etryin electron im pactm ode has been used previously forthe identication ofvolatile

unknownsin adhesivesforfood contact.[4–7]Thespectraobtainedin these analysesare com pared with a spectrum library,and theidentication can beachieved.Nevertheless,databasesarelim itedand the identication isnotalwayspossible.Therefore,additionalanalysesarenecessary.Atm osphericpressuregaschrom atographycoupled to atim e

of ightanalyzer(APGC-M S/Q-TOF)is an interesting toolthatcould be very valuable in thiskind ofanalysis.APGC isa novelanalyticaltechnique,based on APCI,[8]with aplasm ageneratedby a corona pin thatsuppliesreagentionsto ionize the targetm olecules.The ionization processes are driven by a plasm aform edbytheinteractionbetweennitrogenm oleculesandelec-trons.The m ostcom m on ionization processesare chargetrans-ferand protonation;therefore,m assspectra can be dom inatedby M +,M +H+ or M H .This fact,com bined with a Q-TOFanalyzerthatprovidesaccurate m assofthe m olecularion anditsfragm ents,aswellaswith the help ofa chem icaldatabase,could lead to obtain an identication ofcom pounds.[9–12]

* Correspondence to: Cristina Nerín, GUIA Group, Departm ent of AnalyticalChem istry,UniversityofZaragoza,I3A,MaríadeLuna,3,50018Zaragoza,Spain.E-m ail:[email protected]

a GUIA Group,DepartmentofAnalyticalChem istry,University ofZaragoza,I3A,MaríadeLuna,3,50018,Zaragoza,Spain

b Sam tackAdhesivosIndustriales,C/Cerámica,no3,Pol.Ind.MagarolaSud,08292,Esparreguera,Barcelona,Spain

J.MassSpectrom .2014,49,1181–1190 Copyright© 2014John W iley& Sons,Ltd.

Research article

Received:19M ay2014 Revised:9 July2014 Accepted:14 July2014 Published onlinein W ileyOnlineLibrary

(wileyonlinelibrary.com )DOI10.1002/jm s.3445

1181

The aim ofthispaperisto explore the potentialofthe noveltechnique APGC-MS/Q-TOF to study the likely m igrants from acom plexadhesivethatisgoing to bein contactwith food.

Experim ental

M aterials

The adhesive selected forthe study wasa PSA.Thisadhesive isusuallyapplied on labelsused fordirectfood contactwith cheese,cold cured m eatsand fresh m eats.The adhesive wassupplied bySam tack(Barcelona,Spain).Labelsfordirectfoodcontactwerem adewiththeadhesive.Thela-

belsconsisted of20g/m 2 ofPSA applied over36 m polyethyleneterephthalate.ThelabelsweresuppliedbySam tack(Barcelona,Spain).Ricepaperofagram ageof17g/m 2used forthem igration stud-

ieswaspurchased from OCB (France).Naturalporkintestinewassupplied byM elsa(Graus,Spain).Thefollowingreagents,decahydronaphthalene,3-octanol,1-m ethyl-

3-m ethoxy-4-isopropylbenzene and tert-butyldicyclohexylphosphine,werepurchased from Sigm a–Aldrich Quím icaS.A (Madrid,Spain).High-perform ance liquid chrom atography-M S quality m ethanol

and ultrapuried waterweresupplied byJ.T.Baker(Deventer,theNetherlands).Analyticalgrade isoctane wassupplied by ScharlauChem ieS.A.(Sentm enat,Spain).

Sam plepreparation and m igration tests

Firstly,in order to identify the non-target com pounds in theadhesive,solutionsofthe PSA were prepared.PSA iscom pletelydissolved eitherin ethanol95% orisooctane.To facilitate the GCanalysis,isooctane waspreferred.Then,1g ofPSA wasdissolvedin 100g ofisooctane,and 1 lofthis solution was analyzed byGC-M Sand APGC-Q-TOF.In addition,1g ofPSA wasdissolved with 10g m ethanol.Then,

1g ofthissolution wasdiluted in ultrapuried water.The solutionwas analyzed by inductively coupled plasm a m assspectrom etry(ICP-M S).The m igration studies were prepared by two differentproce-

dures.Firstly,the study wascarried outwith poly(2,6-diphenyl-p-phenylene oxide)(Tenax)used assolid sim ulant,initially selectedbecause the adhesive was com pletely dissolved in the organicliquid sim ulants.The labelswere stuckto rice paper.Asa result,alam inate m ade ofpolyethylene terephthalate-PSA-rice paperwasobtained.Then,piecesof2.5×5cm ofthislam inate were placedinPetridishes.Thericepapersideofthelam inatewascoveredwith0.5g ofTenax,taking into accounttherecom m ended 4g ofTenaxperdm 2 oflam inate,in accordance with UNE-EN 14338.[13] Thesam eprocedurewascarried outwith thericepaperused asblank.Theassayswerekeptat40°C for10days.Tenax was extracted following the procedure optim ized by

Vera etal.[14]The nalextraction m ethod wasasfollows:0.5gofTenaxW wasextracted two consecutive tim eswith 1.7m lofm ethanoleach tim e.The two extracts were m ixed,and thenthe nalextractwasconcentrated undera N2 stream to 200 land analyzed byGC-M S.Three replicatesofthe m igration assaywereprepared and analyzed.Thesecond m igration assaywascarried outto sim ulatethereal

application ofthelabelson sausagesorcold cured m eat.Isoctanewasselected thistim easthe worstcase,afterchecking thattherewasno leakage from the system .Five centim etersofnaturalporkintestine were lled with 5.5g of isoctane.The edges of the

intestine were closed with Te on tape (Fig.1),m aking a closesystem .Then,the labelwasapplied on the intestine covering theentiresurface,and thesetwaskeptin theoven for2daysat20°C.Finally,isoctaneinsidetheintestinewasextractedwithasyringe,

and1 lofthissolutionwasanalyzedbyGC-M S.Afterthem igrationtest,pork intestine was cutin sm allpieces and extracted threetim eswith isoctanefor24h at40°C.Theextractswerem ixed andevaporated to 200 lunder nitrogen stream ,and 1 lof thissolution wasanalyzed by GC-M S.Five replicatesofthe m igrationassaywereprepared and analyzed.

Instrum entation

Gaschrom atography-m assspectrom etry singlequadrupole

The equipm entused wasa CTC AnalyticsCom biPalautosam plercoupled to an Agilent 6890N gas chrom atograph with a m assspectrom eterM S 5975B detector.Allofthem were from AgilentTechnologies(Palo Alto,CA,USA).The capillarycolum n used wasaHP-5M S(30m ×0.25 m ×250m )from AgilentTechnologies(Madrid,Spain).Theoven programwasasfollows:40°C for2m in,witharateof10°C/m inupto300°C,m aintained for 2m in.The injection type was splitless,and thehelium ow was 1m l/m in.The acquisition was carried out inelectron im pactionization.The m ass detectorwas setatSCANm ode(in therangem /z45–350).

Atm osphericpressure gaschrom atography-m assspectrom etry_quadrupole-tim e of ight(APGC-M S/Q-TOF)

Theequipm entused forthechrom atographywasaCTC AnalyticsCom biPalautosam plercoupled to an Agilent6890 N de Agilent.The capillary colum n used was a HP-5M S (30m ×0.25 m ×250m )from AgilentTechnologies (Palo Alto,CA,USA).The oven

program wasasfollows:40°C for2m in,with a rate of10°C/m inup to300°C,m aintained for2m in.Theinjection typewassplitless,and thehelium ow was1m l/m in.The detectorwasan atm osphericpressure gaschrom atogra-

phy (APGC) source coupled to a quadrupole-tim e of ightanalyzer(Q-TOF)Xevo G2 from W aters(M ilford,M A,USA).APIpositivepolarityandsensitivityanalyzerm odewereselected.

The m assrange considered wasm /z45–450.The corona voltagewas2.1kV,the sam pling cone voltage wassetat10 and at30V,

Figure 1. M igration assaym adewith porkinstestine lled with isoctane.

E.Canella,P.Veraand C.Nerín

wileyonlinelibrary.com /journal/jm s Copyright© 2014John W iley& Sons,Ltd. J.MassSpectrom.2014,49,1181–1190

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the cone gaswassetat200l/h and the source tem perature was150°C.Thelockm assreferenceused wasper uorotributylam ine.Thetransferlinesettingswereasfollows:m akeup gasused was

nitrogenat400m l/m inandthetransferlinetem peraturewas350°C.M SEm odewasselectedfortheacquisition;collisionram penergy

from 20to 40V wasused.

Inductivelycoupled plasm a m assspectrom etry

ICP-M Sm easurem entswerecarried outwith aquadrupoleICP-M Sinstrum ent,Agilent7500.This instrum entwas equipped with aBabington nebulizerand adoublepassspraycham ber.Analysisofphosphorous(m /z31)wascarried outinthesam ples

andintheblank.Fullquant(3centralpointsperpeak)wasselectedaspeakpattern,theintegrationtim ewas0.1s,theintegrationtim e/m asswas0.3sand thenum berofrepetitionswas10.

Resultsand discussion

Identification

As no inform ation about the com pounds in the adhesive wasavailable,non targeted analysis was carried out.There is notasingle technique thatcan provide allthe necessary data fortheidentication ofthese com pounds.In thiswork,APGC-M S/Q-TOFisexplored asa powerfuland usefultechnique forthe challengeofuntargeted m easurem ents.

Gaschrom atographycoupled with m assspectrom etry

Initially,the identication ofthe com poundsin the adhesive wascarried outbyGC-M S/Q.Thedissolution ofthepureadhesive,withtheappropriatedilution,wasanalyzedfollowingtheproceduresop-tim ized in severalpreviousworksrelated with adhesives.[4,6,7,14,15]

Figure2showsthechrom atogram obtained.TheNIST08m assspectralsearch program version 2.0wasused

forthe identication ofthe com pounds.NIST08 returnsa‘hitlist’ofm atched chem icalcom pounds from the library.In this work,Reverse M atch Factor (RMF) was used for the m atching.Thisparam eteristhe norm alized dotproductwith square-rootscalingofthe subm itted m assspectrum and the library m assspectrum ,buttheelem entsthatarenotpresentin thelibrarym assspectrumare notincluded.ThisNIST 08 toolisespecially usefulwhen thespectrum representsm orethan asinglecom pound.Itcan beseenin Fig.1thattherearem anypeaksbetween theretention tim es18and 21m in.In thisregion ofthe chrom atogram ,clean spectra of

each single com pound are dif cultto obtain so theRM Ftoolwasuseful.Table 1 showsthe listof21 com poundsdetected in thiswork.

Onlyseven com poundsobtained areversem atch over800,whichwasconsidered the value thatcould lead to identication.Thesecom poundswerenaphthalene,decahydro derivativesand isom ersof octadecadienol. These com pounds could com e from thepetroleum -based solventused fortheproduction ofthehydrocar-bon resin[16]ofthePSA.From theGC-M S/Q analysis,14com poundsstillrem ained totally

unknown.ThelackofhighresolutionM Sinthissystem m akesveryalm ostim possible theidentication ofcom poundsbased on theirm assspectra.Therefore,a high resolution M S such asAPGC-M S/Q-TOFwasused in orderto tryto identifythecom pounds.

Atm osphericpressuregaschrom atographycoupled to aquadrupole hyphenated to a tim e of ightm assspectrom eter

Atm ospheric pressure gas chrom atography is a novelanalyticaltechnique that allows a conventionalGC to be coupled to anatm osphericsourceonaQ-TOF.Theionizationprocessesaredrivenbyaplasm aform ed bythe interaction ofnitrogen m oleculesandelectronsgenerated by a corona pin.M assspectra can be dom i-nated byM +,[M +H]+ or[M H] .[17]

TheAPGC cangeneratem assspectradom inated bythem olecu-larionwithreducedfragm entationwhentheconevoltageissetrel-atively low (10V).This fact,com bined with a TOF analyzerthatprovidesaccuratem ass,aswellastheuseofachem icaldatabase,could lead to obtain a m olecularform ula ofthe com pound.W iththe m olecularform ula in handsand the help ofspecicsoftware,theidentication ofthechem icalstructureism uch easier.M oreover,fragm entation com bined with accurate m ass can

provide inform ation about the m olecular structure, and thisinform ation could lead to the identication of the unknowns.Fragm entationwithanAPGC-MS/Q-TOFispossibleintwodifferentways.In the rstplace,fragm entation can be generated with thecone voltage.W hen the cone voltage isincreased,a controllablelevel of fragm entation can be induced.In the second place,fragm entation can be generated in the collision cell,increasingcollision voltage.Conditions used forthe acquisition are explained before.The

chrom atogram wasacquired in M SE m ode.M SEm odeisam ethodofdataacquisition thatinvolvestherapid alternation between twoconditions ofenergy,thus providing the accurate m ass oftheprecursorion,inadditiontofragm entions,forfurthercon rm atorypurposes.Asa result,two functionsare obtained in a single run.

6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.000

20000

40000

60000

80000

100000

120000

140000

Time-->

1

2

3

45

6

78

9

10

11

12

13,14

15

1617

18

19

20

21

Figure2. Chrom atogram oftheisoctaneextractoftheadhesivesam pleobtained byGC-M S/Q.

Identication ofNIASin adhesivesbyAPGC-Q-TOF

J.MassSpectrom .2014,49,1181–1190 Copyright© 2014John W iley& Sons,Ltd. wileyonlinelibrary.com /journal/jm s

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Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

%

0

100

89

1112

13

1417

18

Figure3. Chrom atogram oftheisoctaneextractoftheadhesivesam pleobtained byAPGC-M S/Q-TOF.

Table1. Num berofthecom poundinthechrom atogram ,retentiontim e(m in),com poundnam eanditsToxtreeclass,m atchfactorofspectraobtainedbyGC-Q-M S,m olecularform ula,accuratem assm easured and differencebetween thetheoreticalm assand theaccuratem assexpressed in m Dam ea-sured byAPGC-Q-TOF

Num ber Tr(m in) Com pound nam e(Toxtreeclass)

GC-Q-MS APGC-Q-TOF

M atchfactor

M olecularform ula

Accuratem assm easured

M asserror( m Da)

1 17.70 Unknown com pound

2 17.94 Naphthalene,decahydro-1,6-

dim ethyl-4-(1-m ethylethyl)-(I)

830

3 17.98 Naphthalene,decahydro-2,6-dim ethyl-3-octyl(I) 800

4 18.05 4,7-dim ethyl-1-isopropyl-perhydronaphthalene(I) 803

5 18.24 Z,E-2,13-Octadecadien-1-ol(I) 810

6 18.32 Z,Z-2,13-Octadecadien-1-ol(I) 801

7 18.38 E,Z-2,13-Octadecadien-1-ol(I) 820

8 18.50 Dicyclohexylphosphino isom er*(III) C15H30NP 256.2195 0.1








16 19.52 Naphthalene,decahydro-1,1-dim ethyl-(III) 850

17 19.61 Dicylcohexylphosphino isom er*(III) - C15H30NP 256.2195 0.1

18 20.11 M ethyl-3-(3,5-di-tert-butyl-4-

hydroxyphenyl)propanoate(II)

733 C18H28O3 292.2051 1.0






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Function1,which isthelow-energyfunction.Precursorion spectrathatcanleadtoam olecularform ulaofthecom poundareobtainedin thisfunction.Function 2,thehigh energyfunction,which istheresultofapplying a ram p ofcollision voltage in the collision cell.M assfragm entsfrom theprecursorionsthatcanleadtoastructureelucidation areobtained in thisfunction.Figure3showsthechrom atogram obtainedbyAPGC-MS/Q-TOF.

The peaks pro le obtained were different from those peaksobtained by GC-M S/Q electron im pactionization (Fig.1).These

differencescan be attributed to the differentionization processesthattakeplacein each technique,whatm eansthatin both cases,differentcom poundsareionized and detected.Thespectraofeach peakwerestudied in orderto elucidatethe

com pounds.Initially,thespectracom ingfrom function1withbothcone voltages were studied foreach peak,in orderto obtain am olecularform ula.Consideringtheaccuratem assofthem olecularion in each spectrum ,different possibilities for the m olecularform ulawere established.The criteriataken into accountwere (a)

A

B

Figure 4. (A)Spectrum show the spectra ofthe function 1 obtained at a cone voltage of30V ofthe com pound 1-(dicyclohexylphosphino)-N,N-dim ethylm ethanam ine;(B)spectrum ofthefunction 2ofthecom pound 1-(dicyclohexylphosphino)-N,N-dim ethylm ethanam ine.



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the i-Fit,which is the probability thatthe isotope pattern ofaparticularelem entalcom position in the listofresultsm atchesthepeaksinthem easuredspectrum ,and(b)them asstolerance,whichwassetin thiscaseat3m Da.Table 1 showsthatthe m ostprobable m olecularform ula of

the com pounds 8,9,11,12,13,14 and 17 was C15H30NP.Becausephosphorousisnotaverycom m onelem entinadhesivesam ples, qualitative ICP-M S analysis was carried out. Thepresence ofphosphorousin the sam ple wascon rm ed asthesam plecontainedanam ountofphosphorousthreetim eshigherthan that of the blank. M oreover, following the criteriam entioned previously,the m olecularform ulaofthe com poundnum ber18 wasC18H28O3.Oncethem olecularform ulaofeachaccuratem asswereknown,

itwasnecessaryto useadatabaseofchem icalcom poundsand toknow the typicalcom position ofa PSA in orderto elucidate thepossiblecom poundsthatcould be presentin thesam ple.Forthiswork, the databases [www.chem spider.com ][18] and [www.scinder.org][19]were used in orderto obtain a listofcandidatesforthe identication ofthe com pounds.The m olecularform ulaC15H30NP returned onlyone hitin thedatabaseChem spiderand10hitsinthedatabaseScinder.A bibliographicsearchwascarriedoutaboutifthesecom poundsarecom m onlyused tom anufacturePSAadhesives.Itwasfoundthatthephosphinederivativesareusedas catalyzers in resin polym erizations.[20–22] Therefore, thesecom poundswereproposed asthem ostlikelycandidates.The m olecular form ula C18H28O3 returned 592 hits at

Chem spiderand 2477 hits atScinder.Then,the com poundswere arranged by num ber ofreferences,and a bibliographicsearch aboutifthese com poundsare used to m anufacture PSAadhesives was carried out. Finally, it was found out thatantioxidants such as Irganox are com m only used in adhesiveform ulations.[23] M ethyl-3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate wasone ofthe hitsfound in the databases,and itwas a fragm entofthe Irganox m olecule.This com pound wassetasthe m ain candidatefortheidentication.Then,usingthehighenergyfunction(function2),thefragm enta-

tion spectrawereobtained.Theaccuratem assesofthefragm entswere considered in orderto nd outifthey could be generatedfrom the candidatesobtained in the databasesand then con rmtheiridentication.ThesoftwareM assFragm entfrom W aters(M ilford,M A,USA)was

used in orderto m atch each fragm entobtained by APGC-M S/Q-TOFwithafragm entofthecandidateproposedwiththedatabases.Thissoftware isbased on chem icallyintelligentalgorithm s.Itpro-posesion structurescom ing from the fragm entsofthe candidate

obtained from the databases,along with the exactm ass errorsand scoresforeach proposed structure.The scoresare obtainedbased onthelikelihood ofbreaking certain bondsand consideringhow wellthe resultsagree with the exactm assgenerated.There-fore,the lowerthe score and the betterthe m assaccuracy,thehigherthe con dence isin obtaining the correctstructure forthefragm ention interrogated.As a result, som e com pounds were identied (Table 1).

Figure4A showsthespectraofthefunction 1obtained ataconevoltage of30V ofthe com pound 1-(dicyclohexylphosphino)-N,N-dim ethylm ethanam ine.In thisspectrum ,itcan be observedthatatthisvoltage,fragm entation wasproduced.Nevertheless,them olecularion [M +H]+ wasfound,and itwascon rm ed withthe spectraobtained at10V.M oreover,Fig.4B showsthe spec-trum ofthefunction 2forthiscom pound.Itwasobtained byap-plying aconevoltageof30V and acollision ram p energyin thecollision cellfrom 20to 40V.M aking acom parison ofboth spec-tra,itcan beseen thatin Fig.4A,therearem anyionswithahighm /zbecause fragm entation wasrelatively soft.The m ostabun-dantfragm entwas159.1172 because itsfragm entation did notim plyaringfragm entation.Inthism olecule,ringfragm entationshappened,involving thecleavageoftwobonds,assaturated ali-cyclichydrocarbonsnorm allydecom pose.[24]

In contrast,Fig.4B showsthatthe m ostabundantionsin func-tion 2weretheoneswith alowerm /z,becausethefragm entationwasm oreaggressive,becauseacollision ram p wasapplied.Figure 4 shows the scores obtained for the fragm ents that

rangedfrom 0.5to5.ThevalueS=0.5wasobtainedwhenthefrag-m entation involved onlyone bond and S=5 wasobtained whenfourbondswerebroken,asthiscase islessadvantageous.On theotherhand,thedifferencebetweenthem /zobtainedforeachfrag-m entbyAPGC-M S/Q-TOFand thetheoreticalm /zofthefragm entcandidates proposed ranged between 0.4 and 1.4m Da (Fig.4).Therefore,theywerebelow them assaccuracyoftheQ-TOFequip-m ent,which wassetatam axim um of3m Dain the calibration oftheinstrum ent.Table 1 shows thatseven isom ers ofthis com pound were

identied in the sam ple.Figure 5 showsthe chem icalstructureofthe differentisom ersofthe dicylohexylphosphino derivative.Thespectraofallofthesevenisom ersfound intheadhesivehadahigh abundanceofm /z159.1172,whatim plied thefragm enta-tion ofam ethylgroup bonded to nitrogen.Becauseofthat,theisom er3-(dicyclohexylphospino)propan-1-am ine could not bepresentin the sam ple,because thism olecule doesnothave am ethylgroupbondedtonitrogen.Theothersevenisom erswerepresentin the sam ple.

Figure5. Chem icalstructureofthedifferentisom ersofthedicylohexylphosphino derivative.



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Theoraltoxicityofthese com poundshasnotbeen studied yet,and Toxtreetheoreticalclassication wasused toestim ateitbasedon the chem ical structure (Table 1).These com pounds wereclassied in classIIIofCram ertoxicity,which isthe highestclassof toxicity according to the decision tree.[25,26] This is veryrem arkable inform ation as the adhesive com pany ignored thepresence ofthese com poundsin the resin thatthey used fortheadhesiveform ulation.Figure6A showsthespectraofthefunction1obtainedat30Vof

cone voltage for the com pound m ethyl-3-(3,5-di-tert-butyl-4-

hydroxyphenyl)propanoate.At this voltage, the m olecular ion[M +H]+ wasthem ostabundantion,astherewasnofragm entationduetothepresenceofanarom aticnucleusthatproducesstabiliza-tion bychargedelocalization.[24]

Figure 6B shows fragm entation in function 2,where collisionvoltagewasapplied.Thescoresobtained forthefragm entsrangedfrom 0.5to12.ThevalueS=0.5wasobtainedwhenonlyonebondwas fragm ented,and itdid notinvolve the arom atic ring.Thehighest scores were obtained when the fragm entation im pliedthe bond between the arom atic ring and the tert-butylgroup as

A

B

Figure6. (A)Spectrum showsthespectraofthefunction1obtainedataconevoltageof30Vofthecom poundm ethyl-3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate;(B)spectrum ofthefunction 2ofthecom pound m ethyl-3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate.



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the cleavage of this bond is m ore hindered. M oreover, thedifferencebetween m /zobtained foreach fragm entbyAPGC-M S/Q-TOF and the theoreticalm /zofthe fragm entforthe proposedcandidatesranged between 0.0 and 1.0m Da (Fig.6B).Therefore,theywerebelow them assaccuracyoftheQ-TOFequipm ent,whatwassetatam axim um of3m Dainthecalibrationoftheinstrum ent.Thiscom pound isa NIAS,asitisa degradation productfrom

Irganox 1076 used asantioxidantin the adhesive form ulation.The oraltoxicityofthiscom pound hasnotbeen studied yet,soToxtree theoreticalclassication wasused in orderto estim ateit(Table 1),aswaspreviously m entioned.Thiscom pound wasclassied asclassIIofCram ertoxicity,which ism edium toxicity.Besides,thiscom pound hasam olecularweightof292Da.Itisasem ivolatile com pound that willhave a higher tendency tom igrate than Irganox 1076 with a higher m olecular weight(531Da).The presence ofthis com pound reaf rm s the im por-tance ofidentifying the com pounds in a sam ple intended tobe in directcontactwith food,asthese NIAS could m igrate tothefood and contam inate it.

M igration study

Adhesives are not yet covered by a specic EU legislation.Nevertheless,they m ustcom ply Regulation (EC)No 1935/2004[27]

thatrequires thatm aterials and articles m ustnottransfertheirconstituents to food in quantities,which could endanger thehum an health.Consequently,to nd outifthe adhesive contam i-natesthefood,m igration studiesm ustbecarried out.

The adhesive isnorm allyused forlabelsfordirectfood contactwith cheese,sausages,cold cured m eats and cold fresh m eats.Liquid fatty sim ulants recom m ended by the Com m issionRegulation(EU)10/2011[28]orthesubstitutedliquid sim ulantssuchasethanol95% orisooctane com pletely dissolved the adhesive.Because ofthat,two differentexperim entswere designed forthespecicm igration tests.Firstly,Tenaxwasselected assolid food sim ulant.Becausethe

adhesiveisaPSA forlabelsand itwasverysticky,TenaxW couldnotbe exposed directlyoverthe labelsbecause itcould notberem oved afterthe m igration assay to be analyzed.Because ofthat,the labels were stuck to rice paper,as this paper wasconsidered slim m erand porousenough to letthe com poundsdiffuse quickly through itand reach the TenaxW .In this way,the rice paperwould notactasa barrierbetween the adhesiveand thesolid sim ulant.Table 2 shows the m igration results considering that allthe

packaging wascovered bythe adhesive.M igration ofcom poundsranged from 1.01 to 16.19 g/dm 2.Nevertheless,this m igrationassays underestim ate the m igration because only low m olecularweight substances willdiffuse through the paper.Because theadhesive ispressure-sensitive,the substances willbe transferredto the food both by a diffusion process and also by adhesion/physicaldetachm entfrom thelabel.Becauseofthat,itwasdecidedtodoam orerealisticm igrationassay,thussim ulatingtheintendedcontactofthelabelon sausages.Sausagesin Spain are m ade lling pork intestine with m inced

pork m eat.Then,itwasdecided to llin the pork intestine withisoctane as fat sim ulant.As both ethanol95% and isooctane

Table2. Num berofcom pound in thechrom atogram ,retention tim e,com pound nam e,lim itsofdetection (ng/g)ofthestandardsused forthequan-tication,m igrationvalues( g/dm 2),m igrationvalues( g/g)consideringthesam eproportionsoftheporkintestine lledwithisoctane,m igrationtoporkintestine( g/g)and m igration to isoctane

Num ber Tr(m in)

Com poundnam e

LOD(ng/g)

M igrationTenax( g/dm 2)

M igrationTenax( g/g)

M igrationporkintestine( g/g)

M igrationisoctane( g/g)

2 17.94 Naphthalene.decahydro-1.6-

dim ethyl-4-(1-m ethylethyl)-a(I)

6.1 2.08 0.12 3.01 0.08

3 17.98 Naphthalene.decahydro-2.6-

dim ethyl-3-octyla(I)

6.1 5.93 0.34 1.92 0.11

4 18.05 4.7-dim ethyl-1-isopropyl-

perhydronaphthalenea (I)

6.1 10.42 0.59 3.21 0.42

5 18.24 Z.E-2.13-Octadecadien-1-olb (I) 5.3 1.01 0.06 0.54 0.01

6 18.32 Z.Z-2.13-Octadecadien-1-olb(I) 5.3 5.07 0.29 2.12 0.05

7 18.38 E.Z-2.13-Octadecadien-1-olb (I) 5.3 3.31 0.19 1.71 0.05

8 18.50 Dicyclohexylphosphino isom er*c(III) 8.7 5.53 0.32 15.1 0.45





14 19.17 Dicyclohexylphosphino isom er*c(III) 2.50 0.14 6.91 0.01

16 19.52 Naphthalene.decahydro-1.1-dim ethyl-a(III) 6.1 16.19 0.92 2.55 0.50

17 19.61 Dicylcohexylphosphino isom er*c(III) 8.7 5.73 0.33 6.97 0.60

18 20.11 M ethyl-3-(3.5-di-tert-butyl-4-

hydroxyphenyl)propanoated (II)

7.2 15.14 0.86 6.85 0.40

aQuantied with decahydronaphthalene.bQuantied with 3-octanol.cQuantied with tert-butyldicyclohexylphosphine.dQuantied with 1-m ethyl-3-m ethoxy-4-isopropylbenzene.



1188

dissolved com pletely the adhesive,isooctane wasselected in thiscase to facilitate the furtherGC analysis.Afterthe m igration test,the isoctane used asfood sim ulantand the extractcom ing fromtheporkintestinewereanalyzed separately.Table2showstheresultsofthem igration to porkintestineand

to isoctane.M oreover,m igration to Tenaxexpressed as g/dm 2 isexpressed also as g/g taking into accounttheproportionsofareaandgram sofisoctaneusedin theporkintestinem igrationassayinorderto com parethevaluesin both experim ents.Itcan beseen thatthem igration valuesto porkintestineand

isoctane are m uch higher than m igration values to Tenax.Nevertheless, it was observed that the pork intestine waspartially perm eable to the isoctane asthe externalpartoftheintestine was im pregnated with it.It im plies that isoctane iscapable to diffuse through the pork intestine and reach theadhesive dissolving it.Then,thism igration assayoverestim atedthe m igration values.Itisworth to pointoutthatthe m igrationvaluesin theporkintestinewerem uch higherthan thosefoundin isooctane,what m eans that m ost of the com pounds areconcentrated in thisbarrierlayer,m ade ofproteinsand fat.Thisfactisan additionaladvantage,asthe peelofcold cured m eatcan be rem oved,then rem oving m ostofthe m igrants.Asthem igration conditionsinvolved in thiscase 2daysofexposure at20°C,an additionalstudy could be carried out to con rm iflongerexposure tim e with the intestine enhance the m igrationorthe diffusion to the realm eat.However,thisstudy isoutofthe scope ofthispaper.Thisstudydem onstratesthatTenaxand isooctanerepresentthe

underestim ation and overestim ation m igration values for thisapplication ofPSA adhesives.Thissuggeststhatm igration on realfoodisrequired,asthereisnosim ulantthatcanrepresentafeasiblevalueofm igration in thisparticularcase.Thisworkdem onstratesthatin som escenarios,itisdif cultto

replicate the intended contact of m aterials used in foodpackaging with sim ulants.Adhesivesare notyetcovered by aspeci c EU legislation,and plastics regulations do not coverthesekindsofapplications.Asaresult,theonlystudythatcouldgivecon dentresultsofm igration,toknow ifthelabelcom plieswith the Regulation (EC)No 1935/2004,willbe the m igrationstudy from the labelto the realfood.Thiswork willbe carriedoutin the nearfuture.

Conclusions

This work has dem onstrated thatthe identication ofunknowncom poundsin asam pleintended to be in contactwith food isanessentialstep before the m igration studies.APGC-M S/Q-TOF hasdem onstrated to be a powerful technique,supplem entary totraditionalGC-M S(Q),forthe identication ofunknowns.The factthatthehighresolutionM Sprovidesaccuratem assspectraofbothm olecularion and its fragm ents,com bined with a huge biblio-graphicsearch and suitablesoftware,leadsto the identication ofseveralcom poundspresentin thesam ple,m ostthem NIAS,whichwere notknown by the producercom pany and which were notpreviouslyidentied.It has been proved thatthe NIAS identied in the sam ple

studied here m igrated to the food sim ulantsused in the m igra-tion assays.In addition,the com parison between the m igration results to

Tenax and to isooctane in this particularcase ofPSA adhesivesshowed thatnoneofthesesubstitutesrepresentfeasiblem igration

resultsand m igration to realfood isrequired.Itisinteresting toem phasize thatthe pork intestine used here to sim ulate the realapplicationofthelabelsbehavedasabarrierform igrantsinwhichthe com pounds were concentrated.This fact has never beenstudied beforeand willbethem atterofafutureworkdealing withautoadhesivelabelsand realm eat.

Acknowledgem ents

Authors acknowledge the com pany Sam tack for the sam plessupplied forthiswork and forthe partial nance ofthe research.The APGC-M S/Q-TOF equipm entwas acquired with the help ofFEDER funds.The authorsthank Gobierno de Aragón and FondoSocialEuropeo for nancialhelp given to GUIA group-T-10.

References

[1] E.M .Petrie.Handbook ofAdhesivesand Sealants.M .Graw-Hill,NewYork,2007.

[2] C.Nerin,P.Alfaro,M .Aznar,C.Dom eno.The challenge ofidentifyingnon-intentionallyadded substancesfrom food packaging m aterials:areview.Anal.Chim.Acta.2013,775,14–24.

[3] C Nerin,J.L.Ubeda,P.Alfaro,Y.Dahm ani,M .Aznar,E.Canellas,R Ausejo. Com pounds from m ultilayer plastic bags causereproductive failures in articial insem ination. Scientic Reports.2014,4,4913.DOI:10.1038/srep04913

[4] C.Nerin,E.Canellas,M .Aznar,P.Silcock.Analyticalm ethodsforthescreening ofpotentialvolatile m igrants from acrylic-base adhesivesused in food-contact m aterials.Food Addit.Contam .A.2009,26,1592–1601.

[5] C. Nerin, J. Gaspar, P. Vera, E. Canellas, M . Aznar, P. M ercea.Determ ination ofpartition and diffusion coef cientsofcom ponentsoftwo rubberadhesivesin differentm ultilayerm aterials.Int.J.Adhes.Adhes.2013,40,56–63.

[6] P.Vera,M .Aznar,P.M ercea,C.Nerin.Studyofhotm eltadhesivesused in food packaging m ultilayerlam inates.Evaluation ofthem ain factors affecting m igration to food.J.M ater.Chem .2011,21,420–431.

[7] M .Aznar,P.Vera,E.Canellas,C.Nerin,P.M ercea,A.Stoerm er.Com position of the adhesives used in food packaging m ultilayerm aterials and m igration studies from packaging to food.J.Mater.Chem .2011,21,4358–4370.

[8] E.C.Horning,M .G.Horning,D.I.Carroll,I.Dzidic,Rn.Stillwel.Newpicogram detection system based on a m ass-spectrom eterwith anexternalionization sourceatatm ospheric-pressure.Anal.Chem .1973,45,936–943.

[9] T. Bristow, M . Harrison, M . Sim s. The application of gaschrom atography/atm ospheric pressure chem icalionisation tim e-of-ightm assspectrom etryto im purity identication in Pharm aceuticalDevelopm ent.RapidCom m un.MassSp.2010,24,1673–1681.

[10] E.Canellas,P.Vera,C.Dom eno,A.P.Alfaro,C.Nerin.Atm osphericpressure gas chrom atography coupled to quadrupole-tim e of ightm ass spectrom etry as a powerful tool for identication of nonintentionally added substances in acrylic adhesives used in foodpackaging m aterials.J.Chrom atogr.A.2012,1235,141–148.

[11] D.Stevens,Q.Shi,C.S.Hsu.Novelanalyticaltechniqueforpetroleumbiom arkeranalysis.Ener.Fuel.2013,27,167–171.

[12] C.J.W achsrnuth,M .F.Alm stetter,M .C.W aldhier,M .A.Gruber,N.Nurnberger,P.J.Oefner,K.Dettm er.Perform ance evaluation ofgaschrom atography-atm ospheric pressure chem icalionization-tim e-of- ight m ass spectrom etry for m etabolic ngerprinting andpro ling.Anal.Chem .2011,83,7514–7522.

[13] AENOR.UNE-EN-14338 (2004)AENOR.Papely cartón para contactoalim entario.Condicionesparaladeterm inacióndelam igración desdeelpapelycartón utilizando óxido depolifenileno m odicado (M PPO)com o sim ulante.2004.

[14] P.Vera,E.Canellas,C.Nerin.Identication ofnon-volatile com poundsand theirm igration from hotm eltadhesivesused in food packagingm aterialscharacterized by ultra-perform ance liquid chrom atographycoupled to quadrupole tim e-of- ight m ass spectrom etry. Anal.Bioanal.Chem .2013,405,4747–4754.



1189

[15] E.Canellas,M .Aznar,C.Nerin,P.M ercea.Partition and diffusion ofvolatile com poundsfrom acrylic adhesivesused forfood packagingm ultilayersm anufacturing.J.Mater.Chem .2010,20,5100–5109.

[16] US Patent classication 156334.Aplication Num ber 20130081760.Tokyo OhkaKogyo Co.2013.

[17] K.W orrall,P.Silcock.Addressingchem icaldiversityandexpandinganalyticalcapabilities with atm ospheric pressure GC.W aters application notes.January2010,720003292en.

[18] RoyalSocietyofChem istry.Chem spider,2014,http://www.chem spider.com .[19] Am erican Chem icalSociety.Scinder,2014,https://SciFinder.cas.org.[20] N.Singha,S.Sivaram .Hom ogeneouscatalytichydrogenation ofpoly

(styrene-co-butadiene)using a ruthenium based W ilkinson catalyst.Polym erBulletin.1995,35,121–128.

[21] C.U.Pittm anJr,S.K.W uu,S.E.Jacobson.1,3-Butadieneoligom erizationcatalyzed by polym er-attached palladium com plexes.Com parisonwith hom ogeneouscatalysis.J.Catal.1976,44,87–100.

[22] R.M .Hoff,RobertT.Handbook ofTransition M etalPolym erizationCatalysts.R.M .Hoff,RobertT.,Place,2010.

[23] I.Benedek.Pressure-sensitiveform ulation.B.BV,Place,2000.[24] F. T. M cLafferty, F. Tureček. Interpretation of m ass spectra.

1993.[25] IdeaconsultLtd.Toxtree–ToxicHazard Estim ation bydecision treeap-

proach.(So a,Bulgaria).http://toxtree.sourceforge.net/.Accessed 24M arch 2011.2011.

[26] G.M .Cram er,R.A.Ford,R.L.Hall.Estim ation oftoxic hazard – adecision treeapproach.JCosmetToxicol.1978,16,255–276.

[27] EGULATION (EC)No 1935/2004OFTHEEUROPEAN PARLIAM ENTANDOF THE COUNCIL of 27 October 2004 on m aterials and articlesintended to com e into contactwith food and repealing Directives80/590/EEC and 89/109/EEC.2004.

[28] COMM ISSION REGULATION (EU)No10/2011of14January2011onplas-ticm aterialsandarticlesintendedtocom eintocontactwithfood.2011.



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