Rev. bras. farmacogn. vol.25 número2

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w w w. s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Comparison

and

evaluation

of

two

methods

for

the

pesticide

residue

analysis

of

organophosphates

in

yerba

mate

Lucía

Pareja

a

_,

_Silvina

_Niell

a

_,

_Zisis

_Vryzas

b,1

,

Joaquín

González

c

,

María

Verónica

Cesio

a,c

,

Euphemia

P. Mourkidou

b

_,

_Horacio

_Heinzen

a,c,∗

a_Polo_{Agroalimentario}_y_{Agroindustrial,}_Departamento_de_Química_del_Litoral,_Centro_{Universitario}_de_Paysandú,_Universidad_de_la_República,_Estación_Experimental_Mario_Cassinoni,

Ruta3,Km363,Paysandú,Uruguay

b_Aristotle_University_of_{Thessaloniki,}_Pesticide_Science_Laboratory,_P.O._Box_1678,₅₄₁₂₄_{Thessaloniki,}_Greece

c_{Farmacognosia}_y_Productos_Naturales,_Departamento_de_Química_Orgánica,_Facultad_de_Química,_UdelaR,_General_Flores_2124,₁₁₈₀₀_Montevideo,_Uruguay

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16July2014 Accepted2February2015 Availableonline21March2015

Keywords:

Yerbamate Pesticideresidues QuEChERS MAE GC–FPD

a

b

s

t

r

a

c

t

MicrowaveAssistedExtractionandamodifiedCEN-QuEChERSmethodologywereevaluatedas extrac-tionandcleanupproceduresforthesimultaneousanalysisof42organophosphatepesticidesinyerba mate(Ilexparaguaiensis).Theobtainedextractswereanalyzedbygaschromatographyusingaflame photometricdetector.Linearity,recoverypercentages,relativestandarddeviations,detectionand quan-tificationlimitsandmatrixeffectsweredeterminedaccordingtoDG-SANCOguidelinesforbothmethods. At0.2and0.5mg/kgtheevaluatedmethodsshowedpercentagesrecoveriesbetween70and120%for mostoftheanalytes.UsingMicrowaveAssistedExtractionmethodology,33pesticideresiduescouldbe properlyanalyzedwhereasonly27couldbedeterminedwiththeproposedmodifiedQuEChERS.All rel-ativestandarddeviationwerebelow18%exceptforomethoateanddisulfotonsulfonewhenevaluated bythemodifiedQuEChERS.Thelimitsofdetectioninbothmethodologieswere0.2mg/kgformostof theanalyzedcompounds.TheaveragedetectionlimitforQuEChERSwas0.04mg/kg.For19ofthe ana-lytesdeterminedthroughMicrowaveAssistedExtractionthelowestvalidatedlevelwere0.004mg/kg. Signalsuppression/enhancementwasobservedformostofthepesticides,thusmatrix-matched calibra-tioncurveswereusedforquantification.TheMicrowaveAssistedExtractionandQuEChERSprocedures studiedcoulddetecttheorganophosphatepesticidesabovetheMRLfixedfor“mate”bytheEuropean Union.Theyhavebeensuccessfullyappliedforthedeterminationoforganophosphatepesticideresidues incommercialsamplesandthepositiveswereconfirmedthroughGC–(ITD)-MS.

Introduction

IlexparaguariensisA.St.-Hil.,Aquifoliaceae,isanativetreefrom theRiodelaPlatabasininSouthAmerica.Ithasbeencultivated sincecolonialtimes.Nowadays,300,000tonsofprocessedleaves

areconsumedeach year,which areusedtoprepareaninfusion

calledMate,thenationalbeverageofUruguay,Argentina,southern Brazil,andParaguay.Theartofmatedrinkinghasbeendescribed

∗ Correspondingauthorat: PoloAgroalimentarioy Agroindustrial, Departa-mentodeQuímicadelLitoral,CentroUniversitariodePaysandú,Universidadde laRepública,EstaciónExperimentalMarioCassinoni,Ruta3,Km363,Paysandú, Uruguay.

E-mail:heinzen@fq.edu.uy(H.Heinzen).

1_Present_address:_Democritus_University_of_Thrace,_Faculty_of_Agricultural

Devel-opment,LaboratoryofAgriculturalPharmacology&Ecotoxicology,193,Pantazidou str.68200,N.Orestiada,Greece.

byPérezParadaetal.(2010),Jacquesetal.(2007)andVázquez

andMoyna(1986).Thistraditionalbeverageisreputedtohavea

characteristicbittertasteandhepatoprotective,choleretic, hypoc-holesteremic, antioxidant, antirheumatic, diuretic and lipolitic properties(Filipetal.,2001).

Asanyothercrop,yerbamateisattackedduringfarmingby pests,especially mites,leaf-eatingbeetlesand caterpillars forc-ing theuseof organophosphate insecticides, that left pesticide residues.AsyerbamatehasbeenbeingsoldsteadilyinEuropealone orincombinationwithotherherbsasenergyteaorasaweight reductionaid(Andradeetal.,2012;Hecketal.,2007)the Euro-peanUnionhasestablishedMRLofpesticideresiduesontheleaves

(EuropeanCommission,2005).

Yerbamateisacomplexmatrixforpesticideresiduesanalysis dueitschemicalcomposition(naturalpigments,lipids,vitamins andsecondarymetabolites:polyphenols,saponins,andxanthines likecaffeineand theobromine)(Hecketal., 2007;Vázquez and

http://dx.doi.org/10.1016/j.bjp.2015.02.001

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Moyna, 1986), and only few studieshave beenreported(Pérez

Parada et al., 2010; Jacques et al., 2006). Particularly, caffeine

andsaponinsareco-extractedwithpesticidesastheyhave sim-ilar physicochemical properties. Large amounts of caffeine and saponinscontaminatetheinjectorandthedetectoroftheGC sys-tem,interferingwiththedeterminationofpesticideresidues(Xu

etal.,2011;PérezParadaetal.,2010).Thegaschromatographic

separation of pesticides has been reviewed. Several analytical

strategies and column types have been proposed for pesticide

residueanalysisinmatricessuchastea,tobaccoandherbs(Liuand

Min,2012;Khanetal.,2014).

Theactualtrendforpesticideresiduesdeterminationattrace levelsseeksforvalidatedanalyticalmethodswithshorteranalysis timeandhighersamplethroughput(Chenetal.,2011).Considering matea“tea-like”matrix,thereareseveralmethodologiesreported fortheanalysisofpesticideresiduesinmadetea,teainfusionand spentleaves.Thesemethodsinclude,forexample,extractionwith differentsolventslikeethylacetate(EtOAc),cyclohexaneor ace-tonitrile,combinedwithdifferentcleanupprocedures;suchasgel permeation,andsolidphasecleanup,eitherdispersiveorusing car-tridges,followedbyliquidorgaschromatographyanalysis,coupled tomassdetectors(Huangetal.,2007,2009;Kanraretal.,2010).

Lozanoetal.(2012)andCajkaetal.(2012),describedthe

applica-tionofamodifiedQuEChERSforthedeterminationofpesticides indifferenttypesofteas.TheQuEChERSapproachisavery flex-ibleoneasitisatemplate toadapt theprocedureaccordingto analyteproperties,matrixcomposition,equipmentandanalytical techniquesavailableinthelaboratory(Anastassiadesetal.,2003). QuEChERSbasedmethodshavebeenusedtoassesfoodsafetyand environmentalsustainability.SeveralreportsonQuEChERS appli-cationsinherbshavebeendevelopedbuttherearenoreportson QuEChERSfortheanalysisofpesticideresiduesinyerbamateleaves

(Sadowska-Rocieketal.,2013;Attallahetal.,2012;Lozanoetal.,

2012;Chenetal.,2011,2012a,b;Nguyenetal.,2010;Haywardetal.,

2013).

Some other methodologies employing pressurized liquid

extraction,dispersiveliquid–liquidmicroextractionanddispersive solidphaseextractionhavebeendescribedintheliteratureforthe analysisofpesticideresiduesintea(Nguyenetal.,2010;Moinfar

etal.,2009;Choetal.,2008).Microwaveassistedextraction(MAE)

hasbeenassayedasextractionandclean upprocedurein food

matrices(Vryzasetal.,2007;Papadakisetal.,2006;Vryzasetal., 2002),butthereisnoreportforMAEinherbalteas.Itsmain advan-tagesarelowsolventconsumption,shortextractiontime,andhigh levelof automationwithhighextractionefficiency(Niell etal.,

2011;Papadakisetal.,2006).

ThepresentworkcomparesMAEandQuEChERSperformance

forpesticideresiduesanalysisofyerbamateleaves.

Materialsandmethods

Analyticalstandards andpesticidegrade solventswerefrom

Promochem(Wesel,Germany),Riedel-deHáën(Seelze,Germany)

and Merck (Darmstadt, Germany). Anhydrous magnesium

sul-phate(MgSO4), Graphitized CarbonBlack (GCB) and ENVI-carb

SPE, cartridge and PSA (primary–secondary amine) were from

Sigma–Aldrich(Madrid,Spain).Sep-Paksilicacartridgeswerefrom WatersCorporation(Milford,MA,USA),PSAsodiumcitratedibasic sesquihydrateandsodiumcitratetribasicdihydrateweresupplied fromSupelco(Bellefonte,PA,USA).

Stocksolutionsofindividualanalytesat1mg/mlwereprepared

in EtOAc; three mixed standard stock solutions were prepared

and seriallydilutedwithEtOAc toproducea series ofworking standard solutions of 0.001–20mg/l. The latter solutions were usedfortheconstructionofcalibrationcurvesandthepreparation

of the fortified samples. Stock solutions were stored in deep

freeze(−23◦_C),_while_the_working_standard_solutions_were_stored

refrigerated and renewed at weekly intervals. Matrix-matched

calibrationsolutions(0.05–4␮g/ml)wereprepareddrying0.2ml

yerbamate extractundera N2 streamand fortifiedwith0.2ml workingstandardsolutionsofpesticidesatvariousconcentrations. Thesematrix-matchedsolutionswereusedtopreparecalibration curves,toevaluatethelinearrange,andtocalculaterecoveries.

Apparatus

TheMSP1000laboratorymicrowavesystem(CEM,Matthews,

NC)equippedwith12vesselcarouselwithtemperatureand

pres-sure sensors, operated in the closed mode was used for the

microwaveassistedextraction(MAE)ofyerbamateleaves. PTFE-linedextractionvesselswereused.

PesticideresiduesanalysiswasperformedinaThermoFisher Scientific,modelFinniganTraceGC(Rodano,Milan,Italy),gas chro-matographequippedwithaflamephotometricdetector(FPD),an

autosampler(modelAS3000), and aProgrammed Temperature

Vaporizer(PTV)(initialtemperaturewas60◦_C_(hold_for_1.5_min)

thenincreasedto220◦_C_at_the_rate_of₅◦_C/s_for₃₅_min)._The_GC

ovenhadtwocapillarycolumnsintandem(BP-1,10m,ID0.53mm, 2.65␮m filmthickness respectively) from Agilent Technologies

(Avondale,PH,USA).Thedetectorandinjectortemperatureswere at300and220◦_C,_{respectively.}_Helium_was_used_as_carrier_gas_at

a constant flowrate of7ml/min. For FPDoperationthe

hydro-gen flow was setat 90ml/min and theair one at 115ml/min.

Heliumwasusedasthedetectormakeupgasat30ml/min.The

temperatureprogramoftheGCovenwas:initialT50◦_C_(hold_for

1min),increasedto170◦_C_at₁₆◦_C/min,_ramped_to₂₂₀◦_C_at_the

rateof6◦_C/min_(hold_for₁_min),_increased_to₂₄₀◦_C_at_the_rate_of

4◦_C/min,_finally_to₂₈₀◦_C_at_the_rate_of₅◦_C/min_(hold_for₁₀_min)

andreturnedtoinitialconditionsin5min.Totalruntime40.8min. Theinjectionvolumewas2␮l.Thesoftwareforthecontrolofthe

GC–FPDwasChromCard,ThermoFinnigan(Rodano,Milan,Italy). Residueconfirmationinrealsampleanalysiswereperformedin aTrace2000GCequippedwithaThermoQuestautosampler(model AS2000),asplit/splitlessinjectorconnectedwiththeGCQplus ion-trapmassspectrometer(Thermoquest,Austin,TX,USA),operating ineitherMSn_or_SIM_modes,_injecting₂

␮lofthetestedsolutions.

TheoperationconditionsoftheGCQPlusMS systemwere: the

injectorin splitlessmodeunderisothermalconditionsat220◦_C

andthesplitvalvewasopened1minaftertheinjection.Gas chro-matographywascarriedoutonDB-5MS(J&WScientific)0.25␮m,

30m×0.25mmwitha1m×0.25mmi.d.guardcolumnof

deacti-vatedfusedsilica(Alltech,Augsburg,Germany).Oventemperature gradientwasprogrammedasfollows:theinitialtemperaturewas 50◦_C_for₁_min,_and_increased_to₁₂₀◦_C_at_the_rate_of_22.5◦_C/min,

rampedto250◦_C_at₃◦_C/min_for₁_min_and_then_increased_to₂₈₅◦_C

attherateof15◦_C/min_which_was_held_for₁₀_min_and_returned

totheinitialconditionsin5min.Heliumwasthecarriergasata flowrateof1ml/min.TheMSsystemwasoperatedintheelectron impactionizationwithpositivepolarityionmode. Theemission currentwas250mA,themultipliervoltagewas1700Vandafull

scanrangewassetto50–500amuwithmaximumiontime25ms,

10microscansandAGCtargetvalueof50.Thetransferlineand themanifoldtemperatureweresetat285and220◦_C,_{respectively.}

AnalyteswereidentifiedbycomparingtheirEImassspectrawith home-madelibraries.

Extractionprocedures

MAE

Drymateleaves(5g)wereweighedandputintothe

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vesselandshakedvigorouslybyhandfor30s.Setsof12vessels weremicrowaveextractedaccordingtothefollowingoperational

parameters;magnetronpower800W,maximumpressure100psi,

heatedto80◦_C_in₁₀_min_and_maintained_for₁₅_min.

Afterremovingthevesselsfromthemicrowaveoven,theywere cooledatroomtemperature.Theextractfromeachvesselwas fil-teredundervacuumandrinsedwith15mlMeCN.A15mlaliquot wastransferredtoatubecontaining1mltolueneandevaporated untildrynessunderN2stream.Samplecleanupconsistedintwo stepsfollowingamodificationofthemethoddescribedin2003by Haibetal.First,thedryextractwasre-dissolvedin1mlofMeCN andloadedintoa690mgsilicacartridgefollowedbytheaddition of0.5mloftoluene.Thetargetcompoundswereelutedwith3ml ofanacetone–toluene(8:2)mixture.The3mleluatewasloaded intoa500mgENVI-carbcartridgeandelutedwith3mlofacetone. Eachcartridgewaspre-conditionedwith5mlofacetone.Thefinal eluatewascollected,thesolventevaporatedandtheresiduewas dissolvedin200␮lofEtOAcforGC–FPDanalysis.

QuEChERS

The employed procedure was a modification of the citrate

bufferedQuEChERSmethodCEN15662(www.cen.eu),(Payáetal.,

2007;Anastassiadesetal.,2010).Arepresentative2gsamplewas

weighed in a 50ml PTFE centrifugation tube. Afterwards, 10g

ofchopped ice and 10ml of MeCNwere addedinto each tube

(Haywardetal.,2013;Rajskietal.,2013).Then4gofMgSO4,1gof

NaCl,0.5gofsodiumcitratedibasicsesquihydrateand1gofsodium citratetribasicdihydratewereadded.Thetubewashandshaken for4minandcentrifuged,10minat3000×g.Forthecleanupstep,

a6mlaliquotoftheextractwastransferredtoa15mlPTFE cen-trifugationtubecontaining855mgofMgSO4,150mgofPSAand 45mgofGCB.Thistubewasshakenfor20susingavortexand cen-trifugedfor10minat3000_×g.Afterthat40␮lof5%formicacidin

MeCNwereaddedto4mlofextractanda1mlaliquotwas trans-ferredtoa5mlconictubeandevaporatedundernitrogenstream untildryness.Finally,theextractwasdissolvedin200␮lofEtOAc

forGC–FPDanalysis.

Resultsanddiscussion

Extractionandcleanupoptimization

The analysisof pesticide residues using microwave assisted extractionsystems requirethe optimizationof different opera-tionalparameterssuchasmagnetronpower,temperature,pressure andextractiontime.Theoptimumconditionsfortheextractionof pesticidesbyMAEindifferentmatriceswereselectedtakinginto considerationpreviousreports(Nielletal.,2011;Vryzasetal.,2002,

2007;Papadakisetal.,2006;VryzasandPapadopoulou-Mourkidou,

2002). QuEChERSand MAEprotocolsyielded highly pigmented

extractsandGCBwasused intheclean upsteptoremove the

co-extractedchlorophyll.However,theamountofGCBusedwas

abalancebetweentherecoveriesofthestudiedpesticidesandthe pigmentremoval.IntheMAEprotocol,anENVICARBcartridgewas used,accordingtothemethodproposedbyHaywardetal.forherbs,

whereasQuEChERSusedGCBandPSAinadispersivemode.

Nev-ertheless,PSAwasnotemployedinMAEmethod,aspolyphenols

andshikimicacidanalogs suchaschlorogenicacidpresentinI. paraguaiensiscouldbeanalyteprotectantsforthemostlabile pes-ticidesbyinteractingwiththesilvnolfreeOHintheglasslinerasit hasbeenestablishedintheliterature(Anastassiadesetal.,2003).

Methodsperformanceandvalidation

Allvalidationprocedureswereperformedusingacommercial

yerba mate sample labeled as organic, which was previously

80

70

60

50

40

30

20

10

0 14

22

> 50% 25-50% < 25%

ME QuECHERS ME MAE

% pesticides ₁₉ ₁₉

67 59

Fig.1.CalculatedmatrixeffectsofMAEandQuEChERSmethod.Matrixeffect (%)=(1−(slopematrix/slopesolvent))×100.

analyzedinordertodeterminethepre-existentpesticideresidues content.

Themethodefficiency,expressedasrecoveryratesandrelative standarddeviation(%RSD)ofthetestedpesticides,wasdetermined attwofortificationslevels:0.2and0.5mg/kginspikedsamplesof yerbamate,asitisshowninTable1.

Among the 42 pesticides included in the analytical method

phorate,fenthion,terbufos,fenamiphos,andmetamidofosexhibit recoverieslowerthan50%forbothmethodsandcannotbe

deter-minedaccordingtoDG-SANCOguidelines(EuropeanCommission

DG-SANCO,2014).Theremaininganalytespresenteddifferences

intherecoveryresultsforbothmethods.ParticularlywithMAE extraction,fensulfothionwasnotdetectedatanyfortificationlevel,

whiledichlorvos,phosphamidonanddimefoxpresented

recover-iesbetween19and63%at0.2mg/kg.QuEChERSmethodpresented lowrecoveriesforomethoateat0.2mg/kg,prothiofosatboth lev-elsandchlorpyrifospresentedrecoveriesof65and59%at0.2and 0.5mg/kgrespectively.

Theselowrecoveriescouldbeduetothepossiblevolatilization ordegradationduringGCdetermination(Ingelseetal.,2001)ordue tothelossesduringtheconcentrationprocess.Itwasobservedthat mostofthepesticidesshowinglowrecoveriesarevolatileandhave thesmallestretentiontimes(Table1).ConcerningtheQuEChERS methodmostofthepesticideswithlowrecoverieselutedinthe middleofthechromatogramandaftercaffeine.

AsitisshowninFig.1,QuEChERSmethodshowedlowermatrix

effectthan MAE.Signalenhancement wasobserved for 41 and

33%ofthestudiedpesticidesinMAEandQuEChERS,respectively.

Particularly mevinphos showed 75% of signal enhancement in

QuEChERSmethod,thiscouldleadtooverquantification,aspointed

out by theDG-SANCO guidelines, explaining thehighrecovery

observed.

Matrix-matchedcalibration curves werelinear in the range 0.05–4␮g/mlwithcorrelationcoefficients(r2)higherthan0.99in

mostcases.Onlydichlorvospresentedlinearityproblemsin QuECh-ERSandthiscouldbeattributedtoitshighvolatilityandthermal lability.TheseproblemswerenotobservedinMAE,supportingthe hypothesisoftheanalyteprotectanteffectofmatepolyphenols.

Thelimitsofdetection(LOD),rangedfrom0.004to1mg/kg. TheLOQ,determinedasestablishedinDG-SANCOguidelinesisthe lowestconcentrationoftheanalytethathasbeenvalidatedwith acceptableaccuracybyapplyingthecompleteanalyticalmethod, rangedfrom0.1to0.2mg/kgformostoftheevaluatedpesticides. However,consideringtheLOQastheLOD×10,28/33pesticides

presenteda LOQbelow0.2mg/kginMAEand 11/27in

QuECh-ERS. Somepesticidessuch asphenthoate, prothiofos,parathion

ethyl,omethoate,dimefoxandchlorpyrifosinQuEChERSmethod

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Table1

(%)RecoveryratesandrespectiveRSDobtainedforMAEandQuEChERSmethodat0.2and0.5mg/kgspikinglevels(pesticideswithacceptablerecoveriestooneatleastof thetestedmethodswereonlyincluded).

Pesticide commonname

Stock mix

RT (min)

Spikinglevel (mg/kg)

MAE QuEChERS

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

Acephate I 11.59 0.2_0.5 84₉₂ 4₃ 70₇₇ 4₉

Bromophos

methyl III 21.98

0.2 97 11 75 11

0.5 93 6 65 11

Cadusafos III 15.85 0.2_0.5 84₈₀ 11₄ 99₉₁ 4₉

Chlorfenvinphos I 22.68 0.2_0.5 89₉₄ 5₃ 93₇₄ ₁₂4

Chlorpyrifos III 21.19 0.2 86 11 65 4

0.5 82 5 59 13

Chlorpyrifos

methyl II 19.29

0.2 90 6 76 8

0.5 89 10 73 6

Diazinon I 17.48 0.2_0.5 91₈₈ 1₄ 77₇₆ ₁₅4

Dichlorvos II 9.55 0.2 63 16 99 4

0.5 67 15 109 10

Dimefox II 7.38 0.2 50 15 85 6

0.5 53 14 113 14

Dimethoate II 16.13 0.2_0.5 112₁₀₉ 6₃ ₁₀₇96 ₁₄8

Disulfoton

sulfoxide III 10.50

0.2 91 9 117 12

0.5 90 4 117 9

Disulfotonsulfone I 23.47 0.2 105 2 119 5

0.5 110 1 95 21

Ethion III 26.64 0.2 103 9 76 3

0.5 101 4 65 16

Ethoprophos II 14.91 0.2_0.5 100₁₀₁ 6₄ 78₉₀ 6₉

Fenchlorphos II 19.96 0.2 95 5 70 4

0.5 91 9 67 6

Fenitrothion III 19.99 0.2 118 11 99 5

0.5 111 3 91 13

Fonofos III 17.26 0.2_0.5 86₈₀ 12₄ 85₇₆ ₁₁4

Fensulfothion II 26.67 0.2 ND ND 82 17

0.5 ND ND 89 9

Heptenophos III 13.78 0.2 90 12 119 4

0.5 85 5 116 7

Malathion II 20.52 0.2_0.5 104₉₉ 3₈ 80₈₈ 3₉

Mecarbam III 22.49 0.2_0.5 100₉₇ 10₃ 102₉₃ ₁₂4

Methidathion II 23.28 0.2 107 7 102 10

0.5 103 9 98 7

Mevinphos III 11.69 0.2 88 13 131 3

0.5 83 6 134 4

Omethoate II 13.98 0.2_0.5 93₇₉ 12₁₆ 49₈₁ 18₂₅

Parathion

ethyl II 21.16

0.2 103 4 61 5

0.5 96 9 74 9

Parathionmethyl III 19.14 0.2 118 12 116 3

0.5 117 1 100 11

Phenthoate II 22.76 0.2_0.5 109₁₀₁ 3₈ 66₇₆ 3₈

Phosphamidon I 6.24 0.2_0.5 19₂₂ 12₁₀ 87₈₀ 7₈

Pirimiphosmethyl I 20.30 0.2 100 18 74 6

0.5 91 4 64 14

Profenofos III 24.74 0.2_0.5 118₁₁₃ 13₄ 88₇₇ ₁₄7

Prothiofos II 24.83 0.2_0.5 99₉₄ 5₉ 43₅₀ 7₅

Quinalphos III 22.70 0.2 89 11 93 5

0.5 86 6 85 12

Terbufossulfone II 22.30 0.2 106 3 88 2

0.5 101 8 86 11

Thionazin II 14.41 0.2_0.5 97₉₆ 7₄ 78₉₄ ₁₀6

Tolclofosmethyl I 19.50 0.2 88 2 75 4

0.5 89 3 64 12

Triazophos I 26.73 0.2 98 6 114 3

0.5 104 3 80 14

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Table2

Limitsofdetection(LOD)andlimitsofquantification(LOQ)inmg/kginGC/FPD.

Pesticidecommonname MAEmg/kg QuEChERSmg/kg MRL(EU)mg/kg

LOD LOD×10/LOQ LOD (LOD×10/LOQ

1.Acephate 0.01 0.1/0.2 0.05 0.5/0.2 0.05

2.Bromophosethyl 0.004 0.04/0.2 0.05 0.5/0.2 0.1

3.Cadusafos 0.004 0.04/0.2 0.01 0.1/0.2 0.01

4.Chlorfenvinphos 0.01 0.1/0.2 0.05 0.5/0.2 0.05

5.Chlorpyrifos 0.004 0.04/0.2 0.1 1.0/1.0 0.5

6.Chlorpyrifosmethyl 0.004 0.04/0.2 0.05 0.5/0.2 0.1

7.Diazinon 0.004 0.04/0.2 0.01 0.1/0.2 0.05

8.Dichlorvos 0.01 0.1/0.5 0.05 0.5/0.2 0.02

9.Dimefox 0.05 0.5/1.0 0.05 0.5/0.2 0.01

10.Dimethoate 0.05 0.5/0.2 0.05 0.5/0.2 0.1

11.Disulfotonsulfoxide 0.01 0.1/0.2 0.05 0.5/0.2

0.05

12.Disulfotonsulfone 0.004 0.04/0.2 0.01 0.1/0.2

13.Ethion 0.004 0.04/0.2 0.05 0.5/0.2 0.05

14.Ethoprophos 0.004 0.04/0.2 0.01 0.1/0.2 0.02

15.Fenchlorphos 0.05 0.5/0.2 0.05 0.5/0.2 0.1

16.Fenitrothion 0.01 0.1/0.2 0.05 0.5/0.2 0.05

17.Fonofos 0.004 0.04/0.2 0.01 0.1/0.2 0.01

18.Fensulfothion 1.0 1.0/1.0 0.05 0.5/0.2 0.01

19.Heptenophos 0.004 0.04/0.2 0.01 0.1/0.2 0.01

20.Malathion 0.05 0.5/0.2 0.05 0.5/0.2 0.02

21.Mecarbam 0.01 0.1/0.2 0.05 0.5/0.2 0.1

22.Methidathion 0.004 0.04/0.2 0.05 0.5/0.2 0.1

23.Mevinphos 0.004 0.04/0.2 0.01 0.1/0.2 0.02

24.Omethoate 0.01 0.1/0.2 0.05 0.5/0.5 0.05

25.Parathionethyl 0.01 0.1/0.2 0.05 0.5/0.5 0.1

26.Parathionmethyl 0.004 0.04/0.2 0.01 0.1/0.2 0.05

27.Phenthoate 0.01 0.1/0.2 0.05 0.5/0.5 0.01

28.Phosphamidon 0.01 0.1/1.0 0.05 0.5/0.2 0.02

29.Pirimiphosmethyl 0.004 0.04/0.2 0.01 0.1/0.2 0.3

30.Profenofos 0.01 0.1/0.2 0.05 0.5/0.2 0.1

31.Prothiofos 0.01 0.1/0.2 0.05 0.5/1.0 0.01

32.Quinalphos 0.004 0.04/0.2 0.01 0.1/0.2 0.1

33.Terbufossulfone 0.004 0.04/0.2 0.01 0.1/0.2 0.01

34.Thionazin 0.004 0.04/0.2 0.01 0.1/0.2 0.01

35.Tolclofosmethyl 0.05 0.5/0.2 0.05 0.5/0.2 0.1

36.Triazophos 0.004 0.04/0.2 0.05 0.5/0.2 0.02

37.Trichlorfon 0.004 0.04/0.2 0.01 0.1/0.2 0.05

methodshowedLOQhigherthan0.2mg/kg.astheycouldnotbe validatedwithacceptableaccuracyatthislevel(Table2).

Chromatographicanalysis

Twomegaborecolumnsintandemwereusedinordertoachieve

adequatechromatographicseparation. Megaborecolumns

(typi-cally10m×0.53mm)areadvantageouscomparedtonarrow-or

micro-borecolumnswhenextractsof“difficult”matriceshavetobe analyzedsincemegaborecolumnscanprovidehighloadabilityas filmsupto5␮m(Cajkaetal.,2008;Ravindraetal.,2008.).Theuseof

twomegaborecolumnsintandem(20m×0.53mm×2.65␮m)can

alsoimprovethechromatographicseparationofpesticideswith similarproperties,akeypointwhentheGCisnotconnectedwith aMSdetector(MastovskaandLehotay,2003).Therefore,the selec-tionofacolumnwithhighinternaldiameter(0.53mm)andfilm thickness(2.65␮m)ensurebetterperformancein sampleswith

highmatrixeffect.Alongoventemperaturegradientwasselected (runtime40.8min)toimprovethechromatographicresolutionof theanalyteswhicharedifficulttoresolveundertypicalGC con-ditions.TheOPpesticidesincludedintheanalyticalmethodwere separatedinthreestocksolutionsbasedontheretentiontimeof eachanalyte(Table1).Separationoftargetcompoundswas per-formedinordertoavoidco-elutionofsomepesticides.Fig.2shows thechromatogramobtainedfortheanalysisoffortifiedyerbamate

sampleswithMixIat0.1mg/kgwithbothMAEandQuEChERS

methodsbyGC–FPD.

Asitispresentedinthechromatograms(Fig.2),thereisapeak withretentiontimearound20mincorrespondingtocaffeine.The

cleanupofbothmethodsisnotenoughtoremoveallthecaffeine, althoughMAEcleanupismoreefficientthanQuEChERS.

Realsampleanalysis

Inordertochecktheperformanceof themethodnine

com-mercialsampleswereanalyzed.Thesampleswereextractedusing bothvalidatedmethodsandanalyzedbyGC/FPDandthepositive findingswereconfirmedbyGC/MS.

Acephate, ethoprophos, chlorpyrifos, and cadusafos were

detected in commercial samples and their concentrations are

showninTable3.However,onlychlorpyrifosshowed concentra-tionsabovetheLOQofMAEmethodinfivesamplesandbelowthe correspondingMRL(EuropeanCommission,2005,2014).

MAEandQuEChERScomparison

TheanalyticalresultsofrealsamplesshowninTable3indicate that,undertheexperimentalconditionsemployedinthepresent

communication, MAE provides betterextractability of incurred

residuespresentinrealsamplesasitdetectsnotonlymore pes-ticidesbutalsotheresidueconcentrationsfoundarehigherthan QuEChERS.

Thereasonoftheseresultscouldbebasedintheefficiencyof microwaveenergy,whichishigherthanmanualagitationforthe extractionoftheresiduesfromthematrix.

Concerning matrix effect MAE presented more compounds

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3.500

3.000

2.500

2.000

1.500

1.000

500

0

0 10

1

2 3 4 5

6 7

8

9 10

11

A

B

20 30 40

3.500

3.000

2.500

2.000

1.500

1.000

500

0

0 10

1

2 3 4 5

6 7

8

9 10

11

20 30 40

Fig.2. ChromatogramsoffortifiedmatesampleswithMixIat0.1mg/kgbyGC–FPD.MAE(A)andQuEChERS(B)methods.1:trichlorfon;2:phosphamidon;3:acephate;4: phorate;5:diazinon;6:tolclofosmethyl;7:pirimiphosmethyl;8:fenthion;9:chlorfenvinphos;10:disulfutonsulfone;11:triazophos.

Table3

Pesticides(mg/kg)detectedbyGC–FPDandconfirmedbyGC–MSinrealsamples.ND:notdetected.

Realsample Acephate Ethoprophos Chlorpyrifos Cadusafos

MAE/QuEChERS MAE/QuEChERS MAE/QuEChERS MAE/QuEChERS

1 <LOQ/ND <LOQ/ND ND ND

2 ND ND 0.3/<LOQ ND

3 ND ND <LOQ/ND ND

4 ND ND ND ND

5 ND ND <LOQ/ND ND

6 ND ND 0.2/<LOQ ND

7 ND ND 0.4/<LOQ ND

8 ND ND 0.2/ND <LOQ/ND

9 ND ND 0.2/ND <LOQ/ND

moreeffectiveavoidingtheextractionofcaffeine,whichisthemain detectedinterference.

Ingeneral,MAEmethodensuredlowertosimilarLODforall pes-ticidesexceptforfensulfothion,comparedwithQuEChERSmethod, whiletheLOQ(lowestvalidatedlevel)for28 pesticidesinboth methodsweresimilar.IfLOQarecalculatedasLOD×10,13 pesti-cidescouldbeassessedforMRLcompliancewithMAEmethodand threepesticideswithQuEChERS.

Comparingtheaccuracyandprecisionofbothmethods,MAE presentedbetterperformancethanQuEChERS,sincetherecoveries of33pesticideswerewithintherange70–118%withRSDsfrom1 to18%.QuEChERSmethodpresentedrecoveryratesbetween70 and120%andRSDsintherange3–21%for27pesticides,atthe lowestspikinglevel.Someoftheobtainedresultsinthisstudywith QuEChERSmethodweresimilartothosereportedbyLozanoetal.

(2012),indifferenttypesofteausingGC-QqQ/MS.

QuEChERSmethodologyissimple,cheap,practicallyno glass-wareis needed,and it ismore environmentally friendlyasthe solventconsumptionislowerthanMAE.

MAEpresentedgoodperformance,itallowsthesimultaneous extractionof10samples,buttheequipmentrequiredisnotoften availableinthelaboratories.

Thepresentstudydemonstratedthatalthoughbothmethods

aresuitablefortheanalysisofpesticideresiduesinyerbamate,MAE presentedabetterperformanceundertheexperimentalconditions tested.

Yerbamateisconsumeddailybyalmost50millionpeoplebut therearefewdataontheliteratureconcerningthepersistenceof pesticideresiduesintheprocessedleaves.Thisworkmighthelp

togathertheinformationneededtoperformstudiesonpesticide residueexposureofthepopulationduetoyerbamateintake.

Authors’contributions

JG,SNandLPperformedthelaboratorywork,dataand chro-matographicanalysis.HHandZVranthefirsttrialexperiments withMAE.ZVanalyzedtherealsamplesintheGC–(ITD)-MS,LP,ZV andSNdraftedthepaper.VC,LPandHHgavetheworksconceptual frame,participated intheresultsdiscussionandthemanuscript finalwriting.ZVandEPMsupervisedthelaboratoryworkandZV contributedtocriticalreadingofthemanuscript.

Conflictofinterest

Theauthorshavenoconflictofinteresttodeclare.

Acknowledgment

TheauthorsgratefullyacknowledgetheEuropeanCommission

(Alfa II Programme B-Project EUROLANTRAP, No.

AML/B7-311/97/0666/II0461-FA-FCD-FI).

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