Supercritical fluid chromatography (SFC), combined with mass spectrometry (MS), was employed for the determination of five chiral fungicides, from two different chemical families (acylalanine and triazol) in wine and vineyard soils.
Trang 1journalhomepage:www.elsevier.com/locate/chroma
L Pérez-Mayán, M Ramil, R Cela, I Rodríguez∗
Department of Analytical Chemistry, Nutrition and Food Sciences Research Institute on Chemical and Biological Analysis (IAQBUS) Universidade de
Santiago de Compostela, 15782-Santiago de Compostela, Spain
a r t i c l e i n f o
Article history:
Received 16 November 2020
Revised 22 March 2021
Accepted 25 March 2021
Available online 30 March 2021
Keywords:
Fungicides
Enantiomeric fraction
Wine
Soil
Supercritical fluid chromatography
a b s t r a c t
Supercriticalfluidchromatography(SFC),combinedwithmassspectrometry(MS),wasemployedforthe determinationoffivechiralfungicides,fromtwodifferentchemicalfamilies(acylalanineandtriazol)in wineandvineyardsoils.TheeffectofdifferentSFCparameters(stationaryphase,chiralselector,mobile phasemodifierandadditive)intheresolutionbetweenenantiomersandintheefficiencyofcompounds ionizationattheelectrospraysource(ESI)wasthorouglydescribed.Underfinalworkingconditions,chiral separationsofselectedfungicideswereachievedusingtwodifferentSFC-MSmethods,withananalysis timeof10minandresolutionfactorsfrom1.05to2.45betweenenantiomers.Incombinationwith solid-phaseextractionandpressurizedliquidextraction,theypermittedtheenantiomericdeterminationof tar-getcompoundsinwineandvineyardsoilswithlimitsofquantificationinthelowppbrange(between 0.5and2.5ngmL−1,andfrom1.3to6.5ngg−1,forwineandsoil,respectively),andoverallrecoveries above80%,calculated usingsolvent-based standards.Forazolicfungicides(tebuconazole, myclobutanil andpenconazole)soildissipationandtransferfromvinestowineswerenon-enantioselectiveprocesses Dataobtainedforacylalaninecompoundsconfirmedtheapplicationofmetalaxyl(MET)tovinesas race-mateandas theR-enantiomer.Theenantiomericfractions(MET-S/(MET-S+MET-R))ofthisfungicidein vineyardsoilsvariedfrom0.01to0.96;moreover,laboratorydegradationexperimentsshowedthatthe relativedissipationratesofMETenantiomersvarieddependingonthetypeofsoil
© 2021ElsevierB.V.Allrightsreserved
1 Introduction
Manypesticidesemployedinagriculturehaveachiralstructure;
thus, thepersistenceofthesecompoundsincrops,their
degrada-tion rates in agriculture soils and even their bioaccumulation in
invertebratesandtoxicitiestowardsnon-targetorganismsmightbe
enantioselectiveprocesses[1]
Mildew and botrytis are major diseases impacting the
produc-tivity of vines So, different familiesof fungicideshave been
de-signed and marketed to control these infections Many of these
compounds are chiral molecules Among them, acylalanine and
azoles arewidelyapplied tovineyardsforthepreventionandthe
control of infectionscaused bymildew andbotrytis fungi,
respec-tively.Metalaxyl(MET),andinalesserextentbenalaxyl(BEN),are
the mostpopular acylalanine fungicides Although the fungicidal
activity of the R-enantiomer is much higher than that of the
S-∗ Corresponding author
E-mail address: isaac.rodriguez@usc.es (I Rodríguez)
form[2],currently,bothcompoundsarestillmarketedasthe race-mateinadditiontoformulationsenrichedintheR-form.METhas been oftenreported in wine [3,4] and vineyardsoils [5,6]; how-ever,no data are available regardingthe enantiomeric profiles of the compound in these matrices The group of azolic fungicides gathers a large number of active molecules authorized for agri-culturetreatments.Amongthemmyclobutanil(MYC),tebuconazole (TEB) and penconazole (PEN) are the most popular ones as re-gardsviticultureapplications.Tothebestofourknowledge,these compoundsaremarketedonlyasracemates.Theirtransferfactors fromgrapestowinearelowerthanincaseofMET[7,8];however, they aremorepersistentinsoils[9].Inthisregard,the European Union(EU)hasincludedTEBandPENinthewatchlistof emerg-ingenvironmentalpollutants[10],forwhichdataabouttheir envi-ronmentaldistributionarerequiredinordertoestimatetheirrisk quotients
According to literature, the relative enantiomeric degradation ratesofthe abovefungicidesincropsandsoil are matrix depen-dent.Inthisvein,thedissipationratesofMETisomersinsoils var-iedlargelydependingonsoilmicrobiota[11],withthestabilityof theR-formdecreasingdramaticallyinalkalinesoils[12].Some
re-https://doi.org/10.1016/j.chroma.2021.462124
0021-9673/© 2021 Elsevier B.V All rights reserved
Trang 2MET-S might perturbthe metabolism of mammals ina higherextent
thantheR-enantiomer[13];thus,inadditiontototalconcentration
datatheknowledge ofenantiomericfractionsofthisfungicide,as
well astheir time-courseevolution, isamatter ofconcern.Wang
and co-workers [14] have reported a faster dissipation for the S
formofTEBthantheRenantiomerincabbage,whilsttheopposite
behaviorwasnoticedincucumber.Also,theenantiomeric
degrada-tionratesofMYCandTEBinsoilhavebeenrelatedtotheirorganic
matter content,pHandother physico-chemicalproperties[15].In
summary, non-targeteffectsand dissipation ratesofchiral
fungi-cides mightchangedependingontheinvestigatedorganisms,the
propertiesofeachsoilmatrix,andthespecificmetabolismofeach
crop Tothe bestofour knowledge,littleinformation isavailable
related to the enantioselective accumulation of above fungicides
in viticulturerelatedsamples.Zhangetal.[16]describeda faster
degradationofTEB-RingrapesthanTEB-S;however,nodatahave
beenfoundregardingtheenantiomericfractions(EFs)ofthe
com-poundinwine
Todate,mostmethodsemployedforthedeterminationofchiral
pesticides are basedon liquid chromatography,either under
nor-mal orreversed-phase conditions[1,17] Some limitations of
chi-ral LC-based methods are either the use of isocratic conditions,
often optimized for the separation of the enantiomers of single
compound[15,18,19],or,incaseofmultianalyteprocedures, the
employment ofslowgradientsleadingtoanalysistimesabove 60
minutes [20] Since some years ago, pharmaceutical laboratories
have upgradedtheir chiral separationmethods fromLC to
super-criticalfluidchromatography(SFC).Majoradvantagesofthelatter
techniquearereductionoftheanalysistime,duetothehigher
dif-fusivityandlowerviscosityofsupercritical CO2,andsaveoflarge
volumes of toxic organic solvents used in chiral LC separations
when performedundernormalphaseconditions[21].The
combi-nationbetweenSFCandelectrospraymassspectrometry(ESI-MS)
hasexpandedtheapplicabilityofthetechniquetodeterminetrace
levelcompoundsincomplexextractsobtainedfromenvironmental
andfood samples,eitherusingnon-chiral orchiral columns[22–
25]
Herein, we evaluate the performance of SFC-ESI-MS for the
chiral separation of a selection of five fungicides, belonging to
two different chemicalfamilies, often employed inthe control of
mildew and botrytis infections in vines Theirresidues havebeen
oftenreportednotonlyinviticulturerelatedsamples,butin
gen-eralinagriculturesoilsandother environmentsimpactedby
agri-culture activities [26] Moreover, azolic fungicides are regarded
as an environmental threat and pinpointed as concerning
pol-lutants for which environmental monitorization is recommended
[10].Thereafterthemethodisappliedto theanalysisof
commer-cial winesandvineyardsoils.The enantiomeric fraction(EF)data
are employed todrawconclusionsregardingthe applicationform
of acylalanine fungicides (as racemates oras preparations ofthe
mostactiveRenantiomer),andtoinvestigatetheexistenceof
po-tentialenantioselectivedissipationprocessesduringthewine
mak-ing processandinthesoil ofdifferentvineyards fromthe
North-westofSpain
2 Material and methods
2.1 Standards, solvents and sorbents
Standards ofMET,BEN,TEB, MYCandPEN,asracemates,were
purchasedfromSigma-Aldrich(Milwakee,WI,USA).Isotopically
la-belled compounds(MET-13C6,TEB-d9 andMYC-d4,asracemic
so-lutions)wereobtainedfromthesamesupplier.TheRenantiomers
of METandBENwere also purchasedfromSigma-Aldrich, whilst
R and S forms of TEB were kindly supplied by Shangai
Chiral-wayBiotechCo(MinhangDistrict,Shangai,China).Individual solu-tionsoftheabovecompoundswerepreparedinmethanol(MeOH) Racemicmixturesoffungicides, usedtospikesoilandwine sam-ples processed through this study, were made in the same sol-vent A mix of isotopically labelledcompounds in methanolwas added to soil and wine samples before extraction These com-poundswereemployedassurrogatestandards(SSs)tocompensate non-quantitativerecoveriesand/orchanges oncompounds ioniza-tion yield at the electrospray source (ESI) Calibration standards containing increasing concentrations of native compounds (0.5
100 ng mL−1), and a fixed level of labelled compounds (100 ng
mL−1),werepreparedinMeOH:ACN(50:50)
MeOHand ACN,both LC-MS gradepurity, formicacid(FA, 98
%),NH3 (12%solutioninMeOH),andaceticacidweresuppliedby Merck(Darmstadt,Germany).Ultra-puredeionizedwater(18.2M
cm−1) was obtainedfrom a Milli-Q Gradient A-10system (Milli-pore,Billerica,MA,USA).Carbondioxide(CO2)waspurchasedfrom NipponGases(Madrid,Spain)
OASISHLB200mgcartridges,employedforsolid-phase extrac-tion (SPE)of wine samples,were acquired fromWaters (Milford,
MA,USA).Diatomaceousearth,usedduringpressurizedextraction
ofvineyardsoils,wasprovidedbyVWR(WestChester,PEN,USA)
2.2 Samples and sample preparation
Wineswereeitherpurchasedinlocalsupermarkets,orobtained directlyfromregionalwineproductionassociations.Sampleswere maintainedinthe dark,atroom temperatureandSPEextractions werecarriedoutimmediatelyafteropeningwinebottles
Soils were taken in seven vineyards, corresponding to three different Designations of Origin in Galicia (Spain) Samples used
in thisstudy correspondedto top soil (0-15 cm depth)collected
in polyethylene bags,and transported immediatelyto laboratory After removing coarse materials, samples were freeze-dried and sieved The fraction below 2 mm was storedat -20 °C and em-ployedforanalysis.SamplesusedtomeasuretheEFsoffungicides
invineyardsoilswerecollectedatthebeginningofautumnand/or the endofwinter;thus, fungicideswere in contactwiththe soil since, at least, the end of the previous year summer Soils em-ployed inlaboratory incubationstudies were takenatthe endof spring (middle June), within the year period that fungicides are sprayedonvineyards
Samplepreparationwasperformedusingpreviously published procedures dealingwithpressurizedliquidextraction[9]andSPE [27]ofvineyardsoilsandwines,respectively.Inbrief,soilsamples (2g)werespikedwiththemixtureofSSs(250ngg−1)andpacked
in11mLstainlesssteelcellscontaining1gofdiatomaceousearth Thefree volumeabovethesample, withinthe PLEcell,wasfilled with the same sorbent Cells were pressurized at 1500 psi and compoundswere extractedusingamixtureofMeOH:ACN(70:30)
at80°C,inasinglecyclewithadurationof5min[9].Thisextract wasconcentrated andmadeup to 5 mL,using volumetricflasks, andstoredat4°C.Wines(2mL)weredilutedwiththesame vol-umeofultrapurewater,spikedwithSSs(100ngmL−1)andpassed through a SPEcartridgepreviously conditioned withACN: MeOH (80:20)followed by a mixture ofEtOH: H2O(12:88),2 mL each After loadingthe diluted samples,thesorbent wasrinsedwith3
mLofthe EtOH:H2Osolutionanddriedusinga streamof nitro-gen Compounds were recovered with a mixture of ACN: MeOH (80:20).TheextractfromtheSPEcartridge(2mL)wasmaintained
at 4 °C until analysis.Both sample preparation procedures were previously combined withLC-MS asdetermination technique us-ing a non-chiral column forcompounds separation [9,27] Before injectionintheSFC-MSsystem,allextractswerepassedthrougha 0.22μmsyringefilter
2
Trang 32.3 Soil incubation experiments
In additionto dataobtainedfor fieldsamples (vineyard soils),
thepotentialenantioselectivedegradationoffungicidesinthis
ma-trixwasre-evaluatedinlaboratoryincubationassays.The
physico-chemical properties of the samples used in this series of
exper-iments are given as supplementary information (Table S1)
Frac-tionsof2gfromeachsoil(particlesizebelow2mm)were
trans-ferred to20mL glassvialsandspiked witha racemicmixture of
the five compounds considered in this study (additionlevel 200
ngg−1).Oneofthesoils(samplingpoint2,TableS1)wasfortified
only withBEN andPEN giventhat it containedrelevant residues
ofthe restofcompounds(from50ngg−1 forTEBto250ngg−1
forMYC).Watercontentinincubationvesselswasadjustedto20%
ofsampleweight.After Vortexhomogenization,vialswerecapped
using Teflon lined septa A needle was passed through the
sep-tum anda 0.45μm pore sizefilter wasconnectedon top of the
needle.Thissetuppermitstoassesscompoundsdissipation under
aerobic conditions,whilstit reduceswaterevaporation[28].Vials
weremaintainedat20°C,andretrievedinduplicateatpre-defined
times (from 0 to 66 days) Control experiments were performed
withsterilizedfractionsofeachsoilmatrix,incubatedfor66days
Soilsterilizationwasperformedheatingthesievedsamplesto170
°Cfor90min.Extractionofsoilsampleswascarriedasdefinedin
section2.2afteradditionofSSs
2.4 SFC-ESI-QTOF-MS determination conditions
Separation ofchiralcompounds wascarriedout using an
Agi-lent1260infinity IISFCsystem(Wilmington,DE,USA)connected
to a quadrupole time-of-flight (QTOF) instrument(Agilent model
6550)furnishedwithdualsprayionfunnelESIsource.Themobile
phasefromtheSFCsystemwasmixedwiththemake-upsolution
and divided intwo streams Onereaches the ESIsource through
a 1m x0.050mm i.d.silica capillary.Thesecond stream is
con-nectedtotheback-flushpressureregulator(BPR),whichis
respon-sibletomaintaintheCO2 undersupercriticalconditions
The TOF-MS instrument operated in the 2 GHz mode,
offer-ing a typical spectral resolution of 16000 (calculated as FWHM
at m/z 322.0481).The ESI source wasset in positive mode, and
them/z axiswascontinuouslyrecalibratedusingreferenceionsat
m/z 121.0509 and 922.0098 Nitrogen was employed as
nebuliz-ing (35 PSI) anddrying gas (15 L min−1, 200 °C) in the
ioniza-tion source.TheESI needleandthefragmentorvoltages wereset
at3500Vand380V,respectively.DuringoptimizationofSFC
con-ditions, theinstrument wasrun inthe MS mode,using thepeak
areasforthe[M+H]+ionofeachcompoundasresponsevariable
Analysis ofsoil andwine sampleswascarriedout intheproduct
ionscan acquisitionmode.Inbothcases,quantificationionswere
extractedusingamasswindowof20ppm centredeitherintheir
[M+H]+ion,orinthemostintenseproductionofeachcompound
(Table1)
The polysaccharide-based chiral columns evaluated for
com-poundsseparationwereobtainedfromPhenomenex(Torrance,CA,
USA) Column dimensions were 150 mm (length) x 3 mm (i.d.),
3 μm particle size The tested phases were amylose and
cellu-losewithphenylcarbamatebondedtomethyland/orchlorine
sub-stituentsaschiralselectors Throughthismanuscript,columnsare
termed as amylose-1 (3,5-dimethyl phenyl carbamate),
amylose-3 (3-methyl-5-chloro phenyl carbamate) and cellulose-5
(3,5-dichlorophenyl carbamate) In the former case, the stationary
phaseiscoatedonsilicaparticles,whilstamylose-3andcellulose-5
phasesareimmobilizedonsilica.Theassayedmobilephases
con-sisted ofCO2 (phaseA) combinedwith MeOH,orACN (phaseB)
asmodifiers,containingdifferentadditives,such asFA(0.1%),
am-monium acetate (NH4Ac, 5 mM) or NH3 (0.1%) In all the cases,
the flow of mobile phase was 1.5 mL min−1 and columns were maintainedat40°C.Asmake-upsolution,amixture ofMeOH:FA (99.5:0.5) wasusedto enhancecompounds ionization intheESI source[29].Underfinalconditions,twodifferentchromatographic methods were employed The enantiomers of MET,BEN andTEB were separated using the amylose-1 column The mobile phases consistedofCO2 (A)andMeOH,5mM inNH4Ac,(B) combinedas follows:0-1min(2%B),4-6min(30%B),6.05-10min(2%B).The identityofthe enantiomersofthesefungicideswasconfirmedby injectionofR-formsofMETandBEN,aswellasRandSisomersof TEB.ChiralseparationsofMYCandPEN wereperformedwiththe cellulose-5columnusingACN5mMinNH4Acasorganicmodifier Themobile phasegradient was:0-1min(10%B), 4-6.5 min(30% B),6.51-10min(10%B).Theidentitiesoftheenantiomersforthese twofungicideswerenotelucidated;thus,theyaresimplyreferred
asisomers1and2attendingtotheirelutionorder
2.5 Evaluation of enantiomeric fractions, matrix effects and accuracy
EFs of fungicides in the extracts from wine andsoil samples were calculated as the ratio between the concentration corre-spondingtothe earlierelutingisomer andthe sumof concentra-tionsforbothenantiomers[30]
Matrixeffects(MEs)wereevaluatedwiththeratiobetweenthe slope of calibration curves for matrix-based standards (prepared withspikedextractsfromwineorsoilsamples)andsolvent-based standards Normalized ratios around 100% correspond to similar ionizationefficienciesinbothcases.Valuesbelowandabove100% pointouttosignalsuppressionandenhancement,respectively The accuracy ofthe analytical procedure wasestimated using spikedsamplesofred andwhitewines,andvineyardsoil.Spiked and non-spiked fractions of the above samples were extracted
in triplicate Concentrations of each enantiomer in the obtained extracts were calculated using solvent-based standards Accuracy wasestimated as the ratio betweenthe difference of concentra-tions measured forspiked (samples were fortified before extrac-tion) andnon-spiked fractions of theinvestigated matrix divided
bytheaddedvalueandmultipliedby100
3 Results and discussion
3.1 Optimization of SFC parameters
Enantiomericseparations ofselected compoundswere investi-gatedcombiningthechiralcolumnsdescribedinsection 2.4with MeOHorACNasmodifiersofsupercriticalCO2.Inthissetof pre-liminary experiments, the percentage of modifier in the mobile phasewasvariedasfollows:2%(0-1min),30%(4-7min),2%
(7.1-10 min) The mobile phase flow rate was1.5 mL min−1 andthe temperatureofthecolumnssetat40°C.As ageneraltrend,ACN showedalowersolvationefficiencythanMeOH,leadingtolonger retention times than those observed with the latter modifier In somecases,theseparationefficiencyofthecolumnwasalsolower forACNthanforMeOH,asaconsequenceofwiderpeaksnoticed fortheformersolvent.Asregardsseparationofenantiomers, reso-lutionfactors(Rs)werecolumnandmodifierdependent
The amylose-3 column provided Rs above 1.5 only for the enantiomers ofBEN(obtainedusingMeOH asmodifier),data not shown Table S2 summarizedRs and baseline peak width values obtainedusingamylose-1andcellulose-5columnsincombination with MeOH and ACN as modifiers The latter column separated theenantiomers of BENandPEN withany ofboth organic mod-ifiers;moreover,partialseparationofMYCforms(RS>1) was ob-servedwithACN Ontheother hand,thiscolumndidnot resolve theenantiomersofMETandTEB.Theseparationefficiencyandthe
Trang 4Table 1
Retention times, quantification ions, linearity (R 2 values) and instrument limits of quantification (LOQs) of the SFC-QTOF-MS system
Compound Column Retention
time (min)
Rs Quantification transition (Collision energy, Ev)
Other product ions
Linearity (R 2 , 1-100
ng mL −1 )
LOQ (ng
mL −1 ) Slope ratio (1 st /2 nd
enantiomer)
a MET-S Amylose-
1
2.66 1.05 280.1543 (10) >
220.1332
192.1383;
160.1121;
45.0335
a BEN-S 3.02 2.45 326.1751 (10) >
148.1121
208.1332;
91.0642
c TEB-S 4.19 1.61 308.1524 (20) >
b MYC-1 Cellulose-
5
4.63 1.25 289.1215 (20) >
70.0399
b PEN-1 5.55 1.55 284.0714 (20) >
70.0399
a Met 13 C 6 Amylose-1 2.68; 2.81 1.04 286.175 (10) >
226.1531
198.1583;
166.1319
b MYC-d 4 Cellulose-5 4.49; 4.68 1.26 293.1466 (20) >
70.0399
129.0397
c TEB-d 9 Amylose-1 4.23; 4.40 1.59 317.2089 (20) >
70.0399
125.0153
a Denote the surrogate standard associated to each compound
b Denote the surrogate standard associated to each compound
c Denote the surrogate standard associated to each compound
enantiomeric selectivity ofthe amylose-1columnwasheavily
af-fected by the organic modifier Using ACN, partial resolution (Rs
values from0.76 for MYC to1.0 forPEN) wasobserved between
the pairsofenantiomers ofthe5fungicides However,their peak
widthswere2-3timeslargerthanthosenoticedusingMeOH.The
lattermodifierledtopartialseparationoftheenantiomersofMET
(Rs values around 1), the forms of BEN and TEB were baseline
resolved, and noseparation wasnoticed forPEN and MYC
enan-tiomers
The effectofdifferentadditives(NH3 0.1%,FA0.1% andNH4Ac
5mM) in the performance of SFC separations was assessed
us-ing CO2:MeOH and CO2:ACN as mobile phases combined with
amylose-1 and cellulose-5 columns, respectively Triazolic
fungi-cides are slightly basic compounds, so depending on the mobile
phase pH,secondaryinteractions withthechiralstationaryphase
and/orwiththesilicaparticlesmightaffecttheirSFCretentionand
separation [31] The above additives did not modify the
perfor-mance ofSFCseparations (efficiency, selectivity orresolution
be-tween enantiomers); however, they introduced significant effects
intheefficiencyofcompoundsionization.Relativeresponses
(nor-malizedtothoseobtainedwithoutanymobilephaseadditive)
var-ied dependingonthecompoundandtheSFCcolumn(Fig.1).For
example, NH3 (0.1%) combined with MeOH exerted a minor
ef-fect in the relative response found for MYC with the amylose-1
column, Fig 1A; however, the responses for the enantiomers of
this fungicideincreased bya factor of5when the sameadditive
was combined with ACN(Fig 1B) The adopted compromise
de-cision wasto employ NH4Ac (5 mM)as additive in combination
withMeOH andACN.Thisadditive improvedsignificantly the
re-sponses observed forthe enantiomers ofMET,BEN andMYC On
the other hand,thepeak areasofTEBandPENsuffered a
reduc-tion incomparisontothoseattainedwithout additiveinthe
mo-bile phase.NH4Ac alsopreventeddifferencesintheresponses for
enantiomers of thesame compoundreaching the ESIsource ina
differentenvironment, asregards themobilephase pH.As
exam-ple, the relative intensities of the chromatographicpeaks forthe
enantiomersofBENintheamylose-1column,differedsignificantly
when acid or basic additives are included in the mobile phase,
(Fig.S1)
AnotherparameterconsideredduringoptimizationofSFC
con-ditions wasthe BPRpressure Between90and140 bar, retention
times decreased slightly with increasing the pressure due to a
higher polarity ofthe mobile phase.The effect ofthisparameter
inthe resolutionofenantiomers wasnegligible and, asageneral trend,responses(peakareas)increasedsignificantlywithBPR pres-sure,seeFig.S2.Thus,aworkingvalueof140barwasselectedfor thisparameter
Takingintoaccounttheabovedata,afterslightmodificationsof the mobile phase gradient, two different chromatographic meth-ods were proposed Chiral determinations of MET, BEN and TEB werecarriedoutintheamylose-1column,usingMeOH(5mMin
NH4Ac)asmodifierinthe mobilephase.The percentageof mod-ifier was programmedas follows: 2% (0-1 min), 30 % (4-6 min), 2% (6.05-10 min) Forthesethree compounds, the earliereluting isomerwastheS-form.MYCandPENwere determinedusingthe cellulose-5 column, withACN(5 mM inNH4Ac) asmodifier The contentofmodifierwasvariedasfollows:10%(0-1min),30% (4-6.5min), 2% (6.51-10min) The elutionorder oftheenantiomers
ofthese compounds wasnot established.The cellulose-5 column permittedalsotheseparationofBENenantiomers,withadifferent selectivitytothatreportedfortheamylose-1column.Thatis,
BEN-RelutedfirstthantheS-formofthefungicideinthecellulose col-umn.Underaboveconditions,maintainingchiralcolumnsat40°C, thetotal pressureinthechromatographicsystemvaried withthe chromatographicgradientwithintherangesof210-250bar(ACN), 200-250bar(MeOH);thus, pressureremained100 barbelowthe upperlimit(350bar)establishedfortheemployedchiralcolumns The effect ofthe make-up flow rate (MeOH:FA, 99.5: 0.5) in the responses offungicides wasevaluated inthe range ofvalues from0.1to0.7mLmin−1.Underchromatographicconditions em-ployed with the amylose-1 column, the normalizedresponses of MET enantiomers and that of BEN-S increased significantly be-tween 0.1 and 0.3 mL min−1 of make-up; thus, their ionization efficiencies improvedwith theflow rate ofMeOH: FA (99.5: 0.5) reachingtheESIsource(Fig.S3A).IncaseofBEN-RandTEB enan-tiomers,whichelutefromthecolumnwithahigherpercentageof MeOHin themobile phase,the effectofmake-up flow was neg-ligible A workingvalue of 0.3 mL min−1 wasused in combina-tionwiththiscolumn.Underconditionsemployedinthe
cellulose-5column, responses ofall compounds decreasedwiththe
make-upflowrate,withthemostdramaticeffectobservedforthe enan-tiomersofPEN(Fig.S3B).Thus,avalueof0.1mLmin−1 wasused
incombinationwiththiscolumn Itisworthnotingthat, normal-izedresponsesofBENenantiomersshowedadifferentdependence withmake-up flow rateas function ofthe modifier employed in themobilephase(Fig.S3AandS3B).Thus,thecompositionofthe
4
Trang 5B
0%
100%
200%
300%
400%
500%
0%
100%
200%
300%
400%
500%
600%
Fig 1 Normalized responses as function of the mobile phase additive A, amylose-1 column using methanol as modifier B, cellulose-5 with acetonitrile as modifier, n = 5
replicates
CO2: organicsolventreaching the ESIsourceplays a major effect
intheefficiencyofcompoundsionization
3.2 Characterization of the SFC-ESI-QTOF procedure
Table 1 compiles relevant data relatedto the performance of
SFC-ESI-QTOF-MSmethodsconsideringtheMS/MSdetectionmode
Linearitywasinvestigatedbyinjectionofracemicmixtures ofthe
above compounds prepared in MeOH Within the range of
con-centrations from 1 to 200 ng mL−1 (values referred to the sum
ofenantiomers),linearresponseswereobtainedforallthespecies
withdeterminationcoefficientsabove0.99.Limitsofquantification,
defined as the lowest concentration providing a signal to noise
(S/N) of 10for thequantification product ionvaried from0.5 ng
mL−1 (enantiomers of MET, TEB andMYC) to 2.5 ng mL−1 (PEN
enantiomers).Thesevaluesareonlyslightlyhigherthanthose
ob-tained in a previous study reporting the determination of same
compounds by UPLC-QqQ-MS, using a non-chiral column (LOQs
from0.1to0.4ngmL−1)[27]
3.3 Matrix effects and accuracy assessment
The extraction yield of the sample preparation methods
em-ployed in the current study for wine and soil were
character-ized in previous publications of our group [9,27] Thus,
valida-tionofthemethodologydescribedinthisresearchwaslimitedto
thestudyofMEs,andtheevaluationoftheaccuracywithspiked samples.Bothvariables are affected not onlyby sample prepara-tionconditions, butalsoby thecomposition ofthe mobile phase
inthe ESI source,which differs betweenSFCandreversed-phase
LCmethods TheassessmentofMEs demonstratedsuppressionof the ionization efficiency of certain compounds (Fig 2) Particu-larly, the enantiomers of TEB and BEN showed a moderate sig-nal attenuation for soil extracts and, in a lesser extent, during analysisofred wine.More significantthan themagnitudeof sig-nal attenuation is the lack of differences betweenMEs observed for the enantiomers of the same species This fact, reduces the riskofreportingfalsevariationsintheir EFswhenprocessingreal samples
Therecoveriesoftheprocedure,estimatedusingsolvent-based standards,aregiveninTable2.Thespikedlevelsemployedinthis study were 20 and 50 ng mL−1 (case of wine) and 50 and 100
ngg−1 (soil).These valuesremain inthe rangeofconcentrations reportedincommercialwinesandvineyardsoils[6,9,27] Recov-eries variedin therangefrom80% to117%withRSDsbetween2 and15% The overallLOQsof theprocedure are alsocompiled in Table2 Reported valueswere estimatedfrominstrumental LOQs (Table1),consideringsampleamountandfinalextractvolumefor eachtypeofsample,aswellassignalattenuationeffectsobserved forsome compounds(Fig.2).Inthecaseofwines,theprocedural LOQsareverysimilartoinstrumentalvalues.Forsoils,LOQsvaried
intherangefrom1.3ngg−1to6.3ngg−1
Trang 60.0 20.0 40.0 60.0 80.0 100.0 120.0
MET-S MET-R BEN-S BEN-R TEB-S TEB-R MYC-1 MYC-2 PEN-1 PEN-2
Fig 2 Ratios between slopes of calibration curves for solvent and matrix-matched standards prepared using a pool of extracts from soil and wine samples
Table 2
Overall recoveries, with standard deviations, for soil and wine samples spiked with racemic mixtures of compounds at two different
concentration levels, n = 3 replicates
50 ng g −1 100 ng g −1 20 ng mL −1 50 ng mL −1 20 ng mL −1 50 ng mL −1 (ng g −1 ) (ng mL −1 ) MET-S 98 (7) 108 (4) 103 (10) 91 (3) 117 (4) 107 (12) 1.5 0.5
MET-R 97 (8) 103 (7) 105 (12) 84 (3) 117 (5) 105 (12) 1.5 0.5
BEN-S 99 (8) 112 (5) 102 (15) 85 (3) 104 (5) 87 (12) 3.3 1.4
BEN-R 84 (7) 105(5) 106 (15) 80 (2) 110 (11) 97 (10) 3.3 1.4
TEB-S 93 (7) 107 (5) 104 (14) 89 (6) 111 (5) 107 (11) 2.3 0.7
TEB-R 98 (9) 112 (5) 107 (14) 94 (4) 110 (9) 105 (11) 2.3 0.7
MYC-1 105 (10) 99 (11) 99 (12) 91 (4) 114 (7) 104 (6) 1.3 0.5
MYC-2 100 (14) 110 (11) 97 (13) 91 (4) 108 (5) 108 (8) 1.3 0.5
PEN-2 94 (7) 104 (8) 97 (9) 88 (3) 106 (11) 106 (9) 6.3 2.5
Table 3
Enantiomeric fractions (EFs), with their standard deviations (SD), and average total concentrations of fungicides in commercial wines, n = 3 replicates R, red wine W, white wine
Sample code
EF SD Conc (ng mL −1 ) EF SD Conc (ng mL −1 ) EF SD Conc (ng mL −1 )
W10 0.52 0.01 15
Empty cells correspond to non-detected compounds
3.4 Distribution of fungicides in wine and soil samples
Table 3 shows the total concentrations and the EFs of
fungi-cides in a selection of 17 wines produced in Galicia (Northwest
Spain).BENandPENremainedbelowmethodLOQinall samples,
so thesecompounds are not included inthe table The detection
frequencyforthe restoffungicidesincreasedinthefollowing or-der:MYC <TEB<MET,withresiduesofthelatterspeciesfound
inall samples.Comparedto theEuropean Regulationfor vinifica-tiongrapes,thehighestconcentration ofMETfound inwine(412
ng mL−1, equivalentto 412 ng g−1, since the density of wine is around 0.994g mL−1) wascloseto 50% of its maximumresidue
6
Trang 7level authorized in vinification grapes (1000 ng g−1) [32]
Glob-ally, the EFs of TEB and MYC were equal to 0.5 This fact
con-firms thatboth compounds arecommercializedasracemates and
also, the absence of enantioselective dissipation processes either
atvines,orduringmicrobiologicalprocessesinvolvedinmust
fer-mentation In case of MET,the range of EFs varied from 0.05to
0.57.EFs below0.1, asthatobservedforwine codeR2,likely
cor-respondtograpesfumigatedwiththeR-formofMET
(commercial-ized underthename ofMET-M).On theother hand,EFs slightly,
although significantly, above 0.5 were measured in 4 red wines
Assuming that theywere obtainedfromgrapes treatedonly with
the racemate of this fungicide, it seems that MET-S (the
inac-tive fungicide isomer) is slightly enriched versus the R-form at
vines and/or during wine elaboration Obviously, confirming this
assumptionrequirestotheanalysisofarelevantnumberofwines
elaborated from grapes treated with the racemate of MET, with
vinification developed under controlled conditions to avoid
mix-ing in the same fermentation tank grapes, which received
dif-ferent treatments Finally, in most white wines, EFs below 0.5
(0.37 to 0.43) were observed (Table 3) In this case, without
in-formation ofvineyard treatments, it cannot be concludeda
pref-erential accumulation of the R-form in this wine The reason is
that vines might havebeen fumigated with formulations
includ-ing theracemateandalsowithother preparationscontainingjust
MET-M
AverageconcentrationsandEFs dataforsoil samplesare
sum-marizedinTable4.Sampleswereobtainedfrom7vineyardsfrom
3 Designations of OrigininGalicia (NorthwestSpain).Inthiscase,
allfungicideswerenoticedin,atleast,oneoftheinvestigated
sam-ples Compounds dissipation was noticed in those points where
pairs of samples were taken in autumn and at the end of
win-ter (vineyards 1 to 4) Regarding EFs, those meassured for BEN,
TEBandMYCwereequalto0.50;thus,noenantioselective
degra-dation processeswere identified In caseof PEN, EFvalues
mea-sured in October and March were equivalent in vineyards codes
1 and 2, although in vineyard code 1 a value below 0.5 was
found in both sampling campaigns (Table 4) Finally, the EFs of
MET, and their variation between samples obtained at different
datesfromsamevineyard,differasfunctionofthesamplingpoint
At vineyard code 3, MET-R was the predominant form in
Oc-tober without observing compound enantiomerization in March
EFs obtained for MET at vineyards 4 and 5 show a prevalence
ofMET-S.SincefungicidalpreparationscontainingonlyMET-Sare
not commercially available, EFs above 0.5 are possible
assum-ing a faster degradation of the R-form than that of S-isomer in
these vineyards On the other hand, at vineyards 1,2 and 7, the
R-enantiomer was noticed at higher concentration than the
S-form In the particular case of vineyard 1, faster dissipation of
MET-S compared to theR-formcan be concluded fromEFs
mea-sured in October and March (0.37 ± 0.01 and 0.28 ± 0.01,
re-spectively) The SFCchromatogramsforthemostintense product
ion of MET in soil samples showing different EFs are shown in
Fig.3
3.5 Assessment of EFs under laboratory conditions
The potential existence of enantioselective degradation
pro-cesses at vineyard codes 2, 3 and 4 (Table 4) was further
as-sessed under laboratory conditions Soil from these points were
takeninJune, inorderto mimicmicrobiologicalconditions
exist-ingduringtheapplicationperiodofthesecompounds,spikedwith
selected compounds and incubated under conditions reportedin
section2.3.Table5summarizedthetotalresidualconcentrationof
eachfungicideattheendoftheexperiment,innon-sterilizedand
sterilizedsoils,normalizedtothatmeasuredatdayzero.TEB,MYC
andPEN werehardly degradedduringtheexperiment, whilstthe
)
)
)
)
)
Trang 8Fig 3 Chromatographic profiles for the enantiomers of MET in soil samples obtained from different vineyards at the same date (October 2018) A, vineyard code 4 B,
vineyard code 2 Vineyard code 1
Table 5
Percentage of each fungicide remaining in soil after 66 days of incubation (n = 2 replicates)
Vineyard
soil code
Aerobic Sterilized Aerobic Sterilized Aerobic Sterilized Aerobic Sterilized Aerobic Sterilized
dissipated percentages ofMET andBENvaried depending onthe
vineyardsoil.Inbothcases,thelowestresiduelevelwasfound in
thesamesoil
The average EFs atdays 0 and66 (n= 2replicates) are given
inTableS3.As expected,incaseofcompoundsnotremoved
dur-ing the experiment (TEB, MYC and PEN), EFs measured at days
0 and66 were equivalent ForBEN,the EFs slightlydecreased at
day 66 compared to those calculate at zero time in soils from
vineyards codes 2 and 3, but not in soil from vineyard code 4
The plotshowing evolution of EFvalues for BENand total
com-poundconcentrationinthethreesoilsinvolvedinthestudyis
pro-vided assupplementary information (Fig S4) Finally, thechange
in the EFs of MET depended on the soil matrix Fig 4
summa-rizes the time-course evolution ofMET andthe EFs of the
com-pound duringtheincubationexperiment.Samplesfromvineyards
3 and 4 were spiked with the racemate at 200 ng g−1 at day
0, whereas the initial concentration inthe samplefrom vineyard
code 2corresponds to the native residueofMET existing inthis soil The kineticsof METremovalwassample dependent,with a muchfasterdissipationinsoil number4(Fig.4A),whichmatches the trendobserved forBEN insame sample(Fig S4) The evolu-tionoftheEFsofMETwere alsodifferentbetweensamplesfrom vineyards codes 2 and3, witha faster dissipation ofMET-S (EFs decreasedsteadywithincubationtime),to thatobservedin vine-yardsoil code-4(Fig.4B) Inthelattercase, MET-Rwasdegraded completeleyafter14daysofincubation,leadingtoEFvaluesclose
to 1 Thus, in agreement with data obtained under field condi-tions (Table 4), MET-R was less stable than MET-S in soil from vineyard code 4 Faster degradation of MET-R versus the S-form hasbeen relatedto basicsoils; however,the pH ofsoil obtained from vineyard code 4, and employed in the incubuation experi-ment,was slightlyacidic,andintermediate between those corre-sponding to the other two samples involved in the same study (TableS1)
8
Trang 90 50 100 150 200 250
Incubaon me (days)
0.00 0.20 0.40 0.60 0.80 1.00 1.20
0 10 20 30 40 50 60 70
Incubaon me (days)
A
B
Fig 4 Time-course evolution of MET in soils from 3 different vineyards in laboratory dissipation studies, average values of duplicate assays A, total concentration B, EF
data (MET-S/(MET-S + MET-R) Soil from vineyards 3 and 4 were spiked with the racemic standard of MET (200 ng g −1 ) Soil from vineyard 2 contained significant levels of MET; thus, it was not fortified with this compound in the laboratory dissipation study
4 Conclusions
SFC-ESI-QTOF-MS permittedthe chiral,sensitivedetermination
of five fungicideswidely employed in viticulture and, in general,
in agriculture The modifier added to supercritical CO2 was the
onlyparametershowingasignificantinfluenceontheselectivityof
chiralseparations Ontheotherhand,additivesplayedcompound
and mobile phase dependent effects in the yield of their
ioniza-tionattheESIsource.Dataobtainedforprocessessamples(wines
andsoils)pointouttothefactthatvineyardsarestilltreatedwith
formulationsincludingtheverylowactive enantiomerofMET
(S-form).Thus,withoutarecordofvinestreatments,throughanalysis
ofcommercialwinesishardtoinvestigatepotential
enantioselec-tive removalofMETisomersduringinteractionwithvinesand/or
throughvinificationsteps.Asregardsvineyardsoils,fielddataand
laboratoryexperimentsconfirmedtheenantioselectivedegradation
ofMET.TherelativedissipationratesofRandS-formsdiffered
sig-nificantly among soilsfrom different vineyards Despite BEN
be-longs to the same chemical family as MET, variations of its EFs
duringsoilincubationassaysweremoresubtle
Declaration of Competing Interest
Theauthorsdeclarethattheyhavenoknowncompeting
finan-cialinterestsorpersonalrelationshipsthatcouldhaveappearedto
influencetheworkreportedinthispaper
CRediT authorship contribution statement
L Pérez-Mayán: Investigation, Methodology, Writing review
&editing.M Ramil:Data curation,Formalanalysis, Writing re-view&editing.R Cela:Projectadministration,Fundingacquisition, Writing review&editing.I Rodríguez:Conceptualization, Fund-ingacquisition,Fundingacquisition,Writing originaldraft
Acknowledgments
L.P.MacknowledgesaFPUgranttotheSpanishMinistryof Sci-ence This study was supported by Xunta de Galicia and Span-ish Government through grants GRC-ED431C 2017/36, PGC2018
094613B I00,bothco-fundedbytheEUFEDERprogram
Supplementary materials
Supplementary material associated with this article can be found,intheonlineversion,atdoi:10.1016/j.chroma.2021.462124
References
[1] N Cui, H Xu, S Yao, Y He, H Zhang, Y Yu, Chiral triazole fungicide tebu- conazole: enantioselective bioaccumulation, bioactivity, acute toxicity, and dis- sipation in soils, Environ Sci Pollut Res 25 (2018) 25468–25475, doi: 10.1007/ s11356- 018- 2587- 9
[2] Y Li, F Dong, X Liu, J Xu, X Chen, Y Han, W Liang, Y Zheng, Development
of a multi-residue enanatiomeric analysis method for 9 pesticides in soil and water by chiral liquid chromatography/tandem mass spectrometry, J Hazard Mat 250-251 (2013) 9–18, doi: 10.1016/j.jhazmat.2013.01.071
Trang 10[3] A.R Fontana, I Rodríguez, M Ramil, J.C Altamirano, R Cela, Solid-phase ex-
traction followed by liquid chromatography quadrupole time-of-flight mass
spectrometry for the selective determination of fungicides in wine samples,
J Chromatogr A 128 (2011) 2165–2175, doi: 10.1016/j.chroma.2011.02.025
[4] A Santana-Mayor, R Rodríguez-Ramos, B Socas-Rodríguez, C Díaz-Romero,
M.A Rodríguez-Delgado, Comparison of pesticide residue levels in red wines
from Canary islands, Iberian Peninsula and Cape Verde, Foods 9 (2020) 1555,
doi: 10.3390/foods9111555
[5] A Bermúdez-Couso, M Arias-Estévez, J.C Nóvoa-Muñoz, E López-Periago,
B Soto-González, J Simal-Gándara, Seasonal distributions of fungicides in soils
and sediments of a small river basin partially devoted to vineyards, Water Res
41 (2007) 4515–4525, doi: 10.1016/j.watres.2007.06.029
[6] E Pose-Juan, M.J Sánchez-Martín, M.S Andrades, M.S Rodríguez-Cruz,
E Herrero-Hernández, Pesticide residues in vineyard soils from Spain: spatial
and temporal distributions, Sci Total Environ 514 (2015) 351–358, doi: 10.1016/
j.scitotenv.2015.01.076
[7] P Cabras, A Angioni, Pesticide residues in grapes, wine, and their processing
products, J Agric Food Chem 48 (20 0 0) 967–973, doi: 10.1021/jf990727a
[8] F Cus, H.B Cesnik, S.V Bolta, A Gregoric, Pesticide residues in grapes and
during vinification process, Food Control 21 (2010) 1512–1518, doi: 10.1016/j
foodcont.2010.04.024
[9] L Pérez-Mayán, M Ramil, R Cela, I Rodríguez, Multiresidue procedure to as-
sess the occurrence and dissipation of fungicides and insecticides in vine-
yard soils from Northwest Spain, Chemosphere (2020) 261, doi: 10.1016/j
chemosphere.2020.127696
[10] Decision (EU) 2020/1161 establishing a watch list of substances for Union-wide
monitoring Official journal of the European Union of 6 august 2020 L 257/32
[11] A Monkiedje, M Spiteller, K Bester, Degradation of racemic and enantiopure
metalaxyl in tropical and temperate soils, Environ Sci Technol 37 (2003) 707–
712, doi: 10.1021/es020123e
[12] I.J Buerge, T Poiger, M.D Müller, H.R Buser, Enantioselective degradation of
metalaxyl in soils: chiral preference changes with soil pH, Environ Sci Tech-
nol 37 (2003) 2668–2674, doi: 10.1021/es0202412
[13] J Gu, C Ji, S Yue, D Shu, F Su, Y Zhang, Y Xie, Y Zhang, W Liu, M Zhao,
Enantioselective effects of metalaxyl enantiomers in adolescent rat metabolic
profiles using NMR-based metabolomics, Environ Sci Technol 52 (2018)
5438–5447, doi: 10.1021/acs.est.7b06540
[14] X Wang, X Wang, H Zhang, C Wu, X Wang, H Xu, X Wang, Z Li, Enantios-
elective degradation of tebuconazole in cabbage, cucumber, and soils, Chirality
24 (2012) 104–111, doi: 10.1002/chir.21030
[15] Y Li, F Dong, X Liu, J Xu, Y Han, Y Zheng, Enantioselectivity in tebuconazole
and myclobutanil non-target toxicity and degradation in soils, Chemosphere
122 (2015) 145–153, doi: 10.1016/j.chemosphere.2014.11.031
[16] Q Zhang, X Hua, Y Yang, W Yin, M Tian, H Shi, M Wang, Stereoselective
degradation of flutriafol and tebuconazole in grape, Environ Sci Pollut Res
22 (2015) 4350–4358, doi: 10.1007/s11356- 014- 3673- 2
[17] Z Shen, W Zhu, D Liu, X Xu, P Zhang, Z Zhou, Stereoselective degradation of
tebuconazole in rat liver microsomes, Chirality 24 (2012) 67–71, doi: 10.1002/
chir.21027
[18] B Gámiz, G Facenda, R Celis, Evidence for the effect of sorption enantioselec-
tivity on the availability of chiral pesticide enantiomers in soil, Environ Pollut
213 (2016) 966–973, doi: 10.1016/j.envpol.2016.03.052
[19] Q Zhang, L Zhou, Y Yang, X Hua, H Shi, M Wang, Study on the stereoselec- tive degradation of three triazole fungicides in sediment, Ecotoxicol Environ Saf 117 (2015) 1–6, doi: 10.1016/j.ecoenv.2015.03.014
[20] P Zhao, J Zhao, S Lei, X Guo, L Zhao, Simultaneous enantiomeric analysis of eight pesticides in soils and river sediments by chiral liquid chromatography- tandem mass spectrometry, Chemosphere 204 (2018) 210–219, doi: 10.1016/j chemosphere.2018.03.204
[21] C West, Recent trends in chiral supercritical fluid chromatography, Trends Anal Chem 120 (2019) 115648, doi: 10.1016/j.trac.2019.115648
[22] S Bieber, G Greco, S Grosse, T Letzel, RPLC-HILIC and SFC with mass spectrometry: polarity-extended organic molecule screening in environmental water samples, Anal Chem 89 (2017) 7907–7914, doi: 10.1021/acs.analchem 7b00859
[23] M Ishibashi, Y Izumi, M Sakai, T Ando, E Fukusaki, T Bamba, High- throughput simultaneous analysis of pesticides by supercritical fluid chro- matography coupled with high-resolution mass spectrometry, J Agric Food Chem 63 (2015) 4 457–4 463, doi: 10.1021/jf5056248
[24] V Cutillas, M Murcia-Morales, M Gómez-Ramos, S.M Taha, A.R Fernández- Alba, Supercritical fluid chromatography coupled to tandem mass spectrome- try for the analysis of pesticide residues in dried spices Benefits and draw- backs, Anal Chim Acta 1059 (2019) 124–135, doi: 10.1016/j.aca.2019.01.010 [25] V Cutillas, M García-Valverde, M Gómez-Ramos, F.J Díaz-Galiano, C Ferrer, A.R Fernández-Alba, Supercritical fluid chromatography separation of chiral pesticides: unique capabilities to study cyhalothrin and metalaxyl as examples,
J Chromatogr A 1620 (2020) 461007, doi: 10.1016/j.chroma.2020.461007 [26] V Silva, H.G.J Mol, P Zomer, M Tienstra, C.J Ritsema, V Geissen, Pesticide residues in European agricultural soils- a hidden reality unfolded, Sci Total Environ 653 (2019) 1532–1545, doi: 10.1016/j.scitotenenv.2018.10.441 [27] G Castro, L Pérez-Mayán, T Rodríguez-Cabo, I Rodríguez, M Ramil, R Cela, Multianalyte, high-throughput liquid chromatography tandem mass spec- trometry method for the sensitive determination of fungicides and insec- ticides in wine, Anal Bioanal Chem 410 (2018) 1139–1150, doi: 10.1007/ s00216- 017- 0724- 9
[28] J Masbou, F Meite, B Guyot, G Imfeld, Enantiomer-specific stable carbon iso- tope analysis (ESIA) to evaluate degradation of the chiral fungicide metalaxyl
in soils, J Hazard Mater 353 (2018) 99–107, doi: 10.1016/j.jhazmat.2018.03.047 [29] F Yang, Y Wang, S Liu, C He, X Tao, H Deng, G Tang, Z Bian, Z Fan, A green and effective method for the determination of metalaxyl enantiomers
in tobacco and soil by supercritical fluid chromatography–tandem mass spec- trometry, Chirality 32 (2020) 505–514, doi: 10.1002/chir.23184
[30] T Harner, K Wiberg, R Norstrom, Enantiomer fractions are preferred to enan- tiomer ratios for describing chiral signatures in environmental analysis, Envi- ron Sci Technol 34 (20 0 0) 218–220, doi: 10.1021/es9906958
[31] C Hamman, D.E Schmidt, M Wong, M Hayes, The use of ammonium hydrox- ide as an additive in supercritical fluid chromatography for achirar and chi- ral separations and purifications of small, basic medicinal molecules, J Chro- matogr A 1218 (2011) 7886–7894, doi: 10.1016/j.chroma.2011.08.064 [32] European commision - pesticides database, (n.d.) https://ec.europa.eu/food/ plant/pesticides/eu-pesticides-database/public/?event=homepage&language=
EN
10