Prepared by SASA Photography Unit. All photographs and text © Crown Copyright except where otherwise indicated.
● Routine quantitation is performed using a 4 pointmatrixmatched calibration with internal standardisation(methomyl-D3,carbendazim-D4,pendimethalin-D5)
● Linearityhasbeendemonstratedfrom0.01to0.2mg/kgineachmatrixforallcompounds.
● Recoverydatafor5representativematricesispresented.(n=4,spikedat0.04mg/kg)
● Data for 50 compounds representative of thewide range of pesticides analysed byUPLC-ESI-MS/MS arepresented.
● ThemethodhasbeenvalidatedinaccordancewiththeEUQualityControlProceduresforPesticideResidueAnalysis (EUDocumentNo. SANCO/10232/2006, 24/March/2006) giving good recovery and repeatabilityfor over 200 pesticides and metabolites analysed by GC-MS/MS and LC-MS/MS in a range of fruit andvegetables.
Results for Rapid Analysis of Pesticides in Cabbage
Thereportinglimitforthemajorityofpesticidesis0.02mg/kg.Alimitofquantitationofatleast0.01mg/kgmustbeachieved.
Sample Extraction Procedure
Samplesoffruitandvegetablesarefrozenandcryo-milledonreceiptpriortoextractionbyhomogenisationwithethylacetate.Analiquotofthiscrudeextractissolventexchangedintomethanol,filteredandpresentedforLC-MS/MSanalysis(103analytessought).Theremainderispassedthroughaclean-upstageusinggelpermeationchromatographypriortoanalysisbyGC-MS/MS(97analytessought).
ACQUITY Ultra Performance LC™ (UPLC)
Smallstationaryphaseparticletechnology(1.7µm)andtheabilitytooperateathighbackpressures(15,000psi)allowthissystemtoachievehighersensitivityandpeakcapacityatoptimumflowrates.
The separation and detection of over 100pesticides in complex matrices in less than 4
minutes using UPLC-MS/MSGeorgeAKeenan,KirstyReidandMichaelJTaylorChemistry
ScottishAgriculturalScienceAgency,1RoddinglawRoad,EdinburghEH129FJEmail: [email protected]
Abstract
Ultra Performance Liquid Chromatography (UPLC) with electrospray tandemmass spectrometry (ESI-MS/MS)hasbecomeanestablishedfront-linetechniqueusedbytheScottishAgriculturalScienceAgency(SASA)for thequantitativedeterminationofover100pesticidessoughtinavarietyofcomplexmatrices.ThemethodisappliedroutinelytosupporttheannualUKandEUpesticideresiduesinfoodmonitoringprograms.
Analyticalmethodologies employed in thedeterminationofpesticide residues in foodstuffsmust be capablenotonlyofquantifyingverylowlevelsofincurredresiduesbutalsoofconfirmingtheidentityandmagnitudeoftheseresidues.
Therequirementtoprovideunambiguousevidencebecomesincreasinglychallengingasreportinglimitsandmaximumresiduelevelsaredecreasedandthenumberoftargetpesticidesandmetabolitesisincreased(from125in2006toover200inthe2007UKsurveillanceprogramme).
Agroupof50compoundsfromthesuiteof103pesticidesroutinelysoughtusingLCmethodshasbeenchosentoillustratethepoweroffastanalyticaltechniquesusingUPLC-ESI-MS/MS.Theserepresentativecompoundscoverawiderangeofchemicalfunctionalityandpesticideclass.
Performancedataforvariouscommoditiesandresultsfromcabbagesamplesscreenedaspartofthe2007surveillanceprogramarepresented.
Methodology
PESTICIDE
CLASS
SWEETPOTATO CABBAGE KIWIFRUIT PEACH STRAWBERRYMean
%REC
%RSD
Mean%REC
%RSD
Mean%REC
%RSD
Mean%REC
%RSD
Mean%REC
%RSD
acetamiprid neonicotinoid insecticide 89 8.6 86 8.7 89 4.3 94 6.7 105 15.1atrazine triazine herbicide 86 2.9 93 14.0 94 10.1 95 4.3 98 19.1azoxystrobin strobilurin fungicide 88 7.2 92 13.8 89 7.3 94 6.3 102 21.7bendiocarb carbamate insecticide 88 5.6 93 14.0 95 11.0 96 8.1 103 20.5bitertanol azole fungicide 81 14.5 85 16.3 89 18.4 88 10.5 95 11.1boscalid carboxamide fungicide 86 5.4 90 8.0 96 20.2 98 10.6 83 15.8carbosulfan carbamate insecticide 88 11.3 82 13.9 74 10.5 86 13.7 75 9.9chlorotoluron urea herbicide 90 7.2 97 14.3 92 9.2 93 9.9 87 21.9cymoxanil cyanoacetamide fungicide 93 9.7 98 12.3 89 8.9 96 5.7 91 14.8cyprodinil anilinopyrimidine fungicide 83 9.5 92 11.5 113 10.1 85 5.9 106 15.9diethofencarb phenylcarbamate fungicide 86 9.3 92 8.9 92 9.0 94 9.3 98 20.6difenconazole azole fungicide 86 4.2 85 19.9 83 15.0 90 15.1 82 10.8diflubenzuron benzoylurea insecticide 82 15.9 88 14.5 99 33.1 80 7.8 97 21.9dimethomorph cinnamicacidamide fungicide 91 8.4 95 8.1 92 12.1 94 7.5 99 13.9disulfoton organophosphorus insecticide 72 17.0 84 10.5 85 29.0 91 25.7 96 19.3ethiofencarb carbamate insecticide 80 8.9 78 5.4 74 26.8 80 21.8 91 13.4fenarimol pyrimidine fungicide 91 15.4 79 18.9 126 18.4 93 22.0 76 11.8fenazaquin quinazoline acaricide 90 6.1 82 8.3 88 4.4 103 20.9 89 12.3fenbuconazole azole fungicide 83 6.0 86 7.2 100 10.7 95 16.3 85 10.0fenhexamid hydroxyanilide fungicide 88 4.6 91 7.8 94 8.6 91 4.2 100 16.0fenpropimorph morpholine fungicide 90 7.5 80 15.6 82 8.4 86 21.8 82 17.5fenpyroximate pyrazole acaricide 86 7.3 83 14.3 81 5.0 94 11.6 89 13.4flufenacet oxyacetamide herbicide 91 11.8 90 12.2 92 11.3 96 11.9 99 17.3flusilazole azole fungicide 85 8.9 92 16.7 90 9.7 93 7.9 88 8.9hexythiazox carboxamide acaricide 87 6.9 83 15.2 87 3.3 90 16.0 87 12.8imidacloprid neonicotinoid insecticide 100 8.7 90 9.8 103 13.3 100 15.0 99 27.6indoxacarb oxadiazine insecticide 95 12.0 85 11.8 91 15.7 90 18.7 84 8.9malathion organophosphorus insecticide 85 9.8 94 7.7 93 13.2 94 13.3 102 19.7mepanipyrim pyrimidine fungicide 86 11.6 91 15.8 92 13.4 91 12.5 85 4.4methomyl carbamate insecticide 107 11.7 95 7.7 90 5.9 97 6.2 86 10.9monocrotophos organophosphorus insecticide 93 5.9 74 10.5 87 3.6 97 7.9 84 10.1myclobutanil azole fungicide 87 8.5 91 11.4 92 6.6 94 8.5 107 18.8pendimethalin dinitroaniline herbicide 92 18.3 82 5.3 87 21.4 98 17.1 85 16.7picoxystrobin strobilurin fungicide 91 9.9 90 8.2 92 7.9 94 10.4 96 14.7pirimicarb carbamate insecticide 89 8.3 93 9.5 93 7.0 94 7.8 91 23.2prochloraz azole fungicide 97 10.8 85 12.6 94 6.8 86 11.0 102 21.3pymetrozine azomethine insecticide 82 3.2 66 8.5 75 6.4 92 8.7 63 7.5pyrethrins pyrethroid insecticide 85 8.4 79 5.9 88 22.4 92 10.2 88 16.5pyridaben pyridazinone insecticide 90 11.4 82 11.6 85 5.2 96 11.5 87 9.2pyrimethanil pyrimidine fungicide 87 9.1 91 11.4 89 11.3 97 15.2 91 12.7quinoxyfen phenoxyquinoline fungicide 84 17.9 82 13.3 81 8.8 88 11.1 84 17.3spinosad macrocycliclactone insecticide 94 11.1 81 7.5 85 7.2 91 11.9 88 7.0spiroxamine morpholine fungicide 86 7.9 84 10.3 77 4.9 92 12.0 82 9.6tebufenozide diacylhydrazine insecticide 92 9.4 89 8.7 92 8.9 94 8.3 82 12.3tebufenpyrad pyrazole acaricide 86 9.5 83 10.6 82 3.6 92 16.0 85 8.8tetraconazole azole fungicide 95 8.9 94 20.1 106 22.4 84 10.8 86 18.0thiamethoxam neonicotinoid insecticide 86 4.9 90 8.3 93 5.9 113 8.5 93 7.0thiodicarb carbamate insecticide 62 16.0 70 14.9 86 11.2 96 9.6 96 12.2trifloxystrobin strobilurin fungicide 89 7.7 84 9.8 83 13.4 94 15.0 86 15.0zoxamide benzamide fungicide 86 7.4 90 18.7 93 10.7 93 8.1 85 11.2
UPLC Experimental Parameters
Instrument: Waters ACQUITY UPLC systemColumn: ACQUITYUPLCBEHC181.7µm
(2.1mmi.d.x50mm)
ACQUITYLCPumpInitialConditions
A:H2O/MeOH95/5v/v,5mMammonium acetatesolution
B:MeOH,5mMammoniumacetatesolution
Solvents(gradientelution),Flow0.48ml/min
Mins A% B%0.00 70 300.52 70 300.66 40 601.05 40 603.31 15 854.90 15 854.91 0 1005.5 0 1005.51 70 306.5 70 30
StopTime(mins) 6.5MaxPressure(psi) 15000OvenTemperature(°C) 35.0InjectionVolume(µl) 3.0µl
Mass Spectrometry Experimental Parameters
● To utilise chromatographic performancecommoninUPLC(narrowpeaks1-3sFWHM)the mass spectrometer must be capable ofacquiringMRMdataatasufficientlyhighrate(shortdwelltimes≈10ms).
● The present method comprises 7 consecutiveacquisition functions covering the time from0.1–4.0minutes.EachfunctioncontainsMRMacquisition parameters for between 6 to 27compounds. This single experimentalmethodacquires data for 103 pesticides plus internalstandardsinlessthan4minutes.
Instrument: Waters Quattro Premier-XE
DataSystem MassLynx4.1Acqusition Electrospray±ionisation
MultipleReactionMonitoring(MRM)DwellTimes10-20msCollisionGas Argon
Retention Time (mins)2.00 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 30.00
0
100
%
18.36
27.3225.8220.3524.28
3.912.89
fenpropimorph
azoxystrobin
fenpropimorph
azoxystrobin
HPLC
UPLC
Expanded view of UPLC chromatogram
3.913.393.132.89
UPLCvsHPLCMulti-ResidueAnalysis
Performance Data for Selected Pesticides
Time1.00 2.00 3.00 4.00 5.00
%
0 Time1.40 1.60 1.80 2.00 2.20
%
0
1.40 1.60 1.80 2.00 2.20
%
0
1.55
1.55
Residue 0.38mg/kgazoxystrobinm/z 404<372Screen MRM
Residue 0.38mg/kgazoxystrobinm/z 404<344Confirmation MRM
Sum of MRM Transitions forAcquisition Functions 1-7 for a Calibration Standard Mix
azoxystrobin
Cabbage Sample Extract
Compound name: AzoxystrobinCorrelation coefficient: r = 0.995811, r^2 = 0.991640Calibration curve: 22.0975 * x + -0.00294919Response type: Internal Std ( Ref 22 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None
mg/kg0.000 0.020 0.040 0.060 0.080
Res
pons
e
0.00
1.00
2.00
UPLC-ESI-MS/MS Ion Chromatograms of an Azoxystrobin Residue in Cabbage Extract
UPLC-ESI-MS/MS Calibration Curve for the Quantitation of Azoxystrobin
SASASampleNo. ResidueFound
Conc.mg/kg
ReportingLimit(RL)mg/kg
MaximumResidueLevel(MRL)mg/kg
CAB001 boscalid 0.32 0.02 1CAB001 pyraclostrobin 0.02 0.02 0.2CAB001 difenoconazole 0.03 0.02 NoMRLsetCAB002 tebuconazole 0.07 0.02 0.8CAB007 tebuconazole 0.04 0.02 0.8CAB014 indoxacarb 0.06 0.02 NoMRLsetCAB017 boscalid 0.06 0.02 1CAB017 tebuconazole 0.05 0.02 0.8CAB041 indoxacarb 0.06 0.02 NoMRLsetCAB041 tebufenozide 0.04 0.02 5*CAB047 azoxystrobin 0.38 0.02 0.3CAB047 difenoconazole 0.14 0.02 NoMRLset
* Theresidue levelofazoxystrobin identified incabbagesampleCAB047exceeded theMRL.
Underthesecircumstancesthesampleisre-extractedandtheresiduere-quantifiedbefore
informingthePesticideResidueCommittee. ·Ariskassessmentisundertakentodeterminewhetherconsumerswouldbeatriskfrom
residuespresent ·Thesupplierisnotifiedandaskedtoinvestigatethecause ·Ifresiduesfoundareahealthconcernothermemberstatesareinformed(EUrapid
alerts) ·Enforcementmonitoringmaybeinitiated
Summary
Itishighlylikelythattheupwardtrendinpesticidenumberssoughtinallcommoditieswillcontinue.Thiswillplacegreaterdemandsonthecapabilitiesofanalyticalinstrumentationandmethods.Webelievethatthedevelopmentandvalidationoffast,efficientmethodssuchasthatdescribedwillbeacornerstoneinmeetingthischallenge.
Advantages of Rapid Analysis by UPLC-ESI-MS/MS
● Reduces instrument time and increases efficiency Sample-batch run timesofca.2.5hoursare typicalwithnoobservedadverseeffectson systemstabilityor
chromatographicintegrityduringorbetweenbatchanalyses.Sample-batchruntimesofca.20hoursarecommoninconventionalHPLC.Atypicalbatchcomprisesca.26samplesandincludessampleextracts,matrixmatchedstandards,AQCchecksandblanks.
● Allows a greater throughput of samples
● Reduces solvent usage
● Allows rapid confirmation of identity and quantity as required by the Pesticide Residues Committee
● Allows faster response to identified pesticides
● Minimises possible analyte loss in sample extracts over time
