An Elevated Resolution GC-TOF-MS Method for

the Analysis of Pesticide Residues in Food

OverviewOverview

Pesticide residues in food—EU and worldwide legislation

Analytical challenges—Targeted screening v. untargeted screening

Introduction to exact mass TOF using GCT Premier

Targeted screening in food—Pear, lettuce and fruit-based baby food

—TargetLynx application manager

Untargeted screening in food—ChromaLynx

—Exact mass scoring of library searches

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2000 2001 2002 2003 2004 2005

EU Alert and Information Notifications per Year

Food Safety Alerts in 2005Food Safety Alerts in 2005

EU LegislationEU Legislation

Regulation 396/05/EC amending 91/414/EC —Harmonized for all pesticide/product combinations

—Range from “not detectable” to approximately 50mg/kg

—“Not detectable” defaults to 0.01 mg/kg if no other level is assigned

Recommendation 2006/26/EC of 18th January 2006—Coordinated monitoring program to ensure compliance with

maximum levels of pesticide residues in or on cereals and certain other products of plant origin

Worldwide LegislationWorldwide Legislation

US—Food Quality Protection Act (FQPA) of 1996

ohttp://www.epa.gov/oppfod01/fqpa/backgrnd.htm

—40 CFR 180

ohttp://www.access.gpo.gov/nara/cfr/waisidx_04/40cfr180_04.html

—USDA, Foreign Agricultural Service

ohttp://www.fas.usda.gov/htp/mrl.htm

Japan—Positive List System for Agricultural Chemical Residues in Foods

—Enforced from 29th May 2006

—Uniform limit set to 0.01 mg/kg

ohttp://www.ffcr.or.jp/zaidan/FFCRHOME.nsf/pages/eng.h-page

ohttp://www.m5.ws001.squarestart.ne.jp/foundation/search.html

Analytical ChallengesAnalytical Challenges

Chemically and structurally diverse analytes—Approximately 1000 pesticides registered worldwide

Wide variety of matrices (fruits, vegetables, herbs, spices)

Method must be…—Sensitive, low default reporting limit of 0.01mg/kg—Selective, reduce or eliminate matrix interferences—Multiresidue, multiple targets in a single run—Rugged, complex samples with reduced sample clean-up

But also generic…—‘Food scares’ and changes in legislation require updating of

methods and re-analysis of samples

—Non target components can be detected post acquisition or retrospectively

Trend in Analytical Method Trend in Analytical Method DevelopmentDevelopment

Sample Preparation

Compound Specific

Generic Generic Generic

Separation / Detection

Data Processing

Results

Compound Specific

Compound Specific

Generic Generic

Compound Specific

Compound Specific

Compound Specific

Untargeted

GC-ECD

GC-MS (SIR)

GC-MS (SIR)

GC-MS/MSGC-TOF GC-TOF?

System SolutionSystem Solution

GCT Premier

Orthogonal acceleration GC-TOF-MS high resolution, exact mass

TargetLynx

Advanced quantification with a full range of automatic quality control checks

ChromaLynx

Peak detection, deconvolution, library searching with exact mass scoring

GCT PremierGCT PremierOrthogonal Acceleration TOFOrthogonal Acceleration TOF

Rapid full spectral sampling and acquisition rates—Non skewed spectra

—Narrow chromatographic peaks

—Accurate chromatographic peak profiling enabling deconvolution

Non scanning instrument with a high duty cycle—High full scan sensitivity

Predictable scan law —Routine exact mass calibration with a single lock mass

Elevated Resolution—Enhanced selectivity

GCT PremierGCT Premier

Plug in EI/CI ion volumes for rapid change over

Resolution

—Greater than 7000 (Full width half maximum)

Exact mass

—Less than 5 ppm RMS

Dynamic range

—4 orders linear dynamic range

Spectral acquisition rate

—20 spectra per second

Electron Impact (EI+) with a mass range of m/z 50 → 500

Acquisition Speed = 4 spectra/s

Tris(trifluoromethyl)triazine, 284.9949 single point lock mass

Dynamic Range Enhancement (DRE) On

Low Mass Cut Off = 45Da

Electron Energy = 70eV

Trap Current = 200µA

Source Temperature = 200°C

Interface Temperature = 280°C

Detector Voltage = 2900V

GCT Premier ParametersGCT Premier Parameters

Pesticide ResiduesPesticide Residues

AzoxystrobinBifenthrinBiphenylBromopropylateBupirimateBuprofezinCaptanCarbarylChlorfenvinphosChlorothalonilChlorpyrifosChlorpyrifos-methylChlozolinateCyfluthrinCyhalothrin-lambdaCypermethrinCyprodinilDDDDDE

o,p-DDTp,p-DDTDeltamethrinDiazinonDichlofluanidDicloranDicofolDifenoconazoleDiphenylamineEndosulfanEndosulfan-sulfateEthionEthoprophosFenarimolFenazaquinFenbuconazoleFenhexamidFenitrothionFenpropathrin

FenvalerateFludioxonilFolpetFonofosFuralaxylHCH-gammaHeptenophosIprodioneIsofenphosKresoxim-methylMalathionMecarbamMetalaxylMethidathionMyclobutanilOfuraceOxadixylPaclobutrazolParathion-ethyl

Parathion-methylPendimethalinPentachloroanilinePermethrinPhenthoatePhosalonePhosmetPhosphamidonPirimicarbPirimiphos-ethylPirimiphos-methylProcymidoneProfenofosPropargitePropiconazolePropyzamideProthiofosPyrazophosPyridaphenthion

PyrifenoxPyrimethanilQuinalphosQuintozeneSimazineTebuconazoleTebufenpyradTecnazeneTefluthrinTetrachlorvinphosTetradifonTolclofos-methylTolylfluanidTriazophosTrifloxystrobinTrifluralinVinclozolin

Sample PreparationSample Preparation

Extraction of the baby food, lettuce and pear samples

—Weigh 10g homogenized sample into a centrifuge tube

—Add 10ml acetonitrile with 1% acetic acid, 4g anhydrous MgSO4

and 1.66g sodium acetate and shake immediately

—Centrifuge at 4300g for 5min

—Transfer 1ml aliquot of acetonitrile layer to a vial and then blow down gently with nitrogen to add 0.2ml toluene

—Transfer 1ml extract to a micro-centrifuge vial

—Add 150mg anhydrous MgSO4 + 50mg PSA + 50mg carbon sorbent and vortex mix for 30s

—Centrifuge at 5000g for 1min

—Transfer into a GC vial and submit for GC-TOF-MS analysis

% Recovery% Recovery

Biphenyl 119 (7)

Heptenophos 114 (6)

Tecnazene 90 (4)

Diphenylamine 104 (5)

Ethoprophos 119 (9)

Trifluralin 112 (5)

Dichloran 89 (5)

Simazine 102 (4)

Gamma-HCH 106 (9)

Quintozene 64 (5)

Propyzamide 106 (2)

Fonofos 115 (8)

Pyrimethanil 59 (5)

Diazinon 114 (12)

Phosphamidon-e 93 (12)

Tefluthrin 115 (9)

Chlorothalonil 51 (9)

Pirimicarb 88 (2)

Recovery at 0.01mg/kg from fruit-based baby food (n = 5)

Mean % Recovery and % RSD (in parenthesis)

Target is 70 – 110% with < 30% RSD

Pentachloroaniline 40 (9)

Phosphamidon-z 100 (4)

Vinclozolin 107 (4)

Chlorpyrifos-methyl 97 (8)

Parathion-methyl 103 (5)

Carbaryl 106 (5)

Tolclofos-methyl 107 (8)

Metalaxyl 108 (3)

Fenitrothion 98 (5)

% Recovery% Recovery

Pirimiphos-methyl 102 (5)

Dichlofluanid 111 (7)

Malathion 105 (4)

Chlorpyrifos 85 (5)

Parathion-ethyl 98 (3)

Dicofol BD 105 (9)

Pirimiphos-ethyl 97 (6)

Cyprodinil 52 (2)

Chlorfenvinphos-e 82 (6)

Pendimethalin 85 (4)

Pyrifenox (I) 101 (3)

Chlozolinate 105 (4)

Tolylfluanid 106 (6)

Chlorfenvinphos-z 114 (2)

Isofenphos 104 (4)

Captan 98 (8)

Mecarbam 104 (6)

Quinalophos 89 (4)

Phenthoate 98 (3)

Furalaxyl 106 (1)

Folpet 70 (13)

Procymidone 104 (3)

Methidathion 99 (3)

Pyrifenox (II) 99 (9)

Paclobutrazol 103 (9)

Tetrachlorvinphos 97 (6)

Endosulfan (I) 105 (7)

Prothiofos 81 (7)

Fludioxonil 95 (3)

Profenofos 90 (4)

p,p'-DDE 102 (4)

Myclobutanil 101 (5)

Kresoxim-methyl 101 (5)

Buprofezin 100 (9)

Bupirimate 98 (4)

Endosulfan (II) 99 (5)

o,p-DDT 104 (3)

Oxadixyl 98 (5)

p,p-DDD 102 (7)

Ethion 97 (4)

Triazophos 94 (10)

Ofurace 100 (4)

% Recovery% Recovery

Ofurace 100 (4)

Propiconazole (I) 94 (5)

Endosulfan-sulfate 103 (5)

Trifloxystrobin 100 (6)

Fenhexamid 94 (4)

p,p-DDT 97 (3)

Propiconazole (II) 78 (15)

Tebuconazole 92 (7)

Propargite 100 (9)

Iprodione 99 (7)

Pyridaphenthion 94 (4)

Phosmet 84 (3)

Bromopropylate 99 (4)

Bifenthrin 95 (3)

Fenpropathrin 102 (6)

Tebufenpyrad 91 (3)

Fenazaquin 46 (10)

Tetradifon 89 (6)

Phosalone 73 (4)

Cyhalothrin-λ 99 (4)

Fenarimol 99 (6)

Dicofol 99 (2)

Pyrazophos 51 (9)

Permethrin cis 88 (6)

Permethrin trans 95 (11)

Fenbuconazole 78 (7)

Cyfluthrin (I) 89 (29)

Cyfluthrin (II) 119 (12)

Cyfluthrin (III-IV) 165 (8)

Cypermethrin (I) 130 (11)

Cypermethrin (II) 103 (22)

Cypermethrin (III-IV)

114 (11)

Fenvalerate (I) 99 (6)

Fenvalerate (II) 90 (17)

Difenoconazole (I) 87 (10)

Difenoconazole (II) 87 (6)

Deltamethrin 90 (7)

Azoxystrobin 103 (5)

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% Recovery% Recovery

Agilent 6890N GC

CTC Analytics CombiPal Autosampler

Cryo cooled PTV in solvent vent mode—5μl acetonitrile injected at 50°C

—Multiple baffle liner used to give a large surface area

—Solvent is vented using helium at a pressure of 5kPa and flow of 20ml/min

—Vent closed after 0.5min

—Injector ballistically heated to 300°C

—Remaining material in the liner transferred to column

GC ConditionsGC Conditions

Column—Restek, Rxi-5ms, 30m x 0.25mm i.d., 0.25μm

Constant flow of 1.0 mL/min helium

Oven temperature program

Ramp rate, °C/min Hold, min

50°C -

150°C @ 20°C/min -

280°C @ 6°C/min 7

Run time =34min

GC ConditionsGC Conditions

Bip

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Azo

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Total Ion Chromatogram (TIC)Total Ion Chromatogram (TIC)

Targeted ScreeningTargeted ScreeningTOFTOF

Compound list is known—Exact mass chromatograms, 0.02 or 0.05 Da window

—Full spectrum technique

oAny ion can be chosen for quantification

—Number of residues/ions can be increased without loss in sensitivity

—Screening is based on one or more exact mass chromatograms

—TargetLynx

oHighlight samples that…

… have concentrations greater than the reporting level

… do not pass quality control criteria

Selectivity of Exact Mass Selectivity of Exact Mass ChromatogramsChromatograms

Chlorothalonil, 0.01mg/kg in baby food, m/z = 265.8786

1.0 Da

0.02 Da

Selectivity of Exact Mass Selectivity of Exact Mass ChromatogramsChromatograms

Methidathion, 0.01mg/kg in baby food, m/z = 145.0072

1.0 Da

0.02 Da

Selectivity of Exact Mass Selectivity of Exact Mass ChromatogramsChromatograms

Endosulfan sulfate, 0.01 mg/kg, m/z = 248.0397

Baby food

Lettuce

Pear

1.0 Da 0.02 Da

SensitivitySensitivityIncreasing the Number of IonsIncreasing the Number of Ions

Propyzamide, 0.01 mg/kg in Pear

172.956

172.956

255.022

174.953

Propyzamide SpectrumPropyzamide Spectrum

TargetLynx BrowserTargetLynx Browser

Application to Real SamplesApplication to Real Samples

• Real samples analyzed—Pear, 11 samples

—Lettuce, 12 samples

• Matrix-matched standards used for calibration

• TargetLynx used for automatic quantification with a full range of quality control checks

• Two or three exact mass chromatograms per residue were extracted

For illustration purposes—Reporting Level = 0.005mg/kg

—Maximum Residue Limit = 0.010mg/kg

LettuceLettuceFenitrothionFenitrothion –– 0.023mg/kg0.023mg/kg

PearsPearsChlorpyrifos Chlorpyrifos –– 0.023mg/kg0.023mg/kg

Untargeted ScreeningUntargeted ScreeningTOFTOF

Compound list is unknown—Full spectrum technique with no spectral skew

—Fast acquisition speed enables deconvolution, 5 - 10 spectra/s

—Exact mass facilitates identification, < 5 ppm RMS

—High resolution allowing enhanced selectivity, > 7000 FWHM

—ChromaLynx

oAutomatic peak detection and deconvolution

oAutomatic production of “clean” component spectra

oAutomatic identification via library searching

oExact mass scoring

Deconvolution of LettuceDeconvolution of Lettuce

5 Components?

ChromaLynx found 8 Components!

Deconvolution of Lettuce with Deconvolution of Lettuce with ChromaLynxChromaLynx

Extracted Mass ChromatogramsExtracted Mass Chromatograms3 Components in Lettuce3 Components in Lettuce

Chlorfenvinphos-z

Mecarbam

Isofenphos

PearPearChromaLynxChromaLynx

ChromaLynx found ~ 500 Components

Untargeted ScreeningUntargeted ScreeningExact Mass ScoringExact Mass Scoring

Library searching can be scored by exact

mass in ChromaLynx

Untargeted ScreeningUntargeted ScreeningThiabendazoleThiabendazole in Pearin Pear

Untargeted ScreeningUntargeted ScreeningDCPA in LettuceDCPA in Lettuce

Untargeted ScreeningUntargeted ScreeningDCPA in LettuceDCPA in Lettuce

Targeted ConfirmationTargeted ConfirmationDCPA in LettuceDCPA in Lettuce

Lettuce sample was re-extracted and quantified using the established single quadrupole method

Validation recoveries (n=5) 95-103% at 0.01mg/kg

Confirmation based on 3 ions. literature ion ratios in parenthesis—301 (100%)

—299 (80%)

—332 (30%)

Concentration of DCPA = 0.07mg/kg (measured ratios)—301 (100%)

—299 (78%)

—332 (28%)

SummarySummary

An exact mass GC-TOF-MS method has been developed for the screening of approximately 100 pesticide residues to concentration levels of 0.01 mg/kg or less in pear, lettuce and fruit-based baby food

Good linearity and recoveries for the majority of pesticides were obtained

A targeted approach using TargetLynx and an untargeted approach using ChromaLynx were used for screening

Joint approach led to the successful identification of incurred target and non-target residues in pear and lettuce

The targeted approach correlated well with previous results obtained from established single quadrupole method

AcknowledgementsAcknowledgements

Waters Corporation, Manchester, UK—Peter Hancock

Central Science Laboratory, York, UK—Richard J. Fussell

—Cristiana Leandro

University of York, York, UK—Cristiana Leandro

—Brendan J. Keely