determination of priority pesticide residues in baby food

Determination of Priority Pesticide Residues in Baby Food

Dave Heywood

41st WCTOW

©2006 Waters Corporation

Overview

European Union legislation concerning priority pesticide residues in baby food

Quantitative and confirmatory requirements

Determination of pesticide residues in baby food– Extraction using QuEChERS method– LC/MS/MS application to real samples

HPLC with Quattro PremierAcquity UPLC with Quattro Premier

– GC/MS/MS application to real samplesQuattro micro GC


EU Maximum Residue Limits

Current Situation– Maximum Residue Levels (MRLs) do not always exist and

are not harmonised– Range from not detectable to 25mg/kg– Not detectable defaults to 0.01-0.2mg/kg

EU Proposal 2003/0052 (COD) 14th March 2003– Harmonized MRLs for all pesticide/product combinations– Range from “not detectable” to 25mg/kg– Not detectable defaults to 0.01mg/kg

General target LOD = 0.01mg/kg– Applies to raw commodities of plant and animal origin– Some exceptions, e.g. Baby Food, Medicinal Plants


EU MRLs for Baby FoodPesticide Residue MRL - mg/kg

Cadusafos 0.006 (0.006)

Demeton-s-methyl 0.002 (0.006)

Dieldrin 0.0015 (0.003)

Endrin 0.003 (0.003)

Ethoprophos 0.008 (0.008)

Aldrin 0.0015

Demeton-s-methylsulfone 0.002

Oxydemeton-s-methyl 0.002

Dimethoate 0.01

Disulfoton 0.001 (0.003)

Disulfoton sulfone 0.001

Disulfoton sulfoxide 0.001

Fipronil 0.002 (0.004)

Pesticide Residue MRL - mg/kg

Fipronil-desulfinyl 0.002

HCB 0.003 (0.003)

Heptachlor 0.0015 (0.003)

Heptachlor epoxide 0.0015

Nitrofen 0.003 (0.003)

Fensulfothion 0.00075 (0.003)

Fensulfothion-oxon 0.00075

Fensulfothion-oxon-sulfone

0.00075

Fensulfothion sulfone 0.00075

Omethoate 0.003 (0.003)

Terbufos 0.001 (0.003)

Terbufos sulfone 0.001

Terbufos sulfoxide 0.001


Quantitative Trace AnalysisRequirements

High selectivity – Reduce or eliminate matrix interferences

Reduce sample cleanup

High sensitivity– Low reporting limits for individual components of the MRLs– Quantitative accuracy– Reproducibility, stability, and dynamic range– Accurate quantification of targets at low levels in matrix

Ruggedness– Complex sample matrices, reduced or no sample clean-up

Sample throughput– Multiple targets in one run


Confirmatory Analysis

Tandem quadrupole MS

Multiple Reaction Monitoring (MRM) transitions– Precursor to product ion transition– Argon collision induced reaction that is structure specific– Two transitions were chosen for each pesticide residue

Confirmation criteria– Dependent on relative abundance of the two transitions

>0.5 ± 20% >0.2 < 0.5 ± 25%>0.1 < 0.2 ± 30% < 0.1 ± 50%

– Ratio used to confirm/reject analytes in the extracts


Possible System Solutions

Quattro Premier LC/MS/MS

Alliance 2695

Quattro Premier LC/MS/MS

Acquity UPLC

Quattro micro GC GC/MS/MS


Sample Preparation

Extraction of the baby food samples– Weigh 10g homogenised sample into a centrifuge tube– Add 10ml acetonitrile and shake vigorously for 1min– Add 4g MgSO4 and 1g NaCl and vortex immediately– Centrifuge at 4300g for 5min– Add 150mg anhydrous MgSO4 + 50mg PSA to a micro-centrifuge

vial– Transfer 1ml aliquot of acetonitrile (upper) layer to the vial and

shake for 30s– Centrifuge at 5000g for 1min– Transfer 100μl into a LC vial and add 900μl water– Submit for LC/MS/MS analysis– “QuEChERS” method: Quick, Easy, Cheap, Effective, Rugged & Safe


Sunfire C18 Column, 2.1 x 100mm, 3.5μm at 40°C– Sunfire C18 Guard Column, 2.1 x 10mm, 3.5μm

Flow rate = 0.3ml/min

Mobile phase– Solvent A, 90% Water, 10% MeOH + 20mM CH3CO2NH4

– Solvent B, 10% Water, 90% MeOH + 20mM CH3CO2NH4

Injection volume = 50μl

0min 100% A13min 100% B17min 100% B

HPLC Conditions

Total run time25min


MSDemeton-s-methyl sulfone


MS/MSDemeton-s-methyl sulfone

Quantification Transition

Confirmation Transition


MS Method

MRM functions arranged into 6 time windows– More flexible use of dwell times– Improved signal to noise (S/N) ratios for some analytes– Overlapping allows for small changes in retention time


Total Ion Chromatogram (TIC)

0.01mg/kgPotato-based Baby Food


0.00000 0.00025 0.00050 0.00075 0.00100 0.00125

mg/kg

Omethoate

Oxydemeton-methyl

Demeton-s-methyl sulfone

Dimethoate

Fensulfothion-oxon

Fensulfothion-oxon-sulfone

Demeton-s-methyl

Disulfoton sulfoxide

Disulfoton sulfone

Fensulfothion

Fensulfothion sulfone

Terbufos sulfone

Terbufos sulfoxide

Ethoprophos

Cadusafos

Disulfoton

Terbufos

Cereal Confirmation LODCereal Screening LODPotato Confirmation LODPotato Screening LODFruit Confirmation LODFruit Screening LOD

Instrument Sensitivity

EU MRLs > 0.00075mg/kg


Enhancing the Method

Confirmatory method for all residues?– Disulfoton and terbufos can be screened– Confirmation was not possible in some matrices

Increasing throughput?– HPLC method has a cycle time of 25 minutes


UPLC versus HPLCThroughput

HPLC0.01mg/kg

Cereal Baby Food

UPLC0.01mg/kg

Cereal Baby Food


UPLC versus HPLCSensitivity

HPLCTerbufos

UPLCTerbufos

Determination of Priority Pesticide Residues in Baby Food using GC/MS/MS


Sample Preparation

Extraction of the baby food samples– Weigh 10g homogenised sample into a centrifuge tube– Add 10ml acetonitrile and shake vigorously for 1min– Add 4g MgSO4 and 1g NaCl and vortex immediately– Add 100μl of 1μg/ml δ-HCH and shake for 30s– Centrifuge at 4300g for 5min– Add 150mg anhydrous MgSO4 + 50mg PSA + 200mg

C18 to a micro-centrifuge vial– Transfer 1ml aliquot of acetonitrile (upper) layer to the

vial and shake for 30s– Centrifuge at 5000g for 1min– Submit for GC/MS/MS analysis


% RecoveryFruit and Potato

Recovery at 0.001mg/kg from baby food

Mean % Recovery and % RSD (in parenthesis)Pesticide Residue Fruit (n = 7) Potato (n = 7)

Aldrin 84 (10)

86 (12)

93 (6)

93 (8)

100 (8)

82 (11)

Fipronil 97 (10) 105 (4)

Nitrofen 102 (5) 97 (6)

Fipronil de-sulfinyl 99 (9) 102 (6)

Heptachlor 102 (5) 87 (8)

Heptachlor epoxide 102 (6) 93 (7)

Hexachlorobenzene 77 (7) 71 (4)

83 (19)

77 (7)

Cadusafos 91 (2)

Dieldrin 84 (7)

Dimethoate 91 (6)

Endrin 89 (8)

Ethoprophos 86 (10)

Omethoate 78 (13)


GC Conditions

Agilent 6890

Agilent 7683 Autosampler

Cryo cooled PTV in solvent vent mode, 5μl injected– Injection into a cold injection liner at 50°C – Multiple baffle liner used to give a large surface area– The solvent is vented using helium at a vent pressure of

5kPa and a vent flow of 20ml/min– Vent closed after 0.5min and the injector is ballistically

heated to 300°C– Any remaining material in the liner is transferred to the

head of the column


Column– Varian, FactorFour VF-5ms, 30m x 0.25mm i.d., 0.25μm

Constant flow rate of 1.0ml/min helium

Oven temperature program

0min 50°C1.5min 50°C9min 200°C @ 20°C/min11min 200°C15min 280°C @ 20°C/min

GC Conditions

Total run time20min


MRM ConditionsPesticide Retention Time Quantification Transition Confirmation Transition

Omethoate 9.97min 156 > 110 (10 eV) 156 > 79 (20 eV)

Ethoprophos 10.27min 158 > 114 (5 eV) 200 > 158 (5 eV)

Cadusafos 10.72min 159 > 131 (8 eV) 158 > 114 (5 eV)

Hexachlorobenzene 11.13min 284 > 249 (15 eV) 286 > 251 (15 eV)

Dimethoate 11.22min 125 > 79 (8 eV) 229 > 87 (5 eV)

Fipronil de-sulfinyl 12.46min 388 > 333 (20 eV) 333 > 281 (10 eV)

δ-HCH, Internal Standard 12.14min 219 > 183 (5 eV) 183 > 145 (15eV)

Heptachlor 12.86min 272 > 237 (13 eV) 274 > 239 (15 eV)

Aldrin 13.40min 263 > 193 (25 eV) 263 > 191 (25 eV)

Fipronil 13.69min 367 > 213 (22 eV) 369 > 215 (25 eV)

Heptachlor epoxide 13.99min 183 > 155 (10 eV) 217 > 182 (15 eV)

Dieldrin 14.77min 263 > 193 (25 eV) 263 > 191 (25 eV)

Nitrofen 14.94min 283 > 253 (10 eV) 283 > 162 (20 eV)

Endrin 15.03min 263 > 193 (25 eV) 263 > 191 (25 eV)


MS Method

MRM functions arranged into 6 time windows– More flexible use of dwell times– Improved signal to noise (S/N) ratios for some analytes– Overlapping allows for small changes in retention time


Total Ion Chromatogram (TIC)

0.01mg/kgFruit-based Baby Food


HCB in Fruit

r2 = 0.9975

Ethoprophos in Potato

r2 = 0.9994

Calibration curves were constructed using matrix matched calibration standards

Concentration range was 0.0005 to 0.0100mg/kg equivalents

Good correlation coefficients obtained in both matrices for curves bracketing the samples

Linearity


0.00000 0.00025 0.00050 0.00075 0.00100 0.00125 0.00150

mg/kg

Omethoate

Ethoprophos

Cadusafos

HCB

Dimethoate

Fipronil de-sulfinyl

Heptachlor

Aldrin

Fipronil

Heptachlor epoxide

Dieldrin

Nitrofen

Endrin

Potato Confirmation LODPotato Screening LODFruit Confirmation LODFruit Screening LOD

EU MRLs > 0.0015mg/kg

Instrument Sensitivity


Sensitivity and ConfirmationEndrin in Fruit

263 → 1910.001mg/kg

Blank

Peak Area Ratio 0.674

Expected Ion Ratio0.506 – 0.758

263 → 193


Summary

• Using a combination of LC/MS/MS and GC/MS/MS, methods have been described for the determination and confirmation of 13 priority pesticide residues with 26 components in different baby foods

• The QuEChERS extraction method yielded very good recoveries and precision at the low concentration levels required by legislation

• Sensitivity of Quattro Premier and Quattro micro GC allow the methods to meet the challenge set by the EU Directive with both quantitative and confirmatory data achieved in a single injection


Acknowledgements

Anthony Newton

George Kearney

Martin Green

Amir Farooq

Andrew Baker

Doug Stevens

Peter Hancock

CVUA, Stuttgart, Germany– Carmen Wauschkuhn– Diane Fuegel– Michelangelo Anastassiades

The University of York, York, U.K.

determination of priority pesticide residues in baby food

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