determination of priority pesticide residues in baby food
TRANSCRIPT
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
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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
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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
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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
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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
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Possible System Solutions
Quattro Premier LC/MS/MS
Alliance 2695
Quattro Premier LC/MS/MS
Acquity UPLC
Quattro micro GC GC/MS/MS
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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
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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
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MSDemeton-s-methyl sulfone
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MS/MSDemeton-s-methyl sulfone
Quantification Transition
Confirmation Transition
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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
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Total Ion Chromatogram (TIC)
0.01mg/kgPotato-based Baby Food
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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
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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
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UPLC versus HPLCThroughput
HPLC0.01mg/kg
Cereal Baby Food
UPLC0.01mg/kg
Cereal Baby Food
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UPLC versus HPLCSensitivity
HPLCTerbufos
UPLCTerbufos
Determination of Priority Pesticide Residues in Baby Food using GC/MS/MS
©2006 Waters Corporation
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
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% 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)
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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
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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
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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)
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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
©2006 Waters Corporation
Total Ion Chromatogram (TIC)
0.01mg/kgFruit-based Baby Food
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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
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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
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Sensitivity and ConfirmationEndrin in Fruit
263 → 1910.001mg/kg
Blank
Peak Area Ratio 0.674
Expected Ion Ratio0.506 – 0.758
263 → 193
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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
©2006 Waters Corporation
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.