map-milling national conference, spain salón de actos ita zaragoza, 30 de mayo de 2007 begoña...
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Map-Milling National Conference, Spain
Salón de Actos ITA
Zaragoza, 30 de mayo de 2007
Begoña Alfaro , Alex Barranco, Maria Arestin, Nerea Argarate
AZTI-Tecnalia, Food Research Division Spain
“DEVELOPMENT OF RAPID METHODS FOR DETECCION OF PESTICIDES IN CEREAL PRODUCTS”
Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006)
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Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006)
Map Milling: Objectives of the project Introduction: Pesticides Problem Workplan of Map-Milling (36 months) Development of pesticides measurement systems:
Objectives Target Analytes Methodology: Immunoassay and sampling protocols Results: Analytical characteristics of immunoassays Results: Chlorphyriphos-methly Main conclusions
Acknowledgements
OVERVIEW
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Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006)
To increase the food safety in the grain processing/milling industry according to new regulation and market demands, requiring a high quality products and demonstrated reliability.
GLOBAL OBJECTIVE
OPERATIVE OBJECTIVE
To design and develop new reliable, fast and economic analytical tools able to detect pesticides in the grain milling/processing industry
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Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006)
EU LEGISLATION : COUNCIL DIRECTIVE 86/362/CEE . Fixing the maximum levels for pesticide residues in and on cereals and certain products of plan origin including fruit and vegetables respectively.
Official Journal of the European Communities L221(1986)
Solution:
Rapid/Low cost methods for detection of key pesticides to increase the food safety in the grain processing/milling industry.
Potential risks:
The risk and impact of pesticides residues on the agricultural environment
The risk and impact of pesticides on human health
PROBLEM SOLVING
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Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006)
WP1. Specific analysis of grain processing industry needs
36302418126
WP2. Initial analysis: immunochemical & molecular (PCR) techniques
WP3. Design and development of improved measurement systems:- Mycotoxins- Pesticides- Acrylamide
WP4. Development of a risk management system for grain milling SMEs
WP6. Exploitation of project results:- Patenting- Licensing- Transferring results
WP7. Dissemination:- Awareness- SMEs Technological advice
WP8. Training
WP9. Project Management
“MAP-MILLING” PROJECT
WP5. Integration and validation of
pollutants control in the process at industrial scale
WORKPLAN (36 MONTHS)
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WP3. DESIGN AND DEVELOPMENT OF IMPROVED MEASUREMENT SYSTEMS (IGV AND AZTI)
Objectives:
To develop quick, cheap and reliable techniques for detection of pesticides selected.To assess the performance of the assay with synthetic and real samples (contaminated with pesticides) at laboratory level.
Development of pesticides measurement systems
Immunochemical analytical method using antibodies from commercial and research sources and other immunochemical reagents as appropriate
Validation and test immunochemical methods on real cereals samples
Establish suitable working protocols for rapid determination of pesticides in cereal samples
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Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006)
Target Analytes
N
Cl O
P
OCl
Cl
S
O
CH3
CH3
Cl
ClCl
Cl
Cl
Pesticide EU MRL (mg/kg) Sensitivity (mg/kg)
DDT 0.05a 0.05 Chlorpyrifos-methyl 3b 0.05
2,4-D 0.05c 0.05
Table. Maximum residue limits and sensitivity needed for the target pesticides.
a Directive 86/362/EECb Directive 98/82/ECc Directive 97/02 EC
p-p´-DDT Chlorpyrifos-methyl (2,4-dichlorophenoxy) acetic acid
Cl
Cl
O
OH
O
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ELISA FUNDAMENTALS
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Methodology: Steps in the development of immunoassays to pesticides
1. Antibody production and characterization (Dr. Montoya: Universidad Politécnica de Valencia, Spain; Dr. Fránek:Veterinary Research Institute, Czech Republic)
2. Hapten-protein conjugate synthesis
3. Immunoassay development and optimization( indirect enzyme-linked immunoassay)
4. Sample preparation methods (wheat flour)
5. Immunoassay validation: ELISA methodology versus reference analytical methods (chromatographic methods)
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METHODOLOGY: SAMPLING PROTOCOLS FOR ELISA
Wheat flour Wheat flour spiked with pesticides
Clean-up procedure
Dilution in PBST buffer
No presence of pesticides
Presence of pesticidesSolvents: Methanol, ACN, hexane,
methylene chloride, acetone
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RESULTS: Analytical characteristics of the standard curves of pesticides immunoassays
Table: Immunoassay features of pesticides
Analyte IC50 (nM) Linear Range (µg/L)
LOD (µg/L)
2,4-D 22.62±0.08 1.6-27.3 0.9 DDT 3.68±0.08 0.36-3.50 0.14
Chlorpyrifos-methyl 10.41±3.54 1.02-9.78 0.50
The data presented correspond to the average of 6 calibration curves run in 6 different days. Each curve was build using three-well replicates. The dynamic range is defined by the concentrations corresponding to 20 and 80% of the assay response at zero dose. The limit of detection (LOD) is the analyte concentration corresponding to 90% of
the assay response at zero dose
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METHODOLOGY.Resume of the Chlorpyrifos ELISA Protocol
StepStep Dilution factor, volume/wellDilution factor, volume/well BufferBuffer TempTemp TimeTime
1. Coating (Antigen OVA-TO1)[Antigen] = 0.5 µg/ml
100 µl/wellCoating 4ºC Overnight
2. Washing step 300 µl/well PBST RT 5 times
3. Standards and samples 50 µl/well PBS RT
60 min.
4. Antiserum (Lib-PO Mab)As = 0.2 µg/ml
50 µl/wellPBS RT
5. Washing step 300 µl/well PBST RT 5 times
6. Second antibody, anti-IgG 100 µl/well (1/2000) PBST RT 60 min.
7. Washing step 300 µl/well PBST RT 5 times
8. Substrate 100 µl/wellOPD
solutionRT 20 min.
9. Stop 100 µl/well H2SO4 2,5M RT
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METHODOLOGY: Sample Preparation for Determination of Chlorpyriphos
•An amount of wheat flour (1g) is weighted in a flask and blended with 3 mL of methanol for 2 minutes. •The extract is diluted with water (1:1, v/v)
•A C18 cartridge (500mg) is activated with 3 mL of methanol and conditioned with 3 mL of methanol:water (1:1, v/v)•A 5 mL aliquot of the extract is applied to the cartridges•The cartridge is cleaned with 1 mL of methanol:water (8:2, v/v) and 0.75 mL of methanol:water (8.5:1.5, v/v)•Chlorpyriphos is eluted from the cartridge with 1 mL of methanol:water (9:1, v/v)
Solid phase extraction procedure
C18
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RESULTS:Determination of Chlorpyrifos.Immunoassay features
The data presented correspond to the average of 6 calibration curves run in 6 different days.
Standard Curve CP
10 -3 10 -2 10 -1 100 101 102 103
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
[Chlorpyriphos] (nM)
Ab
s 4
50
Table I. Immunoassay features of Chlorpyrifos.
Amax. Amin. IC50 (nM) Slope Linear range (nM) LOD (nM)
1.18±0.15 0.08±0.08 10.41±3.54 -1.27±0.16 3.18-30.34
1.02-9.78 µg/L 1.57
0.50 µg/L
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RESULTS: Determination of Chlorpyriphos-methyl
Figure: Prototype of ELISA kit for detection of chlorpyriphos-methyl in cereal products
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Mean recovery= 64.5%
Conc.(mg/kg) Recovery (%) % RSD
0.25 72.7 8.9
0.5 62.6 3.3
1 62.8 7.4
1.5 60.5 9.0
Linear Range (mg/kg) LOD (mg/kg) EU MRL (mg/kg)
0.35-2.31 0.19 3
RESULTS: Determination of Chlorpyrifos. Validation
10 -3 10 -2 10 -1 100 101 102 1030.0
0.5
1.0
1.5
Wheat Flour EC50=9.1 nM
STD EC50=10.01nM
[Chlorpyriphos] (nM)
Ab
s 45
0
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RESULTS: Determination of Chlorpyrifos. Validation
0.0 0.2 0.4 0.6 0.8 1.00.0
0.2
0.4
0.6
0.8
1.0
ELISA (mg/kg)
HP
LC
(m
g/k
g) Slope 1.002±0.011
r2 0.9996
Figure: Correlation between ELISA with HPLC with cereals samples. This means that ELISA methodology is performing as HPLC procedure and it can be a good rapid method for chlorpyrifos-methyl evaluation in cereal samples.
Table . Features of HPLC procedure
Slope. r2.
Linear range (µg/L)
LOD (µg/L)
LOQ (µg/L)
1.115±0.010
0.9997
0.5-50 0.15 0.5
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Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006)
Development of ELISA Kits for detection of 2,4-D, chlorophyrifos-methyl and DDT .Evaluation of the optimum combinations and concentrations of Mabs and coating hapten conjugated.
Sample pre-treatment methodologies: Development and optimization of protocols for extraction of pesticides from cereal products. Sample treatment with solid phase extraction are a good approach to solve the problem of the effect of cereals components on the immunoassay.
Suitable working protocols have been established for rapid determination of 2,4-D , chlorophyrifos-methyl and DDT in cereal products by immunoassay. Good recoveries have been obtained with the methodology developed. The final measurement for chlorphyrifos-methyl procedure is able to determine at at concentrations below 3 ppm- mg/kg (MRL set by Directive 98/82/EC).concentrations below 3 ppm- mg/kg (MRL set by Directive 98/82/EC).
MAIN CONCLUSIONS. MAIN CONCLUSIONS. Development of pesticides measurement systems
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AZTI-TECNALIA, SpainAZTI-TECNALIA, SpainFood Safety Group, Food Safety Group, Food Research DivisionFood Research Division
Nerea ArgarateMaria ArestinCarmen Abaroa Alex BarrancoBego Alfaro
2020
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Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006)
Thank you for your attention!!Muchas gracias por su atención!
Begoña Alfaro
AZTI-Tecnalia Unidad de Investigación Alimentaria
Txatxarramendi ugartea z/g
48395 SUKARRIETA (Bizkaia).SPAIN.
AZTI-Tecnalia Unidad de Investigación Alimentaria
Txatxarramendi ugartea z/g
48395 SUKARRIETA (Bizkaia).SPAIN.