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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,

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Presentation on theme: "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,"— Presentation transcript:

1 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 balfaro@suk.azti.es “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)

2 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 2  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

3 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 3 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

4 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 4 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

5 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 5 WORKPLAN (36 MONTHS)

6 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 6 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

7 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 7 Target Analytes Table. Maximum residue limits and sensitivity needed for the target pesticides. a Directive 86/362/EEC b Directive 98/82/EC c Directive 97/02 EC p-p´-DDT Chlorpyrifos-methyl (2,4-dichlorophenoxy) acetic acid

8 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 8 ELISA FUNDAMENTALS

9 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 9 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)

10 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 10 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 pesticides Solvents: Methanol, ACN, hexane, methylene chloride, acetone

11 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 11 RESULTS: Analytical characteristics of the standard curves of pesticides immunoassays 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

12 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 12 METHODOLOGY.Resume of the Chlorpyrifos ELISA Protocol Step Dilution factor, volume/well BufferTempTime 1. Coating (Antigen OVA-TO1) [Antigen] = 0.5 µg/ml 100 µl/well Coating4ºCOvernight 2. Washing step300 µl/wellPBSTRT5 times 3. Standards and samples50 µl/wellPBSRT 60 min. 4. Antiserum (Lib-PO Mab) As = 0.2 µg/ml 50 µl/well PBSRT 5. Washing step300 µl/wellPBSTRT5 times 6. Second antibody, anti-IgG100 µl/well (1/2000)PBSTRT60 min. 7. Washing step300 µl/wellPBSTRT5 times 8. Substrate100 µl/well OPD solution RT20 min. 9. Stop100 µl/wellH 2 SO 4 2,5MRT

13 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 13 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

14 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 14 RESULTS:Determination of Chlorpyrifos.Immunoassay features The data presented correspond to the average of 6 calibration curves run in 6 different days.

15 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 15 RESULTS: Determination of Chlorpyriphos-methyl Figure: Prototype of ELISA kit for detection of chlorpyriphos-methyl in cereal products

16 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 16 Mean recovery= 64.5% Conc.(mg/kg)Recovery (%)% RSD 0.2572.78.9 0.562.63.3 162.87.4 1.560.59.0 Linear Range (mg/kg)LOD (mg/kg)EU MRL (mg/kg) 0.35-2.310.193 RESULTS: Determination of Chlorpyrifos. Validation

17 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 17 RESULTS: Determination of Chlorpyrifos. Validation Slope1.002±0.011 r2r2 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.r2. Linear range (µg/L) LOD (µg/L) LOQ (µg/L) 1.115±0. 010 0.999 7 0.5-500.150.5

18 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 18 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. at concentrations below 3 ppm- mg/kg (MRL set by Directive 98/82/EC). 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 concentrations below 3 ppm- mg/kg (MRL set by Directive 98/82/EC). MAIN CONCLUSIONS. MAIN CONCLUSIONS. Development of pesticides measurement systems

19 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 19 AZTI-TECNALIA, Spain Food Safety Group, Food Research Division Nerea Argarate Maria Arestin Carmen Abaroa Alex Barranco Bego Alfaro

20 © Azti Copyright. Prohibida la reproducción total o parcial de este documento Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) 20 Thank you for your attention! !Muchas gracias por su atención! Begoña Alfaro balfaro@suk.azti.es 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.


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