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Map-Milling National Conference, Spain Salón de Actos ITA Zaragoza, 30 de mayo de 2007

Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006). “DEVELOPMENT OF RAPID METHODS FOR DETECCION OF PESTICIDES IN CEREAL PRODUCTS”. Begoña Alfaro , Alex Barranco, Maria Arestin, Nerea Argarate

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Map-Milling National Conference, Spain Salón de Actos ITA Zaragoza, 30 de mayo de 2007

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  1. Project funded by the European Commission under the research and technological development programme 'Integrating and strengthening the ERA' (2002-2006) “DEVELOPMENT OF RAPID METHODS FOR DETECCION OF PESTICIDES IN CEREAL PRODUCTS” Begoña Alfaro , Alex Barranco, Maria Arestin, Nerea Argarate AZTI-Tecnalia, Food Research Division Spain balfaro@suk.azti.es Map-Milling National Conference, Spain Salón de Actos ITA Zaragoza, 30 de mayo de 2007

  2. OVERVIEW • 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

  3. GLOBAL OBJECTIVE 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. OPERATIVE OBJECTIVE To design and develop new reliable, fast and economic analytical tools able to detect pesticidesin the grain milling/processing industry

  4. PROBLEM SOLVING 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) Potential risks: • The risk and impact of pesticides residues on the agricultural environment • The risk and impact of pesticides on human health • Solution: • Rapid/Low cost methods for detection of key pesticides to increase the food safety in the grain processing/milling industry.

  5. WORKPLAN (36 MONTHS)

  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. 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 (2,4-dichlorophenoxy) acetic acid Chlorpyrifos-methyl

  8. ELISA FUNDAMENTALS

  9. Methodology: Steps in the development of immunoassays to pesticides • Antibody production and characterization (Dr. Montoya: Universidad Politécnica de Valencia, Spain; Dr. Fránek:Veterinary Research Institute, Czech Republic) • Hapten-protein conjugate synthesis • Immunoassay development and optimization( indirect enzyme-linked immunoassay) • Sample preparation methods (wheat flour) • Immunoassay validation: ELISA methodology versus reference analytical methods (chromatographic methods)

  10. Clean-up procedure Dilution in PBST buffer No presence of pesticides Wheat flour Wheat flour spiked with pesticides Solvents: Methanol, ACN, hexane, methylene chloride, acetone Presence of pesticides METHODOLOGY: SAMPLING PROTOCOLS FOR ELISA

  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. METHODOLOGY.Resume of the Chlorpyrifos ELISA Protocol

  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) Solid phase extraction procedure • 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) C18

  14. RESULTS:Determination of Chlorpyrifos.Immunoassay features The data presented correspond to the average of 6 calibration curves run in 6 different days.

  15. RESULTS: Determination of Chlorpyriphos-methyl Figure: Prototype of ELISA kit for detection of chlorpyriphos-methyl in cereal products

  16. RESULTS: Determination of Chlorpyrifos. Validation Mean recovery= 64.5%

  17. RESULTS: Determination of Chlorpyrifos. Validation Table . Features of HPLC procedure 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.

  18. MAIN CONCLUSIONS. Development of pesticides measurement systems • 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-methyland 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).

  19. AZTI-TECNALIA, Spain Food Safety Group, Food Research Division Nerea Argarate Maria Arestin Carmen Abaroa Alex Barranco Bego Alfaro

  20. Thank you for your attention!!Muchas gracias por su atención! AZTI-Tecnalia Unidad de Investigación Alimentaria Txatxarramendi ugartea z/g 48395 SUKARRIETA (Bizkaia).SPAIN. Begoña Alfaro balfaro@suk.azti.es

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