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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 “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' ( )

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' ( ) 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' ( ) 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' ( ) 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' ( ) 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' ( ) 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' ( ) 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' ( ) 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' ( ) 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' ( ) 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' ( ) 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' ( ) 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' ( ) 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' ( ) 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' ( ) 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' ( ) 16 Mean recovery= 64.5% Conc.(mg/kg)Recovery (%)% RSD Linear Range (mg/kg)LOD (mg/kg)EU MRL (mg/kg) 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' ( ) 17 RESULTS: Determination of Chlorpyrifos. Validation Slope1.002±0.011 r2r 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±

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


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