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

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

[email protected]

“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)

mailto:[email protected]

22

© Azti Copyright. Prohibida la reproducción total o parcial de este documento


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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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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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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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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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


6. Second antibody, anti-IgG 100 µl/well (1/2000) PBST RT 60 min.


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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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



Thank you for your attention!!Muchas gracias por su atención!

Begoña Alfaro

[email protected]

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.

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

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