Periodic Reporting for period 1 - MET-PEST (Multi-approach determination of metal based pesticides in food)
Reporting period: 2017-10-01 to 2019-09-30
The industrial and agricultural activities of the modern society lead to significant release of a large number of toxic substances into the environment having an ultimate impact on the food quality and human health. Many of such chemicals are used in agriculture as pesticides, fungicides, etc. to enhance the productivity. This is the case of dithiocarbamates (DTC) that are one of the relatively large used fungicides being effective against a broad spectrum of fungi and plant diseases.
Despite the significant environmental and food chain impact of DTC, the current analytical approaches for their determination suffer from serious drawbacks. The European reference method for this purpose relies on non-selective quantification by indirect determination of the sum of DTC species hence this approach provides incomplete information on the exposure to individual DTC.
The present proposal aims at improving knowledge on the DTCs determination as well as at the development and validation of a method for determination of DTCs and of their degradation products in food by a multi-approach strategy. A combination of analytical methodologies such as those currently employed for trace metals speciation and also for organic contaminants will be developed and fully validated. These methodologies will be critically compared through the analysis of real-life food samples from EU and imported from external markets. By combining powerful separation techniques such as high performance liquid chromatography with elemental and molecular spectrometry, this project will allow obtaining a deeper knowledge on the presence of individual DTCs in various foodstuff and of their degradation mechanisms during food processing. Additionally, it will consistently contribute to the development of a reference method for selective DTC determination in food hence having a great impact on the scientific research in food quality control at European level.
In this context, the overall research objectives of this research proposal can be underlined as follows:
1. Development and validation of new analytical methods for quantitative determination of DTCs in food samples by HPLC hyphenated to either molecular or elemental mass spectrometry (MS) techniques.
2. Application of multi-approach methods to DTCs measurement in real-life food samples
3. Assessment of DTC transformation in different food matrices during various cooking modes
Despite the significant environmental and food chain impact of DTC, the current analytical approaches for their determination suffer from serious drawbacks. The European reference method for this purpose relies on non-selective quantification by indirect determination of the sum of DTC species hence this approach provides incomplete information on the exposure to individual DTC.
The present proposal aims at improving knowledge on the DTCs determination as well as at the development and validation of a method for determination of DTCs and of their degradation products in food by a multi-approach strategy. A combination of analytical methodologies such as those currently employed for trace metals speciation and also for organic contaminants will be developed and fully validated. These methodologies will be critically compared through the analysis of real-life food samples from EU and imported from external markets. By combining powerful separation techniques such as high performance liquid chromatography with elemental and molecular spectrometry, this project will allow obtaining a deeper knowledge on the presence of individual DTCs in various foodstuff and of their degradation mechanisms during food processing. Additionally, it will consistently contribute to the development of a reference method for selective DTC determination in food hence having a great impact on the scientific research in food quality control at European level.
In this context, the overall research objectives of this research proposal can be underlined as follows:
1. Development and validation of new analytical methods for quantitative determination of DTCs in food samples by HPLC hyphenated to either molecular or elemental mass spectrometry (MS) techniques.
2. Application of multi-approach methods to DTCs measurement in real-life food samples
3. Assessment of DTC transformation in different food matrices during various cooking modes
A multi-approach strategy was applied throughout this research project in order to increase knowledge on the DTCs determination from various fruits and vegetable matrices from the following perspectives:
(i) determination of DTCs per class, depending on their chemical structures, as well as of their degradation products, like ethylene- and propylene-thiourea.
The methods are based on hydrophilic interaction liquid chromatography (HILIC) or reverse phase (RP) HPLC hyphenated to either molecular or elemental mass spectrometry (MS) techniques through the detection of organic DTCs, metals and/or sulfur moieties.
(ii) selective and simultaneous determination of the three individually EU regulated DTCs (thiram, ziram and propineb) as well as of the DTCs degradation products by the use of HILIC-MS/MS.
The proposed analytical methodologies were in-house validated for the determination of the target analytes from several fruit and vegetable matrices over a wide concentration range.
The results of the present study show that the analysis of DTCs by class as well as of their degradation products from fruits and vegetables can be achieved by a multi-approach methodology especially when employing soft surface extraction techniques and further separate and detect by the use of HILIC or RP-HPLC coupled to electrospray-MS/MS or to ICP-QQQ-MS.
(i) determination of DTCs per class, depending on their chemical structures, as well as of their degradation products, like ethylene- and propylene-thiourea.
The methods are based on hydrophilic interaction liquid chromatography (HILIC) or reverse phase (RP) HPLC hyphenated to either molecular or elemental mass spectrometry (MS) techniques through the detection of organic DTCs, metals and/or sulfur moieties.
(ii) selective and simultaneous determination of the three individually EU regulated DTCs (thiram, ziram and propineb) as well as of the DTCs degradation products by the use of HILIC-MS/MS.
The proposed analytical methodologies were in-house validated for the determination of the target analytes from several fruit and vegetable matrices over a wide concentration range.
The results of the present study show that the analysis of DTCs by class as well as of their degradation products from fruits and vegetables can be achieved by a multi-approach methodology especially when employing soft surface extraction techniques and further separate and detect by the use of HILIC or RP-HPLC coupled to electrospray-MS/MS or to ICP-QQQ-MS.
The chemical structure for most of the targeted dithiocarbamate (DTCs) molecules possess some particularities which allow for multiple approaches to be tested/implemented in order to fulfill the most important research objective of this project: increasing the method selectivity for the DTCs measurements when compared to the official reference method currently applied worldwide for their determination from fruit and vegetable matrices.
Therefore, the four key research directions were tested through the ongoing of the present research project, while the obtained results aloud for the new analytical methodologies to be developed and in-house validated, as following:
• the use of molecular mass sprectrometry (MS) for the determination of DTC molecules per class;
• the use of molecular mass sprectrometry (MS) for the simultaneous determination of the three individually regulated DTCs, thiram, ziram, propineb;
• determination of the DTC degradation products (ethylene thiourea (ETU) and propylene thiourea (PTU)) from fruit and vegetable samples by targeting the sulphur content by the use of elemental MS.
One of the most important achievements of the present research project is that the method selectivity, for the determination of the dithiocarbamate fungicides from food samples currently based on the Single Residue Method for the analysis of dithiocarbamates via their common degradation product carbon disulphide (CS2), will be significantly increased. Research results obtained through the following of this project evidenced that the DTCs determination from fruit and vegetable matrices might be performed in various modes, depending the scope of the analysis:
• the DTCs determination by their class;
• simultaneously multi-residue determination of the three EU regulated DTCs;
• the inclusion in the analysis of the DTCs degradation products.
These results will have a significant impact on the future development and evolution in the field of food safety measures and control when addressing the DTCs regulation.
By bringing in front of the expert scientists in the particular fields of both analytical chemistry of DTCs and regulatory responsible organizations of the developed knowledge through this project ongoing will allow for setting up the inter-laboratories exchanges and potential organizing of the tests between specialized laboratories in this field, but also will setup new reference methodologies in the field of the food safety regarding the food quality and control of the DTC fungicide residues.
Therefore, the four key research directions were tested through the ongoing of the present research project, while the obtained results aloud for the new analytical methodologies to be developed and in-house validated, as following:
• the use of molecular mass sprectrometry (MS) for the determination of DTC molecules per class;
• the use of molecular mass sprectrometry (MS) for the simultaneous determination of the three individually regulated DTCs, thiram, ziram, propineb;
• determination of the DTC degradation products (ethylene thiourea (ETU) and propylene thiourea (PTU)) from fruit and vegetable samples by targeting the sulphur content by the use of elemental MS.
One of the most important achievements of the present research project is that the method selectivity, for the determination of the dithiocarbamate fungicides from food samples currently based on the Single Residue Method for the analysis of dithiocarbamates via their common degradation product carbon disulphide (CS2), will be significantly increased. Research results obtained through the following of this project evidenced that the DTCs determination from fruit and vegetable matrices might be performed in various modes, depending the scope of the analysis:
• the DTCs determination by their class;
• simultaneously multi-residue determination of the three EU regulated DTCs;
• the inclusion in the analysis of the DTCs degradation products.
These results will have a significant impact on the future development and evolution in the field of food safety measures and control when addressing the DTCs regulation.
By bringing in front of the expert scientists in the particular fields of both analytical chemistry of DTCs and regulatory responsible organizations of the developed knowledge through this project ongoing will allow for setting up the inter-laboratories exchanges and potential organizing of the tests between specialized laboratories in this field, but also will setup new reference methodologies in the field of the food safety regarding the food quality and control of the DTC fungicide residues.