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Dear stakeholder,

The CONffIDENCE project team is proud to present the 9th edition of the CONffIDENCE e-newsletter. In this newsletter you will find recent developments in the CONffIDENCE project and related information in the area of contaminants in food and feed.

If you want to subscribe to CONffIDENCE News, please fill in the registration form on http://www.conffidence.eu/Stakeholders/registration.php

Best regards,

Jacob de Jong, RIKILT - Institute of Food Safety

CONffIDENCE coordinator

CONffIDENCE final stakeholder workshop

CONffIDENCE has just completed the final year of a 56 months research programme. The final stakeholder workshop was held on Tuesday 18th December 2012 in Brussels and was chaired by the Coordinator of the project, Dr. Jacob de Jong.

It was attended by 26 researchers from the CONffIDENCE partners, 2 members of the Advisory Board (from FAO/IAEA and EC-DG SANCO) and 12 stakeholders representative of EC-DG Research, EC-DG SANCO, EFSA, FEFAC, EC JRC-IRMM,and CEN. During this meeting the coordinator and the cluster leaders presented the 56 months activities and achievements in the nine RTD Work packages for organic pollutants, veterinary pharmaceuticals, heavy metals and biotoxins and in the Dissemination Work package.

Over 85 deliverables have been produced during this period. Some of the key outputs are highlighted through the articles of this newsletter.

This project has also been notable for the volume and variety of technology transfer and dissemination activities:

• Website and e-Newsletters
  • ‘CONffIDENCE: contaminants in food and feed: Inexpensive detection for control of exposure’ website was launched on 22nd October 2008. Information and outputs of the project are presented. During the project period, 75000 pages have been visited by 15000 different visitors. (http://www.conffidence.eu )
  • 9 e-Newsletters have been published (2 by year) to inform the stakeholders on scientific progress and upcoming events. (http://www.conffidence.eu/archive/newsletter.php)
  • 120 stakeholders have been registered on-line: 50% from research organisations and 50% from food/feed companies and instrument or kit manufacturers. (http://www.conffidence.eu/Stakeholders/database.php)

• Open Days, workshops and courses
  • 3 Open Days have been organised in 2010 and 2011 in the framework of international food/feed conferences in Netherlands, Brazil and Czech Republic. CONffIDENCE outputs were disseminated to 260 participants through 14 lectures, 55 posters and 12 demonstrations. (http://www.conffidence.eu/Project_output/openday.php)
  • 4 stakeholders workshops have been organised in 2012, in Spain, Netherlands and Belgium, in the framework of 4 international conferences dedicated on one of the 4 topics studied in the project. These workshops aim to present the achievements of the CONffIDENCE partnership for each cluster of workpackages to the stakeholders, allowing them to become familiar with the newly developed methods and their applicability in the food and feed chain. CONffIDENCE outputs were disseminated to 300 participants through 16 lectures, 10 posters and 4 demonstrations. (http://www.conffidence.eu/Project_output/workshops.php)
  • 1 final stakeholder organised in Brussels in December 2012 for invited representatives from DG SANCO, DG RTD, EFSA, industrial food & feed branch organization, ETP-Food for Life, industrial foundations, COST-Actions, CEN-committees and relevant EC-supported projects.
  • An international PhD course on advanced food analysis was organised in 2010 at the VLAG school at Wageningen in the Netherlands. 27 lectures were delivered by CONffIDENCE partners and other experts to 66 PhD students. (http://www.conffidence.eu/Project_output/course.php)
  • BSc education modules organised in 2011 at the CAH Dronten University in the Netherlands were delivered to 75 students in food safety and food chain management. 2 days intensive course comprised theoretical and technical aspects on mycotoxins and plant toxins given by leading specialists in the field from CONffIDENCE project. (http://www.conffidence.eu/Project_output/course2.php)

• Lectures, posters and publications
  • 108 lectures and 110 posters presenting the outputs of the project have been delivered by the CONffIDENCE members to many international conferences over the world. (http://www.conffidence.eu/Project_output/posters.php)
  • Over 30 publications have been published in scientific peer reviewed journals. (http://www.trace.eu.org/library/publications.php)
  • A special issue “CONffIDENCE outputs” Edition of Analytical and Bioanalytical Chemistry journal will be published shortly.

Figure 1a-1: Integrated sample preparation.

Developing detection methods for organic pollutants; POPs, PFCs, Pesticides

WP1a Persistant Organic Pollutants

Achievement 1a-1: Integrated sample preparation.
The integrated sample preparation strategy for the determination of key representatives of polychlorinated biphenyls (PCBs), brominated flame retardants (BFRs) and polycyclic aromatic hydrocarbons (PAHs) in fish was developed, successfully validated and finally the manuscript documenting this work was published by Kalachova et al. in 2011 in Analytica Chimica Acta. To summarize, the sample preparation is based on ethyl acetate extraction of wetted matrix realized by 1 min shaking, transfer of analytes into organic layer was supported by added inorganic salts. Further clean-up is carried out on silica SPE minicolumns and final instrumental analysis is provided using gas chromatography coupled to time of flight mass spectrometry (GC–TOFMS).

Figure 1a-2: GC×GC–TOFMS method.

Achievement 1a-2: GC-MS method.
Following the development of integrated sample preparation strategy for the determination of halogenated persistent organic pollutants (POPs) and PAHs in fish tissue, the two dimensional gas chromatography coupled to time of flight mass spectrometry (GC×GC–TOFMS) was optimized and validated to provide the best analytes separation and, at the same time, low limits of quantification (LOQs) to fulfill the criteria given by legislation. Finally, method for the simultaneous identification and quantification of all 41 analytes including PCBs, BFRs and PAHs was developed and LOQs in the range of 0.01–5 µg/kg were achieved. Paper summarizing this work was published by Kalachova et al. in 2012 in Analytical Bioanalytical Chemistry.

Additionally to GC×GC–TOFMS, an alternative GC–MS method for the determination of a wide range of BFRs including also´emerging´ compounds which were not primarily on the target list of CONffIDENCE project was implemented. For this purpose, gas chromatography coupled to tandem mass spectrometry (GC–MS/MS) with the triple quadrupole ion analyser was optimized. Using this technique, distinctly improved selectivity and sensitivity compared to a routinely used single quadrupole MS can be achieved and accurate determination of even (ultra)trace levels of BFRs, which might be of concern under certain conditions, e.g. within total diet studies, is feasible. Method quantification limits were in the range of 0.005–1 µg kg–1 using GC–MS/MS (EI), which is even lower compared to primarily used GC×GC–TOFMS. The paper summarizing this study was published by Kalachova et al. in 2013 in Talanta.

Figure 1a-3: Imaging platform.

Achievement 1a-3: Screening POPs in fish.
A multiplex immunoassay (IMIA) for selected POPs was developed using superparamagnetic spectrally encoded microspheres (SEMs) and a new imaging platform with a planar readout. The performance of the 3-plex IMIA in buffer and tilapia extracts was critically compared to the previously developed 3-plex FCIA and found to be similar. A preliminary in-house validation with 40 different tilapia fillet samples, blank and spiked with two mixtures of different POPs standards at relevant levels, was performed in both assay platforms. The outcome of the pre-validation study demonstrated the high potential of both 3-plex immunoassays to screen for POPs in tilapia fillet at half of the ML for PCBs and at relevant levels for PAHs and the emerging PBDEs. The 3-plex IMIA has the clear advantage of a relatively low cost and easy transportable system. After further application and validation in a range of different matrices, it can be a useful pre-screening tool for POPs in fish and possibly in other environmental samples.

Figure 1a-4: Fish sampling.

Achievement 1a-4: Monitoring survey.
In a close cooperation with two workpackages dealing with other groups of pollutants – WP1b – PFCs and WP3 – heavy metals, a joint survey focused on a monitoring of these different groups of pollutants in fish samples was realized. Within WP1a, not only halogenated pollutants, but also fat content and profile of polyunsaturated fatty acids (PUFAs) was determined to assess the risk-benefit models of POPs and PUFAs occurring in fish. In order to analyse different European diets through the fish available at the market, the sampling strategy was focused on various fishing areas represented by Mediterranean Sea (Spanish coast), Cantabric Sea, North East of Atlantic Ocean, North Sea and Baltic Sea. Altogether 140 samples were analysed including 18 different species (bivalves, whiting, cod, hake, herring, salmon, trout, tuna from European waters, as well as pangasius fish from Vietnam) both of wild and aquaculture origin.

Three different POP groups were monitored using the previously developed method which enables their simultaneous determination, (i) non-dioxin like PCBs (indicator congeners 28, 52, 101, 138, 153 and 180), non-ortho congeners (No. 77, 81, 126 and 169) and mono-ortho congeners (105, 114, 118, 123, 156, 157, 167 and 189), the latter two groups represent together so called dioxin-like (DL) PCBs; (ii) 16 PAHs (only content of PAH4 – BaA, BaP, BbFA and CHR and BaP is regulated in food) and (iii) PBDEs (congeners No. 28, 47, 99, 100, 154, 153 and 183).

As expected, from the PCBs groups, the highest levels were measured for NDL PCB congeners, mainly for CB-153, CB-138 and CB-180. Their levels were 5-10 times higher compared to mono-ortho congeners and app. 100-times higher than those determined for non-ortho PCBs. From DL-PCB congeners, pentachlorinated CB-105 and CB-118 were detected at highest levels but none of samples exceeded the legislation limits. From the PBDE group, congeners BDE-47, BDE-99 and BDE-100 were the most abundant with levels app. 10-times lower compared to NDL-PCB and comparable to mono-ortho PCBs. As regards PAHs levels in fish, they were also very low, only approaching LOQs, while bivalves were the most contaminated samples by PAHs.

When comparing the contamination of individual fish species, in case of halogenated POPs, the contamination extent might be classified in following order: herring ˜ whiting > salmon ˜ trout > bivalves > hake > pangasius ˜ tuna ˜ cod. Regarding PAHs, as expected, bivalves were much more contaminated than fish since these s organisms do not metabolize such contaminants. When comparing the different region of origin, fish samples from the Baltic Sea were the most contaminated. Nevertheless, none of the examined fish/bivalves samples exceed legislation limit for any of regulated contaminant groups.

Figure 1a-5: DART-MS lipids profiling.

Achievement 1a-5: DART-MS lipids profiling.
The application of a new innovative analytical technique based on ambient mass spectrometry employing Direct Analysis in Real Time (DART) ion source for characterization of fish samples, mainly of lipid fraction including triacylglycerols (TAGs) and fatty acids (FAs), was shown. The main advantage of this new technique for assessment of fish lipids quality is the possibility to measure the metabolomics (lipidomic) profile in the same ethylacetate extract which is, after purification on silica mini-column, examined for levels of organic pollutants, this means no saponification of TAGs is needed. On this account, a high speed of the measurement, e.g. one sample examined in only several seconds can be achieved. Worth to emphasize that the DART-MS technique allows obtaining characteristic mass spectra – metabolomic fingerprints – enabling identification and authentication of individual species, supposing respective libraries are available.

The fingerprints (mass spectra corresponding to both negative and positive ions) obtained on various fish species (herring, hake, tuna, whiting, cod, trout, salmon and pangasius) and bivalves were compared. High variability in TAGs region, i.e. in the m/z range 750–1000 was clearly documented. While in most marine fish species TAGs with higher m/z value i.e. those containing ´long´ PUFA such as arachidonic acid, (eicosapentaenoic acid) EPA, docosahexaenoic acid (DHA) were present, these TAGs were absent in pangasius fish what indicates lower nutritional value.

Recommendations for future research

This WP was focused on the implementation of effective analytical strategies for determination of major classes of POPs in food and feed. The key outcome was development of the new multi-analyte GC-MS method employing a simple and rapid sample preparation step that enabled simultaneous isolation of main representatives of PCBS, PBDEs and PAHs. The performance characteristics of this method comply with regulatory requirements. Significant time, workload and cost savings can be achieved. With respect to these features, the ´CONffIDENCE method´ was selected for international validation by AOAC. Recently, it has been confirmed as ´official method´ for analysis of PAHs in fish and seafood. To assess the nutritional benefits of fish, ambient mass spectrometry employing Direct Analysis in Real Time (DART) ion source was developed for examination of crude extracts. The information on content of polyunsaturated fatty acids (PUFA) can be obtained within one minute.

For future research we propose:

• Testing the possibility to extend the scope of the CONffIDENCE multi-analyte method by (i) other GC-amenable contaminants and (ii) newly emerged compounds such as BFRs
• To enable non-target screening, exploring the potential of recent high-end technologies such as high resolution mass spectrometry employing TOF or Q-TOF mass analyzers
• Development of a complementary LC-MS based method enabling in a single run analysis of (ultra) trace levels of (i) biologically active metabolites / exposure markers of the parent PCBs, BFRs and PAHs and (ii) not GC-amenable pollutants
• Investigation of possible use of DART-TOFMS technique for rapid analysis of fats quality and screening of other lipophilic components contained in fish, seafood and feeds; testing the correlation with ´classic´ approaches based on GC/LC analysis

Figure 1b-1-1: Milk and fish extraction procedures based on dispersive solid phase extraction.

WP1b Perfluorinated Compounds

Achievement 1b-1: The development of simplified analytical methods based on liquid chromatography coupled to mass spectrometry (LC-MS) for the analysis of perfluoroalkyl substances (PFASs) in food and feed.

Under the frame of the project, an innovative and simplified protocol for the analysis of the three selected PFASs (PFOA, PFOS and FOSA) in food and feed samples based on methanol extraction followed by dispersive solid phase extraction with activated charcoal clean-up and, finally, detection by liquid chromatography coupled to mass spectrometry in tandem (LC-MS/MS) was developed. In this context, the methodology was adapted for the extraction of PFASs from fish; fish feed and milk (Figure 1b-1-1).

The work was published in the journal Proceeding of the 6th International Students Conference - Modern Analytical Chemistry and all the validation process can be found in the Czech Journal of Food Science. As an example, a flowchart of the extraction of PFASs from seafood is given in Figure 1b-1-2.

Figure 1b-1-2: Flowchart for the extraction of PFASs in seafood.

Achievement 1b-2: The validation, harmonization and verification of these analytical methods using a step by step approach including the organization of interlaboratory studies at European level.

Two different levels should be differentiated in this section. The first level corresponds to the validation in-house and method transferability between WP1b partners. The second level corresponds to the performance of two different interlaboratory studies for the assessment of the methodology for the extraction of PFASs from fish, fish feed and milk.

Method optimization
The method validation was carried out in 2010 for both matrices (milk and fish) following the 2002/657/EC decision guidelines (2002/657/EC 2002). According to the quality parameters, both methodologies (for fish/fish feed and milk) are suitable for the analysis of PFASs in the matrices of interest. These methods are reproducible, robust, sensitive and fast. Once the methods were validated by the same laboratory that developed the procedures, the method transferability was evaluated by the other WP1b partners. The transferability study presented a low variability which is inherent in the analytical laboratory and technician error. In this context, both methodologies were suitable for the analysis of fish/fish feed and milk samples.

Collaborative study: interlaboratory
Two follow-up interlaboratory studies for the analysis of PFASs in fish, fish feed and milk have been performed among 11 different expert analytical laboratories from different European countries. Good correspondence was observed between the fortified concentrations and the mean values reported by the participants in both cases. Although this high correspondence, the differences observed in the results were unexpected and, sometimes, unacceptable due the same protocol followed by participants as well the same nature of the standards used for the fortification steps and the standards used in the quantification processes. On the other hand, the wide bias (in terms of overestimation or underestimation) has been assessed during the instrumental analysis (1st collaborative study) and during sample analysis. It could indicate a cross contamination during the analysis as well as during the sample preparation.

The statistical studies and the quality parameters concluded that the developed methodology can be applied in routine laboratory analysis for PFASs determination in milk, fish feed and fish at higher levels than 1 µg/L or 1 µg/Kg, respectively. Nonetheless, the transferability of the method cannot be considered completely successful due to the mild results at lowest concentration levels during the statistical studies.

The related journal papers will be submitted in the ABC special issue.

Figure 1b-3: European markets included in this study

Achievement 1b-3: The analysis of real food samples by the developed methodology in order to contribute in the European PFASs data base and the main inputs through the diet.

This achievement was performed by the analysis of European most consumed market sea fish and a small scale study of Mediterranean dairy products. For market sea fish study, different European markets from regions with different dietary habits were selected including samples from Baltic Sea, North Sea, Atlantic Ocean and Mediterranean Sea as well as some river samples. For sampling purposes, four different institutes, all of them partners of the CONffIDENCE project, cooperated during the sampling process into the different markets: ICT (Prague), DTU (Denmark), RIKILT (The Netherlands) and IDAEA-CSIC (Barcelona) (Figure 1b-3). A total number of 124 samples were analyzed including bivalves, salmon, hake, cod, whiting, herring, trout, tuna and pangasius (this last from Vietnam fishing area).

In order to approximate the levels found to a real European diet through the ingestion of fish, the Daily Intake (DI) according to EFSA guidelines (EFSA 2008) for PFOA and PFOS was calculated. The DI was evaluated for a man and woman between 25 and 40 years with a medium weight (70 and 60 Kg, respectively). The proposed diet, among other food that can be taken, was the punctual consumption of 300 g, approximately, of fish fillet. The DI was calculated according to: DI (ng /Kg day) = [(Consumption x PFC concentration) / body weight]. The risk index (RI) was calculated, as in previous published work by the same authors, according to EFSA guidelines (2008): RI = [DI / TDI], where TDI is the Total/TOlerable Daily Intake (150 ng/Kg day for PFOS and 1500 ng/Kg day for PFOA).

The RI may not pose an immediate risk for human health through the consumption of fish although the sum of all perfluoroalkyl substances as well as the long-time exposure should be considered.

Figure 1b-4: Inhibition curves for Vibrio fischeri bioluminescence assay at 30 min of exposure.

Achievement 1b-4: The evaluation of PFASs toxicity using standardized methods.
Toxicity assessment of PFOS, PFOA and FOSA was carried out according to the method based on the bioluminescence inhibition of the standard organism Vibrio fischeri originally described by Bulich. The toxicity of the standard individual PFASs was measured over a wide range of concentration in order to plot a well defined inhibition curve. The 50% effective concentration of each substance (EC50) was calculated. The toxicity units (TU), defined by Sprange and Ramsay equation, were calculated as: TUs = (EC50)^-1 × 100. Figure 1b-4 presents inhibition curves for PFOS, PFOA, and FOSA vs. Vibrio fischeri. A luminometer designed according in full agreement with the DIN/ISO bioluminescence inhibition protocol for toxicity assessment was used to measure the light inhibition during the experiment. Incubations were carried out at a controlled temperature (15 °C) and the incubation times were 15 min and 30 min.

The results showed that these compounds have low acute toxicity levels; however there is a certain degree of acute toxicity for high concentrations. This fact can revert in toxicity at low concentration levels during long expositions. Therefore, taking into account the bioaccumulation and the stability of these analytes, chronic toxicity effects should be considered.

Recommendations for future research
This WP has been working in the study of PFCs in food and feed by a new fast and selective method based on activated charcoal extraction. This method has been evaluated by in-house approaches and by two different interlaboratories. Both studies point out that a harmonized protocol should be implemented in the study of these analytes because of their ubiquity in the laboratory making the cross contamination one of the most important problems.

On the other hand, the study of different food items remarks the presence of PFCs in our diet.

Our recommendations for future research are:

• To harmonize the analytical protocol for PFCs
• Avoid any possible cross contamination due to lab materials
• To evaluate more in deep the daily intake through the diet
• To extend the toxicity studies to other organisms
• The evaluation of chronic toxicities by different organisms because food is eaten every day and the long-term exposure scenario has not been assessed

Figure 1c-1: DESI/DART method

WP1c Pesticides

Achievement 1c-1: Direct detection of Dithiocarbamates in fruit/vegetables by Ambient MS.
Dithiocarbamates are widely used fungicides which are normally analysed through their common hydrolysis product CS2. In CONffIDENCE the feasibility of rapid detection of intact thiram and ziram from fruits and vegetables through ambient mass spectrometry (DART and DESI) has been demonstrated. This enables compliance testing of products with specific maximum residue limits that have been established in the EU for these dithiocarbamates.

Figure 1c-1: Electrochemical sensor.

Achievement 1c-2: Detection of paraquat in potatoes/cereals by electrochemical immunosensor.
Paraquat is a broad spectrum herbicide that is also used as desiccant to facilitate in the harvest of certain crops. Since 2007 this product has been banned in the EU but it is still widely used outside the EU. Paraquat is not amenable to chemical multi-residue methods and rapid screening methods would be highly beneficial for residue monitoring. A new electrochemical immunosensor has been developed. Paraquat is measured through antibodies labeled with semiconductor nanoparticles which are electrochemically detected. Although the primary target analyte was paraquat, the assay has been demonstrated to be suited for multiplex detection by using different types of nanoparticles using deoxynivalenol as a model second target. The assay is extremely sensitive for paraquat. Even when diluting the potato and cereals extracts 100-fold, detection limits of 1 µg/kg can be achieved. The sensor was successfully validated for compliance testing against the EU-MRL (0.02 mg/kg).

Figure 1c-2: Samples analyzed for the survey.

Achievement 1c-3: Survey.
For paraquat and the related herbicide/desiccant diquat, very little data on occurrence of residues is publically available so far. A survey was conducted to gain insight in the occurrence of residues of paraquat and diquat in food and feed commodities. In order to include diquat in the survey, LC-MS/MS was used for detection. A simplified extraction procedure for this method was developed within the CONffIDENCE project. Almost 300 samples were analyzed, including potatoes, cereals, dry pulses and other food/feed commodities. Paraquat was not detected in any of the samples while diquat was detected in 5% of the samples in the range 0.005-0.11 mg/kg. Residue-containing crops were lentils, potatoes, barley and oilseed meal. Organic products were included in the survey. One batch of lentils labeled as organic contained diquat. None of the residues exceeded the EU-MRL.

Recommendations for future research

This WP has been working on development of rapid methods for detection of pesticides not amenable to multi-residue methods (so-called SRM pesticides) with dithiocarbamates and paraquat/diquat as example pesticides. Both instrumental methods based on direct MS-detection and a new type of biochemical assay have been demonstrated to be a feasible option for this. The first option is more suited for use in well-equipped laboratories, the second option requires less skills and laboratory infrastructure.

Our recommendation for future research are:

• Extend the feasibility of direct MS detection to other SRM-pesticides
• Development of better immunoreagents for diquat
• Further development of the multiplex electrochemical immunosensor for dedicated contaminant/matrix combinations
• Continuation of surveys on the occurrence of paraquat/diquat in food/feed commodities (with emphasis on cereals, potatoes, dry pulses and oilseeds)

Figure 2a-1: New screening method.

Developing detection methods for Veterinary Pharmaceuticals: Coccidiostats, Antibiotics

WP2a Coccidiostats

Achievement 2a-1: New relatively fast and inexpensive multiplex method for screening of coccidiostats.
A multiplex assay for the detection of residues of lasalocid A, monensin, salinomycin/narasin, nicarbazin and diclazuril (the latter only in eggs) in laying hens feed and in eggs was successfully developed and single-laboratory validated. Details related to the development of the method were published in 2012 by Bienenmann-Ploum et al. in Analytical and Bioanalytical Chemistry. The assay involves a generic, simple prior extraction procedure and proved to be fit for the purpose of the determination of these residues at the maximum levels set in the latest EU legislation. Additionally, a comparison of the method performance characteristics of the new assay versus existing reference methods confirmed the fitness for screening purposes.

Achievement 2a-2: Ring trial.
The new multiplex assay was further and fully validated through a small-scale inter-laboratory comparison. Validation was achieved for the 6 coccidiostats in eggs and for all coccidiostats except diclazuril in feed. The ring trial allowed to establish robust cut-off values for all analytes in the multiplex assay. The rate of false positives in the blanks showed to be low enough to allow considering using the screening test, hence demonstrating the analytical fitness for purpose of the multiplex assay. Finally, for DNC (nicarbazin) in feed and diclazuril in eggs, the ring trial showed that the final decision for use of the screening test should be based on the cost situation and expected frequency distribution of these target analytes.

Figure 2a-2: Robust cut-off for monensin (Top: eggs; bottom: feed; cut-off: red line).

Figure 2a-3: Correlation between the lasalocid concentration in feed and the lasalocid residue concentration in egg.

Achievement 2a-3: Carry Over study.
Carry-over may occur at all stages of the manufacturing and processing of feedingstuffs as well as during the storage and the transport of feedingstuffs. Since no publication was available to provide us with the indications and parameters, such as partition coefficients, the compartment volumes, dose absorbed over the gut wall needed to build a modelling approach such as the PBPK model this approach was discarded even if rather recommended according to Henri, 2012.

Nevertheless, the correlation between the lasalocid concentration in feed and the lasalocid residue concentration in egg has again been confirmed applying a linear equation. This approach exhibits the major advantage of being rapid and quite reliable. Therefore, in a feeding trial we demonstrated that the relative incidence of lasalocid to accumulate in eggs expressed in ng g-1 can be estimated using the range between 58 and 70 multiplied by the feed concentration (mg kg^-1).

Recommendations for future research
It would be a valuable improvement to include the remaining 5 authorised coccidiostats in the multiplex assay, provided that suitable antibodies would be available. Extended study would also be beneficial to improve the diclazuril assay in feed.

Figure 2b-1: Tetrasensor® for 3 matrices: feed, urine and cooked meat.

WP2b Antibiotics

Achievement 2b-1: Tetrasensor® for feed, urine and cooked meat
The Tetrasensor® is a competitive receptor based lateral flow dipstick assay developed by Unisensor that can detect many tetracycline compounds in different matrices such as milk, honey and raw animal tissues. During the Conffidence project, the application of this kit was extended to feed, urine (http://www.unisensor.be/en/catalog/feed-31/tetrasensor-52.php) and cooked meat. After assay optimisation, a single laboratory validation (CD 2002/657/EC) was performed. This rapid, sensitive and easy to use test is capable of the detection of tetracycline compounds in a range of matrices below the required detection limit of 100 µg kg-1, so that 50+ samples can be analysed in a day (up to 16 samples per h). The results were presented in poster format at ASSET 2011, RAFA 2011 and Euroresidue VII.

Figure 2b-2: Bee4sensor® for honey.

Achievement 2b-2: Bee4sensor® for honey (lab format and field test)
The Bee4sensor® is an indirect competitive immunochromatographic antibody-based dipstick assay for the rapid and simultaneous detection of sulfonamides (SULFA), (fluoro)quinolones (FQs), tylosin A (TYL) and chloramphenicol (CAP) in honey that was developed within the CONffIDENCE project. The kit will be available from Unisensor (http://www.unisensor.be/en/catalog/antibiotics-28/bee4sensor-45.php) in (i) a lab-based format, undergone a single laboratory validation (CD 2002/657/EC) and a mini inter-laboratory validation (ILV) with 7 European laboratories, and (ii) a field test format, undergone a ‘proof of principle’ validation and global field trial with 16 participants from across governmental, industrial organisations and academia. Herewith a rapid test for industry and enforcement authorities has been presented. The results of the successful ILV will be published in the special issue of the ABC journal; while the outcome of the single laboratory validation (lab-based format) and the excellent performance of the Bee4sensor® during the field trial will be reported at a later date.

Recommendations for further research:

There will be scope to expand the Bee4sensor®/multiplex dipstick assay for antimicrobials in honey, to e.g. include tetracyclines (already available as singleplex), streptomycin, lincomycin or fumagillin.

Figure 3-1: Participants at the inorganic arsenic workshop at the regional laboratory in Oldenburg, Germany.

Developing detection methods for Heavy Metals

WP3 Heavy Metals

Achievement 3-1: Inorganic As detection in seafood and rice by SPE-HGAAS and collaborative study.
A novel speciation approach has been developed in the CONffIDENCE project, which allows for the specific determination of inorganic arsenic by solid phase extraction (SPE), which separates inorganic arsenic from organoarsenic compounds, followed by detection by hydride generation atomic absorption spectrometry (HGAAS). The inorganic arsenic was extracted by use of microwave technology at 90°C. In a recent publication in ABC the method development, validation and application to a range of seafood samples has been described (Rasmussen, 2012). This novel separation approach was furthermore tailored for the analysis of rice samples, a sample type, which recently has drawn much attention with regards to its potential high levels of inorganic arsenic and hence the potential high contribution from rice to the dietary exposure of inorganic arsenic (EFSA, 2009). In this approach a simplified extraction procedure was developed by using waterbath and dilute acid, which allowed the simultaneous extraction of many samples. The method was applied to a range of rice samples and rice crackers and the results will be presented in an upcoming publication in the CONffIDENCE special issue of ABC (Rasmussen et al, 2013a). Both sample types (seafood and rice) were successfully tested in collaborative trials following a training session with the participating laboratories (Figure 3-1).

Achievement 3-2: Methyl Mercury detection in seafood by HPLC-ICPMS and collaborative study.
A method for the specific determination of methylmercury in marine food and feed samples was developed. The method procedure included a simplified extraction step with ultrasonification, which allowed for fast extraction of many samples at the same time. The concentration of methylmercury could subsequently be determined in the extracts by a HPLC-ICPMS procedure, where inorganic mercury and methylmercury were separated on a cation-exchange column followed by mercury-specific determination by ICPMS. The method was in-house validated and furthermore tested in three other laboratories on both feed and food samples. The results will be presented in an upcoming publication in the CONffIDENCE special issue of ABC (Rasmussen et al, 2013b). The method was also used to provide data on the methylmercury content (together with other pollutants) in fish feed and fish feed ingredient samples (Granby et al, 2013, to be published). The method addressed the need for speciation methods and speciation data specifically on methylmercury as recently emphasized by EFSA (2012) and JECFA (2011).

Figure 3-3: Methyl mecury in fish and fish feed.

Achievement 3-3: Survey on seafood.
The developed methods were used in a large survey on seafood, which was undertaken in close collaboration with WP1a and Wp1b. Here data on inorganic arsenic and methylmercury in a range of seafood samples, including lean and fatty fish and bivalves from various important fishing regions throughout the world. The dataset has provided valuable input for a better understanding of contaminant level in seafood and will provide an important contribution to risk-benefit assessments of dietary seafood intake in the EU population (Figure 3-3).

Recommendations for future research

The CONffIDENCE project has progressed beyond state-of-the-art in heavy metal analysis, where classically only the total amount of the metal is determined. The CONffIDENCE project has presented novel approaches for speciation analysis of arsenic and mercury in order to allow for specific determination of the species of highest toxicological importance. The methods have shown their applicability on real life samples of marine food and feed as well as for inorganic arsenic in rice samples. These achievements are important in order to enable food and feed control and regulation on the species level rather than on the total amount in the future. A review on arsenic and mercury speciation in feed in BASE provides further information on the subject (Hedegaard, 2011).

It is recommended that future research projects are initiated, which aim at providing a better understanding of the occurrence, metabolism and potential toxicological actions of heavy metals and other elements on a species level rather than only looking at the total amount. An important topic will be studies on the transfer, stability and metabolism of element species throughout the whole feed-to-food chain for improved understanding of the interrelationship between feed and food regulation as well as their impact on human and animal health. The analytical approaches developed in the framework of CONffIDENCE project will form an analytical toolbox that can be used in such studies. The analytical concepts developed may be used directly on arsenic and mercury without further development, but the analytical principles can also be used as starting points for the detection of other element species. It is furthermore suggested to extend these studies to include not only toxic elements, but also elements, which have a beneficial function for human and animal health (e.g. selenium, iodine). Beneficial elements are also used in feed- and food supplements and there is a need to develop analytical techniques to distinguish between the different chemical forms of the element, since bioavailability and nutritional effect (and price) will depend on this parameter. Hence, such studies will include stakeholders from both industry, authorities and consumers.

Figure 4a-1: Dipstick procedure and reading for tropane and ergot alkaloids.

Developing detection methods for Biotoxins: Alkaloids, Marine Biotoxins, Mycotoxins

WP4a Alkaloids

Achievement 4-1: Dipstick methods.
Dipstick methods were developed for the tropane alkaloids scopolamine and hyoscyamine and for the ergot alkaloids ergometrine, ergotamine and ergocristine and were extensively tested and validated in in-house studies and in interlaboratory studies involving 7 participants. For both dipsticks the in-house validations were performed. The tropane alkaloid dipstick performed successfully at the target level of 800 µg/kg for the mixture of L-hyoscyamine and L-scopolamine. The ergot alkaloid dipstick performed successfully at the target level of 200 µg/kg (for individual alkaloids) for ergotamine, ergocristine and ergometrine. Evaluation of the results of the interlaboratory study of the tropane alkaloid dipstick showed very satisfactory results, i.e. there was excellent separation between blank samples and samples containing the tropane alkaloids at 50% and 100% of the target levels. This makes the test quite suitable for screening purposes, since > 95 % of field samples are blank. It was noticed, however, that there exists a significant difference between the cut-off levels derived from the in-house and the interlaboratory studies, which could not yet be explained, and which needs further study. Preliminary evaluation of the results of the interlaboratory study of the ergot alkaloid dipstick also showed very satisfactory results. In this case there was no significant difference between the cut-off levels derived from the in-house and the interlaboratory studies. Regarding the practical use of the ergot alkaloids dipstick it is difficult to draw pertinent conclusions, because ergotamine and ergocristine are detected with different sensitivity on the same test line. This makes it difficult to calculate a combined and realistic cut-off level. Whereas not all relevant ergot alkaloids that can be present in rye ergot are detected, the 3 selected compounds represent a substantial portion of the total ergot alkaloids, normally present in ergot. For both dipstick assays results can be obtained within 30 minutes, and over 60 samples can be analysed per day per person. The method for the determination of the tropane alkaloids will be submitted for the special issue of the ABC journal. The method for the determination of the ergot alkaloids is proposed to be submitted for publication in a special issue of World Mycotoxin Journal about “Rapid Methods for Mycotoxins”.

Figure 4a-2: ELISA for key pyrrolizidine alkaloids.

Achievement 4-2: Multiplex ELISA.
A rapid multiplex method was developed to detect representatives of three important groups of pyrrolizidine alkaloids (senecionine, lycopsamine and heliotrine types) to be used as a screening tool for the detection of these toxic compounds in food and feed. The method was in-house validated for honey and feed matrices and was demonstrated to have a detection capability less than 25 µg/kg (for individual alkaloids) for jacobine, lycopsamine, heliotrine and senecionine. A reduction step was introduced to the extraction procedure to allow for the additional detection of the presence of N-oxides of PAs. With the developed method results can be obtained within 2 hours, and over 50 samples can be analysed per day per person. It is a first assay of its kind for PAs.

A small scale inter-laboratory study was set up to verify the trans-laboratory performance of the developed multiplex assay for the detection of the key PA: senecionine, heliotrine and lycopsamine. The evaluation revealed that for all 3 laboratories acceptable results for the rate of false- positives was obtained, with the exception of the results of 1 laboratory (for senecionine). Because in the inter-laboratory study only three laboratories could participate, it was deemed that a higher number of samples should be analysed to demonstrate the transferability of the assay. The method has been submitted for publication in the special issue of the ABC journal.

Figure 4a-3: Demonstration of NIR hyperspectral method to determine ergot in grains at an industrial setting.

Achievement 4-3: NIR method.
The protocol and validation of the NIR hyperspectral imaging method for the detection of ergot contamination in cereals dedicated for food and feed has been described in detail and published (Food Additives and Contaminants, 2012). The hyperspectral imaging method has been successfully rounded off with testing and demonstrating the system at an industrial site (see figure opposite). Relevant presentation and demonstration about this methodology has been presented at the dedicated CONffIDENCE Cluster workshop at WMFmeetsIUPAC, Rotterdam, the Netherlands in November 2012. It will be published in the special issue of the ABC journal. This technology can be used to detect and quantify ergot contamination in large samples of cereals destined for food or feed in both the laboratory and in the processing industry, where accurate and fast inspection is needed. The classical microscopy method can be used as confirmatory method on reduced samples. The line scan NIR hyperspectral imaging system allows to analyse a sample of 250 g in one minute. In comparison, the existing microscopy method requires 30-60 minutes to analyse the same sample.

Recommendations for future research

Whereas the dipstick methods for tropane alkaloids functions well in practice, further study is needed to investigate the causes of the significant difference between cut-off levels derived from in-house and interlaboratory studies.

For the ergots alkaloids dipstick method the expected rate of false negatives, based on quantitative information about the ergot alkaloid profile in feed cereal grains in the EU, would need to be estimated (similar as was estimated for the Dutch situation to be at approx. 1 %).

The ELISA for pyrrolizidine alkaloids has the potential for lower limits of detection for senecionine, heliotrine and lycopsamine. If this would be desirable, the conditions at which the test is performed would need to be optimised. To get better insight into the between-laboratory variability of the method an interlaboratory exercise with approx. 8 laboratories would be recommended.

Regarding the NIR method, the next challenges will be to demonstrate the potential of the methodology for multicontaminants detection in particular in the organic productions.

Figure 4b-1: EFSA opinions.

WP4b Marine Biotoxins

Achievement 4b-1: Contribution to EFSA opinions on emerging marine toxins.
This Work Package was primarily concerned with conducting the research on a range of regulated and emerging marine biotoxins.

With regards the emerging toxins spirolides and palytoxin; the focus was determining their modes of action so as to assist the European Commission in determining the potential risks of human exposure to these algal metabolites via ingestion of contaminated shellfish. The research conducted at the University of Santiago de Compostela in Spain provided highly important information which was published in the scientific literature and used by the European Food Safety Authority (EFSA) in their producing their scientific opinions on these toxins. EFSA published these opinions in their own journal (2009. 7(12); 1393 and 2010. 8(6) 1628).

Figure 4b-2: The prototype multichannel surface plasmon resonance biosensor used in the CONffIDENCE project.

Achievement 4b-2: Multiplex biosensor assays for multiple shellfish toxins developed and validated.
The second major task for WP4b was to develop a biosensor based tests for a wide range of regulated and emerging biotoxins. Antibodies to the major classes of regulated toxins, Paralytic Shellfish Poisons, Diarrhoeic Shellfish Poisons and Amnesiac Shellfish Poisons along with the emerging biotoxin palytoxin were ultilised on a number of different biosensor platforms to determine if the testing for all compounds could be undertaken simultaneously. In order to do this a substantial amount of research has to be undertaken on developing novel surface chemistries and sample preparation techniques. The results of this research were that the multiplexed analysis of marine toxins was demonstrated for the first time and could be applied to samples to rapidly detect toxin present in samples harvested from coastlines (McNamee et al, Environmental Science and Pollution Research. 2013, in press).

A biosensor assay was also developed and validated for the emerging marine biotoxin tetrodotoxin. The assay based on surface plasmon resonance was shown to be highly effective in detecting the toxin in a range of biological samples. (Campbell et al, Special issue of the ABC journal, under review).

Recommendations for future research

In summary this WP has had a major impact on securing the safety of the European citizens from the consumption of naturally occurring toxins which can accumulate in a variety of marine species. Due to factors such as increasing pollution and climate change topic episodes are occurring more frequently and the need to detect associated toxins in a rapid and reliable manner is of immense importance.

Figure 4c-1: 4 mycosensor.

WP4c Mycotoxins

Achievement 4c-1: Multiplex dipstick tests for fusarium toxins: 4 mycosensor
The aim of mycotoxin research within CONffIDENCE project was to develop multiplex dipstick tests for the determination of the Fusarium toxins DON, ZEA, T-2/HT-2 and FBs in cereals and cereal products.

Prototype multiplex dipsticks were designed and assembled in order to reach detection limits (cut off levels) compatible with the EU legislation in force or expected for Fusarium toxins (EC Regulations No 1881/2006 and 1126/2007). Simplified sample preparation protocols were developed requiring no more than 6 min extraction with a mixture of methanol/water. The immunoassay kit, based on indirect competitive format, is composed by multiplex dipsticks, a set of microwells containing freeze-dried reagents, and an incubator. Results are interpreted by an optical reader measuring the ratio between each test line and a dynamic control line located on the top of the strip. The resulting immunoassay protocol is rapid (total analysis time 50 min for 6 mycotoxins), inexpensive, and easy-to-use.

The kit is now commercially available and distributed by Unisensor, that was active partner in the project.

Achievement 4c-2: Interlaboratory validation
A “single-laboratory validation design” was elaborated and applied to evaluate performances of multiplex dipstick immunoassays for the determination of Fusarium toxins in wheat and maize. Statistical evaluation of the results (ANOVA) showed the ruggedness of the assays, since neither the matrix (i.e. wheat/maize origin) nor the day effect inflated the variation of the results. Rate of false positive of blank samples was generally lower than 10% for all toxins. Results of single-laboratory validation will be published in the special issue of the ABC journal.

The test was subjected to final validation by a collaborative trial involving 13 laboratories. A training phase was included in the experimental design enabling the participants to familiarize with the protocol. The total standard deviation of the response varied from 6 to 24% for the analyte/concentration/matrix combinations included in the study, indicating the assay ruggedness between different laboratories, and therefore, a method transferability. A manuscript describing results and outcomes of the interlaboratory study is in preparation for the special issue of the ABC journal.

Finally, to evaluate the fitness-for-purpose of the test, the performance profile was applied to realistic distribution of the mycotoxins in target materials obtained from European monitoring programmes.

A practical training on determination of Fusarium toxins in maize by multiplex dipstick immunoassays developed in CONffIDENCE project was organized within the ISM-MycoRed Training Course “Detection techniques for mycotoxins and toxigenic fungi in the food chain”, May 28 – June 1, 2012. ISPA-CNR, Bari, Italy.

Furthermore the commercial kit has been presented at the dedicated CONffIDENCE Cluster workshop at WMFmeetsIUPAC, Rotterdam, the Netherlands in November 2012.

Recommendations for future research

In the last years a wide range of commercial test strips have become available reflecting the increasing demand for screening tests that are easy to use and allow rapid decision-making. Most commercial test strips are devoted to mycotoxins that have been regulated for decades (e.g., aflatoxins). However, driven by recent and incoming EU regulations, immunodiagnostic test strips are also attracting interest in Fusarium toxin analysis. The CONffIDENCE project has advanced the state-of-the-art in this area with the development of the first multiplex dipstick for the simultaneous determination of major Fusarium mycotoxins, co-occurring in cereals and derived products.

The kit is now commercially available and the developed method is fully validated in wheat and maize by interlaboratory study. On farm testing demonstrated the transferability of the method, and the fitness-for-the purpose of early monitoring of mycotoxin contamination of incoming materials.

Recommendation for future research are:

• to extend on farm testing and comparison with reference methods;
• to extend the method to further matrices representing a promising field of application of the kit, i.e. other cereals, particularly oats, and feed materials;
• to tailor cut off levels, taking into account, besides the legislation in force, a) monitoring data showing the real mycotoxin contamination levels experienced at farm level, and b) for T-2 and HT-2 toxins, maximum levels indicated in the recent Recommendation 2013/165/EU;
• to move from a “qualitative” to a “quantitative” multiplex dipstick kit.

4th MONIQA INTERNATIONAL CONFERENCE: FOOD SAFETY UNDER GLOBAL PRESSURE OF CLIMATE CHANGE, FOOD SECURITY AND ECONOMIC CRISES

The 4th MoniQA International Conference was organised in Budapest, Hungary, 26 February - 1 March 2013, continuing the successful series of MoniQA International Conferences (Rome - Italy 2008, Krakow - Poland 2010, Varna - Bulgaria 2011). The theme of the conference put various emerging and persistent food safety issues, and the relevant analytical and legal challenges in perspective of globalisation, climate change, food security and economic crises.

A session was dedicated to Rapid methods and sensors chaired by Philippe Delahaut and Jacob de Jong. Several outputs of the CONffIDENCE project were presented.

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4th FEED SAFETY INTERNATIONAL CONFERENCE 2012

On 11, 12 and 13 September 2012, the China Agricultural University (CAU) hosted in Beijing - the People's Republic of China, the 4th International FEED SAFETY Conference “Methods and challenges”. The conference was organized in collaboration with the Walloon Agricultural Research Centre (CRA-W), Gembloux, Belgium, the Institute of Food Safety (RIKILT), Wageningen, The Netherlands and the Queen’s University of Belfast (QUB), Belfast, UK. Former FEED SAFETY Conferences were organized in 2004, 2007 in Namur (Belgium) and in 2009 in Wageningen (The Netherlands) in the framework of different EC projects. The purpose of this 4th Conference was to give an overview of the latest legislative, scientific achievements and industrial progresses regarding the areas such as processed animal proteins, nonprotein nitrogen (NPN), organic contaminants (dioxins, PCB’s, pesticides), feed additives, banned substances, heavy metals, mycotoxins, plant toxins, veterinary drugs, and GMO’s. Results of recent international RTD projects (CONffIDENCE, QSAffE, EURL-AP) were presented. Over 140 participants, including 53 oversea representatives, from 17 countries and 38 organizations, attended the conference. The 31 lectures and the 49 posters presented during this event can be viewed on the FEEDSAFETY website on http://www.feedsafety.org/fs2012/.

The third session cochaired by Jacob de Jong (RIKILT, the Netherlands), coordinator of the CONffIDENCE project was dedicated to “Feed additives, contaminants and undesirable substances.

Several outputs of the European CONffIDENCE project were presented

• Antibody based techniques such as lateral flow and multiplex ELISA to detect a broad range of alkaloids in feed and food (Chris Elliott, QUB, UK),
• GC-MS/MS and GCxGC-TOFMS to detect several PCBs, PBDEs and PAHs in fish feed within one single run (Jana Pulkrabova, ICT Prague, Czech Republic),
• Near infrared hyperspectral imaging to detect and quantify ergot in cereals (Philippe Vermeulen, CRA-W, Belgium),
• Five-plex flow cytometry-based immunoassay (FCIA) for the simultaneous detection of six frequently used coccidiostats in animal poultry feed and eggs (Ursula Vincent, EC-JRC-IRMM, Belgium),
• Multiplex dipstick for the analysis of Fusarium mycotoxins in wheat and maize (Christoph von Holst, EC-JRC-IRMM, Belgium).

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Dust sample collect and measurement using immunosensor

MycoHunt develops a biosensor for rapid detection of mycotoxin in wheat

The project aims at increasing the competitiveness of a large group of SMEs by developing a cost-effective method to detect infection of mycotoxin deoxynivalenol in wheat grains, a major threat to the food and feed sector of the European industry. A group of SME-AGs, covering the two relevant sectors and representing vast number of sector SMEs, have put together this project in order to gain knowledge and resources to further exploit the results of the novel technology proposed by providing a thorough sampling and measurement method of grain. The economic consequences of mycotoxin infection to animal husbandry and the cost of infected crop to farmers as well as mill and storage house owners are of increasing concern while human health and safety effects are and must be addressed additionally.

The main achievements of this project can be summarized as follows:

• The MYCOHUNT system was fully designed reflecting the content of the system specification with the assistance of SME Association and SME partners.
• The MYCOHUNT concept of testing DON from wheat dust has been proven via defining the correlation between DON found in wheat grain samples and in wheat dust samples.
• DON selective antibodies have been developed to be used in the immunosensor for DON detection. Assay development and characterization for the immunosensor is under progress.
• The components of the prototype have been constructed. The physical realization and the integration of the complete system including the sampling unit, the sensor unit and their control system are under development and will be installed at the end-user facilities to continue with the prototype demonstration.

Release of the SAFEED-PAP book: Detection, identification and quantification of processed animal proteins in feedingstuffs.

This book edited in the framework of the SAFEED-PAP project aims to summarize the state of the art of the different issues regarding the detection, identification and quantification of processed animal proteins (PAP) in feedingstuffs. It has been written in such a way that it could be useful for the implementation of the discussed methods in the control and research laboratories. For each method discussed, a short description of the technique is given with references to other relevant books and websites useful to the reader to get more in depth. Special emphasis has been put on the rules and tricks that have to be respected for the correct application of the techniques for the analysis of processed animal proteins in feedingstuffs. The potential of each method (including the fit for purpose and the robustness) as well as future perspectives are discussed.

This book has been written by several experts coming from research, control and feed laboratories and has the ambition to be a reference for the correct implementation and development of methods in laboratories performing or planning to perform the detection, identification and/or quantification of processed animal proteins in feedingstuffs.

This book includes three sections. The first section is devoted to the feed chain specificity, the processed animal proteins history, the legislative aspects as well as the analytical needs. The second section of this book concerns the analytical methods aiming the detection of particles of animal origin (i.e. classical microscopy, NIR infrared microscopy and NIR imaging methods). The third section is focused on methods aiming the detection of DNA (i.e. PCR) and proteins (i.e. Immunology, HPLC/MS) as well as the combination of techniques.

35th MYCOTOXIN WORKSHOP

22 - 24 May 2013

This Mycotoxin workshop will be organized by the Ghent University Association together with the Society for Mycotoxin Research (www.mycotoxin.de). The workshop aims to bring together experts from academia, government and industry to discuss all aspects of mycotoxin research including human and animal health issues, toxicology, mycotoxin production in relation with food and feed safety, analysis of mycotoxins, legislation and regulatory aspects and others.

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FINAL MYCORED INTERNATIONAL CONFERENCE

27 - 30 May 2013

The final MycoRed International Conference will take place in Apulia, Italy, 27 -30 May 2013. Academics, individuals from research institutes, universities, national and international agencies and industry, who are working in this important field of research, will join the last MycoRed appointment. The scientific programme will reflect a spectrum of disciplines (methods of analysis and sampling, toxicity, chemistry, biochemistry, biocontrol, prevention, risk assessment, occurence in foods and feeds), presenting the last advances and results coming from this interdisciplinary project. Finally the conference will represent an opportunity to gain an overview of research and future developments on mycotoxin research.

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RAFA 2013: RECENT ADVANCES IN FOOD ANALYSIS

5 - 8 November 2013

The program will be accompanied by several satellite workshops, interactive and vendor seminars: - 2nd European workshop on Ambient mass spectrometry in food and natural products - Interactive seminar on sample-prep and separation techniques and approaches for mass spectrometry detection in food quality / safety: Step by step strategies for fast development of analytical method- Interactive seminar on data handling and chemometrics, foodomics - Vibrational spectroscopy and chemometrics for monitoring of food and feed products, lab-to-the-sample - Food safety issues beyond the EU – challenges for analytical methods update- Joint Research Centre seminar – research activities and opportunities for collaboration strengthening, with a special focus to young scientists- VENDOR SEMINARS – introducing recent instrumentation and scientific strategies for advanced food quality / safety control

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This work is funded by the European Commission, under the FP7 Food Quality and Safety Priority, within the framework of the Collaborative Project CONffIDENCE - 211326 - entitled "Contaminants in Food and Feed: Inexpensive DEtection for Control of Exposure". This project is carried out by a consortium coordinated by RIKILT - Institute of Food Safety (NL) and includes 17 partners. The information reflects the authors views, the European Commission is not liable for any use of the information contained therein.

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