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Paramagnetic microspheres can be used in planar array fluorescence immunoassays for single or multiplex screening of food contaminants. However, no confirmation of the molecular identity is obtained. Coated blade spray (CBS) is a direct ionization mass spectrometry (MS) technique, and when combined with triple quadrupole MS/MS, it allows for rapid confirmation of food contaminants. The lack of chromatography in CBS, though, compromises the specificity of the measurement for unequivocal identification of contaminants, based on the European Union (EU) regulation. Therefore, a rapid and easy-to-use immuno-magnetic blade spray (iMBS) method was developed in which immuno-enriched paramagnetic microspheres replace the coating of CBS. The iMBS-MS/MS method was fully optimized, validated in-house following the EU 2021/808 regulation, and benchmarked against a commercial lateral flow immunoassay (LFIA) for on-site screening of DA. The applicability of iMBS-MS/MS was further demonstrated by analyzing incurred mussel samples. The combination of immunorecognition and MS/MS detection in iMBS-MS/MS enhances the measurement's selectivity, which is demonstrated by the rapid differentiation between the marine toxin domoic acid (DA) and its structural analog kainic acid (KA), which cannot be achieved with the LFIA alone. Interestingly, this first-ever reported iMBS-MS/MS method is generic and can be adapted to include any other immuno-captured food contaminant, provided that monoclonal antibodies are available, thus offering a complementary confirmatory analysis approach to multiplex immunoassay screening methods. Moreover, thanks to its speed of analysis, iMBS-MS/MS can bridge the logistics gap between future large-scale on-site testings using LFIAs and classical time-consuming confirmatory MS analysis performed in official control laboratories.

On-site testing in food analysis using mass spectrometry (MS) requires miniaturization of vacuum systems, mass analyzers, sample cleanup, and ionization sources. In this study, a simple coated blade spray (CBS) ion source was developed that enables high voltage generation on the blade by ubiquitous certified (micro-)USB On-The-Go devices like smartphones, tablets, and power banks. CBS is capable of performing both analyte enrichment by solid-phase microextraction (SPME) material coated on the metal substrate and direct-spray ionization. The USB-CBS device was used on two different MS systems, a transportable single-quadrupole and a benchtop triple-quadrupole tandem MS. Various characteristics of the USB-CBS device, including high voltage generation and angular positioning, were studied. The potential of the newly developed device for food safety applications is demonstrated by banned and regulated veterinary drugs such as β-agonists and sulfonamide antibiotics, covering a wide range of molecular weights and polarities. The results highlight the potential of the developed, simplified, inexpensive (less than 10 USD), and universal vendor-independent USB-powered CBS ion source coupled with MS(/MS) systems for semiquantitative applications, in laboratories, and in future on-site food quality and safety testing. Apart from that, most likely on-site environmental, biomedical, and forensic testing will also benefit from this USB-CBS instrumental development that is compatible with any atmospheric inlet MS system.

The lack of chromatographic separation in ambient and direct mass spectrometry (MS) ionization techniques jeopardizes the overall selectivity of the developed methods. Incorporating a biosensing element at the ionization source could compensate for that inherent lack of selectivity. Thus, a simplified immunoaffinity-direct MS technique was developed, immunoaffinity blade spray (iBS), featuring a conductive polystyrene blade material. In iBS, the generic coating used in conventional coated blade spray is replaced with a layer of highly specific monoclonal antibodies (mAbs), while the stainless steel is replaced with conductive polystyrene to allow for simple ELISA platelike hydrophobic immobilization of mAbs. Because of its high relevance for climate change-induced food safety issues, the mycotoxin deoxynivalenol (DON) was chosen as a model substance. Following a rapid extraction from wheat flour, DON is immuno-captured, and the blade is positioned in front of the MS for direct iBS-MS/MS analysis. The method's applicability was demonstrated by analyzing spiked and incurred wheat flour samples, omitting the need for time-consuming chromatographic separation. Apart from DON, cross-reacting DON conjugates could be successfully analyzed as well. The direct iBS-MS/MS method is generic and adaptable to detecting any analyte in sample extracts, provided that specific mAbs are available.

Pyrrolizidine alkaloids (PAs) are produced by various plant species and have been detected as contaminants in food and feed. Monitoring programmes should include PAs that are present in relevant matrices and that exhibit a high toxic potential. The aim of the present study was to use a bioassay-directed analysis approach to identify relevant PAs not yet included in monitoring programmes. To that end, extracts of Heliotropium europaeum and H. popovii were prepared and analysed with LC–MS/MS for the presence of 35 PAs included in monitoring programmes, as well as for genotoxic activity in the HepaRG/γH2AX assay. Europine, heliotrine and lasiocarpine were found to be the most abundant PAs. The extracts showed a higher γH2AX activity than related artificial mixtures of quantified known PAs, which might point to the presence of unknown toxic PAs. The H. europaeum extract was fractionated and γH2AX activities of individual fractions were determined. Fractions were further analysed applying LC–Orbitrap-MS analysis and Compound Discoverer software, identifying various candidate PAs responsible for the non-explained genotoxic activity. Altogether, the results obtained show that bioassay-directed analysis allows identification of candidate PAs that can be included in monitoring programmes.

Phycotoxins occur in various marine and freshwater environments, and can accumulate in edible species such as fish, crabs, and shellfish. Human exposure to these toxins can take place, for instance, through consumption of contaminated species or supplements and through the ingestion of contaminated water. Symptoms of phycotoxin intoxication include paralysis, diarrhea, and amnesia. When the cause of an intoxication cannot directly be found, a screening method is required to identify the causative toxin. In this work, such a screening method was developed and validated for marine and freshwater phycotoxins in different matrices: fish, shellfish, water, and food supplements. Two LC methods were developed: one for hydrophilic and one for lipophilic phycotoxins. Sample extracts were measured in full scan mode with an Orbitrap high resolution mass spectrometer. Additionally, a database was created to process the data. The method was successfully validated for most matrices, and in addition, regulated lipophilic phycotoxins, domoic acid, and some paralytic shellfish poisoning toxins could be quantified in shellfish. The method showed limitations for hydrophilic phycotoxins in sea water and for lipophilic phycotoxins in food supplements. The developed method is a screening method; in order to confirm suspected compounds, comparison with a standard or an additional analysis such as NMR is required.

In 2020 is in het kader van het monitoringsprogramma “Contaminanten in vis uit Nederlandse binnenwateren” aal op 18 locaties bemonsterd. Hiervan lagen 10 locaties binnen het voor aalvisserij gesloten gebied en 8 locaties waar de aalvisserij is toegestaan. Voor de bemonstering van grote alen wordt sinds 2016 rekening gehouden met het zwaartepunt van de beroepsmatige vangst, waarbij iets grotere aal is bemonsterd (53-76 cm) dan vóór 2016 (>45 cm). Van de gevangen rode alen zijn mengmonsters samengesteld voor de lengteklassen 30-40 cm en 53-76 cm en deze zijn geanalyseerd op de aanwezigheid van dioxines, dioxineachtige-PCB’s (dl-PCB’s) en niet-dioxineachtige PCB’s (ndl- PCB’s). Voor som dioxines, som dioxines & dl-PCB’s (totaal-TEQ) en som ndl-PCB’s zijn Europese maximumgehalten (zogenaamde maximum levels, ML’s) vastgesteld (EC 1881/2006). De mengmonsters grote aal (53-76 cm) voldeden op de volgende onderzochte locaties aan alle ML’s: Volkerak (Steenbergen), IJsselmeer (Medemblik), IJsselmeer (Urkerbos) en Linge nabij Heukelum. De mengmonsters aal (53-76 cm) van de overige locaties overschreden één of meerdere ML’s. Aanvullend is getoetst of de beleidsregellimieten (ingesteld in 2017) overschreden worden. Dat geldt voor aal (53-76 cm) van de locaties IJsselmeer bij Urk, IJsselmeer nabij Lelystad en Ramsgeul (Ramspolbrug). Voor de locatie IJsselmeer bij Urk is wederom een overschrijding van een beleidsregellimiet waargenomen. Voor de mengmonsters aal in de klasse 30-40 cm overschreed het mengmonster van de Lek de ML voor ndl-PCB’s en dat van de Waal bij Tiel de beleidsregellimiet voor ndl-PCB’s. Van de locaties die nu nog geopend zijn voor visserij, overschreden Schokkerhaven en het IJ (meertje nabij Spaarndam) één of meerdere limieten. Voor de locatie Schokkerhaven is ook twee jaar achtereen een overschrijding van de (beleidsregel)limieten waargenomen. Het huidige rapport geeft de gegevens van voorgaande jaren weer, aangevuld met de resultaten van 2020. Op een enkele locatie is een verhoogd gehalte gevonden ten opzichte van voorgaande jaren, maar over het algemeen passen de gevonden gehalten in het beeld van de voorgaande jaren. De mengmonsters aal zijn ook geanalyseerd op zware metalen (cadmium, lood, kwik en arseen) en perfluoralkylstoffen (PFAS’s). Alle gehalten van cadmium, lood en kwik voldeden aan de geldende ML’s (EC 1881/2006), voor arseen is geen ML vastgesteld. In aanvulling op de vorige jaarlijkse rapportage zijn resultaten van de meting van PFAS’s opgenomen in dit rapport. Dit door de toegenomen aandacht voor deze stoffen in 2019 en 2020, en het recent beschikbaar komen van een risicobeoordeling door de Europese Autoriteit voor Voedselveiligheid (EFSA). De gesommeerde gehalten van de meest voorkomende PFAS’s variëren van circa 5 tot 40 ng/g product, waarbij PFOS de belangrijkste bijdrage leverde. Hoogste gehalten werden aangetroffen in aal uit het IJsselmeer en de Linge bij Heukelum. Voor deze stoffen is geen ML vastgesteld en kunnen de gemeten gehalten dus niet getoetst worden aan de ML.

In current food safety monitoring, lateral flow immunoassays (LFIAs) are widely used for rapid food contaminant screening. Recent advances include smartphone readouts, offering semi-quantitative analysis of LFIAs with time, location, and data transfer in case of on-site testing. Following the screening, the next step in the EU regulations is confirmation by, e.g., liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this work, using direct analysis in real time ambient ionization and triple quadrupole MS/MS (DART-QqQ-MS/MS), we achieved rapid confirmation of the identity of the substance(s) causing the LFIA result. In the workflow proposed, an individual performs the (on-site) smartphone LFIA screening, and when the result is suspect, an identification LFIA (ID-LFIA) strip is developed with the same sample extract. The ID-LFIA can be dissociated and rapidly analyzed in a control laboratory with DART-QqQ-MS/MS. The ID-LFIA consists of multiple lines of monoclonal antibodies against the mycotoxin deoxynivalenol, acting as a bioaffinity trap. The ID-LFIA/DART-QqQ-MS/MS approach has been developed and validated, along with the screening smartphone LFIA, and has demonstrated its applicability by analyzing incurred and spiked samples. The developed approach has been critically compared with our previous direct electrospray ionization MS method and was found to provide highly complementary information on the total deoxynivalenol contamination in the sample.

WFSR report 2021.014 | 9 Summary National Reference Laboratories (NRLs) are part of the system responsible for the control and enforcement of EU food and feed law. Wageningen Food Safety Research (WFSR) has been designated as the NRL for thirteen subjects. The tasks of a NRL depend on its research fields. This report gives an overview of the activities performed by all of NRLs of WFSR in 2020. These NRLs are for: milk and milk products, marine biotoxins, animal proteins, certain substances and residues thereof as laid down in Directive 96/23/EC, additives for use in animal nutrition (feed additives), genetically modified organisms (GMOs) in food and feed, pesticides, metals and nitrogenous substances in feed and food, mycotoxins and plant toxins in food and feed, processing contaminants, halogenated persistent organic pollutants in food and feed, food borne viruses and water content of poultry. This report first gives an overview of relevant legislation and information on the networks of EURLs, NRLs and OLs. For every NRL, a description is then given of all activities performed in the EURL-NRL network such as participation in EURL-NRL workshops, working groups, and proficiency and comparative tests. This is followed by a description of the assistance given to OLs in the form of quality control and/or advice. Finally, the scientific and technical support given to the competent authority is discussed. In some cases, the contact with other NRLs is discussed. An important NRL task is to stay up to date with current developments within its NRL domain. Every EURL organises one or two meetings (workshops) every year for that purpose. Participation in these EURL-NRL workshops is mandatory. In 2020, due to COVID-19, most of these workshops were online. All workshops have been attended by NRLs of WFSR. Additionally, the NRLs have actively participated in EURL working groups, to improve analytical methods. To test the analytical capabilities of NRLs, the EURLs organise proficiency tests. Due to EURL proficiency tests sometimes being limited in their scope, the NRLs have also participated in proficiency tests organised by other organizations if thought to be relevant. Most results (z-scores) in these proficiency tests were good; only a few ‘questionable’ and a few ‘unsatisfactorily’ result were reported. Follow-up actions were implemented in those cases. The performance of the OLs has been assured by checking the results of their performance in proficiency tests (organised by other laboratories or the NRL) or by sending assurance-samples. Some OLs have also received technical support with regard to their analyses.

In recent years, conjugated mycotoxins have gained increasing interest in food safety, as their hydrolysis in human and animal intestines leads to an increase in toxicity. For the production of zearalenone (ZEN) glycosides reference standards, we applied Cunninghamellaelegans and Cunninghamella echinulata fungal strains. A sulphate-depleted medium was designed for the preferred production of ZEN glycosides. Both Cunninghamella strains were able to produce zearalenone-14-β-D-glucopyranoside (Z14G), zearalenone-16-β-D-glucopyranoside (Z16G) and zearalenone-14-sulphate (Z14S). In a rich medium, Cunninghamellaelegans preferably produced Z14S, while Cunninghamellaechinulata preferably produced Z14G. In the sulphate-depleted medium a dramatic change was observed for Cunninghamellaelegans, showing preferred production of Z14G and Z16G. From 2 mg of ZEN in sulphate-depleted medium, 1.94 mg of Z14G and 0.45 mg of Z16G were produced. Following preparative Liquid Chromatography-Mass Spectrometry (LC-MS) purification, both fractions were submitted to 1H and 13C NMR and High-Resolution Mass Spectrometry (HRMS). These analyses confirmed that the purified fractions were indeed Z14G and Z16G. In conclusion, the presented research shows that a single Cunninghamella strain can be an effective and efficient tool for the controlled biotransformation of ZEN glycosides and other ZEN metabolites. Additionally, the biotransformation method was extended to zearalanone, β-zearalenol and other mycotoxins.

Tetrodotoxin (TTX) is a potent neurotoxin responsible for many food poisoning incidents and some fatalities. Although mainly associated with the consumption of pufferfish, in recent years, TTX has been found in shellfish, particularly in Europe. In this work, a magnetic bead (MB)-based colorimetric immunoassay was applied to the detection of TTX in Pacific oysters (Crassostrea gigas), razor clams (Solen marginatus) and mussels (Mytilus galloprovincialis). Effective LODs (eLODs) for TTX of 1 μg/kg in oysters and razor clams and 3.3 μg/kg in mussels, significantly below the EFSA guidance threshold (44 μg/kg), were obtained. The strategy was applied to the analysis of naturally-contaminated Pacific oysters (Crassostrea gigas) and mussels (Mytilus edulis) from the Netherlands, and TTX was detected in all samples. The approach, which takes less than 1.5 h, proved to be useful as a rapid and simple method to detect TTX, support shellfish safety and protect consumers. info:eu-repo/semantics/acceptedVersion

National Reference Laboratories (NRLs) are part of the system responsible for the control andenforcement of EU food and feed law. Wageningen Food Safety Research (WFSR) has been designated as the NRL for twelve subjects. The tasks of a NRL depend on its research fields. This report gives an overview of the activities performed by all of WFSR’s NRLs in 2019. These NRLs are for: halogenated persistent organic pollutants in food and feed, pesticides in products of animal origin, mycotoxins and plant toxins in food and feed, metals and nitrogenous substances in feed and food, p rocessing contaminants, marine biotoxins, certain substances and residues thereof as laid down in Directive 96/23/EC, genetically modified organisms (GMOs) in food and feed, animal proteins, additives for use in animal nutrition (feed additives), milk and milk products, and water content of poultry.This report first gives an overview of relevant legislation and information on the networks of EURLs, NRLs and OLs. For every NRL, a description is then given of all activities performed in the EURL-NRL network such as participation in EURL-NRL workshops, working groups, and proficiency and comparative tests. This is followed by a description of the assistance given to OLs in the form of quality control and/or advice. Finally, the scientific and technical support given to the competentauthority is discussed. In some cases, the contact with other NRLs is discussed.An important NRL task is to stay up to date with current developments within its NRL domain. Every EURL organises one or two meetings (workshops) every year for that purpose. Participation in these EURL-NRL workshops is mandatory. In 2019, 1 5 workshops have been attended by NRLs of WFSR (former name, before June 2019, RIKILT Wageningen University & Research). Additionally, the NRLs have actively participated in EURL working groups to improve analytical methods. To test the analytical capabilities of NRLs, the EURLs organise proficiency tests. Due to EURL proficiency tests sometimes being limited in their scope, the NRLs have also participated in proficiency tests organised by other organizations if thought to be relevant. Most results (z-scores) in these proficiency tests were good; only a few ‘questionable’ and a few ‘unsatisfactorily’ result were reported. Follow-up actions were implemented in those cases. The performance of the OLs has been assured by checking the results of their performance in proficiency tests (organised by other laboratories or the NRL) or by sending assurance-samples. Some OLs have also received technical support with regard to their analyses.

In 2019 zijn monsters Chinese wolhandkrab (Eriocheir sinensis) onderzocht van de locaties Hollands Diep, Nieuwe Maas bij Pernis en Volkerak (Volkeraksluizen en Krammersluizen). Op deze locaties (de zogenaamd gesloten gebieden) is commerciële wolhandkrabvangst verboden. Van de locatie Den Oever, liggend in het gebied dat niet gesloten is voor wolhandkrabvangst, kon geen monster verzameld worden omdat onvoldoende krab gevangen werd, en daarom is uitgeweken naar het Kornwerderzand.De contaminantgehalten in wolhandkrab van deze locaties wordt gemonitord en vergeleken met voorgaande jaren. Naast de dioxine- en polychloorbifenyl (PCB)-gehalten zijn de gehalten aan zware metalen en per- en polyfluoralkylstoffen (PFAS’s) onderzocht. Binnen deze studie is alleen bruin vlees uit het lichaam van de wolhandkrab onderzocht. Vlees uit poten en scharen is in deze studie niet onderzocht omdat daarvan bekend is dat dioxine- en PCB-gehalten niet boven de maximum limiet (ML) uitstijgen (mede omdat dit vlees weinig vet bevat). Voor vlees uit het lichaam gelden geen ML’s. In de wolhandkrab die in 2019 is onderzocht varieerden de gehalten van 8.6-31.8 pg TEQ/g product voor de dioxines, 10.5-3-29.7 pg TEQ/g product voor de dl-PCB’s, 19.1-59.1 pg TEQ/g product voor de totaal-TEQ en 162-945 ng/g product voor het totaal aan ndl-PCB’s. De bijdrage van de dioxines aan de totaal-TEQ varieerde van 44-65%. De wolhandkrab gevangen op de locaties Hollands-Diep en Maas (Pernis) zijn het sterkst gecontamineerd, en de gehalten ontlopen elkaar niet veel. De krab van locatie Kornwerderzand was het minst gecontamineerd. De trendgrafieken van de dioxine-TEQ, PCB-TEQ en totaal-TEQ gehalten voor de locaties Hollands-Diep en Volkerak (Volkeraksluizen en Krammersluizen) zijn stabiel binnen een bandbreedte van 20% (op basis van 4 jaren), terwijl de krab van Maas (Pernis) meer variatie laten zien. De aanwezigheid van meerdere PFAS’s is aangetoond, namelijk PFNA, PFUnDA, PFDoDA en PFOS. De PFOS gehalten waren het hoogst (6.1-20 ng/g). Wolhandkrab van het Kornwerderzand bevatte de hoogste PFAS’s gehalten. Wat betreft de zware metalen in wolhandkrab varieerden de gehalten als volgt: 0.044-0.22 mg/kg voor cadmium

In oral bioavailability studies, evaluation of the absorption and transport of drugs and food components across the intestinal barrier is crucial. Advances in the field of organ-on-a-chip technology have resulted in a dynamic gut-on-a-chip model that better mimics the in vivo microenvironment of the intestine. Despite a few recent integration attempts, ensuring a biologically relevant microenvironment while coupling with a fully online detection system still represents a major challenge. Herein, we designed an online technique to measure drug permeability and analyse unknown product formation across an intestinal epithelial layer of Caco-2 and HT29-MTX cells cultured on a flow-through Transwell system, while ensuring the quality and relevance of the biological model. Chip-based ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was coupled to the dynamic Transwell system via a series of switching valves, thus allowing alternating measurements of the apical and basolateral sides of the in vitro model. Two trap columns were integrated for online sample pre-treatment and compatibility enhancement. Temporal analysis of the intestinal permeability was successfully demonstrated using verapamil as a model drug and ergotamine epimers as a model for natural toxins present in foods. Evidence was obtained that our newly developed dynamic system provided reliable results versus classical static in vitro models, and moreover, for the first time, epimer-specific transport is shown for ergotamine. Finally, initial experiments with the drug granisetron suggest that metabolic activity can be studied as well, thus highlighting the versatility of the bio-integrated online analysis system developed. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-019-02336-6) contains supplementary material, which is available to authorized users.

In 2019 is in het kader van het monitoringsprogramma 'Contaminanten in vis uit Nederlandse binnenwateren' aal op 18 locaties bemonsterd.

A fast screening approach for simultaneous detection of twelve pesticides and seven mycotoxins in food and feed samples using flow injection (FI) mass spectrometry (MS) was developed and evaluated. Ten selected pesticides represented five pairs of structural isomers to challenge their MS differentiation without chromatographic separation. Samples of cereals/grains and animal feed were prepared using QuEChERS extraction, diluted and fortified with the selected contaminants to yield their representative maximum levels (MLs) or maximum residue levels (MRLs). Sensitivity of the developed approach was assessed for qualitative screening of the selected contaminants using triple quadrupole MS/MS and ion mobility (IM) HR-TOFMS. Despite the high complexity of the selected matrices and the absence of chromatography, limits of detection in fortified extracts were below established regulatory levels for 14 and 15 selected contaminants by FI-MS/MS and FI-IM-TOFMS analysis, respectively, in 36–100% samples. Experiments with ion mobility TOFMS did not result in successful separation of pesticide structural isomers. However, for three out of five pairs of pesticide structural isomers: cyproconazole and uniconazole, methiocarb and ethiofencarb, and vernolate and pebulate, unique fragment ions were found and confirmed with HR-QTOFMS for their MS differentiation. The developed approach is sensitive enough for rapid multi-residue screening of some pesticides and mycotoxins in complex samples of food and feed, providing high speed and throughput, simplicity and effective use of high-end instrumentation.

Lateral flow immunoassays (LFIAs) are widely used for rapid food safety screening analysis. Thanks to simplified protocols and smartphone readouts, LFIAs are expected to be increasingly used on-site, even by non-experts. As a typical follow-up in EU regulatory settings, suspect samples are sent to laboratories for confirmatory analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, re-analysis by LC-MS/MS is laborious and time-consuming. In this work, an identification LFIA (ID-LFIA) approach followed by quadrupole-orbitrap MS or triple quadrupole MS/MS analysis is presented. As a proof of concept, a dedicated ID-LFIA strip was developed for the mycotoxin deoxynivalenol (DON) following its initial screening by a commercial smartphone LFIA. The ID-LFIA strip can be simply immersed in the same sample extract used for the smartphone LFIA screening, and next, DON is retrieved from the monoclonal antibody with a dissociation solution consisting of methanol/ammonia. The solution thus obtained was analyzed directly in MS in order to rapidly confirm the presence of DON and any cross-reacting species. The protocol developed is capable of coping with severe ion suppression caused by LFIA buffers and nitrocellulose substrate residues. Initial analysis of blank, spiked, and incurred samples showed that the newly developed ID-LFIA-MS method was able to confirm the presence or absence of mycotoxins in the samples previously analyzed by LFIA and also differentiate between DON and DON 3-glucoside yielding the positive screening result. The concept and technique developed are envisaged to complement on-site screening and confirmation of any low molecular weight contaminant in future food control frameworks. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-020-02890-4) contains supplementary material, which is available to authorized users.

In food analysis, a trend towards on-site testing of quality and safety parameters is emerging. So far, on-site testing has been mainly explored by miniaturized optical spectroscopy and ligand-binding assay approaches such as lateral flow immunoassays and biosensors. However, for the analysis of multiple parameters at regulatory levels, mass spectrometry (MS) is the method of choice in food testing laboratories. Thanks to recent developments in ambient ionization and upcoming miniaturization of mass analyzers, (trans)portable mass spectrometry may be added to the toolkit for on-site testing and eventually compete with multiplex immunoassays in mixture analysis. In this study, we preliminary evaluated a selection of recent ambient ionization techniques for their potential in simplified testing of selected food contaminants such as pesticides, veterinary drugs, and natural toxins, aiming for a minimum in sample preparation while maintaining acceptable sensitivity and robustness. Matrix-assisted inlet ionization (MAI), handheld desorption atmospheric pressure chemical ionization (DAPCI), transmission-mode direct analysis in real time (TM-DART), and coated blade spray (CBS) were coupled to both benchtop Orbitrap and compact quadrupole single-stage mass analyzers, while CBS was also briefly studied on a benchtop triple-quadrupole MS. From the results, it can be concluded that for solid and liquid sample transmission configurations provide the highest sensitivity while upon addition of a stationary phase, such as in CBS, even low μg/L levels in urine samples can be achieved provided the additional selectivity of tandem mass spectrometry is exploited.

The emergence of marine biotoxins in geographical areas where they have never been reported before is a concern of considerable impact on seafood contamination, and consequently, on public health [...]

Over the past decades, accumulation of phycotoxins (toxins from marine algae) in shellfish and other marine biotoxins in seafood has received increasing attention. The latter is largely due to the greater number of algal blooms, the poisoning symptoms in humans resulting from ‘new’ biotoxins in areas where they had until recently not been observed and, finally, the discovery of new, highly potent toxins. The most frequently detected marine biotoxins represent a chemically very heterogeneous group of phycotoxins, the toxicity of which is generally related to a specific interaction of these toxins with ion channels of excitable membranes. Besides these neurotoxins, inhibitors of protein phosphatases have been described. On the basis of the clinical symptoms observed, we distinguish two groups of marine biotoxins: (1) diarrhetic shellfish poisoning toxins; and (2) marine neurotoxic biotoxins that can be found in seafood. In addition, primarily prevalent in fresh water, toxins can be synthesized by cyanobacteria that are also classified as algal toxins. These toxins, which exert hepatotoxic or neurotoxic effects, are mainly relevant as potential contaminants of drinking water and food supplements and are not included in this chapter.

Information on the occurrence and endocrine potencies of analogues of bisphenol A (BPA) and diglycidyl ester derivatives (BDGEs) of BPA and BPF is limited. Such information is, however, important as the current debate on BPA and the lowered BPA migration limit in Europe may provide an incentive for application of structural analogues. A new sensitive multi-analyte LC-ESI-MS/MS method was developed to measure 17 bisphenols (BPs) and 6 BDGEs in food, beverages and drinkware. Yeast based bioassays were used to determine the in vitro (anti)estrogenic and (anti)androgenic properties of these and 7 additional BPs and BDGEs. Drinkware of polycarbonate and other materials were analysed for BPs and BDGEs. Only BPA and BPS and both at trace levels were found in a few containers. A limited number of (canned) foods and beverages were also analysed. BPA was the most frequently detected BP (ranged from 0.03 ng mL−1 in a beverage sample to 68 ng g−1 in food). Other BPs detected were BPS, 2,2-BPF and 4,4-BPF. In addition BADGE, BADGE.HCl, BADGE.H2O and BADGE.2H2O were detected from 0.08 ng mL−1 in a beverage sample to 3.3 ng g−1 in food. In vitro testing showed that most BPs exhibited an equal or higher estrogenic potency than BPA and most of them also showed a higher anti-androgenic potency, i.e. BPB, BPCl, BPC, BPE, 4,4-BPF, BPP, BPAF, and BPTMC. Some BPs and BDGEs were not estrogenic, but showed an anti-estrogenic effect and were anti-androgenic too. BPS was only weakly estrogenic and BADGE.2H2O and BFDGE.2H2O showed no in vitro activity. The present data show that in addition to BPA, other BPs and BDGEs can be present in food and drinks, some displaying in vitro endocrine activities.

There are food and feed safety monitoring programs to protect consumers. These programs however, are strongly focused on known and regulated substances. New or unexpected substances that might be of risk for consumers will thus escape routine controls. These risks are therefore mainly discovered by human or animal intoxications. All kind of analytical chemical methods, in-vitro bioassays, tracking, and chain analysis are then used to reveal the substance(s) responsible for the intoxication. Only in a few occasions (new) risks were revealed in time by analytical chemical methods or cell based in-vitro bioassays. This paper describes some relevant food and feed safety cases and how the causative substances were identified. This overview strongly indicates that more intense monitoring, including the use of cell based effect bioassays, can reduce the number of intoxications. Moreover, registration and follow-up actions should be arranged in a better way, for example by sharing information within the scientific communities or by establishing a national contact point. In addition, a strategy based on broad screening and bioassay directed identification with liquid chromatography high resolution mass spectrometry is proposed to prevent intoxications and identify toxin and toxicants relevant for food and feed safety.

The goal of the present study was to obtain an in vivo relevant prioritization method for the endocrine potencies of different polycarbonate monomers, by combining in vitro bioassay data with physiologically based kinetic (PBK) modelling. PBK models were developed for a selection of monomers, including bisphenol A (BPA), two bisphenol F (BPF) isomers and four different bisphenol A diglycidyl ethers (BADGEs), using in vitro input data. With these models, the plasma concentrations of the compounds were simulated, providing means to estimate the dose levels at which the in vitro endocrine effect concentrations are reached. The results revealed that, whereas the in vitro relative potencies of different BADGEs (predominantly anti-androgenic effects) can be up to fourfold higher than BPA, the estimated in vivo potencies based on the oral equivalent doses are one to two orders of magnitude lower than BPA because of fast detoxification of the BADGEs. In contrast, the relative potencies of 2,2-BPF and 4,4-BPF increase when accounting for the in vivo availability. 4,4-BPF is estimated to be fivefold more potent than BPA in humans in vivo in inducing estrogenic effects and both 2,2-BPF and 4,4-BPF are estimated to be, respectively, 7 and 11-fold more potent in inducing anti-androgenic effects. These relative potencies were considered to be first-tier estimates, particularly given that the potential influence of intestinal metabolism on the in vivo availability was not accounted for. Overall, it can be concluded that both 2,2-BPF and 4,4-BPF are priority compounds.

Paralytic shellfish toxins (PSTs) in bivalve molluscs represent a public health risk and are controlled via compliance with a regulatory limit of 0.8 mg saxitoxin (STX)⋅2HCl equivalents per kilogram of shellfish meat (eq/kg). Shellfish industries would benefit from the use of rapid immunological screening tests for PSTs to be used for regulation, but to date none have been fully validated. An interlaboratory study involving 16 laboratories was performed to determine the suitability of the Neogen test to detect PSTs in mussels and oysters. Participants performed the standard protocol recommended by the manufacturer and a modified protocol with a conversion step to improve detection of gonyautoxin 1&4. The statistical analysis showed that the protocols had good homogeneity across all laboratories, with satisfactory repeatability, laboratory, and reproducibility variation near the regulatory level. The mean probability of detection (POD) at 0.8 mg STX⋅2HCl eq/kg using the standard protocol in mussels and oysters was 0.966 and 0.997, respectively, and 0.968 and 0.966 using the modified protocol. The estimated LOD in mussels was 0.316 mg STX⋅2HCl eq/kg with the standard and 0.682 mg STX⋅2HCl eq/kg with the modified protocol, and 0.710 and 0.734 mg STX⋅2HCl eq/kg for oysters, respectively. The Neogen test may be acceptable for regulatory purposes for oysters in accordance with European Commission directives in which the standard protocol provides, at the regulatory level, a probability of a negative response of 0.033 on 95% of occasions. Its use for mussels is less consistent at the regulatory level due to the wide prediction interval around the POD.

Marine biotoxins in fish and shellfish can cause several symptoms in consumers, such as diarrhea, amnesia, or even death by paralysis. Monitoring programs are in place for testing shellfish on a regular basis. In some countries testing is performed using the so-called mouse bioassay, an assay that faces ethical concerns not only because of animal distress, but also because it lacks specificity and results in high amounts of false positives. In Europe, for lipophilic marine biotoxins (LMBs), a chemical analytical method using LC-MS/MS was developed as an alternative and is now the reference method. However, safety is often questioned when relying solely on such a method, and as a result, the mouse bioassay might still be used. In this study the use of a cell-based assay for screening, i.e., the neuro-2a assay, in combination with the official LC-MS/MS method was investigated as a new alternative strategy for the detection and quantification of LMBs. To this end, samples that had been tested previously with the mouse bioassay were analyzed in the neuro-2a bioassay and the LC-MS/MS method. The neuro-2a bioassay was able to detect all LMBs at the regulatory levels and all samples that tested positive in the mouse bioassay were also suspect in the neuro-2a bioassay. In most cases, these samples contained toxin levels (yessotoxins) that explain the outcome of the bioassay but did not exceed the established maximum permitted levels.

The recent detection of tetrodotoxins (TTXs) in puffer fish and shellfish in Europe highlights the necessity to monitor the levels of TTXs in seafood by rapid, specific, sensitive and reliable methods in order to protect human consumers. A previous immunoassay for TTX detection in puffer fish, based on the use of self-assembled monolayers (SAMs) for the immobilization of TTX on maleimide plates (mELISA), has been modified and adapted to the analysis of oyster and mussel samples. Changing dithiol for cysteamine-based SAMs enabled reductions in the assay time and cost, while maintaining the sensitivity of the assay. The mELISA showed high selectivity for TTX since the antibody did not cross-react with co-occurring paralytic shellfish poisoning (PSP) toxins and no interferences were observed from arginine (Arg). Moreover, TTX-coated maleimide plates stored for 3 months at −20 °C and 4 °C were stable, thus when pre-prepared, the time to perform the assay is reduced. When analyzing shellfish samples, matrix effects and toxin recovery values strongly depended on the shellfish type and the sample treatment. Blank oyster extracts could be directly analyzed without solid-phase extraction (SPE) clean-up, whereas blank mussel extracts showed strong matrix effects and SPE and subsequent solvent evaporation were required for removal. However, the SPE clean-up and evaporation resulted in toxin loss. Toxin recovery values were taken as correction factors (CFs) and were applied to the quantification of TTX contents in the analysis of naturally-contaminated shellfish samples by mELISA. The lowest effective limits of detection (eLODs) were about 20 and 50 µg/kg for oyster extracts without and with SPE clean-up, respectively, and about 30 µg/kg for mussel extracts with both protocols, all of them substantially below the eLOD attained in the previous mELISA for puffer fish (230 µg/kg). Analysis of naturally-contaminated samples by mELISA and comparison with LC-MS/MS quantifications demonstrated the viability of the approach. This mELISA is a selective and sensitive tool for the rapid detection of TTX in oyster and mussel samples showing promise to be implemented in routine monitoring programs to protect human health. info:eu-repo/semantics/acceptedVersion

National Reference Laboratories (NRLs) are part of the system responsible for the control and enforcement of EU food and feed law. RIKILT Wageningen University & Research has been designated as the NRL for twelve subjects. The tasks of a NRL depend on its research field. This report gives an overview of the activities performed by all of RIKILT's NRLs in 2016.

Marine biotoxins are mostly produced by phytoplankton. Proliferation of algae producing marine biotoxins, also known as harmful algal bloom (HAB), occurs worldwide. Such event depends on environmental conditions, including temperature, water pH/salinity, current patterns and anthropogenic nutrient input. Marine biotoxins can accumulate in seafood products and as such present a threat to consumers. This paper reviews and compiles up-to-date literature on reported human intoxications following exposure to marine biotoxins through seafood consumption. The review includes a discussion about prevention of such outbreaks and surveillance programs to identify possible limitations and approaches for limiting the impact of HABs on human health. It is concluded that marine biotoxins represent a threat to human health as thousands of poisonings following consumption of seafood contaminated with marine biotoxins were reported in the 21st century, emphasizing the need for carrying on/developing surveillance programs to detect the presence of HABs, and for development, validation and implementation of sensitive high-throughput methods for detecting these biotoxins in seafood to protect consumers. Regarding the possible presence of unknown toxins and general lack of standards for many known toxins, in vitro effect-based bioassays may play an important role in the monitoring for biotoxins.

Marine biotoxins are natural toxins produced by specific algae species. These toxins can accumulate in seafood such as mussels, oysters and fish. Consumption of contaminated seafood may lead to severe intoxication such as memory loss, paralysis, diarrhoea and even death. In order to protect consumers, a wide variety of methods have been developed. Historically, methods were based on in vivo animal assays. Alternatives for these animal unfriendly and unethical assays have been developed and are still under development. Other alternative methods are based on in vitro cell assays, receptor binding assays, immunological assays and analytical chemical techniques. This chapter gives an overview on the available methodologies and their application for the determination of marine biotoxins in seafood.

The dinoflagellate Alexandrium ostenfeldii is a well-known harmful algal species that can potentially cause paralytic shellfish poisoning (PSP). Usually A. ostenfeldii occurs in low background concentrations only, but in August of 2012 an exceptionally dense bloom of more than 1 million cells L-1 occurred in the brackish Ouwerkerkse Kreek in The Netherlands. The A. ostenfeldii bloom produced both saxitoxins and spirolides, and is held responsible for the death of a dog with a high saxitoxin stomach content. The Ouwerkerkse Kreek routinely discharges its water into the adjacent Oosterschelde estuary, and an immediate reduction of the bloom was required to avoid contamination of extensive shellfish grounds. Previously, treatment of infected waters with hydrogen peroxide (H2O2) successfully suppressed cyanobacterial blooms in lakes. Therefore, we adapted this treatment to eradicate the Alexandrium bloom using a three-step approach. First, we investigated the required H2O2 dosage in laboratory experiments with A. ostenfeldii. Second, we tested the method in a small, isolated canal adjacent to the Ouwerkerkse Kreek. Finally, we brought 50 mg L-1 of H2O2 into the entire creek system with a special device, called a water harrow, for optimal dispersal of the added H2O2. Concentrations of both vegetative cells and pellicle cysts declined by 99.8% within 48 h, and PSP toxin concentrations in the water were reduced below local regulatory levels of 15 µg L-1. Zooplankton were strongly affected by the H2O2 treatment, but impacts on macroinvertebrates and fish were minimal. A key advantage of this method is that the added H2O2 decays to water and oxygen within a few days, which enables rapid recovery of the system after the treatment. This is the first successful field application of H2O2 to suppress a marine harmful algal bloom, although Alexandrium spp. reoccurred at lower concentrations in the following year. The results show that H2O2 treatment provides an effective emergency management option to mitigate toxic Alexandrium blooms, especially when immediate action is required.

Marine biotoxins can accumulate in fish and shellfish, representing a possible threat for consumers. Many marine biotoxins affect neuronal function essentially through their interaction with ion channels or receptors, leading to different symptoms including paralysis and even death. The detection of marine biotoxins in seafood products is therefore a priority. Official methods for control are often still using in vivo assays, such as the mouse bioassay. This test is considered unethical and the development of alternative assays is urgently required. Chemical analyses as well as in vitro assays have been developed to detect marine biotoxins in seafood. However, most of the current in vitro alternatives to animal testing present disadvantages: low throughput and lack of sensitivity resulting in a high number of false-negative results. Thus, there is an urgent need for the development of new in vitro tests that would allow the detection of marine biotoxins in seafood products at a low cost, with high throughput combined with high sensitivity, reproducibility, and predictivity. Mode of action based in vitro bioassays may provide tools that fulfil these requirements. This review covers the current state of the art of such mode of action based alternative assays to detect neurotoxic marine biotoxins in seafood.

A generic high-throughput bioaffinity liquid chromatography-mass spectrometry (BioMS) approach was developed and applied for the screening and identification of known and unknown recombinant human sex hormone-binding globulin (rhSHBG)-binding designer steroids in dietary supplements. For screening, a semi-automated competitive inhibition binding assay was combined with fast ultrahigh-performance-LC-electrospray ionization-triple-quadrupole-MS (UPLC-QqQ-MS). 17β-Testosterone-D3 was used as the stable isotope label of which the binding to rhSHBG-coated paramagnetic microbeads was inhibited by any other binding (designer) steroid. The assay was performed in a 96-well plate and combined with the fast LC-MS, 96 measurements could be performed within 4 h. The concentration-dependent inhibition of the label by steroids in buffer and dietary supplements was demonstrated. Following an adjusted bioaffinity isolation procedure, suspect extracts were injected into a chip-UPLC(NanoTile)-Q-time-of-flight-MS system for full-scan accurate mass identification. Next to known steroids, 1-testosterone was identified in three of the supplements studied and the designer steroid tetrahydrogestrinone was identified in a spiked supplement. The generic steroid-binding assay can be used for high-throughput screening of androgens, estrogens, and gestagens in dietary supplements to fight doping. When combined with chip-UPLC-MS, it is a powerful tool for early warning of unknown emerging rhSHBG bioactive designer steroids in dietary supplements.

A new LC-MS/MS method for the separation and detection of the most prominent marine lipophilic toxin groups comprising okadaic acid, dinophysistoxins, yessotoxins, azaspiracids, pectenotoxins, spirolides and some okadaic acid fatty acid esters has been developed. With this method 28 different marine lipophilic biotoxins can be analysed in a single run. Separation was achieved with an acetonitrile/water gradient containing ammonium hydroxide (pH 11). All toxins were stable under these basic conditions. Compared to chromatography using an acidic gradient, the limit of detection (LODs) for okadaic acid, yessotoxin, gymnodimine and 13-desmethyl spirolide C were improved two- to three-fold, mainly due to better peak shapes. The azaspiracids and pectenotoxins-2 showed comparable LODs under acidic and basic conditions. A major advantage of the developed method is that toxins can be clustered in retention time windows separated for positively and negatively ionized molecular ions. Therefore, there is no need for rapid polarity switching or two separate runs for one sample. The new method showed good repeatability and reproducibility and is an important step in the development of alternatives to the animal tests currently in use for shellfish toxin analysis. © 2009 Elsevier B.V. All rights reserved.

The performances of four different mass spectrometers [triple-quadrupole (TQ), time-of-flight (ToF), quadrupole ToF (Q-ToF) and ion trap (IT)] for the detection of the marine lipophilic toxins pectenotoxin-2 (PTX2) and okadaic acid (OA) were investigated. The spectral data obtained with the different mass spectrometric analyzers were used to propose fragmentation schemes for PTX2 in the positive electrospray mode and for OA in the negative electrospray mode. TQ data were used to obtain product ions, while ToF and Q-ToF-MS produced accurate mass data of the precursor ion and product ions, respectively. IT data provided a better understanding of the fragmentation pathways using MS(n) experiments. With respect to analytical performance, all four mass analyzers showed a good linearity (R(2)0.97) and repeatability (CV20%). Detection limits (LoDs) (S/N = 3) were the lowest on triple-quad MS: 12.2 and 2.9 pg on-column for PTX2 and OA, respectively.

Excited-state intramolecular proton transfer (ESIPT) occurring in the salicylic acid (SA) derivative 5-methoxysalicylic acid (5-MeOSA) in an apolar solvent (cyclohexane) and in the presence of the hydrogen bond accepting agent diethyl ether (DEE) is investigated. Analysis of the directly measured subnanosecond time-resolved emission spectra (TRES) together with conventional steady-state fluorescence and time-correlated single-photon-counting (TCSPC) decays indicates that ESIPT in this system occurs much faster than fluorescence, and that the equilibrium between normal and tautomeric excited states is established before the emission from both states takes place. However, changes in time- and frequency-resolved fluorescence of the 5-MeOSA/DEE complex are observed due to structural relaxation within the complex, which is reflected in the dynamic Stokes shift of the tautomeric fluorescence band. The normal fluorescence band of 5-MeOSA/ DEE does not exhibit marked changes within the investigated time range. A single-exponential relaxation time of 460 ps was determined for the dynamic Stokes shift of the tautomeric band, and it is attributed to a geometric change within the 5-MeOSA/DEE complex upon excitation. Since both tautomeric and normal emission bands are well resolved and exhibit different time-dependent behaviors, a double-well potential appears to be adequate to describe the excited state of the system studied.

In this preliminary study, we evaluated four different types of substrate for the at-line coupling of capillary electrophoresis and surface-enhanced (resonance) Raman spectroscopy, CE-SER(R)S, with emphasis on spectral repeatability. We tested Sub1: etched silver foil, Sub2: a vapour-deposited silver film, Sub3: a silver oxalate-precoated silica TLC plate and Sub4: a silica TLC plate on which colloid and poly(L-lysine) were manually added to the analyte spots, used earlier in at-line CE-SE(R)RS. All substrates were first tested by manual spotting using trans-1,2-bis(4-pyridyl)ethylene (BPE) as a model compound for SERS and crystal violet (CV) as a model compound for SE(R)RS. The spectral features of the SE(R)RS spectra of BPE and CV showed a most satisfactory repeatability on all substrates. As expected, the signal intensities varied considerably between runs; this implies that quantification in at-line CE-SE(R)RS should rather be done by means of an on-line absorbance detector. In addition, the suitability of Sub1, Sub2 and Sub4 as deposition substrates after CE was explored using two cationic dyes: CV and basic fuchsin (BF). Good-quality SERRS spectra could be recorded on all three substrates. Although Sub1 and Sub2 have a poor water-sorptivity, they were found to be good substrates for at-line CE-SERRS. They do not require any post-deposition addition of silver colloid and could therefore become attractive alternatives for Sub4. © 2003 Elsevier B.V. All rights reserved.

The last few years have brought about many changes in the field of marine and freshwater toxins, with advances in analytical technology and the realization that these toxins are a global issue. Offering a complete reference guide, Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection, Third Edition addresses all aspects of the social and scientific influence of phytotoxins, from legislation and monitoring to new drug development. Covering many new topics, the book examines three main aspects: monitoring of toxins; chemical, mechanistic, and toxicological diversity; and detection technologies.

Shellfish products may be contaminated with marine biotoxins which, after consumption, may lead to human illness. The Netherlands has a regular monitoring programme for marine biotoxins and the possible toxic phytoplankton in shellfish production waters. The aim of the current study was to evaluate the presence of potential toxic phytoplankton species and marine biotoxins in Dutch production waters over the last decade, and to analyse the relationship between toxin levels and abundance of possible causative phytoplankton species. The results of the monitoring programme of the period 1999-2009 were used. The presence of Alexandrium spp. were negligible, but Pseudo-nitzschia spp. and phytoplankton causing diarrhetic shellfish poisoning (DSP toxin-producing phytoplankton) were present in nearly all three main production areas and years. The main DSP toxin-producing species was Dinophysis acuminata followed by D. rotundata and Prorocentrum lima. Toxins causing paralytic shellfish poisoning (PSP) and amnesic shellfish poisoning (ASP) were present in only a few individual shellfish samples, all at low levels. At the end of 2002, an episode of DSP toxicity was recorded, based on the rat bioassay results. Of the samples that were chemically analysed for DSP toxins in 2007 and 2008, about half of the samples in 2007 contained these toxins, although levels were low and no positive results were obtained using the rat bioassay. There was a slight positive correlation between concentrations of DSP toxin-producing phytoplankton and levels of DSP toxins in 2007. Increased DSP toxin levels were found up to 5 weeks after the peak in DSP toxin-producing phytoplankton. This positive, but weak, relationship needs to be confirmed in future research using more samples and chemical methods to quantify the presence of DSP toxins. If this relationship is further substantiated and quantified, it could be used within the current monitoring programme in the Netherlands to predict the risk areas regarding DSP toxicity in shellfish.