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Food and feed can be contaminated with microorganisms, parasites and chemical substances, which can have light or severe toxicological effects. Therefore the European Union (EU) has established food and feed safety regulations. Combined with organized control strategies, they help to ensure the safety of food and feed that enter the production chain. Mycotoxins are produced by fungi and can be major food and feed contaminants. Upon ingestion, mycotoxins can cause mycotoxicosis. The symptoms of mycotoxicosis can be both acute and chronic, and range from nausea to cancer, and even death. Grain commodities are the basis for food and feed, and these can be contaminated by what are considered the most important mycotoxins in terms of agriculture and animal production; zearalenone (ZEN), deoxynivalenol (DON), T-2 toxin (T-2), aflatoxins (AFs), ochratoxin A (OTA) and fumonisins (FBs). The main aim of the research in this thesis, was to develop a multiplex immunoassay method capable of detecting the main 6 mycotoxins, and their modified forms, in grain-based feed and food commodities. For the development of the mycotoxin multiplex immunoassays, we implemented the multi-Analyte profiling (xMAP) technology from Luminex. This xMAP technology combines unique color-encoded microspheres in a suspension array format, which can be analysed by dedicated analyser platforms consisting of dedicated flow cytometers and a planar array analyser. The developed multiplex should be able to screen for the main six mycotoxins at food and feed ML levels in a wide range of matrices. This thesis gives a comprehensible look into the main mycotoxins. Their history, toxicity, occurrence and EU recommendations and legislation are summarized. A special focus is on the modified mycotoxins. These are the free forms of mycotoxins that are modified by living organisms like plants, bacteria and fungi. Since antibodies are the key biorecognition molecules for the developed multiplex immunoassays presented in this thesis, they are shortly explained, followed by their implementation in the multiplex mycotoxin detection immunoassays.For the multiplex screening of food and feed matrices a semi-quantitative multiplex competitive immunoassay was developed for the detection of ZEN, DON, T-2, AFs, OTA and FBs. In the intial indirect format, protein-mycotoxin conjugates were coupled to the unique xMAP microspheres, who were then incubated with free mycotoxins (in samples) and mycotoxin specific antibodies. The developed method was critically tested by applying it to contaminated feed reference samples and the results were critically compared to confirmatory instrumental analysis of the same reference samples. The second approach presented is a faster direct detection format, introducing fluorescent mycotoxin-protein conjugates. In the direct format, monoclonal antibodies (mAbs) were coupled to the microspheres and incubated with free mycotoxins and the fluorescent mycotoxin-conjugates. The new 6-plex direct multiplex was applied to barley samples, for a successful in-house validation as a screening method. The cut-off factors were set at 50% of the EU maximum levels (MLs) for food: 2 µg/kg for AFB1, 2.5 µg/kg for OTA, 625 µg/kg for DON, 50 µg/kg for ZEN, 1000 µg/kg for FB1 and 25 µg/kg for T-2. The validation showed very high inter and intra-day precision for all samples. The direct 6-plex was also used to screen available barley and malted barley reference materials. The developed direct 6-plex assay showed potential for future implementation as a semi-quantitative pre-screening method for regulated mycotoxins, prior to instrumental analysis (like LC-MS/MS). Simplifications were introduced that could make the method more suitable for future on-site application. These simplifications comprised; washing steps by a handheld magnetic plate instead of an automated washer, addition of pre-mixed reagents from dropper bottles, instead of laboratory pipettes, addition of samples by disposable fixed volume micropipettes, and a sample extraction time of just 1 minute, additionally omitting centrifugation. Preliminary in-house validation of the simplified multiplex screening assay, using an on-site extraction procedure, was successful at EU maximum levels using the portable MAGPIX planar array analyzer.A major part of this thesis is dedicated to a large global survey for mycotoxin occurence in beer with the aforementioned direct 6-plex. Besides the high number of screened samples, 1000 in total, there was a really strong focus on beers from the emerging craft beer scene. Besides the application of the 6-plex, a confirmatory analysis method (LC-MS/MS) was developed and applied to a selection of samples based on the acquired screening results. The major mycotoxins detected were DON and its plant metabolite deoxynivalenol-3-β-D-glucopyranoside (D3G). The 6-plex immunoassay reported the sum of DON and D3G (DON+D3G) contaminations ranging from 10 to 475 µg/L in 406 beers, of which 73% were craft beers. The popular craft beer style imperial stout, had the highest percentage of samples suspected positive (83%) with 29% of all imperial stout beers having DON+D3G contaminations above 100 µg/L. LC-MS/MS analysis showed that industrial pale lagers from Italy and Spain, predominantly contained FBs (3 – 69 µg/L). Besides FBs, some African traditional beers also contained aflatoxins (0.1 - 1.2 µg/L). The presence of OTA, T-2, HT-2, ZEN, β-zearalenol, 3/15-acetyl-DON, nivalenol and the conjugated mycotoxin zearalenone 14-sulfate were confirmed in some beers. In 27 craft beers, DON+D3G concentrations occurred above (or at) the Tolerable Daily Intake (TDI) level, which may have a health impact upon consumption of these beers.The increased relevance of modified mycotoxins, and their inclusion in the process of the development of the presented mycotoxin multiplexes in this thesis, showed the importance of having access to novel reference substances. Therefore Cunninghamella strains were introduced as bioconjugation models. Liquid media cultures of Cunninghamella were fortified with a range of mycotoxins. The chosen Cunninghamella strains were able to produce a range of ZEN metabolites, including zearalenone-14-β-D-glucopyranoside (Z14G), zearalenone-16-β-D-glucopyranoside (Z16G) and zearalenone-14-sulphate (Z14S).&nbsp

Currently, static in vitro cell culture assays are used in early phases of drug development, food research and hazard identification of chemicals. However, these in vitro models lack organ specific functionality, hampering mechanism-based research needed for novel drug development and next generation risk assessment. Recent advances in microchip- and bio-engineering enabled the development of organ-on-a-chip models. The main technical advantages of organ-on-a-chip technology are the ability to spatiotemporally control the microenvironment and the low reagent consumption. On the other hand, micro-engineered organ-on-a-chip models may still lack robustness for reproducible and bio-relevant studies. In addition, also the low flow rates present a major challenge of organ-on-a-chip technology: how to detect drug uptake and compound metabolism in real-time and at a high temporal resolution at the microscale.In this thesis a dynamic intestinal cell culture device is coupled to highly advanced mass spectrometry equipment, aiming for automated and online analysis of translocation of drugs, natural toxins and nanoparticles, while maintaining the bio-integrity of the cell system. Apart from technical robustness challenges, the fundamental chemical incompatibility between complex biological systems used in cell culturing and the clean sample requirements of sensitive advanced analytical instrumentation need to be resolved. Furthermore, the dynamic cell culture model is placed outside of an incubator for the integration with the mass spectrometer. However, physiological relevant temperatures and pH levels still need to be regulated for a reliable biological experiment. As well as the evaluation of the barrier integrity of the intestinal cell layer an aspect largely overlooked by current literature.In Chapter 2 an overview is provided of the current knowledge regarding analytical techniques integrated with organ-on-a-chip systems. In Chapter 3a a dynamic flow-through in vitro model was developed to predict permeability across the intestine using a well-known model drug, verapamil, to benchmark our results against literature. In Chapter 3b the stereoselective permeability of the natural mycotoxin ergotamine was examined using the model established in chapter 3a. The dynamic flow-through in vitro model system was expanded in Chapter 4. A novel bio-integrated online total analysis system for intestinal absorption and metabolism was developed by successfully interfacing a flow-through transwell with electrospray ionization mass spectrometry. The main advantages of this system being the integration of a relevant biological model with fully automated online analysis, which was missing before. Chapter 5 combines a digestion-on-a-chip with the bio-integrated analysis system developed in chapter 4, including the complexity of digestion creating a more complete bio-availability model. In Chapter 6 the integration of the flow-through transwell with inductively coupled plasma mass spectrometry was realized for the evaluation of the translocation of model gold nanoparticles. Lastly, in Chapter 7 a general discussion on the topics described in this thesis is provided and look to the future of organ-on-a-chip technology, with special emphasis on the integration with mass spectrometry analysis.

Several molecular imaging techniques are available to study and understand biological objects, like positron-emission tomography, fluorescence, and magnetic resonance imaging. These techniques often require chemical probes and image in a targeted approach. As many biological questions can only be answered in a systems approach, molecular imaging methods that can simultaneously measure many molecules are desired. Mass spectrometry imaging (MSI) is capable of measuring many molecules simultaneously without the use of chemical probes. MSI experiments often require sample stage vacuum conditions and extensive sample pretreatment such as matrix application. Vacuum conditions can disrupt or damage biological samples, and sample pretreatment prevents real-time analyses and can cause analyte losses, analyte delocalization and denaturation of proteins. Ambient ionization was introduced to measure samples under ambient conditions without any sample pretreatment, and ambient MSI followed rapidly after that. A next generation of ambient MSI techniques is desired to improve its sensitivity, molecular mass range, and spatial resolution. This work aims to improve the capabilities and broaden the scope of laser ablation electrospray ionization (LAESI) MSI. In order to broaden the scope and increase the understanding of ambient LAESI-MS(I), polymer materials and synthetic fibers were investigated. The direct analysis of synthetic fibers under ambient conditions is highly desired to identify the polymer, the finishes applied and irregularities that may compromise its performance and value. In Chapter 2 LAESI ion mobility MS was used for the analysis of synthetic polymers and fibers. The key to this analysis was the absorption of laser light by aliphatic and aromatic nitrogen functionalities in the polymers. Analysis of polyamide (PA) 6, 46, 66, and 12 pellets and PA 6, 66, polyaramid and M5 fibers yielded characteristic fragment ions, enabling their unambiguous identification. Synthetic fibers are, in addition, commonly covered with a surface layer for improved adhesion and processing. The same setup, but operated in a transient infrared matrix-assisted laser desorption electrospray ionization mode, allowed the detailed characterization of the fiber finish layer and the underlying polymer. Differences in finish layer distribution may cause variations in local properties of synthetic fibers. In Chapter 2, also the feasibility of mass spectrometry imaging (MSI) of the distribution of a finish layer on the synthetic fiber and the successful detection of local surface defects was shown. Reactions in confined compartments like charged microdroplets are of increasing interest, notably because of their substantially increased reaction rates. When combined with ambient MS, reactions in charged microdroplets can be used to improve the detection of analytes or to study the molecular details of the reactions in real time. In Chapter 3, we introduce a reactive LAESI time-resolved MS method to perform and study reactions in charged microdroplets. This approach was demonstrated with so-called click chemistry reactions between substituted tetrazines and a strained alkyne or alkene. Click reactions are high-yielding reactions with a high atom efficiency. Although click reactions are typically at least moderately fast, in a reactive LAESI approach a substantial increase of reaction time is Summary 149 required for these reactions to occur. This increase was achieved using microdroplet chemistry and followed by MS using the insertion of a reaction tube between the LAESI source and the MS inlet, leading to near complete conversions due to significantly extended microdroplet lifetime. This novel approach allowed for the collection of kinetic data for a model click reaction and showed in addition excellent instrument stability, improved sensitivity, and applicability to other click reactions. In Chapter 3, reactive LAESI was also demonstrated in a mass spectrometry imaging setting to show its feasibility in future imaging experiments. In drug discovery it is important to identify phase I metabolic modifications as early as possible to screen for inactivation of drugs and/or activation of prodrugs. As the major class of reactions in phase I metabolism are oxidation reactions, oxidation of drugs with TiO2 photocatalysis can be used as a simple non-biological method to initially eliminate (pro)drug candidates with an undesired phase I oxidation metabolism. Analysis of reaction products is commonly achieved with mass spectrometry coupled to chromatography. However, sample throughput can be substantially increased by eliminating pretreatment steps and exploiting the potential of ambient MS. Furthermore, online monitoring of reactions in a time-resolved way would identify sequential modification steps. In Chapter 4 we introduce a novel (time-resolved) TiO2-photocatalysis LAESI-MS method for the analysis of drug candidates. This method was proven to be compatible with both TiO2-coated glass slides as well as solutions containing suspended TiO2 nanoparticles, and the results were in excellent agreement with studies on biological oxidation of several drugs. Additionally, a time-resolved LAESI-MS setup was developed and results for verapamil showed excellent analytical stability for online photocatalyzed oxidation reactions within the set-up up to at least one hour. Identification and confirmation of (bio)chemical entities in ambient MS mostly involves accurate mass determination, often in combination with MS/MS work flows. However, an accurate mass only provides the elemental composition of the (bio)molecule, still resulting in numerous possible structures. MS/MS procedures are often insufficient in differentiating between the hundreds possible candidate substances in database searches. Obtaining additional information and thereby improving structural assignment as well as reducing the vast number of possible candidates is thus of high importance in any ambient MS(I) study. In Chapter 5 we present an ambient hydrogen/deuterium exchange (HDX) LAESI-MS method for structure elucidation and confirmation of (bio)molecules. The concept was demonstrated with small molecules, peptides, and proteins. Moreover, the same approach could be applied to MSI as shown by the ambient MSI of arginine and oligosaccharides on an orange slice. Eventually, this approach will allow spatially resolved MSI of different protein conformers and may have a major impact in the life sciences. The main achievements that are described in this thesis offer insights on sample compatibility, hardware improvements to enable online time-resolved reactions and structure elucidation approaches. The outcome of the research chapters shows that LAESI-MS(I) is a highly versatile technique applicable to many research areas. Although the technique is highly 150 dependent on endogenous water in samples for analysis of intact molecules, LAESI can also be exploited for the analysis and identification of (water free) polymer materials. Unfortunately, LAESI sensitivity relative to electrospray ionization is weak, and therefore the technique can currently not live up to the status of the next generation of ambient MSI. Analytes that are present in high abundance are feasible for imaging by LAESI-MS. For low abundance analytes, however, several hardware improvements are required to substantially increase the sensitivity of the results. When the hardware improvements are developed and implemented, the road is open for many end users in, e.g., microbiology, pathology, and botany, to make significant breakthroughs in their fields.