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The amalgamation of computer-like capabilities and portability of modern smartphones has fuelled their implementation as detectors and interfaces in emerging smartphone-based (bio)sensors (SbSs) for e.g. healthcare, point-of-need, food safety, environmental science, and forensics systems. SbSs intrinsically carry great potential for consumer diagnostics, and future ‘citizen science’ approaches, which have far-reaching implications for the technological, legal, and ethical aspects associated with the research, development, and deployment of SbSs. In this review (part 2 of a pair of review papers), we evaluated the pertinent literature on issues concerning the development and validation of SbSs, and we address their potential social impact. Finally, insights gleaned are combined in a set of recommendations to guide future ethical, sustainable, and efficient research, development, and deployment of SbSs.

Smartphones are ubiquitous in modern society; in 2021, the number of active subscriptions surpassed 6 billion. These devices have become more than a means of communication; smartphones are powerful, continuously connected, miniaturized computers capable of passively and actively collecting (private) information for us and from us. Their implementation as detectors or instrumental interfaces in emerging smartphone-based (bio)sensors (SbSs) has facilitated a shift towards portable point-of-care platforms for healthcare and point-of-need systems for food safety, environmental monitoring, and forensic applications. These familiar, handheld devices have the capacity to popularize analytical chemistry by simplifying complicated laboratory protocols and automating advanced data handling without requiring expensive equipment or trained analysts. To elucidate the technological, legal, and ethical challenges associated with developing SbSs, we reviewed the existing literature (2016–2021), providing an in-depth critical analysis of state-of-the-art optical and electrochemical SbSs. This analysis revealed the key areas to consider for emerging SbSs, which we will address in a set of review papers. Part I (this review) will consider (i) how the SbS data are acquired and processed and (ii) the implementation of privacy and data protection strategies to keep this data secure. Part II will then focus on (iii) the development and validation of biosensors and (iv) how to assess the usability and (potential) social impact of emerging SbSs. Finally, these insights are applied to generate, for the first time, proposed best practices to help guide the future ethical data handling and development of smartphone-based devices for analytical chemistry applications.

Ultra-performance liquid chromatography combined with time-of-flight mass spectrometry (UPLC-ToF-MS) has been used for screening and quantification of more than 100 veterinary drugs in milk. The veterinary drugs represent different classes including benzimidazoles, macrolides, penicillins, quinolones, sulphonamides, pyrimidines, tetracylines, nitroimidazoles, tranquillizers, ionophores, amphenicols and non-steroidal anti-inflammatory agents (NSAIDs). After protein precipitation, centrifugation and solid-phase extraction (SPE), the extracts were analysed by UPLC-ToF-MS. From the acquired full scan data the drug-specific ions were extracted for construction of the chromatograms and evaluation of the results. The analytical method was validated according to the EU guidelines (2002/657/EC) for a quantitative screening method. At the concentration level of interest (MRL level) the results for repeatability (%RSD20% for 86% of the compounds), reproducibility (%RSD40% for 96% of the compounds) and the accuracy (80-120% for 88% of the compounds) were satisfactory. Evaluation of the CCbeta values and the linearity results demonstrates that the developed method shows adequate sensitivity and linearity to provide quantitative results. Furthermore, the method is accurate enough to differentiate between suspected and negative samples or drug concentrations below or above the MRL. A set of 100 samples of raw milk were screened for residues. No suspected (positive) results were obtained except for the included blind reference sample containing sulphamethazine (88 microg/l) that tested positive for this compound. UPLC-ToF-MS combines high resolution for both LC and MS with high mass accuracy which is very powerful for the multi-compound analysis of veterinary drugs. The technique seems to be powerful enough for the analysis of not only veterinary drugs but also organic contaminants like pesticides, mycotoxins and plant toxins in one single method.

Public concern about the presence of natural and anthropogenic compounds which affect human health by modulating normal endocrine functions is continuously growing. Fast and simple high-throughput screening methods for the detection of hormone activities are thus indispensable. During the last two decades, a panel of different in vitro assays has been developed, mainly for compounds with an estrogenic mode of action. Here we describe the development of an androgen transcription activation assay that is easy to use in routine screening. Recombinant yeast cells were constructed that express the human androgen receptor and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. Compared with other reporters, the yEGFP reporter protein is very convenient because it is directly measurable in intact living cells, i.e., cell wall disruption and the addition of a substrate are not needed. When yeast was exposed to 17beta-testosterone, the concentration where half-maximal activation is reached (EC(50)) was 50 nM. The relative androgenic potencies, defined as the ratio between the EC(50) of 17beta-testosterone and the EC(50) of the compound, of 5alpha-dihydrotestosterone, methyltrienolone, and 17beta-boldenone are 2.3, 1.4, and 0.15 respectively. The results presented in this paper demonstrate that this new yeast androgen bioassay is fast, sensitive, and very specific and also suited to detect compounds that have an antiandrogenic mode of action.

The last 2 years multi-compound methods are gaining ground as screening methods. In this study a high-resolution liquid chromatography combined with time-of-flight mass spectrometry (HRLC-ToF-MS) is tested for the screening of about 100 veterinary drugs in three matrices, meat, fish and egg. While the results are satisfactory for 70-90% of the veterinary drugs, a more efficient sample preparation or extract purification is required for quantitative analysis of all analytes in more difficult matrices like egg. The average mass measurement error of the ToF-MS for the veterinary drugs spiked at concentrations ranging from 4 to 400 microg/kg, is 3.0 ppm (median 2.5 ppm) with little difference between the three matrices, but slightly decreases with increasing concentration. The SigmaFit value, a new feature for isotope pattern matching, also decreases with increasing concentration and, in addition, shows an increase with increasing matrix complexity. While the average SigmaFit value is 0.04, the median is 0.01 indicating some high individual deviations. As with the mass measurement error, the highest deviations are found in those regions of the chromatogram where most compounds elute from the column, be it analytes or matrix compounds. The median repeatability of the method ranges from 8% to 15%, decreasing with increasing concentration, while the median reproducibility ranges from 15% to 20% with little difference between matrices and concentrations. The median accuracy is in between 70% and 100% with a few compounds showing higher values due to matrix interference. The squared regression coefficient is >0.99 for 92% of the compounds showing a good overall linearity for most compounds. The detection capability, CCbeta, is within 2 times the associated validation level for >90% of the compounds studied. By changing a few conditions in the analyses protocol and analysing a number of blank samples, it was determined that the method is robust as well as specific. Finally, an alternative validation strategy is proposed and tested for screening methods. While the results calculated for repeatability, within-lab reproducibility and CCbeta show a good comparison for the matrices meat and fish, and a reasonable comparison for the matrix egg, only 27 analyses are required to obtain these results versus 63 analysis in the traditional, 2002/657/EC, approach. This alternative is suggested as a cost-effective validation procedure for screening methods.

The potential for coupling technologies to deliver new, improved forms of bioanalysis is still in its infancy. We review a number of examples in which coupling has been successful, with special emphasis on combining surface-plasmon-resonance biosensors with mass spectrometry. We give an overview of current progress towards combining biosensor-based bioanalysis with chemical analysis for confirmation of paralytic shellfish poisons that are marine toxins. This comprehensive approach could be an alternative to the official methods currently used (e.g., animal testing and high-performance liquid chromatography with fluorescence detection) and could serve as a model for many more such applications.

Ambient mass spectrometry has been used for the analysis of strobilurin residues in wheat. The use of this novel, challenging technique, employing a direct analysis in a real time (DART) ion-source coupled with a time-of-flight mass spectrometer (TOF MS) and a desorption electrospray ionization (DESI) source coupled with a linear ion trap tandem MS (LIT MS(n)), permitted a direct screen of the occurrence of target fungicides in treated grains in less than 1 min. For quantification purpose by DART-TOF MS, an ethyl acetate extract had to be prepared. With the use of a prochloraz as an internal standard, the performance characteristics obtained by repeated analyses of extract, spiked at 50 microg kg(-1) with six strobilurins (azoxystrobin, picoxystrobin, dimoxystrobin, kresoxim-methyl, pyraclostrobin, and trifloxystrobin), were in the following range: recoveries 78-92%, repeatability (RSD) 8-15%, linearity (R(2)) 0.9900-0.9978. The analysis of wheat with incurred strobilurin residues demonstrated good trueness of data generated by the DART-TOF MS method; the results were in a good agreement with those obtained by the conventional approach, i.e., by the QuEChERS sample handling procedure followed by identification/quantification employing high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Tandem mass spectrometry using DESI-LIT MS(n) provided a sufficient number of product ions for confirmation of the identity of azoxystrobin and pyraclostrobin in incurred wheat samples.

The use of beta-agonists as growth promoters in cattle breeding is forbidden in many countries for reasons of fair trade and consumer protection. In recent years the use of liquid chromatography (LC) tandem mass spectrometry (MS/MS) has been shown to be the method of choice for the control of beta-agonists. In this study an LC-MS/MS multiresidue analysis method is presented for trace analysis of 22 beta-agonists. A truly generic concept has been designed based on mixed-mode solid-phase extraction and positive electrospray ionisation LC-MS/MS operated in the multiple reaction monitoring mode. This method allows application to a wide variety of sample matrices such as urine, feed and hair, following minor modifications to the analysis procedure only. The method features fit-for-purpose sensitivity in urine as shown by CCalpha and CCbeta values of less than 0.2 and less than 0.5 microg/l respectively, for all beta-agonists studied (terbutaline and reproterol, less than 0.3 and less than 1.0 respectively). Similar but semiquantitative application to feed and hair showed CCbeta values of less than 10.0 and less than 5.0 microg/kg, respectively. A further simplification and improvement is demonstrated using Ultra Performance LC (UPLC) and fast-switching MS/MS. The successful validation of this method following the latest EU requirements and its application to real samples demonstrate that a new versatile tool has been achieved for veterinary control of beta-agonists.

Two major trends are observed in the analysis of veterinary drugs and growth-promoting agents. First is the selection of sample material for monitoring the use of registered veterinary drugs. Traditionally meat, kidney and liver were analyzed but, due to the food scandals in which meat was very often involved, the consumption of alternative products (e.g., eggs and fish) has become more popular and therefore more of interest for residue-monitoring programmes. Most food scandals started with contamination of animal feed, so feed has also gained interest for monitoring purposes. For the detection of unauthorized substances (e.g., growth-promoting agents), the matrix hair is frequently used because residues can be detected in hair even a long time after treatment. Second, the techniques used for residue analysis are moving from target-orientated methods, mainly based on liquid chromatography in combination with triple-quadrupole mass-spectrometric detection (LC-QqQ-MS), towards accurate mass full-scan MS techniques (e.g., time-of-flight (ToF)-MS and Fourier-transform(FT)-MS. Full-scan MS techniques enable retrospective analysis (i.e. without re-injecting the sample) and real multi-residue analysis (including different classes of veterinary drugs). The application of full-scan MS also has consequences for separation and extraction techniques, which have to be applicable to a broad range of compounds differing in physical and chemical properties. Experience shows that ToF-MS in combination with ultra-performance LC (UPLC) is very powerful for multi-residue analysis. The introduction of new MS techniques also has consequences for European Union (EU) criteria defined for confirmation of the identity of compounds. It is suggested that both mass-resolution and mass-accuracy data influence identity confirmation and both parameters should be implemented in the revision of European Commission Decision 2002/657/EC. (C) 2007 Elsevier Ltd. All rights reserved.

A liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) method was developed using the latest high-resolution LC column technology, the ultra performance liquid chromatography (UPLC (TM)), and electrospray ionization (ESI) in the positive ion mode. Gradient UPLC separation conditions were optimized for a group of 22 analytes comprising 17 glucocorticosteroids, specific designer steroids such as tetrahydrogestrinone (THG) and specific beta(2)-agonists such as formoterol. The UPLC/TOFMS separation obtained required 5.5 min only for all the substances tested. Even the critical pair of dexamethasone and betamethasone isomers was almost completely resolved. Thanks to the over 10,000 full-width at half maximum (FWHM) mass resolution and high mass accuracy features of TOFMS 50 mDa window accurate mass chromatograms could be reconstructed for the individual analytes. Sensitive screening in human and calf urine samples fortified at the glucocorticosteroids minimum required performance limit (MRPL) of 30 mu g L-1 (human urine, sports doping) and 2 mu g L-1 (calf urine, veterinary control) could be obtained. The potential of UPLC/TOFMS for confirmatory analysis was shown by determining the accurate mass of all compounds and fragment ions upon in-source collision-induced dissociation (CID) at different energies. The exact mass measurement errors for all glucocorticosteroids were found to be within 6 ppm. Considering veterinary control, limits of detection (LOD) and limits of quantification (LOQ) were determined for most of the analytes in calf urine and found to range from 0.1 to 3.3 and from 0.4 to 4.4 mu g L-1, respectively. The method can be easily extended with other banned substances of interest, as demonstrated by the addition of 21 beta(2)-agonists to the original analyte mixture in urine, without causing any interferences. (c) 2006 Elsevier B.V. All rights reserved.

A new combined doping control screening method for the analysis of anabolic steroids in human urine using liquid chromatography/electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (LCoaTOFMS) and gas chromatography/electron ionization orthogonal acceleration time-of-flight mass spectrometry (GCoaTOFMS) has been developed in order to acquire accurate full scan MS data to be used to detect designer steroids. The developed method allowed the detection of representative prohibited substances, in addition to steroids, at concentrations of 10 ng/mL for anabolic agents and metabolites, 30 ng/mL for corticosteroids, 500 ng/mL for stimulants and fl-blockers, 250ng/mL for diuretics, and 200ng/mL for narcotics. Sample preparation was based on liquid-liquid extraction of hydrolyzed human urine, and the final extract was analyzed as trimethylsilylated derivatives in GCoaTOFMS and underivatized in LCoaTOFMS in positive ion mode. The sensitivity, mass accuracy, advantages and limitations of the developed method are presented. Copyright (C) 2007 John Wiley & Sons, Ltd.

New anabolic steroids show up occasionally in sports doping and in veterinary control. The discovery of these designer steroids is facilitated by findings of illicit preparations, thus allowing bioactivity testing, structure elucidation using NMR and mass spectrometry, and final incorporation in urine testing. However, as long as these preparations remain undiscovered, new designer steroids are not screened for in routine sports doping or veterinary control urine tests since the established GC/MS and LC/MS/MS methods are set up for the monitoring of a few selected ions or MS/MS transitions of known substances only. In this study, the feasibility of androgen bioactivity testing and mass spectrometric identification is being investigated for trace analysis of designer steroids in urine. Following enzymatic deconjugation and a generic solid-phase extraction, the samples are analyzed by gradient LC with effluent splitting toward two identical 96-well fraction collectors. One well plate is used for androgen bioactivity detection using a novel robust yeast reporter gene bioassay yielding a biogram featuring a 20-s time resolution. The bioactive wells direct the identification efforts to the corresponding well numbers in the duplicate plate. These are subjected to high-resolution LC using a short column packed with 1.7-microm C18 material and coupled with electrospray quadrupole time-of-flight mass spectrometry (LC/QTOFMS) with accurate mass measurement. Element compositions are calculated and used to interrogate electronic substance databases. The feasibility of this approach for doping control is demonstrated via the screening of human urine samples spiked with the designer anabolic steroid tetrahydrogestrinone. Application of the proposed methodology, complementary to the established targeted urine screening for known anabolics, will increase the chance of finding unknown emerging designer steroids, rather then being solely dependent on findings of the illicit preparations themselves.

A fast non-targeted strategy is described for analysis of formulations—meant for administration to live stock—containing growth-promoting agents or veterinary drugs. The use of 1 H NMR as a first step universal screening method is applied and used in routine analysis. The implementation of this approach has increased the analysis efficiency considerably. Apart from screening on illegal compounds, 1 H NMR information on matrix and thus, indirectly, administration mode, can be present. An ever-growing 1 H NMR database is used containing more than 200 reference substances. Based on the 1 H NMR screening, decisions for further analysis can be made, such as for instance HPLC fractionation of steroid cocktails and subsequent 1 H NMR (and LC–MS) analysis. Examples of unravelling formulations are given in detail including a steroid cocktail containing 15 compounds.

The influence of the ionization efficiency on the measured copolymer sequence distribution is presented. Large differences in ionization efficiency were observed for mixtures of homopolyesters containing dipropoxylated bisphenol A/adipic acid and dipropoxylated bisphenol A/isophthalic acid and the corresponding copolyester dipropoxylated bisphenol A/isophthalic acid/adipic acid. The adipic acid structure has a higher affinity for the sodium cation, which results in more intense peaks for adipic acid containing oligomers. Relative sodium affinities of the oligomers were found to increase with an increasing number of acid end groups in favor of adipic acid containing oligomers. The ESI response of the oligomers depends on the polymer concentration in the sprayed mixture. This makes it impossible to correct for the ionization efficiency necessary for copolymer analysis. If differences in ionization efficiency are not corrected, the ion intensities in the copolymer mass spectra will show large deviations from the real composition and no conclusion can be drawn about the chemical (in)homogeneity of the MWD nor the random or block structure of the copolymer. This will also be valid for other cationization techniques like MALDI and FAB.

The existence of polythiocar- bamatozinc complexes, species pre- sumed to be involved in catalyzing sulfur vulcanization, has been studied by com- putational and mass-spectrometric tech- niques. Density functional calculations reveal that the sulfuration energy of bis(trithiocarbamato)zinc(ii) is compara- ble to that of bis(phenyltrithiolato)- zinc(ii), a stable sulfurated complex. Interestingly, the analogous nonsym- metrically sulfurated complex (dithio- carbamato)(tetrathiocarbamato)zinc(ii) is energetically slightly more favorable. The calculations indicate that polythio- carbamatozinc complexes may indeed be involved in the sulfur vulcanization cascade, a conclusion corroborated by ensuing laser-desorption ionization (LDI) mass-spectrometric measure- ments on mixtures of dithiocarbamato- zinc(ii) complexes and elemental sulfur. These demonstrate the presence of poly- thiocarbamatozinc ions that are sulfu- rated with up to eight additional sulfur atoms.

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.