Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Formation of Taste-Active Pyridinium Betaine Derivatives Is Promoted in Thermally Treated Oil-in-Water Emulsions and Alkaline pH
    Troise, Antonio Dario ; Berton-Carabin, Claire C. ; Vitaglione, Paola ; Fogliano, Vincenzo - \ 2020
    Journal of Agricultural and Food Chemistry 68 (2020)18. - ISSN 0021-8561 - p. 5180 - 5188.
    Amadori products - emulsions - Maillard reaction - mass spectrometry - taste-active molecules

    The oil-water interface can be used as an efficient reaction controller in foods by carrying specific reactants and products in either the hydrophobic or hydrophilic phase. The formation of the taste-active compounds N-(1-carboxyethyl)-6-hydroxymethyl-pyridinium-3-ol inner salt (alapyridaine) and 1-(1-carboxyethyl)-3-hydroxy-pyridinium inner salt is influenced by the presence of a dispersed saturated triglyceride oil phase and by the pH of the aqueous phase. At pH 6.5, the formation of both betaines was 1.24 and 6 times higher in emulsions than in aqueous solution after 4 min at 140 °C. In alkaline emulsions (pH = 9.5, 4 min), the concentrations of alapyridaine and 1-(1-carboxyethyl)-3-hydroxy-pyridinium ion were 6.2 and 3.8 times higher, respectively, than in unbuffered emulsions as a result of the interaction between the polar head group of the surfactant and pyridinium rings. Here, we reported for the first time the effects of multiphase systems on the formation of nonvolatile, taste-active end products.

    Novel COX-2 products of n-3 polyunsaturated fatty acid-ethanolamine-conjugates identified in RAW264.7 macrophages
    Bus, Ian de; Zuilhof, Han ; Witkamp, Renger ; Balvers, Michiel ; Albada, Bauke - \ 2019
    Journal of Lipid Research 60 (2019)11. - ISSN 0022-2275 - p. 1829 - 1840.
    cyclooxygenase - cyclooxygenase 2 - fatty acid amides - fatty acid oxidation - high-performance liquid chromatography - inflammation - mass spectrometry - prostaglandins

    Cyclooxygenase 2 (COX-2) plays a key role in the regulation of inflammation by catalyzing the oxygenation of PUFAs to prostaglandins (PGs) and hydroperoxides. Next to this, COX-2 can metabolize neutral lipids, including endocannabinoid-like esters and amides. We developed an LC-HRMS-based human recombinant (h)COX-2 screening assay to examine its ability to also convert n-3 PUFA-derived N-acylethanolamines. Our assay yields known hCOX-2-derived products from established PUFAs and anandamide. Subsequently, we proved that eicosapentaenoylethanolamide (EPEA), the N-acylethanolamine derivative of EPA, is converted into PGE3-ethanolamide (PGE3-EA), and into 11-, 14-, and 18-hydroxyeicosapentaenoyl-EA (11-, 14-, and 18-HEPE-EA, respectively). Interestingly, we demonstrated that docosahexaenoylethanolamide (DHEA) is converted by hCOX-2 into the previously unknown metabolites, 13- and 16-hydroxy-DHEA (13- and 16-HDHEA, respectively). These products were also produced by lipopolysaccharide-stimulated RAW267.4 macrophages incubated with DHEA. No oxygenated DHEA metabolites were detected when the selective COX-2 inhibitor, celecoxib, was added to the cells, further underlining the role of COX-2 in the formation of the novel hydroxylated products. This work demonstrates for the first time that DHEA and EPEA are converted by COX-2 into previously unknown hydroxylated metabolites and invites future studies toward the biological effects of these metabolites.

    Metaproteomic and 16S rRNA Gene Sequencing Analysis of the Infant Fecal Microbiome
    Cortes, Laetitia ; Wopereis, Harm ; Tartiere, Aude ; Piquenot, Julie ; Gouw, Joost W. ; Tims, Sebastian ; Knol, Jan ; Chelsky, Daniel - \ 2019
    International Journal of Molecular Sciences 20 (2019)6. - ISSN 1661-6596
    fecal - infants - intestinal - mass spectrometry - metabolism - metacluster - microbiome

    A metaproteomic analysis was conducted on the fecal microbiome of eight infants to characterize global protein and pathway expression. Although mass spectrometry-based proteomics is now a routine tool, analysis of the microbiome presents specific technical challenges, including the complexity and dynamic range of member taxa, the need for well-annotated metagenomic databases, and high inter-protein sequence redundancy and similarity. In this study, an approach was developed for assessment of biological phenotype and metabolic status, as a functional complement to DNA sequence analysis. Fecal samples were prepared and analysed by tandem mass spectrometry and a homology-based meta-clustering strategy was used to combine peptides from multiple species into representative proteins. In total, 15,250 unique peptides were sequenced and assigned to 2154 metaclusters, which were then assigned to pathways and functional groups. Differences were noted in several pathways, consistent with the dominant genera observed in different subjects. Although this study was not powered to draw conclusions from the comparisons, the results obtained demonstrate the applicability of this approach and provide the methods needed for performing semi-quantitative comparisons of human fecal microbiome composition, physiology and metabolism, as well as a more detailed assessment of microbial composition in comparison to 16S rRNA gene sequencing.

    Comprehensive mass spectrometry-guided phenotyping of plant specialized metabolites reveals metabolic diversity in the cosmopolitan plant family Rhamnaceae
    Kang, Kyo Bin ; Ernst, Madeleine ; Hooft, Justin J.J. van der; Silva, Ricardo R. da; Park, Junha ; Medema, Marnix H. ; Sung, Sang Hyun ; Dorrestein, Pieter C. - \ 2019
    The Plant Journal (2019). - ISSN 0960-7412
    annotation - classification - mass spectrometry - Rhamnaceae - specialized metabolites - technical advance

    Plants produce a myriad of specialized metabolites to overcome their sessile habit and combat biotic as well as abiotic stresses. Evolution has shaped the diversity of specialized metabolites, which then drives many other aspects of plant biodiversity. However, until recently, large-scale studies investigating the diversity of specialized metabolites in an evolutionary context have been limited by the impossibility of identifying chemical structures of hundreds to thousands of compounds in a time-feasible manner. Here we introduce a workflow for large-scale, semi-automated annotation of specialized metabolites and apply it to over 1000 metabolites of the cosmopolitan plant family Rhamnaceae. We enhance the putative annotation coverage dramatically, from 2.5% based on spectral library matches alone to 42.6% of total MS/MS molecular features, extending annotations from well-known plant compound classes into dark plant metabolomics. To gain insights into substructural diversity within this plant family, we also extract patterns of co-occurring fragments and neutral losses, so-called Mass2Motifs, from the dataset; for example, only the Ziziphoid clade developed the triterpenoid biosynthetic pathway, whereas the Rhamnoid clade predominantly developed diversity in flavonoid glycosides, including 7-O-methyltransferase activity. Our workflow provides the foundations for the automated, high-throughput chemical identification of massive metabolite spaces, and we expect it to revolutionize our understanding of plant chemoevolutionary mechanisms.

    Evaluation of Mycotoxin Screening Tests in a Verification Study Involving First Time Users
    Lattanzio, Veronica M.T. ; Holst, Christoph von; Lippolis, Vincenzo ; Girolamo, Annalisa De; Logrieco, Antonio F. ; Mol, Hans G.J. ; Pascale, Michelangelo - \ 2019
    Toxins 11 (2019)2. - ISSN 2072-6651 - 18 p.
    cereals - immunoassay - mass spectrometry - mycotoxins - screening - validation

    (AFB₁) in maize and wheat using LFD and LC-HRMS, respectively. The results of analyses were used to calculate intermediate precision (RSDip, covering the inter-analyst variability in preparing the analytical samples and the precision under repeatability conditions) cut-off values and false suspect rates. RSDip ranged from 6.5% to 30% for DON, and from 16% to 33% for AFB₁. The highest obtained variances were associated with the AFB₁ analyses due to working with much lower mass fractions. The rate of false suspect results were lower than 0.1% for all tested methods. All methods showed a fit-for-purpose method performance profile, which allowed a clear distinction of samples containing the analytes at the screening target concentration (STC) from negative control samples. Moreover, the first time users obtained method performances similar to those obtained for validation studies previously performed on the screening methods included in the training course.

    Multiclass screening in urine by comprehensive two-dimensional liquid chromatography time of flight mass spectrometry for residues of sulphonamides, beta-agonists and steroids
    Blokland, M.H. ; Zoontjes, P.W. ; Ginkel, L.A. van; Schans, M.G.M. van de; Sterk, S.S. ; Bovee, T.F.H. - \ 2018
    Food Additives & Contaminants. Pt. A, Chemistry, Analysis, Control, Exposure & Risk Assessment 35 (2018)9. - ISSN 1944-0049 - p. 1703 - 1715.
    Antibiotics - comprehensive 2D-LC - growth promoters - LC x LC - mass spectrometry - residues

    Nowadays routine residue monitoring involves the analysis of many compounds from different classes, mainly in urine. In the past two decades, developments heavily focused on the use of mass spectrometers (MS) and faster and more sensitive MS detectors have reached the market. However, chromatographic separation (CS) was rather ignored and the cognate developments in CS were not in line. As a result, residue analysis did not improve to the extent anticipated. CS by LC x LC is a promising technique and will enable a further increase in the range of compounds and compound classes that can be detected in a single run. In the present study, a self-built LC x LC system, using a 10 port valve, was connected to a single quadrupole MS with electrospray interface. Standards containing a mixture of sulphonamides, β-agonists and (steroid) hormones, 53 compounds, in total, were analysed. Results demonstrated that these compounds were well separated and could be detected at low levels in urine, i.e. limit of detection (LOD) from 1 µg L−1 for most β-agonists to 10 µg L−1 for some sulphonamides and most hormones. To enhance the sensitivity, optimisation was performed on an advanced commercial LC x LC system connected to a full scan accurate MS. This ultimately resulted in a fast high throughput untargeted method, including a simple sample clean-up in a 96-well format, for the analysis of urine samples.

    Occurrence of pyrrolizidine alkaloids in animal- and plant-derived food : results of a survey across Europe
    Mulder, Patrick P.J. ; Lopez Sanchez, Patricia ; Castelari, Massimo ; Bodi, Dorina ; Ronczka, Stefan ; Preiss-Weigert, Angelika ; These, Anja - \ 2018
    Food Additives & Contaminants. Pt. A, Chemistry, Analysis, Control, Exposure & Risk Assessment 35 (2018)1. - ISSN 1944-0049 - p. 118 - 133.
    eggs - herbal supplements - mass spectrometry - meat - milk - Pyrrolizidine alkaloids - survey - tea

    Pyrrolizidine alkaloids (PAs) are secondary metabolites of plant families such as Asteraceae or Boraginaceae and are suspected to be genotoxic carcinogens. Recent investigations revealed their frequent occurrence in honey and particularly in tea. To obtain a comprehensive overview of the PA content in animal- and plant-derived food from the European market, and to provide a basis for future risk analysis, a total of 1105 samples were collected in 2014 and 2015. These comprised milk and milk products, eggs, meat and meat products, (herbal) teas, and (herbal) food supplements collected in supermarkets, retail shops, and via the internet. PAs were detected in a large proportion of plant-derived foods: 91% of the (herbal) teas and 60% of the food supplements contained at least one individual PA. All types of (herbal) teas investigated were found to contain PAs, with a mean concentration of 460 µg kg−1 dry tea (corresponding to 6.13 µg L−1 in [herbal] tea infusion). The highest mean concentrations were found in rooibos tea (599 µg kg−1 dry tea, 7.99 µg L−1 tea infusion) and the lowest in camomile tea (274 µg kg−1 dry tea, 3.65 µg L−1 tea infusion). Occurrence of PAs in food supplements was found to be highly variable, but in comparable ranges as for (herbal) tea. The highest concentrations were present in supplements containing plant material from known PA-producing plants. In contrast, only 2% of the animal-derived products, in particular 6% of milk samples and 1% of egg samples, contained PAs. Determined levels in milk were relatively low, ranged between 0.05 and 0.17 µg L−1 and only trace amounts of 0.10–0.12 µg kg−1 were found in eggs. No PAs were detected in the other animal-derived products.

    High-resolution mass spectrometry for the analysis of interfacial kinetics of organic surface reactions
    Sen, Rickdeb - \ 2017
    Wageningen University. Promotor(en): H. Zuilhof. - Wageningen : Wageningen University - ISBN 9789463436243 - 308
    surface chemistry - unimolecular films - chemical reactions - analytical methods - mass spectrometry - oppervlaktechemie - unimoleculaire films - chemische reacties - analytische methoden - massaspectrometrie

    In this thesis, XPS and DART–HRMS have been used in close conjugation to supplement each other, since the latter is a relatively new addition to surface chemist’s repertoire that – after development – needed a firm comparison to build up a reputation of its own. The strength of our approach has been underlined by the high correlation between these two independent analytical techniques. Central to our approach has been the formation of mixed monolayers in case of aluminum oxide substrates. As presented in Chapters 2, 3 and 4, we have succeeded in the rapid formation of range stable, covalently bound mixed monolayers. The subsequent development of a general and fast analytical technique to determine the interfacial reaction kinetics, including the activation parameters DH‡ and DS‡, provided unparalleled insights. We have developed a “MS–ionizable tag” technique, which has been applied for the analysis of surface–bound organic reactions, to the best of our knowledge, for the first time.

    The Strain–Promoted Alkyne–Azide Cycloaddition (SPAAC) reaction was chosen as a model reaction given the fact that its kinetics had been well–studied in solution. As shown in Chapter 2, the microenvironment around the reactive surface group was carefully controlled by the length of the inert alkyl chains surrounding it. We observed a few interesting trends which could be of great interest to future surface chemists. First, the SPAAC reaction – which is a click reaction in solution – does not retain this nature on the surface (It does not proceed to full conversion and converges sluggishly to around 37% yield after significant temporal passage). A partially accessible microenvironment, where the motion of reactive groups is slightly restricted, was found to provide a high rate with the highest surface yield. In contrast, a freely accessible reactive moiety afforded a lower surface yield albeit with the highest overall rate. Finally, a buried microenvironment led to the highest overall rate albeit with a lower surface yield. As a corollary, for the surface–bound SPAAC reaction we can compare the partially accessible microenvironment to a marathon runner who is able to run further but at a pace slower than a sprinter (free microenvironment). This provides the surface chemist with a handle for tuning the monolayer as per her/his reaction goals.

    Harnessing the valuable insights gained from the SPAAC reaction, our concept of ionizable MS tag coupled with DART–HRMS was further extended to a more novel and yet unstudied interfacial reaction in Chapter 3. The Strain–Promoted Oxidation–Controlled cycloalkyne–1,2–Quinone (SPOCQ) cycloaddition was applied for the first time on a surface and afforded a quantitative yield for a free microenvironment in under 4 h. It is to be noted here, that for the first time a 100% (quantitative) metal–free click reaction was observed at a surface. This proved that our approach of engineering the microenvironment around the reactive site provides a distinct edge needed to attain quantitative yields. Quinones are hard to synthesize/store/use in solution given their high propensity to polymerize. However, we demonstrated that on the surface, quinones can be easily generated and stored over–extended period of time by a facile periodate oxidation. Auto–polymerization of surface–bound quinones is precluded by their tether and enforced distal separation by surrounding inert alkyl chains (3:1 ratio). The wider application of this interesting mixture has been further rigorously demonstrated in later chapters too. The bioorthogonality of the SPOCQ reaction coupled with its higher speed and its quantitative yields on the surface are definitely its most salient features.

    After studying strain–promoted click reactions on the surface (culminating for SPOCQ in quantitative conversion within 4 h), the question arose if DART–HRMS could also be used to reproducibly and precisely determine a different class of cycloadditions, for which we selected the interfacial inverse electron demand Diels–Alder (IEDDA) reaction as this reaction was reported to be really fast –at least for click reactions– in solution. This was studied in Chapter 4 extensively and we surpassed our previous kinetic record (SPOCQ) by obtaining a quantitative yield in a mere 15 min. The other interesting observation of this study was that reversing the reaction counterparts on the surface produced a discernible reaction rate difference. We found that one of the reactants when tethered in a particular stereochemistry (exo– form) gave the highest surface coverage (100%) within the shortest amount of time. This was also the first time that the effect of diastereomerism on interfacial reaction rates was studied.

    In Chapter 5, covalent modification of native non–activated mica has been carried out utilizing catechol linkers. Previous studies for mica modification produced poorly defined polymeric structures on the surface or required extensive and tedious organic synthesis. We have addressed both these issues head–on in this thesis. Well–defined and characterized ultrathin layers were constructed on mica using a catechol–based molecule involving a two–step synthesis. Mica being atomically flat provides an ideal surface upon which to study various phenomena by AFM and other forms of microscopy. However, most research until now was restricted to simply drop–casting the pre–fabricated moieties followed by studying their final structures. Our method now allows for the step–wise formation and characterization of these very interesting structures. Along with it, we also performed several click attachment chemistries on these ultrathin layers which can be harnessed by surface chemists to put various functional and structurally complex moieties on the surface. This opens the pathway for the attachment of more complex architectures on the surface with higher functionality along with the ability to study their formation in a step–wise controlled fashion.

    Overall, this thesis wishes to understand organic surface chemistry and several of its intricate mysteries. It clearly outlines several modification techniques and unravels interfacial kinetics of several interesting “metal–free click reactions”. It strives to rationalize the activation parameters in conjunction with classical organic chemistry and gives details on how surrounding “inert” alkyl chains can play a profound role in reaction rates. Lastly, we have striven to and achieved rapid and quantitative reactions on the surface by virtue of optimization of this microenvironment. Personally I believe, we have treaded on a road seldom traveled and unraveled a new understanding about molecular interactions on the ever–interesting and an infinitely–complex surface.

    Identification of methylated GnTI-dependent N-glycans in Botryococcus brauni
    Schulze, Stefan ; Urzica, Eugen ; Reijnders, Maarten J.M.F. ; Geest, Henri van de; Warris, Sven ; Bakker, Linda V. ; Fufezan, Christian ; Martins dos Santos, Vitor A.P. ; Schaap, Peter J. ; Peters, Sander A. ; Hippler, Michael - \ 2017
    New Phytologist 215 (2017)4. - ISSN 0028-646X - p. 1361 - 1369.
    Botryococcus braunii - gene ontology annotation - mass spectrometry - N-glycosylation - post-translational modification
    In contrast to mammals and vascular plants, microalgae show a high diversity in the N-glycan structures of complex N-glycoproteins. Although homologues for β1,2-N-acetylglucosaminyltransferase I (GnTI), a key enzyme in the formation of complex N-glycans, have been identified in several algal species, GnTI-dependent N-glycans have not been detected so far. We have performed an N-glycoproteomic analysis of the hydrocarbon oils accumulating green microalgae Botryococcus braunii. Thereby, the analysis of intact N-glycopeptides allowed the determination of N-glycan compositions. Furthermore, insights into the role of N-glycosylation in B. braunii were gained from functional annotation of the identified N-glycoproteins. In total, 517 unique N-glycosylated peptides have been identified, including intact N-glycopeptides that harbored N-acetylhexosamine (HexNAc) at the nonreducing end. Surprisingly, these GnTI-dependent N-glycans were also found to be modified with (di)methylated hexose. The identification of GnTI-dependent N-glycans in combination with N-glycan methylation in B. braunii revealed an uncommon type of N-glycan processing in this microalgae.
    Leaf phenolics and seaweed tannins : analysis, enzymatic oxidation and non-covalent protein binding
    Vissers, Anne M. - \ 2017
    Wageningen University. Promotor(en): H. Gruppen; W.H. Hendriks, co-promotor(en): J.P. Vincken. - Wageningen : Wageningen University - ISBN 9789463432023 - 154
    phenols - leaves - seaweeds - tannins - beta vulgaris - laminaria - proteins - catechol oxidase - nuclear magnetic resonance spectroscopy - in vitro - mass spectrometry - browning - fermentation - animal feeding - fenolen - bladeren - zeewieren - tanninen - beta vulgaris - laminaria - eiwitten - catechol oxidase - kernmagnetische resonantiespectroscopie - in vitro - massaspectrometrie - bruinkleuring - fermentatie - diervoedering

    Upon extraction of proteins from sugar beet leaves (Beta vulgaris L.) and oarweed (Laminaria digitata) for animal food and feed purposes, endogenous phenolics and proteins can interact with each other, which might affect the protein’s applicability. Sugar beet leaf proteins might become covalently modified by phenolics through polyphenol oxidase (PPO) activity. Oligomeric phenolics from seaweed (so-called phlorotannins (PhT)) might bind non-covalently to protein. The first aim of this thesis was to study factors involved in protein modification by phenolics. The second aim was to investigate the effect of PhT supplementation to feed on in vitro ruminal fermentation.

    Besides PPO activity and the amount of low molecular weight phenolic substrates present, brown colour formation in sugar beet leaves was dependent on the amount of phenolics, which do not serve as a substrate of PPO. These non-substrate phenolics can engage in browning reactions by oxidative coupling and subsequent coupled oxidation of the products formed. Similar reactions might also be involved in covalent protein modification by phenolics, and therewith protein properties.
    High molecular weight PhT from L. digitata could potentially modify protein properties by non‑covalent interactions. L. digitata contained PhT with subunits mainly connected via C‑O-C linkages, as determined using NMR spectroscopy. Further mass spectrometric analysis revealed the presence of a wide range of oligomers with degrees of polymerisation between 3 and 27. The interaction between PhT and proteins (b-casein and bovine serum albumin) was studied using model systems with different pH values, representing the various environments throughout the ruminants digestive tract. Phlorotannins bound to protein independent of pH, and broadened the pH range of protein precipitation from 0.5 to ~1.5 pH unit around the protein’s pI. At the pH of the abomasum of 2-3, the proteins re-solubilised again, presumably by increase in their net charge. Due to their ability to form water insoluble complexes, PhT could improve ruminal fermentation in vitro in a dose dependent manner, resulting in lower methane production and ammonia (NH3) concentration. The decreased NH3 concentration reflected decreased dietary protein breakdown in the rumen, which is considered a nutritional and environmental benefit.

    Nanostructured imaging surface plasmon resonance biosensing
    Joshi, Sweccha - \ 2017
    Wageningen University. Promotor(en): Michel Nielen; Han Zuilhof, co-promotor(en): Teris van Beek. - Wageningen : Wageningen University - ISBN 9789463430203 - 164
    methodology - techniques - biosensors - resonance - mass spectrometry - organic chemistry - physics - methodologie - technieken - biosensoren - resonantie - massaspectrometrie - organische scheikunde - fysica

    The testing and further development of a prototype nanostructured imaging surface plasmon resonance (iSPR) biosensor, with a focus on surface modification and detailed characterization of the biosensor chip and in-field and at-line applicability in the food industry is described. Furthermore, a simplified coupling of SPR and MS is described that allows identification of the mycotoxins of interest along with any other cross-reacting analytes. Chapter 1 describes general information about SPR, SPR instruments along with their components, development of a multiplex SPR biosensor and coupling of SPR to mass spectrometry.

    In Chapter 2, the surface modification, in-depth characterization and the antifouling performance of the nanostructured iSPR chip is described. Different types of polyethylene glycol (PEG) and zwitterionic polymers were chosen as antifouling chemistries. Various surface characterization techniques such as atomic force microscopy, scanning electron microscopy, water contact angle, X-ray photoelectron spectroscopy and direct analysis in real time high resolution mass spectrometry provided complementary information about the chip before and after the modification. Antifouling chemistry, an essential first step in the development of an SPR biosensor, prevents false positive results arising from non-specific binding of sample components to the SPR chip. Upon comparison of the surface modification and antifouling behavior with conventional flat SPR chips, the latter were only slightly better. Zwitterionic polymers and long chain PEG had the best antifouling performance. A proof-of-principle experiment was done to demonstrate the selective detection of streptavidin binding to a surface partially modified with biotin.

    A 6-plex SPR assay for the detection of mycotoxins in barley was developed in Chapter 3. A benchmark double 3-plex assay was developed for the detection of deoxynivalenol (DON), zearalenone (ZEA), T-2 toxin (T-2), ochratoxin A (OTA), fumonisin B1 (FB1) and aflatoxin B1 (AFB1) using benchtop SPR instrument (Biacore). Preliminary in-house validation of the competitive inhibition assay developed using ovalbumin conjugates of the mycotoxins showed that the method is suitable for detection of DON, ZEA, T-2 and FB1 whereas further improvement is required for OTA and AFB1. The method was then transferred to the nanostructured iSPR, which although less sensitive than the benchtop SPR, was able to detect DON, T-2, ZEA and FB1 at the relevant levels.

    In Chapter 4, the assay developed in Chapter 3 was further optimized and an entire assay along with in-house validation and measurement of naturally contaminated was developed using the nanostructured iSPR. The antifouling chemistry used in Chapter 3, PEG, was replaced by carboxymethylated dextran (CMD) that not only allowed direct immobilization of toxins but also helped to improve the stability of the chip whereby the chip could be used for more than 450 cycles. DON could be detected at the relevant levels in beer with minimal sample preparation whereas for OTA an enrichment step using solid phase extraction was required.

    As demonstrated in Chapter 3 and 4, the nanostructured iSPR instrument can be used for screening of different mycotoxins in beer and related ingredients. However, SPR is not able to provide chemical information of the binding analyte especially in cases where the antibodies have cross-reactivity towards conjugates of the analyte. Therefore, a simplified coupling for SPR with ambient mass spectrometry was developed in Chapter 5. The method allowed identification of DON as well as its cross-reacting conjugates such as deoxynivalenol-3-glucoside and 3-acetyl DON.

    The research presented in this thesis is an important step towards the use of the nanostructured iSPR instrument for label free in-field and at-line detection of various analytes. In Chapter 6, discussion of the main achievements of this thesis, challenges and future perspectives of the technology is described.

    Reflectance of botanical, production and geographical origin on the unique compositional traits of purple grape juices
    Granato, Daniel - \ 2016
    Wageningen University. Promotor(en): Saskia van Ruth; Vincenzo Fogliano. - Wageningen : Wageningen University - ISBN 9789462579071 - 151
    grape juice - fruit juices - phenolic compounds - antioxidant properties - taste - provenance - chemometrics - mass spectrometry - druivensap - vruchtensappen - fenolverbindingen - antioxidatieve eigenschappen - smaak - herkomst - chemometrie - massaspectrometrie

    Grape juices represent one of the most consumed fruit juices because of its sensory properties, availability, reasonable price, and more recently because of their functional properties demonstrated by a vast number of in vitro, in vivo, clinical, and epidemiological studies. Although grape juices have been the target of a high number of studies, it is still not fully known how geographical origin and production management system, affect the chemical profile, quality traits related to flavor, and in vitro antioxidant of grape juices. Therefore, the main objective of this study is to elucidate the reflectance of origin (botanical, geographical, production system) in the unique compositional traits of juices from different botanical origins, with emphasis on purple grape juices. Subsequently, chemometric methods were used to try to authenticate the origin of grape juice based on the grape juice’s quality traits. Results showed that it was possible to note that the instrumental taste profile, chemical composition related to phenolic compounds, and antioxidant activity of juices from distinct botanical origins differ considerably. More specifically, pomegranate and elderberry juices presented the highest phenolic content and antioxidant activity, implying that the botanical origin of juices affected remarkably their unique instrumental taste profile and physicochemical parameters, phenolic composition, and in vitro antioxidant activity. The production managements systems, (organic/biodynamic, ORG/BIO, versus conventional, CONV) is influencing the volatile organic composition (VOC) profiles, some phenolic compounds and copper chelating activity. It is not affecting the instrumental taste profile nor the in vitro antioxidant activity results. ORG and BIO purple grape juices can be differentiated by their VOC profiles but not by the other characteristics studied. More specifically, CONV juices had higher mean levels for all ions compared to ORG and BIO juices. More specifically, in fact, BIO juices presented the lowest mean values for almost all ions measured. When European grape juices were studied, no significant difference (p>0.05) between ORG, BIO, and CONV juices was observed for instrumental richness, umami, saltiness, sourness, astringency, bitterness, total phenolic content, total soluble solids, pH, ortho-diphenols, copper chelating activity, and ferric reducing antioxidant activity. For the Brazilian samples, the contents of chlorogenic acid and myricetin were statistically higher in ORG juices, while the in vitro antioxidant activity measured by three assays (DPPH, CUPRAC, and iron chelating ability) were not different between production management systems. For the European juices, some differences were observed: BIO and ORG juices presented higher contents of (-)-epicatechin, quercetin, (+)-catechin, and myricetin compared to the CONV juices. The VOC profile, instrumental taste parameters, phenolic composition, and in vitro antioxidant activity is highly affected between regions, in which Brazilian juices presented higher ion intensities as compared to the European juices. Brazilian juices, regardless of the production management system adopted, presented higher total phenolic content and flavonoids, total anthocyanins, proanthocyanidins, flavonols, and flavanols, except for trans-resveratrol, malvidin-3-glucoside and pelargonidin-3-glucoside. From this work, we can conclude that the geographical and botanical origins affect significantly the VOC profiles, instrumental taste profile, the phenolic composition, and antioxidant activity of grape juices.

    Controlling the self-assembly of protein polymers via heterodimer-forming modules
    Domeradzka, Natalia Eliza - \ 2016
    Wageningen University. Promotor(en): Frans Leermakers, co-promotor(en): Renko de Vries; Frits de Wolf. - Wageningen : Wageningen University - ISBN 9789462578661 - 166
    polymers - nanotechnology - pichia pastoris - modules - mass spectrometry - microscopy - sds-page - rheology - fluorescence emission spectroscopy - protein purification - fermentation - chromatography - polymeren - nanotechnologie - pichia pastoris - modules - massaspectrometrie - microscopie - sds-page - reologie - fluorescentie-emissiespectroscopie - eiwitzuivering - fermentatie - chromatografie

    Supramolecular assemblies formed by protein polymers are attractive candidates for future biomaterials. Ideally, one would like to be able to define the nanostructure, in which the protein polymers should self-assemble, and then design protein polymer sequences that assemble exactly into such nanostructures. Despite progress towards ‘programmability’ of protein polymer self-assembly, we do not yet have such control. This holds especially for hierarchical structures such as self-assembled fibril bundles, where one would like to have independent control over the structures at the different length-scales. In this thesis we explore the use of heterodimerization as a strategy to control self-assembly of protein polymers at multiple length-scales. We tested a selected set of heterodimer-forming peptide modules. The heterodimer-forming modules are genetically incorporated at the C-terminus of protein polymers with a previously characterized self-assembly behavior. Several newly constructed protein polymers were biosynthesized in the yeast Pichia pastoris and, for these new protein polymers we investigated whether the inclusion of the heterodimer-forming blocks improved the control over the assembly of nanostructures.

    The incorporation of heterodimer-forming modules into protein polymers is not the only tool that can be used for improving programmability of assembly. In Chapter 2 we present an overview of several tools that can be use, and we highlighted their advantages and disadvantages.

    In Chapter 3 we test de novo designed heterodimerizing coiled coils DA = LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGK and DB = LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK. These peptides were fused to hydrophilic random coil protein polymer (CP4) and homotrimer forming protein polymer (T9-CP4). We present data on the production, characterization and functionality for four new protein polymers: CP4-DA, CP4-DB, T9-CP4-DA and T9-CP4-DB. When the new protein polymers were produced using the fermentation process established previously for other protein polymers such as CP4 (i.e. standard fermentation), we found the new protein polymers to be partly degraded. The use of a protease deficient strain, as well as changes in aeration or pH were found ineffective in preventing degradation, but nearly intact products were obtained from a fermentation in which the induction was done at 20 ˚C and in which the medium was supplemented with casamino acids. With respect to the physical properties of the new protein polymers, size exclusion chromatography (SEC) showed that an equimolar mixture of CP4-DA and CP4-DB contained mostly dimers, whereas unmixed CP4-DA and CP4-DB contained only monomers. However, we also found that CP4-DB forms homooligomers at concentrations ≥100 µM. A mixture of T9-CP4-DA and T9-CP4-DB forms a hydrogel, most probably due to both homotypic and heterotypic DA/DB associations. We conclude that when used at low concentration, this pair of coiled coils seems to be suitable to control self-assembly of protein polymers produced in Pichia Pastoris.

    Next, in Chapter 4 we test another pair of de novo designed coiled coils. These are much shorter and have lower reported values of the association constant as compared to the DA/DB coiled coils. The systems consist of a peptide DE = (EIAALEK)3 and a peptide DK = (KIAALKE)3. The two peptides were C-terminally fused to protein polymers CP4 and T9-CP4. The standard fermentations resulted in intact CP4-DE and T9-CP4-DE, but protein polymers CP4-DK and T9-CP4-DK were found to be partly degraded. The degradation of variants with DK module could not be readily resolved by fermentation at higher pH or using proteases deficient strain. For CP4-DK, ion exchange chromatography showed that about 40% of protein polymer (by mass) was intact. We find that for this pair of coiled-coils, homotypic interactions are so strong that they can drive gel formation in the case of T9-CP4-DE, and a strong increase in viscosity for T9-CP4-DK. Mixtures of the complimentary triblocks also form hydrogels, but it is not yet clear to what extent this is due to homotypic DE/ DE and DK/ DK associations, and to what extent it is due to DE/ DK heterodimer formation.

    A very different type of heterodimer-forming block is the so-called WW domain that is found in many natural proteins, and which forms heterodimers with proline-rich peptides PPxY. In Chapter 5 we test the interaction between a naturally occurring WW domain (DWW) and its proline-rich ligand (DPPxY). Both were C-terminally fused to the hydrophilic random coil protein polymer CP4. The new protein polymers CP4-DWW and CP4-DPPxY were produced intact during standard fermentations, but CP4-DPPxY was shown to be glycosylated. Using genetic engineering, we mutated the CP4-DPPxY protein polymer sequence by the substitution Ser12→Ala. A standard fermentation resulted in an intact and non-glycosylated protein polymer CP4-DPPxY*. Interaction studies (ITC and steady state tryptophan fluorescence quenching), showed that both CP4-DPPxY and CP4-DPPxY* bind to CP4-DWW with an equilibrium dissociation constant on the order of mM.

    Finally, to demonstrate that heterodimer-forming blocks can be used to independently control protein polymer self-assembly at multiple length-scales, we selected the heterodimer-forming modules DA and DB to control the lateral interactions of fibrils self-assembled from the previously designed triblock protein polymer C2-SH48-C2. In Chapter 6 we construct the protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB. The C2-SH48-C2 protein polymers assemble into long and stiff fibrils at neutral pH. The aim of the C-terminal attachment of the DA/DB blocks was to be able to control subsequent physical cross-linking and bundling of the fibrils. Both protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB were produced intact and with high yield during fermentation at optimal conditions as discussed in Chapter 3. Using Atomic Force Microscopy (AFM) we show that at neutral pH, fibrils consisting of 100% C2-SH48-C2-DA or C2-SH48-C2-DB protein polymers bundle up and cross-link via homotypic DA/DA and DB/DB associations. Control over the degree of cross-linking and bundling can be obtained by using mixed fibrils consisting of C2-SH48-C2 with controlled amounts of the newly developed protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB. While the effect of the heterodimers on the structure of the fibril network as judged from AFM is very strong, oscillation rheology shows that the inclusion of the heterodimer forming blocks merely leads to a moderate increase in gel stiffness.

    In order to place the research discussed in this thesis into the broader perspective, in Chapter 7 we provide a General Discussion. We discuss several general strategies that can be used to control protein polymer self-assembly and discuss why and when there is a need for using heterodimer forming blocks. After providing an overview over results obtained in this thesis, we highlight the most urgent questions that need to be answered next. This is followed by a discussion on the benefits that heterodimer-driven self-assembly may bring to possible future applications of protein polymers as biomaterials. We also discuss the possible risks for human health end environment that might arise from the use of protein polymers technology. Finally we present some speculations about the future of the field of self-assembling protein polymers.

    A critical assessment of the performance criteria in confirmatory analysis for veterinary drug residue analysis using mass spectrometric detection in selected reaction monitoring mode
    Berendsen, Bjorn J.A. ; Meijer, Thijs ; Wegh, Robin ; Mol, Hans G.J. ; Smyth, Wesley G. ; Armstrong Hewitt, S. ; Ginkel, Leen van; Nielen, Michel W.F. - \ 2016
    Drug Testing and Analysis 8 (2016)5-6. - ISSN 1942-7603 - p. 477 - 490.
    confirmatory analysis - ion ratio - liquid chromatography - mass spectrometry - performance criteria

    Besides the identification point system to assure adequate set-up of instrumentation, European Commission Decision 2002/657/EC includes performance criteria regarding relative ion abundances in mass spectrometry and chromatographic retention time. In confirmatory analysis, the relative abundance of two product ions, acquired in selected reaction monitoring mode, the ion ratio should be within certain ranges for confirmation of the identity of a substance. The acceptable tolerance of the ion ratio varies with the relative abundance of the two product ions and for retention time, CD 2002/657/EC allows a tolerance of 5%. Because of rapid technical advances in analytical instruments and new approaches applied in the field of contaminant testing in food products (multi-compound and multi-class methods) a critical assessment of these criteria is justified. In this study a large number of representative, though challenging sample extracts were prepared, including muscle, urine, milk and liver, spiked with 100 registered and banned veterinary drugs at levels ranging from 0.5 to 100 µg/kg. These extracts were analysed using SRM mode using different chromatographic conditions and mass spectrometers from different vendors. In the initial study, robust data was collected using four different instrumental set-ups. Based on a unique and highly relevant data set, consisting of over 39 000 data points, the ion ratio and retention time criteria for applicability in confirmatory analysis were assessed. The outcomes were verified based on a collaborative trial including laboratories from all over the world. It was concluded that the ion ratio deviation is not related to the value of the ion ratio, but rather to the intensity of the lowest product ion. Therefore a fixed ion ratio deviation tolerance of 50% (relative) is proposed, which also is applicable for compounds present at sub-ppb levels or having poor ionisation efficiency. Furthermore, it was observed that retention time shifts, when using gradient elution, as is common practice nowadays, are mainly observed for early eluting compounds. Therefore a maximum retention time deviation of 0.2 min (absolute) is proposed. These findings should serve as input for discussions on the revision of currently applied criteria and the establishment of a new, globally accepted, criterion document for confirmatory analysis.

    Authentication of Closely Related Fish and Derived Fish Products Using Tandem Mass Spectrometry and Spectral Library Matching
    Nessen, Merel A. ; Zwaan, Dennis J. van der; Grevers, Sander ; Dalebout, Hans ; Staats, Martijn ; Kok, Esther ; Palmblad, Magnus - \ 2016
    Journal of Agricultural and Food Chemistry 64 (2016)18. - ISSN 0021-8561 - p. 3669 - 3677.
    food authentication - mass spectrometry - proteomics - species identification - spectral libraries

    Proteomics methodology has seen increased application in food authentication, including tandem mass spectrometry of targeted species-specific peptides in raw, processed, or mixed food products. We have previously described an alternative principle that uses untargeted data acquisition and spectral library matching, essentially spectral counting, to compare and identify samples without the need for genomic sequence information in food species populations. Here, we present an interlaboratory comparison demonstrating how a method based on this principle performs in a realistic context. We also increasingly challenge the method by using data from different types of mass spectrometers, by trying to distinguish closely related and commercially important flatfish, and by analyzing heavily contaminated samples. The method was found to be robust in different laboratories, and 94-97% of the analyzed samples were correctly identified, including all processed and contaminated samples.

    Oligomerization and hydroxylation of green tea catechins by oxidative enzymes
    Verloop, J.W. - \ 2016
    Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Jean-Paul Vincken. - Wageningen : Wageningen University - ISBN 9789462577770 - 146
    green tea - oxidation - theaflavine - peroxidase - oxygenases - flavanols - phenolic compounds - catechol oxidase - mass spectrometry - maldi-tof - process control - groene thee - oxidatie - theaflavine - peroxidase - oxygenasen - flavanolen - fenolverbindingen - catechol oxidase - massaspectrometrie - maldi-tof - procesbewaking

    Black teas are known for their characteristic brown colour, bitter taste and astringent mouth feel. These sensory characteristics are mainly influenced by the phenolic oxidation products present in black tea. The oxidation of phenolics from green tea leaves during black tea manufacturing is an uncontrolled process. With the objective to make tea oxidation a more controlled process, the aim of this thesis was to understand the enzymatic oxidation reactions occurring during tea oxidation, and to enable more rapid analysis of complex mixtures of phenolics. By incubating green tea catechins with an exogenous tyrosinase, a black tea-like phenolic profile was obtained, enriched in theaflavins, which are important for quality of tea. Further oxidation of theaflavins yielded theatridimensins, in which an epicatechin is coupled to the benzotropolone ring of theaflavin. By using MS/MS on selected ions these theatridimensins were shown to occur in black tea. This MS method could also be used to distinguish isomeric procyanidins and dehydrocatechins based on MS2 fragments, as well as the different interflavanic configurations occurring in dehydrodicatechins. The dehydrocatechins were shown to occur in black tea as well. Besides these oligomerization reactions mediated by tyrosinase, oxidation of tea phenolics also comprised hydroxylation. The enzymatic activity from tea leaves responsible for this hydroxylation reaction, was found to be peroxidase. All findings were condensed into a new version of the ‘oxidative cascade hypothesis’, describing the oxidation reactions towards formation of a black tea.

    Oligosaccharides in Urine, Blood, and Feces of Piglets Fed Milk Replacer Containing Galacto-oligosaccharides
    Difilippo, Elisabetta ; Bettonvil, Monique ; Willems, Rianne ; Braber, Saskia ; Fink-Gremmels, Johanna ; Jeurink, Prescilla V. ; Schoterman, Margriet H.C. ; Gruppen, Harry ; Schols, Henk A. - \ 2015
    Journal of Agricultural and Food Chemistry 63 (2015)50. - ISSN 0021-8561 - p. 10862 - 10872.
    absorption - capillary electrophoresis - creatinine - fermentation - GOS - intestine - liquid chromatography - mass spectrometry - pig - prebiotics

    Human milk oligosaccharides (HMOs) are absorbed into the blood (about 1% of the HMO intake) and subsequently excreted in urine, where they may protect the infant from pathogen infection. As dietary galacto-oligosaccharides (GOS) have partial structural similarities with HMOs, this study investigated the presence of GOS and oligosaccharides originating from milk replacer in blood serum, urine, and cecal and fecal samples of piglets, as a model for human infants. Using liquid chromatography-mass spectrometry and capillary electrophoresis with fluorescence detection, oligosaccharides originating from piglet diet including 3′-sialyllactose and specific GOS ranging from degree of polymerization 3 to 6 were detected in blood serum and in urine of piglets. In blood serum, GOS levels ranged from 16 to 23 μg/mL, representing about 0.1% of the GOS daily intake. In urine, approximately 0.85 g of GOS/g of creatinine was found. Cecum digesta and feces contained low amounts of oligosaccharides, suggesting an extensive GOS intestinal fermentation in piglets.

    Modification of Prenylated Stilbenoids in Peanut (Arachis hypogaea) Seedlings by the Same Fungi That Elicited Them : The Fungus Strikes Back
    Aisyah, Siti ; Gruppen, Harry ; Slager, Mathijs ; Helmink, Bianca ; Vincken, Jean Paul - \ 2015
    Journal of Agricultural and Food Chemistry 63 (2015)42. - ISSN 0021-8561 - p. 9260 - 9268.
    Arachis hypogaea - Aspergillus oryzae - detoxification - fungal metabolism - glycosylation - groundnut - mass spectrometry - oxidative cleavage - peanut - Rhizopus oryzae

    Aspergillus oryzae and Rhizopus oryzae were compared for inducing the production of prenylated stilbenoids in peanut seedlings. The fungus was applied at two different time points: directly after soaking (day 1) or after 2 days of germination (day 3). Aspergillus- and Rhizopus-elicited peanut seedlings accumulated an array of prenylated stilbenoids, with overlap in compounds induced, but also with compounds specific to the fungal treatment. The differences were confirmed to be due to modification of prenylated stilbenoids by the fungus itself. Each fungus appeared to deploy different strategies for modification. The content of prenylated stilbenoids modified by fungi accounted for around 8% to 49% (w/w) of total stilbenoids. The contents of modified prenylated stilbenoids were higher when the fungus was applied on day 1 instead of day 3. Altogether, type of fungus and time point of inoculation appeared to be crucial parameters for optimizing accumulation of prenylated stilbenoids in peanut seedlings.

    Single particle ICP-MS combined with a data evaluastion tool as a routine techique for the analysis of nanoparticles in complex matrices
    Peters, R.J.B. ; Herrera-Rivera, Z. ; Undas, A.K. ; Lee, M.K. van der; Marvin, H.J.P. ; Bouwmeester, H. ; Weigel, S. - \ 2015
    Journal of Analytical Atomic Spectrometry 30 (2015). - ISSN 0267-9477 - p. 1274 - 1285.
    inductively-coupled plasma - field-flow fractionation - mass spectrometry - quantitative-determination - silver nanoparticles - gold nanoparticles - chicken meat - food - quantification - nanomaterials
    Detection and characterization of nanoparticles (NPs) in complex media as consumer products, food and toxicological test media is an essential part of understanding the potential benefits and risks of the application of nanoparticles. Single particle ICP-MS (spICP-MS) was studied as a screening tool for the detection and characterization of nanoparticles in complex matrices such as food and biological tissues. A data evaluation tool was created for the calculation of particle size, concentration and size distribution from the raw data. spICP-MS measurements were carried out on a standard quadrupole instrument as well as on a sector-field instrument. Performance characteristics were determined for four types of NPs. For the quadrupole instrument the size detection limits were 20 nm (Au and Ag), 50 (TiO2) and 200 nm (SiO2). For the sector-field instrument size detection limits are lower, 10 nm (Au). Concentration detection limits ranged from 1 ng L-1 for 60 nm Au NPs to 0.1 µg L-1 for 500 nm SiO2 particles. The dynamic range of spICP-MS is limited to two orders of magnitude and as a consequence sample dilution is often required. The precision of the method was found to be
    New analytical approaches for faster or greener phytochemical analyses
    Shen, Y. - \ 2015
    Wageningen University. Promotor(en): Han Zuilhof; B. Chen, co-promotor(en): Teris van Beek. - Wageningen : Wageningen University - ISBN 9789462573307 - 206
    giftige planten - chemische samenstelling - massaspectrometrie - niet-destructief testen - bemonsteren - zuiveren - oplosmiddelen - illicium - poisonous plants - chemical composition - mass spectrometry - nondestructive testing - sampling - purification - solvents - illicium

    Summary

    Chapter 1 provides a short introduction into the constraints of phytochemical analysis. In order to make them faster, less laborious and greener, there is a clear scope for miniaturized and simplified sample preparation, solvent-free extractions and the use of cleaner solvents in preparative HPLC. Possible modern techniques to achieve this, such as microfluidic chips, ambient mass spectrometry, selective magnetic nanoparticles, and use of less toxic but equally efficient solvents are discussed. Clear aims were formulated and research towards fulfilling these aims in the field of phytochemical analysis is carried out in this thesis.

    A first version of a 3-phase liquid-liquid extraction (LLE) chip for the miniaturized sample pretreatment of alkaloids was introduced by our group in 2009. In Chapter 2 more biodegradable and less-toxic solvents for the transport phase and a more suitable pH for the feed phase were evaluated. The extraction efficiency improved. On-line hyphenation of the 3-phase chip to nanoLC-UV/MS was also investigated. This combination saved a lot of time and solvent in comparison with traditional methods for the purification of alkaloids from plant materials.

    A novel Induced Phase Separation Extraction (IPSE) chip was introduced in Chapter 3 for efficient sample pretreatment. The acetonitrile – water (1:1) sample solutions were separated in organic and aqueous phases in this IPSE chip based on their affinity for both phases. In turn this could be correlated with the log D values of the analytes. Some optimization regarding design, operation, flows and solvents was carried out. Extraction efficiencies of several model compounds were determined. A real sample application with a plant used in Traditional Chinese Medicines (TCMs) was carried out to show the usefulness of the IPSE chip in dealing with complex matrixes.

    Chapter 4 presented an unambiguous distinction between toxic Japanese star anise and non-toxic Chinese star anise fruits within seconds without any sample pretreatment by DART-orbitrap MS technology. Both positive and negative mode gave the same result, although the latter mode is preferred because of its higher sensitivity and cleaner spectra. Not only raw plant materials but also a herbal tea containing both Chinese and Japanese star anise could be quickly and accurately distinguished by DART-HRMS.

    In Chapter 5, direct plant spray in combination with orbitrap HRMS allowed, like DART-HRMS, for an unambiguous distinction between toxic Japanese star anise and non-toxic Chinese star anise fruits within seconds without any sample pretreatment in both positive and negative mode. Direct plant spray ionization has the advantage of low cost, simplicity, room temperature and low standard deviations. Neither the DART nor the direct spray method is very suitable for quantitative measurements of solid samples like star anise fruits.

    Chapter 6 describes the purification of eight ginkgolic acids (GAs) from raw plant material (Ginkgo biloba) by using only three steps, namely (1) extraction; (2) selective purification by cheap Fe3O4 magnetic nanoparticles (MNPs); (3) preparative HPLC on a C8 column. The three main constituents occurring at concentrations of 0.15% - 0.60% were enriched to >95% absolute purity without using tedious (gravity) column chromatography with halogenated solvents.

    Preparative RP-HPLC is an efficient but not very green technique for the final purification of fine chemicals and natural products as large volumes of acetonitrile, methanol and tetrahydrofuran (THF) are consumed. In Chapter 7 it was investigated whether less toxic organic solvents could replace them. As a test case the preparative separation of Ginkgo terpene trilactones (TTLs) was selected. By a two-step chromatographic optimization procedure a 30 min gradient using only water, ethanol, acetone and ethyl acetate was developed, which gave a baseline separation of 480 mg of an injected TTLs mixture. All five individual TTLs were > 95% pure.

    Traditional Chinese Medicines (TCMs) are one of the oldest and most used traditional drugs in the world. Many plants are used for their preparation. An overview of HPLC-related methods such as: multicomponent quantitation, fingerprinting, bioaffinity chromatography and on-flow assays for screening and quality control of TCMs was presented and discussed in Chapter 8.

    The final Chapter 9 discusses the major findings of this work and gives further perspectives.

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