El análisis de las heces es una herramienta fundamental para el trabajo de campo, especialmente para identificar la presencia de una determinada especie en un área. Los ácidos biliares fecales y su concentración relativa siguen patrones que son especie-específicos, y pueden ser caracterizados por Cromatografía en Capa Fina (TLC) y Cromatografía Líquida de Alta Performance (HPLC). Estas técnicas han sido utilizadas para diferenciar heces de varias especies de mamíferos, pero nunca en Xenarthra. En esta tesis se identificaron, mediante dichas técnicas, los perfiles de ácidos biliares fecales de 11 especies de Xenarthra, a partir del análisis de 107 heces de diferentes individuos: Zaedyus pichiy (n=10), Chaetophractus vellerosus (n=5), Chaetophractus villosus (n=57), Dasypus hybridus (n=4), Priodontes maximus (n=2), Tamandua tetradactyla (n=14), Myrmecophaga tridactyla (n=4), Tolypeutes matacus (n=8), Euphractus sexcinctus (n=1), Choloepus didactylus (n=1) y C. hoffmanni (n=1). Se encontraron diferencias entre los patrones de ácidos biliares para todas las especies, pero no entre machos y hembras, ni entre animales de cautiverio y silvestres de la misma especie. La TLC resultó ser una técnica útil, de costo relativamente bajo y rápida para el análisis de los perfiles de ácidos biliares fecales. Sin embargo, presentó ciertas limitaciones en la resolución de los ácidos biliares tauroconjugados. La HPLC demostró ser una técnica más resolutiva y sensible que la TLC, permitiendo la separación e identificación de los ácidos biliares tauroconjugados, y de otros compuestos no visualizados por TLC. El 90% o más de los ácidos biliares fueron del tipo VI, con estructura C24. Todas las especies presentaron DHCA, UDCA, CA, GCA, TCA, TDCA y LCA. Los valores de similitud entre los patrones de ácidos biliares fecales reflejaron, en su mayoría, las relaciones filogenéticas establecidas para el Superorden Xenarthra. Se demostró para Xenarthra que la determinación cromatográfica de los ácidos biliares fecales es un método preciso para la identificación específica de heces, por lo que resultaría una valiosa herramienta ecológica. Estos resultados son los primeros para Xenarthra y podrían ser importantes para futuros estudios acerca de la fisiología, ecología y conservación del grupo. / The analysis of feces is a fundamental tool for field work, especially to identify the presence of certain species in an area. Fecal bile acids and their relative concentration follow patterns that are species-specific, and can be characterized by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). These techniques have been used for differentiating feces of several mammal species; however, it has never been used for Xenarthra species. In this thesis we identified, by those techniques, the fecal bile acid profile of 11 Xenarthra species, by the analysis of 107 feces from different individuals: Zaedyus pichiy (n=10), Chaetophractus vellerosus (n=5), Chaetophractus villosus (n=57), Dasypus hybridus (n=4), Priodontes maximus (n=2), Tamandua tetradactyla (n=14), Myrmecophaga tridactyla (n=4), Tolypeutes matacus (n=8), Euphractus sexcinctus (n=1), Choloepus didactylus (n=1) and C. hoffmanni (n=1). There were differences between the bile acid patterns of all the species, but not between males and females, nor between wild and captive animals of the same species. TLC was a useful, low cost and rapid technique to analyze fecal bile acid profiles. However, it showed some limitations in the resolution of tauroconjugated bile acids. HPLC was more resolute and sensitive than TLC, allowing separation and identification of tauroconjugated bile acids and of other compounds which were not visualized by TLC. 90% or more of the bile acids were of the type VI, with a structure C24. All species presented DHCA, UDCA, CA, GCA, TCA, TDCA and LCA. Similitude values among fecal bile acid profiles of all species reflected, in a great majority, phylogenetic relationships established for the Superorden Xenarthra. We established, for Xenarthra, that chromatographic determination of fecal bile acids is a precise method for specific identification of feces, being a useful ecological tool. These results are the first for Xenarthra and would be important for future studies about the physiology, ecology and conservation of the group.

Consumers view natural antioxidants as a safe means to reduce spoilage in foods. In addition, these compounds have been reported to be responsible for human health benefits. Identification of these compounds in peanut skins may enhance consumer interest, improve sales, and increase the value of peanuts. This study evaluated analytical methods which have not been previously incorporated for the analysis of peanut skins. Toyopearl size-exclusion chromatography (SEC) was used for separating phenolic size-classes in raw methanolic extract from skins of Gregory peanuts. This allowed for an enhanced analysis of phenolic content and antioxidant activity based on compound classes, and provided a viable preparatory separation technique for further identification. Toyopearl SEC of raw methanolic peanut skin extract produced nine fractions based on molecular size. Analysis of total phenolics in these fractions indicated Gregory peanut skins contain high concentrations of phenolic compounds. Further studies revealed the fractions contained compounds which exhibited antioxidant activities that were significantly higher than that of butylated hydroxyanisole (BHA), a common synthetic antioxidant used in the food industry. This indicates peanut skin extracts are a viable antioxidant source, and that synthetic antioxidants can be replaced with those naturally-derived from peanut by-products. Structures contained in each fraction were identified using high performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) ion trap mass spectrometry (MSn). Prior to this study, approximately 20 compounds have been identified in peanut skins. The combination of Toyopearl SEC with ESI-HPLC-MSn allowed for the identification of 314 phenolic-based compounds, most of which are newly discovered compounds in peanut skins. Many compounds identified are known to have powerful antioxidant effects, and also have been reported to exhibit numerous beneficial chemical and biological activities, including the treatment of various human health-related conditions. It is evident that peanut skins may be a potential untapped source for the extraction of natural food antioxidants, nutracueticals, and even pharmaceuticals. Because peanut skins are largely a wasted resource to peanut processors, the novel polyphenols identified in this research could have a significant financial impact on the peanut industry. / Ph. D.

Ellagitannins are plant phenolics characterized by biaryl-coupled gallic acid moieties esterified to a D-glucose core. They are widely distributed through higher plants. In the case of oaks, ellagitannin concentrations in heartwood can reach up to 10% (dry wt. basis). These secondary metabolites are not only important physiologically but also influence the economic value and quality of wood products that contain them. Efforts were made to develop and validate the methods used to quantify both soluble and insoluble ellagitannins. First, the efficiencies of the two commonly used extraction solvents, aqueous acetone and aqueous methanol were evaluated. The results showed that aqueous acetone is superior to aqueous methanol in obtaining higher vescalagin and castalagin yields. In a separate study, the method used for determining insoluble ellagitannins was found to under-estimate the contents of insoluble ellagitannins in wood products. Anhydrous methanolic HCl was found to be an excellent reagent for releasing insoluble ellagic acid and gallic acid (as methyl gallate) from biomass substrates. Optimization of both the reaction conditions and the gradient HPLC analysis has led to the development of a robust and reliable protocol. The chemical stability of the two predominant ellagitannins in oaks (vescalagin and castalagin) were evaluated in aqueous methanol and water. It was found that oxygen, pH and higher temperature (60 °C) affect their stability with higher temperature being the most prominent factor. Both vescalagin and castalagin were found unstable in methanolic solutions. Vestalagin, however, is less stable than castalagin. In the course of finding alternative models for ellagitannin biosynthesis study, both callus tissues and suspension cell cultures of white oak (Quercus alba) and sweetgum (Liquidambar styraciflua) were investigated for their possible use as models for ellagitannin biosynthesis. It was found that oak callus tissue cultures (Quercus alba) are capable of producing ellagitannins, and the production and profile of ellagitannins can be modified by adjusting the media composition. Comparison of extracts from the heartwood of Quercus alba with those from callus tissue reveals that they have similar ellagitannin profiles. Through manipulation of the media nitrogen and copper concentrations the callus tissue produced almost 3 times as much castalagin and vescalagin. Suspension cells of Quercus alba and Liquidambar styraciflua were found to be unsuitable for the study of biosynthesis of ellagitannins. These cells either did not produce any detectable level of ellagitannins or the production was unstable. Although the suspension cells could be elicited to produce ellagic acid with glycanases (Driselase), the levels of ellagic acid were too low for quantitative metabolic studies. A method using high performance liquid chromatography – mass spectrometry was developed and optimized with purified ellagitannins. Ellagitannins analyzed under the optimal conditions all provide base peaks of (M-H)- from which the molecular weights of the ellagitannins can be determined. Mild fragmentation was also achieved to give fragments characteristic of ellagitannins (loss of ellagic acid and gallic acid if present). These characteristic peaks allow for rapid identification of ellagitannins from other secondary metabolites present in the samples. Application of the HPLC/ESI-MS in the identification of monomeric ellagitannins in white oak heartwood extracts revealed that it can unambiguously identify the two monomeric ellagitannins, castalagin and vescalagin, and their degradation product, ellagic acid. The key fragmentation pathways of the ellagitannins are also described. Finally, preliminary work using proteomics to study the heartwood formation was conducted. Proteins from transition zone and sapwood were determined and resolved with two-dimensional electrophoresis. It was found that both sapwood and transition woods contain active enzyme(s) capable of catalyzing formation of ellagic acid from pentagalloylglucose. Preliminary results from the 2-D gel separation of sapwood and transition wood proteins showed more protein spots in sapwood than in transition wood, suggesting that sapwood not only had higher protein levels but also a great total number of proteins. The lower complexity of the transition wood proteome suggests that this material may be a good substrate for studying the biaryl-coupling process. / Ph. D.

No / A sensitive and rapid oil-in-water (O/W) microemulsion high performance liquid chromatography (MELC) method has been developed. The water-in-oil (w/o) microemulsion was used as a mobile phase in the determination of salbutamol in aqueous solutions. In addition, the influence of operating parameters on the separation performance was examined. The samples were injected into C18, (250mmx4.6mm) analytical columns maintained at 25(o)C with a flow rate 1 ml/min. The mobile phase was 95.5% v/v aqueous orthophosphate buffer 20 mM (adjusted to pH 3 with orthophosphoric acid), 0.5% ethyl acetate, 1.5% Brij35, and 2.5% 1-butanol, all w/w. The salbutamol and internal standard peaks were detected by fluorescence detection at the excitation and emission wavelengths of 267 and 313 nm respectively. The method had an accuracy of > 97.78% and the calibration curve was linear (r2 = 0.99) over salbutamol concentrations ranging from 25 to 500 ng/mL. The intra-day and inter-day precisions (CV %) were <1.6 and <1.8, respectively. The limit of detection (LOD) and limit of quantitation (LOQ) were 9.61ng/ml and 29.13ng/ml, respectively. The method reported is simple, precise and accurate, and has the capacity to be used for determination of salbutamol in the pharmaceutical preparation.

No / An isocratic oil-in-water microemulsion High Performance Liquid Chromatography (MELC) was developed and validated for the determination of terbutaline in urine samples. A solid phase extraction (SPE) method which used Oasis HLB cartridges was optimised to isolate terbutaline from a urine matrix followed by HPLC with fluorescence detection. The urinary assay was performed in accordance with FDA and ICH regulations for the validation of bioanalytical samples. The method uses the isocratic oil-in-water micro emulsion to separate the terbutaline from the endogenous urine components. The chromatographic separation was carried out on C18-Spherisorb (250mm×4.6mm) analytical column maintained at 30 °C. The mobile phase was 94.4% aqueous orthophosphate buffer (adjusted to pH 3 with orthophosphoric acid), 0.5% ethyl acetate, 1.5% Brij35, 2.5% 1-butanol and 1.1% Octanesulfonic acid (OSA), all w/w. The terbutaline peak was detected by fluorescence detection, using excitation and emission wavelengths of 267 and 313 nm, respectively. The linearity of response was demonstrated at six different concentrations of terbutaline which were extracted from spiked urine, ranging from 60 to 1000ng/ml. The terbutaline was extracted from urine by a solid phase extraction clean-up procedure on Oasis HLB cartridges, and the relative recovery was >87.64% (n = 5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 20.21 and 61.24ng/ml, respectively. The intra-day and inter-day precisions (in term of % coefficient of variation) were <3.56% and <2.87%, respectively. In the method development the influence of the composition of the microemulsion system was also studied and the method was found to be robust with respect to changes of the microemulsion components.

Ubiquitous bacterial species with the potential to serve as reservoir for antibiotic resistance genes are fanning the flames of antimicrobial resistance. Shortly after discovering the high-level daptomycin (DAP) resistant Mammaliicocci sciuri (M. sciuri) strain TS92 this threat was apparent, as first genetic studies of the Ziebuhr group pointed towards a novel resistance mechanism. The elucidation of this resistance mechanism was therefore urgently required and was addressed by investigating the biological and the chemical background. The aim of this work was to explore the chemical background by applying LC-(HR)MS techniques. Non-specific adsorption was identified as the reason for the initial observation of highly deviating results when working with DAP. The extent was therefore evaluated in a study including several solvents and matrices as well as several syringe filter, ultrafiltration, and reaction container materials. It was shown, that the adsorption behavior of DAP is strongly dependent on the solvent and the contact surface but does not follow any general rule. It became apparent that a preliminary empirical study is always necessary to select an individually suitable combination. Moreover, it could be demonstrated that polypropylene containers are entirely unsuitable for DAP in Mueller-Hinton (MH) medium, as the adsorption loss is already > 25% after one transfer. Within the frame of this study, the use of Protein LoBind® or, with reservation, laboratory glass surfaces was highly recommended instead, whenever possible. The most suitable storage solvent for DAP stock solutions was found in 50% methanol. Following these recommendations resulted in precisely reproducible results in both the biological and chromatographic studies. In previous experiments, a degradation of DAP incubated together with M. sciuri TS92 was suspected. To confirm this, a suitable LC-MS/MS method for the quantification of DAP in a bacterial growth medium and in presence of M. sciuri TS92 was therefore developed. The sample preparation consisted of sterile filtration followed by solvent-induced protein precipitation and centrifugation. The reversed phase chromatography assay made use of a gradient elution and covered the concentrations from 1 to 20 μg/mL by a 1/x² weighted linear regression model, that was calculated from eight calibration levels within the range. All requirements of the internationally approved European Medicines Agency (EMA) guideline on bioanalytical method validation were met. It was subsequently applied to study the time-dependent amount of DAP in presence or absence of M. sciuri TS92 in MH medium. The initial suspicion was confirmed, as the amount of DAP decreased significantly after 24 h of incubation only in presence of M. sciuri TS92. Thus, antibiotic inactivation was hypothesized to be the defense mechanism against DAP. The novel two-gene operon drcAB was found by the Ziebuhr group to mediate the resistance of M. sciuri TS92 against DAP. Moreover, the group provided a heterologous expressible clone of this operon on a plasmid vector, which was transformed into DAP-sensitive Staphylococcus aureus (S. aureus), generating S. aureus RN4220_Pxyl/tet-drcAB. The previously developed DAP quantification assay was applied here to monitor the time dependent amount of DAP in a liquid MH medium culture of this strain upon heterologous drcAB expression. The assay remained applicable and valid for the cultures of this artificial S. aureus strain and it could be demonstrated that the presence of DrcAB leads to a significant loss of DAP after 24 h of incubation. Along with the results of the Ziebuhr group, this was evidence that drcAB expression indeed confers DAP resistance to S. aureus, namely by structural modification(s) and thus antibiotic inactivation. For the qualification of these structural modification(s), drcAB expressing and non-expressing S. aureus RN4220_Pxyl/tet-drcAB cultures in MH medium were again spiked with DAP. After 24 h of incubation, their supernatants were subjected to untargeted LC-HRMS analysis. Apart from the detection principle, this method was a slightly modified version of the previously quantification method. HRMS and MS/HRMS data were simultaneously obtained from information-dependent acquisition runs providing comprehensive characterization of the sample compositions. The subsequently performed GUCS approach revealed two substances that emerge in the presence of DrcAB. Under consideration of the corresponding (MS/)HRMS information, the isotope pattern and the in silico plausibility, the data allowed structural elucidation of both substances. The structures found suggested a two-step modification mechanism of DAP, comprising the N-substitution of the arylamine moiety of kynurenine with C3H4NO and subsequently the hydrolysis of the lactone moiety. This resulted in the postulation that DAP is deprived of its antibiotic effect by the enzymatic transfer of dehydroalanine. / Ubiquitär vorkommende bakterielle Spezies, die als Genreservoir für Antibiotikaresistenzen dienen können, sind Öl für das Feuer der antimikrobiellen Resistenz. Kurz nach der Entdeckung des hochgradig Daptomycin (DAP)-resistenten Mammaliicocci sciuri (M. sciuri) TS92, als die ersten genetischen Studien der Arbeitsgruppe Ziebuhr auf einen neuartigen Resistenzmechanismus hindeuteten, war diese Bedrohung erkennbar. Die Aufklärung dieses Resistenzmechanismus war daher dringend erforderlich und wurde durch Untersuchungen des biologischen als auch des chemischen Hintergrunds adressiert. Das Ziel der vorliegenden Arbeit war die Untersuchung des chemischen Hintergrundes unter Anwendung verschiedener flüssigchromatographischer sowie massenspektrometrischer Techniken. Eine unspezifische Adsorption wurde als Grund für die, zu Beginn der Untersuchungen, häufige Beobachtung voneinander abweichender Ergebnisse bei der Arbeit mit DAP identifiziert. Das Ausmaß wurde daher in einer Studie bewertet, in der unterschiedliche Lösungsmittel und Matrices, sowie unterschiedliche Materialien für Spritzenfilter, Ultrafiltrationsmembranen und Reaktionsgefäßen einbezogen wurden. Es wurde gezeigt, dass das Adsorptionsverhalten von DAP stark vom Lösungsmittel und der Kontaktoberfläche abhängig ist, dabei jedoch keiner allgemeinen Regel folgt. Daraus wurde ersichtlich, dass zur Auswahl einer individuell geeigneten Kombination immer eine empirische Voruntersuchung notwendig ist. Darüber hinaus konnte festgestellt werden, dass Polypropylen-Reaktionsgefäße völlig ungeeignet für DAP in Müller-Hinton (MH) Medium sind und dass bei dessen Verwendung bereits nach einem Transferschritt ein Adsorptionsverlust von mehr als 25% auftritt. Im Rahmen dieser Studie wurde stattdessen die Verwendung von Protein LoBind® oder, unter Vorbehalt, Laborglas Oberflächen wann immer möglich empfohlen. Das geeignetste Lösungsmittel zur Lagerung von DAP-Stammlösungen war 50% Methanol. Die Einhaltung dieser Empfehlungen führte sowohl bei den biologischen als auch bei den chromatographischen Studien zu gut reproduzierbaren Ergebnissen. In früheren Experimenten wurde der Abbau von DAP bei Inkubation mit M. sciuri TS92 vermutet. Um dies zu bestätigen, wurde deshalb eine geeignete LC-MS/MS-Methode zur Quantifizierung von DAP in bakteriellen Wachstumsmedien und in Anwesenheit von M. sciuri TS92 entwickelt. Die Probenvorbereitung bestand zunächst aus einer Sterilfiltration mit anschließender lösungsmittelinduzierten Proteinfällung und Zentrifugation. Die Umkehrphasen-chromatographische Prüfung verwendete ein Gradient und umfasste den Konzentrationsbereich von 1 bis 20 μg/mL. Dieser wurde durch ein lineares Regressionsmodell aus acht Kalibrierpunkten mit einer Gewichtung von 1/x² abgedeckt. Alle Anforderungen der international anerkannten European Medicines Agency (EMA)-Leitlinie „Guideline on bioanalytical method validation“ wurden eingehalten. Anschließend wurde die Methode zur zeitabhängigen Untersuchung der Menge an DAP in MH-Medium bei An- bzw. Abwesenheit von M. sciuri TS92 verwendet. Der initiale Verdacht wurde bestätigt, nachdem die Menge an DAP nach einer Inkubationszeit von 24 h nur bei Anwesenheit von M. sciuri TS92 signifikant abfiel. Daher konnte die Inaktivierung des Antibiotikums als Abwehrmechanismus gegen DAP angenommen werden. Die Arbeitsgruppe Ziebuhr zeigte, dass das neuartige und aus zwei Genen bestehende drcAB Operon die DAP-Resistenz an M. sciuri TS92 vermittelt. Außerdem stellte die Gruppe einen heterolog exprimierbaren Klon des Operons auf einem Plasmidvektor bereit, der in einen DAP-sensitiven Staphylococcus aureus (S. aureus) transformiert wurde, wodurch S. aureus RN4220_Pxyl/tet-drcAB entstand. Die zuvor entwickelte DAP-Quantifizierungsmethode wurde verwendet, um die zeitabhängige DAP-Menge in einer flüssigen MH-Kultur dieses Stamms nach heterologer drcAB-Expression zu untersuchen. Die Methode blieb anwendbar und valide für die MH-Kultur dieses künstlichen S. aureus Stamms und es konnte gezeigt werden, dass die Anwesenheit von DrcAB zu einem signifikanten DAP-Verlust nach einer Inkubationszeit von 24 h führte. Zusammen mit den Ergebnissen der Arbeitsgruppe Ziebuhr weist dies daraufhin, dass die Expression von drcAB tatsächlich eine DAP-Resistenz in S. aureus erzeugt, und zwar durch strukturelle Modifikation(en) und somit durch Inaktivierung des Antibiotikums. Zur Qualifizierung dieser strukturellen Modifikation(en) wurden drcAB exprimierende und nicht exprimierende S. aureus RN4220_Pxyl/tet-drcAB Kulturen in MH-Medium erneut mit DAP dotiert. Nach einer Inkubationszeit von 24 h wurden die Überstände einer ungerichteten LC-HRMS Analyse unterzogen. Abgesehen vom Detektionsprinzip wurde eine geringfügig veränderte Methode der Quantifizierungsmethode verwendet. HRMS- und MS/HRMS-Daten wurden simultan durch IDA-Messläufe generiert, wodurch eine umfassende Charakterisierung der Probenbestandteile erhalten wurde. Der im Anschluss durchgeführte GUCS Ansatz offenbarte zwei Substanzen, die bei Anwesenheit von DrcAB auftreten. Unter Berücksichtigung der dazugehörigen (MS/)HRMS-Informationen, der Isotopenmuster und der in silico-Plausibilität, erlaubten diese Daten die Strukturaufklärung beider Substanzen. Diese lassen einen zweistufigen Modifizierungsmechanismus vermuten, in dem zunächst die N-Substitution des Arylaminrestes von Kynurenin mit C3H4NO und anschließend die Hydrolyse des Lactons stattfindet. Dies führte zur Postulierung, dass DAP durch die enzymatische Übertragung von Dehydroalanin seiner antibiotischen Wirkung beraubt wird.

Introduction : La présence des moisissures et de leurs mycotoxines dans les matrices complexes de papiers est peu étudiée. Notre travail a porté sur la recherche de mycotoxines sur les papiers patrimoniaux altérés par le foxing et les papiers peints moisis issus de logements dont les habitants ont été diagnostiqués comme porteurs de symptômes allergiques et du syndrome des bâtiments malsains. Objectifs : Identifier les espèces fongiques de ces deux types de supports papiers et y déterminer la production de métabolites fongiques. Matériels et Méthodes : Le foxing a été caractérisé par des techniques pluridisciplinaires (physiques, biologiques, bioanalytiques, tests de cytotoxicité). Les métabolites fongiques dans les extraits hydro-organiques de ces papiers ont été recherchés et identifiés par spectrométrie de masse afin d'évaluer s'ils pouvaient être reliés aux espèces fongiques détectées par microbiologie. Puis le risque toxique de ces extraits de papiers a été évalué sur un modèle cellulaire in vitro. Résultats : Pour le foxing, nous avons pu en exclure une origine métallique et montrer que les micro-organismes présents dans celui-ci sont essentiellement des espèces fongiques. Pour les papiers peints, des pics relatifs à des métabolites fongiques ont été retrouvés. Grâce à l'ensemencement individuel d'espèces fongiques sur papier peint et à l'aide de la MS2, nous avons pu relier les composés de m/z 401 et 487 à S. chartarum. Les tests de cytoxicité ont montré une augmentation significative de la cytotoxicité des cellules A549 avec certains papiers peints moisis par rapport au papier peint témoin. Les cellules A549 ont montré une surexpression du TNF-α, de l'IL-8 et du CYP 1A1, après contact avec ces mêmes papiers peints. Discussion-Conclusion : Nous n'avons détecté aucune mycotoxine dans le foxing excluant ainsi d'éventuels liens entre inhalation de mycotoxines émanant de vieux manuscrits et symptomatologie respiratoire. Pour les habitants des logements et leurs symptômes respiratoires, il est difficile de les relier à une espèce fongique donnée ou à un métabolite donné, bien que S. chartarum ait pu être mis en cause. Ces premiers résultats doivent être confirmés par des études ultérieures / Introduction: Little study has been carried out on the presence of fungi and their mycotoxins in complex paper matrices. Our work has focused on finding mycotoxins on heritage paper altered by the foxing and wallpapers from moldy homes whose residents have been diagnosed with symptoms of allergies and sick building syndrome. Objectives: To identify the fungal species of these two types of paper and to determine the production of fungal metabolites. Materials and methods: The foxing has been characterized using multi-disciplinary technics (physical, biological, bioanalytical, cytotoxicity assays). The fungal metabolites in the hydro-organic extracts of these papers were sought and identified by mass spectrometry to assess whether they could be related to fungal species detected by microbiology. The toxicological risk of wallpaper extracts was then evaluated on model in vitro cells. Results: For the foxing, we could exclude a metal origine and we showed that the microorganisms present are mainly fungal species. For wallpapers, fungal metabolites were found. By seeding individual fungal species on wallpaper and using MS2, we were able to link the compounds of m/z 401 and 487 to S. chartarum. Cytotoxicity tests showed a significant increase in cytotoxicity of A549 cells with moldy wallpaper in comparison with the control wallpaper. A549 cells showed an overexpression of TNF-α after contact with these wallpapers as well as a significant overexpression of IL-8 and CYP 1A1.Discussion-Conclusion: We detected no mycotoxin in foxing, thus excluding possible links between inhalation of mycotoxins from old manuscripts and respiratory symptoms. For people in their homes and respiratory symptoms, it is difficult to draw any relationships to a given fungal species or a metabolite although S. chartarum has been implicated. These initial results should be confirmed by further study

Charles University in Prague Pharmaceutical Faculty in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Miloslava Ďuráčová Consultant: RNDr. Jitka Vytlačilová, Ph.D. Title of thesis: HPLC-MS determination of active biological compounds in Elbe river Biologically active compounds have a various ways to enter environment. They can occur as pesticides, cosmetics and pharmaceutics and their metabolites. Such compounds are classified as environmental contaminants. There is a increased environmental concentration connected with increasing consumption of biologically active compounds. There is a urgent need to follow the effect on the different parts of ecosystem and levels of biologically active compounds. This work was prepared during the cooperation with Povodí Labe state enterprise. A novel analytical method was described in the experimental part of this thesis. I was employed to evaluate biologically compounds levels in the surface water samples. This method is now routinely used. 10 out of 19 evaluated compounds reached concentrations higher than LOD. Acetaminophene (559 ng/l, Králický stream), gabapentine (457 ng/l Elbe, LB Schmilka), cotinine (139 ng/l, Králický potok) were the biologically active compounds with the highest found concentrations. Keywords: HPLC-MS analysis,...

In der Promotion wird die Entwicklung, Optimierung und Validierung einer Reversed-phase-Chromatography Methode zur Messung des Ribavirinplasmaspiegels beschrieben. Diese wurde mit einer Solid Phase Extraction zur Probenvorbereitung kombiniert. Zudem finden sich zahlreiche Auswertungen von gemessenen Patienenchromatogrammen zu ausgewählten, klinisch relevanten Fragestellungen, wie beispielsweise die Darstellung des Ribavirinplasmaspiegels im Tagesverlauf, im Verlauf der ersten sechs Therapiewochen, im Vergleich von Männern und Frauen, sowie bei einem niereninsuffizienten Patienten. Zu den erhobenen Ergebnissen wird Stellung genommen, und daraus resultierende Schlussfolgerungen bezüglich einer zukünftigen Optimierung der Hepatitis-C-Therapie kommentiert. / The doctorate program will describe the development, optimization and validation of a reversed phase chromatography method for measuring ribavirinplasma levels. This has been combined with solid phase extraction for preparing samples. In addition, numerous analyses of patients’ chromatograms are included relative to clinically relevant questions such as the depiction of ribavirinplasma levels during the course of a day, during the course of the first six weeks of therapy, in comparison to other men and women as well as patients suffering from kidney insufficiencies. The collected data will be commented on, as well as the resulting implications relative to a future optimization of hepatitis-C therapy.

Residuos de fármacos estão presentes em diversas matrizes ambientais e estudos focados na determinação destes tem ganhado grande importância nos últimos anos, devido ao aumento do consumo de medicamentos pela população. A questão do controle de resíduos de compostos farmacologicamente ativos no meio ambiente aquático foi reconhecida como uma das questões emergentes na Química Ambiental, e tem-se dado maior importância visto que os fármacos são encontrados em matrizes em estudos em concentrações de &mu;gL-1 e ngL-1. Nesta pesquisa estudou-se três fármacos antiflamatórios que são amplamente consumidos pela população: diclofenaco, nimesulida e paracetamol. O método analítico foi desenvolvido e validado para a determinação destes fármacos em amostras de águas superficiais da cidade de São Carlos (SP). Inicialmente foi feita a validação do método proposto segundo a Resolução DOQ-CGCRE-008 do INMETRO. Os limites de detecção, e de quantificação e inferior de quantificação do método para a determinação do diclofenaco, nimesulida e paracetamol, foram, respectivamente, 0,5; 1,1 e 1,1 &mu;gL-1. A linearidade, desvio-padrão relativo, exatidão e recuperação média para o diclofenaco foram, respectivamente, R de 0,99, 3,03%, 100,55% e 97,94%. Para a nimesulida, os valores de linearidade, desvio-padrão relativo, exatidão e recuperação, foram, R de 0,98, 2,43%, 101,46% e 100,67%. Já para o paracetamol obteve-se os seguintes valores para linearidade, desvio-padrão relativo, exatidão e recuperação, R de 0,99, 3,50%, 97,94% e 93,17%, respectivamente. Na segunda etapa deste estudo aplicou-se o método validado na análise de amostras de águas coletadas na cidade de São Carlos (SP). Para o método de extração utilizou-se a extração em fase sólida (SPE) e como técnica analítica utilizou-se o HPLC/DAD. Os resultados não indicaram a presença dos fármacos diclofenaco, nimesulida e paracetamol até o limite de detecção do método empregado. / Residues of drugs are present in various environmental matrices and studies focused on the determination of these have gained in importance in recent years, due to increased drug consumption by the population. The issue of control of residues of pharmacologically active compounds in the aquatic environment was recognized as one of the emerging issues in environmental chemistry, and has given greater importance since the drugs are found in studies in matrices at concentrations &mu;gL-1 and ngL-1. In this study was studied three drugs that are widely consumed by the population: diclofenac, nimesulide and acetaminophen. The analytical method was developed for the determination of these drugs in surface water samples from São Carlos (SP). Initially, made a validation of the method proposed second resolution DOQ-008-CGCRE INMETRO. The detection, quantification and lower quantification limits of method for determining of diclofenac, nimesulide and paracetamol were 0.5, 1.1 and 1.1 &mu;gL-1, respectively. The linearity, relative standard , accuracy and average recovery of the method for diclofenac were, respectively, R equal to 0.99, 3.03%, 100.55% and 97.94%. For nimesulide, the values of linearity, relative standard, accuracy and recovery were R equal to 0.98, and 2.43%, 101.46% and 100.67%. For acetaminophen obtained the following values for linearity, relative standard, accuracy and recovery, R equal to 0.99, 3.50%, 97.94% and 93.17% respectively. In the second stage of the study applied the validated method in the analysis of water samples collected in the São Carlos (SP). For extracting the drugs, SPE cartridges were used followed by HPLC / DAD. The results indicate the absense of the studied drugs diclofenac, nimesulide and acetaminophen down to the detection limits of the method employed.