The thesis describes the development of new and improved methods for analyzing degradation markers from organophosphorus Chemical Warfare Agents (CWAs). Paper I and II describes an innovative and significantly improved method for the enrichment, derivatization (trimethysilylation) and GC-MS analysis of a broad range of organophosphorus CWAs degradation markers, namely the alkylphosphonic acids and a zwitterionic compound. That was achieved using solid phase disc extraction in combination with solid phase derivatization. The new method overcomes most limitations observed with existing techniques: it offers almost 100 % recoveries, requires no elution or evaporation steps, facilitates miniaturization of the solid sorbent and reagent, is compatible with in-vial derivatization, and minimizes the chromatographic background due to the use of a highly selective anion exchange sorbent disc. Paper III describes the development of new fluorinated diazomethane derivatization reagents and their evaluation for rapid and high sensitivity screening and identification of nerve agent degradation markers. The reagents are water-tolerant to some extent, which simplifies the derivatization step. The best reagent identified was 3,5-bis(trifluoromethyl)benzyl diazomethane, which outperformed the other reagent isomers tested and also the established commercial alternative, pentafluorobenzylbromide, allowing for the rapid (5 min) and direct derivatization of a 25 μL aqueous sample in acetonitrile. The spectra of the formed derivatives (high-energy collision induced fragmentation MS/MS) were used to construct a database (Paper IV) that proved to be superior in terms of match factor and probability compared to EI data gathered for trimethylsilyl derivatives. The study also focused on efforts towards achieving detailed structure information on the alkyl chains of the compounds in question using diagnostic ion interpretation. The final paper (paper V) describes the first rapid direct derivatization method for analyzing nerve agent metabolites in urine at trace levels. The method is based on the derivative from the paper III and the unambiguous identification was proven using a combination of low resolution and high resolution negative ion chemical ionization selected ion monitoring techniques. Novel results presented in these papers include: the first in-situ derivatization of alkylphosphonic acids on an SPE disc; the first direct derivatization of nerve agent markers in water and biomedical samples; the first high sensitivity GC-MS screening for these markers; and the first highly reproducible high-energy isomer specific CID MS/MS library. Overall, the results presented in this thesis represent significant contributions to the analysis of nerve agent degradation products.

Les neurotoxiques organophosphorés (NOPs) sont des inhibiteurs irréversibles de l’acétylcholinestérase, une enzyme jouant un rôle clé dans la transmission de l’influx nerveux. Une intoxication par ces substances provoque de graves symptômes pouvant aboutir au décès des sujets empoisonnés. Bien qu’interdits par une convention internationale ratifiée par 190 pays, le contexte géopolitique actuel ravive la hantise d’une menace chimique contre des populations civiles, renouvelant l’intérêt de développer de nouveaux remèdes contre une intoxication aux NOP. A l’heure actuelle, les antidotes utilisés contre ces intoxications sont les mono- et bis- pyridinium aldoximes portant une ou deux charge(s) positive(s) permanente(s), qui présentent de nombreuses limitations, dont leur très faible capacité à traverser la barrière hémato-encéphalique et à réactiver efficacement les AChEs inhibées par les NOP dans le système nerveux central (SNC). Par ailleurs, leur efficacité est très dépendante de la nature du NOP utilisé, et il n’existe pas, aujourd’hui, de réactivateur universel. Dans ce contexte, le développement de nouveaux réactivateurs efficaces est un réel défi. Au cours de cette thèse, l’optimisation de la synthèse de cinq réactivateurs non chargés incorporant une amine cyclique en tant que ligand du site périphérique et la 3-hydroxypyridine aldoxime en tant que fonction réactivatrice a été développée pour évaluation in vivo. Dans cette continuité, vingt-et-un réactivateurs non chargés inédits comportant un ligand aminé du site périphérique de l’enzyme associée à la même fonction réactivatrice 3-hydroxypyridine aldoxime ont été conçus, synthétisés et testés sur l’AChE humaine inhibée par différents NOPs mais aussi sur les récepteurs nicotiniques. Certains d’entre eux ont été testés in vivo sur la souris. Parmi les réactivateurs synthétisés, certains possèdent des efficacités de réactivation identiques, voire meilleures que les oximes de référence utilisées de nos jours tout en ayant un effet antagoniste des récepteurs nicotiniques ouvrant ainsi la voie aux réactivateurs dits « Multi-cibles ». Avec comme objectif de traverser la barrière hémato-encéphalique en utilisant les transporteurs GLUTs présents sur les cellules endothéliales constituant cette barrière, quatre réactivateurs incorporant un motif carbohydrate ont été resynthétisés et évalués in vivo et in vitro. Enfin, de nouvelles fonctions réactivatrices constituées d’un motif thiadiazole-aldoxime et naphtaldoxime ont été étudiées ouvrant la voie à de nouvelles familles de réactivateurs et, pour la dernière fonction, remettant en cause le cahier des charges utilisé jusqu’alors pour la conception de nouveaux réactivateurs. / Organophosphorus nerve agents (OPNA) are irreversibly acetylcholinesterase (AChE) inhibitors, a key enzyme in the central nervos system playing a pivotal role in neurotransmission. OPNA poisoning induces to serious symptoms that can lead to death by respiratory failure. Despite an international convention prohibiting the use of chemical weapons, the current geopolitical context is reviving the fear of a chemical threat against civilian populations, and has renewed the interest of developing new remedies for OPNA poisoning. Nowadays, medical countermeasure used against OPNA intoxications are based on mono- and bis-pyridinium aldoximes carrying one or two permanent positive charge(s), which present many limitations, including their very low capacity to cross the blood-brain barrier and to reactivate effectively ChEs nhibited by NOPs in the central nervous system (CNS). Moreover, their effectiveness is highly dependent on the nature of the NOP used, therefore, there is no universal reactivator. In this context, the development of new effective reactivators is a real challenge. In this PhD, an optimized synthetic access to five uncharged reactivators incorporating a cyclic amine as peripheral site ligand and 3-hydroxypyridine aldoxime as reactivating function was developed for in vivo evaluation. Following up on this, twenty-one new uncharged reactivators bearing unprecedented cyclic amines as peripheral site ligand associated with our reactivating function were designed, synthetized and evaluated for reactivation on human AChE inhibited by several NOPs but also in vivo on mice and as antagonists on nicotinic receptors in vitro. Among these reactivators, some have identical or better reactivation efficiencies than the reference oximes used today while having a nicotinic receptor antagonist effect ; thus, paving the way for « Multi-target » reactivators. Aiming at an active BBB crossing using the GLUTs receptors, four reactivators including a carbohydrate moiety have been resynthetized and biologically evaluated. Last but not least, new reactivator functions consisting of a thiadazole-aldoxime and naphtaldoxime moieties have been studied, paving the way to new families of uncharged reactivators and, especially for the last function, calling into question design specifications of new reactivators.

Using questionnaire enquiry, this diploma thesis tries to find out the level of knowledge of single members of basic rescue bodies involved in the Integrated Rescue System of the Czech Republic in the area of nerve agents and subsequently compare this knowledge of single basic bodies. Another aim, which is based on literature examination and information available, is to suggest an appropriate procedure for Integrated Rescue System action in case of misuse of nerve agents by terrorists. The suggested procedure of activities of bodies involved in the Integrated Rescue System could serve as an example for establishing a model plan of activities of bodies involved the Integrated Rescue System in case of events with suspicion of terrorist attack performed by nerve agents or other highly toxic chemical agents. A comprehensive overview on nerve agents could be used for deepening and broadening of knowledge of single Integrated Rescue System bodies. Although such a terrorist attack has not happened in the Czech Republic yet, it cannot be excluded that it will occur in the future. Hence it is very important so that the Integrated Rescue System is perfectly equipped both materially and technically, and single Integrated Rescue System bodies are trained and educated systematically. It is better to be theoretically and practically well prepared, and never to use this knowledge and skills than to improvise and rely on decision carried out only on the place of disaster.

This thesis is dedicated to the topic of the nerve agents with special emphasis on antidotal treatment utilizing application of the auto-injectors. Two methods were used ? literature research and quantitative research. Since this topic covers plenty of information as regards chemical warfare agents and in particular nerve agents these issues were addressed in the theoretical part of the thesis. In the chapter on current status based on literature resources a summary of evolution of the auto-injector is presented; from firsts notes until the situation today. Practical part of the thesis is concentrated on University of Southern Bohemia in ČeskéBudějovice, Faculty of Health and Social Studies students' knowledge in the area of nerve agents. The results of the research were acquired by the method of quantitative research ? questionnaires with 18 questions. With respect to each of the questions a selection of the 2 to 7 answers was offered to the students, whereas in every case only 1 answer was correct. The results were analyzed both with reference to each of the questions and with reference to each of the students. By this method it has been discovered that the respondents have a good knowledge in the area of nerve agents. Furthermore, the practical part of the thesis included an experiment concerning speed with which the respondents were able to apply antidotum with a training auto-injector by themselves with no prior instructions in comparison to how quickly they were able to do the same after being instructed. By this method were tested the quality of the instructions provided to the auto-injectors and its ergonomic qualities (intuitiveness of its application). Recorded times were statistically analyzed and the results, although materially different, were assessed as satisfactory.

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