The quantitative analysis of environmental and toxicological samples must be reliable, rapid, and in some cases field portable. In the United States, the employment of chemical weapons by rogue states and/or terrorist organizations is an ongoing concern. Nerve agent degradative products (methylphosphonic acid) as well as surrogates (glyphosate) must be detected at low quantities in various water matrices. Current methods involve tedious and time-consuming derivatizations for gas chromatography-mass spectrometry and liquid chromatography in tandem with mass spectrometry. Two solid phase extraction (SPE) techniques for the analysis of glyphosate and methylphosphonic acid are described with the utilization of isotopically enriched analytes for quantitation using atmospheric pressure chemical ionization-quadrupole-time of flight-mass spectrometry (APCI-Q-TOF-MS) that does not require derivatization. <br>The use of illicit drugs is also an increasing problem in the United States. Toxicological analysis of illicit drugs is important for death investigation as well as in the treatment of individuals who abuse and misuse drugs. This dissertation describes a newly developed analytical method for the simultaneous quantitative analysis of heroin, 6-acetylmorphine, morphine, cocaine, codeine, methadone, and fentanyl in synthetic urine. The resolution of an electrospray ionization-time of flight-mass spectrometer (ESI-TOF-MS) was utilized for simultaneous analysis of the drugs after extraction from urine using two newly developed SPE procedures. <br>The first SPE technique described in this dissertation is solid phase extraction-isotope dilution mass spectrometry (SPE-IDMS). It involves applying EPA Method 6800 in which a naturally occurring sample is pre-equilibrated with an isotopically enriched standard prior to SPE. The second extraction method, i-Spike, involves loading an isotopically enriched standard onto a SPE column independently from the naturally occurring sample. The sample and the spike are then co-eluted from the column enabling precise and accurate quantitation by molecular IDMS calculations. The SPE methods, in conjunction with IDMS, eliminate concerns of incomplete elution, matrix and sorbent effects, and MS drift. For accurate quantitation with IDMS, the isotopic contribution of all atoms in the target molecule must be statistically taken into account. This dissertation describes two newly developed sample preparation techniques for the analysis of environmental and toxicological samples as well as statistical probability analysis for accurate molecular IDMS. / Bayer School of Natural and Environmental Sciences / Chemistry and Biochemistry / PhD / Dissertation

Nerve agents (NAs) inhibit the essential enzyme acetylcholinesterase. Classified as chemical weapons, NAs are considered a threat to soldiers on the frontlines of warzones. Current treatments can prevent death from NA poisoning, but are not effective in preventing convulsions, seizures, or subsequent brain damage. Butyrylcholinesterase (BChE) binds to NAs, rendering the chemicals harmless to acetylcholinesterase.. Two hundred mg of BChE is the putative prophylactic dose for adult humans, but is difficult to obtain in large quantities from expired human serum. Although recombinant BChE has been expressed in several organisms, the yields are still low. Nicotiana benthamiana is an attractive plant for transient protein production due to its quick growth rate, abundance of tissue, and history of successful recombinant protein production. For this research, N. benthamiana was infiltrated with viral based vectors as well as binary vectors containing the human BChE gene. Multiple assays indicated that binary vector BChE-105-1 + P19 enabled the best expression, producing 26 mg BChE/kg tissue. / Canada Research Chair Program, OMAFRA,

Chemical warfare agents including organophosphorus nerve agents (NA) continue to be a significant threat to both military and civilian populations. The current Canadian Armed Forces (CAF) treatment of NA poisoning includes administration of the oxime HI-6 (used to reactivate inhibited acetylcholinesterase) in combination with atropine contained in an autoinjector, with a benzodiazepine also being administered. Two salts of HI-6 are currently available: HI-6 2Cl (1-[[[4-(Aminocarbonyl)pyridinio]methoxy]methyl]-2-[(hydroxyiminio)methyl] pyridinium dichloride (MW 376.22 g/mol) and HI-6 DMS (1-[[[4-(Aminocarbonyl)pyridinio]methoxy]methyl]-2-[(hydroxyiminio)methyl] pyridinium dimethanesulfonate (MW 477.49 g/mol). Currently HI-6 is available to the Canadian Armed Forces under a special access program. In order to attain licensure of HI-6 numerous studies must be carried out in animal models to ensure its safety (tolerability and toxicity), efficacy and pharmacokinetics prior to human clinical trials. The present experiment aimed to determine and compare the pharmacokinetic parameters of HI-6 in two animal models under various conditions including: direct comparison of salts (HI-6 2Cl compared to HI-6 DMS), comparison of routes of administration (intramuscular compared to intravenous), comparison of effect of anaesthetic, comparison of different concentrations of HI-6, determination of the effect of atropine sulphate co-administration and evaluation of calculated pharmacokinetic parameters when infusing HI-6. Serial plasma samples were collected and HI-6 levels were quantified using a HPLC method. In all studies a significant difference was reported for absorption/distribution parameters when comparing salts. Additionally the absorption/distribution parameters when comparing routes of administration were significantly different however all other parameters were similar. Significant differences in calculated parameters were reported when examining the effect of anaesthetic on the pharmacokinetics of HI-6. Similar to previous ascending dose studies, differences were reported for the absorption/distribution kinetics. Co-administration of HI-6 with atropine sulphate did not have significant effect on the pharmacokinetics of HI-6. The determined pharmacokinetic values for both salts were accurate for the determination of an infusion rate to reach and maintain a target plasma concentration. Finally the calculated animal model pharmacokinetic data was compared to previously published human clinical trial data and the calculated pharmacokinetic values were found to be similar.

No description available.

No description available.

Chemical warfare agents can be currently considered a high risk to humans. Group of nerve agents, is considered the most dangerous. These highly toxic substances have a rapid onset of action, the organism will receive all the entry gates, therapy intoxication is always difficult and death occurs often. The body's resistance to the effects of nerve agents at the expected intoxication increases not only prophylactic administration of antidotes , eg. when working in a contaminated environment after a terrorist attack, but also potentiates the subsequent antidotal therapy after exposure. This thesis is dedicated to gain in depth understanding of nerve agents with aspect of pharmacological prophylaxis. The introduction presents combat toxic substances in general, differentiation due to its effect on human with possible damage to the organism and brief characteristics of every basic type of nerve agents with examples included. Further on the theoretical part focuses on issues of nerve agents. The history of their development and well-known applicationsis breifly mentioned, respectively, abuse in the world. The thesis mentions of the Conventions on the prohibition of chemical weapons in connection with the misuse. Thesis continues dividing type nerve agents, describing the mechanism of action, acute toxicity and its symptoms, diagnosis and therapy of poisoning. Degree, extent and rate of damage to the body caused by nerve agents depends on many factors. A major factor which influences the ultimate toxic effect are taken input. Body damage can be caused, for example. Inhalation administration, contamination of broken or intact skin, ingestion, or hitting the conjunctiva. Since they have nerve agent toxicity and high speed action, protective and preventive measures are an important element to prevent the penetration of substances into the body. The Army of the Czech Republic ensures the protection of personal protective equipment, decontamination of affected skin and pharmacological prophylaxis. Another part of this work is the term prophylaxis and related topics, especially pharmacological prophylaxis. Directions pharmaceutical prophylaxis currently can be divided into three directions - protection against acetylcholinesterase inhibition, administration of antidotes and normal use Scavengers called. "Scavengers". The body's resistance to the effects of nerve agents at the expected intoxication not only increases prophylactic administration of antidotes, but also potentiates the subsequent antidotal therapy after exposure. Prophylactics PANPAL and TRANSANT are in equipment of Czech Army, in the work is devoted more attention to them. Both of these products were developed in the Czech Republic. There are also presented antidotal remedies of first aid, which are available for the Army of the Czech Republic. The practical part of this thesis aims to compare the prophylactic antidotes in the Czech Republic and in selected countries of NATO from four perspectives - onset, prophylactic activities, application forms, and adverse effects. An analysis of the available Czech and foreign professional articles, publications and studies and the subsequent comparative analysis of the various aspects and evaluate various selected countries is used to achieve this goal. These resulting data then point to the position of the Czech Republic in the field of prevention against intoxication by nerve agents - The Army of Czech Republic that has put in service two prophylactic antidotes that achieve the best results investigational products. It may therefore be said that the Army of the Czech Republic currently has a combination of prophylactic best in the world, which is relatively free of side effects has easy application and an unnecessarily long duration of action. This result is even answer the research question - Prophylactic antidotes in the Czech Republic are comparable according to the given parameters with antidotes in selected countries.

Ce projet à caractère dual civil et militaire vise à développer de nouvelles contre-mesures médicales d’urgences hautement efficaces en cas d’intoxications accidentelles ou volontaires aux organophosphorés toxiques (gaz de combat et pesticides). Ces poisons agissent comme des inhibiteurs irréversibles de l’acétylcholinestérase, enzyme clé principalement localisé dans notre système nerveux, par phosphorylation du site actif enzymatique. Cependant, un nucléophile puissant capable de pénétrer jusqu’au site actif de la protéine peut également l’hydrolyser. Notre recherche réside sur l’élaboration de molécules hybrides possédant deux systèmes clés : une partie réactivatrice neutre capable de restaurer l’activité enzymatique et un ligand du site périphérique capable de vectoriser la structure hybride à l’entrée du site actif. Une librairie de nouveaux hybrides bifonctionnels a ainsi été synthétisée, évaluée et décrite dans ces travaux de thèse. L’ensemble de ces hybrides démontre une activité égale ou supérieure à celle des réactivateurs de référence. Nous avons même découvert le premier hybride universel surpassant l’activité de réactivation des antidotes actuellement commercialisés pour des intoxications aux gaz de combat et aux pesticides. / Remediation of both acute and chronic intoxications by organophosphorus nerve agents, both chemical warfare agents and pesticides, continues to be a challenge of paramount importance. These manmade poisons act as covalent and irreversible inhibitors of acetylcholinesterase, a key enzyme mostly located in the nervous system, through phosphylation of its active site. The phosphylated active site residues do not undergo spontaneous hydrolysis. However, hydrolysis can be achieved through the use of strong nucleophiles able to enter the buried active site of the protein. Our research is based on the rational design of hybrid structures containing two key elements: a neutral reactivator to restore the enzyme activity, and a peripheral site ligand giving selectivity by targeted binding to a site at the entrance of the enzyme active site gorge. A library of novel reactivators based on acridine, quinoline and original oxoassoanine analogues was synthesised, evaluated and is herein described. Delightfully, most of these hybrids proved to be equally or more potent than the drugs currently in use. Outstandingly, we have discovered the first broad-spectrum reactivator that outperformed all known reactivators for both chemical warfare agent and pesticide intoxications.

O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX) and O-isobutyl-S-2-diethylaminoethyl methylphosphonothioate (R-VX), are considered chemical warfare agents due to their strong acetylcholinesterase-inhibiting properties. Subsequent to terrorist use of these V-type nerve agents in both Japan and the United States of America (the September 11, 2001 attacks) and the limited capability of anti-terrorist groups to detect such weapons, there has been an increased obligation by the Chemical Weapons Convection for specific detection and identification methods for VX and R-VX. Chemical and/or enzymatic hydrolysis yields sulfhydryl mimic products, diethylaminoethanethiol (DEAET) and dimethylaminoethanethiol (DMAET). This thesis investigates the electrocatalytic parameters of DEAET and DMAET using basal plane pyrolytic graphite electrodes (BPPGEs) modified with: (a) single-wall carbon nanotube (BPPGE-SWCNT); (b) SWCNT functionalised with cobalt (II) tetra-aminophthalocyanine by (i) physical (BPPGE-SWCNT-CoTAPc(mix)), (ii) chemical (BPPGE-SWCNT-CoTAPc(cov)) and (iii) electrochemical adsorption (BPPGE-SWCNT-CoTAPc(ads)) processes; (c) nickel powder (BPPGE-Ni); (d) BPPGE-Ni decorated with SWCNT (BPPGE-Ni-SWCNT), and (e) SWCNT functionalised with nickel (II) tetra-aminophthalocyanine (BPPGE-SWCNT-poly-NiTAPc). Electrochemical studies (performed by voltammetric and electrochemical impedance spectroscopic techniques) revealed that the SWCNT and SWCNT-CoTAPc(mix) films showed comparable electrocatalytic responses towards the detection of DEAET and DMAET whereas competitive electrochemical behaviour was seen between SWCNT and SWCNT-NiTAPc modified BPPGEs. Using the BPPGE-SWCNT-CoTAPc(mix), the estimated catalytic rate constants (k) and diffusion coefficients (D) were higher for DEAET than for the DMAET. Also, the detection limits of approximately 8.0 and 3.0µM for DMAET and DEAET were obtained with sensitivities of 5.0×10−2 and 6.0×10−2 AM−1 for DMAET and DEAET, respectively. Unlike BPPGE-SWCNT-CoTAPc(mix) that detected the two sulfhydryls at slightly different potentials, BPPGE-SWCNT did not. The BPPGE-Ni gave enhanced Faradaic response for the redox probe ([Fe(CN)6]3−/4−) and also displayed enhanced electrocatalytic behaviour towards the detection of DMAET and DEAET with high sensitivity (~23x10−3 AM−1) and low detection limits (4.0 – 9.0 µM range). In comparison to other electrodes reported in the literature, BPPGE-Ni exhibits more promising features required for a simple, highly sensitive, fast and less expensive electrode for the detection of the hydrolysis products of V-type nerve agents in aqueous solution. The efficient response of the BPPGE-Ni is attributed to the high microscopic surface area of the nickel powder. The poor response of the BPPGE-Ni-SWCNT suggests that the nickel impurity in SWCNT did not show any detectable impact on the heterogeneous electron transfer kinetics of SWCNT. Unlike the nickel powder, SWCNT and CoTAPc-SWCNT, the NiTAPc-SWCNT hybrid did not show significant electrocatalysis towards the detection of the sulfhydryls. It is interesting, however, to observe for the first time that SWCNT induced crystallinity on the electropolymer of NiTAPc, and that such electropolymer exhibit charge-storage /-transfer properties that greatly enhance the electrochemical response of nitric oxide. / Dissertation (MSc (Chemistry))--University of Pretoria, 2008. / Chemistry / unrestricted

No description available.

Les organophosphorés (OPs) sont des neurotoxiques. Leur décontamination est difficile et coûteuse. L’utilisation d’enzymes capables de les détoxifier est une solution élégante. Les enzymes bactériennes sont peu stables et chères à produire. Nous avons identifié des enzymes très résistantes, capable de biodégrader lentement ces composés. Nous avons alors développé une stratégie permettant d'améliorer l'activité de l'enzyme très stable en nous basant sur les enzymes les plus actives. Celle-ci fut améliorée de plus de 1000 fois envers ces insecticides. Enfin, nous avons analysé l'origine de ces améliorations et mis en évidence des concepts émergents dans l'évolution des enzymes. / Organophosphorus compounds are neurotoxic. Their decontamination is difficult and cost prohibitive. An appealing solution resides in the use of enzymes capable of degrading such compounds. Bacterial enzymes are poorly stable and expensive. We identified highly resistant enzymes capable of slowly biodegrading these compounds. We have developped a strategy allowing to increase the activity of our enzyme by using the similarities with the highly active enzymes. The activity was increased by a factor of 1000 against OPs. We analyzed the origins of these ameliorations and showed emergent concepts in enzyme evolution.