| Organophosphorus nerve agents (OPs) are a type of chemical warfare agents with high toxicity; they can inhibit the enzymatic activity of cholinestrase (ChE). Nerve agents are distributed and metabolized rapidly in vivo after exposure, existing partly as the intact agent, partly as degradation products or metabolites and partly covalently bound to macromolecule as the adducts. Since these compounds can be used to verify the exposure of nerve agents, monitor the exposure level and evaluate the therapeutic effects, they are defined as the biomarkers of nerve agents. The significant advantages of the nerve agents adduct with biomacromolecule are the specific and retrospective detection of exposure. High sensitivity of adduct detection can be obtained by the use of modern analytical techniques. Nerve agents mainly covalently bind with two types of protein in vivo: cholinesterase and albumin. The adduct of nerve agents with albumin are the focus of recent research because of their significant advantages of high sensitivity, doing not appear to age rapidly and being detected after therapeutic treatment with oximes. The analytical methods are the research foundation of retrospective detection for adduct of nerve agents with albumin. The methods also have important significance to demonstrate the metabolic rule in vivo and evaluate the effection of therapeutic treatment.Recent research was focused on the target molecules produced by protein adducts with nerve agents which were digested with various protease. Novel strategies of protein purification were applied in the research of nerve agents adduct with albumin. However, there was lack of research on the topic of quantitative analysis methods of NAs-albumin adduct and little literature on the dose-effect and time-effect relationships. It is difficult to measure the concentration of NAs-albumin adduct, but it is important to investigate the metabolic rule of NAs-albumin adduct in vivo.In our research, four types of O-(O-alkyl methylphosphonyl) tyrosine adducts were synthesized for the standard compounds and three deuterated products were also synthesized for the first time for the internal standard. The phosphorylated albumin was extracted and concentrated from the exposure human serum by the use of affinity gel column, and the SPE strategy was designed to extract the O-(O-alkyl methylphosphonyl) tyrosine adducts from the pronase digestion matrix of phosphorylation albumin. High sensitivity and low matrix effects were obtained by the sample preparation procedure, which combined the purification of protein and the SPE strategy. The isotope-dilution quantitative method for nerve agent adducts was established for the first time by the use of standard compounds and deuterated internal standards which synthesis by ourselves. The quantitative method was applied to measure the concentration of O-(O-alkyl methylphosphonyl) tyrosine adducts in the sample of animal experiment and the dose-effect relationship was explored.This dissertation consists of six chapters:Chapter one was the preface of the dissertation. The basic information and the toxicologic effection of nerve agents were briefly described. The biomarkers generated by nerve agents in vivo were enumerated and the comparison was made among various biomarkers of nerve agents. The analytical methods were introduced detailly and their advantages and disadvantages were illuminated. Current status and problems of research were pointed out, based on which the purpose and contents of our research were proposed.In chapter two, four types of O-(O-alkyl methylphosphonyl) tyrosine adducts of nerve agents (GA, GB, GD and VX) were synthesised as the standard compounds. Three types of deuterated O-(O-alkyl methylphosphonyl) tyrosine adducts of nerve agents (GA, GB and VX) were synthesized as the internal standards. The purity of these compounds were all higher than 95% and satisfy the demands of quantitative analysis methods.In chapter three, the LC-Q-TOF MS/MS analytical methods were established by the use of standard compounds and deuterated internal standards above synthesized. HiTrap Blue HP affinity column was applied to extract the phosphorylation albumin from human serum exposed to nerve agents. High sensitivity and low matrix effects was obtained by the sample preparation procedure, which combined the purification of protein and the SPE strategy. The sample preparation procedures have been utilized on the qualitative and semi-quantitative detection of human serum which were exposured in vitro by various nerve agents in different dose. The sensitivities of method were 20 ng/mL serum for sarin adduct, 5 ng/mL serum for soman adduct and 2 ng/mL serum for tabun adduct, which were approached to the recent research. A new detection target molecule was discovered, whose structure was approved to be soman-dipeptide adduct. The new detection target molecule covered the disadvantage of soman-tyrosine adduct and provided a new research direction of soman-albumin adduct.In chapter four, LC-Triple quadrupole MS/MS analytical methods were established utilize the standard compounds and deuterated internal standards above synthesized. The isotope-dilution quantitative methods were established for the determination of nerve agent adducts in rat plasma which was concentrated and purified by the HiTrap Blue HP affinity column and SPE. The precision, recovery and linearity of the methods were satisfied and the sensitivities were 10 pg/mL for sarin and soman adducts, 50 pg/mL for VX adduct.In chapter five, the rats were administrated with three dose levels of sarin and soman and with VX in a high dose of 0.7×LD50 to verify the relationship between the agent dose and the concentration of adducts. The isotope-dilution quantitative methods were employed to measure the concentration of adducts in the plasma sample of exposed rats. The inhibition of BuChE was also measured to evaluate the toxicity of agents. The dose-effect relationship between the dose of nerve agents and the concentration of adducts was demonstrated.In chapter six, we have prepared the procainamide-sepharose 4B affinity gel for the purification of nerve agent adduct with BuChE. Human serum was exposed by soman in vitro on different concentration levels. Soman adduct with BuChE was purified by procainamide-sepharose 4B affinity gel. MPA-nonapeptide was obtained by the digestion with pepsin. Utilizing LC-Q-TOF MS/MS, the adduct can be detected at the exposure level of 20μg/mL. According to the principle of procainamide-sepharose 4B affinity gel and HiTrapTM Blue affinity column, we have designed a sample preparation procedure of simultaneous enrichment and purification of nerve agent adducts with BuChE and albumin, with which the serum samples were ultilized efficiently.
|
PDF Full Text Request