|
Capillary electrophoresis (CE) has dual advantages of ordinary electrophoresis and chromatography. Because of its high sensitivity, multi-separation mode, high speed, low-cost, automation, environmental and other characteristics, it has been widely applied in clinical, biochemistry, proteomics, environmental studies, DNA sequencing and drug analysis. Besides, it has gradually become a reliable method for routine analysis. Chemistry and biological science researchers are paying more and more attention to its theory and application, which is developing rapidly now. In this thesis study, the author applied the method of CE-UV, focused on the separation and detection and high-sensitivity analysis methods of neurotransmitter-related substances. the affection of the urine matrix components to the separation of the neurotransmitter-related substances, which provides reference for urine pretreatment; and also, this thesis studied the correlation of urinary metabolites content and sport-fatigue, which can provide a rapid and simple analysis method of fatigue model.The specific work carried out as follows:Chapter one. Introduction briefly reviewed the development of CE technology, introducing its principle, separation modes, methods of injection and features of detection technology, and emphatically introduced the basic theory of CE. According to the research content of this thesis, the author focused on discussing the importance of neurotransmitter substances in the body and the application of CE technology in the detection of neurotransmitter chemicals and analysis of urine.Chapter two. In this study, the capillary electrophoresis (CE) method was developed to separate the mixture of monoamine neurotransmitters (serotonin, dopamine, epinephrine), neurotransmitter metabolites (homovanillic acid,5-hydroxyl indole acetic acid, vanilmandelic acid), and neurotransmitter precursors (arginine, tyrosine). The compositions, pH, and concentration of background electrolyte (BGE) was optimized to separate the standard samples. Meanwhile, the possible interference by matrix components in urine as creatinine, uric acid and acetoacetic acid was investigated. The good quantitative linearity was obtained at the BGE of Na2B4O7-NaOH (pH9.90) and ultraviolet (UV) detection (200nm) for eight neurotransmitter-related substances, which owned the limit of detection (LOD) in the range of0.04～0.60μmol/L. The reproducibility (RSD)(n=5) of migration time and peak area were0.09%～0.48%and0.47%-3.34%, respectively. Eventually, the developed CE method was applied for the qualitative and quantitative analysis of the arginine (Arg) and vanilmandelic acid (VMA) in urine sample. Their concentrations are respectively at95.8±3.8μmol/L and44.6±3.5μmol/L with the recovery at the range of96.65%～104.5%.Chapter three. The of monoamine neurotransmitters (serotonin, dopamine, epinephrine), neurotransmitters metabolites (homovanillic acid,5-hydroxyl indole acetic acid, vanilmandelic acid), neurotransmitter precursors class (arginine, tyrosine), and three kinds of matrix components in urine (creatinine, uric acid, acetoacetic acid) are different, so these substances performed different charge states in running buffer of12.5mM Na2B4O7-NaOH (pH=9.90), and different ion mobility will result in larger electric inject discrimination effects. To simultaneously separate and detect those substances, we use pressure to inject water zone after positive voltage. After that, the polarity of the electrode is converted for inject another substances. The key to the success of this method is to control the ratio of the water zone and the length of sample region. Experimental studies show that the BGE of Na2B4O7-NaOH (pH9.90), and ultraviolet (UV) detection (200nm), at8kv injected30s,0.5psi at20s,-8kv injected40s can simultaneously analysis the eight neurotransmitter-related substances and matrix components in urine as creatinine, uric acid and acetoacetic acid. Which owned the limit of detection (LOD) in a range of0.003～1.0μmol/L, relative standard deviation (RSD)(n=5) of migration time and peak area were0.33%～4.10%and6.41%～37.71%, respectively.Chapter four. To explore the influence of the separation of the mixture and the injected sample volume in different background ionic strength solutions to monoamine neurotransmitters(serotonin, dopamine, epinephrine), neurotransmitters metabolites (homovanillic acid,5-hydroxyl indole acetic acid, vanilmandelic acid), as well as neurotransmitters precursors (arginine, tyrosine), and to establish a rapid and sensitive detection methods by use of CE-UV technology. Through the analysis of the neurotransmitter-related trace substances in simulated urine, to understand what kind of matrix components affect the separation of neurotransmitter-related substances under realistic conditions of urine. The purpose of the method is to explore whether it helps to reduce components discrimination caused by capillary potential drop in high salt content under electrokinetic injection mode, the same mixed standard solution at different ionic strength of the background. What’s more, it provides preliminary research and pretreatment methods of urine analysis of the act for human neurotransmitter levels in the urine. Experimental results show that the organic contents of urine to can reduce by50%to95%of the neurotransmitter related substances injection sample size; Whereas, Na2SO4, K2HPO4, NH4CI and other inorganic components of urine help to reduce the effects of discrimination electrokinetic injection, causing the neurotransmitter substances of pKa <9.90sample volume increased, however, the other urine components were made the sample volume reduced by50%to90%.Chapter five, a novel method of capillary electrophoresis coupled with ultraviolet detection has been developed for the determination of neurotransmitter precursors, neurotransmitters, neurotransmitter metabolites in urine samples. For three models of physiological status:normal, fatigue, relaxed; to establish an athlete model of fatigue, we selected the RPE (subjective feeling of fatigue assessment table) scale, Ventilation ratio and maximal oxygen uptake for evaluation of the extent of fatigue. We did qualitative and quantitative analysis to5athletes which its RPE values are more than18in three models. In this study, we found arginine was lower10.6%～62.2%in urine of exercise fatigue compared with normal and relaxed states, the excretion of homovanillic acid increased2.75%～36.42%after sport fatigue, thus the method could potential applications involving assays of the two biomarker compounds for the purpose of fast diagnose of fatigue model.
|