| In general, biological samples refer to biological tissues, cells, body fluids, metabolites, endogenous substances and biological markers in the body. Biological samples are complex with trace analytes and a large number of interfering substances existing in a large number of sample matrix, which bring huge difficulties to the sample analysis. The biological samples are with characteristics of trace analytes, less sample amount, easy to inactivated, complex matrix, etc. The number of samples in routine analysis is generally large. Therefore, it must establish a simple, rapid, high sensitivity, less sample amount consumption method for the analysis biological samples.Capillary electrophoresis (CE) has the characteristics of efficient and fast, using simple instrument, less reagent consumption, and it becomes one of the active area of research in the science of separation and analysis. These are advantages that other methods is incomparable in biological samples analysis. But because the injection sample volume is small, leading to low detection sensitivity, which limits its extensive applications in trace analysis. In order to improve the detection sensitivity, and make capillary electrophoresis technique suiting for more analysis of trace component in complex samples, online concentration technique are developed. On-line enrichment technique doesn’t need extra instruments, and the enrichment procedure are implemented in the capillary. What’s more, it has low cost, simple operation, high sensitivity, and ease of automation. Researchers in the field of analysis favored it more and more in recent years.On the basis of previous work, this paper studied the application of online concentration methods with capillary electrophoresis in biological sample analysis, and the mainly innovative work were carried out as follows:1. According to the nature of amino acid and weak acid and alkali, combining dynamic pH junction and sweeping technique, and adopting the ultraviolet indirect assay, a new method for determination of trace acidic amino acids was established and its applicability was discussed. The new method was used to determine the acidic amino acids contents in compound amino acid injection and human serum.2. By using the properties that cyclodextrin can form inclusion complex, gamma-cyclodextrin was added to the sample matrix to enhance the affinity of the analytes to the micelle, enhancing sweep effect. Combined field amplification sample stacking and sweeping technique, a method of determination of trace enrofloxacin and ciprofloxacin in biological samples was established. And its applications in six animal origined foods (milk, milk powder, chicken, pork, swine liver, and swine kidney) were discussed.Based on the content, this thesis is divided into three chapters.Chapter 1, this paper expounds the characteristics of biological samples, analysis methods and the pretreatment methods of biological samples. And it reviewed the principle, advantages and disadvantages of online concentration methods with capillary electrophoresis. their application in biological sample analysis are also discussed.Chapter 2, a sensitive and simple method coupled with indirect UV detection, using benzoic acid (BA) as the UV-absorbing probe, was developed and validated for the quantitative determination of glutamic acid (Glu) and aspartic acid (Asp) in human serum and Compound Amino Acid Injection-18 AA. The method combines a dynamic pH junction with a sweeping technique using β-cyclodextrin (β-CD) as the complexing agent for sweeping. Uncoated fused-silica capillaries. The dimensions of the capillary are 50 cm×50 μm I.D×375 μm O.D.. The effective length of the capillary is 42 cm from the injection end of the capillary. The running buffer was composed of 10 mM Tris(hydroxymethyl)aminomethane (Tris),8mM BA and 10 mMβ-CD at pH 7.5. And the sample matrix was composed of 3 mM H3PO4 at pH 2.5. Employing this proposed method, low detection limits of 0.061μg/mL for Glu and 0.032 μg/mL for Asp were obtained. The sensitivity was improved 30- and 55-fold for Glu and Asp compared to conventional CE method. Standard curves were linear (r> 0.999) over the concentration range of 0.1-8.0 μg/mL. To further improve the resolution of Asp from interfering substances in human serum,6% (v/v) methanol was added to the sample matrix, and resulted in the detection limits of 0.125μg/mL for Glu and 0.057 μg/mL for Asp. With a simple precipitation of protein, the method has been successfully applied to the analysis of human serum, and the recoveries (82% for Glu and 87% for Asp) were achieved with relative standard deviations of 1.9% and 2.0%, respectively. The dynamic pH junction-sweeping technique for CE with indirect UV detection is proved to be a rapid and simple online concentration method for analysis of acidic amino acids. The optimized system reduces the analysis time to less than 10 min. And with indirect UV detection, the method can be applied without any requirement for sample derivatization or mass spectrometric detection. It is suitable for the routine analysis of biological samples.Chapter 3, this chapter is divided into two parts.Part 1, a 75 pm ID. and unmodified fused-silica with a total length of 65 cm (effective length of 57 cm) was used as separation capillary. A simple on-line preconcentration method combining field-amplified sample stacking (FASS) with sweeping was developed and validated for the determination of enrofloxacin (ENRO) and ciprofloxacin (CIP) in multiple foods. To improve the efficiency of sweeping, gamma-cyclodextran (y-CD) was introduced to the sample matrix. The optimal experimental conditions were as follows:running buffer (pH 8.8),20 mM Tris and 80 mM sodium deoxycholate (NaDC); sample matrix,300 mM Tris and 5 mM y-CD at pH 10.0; applied voltage,15 kV; detection wavelength,270 nm. The limits of detection (LODs) for ENRO and CIP were 1.87 and 2.21 ng/mL. The sensitivity was improved 376 and 406-fold for ENRO and CIP compared to conventional capillary electrophoresis (CE) method, respectively. Good linearity (r> 0.999) and repeatability (the %RSD values of peak area were 1.46%-2.40% and 2.14%-2.95% for intra-day and inter-day, respectively) were obtained for the analytes.Part 2, In order to make the new method suite for the determination of trace enrofloxacin and ciprofloxacin in biological samples with complex matrix, the sample injection time and sodium deoxycholate concentration was adjusted, namely, the background buffer was composed of 20 mM Tris and 120 mM sodium deoxycholate at pH 8.8. with a sample injection time of 240 s. Under the optimal experimental conditions, the limits of detection (LODs) for ENRO and CIP were 5.70 and 7.39 ng/mL. The sensitivity was improved 124 and 122-fold for ENRO and CIP compared to conventional capillary electrophoresis method, respectively. Good linearity (r=0.9994 and 0.9997, respectively) and repeatability (the%RSD values of peak area were 1.99%-2.40% and 2.14%-2.44% for intra-day and inter-day, respectively) were obtained for the analytes. The method has been successfully applied to the analysis of six kinds of animal foodstuffs(milk, milk powder, chicken muscle, pork, swine liver, and swine kidney). The recoveries (85-102%) were achieved with relative standard deviations of 0.1-4.4%. The LODs were lower than the maximum residue limits (MRLs) of the tested compounds in animal origin foods. What’s more, the method uses simple reagents, with minimum sample preparation procedures, encouraging its application in routine analysis.Chapter 4, the main conclusions and prospect of this paper was summaried.In this paper, the research results showed that the on-line concentration method in capillary electrophoresis is an effective and economic method to improve detection sensitivity of capillary electrophoresis analysis. It is not only with a simple operation procedure and low cost, but also can significantly improve the detection sensitivity without extending the time of analysis. When used for biological sample analysis, the requirements for sample preparation is not as strict as high performance liquid chromatography (HPLC), which can greatly shorten the analysis time. The online enrichment method is applied to the analysis of trace elements in biological samples, which is of important significance to expand the application of capillary electrophoresis in the analysis of trace components. |
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