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京都大学 / 新制・課程博士 / 博士(工学) / 甲第23513号 / 工博第4925号 / 新制||工||1769(附属図書館) / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 大塚 浩二, 教授 松原 誠二郎, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Micellar electrokinetic capillary chromatography (MECC) is a form of capillary zone electrophoresis. Addition of a surfactant produces micelles in an aqueous/organic buffer. Separation of drugs is obtained via differences in the electrophoretic mobilities of the analytes within the capillary, resulting from their electrophoretic velocity and the electroosmotic flow of the buffer in a given electric field. The migration order is determined by the differential partitioning of the drugs between the micelles and the aqueous/organic phase. Barbiturates were extracted from various biological fluids at pH 4.5 with TOXI-TUBES B. MECC analyses were performed using a Waters Quanta 4000 Capillary Electrophoretic System with a 745 Data Module with a 75 μ x 60 cm capillary and an aqueous/organic buffer of 85% 10 mM borate, 10 mM phosphate, 100 mM sodium dodecyl sulfate and 15% acetonitrile at a pH of 8.5 with a voltage of 20 kV using ultraviolet absorption detection at 214 nm. Migration times were: phenobarbital, 7.78 min.; butalbital, 8.01 min.; butabarbital, 8.23 min.; mephobarbital (internal standard), 8.88 min.; amobarbital, 9.41 min.; pentobarbital, 10.03 min. and secobarbital, 10.79 min. Correlation coefficients (r) between peak areas and concentration ranges of 3 to 60 μg/mL were from 0.964 to 0.999. Coefficients of variation (CV) raged from 2.6 to 8.6% between days and 2.3 to 9.8% within day. Application of this methodology to four forensic cases of butalbital intoxication detected concentrations of 0.7 to 12.7 μg/mL in blood; 0.8 to 1.9 μg/mL in vitreous humor and 1.5 to 7.6 μg/mL in urine. MECC is applicable to forensic analysis of barbiturates extracted from biological fluids.

The analysis of proteins in biological fluids by capillary electrophoresis (CE) is of interest in clinical chemistry. However, due to low analyte concentrations and poor concentration limits of detection (CLOD), protein analysis by this technique is frequently challenging. Coupling preconcentration techniques with CE greatly improves the CLOD. An on-line preconcentration-CE method that can selectively preconcentrate any protein for which an antibody is available would be very useful for the analysis of low abundance proteins and would establish CE as a major tool in biomarker discovery. To accomplish this, an on-line protein G monolithic preconcentrator CE system for enrichment and separation of proteins was developed. This system proved effective for on-line sample extraction, clean-up, preconcentration, and CE of IgG in human serum. IgG from diluted (500 and 65,000 times) human serum samples was successfully analyzed using this system. The approach can be applied to the on-line preconcentration and analysis of any protein for which an antibody is available. The desire to separate all proteins present in human tissues, cells and biological fluids has challenged the separation research community for many years. The difficulty of this task resides in the complexity of the sample. Blood serum, for instance, may express up to 10,000 proteins with an estimated dynamic range of 9 orders of magnitude. Additionally, most of these proteins are present at very low concentrations (ng/mL). Identification and quantification of low abundance proteins is hindered by the presence of high abundance proteins, such as human serum albumin (HSA) and immunoglobulins (IgG). Therefore, in most cases, removal of the high abundance proteins or enrichment of low abundance proteins is necessary prior to the analysis of low abundance proteins. To address this, a coupled affinity-hydropobic monolithic column for the simultaneous removal of IgG, preconcentration of low abundance proteins, and separation by capillary zone electrophoresis was designed. The system proved to be very reproducible. The run-to-run %RSD values for migration time and peak area were less than 5%, which is typical of CE. Finally, a new method was developed to prepare monoliths with anion exchange functionality. Polymer monoliths were prepared by in situ polymerization of methacrylate monomers. The monoliths were coated with a water soluble polymer and used for the analysis of proteins. Using this approach, a model monolith was prepared. Subsequent coating yielded a monolith with quaternary ammonium groups on the surface, which was confirmed by strong anodic electroosmotic flow. Analysis of standard proteins by ion exchange LC and CEC was demonstrated. This simple and rapid method for surface modification opened new avenues for the preparation of monoliths with a broad range of functionalities.

Polycyclic aromatic hydrocarbons (PAH) are a class of environmental pollutants consisting of a minimum of two fused aromatics rings originating from the incomplete combustion of organic matter and/or anthropogenic sources. Numerous possible anthropogenic and natural sources make the presence of PAH ubiquitous in the environment. The carcinogenic nature of some PAH and their ubiquitous presence makes their chemical analysis a topic of environmental and toxicological importance. Although environmental monitoring of PAH is an important step to prevent exposure to contaminated sites, it provides little information on the actual uptake and subsequent risks. Parent PAH are relatively inert and need metabolic activation to express their carcinogenicity. Covalent binding to DNA appears to be the first critical step in the initiation of the tumor formation process. To this end, the determination of short term biomarkers – such as monohydroxy-PAH metabolites (OH-PAH) - fills an important niche to interpret actual PAH exposure levels, prevent extreme body burdens and minimize cancer risk. One would certainly prefer an early warning parameter over a toxicological endpoint – such as DNA-adducts – indicating that extensive damage has already been done. Several methods have been developed to determine OH-PAH in specific tissue or excreta and food samples. The general trend for the analysis of OH-PAH follows the pattern of sample collection, sample clean-up and pre-concentration, chromatographic separation and quantification. Popular approaches for sample clean-up and preconcentration include liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Chromatographic separation and quantification has been based on high-performance liquid iv chromatography-room temperature fluorescence detection (HPLC) and gas chromatographymass spectrometry (GC-MS). Although chromatographic techniques provide reliable results in the analysis of OHPAH, their experimental procedures are time consuming and expensive. Elution times of 30-60 minutes are typical and standards must be run periodically to verify retention times. If the concentrations of target species are found to lie outside the detector’s response range, the sample must be diluted and the process repeated. On the other end of the concentration range, many samples are “zeroes,” i.e. the concentrations are below detection limits. Additional problems arise when laboratory procedures are scaled up to handle thousands of samples under mass screening conditions. Under the prospective of a sustainable environment, the large usage of organic solvents is one of the main limitations of the current chromatographic methodology. This dissertation focuses on the development of a screening methodology for the analysis of OH-PAH in urine and milk samples. Screening techniques capable of providing a “yes or no” answer to OH-PAH contamination prevent unnecessary scrutiny of un-contaminated samples via conventional methods, reduce analysis cost and expedite the turnaround time for decision making purposes. The proposed methodology is based on capillary zone electrophoresis (CZE) and synchronous fluorescence spectroscopy (SFS). Metabolites extraction and pre-concentration is achieved with optimized SPE, LLE and/or QuEChERS (quick, easy, cheap, effective, rugged and safe) procedures. The small sample and extracting solvent volumes facilitate the simultaneous extraction of numerous samples via an environmentally friendly procedure, which is well-suited for routine monitoring of numerous samples. Sample stacking is successfully implemented to improve CZE limits of detection by two orders of magnitude. The unique electrophoretic pattern of positional isomers of OH-PAH demonstrates the potential of CZE for v the unambiguous determination of metabolites with similar chromatographic behaviors and virtually similar fragmentation patterns. The direct determination of OH-PAH without chromatographic separation is demonstrated via SFS. The non-destructive nature of SFS provides ample opportunity for further metabolite confirmation via chromatographic techniques

This thesis encompasses a feasibility study of using low-cost materials to manufacture microfluidic chips that can perform the same functions as chips manufactured using traditional methods within an acceptable range of efficiency of chips created with more exotic methods and materials. The major parts of the project are the selection and characterization of the fabrication methods for creating the channels for fluid flow, the methods for sealing the channels to create a usable chip and the electrophoretic separations of carboxylated microspheres of different potentials. In this work we seek to answer the question if laser-etched PMMA microfluidic chips are comparable in functionality to microfluidic chips created with PDMS or glass. In the process of answering this question we will touch on FEA modeling, characterization of the manufacturing process and multiple prototype designs while keeping within the low-cost theme. The purpose of capillary electrophoresis is to separate proteins based on their inherent electric charge. Capillary electrophoresis is a standard chip design used in the microfluidics world to prove a new fabrication method or chip material before branching out to other experiments because it is a fairly simple and robust design. Common problems associated with the manufacturing methods and materials were taken into account such as electroosmotic flow and chip sealing. CZE designs from literature were referenced to create a chip that would separate carboxylated microbeads with reasonable resolution. Wire electrodes were affixed to the chip to induce electric fields for the electrophoresis experiments. The goal of this thesis is to prove the manufacturing methods and attain results within 70% of literature standards.

Capillary electrophoresis (CE) is an analytical chemistry approach that allows for the efficient separation by charge of diverse classes of compounds for analysis, including secondary metabolites. The goal of this work was to optimize a buffer system for plant tissue analysis using micellar electrokinetic chromatography (MEKC), and by doing so to understand the role of buffer components in the performance of this form of capillary electrophoresis. In this experiment we implemented a factorial design to optimize buffer composition for separating plant tissue and secondary metabolites. The results of this experiment will be used to optimize a universal buffer for MEKC analysis that can be used on any variety of plant tissues. To determine the feasibility of this, a diverse set of plant secondary metabolite chemical standards in solution were tested as well as Helianthus annuus tissue to confirm the separation in a real biological sample. The results of this optimization yield insights into the utility of buffer components like electrolyte and pH for MEKC separation.

Exposomics aims to characterize the totality of exposures over the lifespan, and their impact on human health. Currently, chronic exposure to harmful chemicals from air pollution and/or tobacco smoke, along with a suboptimal diet, remain leading causes for preventable mortality and morbidity worldwide. As a result, new analytical methods are needed to measure robust biomarkers of smoke exposure and food intake for improved risk assessment of clinical events. This thesis aims to develop high throughput methods to rapidly quantify urinary biomarkers of environmental smoke in high-risk occupations, and diverse global populations using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) technology. Chapter II outlines an inter-laboratory method comparison for the targeted analysis of urinary 1-hydroxypyrene (HP) when using gas chromatography-high resolution mass spectrometry (GC-HRMS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) on urine samples collected from firefighters. This work revealed the critical role of incomplete enzymatic deconjugation on method bias and underreporting of true smoke exposures. Chapter III introduces a high throughput MSI-CE-MS/MS method (< 3 min/sample) to directly analyze the intact glucuronide conjugate of HP (HP-G) in urine without complex pre-column enzyme deconjugation and derivatization procedures. Importantly, firefighters deployed under emergency conditions at the 2016 Fort McMurray wildfire had creatinine normalized HP-G concentrations below the biological exposure index, likely caused by delays in urine collection under emergency conditions, at early stages of firefighting. Chapter IV extends from targeted biomonitoring of occupational smoke exposure, towards elucidating the relative risk of tobacco smoking in an international cohort of participants (n=1000) from the Prospective Urban and Rural Epidemiological (PURE) study. Comprehensive analysis of nicotine metabolites in urine by MSI-CE-MS allowed for reliable determination of the total nicotine equivalent and nicotine metabolic ratio as robust indicators of recent tobacco smoke exposure and nicotine dependence, respectively. This method also offers a more accurate approach for biochemical verification of smoking status in large-scale epidemiological studies that are prone to social desirability and gender bias when relying on standardized questionnaires. Lastly, Chapter V employs a nontargeted metabolomics workflow using MSI-CE-MS to identify urinary metabolites that may serve as objective dietary biomarkers of food intake in participants across 14 countries from the PURE cohort. A panel of robust and generalizable metabolites were validated for biomonitoring of complex dietary exposures, that may further exacerbate the hazards of tobacco smoking. In summary, this thesis contributes high throughput analytical tools for characterizing the human urine exposome to better decipher the roles of smoke exposure, and suboptimal diet on chronic disease burden among diverse populations and regions worldwide. / Thesis / Doctor of Philosophy (PhD)

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