The initial research of this thesis involves the evaluation of different strategies for developing diverse chemistries of highly stable coatings for the automated 96-blade (thin-film) solid phase microextraction (SPME) system. Thin-film geometry increases the volume of extractive phase, and consequently improves the sensitivity of the analysis. Sol-gel technology was used for the preparation of octadecyl (C18)-silica gel thin-film coating. The evaluation of the C18-silica gel SPME extractive phase resulted in stable physical and chemical characteristics and long-term reusability with a high degree of reproducibility. Biocompatible polyacrylonitrile (PAN) polymer was used for the preparation of particle-based extractive phases in order to improve the biocompatible characteristics of SPME coatings for the extraction from biological samples. Three different immobilization strategies were evaluated for developing highly stable coatings for the automated 96-blade SPME system. The spraying was found to be the optimal method in terms of stability and reusability for long-term use. The optimized C18-PAN coating demonstrated improved biocompatibility, stability, and reusability for the extraction of benzodiazepines from human plasma in comparison with those of C18-silica gel coating. To improve the biocompatible properties of the C18-PAN SPME coating for long-term direct analysis from whole blood, different modification strategies were studied and evaluated. The modification of the coating with an extra layer of biocompatible polyacrylonitrile resulted in significant improvement in the blood compatibility in long-term use. ‘Extracted blood spot’ (EBS) sampling was introduced as a novel approach to overcome the limitations of dried blood spot sampling. EBS includes the application of a biocompatible SPME coating for spot sampling of blood or other biofluids. The compatibility of EBS sampling with different analytical methods was demonstrated. The utilization of EBS as a fast sampling and sample preparation method resulted in a significant reduction of matrix effects through efficient sample clean-up. Modified polystyrene-divinylbenzene (PS-DVB)-PAN and phenylboronic acid (PBA)-PAN 96-blade SPME coatings were developed and evaluated for the extraction of analytes in a wide range of polarity. These coatings demonstrated efficient extraction recovery for both polar and non-polar groups of compounds, and presented chemical and mechanical stabilities and reproducible extraction efficiencies for more than 100 usages in biological sample.

Zirconia is one of the most promising column packing materials for High Performance Liquid Chromatography (HPLC). The perfect HPLC support material should be energetically homogenous, have a high surface area on which different chemical species can reversibly attach and be physically and chemically stable over a wide range of pH, temperature and solvent conditions. Most existing supports do not have all of these properties. This project is also focused on a proteomics study. Zirconia, hafnium oxide and titanium oxide which are some of the more promising materials currently available, can be used for the separation and analysis of phosphorylated proteins. Adenosine triphosphate, Adenosine diphosphate and Adenosine monophosphate were used as prototypes for phosphorylated proteins. Separation, absorption, fluorescence and SEM studies were performed to determine the adsorption of Adenosine phosphates species at a particular pH on Zirconia. Zirconia was also used for the purification of Fibrinogen Growth Factor (FGF) protein, which are a family of growth factors involved in angiogenesis, wound healing, and embryonic development. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) technique was used to analyze the off-column purification and separation of this protein. This research suggests that, at acidic conditions, adenosine monophosphate has more favorable absorption on the Zirconia surface. On the other hand, the separation study suggests that basic conditions are more favorable for the absorption of ATP, ADP and AMP when mixed together on Zirconia 500. Furthermore, it was found that Zirconia is a very promising material for the purification of FGF protein.

A new method for analysis of acidic herbicides, mostly phenoxy acids and their esters, in cereals with liquid chromatography-tandem quadrupole mass spectrometry (LS-MS/MS) has been developed. Samples were hydrolyzed with sodium hydroxide in order to release covalently bound compounds followed by neutralization and finally extraction with acidified ethyl acetate. The extraction efficiency for both ester formulations and acids were studied. Acceptable results (70-120 %) were obtained for 2,4-D, dichlorprop, MCPA and mecoprop for both esters and acids. However, low recoveries were observed for ester formulations of dicamba, fluroxypyr, fluazifop and haloxyfop, possibly due to the complex structure of the compounds in combination with the matrix and/or incomplete hydrolysis step. The limit of quantification (LOQ) for targeted pesticides was 0.01 mg/kg. The method has been tested in the EU Proficiency Test for cereals with good results.

The protic acid-catalyzed polymerization of 1,6-anhydro-6-D-glucopyranose (I) was first reported one-half century ago; however, the mechanism of this reaction has not been resolved and is the topic under investigation in this thesis. In an attempt to resolve this mechanism, a number of 1,6-anhydrides structurally related to 1,6-anhydro-B-D-glucopyranose (I) were prepared and polymerized. The C-2, C-3, or C-4 hydroxyl group was either specifically blocked, replaced by a hydrogen atom or positioned different sterically. The relative rates of disappearance of monomer in the polymerization reaction were measured and this information used to propose a reaction mechanism.

none

We have previously used a two-dimensional fluorescence difference gel electrophoresis protein expression with matrix-assisted laser desorption ionization of mass spectrometry to identify the serum proteomic profiles before and after the Tai Chi exercise in normal adults (Yang & Chang, et al. Clin Chem 56:127, 2010). However, the high abundant serum proteins in seyal samples might interfere the discovery of low abundance proteins in two-dimensional electrophoresis, but these low abundant proteins may play an important role on human physiology. Therefore, we looked for another way to resolve this complex issue. After multiple attempts, we chose a commercial affinity column to exclude 14 kinds of high-abundance proteins before analyses of serum proteomic displays. This column could be fit into a fast liquid chromatography separation of purified proteins and eluted for low abundant proteins. The low abundant proteins were first expressed by one-dimensional gel electrophoresis of proteins followed by a series of gel cut down for in-gel digestion by trypsin and subject to nano-liquid chromatography with electrospray ionization tandem mass spectrometry analysis (nano-LC ESI MS/MS ). The results obtained by software analysis were subject to its functional pathways analysis. We analysed 3 comparisons of the protein displays including differences between normal adults before and after exercise, differences of normal adults and diabetic patients before and after exercise. Experiments were next performed to validate the most significant difference of proteins between each category by enzyme-linked immunoassay. Results showed that dipeptidyl peptidase 4 (DPP4) and neutrophil gelatinase-associated lipocalin (NGAL) proteins were significantly higher in diabetic patients than in normal adults ( P values were 0.011 and less than 0.001), while the prolactin-inducible protein (PIP) was higher in normal adults than in diabetic patients after exercise (P value of 0.042). To our knowledge, decrease of DPP4 in type 2 diabetes has been shown to reduce blood sugar and improve the immunity; and NGAL has been confirmed to be an indicator for early diagnosis of acute kidney injury. Therefore, we have identified certain functional proteomic markers in normal and diabetic patients after Tai Chi exercise. This study model with exclusion of high-abundance serum proteins is a useful mode for identifying immune and metabolic marker with and without.

Fluorescent labeling can improve the detection sensitivity in capillary electrophoretic (CE) separations down to attomolar concentrations. However, most fluorescent labels are not compatible with CE because their fluorescence properties and charge states are pH-dependent, they are often hydrophobic and they have a tendency to significantly change the properties of the analytes after labeling. A group of fluorescent labeling reagents have been prepared whose fluorophores have properties that are optimized for CE separations. These fluorophores have fluorescence properties and charge states that are independent of pH in the 2 < pH < 11 range. Their excitation maxima are also compatible with the 488 nm line of the Argon ion laser. A mono-cationic acridine-based fluorescent label was prepared and was found to not shift the pI of a labeled model protein in capillary isoelectric focusing separation (cIEF). Lower loading, due to increased sensitivity, led to better resolution of closely spaced isoform peaks having a pI = 0.05. A tri-anionic pyrene-based fluorescent labeling reagent was also synthesized and was used in the sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) separation of proteins. The fluorophore led to an LOQ in the nM range, and did not alter the migration behavior of proteins in the sieving matrix. A third fluorescent labeling reagent was developed as a solid phase reagent (SPR) where the fluorophore was immobilized on a solid surface through a cleavable anchor. The fluorophore is di-anionic and is based on pyrene. The SPR was designed to allow the simultaneous capture and labeling of an analyte and the efficient release of the label-analyte conjugate under mild acidic conditions. The use of the SPR allowed the labeling of a diamine whose concentration was in the low nanomolar range. The SPR opens up the possibility for mono-labeling and proportional multiple labeling of proteins.

none

There are two purposes in this research, one is the development of the new method which can be used for detection and quantification of triarylmethanes in fish tissues. The other is that we confirmed validation and utility of triarylmethanes by the method that is according to Commission Decision 2002/657/EC. Homogenized fish tissues were extracted twice with acetonitrile and defatted with n-hexane. HPLC separation was conducted with the RP-18 column. The mobile phases consisted of 0.5 mM ammonium acetate buffer (pH 3.8, adjusted with acetic acid)¡V ACN (contained 0.1% formic acid) solution. Triarylmethane was determined by LC-ESI-MS/MS in positive mode. The correlation coefficients of calibration curves with triarylmethane in fish tissues were 0.998 ~ 0.999. The decision limits (CC£\) were 0.16 ¡Ó 0.07 £gg/kg(MG), 0.15 ¡Ó 0.04 £gg/kg(LMG), 0.20 ¡Ó 0.13 £gg/kg(CV) and 0.23 ¡Ó 0.12 £gg/kg(LCV), and detection capabilities (CC£]) were 0.20 ¡Ó 0.09 £gg/kg(MG), 0.18 ¡Ó 0.05 £gg/kg(LMG), 0.24 ¡Ó 0.16 £ggkg-1(CV) and 0.29 ¡Ó 0.15 £gg/kg(LCV).

Sphingolipids are a highly diverse category of compounds that serve not only as components of biologic structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids influence their biological activity, there is a need for comprehensive methods for quantitation of as many individual subspecies as possible. This dissertation describes methods that have been developed and validated for the extraction, liquid chromatographic separation, identification and quantitation of sphingolipids by electrospray ionization (ESI), tandem mass spectrometry (MS/MS) using an internal standard cocktail developed by the LIPID MAPS Consortium. The compounds that can be readily analyzed are sphingoid bases and sphingoid base 1-phosphates, as well as more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, and mono- and di-hexosylceramides. For broader utility, the methods have been optimized for two categories of tandem mass spectrometers. With minor modifications, these methods can be applied to the analysis of isomers such as glucosylceramide and galactosylceramide, and with the availability of additional internal standards, more complex species such as sulfatides can also be quantified. Using these methods 46 species of these compounds have been quantified in RAW264.7 cells, a macrophage cell line. Quantitation of individual sphingolipid metabolites is possible using liquid chromatography, tandem mass spectrometry, and stable isotope labeling with [13C]palmitic acid can be used to differentiate between metabolites produced by de novo synthesis versus turnover. This approach is more accurate when one knows the isotope enrichment of the precursor pool (in this case, [13C]-palmitoyl-CoA); therefore this dissertation describes methods to analyze both the various isotopic forms of palmitoyl-CoA and sphingolipids through sphingomyelins and monohexosylceramides using two cell models, HEK293 cells and RAW264.7 cells treated with Kdo2-Lipid A. The sphingolipid analysis was simplified by the fragmentation of most of the metabolites to backbone product ions. For example the presence of the isotopic label in the long chain base, N-acyl linked fatty acid, or both was determined via, m/z 264 for [12C]sphingosine (d18:1) and m/z 280 for [13C]sphingosine (m+16, d18:1), versus the m/z of the isotopically labeled precursor, (m+16 versus m+32).