Idiosyncratic drug reactions (IDRs) represent a significant medical problem and pose a great challenge to drug development. Circumstantial evidence suggests that, in most cases, reactive metabolites of the drug are responsible. The major focus of this thesis is the identification of reactive metabolites and the synthesis of analogs required to test several hypotheses related to involvement of metabolism and covalent binding in the mechanisms of IDRs. Minocycline is unique among tetracyclines in causing a significant incidence of a lupus-like syndrome and autoimmune hepatitis. In this study, we demonstrated that minocycline is oxidized to reactive intermediates by myeloperoxidase/H2O2/Cl-, HOCl, horseradish peroxidase/H2O2, or hepatic microsomes. When trapped with N-acetylcysteine (NAC), two adducts with protonated molecular ions at m/z 619 were isolated and analyzed by NMR. One represents attack of the aromatic D ring by NAC meta to the N, N-dimethylamino group, implying that the reactive intermediate was a quinone iminium ion. The other adduct, which was not observed when minocycline was oxidized by hepatic microsomes, indicates that the NAC is attached at the junction of the B and C rings, suggesting that the HOCl added across the double bond of the B ring leading to a reactive molecule, and then NAC displaced the chloride ion. Nevirapine, an anti-HIV drug, is associated with idiosyncratic skin rashes in humans. The goal of this project was to investigate whether the 12-hydroxylation pathway is responsible for the skin rash. To test a part of this hypothesis, 12-trideuteronevirapine, 12-OH-NVP sulfate, and several other analogs of nevirapine were synthesized. D-penicillamine is known to cause idiosyncratic autoimmune reactions in humans. The goal of this project was to test whether D-penicillamine covalently binds to macrophages and triggers downstream events leading to autoimmunity. To test a part of this hypothesis, D-penicillamine conjugated to biotin was synthesized. In summary, reactive metabolites of minocycline were found that likely explain why minocycline has an IDR profile unique among the tetracyclines. In addition, analogs of nevirapine and D-penicillamine required for mechanistic studies of nevirapine and D-penicillamine-induced IDRs were synthesized. These studies provide additional support for the involvement of reactive metabolites in the mechanisms of IDRs.

Previous work by our research group demonstrated that quantitative chemical analysis of analytes, such as mercury and chromium species, in environmental matrices could be successfully carried out without using calibration curves and with correction for species interconversion by using EPA Method 6800A. This method encompasses isotope dilution mass spectrometry (IDMS) and speciated isotope dilution mass spectrometry (SIDMS), both of which are described in detail in chapter 1. Research described in this dissertation expands upon our earlier work by applying the method to the speciation of mercury in biological matrices, the speciation of glutathione in red blood cells and whole blood, and the analysis of enzyme activity in mammalian tissue. / Bayer School of Natural and Environmental Sciences; / Chemistry and Biochemistry; / PhD; / Dissertation;

Metabolites, small endogenous molecules existing in every living cell, tissue or organism, play a vital role for maintaining life. The collective group of all metabolites, the metabolome, is a consequence of the biochemistry and biochemical pathways that a cell or tissue uses to promote survival. Analysis of the metabolome can be done to reveal changes of specific metabolites which can be a manifestation, a reason or a consequence of for example a disease. The physical chemical diversity amongst these components is tremendous and it poses a large analytical challenge to measure and quantify all of them. Targeting sub groups of the meta­bolome such as specific functional classes has shown potential for increasing metabolite coverage. Group selective labeling with biotin-tags followed by high affinity avidin capture is a well established purification strategy for protein purification. The purpose with this project is to explore if it is possible to transfer the avidin biotin approach to metabolomics and use this method for small mole­cules purification. Specifically, this investigation aims to see if it is achievable to make a bio­tinylation of specific functional groups, to increase the sensitivity through reduction of sample complexity in liquid chromatography mass spectrometry metabolomics analyses after high affinity avidin capture. By purifying the analyte of interest and thereby reducing the sample complexity there will be a reduction in ion suppression. The aim is to increase the analytical sensitivity through a reduction in ion suppression during liquid chromatography mass spectrometry analysis. Delimitations have been done to only investigate the possibility to obtain a biotinylation of primary amines and amides. As model compounds phenylalanine, spermi­dine, histamine and nicotinamide have been selected. The result from this study indicates that it is possible to increase metabolite coverage through biotin labeling followed by high affinity avidin capture. It is a gain in analytical sensitivity of selected model compounds when comparing biotinylation strategy with a control non­biotinylation approach in a complex sample. A broader study of additional model compounds and a method development of this strategy are necessary to optimize a potential future method.

A LC-MS/MS method for the analysis of alkylphenol polyethoxylates in environmental waters was developed in this study. Preatment procedures including liquid-liquid extraction and solid phase extraction were compared, it¡¦s concluded that solid phase extraction is the more suitable way due to higher recovery and better stability for the analytical results. The recovery of nonylphenol polyethoxylate and octylphenol polyethoxylate were 62.3-110.7 % and 64.9-112.0 %, limit of detection were 17.60-174.9 ng/L and 7.40-53.56 ng/L. Enviromental water samples were collected from eight sampling sites along Love River in Kaohsiung City to investigate the contents of alkylphenol polyethoxylates. The highest concentration of total alkylphenol polyethoxylates was observed at Ming-Cheng Bridge which located at the upstream of Love River. For all of the analyzed compounds, the concentration of octylphenol tetraethoxylate (40.46 £gg/L) was the highest in all of the sampling sites. It¡¦s also noticed the concentration of octylphenol polyethoxylate (20.11 £gg/L) was higher than that of nonylphenol polyehtoxylate (128.04 £gg/L).

In April and June 2012, environmental water samples were collected from fourteen sampling sites in Hengchun peninsula to investigate the contents of alkylphenol polyethoxylates. A solid phase extraction combined with LC-MS/MS method for the analysis of alkylphenol polyethoxylates in environmental waters was developed in this study. The mobile phase used methanol gradient elution with deionized water. The recovery of nonylphenol polyethoxylate and octylphenol polyethoxylate were 68~94 % and 65~93 %, limit of detection were 1.89~54.20 ng/L and 0.44~39.31 ng/L, limit of quantitative were 6.29~181 ng/L and 1.48~131 ng/L. The SsuChung river contents of NPEO and OPEO were 15.64~36.29 £gg/L and 3.14~7.37 £gg/L. The Paoli river contents of NPEO and OPEO were 16.65~76.41 £gg/L and 5.66~18.80 £gg/L. The Hou Bay contents of NPEO and OPEO were 34.79~66.72 £gg/L and 7.77~19.03 £gg/L. The Shihniou river contents of NPEO and OPEO were 26.67 £gg/L and 6.68 £gg/L. The Wanli Tong, Baisha, Houbi Lake, South Bay, Caesar and Siangjiao Bay contents of NPEO and OPEO were 14.17~48.82 £gg/L and 3.88~14.79 £gg/L. The dry season concentration contents of alkylphenol polyethoxylates were high than the wet season. The concentration of nonylphenol polyethoxylate was higher than that of octylphenol polyehtoxylate.

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).

No description available.

Neurodegenerative diseases have been shown to correlate positively with an ageing population. The most common neurodegenerative diseases are amyotrophic lateral sclerosis (ALS), Parkinson’s disease and Alzheimer’s disease. The cause of these diseases is believed to be the interaction between genetic and environmental factors, synergistically acting with ageing. BMAA (β-methylamino-L-alanine) is one kind of toxin present in our environment and might play an important role in the development of those diseases. BMAA was initially isolated from cycad seeds in Guam, where the incidence of ALS/Parkinsonism-dementia complex among the indigenous people was 50 – 100 times higher than the rest of the world in the 1950’s. BMAA can induce toxic effects on rodents and primates. Furthermore, it can potentiate neuronal injury on cell cultures at concentrations as low as 10 µM. BMAA was reported to be produced by cyanobacteria, and could bio-magnify through the food chain. In this thesis, work was initially focused on the improvement of an existing analytical method for BMAA identification and quantification using liquid chromatography coupled with tandem mass spectrometry.  Subsequently, the refined method was applied to environmental samples for probing alternative BMAA producer(s) in nature and to seafood samples for estimation of human exposure to this toxin. In Paper I, a systematic screening of the isomers of BMAA in a database was performed and seven potential isomers were suggested. Three of them were detected or suspected in natural samples. In Paper II, a deuterated internal standard was synthesized and used for quantifying BMAA in cyanobacteria. In Paper III, Diatoms were discovered to be a BMAA producer in nature. In Paper IV, ten popular species of seafood sold in Swedish markets were screened for BMAA. Half of them were found to contain BMAA at a level of 0.01 – 0.90 µg/g wet weight. In Future perspectives, the remaining questions important in this field are raised.

Vemurafenib is a potent serine/threonine kinase inhibitor and is registered as Zelboraf® for the treatment of metastatic melanomas harboring BRAFV600E mutations. There is a large individual variation in drug response and the side effects observed among patients treated with Zelboraf® has proven to be severe.  LC-MS/MS methods were developed to measure vemurafenib and its metabolites in human plasma for prediction of treatment outcome and side effects in order to individualize treatment with Zelboraf®.  A novel, rapid quantification method was developed for vemurafenib using a stable isotope labeled internal standard. The method was validated according to international guidelines with regard to calibration range, accuracy, precision, carry-over, dilution integrity, selectivity, matrix effects, recovery and stability. All parameters met the set acceptance criteria.  The first method suitable for quantifying vemurafenib metabolites in human plasma is presented. Lacking commercially available reference substances, human liver microsomes were used to produce the metabolites. In patient samples at steady-state five previously in vitro identified metabolites were quantified for the first time.