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.

Malaria is currently one of the most imperative parasitic diseases in developing countries. Artesunate has a short half-life, low aqueous solubility and resultant poor and erratic absorption upon oral administration, which translate to low bioavailability. Mefloquine is eliminated slowly with a terminal elimination half-life of approximately 20 days and has neuropsychiatric side effects. Novel drug delivery systems have been utilised to optimise chemotherapy with currently available antimalarial drugs. Pheroid™ technology is a patented drug delivery system which has the ability to capture, transport and deliver pharmaceutical compounds. Pheroid™ technology may play a key role in ensuring effective delivery and enhanced bioavailability of novel antimalarial drugs. The aim of this study was to evaluate the possible efficacy and bioavailability enhancement of the selected antimalarial drugs, artesunate and mefloquine, in combination with Pheroid™ vesicles. The in vitro efficacy of artesunate and mefloquine co-formulated in the oil phase of Pheroid™ vesicles and entrapped in Pheroid™ vesicles 24 hours after manufacturing were investigated against a 3D7 chloroquine-sensitive strain of Plasmodium falciparum. Parasitemia (%) was quantified with flow cytometry after incubation periods of 48 and 72 hours. Drug sensitivity was expressed as 50% inhibitory concentration (IC50) values. An in vivo bioavailability study with artesunate and mefloquine was also conducted in combination with Pheroid™ vesicles, using a mouse model. A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to analyse the drug levels. C57 BL6 mice were used during this study. The selected antimalarial drugs were administered at a dose of 20 mg/kg with an oral gavage tube. Blood samples were collected by means of tail bleeding. The in vitro drug sensitivity assays revealed that artesunate, co-formulated in the oil phase of Pheroid™ vesicles and evaluated after a 48 hour incubation period, decreased the IC50 concentration significantly by 90%. Extending the incubation period to 72 hours decreased the IC50 concentration of artesunate, also co-formulated in the oil phase of Pheroid ™ vesicles significantly by 72%. No statistically significant differences between the reference and Pheroid™ vesicle groups were achieved when artesunate was entrapped 24 hours after manufacturing of Pheroid™ vesicles. Mefloquine co-formulated in the oil phase of Pheroid™ vesicles and evaluated after a 48 hour incubation period decreased the IC50 concentration by 36%. Extending the incubation period to 72 hours increased the efficacy of the Pheroid™ vesicles and the IC50 concentration was significantly decreased by 51%. In contrast with the results obtained with artesunate, entrapment of mefloquine in Pheroid™ vesicles 24 hours after manufacturing decreased the IC50 concentration significantly by 66%. The LC-MS/MS method was found to be sensitive, selective and accurate for the determination of artesunate and its active metabolite, dihydroartemisinin (DHA) in mouse plasma and mefloquine in mouse whole blood. Most of the artesunate plasma concentrations were below the limit of quantification in the reference group and relatively high outliers were observed in some of the samples. The mean artesunate levels of the Pheroid™ vesicle group were lower compared to the reference group, but the variation within the Pheroid™ vesicle group lessened significantly. The mean DHA concentrations of the Pheroid™ vesicle group were significantly higher. DHA obtained a higher peak plasma drug concentration with the Pheroid™ vesicle group (173.0 ng/ml) in relation to the reference group (105.0 ng/ml) and at a much faster time (10 minutes in Pheroid™ vesicles in contrast to 30 minutes of the reference group). Pharmacokinetic models could not be constructed due to blood sampling per animal limitation. The incorporation of mefloquine in Pheroid™ vesicles did not seem to have improved results in relation to the reference group. No statistical significant differences were observed in the pharmacokinetic parameters between the two groups. The relative bioavailability (%) of the Pheroid™ vesicle incorporated mefloquine was 7% less bioavailable than the reference group. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.