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Although solid-phase microextraction (SPME) technique has gained wide applications from in vitro environmental investigations to in vivo pharmacokinetic studies, there are still challenges for utilizing SPME to track fast concentration change over time at a specific location in a heterogeneous system, such as studying the tissue- specific metabolism or bioaccumulation of pharmaceuticals in a living animal. In this case, the technique must be adaptable for in situ analysis with highly temporal and spatial resolutions. The goal of the research presented was not only to address this issue but also to develop new analytical methods that were more effective for in vivo study using SPME. In order to improve the temporal resolution, fast SPME sampling technique based on pre-equilibrium extraction must be adopted. However, more efforts need to be placed into calibration so as to guarantee the accuracy of the analysis. In this thesis, firstly, the kinetic calibration was proposed for adsorptive SPME fibres that were widely used for biological samples, which paved the way for performing fast sampling for in vivo dynamic monitoring. Secondly, the kinetic calibration was applied for in vivo pharmacokinetic study with beagles, with which not only solid experimental evidence was obtained for the calibration theory, but also an example was shown to address the quantitative capability of in vivo SPME. The developed method showed comparable sensitivity to traditional blood analysis (linear range 5 – 2000 µg/L and limit of detection: 5µg/L). Furthermore, the traditional kinetic calibration based on isotopically labelled standards was simplified to a single time-point calibration, and a single standard calibration was developed for multiple analytes. Therefore, the fast in vivo sampling could be accomplished in a simple but accurate measure; compared to the established equilibrium SPME technique, statistically no significant difference (P<0.05) was observed by using one-way ANOVA and the post-hoc Turkey’s test for multiple comparisons. The second aspect of the thesis addressed the spatial resolution of SPME for in situ analysis. Firstly, the sampling of the SPME with high spatial resolution was modeled with multilayered gel system with the mini-sized SPME fibres. The feasibility of the SPME for in vitro application was demonstrated by sampling in an onion bulb with heterogeneous structure. Afterwards, the miniaturized fibre was successfully applied to the in situ analysis of the concentration distribution of Ochratoxin A in semisolid cheese samples with acceptable sensitivity (Detection limit was 1.5 ng/mL and the linear range was 1.5-500 ng/mL) and comparable accuracy to the standard methods such as liquid extraction and microdialysis. Finally, the in vivo application of the space- and time- resolved SPME was implemented to study the tissue-specific bioaccumulations of pharmaceuticals in fish adipose fins and muscle tissues. The results were validated by the standard method liquid extraction, and they were also comparable to the literature results. The research presented here demonstrated the application potential of the time-and space- resolved SPME for in situ dynamic and static analysis in a living system such as a beagle or fish, and in a non-living system such as a cheese piece or an onion bulb.

Although solid-phase microextraction (SPME) technique has gained wide applications from in vitro environmental investigations to in vivo pharmacokinetic studies, there are still challenges for utilizing SPME to track fast concentration change over time at a specific location in a heterogeneous system, such as studying the tissue- specific metabolism or bioaccumulation of pharmaceuticals in a living animal. In this case, the technique must be adaptable for in situ analysis with highly temporal and spatial resolutions. The goal of the research presented was not only to address this issue but also to develop new analytical methods that were more effective for in vivo study using SPME. In order to improve the temporal resolution, fast SPME sampling technique based on pre-equilibrium extraction must be adopted. However, more efforts need to be placed into calibration so as to guarantee the accuracy of the analysis. In this thesis, firstly, the kinetic calibration was proposed for adsorptive SPME fibres that were widely used for biological samples, which paved the way for performing fast sampling for in vivo dynamic monitoring. Secondly, the kinetic calibration was applied for in vivo pharmacokinetic study with beagles, with which not only solid experimental evidence was obtained for the calibration theory, but also an example was shown to address the quantitative capability of in vivo SPME. The developed method showed comparable sensitivity to traditional blood analysis (linear range 5 – 2000 µg/L and limit of detection: 5µg/L). Furthermore, the traditional kinetic calibration based on isotopically labelled standards was simplified to a single time-point calibration, and a single standard calibration was developed for multiple analytes. Therefore, the fast in vivo sampling could be accomplished in a simple but accurate measure; compared to the established equilibrium SPME technique, statistically no significant difference (P<0.05) was observed by using one-way ANOVA and the post-hoc Turkey’s test for multiple comparisons. The second aspect of the thesis addressed the spatial resolution of SPME for in situ analysis. Firstly, the sampling of the SPME with high spatial resolution was modeled with multilayered gel system with the mini-sized SPME fibres. The feasibility of the SPME for in vitro application was demonstrated by sampling in an onion bulb with heterogeneous structure. Afterwards, the miniaturized fibre was successfully applied to the in situ analysis of the concentration distribution of Ochratoxin A in semisolid cheese samples with acceptable sensitivity (Detection limit was 1.5 ng/mL and the linear range was 1.5-500 ng/mL) and comparable accuracy to the standard methods such as liquid extraction and microdialysis. Finally, the in vivo application of the space- and time- resolved SPME was implemented to study the tissue-specific bioaccumulations of pharmaceuticals in fish adipose fins and muscle tissues. The results were validated by the standard method liquid extraction, and they were also comparable to the literature results. The research presented here demonstrated the application potential of the time-and space- resolved SPME for in situ dynamic and static analysis in a living system such as a beagle or fish, and in a non-living system such as a cheese piece or an onion bulb.

<p>This thesis presents two new concepts for separation of micro particles using dielectrophoresis, demonstrated by calculated examples, as well as a new method for obtaining dielectric data on living cells. The thesis is based on four papers.</p><p>Paper I describes how the trapping efficiency of micro particles may be significantly increased when superpositioned electric fields are employed in a high conductivity medium. Avoiding low conductivity media is important when working with living cells. Calculations were performed to predict trajectories of Escherichia coli bacteria in the system with superpositioned electric fields, and a model was developed which employed two arrays of interdigitated electrodes in a micro channel.</p><p>Paper II proposes a new concept for separation of micro particles, based on repetitive dielectrophoretic trapping and release in a flow system. Calculations show that the resolution increases as a direct function of the number of trap and release steps, and that a difference in size will have a larger influence on the separation than a difference in dielectrophoretic properties. Polystyrene beads in deionized water were used as a model, and calculations were performed to predict the particle behavior and the separation efficiency. It should be possible to separate particles with a size difference of 0.2 % by performing 200 trap-and-release steps. The enhanced separation power of multi step dielectrophoresis could have significant applications, not only for fractionation of particles with small differences in size, but also for measuring changes in surface conductivity.</p><p>Paper III presents a new calculation method for predicting dielectrophoretic motion of micro particles. The method is based on a soft sphere method often used in molecular dynamics. Results from the calculations are in good agreement with theoretical predictions as well as initial experimental results, showing that the method provides good efficiency and accuracy.</p><p>Paper IV describes a new method for measurements of conductivity of living bacteria. To obtain reliable conductivity values, it is important to handle the cells as gently as possible during the measurement process. A standard conductivity meter was used in combination with cross-flow filtration. In this way, repeated centrifugation and resuspension is avoided which otherwise may cause damage to the bacteria. The conductivity of Bacillus subtilis was determined to be 7000 μS/cm by means of the cross-flow filtration method, and the values differ from earlier published values by almost an order of a magnitude.</p><p>In addition to the work presented in the papers, some experimental dielectrophoresis work in chip-based systems was performed. The behavior of Escherichia coli and polystyrene beads at different voltages and frequencies were studied. Separation of beads with different sizes was achieved on an array of interdigitated electrodes. Using electrodes with a pointed shape, alignment in different directions, pearl-chain formation, rotation, and other dielectrophoretic motion of <i>E. coli</i> were observed.</p>

Photon-based tandem mass spectrometry provides a versatile ion activation strategy for the analysis of polypeptides, proteins, and lipids. 351-nm ultraviolet photodissociation mass spectrometry (UVPD-MS) is a facile and selective tandem dissociation technique used to elucidate chromophore-modified peptides within large mixtures. A bis-aryl chromogenic chemical probe was utilized to target solvent exposed primary amine residues within native protein states. Collision-induced dissociation (CID) was employed to indiscriminatly characterize the complete proteolytic digest while chromophore containing peptides were selectively dissociated with 351-nm UVPD; thus streamlining the identification of targeted peptides with structurally informative residues. Protein amine residue reactivities were then compared with predicted solvent exposures to elucidate protein tertiary structures, their mechanistic properties, and ligand-binding interactions. High-energy 193-nm UVPD is a fast, high-energy tandem mass spectrometry method and frequently generates fragment ions typically inaccessible to CID-based methods. Native mass spectrometry was coupled to top-down 193-nm UVPD for the gas phase characterization of non-covalent protein-ligand and protein-protein complexes. This method yielded a unique array of fragment ions for a comprehensive analysis of protein structures. UVPD of non-covalent complexes generated many polypeptide backbone fragments to characterize the primary sequence of proteins. Furthermore, top-down UVPD engendered cleavages with intact electrostatic interactions; this provided insight into the binding interfaces within protein-ligand complexes and the higher order structural architectures of oligomeric complexes. High-resolution 193-nm UVPD was paired with high performance liquid chromatography (LC) for the streamlined structural analysis of amphiphilic glycolipids within complex mixtures. For all glycolipids, UVPD provided the most comprehensive structural analysis tool by affording a diverse array of fragment ions to characterize both hydrophobic and hydrophilic moieties. UVPD based LC-MS separations of gangliosides shed light on the ceramide lipid bases, glycan moieties, and their isobaric structural variants. UVPD activation of lipid A and lipooligosaccharides (LOS) compounds generated a mixture of C-C, C-O, and C-N fragment ions to illustrate the hydrophobic acyl structures, while cleavages within the glycosidic, and cross-ring cleavages allowed the determination of acylation patterns. Novel LC-MS separation strategies were developed to elucidate and structurally characterize complex mixtures of lipopolysaccharide containing compounds. / text

This thesis describes new analytical methods for trace or ultra-trace analysis by molecular absorption and emission spectroscopy. The initial part of the thesis is devoted to an introduction to molecular electromagnetic absorption spectroscopy and molecular fluorescence. The principles, advantages and limitations of spectrophotometric and fluorimetric measurement are discussed. The concepts of enzymes and their applications in analytical chemistry are also expounded. Two organic compounds, 5-( 4-arsonophenylazo )-8-(p-toluenesulfonamido)quinoline (APTSQ) and 5-(p-methoxyphenylazo)-8-(p-toluenesulfonamido) quinoline (MPTSQ), have been synthesised and used as new chromogenic anellor f1uorogenic reagents. Five specific, highly sensitive, simple, precise and inexpensive novel analytical methods have been developed: (1) A spectrophotometric method is described for the determination of cobalt. The maximum absorbance is at 582 nm with a molar absorptivity of 1.18 x 1 as I mor' cm-1 . Beer's law is obeyed for cobalt concentrations in the range 0-0.5 J.l.g mr'. (2) A fluorimetric method for the determination of cobalt is proposed. The fluorescence intensity is measured at Aex 287 nm and Aem 376 nm. The response is linear up to 25 ng mr' and the detection limit is 0.002 ng mr'. The mechanism of the fluorescence reaction has been investigated. (3) A flu~ri~etric method is proposed for the d~ter~i~~tion of H202. The response IS linear up to 12.2 ng mr H:t>2. The detection limit IS 0.16 ng mr'. (4) An enzymatic assay for glucose by spectrofluorimetry is described. The fluorescence intensity is proportional to the concentration of glucose up t0180 ng mr'. A detection limit of 5.4 ng mr' was obtained and allowed the determination of glucose in an extremely small amount of serum (O.5J.1.I) and urine (1 J.l.1). (5) A fluorimetric method for the determination of iron is proposed, based on the reaction between iron(III) and MPTSQ in the presence of cetyltrimethylammonium bromide. The fluorescence intenSity (Aex=317 nm, A.em=534 nm) is linear up to 170 ng mr' with a detection limit of 0.12 n9 mr'. An investigation of the mechanism of the fluorescence reaction has been made. The applications of the proposed methods for the determination of the concerned analytes at low levels in biological, environmental, pharmaceutical or beverage samples are also reported.

This thesis is principally concerned with spectroscopic and computational studies of artists' pigments. Manuscripts, art and archaeological artefacts were examined by Raman microscopy, identifying the pigments and drawing conclusions for historical and conservation purposes. Studies of Anglo Saxon and later manuscripts have shown the Insular palette triumvirate, assumed to be orpiment, red lead and verdigris, to contain red ochre and vergaut, but no verdigris. This remains unchanged until the introduction of lazurite in c. 920 AD and vermilion in the 12th century. Lazurite has been erroneously identified on the Lindisfarne Gospels, by the technique of Roosen-Runge. Raman microscopy shows the blue pigments to be exclusively indigo, casting doubt on analyses performed using Roosen-Runge's technique. The Islamic manuscript palette was found to be remarkably consistent across a substantial geographical area over an extended period. It is also very similar to that of early Western manuscripts. Comparison of these results with existing literary sources has shown the latter to be highly inaccurate. The palette of William Blake was examined and compared to results of analysis by False Colour Infrared Photography (FC-IP). The FC-IP technique was determined to be inappropriate for pigment identification. Two significant artefacts were shown to be modern forgeries: a rare Assyrian fresco contains a modern green pigment, and the world famous Vinland map was found to have significant quantities of anatase in the yellow lines. Density Functional Theory methods were applied to the mechanism of decay isomerisation of As4S4, which was partially clarified, and to the geometries of R2SeX2 (R = CF3, CF2H, CFH2, CH3, CH2CH3, CH(CH3)2, t-Butyl, X=F, Cl, Br, I, At). The most stable geometry was found to be determined by the polarity of the Se-X bonds and the steric and electron-withdrawal effects of the R-group on the C-Se bond strength.

Cyklosporin A (CsA) är en cyklisk polypeptid med molekylvikten är 1202.6 Da. Substansen har svampursprung (Tolypocladium inflatum Gams) och starka immunhämmande egenskaper. CsA används därför som immunsuppressivt läkemedel för att förhindra avstötning av transplanterade organ och benmärg, samt vid behandling av graft-versus-host-disease (transplantat-mot-värd-sjukdom). CsA har ett snävt terapeutiskt fönster, vilket betyder att skillnaden mellan effektivitet och toxicitet är liten. Biverkningarna av substansen är många och en del av dem allvarliga, såsom nedsatt njurfunktion och ökad risk för utvecklande av diabetes och maligna sjukdomar som exempelvis lymfom. Den inter- och intraindividuella variabiliteten i farmakokinetik och farmakodynamik är dessutom stor. Det är därför ytterst viktigt att följa behandlingen med koncentrationsbestämningar av CsA i helblod. Det finns ett flertal olika analysmetoder för CsA tillgängliga, såsom immunoassays, vätskekromatografi (HPLC) och vätskekromatografi-tandem-massspektrometri (LC-MS/MS). Avdelningen för klinisk kemi vid Centralsjukhuset i Karlstad har sedan många år använt en radioimmunoassay, CYCLO-Trac SP®, från DiaSorin för att bestämma CsA i helblod. Laboratoriets önskan är att ersätta denna metod, vilken använder radioaktiva isotoper, med en snabbare och mer selektiv LC-MS/MS-metod.  I detta arbete har en LC-MS/MS-metod för analys av cyklosporin A i helblod utvecklats och validerats. Metoden har snabb provupparbetning och kromatografi och använder positiv elektrospray som joniseringsteknik. Två procedurer för proteinfällning utvärderades som provupparbetningsförfarande under metodutvecklingen och två olika internstandarder testades; CsA analogen cyklosporin D och isotopmärkt CsA (d12-CsA).  Efter den fullständiga valideringen infördes metoden i rutinarbetet 2009-11-01. Resultat från både LC-MS/MS och den radioimmunologiska metoden lämnas ut parallellt under minst fem månader. / Cyclosporine A (CsA) is a cyclic undecapolypeptide of fungal origin (Tolypocladium inflatum Gams). It has a molecular weight of 1202.6 Da and is used as an immunouppressive drug to prevent rejection of transplanted organs and bone marrow, and for the treatment of graft-versus-host disease. CsA exhibits a narrow therapeutic range between efficacy and toxicity. There are many side effects exerted by the drug and some of them are serious, such as renal dysfunction and increased risk of developing diabetes and malignant diseases such as lymphoma. In addition, the inter-individual and intra-individual pharmacokinetic and pharmacodynamic variability is large. Constant monitoring of the CsA-concentration is therefore mandatory.  There are several analytical methods available for the determination of CsA, such as immunoassays, liquid chromatography (HPLC) and tandem mass spectrometry (LC-MS/MS). The department of Clinical Chemistry at the Central Hospital in Karlstad has for many years used a radioimmunoassay, the CYCLO-Trac SP® from DiaSorin, for CsA-determinations. The laboratory wants to replace this method, which uses radioactive isotopes, with a faster and more selective LC-MS/MS method.  In this work a LC-MS/MS method, utilizing positive electrospray, with a fast sample preparation and chromatography for the determination of CsA in whole blood has been developed and validated. Two protein precipitation procedures were evaluated for sample preparation during the method development and two different internal standards were tested; the CsA analog cyclosporine D (CsD) and an isotope labelled CsA (d12-CsA). The LC-MS/MS assay was fully validated and implemented in the routine work at the laboratory on November 1 2009. Results from both the CYCLO-Trac SP® method and the LC-MS/MS assay will be reported for at least five months.

<p> This dissertation details original work focused on the DNA adduct N2-(1-carboxyethyl)-2'-deoxyguanosine (CEdG). This DNA adduct results from the spontaneous reaction of DNA with the endogenous and exogenous formed, carbohydrate-derived, reactive carbonyl species, methylglyoxal. Using <i>in vitro</i> steady state kinetics, we have shown that CEdG in template DNA leads to DNA miscoding effects when the model replicative polymerase, exonuclease-free Klenow fragment (KF-) is used. The development, validation and application of a novel stable isotope dilution, triple quadrupole mass spectrometric method for the quantitation of CEdG is also detailed. This method was used to quantitate CEdG in urine from diabetic rats, urine from human patients, human tumor and adjacent biopsy tissue, diabetic animal tissue and DNA treated with methylglyoxal. Finally, we detail the adaptation, validation and application of a novel, commercially-available microfluidic HPLC-chip for increased sensitivity in the quantitation of CEdG and also apply it to the quantitation of the RNA analogue, CEG. Combined, these studies establish CEdG as a potential biomarker for glycation and point to a viable avenue for connecting chronic glycolytic flux with genetic instability. </p>

<p>Over half of the biosolids produced in wastewater treatment facilities in the United States are land-applied as a nutrient-rich soil amendment. However, these biosolids are not regulated for chemicals of emerging concern that are often present at high concentrations in biosolids. The overall goal of this dissertation is to evaluate the specific chemical fates of these compounds in biosolid-amended soil, including their persistence, degradation pathways, and phytoaccumulation potential. </p><p>As a first step toward this goal, the fate of select brominated flame retardants (BFRs) when exposed to sunlight was examined to evaluate their photodegradation pathways and to identify degradation products that may be used as markers of environmental degradation in future studies. In Chapter 2, the photodegradation of three polybrominated diphenyl ether (PBDE) congeners (i.e., the nonabrominated congeners BDE 206, 207, and 208) was examined individually in three different solvents exposed to natural sunlight. Rapid degradation of nonaBDEs was observed coincident with formation of octa- and heptabrominated PBDEs. The photodegradation pathways of each nonaBDE congener were consistent among the different solvent matrices tested; however, mass balances were found to vary with solvent type. The octabrominated congener, BDE 202, and the ratio of BDE 197 to BDE 201, were identified as degradation products that can serve as environmental markers of debromination. Additional photodegradation studies were conducted with two new BFRs used in replacements for pentaBDE mixtures: 2-ethylhexyltetrabromobenzoate (TBB) and di(2-ethylhexyl)-tetrabromophthalate (TBPH). Both TBB and TBPH underwent photolysis more slowly than nonaBDEs and primarily formed debrominated products. This study is the first to report on the photodegradation of TBB and TBPH via debromination reactions and suggests that these replacement flame retardants may be more photolytically persistent than higher brominated PBDE congeners.</p><p>Chemical analysis of biosolids collected from wastewater treatment plants (WWTPs) can help determine whether these flame retardants are migrating from the indoor environment to the outdoor environment, where little is known about their ultimate fate and effects. In Chapter 3, concentrations of a suite of flame retardants and the antimicrobial compound triclosan were measured in opportunistic samples of municipal biosolids and the domestic sludge Standard Reference Material (SRM) 2781. Grab samples of biosolids were collected from two WWTPs in North Carolina and two in California. Biosolids samples were also obtained during three subsequent collection events at one of the North Carolina WWTPs to evaluate fluctuations in contaminant levels within a given facility over a period of three years. The biosolids and SRM 2781 were analyzed for PBDEs, a suite of alternate brominated and chlorinated flame retardants, and triclosan. PBDEs were detected in every sample analyzed, and &#931;PBDE concentrations ranged from 1750 to 6358 ng/g dry weight (dw). Additionally, the PBDE replacement chemicals TBB and TBPH were detected at concentrations ranging from 120 to 3749 ng/g dw and from 206 to 1631 ng/g dw, respectively. Triclosan concentrations ranged from 490 to 13,866 ng/g dw. The detection of these contaminants of emerging concern in biosolids suggests that these chemicals have the potential to migrate out of consumer products and enter the outdoor environment. Furthermore, land application of these contaminated biosolids may result in soil contamination and enhance the bioaccumulation and long-range transport potential of these compounds. </p><p>In order to fully evaluate the benefits and impacts of biosolids land application, a comprehensive view of the behaviors of chemicals of emerging concern in biosolids is needed. In Chapter 4, the fates of a suite of flame retardants and triclosan in soil were evaluated in a greenhouse experiment utilizing three biosolid amendment levels (control, low, and high) and two vegetation treatments (unplanted and planted with alfalfa (<italic>Medicago sativa</italic>)). BDE 47, BDE 209, TBB, TBPH, and triclosan declined significantly in the high biosolid-amended vegetated soil between Days 0 and 28 (p < 0.05), and then reached a plateau between Days 28 and 90 during which no further significant loss from soil was observed. In contrast, no significant losses of those analytes were observed from soil at the high biosolids amendment in non-vegetated pots. The percent of a given analyte lost from the vegetated soil at the high amendment between Day 0 and the plateau ranged from 43.1% for TBPH to 60.9% for triclosan and was significantly and negatively related to the log octanol-water partition coefficient (log K_OW) of the analyte (p = 0.0103, R<super>2</super> = 0.9178) and marginally significantly and positively related to the log of water solubility (p = 0.0686, R<super>2</super> = 0.7213). Alfalfa root and shoot tissues were monitored for the analytes of interest but no clear evidence of phytoaccumulation was observed. Methyl triclosan formation was observed in the biosolid-amended soils during the study period, indicating that biotransformation played a role in the observed dissipation of triclosan. The results of Chapter 4 demonstrate that PBDEs, selected alternate BFRs and triclosan are highly recalcitrant in biosolid-amended soils but capable of undergoing dissipation in the presence of alfalfa and, in the case of triclosan, biotransformation. </p><p>In conclusion, this dissertation provides a comprehensive view of the fates of flame retardants and triclosan in biosolid-amended soil, identifying markers of degradation that can be used in complex real-world scenarios, developing methods for measurement of a diverse suite of analytes in biosolids and plant tissues, and demonstrating the persistence of these compounds in biosolid-amended soil.</p> / Dissertation