One of the main goals in structural biology is to understand the folding mechanisms and three-dimensional structure of biomolecules. Many biomolecular systems adopt multiple structures as a function of their microenvironment, which makes them difficult to be characterized by traditional structural biology tools (e.g., NMR, X-ray crystallography). As an alternative, complementary tools that can capture and sample multiple conformations needed to be developed. In the present work, we pioneered the application of a new variant of ion mobility spectrometry, trapped ion mobility spectrometry (TIMS), which provides high mobility resolving power and the possibility to study kinetically trapped intermediates as a function of the starting solution (e.g., pH and organic content) and gas-phase conditions (e.g., collisional activation, molecular dopants, hydrogen/deuterium back-exchange). When coupled to mass spectrometry (TIMS-MS), action spectroscopy (IRMPD), molecular dynamics and biochemical approaches (e.g., fluorescence lifetime spectroscopy), a comprehensive description of the biomolecules dynamics and tridimensional structural can be obtained. These new set of tools were applied for the first time to the study of Flavin Adenine Dinucleotide (FAD), Nicotineamide Adenine Dinucleotide (NAD), globular protein cytochrome c (cyt c), the 31 knot YibK protein, 52 knot ubiquitin C terminal hydrolase (UCH) protein, and the 61 knot halo acid dehydrogenase (DehI) protein.

The complexity of biological matrices makes the detection and quantification of compounds of interest challenging. For successful targeted or untargeted identification of compounds within a biological environment, the use of complementary separation techniques is routinely required; in many situations, a single analytical technique is not sufficient. In the present dissertation, a multidimensional analytical technique was developed and evaluated, a combination of new sample preparation/extraction protocols, liquid chromatography, trapped ion mobility and mass spectrometry (e.g., LC-TIMS-MS and LC-TIMS-MS/MS). The performance of these techniques was evaluated for the detection of polybrominated diphenyl ethers metabolites, polychlorinated biphenyls metabolites in human plasma, opioid metabolites in human urine, and lipids in Dictyostelium discoideum cells. The new workflows and methods described in the body of this dissertation allows for rapid, selective, sensitive and high-resolution detection of biomarkers in biological matrices with increased confidence, sensitivity and shorter sample preparation and analysis time.

<p>The objective of this master thesis was to develop an analytical method for the quantification of the cancer drug Imatinib and its main metabolite CGP74588 in plasma. Imatinib is used in the treatment of chronic myeloid leukemia and gastrointestinal stroma tumors. A quantitative analytical method was developed where reversed-phase columns with different stationary phases were studied and the sensitivity was tested with both UV detectors and a mass spectrometric detection. Since the substances were measured in plasma a solid-phase extraction was developed to purify the samples before analysis. The column chosen for the separation was the Max-RP C12 column (100 x 3 mm, 4 μm particle size) manufactured by Phenomenex with a gradient mobile phase with 1% formic acid in methanol and water. The gradient was as follows; 0 min 15:85, 7 min 60:40, 9 min 60:40 with a total runtime of 13.5 min. The internal standard chosen was Opipramol. Mass spectrometric detection using a sonic spray ionization interface in positive mode proved to be about as sensitive as UV detection at 261 nm. The generated (M+H+)+ ions were isolated and fragmented with the use of three mass spectrometric methods; one for Imatinib (transition 494 —› 394), one for CGP74588 (transition 480 —› 394) and one for Opipramol (transition 364 —› 171). For the purification of the plasma samples an Oasis HLB solid-phase extraction cartridge was selected and the recoveries were close to 100%.</p><p>The developed method was partially validated and showed coefficients of variation (CV) for intra-and inter-day precision between 0.4 and 5.4% with UV detection. The validation results for the mass spectrometer were inconclusive.</p>

<p>Gas chromatography (GC) has been utilized for the study of enantiomer resolution of the atropisomers of PCBs, o,p´-DDD and o,p´-DDT. Different substituents and concentrations of cyclodextrin, capillary dimensions and type of stationary phase films have been investigated to achieve the resolution of as many of the atropisomers on one column as possible. The results indicated that the butyl substitution of 6-hydroxyl and the methyl substitution of 2- and 3-hydroxyl were the most promising for the enantiomeric separation. Using Capillary Electrophoresis (CE), the trimers and monomers of PM-β-CDs were compared for enantiomeric resolution, as well as comparing the cationic PMMA-β-CD with the anionic HS-β-CD. In these studies the trimer did not show an improved resolution for mepivacaine, when compared to the equimolar concentration of the monomer. The cationic CD gave increased resolution values for ibuprofen when compared to the anionic CD. A scheme for reversing enantiomeric elution order of both the basic propranolol and acidic ibuprofen is also presented, with the aim of facilitating the detection of impurities in a high sample loading. The detection of 1% of each enantiomer of propranolol, and 1% of R(-)-ibuprofen, was demonstrated, with elution prior to the tailing peak of the corresponding enantiomer. Dimethylacrylamide-coated capillaries were used in this work, and the stability of this coating was demonstrated, giving a highly reduced electroosmotic flow for up to six months. Enantiomeric baseline separations of omeprazole and 5-hydroxyomeprazole have also been achieved with both CE and Open Tubular Capillary Electrochromatography (OT-CEC) methods. With CE-UV, both a non-aqueous method (using HDMS-β-CD) and an aqueous method (using HS-β-CD) were used for enantiomeric resolution of the two racemates. Resolution of omeprazole was also achieved using CE-Electrospray Ionization-Mass Spectrometry (ESI-MS). In OT-CEC, avidin was immobilized on the inside surface of a fused silica capillary and was employed as chiral selector for the enantiomeric baseline resolution of omeprazole and 5-hydroxyomeprazole.</p>

<p>This thesis ties some electrochemical aspects of development and fabrication of an analytical system on a microchip together. These aspects develop through the fundamentals of amperometric detection in microsystems and microfabrication via the interaction of electrochemical detection and electrophoretic separation finally to the interfacing of a microsystem to the macro world.</p><p>Paper <b>I</b> deals with amperometric detection in microscale systems and describes the fabrication of the necessary on-chip microelectrodes together with fluidic channels in silicon. It was furthermore studied, if the interelectrode distance of some μm could be used to improve the sensitivity in amperometric detection by employing redox cycling. </p><p>Papers <b>II</b>, <b>III</b> and <b>IV</b> deal with the effect of a high voltage field on amperometric detection. In analytical microdevices typically an electrophoretic separation step (e.g. capillary electrophoresis, CE) precedes the detection. The interference of the CE high voltage with the amperometric detection potential is often considered one of the main hindrances for an effective combination of these techniques. In paper <b>II</b> one reason for the observed disturbing potential shift was elucidated. It was shown that positioning of working electrode and reference electrode on an equipotiental surface eliminates this problem. Paper <b>III</b> reports an application of this technique. In paper <b>IV</b> it could be shown that this approach could further be used to significantly reduce the instrumental requirements for amperometric detection in CE.</p><p>Papers <b>V</b>, <b>VI</b>, <b>VII</b>, finally discuss the interfacing of low volumetric flows that typically occur on microanalytical devices to other techniques. Both, interfacing from liquid to liquid phase (μLC to CE in paper <b>V</b>) and from liquid to gas phase (CE to MS in paper <b>VI</b> and <b>VII</b>) were discussed. Electrochemical methods are used in this context to evaluate the stability and, in paper <b>VI</b> and <b>VII</b>, to increase the understanding of underlying processes of corrosion.</p>

The objective of this master thesis was to develop an analytical method for the quantification of the cancer drug Imatinib and its main metabolite CGP74588 in plasma. Imatinib is used in the treatment of chronic myeloid leukemia and gastrointestinal stroma tumors. A quantitative analytical method was developed where reversed-phase columns with different stationary phases were studied and the sensitivity was tested with both UV detectors and a mass spectrometric detection. Since the substances were measured in plasma a solid-phase extraction was developed to purify the samples before analysis. The column chosen for the separation was the Max-RP C12 column (100 x 3 mm, 4 μm particle size) manufactured by Phenomenex with a gradient mobile phase with 1% formic acid in methanol and water. The gradient was as follows; 0 min 15:85, 7 min 60:40, 9 min 60:40 with a total runtime of 13.5 min. The internal standard chosen was Opipramol. Mass spectrometric detection using a sonic spray ionization interface in positive mode proved to be about as sensitive as UV detection at 261 nm. The generated (M+H+)+ ions were isolated and fragmented with the use of three mass spectrometric methods; one for Imatinib (transition 494 —› 394), one for CGP74588 (transition 480 —› 394) and one for Opipramol (transition 364 —› 171). For the purification of the plasma samples an Oasis HLB solid-phase extraction cartridge was selected and the recoveries were close to 100%. The developed method was partially validated and showed coefficients of variation (CV) for intra-and inter-day precision between 0.4 and 5.4% with UV detection. The validation results for the mass spectrometer were inconclusive.

Gas chromatography (GC) has been utilized for the study of enantiomer resolution of the atropisomers of PCBs, o,p´-DDD and o,p´-DDT. Different substituents and concentrations of cyclodextrin, capillary dimensions and type of stationary phase films have been investigated to achieve the resolution of as many of the atropisomers on one column as possible. The results indicated that the butyl substitution of 6-hydroxyl and the methyl substitution of 2- and 3-hydroxyl were the most promising for the enantiomeric separation. Using Capillary Electrophoresis (CE), the trimers and monomers of PM-β-CDs were compared for enantiomeric resolution, as well as comparing the cationic PMMA-β-CD with the anionic HS-β-CD. In these studies the trimer did not show an improved resolution for mepivacaine, when compared to the equimolar concentration of the monomer. The cationic CD gave increased resolution values for ibuprofen when compared to the anionic CD. A scheme for reversing enantiomeric elution order of both the basic propranolol and acidic ibuprofen is also presented, with the aim of facilitating the detection of impurities in a high sample loading. The detection of 1% of each enantiomer of propranolol, and 1% of R(-)-ibuprofen, was demonstrated, with elution prior to the tailing peak of the corresponding enantiomer. Dimethylacrylamide-coated capillaries were used in this work, and the stability of this coating was demonstrated, giving a highly reduced electroosmotic flow for up to six months. Enantiomeric baseline separations of omeprazole and 5-hydroxyomeprazole have also been achieved with both CE and Open Tubular Capillary Electrochromatography (OT-CEC) methods. With CE-UV, both a non-aqueous method (using HDMS-β-CD) and an aqueous method (using HS-β-CD) were used for enantiomeric resolution of the two racemates. Resolution of omeprazole was also achieved using CE-Electrospray Ionization-Mass Spectrometry (ESI-MS). In OT-CEC, avidin was immobilized on the inside surface of a fused silica capillary and was employed as chiral selector for the enantiomeric baseline resolution of omeprazole and 5-hydroxyomeprazole.

This thesis ties some electrochemical aspects of development and fabrication of an analytical system on a microchip together. These aspects develop through the fundamentals of amperometric detection in microsystems and microfabrication via the interaction of electrochemical detection and electrophoretic separation finally to the interfacing of a microsystem to the macro world. Paper I deals with amperometric detection in microscale systems and describes the fabrication of the necessary on-chip microelectrodes together with fluidic channels in silicon. It was furthermore studied, if the interelectrode distance of some μm could be used to improve the sensitivity in amperometric detection by employing redox cycling. Papers II, III and IV deal with the effect of a high voltage field on amperometric detection. In analytical microdevices typically an electrophoretic separation step (e.g. capillary electrophoresis, CE) precedes the detection. The interference of the CE high voltage with the amperometric detection potential is often considered one of the main hindrances for an effective combination of these techniques. In paper II one reason for the observed disturbing potential shift was elucidated. It was shown that positioning of working electrode and reference electrode on an equipotiental surface eliminates this problem. Paper III reports an application of this technique. In paper IV it could be shown that this approach could further be used to significantly reduce the instrumental requirements for amperometric detection in CE. Papers V, VI, VII, finally discuss the interfacing of low volumetric flows that typically occur on microanalytical devices to other techniques. Both, interfacing from liquid to liquid phase (μLC to CE in paper V) and from liquid to gas phase (CE to MS in paper VI and VII) were discussed. Electrochemical methods are used in this context to evaluate the stability and, in paper VI and VII, to increase the understanding of underlying processes of corrosion.

O presente trabalho versa sobre a modificação de eletrodos de carbono vítreo por óxidos de molibdênio e estudos referentes à catálise de redução de oxiânions, bem como o emprego do sensor assim preparado para análise de bromato e iodato em amostras sintéticas e naturais. Estudos executados em solução contendo Mo (VI) na faixa de pH de 1,8 a 3,0 indicaram que em meios mais ácidos a resposta catalítica é muito maior, envolvendo a participação de prótons na abstração de oxigênio ao reduzir o substrato. Os resultados dos experimentos voltamétricos realizados com eletrodo de carbono vítreo modificado demonstraram que iodeto é gerado na eletroredução de iodato. O processo de modificação do eletrodo consistiu na execução de experimentos voltamétricos sucessivos com o eletrodo de carbono vítreo em solução contendo Mo (VI), em pH ajustado para otimização da formação do filme e minimização das perdas de depósito por solubilização ácida. O material imobilizado assim obtido foi analisado por técnicas espectrais e microscópicas, observando-se a existência de centros preferenciais de formação de depósito (grumos) ao qual foi atribuída a fórmula mínima de MoO2,78. O óxido de valência mista mostrou grande potencialidade de catálise na redução de oxiânions oxidantes, observando-se antecipação do potencial de redução do iodato, acompanhada de grande intensificação no sinal de corrente. Trabalhando-se em condições de convecção forçada regida pela rotação do eletrodo de trabalho, foram observadas anomalias nos sinais de corrente durante o decorrer dos experimentos eletroquímicos. Explicações para tal fato foram associadas à formação de diferentes fases de óxidos de molibdênio com atividades catalíticas distintas. O sensor desenvolvido para determinação de iodato foi utilizado em sistema em fluxo após otimização de parâmetros como pH do transportador e da amostra, bem como condições relacionadas à modificação do eletrodo (espessura do filme). Amostras de sal de cozinha foram analisadas pelo método proposto e os resultados obtidos foram concordantes com aqueles oriundos do emprego de metodologia padrão. / The electroreduction of both iodate and bromate at glassy carbon discs modified by molybdenum oxide films in sulfuric acid medium is discussed. Studies carried out at the 1.8 - 3.0 pH range with Mo(VI) in solution show that at more acidic conditions the catalytic response is much larger since protons participate in the oxygen abstraction step when the substrate is reduced. The results of voltammetric experiments carried out with a glassy carbon electrode coated by the molybdenum oxide layer established that iodide is generated in the electroreduction of iodate. The electrochemical modification of the glassy carbon electrode was carried out by cycling consecutively the potential in a solution containing Mo(VI) at a convenient pH. At the optimal condition an adherent film is obtained and the acidic solubilisation is minimised. The immobilised material was analysed by spectral techniques and scanning electronics microscopy, indicating the existence of preferential centres of deposition of material whose formula was calculated to be MoO2,78. The electroreduction of the analyte occurs at a glassy carbon disc modified electrode, the film acting as an efficient electrocatalyst for the mass transport controlled reduction. Rotating disc experiments performed in solutions containing both Mo(VI) and iodate at pH 1.8 confirmed that an immobilised form of molybdenum is responsible for the reduction of the substrate, even though at more acidic solutions the film is partially solubilised. The response from chronoamperometric experiments shows unexpected results explained by the existence of different catalytic species in equilibrium as two distinct phases with different catalytical activity. A method for the amperometric determination of iodate with the modified electrode in a flow injection configuration was described and the sensor was applied to the determination of iodate in commercial salt samples in a wall-jet cell coupled to a flow injection apparatus after convenient desoxygenation of the carrier solution.

Please read the abstract in the section 00front of this document / Thesis (PhD (Chemistry))--University of Pretoria, 2007. / Chemistry / unrestricted