Orientador: Marcos Nogueira Eberlin / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-20T20:29:02Z (GMT). No. of bitstreams: 1 Porcari_AndreiadeMelo_M.pdf: 3504713 bytes, checksum: 67c674763a09cf16900c073f27cd5133 (MD5) Previous issue date: 2012 / Resumo: O presente trabalho utiliza a Espectrometria de Massas para caracterização e quantificação de diferentes analitos em matrizes biológicas. Inicialmente é demonstrado o desenvolvimento de uma técnica para análise direta de triacilgliceróis (TAG) em carnes e tecidos, que podem então ser caracterizados por seu perfil lipídico. Nesta técnica, uma etapa de foto aquecimento visa extrair quase instantaneamente TAG da matriz, utilizando pouco ou nenhum solvente. O conteúdo extraído e coletado num papel pardo é então analisado por EASI-MS (Easy Ambient Sonic-spray Ionization - Mass Spectrometry), revelando o perfil de TAG em poucos segundos, sem necessidade de hidrólise, derivação ou outras extrações. Thermally-imprinted EASI-MS (T-EASI-MS) é uma técnica capaz de diferenciar tipos de carnes e seus resultados mostraram-se concordantes com a literatura e com outras técnicas tradicionais para análise de lipídios. Num segundo momento, utilizou-se a espectrometria de massas como ferramenta de quantificação, através do desenvolvimento de dois métodos analíticos para análise de cortisol em plasma e leite bovinos, utilizando LC-MS/MS (cromatografia líquida acoplada à espectrometria de massas seqüencial). Os métodos aqui desenvolvidos foram validados utilizando a metodologia de compatibilização de matriz (para o plasma) e de calibração direta em solvente (curva não extraída) para o leite. Ambos os métodos empregaram pequeno volume de amostra e forneceram baixos limites de quantificação (0,1 ng mL e 0,15 ng mL de cortisol para o plasma e leite, respectivamente). A metodologia desenvolvida foi aplicada para análise de dois experimentos veterinários. No primeiro foi investigada a correlação entre as concentrações de cortisol no plasma e no leite bovino, bem como o efeito da ordenha sobre a concentração do cortisol nesses fluídos biológicos. No segundo nível de cortisol em vacas com e sem mastite sub-clínica foi investigado e os resultados foram comparados aos resultados obtidos por ELISA (Enzime-linked Immunosorbent Assay) para as mesmas amostras / Abstract: This research uses mass spectrometry (MS) as a tool to characterize and quantify different analytes in biological matrices. At first, the development of a technique for direct analysis of triacylglycerols (TAG) in meats and animal tissues is shown. This technique allows sample characterization through its lipid profile. It starts with a photo-heating process which aims to extract, almost instantaneously, TAG from the matrix, using very little amounts of solvent. The extract is collected on a paper which is then analyzed by EASI-MS (easy ambient sonic-spray ionization), thus revealing the TAG profile in a few seconds, without the use of hydrolysis, derivatization or exhaustive extractions. Thermally-imprinted EASI-MS (T-EASI-MS) is able to differentiate kinds of meats and has been shown to be in agreement with previous reported data and results from traditional techniques used for lipid analysis of the same samples. In a second phase, this research uses MS as a tool for quantitative analyses, through the development of two analytical methods for cortisol analysis in bovine plasma and milk, using a LC-MS/MS (liquid chromatography - tandem mass spectrometry) system. These methods were fully validated using the matrix matched methodology for plasma analysis and a non-extracted calibration curve (prepared in solvent) for milk analysis. Both methodologies use small amounts of sample and achieved very low limits of quantification (0.1 ng mL and 0.15 ng mL of cortisol for plasma and milk, respectively). The methods were applied to the analysis of samples from two veterinary experiments. In the first one, the aim was to investigate the correlation between bovine plasma and milk cortisol concentrations, as well as to determine if the milking process can change basal cortisol level in these fluids. In the second experiment, the aim was to evaluate whether milk cortisol concentrations varied or not in cows with or without sub-clinical mastitis. The samples of the second experiment were also analyzed by ELISA (enzime-linked immunosorbent assay) in order to compare the results with those from LC-MS/MS / Mestrado / Quimica Analitica / Mestre em Química

Electrospray ionization mass spectrometry (ESI-MS) is a fast and sensitive technique that is ideal for detecting low concentration species of interest within complex mixtures. Because ESI-MS simply transfers charged species to the gas phase, only ions pre-formed in solution are visible. Accordingly, the charged tag, 3-(4-(bromomethyl)benzyl)-1-methylimidazolium hexafluorophosphate, was designed and synthesized to allow selective detection of phenols in petroleum fractions. Pressurized sample infusion (PSI) was optimized and used for time dependent analyses. PSI ESI-MS was applied to measure O-alkylation of the phenol species leading to rate information about the overall reaction along with dynamic information about reaction progress. The relative intensity of the charged tag was used to determine semi-quantitatively the presence of phenols in different petroleum fractions. Other derivatization methods in petroleomics were also explored. 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide methiodide (EDT) derivatization followed by PSI ESI-MS analysis was applied for the selective measurement and identification of naphthenic acids in petroleum fractions. The reactions of standard naphthenic acids and EDT were studied by PSI ESI-MS to assess the efficiency of EDT and the rate of reactions. The same petroleum fractions were analysed by cold Electron ionization (Cold EI) gas chromatography-mass spectrometry (GC/MS) and classical EI GC/MS. The combination spectra from the subtraction from cold EI spectra to classical EI spectra provide us a new dimension to cold EI analysis of complex matrices. Meanwhile, a Python program was written to rapidly screen cold EI GC/MS data for routine tasks, such as retention time comparison on different instrument parameters for single petroleum sample and spectrum comparison on the same retention time for different petroleum samples. / Graduate

Cell division is a fundamental biological process that occurs in all kingdoms of life. Our understanding of cell division in bacteria stems from studies in the rod-shaped model organisms: Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. The molecular underpinnings of cell division regulation in non-rod-shaped bacteria remain to be studied in detail. Rod-shaped bacteria possess many positive and negative regulatory proteins that are essential to the proper placement of the division septa and ultimately the production of two identical daughter cells, many of which are absent in cocci. Given that essential cell division proteins are attractive antibacterial drug targets, it is imperative for us to identify key cell division factors especially in pathogens, to help counter the emergence of multi-drug resistance. In Staphylococcus aureus, a spherical Gram-positive opportunistic pathogen that causes a range of diseases from minor skin infections to life-threatening sepsis, we have identified the role of an essential protein, GpsB, in the regulation of cell division. We discovered that GpsB preferentially localizes to cell division sites and that overproduction of GpsB results in cell enlargement typical of FtsZ inhibition, while depletion of GpsB results in cell lysis and nucleoid-less minicell formation. The identification of GpsB’s interaction partners will allow us to understand the molecular mechanism by which GpsB regulates cell division.

Cyanobacteria are notorious for their potential to produce toxins with human health effects, particularly the hepatotoxic microcystins (MCs), but cyanobacteria also produce other bioactive compounds. A wide variety of oligopeptides including aeruginosins, cyanopeptolins and cyanobactins may be as toxic as MCs. To investigate the production of these compounds, an UPLC QTOF-MS/MS method was developed to compare the metabolomic profiles of various strains of a common bloom-forming and toxigenic species, Microcystis aeruginosa, as well as those obtained from lakes with mixed cyanobacterial assemblages. Although many compounds could not be confirmed, MCs were rarely the dominant secondary metabolite in any sample. Since the biological role of MCs remains unknown, I tested the hypothesis that MCs provide protection against oxidative stress as induced through exposure to the herbicide atrazine and UV radiation in pure cultures of toxic vs non-toxic strains. Results were inconclusive and varied between strains suggesting other mechanisms exist to counter oxidative stress.

Increasing evidence has shown that abnormal metabolic phenotypes in body fluids reflect the pathogenesis and pathophysiology of Parkinson's disease (PD). However, the relationship between metabolic phenotypes and PD is not fully understood. Mass spectrometry (MS) based metabolomics is a powerful technique, which was frequently used for the sensitive and reproducible detection of hundreds to thousands of metabolites in biofluid samples.. Here we developed and performed MS-based metabolomics studies involving hundreds of human urine samples with data acquired from multiple analytical batches for surveying potential biomarkers of PD. A new software statTarget was developed and introduced. Protocols for liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) were developed, including sample preparation, data acquisition, quality controls, quality assurance and data analysis. Urinary metabolites from a total of 401 clinical urine samples collected from 106 idiopathic PD patients and 104 normal control subjects were profiled by using LC-MS. Quality control (QC) strategy has been performed in MS-based metabolomics for high reproducibility and accuracy of MS data. GC-MS with methyl chloroformate (MCF) derivatization was used for profiling highly polar metabolites in patients with early-, middle- and advanced-stage PD. Our study revealed the significant correlation between clinical phenotypes and urinary metabolite profiles. Comprehensive metabolomics was successfully developed with the goal of identifying urinary metabolite markers that can be used for evaluating the development of PD. A group of 18 metabolites have shown not only a high discriminating ability for the early-stage PD patients but also accurately distinguished the middle- and advanced- stages patients from control subjects. For the evaluation of PD, 18 metabolites showed good potential as metabolite markers with related metabolic pathway variations observed in branched chain amino acid metabolism, glycine derivation, steroid hormone biosynthesis, tryptophan metabolism, and phenylalanine metabolism.. We have further performed targeted analysis of potential biomarkers by using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and GC-MS. The UPLC-MS/MS method was developed and optimized for detecting the concentration variation of metabolites in tryptophan metabolism for alpha-synuclein over-expressed flies (Parkinson's disease model). The altered tryptophan metabolism was proved as one of the common metabolite signatures between PD patients and alpha-synuclein over-expressed fly model of PD, and thus may be used for developing potential markers of the disease and evaluating the efficacy of novel therapeutic agents. An asymmetric labeling strategy and positive chemical ionization gas chromatography-tandem mass spectrometry (PCI-GC-MS-MS) approach was developed for the determination of non-amino organic acids and amino acids, as well as short chain fatty acids. Carboxylic and amino groups could be selectively labelled by propyl and ethyl groups, respectively. The specific neutral losses of C3H8O (60 Da), C3H5O2 (74 Da) and C4H8O2 (88 Da) were useful in the selective identification for qualitative analysis of organic acids and amino acid derivatives. The developed PCI-GC-MS/MS method showed good reproducibility and linear range.. In summary, metabolomics study has its inherent advantage in the characterization of biomarkers for the development of PD and may bring new scientific knowledge as well as impact on the progression of PD and other related neurodegenerative diseases.

Abstract Sensitive chromatographic methods were developed for the quantitative analysis of secondary metabolites in Hypericum perforatum (St. John's wort) and Rhodiola rosea (Golden root, rose root) extracts. Sample preparation methods were developed for plant, cell culture and biotransformation suspension matrixes. High performance liquid chromatography (HPLC) was used for the separation of analytes, and chromatographic data was acquired using photodiode array (PDA) detection or atmospheric pressure ionization mass spectrometry (API-MS). Ionization efficiencies with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were compared under different conditions. Specific mass spectrometric detection methods such as multiple reaction monitoring (MRM) and selective ion monitoring (SIM) were utilized. For identification of known and new secondary metabolites in plant tissues, mass spectrometric methods with triple quadrupole and time-of-flight mass spectrometers were used together with one- and two-dimensional nuclear magnetic resonance spectroscopy (NMR).

Abstract This thesis is focused on the hydrolysis of aluminium, the polymerisation of the hydrolysis products, and how these can be monitored by mass spectrometric methods. The main aim of this research is to figure out how the aqueous speciation of aluminium changes as a function of pH (3.2–10), concentration (1–100 mM), reaction time (1s–14d), and counter anion (Cl-, SO42-, HCOO-). The method used was electrospray mass spectrometry. The results showed more variable speciation than those suggested earlier. The main species were Al2, Al3, and Al13, which were found in all of the conditions under scrutiny. The effect of pH was the most remarkable of all the parameters researched. The formation of large highly charged complexes was strongly dependent on it. Also the Al-concentration in the bulk solution had a clear effect on speciation: in dilute solutions there were more protonated ligands and less attached counter anions. This could mean that the species in more diluted bulk solutions had fewer different states of charge. Reaction time caused only minor changes to speciation in the initial pH: there was slightly more variation of a certain sized species in the aged solution. In elevated pH, the birth of important Al13 oligomers was time dependent. The effect of the counter anion was tremendous. In a chloride environment the speciation was rich and diversified. With sulphate the speciation was limited to solid- like compounds, and the variation of single-sized species was almost lacking. The formate as a counter anion caused most surprising results; the charge of aluminium in some studied complexes was lowered from the common 3+ to 1+. If this reaction also occurs in natural circumstances, the uses of aluminium formate would be wide. The results can be utilised in following the progress of dissolution, the mobilization and toxicity of aluminium in natural waters, as well as in water purification, and in reaching minimal chemical contamination levels in sludge as well as in aqueous waste.

Electrospray ionization (ESI) facilitates the transfer of ions in solution into the gas-phase for analysis by mass spectrometry. The ionization process is intricate and required further investigation, especially because of the lack of in-depth literature on the subject. Furthermore, investigations into the ESI process will serve to assist development of real-time reaction monitoring. To do this, a cationic ionic liquid, butyl methylimidazolium, [BMIM]+, was paired with several counterions and mixed in various solvents. This was analyzed by ESI mass spectrometry to determine the relative response ratio between two observable aggregates. The findings assisted in the elucidation of differential surface activity of chemically distinct ions in ESI, with respect to changes in solvent. Furthermore, the results obtained suggested acetonitrile is an optimal solvent for the analysis of ions of this type due to a reduction in differential effects, whereas other common ESI solvents prove to enhance the surface activity of specific aggregate ions. Further investigations into ESI-MS involved effects of spray head geometry relative to the inlet to the mass spectrometer. The position of the spray-head, the solvent, and additional instrumental parameters were independently adjusted during the analysis of an equimolar mixture of two different ions. It was found that these parameters have dramatic effects on the distribution of signal intensity from one ion to another, and therefore the resulting usefulness of acquired spectra. The sharp contrast in ion intensity, and even differential ion activity, with relatively minor instrument changes (such as temperature programming, gas flow rates and solvent choice) demonstrated the importance of finding the optimal spot for the ESI spray head, especially when signal intensity and a quality analysis is key. Additional ESI-MS work involved working with an industry partner to develop selective charge-tagging reagents for the characterization of petroleum fractions by ESI-MS. A simple chemical derivatization technique was developed in which thiols and disulfides may be selectively analyzed in a complex matrix and easily characterized. These reagents enhanced detection of thiols and disulfides solely due to the nature of the charged tag derivatization agent. The charged disulfides readily and exclusively react with thiols in a complex matrix in a short amount of time. The synthesis of these reagents was simple and resulted in a pure and stable reagent. The efficacy of the reaction was demonstrated using on-line monitoring, while the scope and usefulness of the reaction was demonstrated in petroleum fractions. A combination of UV-Vis spectroscopy and electrospray ionization mass spectrometry was used for real-time monitoring of Pd2(dba)3 activation with sulfonated versions of PPh3 and a Buchwald-type ligand. This provides insight into the effect of ligand and preparation conditions on activation and allows for establishment of rational activation protocols. It is expected that this reaction monitoring technique will be enhanced through the use of tandem mass spectrometry. Finally, an experimental method of visualizing atomic orbitals was developed as a demonstration intended for first year chemistry students. This demonstration involved the examination of nodal and anti-nodal regions of Chladni figures which students could then connect to the concept of quantum mechanical parameters and their relationship to atomic orbital shape. / Graduate

M.Sc. / The understanding of the classification and origin of geological systems is facilitated by the acquisition of accurate and precise analytical data. New instrumentation is rapidly developed for the preparation and quantitative analysis of geological materials using smaller amounts of sample, with lower limits of detection and faster analysis times. The development of new methodologies is crucial for the effective utilisation of new instrumentation. This study was conceptualised because of the relationship between the Department of Geology and the Central Analytical Facility of the Faculty of Science, University of Johannesburg. There is a high demand for accurate and precise chemical data for various lithologies and the availability of high-end analytical equipment, but little practical expertise to utilise this equipment to its full potential. The study is centred round the analysis of four common sedimentary lithological groups that are routinely studied by the Department of Geology namely carbonate rocks, shales, iron ore and manganese ore. A large literature base exists for the decomposition and analysis of geological materials. The bulk of this literature is centred round more established and conventional methods of sample preparation and analysis. The use of microwave digestion instrumentation and methodologies in recent times has revolutionised sample decomposition with shorter decomposition times, smaller sample masses required for decomposition, lower loss of analytes to volatilisation and creating a safer laboratory environment for analysts to work in. Inductively coupled plasma optical emission spectroscopy (ICP-OES) is a more mature method of sample analysis, being commercialised in the mid 1970’s, while inductively coupled plasma mass spectroscopy (ICP-MS) is a newer technique, v being commercialised as of the mid 1980’s. These two techniques are multi-element techniques, with low limits of detection and fast analysis times for a plethora of analytes, over seventy elements for ICP-OES and over eighty elements for ICP-MS. Samples from all four lithological groups were successfully digested in a microwave digestion unit with varying combinations of nitric acid (HNO3), hydrochloric acid (HCl), perchloric acid (HClO4) and hydrofluoric acid (HF) and various digestion programs. Accurate and repeatable methods of analysis were developed for iron, manganese, calcium and potassium for all four lithologies with ICP-OES; aluminium was successfully determined for shale, iron and manganese ore with ICP-OES. Titanium, sodium, arsenic, barium, bismuth, chromium, copper, molybdenum, scandium, strontium and vanadium were determined for all four lithotypes with acceptable accuracy and repeatability using ICP-MS.

M.Tech. / The Inductively Coupled Plasma Mass Spectrometry (ICP-MS) technique showed very high uncertainties associated with the determination of the concentrations of minor elements in the Platinum Group Metal (PGM) smelting plant samples. This project reports on the work done for the identification of, and subsequent minimisation of the sources of uncertainties associated with the measurements of minor elements in the PGM Furnace Matte material by ICP-MS. To perform these studies, Elan 6000, Shimadzu ICPM-8500 and Finnigan Element 2 ICP-M spectrometers were employed. Synthetic Furnace Matte samples (SFMSs) were prepared and used to ascertain the uncertainties associated with the alleged sources at Mintek and Leo-Tech laboratories. The Element 2 spectrometer dominated the other two ICP-M spectrometers in terms of the accuracy for the determination of the concentrations of minor elements in SFMSs. The evidence of spectral interferences from the significant deviations in the measurement results between the isotopes of the same element was observed in the quantification of Zn, Se, Te and Sn in SFMS by the quadrupole Elan 6000 and the Shimadzu spectrometers. It also transpired that the accuracy of the quantitative determination of minor elements in the Furnace Matte (FM) matrix by ICP-MS was hampered by the matrix elements with the severity depending on the specific analyte and the make and model of the ICP-M spectrometer. The Anglo platinum FM material that was analysed in the second round robin was used as a Certified Reference Material (CRM) in the analysis of the Lonmin FM sample. It was revealed that the laboratory standard operating procedures for the preparation, dilution and subsequent analysis of the sample are potential sources of uncertainty in measurement results. The two-fold dilution of the sample for the lessening of the matrix effects was not effective. The use of multi-walled nanotubes for the alleviation of the matrix effects by removal was also not successful. Nevertheless standard addition method (SAM), combined with internal standardisation can be used as an effective calibration method in ICP-MS to achieve less matrix interfered results over the combination of the common external standardisation and internal standardisation methods.