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Development and applications of ambient mass spectrometry techniques
Rejšek, Jan ; Cvačka, Josef (advisor) ; Lemr, Karel (referee) ; Klejdus, Bořivoj (referee)
(EN) Ambient mass spectrometry defines the versatile group of methods providing analysis of solid sample surfaces and liquids in an open atmospheric pressure environment, where the sample is simultaneously accessible to another treatment. Ambient mass spectrometry is a sharply developing research area in the analytical chemistry. It provides fast, direct analysis of objects without any sample pretreatment with the use of the mass spectrometer. Desorption electrospray ionization (DESI) and desorption atmospheric pressure photoionization (DAPPI) equipped with software control of the sample holder were investigated in this doctoral thesis. These methods use a spray of solvents for desorption and ionization molecules from solid substrate and they are suitable tools for mass spectrometry imaging (MSI) of low molecular organic compounds, where the chemical identity of molecules present on a surface is examined as a function of spatial distribution. This project deals with applications and instrumental development. As for the applications, the position of the defense glands on insect bodies, separation of the lipids in complex mixtures on thin-layer chromatography (TLC) plates, or steroid metabolites in woman urine during pregnancy were thus investigated. As for the instrumental development, the most...
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Applications of monolithic columns in capillary liquid chromatography
Holdšvendová, Petra ; Coufal, Pavel (advisor) ; Pacáková, Věra (referee) ; Cvačka, Josef (referee)
INTRODUCTION From the nineties of the twent5r's century [l], when appeared the first references about the new type ofthe stationary phases designated for HPLC called monoliths, these became to be the object of interest of several scientific groups [2-4]. Their uncontested advantage in comparison with classical columns is their high stability in pH range from 2 to 12 where there is no possibility to use the classical columns. other advantages are their easy and cheap preparation and the absence of frits on the ends of the separating medium because the monolith is covalently bound to the inner side of the capillary [5]. On the other hand, their disadvantage remains to be worse in reproducibility of the stationary phase preparation than in case of classical columns. This PhD Thesis is focused on monoliths based on organic polymers, mainly the methacrylate monoliths. For the first time, this ýpe of monoliths was shown by Peters et al. [6] and since then there have been made several monolithic phases based on different separating features 17_101. Methacrylate monoliths are convenient for separation of the low molecular substances L7l as well as the high molecular substances like the proteins, oligonucleotides or saccharides n0-121.
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Structural analysis of natural compounds with aliphatic branched chains using mass spectrometry
Strmeň, Timotej ; Cvačka, Josef (advisor) ; Kozlík, Petr (referee)
The diploma thesis focuses on analysis of fatty alcohols with mass spectrometry. Theoretical part describes distribution, main properties, usage and analytical methods for analysis of fatty alcohols. Experimental work focuses on the search for a proper derivatisation procedure for fatty alcohols, which would enable their detection with soft ionisation techniques of mass spectrometry, as well as their structural analysis. The main aim of the structural analysis is to find the methyl branching in the fragmentation spectra of derivatives of the fatty alcohols.
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Analysis of diol diesters in vernix caseosa using HPLC/APCI-MS2
Šubčíková, Lenka ; Cvačka, Josef (advisor) ; Bosáková, Zuzana (referee)
Vernix caseosa is a natural biofilm which covers the skin of a human fetus from the third trimester of pregnancy. It has hydrating, regenerating and anti-microbial effects. The components responsible for these properties of vernix caseosa could be used in the pharmaceutical and cosmetic industries. For this reason, the total composition is analyzed. The lipid components of vernix caseosa consist of squalene, wax esters, sterol esters, 1,2-diol diesters, triacylglycerides, free fatty acids, fatty alcohols, cholesterol, diacylglycerides, monoacylglyceridesů and phospholipids . This study is focused on structure analysis of the 1,2-diol diesters of vernix caseosa. Conditions were optimized for the HPLC separation. Nova -Pak C18 column was used and a gradient of acetonitrile:ethyl acetate was chosen as a mobile phase. Before entering the APCI source ammonium formate was added; ammonium adducts [M +18]+ were formed. Eight scan events was set in MS method. One for the full scan spectrum, second for MS2 spectrum of the precursor ions and six MS3 data-dependent spectra. The position of the double bonds of unsaturated 1,2-diol diesters was determined by fragmentation of their [M+C3H5N]+ adducts. There were identified over 2200 of 1,2-diol diesters differing in lengths of alcohol and fatty acid chains, and...
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Use of the modern separation techniques for the analysis of insect pheromones
Žáček, Petr ; Jelínek, Ivan (advisor) ; Pacáková, Věra (referee) ; Cvačka, Josef (referee)
(EN) Communication mediated by chemical compounds (semiochemicals) is the most important way of information transfer in insects, especially in the social species. Gas chromatographic methods (one-dimensional, two-dimensional comprehensive, and preparative arrangement) coupled with mass spectrometric and/or electroantennographic detector were used for volatile or semivolatile semiochemicals analysis in various insect species. In this Thesis, biosynthesis of the bumblebee male sex pheromone in species Bombus terrestris, B. lucorum, and B. lapidarius was studied using putative biosynthetic precursors (sodium acetate, fatty acids) labeled with 2 H and 14 C. For the purpose of labeled volatile metabolites analysis, a method of separation and detection of isotopically labeled compounds in two-dimensional comprehensive gas chromatography was studied (2 H, 13 C). An "inverse isotope effects" was confirmed for compounds labeled with both isotopes in all examined types of columns. Concerning the biological samples, analysis of in vitro incubated tissues with labeled sodium acetate showed that pheromone components are synthesized de novo in the labial gland in B. terrestris and B. lucorum. Nevertheless, experiments of in vivo incubation of deuterated fatty acids confirmed biotransformation of the precursors...
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Analysis of reaction products of phthalaldehyde with selected amino acids
Křížová, Věra ; Čabala, Radomír (advisor) ; Cvačka, Josef (referee)
This thesis focuses on analysis of reaction products of phthalaldehyde with selected amino acids using the combination of high-performance liquid chromatography with mass spectrometry. Only one product with dihydroisoindole structure is formed in the case of simple amino acids (glycine, glycine ethyl ester, alanine, α-aminobutyric acid, valine, leucine and isoleucine). Reactions of phthalaldehyde with amino acids with two amino groups (lysine, asparagine, glutamine and arginine) yield different types of compounds. Main products are formed by the interaction of both of the amino groups with one molecule of phthalaldehyde. Apart from this type of product, these reactions also result in formation of the expected analogous structures of dihydroisoindoles. Moreover, the formation of products containing one molecule of amino acid and two molecules of phthalaldehyde is not excluded. The products were structurally analysed by the use of high-performance liquid chromatography and tandem mass spectrometry. Reaction products of phthalaldehyde with α-aminobutyric acid, valine, leucine, isoleucine and lysine were isolated and further analyses such as electron ionisation with double focusing sector analyser and nuclear magnetic resonance were performed. Pieces of information resulting from the analysis of...
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Lipidomic analysis of vernix caseosa
Harazim, Eva ; Cvačka, Josef (advisor) ; Lísa, Miroslav (referee) ; Kuda, Ondřej (referee)
Methods of analytical chemistry are widely used in lipidomics. Separation techniques coupled to mass spectrometry or nuclear magnetic resonance are used very often. They make it possible to identify lipids present in the matrix in very small quantities. This work summarizes the application of modern analytical methods and instrumentation for identifying and characterizing lipids in vernix caseosa. It is shown how I contributed during the Ph.D. studies to the elucidation of the structure and characterization of unknown lipid classes followed by more detailed description of those lipid classes already identified in vernix caseosa. An integral part of my work was the application of the method enabling the localization of double bonds developed by our laboratory in triacylglycerols and 1,2-diol diesters in vernix caseosa. This analytical method is based on the formation of an acetonitrile adduct in an ionization source of a mass spectrometer enabling atmospheric pressure ionization. The complexity of the triacylglycerol class did not allow a complete characterization of the double bonds. However, the fragmentation mostly showed that double bonds up to n-12 position are present, but small peaks in some spectra also indicated double bonds at more distant positions from the chain termini. I have also...
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Study of lipid biosynthesis during hypoxia using chromatography and mass spectrometry
Mušutová, Martina ; Cvačka, Josef (advisor) ; Čabala, Radomír (referee)
5 Abstract The topic of this diploma thesis is the study of lipide biosynthesis in a hypoxic environment with the use of chromatography and mass spectrography. The first part of the thesis explains the theoretical basis for the research stipulated in the study of OSA and its connection to Type 2 diabetes mellitus. I propose the theory that hypoxia leads to the heightened biosynthesis of fatty acids by way of the reductive citric acid cycle. The research of this reductive citric acid cycle was done by means of cultivated cells with added labeled [5-13 C] glutamine and its absorption into the fatty acids has been observed with the help of GC-MS. The lipides have been extracted from the cell samples, from which the fraction of triglyceride has been isolated with the help of thin layer chromatography. Furthermore, the transesterification to fatty acid methyl esters (FAME) has been performed. The collected data showed a 91 % increase in FAME 16:0 and a 102 % increase in FAME 16:1 in a hypoxic environment compared to the control group. Furthermore, it has been found that samples in a hypoxic environment contained 5.9 % more [13 C1] FAME 16:0, 12 % more [13 C1] FAME 16:1 and almost 3 % more [13 C2] FAME 16:1 compared to the control group. Keywords Lipide biosynthesis, Type 2 diabetes mellitus, FAME, GC-MS,...
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