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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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Liquid chromatography with mass-spectrometric detection based on a microfluidic chip
Rumlová, Barbora ; Cvačka, Josef (advisor) ; Jelínek, Ivan (referee)
This diploma thesis deals with hyphenation of liquid chromatography with mass spectrometric detection based on microfluidic chip. Firstly, a miniaturized ion source for atmospheric-pressure chemical ionization (APCI), and atmospheric-pressure photoionization (APPI) was constructed. The main component of this source was a glass microfluidic chip. Geometry and the working conditions of the chip were optimized. Since both ion sources work under the same conditions, possible advantages resulting from APCI/APPI combination were investigated. Furthermore, the performance characteristics of the sources were evaluated, and compared to the conventional high flow-rate sources. The best performing source, APCI, was then hyphenated with HPLC using low flow-rate. A method for separation of fatty acids methyl esters using Supelco 37 standard was developed. The separation conditions were as follows: C18 reversed stationary phase, and acetonitrile containing 0.1 % formic acids was used as the mobile phase. A temperature gradient was used in order to enhance the speed of the separation. The limits of detection and quantitation of for selected analytes using the chip micro-APCI were calculated, and compared to the ones obtained using a commercially available micro-APCI source. The method was used for separation of...
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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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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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Analysis of vernix caseosa lipids by chromatografic methods and mass spectrometry
Míková, Radka ; Cvačka, Josef (advisor) ; Jelínek, Ivan (referee) ; Tůma, Petr (referee)
(EN) Vernix caseosa is a white creamy substance that covers the skin of a newborn. It is produced during the third trimester by the skin of the baby and remains there until the age of one or even two weeks. It is uniquely human. In utero, vernix protects the skin from maceration, during the birth it serves as a lubricant and after the delivery it protects the baby against infection and regulates the temperature. As vernix is produced in third trimester, prematurely born infants lack it and this may lead to, among other things, suffering from desiccation and therefore heat loss. It is important to study it thoroughly and to find a suitable substitute of vernix for the preterm infants. Vernix consists of lipids, proteins and 80 % water. This project is aimed at the lipids. Vernix is composed of 10 % of lipids. Basic analytical methods of pocessing vernix were searched. The methods of isolation, separation and transesterification have been optimized for the lipids. For separation, thin-layer chromatography has been chosen. The method of the lipid analysis of intact molecules by MALDI-TOF MS has been optimized for these lipids. The results were confirmed using fragmentation spectra and transesterification. Esterified lipids were measured by gas chromatography coupled with mass spectrometry detection....
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Development of miniaturized ion source for atmospheric pressure chemical ionization (HPLC/MS)
Rumlová, Barbora ; Cvačka, Josef (advisor) ; Jelínek, Ivan (referee)
ionization in which the main component is a heated glass microfluidic chip. The ion source was assembled from the mirocfluidic chip placed on a micromanipulator and a needle electrode creating a corona discharge. The optimization of geometric arrangement of these components towards to inner heated capillary of the mass spectrometer LCQ Fleet (Thermo) was based on the signal intensity of reserpine. The solution of reserpine with concentration 10 µg/ w y g g y g . Furthermore, the flow rate of the nebulizing gas was optimized. The signal intensity of protonated molecule was two orders of magnitude higher than the signal generated by the commercial ion source for APCI (Thermo) using the same mass flow rate of the analyte. High signal instability of the detected ions is the persisting problem of the miniaturized ion source. Key words: mass spectrometry, micro APCI, microfluidic chip
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