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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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Analysis of bumblebee lipids using chromatographic methods
Kudzejová, Michaela ; Bosáková, Zuzana (advisor) ; Cvačka, Josef (referee)
Lipids from the fat body of queens of Bombus terrestris species in different life stages were studied using chromatographic methods. High-performance liquid chromatography - mass spectrometry with electrospray ionization was used to analyze all lipids from the tissue. Semi- preparative thin-layer chromatography was used to isolate triacylglycerols (TGs) from the fat body tissue. The TGs were subsequently analyzed by high-performance liquid chromatography - atmospheric pressure chemical ionization mass spectrometry. Quantitative differences between different life stages have been found. Qualitative composition has not been changing significantly. Only minor differences have been found in the substances, which were present in amount less than 1%.
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Liquid Chromatography Methods for Analysis of Actinomycete Secondary Metabolites - Potential Antibiotics
Kameník, Zdeněk ; Nesměrák, Karel (advisor) ; Cvačka, Josef (referee) ; Bosáková, Zuzana (referee)
(EN) This dissertation thesis contains scientific results achieved in the field of analytical chemistry, particularly liquid chromatography. The major part of the results has been published in prestigious international journals in five papers. In addition to that, relevant yet unpublished results have been included as well. In general terms, the work presented here contributed to the concerted efforts to tackle the current lack of novel antibiotics. Specifically, high-performance liquid chromatography (HPLC) and ultra high-performance liquid chromatography (UHPLC) techniques coupled to a variety of detection systems have been employed for analysis of antibiotics and actinomycete secondary metabolites. The first thematic part describes the development of liquid chromatography methods for analysis of lincomycin precursors, lincomycin precursor analogues, and lincomycin derivatives. The methods have been applied to study lincomycin biosynthetic pathway and obtain improved lincomycin derivatives by mutasynthesis. The second thematic part aims at investigating alternative approaches for analysis of antibiotics. Firstly, the core-shell particle and the sub-2 μm particle chromatographic columns were compared. The core-shell particle columns compatible with HPLC proved to be a convenient alternative to the...
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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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Localization of double bond positions in triacylglycerols using HPLC/APCI-MS2
Háková, Eva ; Cvačka, Josef (advisor) ; Lísa, Miroslav (referee)
Triacylglycerols are among the most abudant classes of lipid. Their chemical, physical and biological characteristics depend on degree of unsaturation and positions of double bonds in acyls. This thesis was focused on localization of double bonds using tandem mass spectrometry with atmospheric pressure chemical ionization. We studied 14 standards and 44 standard mixtures with different number of double bonds and lenght of hydrocarbob chain in acyls. Standards were obtained from commercial sources and prepared of randomizations reaction in microscale. Localization of double bonds was deduced from fragments of molecular aduct with C3H5N+· ([M+55]+·) created in presence of acetonitrile in the mobile phase. This metod was applied for HPLC/MS analysis of natural mixtures of triacylglycerols. Key words: triacylglycerols, randomization, localization of double bonds, atmospheric pressure chemical ionization, mass spectrometry, high performance liquid chromatography with mass detection
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The use of ambient ionization techniques in mass spectrometry
Rejšek, Jan ; Cvačka, Josef (advisor) ; Coufal, Pavel (referee)
Keywords: ambient ionization techniques; mass spectrometry; desorption electrospray ionization; desorption atmospheric pressure photoionization; thin layer chromatography; lipids, mass spectrometry imaging Ambient ionization technique in mass spectrometry is an ionization, which carries out in open space outside the machine and which does not require any, or only a minimal sample pretreatment. DESI (desorption electrospray ionization) and DAPPI (desorption atmospheric pressure photoionization) equipped with software control of the spray emitter position for analysis of low molecular organic compounds were investigated in this thesis. These methods use a spray of solvents for desorption and ionization molecules from solid substrate. Conditions for the successful analysis of phospholipids, wax esters and some other compounds were developed. Ambient ionization techniques were quantitatively compared. The application was HPTLC/DESI-MS of lipid's mixture and HPTLC/DAPPI-MS of vernix caseosa. DAPPI-MS was applied for the analysis of termites of Prorhinotermes genus (Isoptera, Rhinotermitidae). Pilot experiments of two dimensional analysis and mass spectrometry imaging were realized.
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