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Development of mass spectrometric methods for structural analysis of fatty acids and estolides
Cudlman, Lukáš ; Cvačka, Josef (advisor) ; Kozlík, Petr (referee) ; Lísa, Miroslav (referee)
(EN) This doctoral thesis summarizes the use of modern mass spectrometric methods for the structural identification of fatty acids and triacylglycerol estolides (TG-EST) and their subsequent characterization in the lipidome of vernix caseosa, i.e., the biofilm covering the skin of the human fetus during the last trimester of in-utero development. Vernix caseosa has mainly antimicrobial properties and the ability to heal wounds and burns. The first part of the doctoral thesis focuses on the diversity of fatty acids in the lipidome of vernix caseosa. These acids form the structural subunits of complex lipids, such as the abundant triacylglycerols and wax esters. By studying fatty acids and their structural features, this thesis has provided valuable insights into the structural variability of the vernix caseosa lipidome. Without a full understanding of the structure of these key subunits, it would be impossible to complete the analysis of this lipidome in the future. In this thesis, fatty acids using high-performance liquid chromatography (HPLC) and modern mass spectrometry methods, namely ultraviolet photodissociation (UVPD) and ozone-induced dissociation (OzID), were characterized. The coupling of HPLC and mass spectrometry (HPLC-MS) provided unambiguous information on the positions and geometry of...
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Use of HPLC-HRMS for the study of tacrine group compounds potentially useful in the therapy of Alzheimer's disease.
Novák, Martin ; Kučera, Radim (advisor) ; Hroch, Miloš (referee) ; Lísa, Miroslav (referee)
1 Abstract Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Mgr. Martin Novák Supervisor - specialist: doc. Mgr. et Mgr. Rafael Doležal, Ph.D. Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Title: Use of HPLC-HRMS for the study of tacrine group compounds potentially useful in the therapy of Alzhemier's disease. High-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) is an analytical technique very often used in the new drugs development. This technique is widely used, from the confirmation of the proposed structure and identification of present impurities, through the assessment of absorption, distribution, metabolism and excretion to the monitoring of poisoning and intoxications. In the theoretical part, the presented dissertation thesis is focused on the basic characteristics of Alzheimer's disease, its currently available treatment and new treatment strategies. In addition, attention is paid to areas closely related to the experimental part, namely: the study of pharmacokinetics, analytical tools for determining pharmacokinetic parameters, liquid chromatography, mass spectrometry and the specifics of the bioanalytical methods. The experimental work is in the commented form of...
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Use of HPLC-HRMS for the study of tacrine group compounds potentially useful in the therapy of Alzheimer's disease.
Novák, Martin ; Kučera, Radim (advisor) ; Hroch, Miloš (referee) ; Lísa, Miroslav (referee)
1 Abstract Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Mgr. Martin Novák Supervisor - specialist: doc. Mgr. et Mgr. Rafael Doležal, Ph.D. Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Title: Use of HPLC-HRMS for the study of tacrine group compounds potentially useful in the therapy of Alzhemier's disease. High-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) is an analytical technique very often used in the new drugs development. This technique is widely used, from the confirmation of the proposed structure and identification of present impurities, through the assessment of absorption, distribution, metabolism and excretion to the monitoring of poisoning and intoxications. In the theoretical part, the presented dissertation thesis is focused on the basic characteristics of Alzheimer's disease, its currently available treatment and new treatment strategies. In addition, attention is paid to areas closely related to the experimental part, namely: the study of pharmacokinetics, analytical tools for determining pharmacokinetic parameters, liquid chromatography, mass spectrometry and the specifics of the bioanalytical methods. The experimental work is in the commented form 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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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 LC/MS for the Study of Biotransformation of Xenobiotics in Helminths
Vokřál, Ivan ; Szotáková, Barbora (advisor) ; Lísa, Miroslav (referee) ; Horák, Petr (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate Mgr. Ivan Vokřál Supervisor Doc. Ing. Barbora Szotáková, Ph.D. Title of Doctoral Thesis The use of LC/MS for the study of biotransformation of xenobiotics in helminths Annually, parasites cause many losses regarding not only human lives but also the livestock. One of the most effective weapons that can be used against them are anthelmintics. However, in many cases anthelmintics cease to work and resistance development occurs. One of the possible mechanisms of resistance is the biotransformation of the drug to ineffective or less active products by the parasite. The aim of this work was to bring new knowledge about the biotransformation of drugs in helminths. Emphasis was given to the relationship between biotransformation and the resistance of parasites against anthelmintic drugs. A possible technique to achieve this goal is to study directly the products of biotransformation, i.e. metabolites. As this issue is nowadays most frequently solved by liquid chromatography and mass spectrometry, we used this technique to meet our goals. Biotransformation was studied in representatives of tapeworms, flukes and roundworms. Where possible, economically important representatives as Barber's pole...
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Chromatographic and mass-spectrometric analysis of lipids of vernix caseosa
Vavrušová, Aneta ; Cvačka, Josef (advisor) ; Kuda, Ondřej (referee) ; Lísa, Miroslav (referee)
Lipidomics is a rapidly expanding research field that has captured extensive attention worldwide in the past few years due to the increasing awareness of the crucial roles of lipids in biological systems. The aim of lipidomics is to comprehensively analyze all lipids, to study their structure, biological function within the cell as well as interactions of lipids with other molecules. A combination of advanced analytical techniques, such as extraction, chromatography and mass spectrometry, is an effective tool for studying all aspects of lipidomics. This dissertation thesis is based on two journal publications and presents application of analytical strategies based on chromatography and mass spectrometry for investigation and characterization of new lipid classes of vernix caseosa. Firstly, the applicability of nonaqueous reversed-phase liquid chromatography atmospheric pressure ionization tandem mass spectrometry (LC- APCI-MS2 ) for structural characterization of cholesteryl esters of ω-(O-acyl)-hydroxy fatty acids (Chl-ωOAHFAs) in vernix caseosa was investigated. For this purpose, a TLC chromatography method for the isolation of neutral Chl-ωOAHFAs from vernix caseosa was developed. Their general structure was established using multi-step mass spectrometric approach requiring transesterification...
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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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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 LC/MS for the Study of Biotransformation of Xenobiotics in Helminths
Vokřál, Ivan ; Szotáková, Barbora (advisor) ; Lísa, Miroslav (referee) ; Horák, Petr (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate Mgr. Ivan Vokřál Supervisor Doc. Ing. Barbora Szotáková, Ph.D. Title of Doctoral Thesis The use of LC/MS for the study of biotransformation of xenobiotics in helminths Annually, parasites cause many losses regarding not only human lives but also the livestock. One of the most effective weapons that can be used against them are anthelmintics. However, in many cases anthelmintics cease to work and resistance development occurs. One of the possible mechanisms of resistance is the biotransformation of the drug to ineffective or less active products by the parasite. The aim of this work was to bring new knowledge about the biotransformation of drugs in helminths. Emphasis was given to the relationship between biotransformation and the resistance of parasites against anthelmintic drugs. A possible technique to achieve this goal is to study directly the products of biotransformation, i.e. metabolites. As this issue is nowadays most frequently solved by liquid chromatography and mass spectrometry, we used this technique to meet our goals. Biotransformation was studied in representatives of tapeworms, flukes and roundworms. Where possible, economically important representatives as Barber's pole...
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