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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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Chromatographic and mass-spectrometric analysis of lipids of vernix caseosa
Vavrušová, Aneta
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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Analysis of vernix caseosa lipids by chromatografic methods and mass spectrometry
Míková, Radka
(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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Chromatographic and mass-spectrometric analysis of lipids of vernix caseosa
Vavrušová, Aneta
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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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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Characterization of lipids with four ester bonds in vernix caseosa by HPLC / MS
Tomášková, Sabina ; Cvačka, Josef (advisor) ; Kozlík, Petr (referee)
Diploma thesis is devoted to the analysis of triacylglycerol estolides (TG-est) by HPLC/MS. These lipids contain four ester bonds. TG-est are part of some vegetable oils and in mammals function as a storage form of metabolically important fatty acid estolides. The aim of this thesis was to prove the existence of TG-est in vernix caseosa and explore the possibilities of analysis in this material. Standards of TG-est were studied with using high-resolution and tandem mass spectrometry. Was suggested and optimized HPLC method for TG-est in a reverse nonaqueous system with the use of column Kinetex C18 and gradient elution with acetonitrile and 2-propanol. This method was used for analysis vernix caseosa fraction, and it was shown that TG-est are present in the sample. Attempts to simplify the lipid sample before analysis HPLC/MS by TLC fractionation were not very successful.
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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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Analysis of vernix caseosa lipids by chromatografic methods and mass spectrometry
Míková, Radka
(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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