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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 In addition to that, relevant yet unpublished results have been included as well.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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Study of new synthetic drugs using LC/MS/MS
Obluková, Michaela ; Žabka, Ján (advisor) ; Cvačka, Josef (referee)
In the recent years, many new substances have appeared in the drug market, which are abused for their psychoactive effect. It is used as an alternative to classical drugs (heroin, cocaine, ecstasy ect.) due to their legal status. The great concern is ineffectiveness of the current methods of toxicological screening of biological samples to identify these new compounds. The aim of my diploma thesis was to receive or expand knowledge of chemical properties of 10 standards of new psychoactive substances and its metabolites using MS/MS. Conditions for creation of precursor ions in ion source, optimization of conditions of transport of these ions to the ion trap and optimization of condition of fragments creation in the ion trap were optimized. Subsequently, fragmentation of these ions was studied systematically. Characteristic losses of neutral molecules were determined for each group of these substances which can increase the sensitivity of this method. After that, mephedrone was chosen for detailed study including the study of its metabolites using LC-MS/MS. Standard of mephedrone, spiked urine sample with mephedrone and rat urine dosed with mephedrone were studied. Mephedrone and some its metabolites were identificated in this urine sample. Information received from the investigated substances can...
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Study of substances affecting permeability of the skin barrier
Nováčková, Anna ; Zbytovská, Jarmila (advisor) ; Cvačka, Josef (referee) ; Doležal, Pavel (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Department of Pharmaceutical Technology Candidate Mgr. Anna Nováčková Supervisor doc. Mgr. Jarmila Zbytovská, Dr. rer. nat. Title of Doctoral Thesis Study of substances affecting permeability of the skin barrier The skin barrier plays a vital role in protecting the human body and enables mammals' life on dry land. The epidermis has the primary barrier function due to several cells' layers, which gradually differentiate to their final stage, the stratum corneum (SC). SC is formed by stratified keratinocytes (known as corneocytes) surrounded by a lipid matrix. This intercellular matrix consists of an approximately equimolar ratio of ceramides, free fatty acids and cholesterol. These are particular substances formed in the epidermis from their precursors during the keratinocyte's differentiation, and their arrangement into the multilamellar structure is essential for the impermeability of the skin barrier. However, some substances or factors can disrupt the skin barrier. It is usually an undesirable process of lipid disbalance resulting in disorders or diseases of the skin barrier. On the other hand, specific substances have been developed for a reversible disruption of the skin barrier (so-called enhancers) to allow drug...
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Study of substances affecting permeability of the skin barrier
Nováčková, Anna ; Zbytovská, Jarmila (advisor) ; Cvačka, Josef (referee) ; Doležal, Pavel (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Department of Pharmaceutical Technology Candidate Mgr. Anna Nováčková Supervisor doc. Mgr. Jarmila Zbytovská, Dr. rer. nat. Title of Doctoral Thesis Study of substances affecting permeability of the skin barrier The skin barrier plays a vital role in protecting the human body and enables mammals' life on dry land. The epidermis has the primary barrier function due to several cells' layers, which gradually differentiate to their final stage, the stratum corneum (SC). SC is formed by stratified keratinocytes (known as corneocytes) surrounded by a lipid matrix. This intercellular matrix consists of an approximately equimolar ratio of ceramides, free fatty acids and cholesterol. These are particular substances formed in the epidermis from their precursors during the keratinocyte's differentiation, and their arrangement into the multilamellar structure is essential for the impermeability of the skin barrier. However, some substances or factors can disrupt the skin barrier. It is usually an undesirable process of lipid disbalance resulting in disorders or diseases of the skin barrier. On the other hand, specific substances have been developed for a reversible disruption of the skin barrier (so-called enhancers) to allow drug...
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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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Atmospheric pressure chemical ionization mass spectrometry at low flows: construction and applications
Strmeň, Timotej ; Cvačka, Josef (advisor) ; Sýkora, David (referee) ; Polášek, Miroslav (referee)
(EN) The first part of the thesis describes the ionization reactions in the APCI source in the presence (enclosed configuration) and absence (open configuration) of the ion source housing. Commercial APCI source was modified and used in enclosed, as well as open configuration. All other parameters were kept the same (including the source geometry). In the positive mode, the biggest differences were observed for non-polar analytes dissolved in aprotic solvents like toluene, chloroform and carbon disulfide. Protonated species were dominant in the open configuration, while radical cations were mostly present in the enclosed configuration. The excessive protonation in open ion source was caused by the diffusion of water vapor molecules into the ionization region from the atmosphere. Water vapor molecules were also responsible for the formation of an ion [M + 19]+ from alkynes in the open configuration. The fragmentation study confirmed that the ion [M + 19]+ was a 2-methylketone formed by the addition reaction. The formation of such artifacts can pose problems in qualitative analysis. On the other hand, the ion [M + 55]+ was observed in the enclosed ion source as a reaction product of unsaturated compounds with acetonitrile solvent molecules. Its fragmentation can be used for the double bond...
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