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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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Localization of double and triple donds positions in fatty acids methylesters by HPLC/APCI-MS2
Horká, Petra ; Vrkoslav, Vladimír ; Cvačka, Josef
Poloha a počet dvojných vazeb v alifatickém řetězci ovlivňuje fyzikální, chemické a biologické vlastnosti lipidů, avšak jednoznačné určení poloh těchto násobných vazeb je problematické. V roce 2012 byla publikována metoda kapalinové chromatografie s chemickou ionizací za atmosférického tlaku s hmotnostní detekcí (HPLC/APCI-MS) [1] vhodná pro analýzu polohy dvojných vazeb využívající tvorby molekulových aduktů [M + C3H5N]+. při APCI za přítomnosti acetonitrilu. Methylestery mastných kyselin (FAMEs) byly analyzovány HPLC/APCI-MS2 se 100% acetonitrilem jako mobilní fází. Při APCI docházelo v plynné fázi k tvorbě reaktivních částic, vznikajících ze složek mobilní fáze, které se vázaly na násobné vazby v alifatickém řetězci FAMEs za vzniku aduktu [M + C3H5N]+.. Kolizně indukovanou disociací (CID) aduktu [M + C3H5N]+., se získaly diagnostické fragmenty určující polohu (polohy) násobných vazeb. Zmíněná metoda byla evaluována pro běžně analyzované polynenasycené FAMEs s tradiční polohu dvojných vazeb (methylene-interrupted).
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Structural characterization of fatty acids with triple bond and unusual double bond positions by HPLC/APCI-MS2
Horká, Petra ; Vrkoslav, Vladimír ; Cvačka, Josef
Fatty acid methyl esters (FAMEs) were analyzed by reversed-phase HPLC coupled to atmospheric pressure chemical ionization (APCI) mass spectrometry. Acetonitrile was used as mobile phase. The gas-phase reactions of acetonitrile and unsaturated FAMEs in the APCI source provided [M + C3H5N]+• adducts. Subsequent MS/MS analysis of these ions gave diagnostic fragments. The formation and fragmentation of the acetonitrile-related adducts were utilized for the structural characterization of FAME with isolated double bonds in (Z)-9,19-hexacosadienoic acid methyl ester, FAME with conjugated double bonds in (Z)-9,11-octadecadienoic acid methyl ester and FAME with a triple bond in 9-octadecynoic acid methyl ester.
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