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HPLC of Bioactive Compounds
Lukšanová, Aneta ; Kubíček, Vladimír (advisor) ; Lázníčková, Alice (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of: Biophysics and Physical Chemistry Consultant: Ing. Vladimír Kubíček, CSc. Student: Aneta Lukšanová Title of thesis: HPLC of bioactive compounds This diploma thesis is focused on finding conditions of HPLC analysis of midazolam and it's two metabolites - 1-hydroxymidazolam and 4-hydroxymidazolam, on method validation and on finding an appropriate internal standard. The analysis was carried out under reversed-phase conditions, the chosen column was POROSHELL 120 EC-C18, 3.0 X 100 NM, 2.7 µm, and a mixture of acetonitrile and ammonium acetate buffer with pH 4.5 in a ratio of 35/65 has been proven to be effective mobile phase. Detection was carried out using the diode array detector with wavelength values 223 nm and 272 nm. The column was thermostated at 25 řC, the analysis time was 10 minutes. Flow rate of mobile phase was 0.700 ml/min. Diazepam was found as a suitable internal standard. The method was validated only for 1-hydroxymidazolam and 4-hydroxymidazolam determination in rat plasma due to a submitter's request. Within the analytic method validation the following parameters were tested: precision, accuracy, lower limit of quantitation, linearity and selectivity.
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HPLC of Bioactive Compounds
Lukšanová, Aneta ; Kubíček, Vladimír (advisor) ; Lázníčková, Alice (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of: Biophysics and Physical Chemistry Consultant: Ing. Vladimír Kubíček, CSc. Student: Aneta Lukšanová Title of thesis: HPLC of bioactive compounds This diploma thesis is focused on finding conditions of HPLC analysis of midazolam and it's two metabolites - 1-hydroxymidazolam and 4-hydroxymidazolam, on method validation and on finding an appropriate internal standard. The analysis was carried out under reversed-phase conditions, the chosen column was POROSHELL 120 EC-C18, 3.0 X 100 NM, 2.7 µm, and a mixture of acetonitrile and ammonium acetate buffer with pH 4.5 in a ratio of 35/65 has been proven to be effective mobile phase. Detection was carried out using the diode array detector with wavelength values 223 nm and 272 nm. The column was thermostated at 25 řC, the analysis time was 10 minutes. Flow rate of mobile phase was 0.700 ml/min. Diazepam was found as a suitable internal standard. The method was validated only for 1-hydroxymidazolam and 4-hydroxymidazolam determination in rat plasma due to a submitter's request. Within the analytic method validation the following parameters were tested: precision, accuracy, lower limit of quantitation, linearity and selectivity.
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Oxidation of a psychoactive substance fluoromethoxyacetyl fentanyl
Krejčí, Michaela ; Kubíček, Vladimír (advisor) ; Nováková, Veronika (referee)
7 2. ABSTRACT Fluoromethoxyacetyl fentanyl (FMAcF) is a new psychoactive substance that belongs to the group of newly illegally synthesized fentanyl derivatives that have been entering the European drug market since 2009. The aim was to define the electrochemical behavior of FMAcF and to suggest possible oxidation mechanisms. In this research, the electrochemical methods and in situ UV-Vis spectroelectrochemistry were used to study drug oxidation and reduction mechanism. Electrochemical techniques were combined with high performance liquid chromatography with diode array detector. FMAcF in the aqueous medium provided a single oxidation peak whose potential depended on pH. The substance was not reduced under the studied conditions. UV-Vis spectroelectrochemistry showed the formation of a reaction intermediate. The substance was electrolyzed and the UV-Vis absorption spectra of the products were determined. The oxidation mechanism of the substance has been proposed. In future the incubation with liver microsomes will be performed to detect metabolic pathways. Key words: Cyclic voltammetry, Oxidation, Bioactive substance, Fentanyls, Fluoromethoxyacetyl fentanyl
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Characterization of 32,33-didehydroroflamycoin - secondary metabolite from Streptomyces durmitorensis
Koukalová, Alena ; Černý, Jan (advisor) ; Konopásek, Ivo (referee)
Streptomycetes are soil filamentous Gram-positive bacteria that produce wide variety of pigments and biologically active substances including macrolides. Some of them are used as very efficient antibiotics and strong antifungal agents in medicine, others have became useful tools for staining biomembranes and detecting cholesterol via their internal fluorescence. Actinomycete Streptomyces durmitorensis (wild type strain MS405T ) is a bacteria isolated from Durmitor National Park in Montenegro soil samples. It produces secondary metabolite that has been identified as 32,33-didehydroroflamycoin (DDHR) closely related to the macrolides roflamycoin and generaly used filipin. DDHR exhibits cytototoxic activity against mammalian cells and yeast Saccharomyces cerevisiae strain EGY48. In addition it has interesting fluorescence properties allowing visualization of some membrane components. DDHR interacts with biomembranes, causes their disintegration leading to changes of the actin and tubulin cytoskeleton organization and in higher concentrations it causes cells necrosis. DDHR-sterol interaction in cell membranes decreases fluorescence intensity of DDHR. The compound is able to fluorescently stain aberrant lysosomes and could be therefore potentially used in diagnostics of some lysosomal storage disease.
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Cytostatický efekt nostatinu A a jeho přírodních analogů na buněčnou linii HeLa
VICKOVÁ, Kateřina
Cyanobacterial secondary metabolites are a rich source of bioactive compounds with potential utilization in pharmacology. The aim of this study was to evaluate the effect of the novel compound nostatin A isolated from the cyanobacterium Desmonostoc muscorum. Project was focused on the extraction, purification and characterization of the cytostatic effect caused by this novel compound and its naturally occurred structural analogues. The cytostatic activity of nostatin A and its analogs was evaluated in HeLa cell line. Experiments based on microscopy, flow cytometry and HPLC-HRMS techniques were performed in order to clarify the cytostatic effect of nostatin A in HeLa cells and its mechanism of the action.
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Characterization of 32,33-didehydroroflamycoin - secondary metabolite from Streptomyces durmitorensis
Koukalová, Alena ; Černý, Jan (advisor) ; Konopásek, Ivo (referee)
Streptomycetes are soil filamentous Gram-positive bacteria that produce wide variety of pigments and biologically active substances including macrolides. Some of them are used as very efficient antibiotics and strong antifungal agents in medicine, others have became useful tools for staining biomembranes and detecting cholesterol via their internal fluorescence. Actinomycete Streptomyces durmitorensis (wild type strain MS405T ) is a bacteria isolated from Durmitor National Park in Montenegro soil samples. It produces secondary metabolite that has been identified as 32,33-didehydroroflamycoin (DDHR) closely related to the macrolides roflamycoin and generaly used filipin. DDHR exhibits cytototoxic activity against mammalian cells and yeast Saccharomyces cerevisiae strain EGY48. In addition it has interesting fluorescence properties allowing visualization of some membrane components. DDHR interacts with biomembranes, causes their disintegration leading to changes of the actin and tubulin cytoskeleton organization and in higher concentrations it causes cells necrosis. DDHR-sterol interaction in cell membranes decreases fluorescence intensity of DDHR. The compound is able to fluorescently stain aberrant lysosomes and could be therefore potentially used in diagnostics of some lysosomal storage disease.
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