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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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Influence of Electrolytes on CMC
Šmerdová, Jana ; Kubíček, Vladimír (advisor) ; Lázníčková, Alice (referee)
Souhrn V této diplomové práci bylo použito UV-VIS absorpční spektrofotometrie k měření CMC katonických tenzidů a vlivu elektrolytů na CMC. Dosažené výsledky potvrzují očekávaný trend poklesu hodnot CMC tenzidů se stoupající iontovou silou roztoků. Použitá modifikace metody UV-VIS absorpční spektrofotometrie pro stanovení CMC byla rovněž úspěšná.
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Study of radiolabelling of receptor-specific peptide from the group of gastrin derivatives I
Zeithamová, Jana ; Lázníčková, Alice (advisor) ; Kubíček, Vladimír (referee)
This work studies the radiolabeling of minigastrins using 111 In. For experimental purposes minigastrins 46, 47 and 48 were used with different amino acids sequences. It was made especially for radiochemical purity 111 InCl3 sign, since this parameter is very important for possible future use of these labeled peptides. Another parameter investigated was the monitoring of the stability of radioactively labeled minigastrins. Results were measured using high performance liquid chromatography, the clear evidence of pollution and possible degradation products. The second applied method was performed by instant thin-layer chromatography in several different environments. The objective was to determine the dependence on the purity of the product over time in both short and long time intervals.
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Stability studies of monoclonal antibody nimotuzumab modified with chelator DOTA radiolabelled with lutetium-177 ([177Lu]Lu-hR3(p-SCN-Bn)DOTA)
Petřík, Michal ; Lázníčková, Alice (advisor) ; Kubíček, Vladimír (referee)
The higher expressivity of the gene for the receptor of epidermal growth factor (EGF) and consequently higher number of receptors (EGFR) on the surface of cells is closely linked with the tumour proliferation of these cells and with poor prognosis for patients. For such reason the EGFR became one of the main objectives in the therapy of tumours. Nimotuzumab (also known as h-R3) is humanized monoclonal antibody which blocks receptor for epidermal growth factor. The object of this thesis was to determinate radiochemical purity of this antibody conjugated with bifunctional chelator DOTA and marked by luthecium, possibly purification of this antibody and consequently determination of its stability in different environments. In acetate buffer pH = 7, in an environment of the surplus of competitive ligand EDTA and in plasma of rats. Adequate analytic methods were used for these tasks: high-efficiency liquid chromatography, gel permeation chromatography and chromatography on thin layer. The results of the thesis demonstrated that this way of modification for further radiolabelling is not appropriate for large protein structures such as Nimotuzumab
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HPLC Analysis of Anthelmintics Using Core-shell Columns
Marková, Jitka ; Kubíček, Vladimír (advisor) ; Lázníčková, Alice (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Candidate: Jitka Marková Consultant: Ing. Vladimír Kubíček, CSc. Title of Thesis: HPLC Analysis of Anthelmintics Using Core-shell Columns This diploma thesis deals with HPLC analysis of three anthelmintics included into AAD group (i. e. amino-acetonitrile derivatives). Suitable separation conditions were studied using three core-shell columns (i. e. Ascentis Express RP - Amide, Ascentis Express F5 and Kinetex PFP). Finally, an efficiency of two preextraction methods (SPE, LLE) was tested. The obtained results show the Ascentis Express RP-Amide column to be the suitable choice for the required purpose. The proposed mobile phases are MS compatible. SPE column STRATA X, exhibiting very high recoveries, is the best choice for SPE. The LLE recovery ratio can be taken as satisfactory.
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Hyaluronan oligosacharides and their biological function
Kučerová, Barbora ; Lázníčková, Alice (advisor) ; Netopilová, Miloslava (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biophysic and Physical Chemistry Candidate: Bc. Barbora Kučerová Supervisor: Doc. Ing. Alice Lázníčková, CSc. Title of diploma thesis: Hyaluronan oligosaccharides and their biological function In my diploma thesis I compile informations about hyaluronan oligosaccharides. Although hyaluronan is in the body found mostly in a form of long chains, we can find it also as small fragments - the oligosaccharides. The oligosaccharides embody different properties than hyaluronan polymers. Hyaluronan oligosaccharides can be synthetised, separated and analysed in laborarory in a variety of ways. Large molecules can be broken into fragments chemically, physically or enzymatically. For thein subsequent separation and purification especially chromatographic methods are used. As the most suitable methods for their final analysis electrophoretic techniques or mass spectrometry were shown. Continous metabolism of hyaluronan proceeds inside the body . Hyaluronan synthase takes a part in synthesis, and the biodegradation important for human is supplyed by several hyaluronidases. Interaction of hyaluronan with cells is mediated by several receptors. Specific receptor for hyaluronan is CD44, which can bind either large polymers and...
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Study of radiolabeling of chemically modified bombesines
Saranti, Evangelia ; Lázníčková, Alice (advisor) ; Kubíček, Vladimír (referee)
STUDY OF RADIOLABELING OF CHEMICALLY MODIFIED BOMBESINES Diploma Thesis Evangelia Saranti Charles University in Prague, Faculty of Pharmacy in Hradec Kralove Department of Biophysics and Physical Chemistry The aim of this thesis was to study the radiolabeling of new perspective receptor-specific peptide DOTA-Lys3-bombesin with therapeutic radionuclide Lu-177 and to determine the stability of the labeled product. We have found the optimum conditions for radiolabeling of selected peptide: pH 5.0 and incubated at 42ř C for 35-40 minutes. Under these conditions sufficient radiochemical purity for biological testing of substance was achieved (higher than 97%). We tested the stability of the radiolabeled product when it was stored at 4ř C, and both in the environment by an order of magnitude higher concentration of competing ligand EDTA or DTPA. Stored in the refrigerator peptide was stable for at least four hours after preparation, study of stability in an environment of competing ligands showed good stability of the DOTA-complex with lutetium-177. Comparison of methods for determination of radiochemical purity of the radiolabeled peptide showed greater reliability of HPLC analysis compared to thin-layer chromatography.
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