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Difficulty of urinary neopterin and creatinine determination using high-performance liquid chromatography with focus on clinical practice
Kepka, Zdeněk ; Kujovská Krčmová, Lenka (advisor) ; Matysová, Ludmila (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Zdeněk Kepka Supervisor: Assoc. Prof. RNDr. Lenka Kujovská Krčmová, Ph.D. Title of the diploma thesis: Difficulty of urinary neopterin and creatinine determination using high-performance liquid chromatography with focus on clinical practice This diploma thesis deals with the determination of neopterin and creatinine in urine samples by high performance liquid chromatography. The theoretical part summarizes information about the substances and their analysis. It also includes a review of methods published in the literature. The method of high-performance liquid chromatography including types of stationary phases and chromatographic principles used in the experimental part of this work is briefly introduced. The separation properties of six chromatographic columns for the determination of neopterin and creatinine in urine were verified in the experimental part of the thesis. Columns were compared by the following parameters: repeatability, resolution, peak symmetry, and column efficiency. The composition of the mobile phase was optimized for selected columns. The mobile phase was a phosphate buffer with a concentration of 15 mmol/l and pH 6.5 in most cases. Creatinine detection was performed by...
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Development and validation of method for determination of of Cyclosporine in biorelevant mediums
Tichoň, Matej ; Matysová, Ludmila (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles university Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Matej Tichoň Supervisor: assoc. prof. PharmDr. Ludmila Matysová, Ph.D. Title of diploma thesis: Development and validation of method for determination of cyclosporine A in biorelevant mediums For the determination of cyclosporine A in biorelevant mediums, a new analytical method was developed by using of ultra-high performance liquid chromatography. The analysis was performed on a Kinetex™ 1.7 µm, C18 column, 50 x 2.1 mm (Phenomenex, USA). The mobile phase consisted of ultrapure water acidified with phosphoric acid to pH = 2.40 and mixture of methanol : acetonitrile (90 : 10) in a total ratio of 30 : 70 aqueous to organic phases. The analysis was carried out at 60 řC for 5 minutes with a constant flow rate of 0.5 ml/min. Gradient elution was used in the analysis, starting at 70 % organic phase at 0 minutes and increasing to 100 % at 5 minutes. Cyclosporine A was detected at 210 nm. The retention time of the substance was 3.08 min. Having the optimized conditions in hands, the method was fully validated. Keywords: cyclosporine A, UHPLC, biorelevant mediums
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Development and validation of UHPLC method for determination of miconazole, econazole and its impurities in solid dispersions
Arnoltová, Kristýna ; Matysová, Ludmila (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Kristýna Arnoltová Supervisor: doc. PharmDr. Ludmila Matysová, Ph.D. Title of the diploma thesis: Development and validation of UHPLC method for determination of miconazole, econazole and its impurities in solid dispersions The aim of the submitted thesis was to create a new analytical method for the determination of miconazole, econazole and its three impurities. Ultra-high performance liquid chromatography (UHPLC) was used. A KinetexTM XB C18 column, 1.7 μm particle size, 50 x 2.1 mm was used for the separation. The mobile phase was composed of the acetate buffer pH 7.8 in a mixture with methanol (80:20) and of acetonitrile with methanol (60:40), in a total ratio of 60:40. A gradient elution was used. Flow rate was set to 0.6 ml / min. Butylparaben was chosen as the internal standard. All substances were detected at a wavelength of 225nm. Total analysis time was 9 minutes. After optimization of the separation conditions, the method was partially validated and used for practical measurements to determine the content of miconazole in solid dispersions.
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Separation of tocopherols using HPLC technique
Hutníková, Michaela ; Kujovská Krčmová, Lenka (advisor) ; Matysová, Ludmila (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Michaela Hutníková Supervisor: assoc. prof. RNDr. Lenka Kujovská Krčmová Ph.D. Title of diploma thesis: Separation of tocopherols using HPLC technique Vitamin E represents eight related compounds α, β, γ, δ-tocopherols (saturated phytyl side chain) and α, β, γ, δ-tocotrienols (unsaturated phytyl side chain). α-tocopherol was the most studied isomer in the past and its anti-inflammation and proliferative effect was described. Therefore most of cancer prevention trials have been focused on α-tocopherol. Present research studies have described the important role of γ and δ-tocopherols in cell proliferation, anti-inflammation and tumor burden. Also it has been demonstrated that γ-tocopherol inhibits colon, prostate, mammary and lung tumor genesis in animal models, suggesting to have a high potential in the prevention of human cancer. In this study the novel sensitive method for analysis of α, β, γ, δ-tocopherols was developed using the High Performance Liquid Chromatography (HPLC) technique. All the measurements were carried out on the chromatographic system HPLC Prominence LC 20, Shimadzu (Kyoto, Japan). During the method development core-shell columns-Kinetex (Torrance, USA) with different...
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Analytical and bioanalytical evaluation of novel potential drugs from the group of iron chelators
Bureš, Jan ; Štěrbová, Petra (advisor) ; Kujovská Krčmová, Lenka (referee) ; Hroch, Miloš (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Mgr. Jan Bureš Supervisor: Doc. PharmDr. Petra Štěrbová, Ph.D. Title of Thesis: Analytical and bioanalytical evaluation of novel potential drugs from the group of iron chelators High performance liquid chromatography (HPLC) gained its unprecedented position among bioanalytical techniques due to its effectivity and versatility. If tuned properly, it can separate complex mixtures and can be used for qualitative and quantitative analysis simultaneously, usually within a short time. Reliability of the HPLC methods is proven by their validation performed according to accepted guidelines. Iron chelation is considered to be an effective concept of treatment of various pathologies - from iron overload disease to cancer or neurodegenerative disorders. To develop new chelators for these indications it is important to investigate their structure- activity relationship, the fate of the drug in organism and its relationship to biological effects and toxicity etc. Modern analytical methods are essential tools for these studies. The first part of this work presents validated LC-UV and LC-MS methods for assay of aroylhydrazone iron chelators and related pro-drugs in biological...
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Development and optimization of new chromatographic method for determination of retinol in human urine
Mikanová, Zuzana ; Kujovská Krčmová, Lenka (advisor) ; Sklenářová, Hana (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Zuzana Mikanová Supervisor: assoc. prof. RNDr. Lenka Kujovská Krčmová, Ph.D. Title of the Diploma Thesis: Development and optimization of new chromatographic method for determination of retinol in human urine This diploma thesis is based on the method development for determination of retinol and creatinine using HPLC. The aim of this project was to find optimal HPLC-MS/MS conditions for clinical research. Experiment was carried out using UHPLC set Nexera with mass spectrometer LCMS- 8030, (Shimadzu, Japan). Three stationary phases were tested. The chromatographic separation was achieved using a Kinetex 2.6 µm PFP 100A 100x4.6 mm (Phenomenex, USA). The used mobile phase consisted of acetonitrile (with addition of formic acid 0.005 M) and ammonium acetate (pH 6.69 ) in the ratio 78:22 (v/v). The temperature was maintained at 25řC, flow rate was set at 0.5 ml/min and 3 µl of sample was injected. After optimization of separation conditions, the method was applied to urine samples, and simple sample preparation procedures were tested In these days is the work on method finishing and the new method will be used for determination of renal damage in clinical research and practice. The inclusion of...
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Development and validation of UHPLC method for determination of terbinafine and its impurities
Firleyová, Michaela ; Matysová, Ludmila (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Michaela Firleyová Supervisor: doc. PharmDr. Ludmila Matysová, Ph.D. Title of diploma thesis: Development and validation of UHPLC method for determination of terbinafine and its impurities A new analytical method for the determination of terbinafine and its degradation products - β-terbinafine, Z-terbinafine and 4-methylterbinafine - was developed by using ultra-high performance liquid chromatography. The best separation of the analytes was achieved with a KinetexTM 1.7 μm - Biphenyl 100A column (50 mm x 2.1 mm). The selected mobile phase was composed of the buffer pH 5.0, made up of the citrate and the phosphate buffer, and methanol (25:75, v/v). Propylparaben as the internal standard was detected at the wavelength of 254 nm, whereas the terbinafine and its three degradation products at 222 nm. The retention time of the terbinafine was 4.2 min, the total analysis time was within 6 minutes. After finding the optimal conditions the method was validated.
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Application of liquid chromatography for determination of selected amino-acids and agmatine in biological material
Hazuková, Niké ; Kujovská Krčmová, Lenka (advisor) ; Matysová, Ludmila (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Niké Hazuková Supervisor: Assoc. Prof. RNDr. Lenka Kujovská Krčmová, Ph.D. Title of diploma thesis: High performance liquid chromatogramy determination of selected amino acids and agamtine in biological material Keywords: agmatine, L-arginine, L-citruline, L-ornitine, UHPLC, chronic wounds This thesis follows up the metod development for the determination of agmatine, L- arginine, L-citrulline, L-ornithine in chronic wounds using ultra-high performance liquid chromatography. The aim of this thesis was to optimize suitable chromatographic conditions for the already mentioned analytes with possibility to use this method in clinical research and practice. Four chromatographic columns were tested. Finally the Meteoric Core C18 BIO the dimensions 100 x 4.6 mm, 16 nm, 2.7 μm (YMC, Germany) was chosen, using these conditions: the mobile phase (MF) was 100% 10 mM AMFO (pH 2.5), the temperature was set at 25 řC, the MF flow rate was 0.25 mL / min, and the injection volume was 2 μL. After optimization of the separation conditions, the method was applied to biological material (exudate from chronic wounds), samples were modified by selected extraction procedures as ultrafiltration and protein precipitation....
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Analytical and bioanalytical evaluation of novel potential drugs from the group of iron chelators
Bureš, Jan ; Štěrbová, Petra (advisor) ; Kujovská Krčmová, Lenka (referee) ; Hroch, Miloš (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Mgr. Jan Bureš Supervisor: Doc. PharmDr. Petra Štěrbová, Ph.D. Title of Thesis: Analytical and bioanalytical evaluation of novel potential drugs from the group of iron chelators High performance liquid chromatography (HPLC) gained its unprecedented position among bioanalytical techniques due to its effectivity and versatility. If tuned properly, it can separate complex mixtures and can be used for qualitative and quantitative analysis simultaneously, usually within a short time. Reliability of the HPLC methods is proven by their validation performed according to accepted guidelines. Iron chelation is considered to be an effective concept of treatment of various pathologies - from iron overload disease to cancer or neurodegenerative disorders. To develop new chelators for these indications it is important to investigate their structure- activity relationship, the fate of the drug in organism and its relationship to biological effects and toxicity etc. Modern analytical methods are essential tools for these studies. The first part of this work presents validated LC-UV and LC-MS methods for assay of aroylhydrazone iron chelators and related pro-drugs in biological...
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