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Development of extraction procedure for determination of tocopherols
Klabačková, Sáva ; Kujovská Krčmová, Lenka (advisor) ; Sklenářová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Sáva Klabačková Supervisor: RNDr. Lenka Kujovská Krčmová, Ph.D. Title of diploma thesis: Development of extraction procedure for determination of tocopherols In the present work liquid-liquid extraction for determination of alpha, beta, gamma and delta tocopherol from human serum was developed miniaturized. Individual extraction steps as deproteinization, centrifugation, evaporation and final filtration were optimized. Solid supported liquid extraction (SLE) was also tested for determination of tocopherols and seems to be very promising for use in bioanalysis. During the miniaturization of the extraction procedure the main emphasis was placed on simplicity, speed and low consumption of samples and solvents. Method was also partly validated. The new extraction process is part of an already developed UHPLC method for the determination of individual forms of tocopherols and will serve to determine the antioxidant capacity of cancer patients during the chemotherapy treatment. Key words: tocopherols, LLE, SLE, biological material
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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 of HPLC method for determination of vitamin B1 in clinical research
Chadtová, Anežka ; Kujovská Krčmová, Lenka (advisor) ; Matysová, Ludmila (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Anežka Chadtová Supervisor: RNDr. Lenka Kujovská Krčmová Ph.D. Title of diploma thesis: Development of HPLC method for determination of vitamin B1 in clinical research The present thesis describes the development and partial validation of the new HPLC method for determination of vitamin B1 (thiamine hydrochloride) and its phosphorylated derivatives (thiamine monophosphate, thiamine pyrophosphate). This method is based on the use of liquid chromatography with indirect fluorescence detection. Separation was achieved using modern second generation monolithic stationary phase High Resolution Chromolith RP-18e 100 x 4.6 mm (Merck, Germany) in combination with gradient elution. Mobile phase was consisting of the mixture of 95% phosphate buffer 10 mmol / l pH 6 5 and 5% acetonitrile (ACN), flow rate of the mobile phase was 2.5 ml / min, from 2.1. min 4 ml / min. For fluorescence detection of thiamin hydrochloride (TH), thiamin monophosphate (TMP) and thiamine pyrophosphate (TPP, thiamine diphosphate) was necessary to use chemical oxidation by potassium ferricyanide to thiochrome and its adequate esters having photoluminescence activity. Chromatographic method has been partially validated. Peak...
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Metabolomic analysis of plasma samples using ultra performance liquid chromatography and high resolution mass spectrometry
Machová, Karolína ; Solich, Petr (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Karolína Machová Consultant: Prof. RNDr. Petr Solich, CSc. Diploma Thesis Title: Metabolomic analysis of plasma samples using ultra performance liquid chromatography and high resolution mass spectrometry Metabolomics is a novel type of science which represents a complex methods of metabolite determination in biofluid e.g. plasma, urine, saliva, cerebrospinal fluid and sperm. The method describes cell mechanisms and finds wide assertion in disease diagnostics, biomarkers description, disease, pathophysiology and in clinical practice. It is vital to have a sensitive method because levels of metabolites are quite low. Metabolomic analysis uses mainly high-performance liquid chromatography combined with mass spectrometry for detection. However, other methods such as nuclear magnetic resonance spectroscopy and gas chromatography can also be used. Presented work is mainly focused on method standardization for identifying metabolites in clinical practice with the use of ultra-performance liquid chromatography and mass spectrometry. The primary outcome was to establish searching parameters for the operating software to automatically identify xenobiotics in provided sample and compare them with...
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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 of HPLC method for determination of vancomycine in clinical research
Kučerová, Kateřina ; Kujovská Krčmová, Lenka (advisor) ; Matysová, Ludmila (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Kateřina Kučerová Supervisor: RNDr. Lenka Kujovská Krčmová, Ph.D. Title of diploma thesis: Development of HPLC method for determination of vancomycine in clinical research The aim of this diploma thesis was to develop optimal conditions for the chromatographic determination of vancomycin for clinical routine practice. The chromatographic separation was carried out by KinetexTM C18 column, 2.6 μm particle size, 50 × 4.6 mm (Phenomenex, USA) in combination with potassium phosphate buffer (pH 4.5) and acetonitrile (90 : 10, v/v) as the mobile phase. As internal standard was chosen cefuroxime. For analytes determination UV detection at 220 nm wavelength was applied. After optimization of separation conditions, the method was applied to biological material. Samples were easily modified using the protein precipitation. The method was partially validated. Keywords: vancomycin, HPLC-UV, plasma samples
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Determination of neopterin in biological material
Arnoštová, Veronika ; Kujovská Krčmová, Lenka (advisor) ; Matysová, Ludmila (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Veronika Arnoštová Supervisor: RNDr. Lenka Kujovská Krčmová, Ph.D. Title of diploma thesis: Determination of neopterin in biological material The aim of this diploma thesis is to create an overview of present methods, which can be used for the determination of neopterin in biological material. Neopterin is a pteridine derivative, which belongs to the group of unconjugated pteridines. Its biosynthesis is based on the guanosinetriphosphate and is stimulated by the activity of the IF-γ. Neopterin is a useful marker of the activation of the immune system, it is also an important prognostic indicator of several diseases e.g. in malignant diseases, in HIV-positive patients or in patients after transplantation. Elevated levels of neopterin are found in many pathological states such as in acute and chronic viral infections, in tumor diseases, HIV patients, patients after transplantation with rejection, but also in certain neurological, cardiovascular and autoimmune diseases. The sample preparation procedure is an important part in neopterin analysis. For this reason, sample preparation methods are in this thesis discussed. Chromatographic techniques as HPLC and UHPLC and immunoassays as ELISA...
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Optimization of the HPLC method for determination of propranolol in liquid oral formulations
Koval, Tomáš ; Matysová, Ludmila (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Tomáš Koval Supervisor: doc. PharmDr. Ludmila Matysová, Ph.D. Title of diploma thesis: Optimization of the HPLC method for determination of propranolol in liquid oral formulations The purpose of thesis is the optimization and subsequent validation of HPLC method for determination of propranolol - hydrochloride in liquid oral formulations. A medicinal product containing propranolol - hydrochloride is used in the therapy of infantile haemangiomas as extemporaneous prepared solution. For method optimization were chosen Kinetex® C18 column (150x4,6 mm, size of particles 5 μm) and isocratic elution of mobile phase containing phosphate buffer (c=17,7 mM) and acetonitrile in 30:70 ratio, with pH adjusted to 2,2. Flow rate was set at 1,2 ml/min. According to results of the validation, was found that this developed method for the analysis of the product is suitable, and provides accurate and precise results. Keywords: propranolol hydrochloride, HPLC, optimization, Sodium benzoate, liquid oral formulations
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Testing antioxidant activity using luminol chemiluminescence system
Chládková, Gabriela ; Sklenářová, Hana (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Gabriela Chládková Supervisor: Doc. PharmDr. Hana Sklenářová, Ph.D. Consultant: Doc. RNDr. Miroslav Polášek, Csc. Title of Diploma Thesis: Testing antioxidant activity using luminol chemiluminescence systém The aim of the thesis was to compare various conditions for the testing of the antioxidant activity by means of the luminol chemiluminescent system in the flow system, and evaluation of dependence of the antioxidant efficacy on concentration. The antioxidant efficacy was described as dependency of the decrease of a chemiluminescence signal on the used concentration of an antioxidant. Efficacy of three substances - resveratrol, p-coumaric acid and trolox was observed. Trolox was used as a standard substance in evaluation of the antioxidant activity. The chemiluminescence system was based on a chemiluminescent reagent. In this particular case we used luminol in a sodium hydroxide solution or in a sodium carbonate solution adjusted to pH 10 or without pH adjustment. Luminol demonstrated chemiluminescence emission after oxidation with hydrogen peroxide in the presence of catalysts. As the catalyst, potassium hexacyanoferrate(III) or potassium hexacyanoferrate(II) were used. The working...
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UHPLC analysis of degradation products of tetracaine hydrochloride
Horáková, Michaela ; Matysová, Ludmila (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Michaela Horáková Supervisor: Ludmila Matysová Title of Diploma Thesis: Determination of Degradation Product of Tetracaine hydrochloride by Ultra-High-Performance Liquid Chromatography This diploma thesis deal with using UHPLC for determination of tetracaine hydrochloride and its degradation products, p-aminobenzoic acid and p-n-butylaminobenzoic acid. Separation was achieved by using Kinetex 5 µ XB- C18 100A column and the eluent, acetonitrile-buffer, when buffer containing water with phosphoric acid (pH=2.2) at a flow rate of 1.7 ml/min. Gradient elution was chosen, which began at 35 % of acetonitrile, in the second minute rised to 95 % of organic phase, and then descended to 35 % in the fourth minute. Ethylparaben was used as an internal standard. The wavelengths 300 nm for tetracaine hydrochloride and its degradation products, and 240 nm for internal standard were chosen for detection. Validation approved that this method provides accurate and precise results.
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