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Development of a HPLC-MS/MS method for determination of L-dopa in human blood serum
Neumannová, Johana ; Kozlík, Petr (advisor) ; Křížek, Tomáš (referee)
The diploma thesis is focused on the development of a high-performance liquid chromatography in conjunction with tandem mass spectrometry detection (HPLC-MS/MS) for the determination of L-dopa in human blood serum. Levodopa is a dopamine precursor and is generally used to treat Parkinson's syndrome. The tandem mass spectrometry detection and the high-performance liquid chromatography method were optimized. The optimal conditions were as follows: HALO 90 Å Penta-HILIC column (150 x 2,1 mm; 2,7 µm), Advanced Materials Technology (USA). The mobile phase used was a mixture composed of 0,1% formic acid (component A) and acetonitrile (component B) under gradient elution. The total analysis time was 12,50 minutes, the flow rate was 0,4 ml min-1, a 5 µl sample was injected, the autosampler temperature was 15 řC and the column temperature was 40 řC. The following MRM transitions in positive mode were monitored: 1. levodopa: m/z 198,1 → m/z 152,1 (Q1: -10 V, CE: -16 V, Q3: -28 V) 2. deuterated levodopa (IS): m/z 201,1 → m/z 155,2 (Q1: -10 V, CE: -14 V, Q3: -28 V) The setting of the ion source was as follows: the nebulization gas flow 3 l min-1, the drying gas flow 10 l min-1, the internal temperature 300 řC, the capillary voltage 3 000 V, the desolvation capillary temperature 250 řC. In the end of the work,...
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Analysis and characterization of liposomes using capillary electrophoresis
Orgoníková, Andrea ; Křížek, Tomáš (advisor) ; Coufal, Pavel (referee)
4 Abstract This diploma thesis deals with the analysis of three different types of liposomes using capillary electrophoresis with UV-VIS and laser induced fluorescence detection and the use of liposomes as a pseudostationary phase for analyte separations by liposome electrokinetic chromatography method. In experiments with DSPC-DSPG-PEG2000-DMPE liposomes encapsulating 5-fluoruracil, the peak of liposomes with encapsulated 5-fluoruracil and the peak of free 5-fluoruracil were successfully identified in the electropherograms. Both peaks showed the same absorption spectrum in the UV region, thus confirming their identity. It was proved that capillary electrophoresis with UV-VIS detection is useful for the separation and detection of free and encapsulated drug, which is necessary to determine the efficiency of encapsulation. By monitoring the change of effective analyte mobility after the addition of liposomes to the background electrolyte the applicability of the investigated liposomes in liposome electrokinetic chromatography was evaluated. A change in mobility was observed for negatively charged 5-fluoruracil and positively charged tryptamine and p-toluidine. The absolute value of the effective mobility of negatively charged 5-fluoruracil decreased by 18.2 % due to interactions with liposomes and effective...
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Electrophoretic determination of oxidized and reduced form of glutathion in plant material
Durychová, Eva ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
This work is based on a previously published method for the determination of twenty proteinogenic amino acids by capillary electrophoresis with contactless conductivity detection and deals with possibility of its use for the determination of oxidized (GSSG) and reduced (GSH) forms of glutathione with amino acids. The theoretical part firstly deals with brief introduction of the properties and importance of glutathione and its protection against autooxidation, especially by alkylation with N-ethylmaleimide (NEM), which binds to GSH to form its alkylated form (GSH-NEM). Then, a brief summary of the available methods for determination of glutathione is given, with a focus on the already developed capillary zone electrophoresis methods. Finally, it briefly presents the electrophoretic method for the determination of twenty proteinogenic amino acids on which this work is based. In the experimental part, the integration of the determination of both forms of glutathione into the method for the determination of proteinogenic amino acids is presented. First, a simplified, less time consuming method was tested in shorter 50.0 cm capillary without hydroxyethylcellulose, where both forms of glutathione were separated at baseline within 10 minutes. Relative standard deviations of peak areas (GSH 2.1%; GSSG...
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The study of the electrochemical and chemical oxidative degradation of salicylic and acetylsalicylic acid
Tonnerová, Barbora ; Kubíčková, Anna (advisor) ; Křížek, Tomáš (referee)
The aim of this Thesis was to study the degradation of salicylic acid by the electrochemical oxidation. The electrochemical oxidation of salicylic acid was compared to the electrochemical oxidation of acetylsalicylic acid. Two electrochemical cells were tested - batch cell and thin- layer flow-cell. The newly developed and validated method of ultra-high performance liquid chromatography has been used to analyse the degradation products, salicylic acid and acetylsalicylic acid. The optimal analysis was made by the Kinetex column C18 (2,1 x 100 mm; 1,7 µm) and mobile phase with two components - acetonitrile and water with addition of 0,1% formic acid. These two components of the mobile phase were mixed by the gradient program from 10 % to 60 % (v/v) acetonitrile. The mobile phase flow was set to 0,3 ml min-1 and the volume of injection was 2 µl. The detection was performed by photodiode array detector at the wavelength 240 nm. Total time of the analysis was 11 minutes. The electrochemical degradation has been studied by an anodic oxidation in a flow cell with a boron-doped diamond (BDD) anode and a stainless-steel cathode. The samples of salicylic acid and acetylsalicylic acid was dissolved in the ammonium acetate, concentration 200 mmol dm-3 at pH=4,0. The 20 % oxidation rate of salicylic acid was...
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Separation and determination of possible products of enzymatic cleavage of 4-nitrophenyl-N,N'-diacetyl-β-D-chitobioside using capillary electrophoresis
Velvarská, Romana ; Křížek, Tomáš (advisor) ; Kalíková, Květa (referee)
This work deals with the development and optimization of conditions of a method that can be used to compare the activity of the enzyme β-N-acetylhexosaminidase in hydrolysis of a natural substrate and a chromogenic substrate, which is often used in the study of enzyme kinetics. As a substrate, 4-nitrophenyl-N,N'-diacetyl-β-D-chitobioside was selected for cleavage. This oligosaccharide contains bond, which the enzyme cleaves in the natural substrate, and the bond that occurs in the chromogenic substrate. To determine the products arising from enzymatic hydrolysis of 4-nitrophenyl-N,N'-diacetyl-β-D-chitobioside, capillary zone electrophoresis was used. First, it was necessary to find the optimal composition of the electrolyte, its pH and concentration. The optimal background electrolyte was a solution of sodium tetraborate at a concentration of 25 mmol/l and a pH of 10.25. Subsequently, repeatability, calibration curves and linearity, limit of detection and limit of quantification were investigated. Repeatability of migration times ranged up to 0.6%, the repeatability of peak areas between 2.5 and 6.3%. Limits of detection were ranging from 0.005 to 0.120 mmol/l. Finally, the optimized method was successfully used to monitor the actual enzyme cleavage.
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Czech atomic law
Křížek, Tomáš ; Drobník, Jaroslav (advisor) ; Damohorský, Milan (referee)
The Czech Nuclear law The Nuclear law is an importnat part of the Environmental law. The Nuclear law regulates the peaceful use of nuclear energy and of inonizing radiation, which are the activities potentially dangerous for the environment. This area is a component of a special part of the Environmental law. The ionizing radiation is classified as a particular source of hazard to the environment. This diploma thesis deals with the Czech Nuclear law, and that by means of characterization of a valid legal regulations in the context of international and European law and by means of an analysis of the questions of building and operation of nuclear power stations in Czech republic. The main source of the Czech legal regulation is the Act No. 18/1997 Coll., on peaceful use of nuclear energy and ionizing radiation (Nuclear law) and changes and amendments of some laws. The Nuclear law is a high-quality legal regulation of the peaceful use of nuclear energy and ionizing radiation, which respects the requirements of the international and European law, comparable with the legal regulations of other advanced countries. After a short presentation of the history of development of nuclear energy and ionizing radiation use, the first part of this thesis oulines both the international and European legal regulations of this...
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The club development of american football in the Czech Republic
Dundáček, Jan ; Janák, Vladimír (advisor) ; Křížek, Tomáš (referee)
Title: The club development of american football in the Czech Republic In the first part of this thesis American football is introduced, explaining basic game principles and describing the game so that the reader can imagine what the sport is all about. The second part then focuses on examining the emergence of particular American football clubs in the Czech Republic. The whole period is divided into several stages, with a strong focus on finding a common denominator of individual events. The aim is to map American football in the Czech Republic since the establishment of the first club up to the present with emphasis on main themes and key periods. Recording individual events from the perspective of history is an important element for any future research. Keywords: American football, development, club, origin, history, Czech Republic
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Determination of proteinogenic amino acids by high-performance separation techniques
Hodek, Ondřej ; Křížek, Tomáš (advisor) ; Pacáková, Věra (referee) ; Tůma, Petr (referee)
(EN) Proteinogenic amino acids are key components of living organisms. Thus, the latest metabolomics research has focused on developing fast and sensitive methods for the determination of amino acids. In this context, this thesis contains two studies describing development of high-performance separation techniques for the quantification of amino acids. In the first study, a capillary electrophoresis method was developed for the determination of free amino acids in tobacco plants, particularly focusing on optimizing the extraction of amino acids from solid plant materials. The extraction procedure was optimized using design of experiments (DoE) to obtain the highest possible extraction yield of amino acids. Factors such as volume and concentration of the extraction solvent (hydrochloric acid) were assessed as the most significant. Subsequently, the optimal values of these factors were determined using response surface methodology (RSM). Lastly, proteinogenic amino acids were quantified using capillary electrophoresis with contactless conductivity detection and calibration with internal standard, which improved the precision of the method. The second study aimed at developing a supercritical fluid chromatography method for the determination of free proteinogenic amino acids in human plasma. The most...
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