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Comparison of detection techniques for analysis of saccharides by capillary electrophoresis
Vlčková, Nikol ; Křížek, Tomáš (advisor) ; Kozlík, Petr (referee)
The aim of this work was to compare detection techniques of capillary electrophoresis to determine saccharides. The techniques were contactless conductivity detection, direct and indirect UV detection and fluorescence detection. Determined saccharides were glucose, fructose and sucrose, lactose was employed as internal standard. For conductivity detection the capillary has internal diameter of 20 µm. Voltage applied to capillary was 15 kV and temperature was 25 řC. 40mM NaOH was used as BGE. The calibration dependence was measured between 0,005 - 0,5 mg/ml. Repeteabilityof peak areas and migration times was measured in two concentration levels of 0,02 and 0,2 mg/ml. Detection and quantification limits were between 1,0 - 5,8 µg/ml. RSD values for repeatability of peak areas and migration times were between 0,01- 8,64 %. Difference in recovery from real concentration of analytes was between 7 - 22 %. For other detection methods the capillary had inner diameter of 50 µm. Voltage applied to capillary for indirect UV detection was 28 kV and temperature was 30 řC. Wavelength for detection was 207 nm and 50mM glycylgylycine with pH value of 12,8 was used as BGE. The calibration dependence was measured between 0,1-1 mg/ml. Repeteability of peak areas and migration times was measured in two concentration...
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Development and optimization of HPLC method for determination of peroxide impurities in pharmaceutical excipients
Hrubcová, Kateřina ; Křížek, Tomáš (advisor) ; Sobotníková, Jana (referee)
The chemical stability of drugs can be affected by impurities that occur in commonly used excipients. Hydroperoxides are common trace impurities, especially in polymeric excipients, which can cause oxidative degradation of drugs. Several methods for the quantification of trace levels of hydroperoxides (hydrogen peroxide and organic hydroperoxides) are described in the literature. However, there are few studies dealing with the quantification of trace levels (ppm) of hydroperoxides in drug excipients. The goal of this thesis was the development and optimization of an HPLC method with UV detection for the determination of hydroperoxides in drug excipients, based on methods already described in the literature. The developed HPLC method is based on the determination of triphenylphosphine oxide as a product of the rapid and quantitative reaction of triphenylphosphine with hydroperoxides. Method development also included optimization of sample preparation prior to HPLC analysis. During the experimental work, the influence of individual experimental parameters and sample preparation conditions on the determination of hydroperoxides was determined. The optimized HPLC method used a Kinetex F5 column (100 × 4,6 mm, 2,6 µm) and a mixture of water and acetonitrile as the mobile phase with gradient elution. As...
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Electrokinetic chromatography in nonaqueous and mixed solvents: Determination of critical micellar concentration
Hamráková, Katarína ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
This thesis deals with the investigation of micelle formation following the addition of a surfactant to a background electrolyte in micellar electrokinetic chromatography and the transfer of these systems to nonaqueous and mixed solvents. The addition of organic solvents to the background electrolyte has a significant effect onmicelle formationandhence separation in electrokinetic chromatographyusinga micellar pseudo-stationaryphase. This effect has not yet been studied in depth. Information on the presence and properties of micelles in a given separation system is important for the understanding of separation processes but also for the development and optimization of analytical methods. In this work, the critical micellar concentration (CMC) of two surfactants, sodium dodecyl sulfate (SDS) and cetyltrimethylammoniumbromide (CTAB), was determinedby introducingthem into a mixed environment with non-aqueous solvents, acetonitrile and methanol. The critical micellar concentration was determined from the dependence of three variables on the surfactant concentration in the solution, namely, the effective mobility of the analytes and the viscosity and conductivity of the solution. Prior to the actual measurements of the effective mobility of the analytes, suitable analytes were selected,two which can...
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Oxidative degradation of paracetamol
Tůmová, Michaela ; Křížek, Tomáš (advisor) ; Kozlík, Petr (referee)
Paracetamol is a commonly used drug for newborn babies against increased physical temperature and pain. Therefore, it is necessary to determine the stability of this drug, which is an important factor for its safety and efficiency. One of the methods for determining the stability of drugs is the identification of their degradation products. This bachelor thesis is about the development and optimization of ultra high performance liquid chromatography method for determining paracetamol degradation products, which are formed by chemical or electrochemical oxidative degradation. A column UPLC CSH PHENYL-HEXYL (1,7 μm, 100 × 2,1 mm) was chosen as optimal for this separation. Furthermore, the aqueous component of the mobile phase was a 20 mM ammonium acetate solution, and the organic component was methanol. After optimization, a partial validation of the method was performed, during which the linear dynamic range, the limit of detection, the limit of quantification, repeatability of retention time and peak area, and recovery were evaluated. Chemical and electrochemical oxidative degradation was performed for paracetamol solution with a concentration of 1 mg/ml. Additionally, chemical degradation was performed with the addition of hydrogen peroxide with a concentration of 0,5 % and 3 % at a temperature of...
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Electrochemical oxidation of ibuprofen
Bolíková, Markéta ; Křížek, Tomáš (advisor) ; Kozlík, Petr (referee)
- 4 - Abstract This diploma thesis deals with the electrochemical oxidation of the active pharmaceutical ingredient ibuprofen as one of the parts of the stability studies of pharmaceutical products. The task is to ensure optimal conditions for the course of electrochemical oxidation, which produces the largest percentage of degradation products, to compare the flow and static experimental setup and its effect on the result of electrochemical oxidation and to identify the resulting degradation products. The examined conditions include the pH of the buffer used to dissolve ibuprofen, the potential applied to the working electrode and the duration of oxidation. Ultra-high performance liquid chromatography coupled with mass detection was used to separate and determine degradation products. Acquity UPLC BEH C18 (2.1 × 100 mm; 1.7 µm) was chosen as the separation column. The mobile phase consisted of an aqueous component (10mM ammonium formate with pH 3.0) and an organic component (acetonitrile). Detection was performed using a photodiode array detector at a wavelength of 222 nm and a quadrupole mass detector in the scan range m/z 50-400. The electrochemical oxidation was performed in a radial electrochemical flow cell and the conditions under which the largest percentage of degradation products formed were as...
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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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