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Development of a UHPLC-MS/MS method suitable for the determination of cabozantinib in blood serum
Kleinová, Jana ; Kozlík, Petr (advisor) ; Hraníček, Jakub (referee)
The aim of this Bachelor thesis was to develop a UHPLC-MS/MS method for the determination of Cabozantinib in blood serum. This method was used to monitor the release of the active substance in a model organism - the rat. First, the mass spectrometer setup was optimized, where MRM transitions for Cabozantinib and isotope-labelled Cabozantinib-D4 were found. For Cabozantinib, an MRM transition of 502.2 → 323.1 was found with optimal energy levels of Q1 = -26.0 V, CE = -40.0 V and Q2 = -22.0 V. For Cabozantinib-D4, the MRM transition of 506.2 → 327.0 was found with optimal energy levels being Q1 = -26.0 V, CE = -39.0 V and Q2 = -22 V. The method setup parameters were as follows: The chromatography column used was a Poroshell 120 EC-C18, 2.1x50 mm, 1.9 um, (Agilent Technologies). The mobile phase consisted of acetonitrile (B) and formic acid (A), the flow rate of the mobile phase was 0.350 ml/min, the analysis time was 5.5 min and the injection volume was 1 μl at gradient elution (time: 0; 0.5; 2.0; 3.0; 3.5; 4.0; 5.5 min, B: 10; 10; 60; 100; 10; 10 %). The method was linear (weighted regression 1/x2 ) with a regression coefficient equal to 0.9978, showing an excellent linearity of the method. The precision (relative standard deviation) of the method was within 14 %. The accuracy (relative error) was...
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Comparison of different tandem mass detection scans in the analysis of cannabidiol
Tichá, Tereza ; Kozlík, Petr (advisor) ; Kubíčková, Anna (referee)
The aim of this bachelor thesis was to compare different tandem mass spectrometer scans in order to develop an LC-MS/MS method suitable for the determination of cannabidiol in rat serum. In this bachelor thesis the mass spectrometer and high-performance liquid chromatography method were optimized. The optimum parameters were as follows: chromatographic column Acquity UPLC BEH C18 50 × 2,1 mm, 1,7 μm from Waters (Wexford, Ireland). The mobile phase consisted of methanol and water of LC-MS purity. The flow rate of the mobile phase was 0,4 ml/min, the column temperature 40 řC, the temperature in an autosampler 15 řC, the sample injection volume was 1 μl and the analysis time was 7 minutes under gradient elution conditions. The MRM transitions of highest intensity were monitored. For CBD, the MRM transition in positive mode was 315,2 → 193,1 (Q1 = -10 V, CE = -24 V, Q3 = -20 V), for isotopically labeled cannabidiol D3 was chosen 318,4 → 196,0 (Q1 = -10 V, CE = -23 V, Q3 = -20 V). The calibrations in solvent and rat serum were performed in MRM and SIM mode. No calibration was constructed for the SCAN mode due to its low selectivity, which made it able to detect cannabidiol only from concentration of 312,5 ng/ml. The highly selective MRM was able to operate over the entire observed concentration range of...
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Sample preparation for analyzing oligomeric forms of amyloid beta by mass spectrometry
Mrkvičková, Simona ; Cvačka, Josef (advisor) ; Kozlík, Petr (referee)
Alzheimer's disease is the most widespread chronic and progressive neurodegenerative disease. The main problem of the disease is the reduction and damage of nerve cells. This leads to loss of brain mass and memory impairment. The disease mainly affects the elderly, for whom it is the most common cause of dementia. Around 50 million people suffer from this disease worldwide, and the number is growing. Alzheimer's disease thus represents a serious public health problem. The pathogenesis of this disease is controlled by the production and deposition of amyloid beta peptide. This peptide tends to aggregate. Its lower oligomeric forms are water-soluble and have a toxic effect on nerve cells. Amyloid beta oligomers therefore represent a possible target for the diagnosis of Alzheimer's disease. Oligomeric forms of beta amyloids have a non-covalent character, which can negatively affect their stability during analyzes in model and biological samples. In this bachelor thesis, suitable conditions for the preparation of model amyloid beta samples were sought. It was observed how the distribution of oligomeric forms changes under the influence of solvents, salts and other additives. The effect of pH on the incubation of amyloid beta in ammonium acetate and water was also investigated. Finally, a method for the...
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Development of LC-MS method for the study of topotecan
Mudrová, Barbora ; Širc, Jakub (advisor) ; Kozlík, Petr (referee)
4 Abstract Topotecan (TPT) is a chemotherapeutic agent used to treat retinoblastoma, the most common malignant primary intraocular tumor in children. This drug undergoes a pH-dependent hydrolysis of the lactone ring creating an open-ring carboxylate form, while only the lactone form exhibits an antitumor activity. For clinical studies, it would be useful to develop a highly sensitive analytical method to monitor the concentration of both forms of TPT. In this thesis, the method of determination of the TPT forms by liquid chromatography with fluorescence or tandem mass detection were investigated. A rapid, selective and highly sensitive ultra-high performance liquid chromatography (UHPLC-MS/MS) method was developed for the determination of both forms of TPT in water, porcine plasma and vitreous humor. The separation was performed at 15 řC on a Waters Acquity-UPLC BEH C18 column (2.1 mm × 50 mm, particle size 1.7 µm) with a linear gradient elution. The method showed an excellent linear range of 0.4-120 ng/ml for TPT lactone and 0.8-120 ng/ml for TPT carboxylate in blood plasma and vitreous. In water, the linear range was 0.5-100 ng/ml for the lactone form of TPT and 1.0-100 ng/ml for TPT carboxylate. The effect of pH of TPT aqueous sample and the pH of the aqueous component of the mobile phase on the change...
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Effect of elution solution on the enrichment and purification of glycopeptides using SPE-HILIC
Chobotová, Michaela ; Kozlík, Petr (advisor) ; Křížek, Tomáš (referee)
Changes in protein glycosylation are related to various diseases such as cancer or Alzheimer's disease. Unfortunately, the analysis of glycoproteins remains a difficult task due to the low proportion of glycopeptides compared to peptides obtained after glycoprotein cleavage. As a result, it is almost impossible to analyze glycopeptides without specific enrichment steps. A suitable method for the enrichment of glycopeptides is solid phase extraction (SPE) using the principle of hydrophilic interaction liquid chromatography (HILIC). Since the choice of solvent significantly affects the enrichment of glycopeptides, the aim of this thesis was to compare the effect of different solvents on the efficiency of glycopeptide extraction using SPE-HILIC. The extraction was carried out on a silica column modified with aminopropyl groups. Model analytes were glycopeptides obtained by tryptic digestion of human IgG. During the comparsion of acetonitrile, methanol and isopropanol, acetonitrile was chosen as a suitable elution solvent. During the conditioning and washing step, the content of acetonitrile was changed (65%, 75% and 85%), whereby the elution of glycopeptides is accelerated as the amount of acetonitrile decreases. In the elution step, the effectiveness of different acetonitrile contents (5%, 10% and...
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Use of separation and mass spectrometric techniques in the diagnosis of Alzheimer's disease
Lebertová, Lucie ; Cvačka, Josef (advisor) ; Kozlík, Petr (referee)
This thesis focuses on the preparation of covalently cross-linked oligomeric forms of amyloid beta and their analysis by SDS-PAGE and MALDI-TOF. The covalently cross-linked oligomeric forms of Aβ(1-42) were formed by chemical and enzymatic reactions. The chains of amyloid beta are crossed over the amino acid residue of tyrosine to form a covalent bond. Such covalent cross-linking stabilizes the oligomeric structure, which allows for improved analysis of amyloid beta oligomers. The development of methods for the detection and quantification of amyloid beta oligomers may be of considerable benefit for the diagnosis of Alzheimer's disease, as it is the oligomeric forms of amyloid beta that are most strongly associated with Alzheimer's disease pathology. In the theoretical part, Alzheimer's disease is described with the above-mentioned problems in early diagnosis and therapy. In addition, background information on amyloid beta is given, and analytical methods for the separation and detection of amyloid beta used in research are described. The experimental part deals with the optimization of analytical methods MALDI-TOF and SDS-PAGE. In the MALDI-TOF, 2,5-dihydroxybenzoic acid and sinapic acid matrices were selected as suitable matrices for the determination of Aβ(1-42). The most suitable method of...
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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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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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