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Modification of sorbent for SPE
Mrenková, Nikola ; Lochman, Lukáš (advisor) ; Váňová, Nela (referee)
CHARLES UNIVERSITY FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ Department of Pharmaceutical Chemistry and Drugs Analysis Candidate: Nikola Mrenková Supervisor: PharmDr. Lukáš Lochman, Ph.D. Title of thesis: Modification of sorbent for SPE Sample preparation is one of the most critical points in the chromatographic analysis of biological samples. The success of the chromatographic analysis depends on it. The complex character of biological samples and the low concentrations of target analytes have led to the development of selective sample preparation techniques. Because of its universality and selectivity, solid-phase extraction (SPE) is one of the most widely used methods. Graphene, due to its unique properties (large specific surface area, delocalized π electron system, hydrophobic character), has become a suitable candidate as a substance for the development of a new type of sorbent for SPE. The submitted thesis was focused on the study of the properties of a modified stationary phase by graphene-based sorbent for SPE. Specifically, it involved the coupling of graphene oxide (GO) sheets to aminosilica gel via an amide bond between the carboxyl groups of GO and the amino groups of aminosilica and the reduction of GO by hydrazine to reduced graphene oxide (rGO). Model analytes with different...
Determination of tyrosine metabolites III
Licková, Gabriela ; Kastner, Petr (advisor) ; Váňová, Nela (referee)
CHARLES UNIVERSITY FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ DEPARTMENT OF PHARMACEUTICAL CHEMISTRY AND PHARMACEUTICAL ANALYSIS Candidate: Gabriela Licková Supervisor: PharmDr. Petr Kastner, Ph.D. Consultant: Ing. Daniel Heblík Titile of thesis: Determination of tyrosine metabolites The topic of thesis was determination of tyrosine metabolites (epinephrine, norepinephrine, dopamine, L-DOPA) using derivatization reaction and later analysis using High-performance liguid chromarography with fluorescence detector. The important step of all work was optimalization of conditions of derivatization reaction. As derivatization agent it was used FMOC-Cl (fluorenylmethyloxycarbonyl chloride), which reacted with catecholamines together with borate buffer. Firstly, it was set the ideal concentration of agent (2,7 mM), also the time (15 min) and the temperature (40 řC) of reaction. The concetration (100 mM) and pH (8,0) of borate buffer was optimalized in the end. For effective separation it was necessary to set ideal conditions. As silica gel column was used Kinetex EVO C18 with core-shell particles. The compositions of mobile phase was necessary to optimalize and resulting mobile phase was composed from formic acid (20 mM) and acetonitrile in proportion 32:68. Used flow rate was 0,6 ml/min. The column was tempered...
HPLC determination of oxidative stress
Kalíšková, Tereza ; Váňová, Nela (advisor) ; Lochman, Lukáš (referee)
Reactive oxygen and nitrogen species have a physiological role in the organism, but their extensive production can damage cells and result in many diseases. By interaction of biomolecules with reactive oxygen and nitrogen species are biomolecules damaged. Damaged lipid, protein and DNA molecules then serve as biomarkers of oxidative stress and allow its evaluation. Oxidative stress can be caused by external factors such as drugs and then it may occur as adverse and toxic effects. It is important to monitor a drug's ability to induce oxidative stress to monitor drug safety. A high-performance liquid chromatography method coupled with tandem mass spectrometry has been developed to determine malondialdehyde from the cell pellet as a biomarker of lipoperoxidation. The greatest emphasis was given to the optimization of sample preparation by deproteination, derivatization and solid-phase extraction. The method was validated with acceptable specificity, accuracy, precision, recovery and matrix effect. The method complies with the requirements of the European Medicines Agency for the validation of bioanalytical methods. The method can determine intracellular malondialdehyde in the concentration range 0,1-2 μmol∙dm-3 . This method was successfully applied to the analysis of biological samples from in vitro...
Development and Validation of HPLC Method for Determination of 3-Hydroxykynurenine in Biological Material
Knoblochová, Dominika ; Kastner, Petr (advisor) ; Váňová, Nela (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Dominika Knoblochová Supervisor: PharmDr. Petr Kastner, Ph.D. Title of thesis: Development and Validation of HPLC Method for Determination of 3-Hydroxykynurenine in Biological Material 3-Hydroxykynurenine is one of the metabolites of tryptophan and is studied especially for its oxidative modulatory activity, which provides the ability to protect cells from oxidative stress. It is formed from kynurenine by catalysis of the enzyme kynurenine monooxygenase, whose activity is monitored at the Department of Pharmacology and Toxicology. It is necessary to quantify 3-hydroxykynurenine in a biological matrix for its study. The aim of this diploma thesis was to develop an HPLC method for the determination of 3-hydroxykynurenine in biological material using high performance liquid chromatography and subsequent method validation. During development, several mobile phases with different pH values were tested. The resulting mobile phase consisted of 100 mM formic acid (pH = 2.25) and methanol (95:5). Different types of stationary phases were also tested, from which a YMC TRIART PFP column with dimensions of 15 × 3 mm and a particle size of 5 µm was finally chosen. A UV-VIS...
Optimization of derivatization reaction conditions for GC-MS analysis of selected compounds
Jan, Tomáš ; Kučera, Radim (advisor) ; Váňová, Nela (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Candidate: Tomáš Jan Title of Thesis: Optimization of derivatization reaction conditions for GC-MS analysis of selected compounds Quinolinic acid is an endogenous molecule, downstream product of the kynurenine pathway, through which the amino acid L-tryptophane is metabolised. Quinolinic acid acts as an agonist at the NMDA receptor. Overactivity of this receptor leads to the cell death of neurons. Current research shows the connection between a high level of quinolinic acid and a higher risk of neurodegenerative diseases development and behavioural disorders. The level can be increased by the excessive intake of tryptophane (e.g. food supplements declaring calming effect and mental health support) or ongoing inflammation in the organism. The current work deals with the development of an analytical method for the determination of quinolinic acid in various biological matrices using GC/MS and complements methods analysing other metabolites of tryptophan. Derivatization by alkyl chloroformate was met with success. The selectivity was compared on column RTX-5MS containing non-polar stationary phase and polar column SLB-IL59 using ion...
Assessment of physico-chemical and pharmacokinetic properties of potential antitubercular agents
Nevosadová, Lenka ; Váňová, Nela (advisor) ; Štěrbová, Petra (referee)
This master's thesis focuses on determining the physio−chemical and stability properties of select groups of potential antituberculosis agents such as T, K, LV and G substances. The goal of this work was to expand the portfolio of information necessary as this for subsequent preclinical testing of new potential drugs. The theoretical work introduces the serious global infectious disease, tuberculosis, and the emerging resistance of Mycobacterium tuberculosis strains to current drugs. Furthermore, the developmental directions of new potential antituberculosis drugs and a description of the used analytical methods − spectrophotometry and HPLC analysis − are presented here. The experimental work is dedicated to the description of individual techniques which were used to determine the properties of the selected substances, such as its solubility in 5 % DMSO, stability in plasma, microsomal stability, stability in Šul media, and experimental determination of logP and logD. It is very clear from the experimental results how individual structural changes in the molecules affected some pharmacokinetic or physio−chemical properties of the given substance. For example reducing plasma stability by incorporating sulphur into the carbon liking chain or decrease of microsomal stability by using lacton form. The...
LC-MS/MS study of phase one in vitro biotransformation of potential drugs against Alzheimer's disease
Kuřátková, Aneta ; Kučera, Radim (advisor) ; Váňová, Nela (referee)
No treatment that would completely stop the progression of Alzheimer's disease has not been found yet. Recently used tacrine showed good results in the treatment of Alzheimer's disease, however long-term use led to chronic hepatotoxicity due to its metabolites. This master thesis deals with the compound 7-phenoxytacrine, one of the promising tacrine derivatives, which is one of the candidates for potential use in the therapy of Alzheimer's disease. Due to the formation of hepatotoxic metabolites of tacrine after the biotransformation in human liver, it appears necessary to identify the emerging metabolites of 7-phenoxytacrine molecule. Within this master's thesis in vitro biotransformation study of 7-phenoxytacrine using human liver microsomes was performed. High performance liquid chromatography with tandem mass spectrometry was used to determine the parent substance and the seventeen 7-phenoxytacrine metabolites. The analytical method showed the formation of six monohydroxylated and eleven dehydroxylated metabolites of 7-phenoxytacrine. Thus, we concluded that hydroxylation is the major metabolic reaction after in vitro microsomal biotransformation. In addition to the identification of metabolites, a quantification and microsomal stability study, including the determination of the amount of...
Application of LC-MS/MS methods to in vitro transport experiments
Salvová, Šárka ; Váňová, Nela (advisor) ; Kastner, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department: Deparrment of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Šárka Salvová Supervisor: PharmDr. Nela Váňová, Ph.D. Title of Thesis: Application of LC-MS/MS methods to in vitro transport experiments Gilteritinib, an antineoplastic drug, is used for targeted anticancer therapy. It belongs to the group of receptor tyrosine kinase inhibitors. Its structure is based on a pyrazinecarboxamide skeleton. So far, it has been chromatographically determined in biological material only from plasma. In this diploma thesis, gilteritinib was analyzed from Opti-MEM cell medium to be used in vitro transport experiments. Subsequently the LC-MS/MS method was optimized and validated. During progression and optimization of the LS-MS/MS method, the Thermo Finnigan LCQ Advantage Max Ion Trap was observed that with an increasing number of injections had been shown increasing signal suppression for gilteritinib and its internal standard (maraviroc), which reached up to 75 % after only 30 sprays. Contamination of the inlet to the ion transfer capillary, to which the ion source (electrospray) is placed diagonally, was also visible. Thereafter it was tested whether another construction of the ion source (orthogonal position of electrospray)...
Development and Validation of HPLC Method for Determination of 3-Hydroxykynurenine in Biological Material
Knoblochová, Dominika ; Kastner, Petr (advisor) ; Váňová, Nela (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Dominika Knoblochová Supervisor: PharmDr. Petr Kastner, Ph.D. Title of thesis: Development and Validation of HPLC Method for Determination of 3-Hydroxykynurenine in Biological Material 3-Hydroxykynurenine is one of the metabolites of tryptophan and is studied especially for its oxidative modulatory activity, which provides the ability to protect cells from oxidative stress. It is formed from kynurenine by catalysis of the enzyme kynurenine monooxygenase, whose activity is monitored at the Department of Pharmacology and Toxicology. It is necessary to quantify 3-hydroxykynurenine in a biological matrix for its study. The aim of this diploma thesis was to develop an HPLC method for the determination of 3-hydroxykynurenine in biological material using high performance liquid chromatography and subsequent method validation. During development, several mobile phases with different pH values were tested. The resulting mobile phase consisted of 100 mM formic acid (pH = 2.25) and methanol (95:5). Different types of stationary phases were also tested, from which a YMC TRIART PFP column with dimensions of 15 × 3 mm and a particle size of 5 µm was finally chosen. A UV-VIS...
Optimization of derivatization reaction conditions for GC-MS analysis of selected compounds
Jan, Tomáš ; Kučera, Radim (advisor) ; Váňová, Nela (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Candidate: Tomáš Jan Title of Thesis: Optimization of derivatization reaction conditions for GC-MS analysis of selected compounds Quinolinic acid is an endogenous molecule, downstream product of the kynurenine pathway, through which the amino acid L-tryptophane is metabolised. Quinolinic acid acts as an agonist at the NMDA receptor. Overactivity of this receptor leads to the cell death of neurons. Current research shows the connection between a high level of quinolinic acid and a higher risk of neurodegenerative diseases development and behavioural disorders. The level can be increased by the excessive intake of tryptophane (e.g. food supplements declaring calming effect and mental health support) or ongoing inflammation in the organism. The current work deals with the development of an analytical method for the determination of quinolinic acid in various biological matrices using GC/MS and complements methods analysing other metabolites of tryptophan. Derivatization by alkyl chloroformate was met with success. The selectivity was compared on column RTX-5MS containing non-polar stationary phase and polar column SLB-IL59 using ion...

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2 VAŇOVÁ, Naďa
1 Váňová, Nicol
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