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Application of solvation mocel to retention description of selected compounds in liquid and gas chromatography
Jirkal, Štěpán ; Čabala, Radomír (advisor) ; Feltl, Ladislav (referee) ; Sýkora, David (referee)
(EN) The solvation model based on LSER was applied to study the retention behaviour of analytes in liquid and gas chromatography. In a first chapter, a retention description of 21 solutes was investigated by using the solvation model in a wide range of mobile phase composition methanol-water and acetonitrile-water. Generally, the retention of aromatic compounds was better described by the solvation model, compared to aliphatic compounds. Effect of the particular analytes used to formulate the LSER model on ability of retention description was studied. Different results of a retention estimation was achieved by using the regression set of compounds including aromatic solutes only or by contrast aliphatic solutes only. The solvation model developed on the basis of oxygen derivatives provided distinct results in comparison to model formulated with nitrogen derivatives only. The second chapter of this work, focused on gas chromatography, dealt with a description of retention of 152 isomers C5-C8 alkenes by the LSER model. The solvation descriptor L was obtained by using two estimation methods Havelec-Ševčík (HS) and Platts-Butina (PB), the descriptor E was calculated according to its definition. Two models for retention description of alkenes were constructed, the HS model and the PB model, derived...
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Development of high-performance liquid chromatography methods for determination of major components of tobacco
Rozkovcová, Lucie ; Jelínek, Ivan (advisor) ; Křížek, Tomáš (referee)
The aim of this work was development of high-performance liquid chromatography method with DAD detection for determination of nicotine in tobacco. Standard operating procedure used by World Health Organization was chosen as comparison of the developed method. Optimized high performance chromatography method is suitable for determining nicotine in tobacco. Limit of detection for this method was 0,0003 mg/ml and limit of quantification was 0,0010 mg/ml. Optimization of preparation of samples was significant part of this thesis. Sample preparation procedure was made substantially easier in comparison to other commonly used methods. Nicotine content was determined from real tobacco leaves samples, cigarette tobacco filler, nicotine cartridge for electronic cigarettes and pipe tobacco. Satisfactory relative standard deviation was achieved for all types of samples. Next part of this thesis focused on study of determining polyphenols using high-performance liquid chromatography with diode array detector. Chosen analytes were chlorogenic acid, caffeic acid, rutine, scopoletine and quercitrine. Among the five tested analytes, the highest sensitivity was achieved for chlorogenic acid and caffeic acid. All of the analytes achieved low limits of detection and quantification. Key words Liquid chromatography,...
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Application of solvation mocel to retention description of selected compounds in liquid and gas chromatography
Jirkal, Štěpán
(EN) The solvation model based on LSER was applied to study the retention behaviour of analytes in liquid and gas chromatography. In a first chapter, a retention description of 21 solutes was investigated by using the solvation model in a wide range of mobile phase composition methanol-water and acetonitrile-water. Generally, the retention of aromatic compounds was better described by the solvation model, compared to aliphatic compounds. Effect of the particular analytes used to formulate the LSER model on ability of retention description was studied. Different results of a retention estimation was achieved by using the regression set of compounds including aromatic solutes only or by contrast aliphatic solutes only. The solvation model developed on the basis of oxygen derivatives provided distinct results in comparison to model formulated with nitrogen derivatives only. The second chapter of this work, focused on gas chromatography, dealt with a description of retention of 152 isomers C5-C8 alkenes by the LSER model. The solvation descriptor L was obtained by using two estimation methods Havelec-Ševčík (HS) and Platts-Butina (PB), the descriptor E was calculated according to its definition. Two models for retention description of alkenes were constructed, the HS model and the PB model, derived...
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The analysis of cerebrospinal fluid in patients with hydrocephalus
Chlupáčová, Tereza ; Kolátorová, Lucie (advisor) ; Dušková, Michaela (referee)
Normotensive hydrocephalus (NPH) is a neurodegenerative disease that occurs mainly in patients of high age. The disorder causes accumulation of cerebrospinal fluid (CSF), which leads to enlargement of ventricles and pressure exerted on cerebral structures. Clinical manifestations (gait disorders, development of dementia, incontinence) can be easily confused with symptoms of other neurodegenerative diseases; unlike other such disorders, however, NPH can be treated by surgery, if diagnosed in time. Patients are indicated for the procedure by a lumbar drainage test. There are currently no reliable laboratory biomarkers known that could be the basis of NPH diagnostics. In the past, steroids proved to be linked to neuronal activity in neurodegenerative diseases with the help of specific diagnostic markers. An instrumental method has been developed for the purposes of this thesis and it was used to gauge the level of certain steroids in CSF in a sample group of NPH patients and a control sample group of healthy individuals. A significant difference has been found in levels of aldosterone and cortisone. Aldosterone was higher in NPH sufferers, while cortisone levels were higher in the control group. It is crucial to differentiate patients with NPH from patients with similar clinical manifestations during...
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HPLC-MS determination of active biological compounds in Elbe river
Ďuráčová, Miloslava ; Vytlačilová, Jitka (advisor) ; Sklenářová, Hana (referee)
Charles University in Prague Pharmaceutical Faculty in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Miloslava Ďuráčová Consultant: RNDr. Jitka Vytlačilová, Ph.D. Title of thesis: HPLC-MS determination of active biological compounds in Elbe river Biologically active compounds have a various ways to enter environment. They can occur as pesticides, cosmetics and pharmaceutics and their metabolites. Such compounds are classified as environmental contaminants. There is a increased environmental concentration connected with increasing consumption of biologically active compounds. There is a urgent need to follow the effect on the different parts of ecosystem and levels of biologically active compounds. This work was prepared during the cooperation with Povodí Labe state enterprise. A novel analytical method was described in the experimental part of this thesis. I was employed to evaluate biologically compounds levels in the surface water samples. This method is now routinely used. 10 out of 19 evaluated compounds reached concentrations higher than LOD. Acetaminophene (559 ng/l, Králický stream), gabapentine (457 ng/l Elbe, LB Schmilka), cotinine (139 ng/l, Králický potok) were the biologically active compounds with the highest found concentrations. Keywords: HPLC-MS analysis,...
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Preparation and characterization of cyanide hydratase from Aspergillus niger and nitrilase from Arthroderma benhamiae
Hradilová, Iveta ; Vaněk, Ondřej (advisor) ; Martínek, Václav (referee)
Nitrilases are well known for their unique property to effectively convert nitriles into corresponding carboxylic acids and ammonia. They can also form amides as by-products. In contrast to nitrile hydratases they do not require cofactors or prosthetic groups. The research in this work is focused on nitrilase from filamentous fungus Arthroderma benhamiae and cyanide hydratase from Aspergillus niger K10. Genes of these enzymes were expressed using pET-30a(+) plasmid in the bacterium Escherichia coli strain BL21-Gold (DE3). The products obtained were purified by a series of ion exchange chromatography and gel filtration and subsequently characterized with respect to oligomeric state of the protein and its usability for protein crystallography. To obtain information regarding the structural arrangement of the individual proteins, electrophoretic separation in polyacrylamide gel, gel filtration, analytical ultracentrifugation, mass spectrometry, dynamic light scattering and drop coating deposition Raman spectroscopy were used. Keywords: nitrilase, cyanide hydratase, Aspergillus niger, Arthroderma benhamiae, liquid chromatography (In Czech)
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HPLC and LC-MS/MS methods for diagnosis of inherited metabolic diseases
Bártl, Josef
This dissertation thesis is focused on development and optimization of high- performance liquid chromatography (HPLC) and tandem mass spectrometry (LC-MS/MS) methods, and its utility for diagnosis of inherited metabolic diseases. The first thematic part describes a comprehensive laboratory approach to diagnostics of patients with hereditary xanthinuria by determination of specific markers and enzyme activity. For this purpose HPLC method with diode array detection for measurement of hypoxanthine, xanthine, allopurinol and oxypurinol in urine and plasma and HPLC method with fluorimetric detection for analysis of pterin and isoxanthopterin in plasma were employed. These methods were successfully applied in clinical practice to ascertain two patients with hereditary xanthinuria type I. The second thematic part aims at developing and clinical application of new LC-MS/MS method for simultaneous determination of total homocysteine (tHcy), methionine (Met) and cystathionine (Cysta) in dried blood spots (DBS) and plasma. The results demonstrated the clinical utility of this method for detection of patients with homocystinuria and possibility to distinguish between defects in the remethylation and transsulfuration pathways of homocysteine metabolism. Due to ease of DBS collection and sample transportation...
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Secondary metabolites, allelopathy and their significance in the invasion of Heracleum mantegazzianum
Jandová, Kateřina ; Dostál, Petr (advisor) ; Frouz, Jan (referee) ; Valterová, Irena (referee)
One theory concerning the invasiveness of exotic plants suggests that they exude allelopathic compounds that are novel in areas being invaded. Here, I tested for allelopathic effects of root exudates of the invasive plant Heracleum mantegazzianum (giant hogweed) in a series of experiments both in vitro and in the garden, and compared them with effects of its native congener H. sphondylium (common hogweed) and two less related native species. Moreover, I addressed intraspecific variability of allelopathy by comparing effects of 41 different maternal lines of H. mantegazzianum sampled from several areas and populations within the Czech Republic. Not only the effects, but also the identity of allelopathic compounds was investigated. In order to test also for the interaction of allelochemicals with soil microorganisms, I analysed the composition of soil microbial communities expressed as phospholipid and neutral lipid fatty acids (PLFA and NLFA, respectively) together with nutrient conditions and light availability at invaded sites in the Czech Republic. The results show that H. mantegazzianum is able to exert phytotoxic effects in vitro as well as in the garden experiment; however, its effects did not differ from the inhibition caused by the native species tested. Variation partitioning among areas,...
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HPLC and LC-MS/MS methods for diagnosis of inherited metabolic diseases
Bártl, Josef ; Jelínek, Ivan (advisor) ; Čabala, Radomír (referee) ; Cvačka, Josef (referee)
This dissertation thesis is focused on development and optimization of high- performance liquid chromatography (HPLC) and tandem mass spectrometry (LC-MS/MS) methods, and its utility for diagnosis of inherited metabolic diseases. The first thematic part describes a comprehensive laboratory approach to diagnostics of patients with hereditary xanthinuria by determination of specific markers and enzyme activity. For this purpose HPLC method with diode array detection for measurement of hypoxanthine, xanthine, allopurinol and oxypurinol in urine and plasma and HPLC method with fluorimetric detection for analysis of pterin and isoxanthopterin in plasma were employed. These methods were successfully applied in clinical practice to ascertain two patients with hereditary xanthinuria type I. The second thematic part aims at developing and clinical application of new LC-MS/MS method for simultaneous determination of total homocysteine (tHcy), methionine (Met) and cystathionine (Cysta) in dried blood spots (DBS) and plasma. The results demonstrated the clinical utility of this method for detection of patients with homocystinuria and possibility to distinguish between defects in the remethylation and transsulfuration pathways of homocysteine metabolism. Due to ease of DBS collection and sample transportation...
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