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Analysis of fluoroquinolone antibiotics in the soil
Vdolečková, Monika ; Řezáčová, Veronika (referee) ; Mravcová, Ludmila (advisor)
The thesis dealt with the global problem of the occurrence of pharmaceuticals in the environment, specifically fluoroquinol antibiotics (ciprofloxacin and enrofloxacin), which are among other critically important antibiotics. These drugs enter the soil environment through the application of already contaminated animal waste (e.g. manure) or through wastewater used in irrigation or through the application of treated sewage sludge, which may contain drug residues. Increasing concentrations of these antimicrobial drugs in the environment provide an environment for the emergence and spread of microbial resistance. The accumulation of these drugs then leads to the emergence of resistance to these antibiotics. The experimental part of the thesis focused on the optimization of a method for the extraction and subsequent determination of fluoroquinolones in soil and litter. For this purpose, parameters related to the extraction yield of fluoroquinolones from the soil matrix were optimized, such as sample weighting, selection of a suitable extraction medium, SPE performance, elution method from the SPE, etc. Extraction recoveries of 70 to 140 % were achieved as part of the optimization. Finally, the optimised method for extraction of fluoroquinolones from poultry litter and soil samples provided by the ÚKZÚZ was used. The experimental results confirm the long-term persistence of these drugs in the environment.
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Use of LC/MS technique to analysis of some physiologically active natural compounds
Trčková, Marie ; Čáslavský, Josef (referee) ; Kočí, Radka (advisor)
Presented work is focused on application of combined instrumental method RP-HPLC/ESI-MS in analysis of several groups of natural compounds with positive physiological activities. Especially some antioxidants were studied in commonly and abundantly consumed food. Moreover some other substances than standard compounds were observed in complex dies. In conclusion the HPLC/ESI-MS method is comparatively advantageous in phenolic analysis, while another type of ionisation would be used in case of carotenoid compound.
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Study of phenolic compounds in some biologic materials using method LC/MS
Měřínská, Radana ; Zemanová, Jana (referee) ; Kočí, Radka (advisor)
Presented bachelor thesis was focused on suitable methods for analysis of phenolics, especially modern instrumental methods were studied. In experimental part some standard phenolic compounds were analysed using combined technique HPLC/ESI-MS with off-line and/or on-line detection. Mass spectra of individual stadards and/or mixtures were determined in MS full scan and/or MS/MS full scan mode. Ionisation of chosen standard compounds was done in pozitive and/or negative mode. Parent ions of followed compounds were identified: reserpine (PI 609.9, NI 607.7), acid chlorogenic (NI 353), quercetin (PI 303.5, NI 301.5), rutin (PI 611, NI 609), morin (PI 303.1, NI 301.1). Typical fragments of studied standards were produced using MS/MS full scan mode. By fragmentation characteristic product ions were identified: acid chlorogenic (NI 191), quercetin (PI: 229.1 and 257.1; NI: 179.0 and 151.0), rutin (in MS/MS NI: 301; in MS3 NI: 179, 151.1), morin (PI: 285.1 a 257.1; NI: 283.2, 273.2, 257.1, 229.1). Optimal conditions of phenolic HPLC separation were determined including gradient elution and Restek column use. In conclusion on-line HPLC/MS detection using negative ionization was observed as the best configuration for phenolic analysis.
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Analysis of fluoroquinolone antibiotics in the soil
Vdolečková, Monika ; Řezáčová, Veronika (referee) ; Mravcová, Ludmila (advisor)
The thesis dealt with the global problem of the occurrence of pharmaceuticals in the environment, specifically fluoroquinol antibiotics (ciprofloxacin and enrofloxacin), which are among other critically important antibiotics. These drugs enter the soil environment through the application of already contaminated animal waste (e.g. manure) or through wastewater used in irrigation or through the application of treated sewage sludge, which may contain drug residues. Increasing concentrations of these antimicrobial drugs in the environment provide an environment for the emergence and spread of microbial resistance. The accumulation of these drugs then leads to the emergence of resistance to these antibiotics. The experimental part of the thesis focused on the optimization of a method for the extraction and subsequent determination of fluoroquinolones in soil and litter. For this purpose, parameters related to the extraction yield of fluoroquinolones from the soil matrix were optimized, such as sample weighting, selection of a suitable extraction medium, SPE performance, elution method from the SPE, etc. Extraction recoveries of 70 to 140 % were achieved as part of the optimization. Finally, the optimised method for extraction of fluoroquinolones from poultry litter and soil samples provided by the ÚKZÚZ was used. The experimental results confirm the long-term persistence of these drugs in the environment.
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Methods of monitoring the profile of carnitine metabolites in urine and use in clinical diagnosis
NOVÁKOVÁ, Martina
This study deals with the profiling of carnitine metabolites in the urine of the patients with breast cancer. L-carnitine is a chemical compound that is very important for metabolism of lipids, and therefore some current research suggests that it could serve as a means for the treatment of cancer affecting various organs or tissues. The theoretical part deals with the general characteristics and a description of the L-carnitine and its effect on the body. This part is divided into chapters that summarize various information about L-carnitine and acylcarnitines, for example the metabolism, different effects in the body including a description of responses to the lack of these substances, or methods that can be used for the detection of L-carnitine levels. Methods of monitoring L-carnitine were described in detail and were compared with previously published scientific studies. The authors of scientific studies show procedures, advantages and disadvantages of the proposed methods for determining L-carnitine and acylcarnitines, that were examined in this work in detail and the method for the determination of carnitine metabolites in urine has been proposed. A brief description of the most using methods for the determination of L-carnitine metabolites by liquid chromatography mass spectroscopy (LC / MS) is done the end of this part. I have been motivated with a positive results of studies and I have used this method for analysis in my work. In the research part of my study, the sample preparation protocol and conditions for the application of the method are described. The samples from the hospital of České Budějovice have been obtained from patients with fresh breast cancer diagnosis. In this part, the treatment of samples prior to detection, the used materials, equipment, chemicals, process and device settings for analysis were described. The method provided chromatograms and mass spectra of the individual substances. Samples of patients were compared with samples of control group of healthy women, which enabled us to observe possible differences. For clarity, the graph was drawn up and it was possible to monitor only slight differences between the levels of carnitine metabolites in women with breast cancer versus the control samples. This difference is not statistically significant, especially because the variability between individuals is higher of an order of magnitude than the actual differences between the two groups. This result is due to the fact that the study has not included additional information on patients such as information about their medications or health style, the overall condition of the body, etc. A detailed study requires a systematic and very time consuming work, which was not the aim of my thesis. Liquid chromatography with hydrophilic interactions method associated with mass detector used for monitoring of L-carnitine and its metabolites profiles gives positive results, is not time consuming, affordable and above all allow suitable for the detection of substances that have been selected for my work.
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Use of LC/MS technique to analysis of some physiologically active natural compounds
Trčková, Marie ; Čáslavský, Josef (referee) ; Kočí, Radka (advisor)
Presented work is focused on application of combined instrumental method RP-HPLC/ESI-MS in analysis of several groups of natural compounds with positive physiological activities. Especially some antioxidants were studied in commonly and abundantly consumed food. Moreover some other substances than standard compounds were observed in complex dies. In conclusion the HPLC/ESI-MS method is comparatively advantageous in phenolic analysis, while another type of ionisation would be used in case of carotenoid compound.
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Study of phenolic compounds in some biologic materials using method LC/MS
Měřínská, Radana ; Zemanová, Jana (referee) ; Kočí, Radka (advisor)
Presented bachelor thesis was focused on suitable methods for analysis of phenolics, especially modern instrumental methods were studied. In experimental part some standard phenolic compounds were analysed using combined technique HPLC/ESI-MS with off-line and/or on-line detection. Mass spectra of individual stadards and/or mixtures were determined in MS full scan and/or MS/MS full scan mode. Ionisation of chosen standard compounds was done in pozitive and/or negative mode. Parent ions of followed compounds were identified: reserpine (PI 609.9, NI 607.7), acid chlorogenic (NI 353), quercetin (PI 303.5, NI 301.5), rutin (PI 611, NI 609), morin (PI 303.1, NI 301.1). Typical fragments of studied standards were produced using MS/MS full scan mode. By fragmentation characteristic product ions were identified: acid chlorogenic (NI 191), quercetin (PI: 229.1 and 257.1; NI: 179.0 and 151.0), rutin (in MS/MS NI: 301; in MS3 NI: 179, 151.1), morin (PI: 285.1 a 257.1; NI: 283.2, 273.2, 257.1, 229.1). Optimal conditions of phenolic HPLC separation were determined including gradient elution and Restek column use. In conclusion on-line HPLC/MS detection using negative ionization was observed as the best configuration for phenolic analysis.
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Urine, its composition and the effect of age on the metabolic picture
MENHART, Roman
This work is aimed at monitoring the impact of age on the composition of urine. The correlation of age with the composition of urine was examined by focused research four selected analytes - creatinine, acetylcarnitine, carnitine and alanine in anonymous groups of people aged 15, 25, 35, 45 and 55 years. In the theoretical part the anatomy and function of the kidneys are generally described. The definiton of urine and organic and inorganic substances contained ion are defined. Described are factors that contribute to changes in urine composition such as body mass index, skin color and especially age, which is the main subject of this research. The methods used for analyzing urine in clinical practice but also in research are also stated. Procedures for collection, preservation and preanalytical preparation of urine are included in methodological section. The list of all chemicals, equipment, internal standards, standards, instrumentation used for the preparation and analysis of samples and HPLC/MS methodology are described. The output data from LC/MS method used were processed in a computer program XcaliburTM. There are the chromatograms and mass spectra of creatinine, acetylcarnitine, carnitine and alanine obtained by analyzing the 50 urine samples (in triplicate) of people of different ages. By using the clear graphs and tables peak areas for the individual substances were standardized by creatinine amount. Studied urine issues are very complex and can not be encompassed completely in the context of my thesis.
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Lipidome LC/MS Analysis in the Insect Adaptation and Development Studies
TOMČALA, Aleš
Insects represent very useful experimental model in various branches of biological research. The investigation is driven by economic importance of many insect species, and also by biological features of insects as model organisms such as short period of reproduction, easy breeding and manipulation and, in particular, the minimal regulatory requirements which are associated to the management of vertebrates. Here we report robust and efficient LC/MS/MS methodology for the determination of the physiologically important lipid molecular species in insects. The target metabolites represent polar glycerophos-phopholipids (GPL) and nonpolar lipids diacylglycerols (DG) and triacylglycerols (TG). Combination of the LC/MS data with the subsequent GC fatty acid analysis enables complete structural elucidation of particular lipid species including their fatty acid compositions. The developed methodology was applied to studies of the chill tolerance of the firebug Pyrhocorris appterus. Fields and laboratory experiments were conducted to separate the triggering effects of low temperature, desiccation and diapause progression on the physiological characteristics related to chill tolerance with emphasis on the restructuring of GPL composition. The same effect on the GPL composition was observed during acclimatization in the field and cold acclimation in laboratory. By contrast, the GPL changes related to desiccation and diapause progression were relatively small (Tomčala et al, 2006). In adults of Drosophila melanogaster it has been found that acclimation at 15, 20 and 25°C during preimaginal development affects thermal tolerance and composition of membrane GPLs. Low temperature acclimation was associated with increase in proportion of ethanolamine at the expense of choline in GPLS. Relatively small, but statistically significant changes in lipid molecular compositon were observed with decreasing acclimation temperature (Overgard et al, 2008). Hormonal treatment studies on insect model Locusta migratoria showed a heterogeneous distribution of individual DGs in haemolymph after the hormone application and revealed that mobilization of the DGs is molecular species-specific with the highest proportion of DG 16:0/18:1 and forming in summary about 20% of the total mobilized DG content. Additional analysis of fat body triacylglycerols revealed that the AKH mobilizes the DGs specifically with the preference of those possessing the unsaturated C18 fatty acids (FAs). The fat body FAs with more than 18 carbons did not participate on the mobilization (Tomcala et al, 2009). The LC/MS methodology was further applied to lipid composition studies of several samples with very diverse biological origin (fish, human blood etc.) and was proved to be universally applicable to the wide scope of biological samples.
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