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Comprehensive analysis of warning and defense compounds of true bugs by high-performance separation methods
Krajíček, Jan ; Bosáková, Zuzana (advisor) ; Feltl, Ladislav (referee) ; Ventura, Karel (referee)
Insects have developed many strategies of defence against predators in the course of evolution. The evolutionarily oldest and most widely used type of defence is chemical defence, followed by acoustic or optical defence. However, many species of insects use simultaneously multiple types of warning signals, which affect different sensory receptors of the given predator. Such a complex method of warning signals is called multimodal method. It may consist of a combination of simultaneous chemical and optical signals, or a combination of acoustic and optical signalling. The combination of chemical and optical signalling used against a predator is probably the most common form of multimodal signalling. The presented work deals with the analysis of biologically active substances, which participate in the defence mechanisms of a widespread species of insects - true bugs (Heteroptera). Pterin derivatives represent a large group of natural compounds derived from pteridin, bicyclic heterocycle, and they are found in virtually all living organisms from bacteria to vertebrates. In insects, they primarily serve as pigments, resulting for example in striking coloration of cuticles of Heteroptera. The first part of the dissertation was focused on identification and quantification of pterin derivatives in cuticles...
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Targeted analysis of branched-chain faty acid esters by LC/MS
Březinová, Marie ; Kuda, Ondřej (advisor) ; Vrkoslav, Vladimír (referee)
Lipidomics as a part of metabolomics is a fast-growing area of research due to the improvement in analytical techniques. This master thesis is focused on lipid extraction techniques optimization, using liquid liquid extraction and solid phase extraction as pre-separation methods and ultra performance liquid chromatography coupled with mass spectrometry for extraction and subsequent identification of branched-chain fatty acid esters (FAHFA - branched-chain Fatty Acid esters of Hydroxy Fatty Acids). This newly discovered class of lipid molecules is associated with insulin secretion, which could improve whole body and local glucose metabolism, providing potential for diabetes 2 type treatment. Solid phase extraction of biological samples was optimized on columns regarding to sorbent composition using reversed phase columns with modified styrene divinylbenzene polymer or octadecyl-bonded polymer and normal phase columns packed with silica gel. Column Strata SI-1 Silica was the most effective for FAHFA separation from biological samples. Chromatographic separation of FAHFA was performed on UPLC Ultimate 3000 RSLC equipped with Kinetex C18 1,7 µm, 2,1 x 150 mm column using gradient program. UPLC was coupled to QTRAP 5500/SelexION, a hybrid, triple quadrupole, linear ion trap mass spectrometer equipped...
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Comprehensive analysis of warning and defense compounds of true bugs by high-performance separation methods
Krajíček, Jan
Insects have developed many strategies of defence against predators in the course of evolution. The evolutionarily oldest and most widely used type of defence is chemical defence, followed by acoustic or optical defence. However, many species of insects use simultaneously multiple types of warning signals, which affect different sensory receptors of the given predator. Such a complex method of warning signals is called multimodal method. It may consist of a combination of simultaneous chemical and optical signals, or a combination of acoustic and optical signalling. The combination of chemical and optical signalling used against a predator is probably the most common form of multimodal signalling. The presented work deals with the analysis of biologically active substances, which participate in the defence mechanisms of a widespread species of insects - true bugs (Heteroptera). Pterin derivatives represent a large group of natural compounds derived from pteridin, bicyclic heterocycle, and they are found in virtually all living organisms from bacteria to vertebrates. In insects, they primarily serve as pigments, resulting for example in striking coloration of cuticles of Heteroptera. The first part of the dissertation was focused on identification and quantification of pterin derivatives in cuticles...
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Targeted analysis of eicosanoids by LC/MS
Moravcová, Marie ; Kuda, Ondřej (advisor) ; Kozlík, Petr (referee)
Eicosanoids represent large group of biologically active lipid metabolites syntesized from polyunsaturated fatty acids that play an important role in many physiological processes. To describe complex metabolism of these compounds, analytical methods including extraction from a biological sample using solid phase extraction and liquid chromatography coupled with mass spectrometer detection were used. Solid phase extraction of biological samples was optimized on four types of reverse phase columns of which column Strata X 60 mg/3 ml, 33 um (Phenomenex , USA) was the most effective. Also, alternative solid phase extraction of eicosanoids using columns with ion exchange sorbents and a column with normal phase were tested, but proved to be unsuitable for targeted analysis of eicosanoids. The extraction method yielding the best results - Strata X 60 mg/3 ml, 33 µm (Phenomenex, USA) was used for the separation of eicosanoids from mouse gonadal fat samples. Eicosanoids were analyzed by liquid chromatography and the separation mechanisms were tested on three UPLC core-shell columns of different lengths (50 mm, 100 mm, 150 mm). The most effective separation of prostaglandin E2 and prostaglandin D2 was achieved using the longest column Kinetex 150 mm × 2,1 mm, 2,6 µm. Furthermore, ionization parameters, such...
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Development of analytical methods for determination of phosphorylated components of bacterial cell membranes
Mikulecká, Jana ; Čabala, Radomír (advisor) ; Feltl, Ladislav (referee)
Phospholipids are dominant components of bacterial cell membranes, where they create double layers. Bacteria differ in their phospholipid composition determination of which can help in identification of important groups of microorganisms. Phospholipid composition of bacteria is influenced by many environmental factors, therefore its variation can be observed within one bacterial stem also. Because of its simplicity, thin layer chromatography is usually applied to identification and determination of bacterial phospholipids. Disadvantage of this method are the high demands of time, carefulness and skills of the analytical personnel. The increasing interest in the phospholipid double-layer promotes the detailed investigation of their fatty acid composition because the more detailed analyses allows for more information yield about bacteria. Gas chromatography hyphenated with mass spectrometry seems to be the best choice for these purposes. Fatty acid identity and total fatty acid content in phospholipid molecules could be determined by this method. Additionally, number, position and isomerism of double bonds and presence of other functional groups on hydrocarbon chain could be determined. Whereas a suitable and...
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Electrochemical determination of chlortoluron using carbon paste electrode
Houšková, Lucie ; Zima, Jiří (advisor) ; Vyskočil, Vlastimil (referee)
Chlortoluron is a herbicide from the group of substituted ureas and it is used for weed control in production of cereals and poppy. Substituted ureas are widely used but these substances are known for their high mobility in soil, persistence and contamination of underground water. Therefore, the development of analytical methods suitable for residual concentrations determination is necessary. This thesis deals with the determination of chlortoluron using carbon paste electrode (CPE) by the methods of differential pulse voltammetry and high-performance liquid chromatography with electrochemical (HPLC-ED) and spectrophotometric (HPLC-UV) detection. The aim of the work was to find optimal conditions suitable for chlortoluron determination. Further, the possibility of using miniaturized carbon paste electrode (mCPE) and usability of tested methods for determination of chlortoluron in model samples of river water and soil were studied. Concentration dependences in deionized water were measured by the method of DPV in the Britton-Robinson (BR) buffer (pH = 3) and methanol (10 %, v/v). The limit of detection was 3,7∙10-7 mol∙dm-3 in the case of CPE and 8,7∙10-8 mol∙dm-3 in the case of mCPE. Also, concentration dependences were measured using HPLC with mobile phase containing BR buffer (pH = 4) and...
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Comparison of the fruits of some honeysuckle varieties in terms of content selected biologically active compounds
Veselý, Ondřej ; Diviš, Pavel (referee) ; Vespalcová, Milena (advisor)
This thesis is focused to the analysis of selected varieties of honeysuckle kamtschatica (Lonicera caerula var kamtschatica) and their comparison in terms of the content of biologically active substances. There were analysed only varieties grown in 2014. Theoretical part elaborates and characterized various parameters of biologically active compounds such as phenolic, anthocyanins and ascorbic acid and possibilities of their determination. Further it briefly surveys Kamchatka honeysuckle, its taxonomic and morphological properties and its nutritional properties. The experimental part of the work was focused on the description of the methods used for the analysis of biologically active substances and their analysis. Polyphenolic compounds and anthocyanin’s were determined spectrophotometrically and vitamin C was determined by high-performance liquid chromatography. The results and varieties were compared with each other. Overall, we can say that the variety with the highest content of all three analysed biologically active substances is a variety Valchová, which had the second highest content of polyphenolic compounds (546.3 mg · 100 g-1 fruits), the third highest contend of ascorbic acid (35.1 mg · 100 g-1 fruits) and the sixth highest content of anthocyanin (170.8 mg · 100 g-1 fruits)
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Analysis of drinks sweetened with stevia extracts
Procházka, Václav ; Hrstka, Miroslav (referee) ; Vespalcová, Milena (advisor)
The steviolglycosides are the natural, sweet substances from Stevia rebaudiana Bertoni. It affect human health positively and its sweetness is 300x stronger than the sweetness of sucrose. That's why it's used to sweetening the commercial products. Because of its potential properties it's good to have an appropriate method to isolate it. High performance liquid chromatography ( HPLC) is based on the separation of analyte between two immiscible phases with high pressure pump and appropriately chosen stationary phase in the column. Than the analytes come out from the column in different retention times. This master´s thesis follows up selection of the best HPLC system for isolation of the main steviolglycosides and its analysis in commercial products. In the theoretical part of the thesis is described the origin, basic characteristics, botanical description, cultivation and affect to the human health of Stevia rebaudiana Bertoni and its use in food industry. There are also concisely characterized the sweet substances contained in the plant, so called steviolglycosides. Than there are given the theoretical basics of high performance liquid chromatography, HPLC instrumentation and its specific applications at sleviolglycosides with the basic chromatographic parameters. The object of the first experimental part was to research the optimum conditions for time and separation effective chromatographic analysis and select the best chromatographic system for isolation of steviolglycosides. In the second experimental part, I have compared and defined the main steviolglycosides (stevioside, rebaudioside A) in nine selected products, commercially available in Czech republic, with the best chromatographic method. In these products was the contain of the stevioside or rebaudioside A confirmed or refused.
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Comparison of content anthocyanin pigments in selected cultivars of black elderberry and grape
Becková, Eliška ; Vítová, Eva (referee) ; Vespalcová, Milena (advisor)
The purpose of this thesis is to compare the contents anthocyans in different varieties of European elderberry (Sambucus nigra L.). It was also studied the relationship between the content anthocyans in selected samples of blue grape varieties (Vitis vinifera L.) and position of the track, where the grapes were grown. The theoretical part describes elderberries and grapes in terms of botanical, chemical, and in terms of their using in the food industry. The dominant part of this chapter is to describe anthocyans and possibilities for their determination in the elderberry and grapes. Emphasis has been placed on the pH-differential method and determination by high performance liquid chromatography. The experiments are divided into two parts. The aim of the first experimental part is comparison of the total anthocyanins content in fifteen varieties of elderberry. The performed analysis shows that in terms of anthocyanins content for cultivation of elderberry in the Czech Republic the most suitable varieties are Mammut, Samyl, Reis aus Vossloch and Haschberg. In the second part of the experiments were determined total anthocyanins content in nine varieties of blue grape grown in the Mikulov region. For determination were used HPLC and pH-differential methods, as well as in the first part. The highest content of pigments was found in varieties Alibernet and Neronet. The present study focused on the suitability of the location pointed to vineyard south to southeast orientation plantings. At the end of the experimental part is a comparison of both methods of determination. Method using high performance liquid chromatography to provide very accurate results compared to less demanding pH-differential method of determination. This is recommended only for tentative determination of anthocyanins content in real samples.
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Analysis of Echinacea content compounds
Michalíček, Jan ; Vespalcová, Milena (referee) ; Zemanová, Jana (advisor)
Rostliny rodu Echinacea jsou léčivé byliny stimulující imunitní systém. Používají se zvláště v boji proti chřipce a infekcím horních cest dýchacích. V této práci bylo analyzováno 7 komerčních výrobků obsahujících výtažky z rostliny Echinacea vysokoúčinnou kapalinovou chromatografií. Bylo provedeno kvantitativní stanovení derivátů kyseliny kávové, jež jsou látky zodpovědné za léčivé účinky rostliny. Zjištěné obsahy kyseliny chlorogenové, kávové, cichorové a echinakosidu byly porovnány v jednotlivých výrobcích. Dále byly připraveny extrakty z kořene, listů a květů třapatky nachové pomocí extrakce za zvýšeného tlaku extrakčního rozpouštědla při různých podmínkách, aby se z obsahu účinných látek v nich přítomných zjistilo, které extrakční podmínky jsou nejlepší. Z naměřených dat bylo zjištěno, že největší celková koncentrace obsahových látek je přítomna ve výrobku Imunit Echinaceové kapky firmy Simply You. Jako nejlepší extrakční podmínky se ukázaly teplota 80 °C v kombinaci s rozpouštědlem ve složení EtOH/voda = 40/60, a to jak při extrakci kořene, tak i listů a květů.
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