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New procedures in metabolomic analysis of biofluids
Řimnáčová, Lucie ; Čabala, Radomír (advisor) ; Feltl, Ladislav (referee) ; Sýkora, David (referee)
The aim of this thesis has been the research on reactivity of protic metabolites with chloroformates and its application for GC-MS analysis of biofluids. The research was conducted in three separate studies and the results are three new, original methods for GC-MS determination of low- molecular protic metabolites in biological material especially in biofluids. The first study explores the discovery of fast derivatization of alicyclic hydroxyl groups by fluoroalkyl chloroformates (FCFs) under anhydrous conditions [1]. FCF fully converts the hydroxyl group into a corresponding carbonate and the step can easily be coupled with liquid-liquid microextraction (LLME) of the arising derivatives into organic phase. The reaction of the alicyclic OH group with FCFs was tested on 12 clinically relevant steroids and 4 tocopherols. The analytical properties of determined analytes were described and the method was validated for the GC-MS determination of 6 diagnostic sterols and 4 tocopherols in human serum and amniotic fluid. The new method was further successfully used for determination of sterols and tocopherols in tissues of the bug Pyrrhocoris apterus [2]. The second study was focused on the reactions of protic, particularly urinary metabolites with FCFs, mainly heptafluorobutyl chloroformate (HFBCF)....
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In-matrix esterification of polar carboxylic acids in urine
Anýžová, Petra ; Mráz, Jaroslav (advisor) ; Feltl, Ladislav (referee)
A set of 15 carboxylic acids of various structural types was used to test a new procedure of esterification of these substances in the urine without previous extraction. The acids were first measured by a reference method without the matrix, and then by a procedure, where lyophilized urine was treated with derivatizing reagent alcohol·HCl, which was then evaporated, the residue dissolved in water, esters extracted into an organic solvent and subsequently determined by GC/FID and GC/MS. The work is mainly focused on 2-hydroxyethyl- and carboxymethyl-mercapturic acids, but the procedure was also tested on di-, tri- and hydroxy-carboxylic acids, known as physiological urinary components or markers of metabolic disorders. Key words: carboxylic acids mecapturic acids freeze-dried urine esterification gas chromatography
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Development of separation methods for determination of perfluoroalkyl carboxylic acids in envirnonmental samples
Dufková, Veronika ; Čabala, Radomír (advisor) ; Feltl, Ladislav (referee) ; Kubinec, Róbert (referee)
A complete, sensitive and selective procedure was developed for gas chromatographic determination of perfluoroalkyl carboxylic acids (PFCAs, C5 - C12) in river water samples. A rapid and simple derivatization procedure was developed and optimized at first, using isobutyl chloroformate (IBCF) to convert the acids into the more volatile isobutyl esters. The second task was to find the most suitable stationary phase for separation of perfluoroalkyl carboxylic acids by GC. The retention characteristics of PFCA isobutyl esters were measured and compared on 9 different stationary phases. The physical-chemical parameters of analyte interactions with stationary phase were monitored on selected column Rtx-200MS, and these parameters were compared with a non-fluorinated homologue - octanoic acid. A sensitive GC-MS system was used with negative chemical ionization, which is suitable for the selected type of fluorinated analytes. The developed method exhibits very low limits of detection and determination of PFCA isobutyl esters: 0.05 - 9.7 ng mL-1 (LOD) and 0.16 - 32.2 ng mL-1 (LOQ). An optimum pre-concentration technique was found for real water samples, where SPE SupelTM-Select HLB cartridges were used. In these cartridges, PFCAs were captured as ion- pairs with enrichment factor of 400. The recoveries of...
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Analysis of sensory and toxicologically important compounds in beer
Vrzal, Tomáš ; Čabala, Radomír (advisor) ; ADAM, Martin (referee) ; Feltl, Ladislav (referee)
One of the most relevant group of toxicologically significant compounds in beer are N-nitrosamines. In this thesis, the attention was paid especially on non-volatile nitroso compounds whose concentrations in beer, a chemical structure, and a biological effect has not been known yet. For the reason of the lack of knowledge regarding this compound group, the method for their sensitive detection by chemiluminescence detection after gas chromatographic separation was developed. This method permits a classification of detected nitroso compounds to different groups (N-nitroso, C-nitroso, and combination of C-nitroso and nitro) and distinguish them from interferences. The method is based on recording of a pyrolytic profile of each chromatographic peak, the profiles are then processed by discriminant analysis. The method has been developed for finding and structural identification purposes of these unknown compounds. Its application on an artificially nitrosated beer sample, together with gas chromatographic tandem mass spectrometric analysis, led to structural identification of several representatives of nitroso compounds. Sensory active compounds in this thesis are represented by carbonyl compounds and fatty acids. Carbonyl compounds - furfural and hydroxymethylfurfural - were used during the development...
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Modern microextraction techniques for gas chromatographic analysis
Bursová, Miroslava ; Čabala, Radomír (advisor) ; Feltl, Ladislav (referee) ; Kubinec, Róbert (referee)
The submitted thesis is focused on the development, optimization, testing and practical application of the new microextraction method called Bell Shaped Extraction Device assisted Liquid-Liquid Microextraction (BSED-LLME). The method is based on the application of a miniature bell-shaped extraction tool in which the extraction takes place, so that only minimal solvent losses can occur, and which allows a reproducible dosing and collection of a small volume of the extraction solvent. The BSED- LLME method was used to preconcentrate selected volatile and less volatile analytes from aqueous samples into organic solvents of a density lower than water. After the extraction, the analytes were determined by fast gas chromatography with flame ionization detection and gas chromatography with mass spectrometry. The statistical methods known as Design of Experiment (DOE) were used for determination of the optimal extraction conditions for BSED-LLME procedure. DOE is based on a mathematical description of the system and the prediction of the optimal setting of experimental parameters that may influence extraction efficiency. Factors such as extraction time, volume of extraction solvent, addition of sodium chloride (ionic strength), stirring rate and the diameter of the extraction vessel ect., have been tested....
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New selective adsorbents and sensors based on microporous polymers with releasable links
Hašková, Alena ; Sedláček, Jan (advisor) ; Feltl, Ladislav (referee)
A new concept of Porous Organic Polymers (POP) of the type of hyper-cross- linked networks was implemented, in which the main (polyacetylene) chains were cross-linked both by permanent (biphenylene-type) and releasable (N-benzylideneaniline-type) links. It has been shown that the azomethine groups of the N-benzylideneaniline links of the networks can be readily and efficiently dissociated by means of hydrolysis or transmination. The dissociation of the azomethine groups accompanied by links releasing leads to a change in the covalent structure and texture of the networks. The composition of the networks has been optimized to maintain porous texture after releasing the N-benzylideneaniline links. Two possibilities of potential use of the new POP type for analytical chemistry applications have been studied. It has been shown that newly prepared POPs have a high capacity in the selective and reversible chemisorption of primary aromatic amines and hydrazines from solutions. The networks can be used repeatedly for these purposes, the chemisorption being carried out through transimination in the first use and through condensation of the aldehyde and amine groups in the subsequent uses. Thus, prepared POPs could potentially serve to extract primary aromatic amines from more complex mixtures of analytes....
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Separation and characterizasion of stereoisomers of phosphinic pseudopeptides and serine by capillary electrophoresis
Koval, Dušan ; Jelínek, Ivan (advisor) ; Feltl, Ladislav (referee) ; Tesařová, Eva (referee) ; Stránský, Zdeněk (referee)
CoNcLUstoNs This work demonstrates that capillary electrophoresis is a suitable method for analysis and physico-chemical characterization of biomolecules. lt was exemplified by px" determination of a series of nove| phosphinic pseudopeptides and by separation oÍ stereoisomers of the phosphinic pseudopeptides and serine' A self-consistent procedure for pK" determination by CZE was carried out' ln particular' various background electro|ýes Were tested in the acidic region at pH < 2. A set of background eIectro|ýes Was eVa|uated for reIiab|e determination of eIectrophoretic mobiIities in the 1.S - 12pH interval. The temperature of the solution inside the separation capillary was measured by a simplified approach, thus enabling precise determination of the electrophoretic mobilities' Additionally, a throughput of the CZE method for the pK" determination was substantially improved, while high separation efficiency and precision of the electrophoretic mobilities was retained. The method benefits from adjustable velocity of e|ectroosmotic flow, achieved by modiÍication of the capi||ary inner surface, and efficient determination of electroosmotic mobility. Further, a study was performed which exp|ored the validity of onsager mode| for theoÍetica| description of concentration dependence of the e|ectÍophoretic...
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Development of Methods for High-Throughput Enrichment of Glycoproteins and Glycopeptides Employing Multiple Lectin Affinity Chromatography/Tandem Mass Spectrometry
Maděra, Milan ; Pacáková, Věra (advisor) ; Feltl, Ladislav (referee) ; Foret, František (referee) ; Kučerová, Zdenka (referee)
5 CONCLUSIONS This thesis surrrnrarizes the development of a multimethodological analýical approach employing microcolumn lectin affi1t1,9fuom1tography coupled to highir".otu,to; separation and dete9tion techniques, in order to facilitate the enrichmení of glyco-proteins aná giycopeptioes originating from small quantities of real samples. This is u"toauy a sipificant-ítep towara satis$'ing the demands of contemporary glycoproteomics, which rely orirurt *a small scale analyses, allowing the identification of low abundant protein components' The overď conclusions and contribution to the contemporaÍy scierrce are drawn in tbree followine chaoters. 5.1 combining Lectin Microcolumns with High-Resolution separation Techniques for Enrichment of Glycoproteins and Glycopeptides This section describes coupling small-scďe lectin aÍfinity chromatograpby on-line to high- resolution separation and detection techniques, with the utiliý of a microcolumn loaded with a lectin immobilized onto macroporous silica. Experimental results, involving optimization of coupling procedure, fabrication of lectin microcolumns, verification of tteir Uin&ng efficiency and their possibility of interfacing on-line with nano LC-MS/]víS, me summarizžd into thé following conclusions; o optimized coupling procedure involved resuspending only 125...
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