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Treatment of biological samples before HPLC analysis - determination of alfa-tocoferol in erythrocyte membrane
Pospíchalová, Naďa ; Solich, Petr (advisor) ; Matysová, Ludmila (referee)
α-Tocopherol is the principal membrane antioxidant in mammalian cells, although antioxidant is not the only mechanism of this vitamin. Many studies consider erytrocyte α-tocopherol levels to be more suitable to assess the tocopherol status of organism than its plasma levels. This thesis presents erytrocyte membranes α-tocopherol HPLC-UV analysis with diferential ultracentrigation and solid phase extraction sample pretreatment. Red cell samples were ultracentrifuged (288 000 × g, 3 minutes, 4 řC) in the presence of butylated hydroxytoluene (BHT), D-mannitol, HEPES and CaCl2. Then α-tocopherol was extracted from erytrocyte membranes by solid phase extraction with n-hexane in the presence of ascorbic acid and tocopherol acetate internal standard. The extract was dissolved in methanol and separated on the monolithic column Chromolith Performance RP-18e (100 4.6 mm) using a 100% methanol as the mobile phase. The absorbance of α-tocopherol was measured at 295 nm wavelength.
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Optimization of procedure for isolation of phosphorylated peptides from a peptide mixture by metal oxide affinity chromatography for mass spectrometry analysis
Popovská, Lenka ; Solich, Petr (advisor) ; Zemanová, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Mgr. Lenka Popovská Supervisor: Prof. RNDr. Petr Solich, CSc. Consultant Supervisor: Mgr. Barbora Šalovská, Ph.D. Title of the rigorous thesis: Optimization of procedure for isolation of phosphorylated peptides from a peptide mixture by metal oxide affinity chromatography for mass spectrometry analysis. Protein phosphorylation is the key regulatory mechanism in cellular signalling. It is involved in most cellular processes that are regulated by a complex interplay of protein kinases and protein phosphatases and include proliferation, differentiation, and apoptosis. Defective or altered signalling pathways can lead to various diseases, and therefore it is very important to study protein phosphorylation in detail and try to understand it as much as possible. Technological development of mass spectrometry (MS) in recent years has enabled a significant increase in the number of phosphorylation sites that may be identified in a sample. Increasing scan speed together with commercial availability of high resolution MS have improved quantity and quality of the acquired data, which can be obtained in a relatively short time. However, even high-end devices would not allow to comprehensively identify the...
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Chemiluminiscenční hodnocení celkové antioxidační kapacity potravin
Vlčková, Jitka ; Polášek, Miroslav (advisor) ; Solich, Petr (referee)
Diplomová práce 2009 Abstract_ENG Jitka Vlčková ABSTRACT An investigation presented was concern to measure and evaluate the total antioxidant capacity (TAC) in samples of red wine clarified with four fining agents (polyvinylpolypyrrolidone and three different types of gelatin). All wine samples were treated by concentrations of 10, 20 and 40 g/hL of finings. The main aim was to discover how the particular agents in different dosages influence the TAC. The second part describes relations between changes in TAC in content of single antioxidant components (total polyphenols, anthocyanins, gallic acid and simple phenols). Total antioxidant capacity in red wine samples was evaluated using the Trolox equivalent antioxidant capacity assay (TEAC). A theoretical part of the thesis briefly summarizes basics about luminescence, focused on chemiluminescent reaction. Theory of free radicals and antioxidants, especially those included in red wine are also described. Process of winemaking aimed at fining is also contained in this part. An experimental part describes techniques of determination of the total antioxidant capacity, principle of chemiluminescent assay based on the luminol/peroxidase system. The results show the different behaviour of gelatin from the polyvinylpolypyrrolidone (PVPP). In general gelatin tends to...
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Application of modern separation techniques in the analysis of biological material
Červinková, Barbora ; Solich, Petr (advisor) ; Mikuš, Peter (referee) ; Štěrbová, Petra (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Department of Analytical Chemistry Candidate Mgr. Barbora Červinková Supervisor prof. RNDr. Petr Solich, CSc. Title of Doctoral Thesis Application of modern separation techniques in the analysis of biological material The presented dissertation is focused on development, optimization and validation of new extraction procedures and chromatographic methods for determination of vitamins A and E, inflammation biomarkers, DNA and RNA oxidative stress products in biological matrices. In the theoretical part of this work selected analytes and possibility of their determination in biological materials are described. Moreover, modern trends in liquid chromatography and bioanalytical validation guidelines are included. In this part, special attention is focused on sample handling, transport and pre- treatment. The second part is focused on published methods and divided into two sections. The first section comments published developed analytical methods. As first method, determination of lipophilic vitamins in human serum by miniaturized liquid-liquid extraction with subsequent separation on fluorinated stationary phase using UHPLC system with fluorescent detection in 4 min is discussed. Thereafter, determination of creatinine, DNA and...
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HPLC determination of betamethasone and chloramphenicol using alternative stationary phases
Salabová, Markéta ; Šatínský, Dalibor (advisor) ; Solich, Petr (referee)
A new procedure has been developed for separation and subsequent determination of betamethasone and chloramphenicol in pharmaceutical eye drop BETABIOPTAL ® preparation. The analysis carried out using a mobile phase of acetonitrile / water (25:70, v / v), adjusted to a flow rate of 0.75 ml / min at a temperature of 30 ř C, sample injection 5 Ll in the isocratic mode and the detection wavelength of 241 nm. Internal standard with similar properties as the two active substances has been selected as propylparaben. There was chosen for the separation column Synergi Fusion RP MS 2 cm × 2 mm, with a particle size of 2 m. The peaks have sufficient resolution and symmetrical shape and one analysis lasted up to 2 min while maintaining the quality of measurements (accuracy and reliability). The optimized method was validated before the actual measurement.
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Development of HPLC method for determination of vitamine D in biological material
Vlčková, Alena ; Solich, Petr (advisor) ; Matysová, Ludmila (referee)
In this diploma work the new HLPC method for simultaneous determination of vitamins D2, D3 and their metabolites (1,25(OH)2D3, 25(OH)D3) using the internal standard was developed. During the suggested assessment the monolith column Chromolith Performance RP-18e, 100 x 4,6 mm was used. The detection was carried out with the help of a diode array detector at wavelenght 265 nm for vitamins D and its metabolites, 295 nm for the internal standard tocol. The mixture of methanol : acetonitrile : water in percentual representation 12,5 : 85 : 2,5 was used as the mobile phase. The flow rate of the mobile phase was 1,5 ml.min-1 and the injection volume of the sample was 20 μl. The total time of the analysis was 3,5 minutes including the equilibration of the column. This method was developed and partially optimised with the standards of vitamins D and will be validated for biological material.
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HPLC analýza makrolidových a tetracyklinových antibiotik v nemocničních odpadních vodách
Vrubl, Jaroslav ; Solich, Petr (advisor) ; Polášek, Miroslav (referee)
Analysis of macrolides and tetracyclines in hospital wastewaters by HPLC ABSTRACT Macrolide and tetracycline antibiotics are drugs used for treatment and prevention of infectional illnesses both in human and veterinary medicine. As other environmental contaminats, they can accumulate in environment and induce adverse effects in terrestrial and aquatic organisms. and induce bacterial resistance. The sensitive analytical methods are necessary to quantify their level in wastewaters to rate the risk caused by their presence in environment. The HPLC with MS detection is method mostly used to analysis, because of its high sensitivity. A method using UV or fluorescence detection was tried to be developed as an alternative to HPLC-MS. A method to analyse macrolide using Hichrom Lichrosorb RP 18, 250 x 4.6 mm i. d. and Chromolith Performance column RP 18e, 100 x 4.6 mm i. d. were used. A mobile phase ACN: 0.05M KH2PO4 (pH 7.5) 30:70 (v/v) was used, the detector was set at 215 nm. The method using pre-column derivatization by FMOC-Cl was studied. The analysis was performed on Chromolith Performance column RP 18e, 100 x 4.6 mm i. d. The fluorescence detector was set at excitation wavelength 255 nm and emission wavelength 315 nm. The method to analyse tetracyclines used gradient elution of ACN and 0.02 M oxalic acid....
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Development of New Methods for Monitoring of Occurence of Pharmaceuticals in the Environment
Aufartová, Jana ; Solich, Petr (advisor) ; Fiala, Zdeněk (referee) ; Garcia-Campaña, Ana M. (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate Mgr. Jana Aufartová Supervisor Prof. RNDr. Petr Solich, CSc. Title of Doctoral Thesis DEVELOPMENT OF NEW METHODS FOR DETERMINATION OF SELECTED PHARMACEUTICALS IN ENVIRONMENTAL SAMPLES During last decade, the consumption of pharmaceuticals is increasing substantially. At the same time their occurrence in environment is increasing as well. Pharmaceuticals are released into environment by excretion, as conjugated or unchanged active compounds, as unused pharmaceuticals, which are not disposed according to the recommendations (e.g. thrown away into the rubbish or flushed down the toilet). Other possible sources of pharmaceuticals are in the agriculture, livestock and aquaculture. The current water treatment technologies do not remove all traces of pharmaceuticals in wastewaters. Therefore the monitoring of their occurrence in surface and wastewaters become more important. Antibiotics and steroid hormones are a group of drugs used in human and veterinary medicine. The main problem with these groups of substances is the emergence of bacterial resistance, in the case of antibiotics, and the effect on the endocrine system, including the reproductive cycle, in the case of steroid hormones. The...
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Development of new types of biocompatible hemodialysis membranes for separation of biomolecules
Kohlová, Michaela ; Solich, Petr (advisor) ; Kujovská Krčmová, Lenka (referee) ; Pena, Angelina (referee)
Charles University, Faculty of Pharmacy in Hradec Králové / University of Porto, Faculty of Pharmacy Department of Analytical Chemistry / Department of Chemical Sciences Candidate: Mgr. Michaela Kohlová Supervisor: prof. RNDr. Petr Solich, CSc. Consultants: prof. Maria de Conceição Branco Montenegro assoc. prof. Alice Santos-Silva Title of dissertation thesis: Development of new types of biocompatible haemodialysis membranes for separation of biomolecules This dissertation thesis was focused on the development, preparation, and optimization of flat sheet polysulfone membranes, which would meet the characteristics required for membranes used for haemodialysis treatment. The thin porous membranes were prepared by spin coating method, followed by phase inversion via immersion precipitation. The composition, as well as the preparation process of the membranes, were optimized, to obtain a membrane with good mechanical performance in flow conditions, adequate selective separation ability, and biocompatibility. The prepared membranes with the appropriate characteristics were modified with bioactive compounds to minimize oxidative stress and/or inflammation, which are common complications in haemodialysis treated patients. Three different modification approaches were used to prepare the bioactive...
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HPLC Determination of Neopterin, Tryptophan, Kynurenine and Creatinine in Human Serum
Prokopová, Veronika ; Solich, Petr (advisor) ; Matysová, Ludmila (referee)
In this work the new HPLC method for simultaneous determination of neopterin, tryptophan, kynurenine and creatinine was developed and validated. For separation of analytes two monolithic columns (Chromolith Speed ROD RP-18e, 50 x 4.6 mm and Chromolith Performance RP-18e, 100 x 3.0 mm) were connected together. In front of this connection Chromolith Guard RP-18e, 10 x 4.6 mm disc for better separation and serum proteins elimination was used. Neopterin and tryptophan were monitored using fluorescent detection. Neopterin was monitored at 353 nm excitation and 438 nm emission wavelength, for tryptophan 245 nm excitation and 404 emission wavelength was used. The detection of creatinine and kynurenine was carried out with help of diode array detector at wavelength 235 nm for creatinine and 230 nm for kynurenine. As the mobile phase 15 mmol/l phosphate buffer at pH 4.51 was used with the flow rate 1 ml/min in time 0 - 3 minutes, then the flow rate was changed to 2.3 ml/min. The total time of analysis was 9.5 minutes including the column equilibration. The injection volume of sample was 1 µl. This method was developed and validated using the standards of analytes and also in human serum.
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