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Development and Application of High Performance Separation Methods in Connective Tissue Analysis
Braun, Martin ; Štulík, Karel (advisor) ; Pacáková, Věra (referee) ; Samcová, Eva (referee)
24 Annotation: This work is concerned with the development and application of high performance separation methods suitable for determination of clinically considerable fragments of collagen and elastin as the most important proteins of connective tissues. For their analyses, various chromatographic and electrophoretic methods have been used, including high performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC). This work has aimed at optimization of the developed and modified analytical separation procedures and methods and their utilization for determination of clinically important markers of connective tissue degradation. The key validation parameters of the analytical methods used in this work were evaluated too, which enabled their comparison with other approaches. One of the most important results of this work is thus high sensitivity, precision, repeatability and robustness of the methods which was found in a number of biological sample types. Successful was also very good HPLC separation of the analyzed cross-links in both collagen and elastin tissues. High performance methods are described which have been suggested and optimized for separation and quantitative determination of pentosidine, pyridinolines and...
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Separation of hepcidin using magnetic sorbents with subsequent MALDI-TOF MS analysis
Vávrová, Jana ; Pacáková, Věra (advisor) ; Tichá, Marie (referee)
Hepcidin is cysteine-rich cationic peptide produced by hepatocytes, secreted into blood plasma, and excreted in urine. Hepcidin is proposed to be the key regulator of iron metabolism and an evaluation of changes in the hepcidin level is important for diagnosis of several diseases. However, methods used for the hepcidin detection and determination in urine and serum have certain limitations. At present time MALDI-TOF MS based approaches have been applied for final analysis of urinary and/or serum hepcidin levels. Before MS analysis, separation of hepcidin from analyzed samples is an important and necessary step. The aim of this study was to compare the ability of several magnetic sorbents with different coating matrix and/or different terminal functionalized groups to adsorb hepcidin prior MS analysis. Either commercial magnetic sorbents containing -COOH groups or magnetic hydrophilic IDA-modified polymethacrylate microparticles P(HEMA-co-GMA)-IDA with immobilized metal ions were use for this purpose. Hepcidin was adsorbed to magnetic sorbents containing linked carboxyl groups (i.e. to weak cation exchange magnetic particles) at pH 6.8 independently on a nature of magnetic particle coating layer. Magnetic particles P(HEMA-co- GMA)-IDA with immobilized Cu(II) ions were found to adsorb hepcidin in a...
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Pharmaceutical application of high performance ion chromatography in analysis of organic anions and cations
Čujová, Sabína ; Čabala, Radomír (referee) ; Pacáková, Věra (advisor)
The thesis is focused on application of ion chromatography in pharmaceutical analyses of organic ions. Ion chromatography is increasingly used in the field of pharmaceutical analysis. This includes the analysis of impurities and metabolites. In the first part of this thesis, ion chromatography is compared with common separation techniques used in pharmacy, such as gas chromatography and high performance liquid chromatography. In the second part development and validation of methods of ion chromatography for purity evaluation and quality control of active pharmaceutical substances Rivastigmine hemitartrate and Pramipexole hydrochloride were carried out. Key words: ion chromatography, reversed-phase chromatography, ion-pair chromatography, ion-exclusion chromatography, ion-exchange chromatography, GC, HPLC
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Determination of the lipid profile in biological material by the method HPLC-ELSD
Vaindlová, Petra ; Pacáková, Věra (advisor) ; Feltl, Ladislav (referee)
A method of high-performance liquid chromatography with evaporative light-scattering detector (HPLC-ELSD) has been optimalized for the determination of neutral and polar lipids. Column filled by silica with chemically bonded diol has been used as stationary phase. As mobile phase, a ternary gradient composed from A: hexan-tetrahydrofuran 99:1 (v/v), B: isopropanol-chloroform-acetic acid 82:20:0,01 (v/v/v), C: isopropanol-water-triethylamine 47:47:6 (v/v/v) was used. Calibration curves have been measured in the range 2-200 μg of the injected amount; for individual lipid classes, optimal interlay of experimental data corresponded to the following functions: triacylglycerols - third order polynom (R=0,998), cholesterol esters - exponential dependence (R=0,998), free cholesterol - third order polynom (R=0.9998), ceramid - exponential dependence (R=0,992), cardiolipin - square dependence (R=0,998), phosphatidylethanolamine - exponential dependence (R=0,999), phosphatidylcholine - square dependence (R=0,997), phosphatidylserine - third order polynom (R=0,9985), sphingomyelin - third order polynom (R=0,9997), lysophosphatidylcholine - exponential dependence (R=0,9986). Analysis of the synthetic control sample showed recovery in the range of 82-95%. On the basis od these measurements, concentration of...
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Applications of monolithic columns in capillary liquid chromatography
Holdšvendová, Petra ; Coufal, Pavel (advisor) ; Pacáková, Věra (referee) ; Cvačka, Josef (referee)
INTRODUCTION From the nineties of the twent5r's century [l], when appeared the first references about the new type ofthe stationary phases designated for HPLC called monoliths, these became to be the object of interest of several scientific groups [2-4]. Their uncontested advantage in comparison with classical columns is their high stability in pH range from 2 to 12 where there is no possibility to use the classical columns. other advantages are their easy and cheap preparation and the absence of frits on the ends of the separating medium because the monolith is covalently bound to the inner side of the capillary [5]. On the other hand, their disadvantage remains to be worse in reproducibility of the stationary phase preparation than in case of classical columns. This PhD Thesis is focused on monoliths based on organic polymers, mainly the methacrylate monoliths. For the first time, this ýpe of monoliths was shown by Peters et al. [6] and since then there have been made several monolithic phases based on different separating features 17_101. Methacrylate monoliths are convenient for separation of the low molecular substances L7l as well as the high molecular substances like the proteins, oligonucleotides or saccharides n0-121.
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