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Separation of pharmaceutically active compounds by mixed-mode and chiral chromatography: interaction study
Fojtíková, Sofia ; Bosáková, Zuzana (advisor) ; Kozlík, Petr (referee) ; Fischer, Jan (referee)
During the drug development process, the pharmaceutical industry often relies on chromatographic techniques. High-performance liquid chromatography (HPLC) is among the most commonly used chromatographic techniques for these purposes, with reverse phase and HILIC being the most common choices. However, despite their popularity, these techniques have several drawbacks, such as the contribution of secondary interactions that arise from the nature of the carrier material, and limitations in terms of analyzing molecules with certain properties. In this sense, multimodal (also called mixed-mode) chromatography (MMC) appears to be a promising technique capable of addressing these challenges. The first part of the dissertation is focused on the comparison of the retention of structurally different molecules in different chromatographic systems using seven different multimodal stationary phases and the effort to elucidate the interaction mechanisms applied to the individual studied stationary phases. By observing the trends, obtained under different conditions for molecules with different physicochemical properties, it was at least partially achieved. Chiral chromatography is another key tool in the analysis of pharmaceuticals. Its main purpose in the development of medicinal products is to separate...
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Development and testing of capillary columns based on ion exchanger
Vojta, Jiří ; Coufal, Pavel (advisor) ; Bosáková, Zuzana (referee)
In this work, capillary monolithic columns based on styrene, divinylbenzene and methacrylic acid were prepared by free radical polymerization inside fused sillica capillaries of 320 µm inner diameter. The columns were prepared by very simple process from polymerization mixtures with variable amount of methacrylic acid. Effect of electrostatic interactions between methacrylic acid, as weak cation-exchanger, and analytes on their retention was showed. Based on chromatographic measurements, effect of amount of methacrylic acid in polymerization mixtures on the morphological properties of monoliths and on the retention of small organic amines and benzene was discussed.
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Modification and analysis of low-molecular chitosans
Moravcová, Dagmar ; Bosáková, Zuzana (advisor) ; Strašák, Tomáš (referee)
4 Abstract Chitosan is a natural biopolymer derived from chitin, found in the exoskeletons of crustaceans and fungi. It has gained considerable attention due to its biodegradability, biocompatibility, antimicrobial properties, and potential antitumor activities. Chitosan has cationic amino groups that enable interactions with various anionic compounds. Therefore, it is an ideal candidate for conjugation with monovalent bioactive compounds to form efficient multivalent conjugates. It was found that galectins, especially Gal-1 and Gal-3, are overexpressed in tumor cells. Several monovalent inhibitors derived from the parent inhibitor thiodigalactoside, also those modified with coumaryl groups, have been synthesized, which show a high selectivity and affinity towards Gal-3. The main goal of this work was to synthesize a chitosan-based carrier and conjugate it with ligands exhibiting selectivity and inhibitory effect towards Gal-3. The first step involved the modification of chitosan using a unique effective method based on solid-phase hydrolysis, which significantly reduces the consumption of chemical materials and, in addition, excessive losses during purification. At the same time, the chitosan carrier was functionalized with pentynoic acid. Furthermore, monovalent galectin ligands,...
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Comparison of enantioseparation of selected drugs on commercially available and laboratory-prepared CSP based on amylose.
Sidoryk, Edvard ; Bosáková, Zuzana (advisor) ; Smrček, Stanislav (referee)
This bachelor's thesis compares the performance of two commercially available chiral columns based on derivatized amylose (amylose-tris((3,5-dimethylphenylcarbamate), bound to a silica gel as a chiral selector's linker) with two chiral columns with the same chiral selector, prepared in the laboratory at the Department of Organic Chemistry of the FoS CU, but using electrostatic binding to silica gel. The aim of the work was to evaluate the enantioselectivity and the efficacy in the separation of racemic mixtures of selected chiral drugs and verify the stability and reproducibility of the prepared columns. The results showed that commercially available chiral columns proved acceptable enantioselectivity and enantioseparation in most analyses. However, columns prepared in the laboratory - despite the use of diverse mobile phases - did not show sufficient separation capabilities in any of the analytes tested. Nevertheless, their stability and reproducibility were demonstrated, and the principal usability of electrostatic binding of modifier to silica gel was validated.
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Development of HPLC-MS/MS methods for selected, biologically active compounds
Hrabáková, Kateřina ; Bosáková, Zuzana (advisor) ; Tůma, Petr (referee)
This thesis is focused on the development of an UHPLC-MS/MS method for the determination of lactone and carboxylate form of topotecan in aqueous buffers (Part 1) and on the development of a HPLC-MS/MS method for monitoring the extraction efficiency of 6 organic UV filters from aqueous matrices using deep eutectic solvents and terpenes (Part 2). Topotecan is an important anticancer drug that inhibits topoisomerase I, but only its lactone form is pharmacologically active. In the first part of this work, the change in the ratio of lactone and carboxylate form of topotecan in time was studied as a function of sample pH and the pH of the aqueous component of the mobile phase. For this purpose, a 19-minute UHPLC-MS/MS method with a C18 stationary phase, gradient elution and electrospray ionization in positive mode was developed. It was observed that with an increasing pH of the mobile phase and the pH of the sample, the mass fraction of the carboxylate form of topotecan in the sample increased as well. The same trend was also observed with increasing time since sample preparation. If methanol was added to the sample, a stabilization of the lactone form and a slower transition to the carboxylate form was observed. These findings can be further exploited to optimize the use of topotecan in medicine. The...
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Study of selected mushroom toxins of Amanita muscaria.
Adámek, Michal ; Bosáková, Zuzana (advisor) ; Hložek, Tomáš (referee)
The presented bachelor's thesis was focused on the development of an HPLC-MS/MS method for the separation and eventual quantification of the main toxins of Amanita muscaria (fly agaric). After optimization of tandem mass detection conditions, MRM transitions were found for muscarine (174 → 57), ibotenic acid (159 → 113), muscimol (115 → 98) and cycloserine (103 → 75), which was used as an internal standard. Measurements were performed on Luna Omega 1.6 µm polar C18 column in RP-HPLC mode with an optimized mobile phase composition of 2:98 (v/v) acetonitrile/deionized water with the addition of 0.1% formic acid in both components and Ascentis Express RP- Amide in HILIC mode with an optimized mobile phase composed of 70:30 (v/v) methanol/deionized water with the addition of 0.5% formic acid in both components. Calibration curves with a range of 0.01 to 10 µg/ml for RP-HPLC and 0.01 to 25 µg/ml for HILIC were measured under optimized conditions. Urine and blood serum solutions of a patient with Amanita muscaria poisoning, including diluted versions of these solutions, were measured. Ibotenic acid and muscimol were found in the patient's urine, muscarine, ibotenic acid and muscimol in the blood serum. The RP-HPLC method gave narrower peaks with lower retentions and lower noise levels. The studied toxins...
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Advanced methods of assaying lectin-carbohydrate interaction
Červený, Jakub ; Bosáková, Zuzana (advisor) ; Pavlíček, Jiří (referee)
A reliable and reproducible quantification and detailed characterization of the interactions between a biomedically relevant protein and its synthetic carbohydrate ligand can provide new information for the design of better diagnostic and therapeutic tools. A high sensitivity and specificity are essential requirements for the feasibility of an analytical method. These parameters can be difficult to achieve for highly complex systems such as lectin-saccharide complexes. On the other hand, carbohydrate-based diagnostics and therapeutics offer the great advantage of detecting biologically active lectin receptors, which cannot be achieved with antibodies. In this work we demonstrate the use of the BLI technique to monitor lectin- carbohydrate interactions for a multivalent system. Using functionalization of a novel construct of galectin-1 with an in vivo biotinylated AVI tag, suitable immobilization on a biosensor was achieved without the loss of its lectin activity. Due to the high sensitivity of this technique, kinetic parameters of interaction with prepared multivalent neoglycoproteins based on LacNAc epitopes were obtained in the micro- to nanolar KD range. The complementarity of this method was demonstrated by parallel ITC and competitive ELISA measurements. The neoglycoprotein with a lower LacNAc...
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Determination of estrogen pollutants in real water sample by HPLC-UV after solid phase extraction.
Kozlík, Petr ; Bosáková, Zuzana (advisor) ; Coufal, Pavel (referee)
4 Abstract Estrogens are considered to belong to chemicals that negatively affect the endocrine system, even if present at very low concentrations. They are discharged into environment as a result of an increasing application of drugs etc. This work is focused on the separation and quantification of five estrogens, namely estrone (E1), 17β-estradiol (βE2), 17α-estradiol (αE2), 17α-ethynylestradiol (EE2) and estriol (E3) in natural water samples by HPLC-UV method. The chromatographic system consisted of a C18 stationary phase (SunFire® C18, 150 x 4.6 mm, octadecyl bounded to silica gel, particle size 5 µm) and binary mobile phase of acetonitrile/water in various ratios in isocratic separation mode. The effect of acetonitrile content in the mobile phase and flow rate of the mobile phase on retention and separation parameters was tested. Under the optimized separation conditions (acetonitrile/water 40/60 (v/v), 1.3 ml/min), all the compounds were baseline resolved and eluted within 15 min. These experimental conditions were applied to the calibration measurements which were carried out within the concentration range from 0.001 to 1 mg/ml. Limits of detection (LOD) and limits of quantification (LOQ) for the individual estrogens and their mixture (standards dissolved in methanol) were determinated. The detection...
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Analysis of pigments from integument of Graphosoma semipunctatum.
Krajíček, Jan ; Bosáková, Zuzana (advisor) ; Svobodová, Eva (referee)
Pterines belong to an important group of compounds, acting as pigments in many species. Some of them are probably responsible for characteristic coloration of the insect group (Hereroptera). This coloration is considered to be a visual warning signal for optically orientating predators (birds, lizards etc.). In this work, pterines in the species of Graphosoma semipunctatum have been investigated by high performance liquid chromatography. To develope an appropriate separation method, the reverse-phase separation mode with C18 stationary phase (Spherisorb ODS 2) and binary mobile phase (organic modifier/buffer or water) was used. The effect of type and content of organic modifiers (methanol, ethanol, tetrahydrofuran) and concentration of phosphate buffer pH 3.0 (10 - 30 mM) in the mobile phases on retention and separation behavior of the studied pterines (leukopterin, biopterin, xanthopterin, isoxanthopterin and erythropterin) was studied. Under the optimized separation conditions (5/95 (v/v) methanol/20 mM phosphate buffer, pH 3.0, flow rate 0.7 ml.min-1 , UV detection at 290 nm), the extract from the integument of Graphosoma semipunctatum was analyzed.
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Analysis of adenosine triphosphate and adenosine diphosphate by HPLC-MS/MS
Černá, Martina ; Coufal, Pavel (advisor) ; Bosáková, Zuzana (referee)
In this bachelor thesis, ADP and ATP samples were analysed and detected with HPLC- MS/MS method. Approximate limit of detection (LOD) for these particular substances were found and their values were compared with the LOD values published in the literature obtained via the same methods and under very similar experimental conditions. Our limits of detection for nucleotides were comparable with the limits described in the literature. Mass spectrometry analysis was performed in the positive and the negative mode of multiple reaction monitoring analysis and electrospray was used for the analyte ionization. The optimal conditions for high performance liquid chromatography of ATP and ADP analysis were acquired on a ZIC - HILIC column with the mobile phase of 75:25 (v/v) acetonitrile / 10 mM ammonium acetate. Ammonium acetate buffer was adjusted to pH of 7.15 and the separation was done under the isocratic elution.
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