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Comparison of HPLC-UV and HPLC-MS/MS methods for analysis of selected 1,4-benzodiazepines
Slováček, Jan ; Kozlík, Petr (advisor) ; Křížek, Tomáš (referee)
This work was focused on the optimization of separation of oxazepam and lorazepam in high performance liquid chromatography. Diode array detection and mass spectrometric detection using electrospray ionization and electrospray ionization with Jet Stream technology were optimized and compared. The optimal chromatographic system consisted of a Kinetex C8 100A (75 x 2.1 mm, 2.6 μm) and binary mobile phase of acetonitrile/0.1% formic acid (30/70) (v/v). Under the optimized separation conditions both studied compounds were baseline resolved and eluted within 3 min. Electrospray ionization with Jet Stream technology provided the lowest values of the limit of detection (LOD) and limit of quantification (LOQ). Limits of detection were ranging from 17 to 32 µg mL−1 .
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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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Analysis of degradation of substances of historical pharmaceutical preparations
Vašíčková, Pavla ; Nesměrák, Karel (advisor) ; Kozlík, Petr (referee)
The aim of this thesis is the analysis of transformations of the composition of pharmaceutical preparations from the years 1930-1997. The alkaloids and opium alkaloids are active substances of these pharmaceutical preparations. Pharmaceutical preparations were in form of tablet and injection solution. The presence of active compounds and their degradation products were detected using RP-HPLC with spectrometric detection. The MS/MS fragmentation pathways of compounds found were proposed. The high stability of some active substances was proved (codeine, ethylmorphine and theobromine) while some other compounds were completely decomposed.
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Modern Trends in High Performance Liquid Chromatography and Their Application
Kozlík, Petr
(EN) The dissertation thesis is focused on major trends in high performance liquid chromatography such as miniaturization of separation systems in hyphenation with high- sensitivity detection or characterization of new types of stationary phases for the separation of polar compounds in systems suitable for mass detection. Application of recently developed stationary phases in hydrophilic interaction liquid chromatography (HILIC) is also considered. Capillary liquid chromatography with tandem mass spectrometry (cLC-MS/MS) method was developed for determination of five estrogenic pollutants in samples of water. Several new sorption materials for solid phase extraction (SPE) were compared to obtain sufficient recovery of all the tested analytes. Discovery DSC-18Lt column provided the highest recovery (95 - 100 %). The optimized cLC-MS/MS with SPE was used for determination of estrogens in water samples in the order of units to tens of ng/L. HILIC separation systems with silica gel, cyclofructan and isopropyl cyclofructan modified silica stationary phases were tested and compared. Ability to donate protons and dispersion interactions are the main interactions that affect retention in HILIC with cyclofructan-based columns while they are less important in separation systems with bare silica stationary...
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Analysis of defensive substances of Diving beetles (Dytiscidae) by ultra-high performance liquid chromatography with mass spectrometry
Bosáková, Tereza ; Kozlík, Petr (advisor) ; Nesměrák, Karel (referee)
Obranný sekret potápníků (Dytiscidae) produkovaný jejich prothorakálními žlázami obsahuje řadu steroidních látek. Pro analýzu obranného sekretu potápníka druhu Ilybius fenestratus byla využita ultra- vysokoúčinná kapalinová chromatografie ve spojení s detekcí diodovým polem a hmotnostní detekcí. Analýza methanolického extraktu sekretu probíhala na koloně Acquity BEH C18 za podmínek gradientové eluce s dvousložkovou mobilní fází tvořenou methanolem s 0,1% obsahem amoniaku a 0,1% vodným roztokem amoniaku. V sekretu byla zjištěna přítomnost dvou steroidních hormonů, a to boldenonu a testosteronu a jejich identifikace byla potvrzena na základě shody jejich retenčních časů i hmotnostních spekter s příslušnými standardy.
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Comparison of contactless conductivity detection and direct UV detection sensitivity for electrophoretic determination of N-acetylaminosaccharides
Malý, Michal ; Křížek, Tomáš (advisor) ; Kozlík, Petr (referee)
is work deals with development and optimization of a method for separation and detection of N- acetylglucosamine and its dimer and trimer using capillary zone electrophoresis with contactless conductiv- ity detection. Separation and detection of these substances is possible if sodium hydroxide is used as back- ground electrolyte. Usage of another electrolyte such as disodium phosphate leads to unacceptable baseline noise level. Optimal conditions of this method are a background electrolyte concentration of 40 mmol dm−3 and voltage of 15 kV. e method is very sensitive to background electrolyte pH, used background electrolyte also causes rapid degradation of the inner capillary wall which is why this method has proven difficult to cali- brate properly. e developed method was compared to an already existing method which uses UV detection. e existing method has lower limits of detection and quantification and is less sensitive to working condi- tions. Limits of detection and quantification of the developed method are 34 µmol dm−3 and 104 µmol dm−3 respectively. Limits of detection and quantification of the method based on UV detection are 7 µmol dm−3 and 21 µmol dm−3 respectively. Limits of quantification are that of the analyte whose detection was the least sensitive which was the dimer in both...
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Spectrophotometric Determination of Chondroitine Sulphate by Sequential Injection Analysis
Hrubá, Lucie ; Hraníček, Jakub (advisor) ; Kozlík, Petr (referee)
This thesis deals with the optimization determination of chondroitin sulfate sequential injection analysis with spectrophotometric detection. The reaction proceeds in the presence of phenothiazine cationic dyes (Azure A, Azure B and methylene blue) and measuring the decrease absorbance dyes in their absorption maximum after the addition of chondroitin sulfate. Have found the optimum conditions for this determination: dye concentration 5 10-5 mole dm-3 , the dosing volume 100 µl CS + 50 µl dye + 100 µl CS, reaction time 0 s, flow rate 40 µl s-1 . For the determination of the CS in a static arrangement was found the lowest limit of detection and quantification using metylene blue (LOD = 0,23 mg l-1 a LOQ = 0,76 mg l- 1 ). The best repeatibility was achieved also using methylene blue 2,4 %. On the other side the best sensitivity was achieved using azure A. For the determination of the CS in SIA arrangement was found the lowest limit of detection and quantification using azure (LOD = 0,34 mg l-1 a LOQ =1,01 mg l-1 ). The best repeatibility was achieved also using azure A 1,9 %. And the best sensitivity was achieved using azure B. Based on these findings was chondroitin sulphate determined in food supplements, the more suitable method were the calibration curve .The determination was also carried out...
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Modern Trends in High Performance Liquid Chromatography and Their Application
Kozlík, Petr ; Bosáková, Zuzana (advisor) ; Pacáková, Věra (referee) ; Cajthaml, Tomáš (referee)
(EN) The dissertation thesis is focused on major trends in high performance liquid chromatography such as miniaturization of separation systems in hyphenation with high- sensitivity detection or characterization of new types of stationary phases for the separation of polar compounds in systems suitable for mass detection. Application of recently developed stationary phases in hydrophilic interaction liquid chromatography (HILIC) is also considered. Capillary liquid chromatography with tandem mass spectrometry (cLC-MS/MS) method was developed for determination of five estrogenic pollutants in samples of water. Several new sorption materials for solid phase extraction (SPE) were compared to obtain sufficient recovery of all the tested analytes. Discovery DSC-18Lt column provided the highest recovery (95 - 100 %). The optimized cLC-MS/MS with SPE was used for determination of estrogens in water samples in the order of units to tens of ng/L. HILIC separation systems with silica gel, cyclofructan and isopropyl cyclofructan modified silica stationary phases were tested and compared. Ability to donate protons and dispersion interactions are the main interactions that affect retention in HILIC with cyclofructan-based columns while they are less important in separation systems with bare silica stationary...
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Chiral and achiral separation using in-house coated β-cyclodextrin column, comparison with commercially available β-cyclodextrin column
Procházková, Hana ; Kalíková, Květa (advisor) ; Kozlík, Petr (referee)
The main aim of this work was to prepare a new chiral stationary phase by dynamic coating of an ion-exchange stationary phase by PEMPDA-β-cyclodextrin (N1 , N1 , N3 , N3 , N3 -pentamethylpropane-1,3-diamine-β-cyclodextrin) and to test column selectivity and separation abilities of the newly prepared sorbent for chiral and also achiral sets of analytes and to evaluate stability of the column. Commercial β- cyclodextrin stationary phase (Astec Cyclobond I 2000 column) was used for comparison. The set of chiral analytes contained five structurally different compounds - lorazepam, oxazepam, BNP, HDDP and Cl-HDDP. Six different groups of achiral analytes were used for testing the column selectivity. All measurements were carried out in reversed phase separation mode. Methanol and 10mM ammonium acetate (pH 4.00) were used as mobile phase constituents. Baseline separations of oxazepam enantiomers and five mixtures of achiral analytes were achieved with PEMPDA-β-cyclodextrin stationary phase. Partial separations of two chiral analytes (HDDP and Cl-HDDP) and one mixture of achiral analytes were obtained with this stationary phase. Enantiomers of BNP and oxazepam and four mixtures of achiral analytes were baseline separated on the Astec Cyclobond I 2000 column. Partial separations were obtained for the rest...
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