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Development of HPLC method for evaluating the purity and stability of fesoterodine using a design of experiment approach
Erdeová, Karolína ; Kozlík, Petr (advisor) ; Bosáková, Zuzana (referee)
The aim of this diploma thesis was to develop and validate a high performance liquid chromatography (HPLC) method for purity and stability evaluation of fesoterodine. The HPLC method development was carried out using design of experiments (DOE), which allows to find optimal separation conditions within small number of experimental analysis. Design was done by using L18 linear model. Chromatographic system of the developed method consisted of a C8 stationary phase (SF) XBridge BEH - C8 (100 x 4.6 mm, 2.5 µm), a binary mobile phase (MF) consisting of 10mM borate buffer pH 9.2 and MeOH in various ratios according to the gradient program. Flow rate was 0.7 ml/min, column temperature 35 řC and a diode-array detector (DAD) was applied for the detection at 227 nm. Analysis time was 22 min. The optimized method was validated and the forced degradation study was performed. Studied effects were: the effect of elevated temperature (60 řC), humidity (10 and 75% relative humidity), acidic and basic conditions, oxidation and light. Peak purity of fesoterodine was evaluated for all experiments of forced degradation study. Additionally, the sensitivity of the active substance to hydrolysis was determined within the pH range of 2-10.
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Determination of testosterone esters in injectable dosage form by HPLC-DAD
Folprechtová, Denisa ; Kozlík, Petr (advisor) ; Kubíčková, Anna (referee)
Anabolic steroids are currently very controversial issue not only for its therapeutic use but especially because of the illegal abuse in the sport branch. The aim of this thesis was to develop a rapid, selective, effective and simple method of high performance liquid chromatography suitable for the determination of anabolic steroids in injectable dosage form. The analyzed sample of pharmaceutical product Sustamed contained four testosterone esters: testosterone propionate, testosterone phenylpropionate, testosterone isocaproate and testosterone decanoate. Optimized HPLC-DAD conditions suitable for determination of the selected testosterone esters were as follows: core-shell column Poroshell HPH-C18 (3.0 x 100 mm, 2.7 mm), mobile phase consisted of 10mmol/l ammonium acetate, pH 4.5 and acetonitrile, gradient elution was used. The column was thermostated at 50 řC, injection volume was 1 µl, flow rate was set at 1ml/min and UV detection at 240 nm. Total analysis time was 6 minutes. Key words anabolic steroids, testosterone esters, HPLC-DAD
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Analysis of active pharmaceutical ingredient vemurafenib by LC
Filounová, Barbora ; Kozlík, Petr (advisor) ; Křížek, Tomáš (referee)
The aim of this work was to develop and optimize liquid chromatography method with spectrophotometric detection applicable to assay and purity of vemurafenib in solid dosage form and perform its stability study. The optimized separation conditions consisted of Poroshell HPH-C18 (3 × 100 mm, 2.7 μm) column tempered at 30 žC, mobile phase composed of 10 mM ammonium phosphate, pH 3,0/acetonitrile. Flow rate was set at 0.6 mL/min and gradient elution was performed. Detection wavelength was 250 nm. The calibration curve of vemurafenib was constructed in the concentration range 0.4 - 1.2 mg/mL. Limit of detection was 5.0 µg/mL and limit of quantitation was 16.5 µg/mL. Stability and stress tests of vemurafenib were performed under several conditions: Heat (80 žC), heat combined with humidity (80 žC/75 % relative humidity), hydrochloric acid (0,1 M), sodium hydroxide (0,1 M) and hydrogen peroxide (3% and 0,3% solution). The significant degradation of vemurafenib was observed under acid condition. Vemurafenib also degradated under oxidation condition. No degradation was observed under base condition and under heat and heat combined with humidity. Degradation of vemurafenib was not effected by tested excipients. Judging based on experiments vemurafenib is stable from the point of view of chemical stability.
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Structural analysis of natural compounds with aliphatic branched chains using mass spectrometry
Strmeň, Timotej ; Cvačka, Josef (advisor) ; Kozlík, Petr (referee)
The diploma thesis focuses on analysis of fatty alcohols with mass spectrometry. Theoretical part describes distribution, main properties, usage and analytical methods for analysis of fatty alcohols. Experimental work focuses on the search for a proper derivatisation procedure for fatty alcohols, which would enable their detection with soft ionisation techniques of mass spectrometry, as well as their structural analysis. The main aim of the structural analysis is to find the methyl branching in the fragmentation spectra of derivatives of the fatty alcohols.
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Separation performance of columns packed with fully porous and superficially porous particles in analysis of ambroxol and its three impurities
Slováček, Jan ; Kozlík, Petr (advisor) ; Tirčová, Barbora (referee)
This work was focused on the comparison of the separation of ambroxol and its three impurities with a column contained fully porous particles (Acquity BEH Shield RP18) and with two core-shell columns (Kinetex EVO C18 a Kinetex C18). Different pH values of buffer (borate buffer with pH = 9.70 and a phosphate buffer with pH = 7.00 and pH = 3.00) and different gradient programs were tested for separation of analytes. Compared to the column with fully porous particles (Acquity BEH Shield RP18), core-shell columns provided shorter analysis time. Lower backpressure and different selectivity depend on a separation system. The best separation conditions were obtained with the column containing fully porous particles (BEH Shield RP18) and gradient according to program Method 2 with the borate buffer. Analysis time was 10 minutes and the last peak had the retention time about 9 min. Autosampler was tempered at 10 ř C and injection volume was 2 μl. The column was tempered at 30 řC. Detection was set at wavelength of 247 nm. Component A contained 10 mM borate buffer with pH = 9.70 and component B was acetonitrile. The best separation was achieved by core-shell columns while phosphate buffer with pH = 7.00 was used. Higher efficiency, smaller peak widths and higher sensitivity were observed. Phosphate buffer...
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HPLC study of chemical reactions
Krouská, Hana ; Kubíčková, Anna (advisor) ; Kozlík, Petr (referee)
In this work hydrolysis of phenylisothiocyanate and subsequent decomposition of diphenylthiourea were studied. RP-HPLC with UV detection was selected for monitoring of these reactions. The new method of isocratic elution was developed and mobile phase composition and flow rate were optimized. Hydrolysis of isothiocyanate was studied as a function of pH and reaction temperature. The subsequent decomposition of diphenylthiourea was carried out at 80 řC depending on the pH.
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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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