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Optimization of electrophoretic determination of protamine and insulin
Molnárová, Katarína ; Křížek, Tomáš (advisor) ; Hraníček, Jakub (referee)
This work deals with optimization of a method for separation and detection of protamine and insulin using capillary zone electrophoresis. The composition of background electrolyte, the solution pH and the injection method were optimized. Citric acid in a concentration range of 80 to 240 mmol L-1 and chloroacetic acid ranging from 50 to 150 mmol L-1 were tested sequentially. The optimized method uses a fused silica capillary with inner diameter of 50 μm. The total length of capillary is 50.0 cm, effective length is 8.5 cm. The injection of the sample is performed on the short end of the capillary. The method uses chloroacetic acid of 100 mmol L-1 concentration as the background electrolyte. Driving voltage is 20 kV. Sample is injected hydrodynamically with a pressure of 5 kPa for 3 s. The analytes are detected spectrophotometrically at wavelength of 200 nm. The method allows for determination in case of protamines in concentration range between 4 μg mL-1 and 300 μg mL-1 and insulin from 5 μg mL-1 to 300 μg mL-1 . The limits of detection are 1 μg mL-1 for protamine and 2 μg mL-1 for insulin. Repeatability of migration times and peak areas tested at 30 μg mL-1 and 200 μg mL-1 concentration levels using hydrodynamic injection showed values of relative standard deviation lower than 6 % suggesting...
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Determination of Nadroparine in Fraxiparine injection solution using flow techniques of anlysis
Miklošinová, Aneta ; Hraníček, Jakub (advisor) ; Křížek, Tomáš (referee)
This thesis was focused on a determination of nadroparin using sequential injection analysis and flow injection analysis with fluorimetric and spectrophotometric detection. The principle of determination was based on the interaction of nadroparin with phenothiazine dye. Methylene blue was used as phenothiazine dye. The determination was performed on a laboratory made FIA instrument and on the commercial SIA instrument. Optimal conditions for SIA were concentration of dye 6 ∙ 10-5 mol dm-3 , injected volume 100 µl, flowrate 1,5 ml min-1 , for FIA: concentration of dye 3 ∙10-5 mol dm-3 flowrate 2 ml ∙ min-1 , injected volume= 100 µl, for fluorimetric detection was sensivity of the detector 1000 V, Emission was measured by 2 nm and excitation wavelenght 621 nm. For spectrophotometric detection, absorbance was detected at the absorption maximum wavelength. Repeatability ranged from 2.01 to 2.85%. The limit of detection for FIA was 0.05-0.09 IU ml-1 , for SIA were limits of detection in range 0,25 - 1,63 IU ml-1 , limits of quantification in range 0,83 - 5,44 and linear range was from 0,5 - 20 IU ml-1 . The limits of detection, limits of determination and the linear range could be corrected for the SIA by the injected volume of phenothiazine dye.
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Study of interaction between protamine and heparin and its applicability in capillary electrophoresis
Martínková, Eva ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
Heparin is an acid mixture of glycosaminoglycans with high negative charge density which naturally occurs in human body. Due to its ability to bind antithrombin III and thus accelerate inhibition of thrombin it has anticoagulant effect. This is abundantly used in clinical practice for operations, in case of embolia or heart-attacks. Protamine is a mixture of small basic peptides, which is used in clinical practice as a heparin antidote. The interaction between heparin and protamine is electrostatic and is also used for determination of heparin in human plasma or blood using affinity methods. In my study it was found that if protamine and heparin are mixed in one vial, a complex is formed. Its resulting charge depends on concentration ratio of protamine and heparin. On the other hand, in case the protamine is injected as a sample and heparin is added to background electrolyte as a protein-binding ligand, it is possible to determine heparin from decreasing protamine peak area. Because of the complexity of protamine-heparin interaction, tetraarginine was used as structurally close model of protamine to increase repeatability of measurements. The method for determination of heparin was optimalised. It uses 20 mM or 60 mM ortho-phosphoric acid as background electrolyte, 1 mg/mL solution of tetraarginine...
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Study of vortioxetin complexation by native cyclodextrins and its determination using capillary electrophoresis
Počtová, Žofie ; Křížek, Tomáš (advisor) ; Sobotníková, Jana (referee)
In this master thesis, the formation of supramolecular complexes between vortioxetine active substance and cyclodextrins (α, β, γ) is examined and the stability constants of these complexes are determined. The stoichiometric ratios of cyclodextrin and vortioxetine 1:1 and 2:1 are considered. The results suggest that the 1:1 ratio of cyclodextrin and vortioxetine is more accurate for each of used cyclodextrins and that the strongest interaction is between vortioxetine and β-cyclodextrin. Further, a method for vortioxetine assay in a tablet is developed by using non-aqueous capillary electrophoresis technique. The precision, accuracy, and robustness are validated and the values of detection and quantification limits are determined. This method can be used as a verification method for the results obtained by HPLC as the separation principle is different in capillary electrophoresis. Also, the stability of vortioxetine as a pure powder and as a mixture with excipients is examined under the conditions of elevated temperature and humidity.
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Capillary zone electrophoresis determination of protamines
Malý, Michal ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
This work deals with development and optimization of a method for separation and detection of pro- tamines using capillary zone electrophoresis. The developed method uses a fused silica capillary with inner diameter of 50 µm and effective length of 41,5 cm. Driving voltage is 30 kV. Background electrolyte is aque- ous solution of 45 mmol dm−3 phosphoric acid. Analyte is detected spectrophotometrically at wavelength of 200 nm. Sample is injected hydrodynamically. The method allows for determination of protamines in the concentration range between 11 µg ml−1 to 1000 µg ml−1 , limit of detection is 4 µg ml−1 . Viablity of the method has been verified with a real sample of NPH insulin injection. For sufficiently sensitive detection of protamines in NPH insuline it is necessary to prepare the sample in acidic environment. For the pur- poses of this work the sample was acidified by addition of background electrolyte into the sample so that the background electrolyte concentration in the resulting solution of the sample was 18 mmol dm−3 . The main advantage of the method is the rapid analysis, migration time of the analyte is about 2 min. Disadvantage of the method in comparison to alternate methods using CZE or RP-HPLC is the inability to separate individ- ual protamine peptides. This disadvantage...
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Determination of complexation constants by partial Filling method and Flow Induced Dispersion Analysis and their comparison with values determined by Affinity Capillary Electrophoresis
Ansorge, Martin ; Ušelová, Kateřina (advisor) ; Křížek, Tomáš (referee)
Partial Filling method (PF) and Flow Induced Dispersion Analysis (FIDA) were used for the determination of stability constants of model system of four profens (R-Flurbiprofen, S-Ibuprofen, S-Ketoprofen and S-Naproxen) complexing with β-cyclodextrin. When using PF method, only a part of capillary is filled with the selector and thus the analyte must migrate through the zone of selector first and then through the neat BGE (it has different mobilities in both zones). Dependency of the differences in migation time on the length of the selector zone is then studied. When FIDA is used, the analyte in pushed through the capillary by external pressure and dependency of the rate of peak dispersion of the analyte on the concentration of selector in capillary is observed. Moreover, PF experiments were theoretically studied by Simul 5 Complex computing programme. Values of stability constants obtained by both methods were compared with values obtained by frequently used Affinity Capillary electrophoresis (ACE) method. The comparison of stability constants values determined by PF method and FIDA shows that both investigated methods grants results comparable with those obtained by the ACE.
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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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Development of high-performance liquid chromatography methods for determination of major components of tobacco
Rozkovcová, Lucie ; Jelínek, Ivan (advisor) ; Křížek, Tomáš (referee)
The aim of this work was development of high-performance liquid chromatography method with DAD detection for determination of nicotine in tobacco. Standard operating procedure used by World Health Organization was chosen as comparison of the developed method. Optimized high performance chromatography method is suitable for determining nicotine in tobacco. Limit of detection for this method was 0,0003 mg/ml and limit of quantification was 0,0010 mg/ml. Optimization of preparation of samples was significant part of this thesis. Sample preparation procedure was made substantially easier in comparison to other commonly used methods. Nicotine content was determined from real tobacco leaves samples, cigarette tobacco filler, nicotine cartridge for electronic cigarettes and pipe tobacco. Satisfactory relative standard deviation was achieved for all types of samples. Next part of this thesis focused on study of determining polyphenols using high-performance liquid chromatography with diode array detector. Chosen analytes were chlorogenic acid, caffeic acid, rutine, scopoletine and quercitrine. Among the five tested analytes, the highest sensitivity was achieved for chlorogenic acid and caffeic acid. All of the analytes achieved low limits of detection and quantification. Key words Liquid chromatography,...
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Electrophoretic systems containing charged cyclodextrins
Boublík, Milan ; Riesová, Martina (advisor) ; Křížek, Tomáš (referee)
Cyclodextrins are frequently used in capillary electromigration separation techniques as a complexing agent. Their presence in background electrolyte can enhance selectivity, mobilize neutral analytes or cause separation of chiral analytes. However, cyclodextrins can complex also with buffer constituents. This type of complexation can lead to significant changes of background electrolyte properties and, consequently, may result in distortion of electrophoretic results. In case of neutral additives, complexation can be easily detected by means of pH changes measurement. Situation in background electrolytes containing charged additives is more complicated due to the change of ionic strength. This work is focused on the revealing of unwanted complexations in systems containing charged cyclodextrins and on proposing safe, i.e. noninteracting, buffers for electrophoretic measurements in presence of charged cyclodextrins. Keywords capillary electrophoresis, affinity capillary electrophoresis, cyclodextrin, background electrolyte, complexation
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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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