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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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Indirect determination of heparin by capillary electrophoresis
Filounová, Barbora ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
Heparin is a mixture of sulfonated polysaccharides which is negatively charged. Heparin is a substance which is important in organism and fundamentally affects its physiology. Main attribute of heparin is anticoagulation - it prevents the complete blood coagulation. This anticoagulant effect balances the hemocoagulation by influencing the coagulation pathway. In some cases a pharmacological application of heparin is needed so the heparin is administrated as a injection of physiological solution of sodium or calcium heparine salt. Monitoring of level of the heparin in blood is problematic - methods used today are based on the measurement of a time required for blood clot formation. The result evaluation is done by comparing a sample with reference solution. These methods are relatively imprecise, can not be used in "on-line" setting and are highly influenced by general health condition of patient. In this work some principles of affinity capillary electrophoresis were adapted from another work - heparin was determined indirectly by monitoring of decrease of the peak area of protamine. Protamine is medically used antidote of heparin because they create a stable complex together. In this work protamine was replaced by well defined tetraarginine because the most frequent amino acid in protamine is...
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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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