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HPLC of Bioactive Compounds
Lukšanová, Aneta ; Kubíček, Vladimír (advisor) ; Lázníčková, Alice (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of: Biophysics and Physical Chemistry Consultant: Ing. Vladimír Kubíček, CSc. Student: Aneta Lukšanová Title of thesis: HPLC of bioactive compounds This diploma thesis is focused on finding conditions of HPLC analysis of midazolam and it's two metabolites - 1-hydroxymidazolam and 4-hydroxymidazolam, on method validation and on finding an appropriate internal standard. The analysis was carried out under reversed-phase conditions, the chosen column was POROSHELL 120 EC-C18, 3.0 X 100 NM, 2.7 µm, and a mixture of acetonitrile and ammonium acetate buffer with pH 4.5 in a ratio of 35/65 has been proven to be effective mobile phase. Detection was carried out using the diode array detector with wavelength values 223 nm and 272 nm. The column was thermostated at 25 řC, the analysis time was 10 minutes. Flow rate of mobile phase was 0.700 ml/min. Diazepam was found as a suitable internal standard. The method was validated only for 1-hydroxymidazolam and 4-hydroxymidazolam determination in rat plasma due to a submitter's request. Within the analytic method validation the following parameters were tested: precision, accuracy, lower limit of quantitation, linearity and selectivity.
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HPLC of Bioactive Compounds
Lukšanová, Aneta ; Kubíček, Vladimír (advisor) ; Lázníčková, Alice (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of: Biophysics and Physical Chemistry Consultant: Ing. Vladimír Kubíček, CSc. Student: Aneta Lukšanová Title of thesis: HPLC of bioactive compounds This diploma thesis is focused on finding conditions of HPLC analysis of midazolam and it's two metabolites - 1-hydroxymidazolam and 4-hydroxymidazolam, on method validation and on finding an appropriate internal standard. The analysis was carried out under reversed-phase conditions, the chosen column was POROSHELL 120 EC-C18, 3.0 X 100 NM, 2.7 µm, and a mixture of acetonitrile and ammonium acetate buffer with pH 4.5 in a ratio of 35/65 has been proven to be effective mobile phase. Detection was carried out using the diode array detector with wavelength values 223 nm and 272 nm. The column was thermostated at 25 řC, the analysis time was 10 minutes. Flow rate of mobile phase was 0.700 ml/min. Diazepam was found as a suitable internal standard. The method was validated only for 1-hydroxymidazolam and 4-hydroxymidazolam determination in rat plasma due to a submitter's request. Within the analytic method validation the following parameters were tested: precision, accuracy, lower limit of quantitation, linearity and selectivity.
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Chromatograhic characterization of chiral and achiral separation systems
Kučerová, Gabriela ; Tesařová, Eva (advisor) ; Čabala, Radomír (referee) ; Ševčík, Juraj (referee)
Dissertation thesis is a 5-publications' collection concerning characterization and application potential of cyclodextrins, polysaccharides and macrocyclic antibiotics based chiral stationary phases. The effects of stationary phase and mobile phase are studied. This approach ensures the complex insight into separation systems studied. Systems with different nature of chiral selector were studied by HPLC. Namely, macrocyclic antibiotics and derivatized polysaccharides were used for experiments. Former ones provided better results for enantioseparation of non-coded amino acids than latter ones. Dynamic coating procedure was used for preparation of a new chiral stationary phase. Characterization of new cationic cyclodextrin based chiral stationary phase was performed. Linear free energy relationship method was used for characterization of two different separation systems, i.e. newly prepared stationary phase and commercially available stationary phase. Based on results obtained, newly prepared stationary phase showed better results for separation of different achiral groups of analysts. New stationary phase prepared by dynamic coating was compared with chromatographic system, in which the chiral selector was used as a mobile phase additive. The chiral selector used for the two different approaches was...
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Chromatograhic characterization of chiral and achiral separation systems
Kučerová, Gabriela ; Tesařová, Eva (advisor) ; Čabala, Radomír (referee) ; Ševčík, Juraj (referee)
Dissertation thesis is a 5-publications' collection concerning characterization and application potential of cyclodextrins, polysaccharides and macrocyclic antibiotics based chiral stationary phases. The effects of stationary phase and mobile phase are studied. This approach ensures the complex insight into separation systems studied. Systems with different nature of chiral selector were studied by HPLC. Namely, macrocyclic antibiotics and derivatized polysaccharides were used for experiments. Former ones provided better results for enantioseparation of non-coded amino acids than latter ones. Dynamic coating procedure was used for preparation of a new chiral stationary phase. Characterization of new cationic cyclodextrin based chiral stationary phase was performed. Linear free energy relationship method was used for characterization of two different separation systems, i.e. newly prepared stationary phase and commercially available stationary phase. Based on results obtained, newly prepared stationary phase showed better results for separation of different achiral groups of analysts. New stationary phase prepared by dynamic coating was compared with chromatographic system, in which the chiral selector was used as a mobile phase additive. The chiral selector used for the two different approaches was...
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