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Analysis of biopharmaceuticals using liquid chromatogram. Optimization of sample preparation.
Kolečková, Pavlína ; Khalikova, Maria (advisor) ; Chocholouš, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Pavlína Kolečková Supervisor,consultant: Mgr. Maria Khalikova, CSc. Title of Diploma Thesis: Analysis of biopharmaceuticals by liquid chromatography. Optimalization of sample preparation. This diploma theses is focused on development and optimalization of prepared samples in acidic pH for peptide mapping at the same time with minimization creation of undesirabe peptide modifications during preparation. Within the method of optimalization there were prepared several samples which were different in preparation process, use of chemicals or enzymes and also different condition of incubation. Enzymes used for preparation of samples were trypsin, endoproteinase Lys-C, endoproteinase Glu-C, endoproteinase Asp-N and endoproteinase Sap-9. At diploma theses were also used commercially available sets for preparing samples in acidic pH and then results were compared. Samples were analyzed by UHPLC-UV and LC-MS methods. Results from LC- MS analysis were processed by proteomics softwares like FragPipe and Byonic (Protein Metrics), which could be evaluated processes for digestion and preparation of samples in acidic pH, described their advantages or disadvantages For the complete comparison and evaluation of the...
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The development of method for the determination of atorvastatin and its impurities in tablets using supercritical fluid chromatography
Juchelka, Jan ; Nováková, Lucie (advisor) ; Khalikova, Maria (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Jan Juchelka Supervisor: doc. PharmDr. Lucie Nováková, Ph.D. Title of Diploma Thesis: The development of method for the determination of atorvastatin and its impurities in tablets using supercritical fluid chromatography This diploma thesis was focused on developement and optimization of a method for qualitative and quantitative evaluation of atorvastatin and its impurities using supercritical fluid chromatography. One of the main goals was to optimize chromatographic conditions, which included the selection of a sufficiently selective stationary phase, appropriate modifier and mobile phase additives. Effect of pressure was also explored. Finally this method was validated. A total of seven analytes were evaluated, lately only six of them. One impurity was impossible to separate without using a chiral stacionary phase since it was an enantiomer of atorvastatin. Measurements were performed on UPC2 Acquity system with PDA detector by Waters. Selected wavelength for detection was 245 nm. Tested stationary phases included columns dedicated for SFC, namely Acquity UPC2 BEH 1,7 µm, BEH 2-EP 1,7 µm, HSS C18 SB 1,8 µm a CSH Fluoro-Phenyl 1,7 µm (all of them 3,0 x 100 mm). Insufficient separation...
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Implementation of new analytical system into a routine biochemical laboratory
Berdych, Martin ; Khalikova, Maria (advisor) ; Hyšpler, Radomír (referee)
IN ENGLISH Charles University, Faculty of Pharmacy in Hradec Králové Department: Department of analytical chemistry Candidate: Bc. Martin Berdych Supervisor: Mgr. Maria Khalikova, Ph.D. Consultant: RNDr. Jiří Plíšek, Ph.D. Title of the diploma thesis: Implementation of new analytical system into a routine biochemical laboratory The diploma thesis deals with the procedures in the case of the exchange of a biochemical analyzer under conditions of common laboratory clinical practice in a biochemical laboratory. The experimental part is focused on the comparison of the COBAS Integra 800 and COBAS 6000 analytical systems and the assessment of the degree of conformity in the measurement of biochemical methods using statistical tools (differential analysis according to Bland-Altman and Passing-Bablok regression analysis). With the help of determining the partial uncertainties of the measurement results, it evaluates the verification of individual methods by measuring control and reference materials on the COBAS 6000 analyzer. Comparison of eight methods - alanine aminotransferase, antistreptolysin-O, aspartate aminotransferase, C-reactive protein, HDL cholesterol, magnesium, creatine kinase and LDL cholesterol - a certain degree of difference appeared to have been assessed. Consequently some of the...
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Peptide Mapping of Biopharmaceuticals Using CE
Kosolapov, Dmytro ; Jáč, Pavel (advisor) ; Khalikova, Maria (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Dmytro Kosolapov Supervisor: PharmDr. Pavel Jáč, PhD. Consultant: PharmDr. Juraj Lenčo, Ph.D. Title of graduation thesis: Peptide Mapping of Biopharmaceuticals Using CE This work focuses on optimization of capillary electrophoresis (CE) method for the peptide mapping. and enzymatic digestion of proteins using trypsin. Myoglobin from equine heart and a reference antibody were used as substrates. Two digestion approaches were compared: a conventional tryptic digestion in solution and digestion with a commercial SMART Digest Trypsin Kit that is based on immobilized trypsin. The digestion was monitored with RP-HPLC. After a short optimization of tryptic digestion, a number of experiments were carried out to determine the optimal conditions for peptide mapping by means of CE. All the measurements were carried out using a PVA-coated capillary of 50 µm internal diameter, effective length of 56 cm and total length of 64.5 cm. Samples were applied hydrodynamically at a pressure of 50 mbar, the separation voltage was 30 kV, the temperature of the capillary was set to 20řC. The analytes were detected with UV-VIS detector at 200 nm. The effect of BGE type and concentration (30, 60, 90, 120, 150 mM formic acid...
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Evaluation of the influence of various solvents for column storage in supercritical fluid chromatography
Lidvinová, Kateřina ; Nováková, Lucie (advisor) ; Khalikova, Maria (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Kateřina Lidvinová Supervisor: Assoc. Prof. PharmDr. Lucie Nováková, Ph.D. Title of Diploma Thesis: Evaluation of the influence of various solvents on column storage for supercritical fluid chromatography The diploma thesis deals with the evaluation of the influence of various solvents on column storage for supercritical fluid chromatography. It is focused especially on column aging which is manifested as reduced retention time and the change in selectivity of a stationary phase when stored for a certain period of time. The experiments were performed using the ACQUITY UPC2 SFC system with the PDA detector. The detection took place at wavelength of 225 nm for the mixture of neutral compounds and at wavelength of 230 nm for the mixture of ionisable compounds. The ACQUITY UPC2 BEH 1.7 µm and the ACQUITY UPC2 BEH 2-EP 1.7 µm columns with dimensions of 3.0 x 100 mm (Waters, USA) were used as stationary phases. After the separation, these columns were always flushed with different solvents and stored, namely in CO2, isopropanol, acetonitrile and hexane in which they were stored until the next use. CO2 with methanol as an organic modifier was used as a mobile phase for the mixture of...
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Development of HPLC method for analysis of food supplements based on berberine extract
Zíka, Jan ; Šatínský, Dalibor (advisor) ; Khalikova, Maria (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Jan Zíka Supervisor: prof. RNDr. Dalibor Šatínský, Ph.D. Title of the diploma thesis: Development of HPLC method for analysis of food supplements based on berberine extract In this diploma thesis, an HPLC method for the determination of berberine and its related isoquinoline alkaloids aromoline, berbamine, magnoflorine, tetrahydropalmatine, jatrorrhizine, palmatine, tetrahydroberberine and oxyberberine was developed and validated. The method was subsequently applied to the determination of the relevant analytes in the food supplements Allnature Berberin 98% extract, Ostrovit Berberin, Swanson Berberin and GreenFood Berberine. An Ascentis® Express AQ-C18 (150 x 4.6 mm, particle size 2.7 µm) analytical column was used for chromatography analysis, using an acetonitrile/0.085% aqueous solution of phosphoric acid mobile phase. The flow rate of mobile phase was 1 ml/min with a sample injection volume of 5 µl and a column temperature of 30 řC. The gradient elution was used for analysis and detection was performed with a DAD detector at wavelengths of 225 and 345 nm. Key words: HPLC, berberine, aromoline, magnoflorine, berbamine, palmatine, tetrahydropalmatine, jatrorrhizine, tetrahydroberberine,...
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Peptide Mapping of Biopharmaceuticals Using CE
Kosolapov, Dmytro ; Jáč, Pavel (advisor) ; Khalikova, Maria (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Dmytro Kosolapov Supervisor: PharmDr. Pavel Jáč, PhD. Consultant: PharmDr. Juraj Lenčo, Ph.D. Title of graduation thesis: Peptide Mapping of Biopharmaceuticals Using CE This work focuses on optimization of capillary electrophoresis (CE) method for the peptide mapping. and enzymatic digestion of proteins using trypsin. Myoglobin from equine heart and a reference antibody were used as substrates. Two digestion approaches were compared: a conventional tryptic digestion in solution and digestion with a commercial SMART Digest Trypsin Kit that is based on immobilized trypsin. The digestion was monitored with RP-HPLC. After a short optimization of tryptic digestion, a number of experiments were carried out to determine the optimal conditions for peptide mapping by means of CE. All the measurements were carried out using a PVA-coated capillary of 50 µm internal diameter, effective length of 56 cm and total length of 64.5 cm. Samples were applied hydrodynamically at a pressure of 50 mbar, the separation voltage was 30 kV, the temperature of the capillary was set to 20řC. The analytes were detected with UV-VIS detector at 200 nm. The effect of BGE type and concentration (30, 60, 90, 120, 150 mM formic acid...
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Optimization and validation of UHPSFC-UV methods for quality control of agomelatin and atorvastatin in drug substance and in tablets
Machová, Zdeňka ; Nováková, Lucie (advisor) ; Khalikova, Maria (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Zdeňka Němcová Supervisor: Assoc. Prof. PharmDr. Lucie Nováková, Ph.D. Title of Diploma Thesis: Optimization and validation of UHPSFC-UV methods for quality control of agomelatin and atorvastatin in drug substance and in tablets The aim of this master thesis was to develop two ultra-high performance supercritical fluid chromatography (UHPSFC) methods with PDA detection for determination of agomelatine and atorvastatin and its potential impurities. UHPSFC system Acquity UPC2 with PDA detector was used for measurement. The separation of agomelatine was performed by Torus Diol column (100 × 3.0 mm, 1.7 µm). Gradient elution was performed using CO2 with the mixture of methanol/acetonitrile (1:1) and 31.5 mM (0,1 %) ammonium hydroxide as a mobile phase. Column temperature was set at 40 řC and BPR (back pressure regulator) pressure at 2000 psi. The detection wavelength was set at 225 nm. The method was developed for measurment of API (active pharmaceutical ingredient) and subsequently for tablets. The separation of atorvastatin was performed using Acquity UPLC HSS C18 SB column (100 × 3.0 mm, 1.8 µm). Gradient elution was performed using CO2 with the mixture of methanol/ acetonitrile (2:1), 15 mM...
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Optimization and validation of UHPSFC-UV methods for quality control of agomelatin and atorvastatin in drug substance and in tablets
Němcová, Zdeňka ; Nováková, Lucie (advisor) ; Khalikova, Maria (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Zdeňka Němcová Supervisor: Assoc. Prof. PharmDr. Lucie Nováková, Ph.D. Title of Diploma Thesis: Optimization and validation of UHPSFC-UV methods for quality control of agomelatin and atorvastatin in drug substance and in tablets The aim of this master thesis was to develop two ultra-high performance supercritical fluid chromatography (UHPSFC) methods with PDA detection for determination of agomelatine and atorvastatin and its potential impurities. UHPSFC system Acquity UPC2 with PDA detector was used for measurement. The separation of agomelatine was performed by Torus Diol column (100 × 3.0 mm, 1.7 µm). Gradient elution was performed using CO2 with the mixture of methanol/acetonitrile (1:1) and 31.5 mM (0,1 %) ammonium hydroxide as a mobile phase. Column temperature was set at 40 řC and BPR (back pressure regulator) pressure at 2000 psi. The detection wavelength was set at 225 nm. The method was developed for measurment of API (active pharmaceutical ingredient) and subsequently for tablets. The separation of atorvastatin was performed using Acquity UPLC HSS C18 SB column (100 × 3.0 mm, 1.8 µm). Gradient elution was performed using CO2 with the mixture of methanol/ acetonitrile (2:1), 15 mM...
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