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Method development for determination of 8-hydroxy-2-deoxy guanosine in urine for clinical research
Šestáková, Veronika ; Kujovská Krčmová, Lenka (advisor) ; Matysová, Ludmila (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Veronika Šestáková Supervisor: assoc. prof. RNDr. Lenka Kujovská Krčmová, Ph.D. Title of the Diploma Thesis: Method development for determination of 8-hydroxy-2-deoxy guanosine in urine for clinical research This diploma thesis is based on the method development for determination of 8- hydroxy-2-deoxy guanosine, 8-hydroxyguanosine and creatinine using UHPLC. The aim was to develop optimal conditions for clinical research. Experiments were carried out using UHPLC Nexera with mass spectrometer LCMS- 8030, (Shimadzu, Japan). Two stationary phases were tested. The chromatographic separation was achieved using a Meteoric core C18 BIO 4.6 × 50 mm stationary phase with core-shell particles, particle size 2.7 μm (YMC, Germany) secured with a KrudKatcher Ultra 0.5 μm in-line filtr (Phenomenex, Germany). The used mobile phase consisted of water (pH 3 using acetic acid) and methanol (using formic acid 0.2 mM) in the ratio 90:10 (v/v). The temperature was maintained at 25 řC, a flow rate was set at 0.5 mL/min and 4 µl of sample was injected. After optimization of separation conditions, the method was applied to biological material (urine). The samples were prepared using solid-phase extraction. The method...
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Development and validation of UHPLC method for determination of omeprazole in oral suspensions
Reiská, Veronika ; Matysová, Ludmila (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Anylytical Chemistry Candidate: Veronika Reiská Supervisor: doc. PharmDr. Ludmila Matysová, Ph.D. Title of diploma thesis: Development and validation of UHPLC method for determination of omeprazole in oral suspensions The aim of the diploma thesis was to find out suitable conditions for method which enables to determinate the concentration of omeprazole in oral suspensions. There was used the column Kinetex TM 1.7 µm, C18 100 A, 50 x 2.1 mm, (Phenomenex, USA) for analysis. The mobile phase was 0.025mol/L phosphate buffer and acelonitrile in ratio 74:26. A suitable pH value of 7.6 was chosen. The internal standard method of methylparaben was applied. The whole analysis was detected on the wavelength of 300 nm. After finding of optimal conditions, 6 different samples of oral omeprazole suspensions were measured. The solution of the mobile phase was used for dissolution of samples. The method was validated. Keywords: omeprazole, UHPLC, oral suspensions.
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Validation of bioanalytical methods - EU and USA legislations (review)
Hrochová, Zuzana ; Matysová, Ludmila (advisor) ; Kujovská Krčmová, Lenka (referee)
This thesis was aimed of the legal requirements and the comparison of specific requirements for validation of bioanalytical methods resulting from the requirement administration Food and Drug Administration (FDA) in the US and European medicines Agency (EMA) in the European Union. It was found that both agencies based on a different legal basis. FDA is part of the federal system. Publishes its own laws and the regulations which have binding legal force . In contrast, the EMA is a non governmental agency and only serves as an advisory comitte of the European Union. Both agencies have issued their instruction - Guids. The FDA issued a Guidance for Industry - Bioanalytical Method Validation and EMA issued Guidaline on bioanalytical method validation. Both manuals are only as recommendations and They haven't legal lability. Both manuals contain similar informations. Both agencies differ from the arrangement of the chapters and an emphasis on individual issues, but the basic parameters that are discussed and Theky are the same selectivity, carry over, LLOQ and petting of the calibration curve, accuracy and precision of the method, system suitability, and cross validation. There were no fundamental differences between the two instructions, they only differ from the criteria that recognize the individual...
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Optimization of the HPLC method for determination of propranolol in liquid oral formulations
Koval, Tomáš ; Matysová, Ludmila (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Tomáš Koval Supervisor: doc. PharmDr. Ludmila Matysová, Ph.D. Title of diploma thesis: Optimization of the HPLC method for determination of propranolol in liquid oral formulations The purpose of thesis is the optimization and subsequent validation of HPLC method for determination of propranolol - hydrochloride in liquid oral formulations. A medicinal product containing propranolol - hydrochloride is used in the therapy of infantile haemangiomas as extemporaneous prepared solution. For method optimization were chosen Kinetex® C18 column (150x4,6 mm, size of particles 5 μm) and isocratic elution of mobile phase containing phosphate buffer (c=17,7 mM) and acetonitrile in 30:70 ratio, with pH adjusted to 2,2. Flow rate was set at 1,2 ml/min. According to results of the validation, was found that this developed method for the analysis of the product is suitable, and provides accurate and precise results. Keywords: propranolol hydrochloride, HPLC, optimization, Sodium benzoate, liquid oral formulations
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Determination of neopterin in biological material
Arnoštová, Veronika ; Kujovská Krčmová, Lenka (advisor) ; Matysová, Ludmila (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Veronika Arnoštová Supervisor: RNDr. Lenka Kujovská Krčmová, Ph.D. Title of diploma thesis: Determination of neopterin in biological material The aim of this diploma thesis is to create an overview of present methods, which can be used for the determination of neopterin in biological material. Neopterin is a pteridine derivative, which belongs to the group of unconjugated pteridines. Its biosynthesis is based on the guanosinetriphosphate and is stimulated by the activity of the IF-γ. Neopterin is a useful marker of the activation of the immune system, it is also an important prognostic indicator of several diseases e.g. in malignant diseases, in HIV-positive patients or in patients after transplantation. Elevated levels of neopterin are found in many pathological states such as in acute and chronic viral infections, in tumor diseases, HIV patients, patients after transplantation with rejection, but also in certain neurological, cardiovascular and autoimmune diseases. The sample preparation procedure is an important part in neopterin analysis. For this reason, sample preparation methods are in this thesis discussed. Chromatographic techniques as HPLC and UHPLC and immunoassays as ELISA...
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Development of HPLC method for determination of vitamin B1 in clinical research
Chadtová, Anežka ; Kujovská Krčmová, Lenka (advisor) ; Matysová, Ludmila (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Anežka Chadtová Supervisor: RNDr. Lenka Kujovská Krčmová Ph.D. Title of diploma thesis: Development of HPLC method for determination of vitamin B1 in clinical research The present thesis describes the development and partial validation of the new HPLC method for determination of vitamin B1 (thiamine hydrochloride) and its phosphorylated derivatives (thiamine monophosphate, thiamine pyrophosphate). This method is based on the use of liquid chromatography with indirect fluorescence detection. Separation was achieved using modern second generation monolithic stationary phase High Resolution Chromolith RP-18e 100 x 4.6 mm (Merck, Germany) in combination with gradient elution. Mobile phase was consisting of the mixture of 95% phosphate buffer 10 mmol / l pH 6 5 and 5% acetonitrile (ACN), flow rate of the mobile phase was 2.5 ml / min, from 2.1. min 4 ml / min. For fluorescence detection of thiamin hydrochloride (TH), thiamin monophosphate (TMP) and thiamine pyrophosphate (TPP, thiamine diphosphate) was necessary to use chemical oxidation by potassium ferricyanide to thiochrome and its adequate esters having photoluminescence activity. Chromatographic method has been partially validated. Peak...
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Development of extraction procedure for determination of tocopherols
Klabačková, Sáva ; Kujovská Krčmová, Lenka (advisor) ; Sklenářová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Sáva Klabačková Supervisor: RNDr. Lenka Kujovská Krčmová, Ph.D. Title of diploma thesis: Development of extraction procedure for determination of tocopherols In the present work liquid-liquid extraction for determination of alpha, beta, gamma and delta tocopherol from human serum was developed miniaturized. Individual extraction steps as deproteinization, centrifugation, evaporation and final filtration were optimized. Solid supported liquid extraction (SLE) was also tested for determination of tocopherols and seems to be very promising for use in bioanalysis. During the miniaturization of the extraction procedure the main emphasis was placed on simplicity, speed and low consumption of samples and solvents. Method was also partly validated. The new extraction process is part of an already developed UHPLC method for the determination of individual forms of tocopherols and will serve to determine the antioxidant capacity of cancer patients during the chemotherapy treatment. Key words: tocopherols, LLE, SLE, biological material
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Development of HPLC method for determination of amiloride hydrochloride in pharmaceutical preparation
Sohrová, Barbora ; Matysová, Ludmila (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Barbora Sohrová Consultant: PharmDr. Ludmila Matysová, Ph.D. Diploma Thesis Title: Development of HPLC method for the determination of amiloride hydrochloride in medicinal preparation The purpose of thesis is the development of HPLC methods for determination of amiloride hydrochloride in pharmaceutical preparation. A medicinal product containing amiloride hydrochloride is used in the treatment of cystic fibrosis as a solution for inhalation. During the development, various stationary and mobile phases were tested. For the measurement was selected HS F5 column (150x 2,1 mm, 3 µm) and as the mobile phase was mixture of acetonitrile and 0,085% phosphoric acid to pH adjusted to 3,0 with triethylamine, in the ratio 75:25. Standard solution contained amiloride hydrochloride, Impurity A as a degradation product and butylparaben as an internal standard. The developed method was completely validated. According to results of the validation this method, was found that the developed method for the analysis of the product is suitable, and provides accurate and precise results. One of the findings of validation was the fact that the medicinal product containing amiloride hydrochloride is not stable...
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Development of HPLC method for the determination of homatropine and scopolamine in eye drops
Sohrová, Barbora ; Matysová, Ludmila (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Mgr. Barbora Sohrová Consultant: PharmDr. Ludmila Matysová, Ph.D. Thesis Title: Development of HPLC method for the determination of homatropine and scopolamine in eye drops High performance liquid chromatography (HPLC) method for the determination of homatropine hydrobromide and its degradation product- scopolamine hydrobromide in eye drops at a concentration of 1% (with sodium chloride), 2% (with sodium chloride) and 2% (with buffer F 6.45) active substance was developed. During the development of the method, various mobile and stationary phases were tested. For the validation was chosen column Ascentis Express F5 (100 x 4.6 mm, 2.7 µm). The mobile phase was composed of acetonitrile and phosphate buffer in a ratio of 9:91 (V/V). Phosphate buffer contained 3.12% aqueous solution of sodium dihydrogen phosphate dihydrate and 200 µl of triethylamine. Buffer pH was adjusted with phosphoric acid 85% to the value 2.5. Injection volume was 5 µl, flow rate was 1.1 ml/min and UV detection was performed at a wavelength of 210 nm. Analysis time was less than 6.5 minutes. Analyzed solution contained standard solution homatropine hydrobromide at a concentration of 25 mg/100 ml, and its degradation...
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Developement of HPLC method for monitoring stability of caffeine solutions
Saifrová, Zuzana ; Matysová, Ludmila (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Mgr. Zuzana Saifrová Supervisor: PharmDr. Ludmila Matysová, Ph.D. Title of Rigorous Thesis: Developement of HPLC method for monitoring stability of caffeine solutions This rigorous thesis is aimed on developement of HPLC method for monitoring stability of caffeine and caffeine citrate solution and its consequent validation. Theophylline was chosen as impurity, because of high possibillity of finding it as a product of decomposition in our sample. After consideration of several available methods for HPLC analysis of caffeine (see chapter 3.6 Research) was chosen following method as initial: analysis was carried out in laboratory temperature (20 ± 5 řC), with mobile phase MeOH:H2O 40:60 with flow 1 ml/min, monolithic column C18, 100 × 4,6 mm and sample was injected in volume of 5 µl. During the developement of method several conditions of analysis and composition of mobile phase were adjusted and also proportions of chromatographic column and character of stationary phase were changed. The main pursued parameters were the speed of analysis and capability of providing symetric resoluted peaks separated from the dead volume. The final method was based on isocratic elution using mobile phase...
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