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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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Treatment of biological samples before HPLC analysis - determination of alfa-tocoferol in erythrocyte membrane
Pospíchalová, Naďa ; Solich, Petr (advisor) ; Matysová, Ludmila (referee)
α-Tocopherol is the principal membrane antioxidant in mammalian cells, although antioxidant is not the only mechanism of this vitamin. Many studies consider erytrocyte α-tocopherol levels to be more suitable to assess the tocopherol status of organism than its plasma levels. This thesis presents erytrocyte membranes α-tocopherol HPLC-UV analysis with diferential ultracentrigation and solid phase extraction sample pretreatment. Red cell samples were ultracentrifuged (288 000 × g, 3 minutes, 4 řC) in the presence of butylated hydroxytoluene (BHT), D-mannitol, HEPES and CaCl2. Then α-tocopherol was extracted from erytrocyte membranes by solid phase extraction with n-hexane in the presence of ascorbic acid and tocopherol acetate internal standard. The extract was dissolved in methanol and separated on the monolithic column Chromolith Performance RP-18e (100 4.6 mm) using a 100% methanol as the mobile phase. The absorbance of α-tocopherol was measured at 295 nm wavelength.
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HPLC method development and validation for analysis of anthocyanins content in black currant cultivars
Bobríková, Michaela ; Šatínský, Dalibor (advisor) ; Matysová, Ludmila (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical chemistry Candidate: Michaela Bobríková Supervisor: doc. RNDr. Dalibor Šatínský, Ph.D. Consultant: Mgr. Barbora Šmídová Title of diploma thesis: HPLC method development and validation for analysis of anthocyanins content in black currant cultivars. A high performance liquid chromatography method with diode array detection was used and validated to determine delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-glucoside and cyanidin-3-rutinoside in black currant cultivars and black currant juices. The method used gradient elution with methanol and 2 % formic acid. Supelco Analytical, Ascentis Express C18 column with particle size 2.7 µm, 15 cm x 4.6 mm was used for separation. The analysis was performed using a temperature 50řC and a flow rate 0.8 ml/min. For detection, the wavelength of 520 nm was chosen.
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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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The determination of anticoccidials in chicken meat using QuEChERS and HPLC
Kydlíčková, Jitka ; Chocholoušová Havlíková, Lucie (advisor) ; Matysová, Ludmila (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical chemistry Candidate: Jitka Kydlíčková Supervisor: PharmDr. Lucie Chocholoušová Havlíková, Ph.D. Title of Diploma Thesis: The determination of anticoccidials in chicken meat using QuEChERS and HPLC This diploma thesis is focused on the development of modified QuEChERS method in conjunction with HPLC and UV detection for the determination of five selected anticoccidials - flubendazole, robenidine, diclazuril, toltrazuril and lasalocid A in chicken meat. This thesis deals with the optimization of chromatographic conditions - selection of a suitable stationary and mobile phases, and a suitable wavelength for the UV detection and also deals with optimization of sample preparation using the modified QuEChERS method. At the end of the thesis the method has been partially validated - repeatability, precision, accuracy, linearity, and other parameters were evaluated. For the separation was chosen Ascentis Express Phenyl Hexyl column (4.6 x 100 mm, 2.7 micron), and as the mobile phase after a series of experiments was chosen 0.1% formic acid pH 4 (pH adjusted with triethylamine) with acetonitrile (50:50, v/v). Finally, the test of suitability of the chromatographic system (SST) was performed and the following...
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Interaction of phenylpropionic acids with iron
Mísař, Jakub ; Matysová, Ludmila (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Mgr. Jakub Mísař Consultant: PharmDr. Jana Karlíčková, Ph.D. Title of thesis: Interaction of phenylpropionic acids with iron Iron belongs to a group of important elements in human body, especially due to ability to accept or donate electrons (conversion between the ferric and the ferrous ions). This element is essential part of human body. Under certain circumstances it may even harm (for example participation in the Fenton reaction). Phenolic acids are structurally very simple molecules, which are part of common diet or they are formed during the digestion. In this in vitro study we tested interaction (chelation and reduction) between the phenylpropionic acids and the iron ions. Seven phenylpropionic acids were evaluated at different pH values. For measurement we used spectrophotometric method, based on the indicator ferrozine. The degree of chelation or reduction of iron ions differed between tested compounds. Higher ability to chelate iron ions achieved all dihydroxycompounds and the 3-(4-hydroxyphenyl)propionic acid. The most active ferric reduction achieved by 3-(3,4-dihydroxyphenyl)propionic acid with catechol group. Iron chelation or reduction by phenylpropionic acids could...
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