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Detection of soluble endogline in Lab-in-Syringe
Ilićová, Marie ; Svobodová, Zuzana (advisor) ; Chocholouš, Petr (referee)
The main target of the thesis was to develop an automated method for the isolation of endoglin from a sample using a magnetic immunosorbent in Lab-In-Syringe with subsequent immunodetection by affiblot. The theoretical part deals with endoglin, in its membrane and soluble form, especially with their connection to endothelial dysfunction. The principles of anti-endoglin therapy using the monoclonal antibody carotuximab or TRC105 are also briefly described here. Furthermore, the theoretical part summarizes the basics of the automation in Lab-In-Syringe and of the affiblot technique, their principles, and their applications. The last chapter deals with the biofunctionalization of magnetic particles by antibodies in order to prepare a magnetic immunosorbent which is used for the isolation of proteins from a complex sample. The experimental part describes methods for testing the affiblot prototypes. The main goal was to develop a method for the preparation of anti-endoglin magnetic immunosorbent and to increase the capacity of the method by upscaling it from a 1 ml to a 5 ml syringe. Furthermore, methods for the isolation of the soluble form of human endoglin from the culture medium containing the drug TRC105 were developed for 1 ml and 5 ml syringes. Various experiments were carried out to optimize the...
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Sequential injection chromatography - advanced separations of bioflavonoids in food supplements
Pavelková, Kristýna ; Chocholouš, Petr (advisor) ; Šatínský, Dalibor (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical chemistry Candidate: Kristýna Pavelková Supervisor: doc. PharmDr. Petr Chocholouš, PhD. Title of diploma thesis: Sequential injection chromatography - advanced separations of bioflavonoids in food supplements Two methods were developed in the thesis. A primary method of sequential injection chromatography with UV detection was developed for separation of bioflavonoids rutin, hesperidin and diosmin. A second method was developed for adjustment data, which enables to increase sensitivity of first developed method. Chromatographic column YMC-Triart C18 ExRS; 100 mm 4.6 mm; 5 μ, 8 nm was used for separation. The mobile phase consisted of a mixture of acetonitrile with phosphoric acid solution pH 2.0 prepared in a ratio of 25:75. Isocratic elution was chosen; flowrate was 10 μl/s and total length of analysis was 7.9 minutes. Sequential injection chromatography system and computer software SIAsoft was used for analysis. Programs Microsoft Excel and Origin Pro 9 were used for processing and adjustment of data. Developed method was not succesfull in quantification of amount of flavonoids in Hemostop Probio MAX. Method for data adjustment enabled significant reduction of LOD and LOQ. Keywords: flavonoids, rutin,...
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Automated monitoring of sulisobenzone liberation through model membrane
Marel, David ; Sklenářová, Hana (advisor) ; Chocholouš, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of analytical chemistry Candidate: David Marel Supervisor: assoc. prof. PharmDr. Hana Sklenářová, Ph.D. Title of the diploma thesis: Automated monitoring of sulisobenzone liberation through model membrane This diploma thesis deals with the monitoring of biologically active substance liberation through a model membrane using sequential injection analysis. A UV - VIS spectrophotometer was used for the detection. The tested biologically active substance was sulisobenzone (one of the UV filters used in sunscreens) and the permeation through the polycarbonate membrane was monitored using a Franz cell. At the beginning of the work, a calibration curve was created in two media - water and phosphate buffer of pH 7.4. According to these curves, the concentrations - which were further used in the liberation tests - were selected. The given concentrations of sulisobenzone were prepared in different solvents, namely in water, in 10% ethanol, in 10% propylene glycol and in 5% propylene glycol. The tested solvents were selected based on the assumption of increasing the permeability of the membrane for the liberation of substances with limited solubility in the liberation medium. Each tested solution was measured for 2 hours, with individual...
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HPLC in quality control of food supplements containing gingerols and curcumins
Hujová, Eliška ; Šatínský, Dalibor (advisor) ; Chocholouš, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Eliška Hujová Supervisor: prof. RNDr. Dalibor Šatínský, Ph.D. Title of the diploma thesis: HPLC in quality control of food supplements containing gingerols and curcumins In this diploma thesis, a new HPLC method for determination of 6-, 8- and 10-gingerol, 6-shogaol, curcumin, demethoxycurcumin, bisdemethoxycurcumin, and piperine was developed and validated. The developed method was used for the analysis of food supplements: Kurkumin - piperin plus (+ zázvor) from VIESTE, Bio tumeric + ginger from VANAVITA, Ginger root extract from BioMedical, Shogaol zázvor from EPIGEMIC®, Ginger root extract from SWANSON®, and Ginger root (Zingiber officinale) from SOLGAR®. Just as a matter of interest, this method was applied for the selected tea blends containing ginger and/ or tumeric: Dilmah Orange & Ginger (flavoured ceylon black tea), Loyd Brusinky & Zázvor, Teekanne Dýchací cesty a krk se zázvorem, Pickwick Nos a krk se zázvorem, Tesco Ginger & Lemon, Teekanne Ginger Curcuma. For the analysis YMC-Triart C18 ExRS (150 x 4.6 mm; 3 µm) analytical column was used. Separation was carried out by gradient elution with mobile phases A: acetonitrile, B: ultrapure water at flow rate 1 ml/min. Detection was...
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Sequential injection chromatography - advanced and two-dimensional separations
Gužiňák, Matej ; Chocholouš, Petr (advisor) ; Šatínský, Dalibor (referee)
Department: Department of analytical chemistry Candidate: Matej Gužiňák Supervisor: doc. PharmDr. Petr Chocholouš, Ph.D. Title of the diploma thesis: Sequential injection chromatography - advanced and two- dimensional separations The subject of this work was the development of two analytical methods intended for separating substances contained in silymarin, their comparison and preparing a template in the Microsoft Excel program to simplify data evaluation. In the first developed method, called classical sequential injection chromatography (1D- SIC), a long Ascentis® Express F5 chromatography column (10 cm x 4.6 mm, 5 µm) with an opti-guard® C18 pre-column (1 mm) was used during analysis. The mobile phase consisted of a mixture of acetonitrile with phosphoric acid solution with pH 2.0 prepared in a ratio of 27:73. The isocratic mode was chosen for the analysis; the total length was 16.3 minutes with a flow rate of 5 μL/s. In the second developed method, called two-dimensional sequential injection chromatography (2D-SIC), a Kinetex® 5 µm EVO C18 column (30 x 3.0 mm) with an opti- guard® C18 pre-column (1 mm) was chosen for the first dimension. In the second dimension, an Ascentis® Express F5 column (30 mm x 4.6 mm, 2.7 µm) was used. The analysis mode was chosen isocratic with a total length of 35.3...
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Monitoring of liberation tests for the release of clotrimazole from nanofibers
Ernest, Radovan ; Sklenářová, Hana (advisor) ; Chocholouš, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Kralove Department of Analytical Chemistry Candidate: Radovan Ernest Supervisor: Doc. PharmDr. Hana Sklenarova, Ph.D. Consultant: PhamDr. Martina Hakova, Ph.D. Title of the diploma thesis: Monitoring of liberation tests for the release of clotrimazole from nanofibers The diploma thesis was focused on the analysis of release profiles of clotrimazole from different types of nanofibres using non-separation flow technique, sequential injection analysis (SIA). Simultaneously the diploma thesis dealt with finding ideal conditions for saturation of the fibres with a solution of clotrimazole. Nanofibers were produced at the Technical university of Liberec. The diploma thesis dealt with two types of nanofiber carriers - polymeric (polydioxanone and polycaprolactone) and ceramic (silica). Part of the fibre of polydioxanone and polycaprolactone was saturated directly during production in different ratios of polymer and clotrimazole. The saturated fibres were then analysed in the laboratory. The second part of polydioxanone and polycaprolactone together with ceramic nanofibers was produced in a pure state, i.e., without an active substance. These fibres were tested in the laboratory for saturation with an ethanolic solution of clotrimazole at a certain...
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Automation of liberation tests for clotrimazole released from differently saturated polymer nanofibers
Mukařovská, Sabina ; Sklenářová, Hana (advisor) ; Chocholouš, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of analytical chemistry Candidate: Sabina Mukařovská Supervisor: doc. PharmDr. Hana Sklenářová, Ph.D. Title of the diploma thesis: Automation of liberation tests for clotrimazole released from differently saturated polymer nanofibers This diploma thesis deals with the liberation profiles of clotrimazole released from a nanofiber carrier using an automated non-separation flow technique, sequential injection analysis. Polymer nanofibers polydioxanone and polycaprolactone were chosen as membranes. Nanofiber membranes were manufactured by the Technical University of Liberec. Two nanofiber saturation procedures were successively tested. First, membranes were used which were saturated during production in different ratios of polymer and clotrimazole content. These fiberes were analysed in the laboratory. In the second part, the fiberes without the active substance were tested, it means they originally did not contain clotrimazole. The nanofiber membranes thus prepared were saturated with ethanolic solutions of various concentrations and for a certain period of time in laboratory conditions. These analyses helped to find ideal conditions for nanofiber saturation. Liberation tests were performed under conditions that simulated the...
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HPLC in quality control of food supplements containing gingerols and curcumins
Hujová, Eliška ; Šatínský, Dalibor (advisor) ; Chocholouš, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Eliška Hujová Supervisor: prof. RNDr. Dalibor Šatínský, Ph.D. Title of the diploma thesis: HPLC in quality control of food supplements containing gingerols and curcumins In this diploma thesis, a new HPLC method for determination of 6-, 8- and 10-gingerol, 6-shogaol, curcumin, demethoxycurcumin, bisdemethoxycurcumin, and piperine was developed and validated. The developed method was used for the analysis of food supplements: Kurkumin - piperin plus (+ zázvor) from VIESTE, Bio tumeric + ginger from VANAVITA, Ginger root extract from BioMedical, Shogaol zázvor from EPIGEMIC®, Ginger root extract from SWANSON®, and Ginger root (Zingiber officinale) from SOLGAR®. Just as a matter of interest, this method was applied for the selected tea blends containing ginger and/ or tumeric: Dilmah Orange & Ginger (flavoured ceylon black tea), Loyd Brusinky & Zázvor, Teekanne Dýchací cesty a krk se zázvorem, Pickwick Nos a krk se zázvorem, Tesco Ginger & Lemon, Teekanne Ginger Curcuma. For the analysis YMC-Triart C18 ExRS (150 x 4.6 mm; 3 µm) analytical column was used. Separation was carried out by gradient elution with mobile phases A: acetonitrile, B: ultrapure water at flow rate 1 ml/min. Detection was...
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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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Sequential injection analysis BI-LOV - development of technique for sample treatment using solid phase extraction I
Popovská, Lenka ; Chocholouš, Petr (advisor) ; Sklenářová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical chemistry Candidate: Mgr. Lenka Popovská Supervisor: PharmDr. Petr Chocholouš, Ph.D. Title of diploma thesis: Sequential injection analysis BI - LOV - development of technique for sample treatment using solid phase extraction I Antioxidant activity of polyphenolic compounds can positively affect the therapy and the prevention of diseases associated with oxidative stress, e.g. cardiovascular diseases, atherosclerosis, neurodegenerative diseases (Alzheimer's and Parkinson's diseases), rheumatoid arthritis, virus diseases, neoplasms, benign prostatic hyperplasia (BHP), prostatitis etc. This diploma thesis deals with the analysis of phenolic acids, namely ferulic acid and also protocatechuic acid and o-coumaric acid. The ferulic acid was analysed within five concentrations (10,000 µg/ml, 5,000 µg/ml, 2,500 µg/ml, 1,250 µg/ml and 0,625 µg/ml), four different strong anion exchangers (TMAHP, QAE - A25, SAX - BOND and SAX - DSC), five different pH (5,0, 6,0, 7,0 a 8,5) of washing buffer and three different pH (1,5, 2,0 a 2,5) of elution buffer. Absorbance of the ferulic acid was measured at wavelengths 325 and 240 nm. In this thesis was also assessed handling with each sorbent, especially convenient use within LOV...
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