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Optimization of separation conditions for HPCCC of paclitaxel
Havrlantová, Šárka ; Kučera, Radim (advisor) ; Šatínský, Dalibor (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Šárka Havrlantová Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Name of diploma thesis: Optimization of separation conditions for HPCCC of paclitaxel The aim of this work is a feasibility study of natural substances isolation (in my case it is paclitaxel and its intermediate 10-deacetylbaccatin) using modern technique called High performance countercurrent chromatography (HPCCC) available in Teva in Opava. For this basic research it was necessary to study the literature and test various solvent systems. Chosen solvent systems were then tested together with natural substance in HPCCC, results were processed and discussed. Countercurrent chromatography is considered a modern separation technique based on two immiscible liquids. One of them is called a stationary phase and is held inside the column by centrifugal force, the second liquid is called a mobile phase and is pumped through the column. Then the injected liquid sample is also pumped through the column and depending on distribution coefficients the sample components are either held in the stationary phase or pass through the column and detector into fraction collector. Advantages of this method are e.g. total...
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Modern chromatographic techniques in food contamination analysis
Lhotská, Ivona ; Šatínský, Dalibor (advisor) ; Karlíček, Rolf (referee) ; Malíř, František (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate Mgr. Ivona Lhotská Supervisor doc. RNDr. Dalibor Šatínský, Ph.D. Title of Doctoral Thesis Modern chromatographic techniques in food contamination analysis A compilation of seven publications dealing with chromatographic methods for determination of food contamination (particularly mycotoxins and artificial food colorants) is presented in dissertation thesis. All methods were fully validated, complying with the requirements and applied for real samples analysis. The first part describes the methods for mycotoxins determination based on on-line solid phase extraction (SPE) utilizing column-switching system. Reversed phase columns packed with superficially porous particles were chosen for both extraction and separation step during optimization of on-line SPE-HPLC method for ochratoxin A and citrinin determination in beer. In following work, determination of patulin in juices and zearalenone in beer, more selective molecularly imprinted polymers (MIP) were used for on-line extraction. Due to challenging optimization of on-line MIP SPE, the methods were compared to off-line MIP extraction and on-line extraction using conventional C18 phase to confirm the selectivity. The employed commercial MIP...
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Application of HPLC technique in analysis of food supplements based on plant extracts
Fibigr, Jakub ; Šatínský, Dalibor (advisor) ; Cahlíková, Lucie (referee) ; Horna, Aleš (referee)
ABSTRACT Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Mgr. Jakub Fibigr Supervisor: doc. RNDr. Dalibor Šatínský, Ph.D. Title of the Dissertation Thesis: Application of HPLC technique in analysis of food supplements based on plant extracts The disseration thesis deals with complex issues of food supplements based on plant extracts in terms of the content of bioactive substances, purity assessment, and legislative efficacy and safety requirements. The quality of food supplements available on the market in the Czech Republic was tested in six studies. Two HPLC and four UHPLC methods have been developed and validated to determine the content of bioactive substances in food supplements containing plant extracts and other excipients. In each work, such analytical conditions were found allowing a rapid and accurate determination of the content of the given bioactive substances in food supplements, particulary in the problematic separations of some isomeric substances. Additionally, sample preparation methods for each group of food supplement samples have been developed and optimized to avoid the matrix interferences during the separation and detection of analyzed bioactive substances. A conventional approach to the analytical determination of the...
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Determination of beta-carotene content in sour cherries by HPLC
Svobodová, Markéta ; Šatínský, Dalibor (advisor) ; Sklenářová, Hana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Mgr. Markéta Svobodová Supervisor: Doc. RNDr. Dalibor Šatínský, Ph.D. Title of the rigorous thesis: Determination of beta-carotene content in sour cherries by HPLC In this rigorous thesis, we determined the presence of carotenoids (trans betacarotene, cis beta-carotene and lutein) in 18 cultivars of sour cherries. Cultivars: Rannaja, Meteor, Krasnyj flag, Pandy 45, Muskatnaja pražskaja, Erdi jubileum, Griot Moskovskij, Valkova, Mesabich, Gorsetin, Inga mladi, Naumburger osheimer, Pandy 6039, Körös, Morellen fever, Wolyňská, Heimann's rubín, and Višňa IV/155. The optimized method was taken from the diploma thesis of Štěpán Růžička - HPLC method development for carotenoids determination in fruits (2017). In the chromatographic method, we used a Supelco Analytical RP-Amide column (100 x 4.6 mm, 5 μm) and carotenoids were detected at a wavelength of 450 nm. Measurements were carried out in the isocratic elution mode with a mobile phase of acetonitrile: hexane: dichloromethane (97.5 : 1.25 : 1.25) at a flow rate of 1.8 ml / min at 30 ř C. The analyzed samples were extracted by ultrasound into chloroform. After centrifugation, they were filtered and injected to the HPLC column. The sample volume was 5...
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Development of bioanalytical methods for the determination of diagnostic markers and drugs using chromatographic techniques
Javorská, Lenka ; Solich, Petr (advisor) ; Pohanka, Miroslav (referee) ; Šatínský, Dalibor (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department: Department of Analytical Chemistry Candidate: Mgr. Lenka Javorská Supervisor: prof. RNDr. Petr Solich, CSc. Consultant: assoc. prof. RNDr. Lenka Kujovská Krčmová, Ph.D. Title of dissertation thesis: Development of bioanalytical methods for the determination of diagnostic markers and drugs using chromatographic techniques The presented dissertation is focused on the development and validation of chromatographic method for the determination of glycopeptide antibiotic vancomycin, and L-arginine and its pathway related substances in biofluids. Furthermore, the potential of neopterin, kynurenine and tryptophan as biomarkers of immune response to surgical procedure and prediction of postoperative complications is investigated in connection with oncological diseases. In the theoretical part trends in instrumentation, stationary phases and extraction techniques are described. The issues of separation and retention of polar compounds and biological matrix pretreatment before analysis are also discussed there. Next part deals with selected analytes and their application to the clinical purpose for which the chromatographic methods are developed. It also provides an overview of published methods for their determination using liquid...
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Development of HPLC method for determination of selected phenolic acids and flavonoids in Tokaj wines
Moravcová, Pavlína ; Šatínský, Dalibor (advisor) ; Lhotská, Ivona (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical chemistry Candidate: Bc. Pavlína Moravcová Supervisor: Doc. RNDr. Dalibor Šatínský, Ph.D. Title of the diploma thesis: Development of HPLC method for determination of selected phenolic acids and flavonoids in Tokaj wines The aim of this diploma thesis was to develop and optimize HPLC method for simultaneous determination of 15 phenolic compounds, protocatechic acid, fertaric, coffee, p-hydroxybenzoic, syringic, gallic, p-coumaric, ferulic, chlorogenic, caftaric, 2-hydroxy-4-methoxybenzoic , polydatin, resveratrol, catechin hydrate and epicatechin in Tokaj wines. The quantity and content of selected phenolic compounds was analyzed in 25 samples of Tokaj wines, which come from vineyards from the Slovak part of Tokaj wine region. The work was also focused on the description of the specific characteristics of this unique area, the unique method of Tokaj wine production, its chemical composition, and namely the spectrum of phenolic substances. Analysis of selected phenolic compounds was performed using an Ascentis® Express F5 column (150 x 4.6 mm; 5 µm) using a mobile phase consisting of 0.1% phosphoric acid and acetonitrile in gradient elution. The substances were analyzed at 30 řC and flow rate of 1 ml/min and detected...
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Development of on-line SPE HPLC method for determination of ochratoxin A in Tokaj wines
Uhrová, Adéla ; Šatínský, Dalibor (advisor) ; Chocholouš, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Adéla Uhrová Supervisor, consultant: Doc. RNDr. Dalibor Šatinský, Ph.D. Title of Diploma Thesis: Development of on-line SPE HPLC method for determination of ochratoxin A in Tokaj wines This diploma thesis deals with the optimization and validation of chromatographic method for determination of ochratoxin A in Tokaj wine with subsequent application on real samples. A high- performance liquid chromatography method with fluorescence detection using on-line extraction by column switching has been developed. 50µl of sample was injected directly in the extraction column, where at 1st minute the analyte was extracted on the Ascentis® Express RP-Amide precolumn in the presence of a mobile phase of 40% MeOH in 0.5% aqueous acetic acid. After the extraction, the valve was switched and the extracted substances were separated on a YMC Triart C18 ExRS analytical column. The separation was carried out under gradient elution with an initial acetonitrile ratio of 45% in the mobile phase, which reached a concentration of 80% in the 5th minute. Fluorimeter detection was optimized for excitation and emission wavelengths ʎEx 335 nm and ʎEm 463 nm. In the last part of the work, ochratoxin A was analyzed in real...
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Evaluation of selected substances in fruit by HPLC-DAD-CAD method
Košková, Stanislava ; Sklenářová, Hana (advisor) ; Šatínský, Dalibor (referee)
Charles University Faculty of Pharmacy in Hradci Králové Deparment of Analytical Chemistry Candidate: Bc. Stanislava Košková Supervisor: Doc. PharmDr. Hana Sklenářová, Ph.D. Consultant: Ing. Radek Vávra, Ph.D. (VŠÚO Holovousy) Title of diploma thesis: Evaluation of selected substances in fruit by HPLC-DAD-CAD method Thisdiploma thesis was focused on determination of seven phenolic compounds, namely gallic acid, chlorogenic acid, epicatechin, rutin, phloridzin, quercetin, phloretin in twenty apple cultivars, that have been delivered by the Research and Breeding Institute of Pomology Holovousy s.r.o. For this purpose HPLC separation in reverse mode was used, using the column Omega Polar C 18 (150 mm x 4.6 mm; 5 µm). As the mobile phase the mixture of an ultrapure water acidified with the acetic acid to pH 2.8 and acetonitrile was used. A linear gradient was chosen with a gradual increase of the organic component from the initial 10 % to 50 % during 10 min of separation.This was followed with column equilibration - up to 12.50 min to 10 % organic component. The overall separation time was 12.50 min at a flow rate of 1 ml/min. Column temperature was set to 30 řC. Detection was performed using DAD (254, 280, 320 nm) and CAD detector (nebulizer temperature 25 řC). Part of this thesis is system...
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Development of HPLC method for determination of coumarins in Tokaj wines
Sudová, Tereza ; Šatínský, Dalibor (advisor) ; Sklenářová, Hana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Tereza Sudová Supervisor: Doc. RNDr. Dalibor Šatínský, Ph.D. Title of the diploma thesis: Development of HPLC method for determination of coumarins in Tokaj wines A high performance chromatography method was used for simultaneous determination of coumarins: umbelliferone (7-hydroxycoumarin), coumarin and 7-methoxycoumarin (herniarin) in 60 samples of Tokaj wines and 12 samples of wines and spirits. Before the analysis, HPLC parameters were optimized: detector wavelength, analytical column, mobile phase and gradient elution. The suitability of the HPLC method for the determination of coumarins was confirmed. Then the method was validated by a validation process. The analysis was performed under gradient elution conditions with mobile phase methanol (organic phase) and 0.1% phosphoric acid (aqueous phase) at a flow rate of 1 ml/min. Ascentis® Express F5 - 150 4.6 mm reversed phase column at a temperature of 30 řC was used. The particle size of the stationary phase was 5 µm. Coumarins were detected by a DAD detector at wavelenghts 276 nm and 323 nm. Untreated samples were injected in a volume of 5 µl. The total analysis was 15.51 min.
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