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UHPLC-HRMS in analysis of food supplements containing ginsenosides
Hlaváčová, Veronika ; Nováková, Lucie (advisor) ; Kočová Vlčková, Hana (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Veronika Hlaváčová Supervisor: prof. PharmDr. Lucie Nováková, Ph.D. Title of Diploma Thesis: UHPLC-HRMS in analysis of food supplements containing ginsenosides The aim of this work was to develop an analytical method for the analysis of the main components of a dietary supplement, in which the following plant extracts are contained: fenugreek seed, roseroot root, Asian ginseng root and damiana leaf. The ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC- HRMS) was used. Another goal was to determine whether prohibited testosterone is present in the dietary supplement. Measurements were carried out with ACQUITY UPLC I-Class System. A quadrupole time-of-flight mass analyzer SYNAPT G2-Si was used for mass spectrometric detection. Continuous data were collected. The data independent acquisition method (DIA) was used to collect data. The library of MS spectra of potentially predicted substances was created and ginsenosides were identified as major compounds. In the first step, the preparation of food supplement samples was optimized. Water, acetonitrile, methanol, ethanol and ethyl acetate were tested as solvents. In the next step, the chromatographic...
UHPLC-HRMS in analysis of food supplements containing ginsenosides
Hlaváčová, Veronika ; Nováková, Lucie (advisor) ; Kočová Vlčková, Hana (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Veronika Hlaváčová Supervisor: prof. PharmDr. Lucie Nováková, Ph.D. Title of Diploma Thesis: UHPLC-HRMS in analysis of food supplements containing ginsenosides The aim of this work was to develop an analytical method for the analysis of the main components of a dietary supplement, in which the following plant extracts are contained: fenugreek seed, roseroot root, Asian ginseng root and damiana leaf. The ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC- HRMS) was used. Another goal was to determine whether prohibited testosterone is present in the dietary supplement. Measurements were carried out with ACQUITY UPLC I-Class System. A quadrupole time-of-flight mass analyzer SYNAPT G2-Si was used for mass spectrometric detection. Continuous data were collected. The data independent acquisition method (DIA) was used to collect data. The library of MS spectra of potentially predicted substances was created and ginsenosides were identified as major compounds. In the first step, the preparation of food supplement samples was optimized. Water, acetonitrile, methanol, ethanol and ethyl acetate were tested as solvents. In the next step, the chromatographic...
Development of a sample preparation method for the determination of selected ochratoxins in urine by UHPLC-MS/MS
Šejvlová, Barbora ; Nováková, Lucie (advisor) ; Kočová Vlčková, Hana (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Barbora Šejvlová Supervisor: prof. PharmDr. Lucie Nováková, Ph.D. Title of the diploma thesis: Development of a sample preparation method for the determination of selected ochratoxins in urine by UHPLC-MS/MS The aim of the work was to develop a method for the determination of ochratoxins A, B, C, and  in urine with the lowest possible quantification limit, and subsequently to carry out the validation of this method, which would be applicable in clinical practice. The method of ultra- high-performance liquid chromatography in a reversed-phase mode in coupled with tandem mass spectrometry was selected for the analyse. Optimalization of sample preparation involved: micro-solid phase extraction in pipette tips (-SPE-PT), dilute and shoot (DAS), supported liquid extraction (SLE) and finally salting-out assisted liquid-liquid extraction (SALLE). The method recovery, the interference of the matrix and the limit of quantification of the given method were evaluated. Method validation was carried out on an ACQUITY UPLC BEH C18 1.7 μm, 2.1 x 100 mm column from Waters, using -SPE-PT as sample preparation technique. Method accuracy ranged from 68 to 122 % for all analytes, and method...
Reprodukční analýza plemenic skotu ve stádě s tržní produkcí mléka
Vlčková, Hana
The data for this thesis was collected in ZD Nížkov. In between the years of 2019-2021, monitoring was conducted on an average of 368 dairy cows of Czech fleckvieh cattle and Holstein and brown swiss cattle crossbreeds. Selected reproduction indexes were evaluated – calving-to-first-service interval, calving-to-conception interval, calving-to-calving interval and the number of insemination needed for pregnancy. The measured indicators were analyzed for individual years, within the seasons, according to the order of lactation and breeding affiliation of individuals. The obtained values were evaluated in Microsoft Excel and Statistica12. Basic statistical constants were calculated - arithmetic mean, standard deviation and coefficient of variation. In the Statistica12 program, selected factors were processed by ANOVA and subsequently evaluated by post hoc analysis using Tukey's test. In 2019, the length of the calving-to-first-service interval was 62,52 days, the calving-to-conception interval was 117,17 days, the calving-to-calving interval was 397,17 days and the number of insemination needed for pregnancy were 2,31. In 2020, the length of the calving-to-first-service interval was 55,19 days, the calving-to-conception interval was 118,17 days, the calving-to-calving interval was 398,17 days and the number of insemination needed for pregnancy were 2,61. In 2021, the length of the calving-to-first-service interval was 58,83 days, the calving-to-conception interval was 113,25 days, the calving-to-calving interval was 393,25 days and the number of insemination needed for pregnancy were 2,59. A great value of the breed group influence, as well as a high value of lactation period, was proven in the case of calving-to-first-service interval. For the length of the calving-to-conception interval, the breed group influence has also shown a great statistical significance.
Development of MEPS method for UHPLC-MS/MS determination of entecavir in renal ultrafiltrate
Janák, Jaroslav ; Vlčková, Hana (advisor) ; Chocholoušová Havlíková, Lucie (referee)
1 Abstract Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of analytical chemistry Candidate: Jaroslav Janák Supervisor: RNDr. Hana Vlčková, Ph.D. Title of Thesis: Development of MEPS-UHPLC-MS/MS methods for the determination of entecavir in kidney ultrafiltrate. The thesis deals with the development and validation of sample preparation method for determination of entecavir in kidney ultrafiltrate, using techniques MEPS (microextraction by packed sorbent). The previously developed UHPLC-MS/MS method for the determination of entecavir in rat urine has been used for the analysis. Hydrophilic interaction chromatography by column Acquity BEH Amide and isocratic elution were employed. The composition of the mobile phase was acetonitrile and 5 mM ammonium acetate pH 4.0 in the ratio 75:25 (v:v). Entecavir C2 13 N15 was used as the internal standard. Ionization of the analyte was carried out by electrospray in the positive ion mode and triple quadrupole was employed as the detector. Quantification of the analyte was realized by two SRM transitions of entecavir and internal standard method. Based on the MEPS optimization method, porous graphitic carbon suitable for the analysis of polar compounds was selected as the most suitable sorbent. The mixture of acetonitrile and water in a...
A development of UHPLC-MS/MS method for the determination of pravastatin, rosuvastatin and their metabolites
Mikšová, Alena ; Nováková, Lucie (advisor) ; Vlčková, Hana (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Alena Mikšová Supervisor: Doc. PharmDr. Lucie Nováková, PhD. Title of Graduation Thesis: A development of UHPLC-MS/MS method for the determination of pravastatin, rosuvastatin and their metabolites This graduation thesis is dealing with the development of UHPLC/MS-MS method for the determination of pravastatin, rosuvastatin and their metabolites. The basic parameters were optimized for MS detection - desolvation temperature, ion optics (RF lens and extractor), nitrogen flow at the sample cone, capillary voltage, nitrogen flow and cone voltage, which is specific for each substance. Separation of analytes was performed on BEH C18 analytical column (2.1 x 100 mm, 1.7µm). Triple quadrupole was used for detection. Electrospray ionization was performed in both negative and positive mode. Quantification of analytes was performed using the SRM (selected reaction monitoring). Ion [M+H]+ was chosen for all statins determined in positive ion mode. This ion was the most intense in full scan spectrum. In positive ion mode pravastatin lactone offered also ammonium adduct [M+NH4]+ . All statins determined in negative mode provided a precursor ion [M-H]- . Pravastatin lactone and rosuvastatin lactone...
Development of Stage tips-UHPLC-MS/MS method for determination of statins in human serum
Otoupal, Lukáš ; Vlčková, Hana (advisor) ; Šatínský, Dalibor (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of analytical chemistry Candidate: Lukáš Otoupal Supervisior: RNDr. Hana Vlčková, Ph.D. Title of Thesis: Development of Stage tips-UHPLC-MS/MS method for determination of statins in human serum This diploma thesis deals with development of a method for determination of statins in human serum. Extraction by Stage tips pipette tips was chosen and optimized for their extraction. The measurement was performed by the ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometer. The extraction procedure and important mass spectrometer parameters were optimized. Previously developed LC method for determination of statins was used for the analysis. The optimization of the mass spectrometer parameters was started by the selection of precursor ions, consequently the ion source parameters were tuned and finally appropriate ionization mode, SRM transition and collision energy for each analyte were chosen. The suitability of the LC-MS method was verified by the repeatability method (RSD ≤ 1 for the retention time and RSD ≤ 10 for the peak area), linearity (correlation coefficient ≥ 0,997) and sensitivity (LOQ in the range of 5 x 10-10 - 1 x 10- 9 g/ml). Firstly, the optimization employing mixed standard...
Study of influence of mobile phase composition on selectivity and retention of the analytes on HILIC stationary phases
Štětková, Kateřina ; Vlčková, Hana (advisor) ; Chocholoušová Havlíková, Lucie (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Kateřina Štětková Supervisor: RNDr. Hana Vlčková, Ph.D. Title of Diploma thesis: Study of influence of mobile phase composition on selectivity and retention of the analytes on HILIC stationary phases This graduation thesis deals with an influence of concentration of ACN, pH and buffer concentration in mobile phase on retention of selected analytes and on selectivity of Atlantis HILIC Silica column (3 μm, 2,1 mm x 100 mm) and Luna NH2 column (3 μm, 2,0 mm x 100 mm). A set of 35 analytes including neutral, basic and acidic compounds were chosen for this study. Detection was performed by PDA detector at the wave-length of 245 nm. Measurements were carried out using isocratic flow 0.4 ml.min-1 of mobile phase ACN/water (buffer). The column temperature was set up to 30 řC, auto sampler temperature to 4 řC and injection volume to 2 µl. For easier evaluation of results, measured data are presented in charts and tables. These were used to evaluation of columns in terms of selectivity and retention of the individual groups of analytes. The typical HILIC behavior, an increasing retention of analytes with increasing ACN concentration in mobile phase, was observed for Atlantis HILIC Silica column....
Application of alternative mobile phases in Hydrophilic interaction chromatography method
Münsterová, Barbora ; Nováková, Lucie (advisor) ; Vlčková, Hana (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of analytical chemistry Candidate: Barbora Münsterová Supervisor: doc. PharmDr. Lucie Nováková, Ph.D. Title of Diploma thesis: Application of alternative mobile phases in Hydrophilic interaction chromatography method. This thesis was focused on the possibilities of using 1,3-dioxolan as an alternative organic component of the mobile phase in HILIC. The changes in selectivity and efficiency of the separation were observed when using dioxolan and acetonitrile as the organic components of the mobile phase. 0.5 % acetic acid or 50 mM ammonium acetate pH 3.8, pH 6.8 and pH 9.8 were used as the aqueous components for the conventional HILIC. The mobile phase consisting of acetonitrile-dioxolan, acetonitrile-dioxolan with 0.5 % acetic acid and acetonitrile- dioxolan modified with 50 mM ammonium acetate aqueous solution pH 6.8 were tested for non-aqueous HILIC. For the measurements on column ACQUITY UPLC BEH Amide (1.7 µm, 2.1 mm x 100 mm, Waters) 5 different groups of compounds were used, including of phenolic acids, purine and pyrimidine bases, pyridine derivates, water-soluble vitamins and beta-blockers. Retention, selectivity and the speed of analysis were competed. Measurements were performed using ACQUITY Ultra Performance...

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8 VLČKOVÁ, Hana
2 Vlčková, Hedvika