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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...
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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...
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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...
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Comparison of two types of matrix effect evaluation for LC-MS/MS analysis
Lejnarová, Michaela ; Kočová Vlčková, Hana (advisor) ; Nováková, Lucie (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Michaela Lejnarová Supervisor: RNDr. Hana Kočová Vlčková, Ph.D. Title of Diploma Thesis: Comparison of two types of matrix effect evaluation for LC-MS/MS analysis This diploma thesis deals with a comparison of two methods of matrix effect evaluation in LC-MS/MS analysis using electrospray ionization and triple quadrupole. Specifically, it was a comparison of the method using slopes of a line of the calibration curves and the post-extraction addition method, where the main goal was to assess whether the obtained values of matrix effects correlate with each other or not. Where appropriate, to clarify the reasons for the different outcomes and define the conditions of both methods to obtain correct and accurate results. On the basis of physicochemical properties such as molecular weight, acid-base properties, and partition coefficient, a group of 28 diverse substances was selected. The actual evaluation of matrix effects was preceded by the selection of an appropriate LC method, optimization of the individual parameters of the ion source as well as the selection of appropriate SRM transition and collision energy for each substance. To evaluate the matrix effects, lyophilized serum was chosen as a...
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Development UHPLC-MS/MS method and sample preparation procedure for the determination of steroid compounds in rat plasma
Hromádko, Jiří ; Kočová Vlčková, Hana (advisor) ; Chocholouš, Petr (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Jiří Hromádko Supervisor: RNDr. Hana Kočová Vlčková Ph.D. Title of Diploma Thesis: Development UHPLC-MS/MS method and sample preparation procedure for the determination of steroid compounds in rat plasm The diploma thesis deals with the development of the UHPLC-MS/MS method for the analysis of 37 steroids in plasma and optimization of sample preparation using PP. The analytes include structurally very similar substances with different numbers of hydroxyl or ketonic functional groups, isomeric and mutually isobaric compounds. Since these substances cleave water molecules very easily, precursor ions with one m/z for different substances can be found in the MS spectrum. For these reasons, careful and detailed characterization of the precursors, subsequent scanning of the product ions, and selection of additional SRM transitions were essential. Separation of critical compounds with the same m/z for precursor ions was of importance. The optimization started with the screening of 7 columns using gradient elution of the mobile phase ACN/0.1 % FA in the range of 5 - 98% ACN. Based on the results, an optimization of the mobile phase gradient was performed. The Cortecs C18 column was optimal. Most of the...
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A study of separation conditions and stability of selected phenolic compounds
Jandorová, Zuzana ; Nováková, Lucie (advisor) ; Kočová Vlčková, Hana (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Zuzana Jandorová Supervisor: Prof. PharmDr. Lucie Nováková, Ph.D. Title of the diploma thesis: A study of separation conditions and stability of selected phenolic compounds The goal of this diploma thesis was to determine the most suitable separation conditions for the analysis of ten phenolic compounds by the ultra-high performance liquid chromatography with PDA detection. The thesis is focused mainly on the separation of critical pair tamarixetin and isorhamnetin. Another goal was to determine the short-term stability of all tested compounds for 24 hours. Analyses were performed using the ACQUITY UPLC chromatographic system with PDA detector at a wavelength of 275 nm. The first step in the study of separation conditions was the screening of 17 columns from several different manufacturers. Tested columns included ACE Excel C18, BEH C18, CSH C18, Arion Plus C18, Ascentis Express C18, Atlantis Premier BEH C18 AX, BEH Shield RP18, Cortecs Shield RP18, Ascentis Express RP-Amide, BEH Phenyl, CSH Phenyl-Hexyl, Kinetex Biphenyl, ACE Excel C18-AR, ACE Excel C18-PFP, CSH Fluoro-Phenyl, Ascentis Express F5, and Kinetex PFP. During the screening, 3 organic modifiers (acetonitrile, methanol and acetonitrile...
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Study of influence of mobile phase composition on selectivity and retention of the analytes on HILIC stationary phases
Štětková, Kateřina ; Kočová 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....
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Development UHPLC-MS/MS method and sample preparation procedure for the determination of steroid compounds in rat plasma
Hromádko, Jiří ; Kočová Vlčková, Hana (advisor) ; Chocholouš, Petr (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Jiří Hromádko Supervisor: RNDr. Hana Kočová Vlčková Ph.D. Title of Diploma Thesis: Development UHPLC-MS/MS method and sample preparation procedure for the determination of steroid compounds in rat plasm The diploma thesis deals with the development of the UHPLC-MS/MS method for the analysis of 37 steroids in plasma and optimization of sample preparation using PP. The analytes include structurally very similar substances with different numbers of hydroxyl or ketonic functional groups, isomeric and mutually isobaric compounds. Since these substances cleave water molecules very easily, precursor ions with one m/z for different substances can be found in the MS spectrum. For these reasons, careful and detailed characterization of the precursors, subsequent scanning of the product ions, and selection of additional SRM transitions were essential. Separation of critical compounds with the same m/z for precursor ions was of importance. The optimization started with the screening of 7 columns using gradient elution of the mobile phase ACN/0.1 % FA in the range of 5 - 98% ACN. Based on the results, an optimization of the mobile phase gradient was performed. The Cortecs C18 column was optimal. Most of the...
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Development and validation of UHPLC-MS/MS method for determination of maraviroc in placental perfusion
Čapková Maxová, Eliška ; Nováková, Lucie (advisor) ; Kočová Vlčková, Hana (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of analytical chemistry Candidate: Eliška Čapková Maxová Supervisor: Assoc. Prof. PharmDr. Lucie Nováková, Ph.D. Title of Thesis: Development and validation of UHPLC-MS/MS method for the determination of maraviroc in placental perfusions The goal of this work was to develop fast and selective method for the determination of maraviroc in human placental perfusions. The ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) was used. BEH C18 column and gradient elution with the mobile phase A (water with 0.1% formic acid) and B (acetonitrile) at 0.35 mL/min flow-rate and 40 řC temperature were used for the separation. The mass spectrometry conditions were optimist and set up as follows: electrospray ionization in positive mode, capillary voltage 1.0 kV, cone voltage 35 V, extractor 3.0 V, RF lens 0.1 V, desolvatation gas flow 1000 L/h, temperature 450 žC, cone gas flow 100 l/h, ion source temperature 130 žC. Selected reaction monitoring (SRM) mode was used for quantitation. Liquid-liquid extraction (LLE) was chosen for sample preparation and was optimised. The best results were obtained when dichloromethane was used as the extraction agent (recovery >90%). The optimized method was fully validated in...
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Comparison of two types of matrix effect evaluation for LC-MS/MS analysis
Lejnarová, Michaela ; Kočová Vlčková, Hana (advisor) ; Nováková, Lucie (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Michaela Lejnarová Supervisor: RNDr. Hana Kočová Vlčková, Ph.D. Title of Diploma Thesis: Comparison of two types of matrix effect evaluation for LC-MS/MS analysis This diploma thesis deals with a comparison of two methods of matrix effect evaluation in LC-MS/MS analysis using electrospray ionization and triple quadrupole. Specifically, it was a comparison of the method using slopes of a line of the calibration curves and the post-extraction addition method, where the main goal was to assess whether the obtained values of matrix effects correlate with each other or not. Where appropriate, to clarify the reasons for the different outcomes and define the conditions of both methods to obtain correct and accurate results. On the basis of physicochemical properties such as molecular weight, acid-base properties, and partition coefficient, a group of 28 diverse substances was selected. The actual evaluation of matrix effects was preceded by the selection of an appropriate LC method, optimization of the individual parameters of the ion source as well as the selection of appropriate SRM transition and collision energy for each substance. To evaluate the matrix effects, lyophilized serum was chosen as a...
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