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LC-MS monitoring of selected antimicrobial compounds in waters
Krupka, Dominik ; Kučera, Radim (advisor) ; Kastner, Petr (referee)
5 Abstract Charles University Phaculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Dominik Krupka Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Consultant: Ing. Martin Ferenčík Title of diploma thesis: LC-MS monitoring of selected antimicrobial compounds in waters The monitoring of residues of human and veterinary antibiotics in surface water is gaining more and more importance due to the increasing resistance of bacteria. This work deals with the modificate of an HPLC-MS/MS method to obtain pilot data on the occurrence of antibiotics in surfaces water. The method is focused on the analysis of 30 antibiotics from the groups of β-lactams, macrolides, lincosamides, amphenicols, fluoroquinolones, tetracyclines and sulfonamides. The method was performed on a UHPLC instrument Agilent 1200 Infinity series with MS Agilent model 6495 triple Quad. Separation of antibiotics was performed on reverse phase using gradient elution and tandem mass spectrometry. An electrospray in both positive and negative polarity was used to ionize the analytes. The method was used for the pilot analysis of surface water samples in the Czech Republic. Samples from 15 rivers and streams were analysed. The most frequently found antibiotics were sulfomethoxazole and...
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Retention Behaviour of Catecholamines and Their Precursors under HILIC Conditions
Tibenský, Andrej ; Kastner, Petr (advisor) ; Štěrbová, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Andrej Tibenský Supervisor: PharmDr. Petr Kastner, Ph.D. Title of thesis: Retention behaviour of catecholamines and their precursors under HILIC conditions The aim of this diploma thesis was to study retention behaviour of catecholamines and their precursors under Hydrophilic Interaction Liquid Chromatography by using spectrophotometric and fluorometric detection. 5 analytes were examined - adrenaline, noradrenaline, dopamine, L-DOPA, and tyrosine. Retention behaviour studying began by using LUNA 3 µm (PFP)2 column which possesses both RPLC and HILIC characteristics with 2 different mobile phases. The first one consisted of 25 mM acetic acid in mixture of acetonitrile and water and the second mobile phase was 25 mM acetate buffer (pH 4,50) in mixture of acetonitrile and water. Subsequently, the effect of stationary phase was studied on neutral column XBridge BEH Amide and columns Avantor ACE HILIC-A and Sequant ZIC-HILIC with ionic interactions. Mobile phases consisted of high proportion of acetonitrile (70-90 %) and their composition was the following - 25 mM acetic acid in mixture of acetonitrile and water. Then, the effect of acetic acid concentration as a mobile...
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Use of liquid chromatography in pharmaceutical analysis II.
Sovič, Samuel ; Pilařová, Pavla (advisor) ; Kastner, Petr (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Samuel Sovič Tutor: PharmDr. Pavla Pilařová, Ph.D. Title of Thesis: The use of liquid chromatography in pharmaceutical analysis II The diploma thesis deals with the analysis of the retention behavior of selected antiepileptics: Gabapentin, Levetiracetam, Primidone and Lamotrigine. Three different C18 stationary phases were tested such as Phenyl; PFP; Kinetex, different mobile phase composition, column temperature and suitable wavelengths in the range of 210-220 nm for all analytes. The optimal analysis conditions were on an Agilent Eclipse XDB-Phenyl 5 μm column (4.6 mm x 150 mm), at a temperature of 40 řC and a flow rate of MP at 0.8 ml/min, at a wavelength of 220 nm. By creating a gradient, the analysis was shortened from 15 minutes with isocratic elution to 9 minutes. The method was partially validated in terms of linearity, accuracy, precision, selectivity and robustness. After that, the method was applied for real samples of patients from FN HK.
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Determination of tyrosine metabolites III
Licková, Gabriela ; Kastner, Petr (advisor) ; Váňová, Nela (referee)
CHARLES UNIVERSITY FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ DEPARTMENT OF PHARMACEUTICAL CHEMISTRY AND PHARMACEUTICAL ANALYSIS Candidate: Gabriela Licková Supervisor: PharmDr. Petr Kastner, Ph.D. Consultant: Ing. Daniel Heblík Titile of thesis: Determination of tyrosine metabolites The topic of thesis was determination of tyrosine metabolites (epinephrine, norepinephrine, dopamine, L-DOPA) using derivatization reaction and later analysis using High-performance liguid chromarography with fluorescence detector. The important step of all work was optimalization of conditions of derivatization reaction. As derivatization agent it was used FMOC-Cl (fluorenylmethyloxycarbonyl chloride), which reacted with catecholamines together with borate buffer. Firstly, it was set the ideal concentration of agent (2,7 mM), also the time (15 min) and the temperature (40 řC) of reaction. The concetration (100 mM) and pH (8,0) of borate buffer was optimalized in the end. For effective separation it was necessary to set ideal conditions. As silica gel column was used Kinetex EVO C18 with core-shell particles. The compositions of mobile phase was necessary to optimalize and resulting mobile phase was composed from formic acid (20 mM) and acetonitrile in proportion 32:68. Used flow rate was 0,6 ml/min. The column was tempered...
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Retention Behaviour of Catecholamines and Their Precursors under HILIC Conditions
Tibenský, Andrej ; Kastner, Petr (advisor) ; Štěrbová, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Andrej Tibenský Supervisor: PharmDr. Petr Kastner, Ph.D. Title of thesis: Retention behaviour of catecholamines and their precursors under HILIC conditions The aim of this diploma thesis was to study retention behaviour of catecholamines and their precursors under Hydrophilic Interaction Liquid Chromatography by using spectrophotometric and fluorometric detection. 5 analytes were examined - adrenaline, noradrenaline, dopamine, L-DOPA, and tyrosine. Retention behaviour studying began by using LUNA 3 µm (PFP)2 column which possesses both RPLC and HILIC characteristics with 2 different mobile phases. The first one consisted of 25 mM acetic acid in mixture of acetonitrile and water and the second mobile phase was 25 mM acetate buffer (pH 4,50) in mixture of acetonitrile and water. Subsequently, the effect of stationary phase was studied on neutral column XBridge BEH Amide and columns Avantor ACE HILIC-A and Sequant ZIC-HILIC with ionic interactions. Mobile phases consisted of high proportion of acetonitrile (70-90 %) and their composition was the following - 25 mM acetic acid in mixture of acetonitrile and water. Then, the effect of acetic acid concentration as a mobile...
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LC-MS monitoring of selected antimicrobial compounds in waters
Krupka, Dominik ; Kučera, Radim (advisor) ; Kastner, Petr (referee)
5 Abstract Charles University Phaculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Dominik Krupka Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Consultant: Ing. Martin Ferenčík Title of diploma thesis: LC-MS monitoring of selected antimicrobial compounds in waters The monitoring of residues of human and veterinary antibiotics in surface water is gaining more and more importance due to the increasing resistance of bacteria. This work deals with the modificate of an HPLC-MS/MS method to obtain pilot data on the occurrence of antibiotics in surfaces water. The method is focused on the analysis of 30 antibiotics from the groups of β-lactams, macrolides, lincosamides, amphenicols, fluoroquinolones, tetracyclines and sulfonamides. The method was performed on a UHPLC instrument Agilent 1200 Infinity series with MS Agilent model 6495 triple Quad. Separation of antibiotics was performed on reverse phase using gradient elution and tandem mass spectrometry. An electrospray in both positive and negative polarity was used to ionize the analytes. The method was used for the pilot analysis of surface water samples in the Czech Republic. Samples from 15 rivers and streams were analysed. The most frequently found antibiotics were sulfomethoxazole and...
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HPLC Determination of Selected Antibiotics in Plasma
Sedláčková, Andrea ; Kastner, Petr (advisor) ; Pilařová, Pavla (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Andrea Sedláčková Supervisor: PharmDr. Petr Kastner, PhD. Title of thesis: HPLC Determination of Selected Antibiotics in Plasma The purpose of this diploma thesis was to develop and optimize conditions for determination of selected antibiotics (meropenem, imipenem, ceftazidime, piperacillin and linezolid) in plasma using high performance liquid chromatography with meropenem as the main substance to be determined. Separation was performed on the silica gel column Ascentis® C18 (100Å, 150 × 4,6 mm; 5 µm) using spectrofotometric detection. Selected antibiotics were detected at different wavelenghts (298 nm for meropenem and imipenem, 254 nm for ceftazidime and linezolid, 210 nm for piperacillin) according to maximum absorbance of the antibiotics. Different types of mobile phase were tested as well as different pH of buffer used for the mobile phase. The final mobile phase consisted of two components: mobile phase A: phosphate buffer 50 mM; pH 2,5 mobile phase B: acetonitrile Separation was achieved by gradient elution. The flow rate was set to 1 ml/min and the injection volume was 10 µl. The column oven was tempered to 25řC. Total runtime was 15 min. Validation of...
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Study of Retention Properties of Selected Tyrosine Metabolites under HPLC Conditions
Gieslerová, Michaela ; Kastner, Petr (advisor) ; Pilařová, Pavla (referee)
Charles University Faculty od Pharmacy in Hradec Králové Department od Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Michaela Gieslerová Supervisor: PharmDr. Petr Kastner, Ph.D. Title of thesis: Study of retention properties of selected tyrosine metabolites under HPLC conditions The aim of this diploma thesis was to study the retention properties of selected tyrosine metabolites under the high performance liquid chromatography using spectrofotometric (285 nm) and fluorimetric detection (emission 279 nm, excitation 320 nm). The total of eight analytes was examined - tyrosine, L-DOPA, dopamine, epinephrine, norepinephrine, homovanillic and vanillylmandelic acid. Firstly, we studied the effect of mobile phase composition, buffer concentration and pH for the separation of analytes. Analysis of selected analytes was performed using an KINETEX 5 μm EVO C18 column (150 mm x 3 mm, 5 μm; PHENOMENEX). The best separation showed the mobile phase methanol:100 mM acetic acid (pH adjusted with sodium hydroxide to 4,5); 5:95. Next, the effect of stationary phase for the separation of analytes was studies with the mobile phase described above. A total of 7 different columns were studied, namely stationary phases with modified silica gel C18 group, PFP group, combined stationary phase (C18 group...
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Development and Validation of HPLC Method for Determination of 3-Hydroxykynurenine in Biological Material
Knoblochová, Dominika ; Kastner, Petr (advisor) ; Váňová, Nela (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Dominika Knoblochová Supervisor: PharmDr. Petr Kastner, Ph.D. Title of thesis: Development and Validation of HPLC Method for Determination of 3-Hydroxykynurenine in Biological Material 3-Hydroxykynurenine is one of the metabolites of tryptophan and is studied especially for its oxidative modulatory activity, which provides the ability to protect cells from oxidative stress. It is formed from kynurenine by catalysis of the enzyme kynurenine monooxygenase, whose activity is monitored at the Department of Pharmacology and Toxicology. It is necessary to quantify 3-hydroxykynurenine in a biological matrix for its study. The aim of this diploma thesis was to develop an HPLC method for the determination of 3-hydroxykynurenine in biological material using high performance liquid chromatography and subsequent method validation. During development, several mobile phases with different pH values were tested. The resulting mobile phase consisted of 100 mM formic acid (pH = 2.25) and methanol (95:5). Different types of stationary phases were also tested, from which a YMC TRIART PFP column with dimensions of 15 × 3 mm and a particle size of 5 µm was finally chosen. A UV-VIS...
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HPLC analysis of podophyllotoxin
Nováková, Markéta ; Pilařová, Pavla (advisor) ; Kastner, Petr (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Markéta Nováková Tutor: PharmDr. Pavla Pilařová, Ph.D. Title of Thesis: HPLC Analysis of Podophyllotoxin The diploma thesis is focused on optimalization of HPLC method for the analysis of podophyllotoxin. The method was developed for the analysis of Juniperus virginiana leaves extract. Various stationary phases, mobile phases, temperatures, ultrasonic extraction times were tested. Following conditions were chosen as optimal: 30 minutes ultrasonic extraction time, LiChrospher 100 RP-18 250-4 column, 0,1% CH3COOH:ACN 45:55 mobile phase, temperature 40 řC, flow rate 0,8 ml/min, sample volume 3 μl, fluorescence detection λex 240 nm, λem 320 nm, time of analysis 9 minutes. The method was also developed for the analysis of plant tissue cultures of Juniperus virginiana, for which gradient elution was chosen. This method had following conditions: LiChrospher 100 RP-18 250-4 column, mobile phase 0,1% CH3COOH:ACN, change of acetic acid ratio: 0-8 min 70-10 %, 8-9 min 10 %, 9-10 min 10- 70 %, 10-12 min 70 %, temperature 40 řC, flow rate 0,8 ml/min, sample volume 3 μl, fluorescence detection λex 240 nm, λem 320 nm, time of analysis 12 minutes. Subsequently, both methods were...
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