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Synthesis of azaphthalocyanines with photophysical properties sensitive to metal cations
Lochman, Lukáš ; Zimčík, Petr (advisor) ; Doležal, Martin (referee)
Charles Univerzity in Prague, Faculty of Pharmacy in Hradec Králové Department Department of Pharmaceutical Chemistry and Drug Control Candidate Lukáš Lochman Supervisor Doc. PharmDr. Petr Zimčík, PhD. Consultant PharmDr. Veronika Nováková, PhD. Title of Thesis Synthesis of azaphthalocyanines with photophysical properties sensitive to metal cations Azaphthalocyanines (AzaPc) are macrocyclic planar compounds with interesting photophysical and photochemical properties. They usually absorb light over 650 nm and posses high quantum yields of fluorescence and singlet oxygen. However, we have recently demonstrated that introduction of one alkylamino group on the AzaPc periphery decreases quantum yields to zero due to intramolecular charge transfer (ICT). This property was used in the diploma thesis for design of a fluorescence sensor for metal cations. Detection of cations is based on blocking donor for ICT via its coordination to metal cation leading to restoration of fluorescence. The structure of intended AzaPc was designed to have six bulky tert-butylsulfanyl groups as an antiaggregation part and 1-aza-15-crown-5 moiety, which is suitable for coordination (detection) of sodium and potassium ions. Nucleophilic substitution was used for the synthesis of appropriately 5,6-disubstituted pyrazine-2,3-...
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Development of UHPLC-MS/MS method for analysis of selected drugs in a biological material
Reguli, Adam ; Štěrbová, Petra (advisor) ; Lochman, Lukáš (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Adam Reguli Supervisor - specialist: PharmDr. Hana Bavlovič Piskáčková, Ph.D. Supervisor: doc. PharmDr. Petra Štěrbová, Ph.D. Title of Thesis: Development of UHPLC-MS/MS method for analysis of selected drugs in a biological material Dexrazoxane is a cardioprotective drug clinically used against anthracycline induced cardiotoxicity. ICRF-193 was synthetized as a novel, more effective analogue of dexrazoxan. It was reported that inhibition of topoisomerase II, which seems to be crucial for cardioprotective effect, is mediated by an R,S diastereomer (ICRF-193) while the racemic mixture of R,R and S,S enantiomers is almost ineffective. Nevertheless our in vitro experiments with racemic mixture showed some cardioprotective effect. The aim of this work was 1) to examine the possible role of contamination of standard of racemic mixture with the ICRF-193 and 2) to develop solid phase extraction (SPE) for ICRF-193 and its metabolite (ICRF-193met) from plasma. The UHPLC coupled with a triple quadrupole mass spectrometer with electrospray ionization in positive mode. Bonus-RP column (100 × 3,0 mm, 1,8 μm) and formic acid (0,25%) with methanol as a mobile phase were used for...
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Modification of capillary wall by graphene for separation applications
Ptáčková, Aneta ; Lochman, Lukáš (advisor) ; Kučera, Radim (referee)
CHARLES UNIVERSITY FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Aneta Ptáčková Supervisor: PharmDr. Lukáš Lochman, Ph.D. Title of thesis: Modification of capillary wall by graphene for separation applications Capillary electrophoresis (CE) is a highly efficient separation method. Substances are separated due to their different mobility in an electric field. The CE modes of operation can be modified in different ways, e.g. capillary electrochromatography or micellar electrokinetic capillary chromatography. Modification of the inner wall of the capillary is believed to help improve separation efficiency and selectivity. Graphene is carbon with a hexagonal structure in form of two-dimensional sp2 single-atom-thick sheets. Graphene seems to be a suitable material for separation application due to its excellent properties such as large surface area and affinity to aromatic ring through π-π interactions. Our work is focused on the modification of the capillary wall by graphene. One of the methods of capillary wall modification is the Layer-by-Layer method via layering of differently charged substances bounded by electrostatic forces. Another method is chemical coating employing covalent interactions. Different combinations of polymers...
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Study of α-bromophenylacetic acid suitability as a model analyte for chiral separations using capillary electrophoresis as a separation technique II
Bubáková, Zuzana ; Jáč, Pavel (advisor) ; Lochman, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Zuzana Bubáková Supervisor: PharmDr. Pavel Jáč, Ph.D. Title of diploma thesis: Study of α-bromophenylacetic acid suitability as a model analyte for chiral separations using capillary electrophoresis as a separation technique II Rizvi and Shamsi (Rizvi S.A.A., Shamsi S. A. Anal. Chem. 2006, 78(19), 7061-7069) reported the employment of α-bromophenylacetic acid (BPAA) dissolved in water/methanol mixture (1:1, v/v) as a model analyte for chiral separations in capillary electrophoresis (CE). However, the stability of BPAA in aqueous methanol is questionable (Kováčová G. 2018. Diploma thesis. Charles University, Faculty of Pharmacy). The aim of this work was to determine the reaction order of the decomposition of BPAA (c = 0.47 mM) in 50% aqueous methanol. The reaction kinetic study was performed by capillary electrophoresis in 50 μm (i.d.) poly(vinyl alcohol)-coated capillary (30 cm/24.5 cm) with UV detection. The BPAA and products of nucleophilic substitution (mandelic acid, α-methoxyphenylacetic acid, and Br-) were separated in 60 mM formate buffer (pH 3.0) at -30 kV; the detection was 200 nm. The first order reaction kinetics was confirmed by linear and non-linear regression, yielding the rate...
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Quantitative analysis of tyrosine kinase inhibitors employing liquid chromatography with mass spectrometry detection
Maier, Jan ; Pávek, Petr (advisor) ; Lochman, Lukáš (referee)
The submitted thesis is devoted to the quantitative analysis of tyrosine kinase inhibitors, specifically imatinib and nilotinib, by liquid chromatography-mass spectrometry method. The main purpose of developing this new method of analysis at the Department of Clinical Biochemistry and Diagnostics at the University Hospital in Hradec Králové was measuring and monitoring serum or plasma concentration levels of these drugs in patients with chronic myeloid leukemia, less often in patients with gastrointestinal stromal tumour. The main task during the elaboration of the thesis was to fully optimize and validate the method. Previously, this method for the analysis of tyrosine kinase inhibitors was routinely performed here by high-performance liquid chromatography with spectrophotometric (UV) detection. As part of the modernization of laboratory technology, they started to use high-performance liquid chromatography with mass spectrometry at the workplace. The analytes with their internal standards were obtained by a liquid-liquid extraction process. Then, samples were separated on a C18 reverse phase column using isocratic elution. Subsequently, both analytes were detected by a triple quadrupole tandem mass spectrometer with ESI ion source in a positive mode. As a part of the method validation was to...
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Testing of potential capability of chiral ionic liquids with long alkyl chain in capillary electrophoresis chiral separations II
Müllerová, Kateřina ; Jáč, Pavel (advisor) ; Lochman, Lukáš (referee)
Department: Department of Analytical Chemistry Candidate: Kateřina Műllerová Supervisor: PharmDr. Pavel Jáč, Ph.D. Title of the diploma thesis: Testing of potential capability of chiral ionic liquids with long alkyl chain in capillary electrophoresis chiral separations II Chiral ionic liquid (−)-N-dodecyl-N-methylephedrinium bromide (DMEB) was tested as a selector for chiral separations of selected drugs such as chiral quinolones, ketoprofen, and flurbiprofen by capillary electrophoresis (CE). The effect of several parameters on enantioseparation was examined: (i) type and pH of the separation buffer, (ii) type and amount of organic modifier, and (iii) the concentration of DMEB. When using this chiral selector, only the separation of ofloxacin enantiomers was observed, while the enantiorecognition of other chiral model analytes was not successful under the conditions tested. As a next step, a CE method for the assay of levofloxacin was developed to demonstrate the potential of DMEB as a chiral selector in quality control of single-isomer drugs. The best separation was achieved with 20 mM tris buffer of pH 8.5 containing 100 mM DMEB and 20 % (v/v) of acetonitrile as the background electrolyte. The separation took place in 50 µm id fused silica capillary (80.5 cm / 72 cm) at −30 kV with UV detection...
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The employment of HPLC for separation of bioconjugates of azaphthalocyanines II.
Sčensná, Anna ; Kučera, Radim (advisor) ; Lochman, Lukáš (referee)
7 ABSTRACT Charles University, Faculty of Pharmacy in Hradec Králové Department Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate Anna Sčensná Supervisor doc. PharmDr. Radim Kučera, Ph.D. Title of Thesis The employment of HPLC for separation of bioconjugates of azaphthalocyanines II. Bioconjugates of azaphthalocyanines are studied as dark quenchers in molecular probes to monitor real-time PCR. The detection is based on energy transfer between a fluorophore and a quencher, both labelled to the oligonucleotide chain. The fluorophore emits a radiation and the dark quencher switches it off. The efficiency of fluorescence quenching depends on the distance between fluorophore and quencher. Probes can be mono-labelled, in which two oligonucleotides are used, one attached to a fluorophore and the other one to a quencher. The second type is called double-labelled probes, which contain both (fluorophore and quencher) on the same oligonucleotide chain. An unbound probe is randomly coiled, thus fluorophore and quencher approach each other. The fluorescence is not emitted. After hybridisation with a complementary chain, the quencher and fluorophore move away from each other. The terminal nukleosid at 5'end of Taq-man probe is cleaved after hybridisation, where the fluorophore is bonded. After...
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Biosensors for testing of cholinesterases' inhibitors
Keresteš, Ondřej ; Kučerová, Marta (advisor) ; Lochman, Lukáš (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Student: Bc. Ondřej Keresteš Supervisor: PharmDr. Marta Kučerová, Ph.D. Consultant: pplk. prof. RNDr. Miroslav Pohanka, Ph.D., DSc. Title of diploma's thesis: Biosensors for testing of cholinesterases' inhibitors Acetylcholinesterase (AChE, EC 3.1.1.7) cleaves acetylcholine in the nervous system. Binding of acetylcholine to the receptor and subsequent hydrolytic cleavage at central synapses or neuromuscular junction allows the repeated transmission of nerve impulses. This ensures controlled movement and optimal human cognitive abilities. In case of contact of AChE with the inhibitor, it becomes blocked, thus deteriorating cognitive functions and affects convulsions, with prolonged exposure death can occur. The aim of this work was to test various approaches to measurement of the activity of cholinesterases exposed to two inhibitors, as well as the preparation of biosensors using selected immobilization techniques. Three immobilization procedures were tested. Two of them were using electrochemical detectors and the aim was to optimize the method of quantification activity of AChE by measuring the pH difference (AChE was anchored by the chitosan membrane) and...
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Study of α-bromophenylacetic acid suitability as a model analyte for chiral separations using capillary electrophoresis as a separation technique II
Bubáková, Zuzana ; Jáč, Pavel (advisor) ; Lochman, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Zuzana Bubáková Supervisor: PharmDr. Pavel Jáč, Ph.D. Title of diploma thesis: Study of α-bromophenylacetic acid suitability as a model analyte for chiral separations using capillary electrophoresis as a separation technique II Rizvi and Shamsi (Rizvi S.A.A., Shamsi S. A. Anal. Chem. 2006, 78(19), 7061-7069) reported the employment of α-bromophenylacetic acid (BPAA) dissolved in water/methanol mixture (1:1, v/v) as a model analyte for chiral separations in capillary electrophoresis (CE). However, the stability of BPAA in aqueous methanol is questionable (Kováčová G. 2018. Diploma thesis. Charles University, Faculty of Pharmacy). The aim of this work was to determine the reaction order of the decomposition of BPAA (c = 0.47 mM) in 50% aqueous methanol. The reaction kinetic study was performed by capillary electrophoresis in 50 μm (i.d.) poly(vinyl alcohol)-coated capillary (30 cm/24.5 cm) with UV detection. The BPAA and products of nucleophilic substitution (mandelic acid, α-methoxyphenylacetic acid, and Br-) were separated in 60 mM formate buffer (pH 3.0) at -30 kV; the detection was 200 nm. The first order reaction kinetics was confirmed by linear and non-linear regression, yielding the rate...
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Lochman, Lukáš ; Nováková, Veronika (advisor) ; Musílek, Kamil (referee) ; Kubát, Pavel (referee)
Charles University, Faculty of Pharmacy in Hradec Kralove Department: Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Author: Mgr. Lukas Lochman Supervisor: Doc. PharmDr. Veronika Novakova, Ph.D. Advisor: Doc. PharmDr. Petr Zimcik, Ph.D. Doctoral Thesis: Study of recognition moieties of sensoric azaphthalocyanines Azaphthalocyanines (AzaPcs) are planar macrocyclic compounds with unique photophysical properties like absorption and emission over 650 nm and high quantum yields of fluorescence. Alkylamine substituted AzaPcs have been shown to undergo an ultrafast intramolecular charge transfer (ICT) which is responsible for efficient quenching of excited states (nonfluorescent OFF state). ICT occurs between donor (peripheral amine) and acceptor (macrocyclic core). Blocking of ICT leads to restoration of high fluorescence emission (fluorescent ON state) which is the competitive relaxation pathway. There are two known possible ways how to block ICT in AzaPcs sofar: By protonation of donor in pH sensitive AzaPc sensors or by coordination of metal cation into the recognition moiety resulting in cation sensitive AzaPc sensors. Above-mentioned properties predestinate AzaPcs to become new fluorescent sensors with good brightness and advantageous absorption and emission of red light deep...
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