No description available.

The dissertation thesis is focused on the physico-chemical characterization of interaction mechanisms of chiral stationary phases based on derivatized cyclofructans. Correct interpretation of retention and enantiodiscrimination processes substantially facilitates the development and optimization of new enantioselective methods using cyclofructan-based chiral stationary phases. At first, the interaction mechanisms of three commercially available cyclofructan-based stationary phases were studied in normal-phase mode of liquid chromatography. Namely, systems using chiral stationary phases based on dimethylphenyl carbamate cyclofructan 7, R-naphtylethyl carbamate cyclofructan 6 and isopropyl carbamate cyclofructan 6 were studied. Linear free energy relationship model was used as a basic tool for characterization of interactions on the stationary phases. The mentioned model revealed that the main interactions contributing to retention in cyclofructan-based systems are hydrogen bond acidity and dipolarity/polarizibility, while dispersion interactions cause decrease of retention. The impact of oligosaccharide skeleton of the cyclofructan selector on the enantioselectivity was elucidated by the comparison with seemingly analogous cyclodextrin-based chiral stationary phases. Cyclofructan-based chiral...

Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Chemistry and Pharmaceutical Analysis Student: Bc. Martin Novák Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Consultant: Mgr. et Mgr. Rafael Doležal, Ph.D. Title of diploma thesis: Optimization of chromatographic parameters for chiral separation of biologically active compounds. The diploma thesis was focused on the development of a HPLC-UV method for the determination of K 1277 enantiomers of systematic name N-(2-((6-chloro-1,2,3,4-tetra- hydroacridin-9-yl)amino)hexyl-2-amino-3-(1H-indole-3-yl) propylamide dihydrochloride, which is one of the compounds from the tacrine-tryptophan hybrids group. These tacrine-tryptophan hybrids could be considered as promising candidates of potential drugs against Alzheimer's disease. The thesis brings an explanation of basic characteristic of chiral molecules, principles of chiral separation, pathophysiology, clinical manifestation and treatment of Alzheimer's disease and short characteristic of tacrine-tryptophan hybrids in the theoretical section. The aim of my diploma thesis was to find the optimal chromatographic conditions for separation of K 1277 enantiomers synthesized from tacrine and tryptophan fragments. The experimental part deals with the development of the chiral...

Direct chiral separations of the non-steroidal drugs of 2-methylarylpropionic acids (profens) on the chiral stationary phases (CSPs) of amylose tris(3,5-dimethylphenyl-carbamate), Chiralpak AD, and cellulose tris(3,5-dimethylphenylcarbamate), Chiralcel OD, were investigated. Chiralpak AD and Chiralcel OD are CSPs coated on silica gel and have the same type of constituents. However, they have different higher order structures arising from their different arrangements of the glucose units, i.e., the former has an a-(1,4)-D-glucose linkage and the latter has a b-(1,4)-D-glucose linkage. The orders of optimum enantioselectivity of racemic acids were reversed on the two CSPs which demonstrated that the enantioseparating abilities of these CSPs are complementary. This phenomenon also confirmed that the chiral recognition abilities of both CSPs were dependent on their higher order structures. Mechanisms for retention and chiral recognition for the separation of racemic 2-methylarylpropionic acids on Chiralpak AD and Chiralcel OD were explored. In depth studies of the dependence of retention and enantioselectivity on temperature and mobile phase compositions were made. The thermodynamic parameters, the differences in free energy, enthalpy, and entropy of association between enantiomers and the CSP were evaluated. The results indicated that the retention of racemic acids on both CSPs is mainly dependent on the hydrogen bonding interaction between the acid proton of the carboxyl moiety of the analyte and the carbonyl oxygen of the carbamate moiety of the CSP. The chiral recognition mechanism for Chiralpak AD involves: (1) the formation of transient diastereomeric analyte-CSP complexes through hydrogen bonding interactions between the carboxyl and the carbamate moieties of the acid and CSP, respectively; (2) stabilization of these complexes by insertion of the aromatic portion of the analytes into the chiral cavities of the CSP, as well as pi-pi, dipole-dipole, and additional hydrogen bonding interactions between analyte and CSP; and (3) chiral discrimination between enantiomer analytes arising from the additional hydrogen bond between analyte and CSP. For Chiralcel OD, the chiral recognition mechanisms involve: (1) the formation of transient diastereomeric analyte-CSP complexes through hydrogen bonding interactions between the carboxyl and the carbamate moieties of the acid and CSP, respectively; (2) stabilization of these complexes by insertion of the aromatic portion of the analytes into the chiral cavities of the CSP, as well as pi-pi and dipole-dipole interactions between analyte and CSP; and (3) chiral discrimination due to: (a) the difference in the steric fit of enantiomers into the chiral cavity of the CSP (entropy controlled); and (b) dipole-dipole or p-p interactions between enantiomer analytes and CSP (enthalpy controlled). Chromatographic and quantitative thermodynamic data showed that there are at least two different chiral recognition mechanisms for Chiralcel OD. One mechanism was characterized by negative values for the enthalpy and entropy differences of the association between enantiomers and CSP that classifies the enantioseparation to be enthalpy controlled. This behavior was exhibited by racemic 2-methylarylpropionic acids with fused rings that were favorably separated at low temperatures. The other mechanism was associated with positive values for the enthalpy and entropy differences of the association between enantiomers and CSP, and the enantioseparation is said to be entropy controlled. The analytes with "free" phenyl moieties favored high temperatures for their enantioseparations. Both studies on the effects of temperature and mobile phase composition also indicated that the higher order structures of CSPs influence their chiral recognition abilities. / Ph. D.

In the first study, a new µ-HPLC column was developed using a monolithic silica gel as a column substrate for chiral separation by covalently modifying with (S, S)-Whelk-O1 chiral selector. The monolithic stationary phase was generated through a sol-gel process and prepared in situ in a 100 µm i.d. fused silica capillary tubing. The chromatographic performance was characterized in terms of retention factor, column efficiency, enantioselectivity and resolution, as well as the kinetics parameters affecting the separation. Comparison with a commercial particle packed HPLC column demonstrates a promising enantioselective resolving ability of the monolithic Whelk-O1 capillary column. The second project focuses on characterization of fluorescent sensor for zinc detection. In this work, we have examined the photophysical properties of the fluorescent probe sensor that has been developed in our laboratory for Zn2+ recognition via a photo-induced electron transfer (PET) sensing mechanism. To characterize the fundamental function of sensor, response curves have been conducted, using acetone/methanol (199:1), 1,4-dioxane, acetone, methanol and aqueous buffer as the solvent system. Similar to prior work from our group, the sensor was found to respond selectively to Zn2+ ions with fluorescence enhancement. The fluorescence properties and binding response were evaluated in the presence of water and a Lewis base, which we found to have a marked effect on the fluorescence signal. The selectivity of the sensor for Zn2+ was also observed and compared to other divalent metal such as Ca2+, Mg2+, Cu2+ and Hg2+ with the goal of learning fundamental information on the system that can aid in the development of future PET based sensors.

Charles University Faculty of Pharmacy in Hradec Králové Department: Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Jakub Burda Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Title of Thesis: The employment of HPLC in the field of chiral separations V. Continuously growing requirements of regulatory authorities for the quality and safety of medicines put a high pressure on manufacturers. Last decades are marked by optically pure drugs, whose developement goes hand in hand with developement of chiral syntheses and separations. The most used method in the field of separations are direct separations using chiral stationary phases. The most often used carrier for these stationary phases is silica, which may contain metal impurities on its surface, negatively impacting separation process. The focus of this thesis was testing of influuence of chromatographic conditions on chiral and achiral interactions of seven selected analytes with stationary phase. Column with native β-cyclodextrin as chiral selector was used for the testing. 1

Charles University Faculty of Pharmacy in Hradec Králové Department: Department of Pharmaceutical chemistry and Pharmaceutical analysis Candidate: Jana Marvalová Supervisor: doc. PharmDr. Radim Kučera, PhD. Title of Thesis: The employment of HPLC in field of chiral separations VI. Boron clusters are synthetically prepared substances that are being intensively studied in connection with Boron Neutron Capture Therapy, the substitution of phenyl rings of molecules of already known drugs and as potential inhibitors of HIV protease. Boron clusters are symmetric molecules, however, by endo- or exo-skeletal substitution, the symmetry of the cluster is disrupted and enantiomers are formed. This diploma thesis is focused on the investigation of chromatographic conditions for chiral separations of cosanes (bis(dicarbollides) and 7,8-dicarb-nido-undecaborates derivatives using high performance liquid chromatography and chiral selectors based on cellulose and amylose in reverse-phase liquid chromatography. For this purpose, columns Lux Cellulose-1 and Lux Amylose-1 were selected with chiral selectors cellulose tris(3,5-dimethylphenylcarbamate) and amylose tris(3,5-dimethylphenylcarbamate), respectively. The mobile phases were mixtures of methanol or acetonitrile with sodium perchlorate or sodium chloride...

A diferença na atividade terapêutica, farmacocinética e / ou farmacodinâmica entre os enantiômeros de fármacos quirais impulsionou a necessidade de estudar e desenvolver métodos para determinação exata e precisa da pureza enantiomérica dos produtos farmacêuticos. Inicialmente, os enantiômeros foram separados em escala analítica. As condições analíticas foram adaptadas para a escala semi-preparativa para a obtenção enantiômeros puros. Os enantiômeros do bisoprolol foram separados através de Cromatografia Líquida de Alta Eficiência. Foi adotado o sistema direto de separação, fase normal, utilizando coluna Chiralcel OD (250 x 4,6 mm id). A fase móvel foi composta por hexano: etanol: dietilamina (80:20:0.2, v / v / v), vazão de 1mL/min e detecção em UV a 273 nm. A separação dos enantiômeros (R)-bisoprolol e (S)-bisoprolol foi obtida com sucesso em escala analítica e semi-preparativa. Considerando as características de uma separação quiral, podemos concluir que os resultados são eficazes, por ser uma separação rápida e seletiva. / The difference in therapeutic activity, pharmacokinetics, and / or pharmacodynamics between enantiomers of chiral drugs has raised the need to study and develop methods for accurate and precise determination of enantiomeric purity of pharmaceutical products. Initially, the enantiomers were separation in analytical scale. The analytical conditions were scaled up to semi-preparative level to obtain pure enantiomers. The enantiomers of bisoprolol were separated with high-performance liquid chromatography. Direct separation system was adopted in normal phase mode using Chiralcel OD column (250 x 4.6 mm id). The mobile phase was composed of hexane:ethanol:diethylamine (80:20:0.2, v/v/v), flow rate of 1mL/min and UV detection was made at 273 nm. The separation of enantiomers, (R)-bisoprolol and (S)-bisoprolol was successfully obtained in analytical and semi-preparative scale. Considering the characteristics of a chiral separation, we can conclude that the results are effective, because it is a fast and selective separation.

A diferença na atividade terapêutica, farmacocinética e / ou farmacodinâmica entre os enantiômeros de fármacos quirais impulsionou a necessidade de estudar e desenvolver métodos para determinação exata e precisa da pureza enantiomérica dos produtos farmacêuticos. Inicialmente, os enantiômeros foram separados em escala analítica. As condições analíticas foram adaptadas para a escala semi-preparativa para a obtenção enantiômeros puros. Os enantiômeros do bisoprolol foram separados através de Cromatografia Líquida de Alta Eficiência. Foi adotado o sistema direto de separação, fase normal, utilizando coluna Chiralcel OD (250 x 4,6 mm id). A fase móvel foi composta por hexano: etanol: dietilamina (80:20:0.2, v / v / v), vazão de 1mL/min e detecção em UV a 273 nm. A separação dos enantiômeros (R)-bisoprolol e (S)-bisoprolol foi obtida com sucesso em escala analítica e semi-preparativa. Considerando as características de uma separação quiral, podemos concluir que os resultados são eficazes, por ser uma separação rápida e seletiva. / The difference in therapeutic activity, pharmacokinetics, and / or pharmacodynamics between enantiomers of chiral drugs has raised the need to study and develop methods for accurate and precise determination of enantiomeric purity of pharmaceutical products. Initially, the enantiomers were separation in analytical scale. The analytical conditions were scaled up to semi-preparative level to obtain pure enantiomers. The enantiomers of bisoprolol were separated with high-performance liquid chromatography. Direct separation system was adopted in normal phase mode using Chiralcel OD column (250 x 4.6 mm id). The mobile phase was composed of hexane:ethanol:diethylamine (80:20:0.2, v/v/v), flow rate of 1mL/min and UV detection was made at 273 nm. The separation of enantiomers, (R)-bisoprolol and (S)-bisoprolol was successfully obtained in analytical and semi-preparative scale. Considering the characteristics of a chiral separation, we can conclude that the results are effective, because it is a fast and selective separation.

A malária é a doença que mais mortes causa no mundo. O uso de fármacos antimaláricos representa a solução mais eficaz para o combate e controle da doença e o interesse pelo desenvolvimento de novos fármacos é grande devido a problemas de resistência. Existe uma grande variedade de fármacos antimaláricos, sendo que muitos deles são quirais, vendidos e administrados como mistura racêmica. Nas últimas décadas, houve um aumento no interesse quanto aos aspectos farmacodinâmicos e farmacocinéticos de fármacos quirais, devido ao conhecimento de que um dos isômeros pode ser mais ativo ou mais tóxico que o outro. Sendo assim, tem-se a necessidade do desenvolvimento de métodos analíticos enantiosseletivos, e a eletroforese capilar tem emergido como uma técnica de separação quiral com alto poder de resolução. Além disso, a inexistência de métodos oficiais para fármacos antimaláricos e os poucos estudos que relatam aplicações na análise de formulações farmacêuticas, demandam o desenvolvimento e validação de novos métodos para tal finalidade. Este trabalho tem como objetivo o desenvolvimento de dois métodos analíticos, não quiral e quiral, para a determinação de fármacos antimaláricos em formulações farmacêuticas por eletroforese capilar. O método não quiral utilizou eletroforese capilar de zona, sendo otimizado para a separação de 7 fármacos antimaláricos (cloroquina, hidroxicloroquina, primaquina, quinidina, quinina, quinacrina e mefloquina) com um eletrólito composto por 45 mmol L-1 de tampão citrato, pH 4,50, e apresentou um tempo de análise de 10 minutos, permitindo a separação dos diastereoisômeros quinina e quinidina sem a adição de aditivos. O método quiral utilizou eletroforese capilar de zona modificada por ciclodextrina e foi otimizado para separação enantiosseletiva de cloroquina, mefloquina, hidroxicloroquina, primaquina, quinina e quinidina com um eletrólito composto por 50 mmol L-1 de tampão citrato, e 2% de S-β-CD, pH 2,7. A separação dos fármacos antimaláricos e seus enantiômeros foi alcançada com tempo de análise de 12 minutos. Os métodos desenvolvidos foram validados de acordo com os protocolos oficiais, apresentando características adequadas e foram aplicados na determinação de cloroquina, hidroxicloroquina e mefloquina em formulações farmacêuticas. Como figuras de mérito para o método não quiral, tem-se: linearidade (R2 > 0,99), LD (7,43 - 24,4 µmol L-1), LQ (22,5 - 73,8 µmol L-1), precisão intermediária (0,76 - 1,7% RSD), recuperação (97,8 - 102,2%). Para o método quiral, tem-se: linearidade (R2 > 0,99), LD (7,43 - 9,58 µmol L-1), LQ (22,8 - 29,0 µmol L-1), precisão intermediária (0,50 - 1,8% RSD), recuperação (97,7 - 102,5%). Um ensaio de robustez para ambos os métodos foi realizado para comparar os resultados obtidos aplicando-se pequenas variações de tensão e temperatura. Observou-se que não existe uma diferença significativa entre os resultados, a um nível de confiança de P = 95 %. / Malaria is a disease that causes a large number of deaths worldwide. The use of antimalarial drugs is the most effective solution to combat and control the disease and interest in the development of new antimalarial drugs is still of great importance due to resistance issues. There is a wide variety of antimalarial drugs, many of which are chiral, sold and administered as racemic mixtures. In recent decades there has been an increased interest regarding the pharmacodynamic and pharmacokinetic aspects of chiral drugs, due to the knowledge that one of the isomers can be more active or more toxic than the other. Thus, there is a need for the development of enantioselective analytical methods, and capillary electrophoresis has emerged as a chiral technique separation with high resolving power. Moreover, the lack of official methods for antimalarial drugs and the few studies reporting applications in the analysis of pharmaceutical formulations, demands the development and validation of new methods for this purpose. This work aims at the development of two analytical methods, non-chiral and chiral, for the determination of antimalarial drugs in pharmaceutical formulations by capillary electrophoresis. The non-chiral method used capillary zone electrophoresis, being optimized for the separation of 7 antimalarial drugs (chloroquine, hydroxychloroquine, primaquine, quinidine, quinine, quinacrine and mefloquine) with an electrolyte consisting of 45 mmol L-1 of citrate buffer, pH 4.50, and had an analysis time of 10 minutes, allowing separation of isolated diasteroisomers quinine and quinidine without any additives. The chiral method used capillary zone electrophoresis modified by cyclodextrin and was optimized for enantioselective separation of chloroquine, mefloquine, hydroxychloroquine, primaquine, quinine and quinidine with an electrolyte consisting of 50 mmol L-1 citrate buffer and 2% S-β-CD, pH 2.7. The separation of the antimalarial drugs and their enantiomers was achieved in less than 12 minutes. The proposed methods were validated following official protocols, with adequate results and were used for determination of chloroquine, hydroxychloroquine, and mefloquine in pharmaceutical formulations. Figures of merit for the non-chiral method include: linearity (R2> 0.99), LD (7.43 to 24.4 µmol L-1), LQ (22.5 to 73.8 µmol L-1), intermediary precision (0.76 to 1.7% RSD), and recovery (from 97.8 to 102.2%). For the chiral method, we have: linearity (R2> 0.99), LD (7.43 to 9.58 µmol L-1), LQ (22.8 to 29.0 µmol L-1), intermediary precision (0.50 to 1.8% RSD), and recovery (from 97.7 to 102.5%). For both methods a robustness test was performed to compare the results obtained by applying slight variations in voltage and temperature. It was observed that there is no significant difference between the results, a confidence level P = 95%.