Fluorinated stationary phase in Rtx-200MS column have been characterized by determination of system constants of Abraham equation. Retention on this phase is driven by dispersive and orientation/induction forces. Significant interaction contribution of lone pair electrons or π- electrons provides unique selectivity for analytes with excess of electron density. Unusual behavior of this phase have been determined by study of separation mechanism of polar and nonpolar analytes, in comparison of their separation on polar and nonpolar phases. This behavior is due to medium polarity of the phase (system constant s), which is not so pronounced to cancel separation of nonpolar analytes due to induction forces. In some cases contribution of lone pair electrons or π-electrons can contribute to this separations. Key words fluorinated stationary phase Rtx-200MS, inverse gas chromatography, LFER method, Abraham's equation

A maioria dos agentes terapêuticos, freqüentemente prescritos, são formulados e comercializados sob a forma racêmica, embora para alguns deles, já tenha sido demonstrado que os efeitos farmacológicos e/ou tóxicos estejam relacionados apenas a um dos enantiômeros. Além disso, é conhecido o fato de que os enantiômeros podem apresentar perfis farmacocinéticos e farmacodinâmicos diferentes. Neste trabalho foram selecionados fármacos que fazem parte de dois grupos importantes no uso clínico. São fármacos freqüentemente prescritos, como os β-bloqueadores (atenolol, metoprolol, pindolol, betaxolol e nadolol) e os antiinflamatórios não-esteróides (ibuprofeno e flurbiprofeno ). Existem na literatura científica várias citações que descrevem o uso da cromatografia líquida de alta eficiência com fases estacionárias quirais (CLAE-FEQ) em estudos farmacológicos, mas não na análise quantitativa dos enantiômeros em preparações farmacêuticas. É conhecido o fato de que o método CLAE-FEQs oferece vantagens sobre as técnicas clássicas de separação e análise de estereoisômeros, especialmente para os enantiômeros. As separações enantioméricas diretas do atenolol, metoprolol, nadolol e betaxolol foram obtidas utilizando-se FEQ Chiralcel OD®. Os enantiômeros do pindolol foram separados com FEQ α-Burke 2® e os do ibuprofeno e do flurbiprofeno com FEQ do tipo WheIk-O 1®. Neste trabalho são apresentados métodos rápidos e sensíveis para determinação estereoespecífica do atenolol (AT), do metoprolol (MT) e do flurbiprofeno (FLU) em formulações farmacêuticas. A determinação quantitativa dos enantiômeros do atenolol e do metoprolol nos comprimidos foi realizada através de método cromatográfico validado. As condições analíticas foram padronizadas através do sistema de cromatografia líquida de alta eficiência, usando coluna do tipo carbamato de celulose tris-3,5-dimetilfenil, Chiralcel OD®, (250x4.6 mm, 10µm) como FEQ. As amostras foram cromatografadas à temperatura ambiente, com um volume de injeção de 20µ L. A detecção foi efetuada em 276 nm. Para o atenolol a fase móvel foi constituída de hexano:etanol:dietilamina:ácido acético (60:40:0,2:0,2 v/v/v/v), com vazão de 1,0 rnL/min. As curvas padrões do R-AT e do S-AT apresentaram boa linearidade entre 50,0-130,0µg/rnL, com coeficiente de correlação de 0,9991 e 0,9980 respectivamente. As amostras comerciais A, B, C e D referentes a R-AT analisadas, apresentaram coeficiente de variação e percentual de recuperação de 1,15% e 101,06%; 0,74% e 99,25%; 1,05% e 102,57%; 0,84% e 101,57% respectivamente, já o coeficiente de variação e percentual de recuperação do S- AT nas amostras A, B, C e D foram 1,33% e 98,87%; 0,99% e 100,76%; 1,17% e 101,69%; 1,26% e 100,39%, respectivamente. Para o metoprolol a fase móvel foi constituída de hexano:etanol:dietilamina:ácido acético (40:60:0,2:0,2 v/v/v/v), com vazão de 0,8 rnL/min. As curvas padrões do R-MT e do S-MT apresentaram boa linearidade entre 30,0-110,0µg/rnL, com coeficiente de correlação de 0,9988 e 0,9990 respectivamente. A amostra comercial analisada, apresentou coeficiente de variação e percentual de recuperação de 0,86% e 98,62% para R-MT e de 1,40% e 99,39% para S-MT. Um método cromatográfico foi desenvolvido e validado para separação e quantificação enantiomérica do FLU na forma farmacêutica. As condições analíticas foram padronizadas através do sistema de cromatografia líquida de alta eficiência, usando coluna do tipo Whelk-O 1® (250x4,6 mm, 5,0 µm) como FEQ. As amostras foram cromatografadas à temperatura ambiente, com um volume de injeção de 20µ L. A detecção foi efetuada em 246 nm. A fase móvel foi constituída de hexano:etanol:ácido acético (95:05:0,2 v/v/v), com vazão de 0,9 rnL/min. A curva padrão do S-FLU apresentou boa linearidade entre 2,0-18,0 µg/mL, com coeficiente de correlação de 0,9993. A amostra comercial analisada apresentou coeficiente de variação e percentual de recuperação de 0,16% e 100,1% para R_FLU e 0,14% e 100,4% para S-FLU, respectivamente. Os métodos propostos permitam a separação quantitativa dos enantiômeros de AT, MT e FLU contidos nas formas farmacêuticas analisadas, com precisão e exatidão e que podem ser aplicados no controle de qualidade enantiomérico destes fármacos. / The majority of the therapeutic agents, frequently prescribed, are formulated and commercialized as racemic mixture, even so for some of them, it has been demonstrated that the pharmacological and/or toxic effect are confined only to one of the enantiomer. Besides, it is well known that the enantiomers can present different pharmacokinetic and pharmacodynamic profiles. In the present work we selected drugs belonging to two classes of clínical importance. These pharmaceuticals are widely prescribed in clinical practice such as, the beta-blockers (atenolol, metoprolol, pindoloI, betaxolol and nadolol) and the non-steroid anti-inflammatorydrugs (ibuprofen and flurbiprofen). Several references could be found in scientific literature that describes the use of high performance liquid chromatography with chiral stationary phase (HPLC-CSP) in pharmacological studies, seldom in the quantitative determination of enantiomers in pharmaceutical formulations. It is well known that the HPLC-CSP methods offer distinct advantages over classical techniques of isomeric separation and analysis, especially for the enantiomeric separation. The direct enantiomeric separation of atenolol metoprolol nadolol and betaxolol were obtained using CSP Chiralcel OD®.The enantiomers of pindolol were separate utilizing CSP α-Burke 2® and those of ibuprofen and the flurbiprofen with CSP Whelk-O 1®. In this work are presented efficient and sensitive methods for stereospecific determination of atenolol (AT), metoprolol (MT) and flurbiprofen (FLU) in pharmaceutical formulations. The stereoselective determination of atenolol and metoprolol in pharmaceuticals was performed through validated chromatographic method. The validation of liquid chromatographic methods was done utilizing a cellulose tris- 3,5-dimethylphenyl carbamate, Chiralcel OD®, (250x4.6 mm, 10µm)as CSP. The samples were analyzed at room temperature with injection volume of 20µL and UV detection was made at 276nm. In case of atenolol, the mobile phase was constituted of hexane:ethanol:diethylamine:acetic acid (60:40:0.2:0.2 v/v), with a flow rate of 1.0 mL/min. Separate standard curve for R-AT and S-AT showed good linearity over a concentration range from 50-130 µg/mL, with coefficient of correlation of 0.9991 and 0.998, respectively. The coefficient of variation and average recovery for R-AT in the samples A, B, C, and D were 1.15% and 101.06%; 0.74% and 99.25%; 1.05% and 102.57%; 0.84% and 101.57% respectively. The coefficient of variation and average recovery for S-AT in samples A, B, C and D were 1.33% and 98.87%; 0.99% and 100.76%; 1.17% and 101.69%; 1.26% and 100.39%, respectively. In case of metoprolol, the mobile phase was constituted of hexane:ethanol:diethylamine:acetic acid (40:60:0.2:0.2 v/v), with a flow rate of 0.8 m L/min. Separate standard curve for R-MT and S-MT showed good linearity over a concentration range fIom 30-110 µg/mL, with coefficient of correlation of 0.9988 and 0.9990, respectively. The coefficient of variation and average recovery for R-MT in sample analyzed was 0.86% and 98.62% and for S-MT was 1.40% and 99.39%, respectively. A high performance liquid chromatographic method is developed and validated for enantiomeric separation and quantitative determination of FLU in pharmaceutical preparation. A WheIk-O 1® column (250x4.6 mm, 5µm)was used as chiral stationary phase (CSP). The mobile phase was constituted of hexane:ethanol:acetic acid (95:05:0.2 v/v/v), at a flow rate of 0.9 rnL/min and UV detection at 246nm. All experiments were done at ambient temperature. The S-FLU standard curve showed linearity over a concentration range from 2-18µg/mL, (R2 = 0.9993). The coefficient of variation and average recovery of R-FLU were 0.16% and 100.13% and for S-FLU were 0.14% and 100.4%; respectively. The proposed methods permits quantitative separation of AT, MT and FLU enantiomers in pharmaceutical formulations studied with precision and accuracy. The proposed validated methods can be used in the enantiomeric quality controI of referred pharmaceutical drugs.

A maioria dos agentes terapêuticos, freqüentemente prescritos, são formulados e comercializados sob a forma racêmica, embora para alguns deles, já tenha sido demonstrado que os efeitos farmacológicos e/ou tóxicos estejam relacionados apenas a um dos enantiômeros. Além disso, é conhecido o fato de que os enantiômeros podem apresentar perfis farmacocinéticos e farmacodinâmicos diferentes. Neste trabalho foram selecionados fármacos que fazem parte de dois grupos importantes no uso clínico. São fármacos freqüentemente prescritos, como os β-bloqueadores (atenolol, metoprolol, pindolol, betaxolol e nadolol) e os antiinflamatórios não-esteróides (ibuprofeno e flurbiprofeno ). Existem na literatura científica várias citações que descrevem o uso da cromatografia líquida de alta eficiência com fases estacionárias quirais (CLAE-FEQ) em estudos farmacológicos, mas não na análise quantitativa dos enantiômeros em preparações farmacêuticas. É conhecido o fato de que o método CLAE-FEQs oferece vantagens sobre as técnicas clássicas de separação e análise de estereoisômeros, especialmente para os enantiômeros. As separações enantioméricas diretas do atenolol, metoprolol, nadolol e betaxolol foram obtidas utilizando-se FEQ Chiralcel OD®. Os enantiômeros do pindolol foram separados com FEQ α-Burke 2® e os do ibuprofeno e do flurbiprofeno com FEQ do tipo WheIk-O 1®. Neste trabalho são apresentados métodos rápidos e sensíveis para determinação estereoespecífica do atenolol (AT), do metoprolol (MT) e do flurbiprofeno (FLU) em formulações farmacêuticas. A determinação quantitativa dos enantiômeros do atenolol e do metoprolol nos comprimidos foi realizada através de método cromatográfico validado. As condições analíticas foram padronizadas através do sistema de cromatografia líquida de alta eficiência, usando coluna do tipo carbamato de celulose tris-3,5-dimetilfenil, Chiralcel OD®, (250x4.6 mm, 10µm) como FEQ. As amostras foram cromatografadas à temperatura ambiente, com um volume de injeção de 20µ L. A detecção foi efetuada em 276 nm. Para o atenolol a fase móvel foi constituída de hexano:etanol:dietilamina:ácido acético (60:40:0,2:0,2 v/v/v/v), com vazão de 1,0 rnL/min. As curvas padrões do R-AT e do S-AT apresentaram boa linearidade entre 50,0-130,0µg/rnL, com coeficiente de correlação de 0,9991 e 0,9980 respectivamente. As amostras comerciais A, B, C e D referentes a R-AT analisadas, apresentaram coeficiente de variação e percentual de recuperação de 1,15% e 101,06%; 0,74% e 99,25%; 1,05% e 102,57%; 0,84% e 101,57% respectivamente, já o coeficiente de variação e percentual de recuperação do S- AT nas amostras A, B, C e D foram 1,33% e 98,87%; 0,99% e 100,76%; 1,17% e 101,69%; 1,26% e 100,39%, respectivamente. Para o metoprolol a fase móvel foi constituída de hexano:etanol:dietilamina:ácido acético (40:60:0,2:0,2 v/v/v/v), com vazão de 0,8 rnL/min. As curvas padrões do R-MT e do S-MT apresentaram boa linearidade entre 30,0-110,0µg/rnL, com coeficiente de correlação de 0,9988 e 0,9990 respectivamente. A amostra comercial analisada, apresentou coeficiente de variação e percentual de recuperação de 0,86% e 98,62% para R-MT e de 1,40% e 99,39% para S-MT. Um método cromatográfico foi desenvolvido e validado para separação e quantificação enantiomérica do FLU na forma farmacêutica. As condições analíticas foram padronizadas através do sistema de cromatografia líquida de alta eficiência, usando coluna do tipo Whelk-O 1® (250x4,6 mm, 5,0 µm) como FEQ. As amostras foram cromatografadas à temperatura ambiente, com um volume de injeção de 20µ L. A detecção foi efetuada em 246 nm. A fase móvel foi constituída de hexano:etanol:ácido acético (95:05:0,2 v/v/v), com vazão de 0,9 rnL/min. A curva padrão do S-FLU apresentou boa linearidade entre 2,0-18,0 µg/mL, com coeficiente de correlação de 0,9993. A amostra comercial analisada apresentou coeficiente de variação e percentual de recuperação de 0,16% e 100,1% para R_FLU e 0,14% e 100,4% para S-FLU, respectivamente. Os métodos propostos permitam a separação quantitativa dos enantiômeros de AT, MT e FLU contidos nas formas farmacêuticas analisadas, com precisão e exatidão e que podem ser aplicados no controle de qualidade enantiomérico destes fármacos. / The majority of the therapeutic agents, frequently prescribed, are formulated and commercialized as racemic mixture, even so for some of them, it has been demonstrated that the pharmacological and/or toxic effect are confined only to one of the enantiomer. Besides, it is well known that the enantiomers can present different pharmacokinetic and pharmacodynamic profiles. In the present work we selected drugs belonging to two classes of clínical importance. These pharmaceuticals are widely prescribed in clinical practice such as, the beta-blockers (atenolol, metoprolol, pindoloI, betaxolol and nadolol) and the non-steroid anti-inflammatorydrugs (ibuprofen and flurbiprofen). Several references could be found in scientific literature that describes the use of high performance liquid chromatography with chiral stationary phase (HPLC-CSP) in pharmacological studies, seldom in the quantitative determination of enantiomers in pharmaceutical formulations. It is well known that the HPLC-CSP methods offer distinct advantages over classical techniques of isomeric separation and analysis, especially for the enantiomeric separation. The direct enantiomeric separation of atenolol metoprolol nadolol and betaxolol were obtained using CSP Chiralcel OD®.The enantiomers of pindolol were separate utilizing CSP α-Burke 2® and those of ibuprofen and the flurbiprofen with CSP Whelk-O 1®. In this work are presented efficient and sensitive methods for stereospecific determination of atenolol (AT), metoprolol (MT) and flurbiprofen (FLU) in pharmaceutical formulations. The stereoselective determination of atenolol and metoprolol in pharmaceuticals was performed through validated chromatographic method. The validation of liquid chromatographic methods was done utilizing a cellulose tris- 3,5-dimethylphenyl carbamate, Chiralcel OD®, (250x4.6 mm, 10µm)as CSP. The samples were analyzed at room temperature with injection volume of 20µL and UV detection was made at 276nm. In case of atenolol, the mobile phase was constituted of hexane:ethanol:diethylamine:acetic acid (60:40:0.2:0.2 v/v), with a flow rate of 1.0 mL/min. Separate standard curve for R-AT and S-AT showed good linearity over a concentration range from 50-130 µg/mL, with coefficient of correlation of 0.9991 and 0.998, respectively. The coefficient of variation and average recovery for R-AT in the samples A, B, C, and D were 1.15% and 101.06%; 0.74% and 99.25%; 1.05% and 102.57%; 0.84% and 101.57% respectively. The coefficient of variation and average recovery for S-AT in samples A, B, C and D were 1.33% and 98.87%; 0.99% and 100.76%; 1.17% and 101.69%; 1.26% and 100.39%, respectively. In case of metoprolol, the mobile phase was constituted of hexane:ethanol:diethylamine:acetic acid (40:60:0.2:0.2 v/v), with a flow rate of 0.8 m L/min. Separate standard curve for R-MT and S-MT showed good linearity over a concentration range fIom 30-110 µg/mL, with coefficient of correlation of 0.9988 and 0.9990, respectively. The coefficient of variation and average recovery for R-MT in sample analyzed was 0.86% and 98.62% and for S-MT was 1.40% and 99.39%, respectively. A high performance liquid chromatographic method is developed and validated for enantiomeric separation and quantitative determination of FLU in pharmaceutical preparation. A WheIk-O 1® column (250x4.6 mm, 5µm)was used as chiral stationary phase (CSP). The mobile phase was constituted of hexane:ethanol:acetic acid (95:05:0.2 v/v/v), at a flow rate of 0.9 rnL/min and UV detection at 246nm. All experiments were done at ambient temperature. The S-FLU standard curve showed linearity over a concentration range from 2-18µg/mL, (R2 = 0.9993). The coefficient of variation and average recovery of R-FLU were 0.16% and 100.13% and for S-FLU were 0.14% and 100.4%; respectively. The proposed methods permits quantitative separation of AT, MT and FLU enantiomers in pharmaceutical formulations studied with precision and accuracy. The proposed validated methods can be used in the enantiomeric quality controI of referred pharmaceutical drugs.

Chapter one reviewed the development of sol-gel stationary phase for gas chromatograph (GC). Inorganic sol-gel precursor creates the substrate support for stationary phase bind to silica surface through the condensation of silanol groups, the rough surface with enhanced surface area enlarger the capacity of the sol-gel stationary phase, the porosity of sol-gel structure decreases the mass transferring coefficient, the term C in Van Deemter equation, which makes sol-gel stationary phase thicker coating up to 1 um but keeps the high resolution for gas chromatography. Chemical bound stationary phase significantly improve GC column with better thermal stability and solvent stability. Partial derivatizations of known polymers have not increased the column performance but remained at the same level of 3200 plates per meter. The sol-gel chemistry with essential structure rebuilding will make sol-gel stationary phase to a new level. The chapter two reported the non-silica-based metal alkoxide oxide as the new building block, cooperated with known polymer poly(dimethyldiphenylsilanoxan), developed the original nonpolar feature of the stationary phase to broad the polarity from the nonpolar to extreme polar, overcome the thermal stability for other types of polar column. The theoretical plate number reached the 3200 plates per meter, and the optimized plate number arrived at the top level at 3800 plates per meter. The basic recipe and preparation of sol-gel process were verified by tungsten alkoxide incorporated with poly(dimethyldiphenylsilanoxan) as the stationary phase which arrived the same plate number level at 3800 per meter. Chapter three demonstrated the preparation of core-shell particle for HPLC. Conventional silica core particles were prepared with stÖbe methods. The extended layer of germanium oxide coating was made with acid and alkaline as a catalyst. EDS characterize the extended layers of germanium oxide has been coated at ratio 12/1(Ge/Si). Then the carbon loading with C 18 for surface derivatization were also confirmed with EDS testing. 2 um core-shell particles were successfully prepared from the external composition (1.7 um core, 0.3 um shell). The function of the core-shell particles was slurry packed with 5cm regular steel column and the capillary column with sol-gel frit. The preliminary HPLC testing showed the core-shell particles had more retention ability compare with 4 um commercial core particles. The backpressure of the short steel column and capillary both were beyond the pressure limit of conventional HPLC pump. Chapter three demonstrates the new ideal of the surface sol-gel process for GC stationary phase. Without the catalyst, the sol solution has constant viscosity and gelation time is much longer, the sol-gel reaction was taking on the silica surface only, to accomplish the thinner coating for sol-gel stationary phase. From the retention time of the grob mixture, the surface sol-gel coated 2 meters of column acquired the half retention time, grob mixture analytes were eluted within 7 minutes, compared with conventional sol-gel coated column eluted within 14 minutes. Without the TFA as the catalyst, the sol-gel matrix may not form effective surface area and porosity to support the functional polymer for separation, the column performances were two third of the protocol column, at 1500-2500 plate number per meter. The coating results proved the sol-gel stationary phase could be fulfilled with diluted sol solution by static coating. Basic parameters for dynamic coating and static coating with conventional coating and surface sol-gel were acquired for further development. The germania and niobium precursor is highly active, the water amount in the solvents used as received without drying process can meet the surface sol-gel coating without precipitates and gelation formed before finishing coat. For germania -PDMDPS column, thermal stability is very important because of the temperature for remaining the low residual OH- group in silica and germania film at 350 °C.

<p>Liquid Chromatography (LC) and Capillary Electrophoresis (CE) are modern analytical techniques that play very important roles in many areas of modern science such as life science, biotechnology, biomedicine, environmental studies, and development of pharmaceutics. Even though these two techniques have existed and been subjected to studies for several decades, the developments of new separation materials for them are still very important till now in order to meet the different new demands for improvement from other disciplines in science.</p><p>In this doctoral thesis, several novel covalently bonded sulfobetaine type zwitterionic separation materials are synthesized for the application in LC and CE. These materials carry both positively charged quaternary ammonium groups and negatively charged sulfonic groups, which result in a very low net surface charge compared to conventional separation materials with only anionic or cationic functional groups. Consequently, it is possible to employ these materials for separation of different ionic species under mild conditions. The surface properties have also been characterized, mainly by elemental analysis, sorption isotherm, ζ-potential measurements, and spectroscopic methods.</p><p>By using packed zwitterionic columns for liquid chromatography, small inorganic anions or cations, and acidic or basic proteins can be independently and simultaneously separated in a single run using optimal sets of separation conditions. This is a unique property compared to conventional ionic separation material for LC. When fused silica capillaries coated with zwitterionic polymer are used for capillary electrophoresis, good separations can be achieved for solutes as different as inorganic anions, peptides, proteins, and tryptically digested proteins.</p>

<p>Electrospray ionization (ESI) mass spectrometry (MS) in combination with liquid chromatography (LC) is an excellent tool for the identification of drug metabolites. Utilizing this hyphenated technique in combination with proper sample pretreatment, the metabolic pathways of the analgesic drug ketobemidone were investigated in human urine and rat microdialysate from blood and brain. Two novel phase I metabolites (ketobemidone N-oxide and meta-hydroxymethoxyketobemidone) and three novel phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in human urine. Further, norketobemidone and ketobemidone N-oxide were identified in rat microdialysate from brain after regional distribution of ketobemidone in striatum. This indicates that the brain itself has the possibility to metabolize ketobemidone. </p><p>Synthetic ketobemidone metabolites were used for comparison of retention times and tandem MS spectra with the possible metabolites recovered from the biological samples. The conjugated metabolites were identified by accurate mass measurements and tandem MS spectra of the aglycones. The accuracy of the estimated masses was better than 2.1 ppm for two out of three conjugates in presence of internal standard.</p><p>On-line micro-SPE was successfully used for trapping and desalting of the microdialysates. The small SPE pre-column made it possible to inject approximately 100 times more sample on the analytical column compared to injection without pre-column. Selective trapping was demonstrated for the polar catechol amine metabolite, dihydroxyketobemidone, which forms covalent complexes with phenylboronic acid (PBA). A fluorinated silica type stationary phase was the only column out of several tested that was able to separate ketobemidone and all relevant phase I metabolites. </p><p>Liquid chromatography and mass spectrometry are independently valuable tools in the field of analytical pharmaceutical chemistry. The present study showed that the combination of LC-MS, with its excellent selectivity and sensitivity, offers an outstanding tool in the qualitative analysis of drugs and metabolites in biological fluids. </p>

Liquid Chromatography (LC) and Capillary Electrophoresis (CE) are modern analytical techniques that play very important roles in many areas of modern science such as life science, biotechnology, biomedicine, environmental studies, and development of pharmaceutics. Even though these two techniques have existed and been subjected to studies for several decades, the developments of new separation materials for them are still very important till now in order to meet the different new demands for improvement from other disciplines in science. In this doctoral thesis, several novel covalently bonded sulfobetaine type zwitterionic separation materials are synthesized for the application in LC and CE. These materials carry both positively charged quaternary ammonium groups and negatively charged sulfonic groups, which result in a very low net surface charge compared to conventional separation materials with only anionic or cationic functional groups. Consequently, it is possible to employ these materials for separation of different ionic species under mild conditions. The surface properties have also been characterized, mainly by elemental analysis, sorption isotherm, ζ-potential measurements, and spectroscopic methods. By using packed zwitterionic columns for liquid chromatography, small inorganic anions or cations, and acidic or basic proteins can be independently and simultaneously separated in a single run using optimal sets of separation conditions. This is a unique property compared to conventional ionic separation material for LC. When fused silica capillaries coated with zwitterionic polymer are used for capillary electrophoresis, good separations can be achieved for solutes as different as inorganic anions, peptides, proteins, and tryptically digested proteins.

Electrospray ionization (ESI) mass spectrometry (MS) in combination with liquid chromatography (LC) is an excellent tool for the identification of drug metabolites. Utilizing this hyphenated technique in combination with proper sample pretreatment, the metabolic pathways of the analgesic drug ketobemidone were investigated in human urine and rat microdialysate from blood and brain. Two novel phase I metabolites (ketobemidone N-oxide and meta-hydroxymethoxyketobemidone) and three novel phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in human urine. Further, norketobemidone and ketobemidone N-oxide were identified in rat microdialysate from brain after regional distribution of ketobemidone in striatum. This indicates that the brain itself has the possibility to metabolize ketobemidone. Synthetic ketobemidone metabolites were used for comparison of retention times and tandem MS spectra with the possible metabolites recovered from the biological samples. The conjugated metabolites were identified by accurate mass measurements and tandem MS spectra of the aglycones. The accuracy of the estimated masses was better than 2.1 ppm for two out of three conjugates in presence of internal standard. On-line micro-SPE was successfully used for trapping and desalting of the microdialysates. The small SPE pre-column made it possible to inject approximately 100 times more sample on the analytical column compared to injection without pre-column. Selective trapping was demonstrated for the polar catechol amine metabolite, dihydroxyketobemidone, which forms covalent complexes with phenylboronic acid (PBA). A fluorinated silica type stationary phase was the only column out of several tested that was able to separate ketobemidone and all relevant phase I metabolites. Liquid chromatography and mass spectrometry are independently valuable tools in the field of analytical pharmaceutical chemistry. The present study showed that the combination of LC-MS, with its excellent selectivity and sensitivity, offers an outstanding tool in the qualitative analysis of drugs and metabolites in biological fluids.

Cette thèse décrit quelques développements dans les techniques de calcul des amplitudes de diffusion en théorie supersymétrique de champ de jauge . L'accent est mis sur les relations de récurrence on-shell et sur l'utilisation de méthodes d'unitarité pour des calculs de boucle. En particulier, la récurrence on-shell est liée aux règles BCFW pour calculer les amplitudes de jauge au niveau des arbres. Les combinaisons de techniques de coupe d'unitarité et la récurrence sont utilisées pour calculer les amplitudes de boucle, et finalement, à partir des amplitudes, pour obtenir la fonction de corrélation énergie-énergie en théorie de super-Yang-Mills N = 4 à l'aide de la représentation de Mellin-Barnes. Dans le dernier chapitre, nous tentons de trouver un contour convergent pour les intégrales de Mellin Barnes en multi-dimension obtenu par une certaine approximation d'un contour de phase stationnaire.

The bgl operon of Escherichia coli, involved in the uptake and utilization of aromatic β-glucosides salicin and arbutin, is maintained in a silent state in the wild type organism by the presence of structural elements in the regulatory region. This operon can be activated by mutations that disrupt these negative elements. The fact that the silent bgl operon is retained without accumulating deleterious mutations seems paradoxical from an evolutionary view point. Although this operon appears to be silent, specific physiological conditions might be able to induce its expression and/or the operon might be carrying out function(s) apart from the utilization of aromatic β-glucosides. The experiments described in this thesis were carried out to test these possibilities. In cultures exposed to prolonged stationary phase, majority of the bacterial population dies and a few mutants that have the ability to scavenge the nutrients released by the dying cell mass survive. Bgl+ mutants were found to be enriched in twenty-eight-day-old Luria Broth grown cultures of E. coli that are wild type for bgl but carry the rpoS819 allele. Out of the five Bgl+ mutants that were isolated, four carried a mutation in the hns locus while one of them, ZK819-97, had an activating mutation linked to the bgl operon. Further analysis of ZK819-97 by DNA sequencing revealed the existence of a single C to T transition at the CAP binding site in the regulatory region. ZK819-97 was chosen for further analysis. Competition assays were carried out in which Bgl+ strain, ZK819-97 (Strr), and the parental Bgl- strain, ZK820 (Nalr), were grown independently for twenty-four hours in Luria Broth and then mixed in 1:1,000 (v/v) ratio reciprocally, without addition of fresh nutrients. ZK819-97, when present in minority, was found to increase in number and take over the parental strain, ZK820, i.e. ZK819-97 showed a Growth Advantage in Stationary Phase phenotype. To determine whether the GASP phenotype of ZK819-97 is associated with the bgl locus, the bgl allele from this strain was transferred by P1 transduction to its parental strain, ZK819. The resulting strain, ZK819-97T (Bgl+, Tetr), when competed with the parental strain, ZK819 Tn5 (Bgl-, Kanr), also showed a GASP phenotype when present in minority in the mixed cultures. To reconfirm this further, the bgl locus was deleted from ZK819-97T. The resulting strain, ZK819-97Δbgl, showed a loss of the GASP phenotype. When the bglB locus was disrupted in ZK819-97T, the resulting strain, ZK819-97ΔB, also failed to show a GASP phenotype, indicating that the phospho-β-glucosidase B activity is essential for this phenotype. The strain, ZK819-IS1, carrying an activating IS1 insertion within the bgl regulatory region also showed a GASP phenotype, confirming that this phenotype of the Bgl+ strain is independent of the nature of the activating mutation. All the above mentioned strains used in the competition assays carry a mutant allele of rpoS, rpoS819. Introduction of the wild type rpoS allele in these strains resulted in the loss of the GASP phenotype of the Bgl+ strain, suggesting that the two mutations work in a concerted manner. The Bgl+ strain was found to show the GASP phenotype only when present in minority of 1:1,000 or 1:10,000 in the mixed cultures and showed a slight disadvantage at higher ratios, indicating that the GASP phenotype of the Bgl+ strain is a frequency dependent phenomenon. In competition assays carried out between 24-hour-old cultures of Bgl+ and Bgl- strains resuspended in five-day-old spent medium prepared from a wild type E. coli strain, Bgl+ strain did not show any extra or early GASP phenotype. In addition, a reporter strain, which has a lacZ transcriptional fusion with the activated bgl promoter, was resuspended in spent medium prepared from a five-day-old culture of wild type strain of E. coli and bgl promoter activity was measured by β-galactosidase assay. The bgl promoter did not show any induction in this medium. These experiments suggest the absence of any β-glucoside like molecules in the spent medium within the sensitivity of these assays. A reporter strain that has a lacZ transcriptional fusion to the wild type bgl promoter was used to measure the expression level of this promoter during exponential and stationary phase of growth in LB. Expression of the wild type as well as various activated promoters of bgl was found to be enhanced in stationary phase. To investigate a possible role of the rpoS encoded stationary phase specific sigma factor, RpoS (σs), and another stationary phase factor, Crl, known to be important for the regulation of many genes of the σs regulon, the bgl promoter activity measurements were carried out in the presence or the absence of RpoS and/or Crl. RpoS along with Crl was found to negatively regulate the expression of wild type as well as activated promoters of bgl, both in exponential and stationary phase. In the absence of the negative regulation by RpoS and Crl, the increase in the bgl promoter activity was more pronounced as compared to that in its presence. rpoS and crl mutations are common in nature and it has been suggested that crl deletion gives a growth advantage to the strain in stationary phase. To test this possibility crl deletion was created in wild type as well as in attenuated rpoS allele background. The strain carrying the crl deletion was found to have a growth advantage in stationary phase over the wild type strain in the presence of wild type rpoS allele, while it shows a slight disadvantage in combination with mutant rpoS. Over expression of LeuO or BglJ is known to activate the bgl operon. To study a possible role of these factors in the regulation of the bgl expression in stationary phase, the bgl promoter activity was measured in strains that were deleted for leuO and/or bglJ, in the absence or presence of crl. These studies indicated that BglJ had a moderate effect on the bgl promoter activity in stationary phase in the absence of Crl but not in its presence. LeuO did not have a significant effect on the bgl promoter activity in either condition. Thus under the conditions tested, the physiological increase in the levels of LeuO and BglJ in stationary phase was insufficient to regulate the bgl expression. Preliminary results show that the bgl operon might be involved in the regulation of oppA, an oligopeptide transporter subunit, in stationary phase. Implications of these findings are discussed. The studies reported in this thesis highlight the involvement of the bgl operon of E. coli in stationary phase. This could be mediated by genetic as well as physiological mechanisms. This study also underscores the importance of observing organisms closer to their natural context and the need to reconsider the concept of ‘cryptic genes’.