Dissertations / Theses on the topic 'Chiral analysis'

The analysis of the enantiomeric purity of chiral carboxylic acids requires a reagent to give acceptable NMR chemical shift non-equivalence with a wide range of substrate acids. Extensive studies of the behaviour of N-mono- methyl, N,N-dimethyl and cyclic amines as chiral solvating agents led to the finding that 1,2 diphenyl-1,2-diaminoethane can induce substantial non- equivalence in the diastereomeric salts of chiral a-phenyl and a-halo carboxylic acids. The diastereoisomeric complexes of the diamine with primary carboxylic acids (RCH(_2)CO(_2)H) presents an unusual case in which the internally enantiotopic methylene protons are rendered internally diasteretopic by an external non-covalently bonded reagent. Investigations of the physical parameters determining non-equivalence (stoichiometry, concentration, temperature and substrate enantiomeric purity), combined with NOE observations of the diastereomeric pairs and the crystal structure of the mono- hydrobromide salt were used to suggest the structure for the conformation responsible for shift non-equivalence. The zero valent platinum complex, 3-0-isopropylidene-2,3-dihydroxy-1,4- bis(diphenyl-phosphino)butane-platinum(0)-ethene (DlOP-Pt-ethene) was shown to be a versatile chiral derivatising agent for electron poor and strained η(^2)-donors. This was demonstrated by the enantiomeric purity determinations for alkynes, enones and norbornene derivatives. The crystal structure of DIOP-Pt-ethene was determined and found to be similar to the palladium analogue. If the achiral rhodium complex rhodium(I)-acetylacetone-diethene undergoes a reaction with 2 equivalents of a suitable chiral η(^2)-donor, it will result in the formation of 4 stereoisomers, two meso forms and a pair of enantiomers. The diasteroisomers should display chemical shift non-equivalence in the NMR spectrum of the product, reflecting the enantiomeric purity of the η(^2)-donor (self recognition). The derivatisation of rhodium(l)-acetylacetone-diethene with chiral η(^2)-donors was attempted.

Within the field of depression the “monoamine hypothesis” has been the leading theory to explain the biological basis of depression. This theory proposes that the biological basis of depression is due to a deficiency in one or more of three key neurotransmitter systems, namely noradrenaline, dopamine and serotonin which are thought to mediate the therapeutic actions of virtually every known antidepressant agent. Citalopram is a selective serotonin-reuptake inhibitor (SSRI) used for the treatment of depression and anxiety disorders. Citalopram is a racemic compound, in other words composed of a 50:50 mixture of two enantiomers (S-(+)-citalopram and R-(-)-citalopram) and with one of the enantiomers (S-(+)-citalopram) accounting for the inhibitory effect. At the time of introduction of citalopram the physician needed a therapeutic drug monitoring service to identify patients with interactions, compliance problems and for handling questions concerning polymorphic enzymes and drug metabolism. An achiral analytical separation method based on solid-phase extraction followed by high-performance liquid chromatography (HPLC) was developed for routine therapeutic drug monitoring (TDM) of citalopram and its two main demethylated metabolites. As the data available on citalopram were from achiral concentration determinations and to be able to further investigate citalopram enantiomers effects and distribution, a chiral method for separation of the enantiomers of citalopram and its demethylated metabolites was established. The advances within chiral separation techniques have made measurement of the concentrations of the individual enantiomers in biological fluids possible. The process behind enantioselective separation is however not fully understood and the mechanism behind the separation can be further scrutinized by the use of multivariate methods. A study of the optimization and characterization of the separation of the enantiomers of citalopram, desmethylcitalopram and didesmethylcitalopram on an acetylated ß-cyclodextrin column, by use of two different chemometric programs - response surface modelling and sequential optimization was performed. Sequential optimization can be a quicker mean of optimizing a chromatographic separation; response surface modelling, in addition to enabling optimization of the chromatographic process, also serves as a tool for learning more about the separation mechanism. Studies of the antidepressant effect and pharmacokinetics of citalopram have been performed in adults, but the effects on children and adolescents have only been studied to a minor extent, despite the increasing use of citalopram in these age groups. A study was initiated to investigate adolescents treated for depression, with respect to the steady-state plasma concentrations of the enantiomers of citalopram and its demethylated metabolites. The ratios between the S- and R-enantiomers of citalopram and didesmethylcitalopram were in agreement with studies involving older patients. The concentrations of the S-(+)- and R-(-) enantiomers of citalopram and desmethylcitalopram were also in agreement with values from earlier studies. The results indicate that the use of oral contraceptives may have some influence on the metabolism of citalopram. This might be because of an interaction of the contraceptive hormones with the polymorphic CYP2C19 enzyme. Even though the SSRIs are considered less toxic compared with older monoamine-active drugs like the tricyclic/tetracyclic antidepressants, the risk of developing serious side effects such as ECG abnormalities and convulsions has been seen for citalopram, when larger doses have been ingested. Furthermore, fatal overdoses have been reported where citalopram alone was the cause of death. Data on the toxicity of each of the enantiomers in humans have not been reported and no data on blood levels of the enantiomers in cases of intoxication have been presented. An investigation was initiated on forensic autopsy cases where citalopram had been found at the routine screening and these cases were further analysed with enantioselective analysis to determine the blood concentrations of the enantiomers of citalopram and metabolites. Furthermore the genotyping regarding the polymorphic enzymes CYP2D6 and CYP2C19 were performed. In 53 autopsy cases, we found increasing S/R ratios with increasing concentrations of citalopram. We found also that high citalopram S/R ratio were associated with high parent drug to metabolite ratio and may be an indicator of recent intake. Only 3.8 % were found to be poor metabolizers regarding CYP2D6 and for CYP2C19 no poor metabolizer was found. Enantioselective analysis of citalopram and its metabolites can provide valuable information about the time that has elapsed between intake and death. Genotyping can be of help in specific cases but the possibility of pharmacokinetic interactions is apparently a far greater problem than genetic enzyme deficiency.
On the day of the public defence the status of article IV was: Submitted.

The habilitation thesis covers theoretical investigations on light-induced processes in molecules. The study is focussed on changes of the molecular electronic structure and geometry, caused either by photoexcitation in the event of a spectroscopic analysis, or by a selective control with shaped laser pulses. The applied and developed methods are predominantly based on quantum chemistry as well as on electron and nuclear quantum dynamics, and in parts on molecular dynamics. The studied scientific problems deal with stereoisomerism and the question of how to either switch or distinguish chiral molecules using laser pulses, and with the essentials for the simulation of the spectroscopic response of biochromophores, in order to unravel their photophysics. The accomplished findings not only explain experimental results and extend existing approaches, but also contribute significantly to the basic understanding of the investigated light-driven molecular processes. The main achievements can be divided in three parts: First, a quantum theory for an enantio- and diastereoselective or, in general, stereoselective laser pulse control was developed and successfully applied to influence the chirality of molecular switches. The proposed axially chiral molecules possess different numbers of "switchable" stable chiral conformations, with one particular switch featuring even a true achiral "off"-state which allows to enantioselectively "turn on" its chirality. Furthermore, surface mounted chiral molecular switches with several well-defined orientations were treated, where a newly devised highly flexible stochastic pulse optimization technique provides high stereoselectivity and efficiency at the same time, even for coupled chirality-changing degrees of freedom. Despite the model character of these studies, the proposed types of chiral molecular switches and, all the more, the developed basic concepts are generally applicable to design laser pulse controlled catalysts for asymmetric synthesis, or to achieve selective changes in the chirality of liquid crystals or in chiroptical nanodevices, implementable in information processing or as data storage. Second, laser-driven electron wavepacket dynamics based on ab initio calculations, namely time-dependent configuration interaction, was extended by the explicit inclusion of magnetic field-magnetic dipole interactions for the simulation of the qualitative and quantitative distinction of enantiomers in mass spectrometry by means of circularly polarized ultrashort laser pulses. The developed approach not only allows to explain the origin of the experimentally observed influence of the pulse duration on the detected circular dichroism in the ion yield, but also to predict laser pulse parameters for an optimal distinction of enantiomers by ultrashort shaped laser pulses. Moreover, these investigations in combination with the previous ones provide a fundamental understanding of the relevance of electric and magnetic interactions between linearly or non-linearly polarized laser pulses and (pro-)chiral molecules for either control by enantioselective excitation or distinction by enantiospecific excitation. Third, for selected light-sensitive biological systems of central importance, like e.g. antenna complexes of photosynthesis, simulations of processes which take place during and after photoexcitation of their chromophores were performed, in order to explain experimental (spectroscopic) findings as well as to understand the underlying photophysical and photochemical principles. In particular, aspects of normal mode mixing due to geometrical changes upon photoexcitation and their impact on (time-dependent) vibronic and resonance Raman spectra, as well as on intramolecular energy redistribution were addressed. In order to explain unresolved experimental findings, a simulation program for the calculation of vibronic and resonance Raman spectra, accounting for changes in both vibrational frequencies and normal modes, was created based on a time-dependent formalism. In addition, the influence of the biochemical environment on the electronic structure of the chromophores was studied by electrostatic interactions and mechanical embedding using hybrid quantum-classical methods. Environmental effects were found to be of importance, in particular, for the excitonic coupling of chromophores in light-harvesting complex II. Although the simulations for such highly complex systems are still restricted by various approximations, the improved approaches and obtained results have proven to be important contributions for a better understanding of light-induced processes in biosystems which also adds to efforts of their artificial reproduction.
Die Habilitationsschrift behandelt theoretische Untersuchungen von durch Licht ausgelösten Prozessen in Molekülen. Der Schwerpunkt liegt dabei auf Veränderungen in der Elektronenstruktur und der Geometrie der Moleküle, die durch Bestrahlung mit Licht entweder bei einer spektroskopischen Untersuchung oder bei gezielter Kontrolle durch geformte Laserpulse herbeigeführt werden. Um die dabei auftretende Elektronen- und Kerndynamik zu simulieren, wurden vornehmlich quantentheoretische Methoden eingesetzt und weiterentwickelt. Die wissenschaftlichen Fragestellungen beschäftigen sich mit dem gezielten Verändern und dem Erkennen der räumlichen Struktur von Molekülen ohne Drehspiegelachse, der sog. molekularen Chiralität, sowie mit durch Licht eingeleiteten Prozessen in biologisch relevanten Pigmenten auf sehr kurzen Zeitskalen. Die entwickelten Ansätze und gewonnenen Erkenntnisse lassen sich drei Haupterfolge unterteilen: Erstens gelang die Entwicklung einer generellen Kontrolltheorie für das Ein- und Umschalten von molekularer Chiralität mit geformten Laserpulsen. Dabei wird die räumliche Struktur der vorgeschlagenen molekularen Schalter zwischen ihren stabilen sog. stereoisomeren Formen selektiv geändert, was sich auf ihre optischen und chemischen Eigenschaften auswirkt. Für komplexere Bedingungen, wie z.B. auf einer Oberfläche verankerten molekularen Schaltern verschiedener Orientierung, wurde eine neue Pulsoptimierungsmethode basierend auf Wahrscheinlichkeiten und Statistik entwickelt. Solche laserpulskontrollierten chiralen molekularen Schalter hofft man u.a. in der Nanotechnologie zum Einsatz zu bringen, wo sie z.B. als Informationsspeicher dienen könnten. Zweitens konnte geklärt werden, welche die wesentlichen Einflüsse sind, die das Erkennen von sog. Enantiomeren, das sind spiegelbildliche Moleküle von entgegengesetzter Chiralität, nach Ionisierung durch ultrakurze zirkular polarisierte Laserpulse ermöglichen. Diese Form des sog. Zirkulardichroismus in der Ionenausbeute erlaubt die quantitative und qualitative Unterscheidung von Enantiomeren in der Massenspektrometrie. Durch Simulation der Elektronendynamik während der Laseranregung konnte u.a. erstmals gezeigt werden, dass neben der Zirkularpolarisation der Laserpulse vor allem die schwachen magnetischen Wechselwirkungen für die Unterscheidung entscheidend sind. Drittens wurden die Spektren von in der Natur vorkommenden Pigmenten simuliert, welche u.a. an wichtigen biologischen Funktionen, wie dem Sammeln von Sonnenenergie für die Photosynthese, beteiligt sind. Die Lichtanregung führt dabei zu einer Veränderung der Elektronenstruktur und Geometrie der Pigmente, wobei letzteres wichtige Konsequenzen für die Verteilung der Energie auf die spektroskopisch beobachteten Molekülschwingungen mit sich bringen. Auch der wichtige Einfluss der biochemischen Umgebung auf die Elektronenstruktur der Pigmente bzw. den Energietransfer zwischen solchen wurde untersucht. Neben der Klärung experimenteller Ergebnisse ermöglichen die Untersuchungen neue Einblicke in die fundamentalen Prozesse kurz nach der Lichtanregung -- Erkenntnisse, die auch für die technische Nachahmung der biologischen Funktionen von Bedeutung sein können.

Micellar electrokinetic chromatography (MEKC) and capillary electrochromatography (CEC) are two of the major capillary electrophoresis (CE) modes that have been interfaced to mass spectrometry (MS) for sensitive and selective analysis of chiral compounds. This research combines these two modes and expands their applications in chiral CE analysis. Chapter 1 is a review of amino acid based molecular micelles used in MEKC-MS for enantioselective analysis over the past five years. In this chapter, a typical MEKC-MS experiment setup as well as detailed standard operating procedure in synthesis of molecular micelles and running a typical MEKC-MS experiment using the molecular micelles is discussed. Chapter 2 described a multivariate MEKC-MS optimization for the simultaneous analysis of two negatively charged model chiral compounds in negative ion mode with molecular micelles. In this chapter, a central composite design (CCD) is used to first construct a series of experiments to optimize all the important MEKC-MS parameters. Next, response surface methodology (RSM) was used to analyze the interactions between the factors, picking up the best separation and detection conditions, predicting the result of the chiral separation/MS detection, and finally running the actual experiment and comparing the chromatographic results with the predicted parameters. Chapter 3 demonstrates a similar multivariate MEKC-MS optimization for analysis of a positively charged model chiral compound in a positive ion mode. The same CCD and RSM methods were used to optimize the separations and MS sensitivity. Chapter 4 describes a chiral analysis of four neutral benzoin derivatives (hydrobenzoin, benzoin, benzoin methyl ether, and benzoin ethyl ether) using MEKC coupled to atmospheric pressure photo-ionization mass spectrometry (APPI-MS). The same multivariate experimental design strategy was used to optimize the MEKC as well as APPI-MS parameters. Simultaneous chiral separation of all four benzoin derivatives was achieved with high detection sensitivity compared to UV-detection. Chapter 5 introduces a novel one-pot synthesis scheme for an acryloyl-terminated, carbamate-linked surfactant-bound monolith with leucine head group and different chain lengths. The method promises to open up the discovery of new amino acid based polymeric monoliths for chiral separations and enhanced chemoselectivity for simultaneous chiral separations and enhanced detection in CEC and CEC-MS. In Chapter 6, five amide-linked surfactant-bound monoliths with different chain lengths and head groups (leucine, valine, and phenylalanine) were synthesized and characterized. Enantioseparation of several test compounds was achieved by CEC using the monolithic columns. One of the chiral surfactant, sodium 11-acrylamidoundecanoyl-L-leucinate (SAAUL), was polymerized in aqueous solution under 60Co radiation to form molecular micelle poly-SAAUL. MEKC experiments were carried out with the poly-SAAUL molecular micelle to separate ten cationic chiral compounds. The result was compared with the CEC separation using the AAUL monolithic column. This study is the first comparison of chiral CEC and MEKC with the same surfactant monomer, which has the capability of forming both chiral stationary phase for CEC and chiral pseudophase for MEKC.

Many persistent organic pollutants (POPs) are chiral. These pollutants are generally released into the environment as racemates, but frequently undergo alterations in enantiomeric composition as soon as they are subjected to life chemistry processes. Enantiospecific analysis of chiral POPs is important since enantiomers of chiral compounds often exhibit differences in biological activity, and most biochemical processes in nature are stereospecific. For abiotic processes, such as air-water gas exchange, deposition and long-range air transport, enantiomeric patterns of POPs may be used as chemical markers. The aim of the work described in this thesis was to improve our knowledge about the presence and fate of enantiomers of chiral POPs inthe environment to provide a sound basis for accurate risk assessment. The compounds included were organochlorine (OC) pesticides (α-HCH, chlordanes and o,p’-DDT), atropisomeric PCBs and some of their respective metabolites (heptachlor-exo-epoxide, oxychlordane and MeSO2-PCBs). Analytical methods for chiral PCBs were developed, and the elution sequences of (+) and (−)-enantiomers were determined. Enantiomeric fraction (EF) was proposed as a better reflector of chiral composition than the conventional enantiomeric ratio (ER). Enantioselective bioprocessing in various compartments was studied, with the main emphasis on factors controlling chiral composition in biota Correlations were detected between changes in EFs and differences in trophic levels. The changes were, however, not consistent for all compounds. Instead, the enantiomeric composition was found to be species-specific in the polar bear food chain and in aquatic species from the Baltic Sea. The EFs of some POPs in Baltic seals were related tonutritional status and biotransformation capacity. Enantiomeric and isomeric patterns were used to investigate abiotic processes in the southern Baltic Sea environment and EFs were used tostudy soil as a source of atmospheric heptachlor-exo-epoxide.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第13092号
農博第1597号
新制||農||938(附属図書館)
学位論文||H19||N4218(農学部図書室)
UT51-2007-H365
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 清水 昌, 教授 喜多 恵子, 教授 江﨑 信芳
学位規則第4条第1項該当

The research presented in this dissertation involves the development of chiral and achiral analysis using capillary electrochromatography (CEC) and CEC coupled to mass spectrometry (CEC-MS). Chapter 1 briefly reviews CEC fundamentals and latest development on chiral CEC and CEC-MS. The CEC-UV enantioseparations for several acidic compounds are described in Chapter 2. The optimum resolutions for these acidic enantiomers are achieved in ion-suppression mode, i.e. with an acidic mobile phase. One of major drawback in coupling CEC with MS is the bubble formation at the column outlet end, resulting in irreproducible retention time and erratic baseline, or even current breakdown. By introducing internal tapered columns, the aforementioned limitations of CEC-MS are successfully overcome in Chapter 3. The CEC-MS enantioseparation of warfarin and coumachlor is carefully investigated and applied to quantify R- and S-warfarin in human plasma. For individual enantiomers, a concentration of 25 ng/mL is detectable. To further improve the robustness of CEC-MS column, a new procedure of fabricating internal tapered columns is reported in Chapter 4. These internal tapered columns demonstrate excellent ruggedness, low background noise, and good compatibility in reversed-phase and polar organic modes of CEC-MS. In chapter 5, the feasibility of using internal tapered columns packed with vancomycin chiral stationary phase (CSP) is explored for simultaneous enantioseparation of eight â-blockers using CEC-MS. After a careful optimization of the mobile phase composition, sheath liquid and spray chamber parameter, 15 out of 16 enantiomers could be simultaneously resolved with excellent efficiency and detection sensitivity. The synthesis and characterization of sulfated and sulfonated cellulose phenylcarbamate CSPs is described in Chapter 6. The use of these CSPs, especially the sulfonated one, significantly enhances the EOF profile and sample throughput but maintain high enantiomeric resolving power under various modes of CEC and CEC-MS. By combining CEC and atmospheric pressure photo-ionization (APPI) MS, Chapter 7 demonstrates the separation and detection of mono-methylated benzo[a]pyrene (MBAP) isomers with ~100 times enhancement on detection sensitivity than CEC-UV. In Appedix 2, monolithic columns are synthesized through photopolymerized sol-gel approach and utilized for CEC and CEC-APPI-MS of polyaromatic hydrocarbons, and alkyl phenyl ketones.

Yes
In this work, an analytical study of the electromagnetic propagation in a complex medium-based suspended three-layer coplanar waveguide (CPW) is carried out. The study aims at a numerical calculation of the dominant hybrid mode complex propagation constant in the CPW printed on a bianisotropic substrate. The herein considered bianisotropy is characterized by full 3×3 tensors of permittivity, permeability and magnetoelectric parameters. The study is based on the numerical derivation of the Green's functions of such a complex medium in the spectral domain. The study is carried out using the Full Generalized Exponential Matrix Technique based on matrix- shaped compact mathematical formulations. The Spectral Method of Moments (SMoM) and the Galerkin's procedure are used to solve the resulting homogeneous system of equations. The effect of the chiral and achiral bianisotropy on the complex propagation constant is particularly investigated. Goo d agreements with available data for an anisotropic-medium-based suspended CPW structure are achieved. Various cases of chiral and achiral bianisotropy have been investigated, and particularly, the effect on the dispersion characteristics is presented and compared with cases of isotropic and bianisotropic Tellegen media.
FCT/MEC through national funds and when applicable co-financed by the ERDF, under the PT2020 Partnership Agreement under the UID/EEA/50008/2019 project.

DDT was extensively and globally used as a pesticide in agriculture and for malaria vector control from the 1940’s until the 1970’s. Due to its heavy use, DDT became ubiquitously distributed throughout the environment. DDT and several DDT metabolites are persistent organic pollutants. Two DDT metabolites, 3-MeSO2-DDE and o,p’-DDD have been proved to be tissue specific toxicants in the adrenal cortex. They are bioactivated to reactive intermediates which bind covalently to the adrenal cortex causing cell death. Due to its tissue specific toxicity o,p’-DDD has been used as a chemotherapy drug for adrenal cancer in humans. The efficacy and potency is however low and o,p’-DDD treatment is associated with serious side effects. 3-MeSO2-DDE has been suggested as a potential alternative therapeutic agent. A key aim of this thesis has been to improve the understanding of the kinetics of the two adrenocorticolytic compounds o,p’-DDD, its two enantiomers and 3-MeSO2-DDE. To meet this objective chemical synthesis and enantioselective analysis were required. Furthermore, in vitro toxicity of o,p’-DDD enantiomers and diastereomers were performed. An 11 step synthesis of 3-SH-DDE has been developed to promote both labelled and unlabelled synthesis of 3-alkylsulfonyl-DDE. Toxicokinetic studies showed that 3-MeSO2-DDE and o,p’-DDD were accumulated in tissues and retained in adipose tissue in minipigs. 3-MeSO2-DDE however had a twice as long biological t1/2 and a considerably lower Vd compared to o,p’-DDD. Suckling offspring were more exposed to 3-MeSO2-DDE than their mothers who were given 3-MeSO2-DDE orally. Interindividual differences in enantiomer kinetics in minipigs were observed suggesting polymorphism among the minipigs. Preparative isolation of the o,p’-DDD enantiomers is presented allowing determination of the absolute structures of the o,p’-DDD enantiomers by X-ray. The pure enantiomer of o,p’-DDD showed significant differences in toxicity in human adrenocortical cells.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1993.
Vita. Abstract. Vol. 2 is appendices. Includes bibliographical references (leaves 327-337). Also available via the Internet.

In the present study, mixed systems composed of SDS in the presence of neutral cyclodextrins were considered. Firstly, the effect of the CDs on the CMC of the surfactant was evaluated by CE experiments. Furthermore, a new CE approach based on electric current measurement was developed for the estimation of the stoichiometry as well as of the binding constants of SDS-CDs complexes. The results of these investigations were compared to those obtained with a different technique, electronic paramagnetic resonance (EPR). The obtained results suggested that methylated CDs, in particular (2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD), strongly affect the micellization of SDS in comparison to the other studied CDs. This effect also paralleled the chiral CD-MEKC performance, as indicated by the enantioresolution of (+/-)-Catechin, which was firstly selected as a model compound representative of important chiral phytomarkers. Then a CD-MEKC system, composed of sodium dodecyl sulfate as surfactant (90 mM) and hydroxypropyl-beta-cyclodextrin (25 mM) as chiral selector, under acidic conditions (25 mM borate – phosphate buffer, pH 2.5) was applied to study the thermal epimerisation of epi-structured catechins, (-)-Epicatechin and (-)-Epigallocatechin, to non epi-structured (-)-Catechin and (-)-Gallocatechin. The latter compounds, being non-native molecules, were for the first time regarded as useful phytomarkers of tea sample degradation. The proposed method was applied to the analysis of more than twenty tea samples of different geographical origins (China, Japan, Ceylon), having undergone different storage conditions and manufacturing processes.

In the present study, mixed systems composed of SDS in the presence of neutral cyclodextrins were considered. Firstly, the effect of the CDs on the CMC of the surfactant was evaluated by CE experiments. Furthermore, a new CE approach based on electric current measurement was developed for the estimation of the stoichiometry as well as of the binding constants of SDS-CDs complexes. The results of these investigations were compared to those obtained with a different technique, electronic paramagnetic resonance (EPR). The obtained results suggested that methylated CDs, in particular (2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD), strongly affect the micellization of SDS in comparison to the other studied CDs. This effect also paralleled the chiral CD-MEKC performance, as indicated by the enantioresolution of (+/-)-Catechin, which was firstly selected as a model compound representative of important chiral phytomarkers. Then a CD-MEKC system, composed of sodium dodecyl sulfate as surfactant (90 mM) and hydroxypropyl-beta-cyclodextrin (25 mM) as chiral selector, under acidic conditions (25 mM borate – phosphate buffer, pH 2.5) was applied to study the thermal epimerisation of epi-structured catechins, (-)-Epicatechin and (-)-Epigallocatechin, to non epi-structured (-)-Catechin and (-)-Gallocatechin. The latter compounds, being non-native molecules, were for the first time regarded as useful phytomarkers of tea sample degradation. The proposed method was applied to the analysis of more than twenty tea samples of different geographical origins (China, Japan, Ceylon), having undergone different storage conditions and manufacturing processes.

Nanomatériaux de silice peuvent être facilement fabriqués, façonné et fonctionnalisés comme plates-formes pour le greffage des nanoparticules pour des applications biomédicales et optiques. Ici, nous utilisons une méthodologie basée sur un modèle de préparer une collection variée de hélicoïdale nanoparticules d'or (PNB) superstructures ayant impartialité contrôlable et mesures structurelles en utilisant PNB que les blocs de construction, et les nanohelices de silice que le modèle. Le matériaux présentent synthétisé bien définir Agencement chiral de PNB suivant l'hélicité de nanohelices de silice, montrant des signaux plasmoniques de dichorism circulaire (CD). D'autres observations ont prouvé ce plasmon CD vient de l'arrangement chiral de PNB et cet effet est très taille, l'échelle et dépend du pH. Nous nous attendons à ce que cette stratégie d'assemblage va découvrir une meilleure vue sur les métamatériaux et de susciter la vue vers "bottom-up" des approches en nanosciences
Silica nanomaterials can be easily fabricated, fashioned and functionalized as platforms for grafting of nanoparticles for biomedical and optical applications. Herein, we utilize a template-based methodology to prepare a diverse collection of helical gold nanoparticle (GNPs) superstructures having controllable handedness and structural metrics by using GNPs as the building blocks, and the silica nanohelices as the template. The synthesized materials exhibit well-defined chiral arrangement of GNPs following the helicity of silica nanohelices, showing plasmonic circular dichorism (CD) signals. Further observations proved this plasmon CD comes from the chiral arrangement of GNPs and this effect is highly size, scale and pH dependent. We expect that this assembly strategy will discover a better view towards metamaterials and spark the view towards “bottom-up” approaches in nanoscience

NMR spectroscopy in liquid crystal solutions of polypeptides has been used for almost two decades, and has been shown to be a useful technique for the structure determination and the chiral discrimination of organic solutes. However the underlying mechanism remains largely unknown and there are several open questions. They concern the nature of the forces which control the anisotropic distribution of solutes: are there specific interactions, like hydrogen bonds, which involve selected chemical groups? Also the effects of co-solvent and temperature are scarcely understood: do they control the polypeptide-solute interactions or do they play an indirect role, by affecting the structure of the polypeptide? The lack of knowledge is a limit for the full exploitation of the NMR technique in liquid crystals. A deeper insight would be important to guide the optimization of the working conditions, e.g. by a proper selection of the polypeptide/co-solvent pair, given the nature of the solute. Especially challenging is the phenomenon of chiral discrimination. It occurs because pairs of enantiomers interacting with chiral polypeptides form diastereoisomeric pairs, but the forces relevant to this purpose are not known. In this thesis the behaviour of solutes in ordered solutions of poly(γ-benzyl-glutamate) (PBG) has been investigated using computational methods. The theoretical work has been carried out in close connection with experimentalists, from the laboratory of the Laboratoire de RMN en Milieu Orienté, Institut de Chimie Moléculaire d’Orsay de l’Université de Paris Sud, 11 (ICMMO) where a peculiar expertise in NMR techniques in liquid crystal media has been developed. Theoretical results have been checked against NMR data; moreover, new measurements have been performed in parallel with the computational study, to control the consistency of theoretical and experimental results. A major objective of this thesis has been the development of new computational tools for the analysis of experimental data, with the two-fold purpose of (i) helping the spectral interpretation and (ii) extracting the structural information which is contained in experimental data. In this thesis we have explicitly considered natural abundance deuterium (NAD) 2D-NMR experiments, so we have focussed on 2H-quadrupolar splittings; however the methods used here could be extended to other kinds of magnetic interactions. We have used different approaches to address the problems of chiral discrimination and structure determination. Chiral discrimination has been studied by fully atomistic Molecular Dynamics (MD) simulations. This level is needed, since the difference of orientational order, and then the spectral differences, between enantiomers is extremely small and depends on details of the intermolecular potentials. This is a general feature of chiral properties, which result from a subtle balance of energetically comparable contributions. This study had first of all an explorative purpose: it had to be assessed whether methods based on empirical intermolecular potentials could provide reliable information on chiral properties. A few solutes were investigated, but most of the work focussed on the prochiral molecule benzyl alcohol (BZA), which was found to be particularly appropriate for two main reasons. First, it exhibits a large difference in the 2H quadrupolar splittings of the two enantiotopic directions; moreover, it can be used as co-solvent, and this can be to improve the sampling efficiency in MD simulations. The problem of structural analysis is comparably less delicate, because the experimental differences in orientational order between molecules with different chemical structure are relatively larger. From the theoretical point of view, this means that approximate models can be developed, which are useful to rationalize the mechanism of solute ordering. Moreover, the availability of less demanding approaches, able to provide reliable predictions of order parameters, and then patterns of 2H-NMR splittings, at a relatively low computational cost, is essential for the development of computational tools to be used in connection with experiments. The thesis is divided in two parts. The first part is dedicated to the problem of structure analysis. In chapter 3 the so called tube model will be presented, for the calculation of the deuterium quadrupolar splittings of a solute on the basis of its chemical structure, under the assumption of simple steric repulsions with the polypeptide. The subsequent chapter 4 illustrates the computational methodologies which have been developed to predict the deuterium quadrupolar splitting profiles for flexible solutes, using the tube model coupled with suitable sampling of molecular conformations. Chapter 5 reports the experimental and theoretical study of a series of saturated fatty acid methyl esters (C14 –18). Chapter 6 presents a systematic study of saturated, unsaturated and conjugated fatty acid methyl esters and shows how torsional parameters can be extracted from the 2H quadrupolar splitting profiles. The second part of the thesis deals with chiral discrimination. In chapter 7 the results of a series of NAD 2D-NMR experiments on BZA, carried out at the ICMMO will be summarized. Then, chapter 8 is dedicated to Molecular Dynamics simulations. First the preliminary study of PBLG in chloroform will be presented, which was used to validate the parameterization of the force field for this polypeptide. Then, the results obtained for BZA in binary and ternary mixtures will be reported.
La spettroscopia NMR in soluzioni liquido cristalline di polipeptidi è utilizzata da una ventina d’anni e si è rivelata applicabile con ottimi risultati, sia per la determinazione strutturale, si per la discriminazione chirale di molecole organiche. Tuttavia il meccanismo che ne sta alla base rimane poco chiaro e ci sono molte questioni irrisolte. Queste riguardano la natura delle forze che controllano la distribuzione anisotropica dei soluti; non si conosce, ad esempio, quale possa essere il ruolo di interazioni specifiche, come legami idrogeno. Inoltre gli effetti del cosolvente e della temperatura non sono chiari, non si è capito se incidano direttamente sulle interazioni soluto-peptide oppure se abbiano un effetto indiretto, andando a modificare la struttura del polipeptide stesso. La scarsa comprensione costituisce un limite che impedisce di sfruttare appieno le potenzialità della tecnica NMR in cristalli liquidi. Una maggiore padronanza del fenomeno sarebbe un importante punto di partenza per ottimizzare le condizioni di lavoro; ad esempio si potrebbe scegliere con più cognizione il sistema peptide-cosolvente ideale per un determinato soluto. Un aspetto particolarmente interessante riguarda la discriminazione chirale: si sa che avviene perché l’interazione di coppie di enantiomeri con i polipeptidi chirali origina coppie di diastereoisomeri, ma non è noto quali forze ne siano responsabili. In questa tesi si è studiato il comportamento di soluzioni liquido-cristalline di poli(γ-benzil-glutammato) utilizzando metodi computazionali. Il lavoro teorico è stato svolto in collaborazione con un gruppo sperimentale del laboratorio del Laboratoire de RMN en Milieu Orienté, Institut de Chimie Moléculaire d’Orsay de l’Université de Paris Sud, 11 (ICMMO), che ha sviluppato una competenza specifica nelle tecniche NMR in fasi di cristallo liquido. I risultati teorici sono stati confrontati con quelli dati NMR; inoltre, in parallelo ai calcoli, sono stati eseguiti nuovi esperimenti, in modo tale da controllare la consistenza tra risultati teorici e sperimentali. Uno degli obiettivi principali della tesi è stato quello di sviluppare nuovi strumenti computazionali per l’analisi dei dati sperimentali con il duplice scopo di (i) essere un supporto per l’interpretazione spettrale e (ii) estrarre le informazioni strutturali contenute nei dati sperimentali. Nella tesi si è fatto esplicito riferimento a esperimenti in abbondanza naturale di deuterio (NAD 2D-NMR), perciò ci si è focalizzati su splitting quadrupolari di deuterio; tuttavia i metodi utilizzati possono essere estesi ad altri tipi di interazioni amgnetiche. Si sono utilizzati approcci diversi per affrontare il problema della discriminazione chirale e quello dell’analisi strutturale. I meccanismi che portano a discriminazione chirale sono stati investigati attraverso simulazioni di Dinamica Molecolare (MD) a livello atomistico. La scelta è stata dettata dalla constatazione che le differenze di ordine orientazionale, e quindi le differenze spettrali, tra coppie di enantiomeri sono molto piccole e possono dipendere da dettagli delle interazioni intermolecolari. Questa è una caratteristica fondamentale delle proprietà chirali, che sono il risultato di un bilanciamento delicato di contributi energetici di entità comparabile comparabili. Innanzitutto lo studio ha avuto uno scopo esplorativo: si è voluto valutare se metodi basati su potenziali intermolecolari empirici possano fornire informazioni affidabili di proprietà chirali in fase condensata. Sono stati studiati alcuni soluti, ma la maggior parte del lavoro si è concentrata sull’alcol benzilico (BZA), una molecola prochirale che si è rivelata particolarmente adatta allo scopo per più di un motivo. Innanzitutto essa presenta una differenza relativamente grande tra gli splitting quadrupolari di deuterio per le due direzioni enantiotopiche; inoltre ha il vantaggio di potere essere usata come solvente, e questo permette di aumentare l’efficienza del campionamento nelle simulazioni MD. Per quanto riguarda la determinazione strutturale, il problema è meno delicato in quanto le differenze sperimentali tra i parametri d’ordine di molecole con strutture diverse sono relativamente più grandi. Ciò significa che dal punto di vista teorico è possibile sviluppare modelli approssimati, che sono utili per razionalizzare il meccanismo alla base dell’ordine dei soluti. Inoltre la possibilità di mettere a punto approcci in grado di fornire stime dei parametri d’ordine, e quindi pattern di splitting quadrupolari, a un costo computazionale non troppo elevato, è essenziale per lo sviluppo di metodologie integrate con gli esperimenti. Questa tesi è quindi sviluppata in due parti. Nella prima parte ci si è concentrati sul problema della determinazione strutturale. Nel capitolo 3 viene presentato il modello del cilindro rigido, che permette di calcolare gli splitting quadrupolari di un soluto sulla base della sua struttura, partendo dall’ipotesi che le interazioni soluto-polipeptide siano essenzialmente di tipo sterico. Nel capitolo 4 sono riportate le metodologie computazionali sviluppate per il calcolo dei profili di splitting quadrupolari di soluti flessibili, utilizzando il modello del cilindro rigido accoppiato con un campionamento adeguato dello spazio conformazionale. Nel capitolo 5 si trova l’applicazione di questo metodo ad una serie di esteri metilici di acidi grassi saturi (C14-C18). Il capitolo 6 riporta uno studio sistematico di esteri di acidi grassi saturi, insaturi e insaturi coniugati, nel quale si mostra come i parametri torsionali possono essere estrapolati dal profilo degli splitting quadrupolari. La seconda parte della tesi riguarda la discriminazione chirale. Nel capitolo 7 si riportano i risultati di una serie sistematica di esperimenti NMR sull’alcol benzilico, condotti presso i laboratori dell’ICMMO. Il capitolo 8 è invece dedicato alle simulazioni di Dinamica Molecolare. Innanzitutto viene presentato uno studio preliminare sul PBLG in cloroformio, eseguito per validare la parametrizzazione del campo di forze utilizzato per questo polipeptide. Quindi sono riportati i risultati ottenuti per l’alcol benzilico in miscele liquido-cristalline binarie e ternarie.

A necessidade de metodologias adequadas para análise de fármacos e seus metabólitos em matrizes biológicas complexas levaram a um crescente interesse no desenvolvimento de novas técnicas de preparação de amostras, particularmente as técnicas de microextração, por serem altamente seletivas e requererem o consumo mínimo de solventes orgânicos. Aliado a esses avanços, o emprego de modernas e eficientes tecnologias analíticas, como a eletroforese capilar (CE) e a cromatografia líquida de alta eficiência acoplada à espectrometria de massas (LC-MS-MS), tem resultado em um considerável avanço em qualidade nas metodologias analíticas disponíveis para bioanálises. Dentro desse cenário, destaca-se a utilização dessas técnicas para o desenvolvimento de metodologias enantiosseletivas, permitindo quantificar os enantiômeros de fármacos administrados como racematos. Sendo assim, propusemos o desenvolvimento e a validação de metodologias enantiosseletivas para a análise dos enantiômeros da mirtazapina (MRT) e de seus principais metabólitos em plasma e urina, utilizando a CE e a LC-MS-MS. Para a preparação das amostras foram empregadas a microextração em fase sólida (SPME) e a microextração em fase líquida (LPME). No primeiro método desenvolvido, a LPME foi utilizada para extrair os analitos das amostras de plasma (1 mL), previamente diluídas, alcalinizadas com 3,0 mL de uma solução tampão fosfato 0,5 mol L-1 (pH 8) e adicionadas de 15% (m/v) de cloreto de sódio. Éter n-hexílico e uma solução de ácido acético 0,01 moL L-1 foram utilizados como solvente extrator e fase aceptora, respectivamente. As análises cromatográficas foram feitas em uma coluna Chiralpak AD-RH, empregando acetonitrila:metanol:etanol (98:1:1, v/v/v) mais 0,2% de dietilamina como fase móvel, na vazão de 1 mL min-1. A detecção dos analitos foi conduzida por LC-MS-MS usando um analisador triplo-quadrupolo e ionização por eletrospray positivo. Nessas condições, foram obtidas recuperações de 18,3 a 45,5%, resposta linear na faixa de concentração de 1,25-125 ng mL-1 e limite de quantificação (LQ) de 1,25 ng mL-1 para todos os enantiômeros avaliados. Posteriormente, a CE e a LPME foram utilizadas para a análise da MRT e seus principais metabólitos em urina. Antes da extração, amostras de urina (1 mL) foram submetidas a hidrólise enzimática a 37 ºC por 16 horas. Então, a enzima foi precipitada com ácido tricloroacético, o pH foi ajustado para 8 com uma solução tampão fosfato 0,5 mol L-1 (pH 11) e 10% de NaCl também foi adicionado. Em seguida as amostras foram submetidas a extração de forma similar aquela realizada para as amostras de plasma. As análises eletroforéticas foram obtidas em uma solução tampão fosfato 50 mmol L-1 (pH 2,5) contendo 0,55% (m/v) de carboximetil-b-ciclodextrina (CM-b-CD). O método foi linear na faixa de concentração de 62,5-2500 ng mL-1 para cada enantiômero da MRT e 8-hidroximirtazapina (8-OHM) e 62,5-1250 ng mL-1 para cada enantiômero da desmetilmirtazapina (DMR). O LQ foi 62,5 ng mL-1 para todos os enantiômeros. A SPME também foi utilizada no desenvolvimento de um método para a determinação simultânea do fármaco e seus metabólitos em urina usando CE e LC-MS-MS. Os analitos de interesse foram transferidos da solução aquosa hidrolisada para uma fibra de polidimetilsiloxano-divinilbenzeno (PMDS-DVB) e então foram desorvidos em metanol. As recuperações médias foram de 12 % para os enantiômeros da MRT, 3,8 % para a DMR e 0,72 % para a 8-OHM. O método foi linear na faixa de concentração de 62,5-2500 ng mL-1 com adequado LQ (62,5 ng mL-1) para todos os enantiômeros. A precisão e exatidão foram menores que 15% para todos os métodos desenvolvidos. Além disso, os métodos foram adequadamente aplicados em estudos preliminares de determinação dos enantiômeros da MRT, 8-OHM e DMR em amostras de plasma e urina obtidos após a administração oral de uma dose única de rac-MRT a voluntários sadios.
The need for appropriate methodology for the analysis of drugs and their metabolites in complex biological matrices led to a growing interest in developing new techniques for sample preparation, particularly microextraction techniques because they are highly selective and require a minimum consumption of organic solvents. Allied to these developments, the employment of modern and efficient analytical technologies, such as capillary electrophoresis (CE) and high-performance liquid chromatography coupled to mass spectrometry (LC-MS-MS), has resulted in a considerable improvement in quality in the analytical methodologies available for bioanalysis. In this context, it is worth to mention the use of such techniques to develop enantioselective methodologies, allowing the quantification of the enantiomers of drugs administered as racemates. Therefore, we proposed the development and validation of enantioselective methodologies for the analysis of the enantiomers of mirtazapine (MRT) and of its main metabolites in plasma and urine, using the CE and LC-MS-MS. Solid phase microextraction (SPME) and liquid phase microextraction (LPME) were used for sample preparation. In the first method, LPME was used to extract the analytes from plasma samples (1 ml), previously diluted, alkalinized with 3.0 mL 0.5 mol L-1 pH 8 phosphate buffer solution and supplemented with 15% (w/v) sodium chloride. N-hexyl ether and 0.01 mol L-1 acetic acid solution were used as solvent extractor and acceptor phase, respectively. The analyses were carried out on a CHIRALPAK AD-RH column and acetonitrile: methanol: ethanol (98:1:1, v / v / v) plus 0.2% of diethylamine was used as mobile phase, at a flow rate of 1 mL min-1. The detection was performed by LC-MS-MS equipped with a triple-quadrupole analyzer and ionization by eletrospray positive. Under these conditions, recoveries were from 18.3 to 45.5%; linear response over the 1,25-125 ng ml-1 concentration range and limit of quantification (LOQ) of 1.25 ng ml-1 for all enantiomers evaluated were obtained. CE and LPME were also used for the analysis of MRT and its main metabolites in urine. Before the extraction, urine samples (1 mL) were submitted to enzymatic hydrolysis at 37 ºC for 16 hours, the enzyme was precipitated with trichloroacetic acid, the pH was adjusted to 8 with 0.5 mol L-1 phosphate buffer solution (pH 11) and 10% (w/v) sodium chloride was further added. Then, the LPME extraction was performed according to the procedure previously developed. The electrophoretic analyses were carried out in 50 mmol L-1 phosphate buffer solution (pH 2.5) containing 0.55% (w/v) carboxymethyl-b-cyclodextrin (CM-b-CD). The method was linear over the concentration range of 62.5-2500 ng mL-1 for each MRT and 8-OHM enantiomer and 62.5-1250 ng mL-1 for each DMR enantiomer. The quantification limit (LOQ) was 62.5 ng mL-1 for all the enantiomers. A SPME method was also developed for the simultaneous enantioselective determination of MRT and its metabolites in urine using CE and LC-MS-MS. The target analytes were transferred from the hydrolyzed aqueous solution to the polydimetylsiloxane-divinylbenzene (PMDS-DVB) fiber coating and then desorbed in methanol. The means recoveries were 12 % for the enantiomers of MRT, 3.8 % for DMR and 0.72 % for 8-OHM. The method was linear over the concentration range of 62.5-2500 ng mL-1 with suitable LOQ (62.5 ng mL-1) for all the enantiomers. The precision and accuracy were lower than 15% for all developed methods. Moreover, the methods were successfully employed for the determination of MRT, 8-OHM and DMR enantiomers in plasma and urine samples obtained after oral administration of a single dose of rac-MRT to healthy volunteers.

An efficient two step procedure has been developed using CI GC/MS for analyzing amphetamines and related compounds. The first step allows the analysis of underivatized amphetamines with the necessary sensitivity and specificity to give spectral identification, including differentiation between methamphetamine and phentermine. The second step involves preparing a chiral derivative of the extract to identify d and 1-isomeric composition.

This thesis describes the development of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for the trace analysis of active pharmaceutical ingredients (APIs) and their metabolites in aqueous environmental matrices. The research was focused on the development of chiral LC-MS/MS methods for the analysis of fluoxetine and metoprolol, as well as their chiral metabolites in environmental water samples. A method was also developed for the achiral compounds, diazepam and nordiazepam. The LC-MS/MS methods were validated by the use of the isotope-labeled compounds. As these isotope-labeled compounds were not found in the wastewater samples, the validation could be assessed at trace level concentrations in the actual matrices in which the analytes were detected. The analytes were extracted from the water samples using solid phase extraction methods. Different types of solid phase extraction sorbents were evaluated. Fluoxetine and norfluoxetine were extracted through the use of a mixed mode polymeric based extraction sorbent. A hydrophilic and lipophilic balanced sorbent was employed for the simultaneous extraction of metoprolol and its metabolites, the base α-hydroxymetoprolol and the acidic metabolite deaminated metoprolol. Moreover, silica based C18 extraction discs were applied for the sample preparation of diazepam and nordiazepam. The chromatographic separations were conducted in reversed phase LC with MS compatible mobile phases. The enantiomers of fluoxetine and norfluoxetine were simultaneously separated using the chiral stationary phase (CSP), α1-acid glycoprotein (AGP). The Chiral AGP column was also applied for the separation of the enantiomers of deaminated metoprolol. For the simultaneous separation of the metoprolol enantiomers and the four stereoisomers of α-hydroxymetoprolol, the cellobiohydrolase (CBH) protein based CSP was used. An octadecyl silica based LC column was applied for the separation of diazepam and nordiazepam. The analytes were detected by the use of tandem quadrupole mass spectrometry operating in selective reactive monitoring mode. High resolution MS, employing a quadrupole time-of-flight (QqTOF) mass analyzer, was utilized for the identification of an unknown compound in wastewater samples. The APIs and their metabolites, as well as their respective enantiomers, were quantified in raw and treated wastewater from Uppsala, Sweden along with surface water from the River Fyris in Uppsala.

Thesis (Ph.D.); Submitted to Iowa State Univ., Ames, IA (US); 19 Dec 2004.
Published through the Information Bridge: DOE Scientific and Technical Information. "IS-T 2134" Meera Jay Desai. US Department of Energy 12/19/2004. Report is also available in paper and microfiche from NTIS.

Natural products are teeming with potential therapeutic agents. One group of compounds, polyphenols, from plants, exhibit anti-cancer, anti-inflammatory and anti-oxidant activities. Four polyphenolic compounds and their enantiomers were investigated in this thesis; the stilbene, 3-methoxypterostilbene, a structural analog of resveratrol, and the chiral prenylflavonoids from hops (Humulus lupulus L.); 8-prenylnaringenin, 6-prenylnaringenin and isoxanthohumol. A high performance liquid chromatography method for 3-methoxypterostilbene and enantiospecific liquid chromatography-mass spectrometry assays for the three prenylflavonoids were developed and validated. The methods allowed for quantification of these four polyphenols in biological samples and plant-based materials. Content analysis studies of 3-methoxypterostilbene and the three prenylflavonoids in traditional Chinese medicinal plants and hops-containing nutraceuticals were carried out, respectively. The pharmacokinetics of these four compounds were delineated through intravenous and oral administration in rats. 3-Methoxypterostilbene demonstrated greater bioavailability in rats than reported values for resveratrol. Enantiomeric differences in disposition parameters were observed for the three prenylflavonoids along with differences between compounds despite only small structural differences. The in vitro pharmacodynamics of these four compounds were elucidated including anti-oxidant, anti-inflammatory, anti-diabetic and cytochrome P450 modulation activities. All four compounds demonstrated a range of bioactivities related to chronic diseases and potential drug-botanical interactions. Further studies of polyphenols, especially clinical studies, are needed along with enantiospecific study when applicable to continue delineating the importance of bioactivity, pharmacokinetics and safety. Natural products are further developed into nutraceuticals and sold over-the-counter for both human and veterinary use but are not currently required to demonstrate efficacy prior to marketing. In the final section of this thesis, Phycox®, a multi-component veterinary nutraceutical for joint health was investigated for pharmacological activity in an in vitro model of canine osteoarthritis along with select constituents. A pilot single-dose pharmacokinetic study in dogs was also undertaken. Two liquid chromatography tandem mass spectrometry methods were developed and validated to detect constituents in serum. In vitro study results indicated that Phycox® was able to reduce inflammatory mediators similar to the NSAID, carprofen, and acute pharmacokinetic results revealed that detectable concentrations of glucosamine were evident in serum. It is suggested that further clinical studies of Phycox® are warranted to optimize its usage.
May 2016

La chromatographie en phase liquide bidimensionnelle est une technique à fort potentiel, offrant un grand pouvoir de séparation. Après avoir démontré son intérêt dans l’industrie pharmaceutique et présenté les enjeux liés à l’analyse quantitative, une attention particulière est portée sur le développement de méthodes. Dans l’idée de développer une stratégie d’analyse générique, la première étape est de sélectionner un set de trois systèmes 2D par le biais d’une approche développée au laboratoire. La deuxième étape est d’évaluer le potentiel de ces systèmes pour l’analyse quantitative. Ces deux étapes ont conduit à la proposition d’une stratégie d’analyse applicable à l’analyse pharmaceutique dans un contexte industriel. Enfin le potentiel du couplage RPLCxSFC est envisagé dans deux cas de figure différents. Premièrement, dans le but de comparer ce couplage aux séparations RPLCxRPLC développées dans le cadre d’une stratégie analytique générique, en termes de pouvoir de séparation. Deuxièmement, dans le cadre de l’analyse de composés chiraux, en développant un couplage sRPLCxSFC permettant une analyse achirale/chirale simultanée. Les avantages d’une telle approche ont été mis en avant en la comparant aux approches conventionnelles
Two-dimensional liquid chromatography (2D-LC) is a powerful technique considering its high separation power. After showing the advantage of 2D-LC in the pharmaceutical area and presenting the challenges related to quantitative analysis, special attention was paid to method development. With the aim of developing a generic analytical strategy for pharmaceuticals, the first step of our approach consisted in selecting a set of three 2D-systems with the help of a methodology previously developed. In a second step, the potential of these 2D-systems was evaluated for the purpose of quantitative analysis. An analytical strategy able to be applied to pharmaceutical analysis in an industrial context was proposed. Finally, the potential of RPLCxSFC was investigated in two different cases. Firstly, for comparing this on-line two dimensional technique to on-line RPLCxRPLC with respect of the separation power. Secondly, for chiral compounds by developing a selective RPLCxSFC method for simultaneous achiral-chiral analysis. The advantage of such method was highlighted by comparing to conventional approaches

Stereochemistry plays an important role in nature because biologically important molecules such as amino acids, nucleotides and sugars, only exist in enantiomerically pure forms. Semiochemicals carry messages, between the same species (pheromones) and between different species (allelochemicals). Both pheromones and allelochemicals can be used as environmentally friendly pest management. Many semiochemicals, i.e. behaviour modifying chemicals, consist of pure or well-defined mixtures of stereoisomers, where some of the other stereoisomers can be repellent. It is therefore important to be able to separate them to produce a synthetic pheromone in a mixture that is attractive. Pine sawflies are a family of insects that in some cases can be severe defoliators of conifer trees. Diprion pini, Diprion similis and Neodiprion sertifer are severe pests for these trees and have got the most attention in pine sawfly pheromone studies. The pheromone precursors are stored in the female body as long-chain secondary alcohols, which, when released, are esterified to acetates or propionates. The alcohols are chiral, and normally one of the stereoisomer is the main pheromone component, sometimes possible together with other stereoisomers as essential minor components. Bicyclus is a genus of African butterflies, and especially Bicyclus anynana has become a popular model for the study of life history evolution, morphology, mating choice and genetics. The wing pattern of Bicyclus differs depending on the season, with large eyespots during the rain-season and small or absent spots during the dry season.  Euglossa is one of the genera among the orchid bees in the Neotropics that does not produce its own pheromone. Instead, the males collect fragrances from orchids and other sources and store them in a pocket in their hind legs. Both Bicyclus and Euglossa use semiochemicals similar to pine sawflies, and thus can be analysed by the same methods. Pheromones and other semiochemicals in insects are often present in low amounts in a complex matrix, and purification of the sample before chemical analysis is often required. A common method is gradient elution on a solid phase silica column. Separation of stereoisomers can be achieved either by using a column with a chiral stationary phase (CSP) or with pre-column derivatisation using a column with an achiral stationary phase (ASP) or a combination of both, with mass detection as the dominant detection method. The purpose of this work has been to improve the purification method, find suitable methods to separate the stereoisomers of secondary alcohols, and to apply this on extracts of insects. By selecting the right fractions to collect during gradient elution the purification method was optimised. To reduce plasticizer contamination from ordinary columns, solid phase columns of Teflon or glass were used. For pre-column derivatisation of different chiral alcohols various acid chlorides were tested. For the pine sawfly pheromone precursors enantiopure (2S)-2-acetoxypropionyl chloride was the best choice. To separate some of the stereoisomers achiral 2-naphthoyl chloride was used. For derivatisation of 6,10,14-trimethylpentadecan-2-ol (R)-trans-chrysanthemoyl chloride was the best choice. The derivatised alcohols were separated on different columns, both chiral and non-chiral. Varian FactorFour VF-23ms was chosen as a general-purpose column, the Agilent HP-88 column was the best column with an ASP of those tested, and the Chiraldex B-PA column (CSP) was the only one that could separate all eight stereoisomers of derivatised 3,7-dimethylundecan-2-ol, 3,7-dimethyldodecan-2-ol, and 3,7-dimethyltridecan-2-ol. To determine the stereoisomeric purity of standard solutions used in field experiments and extracts of different species of insects the optimised methods were applied. For extracts from B. anynana, Euglossa and Neodiprion lecontei this work describe the first determination of the stereochemistry of some of their semiochemicals. For the determination of the stereochemistry of chiral semiochemicals the methods for purification and separation presented herein have shown to be of great value. The results will hopefully contribute to a better understanding of the communication among insects, and ultimately to a more environmentally friendly pest control.
Många naturligt förekommande kemiska ämnen finns som två spegelbilder av varandra, ungefär som höger och vänster hand. Dessa kan ha helt olika egenskaper och det är därför viktigt att kunna separera dem. Insekter och andra djur använder olika doftämnen för att kommunicera med varandra, om det är inom samma art kallas de för feromoner. De kan bestå av ett ämne eller en blandning av flera. Dessa doftämnen kan man även använda för att på ett miljövänligt sätt bekämpa skadeinsekter. En fälla med syntetiskt feromon för en viss insekt lockar endast till sig den arten, medan alla andra är opåverkade. Eftersom dessa ämnen ofta finns som spegelbilder där kanske bara den ena är aktiv och den andra rent av frånstötande, måste man kunna separera dem för att framställa ett syntetiskt feromon som är attraktivt. Målet med detta arbete har varit att bestämma feromonet hos olika arter av tallsteklar som kan vara svåra skadedjur på tallskog. De metoder som tagits fram har även tillämpats på några arter av afrikanska fjärilar samt orkidébin från Centralamerika eftersom de använder snarlika doftämnen. Att få fram feromonet från en insekt är lite som att leta efter in nål i en höstack eftersom de ofta bara innehåller några miljarddels gram per individ. Provet behöver först renas, och en del av arbetet i det här projektet har gått ut på att ta fram en lämplig reningsmetod. Huvudfokus har dock varit på att ta fram metoder som kan separera och identifiera det eller de ämnen, och spegelbilder av dessa, som doftämnena består av. När lämpliga metoder tagits fram har extrakt av olika insektsarter analyserats. I några fall är det första gången som deras feromon bestämts i detalj. Resultaten kan förhoppningsvis bidra till en ökad kunskap om insekters sätt att kommunicera, och i slutändan till miljövänligare bekämpning av skadeinsekter.

El proyecto de investigación presentado en esta Tesis tiene como principal objetivo establecer el efecto de las monocapas auto-ensambladas (SAMs) quirales en la nucleación y crecimiento cristalino de compuestos orgánicos, y encontrar las condiciones ideales para favorecer la nucleación heterogénea y su consecuente crecimiento cristalino. La investigación realizada detalla el estudio realizado para conseguir la cristalización controlada sobre superficies funcionalizadas de determinados compuestos quirales y sales diastereoméricas. Para ello se realizaron estudios de cristalización de mencionados compuestos sobre diferentes superficies funcionalizadas. El estudio de la cristalización controlada sobre superficies funcionalizadas comienza por la funcionalización de dicha superficie con phencyphos 4-metilentiol, la cual proporcionara resultados muy prometedores debido a su influencia, demostrada en esta tesis, sobre el proceso de cristalización. La cristalización de phencyphos sobre SAMs de este compuesto, no es controlada y presenta diferentes orientaciones. El disolvente es crucial ya que tiene una enorme influencia en el proceso, según el disolvente empleado en la cristalización de phencyphos sobre superficies funcionalizadas, en el caso de usar isopropanol se obtienen los cristales ramificados, un crecimiento muy peculiar. El método conocido como impresión por microcontacto (microcontact printing en inglés) ofrece un sistema en la superficie que permite favorecer el transporte de masa hacia la zona deseada de la superficie. Las cristalizaciones sobre superficies micropaternadas, tanto de phencyphos como de la sal diastereomérica (p-metilphencyphos y feniletilamina) se han conseguido controlar tras un estudio detallado del efecto del disolvente, el tiempo de la superficie en la disolución, la forma del patrón en superficie, los tioles combinados en superficie y la velocidad de evaporación.
The research presented in this Thesis has as the main objective of the establish the effect of chiral self-assembled monolayers (SAMs) on the nucleation and the crystal growth of organic compound, and find conditions which favour heterogeneous nucleation and subsequent growth. The possibility to control crystallization processes using self-assembled monolayers is an extremely interesting and promising approach in organic materials. This control has achieved by the use of inorganic crystalline substrates where nucleation is induced via epitaxy, although organic single crystals and SAMs have been used to control the polymorphic selectivity of the compound to crystallize, which is based on the lattice match between the molecular cluster and crystalline substrate terraces. According to this concept, SAMs have been used as controlled nucleation centres. This research describes the study in order to achieve the controlled crystallization of the compound phencyphos and diastereomeric salts on functionalized surfaces, and shows the differences between homogenous SAMs and combined SAMs (Microcontact printing method). The controlled crystallisation study starts with the formation of SAMs on gold with a novel chiral thiol, which has potential for nucleating crystal growth, (phencyphos 4-methylenthiol, PMT), and the crystallisation of phencyphos on them. The functionalisation of gold with monolayers of this compound has provided significant results due to its demonstrated influence in the crystallisation process. Thus, the successful functionalisation of the gold substrate by this resolving agent type molecule provided the chiral property to the self-assembled monolayer on gold. Phencyphos crystallises on PMT monolayer following different orientations and grow off the surface; depending on the solvent used these crystals grow as branched crystals (in isopropanol) on the functionalised surface. The microcontact printing method favours the mass transport to the desired thiol, minimizing evaporation effect at small scale. The surface combined of PMT and dodecanethiol has been the key for the development of surfaces which can induce the nucleation process on surface. On micropatterned surfaces, phencyphos crystallizes following a preferential orientation. Crystal growth is highly depending on the solvent used to crystalize phencyphos. The same enantiomer of phencyphos crystallized in different solvent (Chloroform and Isopropanol), yield different crystal growth, because the heterogeneous nucleation is effective when the solvent is allowed to evaporate slowly from the surface, allowing good mass transport to the desired regions. The diastereomeric salt most studied and presented here is the one formed by a phencyphos derivate, p-methyl phencyphos which provides a pair of crystalline salts with chiral amines. X-ray crystal structure of this diastereomeric salt reveal 10-membered rings constructed through hydrogen bonds, in which two ammonium groups formally replace phencyphos molecules seen in the phencyphos hydrate structure. The hydrogen bonds are strong and provide several polar faces to the crystalline structure, thus diastereomeric salts should have their crystals templated easily on polar SAM. There are several parameters that also have a dramatic influence on the crystallisation process such as the pattern shape and size which are critical. Thus the motif size which presents better results of favour the nucleation is for dots of 5 μm diameter spaced by 10 μm, for both crystallisation systems. The Dutch resolution has been also studied on micropatterned surfaces. The complexity of the family type crystallisation will require the development of specific additives to favour heterogeneous nucleation.