Despite the wide range of HPLC stationary phases available for reversed-phase high-performance liquid chromatography (RP-HPLC) and the in-depth studies using probes to highlight differences between them, there is very little in the way of stationary phases which offer selectivity that is substantially different from that offered by the very commonly used alkyl-silicas. Therefore, the primary aim of the research programme was to explore and try to exploit LC stationary phases which offered genuinely different selectivity to alkyl-silicas for typical drug applications. Chiral stationary phases (CSP) potentially had different selectivity and in this context a secondary aim was to explore aspects of the enantioselectivity of CSP as well as their chemical selectivity. Claims of orthogonal selectivity had been made for pentafluorophenyl (PFP) phases and phases exhibiting the hydrophilic interaction liquid chromatography (HILIC) mode. However, the Ultra PFP phase was found to be very similar in selectivity to ACE 5 C18 for both amitriptyline and acemetacin related compounds. The ZIC-HILIC phase was shown to behave as a reversed-phase material at high aqueous content in the mobile phase. There was some indication of selectivity orthogonal to that of ACE 5 C18 with low aqueous content in the mobile phase but this occurred at low retention and with mobile phases unsuitable for use with C18 phases in coupled (column or phase) systems. Nonetheless the work carried out shed more light on the mechanisms taking place in the HILIC mode which is currently attracting so much interest. Also it was possible to put ZIC-HILIC to good use for polar plant metabolites and other applications. Chiral stationary phases (CSP) also offered the prospect of selectivity orthogonal to that of C18 phases. Given the proliferation of such phases though and the fact that it would be useful to use CSP that gave chiral separations for a broad spectrum of compound classes as well as giving orthogonal separations between different compounds, it was decided to carry out comparative studies of CSP classes in order to identify any redundancies and to seek out CSP that were complementary to one another. The Regis Whelk-O1 CSP was shown to be much superior to other higher-generation Pirkle-concept CSP such as DACH-DNB and ULMO. Also it was shown to be complementary to the Chiralcel OD derivatised ii polysaccharide CSP and that both had something to offer alongside the widely used Chiralpak AD derivatised polysaccharide CSP. It was also found that a series of Chiralcel OD clones were virtually identical to Chiralcel OD and similarly for Chiralpak AD clones. Chiralpak IA, an immobilised version of Chiralpak AD, was not markedly less enantioselective than Chiralpak AD. Chiralcel OJ was less enantioselective than Chiralpak AD but the gap in performance was not as wide as between Whelk-O1 and the other Pirkle-concept CSP. The information gathered during these studies should prove to be of enormous value for further work in chiral LC method development screening. Before embarking on applications work utilising the stationary phase selectivity that had been found, a study was carried out on the effectiveness of the high efficiencies obtainable with short run times through ultra-performance liquid chromatography (UPLC). It was found that, for a range of pharmaceutical applications, that it was still necessary in each case to adjust selectivity before increasing speed through working at higher temperatures with faster flow rates. In the course of this work some exceptionally high speed separations for example for paroxetine and related substances, benzodiazepines and flurbiprofen and related substances, were developed. With respect to the evaluation of CSP as orthogonal phases to alkyl silicas under reversed-phase conditions, the Whelk-O1 CSP showed promise. However on closer inspection it was found that the Whelk-O1 CSP had very similar selectivity to the alkyl silica phase, ACE 5 C18, and deviation from this only occurred in instances when there was interaction with the chiral recognition site to give a separation of enantiomers. This prompted the notion that, rather than using Whelk- O1 in a coupled column system with ACE 5 C18, it could be used on its own for the separation of both trace enantiomer and all other related substances. This was shown to be possible using (S)-naproxen, laevokalim and (S)-flurbiprofen as illustrative examples. The evaluation of the enantioselectivity of CSP led to an optimised resolution (suitable for scaling up for preparative work) of the enantiomers of the former ‘legal-high’ drug, mephedrone, on Whelk-O1 under normal phase conditions. It was also shown that the infrequently used Chiralcel OJ derivatised polysaccharide iii CSP was ideal for developing an assay to determine trace amounts of (R)-nicotine in (S)-nicotine. Overall, the information obtained on stationary phase selectivity and retentivity through evaluation and application will be of great value in HPLC and UHPLC column selection and also selection of orthogonal phases for coupled column systems but, ultimately, moving forward, most value may be in aiding the design of two-dimensional LC systems for complex mixture analysis. This would particularly apply to the use of CSP with reversed-phase eluents in achiral-chiral systems.

In pharmaceutical R & D, drug stereochemistry, and consequently the rotation of enantiomers, is very important. Because they act as chiral selectors in vivo, biomacromolecules have been extensively used as chiral selectors for the liquid chromatographic (LC) resolution of enantiomers and more latterly have also been employed in the newer separative technique, capillary electrophoresis (CE). However, at the outset of this research programme, this had generally been restricted to common easily accessible biomacromolecules such as plasma-binding proteins. It was clear that it be would be useful therefore to adapt LC and CE in such a way as would allow the use of a much wider range of biomacromolecules. Accordingly the general aim of this study was to develop LC and CE protocols involving biomacromolecules that would give rise to minimum consumption of the biomacromolecule. To study biomacromolecules in free solution CE, a number of experimental variables had to be established for both optimum chiral discrimination and for investigating biomacromolecule-ligand interactions. The typical and widely used biomacromolecule for chiral discrimination, bovine serum albumin (BSA) was used to study the variables of pH from pH 5.4 to 8.4, concentration of BSA form 0 to 60 mM and concentration of organic modifiers in the range 0 – 20 % v/v for chiral selectivity. This involved an investigation into some unusual artefacts such as ghost peaks and stepped baselines, but ultimately the outcome was a successful free solution CE protocol suitable for the rapid evaluation of chiral discrimination of other biomacromolecules. The conditions were: run buffer (30 mM protein, 67 mM phosphate (pH 7.4) – methanol (97.5 : 2.5, v/v)), capillary CElect p150, 40 cm (35 cm to detector) x 50 mm i.d., temperature of ambient or 25 °C and an applied voltage of 10 kV. The ability of other biomacromolecules, such as human serum albumin (HSA), lactoferrin and protamine, to resolve enantiomers was studied using this protocol including looking at the effect of the addition of modifiers to the buffer such as metal ions like manganese and zinc, competing ligands, e.g. warfarin and ibuprofen, and b-cyclodextrin. As well as using CE, miniaturisation of LC was also studied in view of the success of biomacromolecule-affinity chiral LC. Two different, but similar, microbore LC protocols were employed, i.e. using the protein in free solution or as a pseudo stationary phase. For the former, a Lichrosorb DIOL stationary phase, based on hydroxyl groups immobilised on silica, was chosen in order to minimise the adsorption of protein to the stationary phase. Using this protocol it was demonstrated that free solution microbore LC could be easily be carried out, therefore used to evaluate chiral discrimination and that the use of the system to study in vivo interactions was feasible. The creation of a biomacromolecule pseudo stationary phase, as opposed to conventional chiral stationary phases where the protein is permanently bonded to the stationary phases, involves the biomacromolecule being adsorbed within the pores of the stationary phase. In this way the overall biomacromolecule structure should not be grossly distorted. Three stationary phases were evaluated, viz wide-pore Nucleosil silica, Nucleosil C8 and Lichrosorb DIOL, for optimum biomacromolecule loading and minimal biomacromolecule leakage when mobile phase was pumped through the column. The Nucleosil silica with adsorbed BSA proved the most successful, e.g. a of 3.6 and 4.0 for tryptophan and kynurenine respectively, and robust of the stationary phases with respect to demonstrating the chiral discrimination potential for this system. All the miniaturised systems evaluated were successful, to a greater or lesser degree, for the demonstration of chiral selectivity of biomacromolecules. While CE was better for minimisation of the consumption of the biomacromolecule, it was also important that the biomacromolecule LC systems could be operated in reduced dimensions since these systems have perhaps greater potential for exhibiting enantioselectivity and are more appropriate for the ever increasing need for the study of the interaction of ligands with the biomacromolecule in its ‘natural’ form. With the knowledge gained from this research programme it will now be possible to more easily carry out such studies with much smaller amounts of biomacromolecule, and, accordingly be able to work with biomacromolecules which hitherto it has not been possible to study because of limited availability. While some of the protocols have now been superseded by recent developments the system developed still has potential. The use of such small scale systems offers the potential to study chiral selectivity and drug-biomacromolecule binding of rare or expensive biomacromolecules.

The goal of the project is to design and build a photocalorimeter capable of carrying out photostability testing in the pharmaceutical industry. A current challenge is to develop methods of testing the photostability of solid materials which are non-invasive, non-destructive and allow real time observations to be made. Calorimetry represents one such method. Solution-phase systems are relatively straight-forward and have been applied to calorimetry for many years requiring few assumptions about the reaction system under study to be made. Previous studies have developed iterative methods to fit experimental data to established calorimetric equations. A new approach allowing the direct calculation of reaction parameters is described in chapter two. For solids, the situation is more complicated than that for solutions and equations are more difficult to develop since additional parameters (such as the solid state fitting parameters m and ri) have to be accommodated. New theoretical approaches to data fitting in the solid state are also described in chapter two. Having previously established that the imidazole-catalysed hydrolysis of triacetin reaction is robust and reliable, new applications, such as the effect of fill-volume on the calorimetric output and thus the reaction parameters, are described in chapter three in preparation for use of the system on solid state systems. Chapter four describes the design and development of the photocalorimeter, together with improvements and modifications made to it as the project progressed, whilst chapter five describes the development of actinometric techniques. An actinometer provides a means of measuring the amount of light-energy being delivered to a target sample. Actinometric techniques are described in the literature and studies carried out on candidates for use with the photocalorimeter are outlined. Particular success was achieved with 2-nitrobenzaldehyde, where results within 2% error were achieved for the actinometric experiments. The final stage of the project involves the application of the newly-developed photocalorimeter to the testing of solid samples such as nifedipine. Studies were carried out both on the photodegradation caused by white light and also using monochromatic light. This allowed "causative wavelengths" of photodegradation to be investigated using the photocalorimeter - a significant area of interest in the pharmaceutical industry and the first time quantitative data has been obtained for a solid state material using non-classical techniques. Finally, studies were carried out into establishing the photostability of an unknown solid state test material.

No description available.

Une des principales limites de la chimiothérapie du cancer est le manque de sélectivité des agents cytotoxiques vis-à-vis des cellules tumorales, entrainant de nombreux effets secondaires. L’intérêt de la vectorisation est d’administrer l’agent cytotoxique sélectivement aux cellules cancéreuses et ainsi d’éviter de faire subir l’action du médicament aux cellules saines. Cette thèse a pour but de synthétiser des prodrogues multivalentes qui utilisent la sous unité B de la toxine de Shiga (STxB) comme agent de vectorisation, ciblant préférentiellement les cellules cancéreuses Gb3-positives. Les deux agents cytotoxiques choisis, un dérivé de la camptothécine et de l’auristatine, ont été liés de façon covalente auvecteur via un espaceur bifonctionnel clivable par le glutathion. L’espaceur a également été remplacé par un répartiteur multivalent susceptible de délivrer une quantité plus importante d’agent cytotoxique à l’intérieur des cellules tumorales.Parallèlement à ce projet de vectorisation d’un agent thérapeutique, des sondes IRM capables de marquer in vivo les tumeurs possédant à leur surface le récepteur Gb3 ont été synthétisées. L’IRM a une très bonne définition spatiale, mais souffre d’un manque de sensibilité. Pour augmenter le contraste, nous avons utilisé une stratégie reposant sur lamultivalence des espaceurs liés à des molécules de DOTA-Gd, un agent de contraste classiquement utilisé en IRM. Ces dernières ont été fixées à STxB via un répartiteur multivalent (des nano-bâtonnets d’or). Le signal IRM est plus intense et donc plus facile à détecter. Enfin, nous avons développé une nouvelle méthode chimique basée sur la ligationoxime pour optimiser la création de conjugués entre STxB et des antigènes comme outils de vaccination. Le conjugué STxB-ovalbumine a permis une meilleure présentation de l’antigène par les cellules dendritiques, conduisant chez la souris à une induction efficace de lymphocytes T. / Pas de résumé en anglais

Metabolomics is a discipline where metabolites are assessed, identified and quantified in different samples. Metabolites are crucial components of the biological system and highly informative about its functional state due to the closeness to functional endpoints and to the organism’s phenotypes. 1H NMR and LC-MS, the commonly used metabolomics analytical platforms were used to annotate the metabolites found in Sclerocarya birrea (S. birrea) leaves from five South African provinces, Limpopo (L), Gauteng (G), North West (NW), Mpumalanga (M) and KwaZulu-Natal (KZN). Supervised Orthogonal Partial Least Square – Discriminant Analysis (OPLS-DA) of the full spectra revealed a clear differentiation of S. birrea leaves from five provinces. In addition, the level of common metabolites were measured and compounds previously found to have anti-diabetes potential ((-)-epicatechin 3-0-galloyl ester, myricetin-3-0-α-L-rhamnopyranoside, gallic acid and Kaempferol-3-0-α-L-rhamnopyranoside) were annotated in the samples. The samples from the five provinces showed anti- diabetic activity when exposed to an in-vitro glucose uptake assay, with the highest activity observed in male samples from M. The sample presented high concentrations of (-)-epicathechin 3-0-galloyl ester, one of the metabolites with anti-diabetes activity. Overall 1H NMR and LC-MS metabolic profiling were successfully applied to discriminate all five sources of S. birrea leaves, and obtained qualitative information of many common metabolites / Agriculture, Animal Health and Human Ecology / M. Sc. (Agriculture)