The requirements for the impurity profiling of substances for pharmaceutical use have become greater over time. They can be accomplished by the use of modern instrumental analysis techniques, which have been evolved in the last decades. New types of columns with HILIC, mixed-mode and chiral stationary phases are suitable for the separation of all kinds of substances mixtures, that were previously hardly possible with the use of common reversed phase columns. Modern, almost universal detectors like CAD, ELSD and CNLSD can be applied for a sensitive detection of substances without a chromophore. However, in addition to some small individual disadvantages to these methods, the costs are high and applications are still kind of rare. Thus, the introduction of these devices at a broader level has not yet taken place. While this presumably will change over time, there is a need for methods that enable the impurity profiling of challenging substances with widespread analytics devices. Methionine is a substance with hydrophobic and hydrophilic impurities. With the help of a mixed-mode stationary phase, which is a combination of a reversed phase and a strong cationic exchanger, the separation of all putative impurities was found possible with good sensitivity and selectivity. The method requires apart from the column only standard isocratic HPLC equipment and was successfully validated. The evaluation of the enantiomeric purity of amino acids is challenging. Two approaches were made. The first method utilizes CE by means of in-capillary derivation with OPA and the subsequent separation with a cyclodextrin. With the use of OPA/NAC and γ-cyclodextrin, a simple and cost-effective method for the indirect enantioseparation of 16 amino acids was developed. With the second approach, racemic amino acids can be analyzed with HPLC and in-needle derivatization. For this, different columns and chiral thiols were evaluated and the chromatographic parameters were optimized. A method with OPA/NIBLC, a pentafluorophenyl column made the enantioseparation of 17 amino acids feasible. A LOQ of the minor enantiomer down to 0.04 % can be achieved with UV spectrophotometric detection. A similar method was developed for impurity profiling of L-amino acids. This can be used alternatively for the amino acid analysis performed by the European Pharmacopoeia. A simple, robust, precise and accurate method for the evaluation of impurities in glyceryl trinitrate solution was developed and validated. The four impurities of glyceryl trinitrate are separated by means of an acetonitrile-water gradient and the assay for this substance is also possible. / Die Anforderungen an die Reinheitsanalytik von Substanzen für pharmazeutische Zwecke sind mit der Zeit größer geworden. Diese können durch die Verwendung von modernen instrumentellen Analysetechniken, die sich in den letzten Jahrzehnten entwickelt haben, erfüllt werden. Neue Arten von Säulen mit HILIC, mixed-mode und chiralen Phasen sind geeignet für die Trennung von allen möglichen Substanzgemischen, die vorher mit der Verwendung von gewöhnlichen Umkehrphasensäulen kaum möglich waren. Moderne, fast universelle Detektoren wie CAD, ELSD und CNLSD können für eine sensitive Detektion von Substanzen ohne Chromophor verwendet werden. Allerdings, neben ein paar individuellen Nachteilen von diesen Methoden, sind die Kosten sehr hoch und die Anwendungen noch eher selten. Daher haben sich diese Geräte noch nicht auf breiter Ebene durchgesetzt. Auch wenn sich das mit der Zeit ändern wird, gibt es die Notwendigkeit für Analysemethoden mit denen die Reinheitsanalytik von herausfordernden Substanzen auf verbreiteten analytischen Geräten ermöglicht wird. Methionin ist eine Substanz mit hydrophilen und hydrophoben Verunreinigungen. Mit der Hilfe einer „mixed-mode“-Phase, welche eine Kombination aus Umkehrphase und starker Kationenaustauscher ist, wurde die Trennung von allen mutmaßlichen Verunreinigungen mit guter Sensitivität und Selektivität ermöglicht. Die Methode benötigt abgesehen von der Säule nur eine normale isokratische HPLC und wurde erfolgreich validiert. Die Untersuchung der chiralen Reinheit von Aminosäuren ist anspruchsvoll. Zwei Ansätze wurden durchgeführt. Die erste Methode verwendet CE mittels In-Capillary- Derivatisierung durch OPA und der anschließenden Trennung mit Hilfe von einem Cyclodextrin. Durch den Gebrauch von OPA/NAC und γ-Cyclodextrin, wurde eine einfache und kosteneffektive Methode für die indirekte Enantioseparation von 16 Aminosäuren entwickelt. Mit dem zweiten Ansatz können Aminosäuren mittels HPLC und einer In-Needle- Derivatisierung analysiert werden. Dafür wurden verschiedene Säulen und chirale Thiole getestet und die chromatographischen Parameter optimiert. Eine Methode mit OPA/NIBLC, einer Pentafluorophenyl-Säule ermöglichte die Trennung on 17 Aminosäuren. Ein LOQ des kleinen Enantiomers von 0,04 % kann mit UV-spektroskopischer Detektion erreicht werden. Eine ähnliche Methode wurde für die Reinheitsanalytik von L-Aminosäuren entwickelt. Diese kann alternativ zur Aminosäurenanalyse im Europäischen Arzneibuch verwendet werden. Zusammenfassung 163 Eine einfache, robuste, präzise und richtige Methode zur Untersuchung der Verunreinigungen in Glyceryltrinitratlösung wurde entwickelt und validiert. Die vier Verunreinigungen von Glyceryltrinitrat werden mit Hilfe eines Acetonitril-Wasser-Gradienten getrennt und die Gehaltsbestimmung dieser Substanz ist ebenfalls möglich.

Jusqu’à très récemment, les catalyseurs pour la synthèse asymétrique étaient limités aux enzymes et aux complexes de coordination. Depuis les années 2000, une troisième approche a vu le jour: l’organocatalyse asymétrique. Il est assez surprenant de constater que parmi la multitude de catalyseurs organiques développés chaque année, une minorité d’entre eux seulement est basée sur de nouveaux squelettes chiraux. En effet, l’introduction d’une nouvelle entité chirale pourrait mener à la découverte de nouvelles combinaisons de substrats ainsi qu’à de nouvelles réactions catalytiques hautement stéréosélectives. C’est pourquoi, nous nous sommes proposé de préparer une série de nouveaux catalyseurs organiques bifonctionnels incluant des fonctions thiourées et basée sur le squelette chiral de la base de Tröger. L’accès aux dérivés énantiopures de la base de Tröger reste un défi majeur. C’est pourquoi, nous avons décidé de mettre au point une méthode efficace et prévisible, pour la résolution des analogues de la base de Tröger. Dans la mesure où l’élaboration d’une telle méthode nécessite un grand nombre de molécules, nous avons synthétisé une série de dérivés de la base de Tröger. La condensation d’anilines variablement substituées avec du paraformaldehyde dans de l’acide trifluoroacétique a été étudiée, conduisant à la synthèse d’analogues symétriques de la base de Tröger. L’utilisation de paraformaldehyde n’étant pas compatible avec tous les groupements fonctionnels, d’autres voies de synthèse ont également été explorées. Ainsi, des dérivés amino et cyano ont été préparés par l’intermédiaire de réactions organométalliques. Ensuite, une voie de synthèse menant aux analogues non-symétriques de la base de Tröger a été mise au point. Finalement, une série de dérivés présentant un pont –NCH2CH2N- a été préparée. La résolution de l’ensemble des composés a été systématiquement étudiée par chromatographie sur la phase stationnaire chirale commerciale Whelk O1. Des relations structure vs. énantioséléctivité ont pu être établies permettant de prédire la séparation par notre méthode. Une corrélation entre l’ordre d’élution et la configuration absolue a également pu être mise en évidence. Enfin, l’activité catalytique des dérivés thiourées de la base de Tröger a été évaluée dans la réaction d’addition de Michael de différents dérivés de l’acide malonique au trans-nitrostyrene. Compte tenu de la faible basicité de la base de Tröger, il a été démontré que l’issue de la réaction est fortement dépendante du pKa du nucléophile. De plus, aucune stéréosélectivité n’a pu être mise en évidence dans cette réaction d’addition.

Els micobacteris són un grup de microorganismes que en la seva majoria són sapròfits, malgrat això aquest grup concentra agents causals d’enfermetats tan antigues i conegudes com la tuberculosi (TB) i la lepra juntament amb d’altres de més desconegudes com la úlcera de Buruli. Si deixem de banda la TB i la lepra, trobem un important nombre de micobacteris, anomenats ambientals o micobacteris no tuberculosos (MNT) que, malgrat el seu saprofitisme, poden causar malaties secundàries en els éssers humans principalment a causa d’una immunosupressió. Dels més de 130 micobacteris MNT, aproximadament un terç podrien estar al darrera de les denominades micobacteriosis, dada a tenir en compte sobretot quan tractem amb enfermetats immunosupressores (la SIDA en seria l’exemple més conegut). El grup de micobacteris MNT tenen una gran versatilitat d’ecosistemes on poden sobreviure, podent colonitzar ambients diversos entre els que destaca per la seva importància la xarxa d’aigües de distribució, inclosa la xarxa sanitària, el que fa que puguin estar en contacte amb la majoria de la població. En aquest treball hem identificat primer, per purificar i caracteritzar després, dos components lipídics de la paret cel·lular de dos micobacteris ambientals com són el Mycobacterium gordonae i el Mycobacterium brumae. La característica comuna en ambdós treballs ha estat la identificació/detecció dels components problema mitjançant la tècnica de l’HPLC. Aquesta tècnica cromatogràfica ens ha permès descobrir un patró diferent al ja conegut i a partir d’aqui començar la feina de purificar i caracteritzar els compostos responsables d’aquesta diferència. El fet de ser uns components capaços de modificar el patró cromatogràfic conegut d’aquelles espècies ja era un raó prou important per dur a terme la seva caracterització. En relació amb els components estudiats al M. brumae, en realitat són una família de compostos, en el nostre laboratori es va fer la feina de la detecció, creixement del micobacteri i posterior purificació del compost. La seva caracterització estructural, excepte els estudis de RMN, i els estudis metabòlics es van dur a terme principalment a Toulouse degut a la manca d’experiència tècnica i d’aparells addients a la UAB. / Mycobacteria are a group of microorganisms most of them saprophytes, despite of it concentrates the causal agents of ancient and known diseases as tuberculosis (TB) and leprosy together with other more unknown diseases as Buruli ulcer. With the exceptions of TB and leprosy, it finds an important number of mycobacteria, called environmental or non tuberculous mycobacteria (NMT) that, in spite of their saprophytism, can cause opportunistic diseases in humans mainly because of immunosuppression. Over more than 130 NMT, approximately a third could be responsible for mycobacteriosis, this data takes into account especially when treating with immunosuppresion diseases (AIDS would be the most known example). The group of NMT are capable to grow in a wide range of environmental conditions so they can colonize a great variety of ecosystems between those that highlight are the water supply network so they can be in contact with the majority of the population. In this study we have identified first, for purifying and characterizing next, two lipidic compounds located in the cell wall of two enviromental mycobacteria as Mycobacterium gordonae and Mycobacterium brumae. The common characteristic in both studies has been the identification/detection of unknown compounds by HPLC. This chromatographic technique has allowed us to discover different HPLC patterns to the already known and then to start the characterisation of the compounds that cause this difference. The fact to be some components capable to modify the chromatographic pattern known of those species already was a reason enough important to carry out their characterisation. In regard to the components studied from M. brumae, as fact a family of compounds, we did the work of detection by HPLC, the mycobacterial growth and purification of the compound, but its structural characterisation, except the NMR studies, and the metabolic studies had been carried out mainly in Toulouse owed to the lack of technical experience and devices at UAB.

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The international harbors and fishing ports in Taiwan are nearly not installed the wastewater or oil treatment facilities, which would cause the ocean pollution by the ballast and bilge water from the boats. The purpose of this research is to study the feasibility of treatment of diesel fuel and the polycyclic aromatic hydrocarbons (PAHs) in diesel by aerobic biofiltreation in salty environment. According to the experimental results, the mineral oil and PAHs in the diesel fuel present high good removal efficiencies. Both of the contaminants were decomposed fast at the first 8 hours, and then slow down. The emulsified diesel fuel were found able to be reduced 83% within 8 hours, and the concentrations of phenanthrene would found decreased from 1.5 to 0.1 mg/L. After 8 hours, we observed that the PAHs with high number of rings kept a mild diminish trend, while the PAHs with less rings exhibited arrearage and irregular indicated by some peaks appearing in the chromatography diagram of HPLC, which were not found in the diagram in the initial of test run. In conclusion, the aerobic biofiltreation systems were found able to degrade n-alkanes and PAHs in diesel fuel effectively. Thus we suggested to install the systems at the greasiness accepting facilities in the harbor. However, we still have to keep studying the influence of the interrelationship between the contaminants in diesel fuel and the environment factors.

The purpose of this study was to design a dosing regimen and evaluate the safety of zonisamide (ZNS) following multiple dosing and to determine appropriate monitoring methods. Clinical pharmacokinetics were studied in 8 adult dogs (4 male and 4 female) ranging from 3 to 4 years of age using a randomized crossover design following single intravenous (IV) and oral administration, 6.85 and 10.25 mg/kg, respectively. Samples were collected intermittently for 48 hours. Dogs were then dosed orally (10.17 mg/kg) twice daily for 8 weeks. Blood samples were collected weekly and at discontinuation of the drug. Additionally, urine was collected to determine 24 hour urine ZNS clearance following IV administration. Safety was based on clinical pathology, thyroid and urine testing during both studies. ZNS was measured using high performance liquid chromatography in serum, plasma, erythrocytes (RBC) and whole blood. Data were subjected to standard non-compartmental pharmacokinetic analysis using computer assisted linear regression (WinNonLin?). Comparisons were made in different compartments using one-way ANOVA to identify any differences. Safety parameters at study beginning and end were compared using a Student t-test. ZNS concentrations differed among blood compartments after single dosing, with oral maximum concentration (Cmax) being greatest in RBC (28.73?g/ml) and least (14.36?g/ml) in plasma. Volume of distribution also differed, being greater (1096.05ml/kg) in plasma and least in (379.23ml/kg) RBC. Clearance of ZNS was 57.55ml/hr/kg from plasma and 5.06ml/hr/kg from RBC. Elimination half- life in plasma was 16.4 hr in serum and 57.4 hr in RBC. Bioavailability was 126.8% for RBC and 189.6% for plasma. Following multiple dosing, at steady-state, Cmax averaged 65.8?g/ml with fluctuations of 17.2% between dosings. Accumulation of ZNS was 3.5 (plasma) and 4.3 (RBC). Concentrations did not differ among blood compartments at the end of multiple dosing. Although differences did occur across time in clinical pathology tests, all were within normal limits at study end except for T4. In conclusion, ZNS dosed at 10 mg/kg twice daily for dogs would maintain therapeutic levels (10 to 70?g/ml) recommended in human epileptic patients. Therapeutic monitoring would be best measured in serum or plasma accompanied with thyroid and urine testing.

Described in this thesis is the isolation of two microorganisms containing a nitrile hydratase and amidase to effect the enantioselective hydrolysis of a-substituted nitriles to their corresponding amides and acids. Isolate NP3854 was identified as an atypical Rhodococcus sp. The nitrile hydratase proved to be non-selective for all the substrates tested. However, carboxylic acids with excellent enantiomeric excess were obtained from a large number of amides. X R~CN H nitr-il-e--h-y-d-r.a~tase ~X amidas.e RH CONH2 X R~"""CO H H 2 Optically active acids with an enantiomeric excess of, generally, >98 %, were obtained when X = NH2, Me and Cl, but proved to be racemic for OH and Br. R could be a variety of aromatic, cyclic and acyclic alkyl residues without adversely affecting the enantioselectivity. The pH-activity profile was determined for the amidase of NP3854 using propionamide as the substrate. From this data, coupled with inhibition studies, it may ,.tentatively be suggested that the amidase has a histidine residue in the active site, which may act as a general base for a serine amino acid. The pH-activity profile was determined for 2-amino-2-phenylacetamide 2b, and this suggested that the unprotonated form of the amine acted as the substrate. Within a pH range of 3 - 9 the enantiomeric excess remains high (>98 %) and experimentally invariant. The amidase was found to have a temperature optimum of 60°C and could tolerate 20 % THF with a loss of only 15 % activity. Attempts made to hydrolyse 4,5,6-amino nitriles and amides to the corresponding amino acids and isolate any reaction intermediates failed. This was presumed to be due to the large fraction of the unprotonated amine due to the higher pKa (- 9 - 10).

Anthocyanins have been shown to possess specific antioxidant capacities, which may provide an underlying protective effect against many chronic diseases . Although the antioxidant capacity of anthocyanins has been well established, less is known about the extent to which specific anthocyanin composition affects total antioxidant capacity . The aim of the present study was to compare the antioxidant capacity of two different soft fruits, blackcurrant and grape, which have distinctly different anthocyanin profiles. The anthocyanin profiles of grape and blackcurrant were characterized by HPLC/MS coupled with a diode array detector. Results showed that blackcurrant contained four predominant anthocyanins, cyanidin 3-glucoside, delphinidin 3-glucoside, cyanidin 3-rutinoside, and delphinidin 3-rutinoside . In contrast, malvidin 3-glucoside, delphinidin 3-glucoside, cyanidin 3-glucoside, petunidin 3-glucoside, and peonidin 3- glucoside were the major anthocyanins found in grape . The concentration of individual anthocyanins in all berries was quantified with HPLC/UV using cyanidin 3-glucoside as an external standard . Finally, results showed a greater (p<0.05) antioxidant capacity of blackcurrant compared to grape. The total antioxidant capacity of crude extracts from each was measured by Oxygen Radical Absorbance Capacity (ORAC) and ABTS assays. Anthocyanin antioxidant capacity index (AACI), derived from the product of antioxidant (ORAC) activity for each of major anthocyanin present in blackcurrant and grape, was also used to determine whether the antioxidant capacity of crude anthocyanin fractions represents either the sum total anthocyanin content or, alternatively, a synergy between different anthocyanins components . Our results indicated that a plausible potential synergy between anthocyanin components in regards to ORAC antioxidant capacity existed in blackcurrant and grape semi-purified anthocyanin extracts. Furthermore, it could be concluded that both total anthocyanin content as well as the composition of individual anthocyanins in soft fruits is important to assess total antioxidant capacity of different berry sources .