El bromo es un elemento muy reactivo que se encuentra distribuido en la naturaleza principalmente como bromuros, en pequeñas cantidades. El agua de mar es una fuente virtualmente ilimitada de bromo, cuya evaporación da lugar a la formación de salmueras, sustancias complejas y de gran valor económico debido a la gran concentración de sales disueltas. La determinación de trazas de bromuro, especialmente en muestras de composición y naturaleza compleja como el agua de mar, es un problema difícil de encarar. Los métodos convencionales para la cuantificación de bromuros por gravimetría, yodometría, entre otros, no son confiables debido a la compleja composición del agua de mar cuyos iones provenientes de las sales disueltas actúan como interferentes. El método más utilizado hasta hace unos años fue el que corresponde a la Norma ASTM D3869-09: Métodos estándares de ensayo para iones ioduro y bromuro en aguas salobres, agua de mar y salmueras. Sin embargo, desde hace unos años este no es viable debido al uso de tetracloruro de carbono como solvente de extracción al ser un reactivo prohibido debido a su toxicidad y considerado por el Protocolo de Montreal como una de las sustancias que agotan la capa de ozono. La investigación plantea la optimización y validación de un método cuantitativo para evaluar la concentración de bromuros en el rango de 34 a 200 mg/L, basado en el procedimiento descrito en la Norma ASTM D3869-09. Este nuevo método colorimétrico hace uso de la oxidación de iones bromuro a bromo molecular mediante la reacción de iones bromuro con óxido de cromo (VI) en solución ácida; luego el bromo molecular resultante de la reacción anterior, es extraído con disulfuro de carbono analizando su concentración por espectroscopia ultravioleta-visible a 420 nm. Como resultado de esta investigación, se ha comprobado la validez del método propuesto para evaluar la concentración de bromuro en el rango de 34 a 200 mg/L, se han establecido las condiciones específicas para el tratamiento de las muestras y se ha validado el método siguiendo la guía de IUPAC para validaciones realizadas por un solo laboratorio. Se comprobó que el método es robusto, preciso, veraz y que las concentraciones máximas de sales que pueden estar presentes en la muestra sin interferir en la cuantificación de iones bromuro es de de 224 ppm de sulfato de calcio, 292 ppm de cloruro de potasio, 657 ppm de cloruro de sodio, 1527 ppm de sulfato de magnesio y 40 542 ppm de cloruro de magnesio. Además, se obtuvo la incertidumbre expandida del método igual a ± 0.1418 ppm. / Tesis

The reaction of dimethylcopper lithium and di-n-butylcopper lithium with 3-bromo-2-(2-phenylacetamido) acrylic acid, mp 174 - 175°, produced as major products 3-methyl-2-(2-phenylacetamido) acrylic acid and 3-n-butyl-2-(2-phenylacetamido) acrylic acid, respectively. The dimethylcopper lithium reaction was stereospecific for the E geometrical isomer, mp 198 - 199°, and produced 3-methyl-2-(2-phenylacetamido) acrylic acid and 2-(2-phenylacetamido) acrylic acid in a 3:1 molar ratio, respectively. The di-n-butylcopper lithium reaction produced as the main products 3-n-butyl-2-(2-phenylacetamido) acrylic acid and 2-(2- phenylacetamido) acrylic acid in a 7:1 molar ratio, respectively, with the 3-n-butylacrylic acid comprising 83% of the product. The reaction produced the E isomer, mp 205 - 206°, and the Z isomer, mp 164 - 165°, in the approximate ratio of 2:1, respectively. The configuration of the products of both reactions was determined by the comparison of the nuclear magnetic resonance spectra with spectra of model compounds of known configuration. Nuclear magnetic resonance evidence relating to the stereochemistry of 3-bromo-2-(2-phenylacetamido) acrylic acid based on the single isomers of this acid and the corresponding methyl ester reported in the literature was inconclusive. In order that a comparison of the properties of both isomers could be made, an attempt was made to prepare the uncommon isomer of methyl 3-bromo-2-(2-phenylacetamido) acrylate from (a) the common isomer by hydrobromination-dehydrobromination with triethylamine and (b) from methyl 2-(2-phenylacetamido) acrylate by bromination-dehydrobromination with triethylamine. Both attempts were unsuccessful.

Made available in DSpace on 2016-04-04T12:35:25Z (GMT). No. of bitstreams: 2 loyze_lima_ioc_mest_2014.pdf: 4809047 bytes, checksum: ddb3a58d24cdeff435fb1a250dcb5a46 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Toxoplasma gondii causa uma infecção comumente assintomática, porém pode se apresentar de forma grave durante a gravidez e em pacientes imunocomprometidos. A terapia atual para a toxoplasmose é restrita contra taquizoítos, e possuem pouco ou nenhum efeito sobre bradizoítos, que são mantidos em cistos teciduais como fonte recrudescente da infecção. Com isso, novas alternativas terapêuticas vêm sendo propostas, como o uso da Atovaquona, que apresentou alguma eficácia sobre taquizoítos e bradizoítos em cistos teciduais. Neste trabalho, foi estudado o efeito de 3-BrPA, um composto utilizado em testes sobre células cancerígenas, durante a interação de células LLC-MK2 infectadas com taquizoítos da cepa RH de T. gondii. Quanto à célula hospedeira não se observou efeito do composto sobre a proliferação e viabilidade celular. A avaliação da interferência de 3-BrPA sobre o crescimento in vitro do T. gondii evidenciou uma redução na proliferação intracelular do parasito de cerca de 55% após 24 h de tratamento e 61% após 48 h O desenvolvimento intracelular do parasito, analisado por MEV, apresentou características morfológicas comumente encontradas em cistos teciduais. A incubação das culturas com lectina DBA confirmou o desenvolvimento de cistos e por MET se evidenciou a presença de bradizoítos. Além disso, foram revelados parasitos degradados e a influência do composto sobre a endodiogenia. Outra abordagem adotada foi o tratamento de culturas infectadas com a combinação de 3-BrPA e Atovaquona, que resultou em uma redução de parasitos intracelulares de 73% após 24 h de tratamento e 71% após 48 h, em comparação ao controle, além da ausência da formação de parede cística nessas culturas. Conclui-se, portanto, que a utilização de 3-BrPA pode se apresentar como uma importante ferramenta para o estudo:(i) da cistogênese in vitro;(ii)do metabolismo do parasito, necessitando de maior aprofundamento do alvo de ação do composto em T. gondii;(iii) das vias metabólicas alternativas do parasito, e (iv) dos mecanismos moleculares/celulares desencadeados que levaram a morte dos parasitos / Toxoplasma gondii usually causes an asymptomatic infection, but it can present severity during pregnancy and in immunocompromised patients . Current therapies for toxoplasmosis are restricted only against tachyzoites, and have little or no effect on bradyzoites, which are kept in tissue cysts like source of the infection recrudescent. Consequently, new therapeutic alternatives have been proposed, as the use of Atovaquone that showed some effica cy against tachyzoites and bradyzoites in tissue cysts. In this work, we propose to study the effect of 3 - BrPA, a compound that is being tested against cancer cells, on the infection of LLC - MK 2 cells with tachyzoites of T. gondii (RH strain) . N o effect of 3 - BrPA on host cell proliferation and viability was observed. Evaluation of 3 - BrPA interference on in vitro growth of T. gondii showed a reduction in intracellular parasite proliferation about 55% after 24 h of treatment, and 61% after 48 h. Intracellular d evelopment of parasite, analyzed by SEM, showed morphological characteristics commonly found in tissue cysts. Incubation of cultures with DBA lectin confirmed the development of cysts and TEM showed the presence of bradyzoites. Moreover, we revealed the pr esence of degraded parasites and the influence of compound on endodyogeny. Another approach used was the treatment of infected cultures with combination of 3 - BrPA and Atovaquone. This r esulted in the reduction of intracellular parasites of 73% after 24 h o f treatment and 71% after 48 h, compared to control, besides the absence of cyst wall formation in these cultures. The refore, it is concluded that use of 3 - BrPA may pres ent as an important tool for study of: (i) in vitro c ystogenesis , (ii) the metabolism o f the parasite, requiring deeper understanding of the target of action of the compound in T. gondii , (iii) the alternatives metabolic pathways of the parasite, and (iv) the molecular / cellular triggered that led to death of the parasites mechanisms.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas, Programa de Pós-Graduação em Química, Florianópolis, 2016. / Made available in DSpace on 2016-09-20T04:46:13Z (GMT). No. of bitstreams: 1 340702.pdf: 1405165 bytes, checksum: 4cdf6ad32dede31e89f04ee756ab1183 (MD5) Previous issue date: 2016 / Neste trabalho foi desenvolvido dois métodos analíticos para a determinação de Cl e Br por HR-CS MAS em forno de grafite, utilizando pela primeira vez o cloreto de cálcio (CaCl) para a determinação de cloro no comprimento de onda 593,3887 nm e a molécula brometo de estrôncio (SrBr) na região 666,2985 nm através da análise em suspensão. As otimizações dos métodos foram realizadas utilizando soluções aquosas a partir dos compostos NH4Cl e KBr. Como geradores das moléculas foram empregados Ca(NO3)2 e Sr(NO3)2. A plataforma do forno de grafite foi recoberta com modificador permanente W (520 µg) e Pd (10 µg) foi introduzido a cada ciclo de injeção como modificador químico para a determinação dos dois elementos (Cl e Br). Foram otimizadas as temperaturas de pirólise em 1000 °C, e de vaporização em 2300 e 2400 °C para formação de CaCl e SrBr, respectivamente. A otimização das concentrações dos precursores formadores das moléculas, Ca e Sr, foi realizada através de estudos univariado e multivariado, chegando a uma proporção de 1 Cl:2000 Ca e 1 Br:560 Sr, respectivamente. O tratamento das amostras foi proposto por meio de um preparo simples de uma suspensão com a adição de ácido, surfactante e água. Para análise de Br foram utilizados dois materiais de referência certificados, referentes a cinzas volantes (BCR176R) e material particulado urbano (NIST1648a). Foi necessário uma calibração por adição de analito, obtendo-se LOD de 0,2 e 0,09 mg L-1 de Br para BCR176R e NIST1648a, respectivamente. A verificação da exatidão do método para a determinação do Cl foi realizada utilizando dois materiais de referência certificados, referentes a material particulado urbano (NIST1648a), partículas de escape veicular (NIES 8), material de esgoto doméstico (BCR 144R) e duas amostras reais de resíduos ambientais. Foi necessário o uso da calibração por simulação de matriz somente para uma amostra real (material particulado PM10), apresentando LOD de 0,15 mg L-1 de Cl. Para as demais amostras a determinação foi realizada usando uma calibração externa que mostrou-se apropriada com um LOD de 0,03 mg L-1 de Cl. O método foi eficiente apresentando uma boa precisão, com RSD de 3 % para as determinações de Cl e 3 a 9 % para as determinações de Br, além disso demostrou adequada exatidão para a análise proposta.<br> / Abstract: This work developed two analytical methods for the determination of Cl and Br for HR-CS MAS in graphite furnace, first time using calcium chloride (CaCl) for the determination of chlorine at wavelength 593,3887 nm and the molecule strontium bromide (SrBr) at region 666,2985 nm through analysis the suspension. The optimization of the methods were performed using aqueous solutions from compounds NH4Cl and KBr. As generators of molecules were used Ca(NO3)2 and Sr(NO3)2. The graphite furnace platform was covered with permanent modifier W (520 µg) and Pd (10 µg) was added to each injection cycle as a chemical modifier. Temperatures of pyrolysis have been optimized to 1000 ° C, and vaporization to 2300 and 2400 ° C to CaCl and SrBr, respectively. The optimization of the concentration of Ca and Sr, that were precursors of the molecules, it was performed using univariate and multivariate analysis studies. The proportion obtained for generation maximum of CaCl and SrBr molecules were 1Cl: 2000 and 1 Br Ca: Sr 560, respectively. The treatment of the samples was proposed by means of a simple preparation of a suspension by the addition of acid, surfactant and water. For the Br analysis was used two certified reference materials, relating to fly ash (BCR176R) and urban particulate matter (NIST1648a). For the Br determination was required the calibration by adding standard. The LOD for the BCR176R was 0.2 mg L-1 and for the NIST1648a was 0.09 mg L-1. The verification of accuracy of the method for determination of Cl was conducted using three certified reference material (NIST1648a, NIES 8 CRM144R) and two real samples of environmental waste. For one real sample was necessary the use of calibration by matrix simulation (particulate material PM10), showing LOD of 0.15 mg L-1 Cl. For the other CRM and real sample the aqueous calibration proved to be appropriate reaching a LOD of 0.03 mg L-1 of Cl. The method was effective with a good precision with RSD of 3 % to determination of Cl and 3 to 9 % for determination of Br. Furthermore, it showed adequate accuracy for the analysis proposed.

El bromo es un elemento muy reactivo que se encuentra distribuido en la naturaleza principalmente como bromuros, en pequeñas cantidades. El agua de mar es una fuente virtualmente ilimitada de bromo, cuya evaporación da lugar a la formación de salmueras, sustancias complejas y de gran valor económico debido a la gran concentración de sales disueltas. La determinación de trazas de bromuro, especialmente en muestras de composición y naturaleza compleja como el agua de mar, es un problema difícil de encarar. Los métodos convencionales para la cuantificación de bromuros por gravimetría, yodometría, entre otros, no son confiables debido a la compleja composición del agua de mar cuyos iones provenientes de las sales disueltas actúan como interferentes. El método más utilizado hasta hace unos años fue el que corresponde a la Norma ASTM D3869-09: Métodos estándares de ensayo para iones ioduro y bromuro en aguas salobres, agua de mar y salmueras. Sin embargo, desde hace unos años este no es viable debido al uso de tetracloruro de carbono como solvente de extracción al ser un reactivo prohibido debido a su toxicidad y considerado por el Protocolo de Montreal como una de las sustancias que agotan la capa de ozono. La investigación plantea la optimización y validación de un método cuantitativo para evaluar la concentración de bromuros en el rango de 34 a 200 mg/L, basado en el procedimiento descrito en la Norma ASTM D3869-09. Este nuevo método colorimétrico hace uso de la oxidación de iones bromuro a bromo molecular mediante la reacción de iones bromuro con óxido de cromo (VI) en solución ácida; luego el bromo molecular resultante de la reacción anterior, es extraído con disulfuro de carbono analizando su concentración por espectroscopia ultravioleta-visible a 420 nm. Como resultado de esta investigación, se ha comprobado la validez del método propuesto para evaluar la concentración de bromuro en el rango de 34 a 200 mg/L, se han establecido las condiciones específicas para el tratamiento de las muestras y se ha validado el método siguiendo la guía de IUPAC para validaciones realizadas por un solo laboratorio. Se comprobó que el método es robusto, preciso, veraz y que las concentraciones máximas de sales que pueden estar presentes en la muestra sin interferir en la cuantificación de iones bromuro es de de 224 ppm de sulfato de calcio, 292 ppm de cloruro de potasio, 657 ppm de cloruro de sodio, 1527 ppm de sulfato de magnesio y 40 542 ppm de cloruro de magnesio. Además, se obtuvo la incertidumbre expandida del método igual a ± 0.1418 ppm.

The thesis entitled "Synthesis of Novel Chalcogenides using Acyloxyphosphonium Intermediates and Doubly Activated Cyclopropanes" is divided into six chapters. Chapter 1: Part 1: Synthesis of thioesters from carboxylic acids and alkyl halides using benzyltriethylammonium tetrathiomolybdate In this chapter, we describe the synthesis of thioesters from carboxylic acids and alkyl halides. Aryl carboxylic acids are first activated using PPh3 and NBS to form the corresponding acyloxy phosphonium intermediates which then on further reaction with reagent, 1generate thioaroylate ions in situ. These thioaroylates on further reaction with various electrophiles such as alkyl halides / dihalides in the same pot gives the corresponding functionalized thioesters. This methodology was then extended to carbohydrate based thioesters as they are important synthetic intermediates in various transformations and also they could be deprotected later to synthetically more valuable thiols. For this study, we took 1,2,3,4tetra-O-acetyl-β-D-glucopyranuronic acid which on treatment with PPh3,NBS, reagent, 1 and I-bromo propane (CHCl3, 28°C, 2h) afforded the corresponding thioester in 55% yield. An intramolecular version of the reaction was then performed on a compound containing both anomeric bromide and carboxylic acid functionality. This was achieved by treating tetra acetyl glucuronic acid, with HBr/AcOH to form α-D-bromo-glucopyranuronic acid which on further treatment with PPh3, NBS and reagent, 1 gave the corresponding bicyclic thiolactone in 55% yield. Chapter 1: Part 2: Synthesis of Thioesters by Simultaneous Activation of Carboxylic Acids and Alcohols using PPh3/NBS In this chapter, we have shown the synthesis of thioester from carboxylic acids and alcohols. Both carboxylic acids and alcohols are first activated using PPh3 and NBS to form the corresponding phosphonium salts. Reagent, 1 then reacts selectively with acyloxyphosphonium intermediates to generate thioaroylate ions in situ which then react either with alkoxy phosphonium salts or the corresponding alkyl bromide to give thioesters in good yield. The same methodology was then used for a one pot conversion of N-Boc serine ester to s-protected cysteine using reagent 1 as the key sulfur transfer reagent. Chapter 2: Part 1: Tetrathiomolybdate mediated Michael addition of thioaroylates generated from acyloxyphosphonium salts In this chapter, we have reported an easy and alternative protocol for the Michael addition of thioacids to various Michael acceptors. Acyloxyphosphonium salts and tetrathiomolybdate reacts to generate thioaroylate ions which then undergo Michael additionto givethe corresponding Michael adducts. This methodology was then extended for the synthesis carbohydrate based thiolactone by an intramolecular Michael addition reaction to show the applicability of the methodology. Chapter 2: Part 2: Regioselective and chemoselective ring opening of aziridines and epoxides using thioaroylate ions In this chapter, we have demonstrated nucleophilic ring opening of Aziridines and epoxides using thioaroylate ions generated from acyloxyphosphonium salts and tetrathiomolybdate as a sulfur transfer reagent. We have also demonstrated chemoselective ring opening of azirdines in the presence of an epoxide and tosylate to show the novelty of our method. Chapter 3: Synthesis of bromo esters and bromo thioesters by ring opening of cyclic ethers and thiiranes via acyloxyphosphonium intermediates In this chapter, we report the synthesis of bromo esters and thioesters by the ring opening of epoxides, tetrahydrofuran, and thiiranes with bromide ion to form the corresponding bromo alcohols and thiols followed by the nucleophilic displacement of triphenylphosphine oxide from acyloxyphosphonium salts. At first THF and epoxides were subjected for the ring opening reactions to give the corresponding bromo esters. The methodology was then extended to thiiranes to synthesis bromo thioesters in good to moderate yield. Chapter 4: Synthesis of doubly activated cyclopropranes and their applications to the synthesis of dihydrothiophenes and thiophenes In this chapter we discuss the synthesis and ring opening of doubly activated cyc1opropanes using tetrathiomolybdate and their applications towards the formation of dihydrothiophenes and other bioactive molecules. At first, we synthesized a number of doubly activated cyc1opropanes from dimethyl-α-arylsulfonium bromide,24 a protocol developed by Chow and others. With the doubly activated cyclopropanes in hand, we then attempted the ring opening of cyclopropanes containing a cyano group with tetrathiomolybdate to give the corresponding dihydrothiophene derivatives. Also we have used our methodology for the synthesis of HIV-1 reverse transcriptase inhibitor Chapter 5: Synthesis of unsymmetrical sulfide and disulfide derivatives via ring opening of doubly activated cyclopropanes Here, we describe the synthesis of various monosulfides and mixed disulfides by doubly activated cyclopropane ring opening mediated by tetrathiomolybdate in one pot. Tetrathiomolybdate is known for the reduction of disulfides while diaryl disulfides gives monosulfide, dialkyl disulfides give mixed disulfides with the corresponding doubly activated cyclopropane. Thus diaryl disulfide cleaves readily as the resultant thiolate ion is stable and opens the cyclopropane ring to give a monosulfide. Dibenzyl disulfide on the other hand being less reactive gave a mixed disulfide instead of a monosulfide. We also extended this ring opening reactions for the synthesis of symmetrical disulfides Using tetrathiomolybdate as the key sulfur transfer reagent. Chapter 6: A mild protocol for the nucleophilic ring opening of doubly activated cyclopropanes using selenolates generated in situ Nucleophilic ring opening of doubly activated cyc1opropanes with selenolate ions generated by the reduction of diselenides using NaB14 is discussed in this part of the work. A variety of doubly activated cyc1opropanes have been tested for this reaction giving the corresponding selenium compounds in good yield. This methodology was then extended to other diselenides using nitroester cyclopropane as standard and also to other substituted nitroester cyclopropanes using diphenyl diselenide as standard. This methodology was also then extended to the synthesis of homoselenocysteines by the reduction of nitro group using Sn/HCI for the reduction. (For structural formula pl refer the hard copy)

The ochratoxins, metabolites of certain Aspergillus and Penicillium species are the first group of mycotoxins discovered subsequent to the epoch-making discovery of the aflatoxins. Ochratoxin A (OTA) is a very important mycotoxin owing to its frequent occurrence in nature, its established role in Danish porcine nephropathy and in poultry mycotoxicoses and its implicated role in Balkan endemic nephropathy and urinary system tumors among population groups in North Africa. Chapters 2 and 3 highlight the importance of OTA and the research currently being done on mycotoxins. These efforts are focused on the molecular genetics of toxinogenic fungi; the mechanism of their action; species differences in metabolism and pharmacokinetics; quantification of mycotoxins; risk assessments on the exposure of man and animals to mycotoxins and regulations for the control of mycotoxin contamination. Methods developed to analyse OTA in different matrices by using reversed phase high performance-liquid chromatography with fluorescence detection and tandem liquid chromatography-mass spectrometry techniques are described in Chapter 10. Amino propyl solid phase extraction columns were used for the first time in cleanup steps of ochratoxin analysis. These techniques and methods were applied to the first survey on the levels of OTA in coffee on the South African retail market (Chapter 5). The results suggest that the levels of OT A in the coffee on the South African market are somewhat higher than the levels of OTA in coffees on the European market. The possibility to biologically produce different halogen-ochratoxins by supplementing the growth medium of Aspergillus ochraceus with halogen salts was investigated. Bromoochratoxin A was produced for the first time in this way. Supplementation of inoculated wheat with potassium iodide and -fluoride resulted in the poisoning of the yeast and no iodoor fluoro-ochratoxin B was produced. It was found that Aspergillus ochraceus produced OTA in higher yields at elevated levels of potassium chloride. This finding has important commercial applications in the production ofOTA (Chapter 4). The ochratoxins are hydrolyzed in vivo by carboxypeptidase A. The hydrolysis of the ochratoxins and analogues by carboxypeptidase A was measured in vitro in a structurefunction relation study by employing mass spectrometric techniques. The kinetic data of the ochratoxins were compared to the values of a number of synthesized structural analogues. It was found that the halogen containing analogues had lower turnovers than their des-halo analogues. There were no substantial differences in the kinetic data between the different halogen containing analogues (Chapter 8). The toxicokinetics of OTA in vervet monkeys were determined for the first time. The clearance of OTA from the plasma suggested a two-compartment model and the elimination half-life was determined to be 19-21 days. The half-life of OTA in humans was determined by allometric calculations to be 46 days. We came to the conclusion that the long term consumption of OT A contaminated foods will lead to potentially hazardous levels of the toxin in the body (Chapter 9). This hypothesis can be substantiated by the incidence of OTA in the blood of various population groups. Possible ways to decontaminate OT A contaminated foods by degrading the compound biologically with yeast; moulds or lipases to non-toxic compounds were investigated. Eight moulds, 323 yeasts and 23 lipases were screened for ochratoxin degradation. A lipase from Aspergillus niger is the first lipase that was proven to degrade OTA (Chapter 7). Four yeasts were found to degrade OT A of which one, Trichosporon mucoides degraded OTA substantially within 48 hours in a growing culture (Chapter 6). In addition to this first report of yeasts which have the ability to degrade OTA, the fungi Cochliobolus sativus, Penicillium islandicum and Metarhizium anispoliae also proved to degrade OT A. OT A was degraded in all instances to the non-toxic ochratoxin a and the amino acid phenylalanine. / Thesis (PhD (Chemistry))--Potchefstroom University for Christian Higher Education, 2000

The ochratoxins, metabolites of certain Aspergillus and Penicillium species are the first group of mycotoxins discovered subsequent to the epoch-making discovery of the aflatoxins. Ochratoxin A (OTA) is a very important mycotoxin owing to its frequent occurrence in nature, its established role in Danish porcine nephropathy and in poultry mycotoxicoses and its implicated role in Balkan endemic nephropathy and urinary system tumors among population groups in North Africa. Chapters 2 and 3 highlight the importance of OTA and the research currently being done on mycotoxins. These efforts are focused on the molecular genetics of toxinogenic fungi; the mechanism of their action; species differences in metabolism and pharmacokinetics; quantification of mycotoxins; risk assessments on the exposure of man and animals to mycotoxins and regulations for the control of mycotoxin contamination. Methods developed to analyse OTA in different matrices by using reversed phase high performance-liquid chromatography with fluorescence detection and tandem liquid chromatography-mass spectrometry techniques are described in Chapter 10. Amino propyl solid phase extraction columns were used for the first time in cleanup steps of ochratoxin analysis. These techniques and methods were applied to the first survey on the levels of OTA in coffee on the South African retail market (Chapter 5). The results suggest that the levels of OT A in the coffee on the South African market are somewhat higher than the levels of OTA in coffees on the European market. The possibility to biologically produce different halogen-ochratoxins by supplementing the growth medium of Aspergillus ochraceus with halogen salts was investigated. Bromoochratoxin A was produced for the first time in this way. Supplementation of inoculated wheat with potassium iodide and -fluoride resulted in the poisoning of the yeast and no iodoor fluoro-ochratoxin B was produced. It was found that Aspergillus ochraceus produced OTA in higher yields at elevated levels of potassium chloride. This finding has important commercial applications in the production ofOTA (Chapter 4). The ochratoxins are hydrolyzed in vivo by carboxypeptidase A. The hydrolysis of the ochratoxins and analogues by carboxypeptidase A was measured in vitro in a structurefunction relation study by employing mass spectrometric techniques. The kinetic data of the ochratoxins were compared to the values of a number of synthesized structural analogues. It was found that the halogen containing analogues had lower turnovers than their des-halo analogues. There were no substantial differences in the kinetic data between the different halogen containing analogues (Chapter 8). The toxicokinetics of OTA in vervet monkeys were determined for the first time. The clearance of OTA from the plasma suggested a two-compartment model and the elimination half-life was determined to be 19-21 days. The half-life of OTA in humans was determined by allometric calculations to be 46 days. We came to the conclusion that the long term consumption of OT A contaminated foods will lead to potentially hazardous levels of the toxin in the body (Chapter 9). This hypothesis can be substantiated by the incidence of OTA in the blood of various population groups. Possible ways to decontaminate OT A contaminated foods by degrading the compound biologically with yeast; moulds or lipases to non-toxic compounds were investigated. Eight moulds, 323 yeasts and 23 lipases were screened for ochratoxin degradation. A lipase from Aspergillus niger is the first lipase that was proven to degrade OTA (Chapter 7). Four yeasts were found to degrade OT A of which one, Trichosporon mucoides degraded OTA substantially within 48 hours in a growing culture (Chapter 6). In addition to this first report of yeasts which have the ability to degrade OTA, the fungi Cochliobolus sativus, Penicillium islandicum and Metarhizium anispoliae also proved to degrade OT A. OT A was degraded in all instances to the non-toxic ochratoxin a and the amino acid phenylalanine. / Thesis (PhD (Chemistry))--Potchefstroom University for Christian Higher Education, 2000

O consumo lícito e ilícito de derivados anfetamínicos tem aumentado significativamente nos últimos anos, acentuando a necessidade de métodos analíticos adequados para a determinação dessas substâncias pelos laboratórios de toxicologia forense. Este trabalho teve como objetivo o desenvolvimento de métodos cromatográficos para a análise simultânea de anfepramona (DEP), femproporex (FEM), metilfenidato (MPH), 4-bromo-2,5-dimetoxi-anfetamina (DOB) e 4-bromo-2,5-dimetoxi-fenetilamina (2-CB) em amostras não-biológicas. Foram desenvolvidos métodos de detecção por cromatografia a gás com detector de ionização de chama (CG/DIC), de confirmação por cromatografia a gás com detector de espectrometria de massas (CG/EM) e de detecção e quantificação por cromatografia líquida de alta eficiência com detector ultravioleta (CLAE/UV). Os derivados anfetamínicos estudados foram identificados e quantificados simultaneamente através do método CLAE/UV utilizando uma coluna C-18, comprimento de onda 206 nm e modo isocrático. DEP, FEM, MPH e DOB foram detectados simultaneamente por CG/DIC e CG/EM utilizando colunas capilares, sem derivatização e com um tempo de corrida inferior a 10 minutos. Os métodos desenvolvidos foram validados através da avaliação dos parâmetros de linearidade, especificidade, precisão, exatidão, limite de detecção, limite de quantificação e robustez. Os métodos desenvolvidos são de fácil execução, rápidos e adequados para a utilização na rotina laboratorial. / The consumption of both licit and illicit amphetamine derivatives has grown increasingly in recent years, emphasizing the need for analytical methods suitable for identification and quantification of these substances by forensic toxicology laboratories. This aim of this work was to develop and validate methods for the simultaneous chromatographic analysis of amfepramone (diethylpropione, DEP), fenproporex (FEM), methylphenidate (MPH), 4-bromo-2,5-dimethoxyamphetamine (DOB), and 4-bromo-2,5 dimethoxyphenetylamine (2-CB) in non-biological samples. Methods of detection by gas chromatography with flame ionization detector, (GC/FID), confirmation by gas chromatography with mass spectrometry detection (GC/MS), and detection and quantification by high performance liquid chromatography with ultraviolet detection (HPLC/UV) were developed. The amphetamine derivatives studied were identified and quantified simultaneously by HPLC/UV using a RP-C18, 206 nm wavelength and isocratic conditions. DEP, FEM, MPH and DOB were detected both by GC/FID and GC/MS using capillary columns, without derivatization and running times lower than 10 minutes. The validation parameters accessed were linearity, specificity, precision, accuracy, limit of detection, limit of quantification and robustness. The developed methods are easy to implement, fast and suitable for use in routine laboratory analysis.

O consumo lícito e ilícito de derivados anfetamínicos tem aumentado significativamente nos últimos anos, acentuando a necessidade de métodos analíticos adequados para a determinação dessas substâncias pelos laboratórios de toxicologia forense. Este trabalho teve como objetivo o desenvolvimento de métodos cromatográficos para a análise simultânea de anfepramona (DEP), femproporex (FEM), metilfenidato (MPH), 4-bromo-2,5-dimetoxi-anfetamina (DOB) e 4-bromo-2,5-dimetoxi-fenetilamina (2-CB) em amostras não-biológicas. Foram desenvolvidos métodos de detecção por cromatografia a gás com detector de ionização de chama (CG/DIC), de confirmação por cromatografia a gás com detector de espectrometria de massas (CG/EM) e de detecção e quantificação por cromatografia líquida de alta eficiência com detector ultravioleta (CLAE/UV). Os derivados anfetamínicos estudados foram identificados e quantificados simultaneamente através do método CLAE/UV utilizando uma coluna C-18, comprimento de onda 206 nm e modo isocrático. DEP, FEM, MPH e DOB foram detectados simultaneamente por CG/DIC e CG/EM utilizando colunas capilares, sem derivatização e com um tempo de corrida inferior a 10 minutos. Os métodos desenvolvidos foram validados através da avaliação dos parâmetros de linearidade, especificidade, precisão, exatidão, limite de detecção, limite de quantificação e robustez. Os métodos desenvolvidos são de fácil execução, rápidos e adequados para a utilização na rotina laboratorial. / The consumption of both licit and illicit amphetamine derivatives has grown increasingly in recent years, emphasizing the need for analytical methods suitable for identification and quantification of these substances by forensic toxicology laboratories. This aim of this work was to develop and validate methods for the simultaneous chromatographic analysis of amfepramone (diethylpropione, DEP), fenproporex (FEM), methylphenidate (MPH), 4-bromo-2,5-dimethoxyamphetamine (DOB), and 4-bromo-2,5 dimethoxyphenetylamine (2-CB) in non-biological samples. Methods of detection by gas chromatography with flame ionization detector, (GC/FID), confirmation by gas chromatography with mass spectrometry detection (GC/MS), and detection and quantification by high performance liquid chromatography with ultraviolet detection (HPLC/UV) were developed. The amphetamine derivatives studied were identified and quantified simultaneously by HPLC/UV using a RP-C18, 206 nm wavelength and isocratic conditions. DEP, FEM, MPH and DOB were detected both by GC/FID and GC/MS using capillary columns, without derivatization and running times lower than 10 minutes. The validation parameters accessed were linearity, specificity, precision, accuracy, limit of detection, limit of quantification and robustness. The developed methods are easy to implement, fast and suitable for use in routine laboratory analysis.