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DEVELOPMENT OF ANALYTICAL METHODS AND REFERENCE MATERIALS FOR CYANOBACTERIAL TOXINSHollingdale, Christie 16 May 2013 (has links)
Cyanobacterial toxins present a real and growing threat to humans and animals due to the projected increases in algal blooms resulting from increasing global temperature and pollution. Wild animals, livestock, pet animals and humans can be poisoned from contaminated drinking water. With the discovery of cyanobacterial toxins present in nutritional supplements, a new concern looms over consumers with threats of neurotoxin and hepatotoxin related damage from exposure to these products. To this end, work on the development of a freeze dried algal reference material was pursued for future use in environmental and nutritional supplement analysis. The first stage of the project was to prepare needed calibration standards, starting with homoanatoxin a, a homologue of the highly neurotoxic anatoxin-a compound. The resulting reference material (RM-hATX) had a homoanatoxin-a concentration of 20.2 ± 0.7 ?M, and proved to be stable while stored at temperatures of 80°C. Reference samples for dihydro and epoxy analogues of anatoxin-a and homoanatoxin-a were then prepared by semi-synthesis. The second stage of the project was the development of new analytical methods for the anatoxins. A derivatization reaction in which dansyl chloride was coupled with a novel cleanup step produced anatoxin derivatives suitable for liquid chromatography (LC) with mass spectrometry (MS) or fluorescence detection (FLD). Limits of quantitation were 60 ng L-1 and 1.6 ?g L-1 for the developed LC-MS/MS and LC-FLD methods, respectively, with the limit of quantitation significantly better than that of a previously developed method for the underivatized toxins based on HILIC MS/MS. Quantitative results for anatoxins in various algal samples using all three methods of analysis of were compared and it was found that there were no significant differences between the three methods. Unfortunately, experiments showed that the various toxin analogues did not elicit equimolar responses in either LC-MS/MS or LC FLD, thus indicating the importance of having individual calibration standards for quantitative analysis. The LC-MS/MS and LC-FLD methods were paired with a previously developed method for the analysis of hepatotoxic microcystins to screen a small number of nutritional supplement samples for cyanobacterial toxins. Microcystins were detected in all five Aphanizomenon flos-aquae samples examined. This method involved a fifteen-fold pre-concentration using a solid phase extraction cartridge, which gave a 98% recovery of microcystins. The third phase of the project was the preparation and testing of a preliminary algal matrix reference material as a feasibility study for the eventual production of a CRM. After selecting various algal cultures and samples that could be blended together, a freeze dried algal reference material was prepared and packaged. This material (RM-BGA) was then characterized using several methods including the two new dansylation-based procedures.
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Desenvolvimento de método cromatográfico para quantificação dos níveis de poliaminas e investigação da formação de adutos com naftoquinonasFigueiredo, Eugenia Abrantes de 28 March 2014 (has links)
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Previous issue date: 2014-03-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Polyamines are aliphatic organic bases belonging to the group of bioactive amines that have important metabolic and physiological functions in animals, plants and microorganisms. Amongst the most important polyamines biologically are spermidine [N-(3-aminopropyl) -1,4-butane diamine or tetramethylene diamine-aminopropyl] (SPD) and spermine [N, N'-bis (3-aminopropyl) -1,4-butane diamine or diaminopropil-tetramethylethylenediamine] (SPM). Polyamines are intimately involved in the growth and replication of many cell types, and it has been demonstrated that they are found in high concentrations in rapid growing cells. It is possible that substances such as naphthoquinones, which can react with polyamines have the ability to block its function by depleting its concentration. The direct detection of polyamines by HPLC with UV detection is hampered because they lack structural groups with high molar absorptivity. Thus, the objective of this study was to develop and validate a chromatographic method using fluorescence detection that could be applied for quantifying the cellular levels of polyamines in cell cultures based on the following parameters: selectivity, linearity, precision and accuracy. The polyamines were derivatized pre-column with dansyl chloride and were detected by fluorescence (ex= 365 nm; em= 510 nm) using a chromatographic method using gradient elution with a mobile phase consisting of methanol: water (35-95 v/v), a reversed-phase C18 column (250 x 4.6 mm, 5μm) at a flow rate of 1.0 mL/min., in 35 minutes. The chromatographic method was validated according to Resolution 899 recommended by the National Sanitary Agency of Brazil (ANVISA), and was linear in the concentration range used for SPD (0.0097 to 0.97 pmol/mL) and SPM ( 0.074 to 0.74 pmol/mL), precise (≤ 15% for medium and high concentrations, ≤ 20% for the low concentration), exact for SPD (97-118%) and SPM (92-119%), It also showed selectivity (demonstrated by separation of polyamines and proteins present in cell culture medium). The developed method was also applied to the analysis of polyamines incubated with the naphtoquinones (lapachol and β-lapachone). The concentration of these polyamines was depleted when incubated for 17 hours at ambient temperature (decrease of 67% and 50% for SPD and SPM levels respectively when incubated with lapachol and a decrease of 44% and 37,5% for SPD eand SPM when incubated with β-lapachona). These results demonstrate that the developed method is adequate for the analysis of polyamines in cell culture and that a probable mechanism of antitumoral action for the naphtoquinones may involve the formation of adducts with polyamines / Poliaminas são bases orgânicas alifáticas pertencentes às aminas bioativas que desempenham importantes funções metabólicas e fisiológicas em animais, vegetais e microrganismos. As mais importantes biologicamente são a espermidina [N-(3-aminopropil)-1,4-butano diamina ou aminopropil-tetrametilenodiamina] (SPD) e a espermina [N,N -bis(3-aminopropil)-1,4-butano diamina ou diaminopropil-tetrametilenodiamina] (SPM). Estão intimamente envolvidas no crescimento e replicação de diversos tipos de células, e em especial encontra-se em altas concentrações em células exibindo crescimento rápido. É possível que substâncias tais como as naftoquinonas, que possam reagir com as poliaminas, tenham a capacidade de bloquear as suas funções pela depleção de sua concentração. A sua detecção direta é dificultada por não possuírem grupos que apresentam alta absortividade molar. Desse modo, o objetivo deste trabalho foi desenvolver e validar um método cromatográfico, utilizando detecção por fluorescência que possa ser aplicado a quantificação do nível celular de poliaminas em cultura de células tumorais com base nos seguintes parâmetros: seletividade, linearidade, precisão e exatidão e investigar a formação de adutos com naftoquinonas. Para tanto derivatizou-se as poliaminas fora do sistema cromatográfico (pré-coluna) com cloreto de dansila, formando derivados dansilados que foram detectados por fluorescência (ex= 365 nm; em= 510 nm). A separação se deu por eluição em modo gradiente, utilizando como fase móvel metanol:água (35-95 v/v), uma coluna de fase reversa C-18 (250 x 4,6 mm, 5μm) a um fluxo de 1,0 mL/min, em 35 minutos. O método cromatográfico desenvolvido foi validado segundo o preconizado na Resolução 899 da Agência Nacional de Vigilância Sanitária (ANVISA), e mostrou-se sensível, linear na faixa de concentração utilizada para SPD (97-970 pmol/mL) e para SPM (74-740 pmoL/mL), preciso (≤ 15% para as concentrações média e alta; ≤ 20% para a concentração baixa), exato para SPD (91-112%) e para SPM (92-119%), mostrando-se também seletivo (frente a separação das poliaminas e as proteínas presentes no meio de cultivo celular). O método desenvolvido foi ainda aplicado à análise das poliaminas incubadas com as naftoquinonas (lapachol e β-lapachona), e observou-se que a concentração dessas aminas foi depletada quando incubadas por 17 horas a temperatura ambiente com as naftoquinonas (diminuição de 67% e 50% dos níveis de SPD e SPM respectivamente quando incubadas com o lapachol; e diminuição de 44% e 37,5% de SPD e SPM quando incubadas com β-lapachona). Esses resultados mostram que o método desenvolvido é adequado para análise de poliaminas em cultura de células tumorais e que um provável mecanismo de ação antitumoral das naftoquinonas pode envolver a formação de adutos entre estas e as poliaminas.
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Determination of fumonisins in maize by High Performance Liquid Chromatography with fluorescence and ultraviolet detection of o-phthaldialdehyde, naphthalene-2,3-dicarboxaldehyde and dansyl chloride derivativesNdube, Ncediwe January 2011 (has links)
Fumonisins, carcinogenic mycotoxins produced by various Fusarium species, occur naturally in maize and maize-based food products. They are hazards for animal and human health as they cause cancer in rodents and have been associated with oesophageal cancer and neural tube defects in humans. The most abundant naturally occurring fumonisins analogues in maize are fumonisin B1, B2 and B3 (FB1, FB2 and FB3). For analytical determination, they mostly require suitable extraction, clean-up and pre or post-column derivatization together with reversed-phase HPLC separation. o- Phthaldialdehyde (OPA) had been adopted as the most widely used derivatization reagent for fumonisins as they lack useful chromophores or fluorophores. Alternative derivatization reagents, naphthalene-2,3- dicarboxaldehyde (NDA) and dansyl chloride (DnS-Cl), were investigated in this study
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Determination of fumonisins in maize by High Performance Liquid Chromatography with fluorescence and ultraviolet detection of o-phthaldialdehyde, naphthalene-2,3-dicarboxaldehyde and dansyl chloride derivativesNdube, Ncediwe January 2011 (has links)
Fumonisins, carcinogenic mycotoxins produced by various Fusarium species, occur naturally in maize and maize-based food products. They are hazards for animal and human health as they cause cancer in rodents and have been associated with oesophageal cancer and neural tube defects in humans. The most abundant naturally occurring fumonisins analogues in maize are fumonisin B1, B2 and B3 (FB1, FB2 and FB3). For analytical determination, they mostly require suitable extraction, clean-up and pre or post-column derivatization together with reversed-phase HPLC separation. o- Phthaldialdehyde (OPA) had been adopted as the most widely used derivatization reagent for fumonisins as they lack useful chromophores or fluorophores. Alternative derivatization reagents, naphthalene-2,3- dicarboxaldehyde (NDA) and dansyl chloride (DnS-Cl), were investigated in this study
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Determination of fumonisins in maize by High Performance Liquid Chromatography with fluorescence and ultraviolet detection of o-phthaldialdehyde, naphthalene-2,3-dicarboxaldehyde and dansyl chloride derivativesNdube, Ncediwe January 2011 (has links)
Masters of Science / Fumonisins, carcinogenic mycotoxins produced by various Fusarium species, occur naturally in maize and maize-based food products. They are hazards for animal and human health as they cause cancer in rodents and have been associated with oesophageal cancer and neural tube defects in humans. The most abundant naturally occurring fumonisins analogues in maize are fumonisin B1, B2 and B3 (FB1, FB2 and FB3). For analytical determination, they mostly require suitable extraction, clean-up and pre or post-column derivatization together with reversed-phase HPLC separation. o- Phthaldialdehyde (OPA) had been adopted as the most widely used derivatization reagent for fumonisins as they lack useful chromophores or fluorophores. Alternative derivatization reagents, naphthalene-2,3- dicarboxaldehyde (NDA) and dansyl chloride (DnS-Cl), were investigated in this study. / South Africa
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