Sibiromycin is produced by actinomycetes of Streptosporangium sibiricum and structurally belongs to family of pyrrolo-1,4-benzodiazepines. Sibiromycin is characterized by antibacterial and especially antitumor activity but due to its proved cardiotoxicity it cannot be used. The following research of possible therapeutic sibiromycin utilization is concerned on new nontoxic derivatives produced by genetic manipulated strains of S. sibiricum. For this purpose a new routine chromatographic UPLC-UV method with analyte preconcentration was developed and partially validated. A fermentation broth was extracted by solid phase extraction (SPE) on OASIS MCX columns with the sorbent based on a cation-exchange. Eluted extract was evaporated, reconstituted in methanol and loaded onto UPLC BEH C18 column and analyzed under gradient mode with mobile phases of methanol (A) and trifluoroacetic acid (B). Detection limit of the method was determined as 40 ng/ml, the recovery was 74.75 % and its reproducibility expressed as RSD was 5.18 %. The method was applied to comparison of sibiromycin production by S. sibiricum on 13 different fermentation media. It was found, that a composition of fermentation media influences not only the sibiromycin production but also a synthesis of sibiromycin nature derivatives, which were...

Cowpea (Vigna unguiculata) is a highly nutritious, drought tolerant crop with several agronomic advantages over other legumes. This study demonstrated the association of different cowpea phenotypes with specific phenolic profiles, antioxidants activity, anti-inflammatory properties on non-malignant colonic (CCD18co) cells challenged with a lipopolysaccharide (LPS), and the effect of boiling on their individual and total flavonoid content. Only the black and green phenotypes had detectable anthocyanins; and their levels were highest in the black IT95K-1105-5 variety. The red cowpea phenotypes had the highest level of flavonols (858 - 941 micrograms/g cowpea flour) and white Early Acre variety had the least. Quercetin derivatives were the major flavonols detected, followed by myricetin derivatives. Monomeric, dimeric and polymeric procyanidins also made up a large proportion of cowpea phenolics. The light brown 09FCV-CC27M cowpea variety had the highest average flavan-3-ol content (13,464 micrograms/g cowpea flour); whereas white and green varieties did not contain detectable levels of flavan-3-ols. Thus, seed-coat color was a good indicator of the accumulation of specific flavonoids in cowpeas. The black, red and light-brown cowpeas had the highest antioxidant activity measured by ORAC and ABTS methods, correlating with their higher total phenol content (TPC) and condensed tannin content (CTC); whereas the white and green varieties had the least. Boiling significantly affected the phenolic profiles, TPC and CTC of all cowpea varieties studied, as well as the antioxidant activity associated with these compounds. The net reduction in antioxidant activity after boiling was less than the net TPC reduction, indicating that the heat-induced phenolic products may still have radical scavenging capacity. Overall, proinflammatory genes regulation, intracellular ROS inhibition, and modulation of miR-126 and its target gene VCAM-1 by cowpea were found to be dependent on cowpea variety, phenolic composition and concentrations. The underlying mechanism by which cowpea induced miR-126 may be associated with inhibition of ROS and down-regulation of transcription factor NF-These results emphasize the importance of the cancer inhibitory potential of phenolic compounds from cowpea and their possible role in preventing anti-inflammatory disorders. Further in vivo studies with cowpea diets are required to validate their clinical relevance to human health.

The aim with this project was to evaluate Waters AccQTag method which was ananalytical method that Waters Corporation provides for determination of aminoacids. The amino acid analysis was carried out by reversed-phase AQUITY UPLCH-Class, using a BEH C18-column and a PDA detector.Both primary and secondary amino acids were analyzed after precolumnderivatization with the AccQTag reagent kit by a manual procedure and, detected at260 nm. Fresenius Kabis existing products, Aminoven 10 % and Vamin 18EF wereused as samples. The evaluation was based on external calibration curves withprepared standard solutions from Fresenius Kabi, with the addition of the internalstandard L-Norvaline. Waters amino acid standard was used for the system suitabilitytest. A validation according to the ICH guidelines were performed with the followingparameters: specificity, linearity, precision, LOQ, LOD and robustness.Complementary parameters for system suitability test (SST) and stability of standardand reagent were also evaluated. The validation showed that the % RSD values forspecificity, system suitability test and robustness were within the acceptance criteriaof equal or less than 3 % for amino acids. Although the precision were between1.3-7.5 % which was higher than the acceptance criteria of equal or less than 3 %, thiswas likely due to sensitive sample preparation. The linearity was bad which made itimpossible to quantify the method since the calibration curves were below 0.999. Itwas concluded that the system was robust and can be used to analyze amino acids inAminoven 10 % and Vamin 18EF but the preparation step should if possible beautomatized to achieve better precision in the analytical work. Two amino acids,cysteine and histidine, needs to be studied further; cysteine gave three peaks insteadof one and histidine gave a peak split at higher injection volume than 0.5 uL.

LOPES NETO, José Joaquim. Bioacessibilidade e biodisponibilidade de compostos fenólicos de sementes de Triplaris Gardneriana Wedd (Polygonaceae). 2017. 95 f. Dissertação (Mestrado em Bioquímica)- Universidade Federal do Ceará, 2017. / Submitted by Weslayne Nunes de Sales (weslaynesales@ufc.br) on 2017-07-10T12:20:53Z No. of bitstreams: 1 2017_dis_jjlopesneto.pdf: 1657114 bytes, checksum: 3ec381c68cd046900e25b007ed35226a (MD5) / Approved for entry into archive by Weslayne Nunes de Sales (weslaynesales@ufc.br) on 2017-07-11T11:56:07Z (GMT) No. of bitstreams: 1 2017_dis_jjlopesneto.pdf: 1657114 bytes, checksum: 3ec381c68cd046900e25b007ed35226a (MD5) / Made available in DSpace on 2017-07-11T11:56:07Z (GMT). No. of bitstreams: 1 2017_dis_jjlopesneto.pdf: 1657114 bytes, checksum: 3ec381c68cd046900e25b007ed35226a (MD5) Previous issue date: 2017 / Triplaris gardneriana Wedd is a plant species from Brazilian semiarid region with seeds rich in phenolic compounds and high antioxidant capacity.The consumption of fruits and vegetables has been associated to low incidence of chronic diseases caused by oxidative stress. Among the food components capable of combating excess free radicals arethe phenolic compounds, with antioxidant capacity, which have been extensively studied. However, the total quantity of these substances present in foods does not necessarily reflect the bioactive amount absorbed and metabolized by the body. In this context, the present work aimed the development of an ethanolic extract obtained from the seeds of T. gardneriana (EETg), and from this, to evaluate its antioxidant activity through different methodologies, to investigate the bioaccessibility of EETg by determination of phenolic composition before and after in vitro digestion as well as to estimate its indirect bioavailability by chemical analysis of plasma and urine in rodents after oral administration. In general, EETg presented levels of antioxidant activity by DPPH, ABTS.+, FRAP and TBARS tests comparable to those described in specialized literature. The bioaccessibility indexes of phenolic compounds in EETg were 48.65 and 69.28% in the presence and absence of enzymes, respectively. Among the identified phenolics classes, flavonoids, represented by galloylated procyanidins, proved to be more bioaccessible (81.48 and 96.29% in the post-intestinal phase with and without enzymes, respectively). The oral administration in Wistar rats resulted in a significant decrease in plasma total antioxidant capacity (TAC) by FRAP assay 4 h after beginning the experiment. As to urine analysis, an increase in TAC by DPPH and FRAP was observed from 1 and 4 h after administration, respectively. UPLC-QTOF analysis of urine detected 2 metabolites originated from the degradation of phenolic compounds: hippuric acid and phenylacetil glycine. These results suggest that phenolic compounds in T. gardneriana, despite their antioxidant potential, are unstable under gastrointestinal conditions, being flavonoids the components with higher bioaccessibility; besides that, they showed limited bioavailability due to their rapid biotransformation and urinary elimination. / Triplaris gardneriana Wedd é uma espécie vegetal do semiárido brasileiro com sementes ricas em compostos fenólicos e alta capacidade antioxidante. O consumo de frutas e vegetais tem sido associado à baixa incidência de doenças crônicas causadas por estresse oxidativo. Dentre os componentes alimentares capazes de combater o excesso de radicais livres, os compostos fenólicos, de caráter antioxidante, têm sido bastante estudados.Contudo, a quantidade total destas substâncias presente nos alimentos não reflete necessariamente a porção bioativa absorvida e metabolizada pelo organismo. Neste contexto, o presente trabalho objetivou o desenvolvimento de um extrato etanólico obtido a partir das sementes de T. gardneriana(EETg),e a partir deste,caracterizar sua composição fenólica, avaliar sua atividade antioxidante através de diferentes metodologias, além deinvestigar a bioacessibilidade in vitro dos polifenóis presentes em EETg após digestão gastrointestinal simulada e estimar a biodisponibilidade indireta dos mesmos a partir da análise química do plasma e urina em roedores após sua administração oral. Em linhas gerais, EETg apresentou níveis de atividade antioxidante pelos ensaios DPPH, ABTS.+, FRAP e TBARS comparáveis àqueles descritos naliteratura especializada. Os índices de bioacessibilidade de compostos fenólicos em EETg foram de 48,65 e 69,28% na presença e ausência de enzimas, respectivamente. Dentre as classes fenólicas identificadas, os flavonoides, representados porprocianidinas acrescidas de grupos galoil, mostraram-se mais bioacessíveis (81,48 e 96,29% na fase pós-intestinal com e sem enzimas, respectivamente). A administração oral de EETg em ratos Wistar resultou em uma significativa diminuição da capacidade antioxidante total (CAT) do plasma pelo ensaio FRAP 4 h após o início do experimento. Para as amostras de urina, um aumento naCAT pelos testes DPPH e FRAP foi observado a partir de 1 e 4 h após a administração, respectivamente. Análises por UPLC-QTOF daurina detectou 2 metabólitos oriundos da degradação de compostos fenólicos: ácido hipúrico e fenilacetil glicina. Estes resultados sugerem que os compostos fenólicos deT. gardneriana, apesar do seu potencial antioxidante, são instáveis em condições gastrointestinais, sendo flavonoides os componentes com maior bioacessibilidade. Além disto, mostraram biodisponibilidade limitada devido à sua rápida biotransformação e eliminação urinária.

Phytate is the major storage form of phosphorus in seeds of soybeans. Because phytate chelates mineral cations including calcium, iron, and zinc, these mixed salts are often excreted by non-ruminant animals such as humans, swine, poultry, and fish. While this causes iron and zinc deficiencies, phytate is also considered a water pollutant due to the excess phosphorus excreted in animal waste. These negative environmental and nutritional effects, create a need for low phytate soybeans. While several low phytate soybean lines have been developed, a major drawback is the reduced seedling emergence of these lines resulting in low yields. Therefore, understanding the genetic and molecular bases of the low emergence trait in relation to seed phytate content in major crops such as soybean is of great economic importance. This PhD project worked towards the long term goal of developing low phytate soybean cultivars with good seedling emergence and high-yield. This dissertation focused on metabolomic differences between low and normal phytate lines and how these could relate to the low emergence phenotype. The genetic materials used here include four near isogenic lines that differ in mutations in two multi drug resistance-associated proteins (MRPs). Only the line with both mutations was low phytate. The phytate levels, field- and lab-based emergence rates were determined for these lines, their parents and a control line through replicated field experiments for three consecutive years. The emergence rates of the low phytate lines were not always reduced. This showed that the environment the seeds were produced in is highly important, especially when breeding and commercially growing low phytate lines. A protocol was developed for successful metabolomic discrimination of these closely related soybean lines. The polar and non-polar metabolite profiles were determined using ultra performance liquid chromatography mass spectrometry and metabolomic differences between the low and normal phytate lines were identified. The low phytate double mutant did not contain C22 glucose terminated Group A soyasaponins and almost exclusively contained C22 xylose terminated Group A soyasaponins (A4, A5 and A6). Compared to the normal phytate lines, the low phytate soybean line showed a higher concentration of storage lipids (triacylglycerols and diacylglycerols) and certain phospholipids. / Ph. D.

Este trabalho apresenta a aplicação da Cromatografia Líquida de Ultra Performance acoplada à espectrometria de massas (UPLC-MS/MS) como uma técnica analítica em potencial para a determinação de contaminantes orgânicos em amostras de água para consumo humano. A técnica foi aplicada no desenvolvimento de um método analítico para a determinação de 28 praguicidas listados na Portaria 2914/11 como indicadores da qualidade de águas para o consumo humano. O método analítico apresentou limite de quantificação (LQ) (entre 0,2 e 50 g L-1, considerando todas as substâncias investigadas) capaz de monitorar as substâncias de interesse em concentrações inferiores aos limites máximos permitidos pela legislação, com taxas de recuperação superiores a 80% e coeficiente de variação (CV) menor do que 15% no nível de µg L-1. A filtração da amostra, empregando filtros de seringa de 0,22µm de diâmetro de poro, foi empregada como etapa de preparo de amostra, não sendo necessário o emprego de métodos de extração e concentração da amostra previamente à análise. O método analítico proposto foi aplicado em duas amostras de água potável coletadas na cidade de Paulínia-SP; entretanto, os praguicidas investigados não foram detectados em ambas as amostras analisadas. Adições de padrão foram realizadas sobre as amostras no nível de concentração do Valor Máximo Permitido (VMP) e três vezes o VMP pela Portaria 2914/11. Valores de recuperação entre 80 e 120% foram obtidos, demostrando a aplicabilidade e exatidão do método proposto. / This work describes the application of Ultra Performance Liquid Chromatography tandem mass spectrometry (UPLC-MS/MS) for determination of organic contaminants in drinking water. The tecnique was used for the development of an analytical method for the determination of 28 pesticides, listed in the Brazilian Administrative Rule 2914/11, these compounds are considered as quality indicators for drinking waters. The limit of quantification (LQ) of the analytical method was between 0,1 e 5 µg L-1 for all target substances, being able to quantify the target substances at concentrations lower than the maximum levels of contamination (MLC) established by the Rule, with accuracy above 80% and relative standard deviation below 15%, at g L-1 level. The filtration of the sample, using 0.22 µm syringe filters, was applied as sample preparation step. The analytical method based on UPLCMS/MS was used to analyze two drinking water samples collected in Paulínia-SP city. The target pesticides were not detected in any samples. The two samples were then enriched with analytical standards of the pesticides at the MLC and 3 times the MLC levels and analyzed by the proposed method. Recoveries ranged from 80 to 120%, showing the applicability and efficiency of the proposed analytical method.

A extração por solvente é o método mais eficiente para recuperação do óleo residual de fibra prensada de palma (FPP), o qual pode conter um teor de carotenoides até oito vezes superior ao encontrado no óleo de palma bruto. Neste contexto, a escolha de potenciais solventes ou misturas de solventes torna-se essencial, pois elas podem resultar no efeito sinérgico de extração de óleo e carotenoides promovendo uma maior recuperação destes compostos de interesse. Dessa forma, o presente estudo objetivou a utilização de misturas binárias de solventes hidrocarbonetos e alcoólicos, a fim de promover a maior recuperação de um óleo de FPP rico em carotenoides. Para tanto, as composições das misturas compostas por hidrocarboneto (hexano, heptano e ciclohexano) e álcoois de cadeia curta (etanol e isopropanol) foram definidas a partir do cálculo da distância soluto-solvente (Ra) entre as misturas de solventes e o soluto β-caroteno. Os experimentos de extração foram conduzidos a 60 ± 2°C em um intervalo de tempo de 5 horas em extrator em batelada, utilizando os solventes hexano, heptano, ciclohexano, etanol e isopropanol e suas respectivas misturas, e a 55 ± 3 °C e um intervalo de tempo de 1 hora e 30 minutos, em coluna empacotada de leito fixo, utilizando hexano, etanol, isopropanol e suas respectivas misturas. Os resultados dos experimentos de extração, em batelada na razão mássica sólido:solvente de 1:7, indicaram que hexano e a mistura compreendida por heptano e isopropanol, extraíram o maior conteúdo de lipídeos, em torno de 80 % (em massa), enquanto que os demais solventes puros e misturas de solventes permitiram a obtenção de rendimentos de extração de óleo em torno de 70 % (em massa). Apesar dos solventes ciclohexano e hexano apresentarem a maior extração de carotenoides, o teor deste componente apresentou-se levemente inferior quando foram utilizadas as misturas binárias de solventes. Os maiores rendimentos de extração de óleo de FPP em coluna, na razão mássica sólido:solvente de 1:4, deram-se com o emprego de etanol e da mistura compreendida por hexano e isopropanol, em torno de 66 % (em massa), porém o conteúdo de carotenoides obtido foi o mesmo, independentemente do solvente ou misturas de solventes empregadas. O perfil de carotenoides analisados por UPLC/MS apresentou elevado conteúdo de β-caroteno extraído majoritariamente por hexano, enquanto que o maior conteúdo de α-caroteno foi obtido pela mistura de hexano e etanol, por último, etanol extraiu o maior conteúdo de licopeno. Nesta dissertação não foi observado, de forma expressiva, o sinergismo de extração de óleo de FPP rico em carotenoides com o emprego de misturas de solventes definidas a partir da estimativa dos Parâmetros de Solubilidade de Hansen (PSH) e cálculo da Ra. No entanto, é possível inferir os solventes hidrocarbonetos e suas misturas com os álcoois de cadeia curta, etanol e isopropanol, demonstraram boa habilidade em extrair óleo de FPP rico em carotenoides, mantendo sua composição em ácidos graxos e acidez livre relativamente constantes podendo, assim, serem indicados para operações de extração de óleo de FPP. As misturas de solventes empregadas nos experimentos de extração resultaram em considerável rendimento de óleo de FPP, rico em carotenoides totais mostrando que a substituição parcial de solventes hidrocarbonetos por álcoois de cadeia curta é possível. / Solvent extraction is the most efficient method for recovering palm pressed fiber (PPF) residual oil, which may contain up to eight times the carotenoid content of that found in crude palm oil. In this context, the choice of potential solvents or solvent mixtures becomes essential as they may result in the synergistic effect of extracting oil and carotenoids promoting further recovery of these compounds of interest. Thus, the present study aimed at the use of binary mixtures of hydrocarbon and alcoholic solvents, in order to promote the higher recovery of a carotenoid-rich PPF oil. The compositions of the mixtures composed of hydrocarbons (hexane, heptane, and cyclohexane) and short chain alcohols (ethanol and isopropanol) were defined from the calculation of the solute-solvent distance (Ra) between the solvent mixtures and the solute β-carotene. The extraction experiments were conducted at 60 ± 2 °C during 5 hours in a batch extractor, using the hexane, heptane, cyclohexane, ethanol and isopropanol solvents and their mixtures, and at 55 ± 3 °C and a time interval of 1 hour and 30 minutes in a packed bed column using hexane, ethanol, isopropanol and their mixtures. The results of the batch extraction experiments at the solid: solvent mass ratio of 1:7 indicated that hexane and the mixture comprised of heptane and isopropanol extracted the highest lipid content, around 80 % (by mass), while that the other pure solvents and solvent mixtures allowed to obtain oil extraction yields around 70 % (by mass). Although the solvents cyclohexane and hexane had the highest extraction of carotenoids, the content of this component was slightly lower when the binary solvent mixtures were used. The higher yields of oil extraction of PPF in the column in the solid: solvent mass ratio of 1:4 were given by using ethanol and the mixture comprised of hexane and isopropanol, around 66 % (by mass), but the carotenoids content obtained was the same regardless of the solvent or solvent mixtures employed. The profile of carotenoids analyzed in UPLC/MS showed a high content of β-carotene extracted mainly by hexane, while the highest content of α-carotene was obtained by the mixture of hexane and ethanol, finally, ethanol extracted the highest content of lycopene. In this dissertation, the synergism of extraction of PPF oil rich in carotenoids was not observed within the use of solvent mixtures defined from the estimation of Hansen Solubility Parameters (HSP) and calculation of Ra. However, it is possible to infer that the hydrocarbon solvents and their mixtures with the short-chain alcohols, ethanol, and isopropanol, demonstrated good ability to extract of PPF oil rich in carotenoids, maintaining its composition in fatty acids and free acidity constant being indicated for PPF oil extraction operations. The use of the solvent mixtures resulted in a considerable extraction yield of PPF oil, rich in total carotenoids showing that the partial replacement of hydrocarbon solvents by short chain alcohols is possible.

A extração por solvente é o método mais eficiente para recuperação do óleo residual de fibra prensada de palma (FPP), o qual pode conter um teor de carotenoides até oito vezes superior ao encontrado no óleo de palma bruto. Neste contexto, a escolha de potenciais solventes ou misturas de solventes torna-se essencial, pois elas podem resultar no efeito sinérgico de extração de óleo e carotenoides promovendo uma maior recuperação destes compostos de interesse. Dessa forma, o presente estudo objetivou a utilização de misturas binárias de solventes hidrocarbonetos e alcoólicos, a fim de promover a maior recuperação de um óleo de FPP rico em carotenoides. Para tanto, as composições das misturas compostas por hidrocarboneto (hexano, heptano e ciclohexano) e álcoois de cadeia curta (etanol e isopropanol) foram definidas a partir do cálculo da distância soluto-solvente (Ra) entre as misturas de solventes e o soluto β-caroteno. Os experimentos de extração foram conduzidos a 60 ± 2°C em um intervalo de tempo de 5 horas em extrator em batelada, utilizando os solventes hexano, heptano, ciclohexano, etanol e isopropanol e suas respectivas misturas, e a 55 ± 3 °C e um intervalo de tempo de 1 hora e 30 minutos, em coluna empacotada de leito fixo, utilizando hexano, etanol, isopropanol e suas respectivas misturas. Os resultados dos experimentos de extração, em batelada na razão mássica sólido:solvente de 1:7, indicaram que hexano e a mistura compreendida por heptano e isopropanol, extraíram o maior conteúdo de lipídeos, em torno de 80 % (em massa), enquanto que os demais solventes puros e misturas de solventes permitiram a obtenção de rendimentos de extração de óleo em torno de 70 % (em massa). Apesar dos solventes ciclohexano e hexano apresentarem a maior extração de carotenoides, o teor deste componente apresentou-se levemente inferior quando foram utilizadas as misturas binárias de solventes. Os maiores rendimentos de extração de óleo de FPP em coluna, na razão mássica sólido:solvente de 1:4, deram-se com o emprego de etanol e da mistura compreendida por hexano e isopropanol, em torno de 66 % (em massa), porém o conteúdo de carotenoides obtido foi o mesmo, independentemente do solvente ou misturas de solventes empregadas. O perfil de carotenoides analisados por UPLC/MS apresentou elevado conteúdo de β-caroteno extraído majoritariamente por hexano, enquanto que o maior conteúdo de α-caroteno foi obtido pela mistura de hexano e etanol, por último, etanol extraiu o maior conteúdo de licopeno. Nesta dissertação não foi observado, de forma expressiva, o sinergismo de extração de óleo de FPP rico em carotenoides com o emprego de misturas de solventes definidas a partir da estimativa dos Parâmetros de Solubilidade de Hansen (PSH) e cálculo da Ra. No entanto, é possível inferir os solventes hidrocarbonetos e suas misturas com os álcoois de cadeia curta, etanol e isopropanol, demonstraram boa habilidade em extrair óleo de FPP rico em carotenoides, mantendo sua composição em ácidos graxos e acidez livre relativamente constantes podendo, assim, serem indicados para operações de extração de óleo de FPP. As misturas de solventes empregadas nos experimentos de extração resultaram em considerável rendimento de óleo de FPP, rico em carotenoides totais mostrando que a substituição parcial de solventes hidrocarbonetos por álcoois de cadeia curta é possível. / Solvent extraction is the most efficient method for recovering palm pressed fiber (PPF) residual oil, which may contain up to eight times the carotenoid content of that found in crude palm oil. In this context, the choice of potential solvents or solvent mixtures becomes essential as they may result in the synergistic effect of extracting oil and carotenoids promoting further recovery of these compounds of interest. Thus, the present study aimed at the use of binary mixtures of hydrocarbon and alcoholic solvents, in order to promote the higher recovery of a carotenoid-rich PPF oil. The compositions of the mixtures composed of hydrocarbons (hexane, heptane, and cyclohexane) and short chain alcohols (ethanol and isopropanol) were defined from the calculation of the solute-solvent distance (Ra) between the solvent mixtures and the solute β-carotene. The extraction experiments were conducted at 60 ± 2 °C during 5 hours in a batch extractor, using the hexane, heptane, cyclohexane, ethanol and isopropanol solvents and their mixtures, and at 55 ± 3 °C and a time interval of 1 hour and 30 minutes in a packed bed column using hexane, ethanol, isopropanol and their mixtures. The results of the batch extraction experiments at the solid: solvent mass ratio of 1:7 indicated that hexane and the mixture comprised of heptane and isopropanol extracted the highest lipid content, around 80 % (by mass), while that the other pure solvents and solvent mixtures allowed to obtain oil extraction yields around 70 % (by mass). Although the solvents cyclohexane and hexane had the highest extraction of carotenoids, the content of this component was slightly lower when the binary solvent mixtures were used. The higher yields of oil extraction of PPF in the column in the solid: solvent mass ratio of 1:4 were given by using ethanol and the mixture comprised of hexane and isopropanol, around 66 % (by mass), but the carotenoids content obtained was the same regardless of the solvent or solvent mixtures employed. The profile of carotenoids analyzed in UPLC/MS showed a high content of β-carotene extracted mainly by hexane, while the highest content of α-carotene was obtained by the mixture of hexane and ethanol, finally, ethanol extracted the highest content of lycopene. In this dissertation, the synergism of extraction of PPF oil rich in carotenoids was not observed within the use of solvent mixtures defined from the estimation of Hansen Solubility Parameters (HSP) and calculation of Ra. However, it is possible to infer that the hydrocarbon solvents and their mixtures with the short-chain alcohols, ethanol, and isopropanol, demonstrated good ability to extract of PPF oil rich in carotenoids, maintaining its composition in fatty acids and free acidity constant being indicated for PPF oil extraction operations. The use of the solvent mixtures resulted in a considerable extraction yield of PPF oil, rich in total carotenoids showing that the partial replacement of hydrocarbon solvents by short chain alcohols is possible.

Este trabalho apresenta a aplicação da Cromatografia Líquida de Ultra Performance acoplada à espectrometria de massas (UPLC-MS/MS) como uma técnica analítica em potencial para a determinação de contaminantes orgânicos em amostras de água para consumo humano. A técnica foi aplicada no desenvolvimento de um método analítico para a determinação de 28 praguicidas listados na Portaria 2914/11 como indicadores da qualidade de águas para o consumo humano. O método analítico apresentou limite de quantificação (LQ) (entre 0,2 e 50 g L-1, considerando todas as substâncias investigadas) capaz de monitorar as substâncias de interesse em concentrações inferiores aos limites máximos permitidos pela legislação, com taxas de recuperação superiores a 80% e coeficiente de variação (CV) menor do que 15% no nível de µg L-1. A filtração da amostra, empregando filtros de seringa de 0,22µm de diâmetro de poro, foi empregada como etapa de preparo de amostra, não sendo necessário o emprego de métodos de extração e concentração da amostra previamente à análise. O método analítico proposto foi aplicado em duas amostras de água potável coletadas na cidade de Paulínia-SP; entretanto, os praguicidas investigados não foram detectados em ambas as amostras analisadas. Adições de padrão foram realizadas sobre as amostras no nível de concentração do Valor Máximo Permitido (VMP) e três vezes o VMP pela Portaria 2914/11. Valores de recuperação entre 80 e 120% foram obtidos, demostrando a aplicabilidade e exatidão do método proposto. / This work describes the application of Ultra Performance Liquid Chromatography tandem mass spectrometry (UPLC-MS/MS) for determination of organic contaminants in drinking water. The tecnique was used for the development of an analytical method for the determination of 28 pesticides, listed in the Brazilian Administrative Rule 2914/11, these compounds are considered as quality indicators for drinking waters. The limit of quantification (LQ) of the analytical method was between 0,1 e 5 µg L-1 for all target substances, being able to quantify the target substances at concentrations lower than the maximum levels of contamination (MLC) established by the Rule, with accuracy above 80% and relative standard deviation below 15%, at g L-1 level. The filtration of the sample, using 0.22 µm syringe filters, was applied as sample preparation step. The analytical method based on UPLCMS/MS was used to analyze two drinking water samples collected in Paulínia-SP city. The target pesticides were not detected in any samples. The two samples were then enriched with analytical standards of the pesticides at the MLC and 3 times the MLC levels and analyzed by the proposed method. Recoveries ranged from 80 to 120%, showing the applicability and efficiency of the proposed analytical method.

A zidovudina, conhecida como AZT, é um inibidor da transcriptase reversa, enquanto que a lamivudina é um fármaco antirretroviral que atua na inibição da síntese de ácidos nucléicos. Estes são dois dos 21 fármacos componentes dos medicamentos distribuídas pelo Ministério da Saúde Brasileiro em programas de combate a Síndrome da imunodeficiência Adquirida (Acquired Immunodeficiency Syndrome - AIDS), configurando-se assim uma grande demanda de produção de medicamentos com estes fármacos. Programas de Tecnologia Analítica em Processo (Process Analytical techology - PAT), embasadas por avanços nos guias internacionais da Conferência Internacional sobre a Harmonização dos Requerimentos Técnicos para o Registro de Produtos Farmacêuticos para o uso Humano (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use - ICH) e pela agência norte-americana para a Administração de Alimentos e Medicamentos (Food and Drugs Administration - FDA), estão ganhando força como alternativas para aumentar a eficiência e a segurança na produção de medicamentos, tanto para aqueles já em processo produtivo como também para medicamentos em fase de desenvolvimento. Estes últimos são denominados desenvolvimento em programas de Qualidade por Design (QbD). Métodos de quantificação por espectroscopia (NIR, MID, RAMAM, entre outras) são reconhecidos como ferramentas para a PAT. Neste contexto propôs-se comparar objetivamente o método tradicional de quantificação destes dois fármacos frente a um método de quantificação desenvolvido utilizando-se a espectroscopia no infravermelho médio (MID). Prepararam-se assim 41 amostras de calibração e 23 amostras de validação, compostas por misturas de zidovudina, lamivudina e placebo (qs) em escala laboratorial, na faixa de 80 a 120% da concentração nominal de uma associação comercial dos dois fármacos. As concentrações de referência de todas as preparações foram determinadas empregando-se o método de referência por Cromatografia Líquida de Alta Eficiência (CLAE) da Farmacopeia Americana (United States Pharmacopeia - USP). Subsequentemente, obtiveram-se cinco espectros no infravermelho de cada uma das preparações, na faixa de 450 a 4000 cm-1. Os espectros foram então pré-processados e utilizados para a construção de um modelo de calibração multivariado por PLS (mínimo quadrados parciais), de acordo com a ASTM E1655-05. Adicionalmente, o método de CLAE foi transferido para um método de UPLC de acordo com o Capitulo Geral descrito no volume 37(3) do Fórum da USP (United States Pharmacopeia). O desempenho do método MID foi então comparado com o método tradicional, bem como com o novo método de quantificação por UPLC. Foram definidaLs assim regiões de confiança para embasar a utilização dos métodos desenvolvidos. O método de quantificação por MID apresentou uma grande variabilidade enquanto que o método por UPLC foi totalmente comparável com o método tradicional, reduzindo o tempo de corrida de 60 minutos para 12.55 minutos. / Zidovudine, also known as AZT is a reverse transcriptase inhibitor, whereas lamivudine is an antiretroviral drug that acts on the inhibition of nucleic acid synthesis. These are two of the 21 active ingredients components of medicines distributed by Brazilian Health Ministry in programs against the Acquired Immunodeficiency Syndrome (AIDS), becoming thus a great demand for production of these two drugs. Process Analytical Technology (PAT) programs, supported by advances in international guides from the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and by the FDA (Food and Drugs Administration), are gaining momentum as alternatives to increase efficiency and safety in the production of medicines, both for those medicines already in the production process as well as to those medicines under development. The latter are called Quality by Design (QbD) programs. Spectroscopy quantification methodologies methodologies (NIR, MID, Ramam, among others) are recognized as PAT tools. In this context it was proposed to compare objectively the traditional method for quantification of these two drugs against a quantification method developed using the MID (middle infrared spectroscopy). Thus 41 calibration and 23 validation samples, comprising of laboratorial scale mixtures of lamivudine, zidovudine and placebo (qs), were prepared in the range equivalent to 80 to 120% of the nominal concentration of the commercial tablets product. The concentrations of all calibration and validation samples were determined using the HPLC reference method of USP (United States Pharmacopeia). Subsequently, there were obtained five infrared spectra of each of the preparations in the range 450-4000 cm-1. The spectra were then pre-processed and used to build a multivariate calibration model for PLS (Partial Least Squares) according to ASTM E1655-05. Additionally, the HPLC method was transferred to a UPLC method according to General Chapter described in volume 37 (3) Forum USP (United States Pharmacopeia). The performance of the method MID was then compared with the traditional method and with the new method of quantification by UPLC. Confidence regions were built to support the use of the methods developed. The MID quantification method presented considerable variability, while the method the UPLC method was fully comparable to the traditional method. Another advantage of the UPLC method was the reduction of running time from 60 minutes to 12:55 minutes.