O Capítulo 1 descreve a produção de celulases por 4 linhagens fúngicas de ambiente marinho (Aspergillus sydowii CBMAI 934, A. sydowii CBMAI 935, Penicillium citrinum CBMAI 1186 e Mucor racemosus CBMAI 847) e uma linhagem de ambiente terrestre (Aspergillus sp. CBMAI 1198) cultivados em meio sólido composto por farelo de trigo (5 g) e solução de peptona (0,75 g.L-1) enriquecida com sais inorgânicos. Foram realizadas otimizações da temperatura, pH inicial e umidade do meio de cultura das linhagens obtendo-se maiores atividades celulolíticas na faixa de temperatura entre 25-35 °C, com exceção do fungo A. sydowii CBMAI 935 que foi de 40 °C, e valores diferentes de pH ótimo, desde condições acídicas até alcalinas, bem como valores diferentes de teor de umidade ótima. Quando avaliou-se a influência do pH, da temperatura e do volume de extrato enzimático durante a hidrólise do papel de filtro cada conjunto de celulases produzidas apresentou pontos ótimos diferentes entre elas, e em alguns casos, dois valores ótimos de pH e temperatura. As celulases produzidas nas condições ótimas determinadas foram aplicadas na hidrólise da celulose do bagaço da cana-de-açúcar pré-tratado usando-se 10 U FPU/g de bagaço de cana-de-açúcar. As celulases dos fungos Aspergillus sp. CBMAI 1198 e A. sydowii CBMAI 934 apresentaram a maior capacidade para hidrolisar o bagaço da cana-de-açúcar pré-tratado, 75% e 78% de degradação do material lignocelulósico, respectivamente. No Capítulo 2 foi avaliada a capacidade de 6 bactérias isoladas de turfeira (Bacillus subtilis LQOB-SE1, B. coagulans LQOB-SE2, B. pumilus LQOB-SE3, Brevibacillus brevis LQOB-SE4, Lysinibacillus sp. LQOB-SE5 e Serratia marcescens LQOB-SE6) em produzir 2,3-butanodiol a partir da fermentação de glicerol e a bactéria que apresentou tal capacidade (S. marcescens LQOB-SE6) foi usada para produzir 2,3-butanodiol também a partir da fermentação de glicose visando o reaproveitamento dos resíduos gerados na produção de biodiesel e de etanol. As melhores condições para o uso do glicerol foram: pH inicial 7, Caldo nutriente 8 g.L-1, concentração inicial de glicerol 50 g.L-1 e tempo de cultivo de 7 dias. Foram obtidos bons rendimento (0,30 g.g-1), produtividade (0,13 g.L-1.h-1) e concentração máxima de 2,3-butanodiol (22,4 g.L-1). As melhores condições para a fermentação da glicose foram: pH inicial 7, Caldo nutriente 8 g.L-1, concentração inicial de glicose 75 g.L-1 e tempo de cultivo de 5 dias. Obteve-se um rendimento de 0,42 g.g-1 em 5 dias de fermentação, produtividade de 0,45 g.L-1.h-1 após 2 dias e concentração máxima de 2,3-butanodiol de 31,2 g.L-1. A produção de 2,3-butanodiol a partir do hidrolisado gerado na hidrólise do bagaço de cana-de-açúcar pelas celulases do fungo de ambiente marinho A. sydowii CBMAI 934 não foi observada devido à baixa concentração de açúcares no hidrolisado. Os resultados obtidos nesta tese mostram o potencial biotecnológico da microbiota fúngica e bacteriana isoladas de diferentes biomas brasileiros. / In Chapter 1 it is reported the cellulase production by 4 marine-derived fungi strains (Aspergillus sydowii CBMAI 934, A. sydowii CBMAI 935, Penicillium citrinum CBMAI 1186 and Mucor racemosus CBMAI 847) and 1 terrestrial fungi strain (Aspergillus sp. CBMAI 1198). They were grown in solid state fermentation using wheat straw as substrate (5 g) and with addition of peptone solution (0,75 g.L-1) enriched with inorganic salts. It was performed the enhancement of the growth conditions by changing the temperature, initial pH and moisture. The optimum temperature for all strains varied between 25-35 °C but A. sydowii CBMAI 935 with 40 °C. The optimum pH was different for each strain, varying from acidic to alkaline conditions. The optimum moisture content also varied accordingly the studied strain. In order enhance the cellulose hydrolysis performed by the produced cellulases, it was varied the pH, temperature and amount of the crude cellulase extract during the filter paper hydrolysis reaction. The obtained optimum values were different among strains and, in some cases, there were two optimum pH and temperature for the hydrolysis of the filter paper. Then, the obtained cellulases, using the best conditions for hydrolysis, were used in the sugarcane bagasse hydrolysis (10 FPU/g of sugarcane bagasse). The cellulases from the strains Aspergillus sp. CBMAI 1198 and A. sydowii CBMAI 934 were capable of degrading 75% and 78% of the sugarcane bagasse, respectively, generating reducing sugars. In Chapter 2, the capability of 6 strains (Bacillus subtilis LQOB-SE1, B. coagulans LQOB-SE2, B. pumillus LQOB-SE3, Brevibacillus brevis LQOB-SE4, Lysinibacillus sp. LQOB-SE5 and Serratia marcescens LQOB-SE6), isolated from peat soil, of producing 2,3-butanediol from glycerol fermentation. The only strain that produced 2,3-butanediol was S. marcescens LQOB-SE6, which was also applied in 2,3-butanediol production from glucose fermentation. Therefore, wastes from biodiesel and bioethanol production can be reused in industrial scale. The best conditions for glycerol fermentation: initial pH 7, Nutrient Broth (8 g.L-1), initial glycerol concentration (50 g.L-1) and fermentation time of 7 days. It were obtained good yield (0.30 g.g-1), productivity (0.13 g.L-1.h-1) and 2,3-butanodiol concentration (22.4 g.L-1). The best conditions for glucose fermentation: initial pH 7, Nutrient Broth (8 g.L-1), initial glucose concentration (75 g.L-1) and fermentation time of 5 days. It were also obtained good yield (0.42 g.g-1) and 2,3-butanodiol concentration (31.2 g.L-1) after 5 days and productivity (0.45 g.L-1.h-1) after 2 days. The 2,3-butanediol production from the hydrolysate of sugarcane bagasse, obtained by using cellulases from A. sydowii CBMAI 934, was not observed due the low sugar concentration in the hydrolysate.

Cultivars of Agrostis stolonifera showed weak and strong responsiveness to the systemic acquired resistance (SAR) activator, benzothiadiazole (BTH), or the induced systemic resistance (ISR) activator, 2R, 3R-butanediol (BD). Next Generation RNA sequencing was used to identify 2163 putative transcripts with increased expression in BTH versus water-treated A. stolonifera. Among three BTH-induced genes, AsASP-2 and AsHIR-1 were induced faster, while AsLOX-1 had stronger transient induction, in one out of two strongly BTH-responsive cultivars. Three ISR-responsive genes, AsGNS-5, AsOPR-4 and AsAOS-1, showed no greater induction or priming in the strongly versus weakly BD-responsive cultivars. Cultivars of A. stolonifera vary significantly in their response to defense activators, however this is not consistently related to defense gene expression. To quantify disease severity, chlorophyll fluorescence imaging of the maximum quantum efficiency of photosystem II (Fv/Fm) was tested on Nicotiana benthamiana infected with Colletotrichum orbiculare. Leaf areas of healthy, non-necrotic affected and necrotic tissue could be individually quantified, which demonstrated that BD delayed symptom development by approx. 24-hour and reduced non-necrotic affected tissue compared to controls. Chlorophyll fluorescence imaging can quantify and reveal novel features about induced disease resistance.

Thermoset polymers are widely used polymers in the world, but Increase in global plastic pollution and lack of fossil fuel stimulates intense research towards environmentally sustainable materials. Bio-based unsaturated polyesters (UPs) would be an excellent solution to replace oil-based synthetic polyesters. Most of the unsaturated polyesters have been synthesised by ring opening polymerisation (ROP) of cyclic esters or lactides.  In this study, different resins were developed using different initiators such as isosorbide (IS),1,4 butanediol (BD), and cis-2 butene 1,4 diol (C2BD) with monomers like lactide (L) and alpha angelica lactone (AAL) through the ring opening polymerisation process. The produced resins were further characterised by using Fourier Transform Infrared Spectroscopy (FTIR),Nuclear Magnetic Resonance (NMR), Thermogravimetric Analysis (TGA), Differential Scanning Calorimeter (DSC), and Dynamic Mechanical Analysis (DMA).  Synthesis of resin with lactone monomer was not successful while with lactide monomer it was successful. IS-based resin showed better thermal properties compared to other obtained resins. Tg value of IS containing resin was 63°C, thermal stability up to 235°C and Storage modulus about 3841 MPa. These values are comparable with other bio-based resins produced using the same monomer.

Neste trabalho foi avaliada a possibilidade do emprego de caldo de cana-de-açúcar (Saccharum officinarum) como matéria-prima para obtenção de 2,3-butanodiol por fermentação, utilizando a bactéria Klebsiella pneumoniae NRRL B199. A pesquisa compreendeu o teste de diferentes nutrientes para a composição de um meio de fermentação eficiente com o caldo de cana e experimentos em que a influência da concentração inicial de sacarose (S0) sobre o processo em regime descontínuo foi estudada. Ensaios realizados em frascos agitados, com S0 entre 140 e 150 g/L, mostraram que a fermentação de caldo de cana clarificado não suplementado com nutrientes não é viável. Por outro lado, a adição de 5,0 a 10,0 g/L de fosfato de amônio ao caldo levou a concentrações finais de 2,3-butanodiol de até 64 g/L e rendimentos, em relação ao máximo teórico, da ordem de 81% em cerca de 38 horas. Estes resultados se aproximam daqueles alcançados na fermentação do caldo enriquecido com vários nutrientes, usado como referência, na qual foram produzidos 66,7 g/L de diol com um rendimento de 85,6%. A utilização de concentrações de fosfato de amônio de até 4,0 g/L resultou em fermentações incompletas. A cinética da fermentação foi estudada com o caldo diluído a, aproximadamente, 180 g/L de sacarose e suplementado com 8,0 g/L de fosfato de amônio e, também, com o meio rico, em regime descontínuo em fermentador de bancada. Foram obtidos, nestes ensaios, concentrações de butanodiol de 71,7 e 71,1 g/L, rendimentos de 78,0 e 74,1 %, produtividades de 2,1 e 2,0 g/Lh e tempos de fermentação de 34,5 e 35,7 horas, respectivamente. A principal diferença foi observada na concentração de biomassa que atingiu 9,1 g/L, no meio com fosfato, e 11,8 g/L no meio rico. As máximas velocidades específicas de crescimento (µx, m), 0,61 e 0,45 h-1, observadas com oxigênio dissolvido acima de zero, indicam que o crescimento celular, no meio rico, foi inibido pela alta concentração de nutrientes. Na seqüência das fermentações, com concentrações nulas de oxigênio dissolvido, a produção de diol foi iniciada e as máximas velocidades específicas de formação de produto (µP,m), 0,53 h-1 com fosfato de amônio e 0,45 h-1 com o meio rico, foram atingidas, sugerindo que altas concentrações de nutrientes afetam, também, a formação de produto. Nos dois ensaios foi observada a ocorrência de uma fase estacionária de crescimento que, no caso do meio contendo fosfato de amônio, teve uma duração 12 horas mais longa devido a um insuficiente suprimento de oxigênio ou do esgotamento de algum nutriente. Neste ponto, foi verificada uma queda das velocidades específicas de formação de produto (µP). Com o caldo diluído a cerca de 20 g/L de sacarose e 8,0 g/L de fosfato de amônio, a fermentação transcorreu em um tempo muito longo (23,5 horas) para a baixa concentração de sacarose empregada. Com S0 semelhante e o meio rico, o tempo de processo foi de apenas 6,5 h, o que demonstra que, no primeiro caso, ocorreu uma diluição excessiva dos nutrientes naturalmente presentes no caldo. O estudo do efeito da concentração inicial de sacarose sobre a fermentação, conduzido em fermentador de bancada e com o meio rico em nutrientes, mostrou que este parâmetro influencia fortemente o crescimento celular e a produção de 2,3-butanodiol. O aumento de S0 levou a valores decrescentes dos fatores de conversão de substrato em células, enquanto os rendimentos em diol aumentaram com S0 até 152 g/L. Valores crescentes de S0 provocaram uma inibição do crescimento celular, evidenciada pela diminuição de µx,m de 0,91 a 0,45 h-1, com S0 entre 24 e 181 g/L. Valores aproximadamente constantes de µP,m (0,62 a 0,68 h-1) foram observados com S0 até 152 g/L, enquanto uma queda acentuada foi verificada com S0=181 g/L (µP,m=0,45 h-1). As máximas produtividades (3,2 g/Lh) foram obtidas com valores de S0 de 105 e 152 g/L. Os dados gerados neste trabalho permitem concluir que caldo de cana-de-açúcar é uma matéria-prima adequada para a produção de 2,3-butanodiol por Klebsiella pneumoniae. / The use of sugar cane (Saccharum officinarum) juice, as raw material for the fermentative production of 2,3-butanediol by Klebsiella pneumoniae NRRL B199, was evaluated in this work. The research was focused on testing different nutrients to formulate an efficient fermentation medium with the juice and some experiments to study the effect of the initial sucrose concentration (S0) on the batch process. Shaker flasks experiments with S0 between 140 and 150 g/L have shown that the fermentation of sugar cane juice without added nutrients is not viable. On the other hand, the addition of 5.0 to 10.0 g/L ammonium phosphate to the juice led, after 38 hours, to final 2,3-butanediol concentrations of 64 g/L, with yields of 81% of the theoretical maximum. These results are similar to those found in the fermentation of a reference medium composed of juice enriched with several nutrients in which 66.7 g/L butanediol and a yield of 85.6% were obtained. Ammonium phosphate concentrations up to 4.0 g/L resulted in incomplete fermentations. The fermentation kinetics was studied in a laboratory scale fermentor, with the sugar cane juice diluted to ca. 180 g/L sucrose. Both ammonium phosphate (8.0 g/L) and reference media were tested. In these experiments, after process times close to 35 hours, butanediol concentrations of 71.7 and 71.1 g/L, yields of 78.0 and 74.1 % , and productivities of 2.1 and 2.0 g/Lh, respectively, were obtained. The most important difference was the final biomass concentration that reached 9.1 g/L with the ammonium phosphate medium and 11.8 g/L with the rich medium. The maximum specific growth rates (µx,m=0.61 and 0.45 h-1), measured with dissolved oxygen concentrations above zero, have indicated that cell growth in the rich medium was inhibited by high nutrient concentration. In the sequence, when the oxygen concentration was zero, butanediol production started and maximum specific production rates (µP,m of 0.53 h-1 with the ammonium phosphate medium and 0.45 h-1 with the rich medium were reached. These values suggest that nutrient concentration in the rich medium also affects product formation. ln both runs, a stationary growth phase was noticed. In the experiment with the ammonium phosphate medium, the stationary phase was 12 hours longer due to the insufficient oxygen supply rate or the shortage of some nutrient. At that moment, decreasing specific product formation rates (7#181;P) were observed. With the sugar cane juice diluted to approximately 20 g/L and 8.0 g/L ammonium phosphate, fermentation time was 23.5 hours, too long to such sucrose concentration. With similar S0 and the rich medium, fermentation time was reduced to 6.5 hours. That shows that, in the first case, natural nutrients of the juice were excessively diluted. The study on the effect of the initial sucrose concentration on the process, performed in a laboratory fermentor with the rich medium, has shown that this parameter strongly affects both cell growth and 2,3-butanediol production. Increasing S0 led to decreasing cell yields and, for S0 up to 152 g/L, diol yields increased. Cell growth inhibition was stronger as higher sucrose concentrations were used. This is evidenced by the values of µx,m that decreased from 0.91 to 0.45 h-1 as S0 was augmented from 24 to 181 g/L. Approximately constant µP,m (0.62 to 0.68 h-1) were observed with S0 up to 152 g/L. With S0=181 g/L, µP,m is reduced to 0.45 h-1 . The maximum productivities (3.2 g/Lh) were obtained with S0 between 105 and 152 g/L. From the results of this work, one can conclude that sugar cane juice is a suitable raw material for the production of 2,3-butanediol by Klebsiella pneumoniae.

Neste trabalho foi avaliada a possibilidade do emprego de caldo de cana-de-açúcar (Saccharum officinarum) como matéria-prima para obtenção de 2,3-butanodiol por fermentação, utilizando a bactéria Klebsiella pneumoniae NRRL B199. A pesquisa compreendeu o teste de diferentes nutrientes para a composição de um meio de fermentação eficiente com o caldo de cana e experimentos em que a influência da concentração inicial de sacarose (S0) sobre o processo em regime descontínuo foi estudada. Ensaios realizados em frascos agitados, com S0 entre 140 e 150 g/L, mostraram que a fermentação de caldo de cana clarificado não suplementado com nutrientes não é viável. Por outro lado, a adição de 5,0 a 10,0 g/L de fosfato de amônio ao caldo levou a concentrações finais de 2,3-butanodiol de até 64 g/L e rendimentos, em relação ao máximo teórico, da ordem de 81% em cerca de 38 horas. Estes resultados se aproximam daqueles alcançados na fermentação do caldo enriquecido com vários nutrientes, usado como referência, na qual foram produzidos 66,7 g/L de diol com um rendimento de 85,6%. A utilização de concentrações de fosfato de amônio de até 4,0 g/L resultou em fermentações incompletas. A cinética da fermentação foi estudada com o caldo diluído a, aproximadamente, 180 g/L de sacarose e suplementado com 8,0 g/L de fosfato de amônio e, também, com o meio rico, em regime descontínuo em fermentador de bancada. Foram obtidos, nestes ensaios, concentrações de butanodiol de 71,7 e 71,1 g/L, rendimentos de 78,0 e 74,1 %, produtividades de 2,1 e 2,0 g/Lh e tempos de fermentação de 34,5 e 35,7 horas, respectivamente. A principal diferença foi observada na concentração de biomassa que atingiu 9,1 g/L, no meio com fosfato, e 11,8 g/L no meio rico. As máximas velocidades específicas de crescimento (µx, m), 0,61 e 0,45 h-1, observadas com oxigênio dissolvido acima de zero, indicam que o crescimento celular, no meio rico, foi inibido pela alta concentração de nutrientes. Na seqüência das fermentações, com concentrações nulas de oxigênio dissolvido, a produção de diol foi iniciada e as máximas velocidades específicas de formação de produto (µP,m), 0,53 h-1 com fosfato de amônio e 0,45 h-1 com o meio rico, foram atingidas, sugerindo que altas concentrações de nutrientes afetam, também, a formação de produto. Nos dois ensaios foi observada a ocorrência de uma fase estacionária de crescimento que, no caso do meio contendo fosfato de amônio, teve uma duração 12 horas mais longa devido a um insuficiente suprimento de oxigênio ou do esgotamento de algum nutriente. Neste ponto, foi verificada uma queda das velocidades específicas de formação de produto (µP). Com o caldo diluído a cerca de 20 g/L de sacarose e 8,0 g/L de fosfato de amônio, a fermentação transcorreu em um tempo muito longo (23,5 horas) para a baixa concentração de sacarose empregada. Com S0 semelhante e o meio rico, o tempo de processo foi de apenas 6,5 h, o que demonstra que, no primeiro caso, ocorreu uma diluição excessiva dos nutrientes naturalmente presentes no caldo. O estudo do efeito da concentração inicial de sacarose sobre a fermentação, conduzido em fermentador de bancada e com o meio rico em nutrientes, mostrou que este parâmetro influencia fortemente o crescimento celular e a produção de 2,3-butanodiol. O aumento de S0 levou a valores decrescentes dos fatores de conversão de substrato em células, enquanto os rendimentos em diol aumentaram com S0 até 152 g/L. Valores crescentes de S0 provocaram uma inibição do crescimento celular, evidenciada pela diminuição de µx,m de 0,91 a 0,45 h-1, com S0 entre 24 e 181 g/L. Valores aproximadamente constantes de µP,m (0,62 a 0,68 h-1) foram observados com S0 até 152 g/L, enquanto uma queda acentuada foi verificada com S0=181 g/L (µP,m=0,45 h-1). As máximas produtividades (3,2 g/Lh) foram obtidas com valores de S0 de 105 e 152 g/L. Os dados gerados neste trabalho permitem concluir que caldo de cana-de-açúcar é uma matéria-prima adequada para a produção de 2,3-butanodiol por Klebsiella pneumoniae. / The use of sugar cane (Saccharum officinarum) juice, as raw material for the fermentative production of 2,3-butanediol by Klebsiella pneumoniae NRRL B199, was evaluated in this work. The research was focused on testing different nutrients to formulate an efficient fermentation medium with the juice and some experiments to study the effect of the initial sucrose concentration (S0) on the batch process. Shaker flasks experiments with S0 between 140 and 150 g/L have shown that the fermentation of sugar cane juice without added nutrients is not viable. On the other hand, the addition of 5.0 to 10.0 g/L ammonium phosphate to the juice led, after 38 hours, to final 2,3-butanediol concentrations of 64 g/L, with yields of 81% of the theoretical maximum. These results are similar to those found in the fermentation of a reference medium composed of juice enriched with several nutrients in which 66.7 g/L butanediol and a yield of 85.6% were obtained. Ammonium phosphate concentrations up to 4.0 g/L resulted in incomplete fermentations. The fermentation kinetics was studied in a laboratory scale fermentor, with the sugar cane juice diluted to ca. 180 g/L sucrose. Both ammonium phosphate (8.0 g/L) and reference media were tested. In these experiments, after process times close to 35 hours, butanediol concentrations of 71.7 and 71.1 g/L, yields of 78.0 and 74.1 % , and productivities of 2.1 and 2.0 g/Lh, respectively, were obtained. The most important difference was the final biomass concentration that reached 9.1 g/L with the ammonium phosphate medium and 11.8 g/L with the rich medium. The maximum specific growth rates (µx,m=0.61 and 0.45 h-1), measured with dissolved oxygen concentrations above zero, have indicated that cell growth in the rich medium was inhibited by high nutrient concentration. In the sequence, when the oxygen concentration was zero, butanediol production started and maximum specific production rates (µP,m of 0.53 h-1 with the ammonium phosphate medium and 0.45 h-1 with the rich medium were reached. These values suggest that nutrient concentration in the rich medium also affects product formation. ln both runs, a stationary growth phase was noticed. In the experiment with the ammonium phosphate medium, the stationary phase was 12 hours longer due to the insufficient oxygen supply rate or the shortage of some nutrient. At that moment, decreasing specific product formation rates (7#181;P) were observed. With the sugar cane juice diluted to approximately 20 g/L and 8.0 g/L ammonium phosphate, fermentation time was 23.5 hours, too long to such sucrose concentration. With similar S0 and the rich medium, fermentation time was reduced to 6.5 hours. That shows that, in the first case, natural nutrients of the juice were excessively diluted. The study on the effect of the initial sucrose concentration on the process, performed in a laboratory fermentor with the rich medium, has shown that this parameter strongly affects both cell growth and 2,3-butanediol production. Increasing S0 led to decreasing cell yields and, for S0 up to 152 g/L, diol yields increased. Cell growth inhibition was stronger as higher sucrose concentrations were used. This is evidenced by the values of µx,m that decreased from 0.91 to 0.45 h-1 as S0 was augmented from 24 to 181 g/L. Approximately constant µP,m (0.62 to 0.68 h-1) were observed with S0 up to 152 g/L. With S0=181 g/L, µP,m is reduced to 0.45 h-1 . The maximum productivities (3.2 g/Lh) were obtained with S0 between 105 and 152 g/L. From the results of this work, one can conclude that sugar cane juice is a suitable raw material for the production of 2,3-butanediol by Klebsiella pneumoniae.

In this project a LC-MS-method for the analysis of gamma-hydroxybutyrate, gamma-butyrolactone, 1,4-butanediol, amphetamine and methadone was developed. Initially, the efficiency of the ionisation of the analytes was evaluated with respect to the ionisation technique (ESI, APCI and APPI) and the composition of the mobile phase. In the next step a number of different columns was tested in order to find the one with the greatest potential for separation of the substances in question. Using the selected column, the separation was optimised by means of experimental design and the software The Unscrambler 7.8. The parameters studied were the flow rate, the column temperature and the mobile phase composition. The response variables were the resolution between the target compounds and the retention time of the last eluting compound. These experiments showed that, in order to obtain the best ionisation, the mobile phase should consist of 5 mM formic acid in water and acetonitrile. ESI should be used in the positive mode for all analytes except gamma-hydroxybutyrate, for which the negative mode should be applied. The Hypercarb column exhibited superior retention of the analytes and was therefore selected for further optimisation. The dimensions of this column were 2.1 x 50 mm and the particle size 5 μm, connected to a 2.1 x 10 mm precolumn containing the same packing material. The optimum of the flow rate and the column temperature were 250 μl/min and 20 ºC respectively. For the separation of gamma-hydroxybutyrate, gamma-butyrolactone and 1,4-butanediol the mobile phase consisted of 100% water with 5 mM formic acid. Thereafter a gradient, up to 70% acetonitrile with 5 mM formic acid, was used in order to elute amphetamine and methadone. Efforts were also made to find an internal standard for the method. However, none of the compounds tested was found suitable. In order to get the method usable for routine analysis, which is the goal, further work is required. A suitable internal standard needs to be added to the method and thereafter work remains with validation of the method.

<p>In this project a LC-MS-method for the analysis of gamma-hydroxybutyrate, gamma-butyrolactone, 1,4-butanediol, amphetamine and methadone was developed.</p><p>Initially, the efficiency of the ionisation of the analytes was evaluated with respect to the ionisation technique (ESI, APCI and APPI) and the composition of the mobile phase. In the next step a number of different columns was tested in order to find the one with the greatest potential for separation of the substances in question. Using the selected column, the separation was optimised by means of experimental design and the software The Unscrambler 7.8. The parameters studied were the flow rate, the column temperature and the mobile phase composition. The response variables were the resolution between the target compounds and the retention time of the last eluting compound.</p><p>These experiments showed that, in order to obtain the best ionisation, the mobile phase should consist of 5 mM formic acid in water and acetonitrile. ESI should be used in the positive mode for all analytes except gamma-hydroxybutyrate, for which the negative mode should be applied. The Hypercarb column exhibited superior retention of the analytes and was therefore selected for further optimisation. The dimensions of this column were 2.1 x 50 mm and the particle size 5 μm, connected to a 2.1 x 10 mm precolumn containing the same packing material. The optimum of the flow rate and the column temperature were 250 μl/min and 20 ºC respectively. For the separation of gamma-hydroxybutyrate, gamma-butyrolactone and 1,4-butanediol the mobile phase consisted of 100% water with 5 mM formic acid. Thereafter a gradient, up to 70% acetonitrile with 5 mM formic acid, was used in order to elute amphetamine and methadone. Efforts were also made to find an internal standard for the method. However, none of the compounds tested was found suitable.</p><p>In order to get the method usable for routine analysis, which is the goal, further work is required. A suitable internal standard needs to be added to the method and thereafter work remains with validation of the method.</p>

Hyaluronan (HA) is a substance that is commonly used in biomedical applications in the form of hydrogels. One of these biomedical applications is dermatological fillers where HA is cross-linked with 1,4-Butanediol diglycidyl ether (BDDE) to reduce its rapid turnover within tissue. The filler gives a volumetric effect that can fill out wrinkles. This literature study was conducted in collaboration with Galderma to determine if there is research that explores additives to HA hydrogels that give both volumetric and biological effects when applied as filler. Biological effects that improve the skin's appearance and complexion such as a rejuvenation of the skin was preferable. Both polynucleotides and mannitol show great potential to act as additives in injectable hyaluronan hydrogels.  The main effect of polynucleotides (PN) added in hydrogels is that it is collagen stimulating and provides a more natural tissue regeneration. Rheological properties of the filler change with the addition of PN, where elasticity, viscosity and viscoelasticity have been shown to increase. PNs show no toxicity and are considered safe to inject. The study of mannitol has shown that it does not give a volumetric effect after the injected hydrogel has been broken down. What mannitol can help with, is to prolong the life of the hydrogel and reduce the swelling that is a common side effect after an injection. In addition to this, mannitol is a safe substance to inject.

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