Lisdexanfetamina (LDX) é um pró-fármaco estimulante de longa duração indicado para o tratamento dos sintomas do transtorno do déficit de atenção e hiperatividade e do transtorno da compulsão alimentar periódica. A hidrólise da ligação amida da LDX ocorre in vivo liberando a molécula terapeuticamente ativa d-anfetamina (d-ANF) e o aminoácido l-lisina. Visto que a LDX se biotransforma à d-ANF – um potente estimulante do sistema nervoso central com destaque tanto na clínica quanto na toxicologia – existe potencial para uso inadequado, abuso e desvio para fins não terapêuticos. Nos laboratórios de toxicologia, amostras biológicas com resultados positivos para anfetamina (ANF) são um desafio, uma vez que alguns testes toxicológicos podem detectar ANF devido à utilização de alguns medicamentos, dificultando a sua interpretação. Assim, são necessários métodos bioanalíticos eficientes aliados ao conhecimento farmacocinético, que permite a verificação da possibilidade de detecção, a estimativa da janela de detecção e as concentrações que podem ser alcançadas em diferentes matrizes biológicas. Dessa forma, neste trabalho, foram desenvolvidos métodos bioanalíticos para quantificação simultânea da LDX e de seu produto de biotransformação, a ANF, nas matrizes biológicas fluido oral, plasma e urina utilizando a cromatografia líquida acoplada a detector de massas sequencial (CL-EM/EM). A preparação de amostra é simples, utilizando a precipitação de proteínas para o plasma, com pouca quantidade de solvente orgânico, a diluição para o fluido oral e a filtração para urina, ambas com nenhuma quantidade de solvente orgânico. As curvas de calibração utilizando o padrão interno ANF deuterada apresentaram linearidade entre 1 e 128 ng/mL para o fluido oral e o plasma e entre 4 e 256 ng/mL para a urina. A menor concentração das curvas de calibração é igual ao limite inferior de quantificação. Precisão e exatidão intra e interdia ficaram dentro dos limites de ± 15% para os controles e ± 20% para o limite de quantificação. Os métodos foram seletivos e sem efeito residual, porém apresentaram um leve efeito matriz, frequentemente encontrado em métodos de CL-EM/EM. O método foi aplicado para análise das amostras do estudo farmacocinético da LDX e ANF nas matrizes biológicas fluido oral, plasma e urina após administração oral de LDX. Seis voluntários do sexo masculino coletaram amostras de fluido oral e plasma em tempos pré-determinados durante 72 horas e amostras de urina em intervalos pré-determinados durante 120 horas. Os dados foram avaliados de maneira não-compartimental e compartimental. Considerando a análise não-compartimental, a concentração máxima média da d-ANF foi quase seis vezes inferior no plasma em relação ao fluido oral e ocorreu em 3,8 e 4 horas, respectivamente, após a administração oral. A LDX atingiu a concentração máxima no plasma e no fluido oral em 1,2 e 1,8 horas após a administração oral, respectivamente, com um valor médio de pico de concentração quase duas vezes mais elevado no plasma em comparação com o fluido oral. A eliminação da d-ANF a partir do plasma e a partir do fluido oral foi semelhante, porém para LDX a eliminação a partir do fluido oral foi mais lenta, mesmo com concentrações mais baixas do que no plasma. A detecção da d-ANF ocorreu até 48-72 horas no plasma e fluido oral e até 120 horas em urina. Já para a LDX, a detecção ocorreu até 3, 5 e 12 horas no plasma, fluido oral e urina, respectivamente. LDX intacta e d-ANF foram detectadas nas três matrizes avaliadas. Na análise compartimental, o melhor ajuste de modelo foi observado para 1 compartimento para ambos os analitos tanto no plasma quanto no fluido oral. Houve uma correlação entre as concentrações do fluido oral e do plasma para d-ANF e entre as proporções de LDX intacta/d-ANF pelo tempo no plasma e no fluido oral. O método analítico desenvolvido pode ser aplicado em diferentes áreas do conhecimento a fim de certificar os resultados de uma análise de triagem positiva para ANF. Porém, para interpretação das situações tanto de triagem quanto de confirmação é necessário aliar o conhecimento farmacocinético gerado no trabalho, que demonstra se há a possibilidade de detecção na matriz analisada e por quanto tempo após a administração da LDX. Isto auxilia na diferenciação do uso de outros medicamentos derivados da ANF e do uso ilegal, para que as devidas providências legais e de manejos clínicos de tratamento e controle de dependência sejam tomadas quando necessário. / Lisdexamfetamine (LDX) is a long-acting prodrug stimulant indicated for the treatment of attention-deficit/hyperactivity disorder and binge-eating disorder symptoms. In vivo hydrolysis of lisdexamfetamine amide bond releases the therapeutically active d-amphetamine (d-AMPH) and the amino acid l-lysine. Since LDX biotransformation gives rise to d-AMPH - a potent stimulant of the central nervous system that stands out in clinical and toxicology - there is potential for misuse, abuse and diversion for non-therapeutic purposes. In laboratories of toxicology, biological samples with positive results for amphetamine (AMPH) are a challenge, since some toxicological tests can detect AMPH due to the use of some medications hindering the interpretation. Therefore, we need efficient bioanalytical methods combined with the pharmacokinetic knowledge, which allows to verify the possibility of detection, to assess the detection window and the concentrations that can be reached in different biological matrices. Hence, bioanalytical methods were developed for simultaneous quantification of LDX and its main biotransformation product AMPH in the biological matrices oral fluid, plasma and urine by liquid chromatography-mass spectrometry (LC-MS/MS). The sample preparation is simple, using protein precipitation for plasma, with a small amount of organic solvent, dilution for oral fluid and filtration to urine, both with no amount of organic solvent. Calibration curves using deuterated AMPH internal standard showed linearity between 1 and 128 ng/mL for oral fluid and plasma, and between 4 and 256 ng/mL for urine. The lowest concentration of the calibration curve is the lower limit of quantification. Intra and interday precision and accuracy were within the limits of ± 15% for controls and ± 20% for the limit of quantification. The methods were selective and no carry-over was observed, however with some matrix effect, often found in LC-MS/MS methods. The method was applied to analyze samples from LDX and AMPH pharmacokinetics study in the biological matrices oral fluid, plasma and urine following oral administration of LDX. Six male volunteers collected oral fluid and plasma samples at predetermined times during 72 hours and urine samples at pre-determined intervals during 120 hours. Data were evaluated through non-compartmental and compartmental analysis. Considering the noncompartmental analysis, the mean maximum concentration of d-AMPH was almost 6-fold lower in plasma than in oral fluid and occurred at 3.8 and 4 hours, respectively, after LDX administration. LDX maximum concentration was reached at 1.2 and 1.8 hours after LDX oral administration for oral fluid and plasma, respectively, with a mean peak concentration almost 2-fold higher in plasma when compared with oral fluid. Elimination of d-AMPH from oral fluid and from plasma were similar, albeit for LDX elimination from oral fluid was slower even with lower concentrations than plasma. Detection occurred until 48 to 72 hours in plasma and oral fluid and until 120 hours in urine for d-AMPH. Whereas for LDX, detection could be done for up to 3, 5 and 12 hours in plasma, oral fluid and urine, respectively. Intact LDX and d-AMPH were detected in the three evaluated matrices. In compartmental analysis, the best model fit was observed for 1-compartment model for both analytes in plasma and in oral fluid. There was a correlation between oral fluid and plasma d-AMPH concentrations and between intact LDX/d-AMPH ratios along time in plasma as well as in oral fluid. The bioanalytical methods developed can be applied in different fields of knowledge in order to ensure the results of a positive screening analysis for AMPH. Nevertheless, for interpretation of situations in both screening and confirmation tests is necessary to combine the pharmacokinetic knowledge produced in this study, which shows if there is the possibility of detection in the analyzed matrix and for how long after the administration of LDX. This results aid in the differentiation from other AMPH derived drugs use and from illegal use, so that appropriate legal action and clinical management strategies for treatments and control of dependence be taken when necessary.

As especificações do biodiesel são garantidas por um rigoroso controle de qualidade; entre outros parâmetros, a quantificação de antioxidantes é fundamental para assegurar um produto satisfatório para comercialização. No presente estudo, foram desenvolvidas técnicas voltamétricas e cromatográficas para análise direta de BHA, TBHQ, BHT e PG em biodiesel. Medidas por voltametria de pulso diferencial (DPV) em mistura biodiesel:etanol 1:1 (v/v), contendo perclorato de tetra-hexilamônio como eletrólito suporte e empregando ultramicroeletrodo (ume) de Pt, mostraram picos da corrente diferencial de oxidação bem definidos para TBHQ e PG, sendo obtidos gráficos de calibração lineares na faixa de concentrações de 10-3 mol L-1. Analogamente, empregando um ume de Au em microemulsão de biodiesel livre de surfactante (SFME) contendo tetrafluoroborato de tetra-n-butilamônio, medidas por DPV apresentaram picos da corrente diferencial bem definidos para TBHQ e BHT. Para esses compostos, foram obtidos gráficos de calibração lineares na faixa de concentrações de 10-4 mol L-1. Em ambos os casos, devido à sobreposição dos voltamogramas, os teores de antioxidantes foram calculados por um procedimento matemático de deconvolução empregando o software Origin 8.0®. A quantificação de BHA, BHT e TBHQ por cromatografia GC/qMS em biodiesel diluído em metanol apresentou linearidade entre 5 e 25 mg L-1, para cada um dos antioxidantes. Medidas por HPLC em fase reversa com coluna fenílica apresentaram boa separação dos picos cromatográficos na análise simultânea de BHA, BHT, TBHQ e PG assim como curvas de calibração com linearidade entre 10 e 80 mg L-1, para cada um dos antioxidantes. A identificação dos ésteres metílicos de ácidos graxos (FAMEs) no biodiesel foi realizada por UHPLC-ESI-Orbitrap/MS; os teores de FAMEs e antioxidantes permaneceram estáveis nas amostras fortificadas mesmo após 8 semanas de exposição à luz solar, porém diminuíram significativamente em amostras não-fortificadas. As metodologias propostas são de fácil preparo e baixo consumo de amostra, podendo vir a ser do interesse da indústria para o monitoramento de antioxidantes em biodiesel, na linha de produção. A principal vantagem é não ser necessário nenhum tipo de pré-tratamento da amostra: a simples diluição em metanol ou etanol ou a preparação do biodiesel na forma de SFME conferem rapidez e simplicidade à determinação dos antioxidantes investigados. / Biodiesel features are assured by strict quality control procedures; among those, antioxidants quantification is essential to guarantee a good and satisfactory final product for commercialization. In the present study, voltammetric and chromatographic methodologies were developed for direct analyses techniques of BHA, TBHQ, BHT and PG in biodiesel. Differential pulse voltammetry (DPV) in biodiesel:ethanol 1:1 (v/v), with tetrahexylammonium perchlorate as supporting electrolyte at a Pt ultramicroelectrode (ume), presented well-defined oxidation peaks for TBHQ and PG, with corresponding linear calibration graphs in the range of 10-3 mol L-1. Likewise, DPV at an Au ume in biodiesel surfactant-free microemulsion (SFME), with tetra-n-butylammonium tetrafluoroborate, presented well-defined oxidation peaks for TBHQ and BHT. For these compounds, linear calibration graphs were obtained in the range of 10-4 mol L-1. In both techniques, due to significant voltammograms overlapping, amounts of antioxidants were calculated through mathematical deconvolution process using software Origin 8.0®. BHA, BHT and TBHQ quantification by GC/qMS in biodiesel diluted in methanol presented linearity between 5 and 25 mg L-1 for each antioxidant. Reversed-phase liquid chromatography by HPLC using phenyl column showed good peak separation in simultaneous analysis of BHA, BHT, TBHQ and PG and calibration curves with linearity between 10 and 80 mg L-1. Biodiesel fatty acid methyl esters (FAMEs) identification was carried out by UHPLC-ESI-Orbitrap/MS; both FAMEs and antioxidants contents remained stable in spiked samples even after 8 weeks under sunlight exposure; in non-spiked samples, FAMEs content was significantly reduced. All proposed methodologies are easy to perform and present low sample consumption, which are interesting features for industry to monitor the aforementioned and other antioxidants in biodiesel. Their main advantage lies in the absence of any sample pre-treatment: the simple dilution in methanol or ethanol or biodiesel preparation as SFME deliver speed and simplicity for determination of target antioxidants.

A análise de impurezas é uma etapa importante no controle de qualidade do insumo farmacêutico e do produto final. Provenientes da síntese do medicamento ou dos excipientes, mesmo em pequenas concentrações, as impurezas podem afetar a eficácia e a segurança. No presente trabalho foram desenvolvidos e validados dois métodos empregando cromatografia líquida de alta eficiência (CLAE) para a avaliação do besifloxacino e sua impureza de síntese, um com eluição isocrática e outro com eluição gradiente. As análises por CLAE com modo de eluição isocrática foram executadas utilizando coluna Ciano, mantida a 25 °C. A fase móvel foi constituída por 0,5% de trietilamina (pH 3,0) : acetonitrila (88:12 v/v), eluída na vazão de 1,0 mL/min com detecção a 330 nm. O método com eluição gradiente foi conduzido com a mesma coluna e componentes da fase móvel modificando apenas as proporções entre fase orgânica e aquosa durante as análises. Os procedimentos foram validados de acordo com guias aceitos internacionalmente, observando-se resultados dentro dos limites aceitáveis. Os métodos apresentaram-se lineares na faixa de 140 a 260 μg/mL para o besifloxacino e de 0,3 a 2,3 μg/mL para a impureza A. Com volume de injeção de 20 μL, os limites de detecção e quantificação foram, respectivamente, 0,07 e 0,3 μg/mL. A precisão foi alcançada para todas as análises realizadas, obtendo DPR inter-dia igual a 6,47 e 6,36 para a impureza A com eluição isocrática e gradiente, respectivamente. A exatidão foi superior a 99% e a robustez apresentou resultados satisfatórios. No método isocrático obteve-se tempo de análise de 25 min e no gradiente de 15 min. O número de pratos teóricos no modo isocrático foi na ordem de 5000 enquanto no modo gradiente foi na ordem de 45000, ou seja, obteve-se maior eficiência da coluna com alteração da composição da fase móvel durante a eluição. No insumo besifloxacino e no produto farmacêutico utilizados neste trabalho, impurezas relacionadas estavam presentes, mas a impureza A não foi detectada. Os métodos propostos, considerando-se o limite de quantificação, podem ser aplicados na determinação quantitativa da impureza A na análise da matéria prima do besifloxacino, assim como na suspensão oftálmica. / Analysis of impurities is an important step in quality control of pharmaceutical ingredients and final products. From drug synthesis or excipients, even in small concentrations, impurities may affect efficacy and safety. In the present study two chromatographic methods were developed and validated for high-performance liquid chromatography (HPLC) for the assessment of besifloxacin and its synthesis impurity, one with isocratic and another with gradient elution. Analyses by HPLC in isocratic elution mode were performed using Cyano column maintained at 25 °C. Mobile phase was composed of 0.5% triethylamine (pH 3.0): acetonitrile (88:12 v/v) eluted at a flow rate of 1.0 ml/min with detection at 330 nm. The method with gradient elution was carried out with the same column and mobile phase components modifying only proportion between organic and aqueous phase during analysis. The procedures have been validated according to internationally accepted guidelines, obtaining results within acceptable limits. The methods presented a linear response from 140-260 μg/mL for besifloxacin and from 0.3 to 2.3 mg/mL for impurity. With the injection volume of 20 μL, the limit od detection and limit of quantitation were, respectively, 0.07 and 0.3 μg/mL. Precision was achieved for all analyses, obtaining inter-day RSD equal to 6.47 and 6.36 for impurity A with isocratic and gradient elution, respectively. The accuracy was higher than 99% and robustness exhibited satisfactory results. In the isocratic method was obtained analysis time 25 min and 15 min gradient. The number of theoretical plates in the isocratic mode was of the order of 5000 while in the gradient mode was of the order of 45000, that is, gave greater efficiency of the column by changing the mobile phase composition during elution. In raw material of besifloxacin and pharmaceutical product used in this study, related impurities were present but the impurity A was not detected. The proposed methods, considering the limit of quantitation, can be in quantitative determination of impurity A in the analysis of besifloxacin raw material, as well as in ophthalmic suspension.

A vigabatrina (VGB) é um fármaco anticonvulsivante que apresenta apenas a forma farmacêutica sólida disponível para uso. Na área hospitalar, devido à ausência de medicamentos na forma farmacêutica líquida, são preparadas derivações a partir de comprimidos e cápsulas para adequar a administração da dose prescrita. No entanto, a falta de estudos de estabilidade podem comprometer a eficácia e segurança destas derivações. O objetivo deste trabalho foi analisar a estabilidade química de derivações de comprimidos de vigabatrina em condições de armazenamento sob diferentes temperaturas e variações na embalagem utilizada. A análise das derivações de VGB foi realizada através de cromatografia líquida de alta eficiência (CLAE). O método descrito na Farmacopeia Britânica (2016) foi covalidado quanto a especificidade, linearidade, precisão e exatidão. Amostras de derivação de VGB foram preparadas em triplicata e acondicionadas em frascos de vidro e de PET âmbar. Após, foram armazenadas sob três diferentes condições de temperatura: temperatura ambiente (15 a 30 °C), sob refrigeração (2 a 8 °C) e em estufa (40 °C). Foram coletadas amostras armazenadas nas diferentes embalagens a cada 7 dias, por um período de 35 dias para as amostras conservadas em temperatura ambiente e refrigerada. O mesmo procedimento foi realizado para as amostras conservadas em estufa, porém por um período de 28 dias Também foi analisado o pH das amostras em cada tempo de coleta. As derivações de VGB foram analisadas por CLAE e apresentaram variação dentro dos limites preconizados pela Farmacopeia Britânica 2016, até 21 dias para frascos de vidro e de PET âmbar para as temperaturas ambiente e refrigerada. As amostras de VGB conservadas em estufa, apresentaram redução acima de 10% após 7 dias de estudo. A menor variação de pH ocorreu em frasco de vidro âmbar armazenado sob refrigeração. O resultado deste estudo serve de referência no preparo de derivações de VGB para uso hospitalar, pois apresentou intervalo de tempo confiável e condições de armazenamento adequadas para sua utilização. Desta forma, os pacientes pediátricos que utilizam doses fracionadas ou pacientes em uso de sondas nasogástricas terão as derivações adequadamente preparadas, reduzindo o risco de erros de diluição e contaminação microbiológica, melhorando a eficácia e segurança terapêutica. / Vigabatrin (VGB) is an anticonvulsant drug that has only solid dosage form available for use. In hospital, due to lack of medicines in liquid dosage form, extemporaneous preparations are prepared from tablets and capsules to adapt the administration of the prescribed dose. However, the lack of stability studies may compromise the efficacy and safety of these preparations. The aim of this study was to analyze the chemical stability of VGB extemporaneous preparation from tablets at storage conditions in different temperatures and variations of packaging used. The analysis of VGB extemporaneous preparations were performed by high-performance liquid chromatography (HPLC). The method described in British Pharmacopoeia (2016) was co-validated for specificity, linearity, precision and accuracy. VGB extemporaneous preparations were prepared in triplicate and placed in amber glass and PET bottles, which were stored under three different conditions: at room temperature (15 to 30 °C), under refrigeration (2 to 8 °C), and oven (40 °C). Samples of preparations stored at room temperature and refrigeration were collected every 7 days along 35 days. The same was done for solutions kept at 40 °C, but for a period of 28 days. It was also analyzed the preparations pH for each sampling time VGB extemporaneous preparations were analyzed by HPLC and demonstrated variations within the limits of British Pharmacopoeia (2016) up to 21 days in amber glass and PET bottles at room and refrigerated temperatures. VGB content for preparations kept in oven decreased above 10% after 7 days of study. The lowest pH change occurred in amber glass bottle stored under refrigeration. Results of this study can be applied as a reference for VGB extemporaneous preparation in hospital, once it was demonstrated the reliability of storage time interval and proper conditions for the use. Thus, pediatric patients with fractionated doses or patients using nasogastric probe will have adequately prepared extemporaneous preparations, reducing the risk of dilution errors and microbiological contamination.

The dynamic S-palmitoylation regulates many intracellular events, including protein trafficking, anchoring, targeting, and protein-protein interactions. Direct detection of S-palmitoylation by conventional liquid chromatography-mass spectrometry (LC-MS) methods is challenging because of the tendency of palmitoyl loss during sample preparation and gas phase fragmentation. Additionally, the high hydrophobicity of the palmitoyl group can prevent proper elution of palmitoyl peptides from the commonly used C18 column. Here, we developed a comprehensive strategy tailored for S-palmitoyl detection using three palmitoyl peptide standards. We found that S-palmitoylation was largely preserved in neutral Tris buffer with tris(2-carboxyethyl)phosphine as the reducing agent and that various fragmentation methods provided complementary information for palmitoyl localization. Moreover, S-palmitoyl peptides were efficiently analyzed using a C4 column and the derivatization of free cysteine with a hydrophobic tag allowed relative quantification of palmitoyl peptides and their unmodified counterparts. We further discovered potential complications to S-palmitoylation analysis caused by the use of ProteaseMAXTM, an MS-compatible detergent. The hydrophobic degradation products of ProteaseMAXTM reacted with the free cysteine thiols, generating artifacts that mimic S-acylation and hydroxyfarnesylation. Another MS-compatible detergent, RapiGestTM, did not produce such artifacts, and showed the ability to stabilize S-palmitoylation by preventing thioester hydrolysis and dithiothreitol-induced thioester cleavage. Moreover, we found that the palmitoyl peptide GCpalmLGNAK could undergo intermolecular palmitoyl migration from the cysteine to the peptide N-terminus or the lysine side chain during sample preparation, and this could lead to false discovery of N-palmitoylation. RapiGestTM inhibited such migration, and is thus recommended for S-palmitoyl sample preparation. We then applied the established method to analyze the regulator of G-protein signaling 4 (RGS4) which had been reported to undergo S-palmitoylation by radioactive labeling. It had also been reported that the S-palmitoylation state of RGS4 affects its GTPase activity. With LC-MS/MS analysis, we found that the addition of palmitate to the cell culture medium in metabolic labeling experiments could boost the level of S-palmitoylation, leading to false discovery of new S-palmitoylation site(s). We also noted discrepancies between the S-palmitoylation sites identified by radioactive labeling and by LC-MS/MS analysis. Further studies are needed to evaluate the reliability of S-palmitoyl detection by these two methods.

De nouvelles méthodes de détection et de quantification du VX (O-éthyl S–(2(diisopropylamino)éthyl) (méthylphosphonothioate)) dans le plasma par chromatographie liquide couplée à un spectromètre de masse en tandem (LC-MS/MS) ont été développées. Après avoir été mise en œuvre en milieu HBSS (rendement > 99%), la méthode d'extraction liquide-liquide de notre composé d'intérêt nous permet d'obtenir en milieu plasmatique un rendement de soixante-cinq pour cent (65%). Cette méthode d'extraction permet d'évaluer la présence de VX dans de très faibles volumes d'échantillons de plasma (entre 20 et 1000 µL), donc dans des volumes compatibles avec des expérimentations sur de petits rongeurs. Cette extraction est suivie d'une analyse par LC-MS/MS en phase 100% organique. Des limites de détection de 0,15 et 0,5 pg/mL (pour 5 µL injecté) sont obtenues respectivement avec l'utilisation de la colonne Allure Biphényl (Restek) et Lux Cellulose-1 (Phenomenex). Afin d'étudier la toxicocinétique des énantiomères du VX dans le plasma, nos recherches se sont ensuite focalisées sur la séparation de ces derniers. Des résultats prometteurs ont été obtenus en utilisant une colonne Lux cellulose-2 (Phenomenex). / Innovative analytical methods have been developed to detect and quantify the organophosphorus nerve agent, VX (O-ethyl S–(2(diisopropylamino)ethyl) (methylphosphonothioate)), in plasma using liquid chromatography coupled with mass spectrometry (LC-MS/MS) technique. Liquid-liquid extraction of VX from HBSS environment was achieved with excellent yields (> 99%). Next, extraction from plasma was performed and generated a recovery rate of approximately sixty-five percent (65%). Our distinctive extraction methodology was implemented to evaluate the presence of VX in very small quantities of plasma (between 20 to 1000 µL) as in small rodents' experiments. It was followed by an LC-MS/MS analysis in a 100% organic phase. A Lux Cellulose-1 column (Phenomenex) and an Allure biphenyl column (Restek) were tested with detection limit at 0.15 pg/mL and 0.5 pg/mL in plasma (5 µL injected), respectively. Finally, our study was focused on the separation of VX enantiomers and hopeful results were provided using a Lux cellulose-2 column (Phenomenex).

A planta brasileira Copaifera sp. é uma árvore cujo óleo é tradicionalmente utilizado pela população principalmente como antiinflamatório, analgésico e relaxante muscular justificando o grande interesse científico em seu estudo. Entretanto, estudos tecnológicos, fitoquímicos e biológicos sobre suas folhas são escassos. Assim, este trabalho propôs estudar e otimizar o processo extrativo a partir das folhas de Copaifera langsdorffii Desf., além de determinar as melhores condições de secagem em spray dryer, já que formas farmacêuticas sólidas são mais interessantes do ponto de vista mercadológico e farmacotécnico e ambas as estapas são fundamentais para o desenvolvimento de um futuro medicamento fitoterápico. As condições ótimas de extração e secagem em spray dryer foram obtidas através de diversos planejamentos experimentais como o planejamento Box-Behnken, a inclinação ascendente e a função desejabilidade. Para obtenção do extrato líquido através do processo de maceração dinâmica, objetivou-se maximizar as seguintes respostas: teor de sólidos solúveis e flavonóides totais e dos marcadores químicos, quercetrina e afzelina. As condições ótimas de extração encontradas foram: massa de planta de 41 g, proporção etanol/ água de 79°GL, tempo de extração de 1 h, temperatura de extração de 25°C e velocidade de agitação de 600 rpm. No estudo do processo de secagem em spray dryer, foi realizado inicialmente um teste preliminar onde oito diferentes excipientes disponíveis foram estudados quanto às caracterísitcas físico-químicas entre elas fluidez, compactação e degradação dos marcadores químicos. As condições do equipamento foram: bico atomizador duplo fluido pneumático com orifício de abertura de 1,2 mm que operou com vazão de ar de 40 L/min e pressão de 5,0 bar; vazão do gás de secagem de 0,6 m3/min; temperatura de saída (Ts) de 80°C e fluxo da amostra (Ws) de 5,0 g/min. A proporção sólidos do extrato líquido de C. langsdoffii: excipiente foi de 3:1. Entre os excipientes estudados, o manitol foi o que forneceu o extrato seco de C. langsdorffii com melhores características físicas e físico-químicas e, por isso foi utilizado no estudo seguinte, o planejamento Box-Behnken cujas variáveis estudadas foram concentração da amostra (Cs), fluxo da amostra (Ws) e temperatura de saída (Ts). Foram avaliadas nove respostas das quais: densidade compactada, ângulo de repouso, índice de Carr, umidade absorvida, rendimento, eficiência de secagem, degradação de flavonóides totais, quercetrina e afzelina. Para obter a condição de secagem em que as nove respostas apresentassem valores satisfatórios, utilizou-se a função desejabilidade. Assim, as condições de secagem para se obter o extrato seco de C. langsdorffii com boas características foram: concentração da amostra de 20,7% m/m, fluxo da amostra de 5 g/min e temperatura de saída de 80°C. Como resultado final deste trabalho, obteve-se um extrato líquido de C. langsdorffii otimizado com teores máximos de sólidos solúveis e dos marcadores químicos e um extrato seco com propriedades físicas, físico-químicas e farmacotécnicas adequadas para a obtenção de formas farmacêuticas sólidas. / The Brazilian plant Copaifera sp. is a tree the oil of which is widely used in popular medicine as an anti-inflammatory, analgesic and muscle relaxant, consequently it is the subject of huge scientific interest. However, technological, phytochemical and biological studies regarding its leaves are rare. Thus, this work aimed to study and optimize the extraction process from Copaifera langsdorffii Desf. leaves, as well as to determine the best conditions for the spray drying process, since pharmaceutical solid forms are more marketable and of pharmacotechnical interests, with both steps being fundamental to the development of a future phytomedicine. The best extraction and spray drying conditions were guided by several experimental designs such as the Box-Behnken, the steepest ascent and the desirability function. From liquid extract achievement through dynamic maceration, the purpose was to maximize the following responses: soluble solids and total flavonoids contents and the chemical markers, quercetrin and afzelin contents. The best extraction conditions found were: plant mass of 41 g, ethanol/water proportion of 79°GL, extraction time of 1 h, extraction temperature of 25°C and stirring speed of 600 rpm. In the spray drying study, a preliminary test was accomplished, where eight different excipients were studied with regard to the physicochemical features such as flowability, compaction and chemical marker degradation. The equipment conditions were: a two-fluid pneumatic spray nozzle with an inlet orifice diameter of 1.2 mm that operated on 40 L/min air rate and 5 bar pressure; 0.6 m3/min drying air rate; 80°C outlet temperature (Ts) and 5.0 g/min sample feed rate (Ws). The proportion of C. langsdorffii liquid extract solids content: excipient was 3:1. Among the excipients studied, mannitol provided the best physicochemical feature for C. langsdorffii dried extract, therefore it was used in the following test, the Box-Behnken design, where the studied variables were sample concentration (Cs), sample feed rate (Ws) and outlet temperature (Ts). Nine responses were evaluated: tapped density, angle of repose, Carr index, humidity absorption, yield, drying efficiency, total flavonoids and quercetrin and afzelin degradation. To obtain the drying conditions where the nine responses presented satisfactory values, the desirability function was used. Thus, the drying conditions to obtain a dried extract with good physicochemical features were: sample concentration of 20.7% w/w, sample feed rate of 5.0 g/min and outlet temperature of 80°C. As a final result of this work, an optimized C. langsdorffii liquid extract presenting maximum soluble solids and chemical markers contents and a dried extract presenting the appropriate physical, physicochemical and pharmacotechnical properties to produce pharmaceutical solid forms were obtained.

Metabolism of xenobiotic drug molecules can result in the formation of metabolites which are more chemically reactive than the parent drug from which they are derived. These reactive species have the potential to covalently modify biological macromolecules if they are not detoxified. The formation of drug-protein adducts carries a potential risk of clinical toxicities and idiosyncratic adverse drug reactions which can, in severe cases, result in hospitalisation and even death. Current methods for the evaluation of the risk for a drug to cause adverse drug reactions due to drug-protein binding rely on risk factors such as quantitative covalent binding value, structure, dose etc. The objective of this project was to develop methods for the detection of reactive metabolites directly bound to proteins, which could be used in future evaluations of the mechanisms of binding of candidates in drug development. Three compounds known to produce reactive metabolites, acetaminophen, SB-648969 and amodiaquine, were used as tool substrates. In vitro incubations with human liver microsomes and individual cytochrome P450 enzymes (as Supersomes ) were used to produce reactive metabolite species and binding with the incubation proteins evaluated. Analysis of the intact proteins, peptides generated via trypsin digestion of the incubation protein, and amino acids generated via digestion with pronase were evaluated using a combination of LC/MS and LC-MS/MS. Reactive metabolite trapping experiments with glutathione were used to provide information about the likely structure of the bound species and the specificity of binding, and were useful in the development of sensitive targeted precursor ion scanning and multiple reaction monitoring methods. [14C] radiolabelled acetaminophen and SB-649868 were used to assess the quantitative levels of binding (<5% modification of protein in both cases). Radiodetection using accelerator mass spectrometry (AMS) was used to evaluate the stoichiometry of binding and aid the identification of adducted peptides through retention time comparison. Chemical and electrochemical methods were utilised to produce stable solutions of N-acetyl-p-benzoquinone imine (NAPQI) and amodiaquine quinone imine (AQQI), reactive metabolites of acetaminophen and amodiaquine, respectively, which were bound to selected proteins and used as chromatographic and mass spectrometric standards. These methods were used to successfully identify an acetaminophen-modified peptide (T56) of cytochrome P450 CYP2E1. No modified proteins were observed for the SB-649868 incubations, however, examination of the AMS chromatograms for the incubations with acetaminophen and SB-649868 revealed a difference in the stoichiometry of binding, with one modified peptide observed with acetaminophen, and several for the incubations with SB-649868. The detection and identification of drug-protein adducts remains extremely challenging due to the low levels of any adducts observed, which can be exacerbated by binding on multiple sites of a protein; however this project has demonstrated that sensitive and selective LC/MS methods can be successfully developed to identify drug-protein adducts.

Propolis is an approved food additive containing flavonoids as a major active constituent. Variability has been found in the composition of propolis in distinctive regions and it was noticed that there are limitations in the analysis of propolis. In this study, the identification of ten flavonoids and residual antibiotics in propolis was investigated by using several liquid chromatography techniques, including reversed-phase high-performance liquid chromatography (RP-HPLC), microemulsion LC (MELC) and ultra-performance LC (UPLC). The ten flavonoids that were selected for this research include rutin, myricetin, quercetin, apigenin, kaempferol, pinocembrin, CAPE, chrysin, galangin and acacetin while chlortetracycline, oxytetracycline and doxycycline were selected to examine the residual antibiotics in propolis. For the analysis of the selected flavonoids, routine RP-HPLC method was found to be the best method, while MELC technique was found more efficient for the analysis of the selected antibiotics. Solid phase extraction with HLB sorbent was utilised in the analysis of antibiotics for clean-up of propolis. In method development studies for flavonoids and antibiotics, one-factor-at-a-time (OFAT) approach was followed. The final optimised method for the analysis of flavonoids as well as the method. for the analysis of antibiotics was validated using the ICH guidelines, and various aspects, such as the linearity, selectivity, accuracy, recovery, robustness and stability parameters, were examined. Development of efficient conventional method for the extraction of flavonoids from propolis was studied extensively in the present research work using different extraction techniques such as maceration, hot extraction, ultrasound assisted extraction. Among all extraction experiments, ethanolic extraction using ultrasound extraction method was the best efficient approach. This thesis shows that, in general, the performance of O/W MELC is superior to that of conventional HPLC for the determination of residual antibiotics in propolis. UPLC was not suitable for the analysis of flavonoids and antibiotics. The conventional LC was the only technique to separate the ten flavonoids but MELC was able to separate nine of the flavonoids with faster analysis time. This work also showed that MELC uses cheaper solvents. This considerable saving in both cost and time will potentially improve efficiency within quality control.

A acne vulgaris é uma patologia cutânea crônica que acomete a unidade pilossebácea resultando em hiperqueratinização, aumento da produção de sebo, inflamação e proliferação bacteriana nos folículos pilosos. Diversos fármacos estão disponíveis para o tratamento da acne, entretanto a combinação de tratamentos tópicos, com diferentes mecanismos de ação, apresentam uma melhor eficácia. Desse modo, o presente trabalho visa o desenvolvimento de um sistema nanocarreador contendo adapaleno e dapsona no intuito de promover melhor seletividade, aumentando a eficiência do tratamento e minimizando efeitos adversos. Neste contexto, desenvolveu-se e caracterizou-se físico-quimicamente uma formulação de nanocápsulas de núcleo lipídico contendo adapaleno e dapsona. A suspensão contendo 0,025 % de adapaleno e 0,07 % de dapsona apresentou diâmetro médio de 194,9 ± 0,42 nm, índice de polidispersão de 0,12 ± 0,02, potencial zeta -15 ± 1,2 mV, pH 5,1 ± 0,1 e eficiência de encapsulação próximos a 100 %. A avaliação da morfologia por microscopia eletrônica de transmissão demonstrou partículas esféricas, homogêneas de dimensões nanométricas. Através da análise de retroespalhamento de luz dinâmico (Turbiscan®) não se detectou fenômenos de instabilidade na formulação. Uma nanoemulsão, similarmente preparada, mas sem o uso do polímero, foi desenvolvida para comparação a fim de estabelecer o mecanismo de encapsulação dos fármacos. As suspensões foram incorporadas em veículos semissólidos, hidrogéis de Carpobol® 940, e os seguintes estudos foram realizados: comportamento reológico, pH, perfil de liberação, potencial de irritação (HET-CAM) e permeação cutânea in vitro. A determinação quantitativa dos ativos foi realizada por cromatografia líquida de alta eficiência (CLAE), e os métodos analíticos foram desenvolvidos e validados de acordo com os códigos oficiais vigentes. As formulações apresentaram perfil não-Newtoniano pseudoplástico. No estudo de liberação in vitro, não houve diferença significativa entre as formulações contendo dapsona (p>0,05). Já para o adapaleno, não foi possível estabelecer o perfil de liberação in vitro por membrana de diálise. As formulações foram avaliadas como não irritantes pela técnica de HET-CAM. Através dos estudos de permeação cutânea foi possível quantificar os fármacos na epiderme e na derme, e quantidade não-significativa foi detectada no fluído receptor (p>0,05). Comparando as formulações de nanocápsula de núcleo lipídico e nanoemulsão, as quantidades de adapaleno e dapsona em cada camada da pele foi significativamente diferente (p<0,05). Em conclusão, os resultados obtidos até o presente momento demonstram que as nanocápsulas de núcleo lipídico são um sistema promissor para aplicação cutânea de adapaleno e dapsona. Contudo, estudos clínicos fazem-se necessários a fim de avaliar a efetividade da formulação no tratamento da acne. / Acne vulgaris is a chronic skin condition that affects the pilosebaceous unit resulting in hyperkeratinization, increased sebum production, inflammation and bacterial proliferation in hair follicles. Several drugs are available for treating acne, however the combination of topical treatments with different mechanisms of action presents greater effectiveness. Therefore, this study aims to develop a nanocarrier system containing adapalene and dapsone in order to promote better selectivity, increasing efficiency of treatment and minimizing adverse side effects. In this study, a suspension of lipid-core nanocapsules containing adapalene and dapsone was prepared by interfacial deposition of preformed polymer and characterized physicochemically. The suspension containing 0.025 % of adapalene and 0.07 % of dapsone showed an average diameter of 194.9 ± 0.42 nm, a polydispersity index of 0.12 ± 0.02, zeta potential -15 mV ± 1.2, pH 5.1 ± 0.1 and encapsulation efficiency approaching 100 %. The assessment of morphology by transmission electron microscopy showed spherical particles of homogeneous nanometric dimensions. No detectable instability phenomena in the formulation was observed by dynamic light scattering analysis (Turbiscan®). A nanoemulsion, similarly prepared but omitting the polymer, was developed to establish the mechanism of drug encapsulation. The suspensions were incorporated into semisolid vehicles, hydrogels of Carpobol® 940, and the following studies were performed: rheological behavior, pH, release profile, irritation potential (HET-CAM) and skin permeation in vitro. Drug quantification was carried out by high-performance liquid chromatography (HPLC) and analytical methods were developed and validated according to current official guidelines. The formulations exhibited non-Newtonian pseudoplastic profile. In the in vitro release study, no significant difference was found between the formulations containing dapsone (p>0.05). However, for adapalene, it was not possible to establish the in vitro release profile by dialysis membrane. The formulations were evaluated as non-irritant by HET-CAM technique. Through the skin permeation studies, it was possible to quantify the drug in epidermis and dermis, and non-significant quantity was detected in the receptor fluid (p>0.05). Comparing the lipid-core nanocapsules and nanoemulsion formulations, the amount of adapalene and dapsone in each skin layer was significantly different (p<0.05). In conclusion, the results obtained show that the lipid-core nanocapsules are a promising system for cutaneous application of adapalene and dapsone. However, clinical studies are required in order to evaluate the effectiveness of the formulation in the treatment of acne.