The analysis of low abundant biological molecules is often challenging due to their chemical properties, low concentration and limited sample volumes. Neuropeptides are one group of molecules that fits these criteria. Neuropeptides also play an important role in biological functions, which makes them extra interesting to analyze. A classic chemical analysis involves sampling, sample preparation, separation and detection. In this thesis, an enhanced solid supported microdialysis method was developed and used as a combined sampling- and preparation technique. In general, significantly increased extraction efficiency was obtained for all studied peptides. To be able to control the small sample volumes and to minimize the loss of neuropeptides because of unwanted adsorption onto surfaces, the subsequent analysis steps were miniaturized to a micro total analysis system (µ-TAS), which allowed sample pre-treatment, injection, separation, manipulation and detection. In order to incorporate these analysis functions to a microchip, a novel microfabrication protocol was developed. This method facilitated three-dimensional structures to be fabricated without the need of clean room facilities. The sample pre-treatment step was carried out by solid phase extraction from beads packed in the microchip. Femtomole levels of neuropeptides were detected from samples possessing the same properties as microdialysates. The developed injection system made it possible to conduct injections from a liquid chromatographic separation into a capillary electrophoresis channel, which facilitated for advanced multidimensional separations. An electrochemical sample manipulation system was also developed. In the last part, different electrospray emitter tip designs made directly from the edge of the microchip substrate were developed and evaluated. The emitters were proven to be comparable with conventional, capillary based emitters in stability, durability and dynamic flow range. Although additional developments remain, the analysis steps described in this thesis open a door to an integrated, on-line µ-TAS for neuropeptides analysis in complex biological samples.

Advances in life science require knowledge of active molecules in complex biological systems. These molecules are often only present for a certain time and at limited concentrations. Integrated micro-analytical tools for sampling, separation and mass spectrometric (MS) detection would meet these requests and are therefore continuously gaining interest. An on-line coupling of analytical functions provides shorter analysis time and less manual sample handling. In this thesis, improved compatibility of microdialysis sampling and multidimensional separations coupled to MS detection are developed and discussed. Microdialysis was used in vitro for determination of the non-protein bound fraction of the drug ropivacaine. The sampling unit was coupled on-line to capillary column liquid chromatography (LC) followed by ultraviolet or MS detection. For MS detection, the system was extended with a desalting step and an addition of internal standard. A method for MS screening of microdialysates, collected in vivo, was also developed. The method involved sampling and measurements of the chemical pattern of molecules that generally are ignored in clinical investigations. Chemometric tools were used to extract the relevant information and to compare samples from stimulated and control tissues. Complex samples often require separation in more than one dimension. On-line interfaces for sample transfer between LC and capillary electrophoresis (CE) were developed in soft poly(dimethylsiloxane) (PDMS). MS detection in the LC-CE system was optimised on frequent sampling of the CE peak or on high resolution in mass spectra using time-of-flight (TOF)MS or Fourier transform ion cyclotron resonance (FTICR)MS, respectively. Aspects on electrode positioning in the LC-CE interface led to development of an on-column CE electrode. A successful method for deactivation of the PDMS surface using a polyamine polymer was also developed. The systems were evaluated using peptides and proteins, molecules that are gaining increased attention in bioscience, and consequently also in chemical analysis.

This dissertation describes the advantages and feasibility of newly synthesized near-infrared (NIR) bis-heptamethine cyanine (BHmC) dyes for non-covalent labeling schemes. The NIR BHmCs were synthesized for biomolecule assay. The advantages of NIR BHmCs for biomolecule labeling and the instrumental advantages of the near-infrared region are also demonstrated. Chapter 1 introduces the theory and applications of dye chemistry. For bioanalysis, this chapter presents covalent and non-covalent labeling. The covalent labeling depends on the functionality of amino acids and the non-covalent labeling relies on the binding site of a protein. Due to the complicated binding process in non-covalent labeling, this chapter also discusses the binding equilibria in spectroscopic and chromatographic analyses. Chapter 2 and 3 evaluate the novel BHmCs for non-covalent labeling with human serum albumin (HSA) and report the influence of micro-environment on BHmCs. The interesting character of BHmCs in aqueous solutions is that the dyes exhibit non- or low-fluorescence compared to their monomer counterpart, RK780. It is due to their H-type closed clam-shell form in the solutions. The addition of HSA or organic solvents opens up the clam-shell form and enhances fluorescence. The binding equilibria are also examed. Chapter 4 provides a brief introduction that summaries the use of capillary electrophoresis (CE), and offers a detailed instrumentation that discusses the importance and advantage of a detector in NIR region for CE separation. Chapter 5 focuses on the use of NIR cyanine dyes with capillary electrcophoresis with near-infrared laser induce fluorescence (CE-NIR-LIF) detection. The NIR dyes with different functional groups show that RK780 is a suitable NIR dye for HSA labeling. The use of BHmCs with CE-NIR-LIF reduces signal noises that are commonly caused by the interaction between NIR cyanine dyes and negatively charged capillary wall. In addition, bovine carbonic anhydrase II (BCA II) is applied to study the influence of hydrophobicity on non-covalent labeling. Finally, chapter 6 presents the conformational dependency of BHmCs on the mobility in capillary and evaluates the further possibility of BHmCs for small molecule detection. Acridine orange (AO) is used as a sample and it breaks up the aggregate and enhances fluorescence. The inserted AO into BHmC changes the mobility in capillary, owing to the conformational changes by AO.

Le vieillissement de la peau est l'un des signes extérieurs du passage du temps. Avec l’âge, la peau devient plus sèche et se ride suite à la dégradation des macromolécules de la matrice extracellulaire par des enzymes cutanées telles que l’élastase, l’hyaluronidase et la collagénase. L’objectif de ce travail de thèse est de développer des tests enzymatiques miniaturisés en électrophorèse capillaire pour cribler des extraits de plantes et identifier de nouveaux bioactifs pour la cosmétique et le bienêtre de la peau. Ces essais ont été développés soit en dehors du capillaire (qui sert uniquement de milieu de séparation) ou dans le capillaire (qui sert alors de nanoréacteur enzymatique), puis optimisés pour permettre la détermination des constantes cinétiques (Km, Vmax et IC₅₀). La diffusion transversale des réactifs (TDLFP) a été appliquée pour mélanger les créneaux de réactifs injectés dans le capillaire. Des détecteurs tels que la fluorescence induite par laser ou la spectrométrie de masse à haute résolution ont été couplés à l’électrophorèse capillaire afin d’atteindre de fortes sensibilités de détection et la possibilité d’identifier les produits de la réaction enzymatique. Ces essais miniaturisés ont été appliqués à des algues extraites par électroporation ou à des plantes régionales extraites par des technologies vertes afin d’évaluer leur activité biologique vis-à-vis des enzymes de la peau. Les essais développés sont fiables, robustes et économes en réactifs. Enfin, l’utilisation d’une nouvelle technique d’analyse, la thermophorèse à micro-échelle, s’est montrée très utile et pleine d’espoir pour l’étude des interactions enzyme-effecteur. / Skin aging is one of the exterior/external signs of the passage of time. With age, the skin becomes drier and gets wrinkled due to the degradation of macromolecules of the extracellular matrix by skin enzymes such as elastase, hyaluronidase and collagenase. The aim of this thesis is to develop miniaturized enzymatic assays by capillary electrophoresis to screen plant extracts and identify new bioactives for cosmetics and skin wellbeing. These assays were developed either outside the capillary (which serves only as a separation tool) or in the capillary (which then serves as an enzymatic nanoreactor) then optimized to allow the determination of kinetic constants (Km, Vmax and IC₅₀). Tranvserse diffusion of laminar flow profiles (TDLFP) was applied to mix the reactants injected into the capillary. Detectors such as laser-induced fluorescence or high-resolution mass spectrometry have been coupled to capillary electrophoresis to achieve high sensitivities of detection and the possibility of identifying the products of the enzymatic reaction. These miniaturized assays were applied to algae extracted by electroporation or to regional plants extracted by green technologies in order to evaluate their biological activity towards skin enzymes. The assays developed are reliable, robust and economic in reactants consumption. Finally, the use of a new analytical technique, microscale thermophoresis, was shown to be very useful and hopeful for the study of enzyme-effector interactions.

A análise de fármacos é fundamental nas diversas fases do desenvolvimento farmacêutico, tais como em estudos de formulação, estabilidade e controle de qualidade do produto. O mesilato de gemifloxacino (MGF), liberado para uso clínico no Brasil em novembro de 2006 com o nome comercial de Factive®, é uma fluorquinolona indicada para o tratamento da exacerbação aguda da bronquite crônica e da pneumonia adquirida da comunidade. A literatura pesquisada apresenta poucos relatos de determinação quantitativa e de estudos de estabilidade do fármaco em comprimidos revestidos. Anteriormente aos estudos, foi realizada a caracterização da substância química de referência (SQR) de MGF por espectrofotometria no infravermelho (E IV), ressonância magnética nuclear de hidrogênio (RMN 1H) e carbono (RMN 13C), análise térmica por calorimetria exploratória de varredura (DSC) e determinação da faixa de fusão. Métodos analíticos para determinação qualitativa e quantitativa foram desenvolvidos e validados por espectrofotometria na região do ultravioleta (E UV) e visível (E VIS), cromatografia líquida de alta eficiência (CLAE), eletroforese capilar (EC) e ensaio microbiológico pelo método de cilindros em placas. A validação de um método de dissolução baseado em dados in vivo do fármaco também foi realizada. A elucidação do produto de degradação isolado em condições alcalinas foi realizada por E IV, RMN de 1H, 13C e correlação (COSY, HSQC e HMBC), espectrometria de massas (EM) e emissão atômica. Estudos de citotoxicidade, fototoxicidade, genotoxicidade e fotogenotoxicidade foram empregados para conhecimento da toxicidade dos produtos analisados. / The drug analysis is essential in all areas of the pharmaceutical development, such as during formulation studies, stability and quality control of the product. Gemifloxacin mesylate (GFM), approved for clinical use in Brazil in November of 2007 with the commercial name of Factive®, is a fluoroquinolone prescribed for the treatment of acute exacerbations of chronic bronchitis and community-acquired pneumonia. The research literature shows a few studies of quantitative determination and stabilities studies of the drug in coated tablets. Previously, it was performed the characterization of the reference chemical substance of GFM by infrared spectrometry (IR), nuclear magnetic resonance of 1H (1H NMR) and 13C (13C NMR), thermal analysis by differential scanning calorimetry (DSC) and determination of the melting range. Analytical methods for qualitative and quantitative determination were developed and validated by ultraviolet (UV) and visible (Vis) spectrophotometry, highperformance liquid chromatography (HPLC), capillary electrophoresis (CE) and microbiological assay applying the cylinder–plate method. The validation of the dissolution method based on in vivo data of the GFM was also performed. The elucidation of the isolate degradation product in alkaline conditions was performed by IR, 1H, 13C and correlation (COSY, HSQC and HMBC) NMR, and mass spectrometry (MS). Cytotoxicity, phototoxicity, genotoxicity and photogenotoxicity studies were carried out for the toxicity knowledge of the analyzed products.

Objetivos: os objetivos gerais deste trabalho foram desenvolver, validar e comparar métodos indicativos de estabilidade para a análise do cloridrato de butenafina (BTF), matéria-prima e forma farmacêutica, bem como determinar a cinética de degradação do fármaco em condição de estresse. Adicionalmente, o trabalho visou à validação de método para avaliar e comparar a penetração/permeação cutânea da BTF de diferentes formulações. Métodos: Um método indicativo de estabilidade para análise da BTF por cromatografia líquida de alta eficiência (CLAE) foi desenvolvido e validado, conforme normas do ICH. A cinética de degradação do fármaco (matériaprima e creme) frente à luz UVC foi determinada. Testes para o desenvolvimento de método quantitativo por eletroforese capilar para a análise do fármaco isoladamente e presente na formulação foram realizados. O método analítico cromatográfico previamente desenvolvido para a formulação semi-sólida foi validado para a quantificação de BTF na pele suína. Em seguida, as penetrações/permeações cutâneas do fármaco utilizando células de Franz foram analisadas visando à comparação de duas formulações comerciais (uma delas brasileira e a outra americana). Resultados e Conclusões: O método de CLAE indicativo de estabilidade desenvolvido demonstrou ser adequado para a determinação da substância ativa na formulação mesmo na presença de produtos de degradação, bem como para a quantificação do fármaco na pele suína e no fluído receptor. Os principais fatores extrínsecos que promovem a degradação do fármaco foram estabelecidos: luz, oxidação e meio básico (este último somente na presença de excipientes). A determinação da cinética de fotodegradação como sendo de primeira ordem demonstra que o processo é dependente da concentração do fármaco, reforçando a necessidade de proteção frente à luz. Já o método por eletroforese capilar mostrou-se não específico frente à formulação placebo simulado, inviabilizando seu uso para o creme. Na avaliação da permeação cutânea das formulações brasileira e americana não foi detectada presença considerável do fármaco no fluído receptor para ambos os produtos. Já na verificação da penetração, não houve diferença significativa na retenção do fármaco na epiderme, entretanto, na derme a diferença foi estatisticamente significativa, com maior concentração retida na análise da formulação americana (α = 0,05). / Objectives: The aim of the present work was to develop, validate and compare stability indicating methods to quantify butenafine hydrochloride (BTF), raw material and commercial cream, as well as to establish the degradation kinetics of the drug in a stress condition. Also, the study had the goal of validating a method to assess and compare the cutaneous permeation/penetration of two different formulations. Methods: A stability-indicating method for the analysis of BTF by high performance liquid chromatography (HPLC) was developed and validated according to the ICH guidelines. The degradation kinetics of the drug (raw material and cream) under the UVC light was determined. Analyzes to develop and validate an analytical method by capillary electrophoresis, to quantify the drug by itself and in the cream, were performed. The chromatographic method previously developed for the semi-solid product was validated to quantify the drug in porcine skin. Then, the cutaneous permeation/penetration of BTF, through the Franz-type cell, was analyzed to compare two different commercial formulations (one of them is Brazilian and the other is American). Results and Conclusions: The developed stability-indicating method was adequate to determine the active substance in the formulation even in the presence of the degradation products, as well as to quantify the drug in porcine skin and in the receptor fluid. The main extrinsic factors that promote the drug degradation were established: light, oxidative and basic media (the last in the presence of the excipient ingredients). The photodegradation kinetics was determined as first order showing that the process is dependent on the drug concentration, reaffirming the necessity of protection against light. The method by capillary electrophoresis was not specific considering the placebo formulation, hindering its use to the cream. During the cutaneous permeation analysis, no drug was found in the receptor media of both the Brazilian and the American formulations. No difference was verified to the epidermis, although to the dermis there was a statistical distinction as the concentration of retained drug from the American formulation was higher (α = 0,05).

Les enzymes sont les catalyseurs de toutes les réactions biochimiques. Leur dérégulation peut être impliquée dans de nombreuses pathologies graves. L’étude de ces réactions est importante pour dépister les anomalies, mieux comprendre le fonctionnement des enzymes et rechercher des modulateurs de leur activité. Le présent travail de thèse présente différents types d’essais enzymatiques basés sur l’électrophorèse capillaire pour étudier la cinétique d’enzymes variées. Le mode pré-capillaire où la réaction enzymatique est effectuée à l’extérieur du capillaire et les essais homogènes en-ligne en mode discontinu où la réaction est réalisée dans le capillaire, sont appliqués. Les méthodes développées sont optimisées pour assurer un mélange optimal des réactifs et une bonne séparation électrophorétique. Les constantes cinétiques et d’inhibition (Vmax, Km et IC50) de la réaction enzymatique sont déterminées et comparées à celles obtenues par les techniques classiques. Pour les essais en-ligne, plusieurs types de mélange (par application d’un champ électrique, par diffusion longitudinale ou diffusion transversale) des créneaux de réactifs sont utilisés selon le système enzymatique étudié. Finalement, jusqu’à quatre réactifs injectés successivement dans le capillaire sont mélangés avec succès. De nombreux essais sont effectués sur des matrices complexes (cellules, extraits de plantes). Le criblage d’inhibiteurs de référence et de synthèse est réalisé sur plusieurs kinases humaines : CDK1/cycline B, CDK5/p25, DYRK1A, GSK3!, PI3K, Akt et mTOR. Les essais développés se sont avérés être simples, rapides, quantitatifs, économes en réactifs et répétables. / Enzymes catalyze all enzymatic reactions. Their deregulation can be involved in several severe diseases. The study of these reactions is important to detect anomalies, to better understand enzyme functioning and to seek modulators of their activity. This thesis presents capillary electrophoresis based enzymatic assays developed to study kinetics of various enzymes. The pre-capillary mode in which the enzymatic reaction occurs outside the capillary and the incapillary plug-plug mode of homogenous assays where the reaction is performed inside the capillary are applied. The methods developed are optimized to ensure optimum reactant mixing and a good electrophoretic separation. The kinetic and inhibition constants (Vmax, Km and IC50) of the enzymatic reaction are determined by these assays and compared to the results obtained using conventional techniques. For in-capillary assays, several mixing types (by application of an electric field, by longitudinal diffusion or transverse diffusion) of the reactant plugs are used depending on the enzymatic system studied. Finally, up to four reactants injected successively in the capillary are successfully mixed. Many assays are performed on complex matrices (cells, plant extracts). Screening of referenced and synthesized inhibitors on several human kinases: CDK1/cyclin B, CDK5/p25, DYRK1A, GSK3!, PI3K, Akt and mTOR are performed. Developed assays proved to be quantitative, simple, economic, fast and robust.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This paper describes the development of methodology for capillary electrophoresis with UV detection for the determination of 14 polyphenolic antioxidants in extracts of Connarus perrottetii var. angustifolius Radlk, a native plant of the Amazon forest. The method using capillary zone electrophoresis (CZE) allows simultaneous determination of 3-acetyilcoumarine, resveratrol, 6-hydroxycoumarin, catechin, rutin, ferulic acid, quercitrin, kaempferol, fisetin, myricetin, quercetin, caffeic acid, gallic acid and 4 hydroxicinnamic acid in the optimized conditions: borate buffer 20 mmol L-1 (pH 9.2) as work electrolyte containing 15% methanol (v /v), -20 kV potential separation, temperature 25 °C; hydrodynamic injection gravity 20 cm for 60 s. The method was validated on parameters of linearity, limit of detection, quantification limit, precision and accuracy, and was used in the analysis of aqueous infusion and ethanol, butanol and ethyl acetate plant fractions. The method was capable of identifying the antioxidants present in samples with high selectivity and sensitivity. In infusion and in ethanol and butanol fractions was found the presence of catechin and rutin as major compounds, which makes up about 0.5% of the total mass of medicinal plant. The obtained dry extracts (ethanol and butanol) allowed the concentration of rutin and catechin around 20 times compared to the dry plant. In addition, tests were performed to measure the antioxidant activity of the extracts against DPPH radical, superoxide anion radical, hydroxyl and peroxyl. A sample of aqueous infusion of 10% showed the highest activity against most free radicals. It corroborates the results of characterization by capillary electrophoresis, where catechin and rutin were determined in relatively high concentrations in the aqueous infusion. But all treatments of the samples showed good antiradicalar behavior with the methodologies applied. Against DPPH, for example, the ethyl acetate fraction showed a potential of 94,43%. Against superoxide and hydroxyl radicals, the butanol fraction showed the best performance: 64,23% and 74,97%, respectively. / Este trabalho descreve o desenvolvimento de metodologia em eletroforese capilar com detecção UV para a determinação de 14 antioxidantes polifenólicos em extratos de Connarus perrottetii var. angustifolius Radlk, planta nativa da Amazônia brasileira. O método empregando eletroforese capilar de zona (CZE) permite a determinação simultânea de 3-acetilcumarina, resveratrol, 6-hidroxicumarina, catequina, rutina, ácido ferúlico, quercitrina, canferol, fisetina, miricetina, quercetina, ácido cafeico, ácido gálico e ácido 4-hidroxicinâmco nas condições otimizadas: eletrólito de trabalho tampão borato 20 mmol L-1 (pH 9,2) contendo metanol 15% (v/v), potencial de separação -20 Kv, temperatura 25 °C; injeção hidrodinâmica por gravidade em 20 cm durante 60 s. O método foi validado nos parâmetros de linearidade, limite de detecção, limite de quantificação, precisão e exatidão e foi aplicado na análise da infusão aquosa e frações etanólica, butanólica e acetato de etila da planta. O método foi capaz de identificar os antioxidantes presentes nas amostras com alta seletividade e sensibilidade. Na infusão e nas frações etanólica e butanólica foi encontrada a presença de catequina e rutina como compostos majoritários, os quais compõe cerca de 0,5% da massa total de planta medicinal. Os extratos secos obtidos (etanólico e butanólico) possibilitaram a concentração de rutina e catequina em torno de 20 vezes em relação à planta seca. Além disso, foram realizados testes para mensurar a atividade antioxidante dos extratos frente aos radicais DPPH, ânion radical superóxido, hidroxila e peroxila. A amostra de infusão aquosa a 10% foi a que apresentou maior atividade frente à maioria dos radicais livres. Esse resultado corrobora os resultados de caracterização por eletroforese capilar, onde catequina e rutina foram determinadas em concentrações relativamente altas na infusão aquosa. Porém, todos os tratamentos das amostras apresentaram bom comportamento anti-radicalar com as metodologias aplicadas. Frente ao radical DPPH, por exemplo, a fração acetato de etila demonstrou um potencial de 94,43%. Frente aos radicais superóxido e hidroxila, a fração butanólica apresentou o melhor desempenho: 64,23% e 74,97%, respectivamente.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Dronedarone is a new antiarrhythmic agent and amiodarone analogue developed to reduce the toxic effects related to amiodarone. Dronedarone was approved for the maintenance of the sinus rhythmic in adult patients with atrial fibrillation and to reduce the risk of hospitalization in these patients. It is commercially available as film-coated tablets. There are no official monographs in any pharmacopoeia or analytical methods described in the literature for the analysis of dronedarone in pharmaceutical dosage form or bulk form. In the present study, an analytical methodology was developed for the analysis of dronedarone in pharmaceutical dosage form and bulk form. The reversed-phase liquid chromatography method was performed using a Waters XBridge C18 column (250 mm × 4.6 mm). The mobile phase consisted of buffer solution pH 4.9 / acetonitrile (35:65, v/v), running at a flow rate of 1.0 mL/min, using photodiode array detector set at 289 nm. The chromatographic separation was obtained within 7.0 min and it was linear in the concentration range from 5.0 to 100.0 μg/mL (r = 0.9999). The spectrophotometric method was developed and validated and dronedarone was quantified at 289 nm, using methanol as diluent. The micellar electrokinetic method was also developed and validated, using nimesulide as internal standard. The analyses were performed on a fused-silica capillary (50 μm i.d.; effective length, 40 cm), using electrolyte solution consisted of 40 mm borate buffer and 50 mM SDS at pH 9.2, with detection by photodiode array detector set at 216 nm. The injection was performed using the hydrodynamic mode at 50 mbar for 7 s and a constant voltage of 28 kV was applied during analysis. The electrophoretic separation was obtained within 7.0 min and it was linear in the concentration range from 25 to 150 μg/mL (r = 0.9995). The procedures were validated evaluating parameters such as the specificity, linearity, precision, accuracy, limits of detection and quantitation and robustness, giving results within the acceptable range. The proposed methods were applied for the analysis of the pharmaceutical product, showing significant correlation between the results (p > 0.05). The spectrophotometric method was developed and validated using acetate buffer pH 4.5 as diluent and UV detection at 289 nm, which was applied to evaluate the dissolution test. The dissolution test was developed using 900 mL of acetate buffer pH 4.5 at 37°C, as dissolution medium, and paddle as apparatus at a stirring rate of 75 rpm. / A dronedarona é um novo agente antiarrítmico análogo à amiodarona, desenvolvido com o propósito de reduzir os efeitos adversos relacionados à amiodarona. Foi aprovado para a manutenção do ritmo cardíaco normal em pacientes com fibrilação atrial e, assim, reduzir os riscos de hospitalização. Comercialmente, encontra-se disponível na forma farmacêutica de comprimidos revestidos. Não há monografias descritas em farmacopeias ou métodos na literatura para análise de dronedarona em forma farmacêutica ou matéria-prima. No presente trabalho, foi desenvolvida metodologia para a avaliação de dronedarona em forma farmacêutica e matéria-prima. O método por cromatografia líquida em fase reversa foi realizado utilizando-se coluna Waters XBridge C18 (250 mm × 4,6 mm). A fase móvel foi composta por solução tampão pH 4,9 / acetonitrila (35:65, v/v) eluída no fluxo de 1,0 mL/min e detecção no ultravioleta em 289 nm. A separação cromatográfica foi obtida no tempo de 7,0 min, sendo linear na faixa de concentração de 5-100 μg/mL (r = 0,9999). Paralelamente, desenvolveu-se e validou-se método por espectrofotometria no ultravioleta em 289 nm utilizando metanol como diluente. Também foi desenvolvido e validado método por cromatografia eletrocinética micelar utilizando nimesulida como padrão interno. As análises foram realizadas em capilar de sílica fundida (comprimento efetivo de 40 cm e diâmetro de 50 μm), mantido a 30°C, utilizando solução eletrolítica composta de tampão borato 40 mM e SDS 50 mM, pH 9,2, com detecção no ultravioleta em 216 nm. A injeção foi realizada no modo hidrodinâmico a 50 mbar durante 7 s e voltagem constante de 28 kV foi aplicada durante as análises. A separação eletroforética foi obtida em 7,0 min, sendo linear na faixa de 25-150 μg/mL (r = 0,9995). Os procedimentos foram validados considerando-se os parâmetros especificidade, linearidade, precisão, exatidão, limite de detecção e quantificação e robustez, cujos resultados cumpriram os requisitos preconizados. Os métodos propostos foram aplicados na análise quantitativa de produtos farmacêuticos, demonstrando correlação significativa dos resultados (p > 0,05). Desenvolveu-se e validou-se método por espectrofotometria no UV utilizando tampão acetato pH 4,5 como diluente e detecção em 289 nm, o qual foi aplicado para avaliar a percentagem dissolvida dos comprimidos de dronedarona. O método de dissolução foi desenvolvido utilizando como meio 900 mL de tampão acetato pH 4,5 mantido a 37°C, aparato pá e rotação de 75 rpm.

A análise de fármacos é fundamental nas diversas fases do desenvolvimento farmacêutico, tais como em estudos de formulação, estabilidade e controle de qualidade do produto. O mesilato de gemifloxacino (MGF), liberado para uso clínico no Brasil em novembro de 2006 com o nome comercial de Factive®, é uma fluorquinolona indicada para o tratamento da exacerbação aguda da bronquite crônica e da pneumonia adquirida da comunidade. A literatura pesquisada apresenta poucos relatos de determinação quantitativa e de estudos de estabilidade do fármaco em comprimidos revestidos. Anteriormente aos estudos, foi realizada a caracterização da substância química de referência (SQR) de MGF por espectrofotometria no infravermelho (E IV), ressonância magnética nuclear de hidrogênio (RMN 1H) e carbono (RMN 13C), análise térmica por calorimetria exploratória de varredura (DSC) e determinação da faixa de fusão. Métodos analíticos para determinação qualitativa e quantitativa foram desenvolvidos e validados por espectrofotometria na região do ultravioleta (E UV) e visível (E VIS), cromatografia líquida de alta eficiência (CLAE), eletroforese capilar (EC) e ensaio microbiológico pelo método de cilindros em placas. A validação de um método de dissolução baseado em dados in vivo do fármaco também foi realizada. A elucidação do produto de degradação isolado em condições alcalinas foi realizada por E IV, RMN de 1H, 13C e correlação (COSY, HSQC e HMBC), espectrometria de massas (EM) e emissão atômica. Estudos de citotoxicidade, fototoxicidade, genotoxicidade e fotogenotoxicidade foram empregados para conhecimento da toxicidade dos produtos analisados. / The drug analysis is essential in all areas of the pharmaceutical development, such as during formulation studies, stability and quality control of the product. Gemifloxacin mesylate (GFM), approved for clinical use in Brazil in November of 2007 with the commercial name of Factive®, is a fluoroquinolone prescribed for the treatment of acute exacerbations of chronic bronchitis and community-acquired pneumonia. The research literature shows a few studies of quantitative determination and stabilities studies of the drug in coated tablets. Previously, it was performed the characterization of the reference chemical substance of GFM by infrared spectrometry (IR), nuclear magnetic resonance of 1H (1H NMR) and 13C (13C NMR), thermal analysis by differential scanning calorimetry (DSC) and determination of the melting range. Analytical methods for qualitative and quantitative determination were developed and validated by ultraviolet (UV) and visible (Vis) spectrophotometry, highperformance liquid chromatography (HPLC), capillary electrophoresis (CE) and microbiological assay applying the cylinder–plate method. The validation of the dissolution method based on in vivo data of the GFM was also performed. The elucidation of the isolate degradation product in alkaline conditions was performed by IR, 1H, 13C and correlation (COSY, HSQC and HMBC) NMR, and mass spectrometry (MS). Cytotoxicity, phototoxicity, genotoxicity and photogenotoxicity studies were carried out for the toxicity knowledge of the analyzed products.