Ketoprofen is a potent non-steroidal anti-inflammatory drug which is used for the treatment of rheumatoid arthritis. The oral administration of ketoprofen can cause gastric irritation and adverse renal effects. Transdermal delivery of the drug can bypass gastrointestinal disturbances and provide relatively consistent drug concentrations at the site of administration. The release of ketoprofen from proprietary gel products from three different countries was evaluated by comparing the in vitro release profiles. Twenty extemporaneously prepared ketoprofen gel formulations using Carbopol® polymers were manufactured. The effect of polymer, drug concentration, pH and solvent systems on the in vitro release of ketoprofen from these formulations were investigated. The gels were evaluated for drug content and pH. The release of the drug from all the formulations obeyed the Higuchi principle. Two static FDA approved diffusion cells, namely the modified Franz diffusion cell and the European Pharmacopoeia diffusion cell, were compared by measuring the in vitro release rate of ketoprofen from all the gel formulations through a synthetic silicone membrane. High-performance liquid chromatography and ultraviolet spectrophotometric analytical techniques were both used for the analysis of ketoprofen. The validated methods were employed for the determination of ketoprofen in the sample solutions taken from the receptor fluid. Two of the three proprietary products registered under the same manufacturing license exhibited similar results whereas the third product differed significantly. Among the variables investigated, the vehicle pH and solvent composition were found have the most significant effect on the in vitro release of ketoprofen from Carbopol® polymers. The different grades of Carbopol® polymers showed statistically significantly different release kinetics with respect to lag time. When evaluating the proprietary products, both the modified Franz diffusion cell and the European Pharmacopoeia diffusion cell were deemed adequate although higher profiles were generally obtained from the European Pharmacopoeia diffusion cells. Smoother diffusion profiles were obtained from samples analysed by high-performance liquid chromatography than by ultraviolet spectrophotometry in both diffusion cells. Sample solutions taken from Franz diffusion cells and analysed by ultraviolet spectrophotometry also produced smooth diffusion profiles. Erratic and higher diffusion profiles were observed with samples taken from the European Pharmacopoeia diffusion cell and analysed by ultraviolet spectrophotometry. The choice of diffusion cells and analytical procedure in product development must be weighed against the relatively poor reproducibility as observed with the European Pharmacopoeia diffusion cell.

There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.

The human skin blanching assay (HSBA) is a well-researched and validated method for the bioequivalence assessment of topical corticosteroids. Traditionally, visual assessment of skin blanching has been used. Such testing methods are not conducive for interlaboratory comparisons. Regulatory bodies prefer less subjective methods of analysis. The FDA released guidelines on the assessment of bioequivalence for topical corticosteroids that recommends the use of a chromameter as a reliable method to measure skin blanching although the use of visual assessment with acceptable validation is also provided for. However, the FDA does not elucidate on the manipulation and handling of the chromameter during skin blanching measurements. The purpose of this project was several fold, which included investigations to standardize the manipulation and handling of a chromameter. In particular, measures to avoid skin whitening resulting from the effects of pressure on the skin during chromameter use were investigated. Other methods of analysis should surpass or at least be comparable to the HSBA if such methods are to be used for the assessment of topical corticosteroids. Microdialysis is a relatively new technique for assessing the rate at which drug penetrates the skin. The advantage of using this method is that there are fewer restrictions for selection of an appropriate study population unlike those required for the HSBA where one has to be both a ‘responder’ and a ‘detector’ for their results to be used in data analysis. Microdialysis was investigated by initially conducting experiments in which microdialysis probes were embedded into topical formulations containing mometasone furoate (MF) and the initial results revealed that relatively low drug was released from the formulations. These results indicated that should microdialysis be applied to measure the in vivo release of MF from such topical formulations following application to the skin, even lower concentrations of MF would likely result in the dialysate, necessitating the need for ultra-high sensitive methods of analysis. Typically, the availability of an appropriate analytical technique such as liquid chromatography coupled with mass spectrometry (LCMS) would be a pre-requisite for such in vivo studies. However, only high-pressure liquid chromatography (HPLC) and other less sensitive equipment was available in the laboratories. The study objectives were therefore focussed on in vitro assessment of the release of MF from topical formulations using microdialysis and Franz cells. In addition, the in vivo release of MF was also studied using the HSBA. Data obtained from the microdialysis experiments were compared with the data obtained from the Franz cell diffusion studies in order to provide information on the pharmaceutical availability of MF from the various topical MF dosage forms. Subsequently, pharmaceutical equivalence was investigated from the comparative pharmaceutical availability data using statistical analysis. An additional objective was to attempt to correlate in vitro with in vivo data (IVIVC) to establish a model that could be used to assess safety and efficacy of generic topical drug products. The in vivo data obtained from the HSBA were processed according to the FDA requirements and these pharmacodynamic data were subsequently compared with the microdialysis and Franz cell results. In summary the objectives of this project were: 1. To develop a system to improve the reproducibility of the use of a Minolta® chromameter and compare this with the standard/normal manipulation and handling of such instruments. 2. To develop and validate an HPLC method for the analysis of MF for use with in vitro diffusion studies using microdialysis and Franz cells. 3. To conduct a comparative HSBA on proprietary MF topical creams from two different countries in accordance with the FDA guidance. 4. To assess the pharmaceutical equivalence of topical formulations containing MF using Franz diffusion cells and in vitro microdialysis. 5. To compare the in vivo data obtained from the HSBA with those obtained in vitro using microdialysis and Franz cells.

Orientador: Rodnei Bertazzoli / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica / Made available in DSpace on 2018-08-24T13:08:38Z (GMT). No. of bitstreams: 1 Marques_TamiresValim_M.pdf: 6834346 bytes, checksum: 5eb1bc6d88a438ca9002c92109a6c660 (MD5) Previous issue date: 2014 / Resumo: A crescente preocupação com a poluição das águas por novos poluentes denominados emergentes tem se intensificado, visto que aumentou o número destes compostos detectados em água. Dentre estes compostos encontram-se os fármacos e produtos de higiene pessoal, usados cotidianamente pela população. O presente trabalho teve como objetivo o desenvolvimento de um método simples, rápido e sensível utilizando a microextração líquido líquido (DLLME) combinada com a cromatografia líquida de ultra eficiência (HPLC) com detecção ultravioleta (UV) para a determinação de alguns benzodiazepínicos (bromazepam, clonazepam e diazepam) em amostras de água. A determinação foi realizada em uma coluna C18 de acordo com as condições cromatográficas ótimas (fase móvel acetonitrila:água (60:40, v/v); vazão 1,2 mL min-1; detecção 225 nm). No método de extração, uma mistura apropriada de solvente extrator e dispersor foi injetada rapidamente na amostra aquosa (10 mL) com auxílio de uma seringa. De modo que uma solução turva foi formada, esta solução é caracterizada por conter pequenas partículas do solvente extrator que se dispersa na fase aquosa. Os parâmetros da extração, tais como natureza e volume dos solventes extrator e dispersor, tempo de extração, pH da amostra, força iônica, velocidade e tempo de centrifugação, foram estudados para a otimização. Com as condições ótimas definidas (solvente extrator: clorofórmio, 200 ?L; solvente dispersor: metanol, 700 ?L; pH da amostra 9,0; velocidade e tempo de extração: 5000 rpm, 1 minuto; força iônica: adição de 1% (p/v) de (NH4)2SO4) o método proposto foi validado seguindo as figuras de método preconizadas pela ANVISA na Resolução N° 899 de 2003. A faixa linear para cada fármaco foram 8,0 - 96 ?g L-1 para bromazepam, 4,0 - 48 ?g L-1 para clonazepam e 1,0 - 12 ?g L-1 para diazepam. Todas as curvas obtiveram valores de (r) superiores a 0,999. Os limites de detecção e quantificação obtidos foram 2,4 e 8,0 ?g L-1 para bromazepam, 1,2 e 4,0 ?g L-1 para clonazepam, 0,2 e 1,0 ?g L-1 para diazepam, respectivamente. As recuperações variaram de 50 a 110% com RSD (Desvio Padrão Relativo) inferiores a 12,7 %. Finalmente, o método proposto foi aplicado em amostras coletadas na represa Billings localizada no município de Diadema-SP. / Abstract: The growing concern over water pollution caused by so-called new emerging pollutants has intensified since increased the number of these compounds detected in water. Among these compounds are pharmaceuticals and personal care products used daily by the population. This study aimed to develop a simple, rapid and sensitive liquid using liquid micro extraction (DLLME) combined with ultra-performance liquid chromatography (HPLC ) with ultraviolet detection (UV) for the determination of some benzodiazepines (bromazepam, clonazepam and diazepam) in water samples . The determination was performed on a C18 column in accordance with the optimal chromatographic conditions (mobile phase acetonitrile: water (60:40, v/v), flow rate 1.2 mL min-1, detection 225 nm). In the extraction method, a suitable mixture of extractant and dispersing solvent was injected rapidly into the aqueous sample (10 ml) with a syringe. So that a cloudy solution was formed, this solution is characterized by containing fine drops of the extractor solvent is dispersed in the aqueous phase. The parameters of the extraction, such as the nature and volume of the extractor and disperser solvents, extraction time, sample pH, ionic strength, speed and time of centrifugation, were studied for optimization. With the defined optimal conditions (extracting solvent: chloroform, 200 ?L; disperser solvent: methanol, 700 ?L, sample pH 9.0, extraction time and speed: 5000 rpm, 1 minute; ionic strength: adding 1% (p/v) (NH4)2SO4) the proposed method was validated following the figures of merit recommended by the ANVISA Resolution No. 899 of 2003. The linear ranges for each drug were 8.0 to 96 ?g L- 1 for bromazepam, 4.0 to 48 ?g L- 1 for clonazepam and 1.0 to 12 ?g L- 1 for diazepam. All curves obtained values (r) greater than 0.999. The limits of detection and quantification obtained were 2.4 and 8.0 ?g L- 1 to bromazepam, 1.2 and 4.0 ?g L- 1 to clonazepam, 0.2 and 1.0 ?g L- 1 for diazepam, respectively. Recoveries ranged from 50 to 110% with RSD (Relative Standard Deviation) of less than 12.7%. Finally, the proposed method was applied to samples collected in the Billings dam located in Diadema-SP. / Mestrado / Materiais e Processos de Fabricação / Mestre em Engenharia Mecânica

Rhodococcushas been recognized as a potential antibiotic producer. Recently, a strain of Rhodococcussp. MTM3W5.2 was isolated from a soil sample collected in Morristown, Tennessee and was found to produce an inhibitory compound which is active against other related species. The purpose of this research is to extract, purify and analyze the active metabolite. The compound was extracted from RM broth cultures and purified by preliminary fractionation of crude extract through a Sephadex LH-20 column. Further purification was completed using semi-preparative reversed phase column chromatography. Final purification was obtained using multiple rounds of an analytical C18 HPLC column. Based on the results achieved in the UV-Vis spectroscopy and high-resolution mass spectroscopy, the two desired compounds at a retention time of at 57 and 72 min could be polyketides with the molecular formulas C52H78O13 and C19H32O1N1/C13H34O1N1, respectively.

This thesis deals with sorption of selected drugs from model water by various sorption materials. Contamination of water resources by the pharmaceutical industry is a major problem today. Wastewater treatment plants, whose technological processes are unable to completely remove them, have a significant share in the penetration of these substances into the environment. At present, attention is paid to alternative materials that are capable of eliminating these substances. One of the potential sorption materials is biochar as one of the main pyrolysis products. This work focused on the assessment of the sorption properties of the different types of biochar and commercially used active charcoal. The sorption properties of the individual materials were compared with respect to the non-steroidal anti-inflammatory substance ibuprofen and the sulphonamide antibiotic sulfamethoxazole. The results of vial experiments were analysed on a liquid chromatograph with mass detection.

This diploma thesis deals with the development and validation of an analytical method using high performance liquid chromatography method with fluorescence detection for the simultaneous determination of aflatoxins and ochratoxin A in herbal and fruit tea. The theoretical part describes the most common groups of mycotoxins and the most important methods for their determination in food. The great attention is devoted to HPLC method and the overview of the derivatization techniques for aflatoxins B1 and G1 fluorescence response enhancement. The practical part of this study focuses on the optimization of sample extraction and purification, the settings of the instrumental analysis and the photochemical reactor assembly. The thesis also involves the determination of the basic performance characteristics for the successful method validation.

This diploma thesis deals with sorption properties of biochar produced from wood biomass treated with concentrated hydrochloric acid and activated carbon with respect to the triazine pesticide propazine. Although pesticides are widely used in both agriculture and the private sector worldwide, they pose a significant risk to ecosystems and human health. Propazine belongs to the group of chlorinated triazine herbicides, which pose a risk mainly as endocrine disruptors. The sorption of organic pollutants using pyrogenic carbonaceous materials promises an efficient and economically affordable solution, which has recently received increasing attention from the scientific community. In this work, we used isotherms to describe the equilibria of propazine sorption to given sorbents during vial experiments. Analysis of the propazine content in the model water solutions after reaching equilibrium was performed using high-performance liquid chromatography with mass detection.

This thesis includes NMR studies of free base meso-tetra(otolyl) porphyrin (TTP) and meso -tetraphenylporphyrin (TPP) and their dications protonated by trifluoroacetic acid (TFA). The chemical shift changes of the -NH resonance are very unusual and have never been reported. At the beginning of the titration, the N-H resonances broaden considerably but do not shift; when the ratio of [TFA]/[TTP] or [TFA]/[TPPf are over 2, the N-H resonances shift markedly to lower field by as much as 1.6 ppm. At acid levels well above the equivalence point, the NH resonances moves back to higher field. Moreover upon protonation the NH line of TTP becomes a complex multiplet which changes as a function of [TFA]/[TTP]. The NH line of TPP remains a singlet at all acid levels. We also report here a way of isolating atropisomers of mesotetra( o -tolyl)porphyrin through HPLC by using an analytical C-18 bond pack reverse phase column eluted by a 1 %THF + 99%Me0H solvent combination. A preliminary study by HPLC was also carried out on ZnTTP(II) with aliquots of 2,6-dimethylpyridine, and the retention time of the separation decreased markedly, but this study needs to be repeated and improved. TTP and TPP dications at two different acid levels were studied by VT-NMR, and the downfield shift of -NH resonance of TTP dications was more pronounced at higher temperatures than those of TPP dications.

This diploma thesis deals with the determination of glucosinolates in plant material by capillary electrophoresis and high performance liquid chromatography. The chemical structure, biosynthesis, degradation, and also biological effects of glucosinolates are described. One part of this work also deals with the methods, which glucosinolates in plant materials were determined by. The experimental part describes the separation of intact glucosinolates by capillary electrophoresis and high performance liquid chromatography. Two plant materials were available for the determination of glucosinolates, namely lyophilized rapeseed leaves and broccoli juice. Micellar electrokinetic chromatography using a cationic surfactant was used to determine intact glucosinolates by capillary electrophoresis. After finding the optimal conditions for the separation of intact glucosinolates, it was found that it is impossible to determine these substances in plant samples. The reason was interference from the matrix, which interfered with this determination. While using high performance liquid chromatography under optimal conditions, some of the intact glucosinolates were identified in a rapeseed plant sample. Furthermore, the calibration dependencies of individual glucosinolates were obtained and the recovery and...