Desenvolvimento e validação de método analítico por CLAE para a quantificação simultânea de dipropionato de betametasona e fosfato sódico de betametasona em suspensão injetável

A reversed-phase HPLC method was developed and validated to separate and simultaneously quantify the association of betamethasone sodium phosphate (BP) and betamethasone dipropionate (BD) in injectable suspensions. Chromatographic conditions were ternary gradient elution at 1.6 mL/min on a C18 column with 254 nm. The linearity of the method was established in the range 120 to 280 mg/mL BD, and 48 to 112 mg/mL BP. The RSD of intermediate precision of the method was <1% and recoveries were 99-101% for both drugs. The method proved selective, linear, precise, accurate and robust for quantifying BP and BD in commercial injectable suspensions.

Preparative separation and purification of bufadienolides from ChanSu by high-speed counter-current chromatography combined with preparative HPLC

Eight bufadienolides were successfully isolated and purified from ChanSu by high-speed counter-current chromatography (HSCCC) combined with preparative HPLC (prep-HPLC). First, a stepwise elution mode of HSCCC with the solvent system composed of petroleum ether - ethyl acetate - methanol - water (4:6:4:6, 4:6:5:5, v/v) was employed and four bufadienolides, two partially purified fractions were obtained from 200 mg of crude extract. The partially purified fractions III and VI were then further separated by prep-HPLC, respectively, and another four bufadienolides were recovered. Their structures were confirmed by ESI-MS and ¹H-NMR spectra.

Quantification of beta-lactam antibiotics in veterinary drugs: amoxicillin and ampicillin determination by high performance liquid chromatography

This work focused on the development and validation of an RP-HPLC-UV method for quantification of beta-lactam antibiotics in three pharmaceutical samples. Active principles analyzed were amoxicillin and ampicillin, in 3 veterinary drugs. Mobile phase comprised 5 mmol L-1 phosphoric acid solution at pH 2.00, acetonitrile with gradient elution mode and detection wavelength at 220 nm. The method was validated according to the Brazilian National Health Surveillance regulation, where linear range and linearity, selectivity, precision, accuracy and ruggedness were evaluated. Inter day precision and accuracy for pharmaceutical samples 1, 2 and 3 were: 1.43 and 1.43%; 4.71 and 3.74%; 2.72 and 1.72%, respectively, while regression coefficients for analytical curves exceeded 0.99. The method had acceptable merit figure values, indicating reliable quantification. Analyzed samples had active principle concentrations varying from -12 to +21% compared to manufacturer label claims, rendering the medicine unsafe for administration to animals.

Bioanalytical method for the quantification of the monastrol derivative anticancer candidate lasom 65 in pre-clinical pharmacokinetic investigations

A simple HPLC/UV method was developed for the determination of the anticancer candidate LaSOM 65 in rat plasma. Samples were cleaned by protein precipitation with acetonitrile (recovery > 95%), after which they were subjected to chromatography under the isocratic elution of an acetonitrile:water (45:55, ν/ν) solution with detection at 303 nm. The method was linear (r² > 0.98) over the concentration range (0.05-2 µg mL-1) with intra- and inter-day precision ranging from 9.6% to 13.6% and 4.3% to 5.4%, respectively. The accuracy of the method ranged from 85% to 113.6%, and it showed sufficient sensitivity to determine pharmacokinetic parameters of LaSOM 65 after intravenous administration to Wistar rats.

LC/ESI-MS method applied to characterization of flavonoids glycosides in B. forficata subsp. pruinosa

Bauhinia forficata is used in folk medicine for its hypoglycemiant effect. In the south of Brazil, the subspecies pruinosa is found in a region with the characteristic flora, pampa biome. This species has been consumed by the local population as a tea for diabetes treatment. We studied the chemical composition of hydroethanolic extracts using LC/ESI-MS. The leaf extracts were prepared by percolation with 50% (v/v) ethanol. The chromatographic analyses were performed using a reverse-phase system, gradient elution with acetonitrile:phosphoric acid 0.05%, and ESI-MS in the positive ion mode. The chemical profile of the flavonoids was suggested to involve four quercetin and kaempferol glycosides.

DISPOSITIVOS DGT MODIFICADOS COM MATERIAIS ALTERNATIVOS PARA USO NA ESPECIAÇÃO DE ELEMENTOS TRAÇO

The use of MT-K10 Montmorillonite immobilized onto agarose was investigated in this work as an alternative binding phase in Diffusive Gradient in Thin Film (DGT) devices for the determination of metallic labile species. In addition, agarose itself was also used as the diffusive phase. The percentage of sorption of Zn2+, Cu2+, Cr3+, Mn2+, Cd2+, Pb2+, and Ni2+ onto the binding phase was higher than 80% and the desorption process for all elements was also greater than 75%. Elution factors were determined experimentally, ranging from 0.74 for Zn2+ and 0.90 for Cr3+ and Pb2+. The accumulation of all species was linear with time, in agreement with the Fick's 1st law of diffusion. The deployment of the alternative devices in natural waters was compared to commercial devices. Labile concentrations determined by the alternative devices were slightly superior compared to results obtained with the deployment of original DGT devices due to the less restrictive pores of agarose.

A FTIR study of the metal-support interactions and hydrogen spillover on Pd/TiO2 and Ni/TiO2

Nickel and palladium dispersed on titania support were submitted to reductive treatment, under hydrogen, at 200 and 500 ºC. After the reductive thermal treatment the materials were exposed to carbon monoxide (10 Torr) and analyzed in the infrared region. The increasing of the electronic density in the metallic d subshell, produced by the reductive thermal treatment, was monitored by the infrared stretching band shift of carbon monoxide adsorbed and it was interpreted as a consequence of the metal-support interactions. The highest effect was observed for Pd/TiO2 system. From the FTIR spectra was also observed that the hydrogen spillover was stronger on Pd/TiO2 than Ni/TiO2 system.

Determination of Copper, Iron, Nickel and Zinc in fuel kerosene by FAAS after adsorption and pre-concentration on 2-aminothiazole-modified silica gel

Silica gel chemically modified with 2-Aminotiazole groups, abbreviated as SiAT, was used for preconcentration of copper, zinc, nickel and iron from kerosene, normally used as a engine fuel for airplanes. Surface characteristics and surface area of the silica gel were obtained before and after chemical modification using FT-IR, Kjeldhal and surface area analysis (B.E.T.). The retention and recovery of the analyte elements were studied by applying batch and column techniques. The experimental parameters, such as shaking time in batch technique, flow rate and concentration of the eluent (HCl- 0.25-2.00 mol L-1) and the amount of silica, on retention and elution, have been investigated. Detection limits of the method for copper, iron, nickel and zinc are 0.77, 2.92, 1.73 and 0.097 mg L-1, respectively. The sorption-desorption of the studied metal ions made possible the development of a preconcentration method for metal ions at trace level in kerosene using flame AAS for their quantification.

Use of experimental design in the optimisation of a solid phase preconcentration system for Cobalt determination by GFAAS

In this work is proposed a solid phase preconcentration system of Co2+ ions and its posterior determination by GFAAS in which fractional factorial design and response surface methodology (RSM) were used for optimization of the variables associated with preconcentration system performance. The method is based on cobalt extraction as a complex Co2+-PAN (1:2) in a mini-column of polyurethane foam (PUF) impregnated with 1-(2-pyridylazo)-naphthol (PAN) followed by elution with HCl solution and its determination by GFAAS. The chemical and flow variables studied were pH, buffer concentration, eluent concentration and preconcentration and elution flow rates. Results obtained from fractional factorial design 2(5-1) showed that only the variables pH, buffer concentration and interaction (pH X buffer concentration) based on analysis of variance (ANOVA) were statistically significant at 95% confidence level. Under optimised conditions, the method provided an enrichment factor of 11.6 fold with limit of detection and quantification of 38 and 130 ng L-1, respectively, and linear range varying from 0.13 to 10 µg L-1. The precision (n = 9) assessed by relative standard deviation (RSD) was respectively 5.18 and 2.87% for 0.3 and 3.0 µg L-1 cobalt concentrations.

Electrochemical evaluation of lipophilic antioxidants from Iryanthera juruensis fruits (Myristicaceae)

TLC autographic assays revealed in the hexane extract of Iryanthera juruensis (Myristicaceae) the presence of two compounds, with antioxidant properties towards beta-carotene. They were isolated and identified as 3-methyl-sargachromenol (1) and sargachromenol (2). Further investigation of the hexane extract led to isolations of 3-methyl-sargaquinoic acid (3) and sargaquinoic acid (4). The electrochemical behaviour of these compounds was studied in CH2Cl2/Bu4NBF4 at glassy carbon electrode. The phenolic group in both tocotrienols 1 and 2 are oxidized at +0.23V and +0.32V and their oxidation potentials are correlated with the observed antioxidant activities and oxidation mechanism of alpha-tocopherol. The reductive voltametric behaviour of quinone function in both plastoquinones 3 and 4 is discussed.

Leaf-Targeted Ferredoxin-NADP+-Oxidoreductase (FNR): Electron Transfer Properties and Thylakoid Association in Arabidopsis thaliana

Photosynthetic reactions are divided in two parts: light-driven electron transfer reactions and carbon fixation reactions. Electron transfer reactions capture solar energy and split water molecules to form reducing energy (NADPH) and energy-carrying molecules (ATP). These end-products are used for fixation of inorganic carbon dioxide into organic sugar molecules. Ferredoxin-NADP⁺ oxidoreductase (FNR) is an enzyme that acts at the branch point between the electron transfer reactions and reductive metabolism by catalyzing reduction of NADP+ at the last step of the electron transfer chain. In this thesis, two isoforms of FNR from A rabidopsis thaliana, FNR1 and FNR2, were characterized using the reverse genetics approach. The fnr1 and fnr2 mutant plants resembled each other in many respects. Downregulation of photosynthesis protected the single fnr mutant plants from excess formation of reactive oxygen species (ROS), even without significant upregulation of antioxidative mechanisms. Adverse growth conditions, however, resulted in phenotypic differences between fnr1 and fnr2. While fnr2 plants showed downregulation of photosynthetic complexes and upregulation of antioxidative mechanisms under low-temperature growth conditions, fnr1 plants had the wild-type phenotype, indicating that FNR2 may have a specific role in redistribution of electrons under unfavorable conditions. The heterozygotic double mutant (fnr1xfnr2) was severely devoid of chloroplastic FNR, which clearly restricted photosynthesis. The fnr1xfnr2 plants used several photoprotective mechanisms to avoid oxidative stress. In wild-type chloroplasts, both FNR isoforms were found from the stroma, the thylakoid membrane, and the inner envelope membrane. In the absence of the FNR1 isoform, FNR2 was found only in the stroma, suggesting that FNR1 and FNR2 form a dimer, by which FNR1 anchors FNR2 to the thylakoid membrane. Structural modeling predicted formation of an FNR dimer in complex with ferredoxin. In this thesis work, Tic62 was found to be the main protein that binds FNR to the thylakoid membrane, where Tic62 and FNR formed high molecular weight complexes. The formation of such complexes was shown to be regulated by the redox state of the chloroplast. The accumulation of Tic62-FNR complexes in darkness and dissociation of complexes from the membranes in light provide evidence that the complexes may have roles unrelated to photosynthesis. This and the high viability of fnr1 mutant plants lacking thylakoid-bound FNR indicate that the stromal pool of FNR is photosynthetically active.

A quasi-materialist, quasi-dualist solution to the mind-body problem

If the mental can affect, or be affected by, the physical, then the mental must itself be physical. Otherwise the physical world would not be explanatorily closed. But it is closed. There are reasons to hold that materialism (in both its reductive and non-reductive varieties) is false. So how are we to explain the apparent responsiveness of the physical to the mental and vice versa? The only possible solution seems to be this: physical objects are really projections or isomorphs of objects whose essential properties are mental. (A slightly less accurate way of putting this would be to say: the constitutive - i.e. the non-structural and non-phenomenal - properties of physical objects are mental, i.e. are such as we are used to encountering only in "introspection".) The chair, qua thing that I can know through sense perception, and through hypotheses based strictly thereupon, is a kind of shadow of an object that is exactly like it, except that this other objects essential properties are mental. This line of thought, though radically counterintuitive, explains the apparent responsiveness of the mental to the physical, and vice versa, without being open to any of the criticisms to which materialism, dualistic interaction ism, and epiphenomenalism are open.

Acidic dissolution of iron oxides and regeneration of a ceramic filter medium

The dewatering of iron ore concentrates requires large capacity in addition to producing a cake with low moisture content. Such large processes are commonly energy intensive and means to lower the specific energy consumption are needed. Ceramic capillary action disc filters incorporate a novel filter medium enabling the harnessing of capillary action, which results in decreased energy consumption in comparison to traditional filtration technologies. As another benefit, the filter medium is mechanically and chemically more durable than, for example, filter cloths and can, thus, withstand harsh operating conditions and possible regeneration better than other types of filter media. In iron ore dewatering, the regeneration of the filter medium is done through a combination of several techniques: (1) backwashing, (2) ultrasonic cleaning, and (3) acid regeneration. Although it is commonly acknowledged that the filter medium is affected by slurry particles and extraneous compounds, published research, especially in the field of dewatering of mineral concentrates, is scarce. Whereas the regenerative effect of backwashing and ultrasound are more or less mechanical, regeneration with acids is based on chemistry. The chemistry behind the acid regeneration is, naturally, dissolution. The dissolution of iron oxide particles has been extensively studied over several decades but those studies may not necessarily be directly applicable in the regeneration of the filter medium which has undergone interactions with the slurry components. The aim of this thesis was to investigate if free particle dissolution indeed correlates with the regeneration of the filter medium. For this purpose, both free particle dissolution and dissolution of surface adhered particles were studied. The focus was on acidic dissolution of iron oxide particles and on the study of the ceramic filter medium used in the dewatering of iron ore concentrates. The free particle dissolution experiments show that the solubility of synthetic fine grained iron oxide particles in oxalic acid could be explained through linear models accounting for the effects of temperature and acid concentration, whereas the dissolution of a natural magnetite is not so easily explained by such models. In addition, the kinetic experiments performed both support and contradict the work of previous authors: the suitable kinetic model here supports previous research suggesting solid state reduction to be the reaction mechanism of hematite dissolution but the formation of a stable iron oxalate is not supported by the results of this research. Several other dissolution mechanisms have also been suggested for iron oxide dissolution in oxalic acid, indicating that the details of oxalate promoted reductive dissolution are not yet agreed and, in this respect, this research offers added value to the community. The results of the regeneration experiments with the ceramic filter media show that oxalic acid is highly effective in removing iron oxide particles from the surface of the filter medium. The dissolution of those particles did not, however, exhibit the expected behaviour, i.e. complete dissolution. The results of this thesis show that although the regeneration of the ceramic filter medium with acids incorporates the dissolution of slurry particles from the surface of the filter medium, the regeneration cannot be assessed purely based upon free particle dissolution. A steady state, dependent on temperature and on the acid concentration, was observed in the dissolution of particles from the surface even though the limit of solubility of free iron oxide particles had not been reached. Both the regeneration capacity and efficiency, with regards to the removal of iron oxide particles, was found to be temperature dependent, but was not affected by the acid concentration. This observation further suggests that the removal of the surface adhered particles does not follow the dissolution of free particles, which do exhibit a dependency on the acid concentration. In addition, changes in the permeability and in the pore structure of the filter medium were still observed after the bulk concentration of dissolved iron had reached a steady state. Consequently, the regeneration of the filter medium continued after the dissolution of particles from the surface had ceased. This observation suggests that internal changes take place at the final stages of regeneration. The regeneration process could, in theory, be divided into two, possibly overlapping, stages: (1) dissolution of surface-adhered particles, and (2) dissolution of extraneous compounds from within the pore structure. In addition to the fundamental knowledge generated during this thesis, tools to assess the effects of parameters on the regeneration of the ceramic filter medium are needed. It has become clear that the same tools used to estimate the dissolution of free particles cannot be used to estimate the regeneration of a filter medium unless only a robust characterisation of the order of regeneration efficiency is needed.

Metallien eluointi kelatoivasta ioninvaihtimesta

Kelatoivat ioninvaihtimet ovat yleensä makrohuokoisia hartseja, joiden avulla metalleja poistetaan ja otetaan mahdollisesti talteen teollisuuden puhdistettavista jätevirroista. Ne muodostavat metalli-ionien kanssa komplekseja runkomateriaaliin kovalenttisesti liitettyjen aktiivisten funktionaalisten ryhmiensä välityksellä. Ioninvaihtimen selektiivisyys eri metalli-ioneja kohtaan vaihtelee riippuen siihen liitetystä kelatoivasta ryhmästä. Työssä tutkitaan metallien tarttumista kelatoiviin ioninvaihtimiin, sekä niiden eluoitumista adsorbentistaan. Kelatoivat ioninvaihtimet sitovat metalli-ioneja tehokkaasti ligandiensa monihampaisuudesta johtuen. Metallien tarttuminen kelatoivaan ioninvaihtimeen ei kuitenkaan ole yksiselitteistä, vaan siihen vaikuttaa muun muassa pH yhdessä monen muun tekijän kanssa. Ioninvaihtimien selektiivisyyttä tarkastellaan työssä lähinnä kovien ja pehmeiden happojen ja emästen HSAB -teorian näkökulmasta. Regeneroinnilla ioninvaihdin saadaan jälleen alkuperäiseen muotoonsa, minkä jälkeen se voidaan käyttää uudelleen. Yleensä regenerointi suoritetaan kemiallisesti. Tässä työssä ioninvaihtimien regenerointia ja kohdemetallin eluointia tarkastellaan paitsi teoriassa, myös kokeellisessa osuudessa. Kokeellisessa osuudessa tutkitaan kuparin (Cu2+) eluoitumista kelatoivasta Dowex M-4195 kationinvaihtohartsista. Kokeissa hartsi ladattiin kuparilla erillisessä panoksessa kuparin ollessa syöttöliuoksessa kuparisulfaattina. Eluointiliuoksina käytettiin 2 ja 5 molaarista rikkihappoa, sekä 2 molaarista ammoniumhydroksidia. Eluointi suoritettiin panostoimisena kolonniajona ja eluaatista otetut näytteet analysoitiin atomiadsorptiospektrofotometrillä. Analyysitulokset esitetään läpäisykäyrinä, joiden perusteella 2M ammoniumhydroksidi on kolmesta tutkitusta eluentista tehokkain eluoimaan kuparia Dowex M-4195 hartsista.

Phenolics of the Inner Bark of Silver Birch: Characterization and Intraspecific Variation

This thesis describes work related to the in-depth characterization of the phenolic compounds of silver birch (Betula pendula) inner bark. Phenolic compounds are the most ubiquitous class of plant secondary compounds. The unifying feature of this structurally diverse group is an aromatic ring containing at least one hydroxyl group. Due to the structural diversity, phenolics have various roles in the plant defense against biotic and abiotic stresses. In addition, they can confer several health-promoting properties to humans. Furthermore, the structural diversity of this class of compounds causes challenges for their analysis. The study species in the present work, silver birch, is economically the most important hard wood species in northern Europe. Its inner bark contains a high level of phenolic compounds and it has shown one of the strongest antioxidant activities among 92 Finnish plant materials. The literature review surveys the diversity and organ specific distribution of phenolic compounds in silver birch as well as the proposed ecological functions of phenolic compounds in nature. In addition, the basis for the characterization of phenolics by mass spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR), and circular dichroism spectroscopy (CD) are reviewed. The objective of the experimental work was to extract, purify, characterize, and quantify the inner bark phenolic compounds. Overall 36 compounds were characterized by MS and ultraviolet spectroscopy (UV). 24 compounds were isolated and their structures confirmed by NMR and CD spectroscopy. Five novel natural compounds were identified. Special emphasis was placed on the establishment of a method for the characterization of proanthocyanidins (PAs). Hydrophilic interaction liquid chromatography (HILIC) was utilized because of its high resolution power and predictable elution order of oligomeric and polymeric PAs according to an increasing degree of polymerization. The combination of HILIC and high-resolution MS detection allowed the identification of procyanidin (PC) polymers up to the degree of polymerization of 22. In addition, a series of oligomeric and polymeric PC monoxylosides were observed for the first time in nature. Season and genotype influenced the quantities of the main inner bark phenolics, yet qualitative differences were not observed. However, manual wounding of the inner bark induced the production of ellagitannins (ETs) in the wounded tissues, i.e. callus. Since ETs were not detected in the intact inner bark, this finding may reflect the capacity of silver birch to exploit ellagitannins in its defense.