Spectrophotometric determination of diclofenac in pharmaceutical preparations assisted by microwave oven

In this work, an effective and low-cost method for the determination of sodium or potassium diclofenac is proposed in its pure form and in their pharmaceutical preparations. The method is based on the reaction between diclofenac and tetrachloro-p-benzoquinone (p-chloranil), in methanol medium. This reaction was accelerated by irradiating of reactional mixture with microwave energy (1100 W) during 27 seconds, producing a charge transfer complex with a maximum absorption at 535 nm. The optimal reaction conditions values such as reagent concentration, heating time and stability of the reaction product were determined. Beer's law is obeyed in a concentration range from of 1.25x10-4 to 2.00x10-3 mol l-1 with a correlation coefficient of 0.9993 and molar absorptivity of 0.49 x10³ l mol-1 cm-1. The limit of detection (LOD) was 1.35x10-5 mol l-1 and the limit of quantification (LOQ) was 4.49x10-5 mol l-1. In the presence of the common excipients, such as glucose, lactose, talc, starch, magnesium stearate, sodium sulphite, titanium dioxide, polyethyleneglycol, polyvinylpirrolidone, mannitol and benzilic alcohol no interferences were observed. The analytical results obtained by applying the proposed method compare very favorably with those given by the United States Pharmacopeia standard procedure. Recoveries of diclofenac from various pharmaceutical preparations were within 95.9% to 103.3%, with standard deviations ranging from 0.2% to 1.8%.

Sensitive bromatometric assay methods for finasteride in pharmaceuticals

Three sensitive spectrophotometric methods are presented for the determination of finasteride in bulk and in tablets. The methods rely on the use of bromate-bromide reagent and three dyes namely, methyl orange, indigocarmine and thymol blue as reagents. They involve the addition of a measured excess of bromate-bromide reagent to finasteride in acid medium, and after the bromination reaction is judged to be complete, the unreacted bromine is determined by reacting with a fixed amount of either methylorange and measuring the absorbance at 520 nm (method A) or indigocarmine and measuring the absorbance at 610 nm (method B) or thymol blue and measuring the absorbance at 550 nm (method C). In all the methods, the amount of insitu generated bromine reacted corresponds to the amount of finasteride. The absorbance measured at the respective wavelength is found increase linearly with the concentration of finasteride. Beer's law is obeyed in the ranges 0.25- 2.0, 0.5-6.0 and 1-12 µg mL-1 for method A, method B and method C, respectively. The calculated molar absorptivity values are 5.7x10(4), 3.12x10(4) and 1.77x10(4) L mol-1 cm-1 respectively, for method A, method B and method C, and the corresponding Sandell sensitivity values are 0.0065, 0.012 and 0.021 µg cm-2. The limits of detection (LOD) and quantification (LOQ) are also reported for all the methods. Accuracy and, intra-day and inter-day precisions of the methods were established according to the current ICH guidelines. The methods were successfully applied to the determination of finasteride in commercially available tablets and the results were found to closely agree with the label claim. The results of the methods were statistically compared with those of a reference method by applying Student's t-test and F-test. The accuracy and reliability of the methods were further confirmed by performing recovery tests via standard addition procedure.

Direct determination of calcium in milk by atomic absorption spectrometry using flow-injection analysis

A flow-injection system with sample and reagent addition by the synchronous merging zones approach for calcium determination in milk by flame AAS is proposed. Main parameters were optimized using a factorial design with central point. The optimum conditions were 2.5% (m/v) for La concentration, 8 mL min-1 for the carrier flow-rate, 20 cm for coiled reactor and 250 ìL for sample volume. Different sample preparation procedures were evaluated such as dilution in water or acid and microwave-assisted decomposition using concentrated or diluted acids. The optimized flow system was applied to determine Ca in eleven commercial milk samples and two standard reference materials diluted in water. Similar calcium levels were encountered comparing the results obtained by the proposed method (dilution in water) with those obtained using microwave-oven digestion. Results obtained in two standard reference materials were in agreement at 95% confidence level with those certified. Recoveries of spiked samples were in the 93% - 116% range. Relative standard deviation (n = 12) was < 5.4% and the sample throughput was 150 measurements per hour, corresponding to a consumption of 250 µL of sample and 6.25 mg La per determination.

Spectrophotometric determination of arsenic in soil samples using 2-(5-bromo-2-pyridylazo)-5-di-ethylaminophenol (Br-PADAP)

In this paper, a new, simple and sensitive method for arsenic determination in soil is proposed. This is based on the reduction of silver (I) and iron (III) ions by arsine followed by a complexation reaction of iron (II) with the spectrophotometric reagent Br-PADAP 2-(5-bromo-2-pyridylazo)-5-di-ethylaminophenol. Arsenic determination with a Sandell's sensitivity of 3.1 10-4 cm-2, linear range from 0.1 µg ml-1 to 2.0 µg ml-1 (r560 = 0.9995), molar absorptivity of 2.45 10(5) l mol-1 cm-1 and a concentration detection limit of 1.4 ng ml-1 (3s) were obtained using a 10 ml sample volume. Selectivity was increased with the use of EDTA as a masking agent. The proposed method was applied for arsenic determination in the presence of several ions amounts in digested soil samples. The results revealed that antimony (III), mercury (II), germanium (IV), platinum (IV) interferes at all analyzed proportions. The interferences can be easily removed by the use of EDTA. Precision and accuracy obtained were satisfactory with a R.S.D. < 5 %. Recovery of arsenic in soil samples varied from 95.55 to 102.70 % with a mean of 99.63 %. These results demonstrated that the proposed method is applicable for arsenic analysis in different soil samples.

Flow-batch analyser for preparation of calibration standard mixtures in simultaneous multicomponent spectrometric analysis

The application of multivariate calibration techniques to multicomponent analysis by UV-VIS molecular absorption spectrometry is a powerful tool for simultaneous determination of several chemical species. However, when this methodology is accomplished manually, it is slow and laborious, consumes high amounts of reagents and samples, is susceptible to contaminations and presents a high operational cost. To overcome these drawbacks, a flow-batch analyser is proposed in this work. This analyser was developed for automatic preparation of standard calibration and test (or validation) mixtures. It was applied to the simultaneous determination of Cu2+, Mn2+ and Zn2+ in polyvitaminic and polymineral pharmaceutical formulations, using 4-(2-piridilazo) resorcinol as reagent and a UV-VIS spectrophotometer with a photodiode array detector. The results obtained with the proposed system are in good agreement with those obtained by flame atomic absorption spectrometry, which was employed as reference method. With the proposed analyser, the preparation of calibration and test mixtures can be accomplished about four hours, while the manual procedure requires at least two days. Moreover, it consumes smaller amounts of reagents and samples than the manual procedure. After the preparation of calibration and test mixtures, 60 samples h-1 can be carried out with the proposed flow-batch analyser.

Supersaturation-controlled crystallization

Identification of product requirements and quality, together with the management of production are key issues in chemical engineering. Quality control of crystalline products is part of the quality of many industrially manufactured products like paper, paintings, medicines and fertilizers. In most crystallization cases, quality is described with the size, polymorph, shape and purity of the crystal. The chemical composition, hydrodynamics and driving force, together with the operating temperature are in a key position when the properties of a crystalline product are controlled with the crystallization process. This study concentrates on managing the identified properties of a crystalline product with the control of a driving force. The controlling of the driving force can be based on the change of solubility or the change of concentration. Solubility can be changed with temperature, pressure and an antisolvent. The concentration of crystallizing compound, the solute can be changed with the evaporation of the solvent and with the addition of a reagent. The present study focuses on reagent addition and temperature change as methods of changing the level of the driving force. Three control structures for direct control of supersaturation are built, one for cooling crystallization and two for reactive crystallization. Closed loop feedback control structures are based on the measurement of the solute concentration with attenuated total reflection - Fourier transform infrared spectrometer. The details of the reagent feed are analyzed with experimental studies and with results of computational fluid dynamic simulations of the inert particle pulse in the premixer and inert particle injection to the mixing tank. Nucleation in conditions of controlled reactive crystallization is analyzed with Nielsen’s equation of homogeneous nucleation. The resulting control systems, based on regulation of supersaturation, can be used to produce the desired polymorph of an organic product. The polymorph composition of product crystals is controlled repeatably with the decision of a set value of supersaturation level.

Titrimetric and spectrophotometric assay of bupropion hydrochloride in pharmaceuticals using mercury(II) nitrate

Two simple, rapid and accurate methods for the determination of bupropion hydrochloride (BUP) in pure and in pharmaceutical preparations are described. Both methods are based on the measurement of the chloride of its hydrochloride. In the titrimetric method, the chloride content of bupropion hydrochloride is determined by titrating with mercury(II)nitrate using diphenylcarbazone-bromophenol blue as indicator. Titrimetric method is applicable over a range 2-20 mg of BUP and the reaction stoichiometry is found to be 2:1 (BUP: Hg(NO3)2). The spectrophotometric method involves the addition of a measured excess of mercury(II) nitrate reagent in formate buffer to the drug, and after ensuring the reaction had gone to completion, the unreacted mercury(II) is treated with a fixed amount of diphenylcarbazone, and absorbance measured at 515 nm. The absorbance is found to decrease linearly with increasing concentration of BUP and the calibration curve is linear over 1.0-15.0 µg mL-1 BUP. The proposed methods were successfully applied to the determination of BUP in commercially available dosage forms with good accuracy and precision, and without detectable interference by excipients. The accuracy was further ascertained by placebo blank and synthetic mixture analyses and also by recovery experiments via standard-addition procedure.

New reagents for the spectrophotometric determination of ranitidine hydrochloride

A new spectrophotometric method is proposed for the assay of ranitidine hydrochloride (RNH) in bulk drug and in its dosage forms using ceric ammonium sulphate (CAS) and two dyes, malachite (MAG) green and crystal violet (CV) as reagents. The method involves the addition of a known excess of ceric ammonium sulphate to ranitidine hydrochloride in acid medium, followed by the determination of unreacted CAS by reacting with a fixed amount of malachite green or crystal violet and measuring the absorbance at 615 or 582 nm respectively against the reagent blank. The Beer's law is obeyed in the concentration range of 0.4-8.0 µg/ ml of ranitidine hydrochloride (RNH) for RNH-MAG system and 0.2-1.6µg/ml of ranitidine hydrochloride for RNH-CV system. The molar Absorptivity, Sandell's sensitivity for each system were calculated. The method has been successfully applied to the determination of ranitidine hydrochloride in pure and dosage forms.

A cell spot microarray method for high-throughput biology

High-throughput screening of cellular effects of RNA interference (RNAi) libraries is now being increasingly applied to explore the role of genes in specific cell biological processes and disease states. However, the technology is still limited to specialty laboratories, due to the requirements for robotic infrastructure, access to expensive reagent libraries, expertise in high-throughput screening assay development, standardization, data analysis and applications. In the future, alternative screening platforms will be required to expand functional large-scale experiments to include more RNAi constructs, allow combinatorial loss-of-function analyses (e.g. genegene or gene-drug interaction), gain-of-function screens, multi-parametric phenotypic readouts or comparative analysis of many different cell types. Such comprehensive perturbation of gene networks in cells will require a major increase in the flexibility of the screening platforms, throughput and reduction of costs. As an alternative for the conventional multi-well based high-throughput screening -platforms, here the development of a novel cell spot microarray method for production of high density siRNA reverse transfection arrays is described. The cell spot microarray platform is distinguished from the majority of other transfection cell microarray techniques by the spatially confined array layout that allow highly parallel screening of large-scale RNAi reagent libraries with assays otherwise difficult or not applicable to high-throughput screening. This study depicts the development of the cell spot microarray method along with biological application examples of high-content immunofluorescence and phenotype based cancer cell biological analyses focusing on the regulation of prostate cancer cell growth, maintenance of genomic integrity in breast cancer cells, and functional analysis of integrin protein-protein interactions in situ.

Kullan uutto kloridiliuoksista

Työssä tutkittiin kullan neste-nesteuuttoa kloridiliuoksista kirjallisuustutkimuksen ja laboratoriokokeiden avulla. Uuttoreagenssina käytettiin Outotecin kehittämää kullanuuttoreagenssia. Teoreettisessa osassa tarkastellaan kullan käyttäytymistä happamissa vesiliuoksissa, jotka sisältävät kloridia ja bromidia. Lisäksi työssä käsitellään kultakloridiyhdisteiden käyttöturvallisuutta. Teollisuudessa vuonna 2012 käytettäviä neste-nesteuuttoon perustuvia kullan talteenottomenetelmiä esitellään lyhyesti. Kirjallisuusosassa käsitellään myös joidenkin teollisesti käytettävien polymeeristen rakennemateriaalien ominaisuuksia. Kokeellisessa osassa määritettiin kullanuuttoreagenssin fysikaalisia ominaisuuksia, kuten sen tiheys, viskositeetti, liukoisuus veteen sekä faasien selkeytyminen uuttoprosessissa. Tuloksista havaittiin, että eri modifiointiaineet vaikuttavat huomattavasti kullanuuttoliuoksen fysikaalisiin ominaisuuksiin. Kullan uuttoa tutkittiin pienen mittakaavan laboratoriokokein käyttämällä synteettisiä kultaa sisältäviä lähtöliuoksia. Kokeissa tutkittiin lähtöliuoksen happopitoisuuden ja bromidipitoisuuden, sekä uuttoreagenssin modifiointiaineiden vaikutuksia kullan uuttotasapainoon. Lisäksi tutkittiin joidenkin epäpuhtausmetallien myötäuuttautumista. Materiaalitutkimuksissa tarkasteltiin kullanuuttoreagenssin ja autenttisen kullan halidiliuoksen vaikutuksia polymeerimateriaaleihin pitkäaikaisessa kontaktissa. Uuttoliuosten havaittiin aiheuttavan turpoamaa sekä pinnan pehmenemistä joissakin teollisissa muoveissa.

Production of Mg(OH)2 from Mg-silicate rock for CO2 mineral sequestration

Sequestration of carbon dioxide in mineral rocks, also known as CO2 Capture and Mineralization (CCM), is considered to have a huge potential in stabilizing anthropogenic CO2 emissions. One of the CCM routes is the ex situ indirect gas/sold carbonation of reactive materials, such as Mg(OH)2, produced from abundantly available Mg-silicate rocks. The gas/solid carbonation method is intensively researched at Åbo Akademi University (ÅAU ), Finland because it is energetically attractive and utilizes the exothermic chemistry of Mg(OH)2 carbonation. In this thesis, a method for producing Mg(OH)2 from Mg-silicate rocks for CCM was investigated, and the process efficiency, energy and environmental impact assessed. The Mg(OH)2 process studied here was first proposed in 2008 in a Master’s Thesis by the author. At that time the process was applied to only one Mg-silicate rock (Finnish serpentinite from the Hitura nickel mine site of Finn Nickel) and the optimum process conversions, energy and environmental performance were not known. Producing Mg(OH)2 from Mg-silicate rocks involves a two-staged process of Mg extraction and Mg(OH)2 precipitation. The first stage extracts Mg and other cations by reacting pulverized serpentinite or olivine rocks with ammonium sulfate (AS) salt at 400 - 550 oC (preferably < 450 oC). In the second stage, ammonia solution reacts with the cations (extracted from the first stage after they are leached in water) to form mainly FeOOH, high purity Mg(OH)2 and aqueous (dissolved) AS. The Mg(OH)2 process described here is closed loop in nature; gaseous ammonia and water vapour are produced from the extraction stage, recovered and used as reagent for the precipitation stage. The AS reagent is thereafter recovered after the precipitation stage. The Mg extraction stage, being the conversion-determining and the most energy-intensive step of the entire CCM process chain, received a prominent attention in this study. The extraction behavior and reactivity of different rocks types (serpentinite and olivine rocks) from different locations worldwide (Australia, Finland, Lithuania, Norway and Portugal) was tested. Also, parametric evaluation was carried out to determine the optimal reaction temperature, time and chemical reagent (AS). Effects of reactor types and configuration, mixing and scale-up possibilities were also studied. The Mg(OH)2 produced can be used to convert CO2 to thermodynamically stable and environmentally benign magnesium carbonate. Therefore, the process energy and life cycle environmental performance of the ÅAU CCM technique that first produces Mg(OH)2 and the carbonates in a pressurized fluidized bed (FB) were assessed. The life cycle energy and environmental assessment approach applied in this thesis is motivated by the fact that the CCM technology should in itself offer a solution to what is both an energy and environmental problem. Results obtained in this study show that different Mg-silicate rocks react differently; olivine rocks being far less reactive than serpentinite rocks. In summary, the reactivity of Mg-silicate rocks is a function of both the chemical and physical properties of rocks. Reaction temperature and time remain important parameters to consider in process design and operation. Heat transfer properties of the reactor determine the temperature at which maximum Mg extraction is obtained. Also, an increase in reaction temperature leads to an increase in the extent of extraction, reaching a maximum yield at different temperatures depending on the reaction time. Process energy requirement for producing Mg(OH)2 from a hypothetical case of an iron-free serpentine rock is 3.62 GJ/t-CO2. This value can increase by 16 - 68% depending on the type of iron compound (FeO, Fe2O3 or Fe3O4) in the mineral. This suggests that the benefit from the potential use of FeOOH as an iron ore feedstock in iron and steelmaking should be determined by considering the energy, cost and emissions associated with the FeOOH by-product. AS recovery through crystallization is the second most energy intensive unit operation after the extraction reaction. However, the choice of mechanical vapor recompression (MVR) over the “simple evaporation” crystallization method has a potential energy savings of 15.2 GJ/t-CO2 (84 % savings). Integrating the Mg(OH)2 production method and the gas/solid carbonation process could provide up to an 25% energy offset to the CCM process energy requirements. Life cycle inventory assessment (LCIA) results show that for every ton of CO2 mineralized, the ÅAU CCM process avoids 430 - 480 kg CO2. The Mg(OH)2 process studied in this thesis has many promising features. Even at the current high energy and environmental burden, producing Mg(OH)2 from Mg-silicates can play a significant role in advancing CCM processes. However, dedicated future research and development (R&D) have potential to significantly improve the Mg(OH)2 process performance.

Sparrows (Passer domesticus L.) as intermediary hosts of Toxoplasma gondii in poultry farms from the "agreste" region of Pernambuco, Brazil

This paper aimed to identify Toxoplasma gondii infection in house sparrows (Passer domesticus, Linneaus 1758) coming from poultry farms in the "agreste" region of the Brazilian state of Pernambuco. 151 sparrows (Passer domesticus) captured in eight broiler, egg layer and commercial laying poultry farms, were used. Indirect hemagglutination test was used to research anti-T. gondii antibodies. Animals that presented titration of 1:16 were destined to DNA research through Polymerase Chain Reaction (PCR) technique, followed by Nested-PCR. It was observed that, from 151 analyzed samples. 91 (60.3%) were reagents and 60 (39.7%) were not reagents. It was verified, through analysis of the distribution of infected animals frequency per farm, that in only one farm (12.5%) no animal reagent to T. gondii was captured. It was also observed that three (30.00%) of the ten samples destined to DNA research for T. gondii were positive to PCR and four (40.00%) were positive to Nested-PCR. Anti-T gondii antibodies occurrence and the molecular identification of the agent confirmed natural T. gondii infection in sparrows from poultry farms in Brazil. Other studies must be carried out to highlight the real importance of these animals in the epidemiological chain and their efficiency in the transmission of the parasite to felines. Therefore, researches that use parasite isolation and molecular techniques to determine genomic profile of the agent present in these poultry farms are needed.

Serology, polymerase chain reaction and histopathology for leptospirosis in samples collected at slaughter from dairy cows of Parnaiba region, state of Piauí, Brazil

The presence of anti leptospiral agglutinins (microscopic agglutination test - MAT) and DNA of leptospires was investigated in the kidney and urine (Polymerase Chain Reaction - PCR) in samples collected at the time of slaughter of cattle originating from the dairy basin of Parnaíba, Piauí, Brazil, as also the lesions in kidney, lung, liver, uterus, ovary and placenta (histopathology and immunohistochemistry). In the MAT, Hardjo was the predominant serovar with the highest number of reagent animals for the strain Hardjobovis/Sponselee. Anti-leptospiral antigens were scored in epithelial cells, interstitial vascular endothelium, endothelium of glomerular capillaries and Bowman's capsule of 20 positive animals. Inflammatory cells were more common in the kidney. PCR was positive in urine and kidney tissue

Epidemiology of leptospirosis at Sorocaba Zoo, São Paulo state, Southeastern Brazil

Leptospirosis is considered a worldwide distributed zoonosis, caused by the bacteria Leptospira spp. Since several species of wildlife animals are reportedly reservoirs, the aim of the present study was to know the epidemiology of leptospirosis at the Sorocaba Zoo, Southern Brazil. Serum samples of wild mammals from Artiodactyla, Carnivora, Didelphimorphia, Diprotodontia, Perissodactyla, Pilosa, Primates, Proboscidea and Rodentia orders, kept in captivity as well as from zoological staff were assayed by microscopic agglutination test (MAT). Whole blood, urine and tissue samples from wild mammals and synanthropic animals were assayed by polymerase chain reaction (PCR). An epidemiological survey was applied to evaluate the risk factors for animal infection and staff level of knowledge on leptospirosis. A total of 13/229 (5.68%; CI95% 3.37-9.47%) serum samples from wild mammals were reagent on MAT. Serology from synanthropic animals, zoo staff and molecular analysis of animal samples were all negative. Leptospirosis knowledge of zoo park staff was considered medium. In conclusion, leptospiral infection occurs at the studied zoo but due to the low occurrence found, the lowest reported in literature, wild captive mammals do not act as source of infection of leptospirosis to other animals and human beings.

Prokaryotic expression of a truncated form of bovine herpesvirus 1 glycoprotein E (gE) and its use in an ELISA for gE antibodies

This article describes the expression of a truncated form of bovine herpesvirus 1 (BoHV-1) glycoprotein E (gE) for use as immunodiagnostic reagent. A 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of BoHV-1 gE - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector. A soluble protein of approximately 25 kDa purified from lysates of transformed E. coli was recognized in Western blot (WB) by anti-6xHis-tag and anti-BoHV-1 gE monoclonal antibodies. In addition, the recombinant protein was specifically recognized in WB by antibodies present in the sera of cattle seropositive to BoHV-1 and BoHV-5. An indirect ELISA using the expressed protein as coating antigen performed comparably to a commercial anti-gE ELISA and was able to differentiate serologically calves vaccinated with a gE-deleted BoHV-5 strain from calves infected with BoHV-1. Thus, the truncated gE may be useful for serological tests designed to differentiate BoHV-1/BoHV-5 infected animals from those vaccinated with gE-negative marker vaccines.