Compatibility and stability of valsartan in a solid pharmaceutical formulation

Valsartan (VAL) is a highly selective blocker of the angiotensin II receptor that has been widely used in the treatment of hypertension. Active pharmaceutical ingredient compatibility with excipients (crospovidone, hypromellose, magnesium stearate, microcrystalline cellulose and titanium dioxide) is usually evaluated in solid pharmaceutical development. Compatibility and stability can be evaluated by liquid chromatography. Studies were performed using binary mixtures of 1:1 (w/w) VAL/excipient; samples were stored under accelerated stability test conditions (40 ºC at 75% relative humidity). The results indicate that VAL is incompatible with crospovidone and hypromellose, which reduced the VAL content and gave rise to new peaks in the chromatogram due to degradation products.Valsartana (VAL) é um bloqueador altamente seletivo do receptor da angiotensina II, que tem sido amplamente utilizado para o tratamento da hipertensão. Testes de compatibilidade com excipientes usualmente empregados em formulações sólidas são utilizados no desenvolvimento de formulações sólidas. Neste trabalho, realizaram-se testes utilizando misturas binárias na proporção 1:1 (m/m) de VAL/excipiente e as amostras foram armazenadas em condições de estabilidade acelerada (40 ºC em 75% de umidade relativa). Os resultados obtidos indicam a incompatibilidade de VAL com crospovidona e hipromelose, através da redução do teor de VAL e a presença de novos picos no cromatograma provenientes de produtos de degradação

Evaluation of pancreatin stability through enzyme activity determination

Pancreatin is a biotechnological product containing an enzyme complex, obtained from porcine pancreas, that is employed in treating pancreatic diseases. Experiments regarding the stability of the pharmaceutical formulation containing pancreatin were performed using standard binary mixtures with 6 excipients in a 1:1 ratio 1 (m/m) and a commercial formulation. To accomplish these goals, samples were stored for 1, 3 and 6 months at 40 ± 1 °C and 75 ± 5 % relative humidity (RH) and 40 ± 1 °C and 0 % RH. Stress testing was also performed. All samples were analyzed to evaluate the α-amylase, lipase and protease activities through UV/Vis spectrophotometry. The results revealed that the excipient proprieties and the storage conditions affected enzyme stability. Humidity was a strong influencing factor in the reduction of α-amylase and protease activities. Stress testing indicated that pH 9.0 and UV light did not induce substantial alterations in enzyme activity

LC-MS characterization of valsartan degradation products and comparison with LC-PDA

Valsartan was submitted to forced degradation under acid hydrolysis condition as prescribed by the ICH. Degraded sample aliquots were separated via HPLC using a Hypersil ODS (C18) column (250 x 4.6 mm i.d., 5 µm). Either photodiode array (PDA) detection or mass spectrometry (MS) full scan monitoring of HPLC runs were used. HPLC-PDA failed to indicate Valsartan degradation under forced acid degradation, showing an insignificant peak area variation and that Valsartan apparently remained pure. HPLC-MS using electrospray ionization (ESI) and total ionic current (TIC) monitoring did not reveal any peak variation either, but inspection of the ESI mass spectra showed the appearance of m/z 306 and m/z 352 ions for the same retention time as that of Valsartan (m/z 436). These ions were identified as being protonated molecules of two co-eluting degradation products formed by hydrolysis. These assignments were confirmed by ESI-MS/MS with direct infusion of the degraded samples. The results showed that the use of selective HPLC-MS is essential for monitoring Valsartan degradation. Efficient HPLC separation coupled to selective and structural diagnostic MS monitoring seems therefore mandatory for comprehensive drug degradation studies, particularly for new drugs and formulations, and for method development.Valsartana (VAL) foi submetida à degradação forçada em meio ácido conforme procedimento descrito no ICH. Os produtos de degradação (PDs) foram monitorados ao longo do tempo de degradação pela técnica de Cromatografia Líquida (LC) utilizando uma coluna Hypersil ODS (C18) (250 x 4,6 mm d.i., 5 µm). A detecção foi feita com dois detectores: espectrofotométrico (PDA) e espectrometria de massas (MS) por corrente iônica total. Ambas as técnicas falharam na identificação dos PDs obtidos ao longo do monitoramento, mostrando insignificantes variações na área do pico e permanecendo com pureza de pico ao longo de toda a eluição. Somente depois da avaliação por íon extraído (XIC), foi possível observar o aumento do íon m/z 306 e m/z 352 exatamente no mesmo tempo de retenção do íon molecular (m/z 436). Estes resultados mostram um caso simples e didático em que somente o uso de um método seletivo de LC-MS pode ser utilizado para monitorar produtos de degradação. Neste trabalho, é apresentado um caso real em que a separação por LC deve ser acoplada a métodos seletivos obtidos por MS, especialmente no estudo de PDs para novos fármacos, formulações e no desenvolvimento de métodos

Proteomic analysis of Chromobacterium violaceum and its adaptability to stress

Chromobacterium violaceum (C. violaceum) occurs abundantly in a variety of ecosystems, including ecosystems that place the bacterium under stress. This study assessed the adaptability of C. violaceum by submitting it to nutritional and pH stresses and then analyzing protein expression using bi-dimensional electrophoresis (2-DE) and Maldi mass spectrometry. Chromobacterium violaceum grew best in pH neutral, nutrient-rich medium (reference conditions); however, the total protein mass recovered from stressed bacteria cultures was always higher than the total protein mass recovered from our reference culture. The diversity of proteins expressed (repressed by the number of identifiable 2-DE spots) was seen to be highest in the reference cultures, suggesting that stress reduces the overall range of proteins expressed by C. violaceum. Database comparisons allowed 43 of the 55 spots subjected to Maldi mass spectrometry to be characterized as containing a single identifiable protein. Stress-related expression changes were noted for C. violaceum proteins related to the previously characterized bacterial proteins: DnaK, GroEL-2, Rhs, EF-Tu, EF-P; MCP, homogentisate 1,2-dioxygenase, Arginine deiminase and the ATP synthase β-subunit protein as well as for the ribosomal protein subunits L1, L3, L5 and L6. The ability of C. violaceum to adapt its cellular mechanics to sub-optimal growth and protein production conditions was well illustrated by its regulation of ribosomal protein subunits. With the exception of the ribosomal subunit L3, which plays a role in protein folding and maybe therefore be more useful in stressful conditions, all the other ribosomal subunit proteins were seen to have reduced expression in stressed cultures. Curiously, C. violeaceum cultures were also observed to lose their violet color under stress, which suggests that the violacein pigment biosynthetic pathway is affected by stress. Analysis of the proteomic signatures of stressed C. violaceum indicates that nutrient-starvation and pH stress can cause changes in the expression of the C. violaceum receptors, transporters, and proteins involved with biosynthetic pathways, molecule recycling, energy production. Our findings complement the recent publication of the C. violeaceum genome sequence and could help with the future commercial exploitation of C. violeaceum

Electrophoresis and spectrometric analyses of adaptation-related proteins in thermally stressed Chromobacterium violaceum

Chromobacterium violaceum is a Gram-negative proteobacteria found in water and soil; it is widely distributed in tropical and subtropical regions, such as the Amazon rainforest. We examined protein expression changes that occur in C. violaceum at different growth temperatures using electrophoresis and mass spectrometry. The total number of spots detected was 1985; the number ranged from 99 to 380 in each assay. The proteins that were identified spectrometrically were categorized as chaperones, proteins expressed exclusively under heat stress, enzymes involved in the respiratory and fermentation cycles, ribosomal proteins, and proteins related to transport and secretion. Controlling inverted repeat of chaperone expression and inverted repeat DNA binding sequences, as well as regions recognized by sigma factor 32, elements involved in the genetic regulation of the bacterial stress response, were identified in the promoter regions of several of the genes coding proteins, involved in the C. violaceum stress response. We found that 30°C is the optimal growth temperature for C. violaceum, whereas 25, 35, and 40°C are stressful temperatures that trigger the expression of chaperones, superoxide dismutase, a probable small heat shock protein, a probable phasing, ferrichrome-iron receptor protein, elongation factor P, and an ornithine carbamoyltransferase catabolite. This information improves our comprehension of the mechanisms involved in stress adaptation by C. violaceum. © FUNPEC-RP

Optimization of mixture of coagulants to removal of cadmium, copper and lead of aqueous systems by coprecipitation

Neste trabalho otimizou-se o processo de remoção dos cátions metálicos cádmio(II), cobre(II) e chumbo(II) de sistemas aquosos que utiliza o fenômeno da coprecipitação para arrastar os íons do meio reacional. Misturas em diferentes proporções de três agentes coagulantes, Al2(SO4)3, Fe2(SO4)3 e MnSO4, além de solução de um polímero (PEG 3.500 g mol-1), que reagiram com solução saturada de Ca(OH)2 para elevar o pH, foram utilizadas nos experimentos de otimização. Os experimentos com as referidas misturas foram feitos de acordo com os Planejamentos Experimentais para Mistura do tipo Simplex-Centroid e do tipo Simplex-Lattice. A partir das concentrações dos íons metálicos, obtidas por meio de espectrofotometria de absorção atômica, em cada ponto do planejamento, foi possível fazer a otimização dos sistemas através de programas estatísticos usados para ajustar modelos matemáticos aos dados experimentais. Isto foi feito utilizando o método dos mínimos quadrados ordinários. Com esse procedimento foi possível determinar o comportamento dos componentes da mistura em toda a região experimental. No planejamento do tipo Simplex- Centroid, onde os componentes foram os sulfatos de alumínio, ferro(III) e manganês(II), verificou-se que os efeitos de interação binária entre os componentes Al 2(SO4)3 com MnSO4 ou com Fe2(SO4)3 foram mais eficientes do que os componentes puros na remoção simultânea dos metais estudados. Uma mistura constituída de 70% de Al2(SO4)3 com 30% de MnSO4 a 0,001 mol L-1 foi capaz de remover 99,56% dos metais. Já no planejamento do tipo Simplex- Lattice, onde o Fe2(SO4)3 foi substituído pelo polímero a 0,2% m/v, constatou-se que também as misturas binárias foram as que apresentaram melhores resultados neste planejamento, onde até 98,54% dos metais foram retirados. Em todos os planejamentos, as concentrações do cobre e chumbo ficaram abaixo dos limites máximos estabelecidos pela legislação ambiental para água residual. Já o cádmio foi melhor removido no planejamento do tipo Simplex- Centroid, porém a concentração que ainda permaneceu em solução não alcançou o limite apropriado ficando próximo a ele. Este estudo teve como meta a obtenção de um efluente cujas concentrações de íons metálicos ficassem abaixo dos níveis estabelecido pela legislação ambiental. Com as condições para a coprecipitação otimizadas, a metodologia foi aplicada em efluente de uma indústria de galvanoplastia que possui um elevado teor de cobre(II). O mesmo foi tratado com Al2(SO4)3 comercial e com Al2(SO4)3 de pureza analítica obtendo, após o tratamento, um efluente com características apropriadas segundo a concentração de cobre e pH de acordo com a legislação ambiental vigente. Ao efluente foram adicionados também cádmio(II) e chumbo(II) para deixar o sistema mais complexo, sendo aplicado a ele as proporções das misturas onde as menores concentrações de metais restaram no sobrenadante. Neste caso, foi averiguado que Al2(SO4)3 puro apresentou um melhor resultado onde 99,77% dos metais foram removidos. A difração de raios-X de alguns precipitados mostrou que a presença do cátion metálico induz a formação de novas fases cristalinas e o grau de cristalinidade diminui com a presença do polímero orgânico.In this work the process of removing metallic ions such as cadmium(II), copper(II) and lead(II) in aqueous solution was optimized using the phenomenon of the coprecipitation to drag the ions from the reaction medium. Mixtures in different proportions of three coagulation agents, Al2(SO4)3, Fe2(SO4)3 and MnSO4, and also a solution of a polymer (PEG 3500), that reacted with a saturated solution of Ca(OH)2 to elevate the pH, were used in the experimental optimization. The experiments above were done according to the Experimental Planning for Mixture following two types of procedure: Simplex-Centroid and the Simplex-Lattice. The concentrations of metallic ions, determined by atomic absorption spectroscopy, were used to optimized the system through statistical programs used to adjust mathematical models to the experimental data. This procedure was made using the ordinary square minimal method. In this way, with this procedure it was possible to determine the behavior of the components of the mixture throughout the simplex. In the Simplex-Centroid planning, where the components were the sulfates of aluminum, iron(III) and manganese(II), it was verified that the effects of the binary interaction Al/Mn or Al/Fe were more efficient to remove the ions studied than the pure components themselves. A mixture of 70% of Al2(SO4)3 and 30% of MnSO4 or 30% of Fe2(SO4)3 (0,001 mol L-1) was capable to remove 99,56% of the metals. In the Simplex- Lattice planning, where Fe2(SO4)3 was replaced by the polymer (0,2% m/v), the binary mixtures have also given good results, 98,54% of the ions metallic were removed. In all plannings, the copper and lead concentration were below the maximum limits established by the environmental legislation for wastewater. The cadmium was better removed using the Al2(SO4)3, Fe2(SO4)3 and MnSO4 components, however, the supernatant still have cadmium in a concentration near the limit of 0,2 mg L-1. The aim of this study was to obtain a wastewater whose levels of metallic ions attend the environmental legislation, that is, the removal of those should achieve almost the neutrality of the system. After the conditions of coprecipitation have being optimized, the methodology was applied to a wastewater of galvanizing industry which have a high level of copper(II). The same one was treated with commercial Al2(SO4)3 and also with Al2(SO4)3 of analytical purity. The results, after the treatment, led to a wastewater with appropriate characteristics according to the copper concentration and the pH value, consistent to the effective environmental legislation. Cadmium(II) and lead(II) were also added to the wastewater to leave the system more complex. To the modified wastewater the optimized mixtures were applied to test the efficiency of the procedure developed in this work. The latter, have shown a better result (99,77%) of removing metals when pure Al2(SO4)3 was used. The X- rays diffraction of some precipitates showed that the presence of metallic cation induces the formation of the new crystalline phases and, the crystalline degree decreases with the presence of the organic polymer.Fundação de Amparo à Pesquisa do Estado de Minas Gerai

Microwave-assisted Decomposition Of Polyacrylamide Gels Containing Metalloproteins Using Mini-vials: An Auxiliary Strategy For Metallomics Studies.

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Evaluation of pancreatin stability through enzyme activity determination

Pancreatin is a biotechnological product containing an enzyme complex, obtained from porcine pancreas, that is employed in treating pancreatic diseases. Experiments regarding the stability of the pharmaceutical formulation containing pancreatin were performed using standard binary mixtures with 6 excipients in a 1:1 ratio (m/m) and a commercial formulation. To accomplish these goals, samples were stored for 1, 3 and 6 months at 40 ± 1 °C and 75 ± 5 % relative humidity (RH) and 40 ± 1 °C and 0 % RH. Stress testing was also performed. All samples were analyzed to evaluate the α-amylase, lipase and protease activities through UV/Vis spectrophotometry. The results revealed that the excipient proprieties and the storage conditions affected enzyme stability. Humidity was a strong influencing factor in the reduction of α-amylase and protease activities. Stress testing indicated that pH 9.0 and UV light did not induce substantial alterations in enzyme activity