Development and Validation of a Simple Method for Routine Analysis of Ractopamine Hydrochloride in Raw Material and Feed Additives by HPLC

A simple method was optimized and validated for determination of ractopamine hydrochloride (RAC) in raw material and feed additives by HPLC for use in quality control in veterinary industries. The best-optimized conditions were a C8 column (250 x 4.6 mm id, 5.0 mu m particle size) at room temperature with acetonitrile-100 mM sodium acetate buffer (pH 5.0; 75 + 25, v/v) mobile phase at a flow rate of 1.0 mL/min and UV detection at 275 nm. With these conditions, the retention time of RAC was around 5.2 min, and standard curves were linear in the concentration range of 160-240 mu g/mL (correlation coefficient >= 0.999). Validation parameters, such as selectivity, linearity, limit of detection (ranged from 1.60 to 2.05 mu g/mL), limit of quantification (ranged from 4.26 to 6.84 mu g/mL), precision (relative standard deviation <= 1.87%), accuracy (ranged from 96.97 to 100.54%), and robustness, gave results within acceptable ranges. Therefore, the developed method can be successfully applied for the routine quality control analysis of raw material and feed additives.

A Validated Reverse-phase HPLC Analytical Method for the Quantification of Phenolic Compounds in Baccharis dracunculifolia

Introduction - Baccharis dracunculifolia, which has great potential for the development of new phytotherapeutic medicines, is the most important botanical source of the southeastern Brazilian propolis, known as green propolis on account of its color. Objective - To develop a reliable reverse-phase HPLC chromatographic method for the analysis of phenolic compounds in both B. dracunculifolia raw material and its hydroalcoholic extracts. Methodology - The method utilised a C(18) CLC-ODS (M) (4.6 x 250 mm) column with nonlinear gradient elution and UV detection at 280 nm. A procedure for the extraction of phenolic compounds using aqueous ethanol 90%, with the addition of veratraldehyde as the internal standard, was developed allowing the quantification of 10 compounds: caffeic acid, coumaric acid, ferulic acid, cinnamic acid, aromadendrin-4`-methyl ether, isosakuranetin, drupanin, artepillin C, baccharin and 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran acid. Results - The developed method gave a good detection response with linearity in the range 20.83-800 mu g/mL and recovery in the range 81.25-93.20%, allowing the quantification of the analysed standards. Conclusion - The method presented good results for the following parameters: selectivity, linearity, accuracy, precision, robustness, as well as limit of detection and limit of quantitation. Therefore, this method could be considered as an analytical tool for the quality control of B. dracunculifolia raw material and its products in both cosmetic and pharmaceutical companies. Copyright (C) 2008 John Wiley & Sons, Ltd.

Development and validation of HPLC method for imiquimod determination in skin penetration studies

A simple, rapid and sensitive analytical procedure for the measurement of imiquimod in skin samples after in vitro penetration studies has been developed and validated. In vitro penetration studies were carried out in Franz diffusion cells with porcine skin. Tape stripping technique was used to separate the stratum corneum (SC) from the viable epidermis and dermis. Imiquimod was extracted from skin samples using a 7:3 (v/v) methanol:acetate buffer (100 mm, pH 4.0) solution and ultrasonication. Imiquimod was analyzed by H-PLC using C(8) column and UV detection at 242 ran. The mobile phase used was acetonitrile:acetate buffer (pH 4.0, 100 mM):diethylamine (30:69.85:0.15, v/v) with flow rate 1 mL/min. Imiquimod eluted at 4.1 min and the running time was limited to 6.0 min. The procedure was linear across the following concentration ranges: 100-2500 ng/mL for both SC and tape-stripped skin and 20-800 ng/mL for receptor solution. Intra-day and inter-day accuracy and precision values were lower than 20% at the limit of quantitation. The recovery values ranged from 80 to 100%. The method is adequate to assay imiquimod from skin samples, enabling the determination of the cutaneous penetration profile of uniquimod by in vitro studies. Copyright (C) 2008 John Wiley & Sons, Ltd.

W/O/W multiple emulsions obtained by one-step emulsification method and evaluation of the involved variables

The effects of some composition variables on the development of multiple emulsions by one-step method were evaluated and their morphology characterized. The formulations that remained stable during the period of the test were submitted to centrifugation and thermal stress tests. The stability and the morphology of multiple droplets were affected not only by the type and concentration of the surfactants employed, but also by the water/oil ratios used. The results suggest that the formation of multiple droplets could involve a combination of transitional and catastrophic phase inversions. The results provide improved knowledge about the one-step emulsification method, a simplified process to prepare multiple emulsions when compared to the two-steps method.

Validation of a gas chromatographic method to quantify sesquiterpenes in copaiba oils

Copaifera species (Leguminoseae) are popularly known as ""copaiba"" or ""copaiva"". The oleoresins obtained from the trunk of these species have been extensively used in folk medicine and are commercialized in Brazil as crude oil and in several pharmaceutical and cosmetic products. This work reports a complete validated method for the quantification of beta-caryophyllene, alpha-copaene, and alpha-humulene in distinct copaiba oleoresins available commercially. Thus, essential oil samples (100 mu L) were dissolved in 20 mL of hexanes containing internal standard (1,2,4,5-tetramethylbenzene, 3.0 mM) in a 25 mL glass flask. A 1 mu L aliquot was injected into the GC-FID system. A fused-silica capillary column HP-5, coated with 5% phenylmethylsiloxane was used for this study. The developed method gave a good detection response with linearity in the range of 0.10-18.74 mM. Limits of detection and quantitation variety ranged between 0.003 and 0.091 mM. beta-Caryophyllene, alpha-copaene, and alpha-humulene were recovered in a range from 74.71% to 88.31%, displaying RSD lower than 10% and relative errors between -11.69% and -25.30%. Therefore, this method could be considered as an analytical tool for the quality control of different Copaifera oil samples and its products in both cosmetic and pharmaceutical companies. (C) 2010 Elsevier B.V. All rights reserved.

Relationship between Rheological Properties and One-Step W/O/W Multiple Emulsion Formation

Formation of a normal (not temporary) W/O/W multiple emulsion via the one-step method as a result of the simultaneous occurrence of catastrophic and transitional phase inversion processes has been recently reported. Critical features of this process include the emulsification temperature (corresponding to the ultralow surface tension point), the use of a specific nonionic surfactant blend and the surfactant blend/oil phase ratio, and the addition of the surfactant blend to the oil phase. The purpose of this study was to investigate physicochemical properties in an effort to gain a mechanistic understanding of the formation of these emulsions. Bulk, surface, and interfacial theological properties of adsorbed nonionic surfactant (CremophorRH40 and Span80) films were investigated under conditions known to affect W/O/W emulsion formation. Bulk viscosity results demonstrated that CremophorRH40 has a higher mobility in oil compared than in water, explaining the significance of the solvent phase. In addition, the bulk viscosity profile of aqueous solutions containing CremophorRH40 indicated a phase transition at around 78 +/- 2 degrees C, which is in agreement with cubic phase formation in the Winsor III region. The similarity in the interfacial elasticity values of CremophorRH40 and Span80 indicated that canola oil has a major effect on surface activity, showing the significance of vegetable oil. The highest interfacial shear elasticity and viscosity were observed when both surfactants were added to the oil phase, indicating the importance of the microstructural arrangement. CremophorRH40/Span80 complexes tended to desorb from the solution/solution interface with increasing temperature, indicating surfactant phase formation as is theoretically predicted in the Winsor III region. Together these interfacial and bulk rheology data demonstrate that one-step W/O/W emulsions form as a result of the simultaneous occurrence of phase-transition processes in the Winsor III region and explain the critical formulation and processing parameters necessary to achieve the formation of these normal W/O/W emulsions.

A HPLC method to evaluate the influence of photostabilizers on cosmetic formulations containing UV-filters and vitamins A and E

This paper reports a simple and reliable HPLC method to evaluate the influence of two currently available photostabilizers on cosmetic formulations containing combined UV-filters and vitamins A and E. Vitamins and UV-filters, widely encountered in products of daily use have to be routinely evaluated since photoinstability can lead to reductions in their efficacy and safety. UV-irradiated formulation samples were submitted to a procedure that included a reliable, precise and specific HPLC method employing a C18 column and detection at 325 and 235 nm. Methanol, isopropanol and water were the mobile phases in gradient elution. The method precision was between 0.28 and 5.07. The photostabilizers studied [diethylhexyl 2,6-naphthalate (DEHN) and benzotriazolyl dodecyl p-cresol (BTDC)], influenced the stability of octyl methoxycinnamate (OMC) associated with vitamins A and E. BTDC was considered the best photostabilizer to vitamins and OMC when the UV-filters were combined with both vitamins A and E. (C) 2010 Elsevier B.V. All rights reserved.

Influence of Phase Inversion on the Formation and Stability of One-Step Multiple Emulsions

A novel method of preparation of water-in-oil-in-micelle-containing water (W/O/W(m)) Multiple emulsions using the one-step emulsification method is reported. These multiple emulsions were normal (not temporary) and stable over a 60 day test period. Previously, reported multiple emulsion by the one-step method were abnormal systems that formed at the inversion point of simple emulsion (where there is an incompatibility in the Ostwald and Bancroft theories, and typically these are O/W/O systems). Pseudoternary phase diagrams and bidimensional process-composition (phase inversion) maps were constructed to assist in process and composition optimization. The surfactants used were PEG40 hydrogenated castor oil and sorbitan oleate, and mineral and vegetables oils were investigated. Physicochemical characterization studies showed experimentally, for the First time, the significance of the ultralow surface tension point oil multiple emulsion formation by one-step via phase inversion processes. Although the significance of ultralow surface tension has been speculated previously, to the best of our knowledge, this is the first experimental confirmation. The multiple emulsion system reported here was dependent not only upon the emulsification temperature, but also upon the component ratios, therefore both the emulsion phase inversion and the phase inversion temperature were considered to fully explain their formation. Accordingly, it is hypothesized that the formation of these normal multiple emulsions is not a result of a temporary incompatibility (at the inversion point) during simple emulsion preparation, as previously reported. Rather, these normal W/O/W(m) emulsions are a result of the simultaneous occurrence of catastrophic and transitional phase inversion processes. The formation of the primary emulsions (W/O) is in accordance with the Ostwald theory and the formation of the multiple emulsions (W/O/W(m)) is in agreement with the Bancroft theory.

Simultaneous Analysis of Tramadol, O-Desmethyltramadol, and N-Desmethyltramadol Enantiomers in Rat Plasma by High-Performance Liquid Chromatography-Tandem Mass Spectrometry: Application to Pharmacokinetics

Tramadol (T) is available as a racemic mixture of (+)-trans-T and (-)-trans-T. The main metabolic pathways are O-demethylation and N-demethylation, producing trans-O-desmethyltramadol (M1) and trans-N-desmethyltramadol (M2) enantiomers, respectively. The analgesic effect of T is related to the opioid activity of (+)-trans-T and (+)-M1 and to the monoaminergic action of (+/-)-trans-T. This is the first study using tandem mass spectrometry as a detection system for the simultaneous analysis of trans-T, M1, and M2 enantiomers. The analytes were resolved on a Chiralpak (R) AD column using hexane: ethanol (95.5:4.5, v/v) plus 0.1% diethylamine as the mobile phase. The quantitation limits were 0.5 ng/ml for trans-T and M1 and 0.1 ng/ml for M2. The method developed and validated here was applied to a pharmacokinetic study in rats. Male Wistar rats (n = 6 at each time point) received a single oral dose of 20 mg/kg racemic trans-T. Blood samples were collected up to 12 h after drug administration. The kinetic disposition of trans-T and M2 was enantioselective (AUC((+)/(-)) ratio = 4.16 and 6.36, respectively). The direction and extent of enantioselectivity in the pharmacokinetics of trans-T and M2 in rats were comparable to data previously reported for healthy volunteers, suggesting that rats are a suitable model for enantioselective studies of trans-T pharmacokinetics. Chirality 23: 287-293, 2011. (C) 2010 Wiley-Liss, Inc.

New method for quantification of Trypanosoma cruzi in animal`s tissue in the chronic phase of experimental Chagas` disease

We developed a new method for the quantification of parasites in tissue. Trypanosoma cruzi strain CL parasites were genetically engineered to express the Escherichia coli beta-galactosidase gene, lacZ and this enzyme is able to catalyze a colorimetric reaction with chlorophenol red beta-d galactopyranoside (CPRG) as the substrate. The animals were infected with clone CL Brener strain B5 of T. cruzi and treated with benznidazole in order to verify the reduction in the number of parasites in tissue study by quantifying the enzyme beta-galactosidase. The assay demonstrates a reduction in the number of parasites in the groups treated. Thus, this test can be used to test other substances with the aim of verifying the effectiveness in the chronic phase of experimental Chagas` disease.

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirao Preto, Sao Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTET (TM) QPCR SYBR (R) Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fist and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method. (C) 2009 Elsevier Ltd. All rights reserved.

Is your vision consistent? A method for checking, based on scenario concepts

Common sense tells us that the future is an essential element in any strategy. In addition, there is a good deal of literature on scenario planning, which is an important tool in considering the future in terms of strategy. However, in many organizations there is serious resistance to the development of scenarios, and they are not broadly implemented by companies. But even organizations that do not rely heavily on the development of scenarios do, in fact, construct visions to guide their strategies. But it might be asked, what happens when this vision is not consistent with the future? To address this problem, the present article proposes a method for checking the content and consistency of an organization`s vision of the future, no matter how it was conceived. The proposed method is grounded on theoretical concepts from the field of future studies, which are described in this article. This study was motivated by the search for developing new ways of improving and using scenario techniques as a method for making strategic decisions. The method was then tested on a company in the field of information technology in order to check its operational feasibility. The test showed that the proposed method is, in fact, operationally feasible and was capable of analyzing the vision of the company being studied, indicating both its shortcomings and points of inconsistency. (C) 2007 Elsevier Ltd. All rights reserved.

STRUCTURAL INTERDEPENDENCE AMONG COLOMBIAN DEPARTMENTS

This paper analyzes structural interdependence among Colombian departments. The results show that Bogota has a large influence on the other regional economies through the power of its purchases. Additionally, a center-periphery pattern emerges in the spatial concentration of the effects of the hypothetical extraction of any territory. From a policy point of view, the main findings reaffirm the role played by Bogota in the recent polarization process observed in the regional economies in Colombia. Any policy action oriented to reduce these regional disparities should take into account that, given the structural interdependence among Colombian departments, the effects of new investment in the lagged regions would flow through Bogota and the major regional economies.

Diagnosis of Organizational Soft Problems in a Peruvian Financial Institution by Systemic Thinking

Managing financial institutions in an underdeveloped economic context has become a real challenge nowadays. In order to reach the organization`s planned goals, they have to deal with structural, behavioral and informational problems. From the systemic point of view, this situation gets even worse when the company does not present organizational boundaries and a cohesive identification for their stakeholders. Thus, European countries have some special financial lines in order to help the development of micro credit in Latin communities in an attempt to help the local economy. However, institutions like Caixa dos Andes in Peru present management problems when dealing with this complexity. Based on this, how can the systemic eye help in the diagnosis of soft problems of a Peruvian financial company? This study aims to diagnose soft problems of a Peruvian financial company based on soft variables like identity, communication and autonomy and also intends to identify possible ways to redesign its basic framework. The (VSM--Viable System Model) method from Beer (1967), applied in this diagnostic study, was used in a practical way as a management tool for organizations` analysis and planning. By describing the VSM`s five systems, the creation of a systemic vision or a total vision is possible, showing the organization`s complexity from the inside. Some company`s soft problems like double control, inefficient use of physical and human resources, low information flows, slowness, etc. The VSM presented an organizational diagnosis indicating effective solutions that do integrate its five systems.

A non-invasive method for silencing gene transcription in honeybees maintained under natural conditions

in the Apis mellifera post-genomic era, RNAi protocols have been used in functional approaches. However, sample manipulation and invasive methods such as injection of double-stranded RNA (dsRNA) can compromise physiology and survival. To circumvent these problems, we developed a non-invasive method for honeybee gene knockdown, using a well-established vitellogenin RNAi system as a model. Second instar larvae received dsRNA for vitellogenin (dsVg-RNA) in their natural diet. For exogenous control, larvae received dsRNA for GFP (dsGFP-RNA). Untreated larvae formed another control group. Around 60% of the treated larvae naturally developed until adult emergence when 0.5 mu g of dsVg-RNA or dsGFP-RNA was offered while no larvae that received 3.0 mu g of dsRNA reached pupal stages. Diet dilution did not affect the removal rates. Viability depends not only on the delivered doses but also on the internal conditions of colonies. The weight of treated and untreated groups showed no statistical differences. This showed that RNAi ingestion did not elicit drastic collateral effects. Approximately 90% of vitellogenin transcripts from 7-day-old workers were silenced compared to controls. A large number of samples are handled in a relatively short time and smaller quantities of RNAi molecules are used compared to invasive methods. These advantages culminate in a versatile and a cost-effective approach. (c) 2008 Elsevier Ltd. All rights reserved.