Ultra-Fast Gradient LC Method for Omeprazole Analysis Using a Monolithic Column: Assay Development, Validation, and Application to the Quality Control of Omeprazole Enteric-Coated Pellets

A method was optimized for the analysis of omeprazole (OMZ) by ultra-high speed LC with diode array detection using a monolithic Chromolith Fast Gradient RP 18 endcapped column (50 x 2.0 mm id). The analyses were performed at 30 degrees C using a mobile phase consisting of 0.15% (v/v) trifluoroacetic acid (TFA) in water (solvent A) and 0.15% (v/v) TFA in acetonitrile (solvent B) under a linear gradient of 5 to 90% B in 1 min at a flow rate of 1.0 mL/min and detection at 220 nm. Under these conditions, OMZ retention time was approximately 0.74 min. Validation parameters, such as selectivity, linearity, precision, accuracy, and robustness, showed results within the acceptable criteria. The method developed was successfully applied to OMZ enteric-coated pellets, showing that this assay can be used in the pharmaceutical industry for routine QC analysis. Moreover, the analytical conditions established allow for the simultaneous analysis of OMZ metabolites, 5-hydroxyomeprazole and omeprazole sulfone, in the same run, showing that this method can be extended to other matrixes with adequate procedures for sample preparation.

Box-Behnken design for the optimization of an enantioselective method for the simultaneous analysis of propranolol and 4-hydroxypropranolol by CE

An experimental design optimization (Box-Behnken design, BBD) was used to develop a CE method for the simultaneous resolution of propranolol (Prop) and 4-hydroxypropranolol enantiomers and acetaminophen (internal standard). The method was optimized using an uncoated fused silica capillary, carboxymethyl-beta-cyclodextrin (CM-beta-CD) as chiral selector and triethylamine/phosphoric acid buffer in alkaline conditions. A BBD for four factors was selected to observe the effects of buffer electrolyte concentration, pH, CM-beta-CD concentration and voltage on separation responses. Each factor was studied at three levels: high, central and low, and three center points were added. The buffer electrolyte concentration ranged from 25 to 75 mM, the pH ranged from 8 to 9, the CM-beta-CD concentration ranged from 3.5 to 4.5%w/v, and the applied run voltage ranged from 14 to 20 W. The responses evaluated were resolution and migration time for the last peak. The obtained responses were processed by Minitab (R) to evaluate the significance of the effects and to find the optimum analysis conditions. The best results were obtained using 4%w/v CM-beta-CD in 25 mM triethylamine/H(3)PO(4) buffer at pH 9 as running electrolyte and 17 kV of voltage. Resolution values of 1.98 and 1.95 were obtained for Prop and 4-hydroxypropranolol enantiomers, respectively. The total analysis time was around of 15 min. The BBD showed to be an adequate design for the development of a CE method, resulting in a rapid and efficient optimization of the pH and concentration of the buffer, cyclodextrin concentration and applied voltage.

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.

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.

Methylmercury and inorganic mercury determination in blood by using liquid chromatography with inductively coupled plasma mass spectrometry and a fast sample preparation procedure

Despite the necessity to differentiate chemical species of mercury in clinical specimens, there area limited number of methods for this purpose. Then, this paper describes a simple method for the determination of methylmercury and inorganic mercury in blood by using liquid chromatography with inductively coupled mass spectrometry (LC-ICP-MS) and a fast sample preparation procedure. Prior to analysis, blood (250 mu L) is accurately weighed into 15-mL conical tubes. Then, an extractant solution containing mercaptoethanol, L-cysteine and HCI was added to the samples following sonication for 15 min. Quantitative mercury extraction was achieved with the proposed procedure. Separation of mercury species was accomplished in less than 5 min on a C18 reverse-phase column with a mobile phase containing 0.05% (v/v) mercaptoethanol, 0.4% (m/v) L-cysteine, 0.06 mol L(-1) ammonium acetate and 5% (v/v) methanol. The method detection limits were found to be 0.25 mu g L(-1) and 0.1 mu Lg L(-1) for inorganic mercury and methylmercury, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). The proposed method was also applied to the speciation of mercury in blood samples collected from fish-eating communities and from rats exposed to thimerosal. With the proposed method there is a considerable reduction of the time of sample preparation prior to speciation of Hg by LC-ICP-MS. Finally, after the application of the proposed method, we demonstrated an interesting in vivo ethylmercury conversion to inorganic mercury. (C) 2009 Elsevier B.V. All rights reserved.

Determination of total and inorganic mercury in whole blood by cold vapor inductively coupled plasma mass spectrometry (CV ICP-MS) with alkaline sample preparation

A simple method with a fast sample preparation procedure for total and inorganic mercury determinations in blood samples is proposed based on flow injection cold vapor inductively coupled plasma mass spectrometry (FI-CVICP-MS). Aliquots of whole blood (500 mL) are diluted 1 + 1 v/v with 10.0% v/v tetramethylammonium hydroxide (TMAH) solution, incubated for 3 h at room temperature and then further diluted 1 + 4 v/v with 2.0% v/v HCl. The inorganic Hg was released by online addition of L-cysteine and then reduced to elemental Hg by SnCl(2). On the other hand, total mercury was determined by on-line addition of KMnO(4) and then reduced to elemental Hg by NaBH(4). Samples were calibrated against matrix-matching. The method detection limit was found to be 0.80 mu g L(-1) and 0.08 mu g L(-1) for inorganic and total mercury, respectively. Sample throughput is 20 samples h(-1). The method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). For additional validation purposes, human whole blood samples were analyzed by the proposed method and by an established CV AAS method, with no statistical difference between the two techniques at 95% confidence level on applying the t-test.

Mercury speciation in whole blood by gas chromatography coupled to ICP-MS with a fast microwave-assisted sample preparation procedure

A simple and fast method is described for simultaneous determination of methylmercury (MeHg), ethylmercury (Et-Hg) and inorganic mercury (Ino-Hg) in blood samples by using capillary gas chromatography-inductively coupled plasma mass spectrometry (GC-ICP-MS) after derivatization and alkaline digestion. Closed-vessel microwave assisted digestion conditions with tetramethylammonium hydroxide (TMAH) have been optimized. Derivatization by using ethylation and propylation procedures have also been evaluated and compared. The absolute detection limits (using a 1 mu L injection) obtained by GC-ICP-MS with ethylation were 40 fg for MeHg and Ino-Hg, respectively, and with propylation were 50, 20 and 50 fg for MeHg, Et-Hg and Ino-Hg, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). Additional validation is provided based on the comparison of results obtained for mercury speciation in blood samples with the proposed procedure and with a previously reported LC-ICP-MS method. With the new proposed procedure no tedious clean-up steps are required and a considerable improvement of the time of analysis was achieved compared to other methods using GC separation.

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.

Chlorination treatment of aqueous samples reduces, but does not eliminate, the mutagenic effect of the azo dyes Disperse Red 1, Disperse Red 13 and Disperse Orange 1

The treatment of textile effluents by the conventional method based on activated sludge followed by a chlorination step is not usually an effective method to remove azo dyes, and can generate products more mutagenic than the untreated dyes. The present work evaluated the efficiency of conventional chlorination to remove the genotoxicity/mutagenicity of the azo dyes Disperse Red 1, Disperse Orange 1, and Disperse Red 13 from aqueous solutions. The comet and micronucleus assays with HepG2 cells and the Salmonella mutagenicity assay were used. The degradation of the dye molecules after the same treatment was also evaluated, using ultraviolet and visible absorption spectrum measurements (UV-vis), high performance liquid chromatography coupled to a diode-array detector (HPLC-DAD), and total organic carbon removal (TOC) analysis. The comet assay showed that the three dyes studied induced damage in the DNA of the HepG2 cells in a dose-dependent manner. After chlorination, these dyes remained genotoxic, although with a lower damage index (DI). The micronucleus test showed that the mutagenic activity of the dyes investigated was completely removed by chlorination, under the conditions tested. The Salmonella assay showed that chlorination reduced the mutagenicity of all three dyes in strain YG1041, but increased the mutagenicity of Disperse Red 1 and Disperse Orange 1 in strain TA98. With respect to chemical analysis, all the solutions showed rapid discoloration and a reduction in the absorbance bands characteristic of the chromophore group of each dye. However, the TOC was not completely removed, showing that chlorination of these dyes is not efficient in mineralizing them. It was concluded that conventional chlorination should be used with caution for the treatment of aqueous samples contaminated with azo dyes. (C) 2010 Elsevier B.V. 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.