Effect of different compounds on the induction of laccase production by Agaricus blazei.

Laccases are polyphenol oxidases produced by many fungi and have many applications in textile, food and beverage, and pulp and paper industries. Laccase production can be induced using aromatic or phenolic compounds that mostly affect the transcription of laccase-encoding genes. In this study, we analyzed laccase and biomass production by Agaricus blazei in the presence of different concentrations of nitrogen, copper, and inducers such as pyrogallol, veratryl alcohol, xylidine, vanillin, guaiacol, and ethanol. Laccase production by A. blazei U2-4 reached 43.8 U/mL in the presence of 2.8 g/L nitrogen and 150 µM copper. However, addition of copper to the cultivation medium decreased biomass production. Different compounds differentially induced laccase production by A. blazei. Moreover, different concentrations of these inducers exerted different effects on laccase activity. Ethanol (1.0 mM), guaiacol (0.5 mM), and vanillin (0.5 mM) were the best inducers and increased laccase activity by 120% (A. blazei U2-2), 30% (A. blazei U2-3), and 9% (A. blazei U2-4), respectively. In contrast, pyrogallol and xylidine decreased laccase activity but increased biomass production.

Factorial design of electrolyte systems for the separation of fatty acids by capillary electrophoresis.

In this work, a capillary zone electrophoretic methodology using UV indirect detection (224 nm) for the analysis of fatty acids (FAs) in saponified oils is proposed. The electrolyte consisted of a 5 mmol l(-1) phosphate buffer, pH 7. containing 4 mmol l(-1) sodium dodecylbenzenesulfonate (SDBS) as chromophore, 4 mmol l(-1) dimethyl-beta-cyclodextrin and 45% acetonitrile (ACN). The composition of the electrolyte was optimized by a 2(3) factorial design with triplicate at the central point. The design established practical concentration boundaries for SDBS and ACN. In a defined concentration range of 2-4 l(-1), SDBS can certainly be used as a chromophore for indirect detection without imparting excessive baseline noise. For ACN, a suitable interval of 45-55% was found to enhance FAs solubilization without overflowing the system with bubble formation and current interruption. Additionally, the design revealed the importance of dimethyl-beta-cyclodextrin in the resolution of difficult pairs and its function as a solubilizing agent for long chain FAs. At the optimized conditions, nine FAs from C10 to C20, including mono- di- and tri-unsaturated C18 fatty acids were baseline separated in less than 10 min. The proposed method was applied to the separation of FAs in edible oils and polyunsaturated fatty acid enriched margarine. Additionally, spectral monitoring at 206 nm was used to confirm peak identity in the samples.

Use of capillary electrophoresis in the characterization of sulfonated metallophthalocyanines: a comparative evaluation of purification procedures following synthesis by the condensation method.

Capillary electrophoresis has been presented as a resourceful technique for monitoring the purity of synthetic metallophthalocyanines and evaluating comparatively purification procedures. In this study, the tetrasulfonated cobalt(II) phthalocyanine was synthesized by the condensation method and submitted to several purification procedures such as continuous Soxhlet extraction with ethanol and dioxane, percolation through a gel-filled chromatographic column, and salting-out precipitation in acid medium followed by washing with alkaline solution. The efficiency of each procedure, or combined procedures, was then evaluated by capillary electrophoresis in citrate buffer solution under constant voltage conditions and direct UV-VIS detection at 630 nm and 260 nm. The inorganic anion and cation composition of the purified fractions was also investigated by indirect UV analysis in chromate/cetyltrimethylammonium bromide (CTAB) and imidazole/hydroxyisobutyric acid (HIBA) electrolyte systems, at 254 nm and 214 nm, respectively. The electropherograms obtained at 630 nm showed a high-intensity peak surrounded by satellite peaks of small intensity with migration time of approximately 5-6 min. This profile is indicative of the formation of positional isomers under the synthesis conditions. The electropherograms obtained at 260 nm were useful to monitor contaminants, such as reaction by-products or unreacted starting materials. The salting-out precipitation of the crude product led to a good-quality product with 80% purity (elemental analysis based on nitrogen content), which is comparable to the purity of commercially available tetrasulfonated derivatives. Both UV and infrared (IR) spectra of the purified product resemble that of standards. The capillary electrophoresis ion analysis of the treated product revealed the presence of sodium carbonate as the major contaminant. The combined treatment of salting-out precipitation followed by percolation in a Sephadex column (Pharmacia, São Paulo, Brazil) was successful for the removal of carbonate ion. However, the percolation procedure seemed to cause minor demetallation of the macrocycle ring, as confirmed by the presence of free cobalt(II) in the electropherogram of the column eluted fraction.

Method development strategy for the analysis of anionic solutes by capillary electrophoresis.

A systematic approach to the development of methodology for the analysis of anionic solutes by capillary electrophoresis (CE) has been implemented. The strategy allows a rapid optimization of the experimental conditions and is based on the following steps: 1) A near optimum pH is chosen by inspection of the effective mobility versus pH curves of the sample components. The optimum pH lies in a region where the differences in the mobilities of all solutes are maximal. 2) An electrolyte system is selected by comparing the effective mobility of the electrolyte with the effective mobility of the primary components of the studied mixture. The appropriate electrolyte has a mobility as similar as possible to the mobility of the major components of the sample. 3) An initial experiment is run with the selected electrolyte system adjusted to the chosen pH and input values for other instrumental variables and electrolyte characteristics. 4) Depending on the degree of resolution obtained in the initial run, two important variables, the applied voltage and the electrolyte concentration, are then tuned in the proper direction, favoring the achievement of the highest resolution at the lowest possible analysis time. The proposed method development strategy was validated experimentally with a mixture of short-chained carboxylic acids and inorganic anions.

Capillary electrophoresis determination of urinary muconic acid as a biological marker for benzene in cigarette smoke.

trans,trans-Muconic acid (MA), a benzene metabolite in urine, has currently been indicated as a biological marker for benzene in cigarette smoke. The available methodologies for MA present a few shortcomings, such as lack of specificity and labor-intensive sample pretreatment. In this work, a capillary electrophoresis method for separation, identification, and quantification of urinary muconic acid has been implemented. The electrolyte consisted of a 60-mM phosphate buffer solution (pH 7), containing 0.1 mM cetyltrimethylammonium bromide (CTAB) as an electroosmotic flow modifier. Urine samples from nonsmokers and smokers were filtered through a 0.22-micron membrane prior to injection in a 75 microns i.d. x 80 cm capillary. The analysis was conducted under constant voltage conditions of -30 kV and direct UV detection at 262 nm. The detection capability of the electrophoresis system was enhanced by employing a high-sensitivity optical cell, positioned at 60 cm from the injection port. The CE methodology presented an overall analysis time of less than 10 min, with 5 min spent for capillary conditioning and approximately 5 min for run completion. The method was found to be sensitive for the determination of MA down to 25 micrograms/L, with a percentage recovery of 100 +/- 8%, and suitable for discriminating urinary MA from nonsmokers and smokers.

Comparative evaluation of capillary electrophoresis and high-performance liquid chromatography for the separation of cis-cis, cis-trans, and trans-trans isomers of atracurium besylate.

Atracurium besylate is a highly selective nondepolarizing neuromuscular blocking agent routinely used during anesthetic procedures. The commercial presentation of this drug is a mixture of positional isomers, cis-cis, cis-trans, and trans-trans. Reversed-phase high-performance liquid chromatography has been the technique of choice for the analysis of atracurium besylate formulations at the quality control laboratory of Núcleo de Desenvolvimento Cristália (São Paulo, Brazil), a local pharmaceutical company. HPLC analysis is usually conducted under gradient elution using acetonitrile/0.1 M phosphate buffer eluent mixture as mobile phase and an octadecyl silica (ODS)-packed column. The complete elution of the three isomers takes about 1 hr. In this work, an alternative capillary electrophoresis methodology was developed. The complete resolution of all three isomers was accomplished in about 13 min (+20 kV/72 cm, 211 nm direct detection) using a 60-mM phosphate buffer solution (pH 4) containing 20 mM beta-cyclodextrin and 4 M urea. The isomer ratio was found to be 59.1% cis-cis, 35.9% cis-trans, and 5.02% trans-trans (expected ratio: 59:35:6). Laudanosine, a major metabolite of atracurium besylate, was identified in two commercially available formulations, Tracur (Núcleo de Desenvolvimento Cristália) and Tracrium (Glaxo Wellcome, S.A., Rio de Janeiro, Brazil). Its concentration increases considerably during storage of the product, even if the product is stored at low temperatures.

Method validation for diclofenac sodium in pharmaceuticals by capillary electrophoresis.

A capillary electrophoresis method for the determination of diclofenac sodium in a commercial and simulated tablet formation has been described. The aim of this research was to develop a rapid, simple, efficient, and economical method to determine diclofenac sodium in pharmaceutials. The analysis was carried out in a bare silica capillary (75 pm i.d.) with a total length of 50 cm (28 cm to the detector) with a buffer solution containing 20 mM sodium tetraborate, pH 9.23. The applied voltage was 20 kV. Detection was achieved by ultraviolet absorption at 276 nm. Acetaminophen was used as an internal standard. The calibration curve was linear to the concentration range from 40.0 to 120.0 microg/mL with a correlation coefficient of 0.9992. The percentage recovery was found to be 103.12 +/- 0.91. The results showed that the method allows the determination of diclofenac sodium in commercially available pharmaceutical preparations.