Kinetic and chemical studies on the isomerization of monosaccharides in N-methylmorpholine-N-oxide (NMMO) under Lyocell conditions

The Lyocell process is a modern and environmentally fully compatible industrial fiber-making technology. Cellulosic pulp is dissolved without chemical derivatization in a melt of N-methylmorpholine-N-oxide monohydrate (NMMO). In the present work, the reactions of monosaccharides under Lyocell conditions were investigated in detail, using capillary zone electrophoresis as the analytical technique to clarify the composition of reaction mixtures and to follow the kinetics. Under Lyocell conditions, xylose and glucose undergo two competitive reactions: rapid conversion to nonreducing products, and complete isomerization involving the whole carbohydrate backbone, via ketose intermediates. Sugar acids are present in minor amounts only, as demonstrated by employing isotopically labeled material for NMR techniques.     

Determination of dopamine in the presence of ascorbic acid using poly(3,5-dihydroxy benzoic acid) film modified electrode

A polymerized film of 3,5-dihydroxy benzoic acid (DBA) was prepared on the surface of a glassy carbon electrode (GCE) in neutral solution by cyclic voltammetry (CV). The poly(DBA) film-coated GCE exhibited excellent electrocatalytic activity toward the oxidation of dopamine (DA). A linear range of 1.0 × 10⁻⁷ to 1.0 × 10⁻⁴ M and a detection limit of 6.0 × 10⁻⁸ M were observed in pH 7.4 phosphate buffer solutions. Moreover, the interference of ascorbic acid (AA) was effectively eliminated. This work provides a simple and easy approach to selective detection of DA in the presence of AA.     

Sensitive spectrofluorometric method for the determination of ascorbic acid in pharmaceutical nutritional supplements using acriflavine as a fluorescence reagent

An easily performed, specific, sensitive, rapid, reliable and inexpensive procedure for the spectrofluorometric quantitation of ascorbic acid was proposed using acriflavine as a fluorescence quenching reagent. The procedure was based on the determined quenching effect of ascorbic acid on the natural fluorescence signal of acriflavine and the reaction between ascorbic acid and acriflavine in Britton–Robinson buffer solution (pH 6) to produce an ion‐associated complex. The reduction in acriflavine fluorescence intensity was detected at 505 nm, while excitation occurred at 265 nm. The relationship between quenching fluorescence intensity (?F) and concentration of ascorbic acid was linear (R² = 0.9967) within the range 2–10 μg/ml and with a detection limit of 0.08 μg/ml. No significant interference was detected from other materials often found in pharmaceutical nutritional tablets. The obtained results were compared with those from high‐performance liquid chromatography and appeared in good agreement, with no important differences in precision or accuracy. The proposed spectrofluorimetric method was used to determine the amount of ascorbic acid in a number of commercial pharmaceutical nutritional supplement tablets with a 95% confidence performance.     

A rapid sensitive method for the determination of ascorbic acid in the excess of 2,6-dichlorophenolindophenol using a stopped-flow apparatus

A rapid and sensitive method termed “time difference analysis” for the determination of reduced ascorbic acid even in the presence of excess triose reductone has been developed by using a stopped-flow apparatus in excess 2,6-dichlorophenolindophenol. The lowest limit of the concentration of ascorbic acid was about 2 × 10^?7m. A single stopped-flow trace can be used for both qualitative and quantitative analysis of ascorbic acid. The contamination of triose reductone, which disturbs the analysis in the ordinary static measurement, can be safely distinguished because of the sluggishness of the reaction.     

Colorimetric method for the determination of ketoses using phenol-acetone-boric acid reagent (PABR)

A purplish pink color developed when ketose solution (100 μl) was mixed with phenolacetone-boric acid reagent (0.5 ml 5% phenol, 2% acetone, 4% boric acid) and then treated with 96% sulfuric acid (1.4 ml). The absorbance of the reaction mixture was measured at 568 nm after 60 min at 37°C. This method allowed the simple determination of 3–50 nmol of d-fructose with coefficient of variation 7.8% for 3 nmol and 2.8% for 50 nmol. Carbohydrates other then ketoses did not interfere with this reaction. The influence of various chemicals on the colorimetric reaction and applicability of the method for determination of ketoses in natural products are presented.     

Rapid validated HPTLC method for estimation of betulinic acid in Nelumbo nucifera (Nymphaeaceae) rhizome extract

Introduction – Betulinic acid (pentacyclic triterpenoid) is an important marker component present in Nelumbo nucifera Gaertn. rhizome. N. nucifera rhizome has several medicinal uses including hypoglycaemic, antidiarrhoeal, antimicrobial, diuretic, antipyretic, psychopharmacological activities. Objective – To establish a simple, sensitive, reliable, rapid and validated high‐performance thin‐layer chromatography method for estimation of betulinic acid in hydro‐alcoholic extract of N. nucifera Gaertn. rhizome. Materials and methods – The separation was carried out on a thin‐layer chromatography aluminium plate pre‐coated with silica gel 60F₂₅₄, eluted with chloroform, methanol and formic acid (49 : 1 : 1 v/v). Post chromatographic derivatisation was done with anisaldehyde–sulphuric acid reagent and densitometric scanning was performed using a Camag TLC scanner III, at 420 nm. Results – The system was found to produce a compact spot for betulinic acid (R_f = 0.30). A good linear precision relationship between the concentrations (2–10 µg) and peak areas were obtained with the correlation coefficient (r) of 0.99698. The limit of detection and limit of quantification of betulinic acid were detected to be 0.4 and 2.30 µg per spot. The percentage of recovery was found to be 98.36%. The percentage relative standard deviations of intra‐day and inter‐day precisions were 0.82–0.394 and 0.85–0.341, respectively. Conclusion – This validated HPTLC method provides a new and powerful approach to estimate betulinic acid as phytomarker in the extract. Copyright © 2010 John Wiley & Sons, Ltd.     

A highly sensitive high-performance liquid chromatography method for the estimation of ascorbic and dehydroascorbic acid in tissues, biological fluids, and foods

A highly sensitive procedure for determining ascorbic acid (AA) and dehydroascorbic acid (DHAA) by high-performance liquid chromatography with electrochemical detection in biological fluids, tissues, and foods is described. AA is separated in a C18 reverse-phase column after extraction from the sample with metaphosphoric acid. An aliquot of 20 microliter of diluted extract is injected into the column for the estimation of AA. DHAA is indirectly estimated by converting it to AA after reduction with DL-homocysteine at pH 7.0-7.2 for 30 min at 25 degrees C. After dilution, a 20-microliter aliquot is injected into the column to obtain total vitamin C (AA + DHAA). The concentration of DHAA is calculated by subtraction. AA can be reproducibly quantified at concentrations as low as 50 pg/20 microliter of sample extract. The method described here used a specially designed mobile phase, gave greater stability and a noiseless baseline, and increased substantially the sensitivity and precision. The procedure is rapid, analysis being completed within 10 min after sample preparation, and has been successfully applied to biological fluids, tissues, and foods.     

Self‐quenching in the electrochemiluminescence of Ru(bpy)32+ using ascorbic acid as co‐reactant

In this study, electrochemiluminescence (ECL) of Ru(bpy)₃²⁺ (bpy = 2,2′‐bipyridyl) using ascorbic acid (H₂A) as co‐reactant was investigated in an aqueous solution. When H₂A was co‐existent in a Ru(bpy)₃²⁺‐containing buffer solution, ECL peaks were observed at a potential corresponding to the oxidation of Ru(bpy)₃²⁺, and the intensity was proportional to H₂A concentration at lower concentration levels. The formation of the excited state *Ru(bpy)₃²⁺ was confirmed to result from the co‐reaction between Ru(bpy)₃³⁺and the intermediate of ascorbate anion radical (A⁻•), which showed the maximum ECL at pH = 8.8. It is our first finding that the ECL intensity would be quenched significantly when the concentration of H₂A was relatively higher, or upon ultrasonic irradiation. In most instances, quenching is observed with four‐fold excess of H₂A over Ru(bpy)₃²⁺. The diffusional self‐quenching scheme as well as the possible reaction pathways involved in the Ru(bpy)₃²⁺–H₂A ECL system are discussed in this study. Copyright © 2008 John Wiley & Sons, Ltd.     

Simultaneous voltammetric detection of dopamine and uric acid at their physiological level in the presence of ascorbic acid using poly(acrylic acid)-multiwalled carbon-nanotube composite-covered glassy-carbon electrode

The use of poly(acrylic acid) (PAA)-multiwalled carbon-nanotubes (MWNTs) composite-coated glassy-carbon disk electrode (GCE) (PAA-MWNTs/GCE) for the simultaneous determination of physiological level dopamine (DA) and uric acid (UA) in the presence of an excess of ascorbic acid (AA) in a pH 7.4 phosphate-buffered solution was proposed. PAA-MWNTs composite was prepared by mixing of MWNTs powder into 1 mg/ml PAA aqueous solution under sonication. GCE surface was modified with PAA-MWNTs film by casting. AA demonstrates no voltammetric peak at PAA-MWNTs/GCE. The PAA-MWNTs composite is of a high surface area and of affinity for DA and UA adsorption. DA exhibits greatly improved electron-transfer rate and is electro-catalyzed at PAA-MWNTs/GCE. Moreover, the electro-catalytic oxidation of UA at PAA-MWNTs/GCE is observed, which makes it possible to detect lower level UA. Therefore, the enhanced electrocatalytic currents for DA and UA were observed. The anodic peak currents at approximately 0.18 V and 0.35 V increase with the increasing concentrations of DA and UA, respectively, which correspond to the voltammetric peaks of DA and UA, respectively. The linear ranges are 40 nM to 3 microM DA and 0.3 microM to 10 microM UA in the presence of 0.3 mM AA. The lowest detection limits (S/N=3) were 20 nM DA and 110 nM UA.     

Nanosilica and jasmonic acid as alternative methods for control Tuta absoluta (Meyrick) in tomato crop under field conditions

Recently, Tuta absoluta became one of the major pests that attack commercial tomato globally. Field test was done to evaluate the effect of different concentrations of nanosilica (NS) and jasmonic acid (JA), and compared them with indoxacrb (recommended insecticide) on reduction of damage rate caused by T. absoluta larvae under field conditions. Nanosilica (600 ppm) and indoxacrb (0.25 cm³/L) had the highest efficiency to reduce the rate of mines in the leaves. Jasmonic acid at rate 1.141 μM/plant showed a good reduction of number of fruits damaged. Nanosilica with 600 ppm concentration and Jasmonic acid at rate 1.141 μM/plant is used to control T. absoluta in the field.     

A method for the quantitation of hyaluronan (hyaluronic acid) in biological fluids using a labeled avidin-biotin technique.

An improved method for the detection and quantitation of hyaluronan (hyaluronic acid) (HA) in biological fluids is described. The principle on which the method is based is that HA binds strongly to a biotinylated HA-binding protein (B-HABP) which was prepared from cartilage proteoglycans. HA was immobilized on polyvinyl chloride plates which had been precoated with poly-L-lysine. The unknown sample or HA standards together with excess B-HABP are then added. The B-HABP that binds to the immobilized HA is then incubated with the enzyme-conjugated avidin (e.g., alkaline phosphatase), and the color which develops on addition of enzyme substrate (e.g., p-nitrophenyl phosphate) is determined by light absorption using a microtitration plate reader. The assay is not only convenient and reliable but is capable of measuring HA in solution at the picogram level. The assay was used to determine HA levels in human sera and synovial fluid taken from volunteers and patients with rheumatoid arthritis and osteoarthritis.     

Evaluation of the bioremoval of Cr(VI) and TOC in biofilters under continuous operation using response surface methodology

In the present study, the bioremoval of Cr(VI) and the removal of total organic carbon (TOC) were achieved with a system composed by an anaerobic filter and a submerged biofilter with intermittent aeration using a mixed culture of microorganisms originating from contaminated sludge. In the aforementioned biofilters, the concentrations of chromium, carbon, and nitrogen were optimized according to response surface methodology. The initial concentration of Cr(VI) was 137.35 mg l⁻¹, and a bioremoval of 85.23% was attained. The optimal conditions for the removal of TOC were 4 to 8 g l⁻¹ of sodium acetate, >0.8 g l⁻¹ of ammonium chloride and 60 to 100 mg l⁻¹ of Cr(VI). The results revealed that ammonium chloride had the strongest effect on the TOC removal, and 120 mg l⁻¹ of Cr(VI) could be removed after 156 h of operation. Moreover, 100% of the Cr(VI) and the total chromium content of the aerobic reactor output were removed, and TOC removals of 80 and 87% were attained after operating the anaerobic and aerobic reactors for 130 and 142 h, respectively. The concentrations of cells in both reactors remained nearly constant over time. The residence time distribution was obtained to evaluate the flow through the bioreactors.     

Detection of mRNA degradation intermediates in tissues using the 3'-end poly(A)-tailing polymerase chain reaction method

It has become increasingly clear that mRNA stability is an important determinant of mRNA abundance in virtually all organisms. Although our understanding of prokaryotic lower eukaryotic mRNA stability mechanisms has progressed considerably, little is known about mammalian mRNA stability mechanisms, particularly at the tissue and animal levels. This is due largely to the lack of suitable methods to approach the problem. In this study, we have developed and refined the 3'-end poly(A)-tailing polymerase chain reaction (PCR) method to detect degradation intermediates in vivo. Using an in vitro transcribed RNA as a template, we found that the method could be used to detect a homogeneous pool of RNA down to 0.1 ng. The addition of 10 microg of total RNA from tissues decreased the sensitivity limit to 4 ng. Detection limits of the technique were determined precisely by varying the concentrations of in vitro transcribed RNA in a constant amount of total RNA and varying the concentration of total RNA while maintaining a constant amount of in vitro transcribed RNA. Our overall results showed that the poly(A)-tailing PCR method could be used to detect specific RNA species of approximately 1000 nt in a pool of heterogeneous RNA in the range of 1 in 2500 to 1 in 10,000. To our knowledge, this is the most sensitive method to date for identifying mRNA degradation intermediates. Employing sense strand gene-specific primers in this method, we have discovered the class II and class III P-glycoprotein (Pgp) mRNA degradation intermediates in normal rat tissues. This method should serve as an additional tool to help us understand mRNA decay mechanisms in tissues and at animal levels.     

A colorimetric method for the determination of different functional flavonoids using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and peroxidase

Abstract

In many occasions it is necessary to use fast and simple methods, different to the chromatographic techniques, for the quantification of biomolecules such as flavonoids. Also, the flavonoid levels in some foodstuffs can be influenced by industrial extraction processes such as pressing and squeezing, resulting in modification of their functional value. For this purpose, we have developed a rapid method to analyze flavonoids, based on a coupling reaction between ABTS and flavonoid mediated by peroxidase. The present method can be used to detect and measure flavonoids with hydroxyl moieties on A- or B-rings, not adjacent to methoxy or oxo substitutions. The visible spectrum of the ABTS-flavonoid complex, the calibration curve (within the range 5-50?μM) and the molar absorption coefficients for isosakuranetin, isonaringin, rhoifolin, hyperoside, rutin, hesperetin, quercetin, kaempherol and naringenin are given. The method has been applied to complex culture media and is sensitive, accurate, quick and easy to apply. This method can be used in laboratories that do not have sophisticated and expensive techniques such as liquid chromatography and also as a quick, simple and inexpensive technique for student practice laboratories.     

Direct estimation of the oxygen requirements of Achromobacter xylosoxidans for aerobic degradation of monoaromatic hydrocarbons (BTEX) in a bioscrubber

The O₂ requirements for biomass production and supplying maintenance energy demands during the degradation of both benzene and ethylbenzene by Achromobacter xylosoxidans Y234 were measured using a newly proposed technique involving a bioscrubber. Using this approach, relevant microbial parameter estimates were directly and simultaneously obtained via linear regression of pseudo steady-state data. For benzene and ethylbenzene, the biomass yield on O₂, , was estimated on a cell dry weight (CDW) basis as 1.96 ±.25 mg CDW mgO₂ and 0.98 ±.17 mg CDW mgO₂, while the specific rate of O₂ consumption for maintenance, , was estimated as 0.041 ±.008 mgO₂ mg CDW^h and 0.053 ±.022 mgO₂ mg CDW^h, respectively.